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Title:
TRPA1 ANTAGONIST COMPOUNDS
Document Type and Number:
WIPO Patent Application WO/2014/184248
Kind Code:
A2
Abstract:
Compounds of Formula (I), pharmaceutically acceptable salts thereof, diastereomers, enantiomers, or mixtures thereof: wherein R1, R2, R3, X, Yn and A are as defined in the specification, as well as pharmaceutical compositions including the compounds are prepared. They are useful in therapy, in particular in the management of pain.

Inventors:
SVENSSON, Mats A (1200 NW Marshall Street Apt 417, Portland, Oregon, 97209, US)
WEIGELT, Dirk (Lilldalsvägen 8C, Rönning, S-144 61, SE)
Application Number:
EP2014/059853
Publication Date:
November 20, 2014
Filing Date:
May 14, 2014
Export Citation:
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Assignee:
ACTURUM LIFE SCIENCE AB (P.O. Box 808, Solna, S-169 28, SE)
International Classes:
C07C275/18
Domestic Patent References:
WO2012050512A12012-04-19
Other References:
VALLIN K S ET AL: "N-1-Alkyl-2-oxo-2-aryl amides as novel antagonists of the TRPA1 receptor", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, PERGAMON, AMSTERDAM, NL, vol. 22, no. 17, 1 September 2012 (2012-09-01), pages 5485-5492, XP002716178, ISSN: 0960-894X, DOI: 10.1016/J.BMCL.2012.07.032 [retrieved on 2012-07-14]
Attorney, Agent or Firm:
NEELISSEN-SUBNEL, Marianne (Swea IP Law AB, c/o NellpatBox 138, Hagford, S-683 23, SE)
Download PDF:
Claims:
What is claimed is:

1. A compound of formula (I), a pharmaceutically acceptable salt thereof, diastereomer, enantiomer, or mixture thereof

wherein:

X is selected from

A is selected from halogen or cyano;

Y is independently Ci-4alkyl or halogen;

R1 is hydrogen, Ci-3al kyl or halogenated Ci-3a lkyl;

R2 is hydrogen, Ci-6al kyl or halogenated Ci-6a lkyl;

R3 is Ci-6al kyl or halogenated Ci-6al kyl, wherein said Ci-6al kyl is optionally substituted by C6 ioaryl or C4-iohetereoaryl, wherein said C6 ioa ryl or C4-iohetereoaryl is optionally substituted by one or more halogen, cyano, Ci-6al kyl or halogenated Ci-6a lkyl;

or R2 and R3 together form a 4-7 membered ring, optionally fused to a 5-6 membered ring and optionally substituted with one or more groups selected from C6-ioaryl, C4- lohetereoaryl, C3-6cycloalkyl, C2-ioheterocyclyl, -CN, -SR4, -OR4, -0(CH2)P-OR4, -R4, -C(=0)- R4, -C02R4, -S02R4, -S02N(R4)2, halogen, -N02, -N(R4)2, -(CH2)PN(R4)2, and -C(=0)-N(R4)2; wherein said C6-ioaryl, C4-iohetereoaryl, Cs ecycloalkyl and C2-ioheterocyclyl may themselves optionally be substituted with one or more groups selected from -CN, O-Ci- 6al kyl, halogen and Ci-6al kyl, which in turn may optionally be halogenated;

R4 is independently hydrogen, Ci-6al kyl, C2-6a lkenyl, halogenated Ci-6al kyl or C6-ioaryl, wherein said C6 ioaryl is optionally substituted with one or more halogen; n is 0, 1 or 2; and

p is O, 1 , 2, 3, or 4.

2. A compound according to claim 1, wherein A is halogen or cyano, Y is halogen, and n is 0 or l.

3. A compound according to claim 1 or 2, wherein R1 is hydrogen.

4. A compound according to any one of claim 1-3, wherein R2 and R3 together form a piperazine, piperidine, diazepane or dihydroisoquinoline group, optionally substituted with one or more groups selected from C6-ioaryl, C4-iohetereoaryl, C3-6cycloalkyl, C2- loheterocyclyl, Ci-4alkyl, -0(CH2)i-3, -C(=0)-(CH2)i-3, -C02(CH2)i-3, -halogen; wherein said C6- ioaryl, C4-iohetereoaryl, is optionally substituted with one or more groups selected from cyano, 0-Ci-4alkyl, halogen and Ci-4alkyl, or halogenated Ci-3alkyl.

5. A compound according to any one of claim 1-3, wherein R2 and R3 together form a 5-7 membered ring fused to a 5-6 membered ring and optionally substituted with one or more groups selected -0(CH2)P, -C(=0)- (Chhjp, -C02(CH2)P, - halogen and Ci-4alkyl. 6. A compound according to any one of claims 1-3, wherein R2 is hydrogen or Ci-4alkyl, and R3 is Ci-ealkyl.

7. A compound according to any one of claim 1-3, wherein R1 is hydrogen, R2 is hydrogen or Ci-4alkyl, and R3 is Ci-6alkyl, wherein said Ci-6alkyl is optionally substituted by phenyl, and wherein said phenyl is optionally substituted by one or more halogen, cyano, Ci-4alkyl or halogenated Ci-4alkyl.

8. A compound selected from one or more of the following:

l-ieri-Butyl-3-(l-(4-chlorophenyl)-3-methyl-l-oxobutan-2-yl)-l-methylurea; N-(l-(4-Chlorophenyl)-3-methyl-l-oxobutan-2-yl)-4-(pyrimidin-2-yl)piperazine-l- carboxamide;

l-ieri-Butyl-3-(l-(4-chlorophenyl)-3-methyl-l-oxobutan-2-yl)urea;

Ethyl l-[[l-(4-chlorophenyl)-3-methyl-l-oxobutan-2-yl]carbamoyl]piperidine-4- carboxylate;

N-(l-(4-Chlorophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(pyrazin-2-yl)piperazine-l- carboxamide;

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-3,4-dihydroisoquinoline-2(lH)- carboxamide;

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-4-(4-cyanopyridin-3-yl)piperazine-l- carboxamide;

(3S)-N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-3-methyl-4-(pyrimidin-2- yl)piperazine-l-carboxamide;

(2R)-N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-2-methyl-4-(pyrimidin-2- yl)piperazine-l-carboxamide;

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-2,2-dimethyl-4-(pyrimidin-2- yl)piperazine-l-carboxamide;

N-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-4-phenyl-piperazine-l-carboxamide;

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l-phenethyl-urea;

N-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-4-(2-pyridyl)piperazine-l-carboxamide;

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l-pentyl-urea;

l-Butyl-3-[l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-ethyl-urea;

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l,l-dipropyl-urea;

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-[(4-fluorophenyl)methyl]-l-methyl- urea;

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l-(m-tolylmethyl)urea;

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-3,3-dimethyl-4-(pyrimidin-2- yl)piperazine-l-carboxamide;

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-4-(pyrimidin-2-yl)piperazine-l- carboxamide; N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-methylpyridin-2-yl)piperazine- 1-carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-methylpyridin-2-yl)piperazine- 1-carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-methylpyridin-2-yl)piperazine- 1-carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(3-(trifluoromethyl)pyridin-2- yl)piperazine-l-carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(2-methoxyphenyl)piperazine-l- carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(3-isopropyl-l,2,4-oxadiazol-5- yl)piperidine-l-carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(4-methoxyphenyl)piperazine-l- carboxamide;

4-(2-Cyanophenyl)-N-(l-(4-cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)piperazine-l- carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(4-(trifluoromethyl)pyrimidin-2- yl)-l,4-diazepane-l-carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(pyridin-3-yl)piperazine-l- carboxamide;

N-(l-(4-Cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(3-cyanopyridin-2- yl)piperazine-l-carboxamide;

N-(l-(4-Cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(3-cyanopyridin-2- yl)piperazine-l-carboxamide; and

N-(l-(4-Cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(thiazol-2-yl)piperazine- 1-carboxamide,

or a pharmaceutically acceptable salt, diastereomer or enantiomer thereof.

A compound according to any previous claim, or a pharmaceutically acceptable sal diastereomer or enantiomer thereof, as a medicament in therapy.

10. The use of a compound according to any one of claims 1 - 8 in the treatment of asthma, pertussis, nicotine addiction, various pain conditions such as acute and chronic pain disorders including but not limited to widespread pain, localized pain, nociceptive pain, inflammatory pain, central pain, central and peripheral neuropathic pain, central and peripheral neurogenic pain, central and peripheral neuralgia, chronic tendinitis, low back pain, postoperative pain, peripheral neuropathy, visceral pain, pelvic pain, allodynia, anesthesia dolorosa, causalgia, dysesthesia, fibromyalgia, hyperalgesia, hyperesthesia, hyperpathia, ischemic pain, sciatic pain, pain associated with cystitis, including but not limited to interstitial cystitis, pain associated with multiple sclerosis, pain associated with arthritis, pain associated with osteoarthritis, pain associated with rheumatoid arthritis, and pain associated with cancer.

11. The use of a compound according to any one of claims 1 - 8 in the treatment of Alzheimer's disease.

12. The use of a compound according to any one of claims 1 - 8 in the treatment of schizophrenia. 13. The use of a compound according to any one of claims 1 - 8 in the treatment of GERD.

14. A pharmaceutical composition comprising a compound according to any one of claims 1 - 8 and a pharmaceutically acceptable carrier. 15. A method for the therapy of Alzheimer's disease and/or pain and/or schizophrenia and/or GERD and/or asthma and/or pertussis and/or nicotine addiction, in a warm-blooded animal, comprising the step of administering to said animal in need of such therapy a therapeutically effective amount of a compound according to any one of claims 1 - 8.

Description:
TRPAl ANTAGONIST COMPOUNDS

Field of the invention

The present invention relates to antagonists of the transient receptor potential (TRP) family of ion channels. The present invention also provides compositions comprising such antagonists and methods therewith for treating diseases mediated by the transient receptor potential (TRP) family of ion channels. Particularly, the present invention is related to compounds that may be effective in treating pain and other specific disorders.

Background

The transient receptor potential (TRP) family of ion channels are often found to be sensors of multiple chemical and physical stimuli (temperature, smell, taste and noxious chemicals). Particularly, the non-selective cation channel TRPAl (transient receptor potential ankyrin- repeat 1), originally cloned as an ankyrin-like protein (Jaquemar et al 1999, J Biol Chem 274:7325-7333), is activated by multiple pungent and pro-algesic compounds (Bandell et al 2004, Neuron 41: 849-857). TRPAl is highly expressed in a subset of C-fiber nociceptors in the peripheral nerve system (Kobayashi et al 2005, J Comp Neurol 493(4):596-606). When activated TRPAl permits the conduction of cations (primarily Ca 2+ and Na + ) from the extra cellular environment into the cell, thereby depolarizing the membrane potential and affecting calcium homeostasis in the primary afferents. Depolarization primary nerve terminals lead to action potential firing and consequently increased pain sensation and hyperalgesia in man and rodent experimental models (Jiang and Gebhart 1998, Pain 77(3):305-13; Cervero and Laird 1996, Pain 68(l):13-23). The pungent ingredient in mustard oil: allyl isothiocyanate (AITC), concentration dependently activates TRPAl in vitro measured both on sodium current and calcium influx. Further, AITC also excites small diameter afferent fibers (Reeh 1986, Brain Res 384:42-50) and indeed topical application of AITC induces pain and hyperalgesia in man (Namer et al 2005, Neuroreport 16(9):955-959). Recently TRPAl knock out (KO) mice were described to have lost AITC sensitivity and display severely impaired bradykinin pain response signalling (Kwan 2006, Neuron 50(2):277-289; Bautista 2006, Cell 124(6):1269-1282). Formalin, which is a mixture of methanol, water and formaldehyde is a widely used rodent model for evaluating analgesic compounds in vivo. TRPAl is activated by formaldehyde in vitro and recently it was shown that TRPAl KO mice have almost abolished their response to formalin/formaldehyde (McNamara et al 2007, Proc Natl Acad Sci U S A 104(33):13525-13530). A Hydra Biosciences proprietary compound (HC030031) is a TRPAl antagonist in vitro and has been shown to alleviate the formalin induced pain behaviour in a dose dependant way (McNarama 2007, Proc Natl Acad Sci U S A 104(33):13525-13530 and WO2007/073505 A2). It is therefore suggested that in vitro inhibition of the TRPAl calcium and sodium influx will determine the compounds propensity to work as analgesics in vivo. An object of the present invention is therefore to provide new, improved and useful analgesics.

Definitions and description of the embodiments

The present invention thus aims at providing new antagonists of the non-selective cation channel TRPAl. The new antagonists can be used for, amongst other things, treating pain. Further medical conditions which are mediated by TRPAl antagonists include asthma, pertussis and nicotine addiction.

The term "C m -n" or "C m -n group" refers to any group having m to n carbon atoms.

The term "alkyl" refers to a saturated monovalent straight or branched chain hydrocarbon radical comprising 1 to about 12 carbon atoms. Illustrative examples of alkyls include, but are not limited to, Ci-6alkyl groups, such as methyl, ethyl, propyl, 1-methyl-ethyl, isopropyl, 2-methyl-l-propyl, 2-methyl-2-propyl, 2-methyl-l-butyl, 3-methyl-l-butyl, 2- methyl-3-butyl, 2,2-dimethyl-l-propyl, 2-methyl-l-pentyl, 3-methyl-l-pentyl, 4-methyl-l- pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l-butyl, 3,3- dimethyl-l-butyl, 2-ethyl-l-butyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, and hexyl, and longer alkyl groups, such as heptyl, octyl and n-nonyl. An alkyl can be unsubstituted or substituted with one or two suitable substituents.

The term "alkenyl" refers to a monovalent straight or branched chain hydrocarbon radical having at least one carbon-carbon double bond and comprising at least 2 up to about 12 carbon atoms. The double bond of an alkenyl can be unconjugated or conjugated to another unsaturated group. Suitable alkenyl groups include, but are not limited to C 2 -6alkenyl groups, such as vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2- ethylhexenyl, 2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl. An alkenyl can be unsubstituted or substituted with one or two suitable substituents.

The term "alkynyl" refers to a monovalent straight or branched chain hydrocarbon radical having at least one carbon-carbon triple bond and comprising at least 2 up to about 12 carbon atoms. The triple bond of an alkynyl can be unconjugated or conjugated to another unsaturated group. Suitable alkynyl groups include, but are not limited to C 2 -6alkynyl groups, such as acetylenyl, methylacetylenyl, butynyl, pentynyl, hexynyl. An alkynyl can be unsubstituted or substituted with one or two suitable substituents. The term "cycloalkyi" refers to a saturated monovalent ring-containing hydrocarbon radical comprising at least 3 up to about 12 carbon atoms. Examples of cycloalkyls include, but are not limited to, C3-7cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, and saturated cyclic terpenes. A cycloalkyi can be unsubstituted or substituted by one or two suitable substituents. Preferably, the cycloalkyi is a monocyclic ring.

The term "aryl" refers to a monovalent hydrocarbon radical having one or more polyunsaturated carbon rings having aromatic character, (e.g., 4n + 2 delocalized electrons) and comprising 5 up to about 14 carbon atoms.

The term "heterocycle" refers to a ring-containing structure or molecule having one or more multivalent heteroatoms, independently selected from N, 0, P and S, as a part of the ring structure and including at least 3 and up to about 20 atoms in the ring(s). Heterocycle may be saturated or unsaturated, containing one or more double bonds, and heterocycle may contain more than one ring. When a heterocycle contains more than one ring, the rings may be fused or unfused. Fused rings generally refer to at least two rings share two atoms therebetween. Heterocycle may have aromatic character or may not have aromatic character.

The term "heteroaromatic" refers to a ring-containing structure or molecule having one or more multivalent heteroatoms, independently selected from N, 0, P and S, as a part of the ring structure and including at least 3 and up to a bout 20 atoms in the ring(s), wherein the ring-containing structure or molecule has an aromatic character (e.g., 4n + 2 delocalized electrons).

The term "heterocyclic group," "heterocyclic moiety," "heterocyclic," or "heterocyclo" refers to a radical derived from a heterocycle by removing one or more hydrogens therefrom.

The term "heterocyclyl" refers a monovalent radical derived from a heterocycle by removing one hydrogen therefrom.

The term "heterocyclylene" refers to a divalent radical derived from a heterocycle by removing two hydrogens therefrom, which serves to links two structures together.

The term "heteroaryl" refers to a heterocyclyl having aromatic character.

The term "heterocycloalkyl" refers to a monocyclic or polycyclic ring comprising carbon and hydrogen atoms and at least one heteroatom, preferably, 1 to 3 heteroatoms selected from nitrogen, oxygen, and sulfur, and having no unsaturation. Examples of heterocycloalkyi groups include pyrrolidinyl, pyrrolidino, piperidinyl, piperidino, piperazinyl, piperazino, morpholinyl, morpholino, thiomorpholinyl, thiomorpholino, and pyranyl. A heterocycloalkyi group can be unsubstituted or substituted with one or two suitable substituents. Preferably, the heterocycloalkyi group is a monocyclic or bicyclic ring, more preferably, a monocyclic ring, wherein the ring comprises from 3 to 6 carbon atoms and form 1 to 3 heteroatoms, referred to herein as C3-6heterocycloalkyl.

The term "heteroarylene" refers to a heterocyclylene having aromatic character.

The term "heterocycloalkylene" refers to a heterocyclylene that does not have aromatic character.

The term "seven-membered" refers to a group having a ring that contains seven ring atoms.

The term "six-membered" refers to a group having a ring that contains six ring atoms. The term "five-membered" refers to a group having a ring that contains five ring atoms.

The term "four -membered" refers to a group having a ring that contains four ring atoms.

A five-membered ring heteroaryl is a heteroaryl with a ring having five ring atoms wherein 1, 2 or 3 ring atoms are independently selected from N, 0 and S.

Exemplary five-membered ring heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3- thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4- triazolyl, 1,3,4-thiadiazolyl, and 1,3,4- oxadiazolyl.

A six-membered ring heteroaryl is a heteroaryl with a ring having six ring atoms wherein 1, 2 or 3 ring atoms are independently selected from N, 0 and S.

Exemplary six-membered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl.

Heterocycle includes, for example, monocyclic heterocycles such as: aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazolidine, pyrazolidine, pyrazoline, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran tetrahydrofuran, thiophane, piperidine, 1,2,3, 6-tetrahydro-pyridine, piperazine, morpholine, thiomorpholine, pyran, thiopyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dihydropyridine, 1,4-dioxane, 1,3-dioxane, dioxane, homopiperidine, 2,3,4,7-tetrahydro-l/-/-azepine, homopiperazine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin, and hexamethylene oxide.

In addition, heterocycle includes aromatic heterocycles, for example, pyridine, pyrazine, pyrimidine, pyridazine, thiophene, furan, furazan, pyrrole, imidazole, thiazole, oxazole, pyrazole, isothiazole, isoxazole, 1,2,3-triazole, tetrazole, 1,2,3-thiadiazole, 1,2,3- oxadiazole, 1,2,4-triazole, 1,2,4-thiadiazole, 1,2,4-oxadiazole, 1,3,4-triazole, 1,3,4-thiadiazole, and 1,3,4- oxadiazole.

Additionally, heterocycle encompass polycyclic heterocycles, for example, indole, indoline, isoindoline, quinoline, tetrahydroquinoline, isoquinoline, tetrahydroisoquinoline, 1,4-benzodioxan, coumarin, dihydrocoumarin, benzofuran, 2,3-dihydrobenzofuran, isobenzofuran, chromene, chroman, isochroman, xanthene, phenoxathiin, thianthrene, indolizine, isoindole, indazole, purine, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, phenanthridine, perimidine, phenanthroline, phenazine, phenothiazine, phenoxazine, 1,2-benzisoxazole, benzothiophene, benzoxazole, benzthiazole, benzimidazole, benztriazole, thioxanthine, carbazole, carboline, acridine, pyrolizidine, and quinolizidine.

In addition to the polycyclic heterocycles described above, heterocycle includes polycyclic heterocycles wherein the ring fusion between two or more rings includes more than one bond common to both rings and more than two atoms common to both rings. Examples of such bridged heterocycles include quinuclidine, diazabicyclo[2.2.1]heptane and 7-oxabicyclo[2.2.1]heptane.

Heterocyclyl includes, for example, monocyclic heterocyclyls, such as: aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, pyrazolidinyl, pyrazolinyl, dioxolanyl, sulfolanyl, 2,3-dihydrofuranyl, 2,5-dihydrofuranyl, tetrahydrofuranyl, thiophanyl, piperidinyl, 1,2,3,6-tetrahydro-pyridinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, 2,3-dihydropyranyl, tetrahydropyranyl, 1,4-dihydropyridinyl, 1,4-dioxanyl, 1,3-dioxanyl, dioxanyl, homopiperidinyl, 2,3,4,7- tetrahydro-l/-/-azepinyl, homopiperazinyl, 1,3-dioxepanyl, 4,7-dihydro-l,3-dioxepinyl, and hexamethylene oxidyl.

In addition, heterocyclyl includes aromatic heterocyclyls or heteroaryl, for example, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, thienyl, furyl, furazanyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3- thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4- triazolyl, 1,3,4-thiadiazolyl, and 1,3,4 oxadiazolyl. Additionally, heterocyclyl encompasses polycyclic heterocyclyls (including both aromatic or non-aromatic), for example, indolyl, indolinyl, isoindolinyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, 1,4-benzodioxanyl, coumarinyl, dihydrocoumarinyl, benzofuranyl, 2,3-dihydrobenzofuranyl, isobenzofuranyl, chromenyl, chromanyl, isochromanyl, xanthenyl, phenoxathiinyl, thianthrenyl, indolizinyl, isoindolyl, indazolyl, purinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, phenanthridinyl, perimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxazinyl, 1,2-benzisoxazolyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benzimidazolyl, benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrolizidinyl, and quinolizidinyl.

In addition to the polycyclic heterocyclyls described above, heterocyclyl includes polycyclic heterocyclyls wherein the ring fusion between two or more rings includes more than one bond common to both rings and more than two atoms common to both rings. Examples of such bridged heterocycles include quinuclidinyl, diazabicyclo[2.2.1]heptyl, and 7- oxabicyclo[2.2.1]heptyl.

The term "alkoxy" refers to radicals of the general formula -O-R, wherein R is selected from a hydrocarbon radical. Exemplary alkoxy includes methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, isobutoxy, cyclopropylmethoxy, allyloxy, and propargyloxy.

The term "alkylsulfanyl" refers to radicals of the general formula -S-R, wherein R is selected from a hydrocarbon radical. Exemplary alkoxy includes methylsulfanyl, ethylsulfanyl and propylsulfanyl.

Halogen includes fluorine, chlorine, bromine and iodine.

The following abbreviations are used in the present description:

aq aqueous

Ac acetyl

ACN acetonitrile

CAS Chemical Abstracts Service

coned concentrated

DCM Dichloromethane

DIPEA N,N-Diisopropylethylamine

DMF dimethylformamide DMAP 4-(dimethylamino)pyridine

DMSO Dimethyl sulfoxide

EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

EGTA Ethylene glycol tetraacetic acid

ES electrospray

ESI Electrospray ionization

FLIPR Fluorometric Imaging Plate Reader

GC gas chromatography

h hour(s)

HATU 2-(lH-7-Azabenzotriazol-l-yl)--l,l,3,3-tetramethyl uronium hexafluorophosphate Methanaminium

HBSS Hank's Buffered Salt Solution

HBTU 0-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-pho sphate

HCI Hydrochloride

HOAT l-hydroxy-7-azabenzotriazole

HOBT 1-hydroxybenzotriazole hydrate

HPLC high-performance liquid chromatography

LED Light-Emitting Diode

M Molar (moles per liter)

min minute(s)

MS Mass spectrometry

NMR Nuclear magnetic resonance

RT or rt Room temperature

Rt retention time

SFC Supercritical fluid chromatography

TEA triethylamine

TLC thin layer chromatography temp temperature

THF tetrahydrofuran

UV ultraviolet

vis. visual

In a first aspect, the present invention provides a compound of Formula (I), a pharmaceutically acceptable salt thereof, diastereomer, enantiomer, or mixture thereof

wherein:

X is selected from:

A is selected from halogen or cyano;

Y is independently Ci- 4 alkyl or halogen;

R 1 is hydrogen, Ci-3alkyl or halogenated Ci-3alkyl;

R 2 is hydrogen, Ci-6alkyl or halogenated Ci-6alkyl;

R 3 is Ci-6alkyl or halogenated Ci-6alkyl, wherein said Ci-6alkyl is optionally substituted by C6- ioaryl or C 4 -iohetereoaryl, wherein said C6-ioaryl or C 4 -iohetereoaryl is optionally substituted by one or more halogen, cyano, Ci-6alkyl or halogenated Ci-6alkyl;

or R 2 and R 3 together form a 4-7 membered ring, optionally fused to a 5-6 membered ring and optionally substituted with one or more groups selected from C6-ioaryl, C 4 -iohetereoaryl, Cs-ecycloalkyl, C 2- ioheterocyclyl, cyano, -SR 4 , -OR 4 , -0(CH 2 ) P -OR 4 , -R 4 , -C(=0)-R 4 , -C0 2 R 4 , - S0 2 R 4 , -S0 2 N(R 4 ) 2 , halogen, -N0 2 , -N(R 4 ) 2 , -(CH 2 ) P N(R 4 ) 2 , and -C(=0)-N(R 4 ) 2 ; wherein said C 6 - loaryl, C 4 -iohetereoaryl, C3-6cycloalkyl and C 2 -ioheterocyclyl may themselves optionally be substituted with one or more groups selected from cyano, 0-Ci-6alkyl, halogen and Ci-6alkyl, which in turn may optionally be halogenated;

R 4 is independently hydrogen, Ci-6alkyl, C 2 -6alkenyl, halogenated Ci-6alkyl or C6-ioaryl, wherein said C6 ioaryl is optionally substituted with one or more halogen;

n is 0, 1 or 2; and

p is O, 1 , 2, 3, or 4.

In another embodiment A is halogen or cyano, Y is halogen, and n is 0 or 1, and X, R 1 , R 2 , R 3 , R 4 and p are as defined above. In one embodiment A is chloro or cyano, and n is 0. In another embodiment A is cyano, Y is fluoro and n is 1.

In one embodiment, R 1 is hydrogen, and A, X, Y, R 2 , R 3 , R 4 , n and p are as defined above.

In one embodiment, R 2 is Ci-6alkyl. In another embodiment R 2 is hydrogen or Ci- 4 alkyl, and R 3 is Ci-6alkyl.

In a further embodiment R 1 is hydrogen, R 2 is hydrogen or Ci- 4 alkyl, and R 3 is Ci-6alkyl or halogenated Ci-6alkyl, wherein said Ci-6alkyl is optionally substituted by C6 ioaryl or C 4 - lohetereoaryl, wherein said C6-ioaryl or C 4 -iohetereoaryl is optionally substituted by one or more halogen, cyano, Ci-6alkyl or halogenated Ci-6alkyl, and A, X, Y and n are as defined above. In a further embodiment, R 1 is hydrogen, R 2 is hydrogen or Ci- 4 alkyl, and R 3 is Ci-6alkyl, wherein said Ci-6alkyl is optionally substituted by phenyl, and wherein said phenyl is optionally substituted by one or more halogen, cyano, Ci- 4 alkyl or halogenated Ci- 4 alkyl, and A, X, Y and n are as defined above. In yet a further embodiment, R 1 is hydrogen, R 2 is hydrogen or Ci- 4 alkyl, and R 3 is Ci-6alkyl, wherein said Ci-6alkyl is optionally substituted by phenyl, wherein said phenyl is optionally substituted by one or more fluoro or Ci- 4 alkyl, and A, X, Y and n are as defined above. In another embodiment R 1 is hydrogen, R 2 is Ci- 4 alkyl and R 3 is Ci-6alkyl and A, X, Y and n are as defined above.

In one embodiment, R 2 and R 3 together form a 5-7 membered saturated ring, optionally substituted with one or more groups selected from C6-ioaryl, Cuohetereoaryl, C3-6cycloalkyl, C 2 -ioheterocyclyl, Ci- 4 alkyl, -0(CH 2 ) p , -C(=0)-(CH 2 ) p , -C0 2 (CH 2 ) p , -halogen; wherein said C6- loaryl, C 4 -iohetereoaryl is optionally substituted with one or more groups selected from cyano, 0-Ci- 4 alkyl, halogen and Ci- 4 alkyl, or halogenated Ci-3alkyl, and A, X, Y, R 1 , p and n are as defined above.

In a further embodiment, R 2 and R 3 together form a 5-7 membered saturated ring, optionally substituted with one or more groups selected from phenyl, Cs-6hetereoaryl, Ci-2alkyl, - 0(CH2)i-3, -C(=0)-(CH2)i-3, -C02(CH2)i-3, -halogen; wherein said phenyl, Cs-6hetereoaryl, is optionally substituted with one or more groups selected from cyano, 0-Ci-2alkyl, and Ci- 3alkyl, or fluorinated Ci-3alkyl, and A, X, Y, R 1 , p and n are as defined above.

In another embodiment, R 2 and R 3 together form a 5-7 membered ring fused to a 5-6 membered ring and optionally substituted with one or more groups selected -0(CH2) P , - C(=0)- (CH2)p, -C02(CH2)p, - halogen and Ci- 4 alkyl, and A, X, Y, R 1 , p and n are as defined above.

In one embodiment, R 2 and R 3 together form a piperazine, piperidine, diazepane or dihydroisoquinoline group, optionally substituted with one or more groups selected from C6- l oaryl, C 4 -i 0 hetereoaryl, C 3 - 6 cycloalkyl, C 2 -ioheterocyclyl, -CN, -SR 4 , -OR 4 , -0(CH 2 ) P -OR 4 , -R 4 , - C(=0)-R 4 , -CO2R 4 , -SO2R 4 , -S0 2 N(R 4 ) 2 , halogen, -N0 2 , -N(R 4 ) 2 , -(CH 2 ) P N(R 4 ) 2 , and -C(=0)-N(R 4 ) 2 ; wherein said C6-ioaryl, C 4 -iohetereoaryl, C3-6cycloalkyl and C2-ioheterocyclyl may themselves optionally be substituted with one or more groups selected from -cyano, halogen, halogenated Ci-6alkyl and Ci-6alkyl, and A, X, Y, R 1 , R 4 , p and n are as defined above.

In another embodiment, R 2 and R 3 together form a piperazine, piperidine, diazepane or dihydroisoquinoline group, optionally substituted with one or more groups selected from C6- ioaryl, C 4 -iohetereoaryl, C3-6cycloalkyl, C2-ioheterocyclyl, Ci- 4 alkyl, -0(CH2)i-3, -C(=0)-(CH2)i-3, - C02(CH2)i-3, -halogen; wherein said C6-ioaryl, C 4 -iohetereoaryl, is optionally substituted with one or more groups selected from cyano, 0-Ci- 4 alkyl, halogen and Ci- 4 alkyl, or halogenated Ci-3alkyl, and A, X, Y, R 1 and n are as defined above.

In one embodiment, R 2 and R 3 together form a piperazine, piperidine, diazepane or dihydroisoquinoline group, optionally substituted with one or more groups selected from pyrimidinyl, phenyl, oxadiazolyl, thiazolyl, pyridinyl, -C02-Ci- 4 alkyl and Ci- 4 alkyl, which groups are optionally substituted with one or more groups selected from cyano, 0-Ci- 4 alkyl, halogen and Ci- 4 alkyl, or halogenated Ci-3alkyl, and A, X, Y, R 1 and n are as defined above.

In a further embodiment, R 2 and R 3 together form a piperazine, piperidine, diazepane or dihydroisoquinoline group, optionally substituted with one or more groups selected from methyl, -C02-ethyl, pyrimidinyl, trifluoromethylpyrimidinyl phenyl, methoxyphenyl, cyanophenyl, oxadiazolyl, i-propyloxadiazolyl, thiazolyl, pyridinyl, cyanopyridinyl, methylpyridinyl and trifluoromethylpyridinyl.

In one embodiment, X is selected from:

A is selected from chloro or cyano;

Y is halogen;

R 1 is hydrogen;

R 2 is hydrogen or Ci-6alkyl;

R 3 is phenyl, wherein said phenyl is optionally substituted by one or more fluoro or Ci- 4 alkyl; or R 2 and R 3 together form a 5-7 membered ring, optionally fused to a 5-6 membered ring and optionally substituted with one or more groups selected from C6-ioaryl, C 4 -iohetereoaryl, C 3 -6cycloalkyl, C 2 -ioheterocyclyl, Ci- 4 alkyl, -0(CH 2 )i-3, -C(=0)-(CH 2 )i- 3 , -C0 2 (CH 2 )i- 3 , -halogen; wherein said C 4 -iohetereoaryl, is optionally substituted with one or more groups selected from cyano, 0-Ci- 4 alkyl, halogen and Ci- 4 alkyl, or halogenated Ci-3alkyl;

n is O orl.

As defined above is to be understood as defined any where above. Thus, the present invention is not limited to the specific combinations mentioned but includes all and any possible combination of substituents.

Specific compounds of the invention are:

l-ieri-Butyl-3-(l-(4-chlorophenyl)-3-methyl-l-oxobutan-2- yl)-l-methylurea;

N-(l-(4-Chlorophenyl)-3-methyl-l-oxobutan-2-yl)-4-(pyrimidin -2-yl)piperazine-l- carboxamide;

l-ieri-Butyl-3-(l-(4-chlorophenyl)-3-methyl-l-oxobutan-2- yl)urea;

Ethyl l-[[l-(4-chlorophenyl)-3-methyl-l-oxobutan-2-yl]carbamoyl]pi peridine-4-carboxylate; N-(l-(4-Chlorophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(pyraz in-2-yl)piperazine-l- carboxamide;

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-3,4-dihydrois oquinoline-2(lH)- carboxamide;

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-4-(4-cyano pyridin-3-yl)piperazine-l- carboxamide;

(3S)-N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-3-methyl -4-(pyrimidin-2-yl)piperazine- 1-carboxamide;

(2R)-N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-2-methyl -4-(pyrimidin-2-yl)piperazine- 1-carboxamide;

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-2,2-dimethyl- 4-(pyrimidin-2-yl)piperazine- 1-carboxamide;

N-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-4-phenyl-pipe razine-l-carboxamide;

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l -phenethyl-urea;

N-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-4-(2-pyrid yl)piperazine-l-carboxamide; 3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l-pe ntyl-urea;

l-Butyl-3-[l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l- ethyl-urea;

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l,l-diprop yl-urea;

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-[(4-fluorop henyl)methyl]-l-methyl-urea; 3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l-(m -tolylmethyl)urea;

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-3,3-dimethyl- 4-(pyrimidin-2-yl)piperazine- 1-carboxamide;

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-4-(pyrimidin- 2-yl)piperazine-l- carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-m ethylpyridin-2-yl)piperazine-l- carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-meth ylpyridin-2-yl)piperazine-l- carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-meth ylpyridin-2-yl)piperazine-l- carboxamide; N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxo

yl)piperazine-l-carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(2-meth oxyphenyl)piperazine-l- carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(3-i sopropyl-l,2,4-oxadiazol-5- yl)piperidine-l-carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(4-meth oxyphenyl)piperazine-l- carboxamide;

4-(2-Cyanophenyl)-N-(l-(4-cyanophenyl)-3,3-dimethyl-l-oxobut an-2-yl)piperazine-l- carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(4-(tri fluoromethyl)pyrimidin- 1,4-diazepane-l-carboxamide;

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(pyridi n-3-yl)piperazine-l- carboxamide;

N-(l-(4-Cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-y l)-4-(3-cyanopyridin-2- yl)piperazine-l-carboxamide;

N-(l-(4-Cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-yl)- 4-(3-cyanopyridin-2- yl)piperazine-l-carboxamide; and

N-(l-(4-Cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-yl)- 4-(thiazol-2-yl)piperazine-l- carboxamide

or a pharmaceutically acceptable salt, diastereomer or enantiomer thereof.

It will be understood that when compounds of the present invention contain one or more chiral centers, the compounds of the invention may exist in, and be isolated as, enantiomeric or diastereomeric forms, or as a racemic mixture. The present invention includes any possible enantiomers, diastereomers, racemates or mixtures thereof, of a compound of Formula (I). The optically active forms of the compound of the invention may be prepared, for example, by chiral chromatographic separation of a racemate, by synthesis from optically active starting materials or by asymmetric synthesis based on the procedures described thereafter. One embodiment of the present invention is the S enantiomer of a compound of formula (I).

It will also be appreciated that certain compounds of the present invention may exist as geometrical isomers, for example E and Z isomers of alkenes. The present invention includes any geometrical isomer of a compound of Formula (I). It will further be understood that the present invention encompasses tautomers of the compounds of the Formula (I).

It will also be understood that certain compounds of the present invention may exist in solvated, for example hydrated, as well as unsolvated forms. It will further be understood that the present invention encompasses all such solvated forms of the compounds of the Formula (I).

Within the scope of the invention are also salts of the compounds of the Formula (I). Generally, pharmaceutically acceptable salts of compounds of the present invention may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound, for example an alkyl amine with a suitable acid, for example, HCI or acetic acid, to afford a physiologically acceptable anion. It may also be possible to make a corresponding alkali metal (such as sodium, potassium, or lithium) or an alkaline earth metal (such as a calcium) salt by treating a compound of the present invention having a suitably acidic proton, such as a carboxylic acid or a phenol with one equivalent of an alkali metal or alkaline earth metal hydroxide or alkoxide (such as the ethoxide or methoxide), or a suitably basic organic amine (such as choline or meglumine) in an aq medium, followed by conventional purification techniques.

In one embodiment, the compound of Formula (I) above may be converted to a pharmaceutically acceptable salt or solvate thereof, particularly, an acid addition salt such as a hydrochloride, hydrobromide, phosphate, acetate, fumarate, maleate, tartrate, citrate, methanesulphonate or p-toluenesulphonate.

The present invention also provides the compounds of Formula (I) for use as a medicament in therapy, especially for treatment of Alzheimer's disease, pain, schizophrenia, GERD, asthma, pertussis or nicotine addiction.

The compounds of the present invention may be useful in therapy, especially for treatment of asthma, pertussis, nicotine addiction and of various pain conditions including, but not limited to: acute and chronic pain disorders including but not limited to widespread pain, localized pain, nociceptive pain, inflammatory pain, central pain, central and peripheral neuropathic pain, central and peripheral neurogenic pain, central and peripheral neuralgia, chronic tendinitis, low back pain, postoperative pain, peripheral neuropathy, visceral pain, pelvic pain, allodynia, anesthesia dolorosa, causalgia, dysesthesia, fibromyalgia, hyperalgesia, hyperesthesia, hyperpathia, ischemic pain, sciatic pain, pain associated with cystitis, including but not limited to interstitial cystitis, pain associated with multiple sclerosis, pain associated with arthritis, pain associated with osteoarthritis, pain associated with rheumatoid arthritis, and pain associated with cancer.

The compounds of the present invention may be also useful in treatment of Alzheimer's disease, senile dementia, AIDS-induced dementia, Parkinson's disease, amylotropic lateral sclerosis, Huntington's Chorea, migraine, epilepsy, schizophrenia, depression, anxiety, acute anxiety, ophthalmological disorders such as retinopathies, diabetic retinopathies, glaucoma, auditory neuropathic disorders such as tinnitus, chemotherapy induced neuropathies, post-herpetic neuralgia and trigeminal neuralgia, tolerance, dependency (e.g. nicotine addiction), Fragile X, autism, mental retardation, schizophrenia, Down's Syndrome, stroke, head trauma, anoxic and ischemic injuries, hypoglycemia, cardiovascular diseases, respiratory disorders, itching, epilepsy, gastrointestinal disorders, such as inhibition of transient lower esophageal sphincter relaxations, for the treatment of GERD, for the prevention of gastroesophageal reflux, for the treatment regurgitation, for treatment of asthma, for treatment of laryngitis, for treatment of lung disease, for the management of failure to thrive, for the treatment of irritable bowel syndrome (IBS), for the treatment of pertussis (persisent cough) and for the treatment of functional dyspepsia (FD). The compounds of the present invention may also be useful in treatment of overactive bladder or urinary incontinence.

In use for therapy in a warm-blooded animal such as a human, the compound of the invention may be administered in the form of a conventional pharmaceutical composition by any route including orally, intramuscularly, subcutaneously, topically, intranasally, intraperitoneally, intrathoracially, intravenously, epidurally, intrathecally, transdermal^, intracerebroventricularly and by injection into the joints.

In one embodiment of the invention, the route of administration may be oral, intravenous or intramuscular.

The dosage will depend on the route of administration, the severity of the disease, age and weight of the patient and other factors normally considered by the attending physician, when determining the individual regimen and dosage level at the most appropriate for a particular patient.

The present invention provides the use of a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof, as hereinbefore defined in the manufacture of a medicament for use in therapy.

A further aspect of the invention is a method for the treatment of a subject suffering from any of the disorders/diseases/conditions mentioned above, especially treatment of Alzheimer's disease and/or pain and/or schizophrenia and/or GERD and/or asthma and/or pertussis and/or nicotine addiction whereby an effective amount of a compound according to the Formula (I) above, or pharmaceutically acceptable salt or solvate thereof, is administered to a patient in need of such treatment.

In the context of the present specification, the term "therapy" also includes

"prophylaxis" unless there are specific indications to the contrary. The term "therapeutic" and "therapeutically" should be contrued accordingly. The term "therapy" within the context of the present invention further encompasses to administer an effective amount of a compound of the present invention, to mitigate either a pre-existing disease state, acute or chronic, or a recurring condition. This definition also encompasses prophylactic therapies for prevention of recurring conditions and continued therapy for chronic disorders.

The compounds of Formula (I) have activity as pharmaceuticals, in particular as inhibitors of TRPA1. More particularly, the TRPA1 inhibitors of the present invention are useful in therapy, especially for any of the disorders/diseases/conditions mentioned above, especially treatment of Alzheimer's disease, schizophrenia, GERD, asthma, pertussis or nicotine addiction.

The wording "TLESR", transient lower esophageal sphincter relaxations, is herein defined in accordance with Mittal, R.K., Holloway, R.H., Penagini, R., Blackshaw, LA., Dent, J., 1995; Transient lower esophageal sphincter relaxation. Gastroenterology 109, pp. 601-610.

The wording "reflux" is herein defined as fluid from the stomach being able to pass into the esophagus, since the mechanical barrier is temporarily lost at such times.

The wording "GERD", gastro-esophageal reflux disease, is herein defined in accordance with van Heerwarden, M.A., Smout A.J. P.M., 2000; Diagnosis of reflux disease. Bailliere's Clin. Gastroenterol. 14, pp. 759-774.

For the above-mentioned therapeutic uses the dosage administered will, of course, vary with the compound employed, the mode of administration, the treatment desired and the disorder indicated. The daily dosage of the compound of the invention may be in the range from 0.05 mg/kg to 100 mg/kg.

The compounds of formula (I) and pharmaceutically acceptable salts thereof may be used on their own but will generally be administered in the form of a pharmaceutical composition in which the formula (I) compound/salt (active ingredient) is in association with a pharmaceutically acceptable adjuvant, diluent or carrier. Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, "Pharmaceuticals - The Science of Dosage Form Designs", M. E. Aulton, Churchill Livingstone, 1988.

Depending on the mode of administration, the pharmaceutical composition will preferably comprise from 0.05 to 99 %w (per cent by weight), more preferably from 0.05 to 80 %w, still more preferably from 0.10 to 70 %w, and even more preferably from 0.10 to 50 %w, of active ingredient, all percentages by weight being based on total composition.

The present invention also provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as hereinbefore defined, in association with a pharmaceutically acceptable adjuvant, diluent or carrier.

The invention further provides a process for the preparation of a pharmaceutical composition of the invention which comprises mixing a compound of formula (I) or a pharmaceutically acceptable salt thereof as hereinbefore defined with a pharmaceutically acceptable adjuvant, diluent or carrier.

The pharmaceutical compositions may be administered topically (e.g. to the skin) in the form, e.g., of creams, solutions or suspensions; or systemically, e.g. by oral administration in the form of tablets, capsules, syrups, powders or granules; or by parenteral administration in the form of solutions or suspensions; or by subcutaneous administration; or by rectal administration in the form of suppositories; or transdermally.

For oral administration the compound of the invention may be admixed with an adjuvant or a carrier, for example, lactose, saccharose, sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the like, and then compressed into tablets. If coated tablets are required, the cores, prepared as described above, may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatine, talcum and titanium dioxide. Alternatively, the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.

For the preparation of soft gelatine capsules, the compound of the invention may be admixed with, for example, a vegetable oil or polyethylene glycol. Hard gelatine capsules may contain granules of the compound using either the above-mentioned excipients for tablets. Also liquid or semisolid formulations of the compound of the invention may be filled into hard gelatine capsules.

Liquid preparations for oral application may be in the form of syrups or suspensions, for example, solutions containing the compound of the invention, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such liquid preparations may contain colouring agents, flavouring agents, saccharine and/or carboxymethylcellulose as a thickening agent or other excipients known to those skilled in art.

The compounds of the invention may also be administered in conjunction with other compounds used for the treatment of the above conditions.

In a further embodiment, a compound of the present invention, or a pharmaceutical composition or formulation comprising a compound of Formula (I) is administered concurrently, simultaneously, sequentially or separately with another pharmaceutically active compound or compounds selected from the following:

(i) antidepressants such as agomelatine, amitriptyline, amoxapine, bupropion, citalopram, clomipramine, desipramine, doxepin duloxetine, elzasonan, escitalopram, fluvoxamine, fluoxetine, gepirone, imipramine, ipsapirone, maprotiline, nortriptyline, nefazodone, paroxetine, phenelzine, protriptyline, ramelteon, reboxetine, robalzotan, sertraline, sibutramine, thionisoxetine, tranylcypromaine, trazodone, trimipramine, venlafaxine and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(ii) atypical antipsychotics including for example quetiapine and pharmaceutically active isomer(s) and metabolite(s) thereof.

(iii) antipsychotics including for example amisulpride, aripiprazole, asenapine, benzisoxidil, bifeprunox, carbamazepine, clozapine, chlorpromazine, debenzapine, divalproex, duloxetine, eszopiclone, haloperidol, iloperidone, lamotrigine, loxapine, mesoridazine, olanzapine, paliperidone, perlapine, perphenazine, phenothiazine, phenylbutylpiperidine, pimozide, prochlorperazine, risperidone, sertindole, sulpiride, suproclone, suriclone, thioridazine, trifluoperazine, trimetozine, valproate, valproic acid, zopiclone, zotepine, ziprasidone and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(iv) anxiolytics including for example ainespirone, azapirones,benzodiazepines, barbiturates such as adinazolam, alprazolam, balezepam, bentazepam, bromazepam, brotizolam, buspirone, clonazepam, clorazepate, chlordiazepoxide, cyprazepam, diazepam, diphenhydramine, estazolam, fenobam, flunitrazepam, flurazepam, fosazepam, lorazepam, lormetazepam, meprobamate, midazolam, nitrazepam, oxazepam, prazepam, quazepam, reclazepam, tracazolate, trepipam, temazepam, triazolam, uldazepam, zolazepam and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(v) anticonvulsants including for example carbamazepine, clonazepam, ethosuximide, felbamate, fosphenytoin, gabapentin, lacosamide, lamotrogine, levetiracetam, oxcarbazepine, phenobarbital, phenytoin, pregabaline, rufinamide, topiramate, valproate, vigabatrine, zonisamide and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(vi) Alzheimer's therapies including for example donepezil, rivastigmine, galantamine, memantine, and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(vii) Parkinson's therapies including for example deprenyl, L-dopa, Requip, Mirapex, MAOB inhibitors such as selegine and rasagiline, comP inhibitors such as Tasmar, A-2 inhibitors, dopamine reuptake inhibitors, NMDA antagonists, Nicotine agonists, Dopamine agonists and inhibitors of neuronal nitric oxide synthase and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(viii) migraine therapies including for example almotriptan, amantadine, bromocriptine, butalbital, cabergoline, dichloralphenazone, dihydroergotamine, eletriptan, frovatriptan, lisuride, naratriptan, pergolide, pizotiphen, pramipexole, rizatriptan, ropinirole, sumatriptan, zolmitriptan, zomitriptan, and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(ix) stroke therapies including for thrombolytic therapy with eg activase and desmoteplase, abciximab, citicoline, clopidogrel, eptifibatide, minocycline, and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(x) urinary incontinence therapies including for example darafenacin, falvoxate, oxybutynin, propiverine, robalzotan, solifenacin, tolterodine and and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(xi) neuropathic pain therapies including for example lidocain, capsaicin, and anticonvulsants such as gabapentin, pregabalin, and antidepressants such as duloxetine, venlafaxine, amitriptyline, klomipramine, and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(xii) nociceptive pain therapies such as paracetamol, NSAIDS and coxibs, such as celecoxib, etoricoxib, lumiracoxib, valdecoxib, parecoxib, diclofenac, loxoprofen, naproxen, ketoprofen, ibuprofen, nabumeton, meloxicam, piroxicam and opioids such as morphine, oxycodone, buprenorfin, tramadol, and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(xiii) insomnia therapies including for example agomelatine, allobarbital, alonimid, amobarbital, benzoctamine, butabarbital, capuride, chloral, cloperidone, clorethate, dexclamol, ethchlorvynol, etomidate, glutethimide, halazepam, hydroxyzine, mecloqualone, melatonin, mephobarbital, methaqualone, midaflur, nisobamate, pentobarbital, phenobarbital, propofol, ramelteon, roletamide, triclofos,secobarbital, zaleplon, Zolpidem and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

(xiv) mood stabilizers including for example carbamazepine, divalproex, gabapentin, lamotrigine, lithium, olanzapine, quetiapine, valproate, valproic acid, verapamil, and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

Such combination products employ the compounds of this invention within the dosage range described herein and the other pharmaceutically active compound or compounds within approved dosage ranges and/or the dosage described in the publication reference.

The compounds of Formula (I)

may be synthesised via the following routes: (a) reacting a compound of Formula (II) ,

wherein X, R 1 , Y n and A are as defined in Formula (I),

with a compound of Formula (III),

^CfOjL 2 (III)

wherein L 1 and L 2 are groups independently selected from imidazolyl, chloride, and trichloromethyloxy, and

with a compound of Formula (IV) R 2 R 3 NH (IV)

wherein R 2 and R 3 are as defined in Formula (I), or

(b) if R 1 in Formula (I) is H, by reacting a compound of Formula (IV),

R 2 R 3 NH (IV)

wherein R 3 and R 4 are as defined in Formula (I),

with a compound of Formula (V),

wherein X, Y n and A are as defined in Formula (I), or

c) if one of R 2 or R 3 in Formula (I) is H, by reacting a compound of Formula (II),

wherein X, R 1 , Y n and A are as defined in Formula (I),

with a compound of Formula (VI)

R 2/3 NCO (VI)

wherein one R 2/3 is as defined as the R2 or R3 in Formula (I) which is not H, Compounds (II) and (V) can be synthesised in 3 ways via a common intermediate (VII):

(d) reacting a suitably protected Weinreb amide of formula (VIII)

(VII I)

PG N /X Nx O

H

O wherein X is defined in formula (I), with an organometallic compound of formula (IX) such as Grignard and organolithium reagents wherein M represents e.g. MgBr, MgCI, Mgl, Li; A and Yn are defined in formula (I)

or

(e) reacting formula (X) wherein X is as defined in formula (I) and R may be R 1 as defined in formula (I) or part of a protection group,

with an acid chloride of formula (XI) followed by hydrolysis by an acid such as aq hydrochloric acid.

wherein A and Y n are defined in formula (I)

or

(f) oxidizing a compound of formula (XII).

wherein A, X and Y n are as defined in formula (I) and R may be Rl as defined in formula I.

(g) Compound (II) is then available by necessary deprotection and alkylation or vice versa from compound (VII).

(h) Compound (V) is available via deprotection and isocyanate formation from compound (VII).

In process (a), either compound (II) or compound (IV) may be reacted first with a reagent of formula (III) optionally in the presence of a base, such as TEA, and an inert organic solvent, such as acetonitrile, toluene or dichloromethane, or a mixture of (II) and (IV) may be treated with (III).

In process (b), the reaction can preferably be performed in an inert organic solvent, such as acetonitrile, toluene or dichloromethane, optionally in the presence of a base, such as TEA.

In process (c), the reaction can preferably be performed in an inert organic solvent, such as acetonitrile, toluene or dichloromethane, optionally in the presence of a base, such as TEA.

In process (d), the organometallic compound of formula (IX) may be formed in situ, by reaction of the corresponding aryl halide (e.g., the aryl bromide) with magnesium or an isopropylmagnesiumchloride lithiumchloride complex to form an aryl magnesium species or an alkyl reagent such as butyllithium to form an intermediate aryl lithium species. The organometallic complex is then reacted with a suitable Weinreb amide of formula (VIII). The reaction may conveniently be carried out in an organic solvent such as ether, tetrahydrofuran or dioxane at a temperature, for example, in the range from -78 °C to the boiling point of the solvent.

In process (e), the reaction may conveniently be carried out in an organic solvent such as ether, tetrahydrofuran or dioxane at a temperature, for example, in the range from 0 °C to the boiling point of the solvent. The reaction may be carried out using a suitable base such as TEA, 4-dimethylaminopyridine, pyridine or DIPEA (Ν,Ν-Diisopropylethylamine) and by applying Lewis acid such as MgCI 2 .

In process (f), the reaction may conveniently be carried out in an organic solvent such as acetonitrile, dichloromethane, Ν,Ν-dimethylformamide, tetrahydrofuran or N- methylpyrrolidinone at a temperature, for example, in the range from 0 °C to the boiling point of the solvent. The oxidation reaction may be effected by reagents such as Dess-Martin periodinane or pyridiniumchlorochromate.

In process (g), alkylation of the nitrogen may be achieved with suitable alkyl halides, triflates or similar in an organic solvent such as acetonitrile, dichloromethane, N,N- dimethylformamide, tetrahydrofuran or N-methylpyrrolidinone at a temperature, for example, in the range from 0 °C to the boiling point of the solvent.. Alkylation of the primary amine may also be achieved by a reductive amination process, i.e. formation of an imine with a suitable aldehyde with consequent reduction with agents like NaBH 4 under similar conditions as those stated above.

In process (h), after possibly required deprotection of the primary amino group, formation of the isocyanate moiety may be achieved by reaction with phosgene or similar reagents or with carbon monoxide and a suitable catalyst like Cu(CN) 2 in an organic solvent such as acetonitrile, dichloromethane, Ν,Ν-dimethylformamide, tetrahydrofuran or N- methylpyrrolidinone at a temperature, for example, in the range from 0 °C to the boiling point of the solvent.

In a further embodiment, the present invention also provides intermediate compounds that can be used for producing the compounds of Formula (I). More specifically, these compounds are selected from the group of:

2-Amino-l-(4-chlorophenyl)-3-methylbutan-l-one hydrochloride;

te/t-Butyl l-(methoxy(methyl)amino)-3,3-dimethyl-l-oxobutan-2-ylcarbama te;

te/t-Butyl l-(4-chlorophenyl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate;

2-Amino-l-(4-chlorophenyl)-3,3-dimethylbutan-l-one;

te/t-Butyl l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-ylcarbamate;

4-(2-Amino-3-methylbutanoyl)benzonitrile;

te/t-Butyl 4-(4-cyanopyridin-3-yl)piperazine-l-carboxylate; 3-(Piperazin-l-yl)isonicotinonitrile;

(S)-l-(3-Methyl-4-(pyrimidin-2-yl)piperazin-l-yl)ethanone;

(S)-2-(2-Methylpiperazin-l-yl)pyrimidine;

(R)-ieri-Butyl 2-methyl-4-(pyrimidin-2-yl)piperazine-l-carboxylate;

(R)-2-(3-Methylpiperazin-l-yl)pyrimidine hydrochloride;

te/t-Butyl 2,2-dimethyl-4-(pyrimidin-2-yl)piperazine-l-carboxylate;

2-(3,3-Dimethylpiperazin-l-yl)pyrimidine;

tert-butyl 3,3-dimethyl-4-(pyrimidin-2-yl)piperazine-l-carboxylate; and

2-(2,2-dimethylpiperazin-l-yl)pyrimidine hydrochloride.

Specific processes for the preparation of compounds of Formula (I) are disclosed within the Examples section of the present specification. Such processes form an aspect of the present invention.

The necessary starting materials are either commercially available, are known in the literature or may be prepared using known techniques. Specific processes for the preparation of certain key starting materials are disclosed within the Examples section of the present specification and such processes form an aspect of the present invention.

Compounds of Formula (I) can be converted into further compounds of Formula (I) using standard procedures.

It will be appreciated by those skilled in the art that in the processes of the present invention certain functional groups such as hydroxyl or amino groups may need to be protected by protecting groups. Thus, the preparation of the compounds of Formula (I) may involve, at an appropriate stage, the addition and/or removal of one or more protecting groups.

The protection and deprotection of functional groups is described in 'Protective

Groups in Organic Chemistry', edited by J.W.F. McOmie, Plenum Press (1973) and 'Protective Groups in Organic Synthesis', 3 rd edition, T.W. Greene and P.G.M. Wuts, Wiley-lnterscience (1999). Experimental work

General Methods

All solvents used were analytical grade and commercially available anhydrous solvents were routinely used for reactions. Reactions were typically run under an inert atmosphere of nitrogen or argon.

1 H, 19 F and 13 C NMR spectra were recorded on a Varian Unity+ 400 NMR Spectrometer equipped with a 5mm BBO probe-head with Z-gradients, or a Varian Gemini 300 NMR spectrometer equipped with a 5mm BBI probe head, or a Bruker Avance 400 NMR spectrometer equipped with a 60 01 dual inverse flow probe head with Z-gradients, or a Varian Mercury Plus 400 NMR Spectrometer equipped with a Varian 400 ATB PFG probe, or a Bruker DPX400 NMR spectrometer equipped with a 4-nucleus probe head equipped with Z- gradients, or a Bruker Avance 600 NMR spectrometer equipped with a 5mm BBI probe head with Z-gradients. Unless specifically noted in the examples, spectra were recorded at 400 MHz for proton, 376 MHz for fluorine-19 and 100 MHz for carbon-13.

Alternatively, X H and 13 C NMR spectra were recorded at 400 MHz for proton and 100

MHz for carbon-13 on a Varian Mercury Plus 400 NMR Spectrometer equipped with a Varian 400 ATB PFG probe.

The following reference signals were used: the middle line of DMSO-c/6 δ 2.50 (1H), δ 39.51 (13C); the middle line of CD 3 OD δ 3.31 (1H) or δ 49.15 (13C); CDCI 3 δ 7.26 (1H) and the middle line of CDCU δ 77.16 (13C) (unless otherwise indicated). NMR spectra are reported from low to high field. Alternatively, all deuterated solvents contained typically 0.03% to 0.05% v/v tetramethylsilane, which was used as the reference signal (set at δ 0.00 for both X H and 13 C).

Mass spectra were recorded on a Waters LCMS consisting of an Alliance 2795 (LC), Waters PDA 2996 and a ZQ. single quadrupole mass spectrometer or Waters Micromass ZQ. detector at 120°C. The mass spectrometer was equipped with an electrospray ion source (ESI) operated in a positive or negative ion mode. The capillary voltage was 3 kV and cone voltage was 30 V. The mass spectrometer was scanned between m/z 100-700 or m/z 100- 1000 with a scan time of 0.3s. Separations were performed on either Waters X-Terra MS C8 (3.5 0m, 50 or 100 mm x 2.1 mm i.d.) or an ACE 3 AQ (100 mm x 2.1 mm i.d.) obtained from ScantecLab. Flow rates were regulated to 1.0 or 0.3 mL/min, respectively. The column temperature was set to 40 0C. A linear gradient was applied using a neutral or acidic mobile phase system, starting at 100% A (A: 95:5 10 mM NH 4 OAc:MeCN, or 95:5 8 mM HCOOH:MeCN) ending at 100% B (MeCN). Alternatively, mass spectra were recorded on a Waters LCMS consisting of an Alliance 2690 Separations Module, Waters 2487 Dual 1 Absorbance Detector (220 and 254 nm) and a Waters ZQ. single quadrupole mass spectrometer. The mass spectrometer was equipped with an electrospray ion source (ESI) operated in a positive or negative ion mode. The capillary voltage was 3 kV and cone voltage was 30 V. The mass spectrometer was scanned between m/z 97-800 with a scan time of 0.3 or 0.8 s. Separations were performed on a Chromolith Performance RP-18e (100 x 4.6 mm). A linear gradient was applied starting at 95% A (A: 0.1% HCOOH (aq.)) ending at 100% B (MeCN) in 5 min. Flow rate: 2.0 mL/min.

Alternatively, Ultra Pressure (UP) LCMS analyses were performed on an Waters Acquity UPLC system consisting of a Acquity Autosampler, Acquity Sample Organizer, Acquity Column Manager, Acquity Binary Solvent Manager, Acquity UPLC PDA detector and a Waters SO Detector. The mass spectrometer was equipped with an electrospray ion source (ES) operated in positive and negative ion mode. The capillary voltage was set to 3.0 kV and the cone voltage to 30 V, respectively. The mass spectrometer was scanned between m/z 100- 600 with a scan time of 0.105s. The diode array detector scanned from 200-400 nm. The temperature of the Column Manager was set to 60 °C. Separation was performed on a Acquity column, UPLC BEH, C18 1.7 μΜ run at a flow rate of 0.5 mL/min. A linear gradient was applied starting at 100 % A (A: lOmM NH 4 OAc in 5% CH3CN) ending at 100% B (B: CH3CN) after 1.3 min then 100 % B for 0.6 min. ESpos/ESneg, m/z 100-600.

Alternatively, Compound identification was performed on a GC-MS system supplied by Agilent Technologies, consisting of a 6890N G1530N GC, a G2614A Autosampler, G2613A injector and a G2589N mass spectrometer. The column used was a VF-5 MS, ID 0.25 mm x 30m, 0.25 μιη (Varian Inc.). A linear temperature gradient was applied starting at 70 °C (hold 1 min) and ending at 300 °C (hold 1 min), 25 °C/min. The mass spectrometer was equipped with a chemical ionisation (CI) ion source and the reactant gas was methane. The mass spectrometer scanned between m/z 50-500 and the scan speed was set to 3.21 scan/s. Solvent delay was set from 0 min to 2.0 min.

HPLC analyses were performed on an Agilent HP1000 system consisting of G1379A Micro Vacuum Degasser, G1312A Binary Pump, G1367A Well plate auto-sampler, G1316A Thermostatted Column Compartment and G1315B Diode Array Detector. Column: X-Terra MS, Waters, 3.0 x 100 mm, 3.5 0m. The column temperature was set to 40 0C and the flow rate to 1.0 mL/min. The Diode Array Detector was scanned from 210-300 nm, step and peak width were set to 2 nm and 0.05 min, respectively. A linear gradient was applied, starting at 100 % A (A: 95:5 10 mM NH 4 OAc:MeCN) and ending at 100% B (B: MeCN), in 4 min. Alternatively, HPLC analyses were performed on a Gynkotek P580 HPG consisting of gradient pump with a Gynkotek UVD 170S UV-vis. -detector equipped with a Chromolith Performance RP column (C18, 100 mm x 4.6 mm). The column temperature was set to 25°C. A linear gradient was applied using MeCN/0.1 trifluoroacetic acid in MilliQ water, run from 10% to 100% MeCN in 5 min. Flow rate: 3 mL/min.

Chiral purity analysis was run on a SFC Berger Analytix system with Agilent 1100 PDA detector. The column temperature was set to 50°C. An isocratic condition a mixture of EtOH and C0 2 was applied at flow rate 2.0 mL/min. The PDA was scanned from 190-600nm and 220nm was extracted for purity determination.

Alternatively, chiral HPLC analyses were performed on a Gilson chiral system Column:

Chiralpak AD-H; 4.6*250 mm; 5μιη Mobile phase: 100% EtOH Flow rate: 0.8 mL/min. Optical rotation was determined with a PDR-Chiral laser polarimeter.

Microwave heating was performed in a single-mode microwave cavity producing continuous irradiation at 2450 MHz.

Thin layer chromatography (TLC) was performed on Merck TLC-plates (Silica gel 60

F254) and UV visualized the spots.

Flash chromatography was performed on a Combi Flash * Companion™ using RediSep™ normal-phase flash columns or using Merck Silica gel 60 (0.040-0.063 mm). Typical solvents used for flash chromatography were mixtures of chloroform/methanol, dichloromethane/methanol, heptane/ethyl acetate, chloroform/methanol/ammonia (aq.) and dichlorormethane/methanol/ NH3 (aq.).

Preparative chromatography was run on a Waters auto purification HPLC with a diode array detector. Column: XTerra MS C8, 19 x 300 mm, 10 0m. Narrow gradients with MeCN/(95:5 0.1M NH 4 OAc:MeCN) were used at a flow rate of 20 mL/min.

Alternatively, purification was achieved on a semi preparative Shimadzu LC-8A HPLC with a Shimadzu SPD-10A UV-vis. -detector equipped with a Waters Symmetry * column (C18, 5 0m, 100 mm x 19 mm). Narrow gradients with MeCN/0.1% trifluoroacetic acid in MilliQ. Water were used at a flow rate of 10 mL/min.

Alternatively, purification was achieved on a preparative Gilson 281(Gilson Pump 322) HPLC with a Gilson 156 UV-detector equipped with a Waters Sunfire column (150 mm x 21.2mm). Narrow gradients with MeCN/0.1% formic acid in water were used at a flow rate of 15 mL/min. Chiral preparative chromatography was run on a SFC Berger Multigram system with a Knauer K-2501 UV detector. The column temperature was set to 35°C. An isocratic condition of a mixture of EtOH and C0 2 was applied at flow rate 50.0 mL/min. The UV detector scanned at 220nm. The UV signal determined the fraction collection.

Compounds have been named using either ACD/Name, version 10.06, software from

Advanced Chemistry Development, Inc. (ACD/Labs), Toronto ON, Canada, www.acdlabs. or Lexichem, version 1.4, software from OpenEye.

Abbreviations:

aq aqueous

CAS Chemical Abstracts Service

CDI Ι, -carbonyldiimidazole

coned concentrated

DCM dichloromethane

DIPEA N,N-diisopropylethylamine

DMF dimethylformamide

DMSO dimethyl sulfoxide

EDCI l-ethyl-3-(3-dimethylaminopropyl) carbodiimide

ES electrospray

ESI electrospray ionization

eq equivalent(s)

GC gas chromatography

h hour(s)

HATU 2-(lH-7-Azabenzotriazol-l-yl)--l,l,3,3-tetramethyl uronium hexafluorophosphate methanaminium

HBTU 0-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-pho sphate

HCI hydrochloride

HOBT hydroxybenzotriazole HPLC high-performance liquid chromatography

M Molar (moles per liter)

min minute(s)

MS mass spectrometry

NMR nuclear magnetic resonance

PG protecting group

prep. Preparative

rt room temperature

Rt retention time

SFC Supercritical fluid chromatography

TEA triethylamine

TLC thin layer chromatography

temp temperature

THF tetrahydrofuran

TLC thin layer chromatography

UV ultraviolet

Example 1 l-ieri-Butyl-3-(l-(4-chlorophenyl)-3-methyl-l-oxobutan-2-yl) -l-methylurea

To a solution of CDI (98 mg, 0.60 mmol) and TEA (0.026 mL, 0.19 mmol) in MeCN (3 mL) was added l-(4-chlorophenyl)-3-methyl-l-oxobutan-2-aminium chloride (Example la, 46 mg, 0.19 mmol) and the mixture was stirred for 20 minutes. N,2-dimethylpropan-2-amine (CAS 14610- 37-8, 0.177 mL, 1.48 mmol) was added. After 30 minutes the mixture was purified by preparative HPLC to give l-tert-butyl-3-(l-(4-chlorophenyl)-3-methyl-l-oxobutan-2-yl) -l- methylurea (46.0 mg, 76%).

XH NMR (400 MHz, CDCI 3 ) δ ppm 0.78 (d, 3 H) 1.02 (d, 3 H) 1.41 (s, 9 H) 2.05 - 2.18 (m, 1 H) 2.95 (s, 3 H) 5.15 (d, 1 H) 5.42 (dd, 1 H) 7.43 - 7.49 (m, 2 H) 7.92 - 7.98 (m, 2 H). MS (ESI) m/z 325 [M+H] +

Example la

2-Amino-l-(4-chlorophenyl)-3-methylbutan-l-one hydrochloride

te/t-Butyl l-(methoxy(methyl)amino)-3-methyl-l-oxobutan-2-ylcarbamate (CAS 293329-55-2, 3 g, 11.52 mmol) was dissolved in THF (110 mL). The solution was cooled to -50^C and 4- chlorophenylmagnesium bromide (CAS 873-77-8, 1.0 M in THF, 46.1 mL, 46.1 mmol) was added. After 10 min the reaction was allowed to reach rt. After 5 h the reaction was quenched with NH 4 CI at O^C. The slurry was diluted with water and extracted with EtOAc, dried over MgS0 4 , filtered and solvent was removed in vacuo. The crude product was purified by flash chromatography to give te/t-butyl l-(4-chlorophenyl)-3-methyl-l-oxobutan- 2-ylcarbamate (3.6 g).

MS (ESI) m/z 311.8 [M+H] + ieri-Butyl l-(4-chlorophenyl)-3-methyl-l-oxobutan-2-ylcarbamate (3.6 g, 11.6 mmol) was dissolved in methanol (91 mL). Hydrochloric acid in isopropanol (5 M, 91 mL, 454 mmol) was added and the reaction mixture was stirred at ambient temperature for 3.5 h, then acid and solvent were evaporated. The product was diluted in diethyl ether and co-evaporated in vacuo to give 2-amino-l-(4-chlorophenyl)-3-methylbutan-l-one hydrochloride (2.23 g, 92 %).

X H NMR (400 MHz, DMSO-de) δ ppm 0.79 (d, 3 H) 1.01 (d, 3 H) 2.20 (td, 1 H) 5.09 (d, 1 H) 7.64 - 7.72 (m, 2 H) 8.03 - 8.17 (m, 2 H) 8.41 (s, 3 H). MS (ESI) m/z 211.8 [M+H] +

Example 2

N-(l-(4-Chlorophenyl)-3-methyl-l-oxobutan-2-yl)-4-(pyrimi din-2-yl)piperazine-l- carboxamide

2-Amino-l-(4-chlorophenyl)-3-methylbutan-l-one hydrochloride (Example la, 40 mg, 0.16 mmol) in MeCN (2.0 mL) was added to a solution of CDI (78 mg, 0.48 mmol) and TEA (0.025 mL, 0.18 mmol) in MeCN (2.0 mL). The reaction mixture was stirred at rt for 90 minutes before 2-(piperazin-l-yl)pyrimidine (CAS 20980-22-7, 132 mg, 0.81 mmol) was added. The mixture was stirred for 1 h and the solvent was evaporated under reduced pressure. The crude was dissolved in methanol, filtered and purified by prep. HPLC to yield N-(l-(4- chlorophenyl)-3-methyl-l-oxobutan-2-yl)-4-(pyrimidin-2-yl)pi perazine-l-carboxamide (32.2 mg, 50%).

XH NMR (400 MHz, CDCI 3 ) δ ppm 0.79 (d, 3 H) 1.03 (d, 3 H) 2.10 - 2.19 (m, 1 H) 3.48 - 3.64 (m, 4 H) 3.82 - 3.96 (m, 4 H) 5.37 - 5.42 (m, 1 H) 5.43 - 5.49 (m, 1 H) 6.54 (t, 1 H) 7.41 - 7.53 (m, 2 H) 7.89 - 8.00 (m, 2 H) 8.34 (d, 2 H). MS (ESI) m/z 402.2 [M+H] +

Example 3

l-ieri-Butyl-3-(l-(4-chlorophenyl)-3-methyl-l-oxobutan-2- yl)urea

2-Amino-l-(4-chlorophenyl)-3-methylbutan-l-one (Example la, 30 mg, 0.14 mmol) dissolved in toluene (1 mL) with TEA (10 μί, 0.07 mmol) was cooled to 0°C and treated with 2- isocyanato-2-methylpropane (CAS 1609-86-5, 0.018 mL, 0.16 mmol). The mixture was stirred at rt. After 2 h, solvent was replaced with DMSO and the mixture was purified by preparative HPLC to give l-ieri-butyl-3-(l-(4-chlorophenyl)-3-methyl-l-oxobutan-2-yl) urea (8.0 mg, 18%).

X H NMR (500 MHz, CDCI 3 ) δ ppm 0.73 (d, 3 H) 1.03 (d, 3 H) 1.35 (s, 9 H) 2.09 (dt, 1 H) 4.33 (br. S., 1 H) 4.98 (d, 1 H) 5.36 (dd, 1 H) 7.47 (m, 2 H) 7.93 (m, 2 H). MS (ESI) m/z 311 [M+H] +

Example 4

Ethyl l-[[l-(4-chlorophenyl)-3-methyl-l-oxobutan-2-yl]carbamoyl]pi peridine-4-carboxylate

To a suspension of 2-amino-l-(4-chlorophenyl)-3-methylbutan-l-one (Example la, 0.2 mmol, 1.0 eq) in MeCN (2 mL) was added CDI (0.6 mmol, 3.0 eq) and TEA (0.8 mmol, 4.0 eq). The mixture was stirred for 1 h at room temperature and ethyl piperidine-4-carboxylate (CAS 1126-09-6, 1.0 mmol, 5.0 eq) was added. The resulting mixture was heated at 80°C for another 2 h. The crude product was purified by preparative TLC (petroleum ethenethyl acetate 1:1) to give ethyl l-[[l-(4-chlorophenyl)-3-methyl-l-oxobutan-2- yl]carbamoyl]piperidine-4-carboxylate (110 μιηοΙ, 55%).

X H NMR (500 MHz, CDCI3) δ ppm 0.78 (d, 3 H) 1.02 (d, 3 H) 1.27 (t, 3 H) 1.65 - 1.79 (m, 2 H) 1.91 - 2.00 (m, 2 H) 2.12 (td, 1 H) 2.44 - 2.55 (m, 1 H) 2.98 (dddd, 2 H) 3.89 - 4.02 (m, 2 H) 4.16 (q, 2 H) 5.33 - 5.47 (m, 2 H) 7.44 - 7.50 (m, 2 H) 7.92 - 7.98 (m, 2 H). MS (ESI) m/z 395.1 [M+H] +

Example 5

N-(l-(4-Chlorophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(py razin-2-yl)piperazine-l- carboxamide

2-Amino-l-(4-chlorophenyl)-3,3-dimethylbutan-l-one hydrochloride (Example 5c, 42 mg, 0.16 mmol) in MeCN (2 mL) was added to a solution of CDI (78 mg, 0.48 mmol) and TEA (0.025 mL, 0.18 mmol) in MeCN (2 mL). The reaction mixture was stirred at room temperature for 90 minutes before 2-(piperazin-l-yl)pyrazine (CAS 34803-68-4, 132 mg, 0.80 mmol) was added. The mixture was stirred for 1 h and the solvent was evaporated under reduced pressure. The crude was dissolved in methanol, filtered and purified by prep. HPLC to yield N-(l-(4-chlorophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(pyraz in-2-yl)piperazine-l- carboxamide (34.3 mg, 52%).

XH NMR (400 MHz, CDCI 3 ) δ ppm 0.95 (s, 9 H) 3.55 - 3.64 (m, 4 H) 3.64 - 3.75 (m, 4 H) 5.32 - 5.38 (m, 1 H) 5.38 - 5.43 (m, 1 H) 7.42 - 7.51 (m, 2 H) 7.90 (br. S., 1 H) 7.95 - 8.01 (m, 2 H) 8.13 (br. S., 2 H). MS (ESI) m/z 416.2 [M+H] +

Example 5a

te/t-Butyl l-(methoxy(methyl)amino)-3,3-dimethyl-l-oxobutan-2-ylcarbama te

To a solution of 2-(ieri-butoxycarbonylamino)-3,3-dimethylbutanoic acid (CAS 102185-35-3, 500 mg, 2.16 mmol) in DCM (5 mL) was added EDCI (CAS 25952-53-8, 497 mg, 2.59 mmol) and stirred for 10 min at rt. A solution of Ν,Ο-dimethylhydroxylamine hydrochloride (CAS 6638-79-5, 253 mg, 2.59 mmol) and DIPEA (0.429 mL, 2.59 mmol) in DCM (5 mL) was added and the reaction mixture was left to stir at rt over weekend. The mixture was diluted with DCM (30 mL) and extracted with saturated aqueous NaHC03 (50 mL). The organic phase was washed with water before dried over MgS0 4 and filtered. The solvent was removed in vacuo to give te/t-butyl l-(methoxy(methyl)amino)-3,3-dimethyl-l-oxobutan-2-ylcarbama te (466 mg, 79%). X H NMR (500 MHz, CDCI 3 )060ppm 0.98 (s, 9 H) 1.43 (s, 9 H) 2.19 - 2.28 (m, 1 H) 3.21 (s, 3 H) 3.78 (s, 3 H) 4.66 (d, 1 H). GC-MS (unprotected amino acid) m/z 130, Rt: 5.82 min.

Example 5b te/t-Butyl l-(4-chlorophenyl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate

te/t-Butyl l-(methoxy(methyl)amino)-3,3-dimethyl-l-oxobutan-2-ylcarbama te (Example 5a, 466 mg, 1.70 mmol) was dissolved in dry THF (10 mL). (4-Chlorophenyl)magnesium bromide (CAS 873-77-8, 1.0 M in THF, 6.79 mL, 6.79 mmol) was then added dropwise at rt. After stirring at rt over night The reaction was quenched with NH 4 CI and extracted with EtOAc (2x50 mL). The combined organic phases were washed once with water before dried over MgS0 4 and filtered. After removal of the solvent the crude was purified on a silica gel column eluted with heptane:EtOAc 10-20% to give te/t-butyl l-(4-chlorophenyl)-3,3-dimethyl-l- oxobutan-2-ylcarbamate (137 mg, 25%).

X H NMR (500 MHz, CDCI 3 )060ppm 0.93 (s, 9 H) 1.44 (s, 9 H) 5.12 (d, 1 H) 5.40 (d, 1 H) 7.46 (d, 2 H) 7.95 (d, 2 H). MS (ESI) m/z 325.8 [M+H] +

Example 5c

2-Amino-l-(4-chlorophenyl)-3,3-dimethylbutan-l-one hydrochloride

te/t-Butyl l-(4-chlorophenyl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (Example 5b, 87 mg, 0.27 mmol) was dissolved in MeOH (10 mL). Hydrogen chloride (5 M in MeOH, 2.136 mL, 10.68 mmol) was added and the reaction mixture was stirred at rt over night. The solvent was evaporated to give 2-amino-l-(4-chlorophenyl)-3,3-dimethylbutan-l-one hydrochloride (79 mg).

MS (ESI) m/z 225.8 [M+l] + Example 6

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-3,4-dihydr oisoquinoline-2(lH)-carboxamid

To a solution of CDI (70 mg, 0.43 mmol) and TEA (24 μί, 0.17 mmol) in MeCN (1 mL) was added l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-aminium chloride (Example 6b, 36 mg, 0.15 mmol). After stirring for 90 min, 1,2,3,4-tetrahydroisoquinoline (CAS 91-21-4, 94 μί, 0.75 mmol) was added and the mixture was stirred for 90 min. Purification by prep. HPLC gave N- (l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl)-3,4-dihydroisoq uinoline-2(lH)-carboxamide (29.2 mg, 54%).

XH NMR (500 MHz, CDCI 3 ) δ ppm 0.84 (d, 3 H) 1.03 (d, 3 H) 2.09 - 2.17 (m, 1 H) 2.86 - 2.98 (m, 2 H) 3.63 (ddd, 1 H) 3.75 (ddd, 1 H) 4.63 (s, 2 H) 5.29 (d, 1 H) 5.47 (dd, 1 H) 7.15 - 7.25 (m, 4 H) 7.79 - 7.83 (m, 2 H) 8.11 - 8.14 (m, 2 H). MS (ESI) m/z 362.1 [M+l] +

Example 6a te/t-But l l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-ylcarbamate

4-lodobenzonitrile (CAS 3058-39-7, 3.67 g, 16.02 mmol) was dissolved in THF (40 mL) under argon. The solution was cooled to -50°C. Isopropylmagnesium chloride lithium chloride complex (1.3 M in THF, 30.8 mL, 40.06 mmol) was added dropwise at -50°C. The reaction was stirred at -50°C for 50 min before ieri-butyl l-(methoxy(methyl)amino)-3-methyl-l- oxobutan-2-ylcarbamate (CAS 293329-55-2, 3.48 g, 13.35 mmol) in THF (20 mL) was added during 5 min at -50°C. The mixture was left to slowly reach rt for 16 h before cooled to -25°C and quenched with NH 4 CI and extracted with EtOAc (2x100 mL). The combined organic phases were washed with brine, dried over MgS0 4 , filtered and evaporated. The resulting crude (5.16 g) was evaporated onto silica gel and purified on a silica gel column (80 g, heptane:EtOAc 0-60%) to give ieri-butyl l-(4-cyanophenyl)-3-methyl-l-oxobutan-2- ylcarbamate (2.57 g, 64%).

X H NMR (500 MHz, DMSO-de) δ ppm 0.85 (dd, 6 H) 1.34 (s, 9 H) 2.08 (dq, 1 H) 4.82 (dd, 1 H) 7.37 (d, 1 H) 8.01 (d, 2 H) 8.10 (d, 2 H). MS (ESI) m/z 301.2 [M-H] "

Example 6b

-Amino-3-meth lbutanoyl)benzonitrile hydrochloride

ieri-Butyl l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-ylcarbamate (Example 6a, 1.136 g, 3.76 mmol) was dissolved in methanol (29.5 mL) . Hydrochloric acid in isopropanol (5 M, 29.5 mL, 147.50 mmol) was added and the reaction mixture was stirred at rt for 2 h and then evaporated to dryness to give 4-(2-amino-3-methylbutanoyl)benzonitrile hydrochloride (893 mg, 100%).

X H NMR (400 MHz, DMSO-cf 6 ) 60ppm 0.78 (d, 3 H) 1.01 (d, 3 H) 2.14 - 2.27 (m, 1 H) 5.16 (d, 1 H) 8.04 - 8.16 (m, 2 H) 8.19 - 8.29 (m, 2 H) 8.47 (s, 3 H). MS (ESI) m/z 202.9 [M+H] +

Example 7

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-4-(4-cyanopyr idin-3-yl)piperazine-l- carboxamide

Bis(trichloromethyl)carbonate (24.01 mg, 0.08 mmol) was added portionwise to l-(4- cyanophenyl)-3-methyl-l-oxobutan-2-aminium chloride (Example 6b, 48.3 mg, 0.20 mmol) and TEA (0.113 mL, 0.81 mmol) in DCM (2 mL) under 1 minute at rt. After stirring for 30 min, a solution of 4-(4-cyanopyridin-3-yl)piperazin-l-ium chloride (Example 7b, 50 mg, 0.22 mmol) and TEA (0.056 mL, 0.40 mmol) in DCM (2 mL) was added dropwise and the mixture was stirred over night. The crude reaction mixture was purified by prep. HPLC to give N-(l-(4- cyanophenyl)-3-methyl-l-oxobutan-2-yl)-4-(4-cyanopyridin-3-y l)piperazine-l-carboxamide (17.7 mg, 21%).

1 H NMR (400 MHz, CDCI 3 0060ppm 0.82 (d, 3 H) 1.03 (d, 3 H) 2.07 - 2.18 (m, 1 H) 3.66 (q, 4 H) 3.80 (t, 4 H) 5.30 (d, 1 H) 5.44 (dd, 1 H) 6.85 (dd, 1 H) 7.81 (d, 2 H) 7.85 (dd, 1 H) 8.11 (d, 2 H) 8.39 (dd, 1 H). MS (ESI) m/z 417.1 [M+H] +

Example 7a

tert-Butyl 4-(4-cyanopyridin-3-yl)piperazine-l-carboxylate

3-Fluoroisonicotinonitrile (CAS 113770-88-0, 68.4 mg, 0.56 mmol) and ieri-butyl piperazine- 1-carboxylate (CAS 76535-74-5, 219 mg, 1.18 mmol) were dissolved in toluene in a microwave vial before flushed with Ar(g) and heated in a microwave reactor for 15 min at 100°C. The reaction was diluted with EtOAc and filtered through a plug of silica. The filtrate was reduced in vacuo to give ieri-butyl 4-(4-cyanopyridin-3-yl)piperazine-l-carboxylate (153 mg, 95%).

MS (ESI) m/z 189.0 [(M-Boc)+H] +

Example 7b 3-(Piperazin-l-yl)isonicotinonitrile hydrochloride HCI

te/t-Butyl 4-(4-cyanopyridin-3-yl)piperazine-l-carboxylate (Example 7a, 153 mg, 0.53 mmol) was dissolved in MeOH (5 mL). Hydrochloric acid in 2-propanol (5 M, 2 mL, 10 mmol) was added and the reaction mixture was stirred at rt over the weekend and evaporated to give 3- (piperazin-l-yl)isonicotinonitrile hydrochloride (100 mg, 100%).

MS (ESI) m/z 189.1 [M+H] +

Example 8

(3S)-N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-3-met hyl-4-(pyrimidin-2-yl)piperazine- 1-carboxamide

Bis(trichloromethyl)carbonate (23.37 mg, 0.08 mmol) was added portionwise to l-(4- cyanophenyl)-3-methyl-l-oxobutan-2-aminium chloride (Example 6b, 47 mg, 0.20 mmol) and TEA (0.110 mL, 0.79 mmol) in DCM (2 mL) under 1 minute at rt. After stirring for 30 min, a solution of (S)-3-methyl-4-(pyrimidin-2-yl)piperazin-l-ium chloride (Example 8b, 46.5 mg, 0.22 mmol) and TEA (0.055 mL, 0.39 mmol) in DCM (2 mL) was added dropwise and the reaction was stirred for 30 min. The crude reaction mixture was filtered and diluted with MeOH before purification by prep. HPLC to give (3S)-N-(l-(4-cyanophenyl)-3-methyl-l- oxobutan-2-yl)-3-methyl-4-(pyrimidin-2-yl)piperazine-l-carbo xamide (29.8 mg, 37%).

X H NMR (500 MHz, DMSO-cfe) δ ppm 0.84 (dd, 3 H) 0.94 (m, 6 H) 2.16 (td, 1 H) 2.83 (tt, 1 H) 3.01 (m, 2 H) 3.90 (d, 1 H) 4.02 (t, 1 H) 4.31 (d, 1 H) 4.70 (qd, 1 H) 4.93 (dt, 1 H) 6.63 (td, 1 H) 6.87 (dd, 1 H) 7.99 (d, 2 H) 8.14 (dd, 2 H) 8.36 (dd, 2 H). MS (ESI) m/z 407.1 [M+H] +

Example 8a

(S)-l-(3-Methyl-4-(pyrimidin-2-yl)piperazin-l-yl)ethanone

(S)-l-(3-Methylpiperazin-l-yl)ethanone hydrochloride (CAS 612493-89-7, 252 mg, 1.41 mmol), 2-chloropyrimidine (CAS 1722-12-9, 162 mg, 1.41 mmol) and TEA (0.590 mL, 4.23 mmol) were dissolved in ethanol (3 mL) before heated in a microwave reactor for 60 min at 150°C. The solvent was removed in vacuo and the crude re-dissolved in EtOAc before evaporated onto silica gel and purified on a silica gel column (12 g ,heptane:EtOAc with 1% TEA 40-100%) to give (S)-l-(3-methyl-4-(pyrimidin-2-yl)piperazin-l-yl)ethanone (58.0 mg, 19%).

MS (ESI) m/z 221.1 [M+H] +

Example 8b

(S)-2-(2-Methylpiperazin-l-yl)pyrimidine hydrochloride

(S)-l-(3-methyl-4-(pyrimidin-2-yl)piperazin-l-yl)ethanone (Example 8a, 81 mg, 0.37 mmol) was suspended in aquaeous hydrochloric acid (6 M, 5 mL, 30 mmol). The mixture was refluxed at 95°C for 14 h. The reaction was left to cool before filtered. The residue was washed with water and diethyl ether to yield (S)-2-(2-methylpiperazin-l-yl)pyrimidine hydrochloride (92 mg, quantitative).

X H NMR (500 MHz, MeOD) δ ppm 1.51 (d, 3 H) 3.47 (m, 5 H) 3.69 (m, 1 H) 4.73 (d, 1 H) 5.12 (m, 1 H) 7.10 (t, 1 H) 8.69 (d, 2 H). MS (ESI) m/z 179.1 [M+H] +

Example 9

(2R)-N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-2-met hyl-4-(pyrimidin-2-yl)piperazine- 1-carboxamide

Bis(trichloromethyl)carbonate (25.1 mg, 0.08 mmol) was added portionwise to 4-(2-amino-3- methylbutanoyl)benzonitrile hydrochloride (Example 6b, 50.5 mg, 0.21 mmol) and TEA (0.118 mL, 0.85 mmol) in DCM (2 mL) under 1 min at rt. After stirring for 25 min, a solution of (R)-2- (3-methylpiperazin-l-yl)pyrimidine hydrochloride (Example 9b, 50 mg, 0.23 mmol) and TEA (0.059 mL, 0.42 mmol) in DCM (2 mL) was added dropwise and the mixture was stirred for 10 min. The crude reaction mixture was filtered and diluted with MeOH before purification by prep. HPLC to give (2R)-N-(l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl)-2-methyl -4- (pyrimidin-2-yl)piperazine-l-carboxamide (45.0 mg, 52%).

XH NMR (500 MHz, DMSO-ck) δ ppm 0.83 (dd, 3 H) 0.93 (m, 6 H) 2.14 (dq, 1 H) 2.87 (m, 1 H) 2.97 (m, 1 H) 3.07 (td, 1 H) 3.81 (m, 1 H) 4.30 (m, 1 H) 4.43 (m, 2 H) 4.93 (m, 1 H) 6.62 (t, 1 H) 6.84 (m, 1 H) 7.99 (d, 2 H) 8.14 (m, 2 H) 8.35 (m, 2 H). MS (ESI) m/z 407.1 [M+H] +

Example 9a

(R -tert-Butyl 2-methyl-4-(pyrimidin-2-yl)piperazine-l-carboxylate

(R)-l-N-Boc-2-Methyl piperazine (CAS 1000853-53-1, 227 mg, 1.13 mmol), 2- chloropyrimidine (CAS 1722-12-9, 143 mg, 1.25 mmol) and TEA (0.395 mL, 2.83 mmol) were dissolved in EtOH (3 mL) in a vial, flushed with argon, capped and heated in a microwave reactor for 30 min at 100°C. The solvent was evaporated, the solid was filtered off and the organic phase was evaporated onto silica gel. The crude was purified on a silica column (EtOAc with 1% TEA:n-heptane) to give (R)-tert-butyl 2-methyl-4-(pyrimidin-2-yl)piperazine- 1-carboxylate (154 mg, 49%). MS (ESI) m/z 279.1 [M+H] +

Example 9b

(R)-2-(3-Methylpiperazin-l-yl)pyrimidine hydrochloride

(R)-ieri-Butyl 2-methyl-4-(pyrimidin-2-yl)piperazine-l-carboxylate (Example 9a, 154 mg, 0.55 mmol) was dissolved in MeOH (1 mL), HCI in 2-propanol (5 M, 4.43 mL, 22.13 mmol) was added and the reaction mixture was stirred at rt for 2 h. The solvent was evaporated and the residue was dried in a vacuum desiccator to give (R)-2-(3-methylpiperazin-l-yl)pyrimidine hydrochloride (123 mg, quantitative).

MS (ESI) m/z 179.4 [M+H] +

Example 10

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-2,2-dimeth yl-4-(pyrimidin-2-yl)piperazine- 1-carboxamide

Bis(trichloromethyl)carbonate (47.2 mg, 0.16 mmol) was added portionwise to 4-(2-amino-3- methylbutanoyl)benzonitrile hydrochloride (Example 6b ,95 mg, 0.40 mmol) and TEA (0.222 mL, 1.59 mmol) in DCM (3 mL) under 1 min at rt. After stirring for 20 min, a solution of 2-(3,3- dimethylpiperazin-l-yl)pyrimidine hydrochloride (Example 10b, 100 mg, 0.44 mmol) and TEA (0.110 mL, 0.79 mmol) in DCM (2 mL) was added dropwise and the mixture was stirred for 10 min. The crude mixture was filtered and diluted with MeOH before purification by prep. HPLC to give N-(l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl)-2,2-dimethyl- 4-(pyrimidin-2- yl)piperazine-l-carboxamide (50.0 mg, 30%).

X H NMR (500 MHz, DMSO-de) δ ppm 0.84 (d, 3 H) 0.91 (d, 3 H) 1.24 (d, 6 H) 2.11 (dq, 1 H) 3.59 (m, 5 H) 3.84 (d, 1 H) 4.84 (t, 1 H) 6.62 (t, 1 H) 6.71 (d, 1 H) 7.99 (d, 2 H) 8.13 (d, 2 H) 8.35 (d, 2 H). MS (ESI) m/z 421.0 [M+H] +

Example 10a te/t-Butyl 2,2-dimethyl-4-(pyrimidin-2-yl)piperazine-l-carboxylate

te/t-Butyl 2,2-dimethylpiperazine-l-carboxylate (CAS 674792-07-5, 423 mg, 1.97 mmol), 2- chloropyrimidine (CAS 1722-12-9, 339 mg, 2.96 mmol), potassium fluoride (172 mg, 2.96 mmol) and TEA (0.549 mL, 3.95 mmol) were dissolved in DMF (3 mL) before heated in a microwave reactor for 3 h at 150°C. After cooling to rt the mixture was partitioned between DCM (40 mL) and water (15 ml). The organic layer was washed with water, brine, dried over MgS0 4 and evaporated to give crude te/t-butyl 2,2-dimethyl-4-(pyrimidin-2-yl)piperazine-l- carboxylate (1.04 g).

MS (ESI) m/z 293.1 [M+H] +

Example 10b

2-(3,3-Dimethylpiperazin-l-yl)pyrimidine hydrochloride

te/t-Butyl 2,2-dimethyl-4-(pyrimidin-2-yl)piperazine-l-carboxylate (Example 10a, 577 mg, 1.97 mmol) was dissolved in MeOH (5 mL). Hydrochloric acid in 2-propanol (5 N, 13.16 mL, 78.94 mmol) was added and the reaction mixture was stirred at rt for 16 h. A solid was isolated via decanting to give 2-(3,3-dimethylpiperazin-l-yl)pyrimidine hydrochloride (236 mg, 52%).

X H NMR (500 MHz, DMSO-de) δ ppm 1.32 (s, 6 H) 3.16 (d, 2 H) 3.82 (s, 2 H) 3.99 (t, 2 H) 6.73 (t, 1 H) 8.42 (D, 2 H) 9.63 (br. s., 1 H). MS (ESI) m/z 193.1 [M+H] +

Example 11

N-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-4-phenyl-p iperazine-l-carboxamide

To a suspension of 4-(2-amino-3-methylbutanoyl)benzonitrile hydrochloride (Example 6b, 0.2 mmol, 1.0 eq) in MeCN (2 mL) was added CDI (0.6 mmol, 3.0 eq) and TEA (0.8 mmol, 4.0 eq). The mixture was stirred for 1 h at room temperature and 1-phenylpiperazine (CAS 92-54-6, 1.0 mmol, 5.0 eq) was added. The resulting mixture was heated at 80°C for another 2 h. The crude product was purified by preparative TLC (petroleum ethenethyl acetate 1:1) to give N- [l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl]-4-phenyl-pipera zine-l-carboxamide (89 μιηοΙ, 44%).

XH NMR (500 MHz, -DCI 3 ) δ ppm 0.75 - 0-90 (m, 3 H) 0.98 - 1-14 (m, 3 H) 2.05 - 2-20 (m, 1 H) 3.14 - 3-36 (m, 4 H) 3.55 - 3.76 (m, 4 H) 5.31 (d, 1 H) 5.44 (ddd, 1 H) 6.94 (d, 3 H) 7-31 (t, 2 H) 7.76 - 7-90 (m, 2 H) 8.06 - 8.20 (m, 2 H). MS (ESI) m/z 391.2 [M+H] +

Example 12

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l -phenethyl-urea

To a suspension of 4-(2-amino-3-methylbutanoyl)benzonitrile hydrochloride (Example 6b ,0.2 mmol, 1.0 eq) in MeCN (2 mL) was added CDI (0.6 mmol, 3.0 eq) and TEA (0.8 mmol, 4.0 eq). The mixture was stirred for 1 h at room temperature and N-methyl-2-phenyl-ethanamine (CAS 124774-52-3, 1.0 mmol, 5.0 eq) was added. The resulting mixture was heated at 80°C for another 2 h. The crude product was purified by preparative TLC (petroleum ethenethyl acetate 1:1) to give 3-[l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l-ph enethyl- urea (67 μιηοΙ, 33%).

X H NMR (500 MHz, CDCI 3 ) δ ppm 0.78 (d, 3 H) 1-00 (d, 3 H) 2.01 - 2-14 (m, 1 H) 2.85 - 2.89 (m, 2 H) 2.89 (s, 3 H) 3.53 (m, 2 H) 5.08 (d, 1 H) 5.39 (dd, 1 H) 7-22 (d, 3 H) 7.28 - 7-33 (m, 2 H) 7.79 - 7-82 (m, 2 H) 8.07 - 8.14 (m, 2 H). MS (ESI) m/z 364.1 [M+H] +

Example 13

N-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-4-(2-pyridyl) piperazine-l-carboxamide

To a suspension of 4-(2-amino-3-methylbutanoyl)benzonitrile hydrochloride (Example 6b ,0.2 mmol, 1.0 eq) in MeCN (2 mL) was added CDI (0.6 mmol, 3.0 eq) and TEA (0.8 mmol, 4.0 eq). The mixture was stirred for 1 h at room temperature and l-(2-pyridyl)piperazine (CAS 34803- 66-2, 1.0 mmol, 5.0 eq) was added. The resulting mixture was heated at 80°C for another 2 h. The crude product was purified by preparative TLC (petroleum ethenethyl acetate 1:1) to give N-[l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl]-4-(2-pyridyl) piperazine-l-carboxamide (84 μιηοΙ, 42%).

X H NMR (500 MHz, CDCI 3 ) δ ppm 0.83 (d, 3 H) 1.03 (d, 3 H) 2.-2 (td, 1 H) 3.55 - 3.73 (m, 8 H) 5.29 (d, 1 H) 5.-4 (dd, 1 H) 6.62 - 6.72 (m, 2 H) 7-53 (t, 1 H) 7.79 - 7-83 (m, 2 H) 8.07 - 8-14 (m, 2 H) 8.19 - 8.24 (m, 1 H). MS (ESI) m/z 392.2 [M+H] +

Example 14

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l -pentyl-urea

To a suspension of 4-(2-amino-3-methylbutanoyl)benzonitrile hydrochloride (Example 6b ,0.2 mmol, 1.0 eq) in MeCN (2 mL) was added CDI (0.6 mmol, 3.0 eq) and TEA (0.8 mmol, 4.0 eq). The mixture was stirred for 1 h at room temperature and N-methyl-pentan-l-amine (CAS 25419-06-1, 1.0 mmol, 5.0 eq) was added. The resulting mixture was heated at 80°C for another 2 h. The crude product was purified by preparative TLC (petroleum ethenethyl acetate 1:1) to give 3-[l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l-pe ntyl-urea (139 μιηοΙ, 70%).

X H NMR (500 MHz, CDCI 3 ) δ ppm 0.81 (d, 3 H) 0.91 (t, 3 H) 1-02 (d, 3 H) 1.25 - 1-40 (m, 4 H) 1.52 - 1.62 (m, 2 H) 2.10 (M, 1 H) 2.96 (br. s., 3 H) 3.29 (T, 2 H) 5.14 (br. s., 1 H) 5.41 (br. s., 1 H) 7.80 (d, 2 H) 8.11 (d, 2 H). MS (ESI) m/z 330.2 [M+H] +

Example 15 l-But l-3-[l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-ethyl-ure a

To a suspension of 4-(2-amino-3-methylbutanoyl)benzonitrile hydrochloride (Example 6b ,0.2 mmol, 1.0 eq) in MeCN (2 mL) was added CDI (0.6 mmol, 3.0 eq) and TEA (0.8 mmol, 4.0 eq). The mixture was stirred for 1 h at room temperature and N-ethyl-butan-l-amine (CAS 13360- 63-9, 1.0 mmol, 5.0 eq) was added. The resulting mixture was heated at 80°C for another 2 h. The crude product was purified by preparative TLC (petroleum ethenethyl acetate 1:1) to give l-butyl-3-[l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-eth yl-urea (121 μιηοΙ, 60%). X H NMR (500 MHz, CDCI3) δ ppm 0.81 (d, 3 H) 0.96 (t, 3 H) 1.02 (d, 3 H) 1.20 (t, 3 H) 1.-6 (dq, 2 H) 1.53 - 1.63 (m, 2 H) 2.1- (dtd, 1 H) 3.22 - 3.27 (m, 2 H) 3.32 (qd, 2 H) 5.20 (d, 1 H) 5.43 (dd, 1 H) 7.80 (d, 2 H) 8.10 (d, 2 H). MS (ESI) m/z 330.2 [M+H] +

Example 16

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l,l-diprop yl-urea

To a suspension of 4-(2-amino-3-methylbutanoyl)benzonitrile hydrochloride (Example 6b ,0.2 mmol, 1.0 eq) in MeCN (2 mL) was added CDI (0.6 mmol, 3.0 eq) and TEA (0.8 mmol, 4.0 eq). The mixture was stirred for 1 h at room temperature and N-propyl-propane-l-amine (CAS 142-84-7, 1.0 mmol, 5.0 eq) was added. The resulting mixture was heated at 80°C for another 2 h. The crude product was purified by preparative TLC (petroleum ethenethyl acetate 1:1) to give 3-[l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l,l-dipropyl- urea (81 μιηοΙ, 41%).

X H NMR (500 MHz, CDCI 3 ) δ ppm 0.80 (d, 3 H) 0.93 (t, 6 H) 1.02 (d, 3 H) 1.62 (sxt, 4 H) 2.10 (td, 1 H) 3.22 (td, 4 H) 5.14 (d, 1 H) 5.-2 (dd, 1 H) 7.78 - 7-82 (m, 2 H) 8.08 - 8.12 (m, 2 H). MS (ESI) m/z 330.2 [M+H] +

Example 17

3- l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-[(4-fluorophen yl)methyl]-l-methyl-urea

To a suspension of 4-(2-amino-3-methylbutanoyl)benzonitrile hydrochloride (Example 6b ,0.2 mmol, 1.0 eq) in MeCN (2 mL) was added CDI (0.6 mmol, 3.0 eq) and TEA (0.8 mmol, 4.0 eq). The mixture was stirred for 1 h at room temperature and l-(4-fluorophenyl)-N-methyl- methanamine (CAS 405-66-3, 1.0 mmol, 5.0 eq) was added. The resulting mixture was heated at 80°C for another 2 h. The crude product was purified by preparative TLC (petroleum ethenethyl acetate 1:1) to give 3-[l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-[(4- fluorophenyl)methyl]-l-methyl-urea (133 μιηοΙ, 67%).

X H NMR (500 MHz, CDCI3) δ ppm 0.76 (d, 3 H) 1-01 (d, 3 H) 2.04 - 2.16 (m, 1 H) 2.96 (s, 3 H) 4.50 (m, 2 H) 5.20 (d, 1 H) 5.-4 (dd, 1 H) 7.01 - 7-06 (m, 2 H) 7.20 - 7-25 (m, 2 H) 7.79 - 7-83 (m, 2 H) 8.06 - 8.16 (m, 2 H). MS (ESI) m/z 368.1 [M+H] + Example 18

3-[l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l -(m-tolylmethyl)urea

To a suspension of 4-(2-amino-3-methylbutanoyl)benzonitrile hydrochloride (Example 6b ,0.2 mmol, 1.0 eq) in MeCN (2 mL) was added CDI (0.6 mmol, 3.0 eq) and TEA (0.8 mmol, 4.0 eq). The mixture was stirred for 1 h at room temperature and N-methyl-l-(m-tolyl)methanamine (CAS 39180-84-2, 1.0 mmol, 5.0 eq) was added. The resulting mixture was heated at 80°C for another 2 h. The crude product was purified by preparative TLC (petroleum ethenethyl acetate 1:1) to give 3-[l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl]-l-methyl-l-(m - tolylmethyl)urea (88 μηηοΙ, 44%).

X H NMR (500 MHz, CDCI 3 ) δ ppm 0.74 (d, 3 H) 1.00 (d, 3 H) 2.08 (td, 1 H) 2.35 (s, 3 H) 2.98 (s, 3 H) 4.50 (s, 2 H) 5.18 (d, 1 H) 5.-3 (dd, 1 H) 7.03 - 7-12 (m, 3 H) 7.22 - 7-26 (m, 1 H) 7.78 - 7- 82 (m, 2 H) 8.08 - 8.14 (m, 2 H). MS (ESI) m/z 364.2 [M+H] +

Example 19

N-(l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-3,3-dimeth yl-4-(pyrimidin-2-yl)piperazine- 1-carboxamide

Bis(trichloromethyl)carbonate (19.89 mg, 0.07 mmol) was added portionwise to 4-(2-amino- 3-methylbutanoyl)benzonitrile hydrochloride (Example 6b, 40 mg, 0.17 mmol) and TEA (0.093 mL, 0.67 mmol) in DCM (3 mL) under 1 min at rt. After stirring for 20 min, a solution of 2-(2,2-dimethylpiperazin-l-yl)pyrimidine hydrochloride (Example 19b, 22 mg, 0.10 mmol) and TEA (0.047 mL, 0.34 mmol) in DCM (2 mL) was added dropwise and the reaction stirred for 14 min. The crude reaction mixture was filtered and diluted with MeOH before purified by prep. HPLC to give N-(l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl)-3,3-dimethyl- 4- (pyrimidin-2-yl)piperazine-l-carboxamide (1.7 mg, 2.4%).

X H NMR (500 MHz, MeOD) δ ppm 0.95 (m, 6 H) 1.51 (d, 6 H) 2.92 (s, 2 H) 3.55 (t, 2 H) 3.62 (m, 2 H) 4.17 (t, 2 H) 5.15 (d, 1 H) 6.56 (t, 1 H) 7.87 (m, 2 H) 8.16 (d, 2 H) 8.30 (d, 2 H). MS (ESI) m/z 421.2 [M+H] +

Example 19a

Tert-butyl 3,3-dimethyl-4-(pyrimidin-2-yl)piperazine-l-carboxylate

Tert-butyl 3,3-dimethylpiperazine-l-carboxylate (CAS 259808-67-8, 300 mg, 1.40 mmol), 2- chloropyrimidine (CAS 1722-12-9, 240 mg, 2.10 mmol), potassium fluoride (122 mg, 2.10 mmol) and TEA (0.389 mL, 2.80 mmol) were dissolved in DMF (3 mL). After flushing with argon, the solution was heated in a microwave reactor for 3 h at 80°C, 6h at 100°C, 6 h at 120°C and 10 h at 140°C.After cooling to rt the crude mixture was extracted with EtOAc (2x10 mL) and water. The combined organic phases were washed with 1M HCI (aq), dried over MgS04 and evaporated to give crude tert-butyl 3,3-dimethyl-4-(pyrimidin-2-yl)piperazine-l- carboxylate.

Example 19b

2-(2,2-dimethylpiperazin-l-yl)pyrimidine hydrochloride

tert-butyl 3,3-dimethyl-4-(pyrimidin-2-yl)piperazine-l-carboxylate (Example 19a, 130 mg, 0.44 mmol) was dissolved in MeOH (3 mL). Hydrochloric acid in 2-propanol (5 M, 2 mL, 12.00 mmol) was added and the reaction mixture was stirred at room temperature for 16 h. The formed solid was isolated via decanting to give crude 2-(2,2-dimethylpiperazin-l- yl)pyrimidine hydrochloride.

Example 20

N- l-(4-Cyanophenyl)-3-methyl-l-oxobutan-2-yl)-4-(pyrimidin-2-y l)piperazine-l-carboxamide

Triphosgene (35.8 mg, 0.12 mmol) was added portionwise to a solution of 4-(2-amino-3- methylbutanoyl)benzonitrile hydrochloride (Example 6b, 72 mg, 0.30 mmol) and TEA (0.126 mL, 0.90 mmol) in DCM (5 mL) under 1 min at rt. After stirring 15 min, a solution of 2- (piperazin-l-yl)pyrimidine (CAS 20980-22-7, 49.5 mg, 0.30 mmol) and TEA (0.042 mL, 0.30 mmol) in DCM (2 mL) was added dropwise and the mixture was stirred for 90 min. The crude mixture was extracted with DCM (2x50 mL) and water, combined org. phases washed with 1M HCI (aq) before dried over MgS0 4 and evaporation. The crude was diluted with MeOH before purified by prep. HPLC to give N-(l-(4-cyanophenyl)-3-methyl-l-oxobutan-2-yl)-4- (pyrimidin-2-yl)piperazine-l-carboxamide (25.0 mg, 21%).

X H NMR (500 MHz, DMSO-cfe) δ ppm 0.83 (d, 3 H) 0.93 (d, 3 H) 2.14 (m, 1 H) 3.39 (q, 4 H) 3.64 (t, 4 H) 4.93 (t, 1 H) 6.65 (t, 1 H) 6.93 (d, 1 H) 8.00 (m, 2 H) 8.15 (m, 2 H) 8.37 (d, 2 H). MS (ESI) m/z 393.1 [M+H] +

Example 21 N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-meth ylpyridin-2-yl)piperazine-l- carboxamide

Method 1

4-(2-Amino-3,3-dimethylbutanoyl)benzonitrile (Example 21a, 50 mg, 0.23 mmol) was added to a solution of CDI (112 mg, 0.69 mmol) and TEA (0.035 mL, 0.25 mmol) in MeCN (5 mL). The reaction mixture was stirred at rt for 90 minutes before l-(6-methylpyridin-2-yl)piperazine (CAS 55745-89-6, 205 mg, 1.16 mmol) was added. The mixture was stirred for 1 h and the solvent was evaporated under reduced pressure. The crude was dissolved in methanol, filtered and purified by prep. HPLC yielding N-(l-(4-cyanophenyl)-3,3-dimethyl-l-oxobutan-2- yl)-4-(6-methylpyridin-2-yl)piperazine-l-carboxamide (36.3 mg, 37%).

XH NMR (400 MHz, CDCI 3 ) δ ppm 0.94 (s, 9 H) 2-40 (s, 3 H) 3.48 - 3-68 (m, 8 H) 5.25 - 5-31 (m, 1 H) 5.33 - 5.39 (m, 1 H) 6.43 (d, 1 H) 6.53 (d, 1 H) 7.40 (dd, 1 H) 7.78 (m, 2 H) 8.14 (m, 2 H). MS (ESI) m/z 418.3 [M-H] "

Method 2

Bis(trichloromethyl)carbonate (60.6 mg, 0.20 mmol) was added portionwise to 4-(2-amino- 3,3-dimethylbutanoyl)benzonitrile hydrochloride (Example 21a, 129 mg, 0.51 mmol) and TEA (0.285 mL, 2.04 mmol) in DCM (2 mL) under 1 minute at rt. After stirring for 20 min, a solution of l-(6-methylpyridin-2-yl)piperazine (CAS 55745-89-6, 90 mg, 0.51 mmol) and TEA (0.142 mL, 1.02 mmol) in DCM (2 mL) was added dropwise and the reaction was stirred for 90 min. The volatiles were removed before the crude reaction mixture was diluted with MeOH, filtered and purified by prep. HPLC. Fractions were pooled and freeze-dried to give N- (l-(4-cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-methyl pyridin-2-yl)piperazine-l- carboxamide (76 mg, 36%).

X H NMR (500 MHz, MeOD) δ ppm 0.99 (s, 9 H) 2.36 (s, 3 H) 3.53 (m, 8 H) 5.30 (m, 1 H) 6.34 (m, 1 H) 6.57 (t, 3 H) 7.44 (dd, 1 H) 7.88 (m, 2 H) 8.17 (m, 2 H). MS (ESI) m/z 420.2 [M+H] + Example 21a

4-(2-Amino-3,3-dimethyl-butyryl)-benzonitrile hydrochloride

te/t-Butyl l-(4-chlorophenyl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (Example 5b, 2.30 g, 7.06 mmol) and Zn(CN) 2 (0.87 g, 7.4 mmol) were dissolved in DMF (20 mL) under N 2 (g). Pd(PPh3) 4 (0.86 g, 0.74 mmol) was added and the mixture was heated at 130°C over night. The mixture was cooled to rt, diluted with water and extracted with EtOAc. The organic phase was washed with brine, dried over Na 2 S0 4 and evaporated. The crude product was purified on a silica column (EtOAc:hexane 1:10) to give ieri-butyl l-(4-cyanophenyl)-3,3- dimethyl-l-oxobutan-2-ylcarbamate (961 mg, 43%).

te/t-Butyl l-(4-cyanophenyl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (961 mg, 3.04 mmol) was dissolved in 1.25 M HCI in MeOH at 0°C and the mixture was stirred at rt for 8 h. The solvent was removed and the residue dried under vacuum to give 4-(2-amino-3,3-dimethyl- butyryl)-benzonitrile hydrochloride (110 mg, 93%).

XH NMR (400 MHz, MeOD) δ ppm 1.02 (s, 9 H) 5.05 (s, 1 H) 7.96 (d, 2 H) 8.20 (d, 2 H). MS (ESI) m/z 217.1 [M+H] +

Example 22

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-m ethylpyridin-2-yl)piperazine-l- carboxamide

19 mg of the racemate from Example 21 was separated by chiral chromatography to yield N- (l-(4-cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-methyl pyridin-2-yl)piperazine-l- carboxamide (5.6 mg, 30 %).

Chiral purity 100%, Rt 4.42 min on the following system : SFC Berger Analytix, Column: Chiralcel OD-H; 4.6*250 mm; 5μιη, mobile phase: 20% EtOH; 80% C02, flow: 2 mL/min.

X H NM R (500 MHz, DMSO-de) δ ppm 0.93 (s, 9 H) 2.29 (s, 3 H) 3.43 (m, 8 H) 5.11 (d, 1 H) 6.52 (d, 1 H) 6.59 (m, 2 H) 7.42 (dd, 1 H) 7.98 (d, 2 H) 8.14 (d, 2 H). MS (ESI) m/z 420.3 [M+H] +

Example 23

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-m ethylpyridin-2-yl)piperazine-l- carboxamide

19 mg of the racemate from Example 21 was separated by chiral chromatography to yield N- (l-(4-cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-methyl pyridin-2-yl)piperazine-l- carboxamide (4.2 mg, 22%).

Chiral purity 99.8%, Rt 6.34 min on the following system : SFC Berger Analytix, Column: Chiralcel OD-H; 4.6*250 mm; 5μιη, mobile phase: 20% EtOH; 80% C02, flow: 2 mL/min.

X H N MR (500 M Hz, DMSO- d 6 ) δ ppm 0.94 (s, 9 H) 2.29 (s, 3 H) 3.42 (m, 8 H) 5.12 (d, 1 H) 6.52 (d, 1 H) 6.60 (dd, 2 H) 7.42 (dd, 1 H) 7.99 (m, 2 H) 8.15 (m, 2 H). MS (ESI) m/z 420.3 [M+H] +

Example 24 N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(3-(tri fluoromethyl)pyridin-2- yl)piperazine-l-carboxamide

4-(2-Amino-3,3-dimethylbutanoyl)benzonitrile (Example 21a, 31 mg, 0.14 mmol) in MeCN (2 mL) was added to a solution of CDI (69.7 mg, 0.43 mmol) and TEA (0.022 mL, 0.16 mmol) in MeCN (2 mL). The reaction mixture was stirred at room temperature for 90 minutes before l-(3-(trifluoromethyl)pyridin-2-yl)piperazine (CAS 87394-63-6, 166 mg, 0.72 mmol) was added. The mixture was stirred for 1 h and then the solvent was evaporated. The crude was dissolved in methanol, filtered and purified by prep. HPLC to give N-(l-(4-cyanophenyl)-3,3- dimethyl-l-oxobutan-2-yl)-4-(3-(trifluoromethyl)pyridin-2-yl )piperazine-l-carboxamide (22.4 mg, 33%).

XH NMR (400 MHz, CDCI 3 ) δ ppm 0.95 (s, 9 H) 3-29 (t, 4 H) 3.49 - 3-64 (m, 4 H) 5.25 - 5-32 (m, 1 H) 5.34 - 5.40 (m, 1 H) 7.05 (dd, 1 H) 7.78 (m, 2 H) 7.89 (dd, 1 H) 8.14 (m, 2 H) 8.45 (dd, 1 H). MS (ESI) m/z 474.3 [M+H] +

Example 25

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(2-m ethoxyphenyl)piperazine-l- carboxamide

4-(2-Amino-3,3-dimethylbutanoyl)benzonitrile (Example 21a, 34 mg, 0.16 mmol) in MeCN (2 mL) was added to a solution of CDI (76 mg, 0.47 mmol) and TEA (0.024 mL, 0.17 mmol) in MeCN (2 mL). The reaction mixture was stirred at room temperature for 90 minutes before l-(2-methoxyphenyl)piperazine (CAS 35386-24-4, 151 mg, 0.79 mmol) was added. The mixture was stirred for 1 h and then the solvent was evaporated. The crude was dissolved in methanol, filtered and purified by prep. HPLC to give N-(l-(4-cyanophenyl)-3,3-dimethyl-l- oxobutan-2-yl)-4-(2-methoxyphenyl)piperazine-l-carboxamide (32.4 mg, 47%).

X H NMR (400 MHz, CDCI 3 ) δ ppm 0-95 (s, 9 H) 3.00 - 3-13 (m, 4 H) 3.54 - 3.68 (m, 4 H) 3-88 (s, 3 H) 5.26 - 5-32 (m, 1 H) 5.35 - 5-40 (m, 1 H) 6.86 - 6-98 (m, 3 H) 7.00 - 7.09 (m, 1 H) 7.79 (m, 2 H) 8.15 (m, 2 H). MS (ESI) m/z 435.3 [M+H] +

Example 26

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(3-i sopropyl-l,2,4-oxadiazol-5- l)piperidine-l-carboxamide

4-(2-Amino-3,3-dimethylbutanoyl)benzonitrile (Example 21a, 29 mg, 0.13 mmol) in MeCN (2 mL) was added to a solution of CDI (65.2 mg, 0.40 mmol) and TEA (0.021 mL, 0.15 mmol) in MeCN (2 mL). The reaction mixture was stirred at room temperature for 90 minutes before 3-isopropyl-5-(piperidin-4-yl)-l,2,4-oxadiazole (CAs 733748-92-0, 131 mg, 0.67 mmol) was added. The mixture was stirred for 1 h and then the solvent was evaporated. The crude was dissolved in methanol, filtered and purified by prep. HPLC to yield N-(l-(4-cyanophenyl)-3,3- dimethyl-l-oxobutan-2-yl)-4-(3-isopropyl-l,2,4-oxadiazol-5-y l)piperidine-l-carboxamide (32.5 mg, 55%).

X H NMR (400 MHz, CDCI3) δ ppm 0.94 (s, 9 H) 1-33 (d, 6 H) 1.81 - 1-95 (m, 2 H) 2.08 - 2-18 (m, 2 H) 2.98 - 3-19 (m, 4 H) 3.90 - 4-07 (m, 2 H) 5.22 - 5-28 (m, 1 H) 5.31 - 5.36 (m, 1 H) 7.79 (m, 2 H) 8.13 (m, 2 H). MS (ESI) m/z 438.3 [M+H] +

Example 27

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(4-m ethoxyphenyl)piperazine-l- carboxamide

4-(2-Amino-3,3-dimethylbutanoyl)benzonitrile (Example 21a, 36 mg, 0.17 mmol) in MeCN (2 mL) was added to a solution of CDI (81 mg, 0.50 mmol) and TEA (0.026 mL, 0.18 mmol) in MeCN (2 mL). The reaction mixture was stirred at room temperature for 90 minutes before l-(4-methoxyphenyl)piperazine (CAS 38212-30-5, 160 mg, 0.83 mmol) was added. The mixture was stirred for 1 h and then the solvent was evaporated. The crude was dissolved in methanol, filtered and purified by prep. HPLC to yield N-(l-(4-cyanophenyl)-3,3-dimethyl-l- oxobutan-2-yl)-4-(4-methoxyphenyl)piperazine-l-carboxamide (42.1 mg, 58%).

X H NMR (400 MHz, CDCI 3 ) δ ppm 0-95 (s, 9 H) 3.03 - 3-12 (m, 4 H) 3.50 - 3.65 (m, 4 H) 3-78 (s, 3 H) 5.25 - 5-31 (m, 1 H) 5.34 - 5.40 (m, 1 H) 6.88 (m, 4 H) 7.79 (m, 2 H) 8.14 (m, 2 H). MS (ESI) m/z 433.3 [M-H] "

Example 28

4-(2-Cyanophenyl)-N-(l-(4-cyanophenyl)-3,3-dimethyl-l-oxo butan-2-yl)piperazine-l- carboxamide

4-(2-Amino-3,3-dimethylbutanoyl)benzonitrile (Example 21a, 46 mg, 0.21 mmol) in MeCN (2 mL) was added to a solution of CDI (103 mg, 0.64 mmol) and TEA (0.033 mL, 0.23 mmol) in MeCN (2 mL). The reaction mixture was stirred at room temperature for 90 minutes before 2-(piperazin-l-yl)benzonitrile (CAS 111373-03-6, 199 mg, 1.06 mmol) was added. The mixture was stirred for 1 h and then the solvent was evaporated. The crude was dissolved in methanol, filtered and purified by prep. HPLC to yield 4-(2-cyanophenyl)-N-(l-(4- cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)piperazine-l-carbo xamide (32.6 mg, 36%).

X H NMR (400 MHz, CDCI 3 ) δ ppm 0-95 (s, 9 H) 3.16 - 3-26 (m, 4 H) 3.60 - 3-68 (m, 4 H) 5.27 - 5.33 (m, 1 H) 5.33 - 5.39 (m, 1 H) 7.00 (d, 1 H) 7.07 (td, 1 H) 7.46 - 7.55 (m, 1 H) 7.60 (dd, 1 H) 7.79 (m, 2 H) 8.14 (m, 2 H). MS (ESI) m/z 430.2 [M+H] +

Example 29

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(4-( trifluoromethyl)pyrimidin-2-yl)- 1,4-diaze ane-l-carboxamide

4-(2-Amino-3,3-dimethylbutanoyl)benzonitrile (Example 21a, 50 mg, 0.23 mmol) was added to a solution of CDI (112 mg, 0.69 mmol) and TEA (0.035 ml_, 0.25 mmol) in MeCN (5 ml_). The reaction mixture was stirred at room temperature for 90 minutes before l-(4- (trifluoromethyl)pyrimidin-2-yl)-l,4-diazepane (CAS 238403-48-0, 250 mg, 1.02 mmol) was added. The mixture was stirred for 1 h and then the solvent was evaporated. The crude was dissolved in methanol, filtered and purified by prep. HPLC to yield N-(l-(4-cyanophenyl)-3,3- dimethyl-l-oxobutan-2-yl)-4-(4-(trifluoromethyl)pyrimidin-2- yl)-l,4-diazepane-l- carboxamide (47.0 mg, 42%).

X H NMR (400 MHz, CDCI 3 ) δ ppm 0.90 (s, 9 H) 2.05 (br. s., 2 H) 3.37 (br. s., 1 H) 3.44 (br. s., 1 H) 3.63 (br. s., 2 H) 3.70 (br. s., 1 H) 3.83 (br. s., 1 H) 3.95 (br. s., 2 H) 5.19 (d, 1 H) 5.30 (d, 1 H) 6.76 (d, 1 H) 7.77 (m, 2 H) 8.11 (m, 2 H) 8.48 (br. s., 1 H). MS (ESI) m/z 487.3 [M-H] "

Example 30

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(pyr idin-3-yl)piperazine-l- carboxamide

4-(2-Amino-3,3-dimethylbutanoyl)benzonitrile (Example 21a, 37 mg, 0.17 mmol) in MeCN (2 mL) was added to a solution of CDI (83 mg, 0.51 mmol) and TEA (0.026 mL, 0.19 mmol) in MeCN (2 mL) . The reaction mixture was stirred at room temperature for 90 minutes before l-(pyridin-3-yl)piperazine (CAS 67980-77-2, 140 mg, 0.86 mmol) was added. The mixture was stirred for 1 h and then the solvent was evaporated. The crude was dissolved in methanol, filtered and purified by prep. HPLC to yield N-(l-(4-cyanophenyl)-3,3-dimethyl-l-oxobutan-2- yl)-4-(pyridin-3-yl)piperazine-l-carboxamide (22.0 mg, 32%).

X H NMR (400 MHz, CDCI 3 ) δ ppm 0.94 (s, 9 H) 3.19 - 3.29 (m, 4 H) 3.53 - 3.66 (m, 4 H) 5.28 - 5.39 (m, 2 H) 7.21 (br. s., 2 H) 7.75 - 7.82 (m, 2 H) 8.09 - 8.16 (m, 3 H) 8.32 (br. s., 1 H). MS (ESI) m/z 406.2 [M+H] +

Example 31

N-(l-(4-Cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-y l)-4-(3-cyanopyridin-2- yl)piperazine-l-carboxamide

4-(2-Amino-3,3-dimethylbutanoyl)-2-fluorobenzonitrile (Example 31b, 25 mg, 0.11 mmol) in MeCN (2 mL) was added to a solution of CDI (51.9 mg, 0.32 mmol) and TEA (0.016 mL, 0.12 mmol) in MeCN (2.000 mL). The reaction mixture was stirred at room temperature for 90 minutes before 2-(piperazin-l-yl)nicotinonitrile (CAS 84951-44-0, 100 mg, 0.53 mmol) was added. The mixture was stirred for 1 h and then the solvent was evaporated under reduced pressure. The crude was dissolved in methanol, filtered and purified by prep. HPLC to yield N- (l-(4-cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4- (3-cyanopyridin-2-yl)piperazine- 1-carboxamide (25.0 mg, 52%).

X H NMR (400 MHz, CDCI 3 ) δ ppm 0.96 (s, 9 H) 3.61 (q, 4 H) 3.69 - 3.82 (m, 4 H) 5.14 - 5.32 (m, 2 H) 6.83 (dd, 1 H) 7.78 (dd, 1 H) 7.82 (dd, 1 H) 7.86 (dd, 1 H) 7.96 (dd, 1 H) 8.37 (dd, 1 H). MS (ESI) m/z 449.2 [M+H] +

Example 31a te/t-Butyl l-(4-cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-ylcarba mate

2-Fluoro-4-iodobenzonitrile (CAS 137553-42-5, 0.5 g, 2.02 mmol) was dissolved in THF (10 mL) under Ar(g) to give a colorless solution. Isopropyl magnesium chloride lithium chloride complex (1.3 M in THF, 2.83 mL, 3.68 mmol) was added dropwise at -25^C. The mixture was stirred at -20^C for 90 min before ieri-butyl l-(methoxy(methyl)amino)-3,3-dimethyl-l- oxobutan-2-ylcarbamate (Example 5a, 0.505 g, 1.84 mmol) in THF (5 mL) was added dropwise. The mixture was left to reach rt and stirred for 18 h. The reaction was quenched with NH 4 CI and extracted with EtOAc (2x50 mL). The combined organic phases were washed with water (xl) before dried over MgS0 4 , filtered and evaporated to give the crude product (608 mg), which was evaporated onto silica gel and purified on a silica gel column (12 g, heptane:EtOAc 0-20%) to give ieri-butyl l-(4-cyano-3-fluorophenyl)-3,3-dimethyl-l- oxobutan-2-ylcarbamate (145 mg, 24%).

MS (ESI) m/z 333.0 [M-H] "

Example 31b

4-(2-Amino-3,3-dimethylbutanoyl)-2-fluorobenzonitrile hydrochloride

ieri-Butyl l-(4-cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-ylcarba mate (Example 31a, 145 mg, 0.43 mmol) was dissolved in MeOH (10 mL). Hydrochloric acid in 2-propanol (5 M, 0.696 mL, 3.5 mmol) was added and the reaction mixture was stirred at rt for 72 h, then evaporated and re-dissolved in diethyl ether before being evaporated again to give 4-(2- amino-3,3-dimethylbutanoyl)-2-fluorobenzonitrile hydrochloride (122 mg).

MS (ESI) m/z 235.0 [M+H] +

Example 32

N-(l-(4-Cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-y l)-4-(3-cyanopyridin-2- yl)piperazine-l-carboxamide

18 mg of the racemate from Example 31 was separated by chiral chromatography to yield N- (l-(4-cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4- (3-cyanopyridin-2-yl)piperazine- 1-carboxamide (4.9 mg, 27%).

Chiral purity 100%, Rt 4.22 min on the following system: SFC Berger Analytix, Column: Chiralcel OD-H; 4.6*250 mm; 5μιη, mobile phase: 20% EtOH; 80% C0 2 , flow: 2 mL/min.

X H NMR (400 MHz, CDCI 3 ) δ ppm 0.96 (s, 9 H) 3.61 (q, 4 H) 3.69 - 3.82 (m, 4 H) 5.14 - 5.32 (m, 2 H) 6.83 (dd, 1 H) 7.78 (dd, 1 H) 7.82 (dd, 1 H) 7.86 (dd, 1 H) 7.96 (dd, 1 H) 8.37 (dd, 1 H). MS (ESI) m/z 449.2 [M+H] + Example 33

N-(l-(4-Cyano-3-fluorophenyl)-3,3-dimethyl-l-oxobutan-2-y l)-4-(thiazol-2-yl)piperazine-l- carboxamide

4-(2-Amino-3,3-dimethylbutanoyl)-2-fluorobenzonitrile (Example 31b, 30 mg, 0.13 mmol) in MeCN (2 mL) was added to a solution of CDI (62.3 mg, 0.38 mmol) and TEA (0.020 ml_, 0.14 mmol) in MeCN (2 mL). The reaction mixture was stirred at room temperature for 90 minutes before 2-(piperazin-l-yl)thiazole (CAS 42270-37-1, 108 mg, 0.64 mmol) was added. The mixture was stirred for 1 h and then the solvent was evaporated. The crude was dissolved in methanol, filtered and purified by prep. HPLC to yield N-(l-(4-cyano-3-fluorophenyl)-3,3- dimethyl-l-oxobutan-2-yl)-4-(thiazol-2-yl)piperazine-l-carbo xamide (29.8 mg, 54%).

X H NMR (400 MHz, CDCI 3 ) δ ppm 0.96 (s, 9 H) 3.60 (br. s., 8 H) 5.19 - 5.27 (m, 2 H) 6.64 (d, 1 H) 7.24 (d, 1 H) 7.78 (dd, 1 H) 7.86 (dd, 1 H) 7.95 (dd, 1 H). MS (ESI) m/z 430.2 [M+H] + Solid state characterisation

N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-meth ylpyridin-2-yl)piperazine-l- carboxamide (example 22)

X-Ray Powder Diffraction (XRPD) patterns were collected on a PANalytical X ' Pert PRO MPD theta-theta system using long-fine-focus Cu Κα-radiation, wavelength of X-rays 1.5418 A, at 45 kV and 40 mA . A programmable divergence slit and a programmable anti-scatter slit giving an irradiated length of 10 mm were used. 0.02 radian Soller slits were used on the incident and on the diffracted beam path. A 20 mm fixed mask was used on the incident beam path and a Nickel-filter was placed in front of a PIXcel-detector using 255 active channels. Thin flat samples were prepared on flat silicon zero background plates using a spatula. The plates were mounted in sample holders and rotated in a horizontal position during measurement. Diffraction patterns were collected between 2°2theta and 40°2theta in a continuous scan mode. Total time for a scan between 2 and 40°2theta was approximately 10 minutes.

A diffractogram for N-(l-(4-Cyanophenyl)-3,3-dimethyl-l-oxobutan-2-yl)-4-(6-meth ylpyridin- 2-yl)piperazine-l-carboxamide is provided in figure 1.

Biolofiical evaluation

Assay for Determining Biological Activity

The assay was designed to detect compounds that act on hTRPAl by monitoring intracellular Ca2+ levels in whole-cells. To this end, a dual addition step FLIPR (Fluorescence Imaging Plate Reader) assay for TRPAl activity has been designed. In brief, HEK293 cells expressing TRPAl were grown in a 384 well microtitre plate and loaded with Fluo-4, a fluorescent probe that reports changes in intracellular calcium. TRPAl channel activity was assayed by measuring a baseline signal in assay buffer, followed by application of an EC80-concentration of the TRPAl-agonist zinc. The subsequent influx of calcium through TRPAl channels was detected as a rise in cytoplasmic calcium, which in turn was reported as an increase in the Fluo-4 fluorescence. The activity of test compounds was assessed by adding compounds five minutes prior to the addition of zinc. TRPAl blockers (antagonists) inhibited the calcium influx elicited by the zinc addition and hence no increase in Fluo-4 fluorescence occurred. TRPAl openers (agonists) gave by themselves rise to calcium influx and an increase in the Fluo-4 fluorescence was detected immediately after the compound addition.

Expression of TRPAl in the HEK293 cell line is under the control of an inducible promoter. Therefore, it is possible to establish the specificity of the TRPAl signal by comparing the signal from the zinc stimulus on induced and non-induced cells.

TRPAl is activated by a number of irritants that cause pain. Zinc is an essential biological trace element that excites nociceptive sensory neurons in mice in a TRPAl-dependent matter. Zinc activates TRPAl through a unique mechanism that requires zinc influx through TRPAl channels and subsequent activation via specific intracellular cystein and histidine residues. hTRPAl-HEK293-TREx cells were seeded in Poly-D-Lysine coated plate and allowed to grow a confluent monolayer in cell medium. Prior to the experiment the medium was discarded and cells loaded with fluo-4 NW (Molecular Probes) in assay buffer for 1 hour in room temperature. Compounds were added to the cell-plate and pre-incubated for 5 minutes in assay buffer without Ca 2+ . 200mM zinc 2+ in assay buffer with Ca 2+ was subsequently added to the cells and raw fluorescent counts measured using excitation LED-banks with wavelength 470-495 nm and emission filter with wavelength 515-575nm. The typical assay conditions were: Test compounds: 30 μΜ to 0.001 μΜ, or zero in positive and negative controls; Assay buffer pH 7.4: HBSS with or without Ca 2+ and Mg 2+ , lOmM Hepes, ImM Glucose, 0.4% NaHC0 3 , Agonist; 200uM zinc chloride, Compounds to be tested were diluted in 100% DMSO and prior to the experiment further diluted 50 times in assay buffer.

In the assay 100% activity was defined as the peak fluorescence level aroused from 200 uM zinc 2+ in absence of test compound. IC50s represented the concentration of test compound required to inhibit this response with 50%.

Data from this assay for representative compounds is shown in the table below. The potency is expressed as IC50 (concentration needed for 50% inhibition relative to the mean signal in calcium buffer) and the value indicated is an average of at least two individual experiments.

TABLE 1: IC50 VALUES FOR THE EXEMPLIFIED COMPOUNDS

Example Mean 50 (μΜ) 9 11

1 0.57 10 1.4

2 0.94 11 1.4

3 2.7 12 2.3

4 4.1 13 2.8

5 1 14 3.6

6 3.8 15 5.9

7 8.2 16 5.9

8 10 17 6.8 13 26 1.8

1.8 27 1.9

11 28 2.6

0.2 29 2.7

0.06 30 12

6.8 31 0.71

0.41 32 0.32

1.5 33 0.85