CA2933811C

P2x3 and/or p2x2/3 compounds and methods

Abstract

The present application provides novel compounds and methods tor preparing and using these compounds. In one embodiment, the compounds are of the structure of formula (I), wherein R1-R4 are defined herein. In a further embodiment, these compounds are useful in method for regulating one or both of the P2X3 or P2X2/3 receptors. In another embodiment, these compounds are useful for treating pain in patients by administering one or more of the compounds to a patient.

CA2933811C, drawing sheet 1 — Sheet 1 of 59

Term

8.2 yearsleft in the term

Expires 16 December 2034.

71 claims: 8 independent, 63 dependent

1
A compound of the structure of formula (I):R 1 • H rM r 3 S r2 (I) wherein: R 1 is optionally substituted heteroaryl or NR 5 R 6 ;R 2 is optionally substituted heteroaryl;R 3 is H or Ci to Cô alkyl;R 4 is optionally substituted heteroaryl;R 5 and R 6 are, independently, selected from the group consisting of H, optionally substituted Ci to Ce alkyl, C3 to Cô cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and CO(Ci to Ce alkyl);or R 5 and R 6 are joined with the N of NR 5 R 6 to form a 5 or 6-membered heterocyclic ring optionally substituted by one or more of halogen, Ci to Cô alkyl, Ci to Cô alkoxy, Ci to Cô hydroxyalkyl, C3 to Cô cycloalkyl, Ci to Cô alkyl containing 1 to 3 fluorine atoms, C3 to Cô cycloalkyl-Ci to Cô alkyl, or CONHz;or a pharmaceutically acceptable salt or prodrug thereof, and wherein the prodrug comprises one or more of an acetyl analog, ester analog, amide, (alkoxycarbonyloxy)alkyl carbamate, (acyloxy)alkyl carbamate, and (oxodioxolenyl)alkyl carbamate.
3
A compound according to claim I which is of the structure of formula (III):170 CA 2933811 2019-09-12
43
A pharmaceutical composition comprising a compound of any one of claims 1 to 42 and a pharmaceutically inert or inactive ingredient. 186 CA 2933811 2019-09-12
44
A kit comprising a compound of any one of claims 1 to 42 and instructions for administering the compound.
45
Use of a compound of any one of claims 1 to 42 to regulate one or both of the P2X3 or P2X 2 /3 receptors.
47
Use of a compound of any one of claims 1 to 42 to treat pain in a patient.
65
The use a compound of any one of claims 1 to 42 for modulating one or both of the P2X3 or P2X2/3 pathways.
70
The use of a compound of any one of claims 1 to 42 to treat a respiratory disorder in a patient.

Independent claims8

1,307 paragraphs in 432 sections, as filed

P2Xj and/or P2Xm Compounds and Methods

BACKGROUND

[0002] Adenosine triphosphate (ATP) is well-recognized as the primary energy currency of living cells, but has also emerged as a significant signaling molecule that can shape physiological and pathophysiological processes by interacting with any of several 'purinergic* membrane-associated receptor molecules. The purinergic receptor family comprises both G-protein-coupled (GPCR) receptors (assigned a P2Y nomenclature) and ligand-gated ion channel (P2X) variants. ATP elicits an excitatoiy effect on afferent sensory nerves via an interaction with receptors of the P2X subfamily. The consequence of such hyperexcitability may be interpreted as pain when the ATP effect is elicited in skin, bone or visceral tissues, as pain and/or cough in airway tissues, or as pain and/or instability when it occurs in the bladder. Two particular receptor variants within the P2X subfamily, designated. P2Xj and P2X2/3, have emerged as targets of particular interest in a variety of studies designed to measure nociception, airway or bladder function in rodents, since activation of these receptors by ATP is capable of generating the adverse events cited above.

[0003] Accordingly, there is a need for more potent and selective P2X3/P2X2/3 modulators.

SUMMARY OF THE INVENTION

[0004] In one aspect, a compound of tire structure of formula (I) is provided, wherein R1 -R4 are described herein, or a pharmaceutically acceptable salt thereof.

R3 O R (I)

[0005] In another aspect, a compound of the structure of formula (II) is provided, which is embraced by formula (I), wherein R‘, R2, and R4 are described herein, or a pharmaceutically acceptable salt thereof.

CA 2933811 2018-01-11

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

(Π)

[0006] In a further aspect, a compound of the structure of formula (ΠΙ) is provided, which is embraced by formula (I), wherein R1, R2 *, and R4 * are described herein, or a pharmaceutically acceptable salt or prodrug thereof.

R’

I 0 R (ΠΙ)

PCT/US2014/070497 embodiment, R2 is Ci to Ce alkyl. In another embodiment, R2 is butyl. In still a further embodiment, R2 is -S-(Ci to Ce alkyl). In yet another embodiment, R2 is -SCH2CH2CH3. In a further embodiment, R2 is optionally substituted heteroaryl. In still another embodiment, R2 is heteroaryl substituted with one or more halogen, cyano, Ci to Ce alkyl or C] to Ce alkyl containing 1-3 fluorine atoms. In yet a further embodiment, R2 is thiazole, thiophene, or furan. In another embodiment, R2 is thiazole substituted with Ci to Ce alkyl. In still a further embodiment, R2 is thiophene substituted with one or more of halogen, cyano, C] to Co alkyl or Cj to Ce alkyl containing 1-3 fluorine atoms. In yet another embodiment, R2 is furan substituted with Cj to Ce alkyl. In a further embodiment, R2 is 2-chloro-thiophene, 2-methylthiophene, 2-cyano-thiophene, 2-trifluoromethyl-thiophene, 5-methyl-thiazole, 2-methylthiazole, or 2-methyl-furan.

[0027] R3 is H or C] to C« alkyl. In one embodiment, R3 is H. In another embodiment, R3 is Ci to Cô alkyl.

[0028] R4 is optionally substituted heteroaryl. In one embodiment, R4 is optionally substituted triazole, optionally substituted pyridine, optionally substituted pyridone, optionally substituted oxadiazole, optionally substituted pyrazine, or optionally substituted pyrimidine. In another embodiment, R4 is heteroaryl optionally substituted with Ci to Ce alkyl, C3 to Ce cycloalkyl, Ci to Ce alkoxy, or Ci to Ce trifluoroalkyl. In a further embodiment, R4 is pyridine substituted with one or more Ci to Ce alkyl, C3 to Ce cycloalkyl, Ci to Ce alkoxy, or Ci to Ce trifluoroalkyl. In yet another embodiment, R4 is pyridine and the nitrogen atom of said pyridine is bound to an 0-atom. In still a further embodiment, R4 is pyrazine substituted with one or more Ci to Ce alkyl. In another embodiment, R4 is pyrimidine substituted with one or more Ci to Ce alkyl or Ci to Ce alkoxy. In yet a further embodiment, R4 is pyridone substituted with one or more Ci to Ce alkyl. In still another embodiment, R4 is 1,2,4-triazole, 2-methyl-pyridine, 2-methoxy-pyridine, 1-oxo-pyridine, 1 -oxo-2-methyl-pyridine, 1 -oxo-2-trifluoromethyl-pyridine, 2-trifluoromethyl-pyridine, 2-cyclopropyl-pyridine, 1,3,4-oxadiazole, 1,2,4-oxadiazole, 3-methyl-l,2,4-oxadiazole, 2-methyl-l,3,4-oxadiazole, 2-methyl-pyrazine, 2-methyl-pyrimidine, 2-methoxy-pyrimidine, or 1-methyl-pyridone.

[0029] In one embodiment, the compound is of the structure of formula (II), wherein R1, R2, and R4 are defined above.

H8324I17CA

R1

O R (H)

[0030] In another embodiment, the compound is of the structure of formula (III), wherein R*, R2, and R4 are defined above.

R1

(III)

[0031] In a further embodiment, the compound is of the structure of formula (IV), wherein R2 and R4-R6 are defined above.

NR5R6

(IV)

[0032] Representative pharmaceutically acceptable salts include but are not limited to those of an acid or base. In one embodiment, the pharmaceutical salt is selected from among water-soluble and water-insoluble salts. Tie salt may be of an acid selected from, e.g., among acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic, trifluoroacetic, and camphorsulfonic. The salt can also be of a base selected from, e.g., sodium, potassium, calcium, and ammonium. In some embodiments, a composition of the invention may contain both a pharmaceutically acceptable salt and the free base form of a compound of the invention.

[0033] A compound of the invention may also be a prodrug of formula (I). Prodrugs of compounds of formula (I) may be prepared using various methods known to those skilled in the art. See, e.g., Rautio, Nature Reviews Drug Discovery, 7:255-270 (2008) and Ettmayer, J. Med. Chern., 47:2393-2404 (2004). In the case of drugs containing a hydroxy moiety, acetyl

CA 2933811 2018-01-11

H8324117CA and other ester analogs are contemplated for use as prodrugs. See, e.g., Beaumont, Current Drug Metabolism, 4:461-485 (2003). In the case of drugs containing an amine moiety, prodrugs containing amides and carbamates are contemplated. See, e.g., Simplicio, Molecules, 13:519-547 (2008). As specific examples, (alkoxycarbonyloxy)alkyl carbamates, (acyloxy)alkyl carbamates, and (oxodioxolenyl)alkyl carbamates may be utilized as effective prodrug strategies for amines. See, e.g., Li, Bioorg. Med. Chern. Lett, 7:2909-2912 (1997); Alexander, J. Med. Chern., 34:78-81 (1991); Alexander, J. Med. Chern., 31:318-322 (1988); and Alexander, J. Med. Chern., 39:480-486 (1996).

[0034] Some compounds described herein possess one or more chiral centers. Therefore, the disclosure of each compound includes its separate enantiomers as well as mixtures of the enantiomers. Where multiple chiral centers exist in the compounds, also provided are each possible combination of chiral centers within a compound, as well as all possible enantiomeric and diastereomeric mixtures thereof. All chiral, diastereomeric, and racemic forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials.

[0035] The following definitions are used in connection with the compounds described herein. In general, the number of carbon atoms present in a given group is designated Cx to Cy, where x and y are the lower and upper limits, respectively. The carbon number as used in the definitions herein refers to carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like. Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein is determined by naming from left to right the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. As used herein, optionally substituted means that at least 1 hydrogen atom of the optionally substituted group has been replaced.

[0036] Alkyl refers to a hydrocarbon chain that may be straight or branched. In one embodiment, an alkyl contains 1 to 6 (inclusive) carbon atoms. In another embodiment, an alkyl contains 1 to 5 (inclusive) carbon atoms. In a further embodiment, an alkyl contains 1 to 4 (inclusive) carbon atoms. In yet another embodiment, an alkyl contains 1 to 3 (inclusive)

CA 2933811 2018-01-11

2,5-diazabicyclo[2.2.1 ]heptane-5-yl, 6-azabicyclo[3.2.1 ]oct-6-yl, 8-azabicyclo[3.2.1 ]octan-8yl, 3-oxa-7,9-diazabicyclo[3.3.1]nonan-9-yl, 9-oxa-3-azabicyclo[3.3.1]nonan-3-yl, 3-oxa-9azabicyclo[3.3.1]nonan-9-yl, 3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl, 3,4-dihydro-2II-l,4benzoxazin-7-yl, thiazine, dithiane, and dioxane. In another embodiment, the heterocycle contains 1 or 2 nitrogen atoms. In a further embodiment, the heterocycle contains 1 or 2 nitrogen atoms and 3 to 6 carbon atoms. In still a further embodiment, the heterocycle

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 contains 1 or 2 nitrogen atoms, 3 to 6 carbon atoms, and 1 oxygen atom. In a still a further embodiment, the heterocycle is 5- to 8-membered. In still a further embodiment, the heterocycle is 5-membered. In still a further embodiment, the heterocycle is 6-membered. In still a further embodiment, the heterocycle is 8-membered. A heterocycle may be unsubstituted or substituted with one or more groups including, without limitation, halogen, CN, NO2, Ci to Cô alkyl, OH, Ci to C& alkoxy, Ci to Cô alkoxy-Ci to Cô alkoxy, Ci to Cô alkoxy-C] to Ci alkoxy-C] to Cô alkoxy, C] to Co hydroxyalkyl, C3 to Co cycloalkyl, C] to Co alkyl containing 1 to 3 fluorine atoms, C3 to Cô cycloalkyl-C] to Cô alkyl, heterocyclyloxy, C] to Cô alkylthio, aryl, heterocycle, heteroaryl, C(O)(Ci to Cô alkyl), C(O)(heterocycle), C(O)O(Ci to C6 alkyl), C(0)NH2, C(0)NH(Ci to C6 alkyl), C(O)N(Ci to C6 alkyl)(Ci to C6 alkyl), SO2(C] to Co alkyl), SO2(C2 to Co alkynyl), SO2NH(C] to Co alkyl), SO2(heterocycle), NHC(O)(Cj to C6 alkyl), NHSO2(Ci to C6 alkyl), N(Cj to C6 alkyl)SO2(C] to C6 alkyl), NH2, NH(aryl), N(Ci to C6 alkyl)(Ci to C6 alkyl) and NHC(0)NH2.

[0043] Alkylamino refers to an NH or N group, the nitrogen atom of the group being attached to 1 or 2 alkyl substituents, respectively, wherein the alkyl is optionally substituted and defined above. The alkylamino is bound through the nitrogen atom of the group. In one embodiment, alkylamino refers to NH(alkyl). In another embodiment, alkylamino refers to — N(alkyl)(alkyl), i.e., a dialkylamino. In a further embodiment, alkylamino refers to N(Ci to Cô alkyl)(Ci to Cô alkyl). In still a further embodiment, alkylamino refers to ·~“ N(alkyl)(heterocycle). In still a further embodiment, alkylamino refers to N(alkyl)(aryl). In still a further embodiment, alkylamino refers to ·»~ N(alkylXheteroaryl). In still a further embodiment, alkylamino refers to N(alkyl)(alkenyl). When the nitrogen atom is bound to two alkyl groups, each alkyl group may be independently selected. In still a further embodiment, two alkyl groups on the nitrogen atom may be taken together with the nitrogen to which they are attached to form a 3- to 4-membered nitrogen-containing heterocycle where up to two of the carbon atoms of the heterocycle can be replaced with N(H), N(Ci to Cô alkyl), N(aryl), N(heteroaryl), O, S(O), or S(O)2. Examples of alkylamino include, but are not limited to N(CH3)2, N(CH2CH3)(CH3), N(CH2CH3)2, N(CH2CH2CH3)2,

N(CII2CII2CII2CIl3)2, and N(CII(CIl3)2XCIl3).

[0044] Arylamino refers to an NH or N group, the nitrogen atom of the group being attached to 1 or 2 aryl substituents, respectively, wherein the aryl is optionally substituted and defined above. The arylamino is bound through the nitrogen atom of the group. In one

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 embodiment, arylamino refers to NH(aryl). In another embodiment, aryl ami no refers to N(aryl)(aryl), i.e., a diarylamino. When the nitrogen atom is bound to two aryl groups, each aryl may be independently selected.

[0045] Alkylcarbonylamino refers to -v NHC(O)(alkyl) or — N(alkyl)C(O)(alkyl) where the alkyl groups are independently defined and independently optionally substituted as described above. Examples of alkylcarbonylamino include, but are not limited to, CH3CONH, CH3CH2CONH, CH3CH2CH2CONH, and CH3CH(CH3)CONH.

[0046] Ester refers to C(O)O(alkyl), which is bound through the carbon atom. The alkyl group is defined and optionally substituted as described above. Examples of ester include, without limitation, C(O)OCH3, C(O)O(CH2CH3), C(O)O(CH2CH2CH3), and C(O)(O)(CH2CH2CH2CH3).

[0047] Urea refers to a group having a ^NHCXOjNH·^ where one of the nitrogen atoms is bound to an alkyl or heteroaryl group. The alkyl or heteroaryl groups are defined and optionally substituted as described above. Examples of urea include, without limitation, NHC(O)NHCH3, NHC(O)NHCH2CH3, NHC(O)NHCH2CH2CH3, and NHC(O)NHCH2CH2CH2CH3.

[0048] Alkylaminocarbonyl refers to C(O)NH(alkyl) or -w C(O)N(alkyl)(alkyl) where the alkyl groups are independently defined and independently optionally substituted as described above. Examples of alkylaminocarbonyl include, but are not limited to, CH3NHCO, CH3CH2NHCO, CH3CH2CH2NHCO, and CH3CH(CH3)NHCO.

[0049] It is to be noted that the term a or an refers to one or more. As such, the terms a (or an), one or more, and at least one are used interchangeably herein.

[0050] The words comprise, comprises, and comprising are to be inteipreted inclusively rather than exclusively. The words consist, consisting, and its variants, are to be interpreted exclusively, rather than inclusively.

[0051] As used herein, the term about means a variability of 10 % from the reference given, unless otherwise specified.

[0052] The term regulation or variations thereof as used herein refers to the ability of a compound of formula (I) to inhibit one or more components of a biological pathway.

[0053] As used herein, the terms subject and patient are used interchangeably and include a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or gorilla.

CA 02933811 2016-06-13

[0064] In one embodiment, the compound of formula (I) may be administered topically to the eye, e.g., as solutions, suspensions or ointments. Examples of ophthalmically compatible carriers which may be used include, without limitation, an aqueous solution, such as saline

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 solution, oil solution or ointments containing ophthalmically compatible preservatives, surfactants, buffers, and viscosity regulators. These compositions may also contain stabilizing agents, antibacterial agents, and may be manufactured in different dosage units, suitable for ocular administration. Drug inserts, either soluble or insoluble, may also be used.

[0065] In another embodiment, the compound of formula (I) may be administered by injection. Solutions for injection or infusion may be prepared as aqueous solutions. In one embodiment, the compound of formula (I) is present in a concentration of about 0.1% w/w to about 3% w/w. These solutions may also contain stabilizing agents, antibacterial agents, buffers and may be manufactured in different dosage unit ampoules or bottles.

[0066] In a further embodiment, the compound of formula (I) may be administered rectally. Dosage units for rectal administration may be prepared in the form of ointments or suppositories, which contain the compound of formula (I) in a mixture with a neutral fat base, or they may be prepared in the form of gelatin-rectal capsules that contain the compound of formula (I) in a mixture with, e.g., a vegetable oil or paraffin oil. Ointments, suppositories or creams containing at least one compound of formula (I) are useful for the treatment of hemorrhoids.

[0067] In still another embodiment, the compound of formula (I) may be administered transdermally. A variety of transdermal delivery systems are known. For use in these systems, a compound of formula (I) may be admixed with a variety of excipients which may include, e.g., pH adjusters, preservatives, and/or penetration enhancers in order to form a solution, ointment, cream, lotion, or gel. Such a composition may form a constituent of a transdermal delivery system (patch etc.).

[0068] A transdermal delivery system may be selected which permits or assists a compound of the invention in passing through the dermal layer and to the targeted area, such as muscular tissues or a perineural space. Such systems may include formulation with skin penetration enhancers. Examples of skin penetration enhancers include physical enhancers (ultrasound, iontophoresis, electroporation, magnetophoresis, microneedle), vesicles, particulate systems (liposome, niosome, transfersome, microemulsion, solid lipid nanoparticle), and chemical enhancers (sulfoxides, azones, glycols, alkanols, terpenes, etc.). Further examples of chemical enhancers include, e.g., propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, which increase the permeability of the skin to the compounds, and permit the compounds to penetrate through the skin to deeper

H8324117CA tissues. See, Sagie & Kohane, Prolonged Sensory-Selective Nerve Blockade, PNAS, 2010(8): 3740-3745,2010for additional examples of chemical enhancers.

[0069] As a further embodiment, the compound of formula (I) may be instilled via direct instillation into the bladder and/or urothelium. In one example, a pharmaceutical composition containing a compound of formula (I) and one or more carriers or excipients is formulated for instillation. For example, the compound of formula (I) may be instilled as a solution. In a further example, the compound instilled may be placed into said bladder or urothelium as an extended-release formulation. A variety of extended-release formulations may be utilized for this purpose and include, without limitation, solution, suspension, gel or other solid dosage form containing reservoirs, a drug coated material, a drug impregnated material, a liposomal-drug formulation, among others.

[0070] The pharmaceutical compositions containing a compound of formula (I) may be formulated neat or with one or more pharmaceutical carriers and/or excipients for administration. The amount of the pharmaceutical carrier(s) is determined by the solubility and chemical nature of the compound of formula (I), chosen route of administration and standard pharmacological practice. The pharmaceutical carrier(s) may be solid or liquid and may incorporate both solid and liquid camers/matrices. A variety of suitable liquid carriers isknown and may be readily selected by one of skill in the art. Such carriers may include, e.g., dimethylsulfoxide (DMSO), saline, buffered saline, cyclodextrin, hydroxypropylcyclodextrin (HPpCD), n-dodecyl-p-D-maltoside (DDM) and mixtures thereof. Similarly, a variety of solid (rigid or flexible) carriers and excipients are known to those of skill in the art. Such carriers may also be designed so as to undergo a state transition when injected into the bladder (e.g., liquid to gel, liquid to solid, gel to solid); such materials are known to those skilled in the art. Such carriers may also comprise a membrane, for example comprising a thermoelastic polymer, which defines a reservoir containing a solid or liquid composition. Such carriers may also comprise a thermoelastic polymer matrix, in which a composition which contains a compound of formula (I) is embedded.

[0071] The compounds of formula (1) can also be administered together with other-membrane stabilizers (local anesthetics), for example to form eutectic mixtures.

[0072] Although the compound of formula (1) may be administered alone, it may also be administered in the presence of one or more pharmaceutical carriers that are physiologically

CA 2933811 2018-01-11

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 compatible. The carriers may be in dry or liquid form and must be pharmaceutically acceptable. Liquid pharmaceutical compositions may be sterile solutions or suspensions. When liquid carriers are utilized, they may be sterile liquids. Liquid carriers may be utilized in preparing solutions, suspensions, emulsions, syrups and elixirs. In one embodiment, the compound of formula (I) is dissolved a liquid carrier. In another embodiment, the compound of formula (I) is suspended in a liquid carrier. One of skill in the art of formulations would be able to select a suitable liquid carrier, depending on the route of administration. The compound of formula (I) may alternatively be formulated in a solid carrier. In one embodiment, the composition may be compacted into a unit dose form, Le., tablet or caplet. In another embodiment, the composition may be added to unit dose form, i.e., a capsule. In a further embodiment, the composition may be formulated for administration as a powder. The solid carrier may perform a variety of functions, i.e., may perform the functions of two or more of the excipients described below. For example, a solid carrier may also act as a flavoring agent, lubricant, solubilizer, suspending agent, filler, glidant, compression aid, binder, disintegrant, or encapsulating material. In one embodiment, a solid carrier acts as a lubricant, solubilizer, suspending agent, binder, disintegrant, or encapsulating material. In another embodiment, the carrier comprises a thermoelastic polymer defining a reservoir containing at a minimum, at least one compound of formula (I) as a solid or liquid composition. In a further embodiment, such carriers comprise a thermoelastic polymer matrix, in which a composition described herein is embedded.

[0073] The composition may also be sub-divided to contain appropriate quantities of the compound of formula (I). For example, the unit dosage can be packaged compositions, e.g., packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids.

[0074] In one embodiment, compositions described herein optionally contain one or more carriers and one or more excipients, and one or more compounds of formula (I). Examples of suitable excipients include without limitation, surfactants, adjuvants, antioxidants, binders, buffers, coatings, coloring agents, compression aids, diluents, disintegrants, emulsifiers (e g., polyoxyethylene fatty acid esters), emollients, encapsulating materials, fillers, flavoring agents, glidants, granulating agents, lubricants, metal chelators, osmo-regulators, pl I adjustors (e.g., sodium hydroxide), preservatives, solubilizers, sorbents, stabilizing agents, sweeteners (such as saccharin), surfactants, suspending agents, syrups, thickening agents (e.g., carboxypolymethylene or hydroxypropylmethylcellulose), penetration enhancers (e.g.,

H8324I17CA hydroxypolyethoxydodecane, DMSO, DMAC, DDM, etc.) or viscosity regulators (such as polymers to increase viscosity). See, for example, the excipients described in the Handbook of Pharmaceutical Excipients, 5,h Edition, Eds.: Rowe, Sheskey, and Owen, APhA Publications (Washington, DC), December 14,2005.

[0075] In one embodiment, the compositions may be utilized as inhalants. For this route of administration, compositions may be prepared as fluid unit doses using a compound of formula (I) and a vehicle for delivery by an atomizing spray pump or by dry powder for insufflation.

[0076] In another embodiment, the compositions may be utilized as aerosols, i.e., oral or intranasal. For this route of administration, the compositions are formulated for use in a pressurized aerosol container together with a gaseous or liquefied propellant, e.g., dichlorodifluoromethane, carbon dioxide, nitrogen, propane, and the like. Also provided is the delivery of a metered dose in one or more actuations.

[0077] In another embodiment, the compositions may be administered by a modified-release delivery device. Modified-release as used herein refers to delivery of a compound of formula (I) which is controlled, for example over a period of at least about 8 hours (e.g., extended delivery) to at least about 12 hours (e.g., sustained delivery). Such devices may also permit immediate release (e.g., therapeutic levels achieved in under about 1 hour, or in less than about 2 hours). Those of skill in the art know suitable modified-release delivery devices. For use in such modified-release delivery devices, the compound of formula (I) is formulated as described herein.

[0078] Suitable modified release delivery devices include drug-eluting implants. Such implants can comprise a thermoelastic polymer matrix, such as a silicon or ethylene vinyl acetate matrix, wherein one or more compounds of formula (I), optionally with one or more excipients, is embedded. See, e.g., US Patent No. 7,736,665 and US Patent Publication No. US-2011/0280922. Other drug-eluting implants can comprise an osmotic pump or other mechanism by which a solution comprising one or more compounds of formula (1) (optionally with one or more excipients) contained within the device is forced out, for example through the implant walls or through one or more apertures, by osmotic pressure which builds within the device once it is implanted into a subject. See, e.g., US Patent Nos. 5,035,891 and 6,464,688. Still other drug-eluting implants can comprise a hydrogel such as a

CA 2933811 2018-01-11

H8324117CA poly methacrylate-based polymer (e.g., US Patent Nos. 5,292,515 and 5,266,325), or a thermoelastic polymer, such as a polyurethane (e.g., US Patent Nos. 7,858,110 and 7,842,303), which define a reservoir containing a solid or liquid composition comprising one or more compounds of formula (I) optionally with one or more excipients. Still other drug-eluting implants can comprise a bio-degradable or bio-erodable polymer and at least one or more compounds of formula (I), optionally with one or more excipients. See, e.g., US Patent Nos. 4,906,474 and 5,633,002.

{0079] Modified release of the compounds of formula (I) may also be achieved by injecting a composition comprising one or more of these compounds into the bladder tissue (e.g., into the urothelium or muscularis propria) with a device that can be employed via an endoscope inserted into the bladder or percutaneously. For example, one or more compounds of formula (I) can be injected into the bladder tissue via a needle, or a needleless device as described in US Patent Publication No. US-2011/0046600. A suitable needleless injection device includes the JetTouch™ platform (American Medical Systems; Minnetonka, Minnesota). The injected compounds can form a depot, and in certain embodiments, the one or more compounds of formula (1) can be encapsulated in a bio-degradable or bio-erodable polymer, for example as described in US Patent Nos. 5,480,656 and 6,036,976.

[0080] Modified release of the compounds of formula (I) may also be achieved by instilling a composition comprising one or more compounds of formula (I) and a material which solidifies or gels, for example once instilled into the bladder or upon contact with the bladder urothelium, to coat at least a portion of the bladder wall. The one or more compounds of formula (I) can then elute from the solidified or gelled material. See, e.g., US Patent Nos. 6,894,071; 5,575,815 and 6,039,967.

[0081] In still a further embodiment, the compositions may be administered transdermally, i.e., via the use of a drug-eluting patch. In one embodiment, the patch is an iontophoretic transdermal patch in which one or more medication(s) is delivered using a simple or more sophisticated, e.g., microprocessor-controlled, electrical current using, for

WO 2015/095128 PCT/US2014/070497 compositions may be further diluted. In one embodiment, these compositions may be diluted 2-fold. In another embodiment, these compositions may be diluted 4-fold.

[0088] Also contemplated for use in the pharmaceutical combinations and methods described below are inhibitors of voltage-gated ion channels. In one embodiment, the voltage-gated ion channels are sodium or calcium ion channels. In a further embodiment, the voltage-gated sodium channel inhibitor includes, without limitation, QX-314, N-methylprocaine (QX-222), N-octyl-guanidine, 9-aminoacridine, and pancuronium. In another embodiment, the inhibitor of voltage-gated calcium channels includes, but is not limited to, D-890 (quaternary methoxy verapamil) and CERM 1 1888 (quaternary bepridil). In a further embodiment, voltage-gated ion channel inhibitors such as riluzole, mexilitine, phenytoin, carbamazepine, procaine, tocainide, prilocaine, diisopyramide, bencyclane, quinidine, bretylium, lifarizine, lamotrigine, flunarizine, articaine, bupivicaine, mepivicaine, tluspirilene, orphenadrine, phenbenzamine, bepridil, pimozide, penfluridol, fluspirilene, propiverine, disopyramide, methadone, tolterodine, tridihexethyl salts, tripelennamine, mepyramine, brompheniramine, chlorpheniramine, dexchlorpheniramine, carbinoxamine, levomethadyl acetate, gallopamil, verapamil, devapamil, tiapamil, emopamil, dyclonine, pramoxine, lamotrigine, mibefradil, gabapentin, amiloride, diltiazem, nifedipine, nimodipine, nitrendipine, cocaine, mexiletine, propafenone, quinidine, oxethazaine, articaine, riluzole, bencyclane, lifarizine, and strychnine may be combined with the compound of formula (I).

[0089] Membrane permeable inhibitors of voltage-gated ion channels may also be utilized in combination with the compound of formula (I) in the compositions, combinations, or methods described herein. In one embodiment, the membrane permeable inhibitor of voltage-gated ion channels includes, but is not limited to, cocaine, carbamazepine, disopyramide, lamotrigine, procainamide, phenytoin, oxcarbazepine, topiramate, zonisamide, tetracaine, ethyl aminobenzoate, prilocaine, disopyramide phosphate, flecainide acetate, mexiletine, propafenone, quinidine gluconate, quinidine polygalacturonate, chloroprocaine, dibucaine, dyclonine, mepivacaine, pramoxine, procaine, tetracaine, oxethazaine, propitocaine, levobupivacaine, bupivacaine, lidocaine, moricizine, tocainide, proparacaine, ropivacaine, quinidine sulfate, encainide, ropivacaine, etidocaine, moricizine, quinidine, encainide, flecainide, tocainide, fosphenytoin, chloroprocaine, dyclonine, L-(-)-l-butyl-2’,6'pipecoloxylidide, and pramoxine.

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

[0090] Additionally, one or more agents may be used to treat pain, i.e., analgesics, may be used in conjunction with a combination of the invention in the methods, compositions, and kits described herein. Such agents include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs), opioids, tricyclic antidepressants, amine transporter inhibitors, and anticonvulsants (such as gabapentinoids).

[0091] The compound of formula (I) may be administered together with a vasoconstrictor, e.g., epinephrine or vasopressin, when utilized in injectable solutions.

[0092] The compound of formula (I) may be combined with glucose or dextrose when utilized for infusion or as a regional analgesic or anti-pruritic.

WO 2015/095128

PCT/US2014/070497 with cancer, which may be due to multiple causes, such as inflammation, nerve compression, and mechanical forces resulting from tissue distension as a consequence of invasion by a tumor and tumor metastasis into bone or other tissues. In one embodiment, the cancer is bone cancer.

[0108] In one embodiment, the pain is neuropathic pain, such as post-herpetic neuralgia. In another embodiment, the pain is inflammatory' pain. In a further embodiment, the pain is nociceptive pain. In still another embodiment, the pain is procedural pain. In yet a further embodiment, the pain is caused by esophageal cancer, colitis, cystitis, irritable bowel syndrome, colitis or idiopathic neuropathy. In still another embodiment, the pain is caused by airway, bladder or visceral organ dysfunction.

[0109] Somatic pain includes pain from bone, joint, muscle, skin, or connective tissue.

[0110] Central pain includes pain arising as a consequence of brain trauma, stroke, or spinal cord injury'.

[0111] Visceral pain includes pain from visceral organs, such as the respiratory or gastrointestinal tract and pancreas, the urinary tract and reproductive organs. In one embodiment, visceral pain results from tumor involvement of the organ capsule. In another embodiment, visceral pain results from obstruction of hollow viscus. In a further embodiment, visceral pain results from inflammation as in cystitis or reflux esophagitis.

[0112] Idiopathic pain refers to pain which has no underlying cause or refers to pain caused by condition which remains undiagnosed.

[0113] Dysfunctional pain refers to pain which occurs in the absence of a noxious stimulus, tissue damage or a lesion to the nervous system. In one embodiment, dysfunctional pain results from rheumatologic conditions such as arthritis and fibromyalgia, tension type headache, irritable bowel disorders and erythermalgia.

[0114] Nociceptive pain includes pain caused by noxious stimuli that threaten to or actually injure body tissues. In one embodiment, nociceptive pain results from a cut, bruise, bone fracture, crush injury, bum, trauma, surgery, labor, sprain, bump, injection, dental procedure, skin biopsy, or obstruction. In another embodiment, nociceptive pain is located in the skin, musculoskeletal system, or internal organs.

[0115] Neuropathic pain is pain due to abnormal processing of sensory input by the peripheral or central nervous system consequent on a lesion to these systems. In one embodiment, neuropathic pain is chronic and non-malignant. In one embodiment,

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 neuropathic pain is due to trauma, surgery, herniation of an intervertebral disk, spinal cord injury, diabetes, infection with herpes zoster (shingles), HIV/AIDS, late-stage cancer, amputation (such as mastectomy), carpal tunnel syndrome, chronic alcohol use, exposure to radiation, and as an unintended side-effect of neurotoxic treatment agents, such as certain anti-HIV and chemotherapeutic drugs. In another embodiment, neuropathic pain is may be described as burning, electric, tingling, or shooting.

[0116] The phrase inflammatory' pain includes pain resulting from inflammation caused by any number of factors. In one embodiment, inflammatory' pain occurs due to tissue damage or inflammation. In another embodiment, inflammatory pain is due to injury (including joints, muscle, and tendons injuries), surgical procedures, infection, and/or arthritis.

[0117] Procedural pain includes refers to pain arising from a medical procedure. The medical procedure may include any type of medical, dental or surgical procedure. In one embodiment, the procedural pain is postoperative. In another embodiment, the pain is associated with an injection, draining an abscess, surgery, dermatological, dental procedure, ophthalmic procedure, arthroscopy and use of other medical instrumentation, and/or cosmetic surgery.

[0118] A migraine is a headache due to activation of sensory fibers innervating the meninges of the brain.

[0119] The term treat, treating, or any variation thereof is meant to include therapy utilized to remedy a health problem or condition in a patient or subject. In one embodiment, the health problem or condition may be eliminated permanently or for a short period of time. In another embodiment, the severity of the health problem or condition, or of one or more symptoms characteristic of the health problem or condition, may be lessened permanently, or for a short period of time. The effectiveness of a treatment of pain can be determined using any standard pain index, such as those described herein, or can be determined based on the patient's subjective pain. A patient is considered treated if there is a reported reduction in pain or a reduced reaction to stimuli that should cause pain.

[0120] In order to measure the efficacy of any of the methods, compositions, or kits described herein, a measurement index may be used. Indices that are useful for the measurement of pain associated with musculoskeletal, immunoinflammatory and neuropathic disorders include a visual analog scale (VAS), a Likert scale, categorical pain scales,

H8324117CA descriptors, the Lequesne index, the WOMAC index, and the AUSCAN index, each of which is well known in the art. Such indices may be used to measure pain, function, stiffness, or other variables.

[0121] Indices that are useful of the measurement of pain associated with interstitial cystitis include the interstitial cystitis symptom index (ICSI), the interstitial cystitis problem index (ICP1), the pain-urgency-frequency score (PUF), the Wisconsin Symptom Instrument (UWI) and a visual analog scale (VAS) such as the Likert scale and other categorical pain scales.

[0122] A visual analog scale (VAS) provides a measure of a one-dimensional quantity. A VAS generally utilizes a representation of distance, such as a picture of a line with hash marks drawn at regular distance intervals, e.g., ten 1-cm intervals. For example, a patient can be asked to rank a sensation of pain by choosing the spot on the line that best corresponds to the sensation of pain, where one end of the line corresponds to no pain (score of 0 cm) and the other end of the line corresponds to unbearable pain. This procedure provides a simple and rapid approach to obtaining quantitative information about how the patient is experiencing pain. VAS scales and their use are described, e.g., in US Patent Nos. 6,709,406 and 6,432,937.

[0123] A Likert scale similarly provides a measure of a one-dimensional quantity. Generally, a Likert scale has discrete integer values ranging from a low value, e.g., 0, meaning no pain, to a high value, e.g., 7, meaning extreme pain. A patient experiencing pain is asked to choose a number between the low value and the high value to represent the degree of pain experienced. Likert scales and their use are described, e.g., in US Patent Nos. 6,623,040 and 6,766,319.

[0124] The Lequesne index and the Western Ontario and McMaster Universities (WOMAC) osteoarthritis (OA) index assess pain, function, and stiffness in the knee and hip of OA patients using self-administered questionnaires. Both knee and hip are encompassed by the WOMAC, whereas there is one Lequesne questionnaire for the knee and a separate one for the hip. These questionnaires are useful because they contain more information content in comparison with VAS or Likert scale. Both the WOMAC index and the Lequesne index questionnaires have been extensively validated in OA, including in surgical settings, e.g., knee and hip arthroplasty. Their metric characteristics do not differ significantly.

CA 2933811 2018-01-11

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

[0125] The AUSCAN (Australian-Canadian hand arthritis) index employs a valid, reliable, and responsive patient self-reported questionnaire. In one instance, this questionnaire contains 15 questions within three dimensions (Pain, 5 questions; Stiffness, 1 question; and Physical function, 9 questions). An AUSCAN index may utilize, e.g., a Likert or a VAS scale.

[0126] The O’Leary-Sant score and IC Problem Index are self-administered indices for measuring lower urinary tract symptoms.

[0127] The Pain-Urgency-Frequency symptom scale is balanced assessment of urinary dysfunction, pelvic pain and symptoms associated with sexual intercourse and frequently used in conjunction with intravesical potassium chloride administration.

[0128] The UWI utilizes seven IC-related questions about frequency, urgency, noctuira and pain.

[0129] Other suitable indices that are useful for the measurement of pain include the Pain Descriptor Scale (PDS), the Verbal Descriptor Scales (VDS), the Numeric Pain Intensity Scale (NPIS), the Neuropathic Pain Scale (NPS), the Neuropathic Pain Symptom Inventory (NPSI), the Present Pain Inventory' (PPI), the Geriatric Pain Measure (GPM), the McGill Pain Questionnaire (MPQ), mean pain intensity (Descriptor Differential Scale), numeric pain scale (NPS) global evaluation score (GES) the Short-Form McGill Pain Questionnaire, the Minnesota Multiphasic Personality Inventory, the Pain Profile and Multidimensional Pain Inventory, the Child Heath Questionnaire, and the Child Assessment Questionnaire.

[0130] In one embodiment, the treatment methods described herein include administering a compound of formula (I) to a patient. Additional, optional agents, such as those described above for use in the combination, may be administered to the patient prior to, concurrently with, or subsequent to the compound of formula (I).

[0131] A variety of in vivo assays and animal models are useful for assessing the ability of compounds to inhibit pain via internal sodium channel inhibition. These models may or may not involve opening (activation) of TRPV1 channels via inducing pain through physical, mechanical, or chemical, e.g., capsaicin, means. Examples of suitable models include, e.g., those described in Binshtok, Anesthesiology, July 2009, 111(1):127-137: Reis, Anesthesiology, July 2009, 111(1): 122-126; Gemer, Anesthesiology, November 2008, 109(5):872-878; and Binshtok, Nature, October 2007, 449:607-610, the use of isolated bladder detrusor muscle preparations (Witte, Naunyn-Schmeideberg's Arch. Pharmacol.

H8Î24117CA

2011,384:555-563), measurement of voiding frequency and volume in freely moving animals (Clouse, 2012. Urology 79:1410el-1410e6), measurement of bladder urodynamics using cystometry in anesthetized animals (Shimizu, 2000, British Journal of Pharmacology 131:610-616). However, for a variety of reasons which will be readily apparent to those of ordinary skill in the art, one may provide in vitro assays which allow for the identification of compounds with the properties. Described herein are several such in vitro assays.

[0132] In one embodiment, a modified FL1PR® (Fluorometric imaging Plate Reader) based assay system was developed which is capable of discriminating between non-specific versus hTRPVl -mediated entry of test compounds. Advantageously, the assay system utilizes heat activated opening of hTRPVl channels followed by an assessment of internal sodium channel block. The assay allows a permanently charged compound to selectively enter through opened hTRPV 1 channels and that compound's potency in inhibiting sodium channels from the cytoplasm side of the same cell can be assessed and quantified.

[0133] The modified FLIPR® assay utilizes cells which functionally express hTRPV 1. As used herein, the term functionally express includes those cells which express the human TRPV1 protein and which respond to stimuli which naturally open this channel, including, e.g., thermal (heat) or chemical (capsaicin, lidocaine, among others) means described herein. Suitable assays may include the calcium or membrane potential assays described herein, e.g., Example 49. However, other functional assays are known in the art, e.g., voltage-clamp electrophysiology such as used by Binshtok, Nature 449(4) 607-610,2007.

[0134] A suitable cell may be selected for expression of TRPV1 in cis or in trans and constructed using known techniques. In one embodiment, a neuroblastoma cell line such as N1E115 [CRL-2263] or ND7/23 [ECACC catalog code: 92090903] is selected for expression of the hTRPVl. However, another neuroblastoma cell line may be selected, e.g., such as IMR-32 [CRL-127]; Neuro-2a [CRL-131]; NB41A3 [CRL-147]; B104-1-1 [CRL-1887]; SK-N-AS [CRL-2137]; SK-N-F1 [CRL-2142]; SK-N-DZ [CRL-2149]; SH-SY5Y [CRL-2266]; BE(2)-M17 [CRL-2267]; BE(2)-C [CRL-2268]; MC-IXC [CRL-2270]; SK-NBE(2) (CRL-2271); CHP-212 (CRL-2273]; B35 [CRL-2754], which are available from the American Type Culture Collection, Manassas, Virginia (US). Still other cell lines may be selected.

CA 2933811 2018-01-11

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

[0135] For a generation description of how the cells are produced, see generally, e.g.,

Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (US) 2001. In one embodiment, a stable cell line may be prepared using the techniques in Sambrook, using wild-type (wt) or recombinant hTRPVl coding sequences. For example, preparation of one such cell line is described in detail herein (see Example 32). Preparation of another cell line is described in International Patent Publication N. WO 2007/0066068; the Lipofectamine® method may be employed for transfection of TRPV1 and hTRPVl into Human Embryonic Kidney cells (HEK293) according to the manufacturers protocol (Gibco). To create a permanently expressing cell line, wt-TRPVl transfected HEK cells can be subcloned in geneticin (0.6 mg/mL) containing medium (DMEM containing 10% FCS, 100 U/mL penicillin, 100 pg/mL streptomycin, and 250 ng/mL amphotericin B) and propagated for two weeks to allow selection. To obtain a TRPV1 permanently expressing single cell line, transfected cells can be plated in 96 well plates (1 cell per well) and colonies grown from single cells were subsequently tested for capsaicin responsiveness by measuring increases in intracellular calcium. The final clones selected, are taken through three further rounds of single cell cloning to ensure the cell lines are derived from a single cell. Variations on this methodology will be readily apparent to one of skill in the art. In another embodiment, cells may be selected from a stable cell line to express the hTRPVl, in trans, e.g., from a viral vector or another suitable genetic element.

[0136] The following examples are illustrative only and are not intended to limit the present invention.

[0137] EXAMPLES

[0138] Unless otherwise stated, all the raw materials are purchased from commercially available common suppliers. ’H-NMR spectra were recorded using trimethylsilane (TMS) as the internal reference for CDCI3 dissolved compounds. For DMSO-d(, and CD3OD dissolved compounds the instrument was calibrated at δ 2.5 and 3.3 ppm respectively. The chemical shift values are quoted in δ (parts per million).

[0139] For LCMS analysis LCMS/MS API 2000 (Applied Biosystem) instrument was used. The columns included:

Column U: YMC, 4.6 x 50 mm, 5μ

Column V: Zorbax® C18 column, 4.6 x 50 mm, 5μ

Column W: Zorbax® Extend C18 column, 4.6 x 50 mm, 5μ

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

Column X: Gemini® NX C18 column, 4.6 x 50 mm, 5μ

Column Y: Xbridge® C18 column, 4.6 x 50 mm, 5μ

Column Z: Reprosil® column, 4.6 x 50 mm, 5μ

The eluent (solvent) typically included (acidic or basic buffer as aqueous phase):

A channel: (i) 0.05% formic acid in water;

(ii) 10 mM ammonium acetate in water; or (iii) 0.05% TFA in water.

B channel: acetonitrile (organic phase).

The detector was UV measured at dual wavelengths: 220 and 260 nni.

[0140] The LCMS gradients were one of the following:

[0141] 1. LCMS reaction monitoring and final compound analysis method (for general polarity compounds)

Gradient condition: 5 min run time

Time Programs: Pl:10 mM ammonium acetate in water/acetonitrile

QI: 0.05%TFA in water/acetonitrile,

RI: 0.05% formic acid in water/acetonitrile.

'Ihc gradient varied acetonitrile from 10% to 90% to 10%.

Flow rate: 1.2 mL/min.

[0142] 2. LCMS reaction monitoring and final compound analysis method in 12 min run (for close eluting compounds):

Gradient condition: 12 min run time

Time Programs: P2: 10 mM ammonium acetate in water/acetonitrile

Q2:0.05%TFA in water/acetonitrile

R2: 0.05% formic acid in water/acetonitrile

The gradient varied acetonitrile from 5% to 90% to 5%

Flow rate: 1.0 mL/min.

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

[0143] 3. LCMS after method development in HPLC - gradient conditions are as per

HPLC.

Mass spectral data was obtained using the following:

Ionization technique: ESI (Electron Spray Ionization) using API (Atmospheric pressure Ionization) source

Declustering Potential: 10-70 V depending on the ionization of compound

Mass range: 100-800 amu

Scan type:QI

Polarity:+/-ve

Ion Source: Turbo spray

Ion spray voltage: +5500 for +ve mode and -4500 for -ve mode

Mass Source temperature:200°C

[0144] Preparation of Intermediates — few of the intermediates were used for the synthesis of the compounds which were synthesized by following sixteen (16) routes which are listed as examples below.

[0145] Preparation 1: l-Pyrazin-2-yl-ethylamine

Scheme 1

[0146] To a stirred solution of compound I (2 g; 16.4 mmol; 1 eq) in methanol (20 mL) were added ammonium acetate (12.6 g; 164 mmol; 10 eq) and sodium cyanoborohydride (1 g; 16.4 mmol; I eq) and the resulting mixture was stirred at 23 °C for 17 h. The mixture was quenched with water (50 mL) and the organic components were extracted with ethyl acetate (2 x 200 mL). The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered and the solvents were removed in vacuo to afford crude compound, which was purified by silica gel (230-400 mesh) column chromatography, eluting

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 with 10% MeOH/CH2Cl2, to afford the title compound (0.8 g, 40%). ’H NMR (CDCI3) δ 8.60 (s, III), 8.49 (m. Ill), 8.43 (d, III, J = 2 IIz), 4.20 (m, III), 1.45 (d, 311, J = 7 Ik).

[0147] Preparation 2: 4-Aminomethyl-l-methyl-lH-pyridin-2-one

I II HI

PCT/US2014/070497 organic layer was then dried over anhydrous Na2SÜ4 and evaporated to dryness to obtain a crude material. The crude product was purified by silica gel (100-200 mesh) column chromatography, eluting with 50% EtOAc/hexanes to obtain the title compound (3.5 g, 100%). *H NMR (DMSO-î/ô) δ 9.11 (s, 2H), 4.00 (s, 3H). LCMS: m/z = 136.0 [M+H]+, RT =

1.74 minutes, (Program Pl, Column V).

[0173] Step 2: N-((2-Methoxy-pyrimidin-5-yl)methyl)carbamic acid tert-butyl ester [0174] To a stirred solution of compound II (4.5 g; 33.3 mmol; 1 eq) in methanol (230 mL) were added nickel chloride hexahydrate (3.15 g; 13.3 mmol; 0.4 eq) and boc anhydride (14.4 g; 66.66 mmol; 2 eq) at 0 °C. To the mixture was then added powdered NaBHj (8.7 g; 233 mmol; 7 eq) in portions and stirred at 23 °C for 17 h. The mixture was evaporated to dryness, diluted with water and the organic components were extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and the solvents were removed in vacuo to obtain a dry residue which was purified by silica gel (230-400 mesh) column chromatography using 2-5% methanol/dichloromethane as the eluent to afford the title compound (3.8 g, 48%). 'H NMR (DMSO-dfi) δ 8.46 (s, 2H), 4.06 (d, 2H, J = 6 Hz), 3.88 (s, 3H), 1.36 (s, 9H). LCMS: m/z = 240.0 [M+H]+, RT = 2.67 minutes, (Program Pl, Column W).

[0175] Step 3: 2-Methoxypyrimidin-5-yl)methylamine hydrochloride

[0176] To a stirred solution of compound III (4 g; 28.98 mmol; 1 eq) in 1,4-dioxane (20 mL) was added 4M HC1 in 1,4-dioxane (30 mL) and the mixture was stirred at 23 °C for 4 h. The solvent was removed in vacuo to afford the title compound (2.8 g, 95%). *H NMR (DMSO-i/0) δ 8.73 (s, 2H), 4.03 (m, 2H), 3.89 (s, 3H). LCMS: m/z = 140.0 [M+H]+, RT = 0.59 minutes, (Program Pl, Column V).

[0177] Preparation 5: (2-Cyclopropylpyridin-5-yl)methylamine hydrochloride

Scheme 5

[0178] Step 1: 2-Cyclopropyl-5-cyanopyridine

[0179] To a stirred solution of compound I (0.5 g; 3.62 mmol; 1 eq) in toluene (20 mL) and water (1 mL) in a reaction tube, was added cyclopropyl boronic acid (0.46g; 5.43 mmol;

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

1.5 eq) and the resulting mixture was degassed with argon tor 5 min. To the mixture was then added K3PO4 (2.7 g; 12.7 mmol; 3.5 eq), Pd(OAc)2 (0.04 g; 0.18 mmol: 0.05 eq), tricyclohexyl phosphine (0.10 g; 0.36 mmol; 0.1 eq), and the resulting mixture was degassed with argon for another 5 min, then the reaction tube was sealed and heated at 110 °C for 3 h. The mixture was filtered through a Celite® pad and the filtrate was washed with saturated aqueous NaHCOs solution, followed by brine. The organic layer was then dried over anhydrous Na2SO4 and evaporated to diyness to obtain the crude material product. The crude materia] was purified by silica gel (100-200 mesh) column chromatography, eluting with 5-30% F.tOAc/hexanes to obtain the title compound (0.28 g, 56%). *H NMR (CDCI3) δ 8.67 (s, 1H), 7.75 (dd, 1H, ./ = 2,8 Hz), 7.23 (m, 1H), 2.06 (m, 1H), 1.09 (m, 4H).

[0180] Step 2: (2-Cyclopropylpyridin-5-yl)methylamine hydrochloride

[0181] To a stirred solution of compound II (1.5 g; 11.11 mmol; 1 eq) in methanol (80 mL) was added concentrated HC1 (1 mL) followed by 10% Pd/C (1.6 g) and stirred under a hydrogen atmosphere at a pressure of 50 psi for 4 h in a Pair autoclave. The mixture was filtered through a Celite® pad, washed with methanol, and solvent from the filtrate was removed in vacuo to afford the title compound (1.2 g, 78%). *H NMR (DMSO-ί/ή) δ 8.33 (s, 1H), 7.64 (dd, 1H, J = 2, 8 Hz), 7.27 (d, 1H, J= 8 Hz), 1.89 (m, 1H), 0.92 (m, 4H).

[0182] Preparation 6: l-(2-Methyl-pyrimidin-5-yl)-ethylamine

II III

Scheme 6

[0183] Step 1: 3,3-Dimethoxy-2-methoxycarbonyl-1-propen-l-ol sodium salt

[0184] To a stirred solution of compound I (50 g; 337 mmol; 1 eq) in 1,2-dimethoxy ethane (0.2 L) was added methyl formate (50 mL; 809 mmol; 2.4 eq) and the mixture was cooled to -5 °C. To the mixture was added sodium hydride (60% in mineral oil, 17.5 g; 438 mmol; 1.3 eq) in portions and the resulting mixture was stirred at 50 °C until effervescence had ceased. The mixture was then allowed to stir at 23 °C for 17 h. The mixture was filtered and the solid residue was collected, washed with ether and dried under reduced pressure to

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 afford the tide compound (63 g, 100%) as an off-white solid. 'H NMR (CD3OD) δ 8.88 (s, III), 8.54 (s, III), 5.30 (s. III), 3.51 (s, 311), 3.30 (s, 611).

[0185] Step 2: 2-Methylpyrimidine-5-carboxylic acid methyl ester

[0186] To a stirred solution of compound II (60 g; 340 mmol; 1 eq) in dry DMF (IL) was added acetamidine hydrochloride (38.6 g; 409 mmol; 1.2 eq) and the resulting mixture was heated at 100 °C for 3 h. The mixture was quenched with water (500 mL) and the organic components were extracted with ethyl acetate (2 x 200 mL). The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to obtain the title compound (27 g, 53%). JH NMR (CDCI3) δ 9.13 (s, 2H), 3.93 (s, 3H), 2.77 (s, 3H).

[0187] The following amine was synthesized from compound III following the procedure of preparation 3, steps 2-5.

[0202] Step 4: (S)-2-Methyl-l-(4H-[l,2,4]triazol-3-yl)-propylamine

PCT/US2014/070497

[0222] Preparation 11: 2-[4-(4,4,5,5-Tetramethyl-[l,3,2]dioxaborolan-2-yl)-pyrazol-lyl]-acetamide

HN-N

Scheme 11

[0223] To a solution of compound I (5 g, 25.7 mmol; 1 eq) in acetonitrile in a reaction lube (150 mL) was added bromo acetamide (5.68 g, 41.2 mmol; 1.6 eq) and cesium carbonate (33.5 g, 102 mmol; 4 eq), and the reaction tube was sealed and heated at 90 °C for 3 h. The mixture was cooled to room temperature and filtered through a Celite® pad and the filtrate was concentrated in vacuo to obtain the title compound (4.2 g, 65%), as a crude product which was directly used in next step without further purification. 'H NMR (400 MHz, DMSO-ί/ό) δ 7.67 (d, 1H, J = 2 Hz), 7.57 (m, 1H), 7.48 (brs, 1H), 7.42 (s, 1H), 4.76 (s, 2H), 0.89 (s, I2H)

[0224] Preparation 12: 5,5,5-Trifluoro-3-oxo-pentanoic acid methyl ester synthesis c F O F 1 î Î pkAd '

I II

Scheme 12

[0225] To a stirred solution of Meldrum's acid (5 g, 34.7 mmol; 1 eq) in dry CHjCh (5 mL) was added pyridine (3.07 mL; 38.2 mmol; 1.1 eq) at 0°C followed by 3,3,3-trifluoropropionyl chloride T (5.5 g; 38.2 mmol; 1.1 eq) and the resulting mixture was allowed to stir at 0 °C for 1 h. The temperature of the reaction was then increased to 23 °C and stirring was continued for another 2 h. The mixture was concentrated under reduced pressure to remove volatiles and the resulting paste-like material was dissolved in methanol and heated at 80°C for 5 h. The mixture was cooled to room temperature, concentrated under

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 reduced pressure and diluted with aqueous ammonium chloride solution. The organic components were extracted with EtOAc, washed with IN HC1 solution, and brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to dryness to obtain the title compound (3.1 g, 50%), as a crude product which was directly used in next step without purification. 'H NMR (DMSO-r/0) 8 3.87 (m, 2H), 3.70 (s, 3H), 3.64 (s, 3H).

[0226] Preparation 13: 4,4-Difluoro-3-oxo-pentanoic acid methyl ester

II Ila

Scheme 13

[0227] To a stirred solution of diisopropyl amine in dry THF (150 mL) was added n-butyllithium (2.3 M in hexane, 161 mL; 370.9 mmol; 2 eq) at -78°C and the resulting mixture was allowed to stir at 0 °C for 40 min. The mixture was cooled to -78 °C, ethyl acetate in dry THF was added drop wise and stirring was continued at -78°C for 1 h. To the mixture, compound I (23 g; 185.4 mmol; 1 eq) in dry THF was added slowly and the resulting mixture was stirred for another I h at -78°C. The mixture was allowed to stir at 23 °C for 16 h. The mixture was quenched with aqueous ammonium chloride solution (200 mL) and the organic components were extracted with EtOAc (2 x 700 mL). The combined organic layers were washed with IN HC1 solution, brine and dried over anhydrous sodium sulfate, then filtered. The filtrate was then concentrated under reduced pressure to obtain the title compound (23 g, 75%) as a crude material which was directly used for the next step without any purification. ’H NMR (DMSO-tk) δ 4.21 (m, 4H), 3.93 (s, 4H), 2.17 (s, 2H), 1.77 (t, 3H), 1.187 (m, 6H).

[0228] Preparation 14: Tributyl-(5-methyl-thiophen-2-yl)stannane

Scheme 14

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

[0229] To a stirred solution of 2-bromo-5-methyl thiophene I (6 g, 33.9 mmol, leq) in dry THF (60 mL) cooled to -78 °C, was added n-BuLi (14.7 mL, 2.3 M in THF, 1 eq) and the resulting mixture was stirred for 45 min at -78 °C. To the mixture was added tributyltin chloride (11g, 33.9 mmol, 1 eq) and the resulting mixture was allowed to stir at -78 °C for another 1 h. The mixture was then diluted with saturated aqueous ammonium chloride solution, and the organic components were extracted with CH2CI2 (2 x 50 mL). The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness to obtain crude tributyl-(5-methyl-thiophen-2yl)stannane (13.1 g) which was directly used in subsequent reactions without any purification. ]H NMR (CDC13) δ 6.95 (d, 1H, J = 3 Hz), 6.87 (d, 1H, J = 2 Hz), 2.53 (s, 3H), 1.54 (m, 6H), 1.30 (m, 6H), 1.06 (m, 6H), 0.88 (m, 9H).

[0230] Following the same protocol, the below compounds were also prepared.

NC

Preparation 15: Tributyl-(5-methyl-furan-2-yl)-stannane

[0231] .0.

Scheme 15

[0232]

To a stiired solution of 2-methylfuran I (3 g, 36.5 mmol, 1 eq) in dry THF (20 mL) cooled to -78 °C was added n-butyllithium (15.9 mL, 2.3 M in THF, 1 eq) and the resulting mixture was stirred for 1 h at 0°C. The mixture was then cooled to -78°C and tributyltin chloride was added (11.9 g, 36.5 mmol, 1 eq) and the resulting mixture was allowed to stir at 23°C for 18 h. The mixture was then diluted with saturated aqueous ammonium chloride solution, and the organic components were extracted with ethyl acetate

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 (2 x 50 mL). The combined organic layers were washed with water, brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness to obtain the title compound as a crude product (4.5 g) which was directly used for the next reaction without any purification 'H NMR (400 MHz, CDC13) Ô 6.43 (d, 1H, J = 3 Hz), 5.97 (d, 1H, J = 3 Hz),

2.32 (s, 3H), 1.70-1.40-1.28 (m, 6H), 1.32 (m, 12H), 0.91 (m, 9H).

[0233] Preparation 16: 8-(Cyclopropyl-propionyl-amino)-imidazo[l,2-a]pyridine-6carboxylic acid ethyl ester

PCT/US2014/070497 was stirred at 23 °C for 18 h. The mixture was poured into ice-cold saturated aqueous NaHCOs solution and the organic components were extracted with ethyl acetate (3 x 200 mL). The combined organic layers were then washed with brine solution, dried over anhydrous sodium sulfate, filtered and evaporated to dryness to afford the title compound (22 g, 100%) as an off white solid. 'H NMR (DMSO-ffc) δ 8.29 (d, 1H, J = 3 Hz), 8.12 (d, 1H, J = 2 Hz), 7.31 (s, 211), 4.29 (q, 211, J = 7 IIz), 1.30 (t, 311, J = 1 Ik). LCMS: m/z = 245.0 [M+], 247.0 [M+2], RT = 3.34 minutes, (Program Pl, Column W).

[0244] Step 3: 6-Bromo-imidazo[1.2-a]pyridine-8-carboxylic acid ethyl ester

[0245] To a stirred solution of compound III (22 g; 90 mmol; 1 eq) in ethanol (500 mL) was added sodium bicarbonate (13 g; 179 mmol; 2 eq) and chloroacetaldehyde solution (-50% aqueous solution, 60 mL; 449 mmol; 5 eq) drop wise and the mixture was heated at reflux for 8 h. The mixture was then evaporated to dryness and the pH was adjusted to 7 using ice-cold saturated aqueous NaHCOi solution and solid NaHCOs. The organic components were extracted from the aqueous layer with ethyl acetate (3 x 700 mL) and the combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and the solvents were removed in vacuo to obtain a dry residue which was purified by silica gel (230-400 mesh) column chromatography using 10-50% ethyl acetate/hexanes to afford the title compound (14.5 g, 60%) as a brown solid. 'H NMR (DMSO-Jô) δ 9.16 (s, 1H), 8.02 (s, 1H), 7.84 (s. 1H), 7.70 (s, 1H), 4.38 (q, 2H, J = 7 Hz), 1.30 (t, 3H, J = 7 Hz). LCMS: m/z = 268.8 [M+], 270.8 [M+2], RT = 2.77 minutes, (Program Pl, Column Y).

[0246] Step 4: 6-Bromo-imidazo[l,2-a]pyridine-8-carboxylic acid hydrazide

[0247] To a stirred solution of compound IV (5 g; 18.6 mmol; 1 eq) in ethanol (50 mL) was added hydrazine hydrate (9.3 g; 186 mmol; 10 eq) and the resulting mixture was heated at reflux for 5 h. The mixture was cooled, filtered and the solid residue was washed with hexane and dried under reduced pressure to obtain the title compound (4.5 g, 95%) as white solid. ’H NMR (DMSO-</6) δ 10.91 (s, 1H), 9.13 (d, 1H, J= 2 Hz), 8.07 (s, 1H), 7.96 (d, 1H, J = 2 Hz), 7.74 (s, 1H) 4.86 (s, 2H).

[0248] Step 5: 6-Bromo-imidazo [1,2-a]pyridine-8-carboxylic acid N'-propionylhydrazidc

[0249] To a stirred solution of compound V (4.5 g; 17.6 mmol; 1 eq) in dry CH2CI2 (50 mL) at 0 °C were added TEA (7.6 mL; 52.9 mmol; 3 eq) and propionyl chloride dropwise and the resulting mixture was allowed to stir at 23 °C for 2 h. The mixture was poured into

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 saturated ice-cold aqueous NaHCOj solution and the organic components were extracted with ethyl acetate (3 x 250 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and the solvents were removed in vacuo to obtain a dry residue which was purified by silica gel (230-400 mesh) column chromatography using 0-5% methanol/dichloromethane as eluent to afford the title compound (1.2 g, 23%) as an off white solid. ’H NMR (DMSO-d6) Ô 11.97 (d, 1H, J = 4 Hz), 10.73 (d, 1H, J = 4 Hz), 9.19 (d, 1H, J = 2 Hz), 8.10 (s, 1H), 8.01 (d, 1H, J = 2 Hz), 7.77 (s, 1H), 2.27 (q, 2H, J = 8 Hz), 1.07 (t, 3H, J = 8 Hz). LCMS: m/z = 311.0 [M+], 313.0 [M+2], RT = 2.29 minutes, (Program Pl, Column W).

[0250] Step 6: 6-Bromo-8-(5-ethyl-[l,3,4]oxadiazol-2-yl)-imidazo[l,2-a]pyridine

[0251] Λ stirred solution of compound VI (1.2 g; 3.8 mmol; 1 eq) in dry POOa (20 mL) was heated at reflux for 2 h. The mixture was evaporated to dryness and diluted with saturated aqueous NaHCOj solution. The organic components were extracted with ethyl acetate (3 x 100 mL) and the combined extracts were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to obtain a dry residue. The crude material was purified by silica gel (230-400 mesh) column chromatography using 0-5% methanol/dichloromethane as the eluent to obtain the title compound (0.6 g, 53%) as a light brown solid. ]H NMR (DMSO-</6) δ 9.18 (s, 1H), 8.09 (s, 1H), 7.99 (s, 1H), 7.76 (s, 1H), 3.32 (s, 3H), 3.02 (q, 2H), 1.36 (t, 3H). LCMS: m/z = 293.0 [M+], 295.0 [M+2], RT = 2.46 minutes, (Program Pl, Column W).

[0252] Step 7: 8-(5-Ethyl-[l,3,4]oxadiazol-2-yl)-imidazo[l,2-a]pyridine-6-carboxylic acid methyl ester

[0253] To a solution of compound VII (0.6 g; 2.1 mmol; 1 eq) in methanol (20 mL) was added diisopropylethyl amine (1.05 mL; 6.0 mmol; 3 eq), and the resulting solution was degassed with argon for 5 min, followed by an addition of PdChfdppf) (0.16 g; 0.21 mmol; 0.1 eq). The mixture was then degassed again with argon for 5 min, and healed to 90 °C for 16 h in an autoclave at a pressure of 50 psi under carbon monoxide atmosphere. The mixture was filtered through a Celite® pad and the filtrate was washed with saturated aqueous NaHCOj solution, followed by brine. The organic layer was dried over anhydrous NajSC^ and finally evaporated to dryness to obtain a crude material. The crude product was purified by silica gel (100-200 mesh) column chromatography, eluting with 0-10%

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 methanol/dichloromethane as the eluent to obtain the title compound (0.3 g, 55%) as a light brown solid. LCMS: m/z = 272.8 [M+H]+, RT = 2.46 minutes, (Program Pl, Column Y).

[0254] Step 8: 3-Bromo-8-(5-ethyl-[l,3,4]oxadiazol-2-yl)-imidazo[l,2-a]pyridine-6carboxylic acid methyl ester

[0255] To a stirred solution of compound VIII (0.32 g; 1.17 mmol; 1 eq) in dry THF (15 mL) was added NBS (0.12 g; 0.71 mmol; 0.6 eq) in portions at 0 °C and the mixture was stirred for 30 min. The mixture was poured into ice-cold saturated aqueous NaHCOj solution and the organic components were extracted with ethyl acetate (3 x 50 mL). The organic layer was then washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated to dryness to obtain a crude material. The crude product was purified by silica gel (100-200 mesh) column chromatography, eluting with 0-5% methanol/dichloromethane to obtain the title compound (0.21 g, 51%) as a brown solid. 'H NMR (DMSO-</6) δ 8.94 (s, 1H), 8.36 (s, 1H), 8.04 (s, 1H), 3.97 (s, 3H), 3.03 (q, 2H, J = 8 Hz), 1.36 (t, 3H). LCMS: m/z = 350.8 [M+], 352.8 [M+2], RT = 2.70 minutes, (Program Pl, Column Y).

[0256] Step 9: 8-(5-Ethyl-[l,3,4]oxadiazol-2-yl)-3-(5-methyl-thiophen-2-yl)imidazo[l,2-a]pyridine-6-carboxylic acid methyl ester

[0257] To a stirred solution of compound IX (0.21 g; 0.6 mmol; 1 eq) in dry toluene in a reaction tube was added compound Via (0.27g; 0.72 mmol; 1.2 eq) and the resulting mixture was degassed with argon for 5 min. To the mixture was then added Pd(PPhO4 (0.06g; 0.06 mmol; 0.1 eq) and the mixture was degassed further with argon for 5 min. The reaction tube was sealed and healed al 115 °C with sliiring for 4 h. The mixture was filleted through a Celite® ad and the filtrate was washed with saturated aqueous NallCOj solution, followed by brine. The organic layer was dried over anhydrous Na2SO4 and the solvent was evaporated to diyness to obtain a crude material. The crude product was purified by silica gel (230-400 mesh) column chromatography eluting with 0-5% methanol/dichloromethane to obtain the title compound (0.22 g, 100%) which was used for the next step without further purification. LCMS: m/z = 368.8 [M+H]+, RT = 3.07 minutes, (Program Pl, Column Y).

[0258] Step 10: 8-(5-Ethyl-[l,3,4]oxadiazol-2-yl)-3-(5-methyl-thiophen-2-yl)imidazo[l,2-a]pyridine-6-carboxylic acid

[0259] To a solution of compound X (0.26 g, 0.7 mmol, 1 eq) in TIIF:methanol:II2O (12 mL, 3:2:1) was added an aqueous solution (1 mL) of lithium hydroxide monohydrate (0.09 g, 2.12 mmol, 3 eq) at 0 °C and the resulting mixture was stirred at 23 °C for 3 h. The solvent of

WO 2015/095128

PCT/US2014/070497 by silica gel (230-400 mesh) column chromatography using 10-50% ethyl acetate/hexanes as the eluent to afford the title compound (3.46 g, 100%) as a solid. ’H NMR (DMSO-dô) δ 8.79 (s, 1H), 7.88 (s, 1H), 7.68 (s, 1H), 3.93 (s, 3H), 1.34 (s, 18H). LCMS: m/z = 469.8 [M+], 471.8 [M+2], RT = 3.54 minutes, (Program Pl, Column Y).

PCT/US2014/070497 ]H NMR (DMSO-î/6) δ 9.65 (s, 1H), 8.33 (s, 1H), 8.10 (s, 1H), 7.74 (s, 1H), 7.46 (m, 1H), 4.43 (q, J = 7 Πζ, 211), 3.96 (s, 311), 1.36 (t, J = 7 Hz, 311). LCMS: m/z = 365.8 [M+II]+, RT = 2.97 minutes. (Program Pl, Column Y).

[0302] Step 5: 3-Bromo-8-(5-difluoromethyl-4-ethoxycarbonyl-[l,2,3]triazol-l-yl)imidazo[l,2-a]pyridine-6-carboxylic acid methyl ester

[0303] To a stirred solution of compound V (10 g; 27.4 mmol; 1 eq) in dry THF (150 mL) at 0 °C was added NBS (7.4 g; 41.1 mmol; 1.5 eq) in portions and the resulting mixture was stirred at 0 °C for 30 min. The mixture was poured into ice-cold saturated aqueous NaHCOj solution and the organic components were extracted with ethyl acetate (3 x 500 mL). The organic layer was then washed with brine, dried over anhydrous sodium sulfate and the solvents were removed in vacuo to obtain a dry residue which was chromatographically purified by silica gel (230-400 mesh) gravity column using 10-50% ethyl acetate/hexanes as the eluent to afford the title compound (10 g, 82%) as a solid. *H NMR (DMSO-î/6) δ 9.00 (s, 1H), 8.27 (s, 1H), 7.96 (s, 1H), 7.44 (m, 1H), 4.43 (q, 2H, J = 5 Hz), 3.96 (s, 3H), 1.35 (t, 3H, ./ = 7 Hz). LCMS: m/z = 443.8 [Μ+], 445.8 [Μ+2], RT = 3.20 minutes, (Program PI, Column Y).

[0304] Step 6: 3-Bromo-8-(4-carboxy-5-difluoromethyl-[l,2,3]triazol-l-yl)-imidazo[l,2a]pyridine-6-carboxylic acid

[0305] To a solution of compound VI (7 g, 15.8 mmol, 1 eq) in THF: methanol :Η2θ (115 mL, 3:2:1) at 0 °C was added a solution of lithium hydroxide monohydrate (2 g, 47.3 mmol, 3 eq) in water (5 mL) and the resulting mixture was stirred at 23 °C for 3 h. The solvent was removed under reduced pressure and the residue was diluted with water and acidified with IN HC1 to adjust the pH to 3. The precipitated solid was collected by filtration and dried under vacuum to afford the title compound (5 g, 80 %). ’ll NMR (DMS0-</6) δ 14.01 (s, 211), 8.97 (s, 1H), 8.19 (s, 1H), 7.93 (s, 1H), 7.46 (m, 1H). LCMS: m/z = 402.0 [M+J, 403.8 [M+2J, RT = 0.84 minutes, (Program Pl, Column Y).

[0306] Step 7: 3-Bromo-8-(5-difluoromethyl-[l,2,3]triazol-l-yl)-imidazo[l,2-a]pyridine6-carboxylic acid

[0307] Compound Vil (5 g; 12.4 mmol; 1 cq) was heated at 200 °C for 1 h to obtain the title compound (3.5 g; 80%). 'H NMR (DMSO-d6) Ô 14.01 (s, 1H), 8.96 (s, 1H), 8.43 (s, 1H), 8.15 (s, 1H), 7.94 (s, 1H), 7.38 (m, 1H). LCMS: m/z = 357.8 [M+], 359.8 [M+2], RT = 1.71 minutes, (Program Pl, Column Y).

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

[0308] Step 8: 3-Bromo-8-(5-difluoromethyl-[l,2,3]triazol-l-yl)-imidazo[l,2-a]pyridine6-carboxylic acid [(S)-l-(4H-[l,2,4]triazol-3-yl)-ethyl]-amide

[0309] To a stirred solution of compound VIII (2 g, 5.6 mmol, 1 eq) in DMF (20 mL) were added DIPEA (3 mL, 16.8 mmol, 3 eq) and HATU (3.2 g, 8.40 mmol, 1.5 eq) at 0 °C and the resulting mixture was stirred for 15 min. To the mixture was added (S)-1-(4H[l,2,4]triazol-3-yl)-ethylamine hydrochloride (0.94 g, 8.40 mmol, 1.2 eq) and the resulting mixture was allowed to stir at 23 °C for another 16 h. From the mixture, solvent was removed in vacuo and the residue was diluted with EtOAc. The organic layer was washed with saturated aqueous sodium bicarbonate solution, aqueous ammonium chloride solution and brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The crude product was purified by silica gel (230-400 mesh) column chromatography eluting with 0-5% methanol/CHjCh to obtain the title compound (1 g, 49%) as brown solid. ]H NMR (DMSO-<4) δ 13.94 (d, IH), 9.48 (dd, IH, J = 8 Hz), 9.36 (d, 1H), 8.45 (d, 2H), 8.29 (d, IH, ./= 12 Hz), 7.93 (m, IH), 7.52 (m, IH), 5.35 (q, IH, ./ = 8 Hz), 1.55 (d, 3H, J = 7 Hz). LCMS: m/z = 452.2 [M+], 454.2 [M+2], RT = 2.40 minutes, (Program Pl, Column Y).

[0310] Step 9: (S)-N-(l-(4H-l,2,4-Triazol-3-yl)ethyl)-8-(5-(difluoromethyl)-lH-l,2,3triazol-l-yl)-3-(5-methylthiophen-2-yl)imidazo[l,2-a]pyridine-6-carboxamide

[0311] To a stirred, dry DMF solution of compound IX (0.7 g; 1.55 mmol; 1 eq) in a reaction tube was added compound Via (0.9g; 2.32 mmol; 1.5 eq) and the resulting mixture was degassed with argon for 5 min. To the mixture was added Pd(PPh3)4 (0.2 g; 0.15 mmol; 0.1 eq) and degassing with argon was repeated for about 5 min. The reaction tube was sealed and heated at 115 °C for 4 h. The mixture was filtered through a Celite® pad and the filtrate was washed with saturated aqueous NaHCOj solution, followed by brine. The organic layer was then dried over anhydrous Na2SO4, filtered and solvent was removed in vacuo to obtain a crude material. The crude product was purified by silica gel (230-400 mesh) column chromatography eluting with 0-5% inethanol/CH^Cb to obtain the title compound (0.29 g, 40%) as light brown solid. *H NMR (DMSO-^) δ 13.56 (s, IH), 9.20 (s, IH), 9.00 (d, IH), 8.32 (s, IH), 8.23 (s, IH), 7.85 (s, IH), 7.41 (m, 3H), 7.03 (s, IH), 5.37 (q, IH), 2.50 (s, 3H), 1.60 (s, 3H). LCMS: m/z = 470.2 [M+H]+, RT = 2.87 minutes, (Program Pl, Column Y).

[0312] EXAMPLE 5: 8-(5-(Difluoromethyl)-lH-l,2,3-triazol-l-yl)-3-(5-methyl-l,3,4thiadiazol-2-yl)-N-((6-methylpyridin-3-yl)methyl)imidazo[l,2-a]pyridine-6-carboxamide

CA 02933811 2016-06-13

[0324] To a stirred solution of compound III (3 g; 15.6 mmol; 1 eq) in dry THF (200 mL) at 0 °C were added TEA (6.52 mL; 46 mmol; 3 eq), boc anhydride (4.32 mL, 18.8 mmol; 1.2 eq) dropwise and a catalytic amount of DMAP, and the resulting mixture was heated al reflux for 17 h. The mixture was diluted with water (300 mL) and the organic components were extracted with ethyl acetate (3 x 200 mL). The combined organic layers were washed with

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 brine, dried over anhydrous sodium sulfate, filtered and the solvents were removed in vacuo to obtain a dry residue which was purified by silica gel (230-400 mesh) column chromatography using 10-50% ethyl acetate/hexanes as the eluent to afford the title compound (2.9 g, 49%) as solid. *H NMR (DMSO-i/6) Ô 9.36 (s, 1H), 8.15 (s, 1H), 7.67 (s, 1H), 7.50 (s, 1H), 3.89 (s, 3H), 1.33 (s, 18H). LCMS: m/z = 391.8 [M+H] +, RT = 3.18 minutes, (Program Pl, Column Y).

[0325] Step 4: 3-Bromo-8-[to-(tert-Butoxycarbonyl)amino]-imidazo[l,2-a]pyridine-6carboxylic acid methyl ester

[0326] To a stirred solution of compound IV (2.88 g; 7.3 mmol; 1 eq) in dry THF (80 mL) was added NBS (1.31 g; 7.3 mmol; 1 eq) in portions at 0 °C and the resulting mixture was stirred for 30 min at 0 °C. The temperature of the mixture was then slowly raised to 23 °C and the mixture was stirred for 2 h. The mixture was poured into ice-cold saturated aqueous NaHCO3 solution and the organic components were extracted with ethyl acetate (3 x 50 mL). The combined organic layers were then washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo to obtain a dry residue which was purified by silica gel (230-400 mesh) column chromatography using 10-50% ethyl acetate/hexanes as the eluent to afford the title compound (3.46 g, 100%) as a solid. JH NMR (DMSO-Jg) δ 8.79 (s, 1H), 7.88 (s, 1H), 7.68 (s, 1H), 3.93 (s, 3H), 1.34 (s, 18H). LCMS: m/z = 469.8 [M+], 471.8 [M+2], RT = 3.54 minutes, (Program Pl, Column Y).

[0327] Step 5: 8-Amino-3-bromo-imidazo[l,2-a]pyridine-6-carboxylic acid methyl ester [0328] To a stirring solution of compound V (3.5 g; 7.4 mmol; 1 eq) in methanol (50 mL) at 0 °C was added 4M methanolic HC1 (100 mL) dropwise over a period of 30 min and the resulting solution was stirred at 23 °C for 24 h. The mixture was poured into ice-cold saturated aqueous NaHCO3 solution and the organic components were extracted with ethyl acetate (3 x 500 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo to obtain the title compound (2 g, 100%) as a solid. LCMS: m/z = 269.8 [M+], 271.8 [M+2], RT = 2.91 minutes, (Program Pl, Column Y).

[0329] Step 6: 3-Bromo-8-hydrazino-imidazo[l,2-a]pyridine-6-carboxylic acid methyl ester

[0330] To a stirred, cooled (-10°C) solution of compound VI (0.7 g; 2.5 mmol; 1 eq) in concentrated HC1 (7 mL) was added an aqueous solution of sodium nitrite (0.35 g; 5.1 mmol;

CA 02933811 2016-06-13

WO 2015/095128

[0348] Step 4: 3-Bromo-8-iodo-imidazo[l,2-a]pyridine-6-carboxylic acid ethyl ester [0349] To a stirred solution of compound IV (30 g; 95 mmol; 1 eq) in dry THF (200 mL) at 0 °C was added NBS (16.8 g: 94.9 mmol; 1 eq) in portions and the resulting mixture was stirred at 23 °C for 1 h. The mixture was poured inlo ice-cold saturated NaHCOj aqueous solution and the organic components were extracted with ethyl acetate (3 x 500 mL). The combined organic layers then washed with brine, dried over anhydrous sodium sulfate, filtered and the solvents were removed in vacuo to obtain a dry residue. The crude material was purified by silica gel (230-400 mesh) column chromatography using 10-50% ethyl acetate/hexanes as the eluent to afford the compound (38 g, 100%) as a brown solid, ’ll NMR (DMSO-dé) δ 8.75 (s, 1H), 8.16 (s, 1H), 7.93 (s, 1H), 4.39 (q, 2H, J = 7 Hz), 1.36 (t, 3H, J =

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

Hz). LCMS: m/z = 395.0 [M+], 397.0 [M+2], RT = 3.35 minutes, (Program PI, Column Y).

[0350] Step 5: 3-Bromo-8-phenylamino-imidazo[l,2-a]pyridine-6-carboxylic acid ethyl ester

[0351] To a stirred 1,4-dioxane solution of compound V (1 g; 2.55 mmol; 1 eq) in a reaction tube were added aniline (280 mg; 3.03 mmol; 1.2 eq) and the resulting mixture was degassed with argon for 5 min, followed by an addition of K2CO3 (1.05 g; 7.59 mmol; 3 eq), Pd(OAc>2 (57 mg; 0.25 mmol; 0.1 eq), xantphos (0.46g; 0.51 mmol; 0.2 eq). The mixture was then degassed again with argon for 5 min, the reaction tube was sealed and heated at 110 UC for 5 h. The mixture was filtered through a Celite® pad and the filtrate was washed with saturated aqueous NaHCO? solution, followed by brine. The organic layer was then dried over anhydrous Na2SÛ4 and evaporated to dryness to obtain a crude material. The crude product was purified by silica gel (100-200 mesh) column chromatography, eluting with 5-30% EtOAc/hexanes to obtain the title compound (1.5 g, 82%). LCMS: m/z = 360.2 [M+], 362.0 [M+2], RT = 3.84 minutes, (Program Pl, Column W).

[0352] Step 6: 3-(5-Methyl-thiophen-2-yl)-8-phenylamino-imidazo[l,2-a]pyridine-6carboxylic acid ethyl ester

[0353] To a stirred dry toluene solution of compound VI (0.75 g; 2.1 mmol; 1 eq) in a reaction tube was added compound Via (0.96 g; 2.5 mmol; 1.2 eq) and the resulting mixture was degassed with argon for 5 min. To the mixture was added Pd(PPh3)4 (0.24 g; 0.21 mmol; 0.1 eq) and the resulting mixture was degassed with argon for an additional 5 min. The reaction tube was sealed and heated at 115 °C for 4 h. The mixture was filtered through a Celite® pad and the filtrate was washed with saturated aqueous NaHCO3 solution, followed by brine, and the organic layer was dried over anhydrous Na2SO4, filtered and evaporated to dryness to obtain a crude material. The crude product was purified by silica gel (230-400 mesh) column chromatography eluting with 50% EtOAc/hexanes to obtain the title compound (0.48 g, 61%) as a light brown solid. ]H NMR (CDC13) δ 8.63 (s, 1H), 7.63 (s, 1H), 7.42 (m, 1H), 7.38 (d, 2H, J = 7 Hz), 7.34 (d, 2H, J = 8 Hz), 7.20 (m, 1H), 6.86 (tn, 1H), 6.68 (d, 2H, J = 8 Hz),4.38 (q, 2H, J = 7 Hz), 2.57 (s, 3H) 1.37 (t, 3H, J = 7 Hz). LCMS: m/z = 378.2 [M+II]+, RT - 4.20 minutes, (Program Pl, Column W).

[0354] Step 7: 3-(5-Methyl-thiophen-2-yl)-8-phenylamino-imidazo[l,2-a]pyridine-6carboxylic acid

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

[0367] Step 5: 3-Bromo-8-(l-methyl-lH-pyrazol-4-y l)-imidazo[l,2-a]pyridine-6carboxylic acid ethyl ester

[0368] To a stirred solution of compound V (0.8 g; 2.90 mmol; 1 eq) in dry THF (20 mL) at 0 °C was added NBS (520 mg; 2.9 mmol; 1 eq) in portions and the resulting mixture was stirred at 0 °C for 30 min. The mixture was poured into ice-cold saturated aqueous NaHCO3 solution and the organic components were extracted with ethyl acetate (3 x 50 mL). The combined organic layers were then washed with brine, dried over anhydrous sodium sulfate and evaporated to dryness to afford the title compound (0.5 g, 50%) as an off white solid.

NMR (DMSCMi) Ô 8.75 (s, 1H), 8.64 (s, 1H), 8.36 (s, 1H), 7.97 (s, 1H), 7.93 (s, 1H), 4.40 (q, 2H, J = 7 Hz), 3.94 (s, 3H), 1.38 (t, 3H, J = 7 Hz). LCMS: m/z = 349.0 [M+], 351.0 [M+2], RT = 3.28 minutes, (Program Pl, Column Y).

[0369] Step 6: 8-(l-Methyl-lH-pyrazol-4-yl)-3-(5-methyl-thiophen-2-yl)-imidazo[l,2a]pyridine-6-caiboxylic acid ethyl ester

[0370] To a stirred solution of compound VI (0.5 g: 1.49 mmol; 1 eq) in dry toluene in a reaction tube was added compound Via (0.63 g; 1.64 mmol; 1.1 eq) and the resulting mixture was degassed with argon for 5 min. To the mixture was added Pd(PPh3)4 (0.17 g; 0.14 mmol; 0.1 eq) and the resulting mixture was degassed with argon again for about 5 min. The reaction tube was then sealed and heated at 115 °C for 4 h. The mixture was filtered through a Celite® pad and the filtrate was washed with saturated aqueous NaHCO3 solution, followed by brine. The organic layer was then dried over anhydrous Na2SO4, filtered and solvents were removed in vacuo to obtain a crude material. The crude product was purified by silica gel (230-400 mesh) column chromatography eluting with 50% EtOAc/hexanes to obtain the title compound (0.32g, 63%). JH NMR (DMSO-d6) δ 8.87 (s, 1H), 8.78 (s, 1H), 8.37 (s, 1H), 7.94 (s, 1H), 7.90 (s, 1H), 7.34 (d, 1H, J = 4 Hz), 7.02 (d, 1H, J = 3 Hz), 4.38 (q, 2H, J = 7 Hz), 3.95 (s, 3H), 2.56 (s, 3H), 1.35 (t, 3H, J = 7 Hz). LCMS: m/z = 367.0 [M+H] +, RT = 3.58 minutes, (Program Pl, Column Y).

[0371] Step 7: 8-(1 -Methyl-lH-pyrazol-4-yl)-3-(5-methyl-thiophen-2-yl)-imidazo[ 1,2a]pyridine-6-carboxylic acid

[0372] To a solution of compound VII (0.25 g, 0.68 mmol, 1 eq) in THF:methanol:H2O (12 mL, 3:2:1) at 0 °C was added a solution of lithium hydroxide monohydrate (57 mg, 1.30

WO 2015/095128

PCT/US2014/070497 (m, 5H), 0.9 (t, 3H, J = 4 Hz). LCMS: m/z = 288 [M+H]+, RT = 3.56 minutes, (Program Pl, Column W).

[0384] Step 5: 3-((E)-But-l-enyl)-8-(4-methyl-piperidin-l-yl)-imidazo[l,2-a]pyridine-6carboxylic acid ethyl ester

PCT/US2014/070497

1.56 (m, 1H), 1.3 (m, 5H), 0.9 (t, 3H, J = 4 Hz). LCMS: m/z = 288 [M+H] +, RT = 3.56 minutes, (Program Pl, Column W).

[0401] Step 5: 3-Bromo-8-(4-methyl-piperidin-l-yl)-imidazo[l,2-a]pyridine-6carboxylic acid ethyl ester

PCT/US2014/070497 (m, 5H), 0.9 (t, 3H, J = 4 Hz). LCMS: m/z = 288 [M+H]+, RT = 3.56 minutes, (Program Pl, Column W).

[0418] Step 5: 3-Bromo-8-(4-methyl-piperidin-l-yl)-imidazo[l,2-a]pyridine-6carboxylic acid ethyl ester

[0419] To a stirred solution of compound (1.6 g; 5.5 mmol; 1 eq) in dry THF (30 mL) at 0 °C was added NBS (0.99 g; 5.5 mmol; 1 eq) in portions, and the resulting mixture was stirred at 0 °C for 30 min. The mixture was poured into ice-cold saturated aqueous NaHCOj solution and the organic components were extracted with ethyl acetate (3 x 500 mL). The combined organic layers were then washed with brine solution, dried over anhydrous sodium sulfate, filtered and evaporated to dryness to afford the title compound (1.7 g, 83%) as an off white solid. 'H NMR (DMSO-J6) Ô 8.34 (s, IH), 7.75 (s, IH), 6.87 (s, IH), 4.35 (m, 4H), 2.79 (q, 2H, J = 12 Hz), 1.73 (d, 2H, J = 12 Hz), 1.58 (s, IH), 1.3 (m, 5H), 0.95 (t, 3H, J = 4 Hz). LCMS: m/z = 366.2 [M+], 368.2 [M+2], RT = 2.51 minutes, (Program Pl, Column W).

WO 2015/095128

PCT/US2014/070497

[0444] Step A: 3-(5-Methyl-thiophen-2-yl)-8-piperazin-l-yl-imidazo[l,2-a]pyridine-6carboxylic acid [(S)-l-(lH-[l,2,4]triazol-3-yl)-ethyl]-amide

[0445] To the solution of IXb (240 mg, 0.44 mmol, 1 eq) in CH2CI2 (8 mL) was added TFA (0.33 mL, 4.4 mmol, 10 eq) and the resulting mixture was stirred at 23 °C for 16 h. The mixture was concentrated in vacuo to remove TFA and diluted with water. Aqueous NaHCO3 was added and the organic components were extracted with 5% MeOH/CfLCh- The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain the title compound (140 mg, 72%) as an off white solid. 'H NMR (DMSO-rfé) Ô 9.24 (t, 1H, J = 5 Hz), 8.60 (s, 1H), 8.42 (s, 1H), 7.69 (s, 1H), 7.62-7.60 (m, 1H), 7.30 (d, 1H, J = 3 Hz), 7.21-7.20 (m, 1H), 6.99-6.97 (m, 2H), 4.45 (d, 2H, .7 = 5 Hz), 3.57 (s, 4H), 3.02 (s, 4H), 2.53 (s, 3H), 2.43 (s. 3H). LCMS: m/z = 447.3 [M+H] +, RT = 2.54 minutes, (Program Pl, Column V).

[0446] Step B: 8-(4-Caibamoylpiperazin-l-yl)-N-((6-methylpyridin-3-yl)methyl)-3-(5methylthiophen-2-yl)imidazo[ 1,2-a]pyridine-6-carboxamide

[0447] To the solution of Xb (80 mg, 0.18 mmol, 1 eq) in THF (5 mL) was added TMSisocyanate (0.05 ml, 0.36 mmol, 2 eq) at 23 °C and the resulting mixture was stirred for 4 h. The mixture was then diluted with aqueous NaHCO3 and the organic components were extracted with 15% MeOH/CH2Cl2, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by flash Combiflash™ chromatography using 100-200 mesh silica gel eluting with 17% MeOH/CH2C12 to obtain the title compound (14 mg, 16 %) as an off white solid. *H NMR (DMSO-i/5) δ 9.20 (t, 1H, J = 6 Hz), 8.60 (s, 1H), 8.42 (s, 1H), 7.70 (s, 1H), 7.62-7.60 (m, 1H), 7.30 (d, 1H, J = 3 Hz), 7.227.20 (m, 1H), 6.98 (m, 2H), 4.46 (d, 2H, .7 = 6 Hz), 3.51 (s, 8H), 2.53 (s, 3H), 2.43 (s, 3H). LCMS: m/z = 490.2 [M+H]+, RT = 2.65 minutes, (Program Pl, Column W).

[0448] EXAMPLE 14: (S)-N-(l-(l,3,4-Oxadiazol-2-yl)ethyl)-8-(5-(difluoromethyl)-lH1,2,3-lriazol-1 -yl)-3-(5-melhylthiophen-2-yl)imidazo[ 1,2-a]pyridine-6-carboxamide

101

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

[0463] Step 8: 8-(5-Difluoromethyl-[l,2,3]triazol-l-yl)-3-(5-methyl-thiophen-2-yl)imidazof l,2-a]pyridine-6-carboxylic acid

PCT/US2014/070497

NMR (DMSO-Jÿ) δ 8.49 (s, 1H), 8.07 (s, 1H), 7.15 (s, 2H), 4.22 (q, 2H, J = 8 Hz), 1.30 (t, 311, J = 8 Hz). LCMS: m/z = 245 [M+], 247 [M+2], RT = 2.97 minutes, (Program PI, Column W).

[0476] Step 3: 8-Bromo-imidazo[ 1,2-a]pyridine-6-carboxylic acid ethyl ester

[0477] To a stirred solution of compound III (80 g; 326 mmol; 1 eq) in ethanol (1 L) were added sodium bicarbonate (54.8 g; 652 mmol; 2 eq) and aqueous chloroacetaldehyde solution (-50% aqueous solution, 212 mL; 1.63 mol; 5 eq) dropwise and the mixture was heated at reflux for 17 h. The mixture was then evaporated to dryness and the pH was adjusted to 7 using ice-cold saturated aqueous NaHCO3 solution and solid NaHCO3. The organic components were extracted from the aqueous phase with ethyl acetate (3 x 1000 mL) and the combined organic layers were washed with brine, dried over anhydrous sodium sulfate. The solvent from the organic layer was removed in vacuo to obtain a dry residue which was purified by silica gel (230-400 mesh) column chromatography using 10-50% ethyl acetate/hexanes as the eluent to afford the title compound (55 g, 63%) as a brown solid. *H NMR (DMSO-d6) δ 9.33 (s, 1H), 8.24 (s, 1H), 7.84 (s, 1H), 7.73 (s, 1H), 4.34 (q, 2H, J = 8 Hz), 1.34 (t, 3H, J = 8 Hz). LCMS: m/z = 268.8 [M+], 270.8 [M+2], RT = 2.90 minutes, (Program Pl, Column W).

[0478] Step 4: 8-(4-Methyl-piperidin-l-yl)-imidazo[l,2-a]pyridine-6-carboxylic acid ethyl ester (0479] To a stirred 1,4-dioxane solution of compound IV (1 g; 3.71 mmol; 1 eq) in a reaction tube was added 4-methyl piperidine (0.73g; 7.43 mmol; 2 eq) and the resulting mixture was degassed with argon for 5 min, followed by an addition of Cs2CO3 (1.81 g; 5.56 mmol; 1.5 eq), Pd2(dba)3 (0.16g; 0.018 mmol; 0.05 eq) and xantphos (0.21g; 0.37 mmol; 0.1 eq). The mixture was then degassed again with argon for 5 min, and the reaction tube was sealed and healed at 115 °C for 5 h. The mixture was filtered through a Celite® pad and the filtrate was washed with saturated aqueous NaHCO3 solution, followed by brine. The organic layer was then collected and dried over anhydrous Na2SO4, filtered and evaporated to dryness to obtain a crude material. The crude product was purified by silica gel (100-200 mesh) column chromatography, eluting with 50% EtOAc/ hexanes to obtain the title compound (0.25 g, 23%) as a light brown solid. *H NMR (DMSO-r/6) δ 8.84 (s, 1H), 8.03 (s, 1H), 7.55 (s, 1H), 6.75 (s, 1H), 4.31 (m, 4H), 2.73 (q, 2H, J = 8 Hz), 1.73 (d, 2H, J = 12 Hz), 1.56 (m,

108

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

1H), 1.3 (m, 5H), 0.9 (t, 3H, J = 4 Hz). LCMS: m/z = 288 [M+H] +, RT = 3.56 minutes, (Program Pl, Column W).

[0480] Step 5: 3-Bromo-8-(4-methyl-piperidin-l-yl)-imidazo[l,2-a]pyridine-6carboxylic acid ethyl ester

[0517] Step 2: Acetic acid l-(2-tert-butylamino-pyridin-3-yl)-lH-tetrazol-5-ylmethyl ester

[0536] To a solution of compound XIII (0.35 g, 0.98 mmol, 1 eq) in DMF (30 mL) were added HATU (0.48 g, 1.27 mmol, 1.3 eq) and DIPEA (2 mL) at 0 °C under an argon atmosphere. After 10 minutes, a DMF solution of C-(6-methyl-pyridin-3-yl)-methylamine (0.18 g, 1.47 mmol, 1.5 eq) was added and the resulting mixture was stirred at 23 °C for 16 h. The mixture was diluted with ice cold water (30 mL) and the organic components were extracted with ethyl acetate (2 x 50 mL) and 10% MeOH/CH2C12 (50 mL). The combined organic layers were washed with brine (80 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain crude compound. The crude product was purified by flash Combiflash™ chromatography using 100-200 mesh silica gel eluting with 9% MeOH/CH2C12 to obtain the title compound (0.25 g, 55%) as an off-white solid. *H NMR (400 MHz, DMSO-Jô) Ô 9.46-9.38 (m, IH), 9.21 (s, IH), 8.45 (s, IH), 8.18 (s, IH), 7.90 (s,

DMSO-î/6) δ 9.53 (t, IH, J = 6 Hz), 9.18 (s, IH), 8.47 (s, IH), 8.37 (s, IH), 7.94 (s, IH), 7.70-7.44 (m, 211), 7.23 (d, III, 7 = 8 Hz), 4.53 (d, 211,7=5 Hz), 2.45 (s, 311). LCMS: m/z = 463.0 [M+], 465.0 [M+2], RT = 1.94 minutes; (Program RI, Column W).

[0558] Step 8: 8-(5-(Difluoromethyl)-lH-tetrazol-l-yl)-N-((6-methylpyridin-3yl)methyl)-3-(5-methylthiophen-2-yl)imidazo[l,2-a]pyridine-6-carboxamide

[0559] To a DMF (20 mL) solution of compound VIII (0.4 g, 0.86 mmol, 1 eq), degassed with argon, in a reaction tube was added compound Via (0.50 g, 1.29 mmol, 1.5 eq) and the resulting mixture was degassed with argon for 10 min, followed by the addition of Pd(PPh3)4 (0.1 g, 0.086 mmol, 0.1 eq). Further degassing with argon was done for another 15 min and the reaction tube was sealed then heated at 120 °C for 5 h. The mixture was filtered through a Celite® pad and the filtrate was concentrated under reduced pressure to provide a solid residue. The organic components were then extracted with EtOAc (2 x 40 mL) and 10% MeOH/CH2C12 (50 mL) and the combined extracts were washed with brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to provide crude compound. The crude product was purified by flash Combiflash™ chromatography using 100-200 mesh silica gel eluting wdth 4% MeOH/CH2C12 to obtain the title compound (0.18 g, 44%) as an off-white solid. 'H NMR (400 MHz, DMSO-Tg) δ 9.45-9.36 (m, IH), 9.21 (s, IH), 8.42 (s, IH), 8.30 (s, IH), 7.88 (s, IH), 7.73-7.46 (m, 3H), 7.19 (d, IH, 7 = 8 Hz), 7.03 (s, IH), 4.48 (d, 2H, 7 = 5 Hz), 2.53 (s, 3H), 2.41 (s, 3H). LCMS: m/z = 481.0 [M+H]+, RT = 2.80 minutes; (Program RI, Column W).

[0560] EXAMPLE 19: (R)-8-(5-(Difluoromethyl)-lH-tetrazol-l-yl)-N-(l-(3-methyl1,2,4-oxadiazol-5-yl)ethyl)-3-(5-methylthiophen-2-yl)imidazo[ 1,2-a]pyridine-6-carboxamide

N-N

Scheme 35

[0561] Following the experimental procedure described for Example 18, compound VI was prepared.

127

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497

[0562] Step 6: 8-(5-Difluoromethyl-tetrazol-l-yl)-3-(5-methyl-thiophen-2-yl)imidazo[l,2-a]pyridine-6-carboxylic acid methyl ester

WO 2015/095128

PCT/US2014/070497 another 2 h. The mixture was quenched with water (100 mL) and the organic components were extracted with ethyl acetate (2 x 200 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and the solvent was removed under reduced pressure to provide crude compound (0.8 g, 76%) as a solid. The crude product was directly used in the next step without further purification. *H NMR (DMSO-de) δ 9.37 (s, IH), 8.20 (s, IH), 7.71 (s, IH). 7.62 (s, IH), 3.90 (s, 3H), 3.21 (s, 3H), 2.30 (m, IH), 0.90 (m, 6H). LCMS: m/z = 276.2 [M+], RT = 2.40 minutes, (Program Pl, Column V).

[0574] Step 5: 3-Bromo-8-(isobutyryl-methyl-amino)-imidazo[l,2-a]pyridine-6carboxylic acid methyl ester

[0575] To a stirred solution of compound V (0.8 g, 2.9 mmol, 1 eq) in THF (20 mL) was added N-bromosuccinamide (0.51 g, 2.9 mmol, 1 eq) at 0 °C and the resulting mixture was stirred for 30 min. The mixture was quenched with w'ater (100 mL) and the organic components were extracted with ethyl acetate (2 x 200 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and solvents were removed under reduced pressure to provide crude compound. The crude product was purified by flash chromatography using 100-200 mesh silica gel eluting with 30% EtOAc/hexanes to obtain the title compound (1 g, 98%) as a brown solid. ]H NMR (DMSO-tfc) δ 8.77 (s, IH), 7.91 (s, IH), 7.77 (s, IH), 3.93 (s, 3H), 3.21 (s, 3H), 2.38 (m, IH), 090 (m, 6H). LCMS: m/z = 354.0 [M +], 356.0 [M + 2], RT = 2.86 minutes; (Program PI, Column V).

[0576] Step 6: 8-(Isobutyryl-methyl-amino)-3-(5-methyl-thiophen-2-yl)-imidazo[l,2a]pyridine-6-carboxylic acid methyl ester

[0577] To a stirred, dry DMF solution of compound VI (1 g; 2.82 mmol; 1 eq) in a reaction tube was added compound Via (1.6g; 4.23 mmol; 1.5 eq) and the resulting mixture was degassed with argon for 5 min. To the mixture was added Pd(PPh3)4 (0.32 g; 0.28 mmol; 0.1 eq) and degassing with argon was repeated for about 5 min, then the reaction tube was sealed and heated at 140 °C for 2 h. The mixture was cooled to room temperature, quenched with water (100 mL) and the organic components were extracted with ethyl acetate (2 x 200 mL). The combined organic layers were washed with waler (200 mL), brine and dried over anhydrous Na2SÛ4, then filtered. The filtrate was evaporated under vacuum to obtain a crude material which was purified by silica gel (230-400 mesh) column chromatography eluting

131

CA 02933811 2016-06-13

WO 2015/095128

PCT/US2014/070497 with 10-50% ethyl acetate/hexane to obtain the title compound (0.95 g, 91%) as an off white solid. ’H NMR (DMSO-</«) Ô 8.98 (s, IH), 7.89 (s, IH), 7.72 (s, IH), 7.38 (d, IH, J = 3 Hz,), 7.03 (d, IH, J = 3 Hz), 3.90 (s. 3H), 3.23 (s, 3H), 2.55 (s, 3H), 2.50 (m, IH), 0.93 (m, 6H). LCMS: m/z = 372.4 [M+], RT = 3.24 minutes, (Program Pl, Column V).

[0578] Step 7: 8-(Isobutyryl-methyl-amino)-3-(5-methyl-thiophen-2-yl)-imidazo(l,2a]pyridine-6-carboxylic acid

[0579] To a solution of compound VII (0.95 g, 2.56 mmol, 1 eq) in THF:methanol:H2O (40 mL, 5:1:1) at 23 °C was added a solution of lithium hydroxide monohydrate (0.16 g, 3.84 mmol, 1.5 eq) in water (5 mL) and the resulting mixture was stirred at 23 °C for 1 h. The organic solvent was removed under reduced pressure and the residue was diluted with water and acidified with 6N HC1 to adjust the pH to about 5-6. The precipitated solid was collected by filtration and the solid was dried under vacuum to afford the title compound (0.8 g, 82 %) as an off white solid. *H NMR (DMSO-ô/6) δ 13.5 (brs, IH), 8.97 (s, IH), 7.88 (s, IH), 7.69 (s, IH), 7.37 (d, IH, J = 4 Hz), 7.02 (s, IH), 3.23 (s, 3H), 2.55 (s, 3H), 2.50 (m, IH), 0.93 (m, 6H). LCMS: m/z = 358.0 [M+], RT = 2.35 minutes, (Program Pl, Column Y).

(0580] Step 8: (S)-N-(l-(4H-l,2,4-Triazol-3-yl)ethyl)-8-(N-methylisobutyramido)-3-(5methylthiophen-2-yl)imidazo[ 1,2-a]pyridine-6-carboxamide

[0581] To a stirred solution of compound VIII (0.18 g, 0.50 mmol, leq) in DMF (2 mL) were added TEA (0.21 mL, 1.51 mmol, 3 eq), a DMF (2 mL) solution of (S)-1-(4H[l,2,4]triazol-3-yl)-ethylamine hydrochloride (0.09 g, 0.60 mmol; 1.2 eq) and T3P (0.23 mL, 0.75 mmol, 1.5 eq) at 23 °C and the resulting mixture was stirred at 120 °C for 2 h. The mixture was cooled to room temperature and quenched with water. The organic components were extracted with ethyl acetate (2 x 100 mL) and the combined extracts were washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to dryness to provide crude material which was purified by silica gel (100-200 mesh) column chromatography eluting with 0-5% melhanol/CH2Cl2 to obtain the title compound (0.12 g, 53%) as an off while solid. *H NMR (DMSO-ώ,) δ 13.85 (brs, IH), 9.13 (brs, IH), 9.01 (s, IH), 8.48 (s, IH), 7.84 (m, 2H), 7.40 (m, IH), 7.02 (s, IH), 5.32 (m, IH), 3.17 (s, 3H), 2.54 (s, 3H), 2.43 (m. IH). 1.55 (m, 3H), 0.95 (m, 6H). LCMS: m/z = 452.3 [M+J, RI' = 2.61 minutes, (Program Pl, Column V).

132