TITLE OF THE INVENTION

4-OXO- 1 ,4-DIH YDROQUINOLINE Ml RECEPTOR POSITIVE ALLOSTERIC

MODULATORS

FIELD OF THE INVENTION

The invention is directed to a class of aryl pyridine compounds, their salts, pharmaceutical compositions comprising them and their use in therapy of the human body. In particular, the invention is directed to a class of quinolizidinone compounds which are muscarinic Ml receptor positive allosteric modulators, and hence are useful in the treatment of Alzheimer's Disease and other diseases mediated by the muscarinic Ml receptor.

BACKGROUND OF THE INVENTION

Alzheimer's Disease is a common neurodegenerative disease affecting the elderly, resulting in progressive memory impairment, loss of language and visuospatial skills, and behavior deficits. Characteristics of the disease include degeneration of cholinergic neurons in the cerebral cortex, hippocampus, basal forebrain, and other regions of the brain, neurofibrillary tangles, and accumulation of the amyloid β peptide (Aβ). Aβ is a 39-43 amino acid produced in the brain by processing of the beta-amyloid precursor protein (APP) by the beta-amyloid protein cleaving enzyme ("beta secretase" or "BACE") and gamma-secretase. The processing leads to accumulation of Aβ in the brain.

Cholinergic neurotransmission involves the binding of acetylcholine either to the nicotinic acetylcholine receptor (nAChR) or to the muscarinic acetylcholine receptor (mAChR). It has been hypothesized that cholinergic hypofunction contributes to the cognitive deficits of patients suffering from Alzheimer's Disease. Consequently, acetyl cholinesterase inhibitors, which inhibit acetylcholine hydrolysis, have been approved in the United States for use in the treatment of the cognitive impairments of Alzheimer's Disease patients. While acetyl cholinesterase inhibitors have provided some cognitive enhancement in Alzheimer's Disease patients, the therapy has not been shown to change the underlying disease pathology.

A second potential pharmacotherapeutic target to counteract cholinergic hypofunction is the activation of muscarinic receptors. Muscarinic receptors are prevalent throughout the body. Five distinct muscarinic receptors (M1-M5) have been identified in mammals. In the central nervous system, muscarinic receptors are involved in cognitive, behavior, sensory, motor and autonomic functions. The muscarinic Ml receptor, which is prevalent in the cerebral cortex, hippocampus and striatum, has been found to have a major role in cognitive processing and is believed to have a role in the pathophysiology of Alzheimer's Disease. See Eglen et al, TRENDS in Pharmacological Sciences, 2001 , 22:8, 409-414. In addition, unlike acetyl cholinesterase inhibitors, which are known to provide only symptomatic

treatment, Ml agonists also have the potential to treat the underlying disease mechanism of Alzheimer's Disease. The cholinergic hypothesis of Alzheimer's Disease is linked to both β- amyloid and hyperphosphorylated tau protein. Formation of β-amyloid may impair the coupling of the muscarinic receptor with G-proteins. Stimulation of the Ml muscarinic receptor has been shown to increase formation of the neuroprotective αAPPs fragment, thereby preventing the formation of the Aβ peptide. Thus, Ml agonists may alter APP processing and enhance αAPPs secretion. See Fisher, Jpn J Pharmacol, 2000, 84:101-112.

However, Ml ligands that have been developed and studied for Alzheimer's Disease have produced side effects common to other muscarinic receptor ligands, such as sweating, nausea and diarrhea. See Spalding et al, MoI Pharmacol, 2002, 61 :6, 1297-1302.

The muscarinic receptors are known to contain one or more allosteric sites, which may alter the affinity with which muscarinic ligands bind to the primary binding or orthosteric sites. See, e.g., S. Lazareno et al, MoI Pharmacol, 2002, 62:6, 1491-1505; S. Lazareno et al, MoI Pharmacol, 2000, 58, 194-207. Thus the compounds of the invention, which are muscarinic Ml receptor positive allosteric modulators, are believed to be useful in the treatment of Alzheimer's Disease and other diseases mediated by the muscarinic Ml receptor.

SUMMARY OF THE INVENTION The present invention is novel 4-oxo-l ,4-dihydroquinoline compounds of generic formula (I)

(I) or a pharmaceutically acceptable salt thereof, which is useful as an Ml receptor positive allosteric modulator.

The invention is further directed to methods of treating a patient (preferably a human) for diseases or disorders in which the Ml receptor is involved, such as Alzheimer's disease, cognitive impairment, schizophrenia, pain disorders and sleep disorders, by administering to the patient a therapeutically effective amount of a compound of general formula

(I), or a pharmaceutically acceptable salt thereof. The invention is also directed to pharmaceutical compositions which include an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, and the use of the compounds and pharmaceutical compositions of the invention in the treatment of such diseases.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the invention is directed to compounds of general formula (I)

and pharmaceutically acceptable salts thereof, wherein

one or two of X ¹ , X ² , X ³ , X ⁴ and X ⁵ is NH, and the others are each CR ³ ;

Rl and R2 are each selected from the group consisting of (1) hydrogen, or

(2) fluoro;

R3 is selected from the group consisting of

(1) hydrogen, (2) halogen,

(3) cyano,

(4) -Ci-6-alkyl,

(5) -OCi-6-alkyl,

(6) -C3-8 cycloalkyl, (7) -C6-10 aryl,

(8) heteroaryl group having 5 to 12 ring atoms,

(9) -NR6AR6B _; or

(10) _S(O)m- R5A

wherein any alkyl, aryl, heteroaryl, and cycloalkyl R3 moiety is optionally substituted with one or more

(a) halogen,

(b) hydroxy, (c) cyano,

(d) -(CH ₂ )q-aryl,

(e) -O-C 1-6 alkyl.

(f) -C 1-6 alkyl,

(g) -(CH2) _P -NR6CR6D, (h) -C3-8 cycloalkyl,

(i) heteroaryl group having 5 to 12 ring atoms,

0) -C(=O)-OR5A,

. (k) -C(=O)- R5A,

(D -S(0)m- R5A (m) -C(=O)-NR6AR6B _}

(n) -NR5ASO2- R5B,

( ₀ ) -NR5AC(=O)R5B,

(p) -NR5AC(=O)-O-R5B, or and wherein said alkyl, aryl or heteroaryl moiety is optionally substituted with one or more

(1) halogen,

(ii) hydroxy, or (iii) cyano;

R4 is selected from the group consisting of

(1) hydrogen,

(2) -C 1-6 alkyl, or

(3) -CH ₂ -aryl, wherein said R3 alkyl or aryl moiety is optionally substituted with one or more (a) halogen,

(b) cyano, and

(c) -O-Ci-6 alkyl, wherein said alkyl is optionally substituted with one or more halo;

R5A and R5B are independently selected from the group consisting of

(1) hydrogen,

(2) -C 1-6 alkyl, or

(3) -CCH2)n-aryl, wherein said R5A or R5B alkyl or aryl moiety is optionally substituted with one or more

(a) halogen, (b) cyano, or

(c) -O-Ci-6 alkyl, wherein said alkyl is optionally substituted with one or more halo;

ROA ₄ ROB ₅ R6C and R.6D are selected from the group consisting of (1) hydrogen,

(2) -C 1-6 alkyl, or

(3) -C3-6 cycloalkyl, or R.6A. and R.6B, or R6C and R6D are linked together with the nitrogen to which they are both attached to form a 3-6 membered carbocyclic ring, wherein one or two of the ring carbon atoms is replaced by a nitrogen, oxygen or sulfur;

m is 0, 1 or 2; n is O or 1; p is 0 or 1 ; and q is 0 or 1.

In particular embodiments of compounds of formula (I), one of X ¹ , X ² , X ³ , X ⁴ and X ⁵ is NH, and the others are each CR ³ . Typically, three of the CR ³ groups are CH and the CR ³ in the remaining CR ³ group is other than hydrogen.

In other embodiments of compounds of formula (I), two of X ¹ , X ² , X ³ , X ⁴ and X ⁵ is NH, and the others are each CR ³ . Typically, two of the CR ³ groups are CH and the R ³ in the remaining CR ³ group is other than hydrogen.

In preferred embodiments, when R ³ is other than hydrogen, R ³ is selected from

( 1 ) -C6- 10 aryl (preferably phenyl), or

(2) heteroaryl group having 5 to 12 ring atoms. In one subgroup, the heteroaryl R ³ groups include groups having 5 or 6 ring atoms and a single heteroatom, which is nitrogen. Exemplary heteroaryl groups in this embodiment are pyridyl and pyrrolyl.

Another subgroup of R3 heteroaryl groups have 5 or 6 ring atoms and two heteroatoms, which are selected from sulfur and nitrogen. Exemplary heteroaryl groups in this embodiment are pyrazolyl, pyrimidinyl, imidazolyl, thienyl and isothiazolyl.

Another subgroup of R3 heteroaryl groups have 9 or 10 ring atoms and two heteroatoms, which are selected from oxygen, sulfur and nitrogen. Exemplary heteroaryl groups in this embodiment are benzoxazolyl, benzothiazolyl and quinoxalinyl.

When R ³ is aryl or heteroaryl, in certain embodiments the aryl or heteroaryl moiety is substituted with one or more

(a) halogen,

(b) cyano,

(c) -O-C i -6 alkyl (preferably -O-C I -3 alkyl), wherein the alkyl is optionally substituted with one or more fluoro, (d) -Cl -6 alkyl (preferably -Ci -3 alkyl), wherein the alkyl is optionally substituted with one or more fluoro,

(e) -NR6CR6D,

(0 -C(=O)-OR5A,

(g) -C(=O)- R5A, (h) -S(COm- RSA,

(i) -C(=O)-NR6AR6B,

(j) -NR5ASO2- R5B,

(k) -NR5AC(=O)R5B, or

(1) -NR5AC(=O)-O-R5B. In particular embodiments of compounds of formula (I), one of Rl and R2 is fluoro and the other is hydrogen. In other embodiments of compounds of formula (I), both of Rl and R2 are fluoro. In other embodiments of compounds of formula (I), both of Rl and R2 are hydrogen.

In particular embodiments of the compounds of formula (I), R4 is hydrogen. In one embodiment, the invention is directed to methods of treating a patient

(preferably a human) for diseases in which the Ml receptor is involved, such as Alzheimer's Disease, cognitive impairment, schizophrenia, pain disorders and sleep disorders, by administering to the patient a therapeutically effective amount of a compound of general formula

(D- The invention is also directed to the use of a compound of formula (I) for treating diseases or disorders in which the Ml receptor is involved, such as Alzheimer's disease, cognitive impairment, schizophrenia, pain disorders and sleep disorders.

The invention is also directed to medicaments or pharmaceutical compositions for treating diseases or disorders in which the Ml receptor is involved, such as Alzheimer's disease, cognitive impairment, schizophrenia, pain disorders and sleep disorders, which comprise a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The invention is further directed to a method for the manufacture of a medicament or a composition for treating diseases or disorders in which the Ml receptor is involved, such as Alzheimer's disease, cognitive impairment, schizophrenia, pain disorders and sleep disorders, comprising combining a compound of formula (I) with one or more pharmaceutically acceptable carriers.

Within the genus of compounds of formula (I), there is a sub-genus of compounds of formula (II):

(H) and pharmaceutically acceptable salts thereof, wherein R ¹ , R ² , R ³ and R ⁴ are as defined above. Preferably, R ³ is other than hydrogen.

Within the genus of compounds of formula (I) ₁ there is a sub-genus of compounds of formula (III):

(III) and pharmaceutically acceptable salts thereof, wherein R ¹ , R ² and R ⁴ are as defined above, and R ^3A and R ^3B group are from the group described above for R ³ . Preferably, at least one ^' of R ^3A and R ^3B is other than hydrogen.

Within the genus of compounds of formula (I), there is a sub-genus of compounds of formula (IV):

(IV) and pharmaceutically acceptable salts thereof, wherein R ¹ , R ² , R ³ and R ⁴ are as defined above. Preferably, R ³ is other than hydrogen. Within the genus of compounds of formula (I), there is a sub-genus of compounds of formula (V):

(V) and pharmaceutically acceptable salts thereof, wherein R ¹ , R ² and R ⁴ are as defined above, and R ^3A and R ^3B group are from the group described above for R ³ . Preferably, at least one of R ^3A and R ^3B is other than hydrogen.

Within the genus of compounds of formula (I), there is a sub-genus of compounds of formula (VI):

(VI) and pharmaceutically acceptable salts thereof, wherein R ¹ , R ² , R ³ and R ⁴ are as defined above. Preferably, R ³ is other than hydrogen.

Within the genus of compounds of formula (T), there is a sub-genus of compounds of formula (Vn):

(VlI) and pharmaceutically acceptable salts thereof, wherein R ¹ , R ² , R ³ and R ⁴ are as defined above. Preferably, R ³ is other than hydrogen. Within the genus of compounds of formula (I), there is a sub-genus of compounds of formula (VIII):

(VIII) and pharmaceutically acceptable salts thereof, wherein R . 1 , D R2 , D R3 and R are as defined above. Preferably, R ³ is other than hydrogen.

Within the genus of compounds of formula (I), there is a sub-genus of compounds of formula (IX):

(IX) and pharmaceutically acceptable salts thereof, wherein R ¹ , R ² , R ³ and R ⁴ are as defined above. Preferably, R ³ is other than hydrogen. Specific embodiments of formula (I) are described herein as Examples 1 -373, or a pharmaceutically acceptable salt thereof.

The invention is also directed to methods of treating a patient (preferably a human) for diseases or disorders in which the Ml receptor is involved, such as Alzheimer's Disease, cognitive impairment, schizophrenia, pain disorders and sleep disorders, by administering to the patient a therapeutically effective amount of a compound of formulae (IT) to (IX), or a pharmaceutically acceptable salt thereof.

The invention is also directed to the use of a compound of formulae (II) to (IX) for treating a disease or disorder in which the Ml receptor is involved, such as Alzheimer's Disease, cognitive impairment, schizophrenia, pain disorders and sleep disorders, by administering to the patient a compound of formulae (II) to (IX), or a pharmaceutically acceptable salt thereof.

The invention is also directed to medicaments or pharmaceutical compositions for the treatment of diseases or disorders in a patient (preferably a human) in which the Ml receptor is involved, such as Alzheimer's Disease, cognitive impairment, schizophrenia, pain disorders, and sleep disorders, which comprise a compound of formulae (II) to (IX), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The invention is also directed to a method for the manufacture of a medicament or a pharmaceutical composition for treating diseases in which Ml receptor is involved, such as Alzheimer's Disease, cognitive impairment, schizophrenia, pain disorders, and sleep disorders, by combining a compound of formulae (II) to (IX), or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier.

Where a variable occurs more than once in any of Formulas (I) to (DC) or in a substituent thereof, the individual occurrences of that variable are independent of each other, unless otherwise specified.

As used herein, in particular in the definitions of R2, R3, R4A and R^B ₁ the term "alkyl," by itself or as part of another substituent, means a saturated straight or branched chain

hydrocarbon radical having the number of carbon atoms designated (e.g., C]. \Q alkyl means an alkyl group having from one to ten carbon atoms). Preferred alkyl groups for use in the invention are Cl-6 alkyl groups, having from one to six atoms. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like. Co alkyl means a bond.

As used herein, in particular in the definition of R.2, the term "alkenyl," by itself or as part of another substituent, means a straight or branched chain hydrocarbon radical having a single carbon-carbon double bond and the number of carbon atoms designated (e.g., C2-10 alkenyl means an alkenyl group having from two to ten carbon atoms). Preferred alkenyl groups for use in the invention are C2-6 alkenyl groups, having from two to six carbon atoms.

Exemplary alkenyl groups include ethenyl and propenyl.

As used herein, in particular in the definition of R.2, the term "alkynyl," by itself or as part of another substituent, means a straight or branched chain hydrocarbon radical having a single carbon-carbon triple bond and the number of carbon atoms designated (e.g., C2-10 alkynyl means an alkynyl group having from two to ten carbon atoms). Preferred alkynyl groups for use in the invention are C2-6 alkynyl groups, having from two to six carbon atoms. Exemplary alkenyl groups include ethynyl and propynyl.

As used herein, in particular in the definitions of R2, R3, R4A and R4B ₍ the term

"aryl," by itself or as part of another substituent, means an aromatic cyclic hydrocarbon radical. Preferred aryl groups have from six to ten carbons atoms. The term "aryl" includes multiple ring systems as well as single ring systems. Preferred aryl groups for use in the invention include phenyl and naphthyl.

The term "aryl" also includes fused cyclic hydrocarbon rings which are partially aromatic (i.e., one of the fused rings is aromatic and the other is non-aromatic). An exemplary aryl group which is partially aromatic is indanyl.

As used herein, the term "halo" or "halogen" includes fluoro, chloro, bromo and iodo.

As used herein, in particular in the definition of R2, the term "heteroaryl," by itself or as part of another substituent, means a cyclic or polycyclic group having ring carbon atoms and at least one ring heteroatom (O, N or S), wherein at least one of the constituent rings is aromatic. Exemplary heteroaryl groups for use in the invention include carbazolyl, carbolinlyl, chromenyl, cinnolinyl, furanyl, benzofuranyl, benzofurazanyl, isobenzofuranyl, imidazolyl, benzimidazolyl, benzimidazolonyl, indazolyl, indolyl, isoindolyl, indolinyl, indolazinyl, indynyl, oxadiazolyl.oxazolyl, benzoxazolyl, isoxazolyl, pyranyl, pyrazinyl, pyrazolyl, benzopyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinolyl, isoquinolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, thienyl, benzothioenyl, benzothiazolyl, quinoxalinyl, triazinyl and triazolyl, and N-oxides thereof.

Preferred R.2 heteroaryl groups have from 5 to 12 ring atoms. In one such embodiment, the heteroaryl groups have 5 or 6 ring atoms.

For example, one subgroup of R2 heteroaryl groups have 5 or 6 ring atoms and a single heteroatom, which is nitrogen. Exemplary heteroaryl groups in this embodiment are pyridyl and pyrrolyl.

Another subgroup of R ² heteroaryl groups have 5 or 6 ring atoms and two heteroatoms, which are selected from sulfur and nitrogen. Exemplary heteroaryl groups in this embodiment are pyrazolyl, imidazolyl, thienyl and isothiazolyl.

Another subgroup of R2 heteroaryl groups have 7or 8 ring atoms and two heteroatoms, which are selected from oxygen, sulfur and nitrogen. Exemplary heteroaryl groups in this embodiment are benzoxazolyl, benzothiazolyl and quinoxalinyl.

The term "heteroaryl" also includes fused cyclic heterocyclic rings which are partially aromatic (i.e., one of the fused rings is aromatic and the other is non-aromatic). An exemplary heteroaryl group which is partially aromatic is benzodioxol. When a heteroaryl group as defined herein is substituted, the substituent may be bonded to a ring carbon atom of the heteroaryl group, or on a ring heteroatom (i.e., a nitrogen, oxygen or sulfur), which has a valence which permits substitution. Preferably, the substituent is bonded to a ring carbon atom. Similarly, when a heteroaryl group is defined as a substituent herein, the point of attachment may be at a ring carbon atom of the heteroaryl group, or on a ring heteroatom (i.e., a nitrogen, oxygen or sulfur), which has a valence which permits attachment. Preferably, the attachment is at a ring carbon atom.

The compounds of the invention may have one or more asymmetric centers. Compounds with asymmetric centers give rise to enantiomers (optical isomers), diastereomers (configurational isomers) or both, and it is intended that all of the possible enantiomers and diastereomers in mixtures and as pure or partially purified compounds are included within the scope of this invention. The present invention is meant to encompass all such isomeric forms of the compounds of formulae (T) to (IX).

Formulae (I) to (IX) are shown above without a definite stereochemistry at certain positions. The present invention includes all stereoisomers of formulae (I) to (VI) and pharmaceutically acceptable salts thereof.

The independent syntheses of the enantiomerically or diastereomerically enriched compounds, or their chromatographic separations, may be achieved as known in the art by appropriate modification of the methodology disclosed herein. Their absolute stereochemistry may be determined by the x-ray crystallography of crystalline products or crystalline intermediates that are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.

If desired, racemic mixtures of the compounds may be separated so that the individual enantiomers or diastereomers are isolated. The separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography. The coupling reaction is often the formation of salts using an enantiomerically pure acid or base. The diastereomeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue. The racemic mixture of the compounds can also be separated directly by chromatographic methods using chiral stationary phases, which methods are well known in the art.

Alternatively, any enantiomer or diastereomer of a compound may be obtained by stereoselective synthesis using optically pure starting materials or reagents of known configuration by methods well known in the art.

The compounds of the invention may be prepared according to the following reaction Schemes, in which variables are as defined before or are derived, using readily available starting materials, from reagents and conventional synthetic procedures. It is also possible to use variants which are themselves known to those of ordinary skill in organic synthesis art, but are not mentioned in greater detail.

Ethyl 8-fluoro-4-oxo-l,4-dihydroquinoline-3-carboxylate 1 is a known compound (Scheme 1 ) and can be alkylated with the appropriate halide or mesylate such as 2 in a solvent like DMF using a base like potassium carbonate. Ethyl ester 3 can undergo hydrolysis using a base like lithium hydroxide in a solvent like dioxane. Acid 4 can undergo a cross coupling with boronic acid 5 using a metal like palladium (0), a phosphine ligand such as tri fert-butyl phosphine, with a base like cesium carbonate in a solvent like THF, with conventional or microwave heating to afford Example 1.

Scheme 1

Example 1

As shown in Scheme 2, ethyl ester 6 can undergo a cross-coupling similar to Scheme 1 with a boronate ester such as 7 as shown to afford compound 8. In this case, the tert- butoxycarbonyl group present in 7 may fall off under the reaction conditions, or can be removed using an acid such as HCl in a solvent like ethyl acetate. Pyrazole 8 may be alkylated with an alkyl halide such a ethyl bromide using a base like sodium hydride in DMF. Subsequent hydrolysis of the ethyl ester using a base like sodium hydroxide affords Example 2.

Carboxylic acid 9 can undergo an N-arylation reaction with a heterocycle like imidazole in the presence of a copper catalyst like copper iodide, a ligand such as 10, employing a base like cesium carbonate, in a solvent like N-methyl pyrrolidone, with conventional or microwave heating to afford Example 3.

Scheme 3

The chloride in acid 11 may be directly displaced by nucleophiles as shown in Scheme 4. Heating 11 in a solvent like DMSO with an amine such as piperidine affords Example 372. Displacement of the chloride with a sulfur nucleophile such as sodium thiomethoxide in DMSO, followed by subsequent oxidation of 12 with a reagent like oxone in methanol-water affords Example 373.

Scheme 4

11 Example 372

NaSMe DMSO

12 Example 373

The present invention also provides a method for the synthesis of compounds useful as intermediates in the preparation of compounds of the invention During any of the above synthetic sequences it may be necessary 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, 1991. The protecting groups may be removed at a convenient sequent stage using methods known from the art.

Specific embodiments of the compounds of the invention, and methods of making them, are described in the Examples herein.

The term "substantially pure" means that the isolated material is at least 90% pure, and preferably 95% pure, and even more preferably 99% pure as assayed by analytical techniques known in the art.

As used herein, the term "muscarinic Ml receptor" refers to one of the five subtypes of the muscarinic acetylcholine receptor, which is from the superfamily of G-protein coupled receptors. The family of muscarinic receptors is described, for example, in Pharmacol Ther, 1993, 58:319-379; Eur J Pharmacol, 1996, 295:93-102, and MoI Pharmacol, 2002, 61 :1297-1302. The muscarinic receptors are known to contain one or more allosteric sites, which may alter the affinity with which muscarinic ligands bind to the primary binding or orthosteric sites. See, e.g., S. Lazareno et al, MoI Pharmacol, 2002, 62:6, 1491-1505.

As used herein, the terms "positive allosteric modulator" and "allosteric potentiator" are used interchangeably, and refer to a ligand which interacts with an allosteric site of a receptor to activate the primary binding site. The compounds of the invention are positive allosteric modulators of the muscarinic Ml receptor. For example, a modulator or potentiator may directly or indirectly augment the response produced by the endogenous ligand (such as acetylcholine or xanomeline) at the orthosteric site of the muscarinic Ml receptor in an animal, in particular, a human.

The actions of ligands at allosteric receptor sites may also be understood according to the "allosteric ternary complex model," as known by those skilled in the art. The allosteric ternary complex model is described with respect to the family of muscarinic receptors in Birdsall et al, Life Sciences, 2001 , 68:2517-2524. For a general description of the role of allosteric binding sites, see Christopoulos, Nature Reviews: Drug Discovery, 2002, 1 :198-210.

It is believed that the compounds of the invention bind to an allosteric binding site that is distinct from the orthosteric acetylcholine site of the muscarinic Ml receptor, thereby augmenting the response produced by the endogenous ligand acetylcholine at the orthosteric site of the Ml receptor. It is also believed that the compounds of the invention bind to an allosteric site which is distinct from the xanomeline site of the muscarinic Ml receptor, thereby augmenting the response produced by the endogenous ligand xanomeline at the orthosteric site of the Ml receptor. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. The compounds of the invention may be mono, di or tris salts, depending on the number of acid functionalities present in the free base form of the compound. Free bases and salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like.

Salts in the solid form may exist in more than one crystal structure, and may also be in the form of hydrates. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N.N'-dibenzylethylene-diamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids

include acetic, trifluoroacetic, benzenesυlfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. The present invention is directed to the use of the compounds of formulas (I) to

(IX) disclosed herein as Ml allosteric modulators in a patient or subject such as a mammal in need of such activity, comprising the administration of an effective amount of the compound. In addition to humans, a variety of other mammals can be treated according to the method of the present invention. The compounds of the present invention have utility in treating or ameliorating

Alzheimer's disease. The compounds may also be useful in treating or ameliorating other diseases mediated by the muscarinic Ml receptor, such as schizophrenia, sleep disorders, pain disorders (including acute pain, inflammatory pain and neuropathic pain) and cognitive disorders (including mild cognitive impairment). Other conditions that may be treated by the compounds of the invention include Parkinson's Disease, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), asthma, urinary incontinence, glaucoma, schizophrenia, Trisomy 21 (Down Syndrome), cerebral amyloid angiopathy, degenerative dementia, Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D), Creutzfeld-Jakob disease, prion disorders, amyotrophic lateral sclerosis, progressive supranuclear palsy, head trauma, stroke, pancreatitis, inclusion body myositis, other peripheral amyloidoses, diabetes, autism and atherosclerosis.

In preferred embodiments, the compounds of the invention are useful in treating Alzheimer's Disease, cognitive disorders, schizophrenia, pain disorders and sleep disorders. For example, the compounds may be useful for the prevention of dementia of the Alzheimer's type, as well as for the treatment of early stage, intermediate stage or late stage dementia of the Alzheimer's type.

Potential schizophrenia conditions or disorders for which the compounds of the invention may be useful include one or more of the following conditions or diseases: schizophrenia or psychosis including schizophrenia (paranoid, disorganized, catatonic or undifferentiated), schizophreniform disorder, schizoaffective disorder, delusional disorder, brief psychotic disorder, shared psychotic disorder, psychotic disorder due to a general medical condition and substance-induced or drug-induced (phencyclidine, ketaniine and other dissociative anaesthetics, amphetamine and other psychostimulants and cocaine) psychosispsychotic disorder, psychosis associated with affective disorders, brief reactive psychosis, schizoaffective psychosis, "schizophrenia-spectrum" disorders such as schizoid or schizotypal personality disorders, or illness associated with psychosis (such as major depression, manic depressive (bipolar) disorder, Alzheimer's disease and post-traumatic stress syndrome),

including both the positive and the negative symptoms of schizophrenia and other psychoses; cognitive disorders including dementia (associated with Alzheimer's disease, ischemia, multi- infarct dementia, trauma, vascular problems or stroke, HIV disease, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jacob disease, perinatal hypoxia, other general medical conditions or substance abuse); delirium, amnestic disorders or age related cognitive decline.

In another specific embodiment, the present invention provides a method for treating schizophrenia or psychosis comprising: administering to a patient in need thereof an effective amount of a compound of the present invention. Particular schizophrenia or psychosis pathologies are paranoid, disorganized, catatonic or undifferentiated schizophrenia and substance-induced psychotic disorder. At present, the text revision of the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR) (2000, American Psychiatric Association, Washington DC) provides a diagnostic tool that includes paranoid, disorganized, catatonic or undifferentiated schizophrenia and substance-induced psychotic disorder. As used herein, the term "schizophrenia or psychosis" includes treatment of those mental disorders as described in DSM-IV-TR. The skilled artisan will recognize that there are alternative nomenclatures, nosologies and classification systems for mental disorders, and that these systems evolve with medical and scientific progress. Thus the term "schizophrenia or psychosis" is intended to include like disorders that are described in other diagnostic sources. Examples of combinations of the compounds include combinations with agents for the treatment of schizophrenia, for example in combination with sedatives, hypnotics, anxiolytics, antipsychotics, antianxiety agents, cyclopyrrolones, imidazopyridines, pyrazolopyrimidines, minor tranquilizers, melatonin agonists and antagonists, melatonergic agents, benzodiazepines, barbiturates, 5HT-2 antagonists, and the like, such as: adinazolam, allobarbital, alonimid, alprazolam, amisulpride, amitriptyline, amobarbital, amoxapine, aripiprazole, bentazepam, benzoctamine, brotizolam, bupropion, busprione, butabarbital, butalbital, capuride, carbocloral, chloral betaine, chloral hydrate, clomipramine, clonazepam, cloperidone, clorazepate, chlordiazepoxide, clorethate, chlorpromazine, clozapine, cyprazepam, desipramine, dexclamol, diazepam, dichloralphenazone, divalproex, diphenhydramine, doxepin, estazolam, ethchlorvynol, etomidate, fenobam, flunhrazepam, flupentixol, fluphenazine, flurazepam, fluvoxamine, fluoxetine, fosazepam, glutethimide, halazepam, haloperidol, hydroxyzine, imipramine, lithium, lorazepam, lormetazepam, maprotiline, mecloqualone, melatonin, mephobarbital, meprobamate, methaqualone, midafiur, midazolam, nefazodone, nisobamate, nitrazepam, nortriptyline, olanzapine, oxazepam, paraldehyde, paroxetine, pentobarbital, perlapine, perphenazine, phenelzine, phenobarbital, prazepam, promethazine, propofol, protriptyline, quazepam, quetiapine, reclazepam, risperidone, roletamide, secobarbital, sertraline, suproelone, temazepam, thioridazine, thiothixene, tracazolate, tranylcypromaine,

trazodone, triazolam, trepipam, tricetamide, triclofos, trifluoperazine, trimetozine, trimipramine, uldazepam, venlafaxine, zaleplon, ziprasidone, zolazepam, Zolpidem, and salts thereof, and combinations thereof, and the like, or the subject compound may be administered in conjunction with the use of physical methods such as with light therapy or electrical stimulation. In another embodiment, the subject compound may be employed in combination with levodopa (with or without a selective extracerebral decarboxylase inhibitor such as carbidopa or benserazide), anticholinergics such as biperiden (optionally as its hydrochloride or lactate salt) and trihexyphenidyl (benzhexol) hydrochloride, COMT inhibitors such as entacapone, MOA-B inhibitors, antioxidants, A2a adenosine receptor antagonists, cholinergic agonists, NMDA receptor antagonists, serotonin receptor antagonists and dopamine receptor agonists such as alentemol, bromocriptine, fenoldopam, lisuride, naxagolide, pergolide and pramipexole. It will be appreciated that the dopamine agonist may be in the form of a pharmaceutically acceptable salt, for example, alentemol hydrobromide, bromocriptine mesylate, fenoldopam mesylate, naxagolide hydrochloride and pergolide mesylate. In another embodiment, the subject compound may be employed in combination with a compound from the phenothiazine, thioxanthene, heterocyclic dibenzazepine, butyrophenone, diphenylbutylpiperidine and indolone classes of neuroleptic agent. Suitable examples of phenothiazines include chlorpromazine, mesoridazine, thioridazine, acetophenazine, fluphenazine, perphenazine and trifluoperazine. Suitable examples of thioxanthenes include chlorprothixene and thiothixene. An example of a dibenzazepine is clozapine. An example of a butyrophenone is haloperidol. An example of a diphenylbutylpiperidine is pimozide. An example of an indolone is molindolone. Other neuroleptic agents include loxapine, sulpiride and risperidone. It will be appreciated that the neuroleptic agents when used in combination with the subject compound may be in the form of a pharmaceutically acceptable salt, for example, chlorpromazine hydrochloride, mesoridazine besylate, thioridazine hydrochloride, acetophenazine maleate, fluphenazine hydrochloride, flurphenazine enathate, fluphenazine decanoate, trifluoperazine hydrochloride, thiothixene hydrochloride, haloperidol decanoate, loxapine succinate and molindone hydrochloride. Perphenazine, chlorprothixene, clozapine, haloperidol, pimozide and risperidone are commonly used in a non-salt form. Thus, the subject compound may be employed in combination with acetophenazine, alentemol, aripiprazole, amisuipride, benzhexol, bromocriptine, biperiden, chiorpromazine, chlorprothixene, clozapine, diazepam, fenoldopam, fluphenazine, haloperidol, levodopa, levodopa with benserazide, levodopa with carbidopa, lisuride, loxapine, mesoridazine, molindolone, naxagolide, olanzapine, pergolide, perphenazine, pimozide, pramipexole, quetiapine, risperidone, sulpiride, tetrabenazine, frihexyphenidyl, thioridazine, thiothixene, trifluoperazine or ziprasidone.

Potential sleep conditions or disorders for which the compounds of the invention may be useful include enhancing sleep quality; improving sleep quality; augmenting sleep

41493

maintenance; increasing the value which is calculated from the time that a subject sleeps divided by the time that a subject is attempting to sleep; decreasing sleep latency or onset (the time it takes to fall asleep); decreasing difficulties in falling asleep; increasing sleep continuity; decreasing the number of awakenings during sleep; decreasing nocturnal arousals; decreasing the time spent awake following the initial onset of sleep; increasing the total amount of sleep; reducing the fragmentation of sleep; altering the timing, frequency or duration of REM sleep bouts; altering the timing, frequency or duration of slow wave (i.e. stages 3 or 4) sleep bouts; increasing the amount and percentage of stage 2 sleep; promoting slow wave sleep; enhancing EEG-delta activity during sleep; increasing daytime alertness; reducing daytime drowsiness; treating or reducing excessive daytime sleepiness; insomnia; hypersomnia; narcolepsy; interrupted sleep; sleep apnea; wakefulness; nocturnal myoclonus; REM sleep interruptions; jet- lag; shift workers' sleep disturbances; dyssomnias; night terror; insomnias associated with depression, emotional/mood disorders, as well as sleep walking and enuresis, and sleep disorders which accompany aging; Alzheimer's sundowning; conditions associated with circadian rhythmicity as well as mental and physical disorders associated with travel across time zones and with rotating shift-work schedules, conditions due to drugs which cause reductions in REM sleep as a side effect; syndromes which are manifested by non-restorative sleep and muscle pain or sleep apnea which is associated with respiratory disturbances during sleep; and conditions which result from a diminished quality of sleep. Pain disorders for which the compounds of the invention may be useful include neuropathic pain (such as postherpetic neuralgia, nerve injury, the "dynias", e.g., vulvodynia, phantom limb pain, root avulsions, painful diabetic neuropathy, painful traumatic mononeuropathy, painful polyneuropathy); central pain syndromes (potentially caused by virtually any lesion at any level of the nervous system); postsurgical pain syndromes (eg, postmastectomy syndrome, postthoracotomy syndrome, stump pain); bone and joint pain (osteoarthritis), repetitive motion pain, dental pain, cancer pain, myofascial pain (muscular injury, fibromyalgia); perioperative pain (general surgery, gynecological), chronic pain, dysmennorhea, as well as pain associated with angina, and inflammatory pain of varied origins (e.g. osteoarthritis, rheumatoid arthritis, rheumatic disease, teno- synovitis and gout), headache, migraine and cluster headache, headache, primary hyperalgesia, secondary hyperalgesia, primary allodynia, secondary allodynia, or other pain caused by central sensitization.

Compounds of the invention may also be used to treat or prevent dyskinesias. Furthermore, compounds of the invention may be used to decrease tolerance and/or dependence to opioid treatment of pain, and for treatment of withdrawal syndrome of e.g., alcohol, opioids, and cocaine.

The subject or patient to whom the compounds of the present invention is administered is generally a human being, male or female, in whom Ml allosteric modulation is is

9 041493

desired, but may also encompass other mammals, such as dogs, cats, mice, rats, cattle, horses, sheep, rabbits, monkeys, chimpanzees or other apes or primates, for which treatment of the above noted disorders is desired.

The compounds of the present invention may be used in combination with one or more other drugs in the treatment of diseases or conditions for which the compounds of the present invention have utility, where the combination of the drugs together are safer or more effective than either drug alone. Additionally, the compounds of the present invention may be used in combination with one or more other drugs that treat, prevent, control, ameliorate, or reduce the risk of side effects or toxicity of the compounds of the present invention. Such other drugs may be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with the compounds of the present invention. Accordingly, the pharmaceutical compositions of the present invention include those that contain one or more other active ingredients, in addition to the compounds of the present invention. The combinations may be administered as part of a unit dosage form combination product, or as a kit or treatment protocol wherein one or more additional drugs are administered in separate dosage forms as part of a treatment regimen.

Examples of combinations of the compounds of the present invention include combinations with anti-Alzheimer's Disease agents, for example beta-secretase inhibitors; alpha 7 nicotinic agonists, such as ABT089, SSRl 80711 and MEM63908; ADAM 10 ligands or activators; gamma-secretase inhibitors, such as LY450139 and TAK 070; gamma secretase modulators; tau phosphorylation inhibitors; glycine transport inhibitors; LXR β agonists; ApoE4 conformational modulators; NR2B antagonists; androgen receptor modulators; blockers of Aβ oligomer formation; 5-HT4 agonists, such as PRX-03140; 5-HT6 antagonists, such as GSK 742467, SGS-518, FK-962, SL-65.0155, SRA-333 and xaliproden; 5-HTla antagonists, such as lecozotan; p25/CDK5 inhibitors; NK1/NK3 receptor antagonists; COX-2 inhibitors; HMG-CoA reductase inhibitors; NSAIDs including ibuprofen; vitamin E; anti-amyloid antibodies (including anti-amyloid humanized monoclonal antibodies), such as bapineuzumab, ACCOOl , CAD 106, AZD3102, Hl 2Al IVl; anti-inflammatory compounds such as (R)-flurbiprofen, nitroflurbiprofen, ND-1251, VP-025, HT-0712 and EHT-202; PPAR gamma agonists, such as pioglitazone and rosiglitazone; CB-I receptor antagonists or CB-I receptor inverse agonists, such as AVE 1625; antibiotics such as doxycycline and rifampin; N-methyl-D-aspartate (NMDA) receptor antagonists, such as memantine, neramexane and EVTlOl ; cholinesterase inhibitors such as galantamine, rivastigmine, donepezil, tacrine, phenserine, ladostigil and ABT-089; growth hormone secretagogues such as ibutamoren, ibutamoren mesylate, and capromorelin; histamine H3 receptor antagonists such as ABT-834, ABT 829, GSK 189254 and CEP 16795;

AMPA agonists or AMPA modulators, such as CX-717, LY 451395, LY404187 and S-18986; PDE IV inhibitors, including MEM1414, HT0712 and AVE8112; GABAA inverse agonists;

GSK3β inhibitors, including AZD1080, SAR502250 and CEPl 6805; neuronal nicotinic agonists; selective Ml agonists; HDAC inhibitors; and microtubule affinity regulating kinase (MARK) ligands; or other drugs that affect receptors or enzymes that either increase the efficacy, safety, convenience, or reduce unwanted side effects or toxicity of the compounds of the present invention.

Examples of combinations of the compounds include combinations with agents for the treatment of pain, for example non-steroidal anti-inflammatory agents, such as aspirin, diclofenac, duflunisal, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, naproxen, oxaprozin, piroxicam, sulindac and tolmetin; COX-2 inhibitors, such as celecoxib, rofecoxib, valdecoxib, 406381 and 644784; CB-2 agonists, such as 842166 and SAB378; VR-I antagonists, such as AMG517, 705498, 782443, PAC20030, Vl 14380 and A425619; bradykinin B 1 receptor antagonists, such as SSR240612 and NVPSAA 164; sodium channel blockers and antagonists, such as VX409 and SPI860; nitric oxide synthase (NOS) inhibitors (including iNOS and nNOS inhibitors), such as SD6010 and 274150; glycine site antagonists, including lacosamide; neuronal nicotinic agonists, such as ABT 894; NMDA antagonists, such as

AZD4282; potassium channel openers; AMPA/kainate receptor antagonists; calcium channel blockers, such as ziconotide and NMED160; GABA-A receptor IO modulators (e.g., a GABA- A receptor agonist); matrix metalloprotease (MMP) inhibitors; thrombolytic agents; opioid analgesics such as codeine, fentanyl, hydromorphone, levorphanol, meperidine, methadone, morphine, oxycodone, oxymorphone, pentazocine, propoxyphene; neutrophil inhibitory factor (NlF); pramipexole, ropinirole; anticholinergics; amantadine; monoamine oxidase B15 ("MAO- B") inhibitors; 5HT receptor agonists or antagonists; mGlu5 antagonists, such as AZD9272; alpha agonists, such as AGNXX/YY; neuronal nicotinic agonists, such as ABT894; NMDA receptor agonists or antagonists, such as AZD4282; NKI antagonists; selective serotonin reuptake inhibitors ("SSRI") and/or selective serotonin and norepinephrine reuptake inhibitors ("SSNRI"), such as duloxetine; tricyclic antidepressant drugs, norepinephrine modulators; lithium; valproate; gabapentin; pregabalin; rizatriptan; zolmitriptan; naratriptan and sumatriptan. The compounds of the present invention may be administered in combination with compounds useful for enhancing sleep quality and preventing and treating sleep disorders and sleep disturbances, including e.g., sedatives, hypnotics, anxiolytics, antipsychotics, antianxiety agents, antihistamines, benzodiazepines, barbiturates, cyclopyrrolones, orexin antagonists, alpha- 1 antagonists, GABA agonists, 5HT-2 antagonists including 5HT-2A antagonists and 5HT- 2A/2C antagonists, histamine antagonists including histamine H3 antagonists, histamine H3 inverse agonists, imidazopyridines, minor tranquilizers, melatonin agonists and antagonists, melatonergic agents, other orexin antagonists, orexin agonists, prokineticin agonists and antagonists, pyrazolopyrimidines, T-type calcium channel antagonists, triazolopyridines, and the like, such as: adinazolam, allobarbital, alonimid, alprazolam, amitriptyline, amobarbital,

amoxapine, armodafinil, APD- 125, bentazepam, benzoctamine, brotizolam, bupropion, busprione, butabarbital, butalbital, capromorelin, capuride, carbocloral, chloral betaine, chloral hydrate, chlordiazepoxide, clomipramine, clonazepam, cloperidone, clorazepate, clorethate, clozapine, conazepam, cyprazepam, desipramine, dexclamol, diazepam, dichloralphenazone, divalproex, diphenhydramine, doxepin, EMD-281014, eplivanserin, estazolam, eszopiclone, ethchlorynol, etomidate, fenobam, flunitrazepam, flurazepam, fluvoxamine, fluoxetine, fosazepam, gaboxadol, glutethimide, halazepam, hydroxyzine, ibutamoren, imipramine, indiplon, lithium, lorazepam, lormetazepam, LY-156735, maprotiline, MDL-100907, mecloqualone, melatonin, mephobarbital, meprobamate, methaqualone, methyprylon, midaflur, midazolam, modafinil, nefazodone, NGD-2-73, nisobamate, nitrazepam, nortriptyline, oxazepam, paraldehyde, paroxetine, pentobarbital, perlapine, perphenazine, phenelzine, phenobarbital, prazepam, promethazine, propofol, protriptyline, quazepam, ramelteon, reclazepam, roletamide, secobarbital, sertraline, suproclone, TAK-375, temazepam, thioridazine, tiagabine, tracazolate, tranylcypromaine, trazodone, triazolam, trepipam, tricetamide, triclofos, trifluoperazine, trimetozine, trimipramine, uldazepam, venlafaxine, zaleplon, zolazepam, zopiclone, Zolpidem, and salts thereof, and combinations thereof, and the like, or the compound of the present invention may be administered in conjunction with the use of physical methods such as with light therapy or electrical stimulation.

In another embodiment, the subject compound may be employed in combination with levodopa (with or without a selective extracerebral decarboxylase inhibitor such as carbidopa or benserazide), anticholinergics such as biperiden (optionally as its hydrochloride or lactate salt) and trihexyphenidyl (benzhexol) hydrochloride, COMT inhibitors such as entacapone, MOA-B inhibitors, antioxidants, A2a adenosine receptor antagonists, cholinergic agonists and dopamine receptor agonists such as alentemol, bromocriptine, fenoldopam, lisuride, naxagolide, pergolide and pramipexole.

The term "composition" as used herein is intended to encompass a product comprising specified ingredients in predetermined amounts or proportions, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. This term in relation to pharmaceutical compositions is intended to encompass a product comprising one or more active ingredients, and an optional carrier comprising inert ingredients, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. In general, pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the

pharmaceutical composition the active compound, which is a compound of formulae (1) to (IX), is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.

The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil- in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the invention, or pharmaceutically acceptable salts thereof, may also be administered by controlled release means and/or delivery devices. Pharmaceutical compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically palatable preparations. Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

A tablet containing the composition of this invention may be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Each tablet preferably contains from about 0.1 mg to about 500 mg of the active ingredient and each cachet or capsule preferably containing from about 0.1 mg to about 500 mg of the active ingredient.

Compositions for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Other pharmaceutical compositions include aqueous suspensions, which contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. In addition, oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may also contain various excipients. The pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions, which may also contain excipients such as sweetening and flavoring agents.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension, or in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi.

Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, or the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations may be prepared via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency. Pharmaceutical compositions of this invention can also be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art.

By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The terms "administration of or "administering a" compound should be understood to mean providing a compound of the invention to the individual in need of treatment in a form that can be introduced into that individual's body in a therapeutically useful form and therapeutically useful amount, including, but not limited to: oral dosage forms, such as tablets, capsules, syrups, suspensions, and the like; injectable dosage forms, such as IV, IM, or IP, and

the like; transdermal dosage forms, including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and the like; and rectal suppositories.

The terms "effective amount" or "therapeutically effective amount" means the amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

As used herein, the term "treatment" or "treating" means any administration of a compound of the present invention and includes (1) inhibiting the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., arresting further development of the pathology and/or symptomatology), or (2) ameliorating the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., reversing the pathology and/or symptomatology).

The compositions containing compounds of the present invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The term "unit dosage form" is taken to mean a single dose wherein all active and inactive ingredients are combined in a suitable system, such that the patient or person administering the drug to the patient can open a single container or package with the entire dose contained therein, and does not have to mix any components together from two or more containers or packages. Typical examples of unit dosage forms are tablets or capsules for oral administration, single dose vials for injection, or suppositories for rectal administration. This list of unit dosage forms is not intended to be limiting in any way, but merely to represent typical examples of unit dosage forms.

The compositions containing compounds of the present invention may conveniently be presented as a kit, whereby two or more components, which may be active or inactive ingredients, carriers, diluents, and the like, are provided with instructions for preparation of the actual dosage form by the patient or person administering the drug to the patient. Such kits may be provided with all necessary materials and ingredients contained therein, or they may contain instructions for using or making materials or components that must be obtained independently by the patient or person administering the drug to the patient. When treating or ameliorating a disorder or disease for which compounds of the present invention are indicated, generally satisfactory results are obtained when the compounds of the present invention are administered at a daily dosage of from about 0.1 mg to about 100 mg per kg of animal body weight, preferably given as a single daily dose or in divided doses two to six times a day, or in sustained release form. The total daily dosage is from about 1.0 mg to about 2000 mg, preferably from about 0.1 mg to about 20 mg per kg of body weight. In the case of a 70 kg adult human, the total daily dose will generally be from about 7 mg to about 1 ,400 mg. This dosage regimen may be adjusted to provide the optimal therapeutic response. The

compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration to humans may conveniently contain from about 0.005 mg to about 2.5 g of active agent, compounded with an appropriate and convenient amount of carrier material. Unit dosage forms will generally contain between from about 0.005 mg to about 1000 mg of the active ingredient, typically 0.005, 0.01 mg, 0.05 mg, 0.25 mg, 1 mg, 5 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg or 1000 mg, administered once, twice or three times a day.

It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

Several methods for preparing the compounds of this invention are illustrated in the schemes and examples herein. Starting materials are made according to procedures known in the art or as illustrated herein. The following examples are provided so that the invention might be more fully understood, and are not to be construed as limiting the scope of the invention in any manner.

Intermediate A: (5-Bromopyridin-3-yl)methanol

A solution of 5-bromonicotinic acid (6.65 g, 32.9 mmol) in 75 mL of MeOH was heated to reflux for 4 hours, cooled to room temperature, and concentrated in vacuo. The residue was diluted with aqueous sodium bicarbonate and EtOAc, and the organic fraction was washed with brine, dried, filtered, and concentrated in vacuo to provide crude methyl 5-bromonicotinate. To a solution of the above compound in 100 mL of THF at -70 ⁰ C was added LAH (1 M in THF, 0.036

mL, 0.036 mmol). After 1 hour, 1 mL of water and 1 mL 1 NNaOH were added, followed by 2 mL of aqueous ammonium chloride. The mixture was diluted with ether and sodium sulfate, filtered, and concentrated in vacuo to provide the title compound as a yellow oil.

Intermediate B: 5-Broτno-2-(brornomethyl)pyrimidine

A solution of 5-bromo-2-iodopyrimidine (5.00 g, 17.6 mmol) in 25 mL of THF was cooled to 0 ⁰ C under nitrogen. Dimethyl zinc (2.0 M in toluene, 13.2 mL, 26.3 mmol) and palladium tetrakis (5 mol%) were added. After 5 hours, aqueous ammonium chloride (5 mL) was added, and the reaction mixture was warmed to room temperature. The mixture was diluted with ethyl acetate (30 mL) and washed with aqueous sodium bicarbonate (2 x 30 mL) and brine (30 mL). The combined organic extracts were dried, filtered and concentrated in vacuo to provide 5- bromo-2-methylpyridine that gave a proton NMR spectra consistent with theory and a mass ion (ES+) of 173.1 for M+H* ( ⁷⁹ Br). To a solution of the above compound (3.40 g, 19.6 mmol) in 100 mL of CCl ₄ (25 mL) was added N-bromosuccinimide (6.98 g, 39.2 mmol) and 2,2'-azobisisobutyronitrile (2.30 g, 14.0 mmol). The reaction was heated to 100 ⁰ C for 24 hours, then cooled to room temperature and washed with aqueous sodium bicarbonate and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The crude material was subjected to chromatography on silica gel eluting with 0-5% EtOAc in hexanes to yield the title compound as a pale yellow needles which gave a proton νMR spectrum consistent with theory and a mass ion (ES+) of 252.8 for M+H+.

Intermediate C: (5-Chloropyrzin-2-yl)methanol

A solution of 5-chloropyrazine-2-carbaldehyde (5.20 g, 36.5 mmol) in 50 mL of methanol was cooled to O ⁰ C. Sodium borohydride (1.45 g, 38.3 mmol) was added and the reaction mixture was allowed to stir at ambient temperature for 3 hour. Saturated aqueous ammonium chloride (5 mL) was added, and the methanol was removed in vacuo. The resulting residue was dissolved in ethyl acetate (50 mL) and washed with aqueous sodium bicarbonate (2 x 50 mL) and brine (50 mL).

The combined organic extracts were dried, filtered and concentrated in vacuo to provide the title compound that gave a proton NMR spectra consistent with theory.

Intermediate D: (6-Chloropyridazin-3-yl)methyl methanesulfonate

To a suspension of ό-chloropyridazine-S-carbaldehyde (2.50 g, 17.5 mmol) in 30 mL of methanol (30 mL) at 0°C was added sodium borohydride (0.26 g, 7.02 mmol). The reaction mixture was warmed to room temperature. After 2 hours, the reaction mixture was concentrated in vacuo and triturated with 20% MeOH in dichloromethane to provide (6-chloropyridazin-3-yl)methanol. To a solution of the above compound (0.30 g, 2.07 mmol) and triethylamine (0.87 mL, 6.23 mmol) in 5 mL of dichloromethane (5 mL) at O ⁰ C was added methanesulfonyl chloride (0.32 mL, 4.15 mmol) dropwise. The reaction was stirred for 20 minutes and concentrated in vacuo to provide the title compound that gave a mass ion (ES+) of 223.2 for M+H ⁺ .

EXAMPLE 1

5-{6-[(3-Carboxy-8-fluoro-4-oxoquinolin-l(4H)-yl)methyl]p yridine-3-yl}-2-fluoropyridinium

A solution of (5-bromo-pyridin-2-yl)-methanol (20.0 g, 106 mmol) in 150 mL of dichloromethane was cooled to 0 ⁰ C under nitrogen. Triethylamine (19.3 mL, 138 mmol) was added, followed by methanesulfonyl chloride (9.95 ml, 128 mmol). After 2.5 hours, the mixture was quenched with 50 mL of water and diluted with 100 mL of dichloromethane. The organic layer was washed with an additional 50 mL of water and then the aqueous layer was extracted

with 100 mL of methylene chloride. The organic extracts were dried over sodium sulfate, filtered and concentrated to provide (5-bromopyridin-2-yl)methyl methanesulfonate as a red solid that gave a proton NMR spectra consistent with theory and a mass ion (ES+) of 268.1 for MH-H ⁺ ( ⁸¹ Br).

5 To a solution of ethyl-8-fluoro-4-oxo-l,4-dihydroquinoline-3-carboxylate (5.00 g,

21.3 mmol) in 50 mL of DMF under nitrogen was added the above compound (8.48 g, 31.9 mmol), potassium iodide (0.176 g, 1.06 mmol), and potassium carbonate (3.53 g, 25.5 mmol). The reaction mixture was stirred at 6O ⁰ C for 15 hours, cooled to room temperature, and diluted with 200 mL of water. The resulting solid was washed with water (2 x 100 mLl) and diethyl0 ether (2 x 75 mL) and dried in vacuo to provide ethyl l-[(5-bromopyridin-2-yl)methyl]-8-fluoro- 4-oxo- l,4-dihydroquinoline-3-carboxylate that gave a proton NMR spectra consistent with theory and a mass ion (ES+) of 407.0 for M+H ⁺ ( ⁸¹ Br).

To a solution of the above compound (7.28 g, 17.97 mmol) in 40 mL of dioxane was added aqueous saturated LiOH to pH ~10.5. After 24 hours, the mixture was acidified with S 1 NHCl to pH 4. The resulting solid was washed with water (3 x 80 mL) and dried in vacuo to provide 1 -[(5-bromopyridin-2-yl)rnethyl]-8-fluoro-4-oxo-l ,4-dihydroquinoline-3-carboxylic acid that gave a proton NMR spectra consistent with theory and a mass ion (ES+) of 379.0 for M+H ⁺ ( ^8l Br).

To a solution of the above compound (0.200 g, 0.530 mmol), 2-fluoropyridine-5-0 boronic acid (0.082 g, 0.583 mmol), and 1 N cesium carbonate (1.06 ml, 1.06 mmol) in 2 mL THF under nitrogen was added bis(tri-/erM>utyl-phosphine)palladium(0) (10 mol%). The mixture was heated for 10 minutes in the microwave at 16O ⁰ C. The reaction was cooled to room temperature, and additional bis(trWerf-butyl-phosphine)palladium(0) (10 mol% ) was added. The mixture was heated again for 10 minutes in the microwave at 160 ⁰ C, then cooled to room5 temperature and stirred overnight with Quadrapure TU resin. The sample was filtered with methanol (10 mL) and concentrated in vacuo. The resulting residue was purified via reverse phase HPLC to provide the title compound which gave a proton νMR consistent with theory and a mass ion (ES+) of 394.0 for M+H ⁺ . ¹ H νMR (400 MHz, DMSO) δ 9.21 (s, 1 H), 8.79 (d, J= 1.7 Hz, IH), 8.59 (d, J = 2.6 Hz, IH), 8.33 (dt, 7= 8.2 Hz, 2.6 Hz, IH), 8.27 (dd, J= 8.1 Hz, 1.00 Hz, IH), 8.21 (dd, J = 8.2 Hz, 2.4 Hz, IH), 7.74 (m, IH), 7.65-7.58 (m, 2H), 7.31 (dd, J= 8.6 Hz, 2.6 Hz, IH), 6.09 (d, J = 4.0, 2H).

EXAMPLE 2

Ethyl 1 - { [5-( 1 -ethyl- 1 H-pyrazol-4-yl)pyridin-2-yl]methyl } -5 ,8-difiuoro-4-oxo- 1 ,4-5 dihydroquinoline-3-carboxylate

To a solution of ethyl l-[(5-bromopyridin-2-yl)methyl]-5,8-difluoro-4-oxo-l,4- dihydroquinoline-3-carboxylate (3.00 g, 7.09 mmol), fert-butyl 4-(4,4,5,5-tetramethyl- 1,3,2- dioxaborolan-2-yl)-lH-pyrazole-l-carboxylate(5.42 g, 18.4 mmol), and 1 N cesium carbonate (14.2 mL, 14.2 mmol) in 50 mL of TηF under nitrogen was added bis(tri-rm- butylphosphoranyl)palladium (10 mol%). The reaction mixture was heated to 100 ⁰ C for 20 hours, then cooled to room temperature. Ethyl acetate (100 mL) and water (100 mL) were added, and the resultant brown solid was collected to provide ethyl 5,8-difluoro-4-oxo-l-{[5-(lH- pyrazol-4-yl)pyridin-2-yl]methyl}-l,4-dihydroquinoline-3-car boxylate that gave a proton NMR spectra consistent with theory and a mass ion (ES+) of 411.0 for M+η*.

To a solution of the above compound (0.100 g, 0.244 mmol) in 1 mL of DMF was added sodium hydride (0.010 g, 0.26 mmol) and ethylbromide (0.053g, 0.49 mmol). After 1.5 hours, 1 N NaOH (0.40 mL, 0.40 mmol) was added and the reaction was stirred for an additional 16 hours. The reaction mixture was acidified with 6 N HCl to pη ~2, diluted with ethyl acetate (30 mL), and washed with aqueous sodium bicarbonate (2 x 30 mL) and brine (30 mL). The organic extract was dried, filtered and concentrated in vacuo to provide the title compound which gave a proton NMR spectrum consistent with theory and a mass ion (ES+) of 411.0 for M+η ⁺ : ¹ H NMR(400 MHz, d ⁶ -DMSO) δ 9.35 (s, IH), 8.90 (s, IH), 8.46 (s, IH), 8.20-8.17 (m, 2H), 8.14 (s, IH), 8.00-7.93 (m, IH), 7.65-7.59 (m, 2H), 6.18 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H), 1.60 (t, J = 7.1 Hz, 3H).

EXAMPLE 3 5,8-difluoro-l-{[2-(lH-imidazol-l-yl)pyridin-4-yl]methyl}-4- oxo-l ,4-dihydroquinoline-3- carboxylic acid

In a 0.5-2 mL Emrys™ process vial, a mixture of the l-[(2-bromopyridin-4-yl)methyl]-5,8- difluoro-4-oxo-l,4-dihydroquinoline-3-carboxylic acid (0.015 g, 0.37 mmol), imidazole (0.035 g, 0.52 mmol), cesium carbonate (0.241 g, 0.740 mmol), racemic trans-N.N'-dimethylcyclohexane- 1,2-diamine (5.3 mg, 0.037 mmol), copper (I) iodide (0.704 mg, 0.0037 mmol) and l-methyl-2- pyrrolidinone (1 ,8 mL) were combined, purged with a stream of nitrogen and heated via Emrys Optimizer™ microwave to 100 ⁰ C for 30 minutes. The mixture was cooled to room temperature, filtered, and concentrated in vacuo. The resulting residue was purified via reverse phase HPLC to provide the title compound as the trifluoroacetate salt, which gave a proton νMR spectrum consistent with theory and a high resolution mass spectrum (ES+) m/z of 383.0976 calculated for M+H ⁺ [Ci ₉ Hi ₂ F ₂ N ₄ O ₃ : 383.095]: ¹ H NMR (500 MHz, DMSO-c?6) δ 14.76 (bs, CO ₂ H), 9.36 (bs, IH), 9.16 (s, IH), 8.54 (d, J= 5.4 Hz, IH), 8.26 (bs, IH), 7.72-7.78 (m, 2H), 7.60 (bs, IH), 7.38- 7.44 (m, 2H), 6.01 (s, 2H).

The compounds in Table 1 below (of formula (H)) were prepared according to the general procedure provided in Examples 1-3. The starting materials are either commercially available or may be prepared from commercially available reagents using conventional reactions well known to those skilled in the art.

(II)

Table 1 - 4-Oxo-l,4-Dihydroquinoline Compounds of General Formula (II)

The compounds in Table 2 below (of General Formula (V)) were prepared according to the general procedure provided in Examples 1-3. The starting materials are either commercially available or may be prepared from commercially available reagents using conventional reactions well known to those skilled in the art.

(V) Table 2 - 4-Oxo-l,4-Dihydroquinoline Compounds of General Formula (V)

The compounds in Table 3 below (of General Formula (IH)) were prepared according to the general procedure provided in Examples 1 -3 The starting materials are either commercially available or may be prepared from commercially available reagents using conventional reactions well known to those skilled in the art.

(in)

Table 3 - 4-Oxo-l,4-Dihydroquinoline Compounds of General Formula (III)

Compound 277 is a mixture of two isomers (as depicted for the R ^3B group).

The compounds in Table 4 below were prepared according to the general procedure provided in Examples 1 -3 The starting materials are either commercially available or may be prepared from commercially available reagents using conventional reactions well known to those skilled in the art.

Table 4 - 4-Oxo-l,4-Dih dro uinoline Compounds

The compounds in Table 5 (of General Formula (VI)) below were prepared according to the general procedure provided in Examples 1-3. The starting materials are either commercially available or may be prepared from commercially available reagents using conventional reactions well known to those skilled in the art.

(VI) Table 5 - 4-Oxo-l,4-Dihydroquinoline Compounds of General Formula (VI)

The compounds in Table 6 below (of General Formula (VII)) were prepared according to the general procedure provided in Examples 1-3. The starting materials are either commercially available or may be prepared from commercially available reagents using conventional reactions well known to those skilled in the art.

(VII) Table 6 -4-Oxo-l ,4-Dihydroquinoline Compounds of General Formula (VII)

The compounds in Table 7 below (of General Formula (VIII)) were prepared according to the general procedure provided in Examples 1-3. The starting materials are either commercially available or may be prepared from commercially available reagents using conventional reactions well known to those skilled in the art.

Table 7 - 4-Oxo-l ,4-Dihydroquinoline Compounds of General Formula (VIII)

The compounds in Table 8 below (of General Formula (IX)) were prepared according to the general procedure provided in Examples 1-3. The starting materials are either commercially available or may be prepared from commercially available reagents using conventional reactions well known to those skilled in the art.

(IX)

Table 8 -4-Oxo-l,4-Dihydroquinoline Compounds of General Formula (IX)

EXAMPLE 372

S-Flυoro^-oxo-l-fCδ-piperidin-l-ylpyridazin-S-y^methylj -l^-dihydroquinoline-S-carboxylic acid.

To a solution of l-[ (6-chloropyridazin-3-yl)methyl]-8-fluoro-4-oxo-l,4-dihydroqu inoline-3- carboxylic acid (0.040 g, 0.12 mmol) in 1 mL of DMSO was added piperidine (0.015 πiL, 0.16 mmol). The reaction mixture was irradiated in the microwave at 100 ⁰ C for 10 minutes, and purified via reverse phase HPLC to provide the title compound that gave a proton NMR spectra consistent with theory and a mass ion (ES+) of 383.1 for M+HT ^1" .

EXAMPLE 373

8-Fluoro-l-{[6-(methylsulfonyl)pyridazin-3-yl]methyl}-4-o xo-l,4-dihydroquinoline-3-carboxylic acid

To a solution of l-[ (6-chloropyridazin-3-yl)methyl]-8-fluoro-4-oxo-l,4- dihydroquinoline-3-carboxylic acid (0.050 g, 0.15 mmol) in 1 mL of DMSO was added sodium thiomethoxide (0.032 g, 45 mmol). The reaction mixture was irradiated in the microwave at 100 ⁰ C for 10 minutes, and purified via reverse phase HPLC to provide 8-fluoro-l-{[6- (methylthio)pyridazin-3-yl]methyl}-4-oxo-l ,4-dihydroquinoline-3-carboxylic acid that gave a proton NMR spectra consistent with theory and a mass ion (ES+) of 346.1 for M+H ⁺ .

To a solution of the above compound in 1 mL of MeOH was added oxone (0.160 g, 0.261 mmol) dissolved in 0.5 mL of water. After 1 hour, the reaction mixture was poured into water (15 mL) and extracted with ethyl acetate (3 x 15 mL). The combined organics were washed with brine (15 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified via reverse phase HPLC to provide 8-fluoro-l-{[6-(methylsulfinyl)pyridazin-3- yl]methyl}-4-oxo-l,4-dihydroquinoline-3-carboxylic acid that gave a proton NMR spectra consistent with theory and a mass ion (ES+) of 362.1 for M+H ⁺ , and the title compound that gave a proton NMR spectra consistent with theory and a mass ion (ES+) of 378.1 for M+H ⁺ .

Ml Receptor Positive Allosteric Modulator Properties

The utility of the compounds as Ml receptor positive allosteric modulators may be demonstrated by methodology known in the art, including by the assay described below. The assay is designed to select compounds that possess modulator activity at the acetylcholine muscarinic Ml receptor or other muscarinic receptors expressed in CHOnfat cells by measuring the intracellular calcium with a FLIPR384 Fluorometric Imaging Plate Reader System. The assay studies the effect of one or several concentrations of test compounds on basal or acetylcholine-stimulated Ca2+ levels using FLIPR.

Compounds are prepared and subjected to a preincubation period of 4 min. Thereafter, a single EC20 concentration of acetylcholine is added to each well (3nM final). The

intracellular Ca2+ level of each sample is measured and compared to an acetylcholine control to determine any modulatory activity.

Cells: CHOnfat/hMl, hM2, hM3 or hM4 cells are plated 24 hr before the assay at a density of 18,000 cells/ well (100 μL) in a 384 well plate. CHOnfat/hMl and CHOnfat/hM3 Growth Medium: 90% DMEM (Hi Glucose); 10% HI FBS; 2mM L-glutamine; 0.1 mM NEAA; Pen-Strep; and lmg/ ml Geneticin, are added. For M2Gqi5CHOnfat and M4Gqi5CHOnfat cells, an additional 600 μg/ml hygromycin is added.

Equipment: 384 well plate, 120 μL addition plate; 96-well Whatman 2ml Uniplate Incubator, 37°C, 5% CO2; Skatron EMBLA-384 Plate Washer; Multimek Pipetting System; Genesis Freedom 200 System; Mosquito System; Temo Nanolitre Pipetting System; and FLIPR.384 Fluorometric Imaging Plate Reader System are used.

Buffers. Assay Buffer: Hanks Balanced Salt Solution, with 2OmM Hepes, 2.5mM Probenecid (Sigma P-8761) first dissolved in IN aqueous NaOH, 1% Bovine Serum Albumin (Sigma A-9647). Dye Loading Buffer: Assay Buffer plus 1% Fetal Bovine Serum and Fluo- 4AM/Pluronic Acid Mixture. 2mM Fluo-4 AM ester stock in DMSO (Molecular Probes F-

14202) Concentration of 2μM in buffer for a final concentration of lμM in Assay. 20% Pluronic Acid Solution stock, with a concentration of 0.04% in Buffer, 0.02% in Assay.

65 μL of 2 mM Fluo-4AM are mixed with 130 μL of 20% Pluronic Acid. The resulting solution and 650 μL FBS is added to the assay buffer for a total volume of 65 mL. Positive Controls: 4-Br-A23187: 10 mM in DMSO; final concentration 10 μM. Acetylcholine: 1OmM in water, working stock at both 20 μM and 30 μM in assay buffer, final concentration of 10 μM. This is used to check the maximum stimulation of the CHOKl/hMl cells. 20 μM (2x) acetylcholine is added in the preincubation part of the assay, and the 30 μM (3x) stock is added in the second part. (EC20)Acetylcholine: 1OmM in water, working stock of 9 nM (3x) ₍ and final concentration in assay is 3nM. This is used after the preincubation with test compounds. Addition of the EC20 Acetylcholine to each well with a test compound will ascertain any modulator activity. 24 wells contain 3nM Acetylcholine alone as a control. Determining Activity of Putative Compounds: .

Screening Plate: Compounds are titrated in 96-well plates (columns 2-11), 100% DMSO, started at a concentration of 15 mM (15Ox stock concentration), and 3-fold serial dilutions using Genesis

Freedom200 System. Four 96-well plates are combined into a 384-well plate using Mosquito

Nanolitre Pipetting System by transferring 1 μl of serial diluted compounds to each well, and

ImM acetylcholine (10Ox stock concentration) were added as a control. Using Temo, 49 μl assay buffer is added to each well of the 384- well plate right before assay. In a 96-well Whatman 2ml Uniplate, 9nM Acetylcholine (3x) is pipetted into wells corresponding to the screening compounds, and into control wells. The 30 μM

acetylcholine control (3x) is added into control wells, and the 3x agonist plate is transferred into a 384-well plate.

Cells are washed three times with 100 μL of buffer, leaving 30μL of buffer in each well. Using Multimek, 30 μL of Dye Loading Buffer is added into each well and incubated at 37 ⁰ C, 5% CO2 for up to one hr.

After 60 min, the cells are washed three times with 100 μL of buffer, leaving 30 μL of buffer in each well. The cell plate, screening plate, and agonist addition plates are placed on the platform in the FLIPR and the door is closed. A signal test to check background fluorescence and basal fluorescence signal is performed. Laser intensity is adjusted if necessary. 4 min of preincubation with the test compounds is provided to determine any agonist activity on the Ml receptor by comparison to the 1 mM acetylcholine control. After preincubation, the EC20 value of acetylcholine (3nM final) is added to determine any modulator activity.

A further description of the muscarinic FLIPR assay can be found in International patent application WO2004/073639.

In particular, the compounds of the following examples had activity in the aforementioned assay, generally with an IP (inflection point) of 30μM (30,000 nM) or less. The inflection point is calculated from the FLIPR values, and is a measure of activity. Such a result is indicative of the intrinsic activity of the compounds in as Ml allosteric modulators. IP values from the aforementioned assay for representative exemplary compounds of the invention (as described herein) are provided below in Table 9 below:

The following abbreviations are used throughout the text:

Me: methyl

Et: ethyl t-Bu: /er/-butyl

Ar: aryl

Ph: phenyl

Bn: benzyl

BOC: t-butyloxycarbonyl

LAM: lithium aluminum hydride

DMF: dimethylformamide

HMDS: hexamethyldisilazane

THF: tetrahydrofuran

Ac: acetyl or acetate

DMSO: dimethylsulfoxide

DMEM: Dulbecco's Modified Eagle Medium (High Glucose)

FBS: fetal bovine serum it: room temperature aq: aqueous

HPLC: high performance liquid chromatography

MS: mass spectrometry

LRMS: low resolution mass spectrometry

HRMS: high resolution mass spectrometry

While the invention has been described and illustrated with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additions of procedures and protocols may be made without departing from the spirit and scope of the invention. It is intended, therefore, that the invention be defined by the scope of the claims that follow and that such claims be interpreted as broadly as is reasonable.