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Title:
PDZ DOMAIN MODULATORS
Document Type and Number:
WIPO Patent Application WO/2008/148747
Kind Code:
A2
Abstract:
This invention relates to compounds useful as PDZ domain modulators, in particular the PDZ domain of PICK1. In other aspects the invention relates to the use of these compounds in a method for therapy and to pharmaceutical compositions.

Inventors:
GETHER ULRIK (DK)
MADSEN KENNETH (DK)
THORSEN THOR SENECA (DK)
PETERS DAN (DK)
DYHRING TINO (DK)
ROENN LARS CHRISTIAN (DK)
Application Number:
PCT/EP2008/056814
Publication Date:
December 11, 2008
Filing Date:
June 03, 2008
Export Citation:
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Assignee:
UNIV COPENHAGEN (DK)
NEUROSEARCH AS (DK)
GETHER ULRIK (DK)
MADSEN KENNETH (DK)
THORSEN THOR SENECA (DK)
PETERS DAN (DK)
DYHRING TINO (DK)
ROENN LARS CHRISTIAN (DK)
International Classes:
A61K31/11; A61K31/325; A61K31/35; A61K31/4155; A61K31/42; A61K31/515; A61K31/655; A61P25/00; C07C47/575; C07C271/64; C07D213/30; C07D307/46; C07D307/54; C07D311/76; C07D333/22; C07D333/60; C07D407/04; C07D409/04
Domestic Patent References:
WO2006119736A22006-11-16
WO2005047275A12005-05-26
WO2005040132A12005-05-06
WO2004078126A22004-09-16
WO2003050098A12003-06-19
Foreign References:
US7074836B12006-07-11
DE1112282B1961-08-03
Other References:
DATABASE CHEMCATS [Online] 6 March 2007 (2007-03-06), XP002493926 retrieved from STN Database accession no. 2033031312
DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; OCHESKEY, JOSEPH A. ET AL: "Metalloantimalarials: Synthesis and characterization of a novel agent possessing activity against Plasmodium falciparum" XP002493927 retrieved from STN Database accession no. 2005:342921 & CHEMICAL COMMUNICATIONS (CAMBRIDGE, UNITED KINGDOM) , (12), 1622-1624 CODEN: CHCOFS; ISSN: 1359-7345, 2005,
DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; ISHII, HISASHI ET AL: "A convenient preparation of salicylaldehydes from 2-methylbenzofurans by ozonolysis" XP002493928 retrieved from STN Database accession no. 1994:76974 & CHEMICAL & PHARMACEUTICAL BULLETIN , 41(6), 1166-8 CODEN: CPBTAL; ISSN: 0009-2363, 1993,
DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; OHMORI, HIDENOBU ET AL: "Anodic pyridination of 2-hydroxy-3-methoxy-5-methylbenzaldehyde and its Schiff base derivatives" XP002493929 retrieved from STN Database accession no. 1980:407313 & JOURNAL OF THE CHEMICAL SOCIETY, PERKIN TRANSACTIONS 2: PHYSICAL ORGANIC CHEMISTRY (1972-1999) , (2), 347-57 CODEN: JCPKBH; ISSN: 0300-9580, 1980,
DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; WANG, WENHUA ET AL: "Antifertility actions of gossypol derivatives and analogs" XP002493930 retrieved from STN Database accession no. 1990:584883 & ZHONGGUO YAOLI XUEBAO , 11(3), 268-71 CODEN: CYLPDN; ISSN: 0253-9756, 1990,
LO WING-KIT ET AL: "Heterobimetallic Zn(II)-Ln(III) phenylene-bridged schiff base complexes, computational studies, and evidence for singlet energy transfer as the main pathway in the sensitization of near-infrared Nd3+ luminescence." INORGANIC CHEMISTRY 13 NOV 2006, vol. 45, no. 23, 13 November 2006 (2006-11-13), pages 9315-9325, XP002493920 ISSN: 0020-1669
GALLANT AMANDA J ET AL: "Synthesis, structure, and computational studies of soluble conjugated multidentate macrocycles." THE JOURNAL OF ORGANIC CHEMISTRY 30 SEP 2005, vol. 70, no. 20, 30 September 2005 (2005-09-30), pages 7936-7946, XP002493921 ISSN: 0022-3263
AKINE S ET AL: "Synthesis and structure of polyhydroxyl rigid triangular nano-macrocyclic imine having multiple hydrogen-bonding sites" TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM, vol. 45, no. 22, 24 May 2004 (2004-05-24), pages 4225-4227, XP004506328 ISSN: 0040-4039
BOTELLA L ET AL: "Cross-coupling reactions with boronic acids in water catalysed by oxime-derived palladacycles" JOURNAL OF ORGANOMETALLIC CHEMISTRY, ELSEVIER-SEQUOIA S.A. LAUSANNE, CH, vol. 663, no. 1-2, 2 December 2002 (2002-12-02), pages 46-57, XP004402850 ISSN: 0022-328X
BACKSTROM R ET AL: "SYNTHESIS OF SOME NOVEL POTENT AND SELECTIVE CATECHOL O METHYLTRANSFERASE INHIBITORS" JOURNAL OF MEDICINAL CHEMISTRY, vol. 32, no. 4, 1989, pages 841-846, XP002493922 ISSN: 0022-2623
PEREZ R A ET AL: "Dihydroxynitrobenzaldehydes and Hydroxymethoxynitrobenzaldehydes: Synthesis and Biological Activity as Catechol-O-methyltransferase Inhibitors" JOURNAL OF MEDICINAL CHEMISTRY, US AMERICAN CHEMICAL SOCIETY. WASHINGTON, vol. 35, no. 24, 1 January 1992 (1992-01-01), pages 4584-4588, XP000925996 ISSN: 0022-2623
HA JEOUNG-HEE ET AL: "In vitro effects of hydroxybenzaldehydes from Gastrodia elata and their analogues on GABAergic neurotransmission, and a structure-activity correlation" PLANTA MEDICA, vol. 67, no. 9, December 2001 (2001-12), pages 877-880, XP002493923 ISSN: 0032-0943
DE FELICE FERNANDA G ET AL: "Targeting the neurotoxic species in Alzheimer's disease: inhibitors of Abeta oligomerization" FASEB JOURNAL, vol. 18, no. 12, September 2004 (2004-09), pages 1366-1372, XP002493924 ISSN: 0892-6638
DEV KUMLESH K: "PDZ domain protein-protein interactions: A case study with PICK1" CURRENT TOPICS IN MEDICINAL CHEMISTRY, vol. 7, no. 1, 2007, pages 3-20, XP002493925 ISSN: 1568-0266
Attorney, Agent or Firm:
ABILDGREN, Michael (Patent Department93 Pederstrupvej, Ballerup, DK)
Download PDF:
Claims:

CLAIMS

The use of a compound of Formula 1 a, 1 b, 1 c, 1d, 1e or 1f:

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; for the manufacture of a pharmaceutical composition for the treatment, prevention or alleviation of a disease or a disorder or a condition of a mammal, including a human, which disease or disorder or condition is responsive to modulation of a PDZ domain;

where in Formula 1 a, 1 b, 1 c, 1d, 1 e and 1f:

R 1 , R 2 and R 3 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy, formyl, alkylcarbonyl and -(C≡C) n -R a ; wherein n is O or 1 ; and

R a represents an aryl or a heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkyl alkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR'R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R' and R" independent of each other are hydrogen or alkyl; R 4 represents hydrogen or alkyl;

R 5 , R 6 , R 7 and R 8 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy, formyl, alkylcarbonyl or an aryl or a heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR'R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R' and R" independent of each other are hydrogen or alkyl;

R 9 and R 10 together form -(0-(C=O))-, -O- or -S-; or

R 9 represents hydrogen or alkyl; and R 10 represents hydrogen, cyano or alkyl;

R 11 and R 12 together form -(CHR'-CH 2 )-; wherein R' represents hydrogen, alkyl or phenyl; or

R 11 represents hydrogen or alkyl; and

R 12 represents hydrogen, alkyl, alkenyl or alkynyl; which alkyl, alkenyl or alkynyl is optionally substituted with an aryl or heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR'R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R' and R" independent of each other are hydrogen or alkyl;

R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 and R 22 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy, formyl, alkylcarbonyl, hydroxycarbonyl, alkoxycarbonyl, R b and - C=N-R b ; wherein

R b represents an aryl or a heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR'R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R' and R" independent of each other are hydrogen or alkyl; -X 1 -X 2 - represents -N=(C-R')- or -NR"-(C=O)-; wherein wherein R' and R" independent of each other are hydrogen or alkyl;

-Y 1 -Y 2 - represents ; wherein

R 23 , R 24 , R 25 and R 26 are independently selected from the group consisting of:

hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy, formyl, alkylcarbonyl, and R d ;

R c and R d independent of each other represents an aryl or a heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, R e -alkoxy, -NR'R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R' and R" independent of each other are hydrogen or alkyl;

R e represents an aryl group; which aryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR'"R"", -(C=O)NR 111 R"" or -NR'"(C=O)R""; wherein R'" and R"" independent of each other are hydrogen or alkyl;

-Z 1 -Z 2 - represents -NR'-C(COOR")- or -(C=O)-(C=O)-; wherein wherein R' and R" independent of each other are hydrogen or alkyl; -W 1 -W 2 - represents -C(R 27 R 28 )- or -CR 27 =CR 28 -; wherein wherein R 27 and R 28 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy and alkoxy; the bond ^^ represents a single or a double bond.

2. The use according to claim 1 wherein the compound is a compound of Formula 1a

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 1 , R 2 , R 3 and R 4 are as defined in claim 1.

3. The use according to claim 1 wherein the compound is a compound of Formula 1 b

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are as defined in claim 1.

The use according to claim 1 wherein the compound is a compound of Formula 1 c

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 and R 22 are as defined in claim 1.

The use according to claim 1 wherein the compound is a compound of Formula 1d

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and -X 1 -X 2 - are as defined in claim 1 .

The use according to claim 1 wherein the compound is a compound of Formula 1 e

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R c and -Y 1 -Y 2 - are as defined in claim 1 .

7. The use according to claim 1 wherein the compound is a compound of Formula 1f

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , -Z 1 -Z 2 - and -W 1 -W 2 - and are as defined in claim 1 .

8. The use according to claim 1 wherein the compound of Formula 1 a-1f is 2,3-Dibromo-5-ethoxy-6-hydroxy-benzaldehyde (a); 3-Ethoxy-2-hydroxy-benzaldehyde (b); 5-Chloro-3-ethoxy-2-hydroxy-benzaldehyde (c);

2,3-Dichloro-5-ethoxy-6-hydroxy-benzaldehyde (d); 5-Bromo-3-ethoxy-2-hydroxy-benzaldehyde (e); 6-Bromo-3-ethoxy-2-hydroxy-benzaldehyde (f); 2-Bromo-3-chloro-5-ethoxy-6-hydroxy-benzaldehyde (g); 3-Ethoxy-2-hydroxy-5-nitro-benzaldehyde (h);

((Z)-2-Cyano-3-phenyl-acryloyl)-carbamic acid ethyl ester (i); [(Z)-2-Cyano-3-(3,4-dichlorophenyl)-acryloyl]-carbamic acid ethyl ester (j); 3-[(E)-(3-Phenyl-acryloyl)]-oxazolidin-2-one (k); (Benzo[b]thiophene-2-carbonyl)-carbamic acid prop-2-ynyl ester (I); 4-Phenyl-3-[(E)-3-phenyl-acryloyl)]-oxazolidin-2-one (m);

(6-Bromo-2-oxo-2H-chromene-3-carbonyl)-carbamic acid ethyl ester (n); (6,8-Dichloro-2-oxo-2H-chromene-3-carbonyl)-carbamic acid butyl ester (o); (6,8-Diiodo-2-oxo-2H-chromene-3-carbonyl)-carbamic acid ethyl ester (p); 4-tert-Butyl-2-{[1 -[5-(4-chloro-phenylazo)-2-hydroxy-phenyl]-meth-(E)-ylidene]- amino}-phenol (q);

5-(4-Bromo-phenylazo)-2-hydroxy-3-methoxy-benzaldehyde (r); 5-{5-[1 -(3-Carboxy-phenyl)-3-methyl-5-oxo-1 ,5-dihydro-pyrazol-(4Z)-ylidene- methyl]-furan-2-yl}-2-chloro-benzoic acid butyl ester (s); 4-{5-[1 -(3-Chloro-4-methyl-phenyl)-3,5-dioxo-pyrazolidin-(4Z)-ylidenemethyl]- furan-2-yl}-benzoic acid ethyl ester (t);

1 -(2-Chloro-phenyl)-5-[1 -furan-2-yl-meth-(E)-ylidene]-pyrimidine-2,4,6-trione (u); 2-(2-Benzyloxy-5-bromo-benzylidene)-indan-1 ,3-dione (v); 2-Nitro-phenanthrene-9,10-dione (w);

8-Chloro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-4-carboxylic acid (x); or a pharmaceutically acceptable salt thereof.

9. The use according to claim 1 wherein the compound of Formula 1 a or 1 b is a compound according to any one of claims 13-20.

10. The use according to any one of claims 1 -9, wherein the disease or disorder or condition is responsive to modulation of a PDZ domain is disease or disorder or condition is responsive to modulation of the PDZ domain of PICK1 .

1 1 . The use according to any one of claims 1 -10, wherein the disease or disorder or condition is responsive to modulation of a PDZ domain is acute pain, chronic pain, neuropathic pain, intractable pain, migraine, neurological and psychiatric disorders, depression, anxiety, psychosis, schizophrenia, excitatory amino acid- dependent psychosis, cognitive disorders, dementia, senile dementia, AIDS-

induced dementia, stress-related psychiatric disorders, stroke, global ischaemic, focal ischaemic, haemorrhagic stroke, cerebral hypoxia, cerebral ischaemia, cerebral infarction, cerebral ischaemia resulting from thromboembolic or haemorrhagic stroke, cardiac infarction, brain trauma, brain oedema, cranial trauma, brain trauma, spinal cord trauma, bone-marrow lesions, hypoglycaemia, anoxia, neuronal damage following hypoglycaemia, hypotonia, hypoxia, perinatal hypoxia, cardiac arrest, acute neurodegenerative diseases or disorders, chronic neurodegenerative diseases or disorders, brain ischaemia, CNS degenerative disorders, Parkinson's disease, Alzheimer's disease, Huntington's disease, idiopathic Parkinson's Disease, drug induced Parkinson's Disease, amyotrophic lateral sclerosis (ALS), post-acute phase cerebral lesions, chronic diseases of the nervous system, cerebral deficits subsequent to cardiac bypass surgery, cerebral deficits subsequent to grafting, perinatal asphyxia, anoxia from drowning, anoxia from pulmonary surgery, anoxia from cerebral trauma, hypoxia induced nerve cell damage, epilepsy, status epilepticus, seizure disorders, cerebral vasospasm,

CNS mediated spasms, motility disorders, muscular spasms, urinary incontinence, convulsions, disorders responsive to anticonvulsants, autoimmune diseases, emesis, nausea, obesity, chemical dependencies, chemical addictions, addictions, withdrawal symptoms, drug induced deficits, alcohol induced deficits, drug addiction, ocular damage, retinopathy, retinal neuropathy, tinnitus, and tardive dyskinesia, inflammatory pain, neurogenic pain, fibromyalgia, chronic fatigue syndrome, nociceptive pain, cancer pain, postoperative pain, migraine, tension-type headache, pain during labour and delivery, breakthrough pain, stroke, drug abuse and cocaine abuse.

12. A method for treatment, prevention or alleviation of a disease or a disorder or a condition of a living animal body, including a human, which disorder, disease or condition is responsive to modulation of a PDZ domain, which method comprises the step of administering to such a living animal body in need thereof a therapeutically effective amount of a compound according to any one of the claims 1 -8 or 13-20, any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof.

13. A compound of Formula 1 a or 1 b:

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein

one of R 1 , R 2 and R 3 represents -(C≡C) n -R a ; wherein wherein n is O or 1 ; and R a represents an aryl or a heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR'R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R' and R" independent of each other are hydrogen or alkyl; the remaining two of R 1 , R 2 and R 3 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy formyl and alkylcarbonyl; R 4 represents hydrogen or alkyl;

R 5 , R 6 , R 7 and R 8 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy or an aryl or a heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR'R",

-(C=O)NR 1 R" or -NR'(C=0)R"; wherein R' and R" independent of each other are hydrogen or alkyl;

R 9 and R 10 together form -(0-(C=O))-, -O- or -S-; or

R 9 represents hydrogen or alkyl; and R 10 represents hydrogen, cyano or alkyl;

R 11 and R 12 together form -(CHR'-CH 2 )-; wherein R' represents hydrogen, alkyl or phenyl; or

R 11 represents hydrogen or alkyl; and

R 12 represents hydrogen, alkyl, alkenyl or alkynyl; which alkyl, alkenyl or alkynyl is optionally substituted with an aryl or heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR'R", -(C=O)NR 1 R" or -NR'(C=0)R"; wherein R' and R" independent of each other are hydrogen or alkyl.

14. The compound of claim 13 being a compound of Formula 1a

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 1 , R 2 , R 3 and R 4 are as defined in claim 13.

15. The compound of claim 14 wherein one of R 1 , R 2 and R 3 represents -C≡C-R a .

16. The compound of claim 14 wherein one of R 1 , R 2 and R 3 represents a monocyclic heteroaryl group.

17. The compound of claim 13 being a compound of Formula 1 b

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are as defined in claim 13.

18. The compound of claim 17 wherein R 12 represents substituted alkynyl.

19. The compound of claim 17 being a compound of Formula 1 b1 , 1 b2, 1 b3 or 1 b4:

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein one of R 5 , R 6 , R 7 and R 8 is an optionally substituted aryl or a heteroaryl group; and the remaining three of R 5 , R 6 , R 7 and R 8 and R 11 and R 12 are as defined in claim 9.

20. The compound of claim 13, which is

4-Ethoxy-3-hydroxy-biphenyl-2-carbaldehyde; 4-Ethoxy-2'-fluoro-3-hydroxy-biphenyl-2-carbaldehyde;

4-Ethoxy-3'-fluoro-3-hydroxy-biphenyl-2-carbaldehyde;

4-Ethoxy-4'-fluoro-3-hydroxy-biphenyl-2-carbaldehyde;

4-Ethoxy-3-hydroxy-2'-methoxy-biphenyl-2-carbaldehyde;

4-Ethoxy-3-hydroxy-3'-methoxy-biphenyl-2-carbaldehyde; 4-Ethoxy-3-hydroxy-4'-methoxy-biphenyl-2-carbaldehyde;

3-Ethoxy-2-hydroxy-6-pyridin-3-yl-benzaldehyde;

3-Ethoxy-6-furan-2-yl-2-hydroxy-benzaldehyde;

3-Ethoxy-6-furan-3-yl-2-hydroxy-benzaldehyde;

3-Ethoxy-2-hydroxy-6-thiophen-2-yl-benzaldehyde; 3-Ethoxy-2-hydroxy-6-thiophen-3-yl-benzaldehyde;

5-Ethoxy-4-hydroxy-biphenyl-3-carbaldehyde;

3-Ethoxy-5-furan-2-yl-2-hydroxy-benzaldehyde;

3-Ethoxy-5-furan-3-yl-2-hydroxy-benzaldehyde;

3-Ethoxy-2-hydroxy-5-thiophen-2-yl-benzaldehyde; 3-Ethoxy-2-hydroxy-5-thiophen-3-yl-benzaldehyde;

6-Chloro-4-ethoxy-3-hydroxy-biphenyl-2-carbaldehyde;

((Z)-3-Biphenyl-3-yl-2-cyano-acryloyl)-carbamic acid isopropyl ester;

[(Z)-2-Cyano-3-(3-furan-2-yl-phenyl)-acryloyl]-carbamic acid isopropyl ester;

((Z)-3-Biphenyl-4-yl-2-cyano-acryloyl)-carbamic acid ethyl ester; [(Z)-2-Cyano-3-(4-furan-2-yl-phenyl)-acryloyl]-carbamic acid ethyl ester;

(2-Oxo-6-phenyl-2H-chromene-3-carbonyl)-carbamic acid ethyl ester;

(6-Furan-2-yl-2-oxo-2H-chromene-3-carbonyl)-carbamic acid ethyl ester;

(6-Furan-3-yl-2-oxo-2H-chromene-3-carbonyl)-carbamic acid ethyl ester;

(2-Oxo-6-thiophen-2-yl-2H-chromene-3-carbonyl)-carbamic acid ethyl ester;

(2-Oxo-6-thiophen-3-yl-2/-/-chromene-3-carbonyl)-carbamic acid ethyl ester;

3-Ethoxy-2-hydroxy-6-phenylethynyl-benzaldehyde;

3-Ethoxy-2-hydroxy-6-pyridin-3-ylethynyl-benzaldehyde;

3-Ethoxy-2-hydroxy-6-(4-methoxy-phenylethynyl)-benzaldehyde; 5 3-Ethoxy-2-hydroxy-6-thiophen-3-ylethynyl-benzaldehyde;

3-Ethoxy-2-hydroxy-5-phenylethynyl-benzaldehyde;

3-Chloro-5-ethoxy-6-hydroxy-2-phenylethynyl-benzaldehyde;

(Benzo[ib]thiophene-2-carbonyl)-carbamic acid phenylethynyl ester;

(Benzo[ib]thiophene-2-carbonyl)-carbamic acid furan-2-ylethynyl ester; 10 (Benzo[ib]thiophene-2-carbonyl)-carbamic acid thiophen-2-ylethynyl ester;

(Benzo[ib]thiophene-2-carbonyl)-carbamic acid furan-3-ylethynyl ester;

(Benzo[ib]thiophene-2-carbonyl)-carbamic acid thiophen-3-ylethynyl ester;

[(Z)-2-Cyano-3-(3,4-dichloro-phenyl)-acryloyl]-carbamic acid methyl ester;

[(Z)-2-Cyano-3-(3,4-dichloro-phenyl)-acryloyl]-carbamic acid isopropyl ester; 15 [(Z)-2-Cyano-3-(3,4-dichloro-phenyl)-acryloyl]-carbamic acid propyl ester;

((Z)-2-Cyano-3-phenyl-acryloyl)-carbamic acid methyl ester;

((Z)-2-Cyano-3-phenyl-acryloyl)-carbamic acid isopropyl ester;

(Benzo[ib]thiophene-2-carbonyl)-carbamic acid ethyl ester;

(Benzo[ib]thiophene-2-carbonyl)-carbamic acid vinyl ester; 20 or a pharmaceutically acceptable salt thereof.

21 . A pharmaceutical composition, comprising a therapeutically effective amount of a compound of any one of claims 13-20, any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof, together with at

25 least one pharmaceutically acceptable carrier, excipient or diluent.

22. Use of a compound of any one of claims 13-20, any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament.

30

23. A compound according to any one of claims 13-20, any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof, for use as a medicament.

35 24. A compound according to any one of claims 1 -8 or 13-20, any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof, for use in the treatment, prevention or alleviation of a disease or a disorder or a condition of a mammal, including a human, which disease or disorder or condition is responsive to modulation of a PDZ domain.

Description:

PDZ DOMAIN MODULATORS

TECHNICAL FIELD

This invention relates to compounds useful as PDZ domain modulators, in particular the PDZ domain of PICK1.

In other aspects the invention relates to the use of these compounds in a method for therapy and to pharmaceutical compositions.

BACKGROUND ART

PDZ (PSD-95/Discs-large/ZO-1 homology) domains are one of the most common protein domains in the human genome with over 540 domains in more than 300 different proteins. They mediate cellular protein-protein interactions and serve important roles in protein targeting and in the assembly of protein complexes. PICK1 (Protein Interacting with C Kinase 1 ) contains a single N-terminal PDZ domain and was originally identified as a protein interacting with protein kinase Ca (PKCa). In addition to its N-terminal PDZ domain, PICK1 contains a coiled-coil domain (residue 145-165), which is believed to mediate dimehzation of PICK1. This is followed by a region with homology to Arfaptin 1 and 2 (residue 152-362), and a C-terminal acidic cluster (residue 381-389).

Functionally, PICK1 protein has been shown to be important for regulation of signaling through the AMPA receptor. PICK1 interacts with the AMPA receptor via binding of the C-terminal 3-4 residues of the GluR2 subunit in its single N-terminal PDZ domain. This interaction has, depending on cell type, been shown to be a positive and a negative regulator of the levels of GluR2 at the plasma membrane, thus affecting the molecular composition and gating properties of the AMPA receptor. Most importantly a recent study has shown that disruption of the PICK1 interaction with the GluR2 subunit by intrathecal injection of membrane permeable peptides that specifically bound to the PDZ domain of PICK1 showed efficacy in an animal model for neuropathic pain. These data suggest that the PDZ domain of PICK1 might in particular be a relevant target for treatment of pain, such as neuropathic pain.

SUMMARY OF THE INVENTION

It is the object of the invention to provide small molecule inhibitors that target the PDZ domain, and in particular the PDZ domain of PICK1 , preferably compounds which bind with high affinity and high specificity to the PDZ domain of PICK1.

In its first aspect, the invention provides the use of a compound of Formula 1 a, 1 b, 1 c, 1d, 1 e or 1f:

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; for the manufacture of a pharmaceutical composition for the treatment, prevention or alleviation of a disease or a disorder or a condition of a mammal, including a human, which disease or disorder or condition is responsive to modulation of a PDZ domain; where in Formula 1a, 1 b, 1c, 1d, 1e and 1f, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R c , X 1 , X 2 , Y 1 , Y 2 , Z 1 , Z 2 , W 1 and W 2 are as defined below. In a further aspect, the invention relates to a method for treatment, prevention or alleviation of a disease or a disorder or a condition of a living animal body, including a human, which disorder, disease or condition is responsive to modulation of a PDZ

domain, which method comprises the step of administering to such a living animal body in need thereof a therapeutically effective amount of a compound for use according to the invention, any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof.

In a still further aspect, the invention relates to novel compounds of Formula Ia and Ib, any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof.

Other objects of the invention will be apparent to the person skilled in the art from the following detailed description and examples.

DETAILED DISCLOSURE OF THE INVENTION

In its first aspect the present invention provides the use of a compound of Formula 1a, 1 b, 1c, 1d, 1 e or 1f:

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; for the manufacture of a pharmaceutical composition for the treatment, prevention or alleviation of a disease or a disorder or a condition of a mammal, including a human, which disease or disorder or condition is responsive to modulation of a PDZ domain; where in Formula 1 a, 1 b, 1 c, 1d, 1 e and 1f,

R 1 , R 2 and R 3 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy, formyl, alkylcarbonyl and -(C≡C) n -R a ; wherein n is O or 1 ; and R a represents an aryl or a heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR 1 R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R' and R" independent of each other are hydrogen or alkyl; R 4 represents hydrogen or alkyl;

R 5 , R 6 , R 7 and R 8 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy, formyl, alkylcarbonyl, or an aryl or a heteroaryl group;

which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR 1 R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R 1 and R" independent of each other are hydrogen or alkyl; R 9 and R 10 together form -(0-(C=O))-, -O- or -S-; or

R 9 represents hydrogen or alkyl; and R 10 represents hydrogen, cyano or alkyl; R 11 and R 12 together form -(CHR'-CH 2 )-; wherein R' represents hydrogen, alkyl or phenyl; or R 11 represents hydrogen or alkyl; and R 12 represents hydrogen, alkyl, alkenyl or alkynyl; which alkyl, alkenyl or alkynyl is optionally substituted with an aryl or heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR 1 R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R 1 and R" independent of each other are hydrogen or alkyl

R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 and R 22 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy, formyl, alkylcarbonyl, hydroxycarbonyl, alkoxycarbonyl, R b and -C=N-R b ; wherein

R b represents an aryl or a heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl,

cycloal kylal kyl , alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR 1 R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R 1 and R" independent of each other are hydrogen or alkyl; -X 1 -X 2 - represents -N=(C-R 1 )- or -NR"-(C=O)-; wherein wherein R 1 and R" independent of each other are hydrogen or alkyl;

-Y 1 -Y 2 - represents or ; wherein

R 23 , R 24 , R 25 and R 26 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy, formyl, alkylcarbonyl, and R d ; wherein

R c and R d independent of each other represents an aryl or a heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloal koxy, alkoxyalkyl, cycloal koxyal kyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloal kyl, cycloal kylal kyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, R e -alkoxy, -NR 1 R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R 1 and R" independent of each other are hydrogen or alkyl;

R e represents an aryl group; which aryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloal koxyal kyl, formyl, alkylcarbonyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloal kyl alkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR 111 R"", -(C=O)NR 111 R"" or -NR 11 XC=O)R""; wherein R 1 " and R"" independent of each other are hydrogen or alkyl;

-Z 1 -Z 2 - represents -NR'-C(COOR")- or -(C=O)-(C=O)-; wherein wherein R 1 and R" independent of each other are hydrogen or alkyl; -W 1 -W 2 - represents -C(R 27 R 28 )- or -CR 27 =CR 28 -; wherein wherein R 27 and R 28 are independently selected from the group consisting of:

hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy alkoxy, formyl and alkylcarbonyl; the bond 1 ^ * = represents a single or a double bond.

In one embodiment, the compound for use is a compound of Formula 1 a

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 1 , R 2 , R 3 and R 4 are as defined above.

In a special embodiment of the compound of Formula 1a, R 1 , R 2 and R 3 independent of each other represent hydrogen, halo or nitro. In a further embodiment, R 1 represents hydrogen. In a still further embodiment, R 1 represents halo, such as bromo or chloro. In a further embodiment, R 2 represents hydrogen. In a still further embodiment, R 2 represents halo, such as bromo or chloro. In a further embodiment, R 2 represents nitro. In a still further embodiment, R 3 represents hydrogen.

In a further special embodiment of the compound of Formula 1a, R 4 represents alkyl, such as ethyl.

In a further embodiment, the compound for use is a compound of Formula 1 b

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are as defined above.

In a special embodiment of the compound of Formula 1 b, R 5 , R 6 , R 7 and R 8 independent of each other represent hydrogen or halo. In a further embodiment, R 5 represents hydrogen. In a further embodiment, R 6 represents hydrogen. In a further embodiment, R 7 represents hydrogen. In a further embodiment, R 7 represents halo, such as chloro. In a further embodiment, R 8 represents hydrogen. In a further embodiment, R 8 represents halo, such as chloro.

In a further special embodiment of the compound of Formula 1 b, R 9 and R 10 together form -(0-(C=O))-. In a further embodiment, R 9 and R 10 together form -O-. In a still further embodiment, R 9 and R 10 together form -S-.

In a still further special embodiment of the compound of Formula 1 b, R 9 represents hydrogen or alkyl; and R 10 represents hydrogen, cyano or alkyl. In a further embodiment, R 9 represents hydrogen and R 10 represents hydrogen. In a still further embodiment, R 9 represents hydrogen and R 10 represents cyano.

In a further special embodiment of the compound of Formula 1 b, R 11 and R 12 together form -(CHR'-CH 2 )-, wherein R' represents hydrogen or phenyl. In a further embodiment, R' represents hydrogen. In a still further embodiment, R' represents phenyl.

In a further special embodiment of the compound of Formula 1 b, R 11 represents hydrogen and R 12 represents optionally substituted alkyl, such as ethyl or n-butyl.

In a still further embodiment, the compound for use is a compound of Formula 1 c

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 and R 22 are as defined above.

In a special embodiment of the compound of Formula 1c, four of R 16 , R 17 , R 18 , R 19 , R 20 , R 21 and R 22 represent hydrogen. In a further embodiment, R 20 represents halo, such as chloro.

In a further special embodiment of the compound of Formula 1 c, two of R 16 , R 17 , R 18 , R 19 , R 20 , R 21 and R 22 represent hydrogen. In a further embodiment, three of R 16 , R 17 , R 18 , R 19 , R 20 , R 21 and R 22 , such as R 19 , R 20 and R 21 , independent of each other represent hydroxy, alkoxy, or formyl. In a still further embodiment, R 19 represents alkoxy, such methoxy. In a further embodiment, R 20 represents hydroxy. In a still further embodiment, R 21 represents formyl.

In a still further special embodiment of the compound of Formula 1 c, four of R 13 , R 14 , R 15 , R 16 and R 17 represent hydrogen. In a further embodiment, one of R 13 , R 14 , R 15 , R 16 and R 17 , such as R 15 , represents halo, such as bromo.

In a further special embodiment of the compound of Formula 1 c, three of R 13 , R 14 , R 15 , R 16 and R 17 represent hydrogen. In a further embodiment, two of R 13 , R 14 , R 15 , R 16

and R 17 , such as R 14 and R 15 , independent of each other represent hydroxy or -C=N-R b ; wherein R b represents optionally substituted phenyl, such as alkyl-hydroxy-phenyl, such as 5-tertbutyl-2-hydroxy-phenyl. In a still further embodiment, R 14 represents -C=N-R b and R 15 represents hydroxy.

In a further embodiment, the compound for use is a compound of Formula 1d

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and -X 1 -X 2 - are as defined above.

In a special embodiment of the compound of Formula 1d, four of R 13 , R 14 , R 15 , R 16 and R 17 represent hydrogen. In a further embodiment, one of R 13 , R 14 , R 15 , R 16 and R 17 , such as R 14 , represents hydroxycarbonyl.

In a further special embodiment of the compound of Formula 1d, three of R 13 , R 14 , R 15 , R 16 and R 17 represent hydrogen. In a further embodiment, two of R 13 , R 14 , R 15 , R 16 and R 17 , such as R 14 and R 15 , independent of each other represent halo or alkyl. In a still further embodiment, R 14 represents halo, such as chloro, and R 15 represents alkyl, such as methyl.

In a still further special embodiment of the compound of Formula 1 d, four of R 18 , R 19 , R 20 , R 21 and R 22 represent hydrogen. In a further embodiment, one of R 18 , R 19 , R 20 , R 21 and R 22 , such as R 20 , represents alkoxycarbonyl, such as ethoxycarbonyl.

In a further special embodiment of the compound of Formula 1d, three of R 18 , R 19 , R 20 , R 21 and R 22 represent hydrogen. In a further embodiment, two of R 18 , R 19 , R 20 , R 21 and R 22 , such as R 19 and R 20 , independent of each other represent halo or alkoxycarbonyl, such as butoxycarbonyl. In a still further embodiment, R 19 represents alkoxycarbonyl, such as butoxycarbonyl and R 20 represents halo such as chloro.

In a still further special embodiment of the compound of Formula 1d, -X 1 -X 2 - represents -N=(C-R')-, such as -N=(C-R')- wherein R' represents alkyl, such as methyl. In a further special embodiment, -X 1 -X 2 - represents -NR"-(C=O)-, such as -NH-(C=O)-. In a still further embodiment, the compound for use is a compound of Formula

1e

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R c and -Y 1 -Y 2 - are as defined above.

In a special embodiment of the compound of Formula 1e, R c represents an optionally substituted heteroaryl group, such as optionally substituted furanyl. In a further embodiment, R c represents furanyl, such as furan-2-yl.

In a further special embodiment of the compound of Formula 1e, R c represents an optionally substituted aryl group, such as optionally substituted phenyl. In a further embodiment, R c represents phenyl substituted with R e -alkoxy and halo, such as benzyloxy and bromo. In a still further embodiment, R c represents 2-benzyloxy-5- bromo-phenyl.

In a still further special embodiment of the compound of Formula 1e, -Y 1 -Y 2 -

represents . In a further embodiment, R 23 , R 24 , R 25 and R 26 represent hydrogen. In a further special embodiment of the compound of Formula 1e, -Y 1 -Y 2 -

represents . In a further embodiment, R 23 represents hydrogen. In a still further embodiment, R 24 represents an optionally substituted aryl group, such as halophenyl. In a further embodiment, R 24 represents chlorophenyl, such as 2- chlorophenyl.

In a further embodiment, the compound for use is a compound of Formula 1f

(1f)

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , -Z 1 -Z 2 - and -W 1 -W 2 - and are as defined above.

In a special embodiment of the compound of Formula 1 e, R 13 , R 14 , R 15 , R 16 , R 17 , R 18 independent of each other represent hydrogen or halo. In a further embodiment, all of R 13 , R 14 , R 15 , R 16 , R 17 , R 18 represent hydrogen. In a still further embodiment, one of R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , such as R 14 , represent halo, such as chloro.

In a further special embodiment of the compound of Formula 1 e, -Z 1 -Z 2 - represents -NR'-C(COOR")-, such as -NH-C(COOH)-. In a still further special embodiment of the compound of Formula 1 e, -Z 1 -Z 2 - represents or -(C=O)-(C=O)-.

In a further special embodiment of the compound of Formula 1 e, -W 1 -W 2 - represents -C(R 27 R 28 )-, such as -CH 2 -.

In a still further special embodiment of the compound of Formula 1 e, -W 1 -W 2 - represents -CR 27 =CR 28 -, such as -CH=C(NO 2 )-.

In a further embodiment of the compound of Formula 1 e, the bond 1 ^ * = represents a single bond. In a further embodiment, the bond ^^ represents a double bond.

In a special embodiment, the compound of Formula 1 a-1f for use is

2,3-Dibromo-5-ethoxy-6-hydroxy-benzaldehyde (a);

3-Ethoxy-2-hydroxy-benzaldehyde (b) ;

5-Chloro-3-ethoxy-2-hydroxy-benzaldehyde (c);

2,3-Dichloro-5-ethoxy-6-hydroxy-benzaldehyde (d); 5-Bromo-3-ethoxy-2-hydroxy-benzaldehyde (e);

6-Bromo-3-ethoxy-2-hydroxy-benzaldehyde (f);

2-Bromo-3-chloro-5-ethoxy-6-hydroxy-benzaldehyde (g);

3-Ethoxy-2-hydroxy-5-nitro-benzaldehyde (h);

((Z)-2-Cyano-3-phenyl-acryloyl)-carbamic acid ethyl ester (i); [(Z)-2-Cyano-3-(3,4-dichlorophenyl)-acryloyl]-carbamic acid ethyl ester (j);

3-[(E)-(3-Phenyl-acryloyl)]-oxazolidin-2-one (k);

(Benzo[b]thiophene-2-carbonyl)-carbamic acid prop-2-ynyl ester (I);

4-Phenyl-3-[(E)-3-phenyl-acryloyl)]-oxazolidin-2-one (m);

(6-Bromo-2-oxo-2H-chromene-3-carbonyl)-carbamic acid ethyl ester (n); (6,8-Dichloro-2-oxo-2H-chromene-3-carbonyl)-carbamic acid butyl ester (o);

(6,8-Diiodo-2-oxo-2H-chromene-3-carbonyl)-carbamic acid ethyl ester (p);

4-tert-Butyl-2-{[1 -[5-(4-chloro-phenylazo)-2-hydroxy-phenyl]-meth-(E)-ylidene] -amino}- phenol (q);

5-(4-Bromo-phenylazo)-2-hydroxy-3-methoxy-benzaldehyde (r);

S-IS-ti ^S-Carboxy-phenyO-S-methyl-δ-oxo-I .S-dihydro-pyrazol^ZJ-ylidene-methyl]- furan-2-yl}-2-chloro-benzoic acid butyl ester (s);

^{δ-ti ^S-Chloro^-methyl-phenyO-S.δ-dioxo-pyrazolidin^ZJ-ylideneme thyll-furan^- yl}-benzoic acid ethyl ester (t);

1 -(2-Chloro-phenyl)-5-[1 -furan-2-yl-meth-(E)-ylidene]-pyrimidine-2,4,6-trione (u); 2-(2-Benzyloxy-5-bromo-benzylidene)-indan-1 ,3-dione (v); 2-Nitro-phenanthrene-9,10-dione (w);

8-Chloro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-4 -carboxylic acid (x); or a pharmaceutically acceptable salt thereof.

In a further embodiment, the compound of Formula 1 a or 1 b for use is a compound of the invention as described below.

Compounds of the invention

A further aspect of the invention provides compounds of Formula 1 a or 1 b:

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein one of R 1 , R 2 and R 3 represents -(C≡C) n -R a ; wherein wherein n is O or 1 ; and R a represents an aryl or a heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR'R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R' and R" independent of each other are hydrogen or alkyl; the remaining two of R 1 , R 2 and R 3 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy, formyl and alkylcarbonyl;

R 4 represents hydrogen or alkyl;

R 5 , R 6 , R 7 and R 8 are independently selected from the group consisting of: hydrogen, halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, alkyl, hydroxy, alkoxy or an aryl or a heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR 1 R", -(C=O)NR 1 R" or -NR'(C=O)R"; wherein R' and R" independent of each other are hydrogen or alkyl;

R 9 and R 10 together form -(0-(C=O))-, -O- or -S-; or R 9 represents hydrogen or alkyl; and R 10 represents hydrogen, cyano or alkyl; R 11 and R 12 together form -(CHR'-CH 2 )-; wherein R' represents hydrogen, alkyl or phenyl; or R 11 represents hydrogen or alkyl; and R 12 represents hydrogen, alkyl, alkenyl or alkynyl; which alkyl, alkenyl or alkynyl is optionally substituted with an aryl or heteroaryl group; which aryl or heteroaryl group is optionally substituted with one or more substituents independently selected from the group consisting of: halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxy, alkoxy, cycloalkoxy, alkoxyalkyl, cycloalkoxyalkyl, methylenedioxy, ethylenedioxy, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, sulfanyl, thioalkoxy, -NR 1 R", -(C=O)NR 1 R" or -NR'(C=0)R"; wherein R' and R" independent of each other are hydrogen or alkyl.

In a special embodiment, the compound of Formula 1 a or 1 b is a compound of

Formula 1a

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 1 , R 2 , R 3 and R 4 are as defined above. In a further

embodiment, one of R 1 , R 2 and R 3 represents -C≡C-R a . In a still further embodiment, one of R 1 , R 2 and R 3 represents a monocyclic heteroaryl group.

In a further embodiment, the compound of Formula 1 a or 1 b is a compound of Formula 1 b

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are as defined above. In a further embodiment, R 12 represents substituted alkynyl. In a still further embodiment, R 9 represents hydrogen and R 10 represents cyano.

In a still further embodiment, the compound of formula 1 b is a compound of Formula 1 b1 , 1 b2, 1 b3 or 1 b4:

any of its stereoisomers or any mixture of its stereoisomers, or a pharmaceutically acceptable salt thereof; wherein one of R 5 , R 6 , R 7 and R 8 is an optionally substituted aryl or a heteroaryl group; and the remaining three of R 5 , R 6 , R 7 and R 8 and R 11 and R 12 are as defined above.

In a special embodiment, the compound of Formula 1 a or 1 b is

4-Ethoxy-3-hydroxy-biphenyl-2-carbaldehyde;

4-Ethoxy-2'-fluoro-3-hydroxy-biphenyl-2-carbaldehyde;

4-Ethoxy-3'-fluoro-3-hydroxy-biphenyl-2-carbaldehyde; 4-Ethoxy-4'-fluoro-3-hydroxy-biphenyl-2-carbaldehyde;

4-Ethoxy-3-hydroxy-2'-methoxy-biphenyl-2-carbaldehyde;

4-Ethoxy-3-hydroxy-3'-methoxy-biphenyl-2-carbaldehyde;

4-Ethoxy-3-hydroxy-4'-methoxy-biphenyl-2-carbaldehyde;

3-Ethoxy-2-hydroxy-6-pyridin-3-yl-benzaldehyde; 3-Ethoxy-6-furan-2-yl-2-hydroxy-benzaldehyde;

3-Ethoxy-6-furan-3-yl-2-hydroxy-benzaldehyde;

3-Ethoxy-2-hydroxy-6-thiophen-2-yl-benzaldehyde;

3-Ethoxy-2-hydroxy-6-thiophen-3-yl-benzaldehyde;

5-Ethoxy-4-hydroxy-biphenyl-3-carbaldehyde; 3-Ethoxy-5-furan-2-yl-2-hydroxy-benzaldehyde;

3-Ethoxy-5-furan-3-yl-2-hydroxy-benzaldehyde;

3-Ethoxy-2-hydroxy-5-thiophen-2-yl-benzaldehyde;

3-Ethoxy-2-hydroxy-5-thiophen-3-yl-benzaldehyde;

6-Chloro-4-ethoxy-3-hydroxy-biphenyl-2-carbaldehyde; ((Z)-3-Biphenyl-3-yl-2-cyano-acryloyl)-carbamic acid isopropyl ester;

[(Z)-2-Cyano-3-(3-furan-2-yl-phenyl)-acryloyl]-carbamic acid isopropyl ester;

((Z)-3-Biphenyl-4-yl-2-cyano-acryloyl)-carbamic acid ethyl ester;

[(Z)-2-Cyano-3-(4-furan-2-yl-phenyl)-acryloyl]-carbamic acid ethyl ester;

(2-Oxo-6-phenyl-2H-chromene-3-carbonyl)-carbamic acid ethyl ester; (6-Furan-2-yl-2-oxo-2H-chromene-3-carbonyl)-carbamic acid ethyl ester;

(6-Furan-3-yl-2-oxo-2H-chromene-3-carbonyl)-carbamic acid ethyl ester;

(2-Oxo-6-thiophen-2-yl-2H-chromene-3-carbonyl)-carbamic acid ethyl ester;

(2-Oxo-6-thiophen-3-yl-2H-chromene-3-carbonyl)-carbamic acid ethyl ester;

3-Ethoxy-2-hydroxy-6-phenylethynyl-benzaldehyde; 3-Ethoxy-2-hydroxy-6-pyridin-3-ylethynyl-benzaldehyde;

3-Ethoxy-2-hydroxy-6-(4-methoxy-phenylethynyl)-benzaldehy de;

3-Ethoxy-2-hydroxy-6-thiophen-3-ylethynyl-benzaldehyde;

3-Ethoxy-2-hydroxy-5-phenylethynyl-benzaldehyde;

3-Chloro-5-ethoxy-6-hydroxy-2-phenylethynyl-benzaldehyde;

(Benzo[ib]thiophene-2-carbonyl)-carbamic acid phenylethynyl ester; (Benzo[ib]thiophene-2-carbonyl)-carbamic acid furan-2-ylethynyl ester; (Benzo[ib]thiophene-2-carbonyl)-carbamic acid thiophen-2-ylethynyl ester; (Benzo[ib]thiophene-2-carbonyl)-carbamic acid furan-3-ylethynyl ester; (Benzo[ib]thiophene-2-carbonyl)-carbamic acid thiophen-3-ylethynyl ester; [(Z)-2-Cyano-3-(3,4-dichloro-phenyl)-acryloyl]-carbamic acid methyl ester; [(Z)-2-Cyano-3-(3,4-dichloro-phenyl)-acryloyl]-carbamic acid isopropyl ester; [(Z)-2-Cyano-3-(3,4-dichloro-phenyl)-acryloyl]-carbamic acid propyl ester; ((Z)-2-Cyano-3-phenyl-acryloyl)-carbamic acid methyl ester; ((Z)-2-Cyano-3-phenyl-acryloyl)-carbamic acid isopropyl ester; (Benzo[ib]thiophene-2-carbonyl)-carbamic acid ethyl ester; (Benzo[ib]thiophene-2-carbonyl)-carbamic acid vinyl ester; or a pharmaceutically acceptable salt thereof.

Any combination of two or more of the embodiments as described above is considered within the scope of the present invention.

Definition of Substituents

In the context of this invention halo represents fluoro, chloro, bromo or iodo. In the context of this invention an alkyl group designates a univalent saturated, straight or branched hydrocarbon chain. The hydrocarbon chain preferably contains of from one to six carbon atoms (Ci -6 -alkyl), including pentyl, isopentyl, neopentyl, tertiary pentyl, hexyl and isohexyl. In a preferred embodiment alkyl represents a Ci -4 -alkyl group, including butyl, isobutyl, secondary butyl, and tertiary butyl. In another preferred embodiment of this invention alkyl represents a Ci -3 -alkyl group, which may in particular be methyl, ethyl, propyl or isopropyl.

In the context of this invention an alkenyl group designates a carbon chain containing one or more double bonds, including di-enes, tri-enes and poly-enes. In a preferred embodiment the alkenyl group of the invention comprises of from two to six carbon atoms (C 2-6 -alkenyl), including at least one double bond. In a most preferred embodiment the alkenyl group of the invention is ethenyl; 1 - or 2-propenyl; 1 -, 2- or 3- butenyl, or 1 ,3-butadienyl; 1 -, 2-, 3-, 4- or 5-hexenyl, or 1 ,3-hexadienyl, or 1 ,3,5- hexatrienyl.

In the context of this invention an alkynyl group designates a carbon chain containing one or more triple bonds, including di-ynes, tri-ynes and poly-ynes. In a preferred embodiment the alkynyl group of the invention comprises of from two to six carbon atoms (C 2-6 -alkynyl), including at least one triple bond. In its most preferred embodiment the alkynyl group of the invention is ethynyl; 1 -, or 2-propynyl; 1 -, 2-, or 3-

butynyl, or 1 ,3-butadiynyl; 1 -, 2-, 3-, 4-pentynyl, or 1 ,3-pentadiynyl; 1 -, 2-, 3-, 4-, or 5- hexynyl, or 1 ,3-hexadiynyl or 1 ,3,5-hexatriynyl.

In the context of this invention a cycloalkyl group designates a cyclic alkyl group, preferably containing of from three to seven carbon atoms (C 3-7 -cycloalkyl), including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

Alkoxy is O-alkyl, wherein alkyl is as defined above.

Cycloalkoxy means O-cycloalkyl, wherein cycloalkyl is as defined above.

Cycloalkylalkyl means cycloalkyl as above and alkyl as above, meaning for example, cyclopropyl methyl. In the context of this invention an aryl group designates a carbocyclic aromatic ring system such as phenyl, naphthyl (1 -naphthyl or 2-naphthyl) or fluorenyl.

In the context of this invention a heteroaryl group designates an aromatic mono- or bicyclic heterocyclic group, which holds one or more heteroatoms in its ring structure. Preferred heteroatoms include nitrogen (N), oxygen (O), and sulphur (S). Preferred monocyclic heteroaryl groups of the invention include aromatic 5- and

6-membered heterocyclic monocyclic groups, including for example, but not limited to, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, tetrazolyl, 1 ,2,4-oxadiazolyl, 1 ,2,5- oxadiazolyl, 1 ,3,4-oxadiazolyl, triazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,5-thiadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, furanyl, thienyl, pyridyl, pyrimidyl, or pyridazinyl. Preferred bicyclic heteroaryl groups of the invention include for example, but not limited to, indolizinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzo[ιb]thienyl, benzimidazolyl, benzoxazolyl, benzooxadiazolyl, benzothiazolyl, benzo[c/]isothiazolyl, purinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 1 ,8- naphthyridinyl, pteridinyl, and indenyl.

Pharmaceutically Acceptable Salts

The chemical compound or the compound for use according to the invention may be provided in any form suitable for the intended administration. Suitable forms include pharmaceutically (i.e. physiologically) acceptable salts, and pre- or prodrug forms of the chemical compound or the compound for use according to the invention.

Examples of pharmaceutically acceptable addition salts include, without limitation, the non-toxic inorganic and organic acid addition salts such as the hydrochloride, the hydrobromide, the nitrate, the perchlorate, the phosphate, the sulphate, the formate, the acetate, the aconate, the ascorbate, the benzenesulphonate, the benzoate, the cinnamate, the citrate, the embonate, the enantate, the fumarate, the glutamate, the glycolate, the lactate, the maleate, the malonate, the mandelate, the methanesulphonate, the naphthalene-2-sulphonate, the phthalate, the salicylate, the sorbate, the stearate, the succinate, the tartrate, the toluene-p-sulphonate, and the like. Such salts may be formed by procedures well known and described in the art.

Examples of pharmaceutically acceptable cationic salts of a chemical compound or the compound for use according to the invention include, without limitation, the sodium, the potassium, the calcium, the magnesium, the zinc, the aluminium, the lithium, the choline, the lysinium, and the ammonium salt, and the like, of a chemical compound or the compound for use according to the invention containing an anionic group. Such cationic salts may be formed by procedures well known and described in the art.

In the context of this invention the "onium salts" of N-containing compounds are also contemplated as pharmaceutically acceptable salts. Preferred "onium salts" include the alkyl-onium salts, the cycloalkyl-onium salts, and the cycloalkylalkyl-onium salts.

Examples of pre- or prodrug forms of the chemical compound or the compound for use according to the invention include examples of suitable prodrugs of the substances for use or according to the invention including compounds modified at one or more reactive or derivatizable groups of the parent compound. Of particular interest are compounds modified at a carboxyl group, a hydroxyl group, or an amino group. Examples of suitable derivatives are esters or amides.

The chemical compound or the compound for use according to the invention may be provided in dissoluble or indissoluble forms together with a pharmaceutically acceptable solvent such as water, ethanol, and the like. Dissoluble forms may also include hydrated forms such as the monohydrate, the dihydrate, the hemihydrate, the trihydrate, the tetrahydrate, and the like. In general, the dissoluble forms are considered equivalent to indissoluble forms for the purposes of this invention.

Steric Isomers

It will be appreciated by those skilled in the art that the compounds or the compounds for use according to the present invention may exist in different stereoisomeric forms - including enantiomers, diastereomers and cis-trans-isomers. The invention includes all such stereoisomers and any mixtures thereof including racemic mixtures.

Racemic forms can be resolved into the optical antipodes by known methods and techniques. One way of separating the enantiomeric compounds (including enantiomeric intermediates) is - in the case the compound being a chiral acid - by use of an optically active amine, and liberating the diastereomeric, resolved salt by treatment with an acid. Another method for resolving racemates into the optical antipodes is based upon chromatography on an optical active matrix. Racemic compounds of the present invention can thus be resolved into their optical antipodes, e.g., by fractional crystallisation of D- or L- (tartrates, mandelates, or camphor- sulphonate) salts for example.

The chemical compounds of the present invention may also be resolved by the formation of diastereomeric amides by reaction of the chemical compounds of the present invention with an optically active activated carboxylic acid such as that derived from (+) or (-) phenylalanine, (+) or (-) phenylglycine, (+) or (-) camphanic acid or by the formation of diastereomeric carbamates by reaction of the chemical compound of the present invention with an optically active chloroformate or the like.

Additional methods for the resolving the optical isomers are known in the art. Such methods include those described by Jaques J, Collet A, & Wilen S in "Enantiomers, Racemates, and Resolutions", John Wiley and Sons, New York (1981 ). Optical active compounds can also be prepared from optical active starting materials or intermediates.

Labelled Compounds

The compounds or the compounds for use according to the invention may be used in their labelled or unlabelled form. In the context of this invention the labelled compound has one or more atoms replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. The labelling will allow easy quantitative detection of said compound.

The labelled compounds or the compounds for use according to the invention may be useful as diagnostic tools, radio tracers, or monitoring agents in various diagnostic methods, and for in vivo receptor imaging.

The labelled compound or the compound for use according to the invention preferably contains at least one radionuclide as a label. Positron emitting radionuclides are all candidates for usage. In the context of this invention the radionuclide is preferably selected from 2 H (deuterium), 3 H (tritium), 11 C, 13 C, 14 C, 131 1, 125 1, 123 I, and 18 F.

The physical method for detecting the labelled compound of the present invention may be selected from Position Emission Tomography (PET), Single Photon Imaging Computed Tomography (SPECT), Magnetic Resonance Spectroscopy (MRS), Magnetic Resonance Imaging (MRI), and Computed Axial X-ray Tomography (CAT), or combinations thereof.

Methods of Preparation

Some of the compounds for use according to the invention are known compounds that are commercially available.

Other chemical compounds of the invention may be prepared by conventional methods for chemical synthesis, e.g. those described in the working examples. The starting materials for the processes described in the present application are known or

may readily be prepared by conventional methods from commercially available chemicals.

Also one compound of the invention can be converted to another compound of the invention using conventional methods. The end products of the reactions described herein may be isolated by conventional techniques, e.g. by extraction, crystallisation, distillation, chromatography, etc.

Biological Activity Compounds of the invention or for use according to the invention may be tested for their ability to modulate a PDZ domain, such as the PDZ domain of PICK1 e.g. as described in the "TEST METHODS" paragraph. Further, in particular in relation to pain disorders, compounds of the invention or for use according to the invention may be tested in various in vivo pain models well known in the art, such as the hot plate test, the formalin test, capsaicin-induced sensitization, the CFA test, the CCI test and the SNI model.

Based on the PICK1 inhibition and PICK1 interaction with GluR2 the compounds of the invention or for use according to the invention are considered useful for the treatment, prevention or alleviation of a disease or a disorder or a condition of a mammal, including a human, which disease, disorder or condition is responsive to modulation of a PDZ domain. In a special embodiment, the disease or disorder or condition is responsive to modulation of a PDZ domain is disease or disorder or condition is responsive to modulation of the PDZ domain of PICK1.

In a further embodiment, the compounds of the invention are considered useful for the treatment, prevention or alleviation of a variety of disorders of the CNS and PNS and disorders of other origin, including acute pain, chronic pain, neuropathic pain, intractable pain, migraine, neurological and psychiatric disorders, depression, anxiety, psychosis, schizophrenia, excitatory amino acid-dependent psychosis, cognitive disorders, dementia, senile dementia, AIDS-induced dementia, stress-related psychiatric disorders, stroke, global ischaemic, focal ischaemic, haemorrhagic stroke, cerebral hypoxia, cerebral ischaemia, cerebral infarction, cerebral ischaemia resulting from thromboembolic or haemorrhagic stroke, cardiac infarction, brain trauma, brain oedema, cranial trauma, brain trauma, spinal cord trauma, bone-marrow lesions, hypoglycaemia, anoxia, neuronal damage following hypoglycaemia, hypotonia, hypoxia, perinatal hypoxia, cardiac arrest, acute neurodegenerative diseases or disorders, chronic neurodegenerative diseases or disorders, brain ischaemia, CNS degenerative disorders, Parkinson's disease, Alzheimer's disease, Huntington's disease, idiopathic Parkinson's Disease, drug induced Parkinson's Disease, amyotrophic lateral sclerosis (ALS), post-acute phase cerebral lesions, chronic

diseases of the nervous system, cerebral deficits subsequent to cardiac bypass surgery, cerebral deficits subsequent to grafting, perinatal asphyxia, anoxia from drowning, anoxia from pulmonary surgery, anoxia from cerebral trauma, hypoxia induced nerve cell damage, epilepsy, status epilepticus, seizure disorders, cerebral vasospasm, CNS mediated spasms, motility disorders, muscular spasms, urinary incontinence, convulsions, disorders responsive to anticonvulsants, autoimmune diseases, emesis, nausea, obesity, chemical dependencies, chemical addictions, addictions, withdrawal symptoms, drug induced deficits, alcohol induced deficits, drug addiction, ocular damage, retinopathy, retinal neuropathy, tinnitus, and tardive dyskinesia.

In a special embodiment, the compounds of the invention are considered useful for the treatment, prevention or alleviation of: pain, acute pain, chronic pain, neuropathic pain, intractable pain, inflammatory pain, neurogenic pain, fibromyalgia, chronic fatigue syndrome, nociceptive pain, cancer pain, postoperative pain, migraine, tension-type headache, pain during labour and delivery, breakthrough pain, stroke, drug abuse and cocaine abuse.

It is at present contemplated that a suitable dosage of the active pharmaceutical ingredient (API) is within the range of from about 0.1 to about 1000 mg API per day, more preferred of from about 10 to about 500 mg API per day, most preferred of from about 30 to about 100 mg API per day, dependent, however, upon the exact mode of administration, the form in which it is administered, the indication considered, the subject and in particular the body weight of the subject involved, and further the preference and experience of the physician or veterinarian in charge.

Preferred compounds of the invention show a biological activity in the sub- micromolar and micromolar range, i.e. of from below 1 to about 100 μM.

Pharmaceutical Compositions

In another aspect the invention provides novel pharmaceutical compositions comprising a therapeutically effective amount of the chemical compound of the invention.

While a chemical compound of the invention for use in therapy may be administered in the form of the raw chemical compound, it is preferred to introduce the active ingredient, optionally in the form of a physiologically acceptable salt, in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries.

In a preferred embodiment, the invention provides pharmaceutical compositions comprising the chemical compound of the invention, or a pharmaceutically acceptable salt or derivative thereof, together with one or more pharmaceutically acceptable carriers, and, optionally, other therapeutic and/or prophylactic ingredients, known and

used in the art. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof. The pharmaceutical composition of the invention may be administered by any convenient route, which suits the desired therapy. Preferred routes of administration include oral administration, in particular in tablet, in capsule, in drage, in powder, or in liquid form, and parenteral administration, in particular cutaneous, subcutaneous, intramuscular, or intravenous injection. The pharmaceutical composition of the invention can be manufactured by any skilled person by use of standard methods and conventional techniques appropriate to the desired formulation. When desired, compositions adapted to give sustained release of the active ingredient may be employed.

Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA). The actual dosage depends on the nature and severity of the disease being treated, and is within the discretion of the physician, and may be varied by titration of the dosage to the particular circumstances of this invention to produce the desired therapeutic effect. However, it is presently contemplated that pharmaceutical compositions containing of from about 0.1 to about 500 mg of active ingredient per individual dose, preferably of from about 1 to about 100 mg, most preferred of from about 1 to about 10 mg, are suitable for therapeutic treatments.

The active ingredient may be administered in one or several doses per day. A satisfactory result can, in certain instances, be obtained at a dosage as low as 0.1 μg/kg i.v. and 1 μg/kg p.o. The upper limit of the dosage range is presently considered to be about 10 mg/kg i.v. and 100 mg/kg p.o. Preferred ranges are from about 0.1 μg/kg to about 10 mg/kg/day i.v., and from about 1 μg/kg to about 100 mg/kg/day p.o.

Methods of Therapy

In another aspect the invention provides a method for the treatment, prevention or alleviation of a disease or a disorder or a condition of a living animal body, including a human, which disease, disorder or condition is responsive to modulation of a PDZ domain, and which method comprises administering to such a living animal body, including a human, in need thereof an effective amount of a chemical compound of the invention or for use according to the invention. It is at present contemplated that suitable dosage ranges are 0.1 to 1000 milligrams daily, 10-500 milligrams daily, and especially 30-100 milligrams daily, dependent as usual upon the exact mode of administration, form in which administered, the indication toward which the administration is directed, the subject

involved and the body weight of the subject involved, and further the preference and experience of the physician or veterinarian in charge.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further illustrated by reference to the accompanying drawing, in which:

Fig. 1 shows PICK1 saturation binding. Fluorescently labeled peptides (40 nm) corresponding to the C-terminal 13 residues of DAT, PKC α 13, and β 2 AR (DAT13 OrG, PKC α 13 OrG, and β2AR13 OrG) were titrated with increasing amounts of purified WT PICK1 protein. After 15 min of incubation, FP values were determined as a direct readout of peptide binding to PICK1. Data are representative of at least five similar experiments.

Fig. 2 shows PICK1 competition binding. A, competition binding to PICK1 of Oregon Green-labeled DAT peptide (DAT13 OrG) with unlabeled peptides corresponding to the 13 C-terminal residues of DAT, PKC 0 , and β2AR. B, competition binding to PICK1 of Oregon Green-labeled DAT peptide (DAT13 OrG) with unlabeled peptides corresponding to the 13 C-terminal residues of GluR2. Data are representative of at least three similar experiments. For the experiments, tracer (40 nM) was incubated with a fixed subsaturating amount of PICK1 and titrated with increasing amounts of the indicated unlabeled peptides. After 15 min of incubation, FP values were determined.

Fig. 3 shows screening setup and identification of an active well. 1B-C: Oregon Green-labeled DAT peptide (40 nM) was incubated with a fixed subsaturating amount of PICK1 to estimate top of the window. 1 D-E: Oregon Green-labeled DAT peptide (40 nM) alone to estimate bottom of window. 1F-G: Oregon Green-labeled DAT peptide (40 nM) was incubated with a fixed subsaturating amount of PICK1 and 20μM of non- labeled DAT as a positive control for competition. 2A-12H: Oregon Green-labeled DAT peptide (40 nM) was incubated with a fixed subsaturating amount of PICK1 and 100μM of the small molecule drug to be screened. All wells contained 10% DMSO. After 15 min of incubation, FP values were determined. 'Active wells' (FP<80% of 1 B-C), wells showing increased FP and wells showing increased fluorescence (indicative of fluorescent drugs) are highlighted.

Fig. 4 shows PICK1 competition binding. Competition binding to PICK1 of Oregon Green-labeled DAT peptide (DAT13 OrG) with compound (j). Data are representative of at least three similar experiments. For the experiments, tracer (40 nM) was incubated with a fixed subsaturating amount of PICK1 and titrated with increasing amounts of compound (j). After 15 min of incubation, FP values were determined. The K 1 of compound (j) was shown to be 40 μM [39.3-41.7 μM].

Fig. 5A-E show additional competition curves for five additional compounds. Competition binding to PICK1 of Oregon Green-labeled DAT peptide (DAT13 OrG). Data are represent-tative of at least three similar experiments. For the experiments, tracer (40 nM) was incubated with a fixed subsaturating amount of PICK1 and titrated with increasing amounts of compound. After 15 min of incubation, FP values were determined.

Fig. 6 shows representative experiment showing the inhibition of GST-DAT pulldown of PICK by the compound compound (j) and the peptide GITI b.

Fig. 7 shows densitometry analysis of GST-DAT pull-down assay of PICK1 . Indicated compounds or the C-terminal peptide of GIT1 b were added at a concentration of 50 μM prior to performing the pull-down. Data are means ± SE of three independent experiments and expressed in percent of the pull-down seen in the presence of vehicle.

Fig 8 shows that FRET can be observed between eCFP-GluR2 C29 and eYFP- PICK1 . Indicated constructs were expressed transiently in COS7 cells and FRET values measured as described in Methods. Data are means ± S. E. of N=15-46.

Fig. 9A and 9B shows FRET between eCFP-GluR2 C29 and eYFP-PICK1 was specifically reduced by compound (j). Indicated constructs were expressed transiently in COS7 cells and FRET values measured as described in Methods. Compound or vehicle (1 % DMASO) was added 20 min prior to image acquisition. Data are means ± S. E. of N=8-41 , **p<0.01 as compared to vehicle.

EXAMPLES

The invention is further illustrated with reference to the following examples, which are not intended to be in any way limiting to the scope of the invention as claimed.

Synthetic examples

All reactions involving air sensitive reagents or intermediates are performed under nitrogen and in anhydrous solvents. Sodium sulphate is used as drying agent in the workup-procedures and solvents are evaporated under reduced pressure.

Method A 4-Ethoxy-3-hydroxy-biphenyl-2-carbaldehyde Is prepared by the Suzuki-reaction, by stirring a mixture of 6-bromo-3-ethoxy-2- hydroxy-benzaldehyde, benzenebononic acid (1 .5 eq), Pd(PPh 3 ) 4 (2%), 1 ,3- dihydroxypropane (3 eq), potassium carbonate (3eq), water and DMT at reflux for 15 h. Water is added and the mixture is extracted twice with dichloromethane.

Chromatography on silica gel with dichloromethane and methanol solvent gives the title compound.

4-Ethoxy-2'-fluoro-3-hydroxy-biphenyl-2-carbaldehyde Is prepared according to method A from 2-flourobenzeneboronic acid.

4-Ethoxy-3'-fluoro-3-hydroxy-biphenyl-2-carbaldehyde

Is prepared according to method A from 3-flourobenzeneboronic acid.

4-Ethoxy-4'-f luoro-3-hydroxy-biphenyl-2-carbaldehyde

Is prepared according to method A from 4-flourobenzeneboronic acid.

4-Ethoxy-3-hydroxy-2'-methoxy-biphenyl-2-carbaldehyde

Is prepared according to method A from 2-methoxybenzeneboronic acid.

4-Ethoxy-3-hydroxy-3'-methoxy-biphenyl-2-carbaldehyde

Is prepared according to method A from 3-methoxybenzeneboronic acid.

4-Ethoxy-3-hydroxy-4'-methoxy-biphenyl-2-carbaldehyde Is prepared according to method A from 4-methoxybenzeneboronic acid.

3-Ethoxy-2-hydroxy-6-pyridin-3-yl-benzaldehyde

Is prepared according to method A from diethyl-3-pyridylborane.

3-Ethoxy-6-furan-2-yl-2-hydroxy-benzaldehyde

Is prepared according to method A from 2-furaneboronic acid.

3-Ethoxy-6-furan-3-yl-2-hydroxy-benzaldehyde

Is prepared according to method A from 3-furaneboronic acid.

3-Ethoxy-2-hydroxy-6-thiophen-2-yl-benzaldehyde

Is prepared according to method A from 2-thiopheneeboronic acid.

3-Ethoxy-2-hydroxy-6-thiophen-3-yl-benzaldehyde Is prepared according to method A from 3-thiopheneboronic acid.

5-Ethoxy-4-hydroxy-biphenyl-3-carbaldehyde

Is prepared from 5-bromo-3-ethoxy-2-hydroxy-benzaldehyde and benzeneboronic acid according to method A.

3-Ethoxy-5-furan-2-yl-2-hydroxy-benzaldehyde

Is prepared from 5-bromo-3-ethoxy-2-hydroxy-benzaldehyde and 2-furanboronic acid according to method A.

3-Ethoxy-5-furan-3-yl-2-hydroxy-benzaldehyde Is prepared from 5-bromo-3-ethoxy-2-hydroxy-benzaldehyde and 3-furanboronic acid according to method A.

3-Ethoxy-2-hydroxy-5-thiophen-2-yl-benzaldehyde

Is prepared from 5-bromo-3-ethoxy-2-hydroxy-benzaldehyde and 2-thiophenboronic acid according to method A.

3-Ethoxy-2-hydroxy-5-thiophen-3-yl-benzaldehyde

Is prepared from 5-bromo-3-ethoxy-2-hydroxy-benzaldehyde and 3-thiophenboronic acid according to method A.

θ-Chloro^-ethoxy-S-hydroxy-biphenyl^-carbaldehyde

Is prepared from 2-bromo-3-chloro-5-ethoxy-6-hydroxy-benzaldehyde and benzeneboronic acid according to method A.

((Z)-3-Biphenyl-3-yl-2-cyano-acryloyl)-carbamic acid isopropyl ester

Is prepared according to method A from [(Z)-3-(3-bromo-phenyl)-2-cyano-acryloyl]- carbamic acid ethyl ester and benzeneboronic acid.

[(Z)-2-Cyano-3-(3-furan-2-yl-phenyl)-acryloyl]-carbamic acid isopropyl ester Is prepared according to method A from [(Z)-3-(3-bromo-phenyl)-2-cyano-acryloyl]- carbamic acid ethyl ester and 2-furanboronic acid.

((Z)-3-Biphenyl-4-yl-2-cyano-acryloyl)-carbamic acid ethyl ester

Is prepared according to method A from [(Z)-3-(4-bromo-phenyl)-2-cyano-acryloyl]- carbamic acid ethyl ester and benzeneboronic acid.

[(Z)-2-Cyano-3-(4-furan-2-yl-phenyl)-acryloyl]-carbamic acid ethyl ester

Is prepared according to method A from [(Z)-3-(4-bromo-phenyl)-2-cyano-acryloyl]- carbamic acid ethyl ester and 2-furanboronic acid.

(2-Oxo-6-phenyl-2H-chromene-3-carbonyl)-carbamic acid ethyl ester

Is prepared according to method A from (6-bromo-2-oxo-2H-chromene-3-carbonyl)- carbamic acid ethyl ester and benzeneboronic acid.

(6-Furan-2-yl-2-oxo-2H-chromene-3-carbonyl)-carbamic acid ethyl ester Is prepared according to method A from (6-bromo-2-oxo-2H-chromene-3-carbonyl)- carbamic acid ethyl ester and 2-furanboronic acid.

(6-Furan-3-yl-2-oxo-2H-chromene-3-carbonyl)-carbamic acid ethyl ester

Is prepared according to method A from (6-bromo-2-oxo-2H-chromene-3-carbonyl)- carbamic acid ethyl ester and 3-furanboronic acid.

(2-Oxo-6-thiophen-2-yl-2H-chromene-3-carbonyl)-carbamic acid ethyl ester

Is prepared according to method A from (6-bromo-2-oxo-2H-chromene-3-carbonyl)- carbamic acid ethyl ester and 2-furanboronic acid.

(2-Oxo-6-thiophen-3-yl-2H-chromene-3-carbonyl)-carbamic acid ethyl ester

Is prepared according to method A from (6-bromo-2-oxo-2H-chromene-3-carbonyl)- carbamic acid ethyl ester and 3-furanboronic acid.

Method B

3-Ethoxy-2-hydroxy-6-phenylethynyl-benzaldehyde

Is prepared by the Sonogashira reaction, by stirring a mixture of 6-bromo-3-ethoxy-2- hydroxy-benzaldehyde, phenylacetylene (3 eq), diisopropylethylamine (2 eq), CuI (0.1 eq), Pd(PPh 3 ) 4 (2 %) and dioxane at reflux for 15 h. Water is added and the mixture is extracted twice with dichloromethane. Chromatography on silica gel with dichloromethane and methanol solvent gives the title compound.

3-Ethoxy-2-hydroxy-6-pyridin-3-ylethynyl-benzaldehyde

Is prepared according to method B from 3-pyridylacetylene.

3-Ethoxy-2-hydroxy-6-(4-methoxy-phenylethynyl)-benzaldehy de

Is prepared according to method B from 4-methoxyphenylacetylene.

3-Ethoxy-2-hydroxy-6-thiophen-3-ylethynyl-benzaldehyde

Is prepared according to method B from 3-thienylacetylene.

3-Ethoxy-2-hydroxy-5-phenylethynyl-benzaldehyde Is prepared according to method B from 5-Bromo-3-ethoxy-2-hydroxy-benzaldehyde.

S-Chloro-δ-ethoxy-β-hydroxy^-phenylethynyl-benzaldehyde

Is prepared according to method B from 2-bromo-3-chloro-5-ethoxy-6-hydroxy- benzaldehyde.

(Benzo[ϋ]thiophene-2-carbonyl)-carbamic acid phenylethynyl ester

Is prepared according to method B from (Benzo[;b]thiophene-2-carbonyl)-carbamic acid ethynyl and iodobenzene.

(Benzo[ϋ]thiophene-2-carbonyl)-carbamic acid furan-2-ylethynyl ester

Is prepared according to method B from (Benzo[;b]thiophene-2-carbonyl)-carbamic acid ethynyl and 2-bromofuran.

(Benzo[ϋ]thiophene-2-carbonyl)-carbamic acid thiophen-2-ylethynyl ester Is prepared according to method B from (Benzo[;b]thiophene-2-carbonyl)-carbamic acid ethynyl and 2-bromothiophene

(Benzo[ϋ]thiophene-2-carbonyl)-carbamic acid furan-3-ylethynyl ester

Is prepared according to method B from (Benzo[;b]thiophene-2-carbonyl)-carbamic acid ethynyl and 3-bromofuran

(Benzo[ϋ]thiophene-2-carbonyl)-carbamic acid thiophen-3-ylethynyl ester

Is prepared according to method B from (Benzo[;b]thiophene-2-carbonyl)-carbamic acid ethynyl and 3-bromothiophene

Method C

[(Z)-2-Cyano-3-(3,4-dichloro-phenyl)-acryloyl]-carbamic acid methyl ester Is prepared by solvolysis under acidic conditions from [(Z)-2-Cyano-3-(3,4-dichloro- phenyl)-acryloyl]-carbamic acid ethyl ester and methanol.

[(Z)-2-Cyano-3-(3,4-dichloro-phenyl)-acryloyl]-carbamic acid isopropyl ester

Is prepared according to method C from [(Z)-2-Cyano-3-(3,4-dichloro-phenyl)-acryloyl]- carbamic acid ethyl ester and 2-propanol.

[(Z)-2-Cyano-3-(3,4-dichloro-phenyl)-acryloyl]-carbamic acid propyl ester

Is prepared according to method C from [(Z)-2-Cyano-3-(3,4-dichloro-phenyl)-acryloyl]- carbamic acid ethyl ester and n-propanol.

((Z)-2-Cyano-3-phenyl-acryloyl)-carbamic acid methyl ester

Is prepared according to method C from ((Z)-2-Cyano-3-phenyl-acryloyl)-carbamic acid ethyl ester and methanol.

((Z)-2-Cyano-3-phenyl-acryloyl)-carbamic acid isopropyl ester

Is prepared according to method C from ((Z)-2-Cyano-3-phenyl-acryloyl)-carbamic acid ethyl ester and 2-propanol.

Method D (Benzo[ϋ]thiophene-2-carbonyl)-carbamic acid ethyl ester

Is prepared from (benzo[;b]thiophene-2-carbonyl)-carbamic acid ethynyl ester by stirring with Pd/C under an atmosphere of hydrogen. Workup: filtration through celite.

(Benzo[ϋ]thiophene-2-carbonyl)-carbamic acid vinyl ester Is prepared from benzo[;b]thiophene-2-carbonyl)-carbamic acid ethynyl, using Lindlars poisoned Pd-catalyst.

TEST METHODS

Purification of PICK1 for fluorescence polarization assay

The entire coding region of rat PICK1 (residues 2-416) is amplified from a pCINEO vector by PCR using pfu polymerase according to the instructions by the manufacturer (Stratagene, La JoIIa, CA). The primers used introduce a 5' restriction site for Muni and 3' restriction site for Avrll. The PCR fragment is cleaved with Muni and Avrll and cloned into the reading frame of the pET41 a vector (Novagen, Madison, Wl) producing an N-terminally glutathione-S-transferase (GST) fusion of PICK1. The GST-PICK1 fusion was expressed in E. coli BL21 (DE3) pLysS (Novagen). The transformed bacteria are grown to OD 6 oo 0.6 and expression of the fusion protein is induced with isopropyl-β-D1 -thiogalactopyranoside (105μM) overnight at 3O 0 C. The bacteria are lysed by freezing and thawing in TBS buffer containing [5OmM Tris pH 7.4, 125mM NaCI, 1 % TX-100, 20μg/ml_ DNAse I, 1 mM DTT (Sigma)]. The lysate is cleared by centrifugation (rotor SS-34, 18000 rpm, 48000 x g, 30 min). The supernatant is incubated with glutathione-sepharose beads (Amersham Biosciences) under slow

rotation for 90 minutes at 4 0 C. The beads are pelleted at 3500 g for 5 minutes and washed in TBS buffer containing [50 mM Tris pH 7.4, 125 mM NaCI, 0.1 % TX-100, 1 mM DTT] by three batch washes. The protein is separated from the GST domain by cleavage with thrombin protease (Novagen) in the above wash buffer at 4 0 C overnight. Samples of 10 μl are taken from the protein solution for determination of protein concentration and for analysis by SDS-PAGE. Protein determinations are performed using the BCA Protein Assay Reagent kit (Pierce Biotechnology, Inc, Rockford, III) according to manufactures protocol using bovine serum albumin as standard. Gels are stained with GelCode Blue Stain Reagent (Pierce Biotechnology) in order to inspect size, integrity and purity of the protein (Madsen et al., JBC, 280, 20539-48, 2005).

Fluorescence polarization assay

This assay is used to screen for compounds binding to the PDZ binding groove in PICK1. The assay is based on the predicted decrease in rotational diffusion of a fluorescently labeled peptide upon its binding to a larger protein. The decrease in rotational diffusion upon binding of a fluorescent labeled peptide to PICK1 can be detected as an increase in fluorescence polarization (FP). Binding of a small-molecule compound to the PDZ binding groove can be detected by its ability to displace the fluorescently labeled peptide resulting in a decreased in FP (Madsen et al., JBC, 280, 20539-48, 2005).

To perform the assay, compounds are loaded (10 μl of each) in microtiter-plates (88 compounds per 96 well plate) (Corning) at a concentration of 1 mM in DMSO. To each well is added 90 μl buffer containing Tris base (50 mM), NaCI (125 mM), TX-100 (0.1 %), PICK1 (0.45 μM) and fluorescently labeled peptide. This peptide corresponds to the C-terminus of a protein that binds to the PICK1 PDZ domain. For example, the peptide can correspond to the last 13 C-terminal residues of the DAT, of protein kinase Ca or of the GluR2 subunit of the AMPA reaceptor. The peptide can be labeled with any fluorophore. For example it can be labeled with a sulfhydryl-reactive derivative of Oregon Green via a cysteine introduced in the peptide. The peptide is used in a concentration of about 40 nM. The final concentration of the compounds in the initial screen is 100 μM. After approximately 15 min of incubation at 3 0 C the plates are analyzed in a Chameleon plate-reader (HIDEX) in the FP mode using a 488 nm excitation filter and a 535nm long pass emission filter. Each measurement is an average of 100 flashes and is carried out four times. FP is calculated according to the equation FP = (l v - g * I H V(I V + g * I H )- AS negative and positive controls, respectively, pure DMSO and DMSO with unlabeled DAT peptide (20 μM final cone.) are measured in parallel. All compounds are tested twice on separate plates. Active compounds are

recognized by a decrease in (below 80%) depolarization compared to the control wells with pure DMSO.

To determine the affinity of identified compounds (K 1 values) the K d value for the fluorescently labeled peptide is first determined by performing saturation binding isotherms using a fixed amount of Oregon Green labeled peptide (4OnM) with an increasing amount of PICK1 in a final volume of 100 μl. An equilibrium saturation binding isotherms is constructed by plotting FP versus the concentration of PICK1. To determine K d , a curve is fitted with the equation Y=FP b *X/(K d *X), where FP b is the maximal value of FP reached by complete saturation. Subsequently, competition binding experiments are carried out in the same format as the saturation binding experiments using a fixed concentration of fluorescently labeled peptide (4OnM) and a fixed non-saturating concentration of purified PICK1 , and an increasing concentration of the compounds to be tested (up to 1 mM). Equilibrium competition binding isotherms are constructed by plotting FP versus the concentration of compound. To determine K 1 , a curve was fitted to the equation FP = FP f + ((FP f - FP b ) * [R t ])/(K d * (1 + XJK 1 ) + [RJ), with FP f and FP b being the FP value of the free and bound peptide, [R t ] the concentration of PICK1 and K d the apparent dissociation constant determined from parallel saturation experiments. K 1 , FP b, and FP f were treated as free parameters.

Pull Down

To verify binding of active compounds identified in the FP screen, a biochemical pulldown assay can be employed.

A fusion between GST and the 24 C-terminal amino acids of the dopamine transporter (GST DAT C24) is expressed in BL21 (DE3)pLysS (Novagen) using the pET41 a vector (Novagen, Madison, Wl) and purified as described for PICK1 but without digestion with thrombin.

5 μl dry volume of glutathione-coated sepharose beads (Pharmacia) with bound GST alone or GST DAT C24 are suspended in 200 μl TBS buffer containing as follows [5OmM Tris pH 7.4, 125 mM NaCI, 0.1 % TX-100, 1 mM DTT (Sigma) and PICK1 0.5 μM]. The compounds to be tested are added in DMSO to a final concentration of 200 μM (2% DMSO final concentrtaion). The last 11 amino acids of the C-terminus of the GLT1 b glutamate transporter (60 μM final) and pure DMSO (2%) can be used as a positive and negative control, respectively.

The samples are incubated at 4 0 C under slow rotation for 30 min. The beads are centrifuged at 4000 g for 5 min and subsequently washed in TBS buffer and recentrifuged. The beads containing bound protein are eluted in loading buffer and analyzed by 12% SDS-PAGE and proteins are stained with Gelcode blue stain reagent 5 (Pierce). Active compounds i.e. a blocked pull-down are recognized by a reduced PICK1 band on the SDS-PAGE gel compared to the control pull-down.

Fluorescence Resonance Energy Transfer (FRET)

To determine whether a compound is able to block binding of a PD ligand, such as the 10 c-terminus of the AMPA receptor GluR2 subunit, to the PICK1 PDZ domain in a cellular system a Fluorescent Resonance Energy Transfer (FRET) assay can be employed. PICK1 is fused to eYFP (eYFP-PICK1 ) and the C-terminal 29 residues of the AMPA receptor subunit GluR2 is fused to eCFP (eCFP-GluR2 C29). As a control for specificity of the FRET signal an alanine is added to the C-terminal 29 residues of 15 GluR2 (eCFP-GluR2 C29+A) to disrupt the PDZ interaction. Coexpression of eYFP- PICK + eCFP-GluR2 C29 (but not eCFP-GluR2 C29+A) will provide a FRET signal that are reduced by a small molecule compound if it can pass the plasma membrane and bind to the PICK1 PDZ domain.

20 Fluorescence resonance energy transfer (FRET; see Schmid and Sitte, 2003) is measured with an epi-fluorescence microscope (Carl Zeiss TM210, Germany) using the "three-filter method" according to Xia and Liu (2001 ). COS7 cells (3 x 10 5 /well) are seeded on to poly-D-lysine-coated glass coverslips (24mm diameter). The next day, cells are transiently transfected, using the calcium phosphate precipitation method.

25 Briefly, 1 -3μg cDNA was mixed with CaCI 2 and HBS buffer (28OmM NaCI / 1 OmM KCI / 1 ,5mM Na 2 HPO 4 is 12mM dextrose / 5OmM HEPES); after 6-10 minutes, the calcium phosphate-DNA precipitate was added to the cells. After 4-5 hours, the cells are washed twice with PBS and briefly treated with glycerol, followed by the addition of FCS-containing medium.

30

Media are replaced by Krebs-HBS buffer (1 OmM HEPES / 12OmM NaCI / 3mM KCI / 2mM CaCI 2 / 2mM MgCI 2 ), and images are taken using a 63x oil objective and a LUDL filter wheel that allows for rapid exchange of filters (less than 100 ms). The system is equipped with the following fluorescence filters: CFP filter (I CFP ; exc: 436 nm, dichr.: 35 455nm, em.: 480nm), YFP filter (l YF p; exc: 500nm, dichr.: 515nm, em.: 535nm) and FRET filter (I FRET : excitation = 436 nm, dichroic mirror = 455nm, emission = 535 nm). The acquisition of the images is done with MetaMorph (Meta Imaging, Universal Imaging Corporation, V. 4.6.). Background fluorescence is subtracted from all images

and fluorescence intensity was measured in cytosolic regions in all images. To calculate a normalized FRET signal (nFRET), the following equation is used:

N FRET = - JML a . YFP — — , where a and b represents the bleed-through values

S ΎFP ^- * CFP for YFP and CFP.

TEST RESULTS

Example 1

Fluorescence polarization To perform the initial large scale screen of compounds we used a fluorescence polarization (FP) assay as described in Methods. The assay is based on the capability of purified PICK1 to bind a fluorescently conjugated peptide in its PDZ domain binding pocket. In the assay we used peptides corresponding to the 13 C-terminal residues of PKCα, which has a type I PDZ binding sequence -QSAV, and of the human dopamine transporter (hDAT), which has a type Il PDZ binding sequence -WLKV, both of which are known to bind PICK1. A peptide corresponding to the 13 C-terminal residues of the β 2 AR was included as a control for the specificity of the saturation binding assay. Like the PKCα sequence, the β 2 AR sequence contains a type I PDZ binding sequence (- DSLL), but unlike the PKCα sequence it was believed not to bind PICK1. The 13-mer peptides used for saturation binding experiments all had an N-terminal cysteine that allowed fluorescent labeling with the sulfhydryl-reactive fluorophore Oregon Green maleimide. In the binding assay we took advantage of the predicted decrease in rotational diffusion of the fluorescently labeled peptides upon binding to a larger protein. Thus, we could detect the decrease in rotational diffusion upon binding of the peptides to PICK1 as an increase in FP. The increase in FP is illustrated by the saturation binding experiments shown in Fig. 1 in which a fixed concentration of fluorescently labeled peptide was titrated with an increasing amount of PICK1 (Fig. 1 ). The saturation binding experiments suggested that the DAT peptide bound with highest affinity to the purified preparation of PICK1 with an apparent K d value of ~1 μM. The PKCα peptide bound with an almost 10-fold lower apparent affinity than the DAT peptide, whereas the β 2 AR peptide bound with very low apparent affinity (Fig. 1 ).

Next, we carried out competition binding experiments in which fixed concentrations of PICK1 and of the fluorescently labeled peptide were titrated with an increasing amount of non- labeled peptide (Fig. 2A). In agreement with the saturation binding experiments, the unlabeled DAT peptide was more than one order of magnitude more potent in displacing the fluorescently tagged tracer than was the unlabeled PKCα peptide. In further agreement with the saturation binding experiments, the β 2 AR

peptide was much less potent than the two other peptides. From the competition binding experiments it was possible to calculate K 1 values for the interaction of the peptides with PICK1. We should note that the calculated K 1 values represent the most accurate estimate of the actual affinities. Thus, the absolute affinities obtained in the saturation binding assay might be affected both by the attached fluorophore and by the ratio between functional and non-functional protein in different purified preparations.

To further verify the specificity of the assay the unlabeled C13 terminal residues of the GluR2 AMPA receptor subunit, which is expected to bind PICK in a similar manner as DAT, was titrated against a fixed concentration of DAT-ORG and PICK, showing the expected sigmoid curve for a one-site competition (Fig. 2B).

Binding of a small-molecule compound to the PDZ binding groove can be detected by its ability to displace the fluorescently labeled peptide resulting in a decreased in FP just as the unlabelled GluR2 peptide.

Using the FP assay we screened -40,000 compounds using a setup in which 88 compounds were being tested in one reading session for the capability to block the interaction between PICK1 and the Oregon Green labeled DAT peptide. We used a concentration of 100 μM (10 %DMSO final) of each compound. Positive or active wells were recognized by comparing to wells containing DMSO (negative control/mP top level) and wells containing no PICK1 (basal mP level). Active wells were defined as wells giving an mP value below 80 % of the negative controls. (Fig. 3)

The screening strategy led to identification of a number of active wells. To confirm these active wells, a competition curve, similar to the above with GluR2 peptide, was conducted and the K 1 values were determined (Fig. 4 and 5) and Table 1.

Table 1. Calculated K 1 values for all the six compounds issued in this patent. K 1 values were calculated based on the FP assay experiments as described in Methods. Data are means of three independent experiments. The IC 50 values used in the estimation of

the K 1 values were calculated from the means of plC 50 values and the SE interval from the pICso ±SE.

Example 2 Pull down assay

To confirm the specific activity of the compounds found in the FP screen a pull down assay was developed.

In this assay the compounds are tested for the capability to block the interaction between PICK1 and a DAT-peptide fused to GST. Most importantly this assay is not influenced be fluorescent properties of the small molecules.

PICK1 and the GST-DAT fusion protein bound to glutathione-coated sepharose beads were allowed to interact in a buffer containing the compound to be tested. After 30 min. of rotation at 4 0 C, the beads with bound GST-DAT and possibly bound PICK were pelleted and washed before elution in loading buffer and analysis by SDS-PAGE. Proteins were visualized using Gelcode blue stain.

As shown in Fig. 6, GST-DAT efficiently pulled down PICK1 and, as expected, the pull- down was blocked by a peptide corresponding to the C-terminus (13 a. a.) of the GIT1 b glutamate transporter. Moreover, we observed that the pull-down of PICK1 by GST- DAT was inhibited by compound (j) as reflected in the weaker PICK1 band and consistent with inhibition of the PICK1 GST-DAT interaction.

Additional compounds were tested in the same assay using the same concentration of each compound (50 μM) and the results were analyzed by densitometry of the gels. The results are summarized in Fig. 7.

Example 3 Disruption of the PICK1 PDZ interaction in intact cellular system

A functional FRET pair dependent on PDZ mediated interaction of PICK1 with GluR2 was established by fusing PICK1 to eYFP (eYFP-PICK1 ) and the C-terminal 29 residues of GluR2 to eCFP (eCFP-GluR2 C29) as described in Methods. When transiently transfected into COS-7 cells significant FRET was observed from eCFP- GluR2 C29 to eYFP-PICK1 , suggesting an interaction between the two proteins (Fig. 8). By introducing an alanine at the C-terminal of GluR2 (eCFP-GluR2 C29+A), which is believed to disrupt the PDZ interaction, the FRET value was reduced to value of the negative control (eCFP + eYFP expressed independently and predicted not to

interact). We also tested a fusion protein between eCFP and eYFP (eCFP-eYFP) as a positive control providing a FRET value of 0.361 ± 0.008. Altogether, the data suggest that we are able to monitor the PICK1 GluR2 interaction by FRET in intact living cells (Fig. 8).

In order to test compounds identified in our screen, cells expressing eYFP-PICK1 together with eCFP-GluR2 C29 and eCFP-GluR2 C29+A were incubated with compound (j) for 20 min prior to image acquisition. Incubation with the compound significantly reduced FRET between eYFP-PICK1 and eCFP-GluR2 C29 (Fig. 9A). No effect was observed of the vehicle (1 %DMSO) (Fig. 9A) and no effect was seen for FRET between eYFP-PICK1 and eCFP-GluR2 C29+A (Fig. 9B).