# Patent

## Title
Piperidine derivatives for treatment of Alzheimer's disease

## Abstract

Compounds of formula (I) modulate the activity of gamma secretase and hence find use in treatment or prevention of Alzheimer's disease and related conditions.

## Background
RELATED APPLICATION DATA
This is a National filing under 35 U.S.C. 371 of PCT/GB2007/050176, filed Apr. 2, 2007, which claims priority under 35 U.S.C. 119(a) and 365(b) to GB0606985.0, filed Apr. 7, 2006.
This invention relates to compounds for use in therapeutic treatment of the human body. In particular, it provides carboxy-functional 1,2-disubstituted piperidines and related compounds useful for treating diseases associated with the deposition of β-amyloid peptide in the brain, such as Alzheimer's disease, or of preventing or delaying the onset of dementia associated with such diseases.
Alzheimer's disease (AD) is the most prevalent form of dementia. Its diagnosis is described in the Diagnostic and Statistical Manual of Mental Disorders, 4th ed., published by the American Psychiatric Association (DSM-IV). It is a neurodegenerative disorder, clinically characterized by progressive loss of memory and general cognitive function, and pathologically characterized by the deposition of extracellular proteinaceous plaques in the cortical and associative brain regions of sufferers. These plaques mainly comprise fibrillar aggregates of β-amyloid peptide (Aβ). Aβ is formed from amyloid precursor protein (APP) via separate intracellular proteolytic events involving the enzymes β-secretase and γ-secretase. Variability in the site of the proteolysis mediated by γ-secretase results in Aβ of varying chain length, e.g. Aβ(1-38), Aβ(1-40) and Aβ(1-42). N-terminal truncations such as Aβ(4-42) are also found in the brain, possibly as a result of variability in the site of proteolysis mediated by β-secretase. For the sake of convenience, expressions such as “Aβ(1-40)” and “Aβ(1-42)” as used herein are inclusive of such N-terminal truncated variants. After secretion into the extracellular medium, Aβ forms initially-soluble aggregates which are widely believed to be the key neurotoxic agents in AD (see Gong et al, PNAS, 100 (2003), 10417-22), and which ultimately result in the insoluble deposits and dense neuritic plaques which are the pathological characteristics of AD.
Other dementing conditions associated with deposition of Aβ in the brain include cerebral amyloid angiopathy, hereditary cerebral haemorrhage with amyloidosis, Dutch-type (HCHWA-D), multi-infarct dementia, dementia pugilistica and Down syndrome.
Various interventions in the plaque-forming process have been proposed as therapeutic treatments for AD (see, for example, Hardy and Selkoe, Science, 297 (2002), 353-6). One such method of treatment that has been proposed is that of blocking or attenuating the production of Aβ for example by inhibition of β- or γ-secretase. It has also been reported that inhibition of glycogen synthase kinase-3 (GSK-3), in particular inhibition of GSK-3α, can block the production of Aβ (see Phiel et al, Nature, 423 (2003), 435-9).
Other proposed methods of treatment include administering a compound which blocks the aggregation of Aβ, and administering an antibody which selectively binds to Aβ.
Another proposed treatment is that of modulation of the action of γ-secretase so as to selectively attenuate the production of Aβ(1-42). This results in preferential secretion of the shorter chain isoforms of Aβ, which are believed to have a reduced propensity for self-aggregation and plaque formation, and hence are more easily cleared from the brain, and/or are less neurotoxic. Compounds showing this effect include certain non-steroidal antiinflammatory drugs (NSAIDs) and their analogues (see WO 01/78721 and US 2002/0128319 and Weggen et al Nature, 414 (2001) 212-16; Morihara et al, J. Neurochem., 83 (2002), 1009-12; and Takahashi et al, J. Biol. Chem., 278 (2003), 18644-70). Compounds which modulate the activity of PPAR(X and/or PPARδ are also reported to have the effect of lowering Aβ(1-42) (WO 02/100836). NSAID derivatives capable of releasing nitric oxide have been reported to show improved anti-neuroinflammatory effects and/or to reduce intracerebral Aβ deposition in animal models (WO 02/092072; Jantzen et al, J. Neuroscience, 22 (2002), 226-54). US 2002/0015941 teaches that agents which potentiate capacitative calcium entry activity can lower Aβ(1-42).
It has now been found that certain carboxy-functional 1,2-disubstituted piperidines and related compounds have the desirable property of selectively inhibiting production of Aβ(1-42).
According to the present invention there is provided a compound of formula I:




or a pharmaceutically acceptable salt or hydrate thereof, wherein:

p is 0 or 1;
q is 0, 1, 2 or 3;
V represents a bond or a carbon atom whose remaining valencies are satisfied via bonding to H, R2 or X—Z or to any combination thereof,
W represents a nitrogen atom or a carbon atom whose remaining valencies are satisfied via bonding to H, R2 or X—Z or to any combination thereof, provided that when W represents a nitrogen atom, V represents a carbon atom and the moiety X—Z is attached to W;
X represents a bond or C(R1)2 or CH2C(R1)2, provided that when W represents N, X does not represent a bond;
Y represents a bond or CH2 or CH2CH2;
Z represents CO2H or a tetrazole ring;
each R1 independently represents H or a non-aromatic hydrocarbon group of up to 6 carbon atoms; or the two R1 groups complete a C3-6alicyclic group;
R2 represents a non-aromatic hydrocarbon group of up to 6 carbon atoms;
R3 and R4 each represents H, or when V and W each represents a carbon atom, R3 and R4 may together represent a CH2CH2 bridge;
each R5 independently represents halogen, C1-6alkyl bearing 0-3 fluorine substituents, C1-6alkoxy bearing 0-3 fluorine substituents, or C2-6alkenyl; and
R6 and R7 independently represent hydrocarbon groups each containing up to 10 carbon atoms and optionally bearing a substituent selected from halogen, perfluoroC1-4alkyl, OH, C1-4alkoxy and OCF3.
Where a variable occurs more than once in formula I, the identity taken by said variable at any particular occurrence is independent of the identity taken at any other occurrence.
As used herein, the expression “hydrocarbon group” refers to groups consisting solely of carbon and hydrogen atoms. Unless indicated otherwise, such groups may comprise linear, branched or cyclic structures, singly or in any combination consistent with the indicated maximum number of carbon atoms, and may be saturated or unsaturated, and may include aromatic structures.
As used herein, the expression “C1-xalkyl” where x is an integer greater than 1 refers to straight-chained and branched alkyl groups wherein the number of constituent carbon atoms is in the range 1 to x. Particular alkyl groups are methyl, ethyl, n-propyl, isopropyl and t-butyl. Derived expressions such as “C2-6alkenyl”, “hydroxyC1-6alkyl”, “heteroarylC1-6alkyl”, “C2-6alkynyl” and “C1-6alkoxy” are to be construed in an analogous manner.
The expression “perfluoroC1-4alkyl” refers to linear or branched alkyl groups of up to 4 carbon atoms in which all the hydrogen atoms are replaced by fluorine atoms.
The expression “C3-6alicyclic” refers to cyclic non-aromatic hydrocarbon groups containing from 3 to 6 ring carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentenyl, cyclopentyl and cyclohexyl.
The term “halogen” as used herein includes fluorine, chlorine, bromine and iodine, of which fluorine and chlorine are preferred unless otherwise indicated.
For use in medicine, the compounds of formula I may be in the form of pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds of formula I or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methanesulphonic acid, benzenesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Alternatively, a pharmaceutically acceptable salt may be formed by neutralisation of the carboxylic acid group with a suitable base. Examples of pharmaceutically acceptable salts thus formed include alkali metal salts such as sodium or potassium salts; ammonium salts; alkaline earth metal salts such as calcium or magnesium salts; and salts formed with suitable organic bases, such as amine salts (including pyridinium salts) and quaternary ammonium salts.
It is to be understood that all the isomeric forms encompassed by formula I, both optical and geometrical, fall within the scope of the invention, singly or as mixtures in any proportion. Thus the moieties:




may be in a cis- or trans-configuration with respect to the ring completed by V—W. Furthermore, a given compound in the cis- or trans-configuration has two enantiomeric forms, both of which are within the scope of the invention, whether as single homochiral compounds or as racemic mixtures in any proportion. For the avoidance of any doubt, structural formulae such as (A) and (B):





as used herein shall be taken to be definitive of the relative configurations of the carbon atoms marked with asterisks, but not their absolute configurations, unless expressly stated otherwise.

In formula I, V represents a bond or a carbon atom and W represents a nitrogen atom or a carbon atom, with the proviso that when W represents a nitrogen atom, V cannot be a bond and the moiety X—Z must be attached to W. Formula I therefore encompasses pyrrolidine, piperidine and piperazine derivatives, depending on the identities of V and W. In preferred embodiments, V represents a carbon atom, and hence completes a piperidine or piperazine ring. Most preferably, V and W both represent carbon atoms, and hence complete a piperidine ring. When one or both of V and W represents a carbon atom, the remaining valencies of said carbon atom(s) are satisfied via bonding to H, R2 or X—Z, or to any combination thereof.
The moiety X—Z may be attached at any available ring position, including ring positions represented by V and W, but when W represents a nitrogen atom, X—Z must be attached to W. Preferably, X—Z is not attached at either of the positions adjacent to the nitrogen atom which is bonded to the moiety —CHR6R7. Thus, in the case of the preferred piperidine rings, X—Z is typically attached at the 3-, 4- or 5-position, preferably the 3- or 4-position, and most preferably at the 4-position. For the avoidance of doubt, the nitrogen atom of the piperidine ring shall be taken as the 1-position, and the carbon atom bonded to Y and R4 as the 2-position.
Z represents CO2H or a tetrazole ring, in particular, Z represents CO2H or 1,2,3,4-tetrazol-5-yl, but preferably represents CO2H.
The group X represents a bond, C(R1)2 or CH2C(R1)2, where each R1 independently represents H or a non-aromatic hydrocarbon group of up to 6 carbon atoms; or the two R1 groups complete a C3-6alicyclic group (such as cyclopropyl, cyclobutyl, cyclopentenyl or cyclopentyl). Preferably, one R1 group is H and the other is H or C1-6alkyl such as methyl, ethyl, propyl or butyl. Particular identities for X include a bond, CH2, CH(CH3), CH2CH2, cyclopentan-1,1-diyl and cyclopent-3-en-1,1-diyl, with the proviso that when W represents a nitrogen atom, X cannot be a bond. Preferably, X represents a bond or CH2, and most preferably X represents CH2.
Y represents a bond, CH2 or CH2CH2, preferably a bond or CH2, and most preferably a bond.
The group R2 (when present) may be attached at any available position on the ring, including a carbon atom represented by V or W and including a carbon atom to which the moiety X—Z is attached. In one particular embodiment, p is 0 and R2 is absent. In another particular embodiment, p is 1, V and W complete a piperidine ring and R2 and the moiety X—Z are both attached at the 4-position thereof. In another particular embodiment, p is 1, V and W complete a piperidine ring, R2 is attached at the 3-position and the moiety X—Z is attached at the 4-position. In another particular embodiment, p is 1, R3 is H, V and W complete a piperidine ring, R2 is attached at the 6-position and the moiety X—Z is attached at the 4-position. Typical identities for R2 include C1-6alkyl, such as methyl, ethyl or n-propyl, and C2-6alkenyl, such as allyl.
R3 and R4 each represent H, or when V and W each represents a carbon atom, R3 and R4 may together represent —CH2CH2—, thereby completing a bridged bicyclic structure. When R3 and R4 together represent —CH2CH2—, p is preferably 0, and Y is preferably a bond. In a particular embodiment, R3 and R4 both represent H.
In formula I, q is preferably 1 or 2, most preferably 1. Each R5 independently represents halogen (especially F), C1-6alkyl bearing 0-3 fluorine substituents, C1-6alkoxy bearing 0-3 fluorine substituents or C2-6alkenyl. When one R5 is present, it is very suitably (but not necessarily) attached in the 4-position. Typical identities for (R5)q include 2-CF3, 3-CF3, 4-CF3, 2,4-di(CF3), 2-F-4-CF3, 4-OCF3, 4-allyl, 4-n-propyl, 4-isopropyl and 4-tert-butyl. In one embodiment, (R5)q represents 4-CF3 or 4-n-propyl, in particular 4-CF3.
R6 and R7 independently represent hydrocarbon groups containing up to 10 carbon atoms optionally bearing a substituent as defined previously. Said hydrocarbon groups may be fully saturated or may comprise one or more double or triple bonds or combinations thereof, and may comprise a phenyl ring. Typically, said hydrocarbon groups contain up to 8 carbon atoms, e.g. up to 6 carbon atoms. Typically at least one of R6 and R7 comprises 4 or more carbon atoms. Examples of unsubstituted hydrocarbon groups represented by R6 or R7 include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, 3-methylbutyl, 2-ethylbutyl, 4-methylpentyl, 3,3,-dimethylbutyl, allyl, cyclohexyl, cyclopropylmethyl, phenyl, benzyl and 2-phenylethyl. Examples of substituted hydrocarbon groups represented by R6 or R7 include 2,2,2-trifluoroethyl, 3,3,3-trifluoropropyl, 4,4,4-trifluorobutyl, 1-methyl-3,3,3-trifluoropopyl, 2,2,3,3,3-pentafluoropropyl, hydroxymethyl, isopropoxymethyl, 4-hydroxy-3-methylbutyl, fluorophenyl, chlorophenyl, trifluoromethylphenyl and trifluoromethoxyphenyl.
In a particular embodiment, R6 represents phenyl or substituted phenyl (e.g. 4-trifluoromethylphenyl) and R7 represents C1-6alkyl, in particular a branched alkyl group such as t-butyl.
A first subset of the compounds according to the invention consists of the compounds of formula II:




and the pharmaceutically acceptable salts and hydrates thereof,

wherein p, R2, R5, R6 and R7 have the same definitions and preferred identities as before.

In a particular embodiment of this subset, p is 0 and Y is a bond.
A second subset of the compounds according to the invention consists of the compounds of formula III:




and the pharmaceutically acceptable salts and hydrates thereof,

wherein p, R2, Y, R5, R6 and R7 have the same definitions and preferred identities as before.

Within this subset, Y is preferably a bond, and p is preferably O.
A third subset of the compounds according to the invention consists of the compounds of formula IV:




and the pharmaceutically acceptable salts and hydrates thereof, wherein:

Ra represents H, halogen or CF3;
and R6 and R7 have the same definitions and preferred identities as before.
The substituted phenyl group attached to the 2-position of the piperidine ring and the CH2CO2H group attached in the 4-position are advantageously (but not necessarily) in the cis-configuration with respect to the piperidine ring.
In a particular embodiment, Ra represents H.
Specific compounds in accordance with the invention are described in the Examples section appended hereto.
The compounds of formula I in which Z is CO2H are typically obtained by hydrolysis of the corresponding esters (1):




where R represents C1-6alkyl such as methyl or ethyl and p, q, V, W, X, Y. R2, R3, R4, R5, R6 and R7 have the same meanings as before, e.g. by refluxing with LiOH in aqueous THF.

Corresponding compounds in which Z represents 1H-tetrazol-5-yl are obtainable by conversion of the esters (1) to the corresponding nitriles, followed by treatment with azidotrimethylsilane in refluxing toluene in the presence of tributyltin oxide. The conversion to the nitrile may be carried out by adding trimethylaluminium to a suspension of ammonium chloride in toluene, then adding the ester (1), refluxing the mixture, and treating with solid potassium sodium tartrate.
Compounds (1) may be obtained by reaction of compounds (2) with diazoesters R6—C(═N2)—CO2R7:




where R, p, q, V, W, X, Y, R2, R3, R4, R5, R6 and R7 have the same meanings as before. The reaction may be carried out in refluxing toluene in the presence of Rh(OAc)4 as catalyst.

The relevant diazoesters may be obtained by treatment of esters R6—CH2—CO2R7 with p-nitrobenzenesulfonylazide, where R6 and R7 have the same meanings as before.
Piperidines (2) in which V and W are both carbon atoms and R3 and R4 are H may be obtained by hydrogenation of the corresponding pyridines (3):




where R, p, q, X, Y, R2 and R5 have the same meanings as before, e.g. in methanolic HC1 over a PtO2 catalyst.

Pyridines (3) in which X is a bond and Y is a bond are obtainable by coupling of chloropyridines (4a) with arylboronic acids (5a):




where R, p, q, R2 and R5 have the same meanings as before. The reaction takes place under standard Suzuki coupling conditions, e.g. in aqueous dimethoxyethane in the presence of sodium carbonate and Pd(PPh3)4.

Pyridines (3) in which X is a bond and Y is CH2 are obtainable by coupling of chloropyridines (4a) with benzylzinc derivatives (5b). The reaction may be carried out at 0° C. to ambient temperature in THF in the presence of a nickel catalyst such as (Ph3P)2NiCl2.
Pyridines (3) in which X is a bond and Y is CH2CH2 are obtainable by coupling of chloropyridines (4a) with alkynes (5c) followed by hydrogenation. The coupling may be carried out in the presence of CuI and a Pd(0) catalyst such as Pd(Ph3)4, e.g. in a mixture of dioxan and triethylamine with microwave heating. The hydrogenation takes place under similar conditions to the conversion of pyridines (3) to piperidines (2), and indeed is preferably combined with that process.
Pyridines (3) in which X is CH2 may be obtained by elaboration of chloropyridines (4b) with (5a), (5b) or (5c) as described above, then treating the product with CO(OR)2 in the presence of strong base such as lithium diisopropylamide, where R has the same meaning as before. Alternatively, the chloropyridines (4b) may be treated with CO(OR)2 prior to the reaction with (5a), (5b) or (5c).
Piperidines of formula (2) in which V and W are both carbon atoms and R3 and R4 are H, X is a bond, p is 1 and R2 is attached to the same ring position as the CO2R group are obtained by alkylation of the corresponding compounds in which p is 0 with R2-L, where L has the same meaning as before. The reaction may be carried out in THF solution in the presence of strong base such as lithium hexamethyldisilazide (HMDS). During this procedure, it is preferable to protect the 1-position of the piperidine ring, e.g. as the BOC derivative.
An alternative route to piperidines (2) in which V and W are both carbon atoms and R3 and R4 are H, p is 0, X is CH2 and the CH2CO2R group is attached to the 4-position involves reduction of the dihydropyridones (6):




(e.g. using a borohydride reductant such as L-Selectride in THF at −78° C.), condensation of the resulting piperidone with (RO)2P(O)CH2CO2R, hydrogenation of the resulting olefin, then removal of the benzyloxycarbonyl protecting group (e.g. by treatment with acid), where R, R5, Y and q have the same meanings as before. The condensation takes place in THF in the presence of NaH, while the hydrogenation may be carried out over a Pd/C catalyst in ethanol. Compounds (6) are available by a one-pot reaction of 4-methoxypyridine with benzyl chloroformate and organometallic derivatives (7), where M1 is Li or Mg-halide, and Y, q and R5 have the same meanings as before. The reaction takes place at reduced temperature (e.g. −25° C.) in a solvent such as THF.

In the above-described route involving dihydropyridones (6), Y is preferably a bond.
The above-described route involving dihydropyridones (6) may be adapted to provide products having an R2 in the 3-position of the piperidine ring via alkylation of the compounds (6) with R2-L, where L represents a leaving group (such as halide, tosylate or mesylate) and R2 has the same meaning as before. Said alkylation takes place under conventional conditions (e.g. in THF in the presence of lithium diisopropylamide).
Alternatively, the aforesaid route may be adapted to provide products having an R2 group in the 6-position of the piperidine ring via treatment of compounds (6) with R2—Mg-halide in the presence of CuI, where R2 has the same meaning as before. The reaction may be carried out in THF at reduced temperature (e.g. −78° C. to −10° C.).
In a further alternative, the aforesaid route may be adapted to provide products in which the —CH2CO2R group is attached at the 3-position of the piperidine ring via alkylation of compounds (6) with L-CH2CO2R, where L and R have the same meanings as before, followed by reduction of the keto group to CH2. The alkylation takes place under conventional conditions, and reduction of the keto group is readily effected via treatment with 1,2-ethanedithiol to form the dithioketal, followed by treatment with Raney nickel.
Piperidines of formula (2) in which R3 and R4 complete a —CH2CH2— bridge and Y is a bond may be obtained by elaboration of bicyclic ketones (8):




wherein q and R5 have the same meanings as before, by the methods described above for the conversion of ketones (6) to piperidines (2), with removal of the protecting group 4-methoxybenzyl by hydrogenation.

The bicyclic ketones (8) are available by reaction of 4-methoxybenzylamine with acetonedicarboxylic acid, then treatment of the product in situ with a ketoaldehyde (9):




where R5 and q have the same meanings as before.

A suitable route to piperazines of formula (1) in which V is a carbon atom and W is a nitrogen atom involves alkylation of compounds (10) with L-X′—CO2R:




where X′ is X that is other than a bond and all other variables are as defined previously.

Compounds (10) are available from compounds (11):





using the methods described previously for conversion of compounds (2) to compounds (1), followed by removal of the trityl group (e.g. by treatment with methanolic HC1). Compounds (11) are available via coupling of compounds (5a), (5b) or (5c) with the appropriate pyrazine derivatives in the manner described above for the synthesis of pyridines (3), followed by hydrogenation (as in the conversion of compounds (3) to compounds (2)) and tritylation by standard methods.

A preferred route to pyrrolidines of formula (1) in which V is a bond, p is 0 and X represents CH2 involves the steps of (a) condensing an aldehyde (12) with t-butylsulphinamide and reacting the resulting imine with allylMgBr to provide the adduct (13a):




    (b) N-alkylation to provide compounds (13b) (e.g. by the methods described previously for converting (2) to (1));
    (c) reaction with CH2═CH—CO2R in the presence of a ruthenium catalyst (e.g. Zhan I), followed by treatment with methanolic HCl, to provide compounds (14a):






    (d) reaction with 1-hydroxymethylbenzotriazole (e.g. in refluxing benzene with molecular sieves) to provide compounds (14b); and
    (e) cyclisation by treatment with SmI2 and t-butanol in THF at −78° C.


Carboxylic acids of formula I in which X is a bond may be converted to the corresponding compounds in which X is CH2 by standard methods of homologation, for example sequential treatment with oxalyl chloride; trimethylsilyldiazomethane and acetonitrile; ROH and silver benzoate; then hydrolysis of the resulting ester. Similar treatment of the corresponding compounds in which X is CH2 provides the compounds in which X is CH2CH2. Esters of formula (1) in which X is C(R1)2 or CH2C(R1)2 and at least one R1 is other than H may be prepared by alkylation of the corresponding compounds in which each R1 is H by standard methods.
Similarly, a given compound in accordance with formula I may be converted to a different compound in accordance with formula I by means of the standard techniques of bond formation or cleavage known to those skilled in the art of organic synthesis.
Where they are not themselves commercially available, the starting materials for the synthetic schemes described above are available by straightforward chemical modifications of commercially available materials.
Certain compounds according to the invention may exist as optical isomers due to the presence of one or more chiral centres or because of the overall asymmetry of the molecule. Such compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution. The novel compounds may, for example, be resolved into their component enantiomers by standard techniques such as preparative HPLC, or the formation of diastereomeric pairs by salt formation with an optically active acid, such as di-p-toluoyl-D-tartaric acid and/or di-p-toluoyl-L-tartaric acid, followed by fractional crystallisation and regeneration of the free base. The novel compounds may also be resolved by formation of diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, racemic intermediates in the preparation of compounds of formula I may be resolved by the aforementioned techniques, and the desired enantiomer used in subsequent steps. For example, racemic piperidine derivatives (2a) may be resolved via salt formation with L-mandelic acid.
During any of the above synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 3rd ed., 1999. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
The compounds of the invention have the useful property of modifying the action of γ-secretase on amyloid precursor protein so as to selectively reduce the formation of the 1-42 isoform of Aβ, and hence find use in the development of treatments for diseases mediated by Aβ(1-42), in particular diseases involving deposition of β-amyloid in the brain.
According to a further aspect of the invention there is provided the use of a compound according to formula I as defined above, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treatment or prevention of a disease associated with the deposition of β-amyloid in the brain.
The disease associated with deposition of Aβ in the brain is typically Alzheimer's disease (AD), cerebral amyloid angiopathy, HCHWA-D, multi-infarct dementia, dementia pugilistica or Down syndrome, preferably AD.
In a further aspect, the invention provides the use of a compound of Formula I as defined above, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating, preventing or delaying the onset of dementia associated with Alzheimer's disease, cerebral amyloid angiopathy, HCHWA-D, multi-infarct dementia, dementia pugilistica or Down syndrome.
The invention also provides a method of treating or preventing a disease associated with deposition of Aβ in the brain comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I as defined above or a pharmaceutically acceptable salt thereof.
In a further aspect, the invention provides a method of treating, preventing or delaying the onset of dementia associated with Alzheimer's disease, cerebral amyloid angiopathy, HCHWA-D, multi-infarct dementia, dementia pugilistica or Down syndrome comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I as defined above or a pharmaceutically acceptable salt thereof.
The compounds of Formula I modulate the action of γ-secretase so as to selectively attenuate production of the (1-42) isoform of Aβ without significantly lowering production of the shorter chain isoforms such as Aβ(1-40). This results in secretion of Aβ which has less tendency to self-aggregate and form insoluble deposits, is more easily cleared from the brain, and/or is less neurotoxic. Therefore, a further aspect of the invention provides a method for retarding, arresting or preventing the accumulation of Aβ in the brain comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula I as defined above or a pharmaceutically acceptable salt thereof.
Because the compounds of formula I modulate the activity of γ-secretase, as opposed to suppressing said activity, it is believed that the therapeutic benefits described above will be obtained with a reduced risk of side effects, e.g. those that might arise from a disruption of other signalling pathways (e.g. Notch) which are controlled by γ-secretase.
In one embodiment of the invention, the compound of Formula I is administered to a patient suffering from AD, cerebral amyloid angiopathy, HCHWA-D, multi-infarct dementia, dementia pugilistica or Down syndrome, preferably AD.
In an alternative embodiment of the invention, the compound of Formula I is administered to a patient suffering from mild cognitive impairment or age-related cognitive decline. A favourable outcome of such treatment is prevention or delay of the onset of AD. Age-related cognitive decline and mild cognitive impairment (MCI) are conditions in which a memory deficit is present, but other diagnostic criteria for dementia are absent (Santacruz and Swagerty, American Family Physician, 63 (2001), 703-13). (See also “The ICD-10 Classification of Mental and Behavioural Disorders”, Geneva: World Health Organisation, 1992, 64-5). As used herein, “age-related cognitive decline” implies a decline of at least six months' duration in at least one of: memory and learning; attention and concentration; thinking; language; and visuospatial functioning and a score of more than one standard deviation below the norm on standardized neuropsychologic testing such as the MMSE. In particular, there may be a progressive decline in memory. In the more severe condition MCI, the degree of memory impairment is outside the range considered normal for the age of the patient but AD is not present. The differential diagnosis of MCI and mild AD is described by Petersen et al., Arch. Neurol., 56 (1999), 303-8. Further information on the differential diagnosis of MCI is provided by Knopman et al, Mayo Clinic Proceedings, 78 (2003), 1290-1308. In a study of elderly subjects, Tuokko et al (Arch, Neurol., 60 (2003) 577-82) found that those exhibiting MCI at the outset had a three-fold increased risk of developing dementia within 5 years.
Grundman et al (J. Mol. Neurosci., 19 (2002), 23-28) report that lower baseline hippocampal volume in MCI patients is a prognostic indicator for subsequent AD. Similarly, Andreasen et al (Acta Neurol. Scand, 107 (2003) 47-51) report that high CSF levels of total tau, high CSF levels of phospho-tau and lowered CSF levels of Aβ42 are all associated with increased risk of progression from MCI to AD.
Within this embodiment, the compound of Formula I is advantageously administered to patients who suffer impaired memory function but do not exhibit symptoms of dementia. Such impairment of memory function typically is not attributable to systemic or cerebral disease, such as stroke or metabolic disorders caused by pituitary dysfunction. Such patients may be in particular people aged 55 or over, especially people aged 60 or over, and preferably people aged 65 or over. Such patients may have normal patterns and levels of growth hormone secretion for their age. However, such patients may possess one or more additional risk factors for developing Alzheimer's disease. Such factors include a family history of the disease; a genetic predisposition to the disease; elevated serum cholesterol; and adult-onset diabetes mellitus.
In a particular embodiment of the invention, the compound of Formula I is administered to a patient suffering from age-related cognitive decline or MCI who additionally possesses one or more risk factors for developing AD selected from: a family history of the disease; a genetic predisposition to the disease; elevated serum cholesterol; adult-onset diabetes mellitus; elevated baseline hippocampal volume; elevated CSF levels of total tau; elevated CSF levels of phospho-tau; and lowered CSF levels of Aβ(1-42), A genetic predisposition (especially towards early onset AD) can arise from point mutations in one or more of a number of genes, including the APP, presenilin-1 and presenilin-2 genes. Also, subjects who are homozygous for the ε4 isoform of the apolipoprotein E gene are at greater risk of developing AD.
The patient's degree of cognitive decline or impairment is advantageously assessed at regular intervals before, during and/or after a course of treatment in accordance with the invention, so that changes therein may be detected, e.g. the slowing or halting of cognitive decline. A variety of neuropsychological tests are known in the art for this purpose, such as the Mini-Mental State Examination (MMSE) with norms adjusted for age and education (Folstein et al., J. Psych. Res., 12 (1975), 196-198, Anthony et al., Psychological Med., 12 (1982), 397-408; Cockrell et al., Psychopharmacology, 24 (1988), 689-692; Crum et al., J. Am. Med. Assoc'n. 18 (1993), 2386-2391). The MMSE is a brief quantitative measure of cognitive status in adults. It can be used to screen for cognitive decline or impairment, to estimate the severity of cognitive decline or impairment at a given point in time, to follow the course of cognitive changes in an individual over time, and to document an individual's response to treatment. Another suitable test is the Alzheimer Disease Assessment Scale (ADAS), in particular the cognitive element thereof (ADAS-cog) (See Rosen et al., Am. J. Psychiatry, 141 (1984), 1356-64).
The compounds of Formula I are typically used in the form of pharmaceutical compositions comprising one or more compounds of Formula I and a pharmaceutically acceptable carrier. Accordingly, in a further aspect the invention provides a pharmaceutical composition comprising a compound of formula I as defined above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, transdermal patches, auto-injector devices or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. The principal active ingredient typically is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate and dicalcium phosphate, or gums, dispersing agents, suspending agents or surfactants such as sorbitan monooleate and polyethylene glycol, and other pharmaceutical diluents, e.g. water, to form a homogeneous preformulation composition containing a compound of the present invention, or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Typical unit dosage forms contain from 1 to 100 mg, for example 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient. Tablets or pills of the composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The liquid forms in which the compositions useful in the present invention may be incorporated for administration orally or by injection include aqueous solutions, liquid- or gel-filled capsules, suitably flavoured syrups, aqueous or oil suspensions, and flavoured emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, poly(ethylene glycol), poly(vinylpyrrolidone) or gelatin.
For treating or preventing Alzheimer's disease, a suitable dosage level is about 0.01 to 250 mg/kg per day, preferably about 0.01 to 100 mg/kg per day, and more preferably about 0.05 to 50 mg/kg of body weight per day, of the active compound. The compounds may be administered on a regimen of 1 to 4 times per day. In some cases, however, a dosage outside these limits may be used.
The compounds of Formula I optionally may be administered in combination with one or more additional compounds known to be useful in the treatment or prevention of AD or the symptoms thereof. Such additional compounds thus include cognition-enhancing drugs such as acetylcholinesterase inhibitors (e.g. donepezil and galanthamine), NMDA antagonists (e.g. memantine) or PDE4 inhibitors (e.g. ArifloTM and the classes of compounds disclosed in WO 03/018579, WO 01/46151, WO 02/074726 and WO 02/098878). Such additional compounds also include cholesterol-lowering drugs such as the statins, e.g. simvastatin. Such additional compounds similarly include compounds known to modify the production or processing of Aβ in the brain (“amyloid modifiers”), such as compounds which inhibit the secretion of Aβ (including γ-secretase inhibitors, β-secretase inhibitors, and GSK-3α inhibitors), compounds which inhibit the aggregation of Aβ, and antibodies which selectively bind to Aβ. Such additional compounds also include growth hormone secretagogues, as disclosed in WO 2004/110443.
In this embodiment of the invention, the amyloid modifier may be a compound which inhibits the secretion of Aβ, for example an inhibitor of γ-secretase (such as those disclosed in WO 01/90084, WO 02/30912, WO 01/70677, WO 03/013506, WO 02/36555, WO 03/093252, WO 03/093264, WO 03/093251, WO 03/093253, WO 2004/039800, WO 2004/039370, WO 2005/030731, WO 2005/014553, WO 2004/089911, WO 02/081435, WO 02/081433, WO 03/018543, WO 2004/031137, WO 2004/031139, WO 2004/031138, WO 2004/101538, WO 2004/101539 and WO 02/47671), or a β-secretase inhibitor (such as those disclosed in WO 03/037325, WO 03/030886, WO 03/006013, WO 03/006021, WO 03/006423, WO 03/006453, WO 02/002122, WO 01/70672, WO 02/02505, WO 02/02506, WO 02/02512, WO 02/02520, WO 02/098849 and WO 02/100820), or any other compound which inhibits the formation or release of Aβ including those disclosed in WO 98/28268, WO 02/47671, WO 99/67221, WO 01/34639, WO 01/34571, WO 00/07995, WO 00/38618, WO 01/92235, WO 01/77086, WO 01/74784, WO 01/74796, WO 01/74783, WO 01/60826, WO 01/19797, WO 01/27108, WO 01/27091, WO 00/50391, WO 02/057252, US 2002/0025955 and US2002/0022621, and also including GSK-3 inhibitors, particularly GSK-3α inhibitors, such as lithium, as disclosed in Phiel et al, Nature, 423 (2003), 435-9.
Alternatively, the amyloid modifier may be a compound which inhibits the aggregation of Aβ or otherwise attenuates is neurotoxicicity. Suitable examples include chelating agents such as clioquinol (Gouras and Beal, Neuron, 30 (2001), 641-2) and the compounds disclosed in WO 99/16741, in particular that known as DP-109 (Kalendarev et al, J. Pharm. Biomed. Anal., 24 (2001), 967-75). Other inhibitors of Aβ aggregation suitable for use in the invention include the compounds disclosed in WO 96/28471, WO 98/08868 and WO 00/052048, including the compound known as ApanTM (Praecis); WO 00/064420, WO 03/017994, WO 99/59571 (in particular 3-aminopropane-1-sulfonic acid, also known as tramiprosate or AlzhemedTM); WO 00/149281 and the compositions known as PTI-777 and PTI-00703 (ProteoTech); WO 96/39834, WO 01/83425, WO 01/55093, WO 00/76988, WO 00/76987, WO 00/76969, WO 00/76489, WO 97/26919, WO 97/16194, and WO 97/16191. Further examples include phytic acid derivatives as disclosed in U.S. Pat. No. 4,847,082 and inositol derivatives as taught in US 2004/0204387.
Alternatively, the amyloid modifier may be an antibody which binds selectively to Aβ. Said antibody may be polyclonal or monoclonal, but is preferably monoclonal, and is preferably human or humanized. Preferably, the antibody is capable of sequestering soluble Aβ from biological fluids, as described in WO 03/016466, WO 03/016467, WO 03/015691 and WO 01/62801. Suitable antibodies include humanized antibody 266 (described in WO 01/62801) and the modified version thereof described in WO 03/016466.
As used herein, the expression “in combination with” requires that therapeutically effective amounts of both the compound of Formula I and the additional compound are administered to the subject, but places no restriction on the manner in which this is achieved. Thus, the two species may be combined in a single dosage form for simultaneous administration to the subject, or may be provided in separate dosage forms for simultaneous or sequential administration to the subject. Sequential administration may be close in time or remote in time, e.g. one species administered in the morning and the other in the evening. The separate species may be administered at the same frequency or at different frequencies, e.g. one species once a day and the other two or more times a day. The separate species may be administered by the same route or by different routes, e.g. one species orally and the other parenterally, although oral administration of both species is preferred, where possible. When the additional compound is an antibody, it will typically be administered parenterally and separately from the compound of Formula I.


EXAMPLES
The ability of the compounds of Formula I to selectively inhibit production of Aβ(1-42) was determined using the following assay:
Cell-Based γ-Secretase Assay
Human SH-SY5Y neuroblastoma cells overexpressing the direct γ-secretase substrate SPA4CT were induced with sodium butyrate (10 mM) for 4 hours prior to plating. Cells were plated at 35,000 cells/well/100 μl in 96-well plates in phenol red-free MEM/10% FBS, 50 mM HEPES, 1% Glutamine and incubated for 2 hrs at 37° C., 5% CO2.
Compounds for testing were diluted into Me2SO to give a ten point dose-response curve. Typically 10 μl of these diluted compounds in Me2SO were further diluted into 182 μl dilution buffer (phenol red-free MEM/10% FBS, 50 mM HEPES, 1% Glutamine) and 10 μl of each dilution was added to the cells in 96-well plates (yielding a final Me2SO concentration of 0.5%). Appropriate vehicle and inhibitor controls were used to determine the window of the assay.
After incubation overnight at 37° C., 5% CO2, 10 μl and 50 μl media were transferred into a fresh Costar round-bottom 96-well plate for detection of Aβ(40) and Aβ(42) peptides, respectively. 40 μl Origen buffer (PBS, 2% BSA, 0.2% Tween-20) was added to the Aβ(40) wells followed by the addition of 25 μl the respective antibody premixes to the wells:

    
    
        Aβ(40) premix: 1 μg/ml ruthenylated G2-10 antibody, 4 μg/ml biotinylated 4G8 antibody diluted in Origen buffer
        Aβ(42) premix: 0.5 μg/ml ruthenylated G2-11 antibody, 4 μg/ml biotinylated 4G8 antibody diluted in Origen buffer
    
    


(Biotinylated 4G8 Antibody Supplied by Signet Pathology Ltd; G2-10 and G2-11 Antibodies Supplied by Chemicon)
After overnight incubation of the assay plates on a shaker at 4° C., the Origen M8 Analyser (Igen Inc.) was calibrated according to the manufacturer's instructions. 25 μl of streptavidin magnetic bead (Dynal) premix (400 μg/ml streptavidin beads/ml in Origen buffer) was added to the assay plates and incubated on a shaker for 15 minutes. 150 μl Origen buffer was added to each well and the plates were read on the Origen M8 Analyser according to the manufacturer's instructions.
Cell viability was measured in the corresponding cells after removal of the media for the Aβ assays by a colorimetric cell proliferation assay (CellTiter 96TM AQ assay, Promega) utilizing the bioreduction of MTS (Owen's reagent) to formazan according to the manufacturer's instructions. Briefly, 5 μl of 10×MTS/PES was added to the remaining 50 μl of media before returning to the incubator. The optical density was read at 495 nm after  ̃4 hours.
LD50 and IC50 values for inhibition of Aβ(40) and Aβ(42) were calculated by nonlinear regression fit analysis using the appropriate software (eg. Excel fit). The total signal and the background were defined by the corresponding Me2SO and inhibitor controls.
The compounds listed in the following examples all gave IC50 values for Aβ(1-42) inhibition that were at least 2-fold lower than the corresponding IC50 values for Aβ(1-40) inhibition, typically at least 5-fold lower, and in the preferred cases at least 50-fold lower.
Intermediate 1: (±)-Methyl {2-[4-(trifluoromethyl)phenyl]piperidin-4-yl}acetate Hydrochloride




Step 1: 4-Methyl-2-[4-(trifluoromethyl)phenyl]pyridine





A mixture of 2-chloro-4-methylpyridine (1.9 ml, 21.6 mmol) and 4-(trifluoromethyl)benzeneboronic acid (5.0 g, 26 mmol) in DME (40 ml) and aqueous Na2CO3 (2M, 40 ml) was degassed (Firestone® valve×3). Tetrakis(triphenylphosphine) palladium (0) (1.15 g, 1.0 mmol, 5 mol %) was added and following a further degassing (Firestone® valve×3) the mixture was heated at reflux for 16 hours. The reaction was cooled to room temperature diluted with H2O (100 ml) and EtOAc (150 ml). The mixture was filtered through a Celite® pad, washing through with EtOAc. The phases were separated and the aqueous layer was extracted with EtOAc (200 ml). The combined extracts were washed with H2O (100 ml) and brine (×1), then dried (MgSO4), filtered and evaporated. The residue was purified by chromatography on silica, eluting with 10% EtOAc/isohexane to give the ester (3.5 g, 68%) as a white solid. 1H NMR (360 MHz, CDCl3) δ: 2.44 (3H, s), 7.13 (2H, d, J 5.0), 7.58 (1H, s), 7.72 (2H, d, J 8.2), 8.09 (2H, d, J 8.2), 8.57 (1H, d, J 5.0).
Step 2: Methyl {2-[4-(trifluoromethyl)phenyl]pyridin-4-yl}acetate




A solution of LDA (2M in THF/heptane/ethyl benzene, 44 ml, 88 mmol) was added dropwise to a stirred solution of 4-methyl-2-[4-(trifluoromethyl)phenyl]pyridine (10.5 g, 44 mmol) in dry THF (300 ml) under N2, such that the internal temperature remained <−70° C. After 1 hour at this temperature, dimethyl carbonate (8.9 ml, 106 mmol) was added. After 30 minutes the cooling bath was removed. When the internal temperature had reached −20° C. the reaction was transferred to a cold bath at −10° C., and then allowed to warm slowly to 0° C. After 1 hour at 0° C. the reaction was quenched with aqueous NH4Cl (half saturated, 100 ml). The reaction mixture was concentrated in vacuo. The residue was diluted with H2O (200 ml) and extracted with EtOAc (2×200 ml). The combined extracts were washed with brine (×1), then dried (MgSO4), filtered and evaporated. The residue was purified by chromatography on silica, eluting with 10-30% EtOAc/isohexane to give the ester (9.2 g, 71%) as a pale yellow liquid. 1H NMR (400 MHz, CDCl3) δ: 3.72 (2H, s), 3.75 (3H, s), 7.24 (1H, dd, J 1.4, 5.0), 7.72 (3H, t, J 8.4), 8.11 (2H, d, J 8.2), 8.68 (1H, d, J 5.0).
Step 3: (±)-Methyl {2-[4-(trifluoromethyl)phenyl]piperidin-4-yl}acetate Hydrochloride
A mixture of methyl {2-[4-(trifluoromethyl)phenyl]pyridin-4-yl}acetate (6.2 g, 21 mmol), PtO2 (200 mg, 0.9 mmol) and HCl solution (4N in dioxane, 5.8 ml, 23 mmol) in MeOH (100 ml) was hydrogenated at 20 psi on a Parr® apparatus for 5 hours. The catalyst was removed by filtration and the filtrate evaporated in vacuo to give the desired piperidine as white solid (7.1 g, quant). 1H NMR (400 MHz, CD3OD) δ: 1.58-1.72 (1H, m), 1.75-1.85 (1H, m), 2.08 (1H, d, J 14.2), 2.19 (1H, t, J 14.2), 2.28-2.38 (1H, m), 2.45 (2H, d, J 6.9), 3.24-3.32 (1H, m), 3.51-3.57 (1H, m), 3.67 (3H, s), 4.46 (1H, d, J 10.2), 7.72 (2H, d, J 8.3), 7.79 (2H, d, J 8.4).
The free base was obtained by treatment with NaHCO3 (aq) and extraction in to DCM. The organic extracts were dried, filtered and evaporated.
Intermediate 2: (+)-Methyl {(2S,4R)-2-[4-(trifluoromethyl)phenyl]piperidin-4-yl}acetate




(±)-cis Methyl 4-(trifluoromethyl)phenylpiperidin-4-yl)acetate (Intermediate 1 [free base], 32.6 g, 0.108 mol), was dissolved in hot isopropanol (100 ml) and the solution was added to a solution of L-(+)-mandelic acid (9 g, 0.054 mol) in hot isopropanol (170 ml) and the resulting solution was allowed to stand at room temperature overnight. A white crystalline solid was deposited (17.55 g, 36%) and was filtered. The mother liquors were evaporated and the residue was neutralized with sodium carbonate (2M, 100 ml) and extracted with dichloromethane (3×100 ml). The combined extracts were washed with brine, dried (MgSO4), filtered and evaporated. This extract was dissolved in hot isopropanol (100 ml) and was added to a solution of D-(−)-mandelic acid (9 g, 0.054 mol) in hot isopropanol (170 ml); immediate crystallization occurred and the mixture was allowed to stand for 2 h. The white crystalline solid was isolated by filtration (21 g, 44%) and was recrystallised from isopropyl acetate (250 ml) to give the product (19.8 g, 40%) as a white crystalline material, ee>99.5%. This material was neutralized with sodium carbonate (2M, 100 ml and extracted with dichloromethane (3×100 ml). The combined extracts were washed with brine, dried (MgSO4), filtered and evaporated to give the free base: αD (c=1, MeOH)+23°; 1H NMR (360 MHz, CDCl3) δ: 1.23 (6H, d, J 6.9), 2.88 (1H, qn, J 6.9), 4.27 (2H, s), 7.15-7.21 (4H, m), 7.71 (2H, d, J 8.2), 8.10 (2H, d, J 8.2).
Intermediate 3: (±)-Methyl {(2R*,3S*)-2-[4-(trifluoromethyl)phenyl]piperidin-3-yl}acetate




Step 1: (±)-Benzyl 4-oxo-2-[4-(trifluoromethyl)phenyl]-3,4-dihydropyridine-1(2H)-carboxylate





4-Trifluoromethyl bromobenzene (4.2 ml, 30 mmol) was added portionwise to magnesium turnings (0.729 g, 30 mmol) in dry THF (30 ml), and two drops of 1,2-dibromoethane were added to initiate the reaction. The resulting brown solution was cooled to −25° C. 4-Methoxypyridine (3.0 ml, 30 mmol) was added followed by benzyl chloroformate (4.3 ml, 30 mmol). The reaction was stirred for 30 mins at −20° C. then quenched with 2N HCl. After stirring for 10 mins the mixture was extracted with EtOAc (×3). The combined extracts were washed with brine, dried (MgSO4), filtered and evaporated. The residue was purified by chromatography (silica, 10-40% EtOAc/hexanes) to give the dihydropyridine (9.30 g, 83%). 1H NMR (500 MHz, CDCl3) δ: 2.77 (1H, d, J 16.6), 3.18 (1H, dd, J 7.7, 16.6), 5.20 (1H, d, J 12.0), 5.27 (1H, d, J 12.0), 5.42 (1H, d, J 8.3), 5.77 (1H, d, J 6.3), 7.24-7.37 (7H, m), 7.54 (2H, d, J 8.2), 8.00 (1H, m).
Step 2: (±)-Benzyl (2R*,3S*)-3-(2-methoxy-2-oxoethyl)-4-oxo-2-[4-(trifluoromethyl)phenyl]-3,4-dihydropyridine-1(2H)-carboxylate




A solution of the dihydropyridine from Step 1 (3.00 g, 8.0 mmol) in dry THF (80 ml) was cooled to −78° C. and lithium bis(trimethylsilyl)amide (1.0M in THF, 9.6 ml, 9.6 mmol) was added dropwise. After stirring at −78° C. for 1 hr, methyl bromoacetate (2.2 ml, 24 mmol) was added. The reaction was stirred at −78° C. for 1 hr then at 0° C. for 1.5 hrs. The reaction was quenched with saturated NH4Cl solution. The mixture extracted with EtOAc (×3), the combined extracts were washed with brine, dried (MgSO4), filtered and evaporated. The residue was purified by chromatography (silica, 40% Et2O/hexanes) to give the ester (2.74 g, 77%). 1H NMR (360 MHz, CDCl3) δ: 2.61-2.69 (2H, m), 3.15 (1H, dd, J 5.0, 9.8), 3.74 (3H, s), 5.18-5.28 (2H, m), 5.39 (1H, d, J 8.5), 5.70 (1H, s), 7.18-7.33 (7H, m), 7.54 (2H, d, J 8.3), 8.08 (1H, d, J 8.6).
Step 3: (±)-Benzyl (2R*,3S*)-3-(2-methoxy-2-oxoethyl)-4-oxo-2-[4-(trifluoromethyl)phenyl]piperidine-1-carboxylate




A solution of the enone from Step 2 (1.650 g, 3.7 mmol) in dry THF (40 ml) was cooled to −78° C. and L-Selectride® (1.0M in THF, 4.6 ml, 4.6 mmol) was added. The reaction was stirred for 2 mins then quenched with saturated NH4Cl solution. The mixture extracted with EtOAc (×3), the combined extracts were washed with brine, dried (MgSO4), filtered and evaporated. The residue was purified by chromatography (silica, 40-50% Et2O/hexanes) to give the ketone (1.473 g, 89%). 1H NMR (360 MHz, CDCl3) δ: 2.24 (1H, dd, J 4.9, 16.8), 2.57-2.70 (3H, m), 3.46-3.52 (1H, m), 3.58 (3H, s), 3.80-3.89 (1H, m), 4.57 (1H, dd, J 5.2, 14.2), 4.93-4.98 (2H, m), 5.10 (1H, d, J 12.1), 7.07 (2H, s), 7.26-7.41 (5H, m), 7.57 (2H, d, J 8.1).
Step 4: (±)-Methyl {(6S*,7R*)-7-[4-(trifluoromethyl)phenyl]-1,4-dithia-8-azaspiro[4.5]dec-6-yl}acetate




A solution of the ketone from Step 3 (0.772 g, 1.7 mmol) in DCM (20 ml) was cooled to 0° C. and 1,2-ethanedithiol (0.43 ml, 5.1 mmol) and BF3.OEt2 (1.27 ml, 10 mmol) were added. The reaction was stirred at RT for 48 hrs then diluted with EtOAc. The mixture was washed with 1N NaOH solution then brine, dried (MgSO4), filtered and evaporated. The residue was purified by chromatography (silica, 30% EtOAc/hexanes) to give the dithiane (0.420 g, 63%). 1H NMR (360 MHz, CDCl3) δ: 2.03-2.11 (1H, m), 2.20-2.26 (1H, m), 2.35-2.43 (1H, m), 2.73-2.83 (2H, m), 3.06-3.16 (2H, m), 3.24 (3H, s), 3.25-3.32 (4H, m), 3.50 (1H, d, J 9.9), 7.47-7.58 (4H, m).
Step 5: (±)-Methyl {(2R*,3S*)-2-[4-(trifluoromethyl)phenyl]piperidin-3-yl}acetate
Raney nickel (slurry in water) was added portionwise to a solution of the dithiane from Step 4 (0.418 g, 1.1 mmol) in MeOH (20 ml). The reaction was heated under reflux for 3 hrs then allowed to cool. The reaction was filtered through Hyflo® and the solvent was evaporated. The resulting mixture was extracted with DCM, dried (MgSO4), filtered and evaporated. The residue was purified by chromatography (silica, 2% MeOH/DCM) to give the piperidine (0.168 g, 52%). 1H NMR (360 MHz, CDCl3) δ: 1.21-1.26 (1H, m), 1.69-1.75 (2H, m), 1.90-2.10 (4H, m), 2.72-2.80 (1H, m), 3.16 (1H, m), 3.36 (1H, d, J 9.7), 3.50 (3H, s), 7.48 (2H, d, J 8.1), 7.57 (2H, d, J 8.2).
Example 1
{(2S,4R)-1-{(1S)-2-[(1,1-dimethylethyl)oxy]-2-oxo-1-[4-(trifluoromethyl)phenyl]ethyl}-2-[4-(trifluoromethyl)phenyl]piperidin-4-yl}acetic acid




Step 1





A mixture of 2-diazo-2-(4-trifluoromethyl)phenylacetic acid, t-butyl ester (3.2 g 11.18 mmol), Intermediate 2 (3.37 g, 11.18 mmol) and Rh(OAc)4 (99 mg, 0.224 mmol) in toluene (40 ml) was degassed and refluxed for 16 hours. After cooling and evaporation, the residue was purified by column chromatography (silica, dichloromethane/isohexane 1:1) to provide the desired methyl ester intermediate as a single diastereoisomer (beige foam, 1.24 g).
Step 2
A solution of the methyl ester from Step 1 (240 mg, 0.429 mmol) and lithium hydroxide (10.27 mg, 0.429 mmol) in a mixture of THF (3 ml) and water (2 ml) was stirred at ambient temperature until TLC (5% MeOH in dichloromethane) showed reaction to be complete. The mixture was diluted with HCl (1N), extracted with dichloromethane, and the extracts dried (MgSO4) and evaporated. Purification of the residue by column chromatography (silica, 5% MeOH in dichloromethane) afforded the desired acid (pale foam, 210 mg). m/z ES+ 546.
Further compounds according to the invention may be prepared by analogous procedures using the appropriate diazoester and Intermediate 1, Intermediate 2, Intermediate 3, or analogue thereof.

## Claims

1. A compound of formula I:




or a pharmaceutically acceptable salt thereof; wherein:
p is 0 or 1;
q is 0, 1, 2 or 3;
V represents a bond or a carbon atom whose remaining valencies are satisfied via bonding to H, R2 or X—Z or to any combination thereof;
W represents a nitrogen atom or a carbon atom whose remaining valencies are satisfied via bonding to H, R2 or X—Z or to any combination thereof, provided that when W represents a nitrogen atom, V represents a carbon atom and the moiety X—Z is attached to W;
X represents a bond or C(R1)2 or CH2C(R1)2, provided that when W represents N, X does not represent a bond;
Y represents a bond or CH2 or CH2CH2;
Z represents CO2H or a tetrazole ring;
each R1 independently represents H or a non-aromatic hydrocarbon group of up to 6 carbon atoms; or the two R1 groups complete a C3-6alicyclic group;
R2 represents a non-aromatic hydrocarbon group of up to 6 carbon atoms;
R3 and R4 each represents H, or when V and W each represents a carbon atom, R3 and R4 may together represent a CH2CH2 bridge;
each R5 independently represents halogen, C1-6alkyl bearing 0-3 fluorine substituents, C1-6alkoxy bearing 0-3 fluorine substituents, or C2-6alkenyl; and
R6 and R7 independently represent hydrocarbon groups each containing up to 10 carbon atoms and optionally bearing a substituent selected from halogen, perfluoroC1-4alkyl, OH, C1-4alkoxy and OCF3.

2. A compound according to claim 1 wherein V and W both represent carbon atoms.

3. A compound according to claim 1 wherein R3 and R4 both represent H.

4. A compound according to any previous claim 1 wherein (R5)q represents 2-CF3, 3-CF3, 4-CF3, 2,4-di(CF3), 2-F-4-CF3, 4-OCF3, 4-allyl, 4-n-propyl, 4-isopropyl or 4-tert-butyl.

5. A compound according to claim 1 which is a compound of formula IV:




or a pharmaceutically acceptable salt thereof; wherein:
Ra represents H, halogen or CF3.

6. A compound according to claim 5 wherein the substituted phenyl group attached to the 2-position of the piperidine ring and the CH2CO2H group attached in the 4-position are in the cis-configuration with respect to the piperidine ring.

7. A compound according to claim 1 wherein R6 represents phenyl or substituted phenyl and R7 represents C1-6alkyl.

8. A compound according to claim 7 wherein R6 represents 4-trifluoromethylphenyl and R7 represents t-butyl.

9. A pharmaceutical composition comprising a compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.