TrkA Inhibitor

Abstract

The present invention relates to compound of the following structure ##STR00001##
for use as an inhibitor of TrkA, as well as compositions including this compound, and methods of using this compound for the treatment of pain including post-surgical pain, rheumatoid arthritis pain, neuropathic pain and osteoarthritis pain.

Claims

1. A compound of the Formula: ##STR00020## or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 which is: ##STR00021##

3. A pharmaceutical composition comprising a compound according to claim 1, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

4. A pharmaceutical composition comprising a compound according to claim 2 and one or more pharmaceutically acceptable carriers, diluents or excipients.

5. A method of treating pain comprising administering to a patient in need thereof an effective amount of a compound according to claim 1, or pharmaceutically acceptable salt thereof.

6. The method of claim 5, wherein the pain is selected from the group consisting of post-surgical pain, neuropathic pain, rheumatoid arthritis pain and osteoarthritis pain.

7. The method of claim 5, wherein the pain is chronic pain.

8. A method of treating pain comprising administering to a patient in need thereof the composition of claim 3.

9. The method of claim 8, wherein the pain is selected from the group consisting of post-surgical pain, neuropathic pain, rheumatoid arthritis pain and osteoarthritis pain.

10. The method of claim 8, wherein the pain is chronic pain.

11. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, for use in the treatment of pain.

12. The compound for use in the treatment of pain according to claim 11, wherein the pain is selected from the group consisting of post-surgical pain, neuropathic pain, rheumatoid arthritis pain and osteoarthritis pain.

13. The compound for use in the treatment of pain according to claim 11, or a pharmaceutically acceptable salt thereof, wherein the pain is chronic pain.

14. A process for preparing a pharmaceutical composition, comprising admixing a compound according to claim 1, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

15. A compound which is a hemisuccinic acid salt of the following structure: ##STR00022##

16. The hemisuccinic acid salt according to claim 15 which is crystalline.

17. The hemisuccinic acid salt according to claim 16 which is characterized by a substantial peak in the X-ray diffraction spectrum, at diffraction angle 2-theta of 10.5° in combination with one or more of the peaks selected from the group consisting of 12.6, and 22.2; with a tolerance for the diffraction angles of 0.2 degrees.

18. A pharmaceutical composition comprising a compound according to either claim 15 and one or more pharmaceutically acceptable carriers, diluents or excipients.

19. A method of treating pain comprising administering to a patient in need thereof an effective amount of a compound according to either claim 15.

20. The method of claim 19, wherein the pain is selected from the group consisting of post-surgical pain, neuropathic pain, rheumatoid arthritis pain and osteoarthritis pain.

21. The method of claim 19, wherein the pain is chronic pain.

Description

PREPARATIONS AND EXAMPLES

(1) The following Preparations and Examples further illustrate the invention and represent typical synthesis of various compounds of the invention. The reagents and starting materials are readily available, or may be readily synthesized by one of ordinary skill in the art. It should be understood that the Preparations and Examples are set forth by way of illustration and not limitation, and that various modifications may be made by one of ordinary skill in the art.

(2) LC-ES/MS is performed on an AGILENT® HP1100 liquid chromatography system. Electrospray mass spectrometry measurements (acquired in positive and/or negative mode) are performed on a Mass Selective Detector quadrupole mass spectrometer interfaced to the HP1100 HPLC. LC-MS conditions (low pH): column: PHENOMENEX® GEMINI® NX C18 2.1 mm×50 mm, 3.0μ; gradient: 5-100% B in 3 min, then 100% B for 0.75 min column temperature: 50° C.+/−10° C.; flow rate: 1.2 mL/min; Solvent A: deionized water with 0.1% HCOOH; Solvent B: ACN with 0.1% formic acid; wavelength 214 nm. Alternate LC-MS conditions (high pH): column: XTERRA® MS C18 columns 2.1×50 mm, 3.5 μm; gradient: 5% of solvent A for 0.25 min, gradient from 5% to 100% of solvent B in 3 min and 100% of solvent B for 0.5 min or 10% to 100% of solvent B in 3 min and at 100% of solvent B for 0.75 min; column temperature: 50° C.+/−10° C.; flow rate: 1.2 mL/min; Solvent A: 10 mM NH.sub.4HCO.sub.3 pH 9; Solvent B: ACN; wavelength: 214 nm.

(3) Unless otherwise specified, preparative reversed phase chromatography is performed on an AGILENT® 1200 LC-ES/MS equipped with a Mass Selective Detector mass spectrometer and a LEAP® autosampler/fraction collector. High pH methods are run on a 75×30 mm PHENOMENEX® GEMINI®-NX, 5μ particle size column with a 10×20 mm guard. Flow rate of 85 mL/min. Eluent is 10 mM ammonium bicarbonate (pH 10) in acetonitrile.

(4) NMR spectra are performed on a Bruker AVIII HD 400 MHz NMR Spectrometer, obtained as CDCl.sub.3 or (CD.sub.3).sub.2SO solutions reported in ppm, using residual solvent [CDCl.sub.3, 7.26 ppm; (CD.sub.3).sub.2SO, 2.05 ppm] as reference standard.

(5) When peak multiplicities are reported, the following abbreviations may be used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br-s (broad singlet), dd (doublet of doublets), dt (doublet of triplets). Coupling constants (J), when reported, are reported in hertz (Hz).

Preparation 1

Methyl 2-fluoro-4-(trifluoromethyl)benzoate

(6) ##STR00003##

(7) 2-Fluoro-4-(trifluoromethyl)benzoic acid (5 g, 23 mmol) is dissolved in MeOH (100 mL) and a concentrated aqueous solution of H.sub.2SO.sub.4 (200 μL, 4 mmol) is added. The resulting mixture is heated to reflux with stirring for 48 h. The reaction mixture is concentrated under reduced pressure, the residue is diluted with water, and extracted with EtOAc. The organic extracts are washed sequentially with saturated aqueous NaHCO.sub.3 and saturated aqueous NaCl, dried over Na.sub.2SO.sub.4, filtered, and the filtrate is concentrated under reduced pressure to obtain the title compound (5.2 g, 99% yield). .sup.1H nmr (CDCl.sub.3): δ 3.993 (s, 3H), 7.447 (d, 1H), 7.507 (d, 1H), 8.091 (t, 1H).

Preparation 2

Methyl 2-fluoro-5-nitro-4-(trifluoromethyl)benzoate

(8) ##STR00004##

(9) Methyl 2-fluoro-4-(trifluoromethyl)benzoate (5.1 g, 23 mmol) is dissolved in concentrated aqueous H.sub.2SO.sub.4 (25 mL) and the resulting mixture is cooled to 0° C. with stirring. An aqueous solution of 7M HNO.sub.3 (2.5 mL, 40 mmol) is added dropwise over 15 min at 0° C., and the resulting mixture is allowed to warm to RT with stirring for 1 h. The reaction mixture is poured over ice, and the resulting precipitate is collected by filtration, washed with water, and air-dried with vacuum suction. The filtercake is dissolved in EtOAc (100 mL), and the organic mixture is washed sequentially with saturated aqueous NaHCO.sub.3 and saturated aqueous NaCl. The organic extract is dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to obtain the title compound (5.8 g, 95% yield) as a yellow solid. .sup.1H nmr (CDCl.sub.3): δ 4.043 (s, 3H), 7.672 (d, 1H), 8,598 (d, 1H).

Preparation 3

Methyl 5-amino-2-fluoro-4-(trifluoromethyl)benzoate

(10) ##STR00005##

(11) Methyl 2-fluoro-5-nitro-4-(trifluoromethyl)benzoate (5.8 g, 21.7 mml) is dissolved in MeOH (150 mL), and the mixture is sparged with N2. Pd/C (500 mg) is added, the reaction mixture is sealed, and the resulting mixture is stirred under a balloon of H.sub.2 at ambient temperature and pressure for 4 h. The reaction mixture is purged with N2, filtered over a bed of diatomaceous earth, and the methanolic filtrate is concentrated under reduced pressure. The resulting crude residue is purified by chromatography over silica gel, eluting with a gradient of 5-60% EtOAc in hexanes) to obtain the title compound (3.5 g, 68% yield), after evaporation of the desired chromatographic fractions. ESMS (m/z): 236 (M−H).

Preparation 4

Methyl 5-bromo-2-fluoro-4-(trifluoromethyl)benzoate

(12) ##STR00006##

(13) CuBr.sub.2 (3.15 g, 14.1 mmol) and isoamylnitrite (2.2 mL, 16 mmol) are added to a solution of methyl 5-amino-2-fluoro-4-(trifluoromethyl)benzoate (3 g, 12.7 mmol) dissolved in ACN (30 mL) and the resulting mixture is stirred at RT for 2 h. The reaction mixture is diluted with hexanes, filtered through a bed of diatomaceous earth, and the collected filtrate is concentrated under reduced pressure. The resulting residue is purified by chromatography over silica gel, eluting with a gradient of 5-40% EtOAc in hexanes, to obtain the title compound (3.8 g, 71% yield) after evaporation of the desired chromatographic fractions. .sup.1H nmr (CDCl.sub.3): δ 3.996 (s, 3H), 7.521 (dd, 1H), 8.272 (t, 1H).

Preparation 5

Methyl 2-fluoro-5-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzoate

(14) ##STR00007##

(15) 1-Methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.0 g, 4.8 mmol) and methyl 5-bromo-2-fluoro-4-(trifluoromethyl)benzoate (1.3 g, 4.3 mmol) are suspended in THF (6 mL) containing 1M aqueous K.sub.3PO.sub.4 (2 mL, 2 mmol) in a microwave vial equipped with a stir bar and the mixture is sparged with N2 for 5 min. Methanesulfonato(2-dicyclohexylphosphino-2′,4′,6′-tri-iso-propyl-1,1′-biphenyl)(2′-methylamino-1,1′-biphenyl-2-yl)palladium(II) (190 mg, 0.2 mmol) is added and the vial is sealed with a Teflon cap. The resulting mixture is irradiated in a microwave reactor at 100° C. for 4 h. The reaction mixture is cooled to RT and loaded directly onto silica gel for purification, eluting with a gradient of 5-100% MeOH (containing 10% v/v 2 M NH.sub.3 in MeOH) in DCM to obtain, after evaporation of the desired chromatographic fractions, the title compound (1.1 g, 85% yield) as a white solid. ESMS (m/z): 303 (M+H).

Preparation 6

2-fluoro-5-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzoic acid

(16) ##STR00008##

(17) Solid LiOH (1.0 g, 41 mmol) is added to a solution of methyl 2-fluoro-5-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzoate (1.1 g, 3.7 mmol) in EtOH (50 mL) and H.sub.2O (10 mL) and the resulting reaction mixture is stirred at RT for 2 h. The mixture is acidified with 5N HCl and extracted with EtOAc. The organic extracts are washed with saturated aqueous NaCl, dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to obtain the title compound (1.1 g, 99% yield) as a white solid. ESMS (m/z): 298 (M+H).

Preparation 7

5-bromo-4-iodo-pyridine-3-carboxylic acid hydrochloride

(18) ##STR00009##

(19) A mixture of THF (350 mL) and 2,2,6,6-tetramethylpiperidine (41 mL, 240 mmol) is cooled with stirring to −50° C. under N2. A 2.5 M solution of n-BuLi in hexanes (81 mL, 220 mmol) is added portion wise, maintaining temperature below −40° C., and the resulting mixture is stirred at −50° C. for 10 min. 5-Bromopyridine-3-carboxylic acid (20.0 g, 99.0 mmol) is added portion wise, maintaining temperature below −40° C. The resulting reaction mixture is stirred at −50° C. and added to a solution of 12 (30.2 g, 119 mmol) in THF (300 mL) via cannula under N2 with slight pressure at −50° C. with stirring. The resulting mixture is warmed to RT with stirring under N2 for 4 h. The reaction mixture is quenched with water (20 mL), and most of the solvent is evaporated under a stream of N2. The resulting residue is poured into 1 N aqueous solution of NaOH (250 mL). The resulting basic mixture is washed with Et.sub.2O:EtOAc (1:1, ˜250 mL), and the separated aqueous layer is acidified to pH ˜1 with 5 N aqueous HCl. The resulting precipitate is collected by filtration, triturated with MeOH, collected by filtration, and the filter cake is washed with Et.sub.2O and hexanes and air-dried, to obtain the title compound (20.0 g, 55% yield) as a tan solid, sufficient for use without additional purification and used as is in the next step. ESMS (m/z): (.sup.79Br/.sup.81Br) 328/330 (M+H).

Preparation 8

Methyl 5-bromo-4-iodo-pyridine-3-carboxylate

(20) ##STR00010##

(21) In a 50 mL RBF is added 5-bromo-4-iodo-pyridine-3-carboxylic acid (2.8 g, 8.5 mmol), acetone (23 mL), K.sub.2CO.sub.3 (1.8 g, 12.8 mmol), and dimethyl sulfate (891 μL, 9.3 mmol). The mixture is allowed to stir at RT overnight. The reaction mixture is filtered through diatomaceous earth, rinsed with acetone and EtOAc, and the filtrate is concentrated under reduced pressure. The resulting residue is purified by flash chromatography on silica gel, eluting with a gradient of 0-100% EtOAc (containing 2% Et.sub.3N) in isohexane, to give the title compound (2.15 g, 70.5% yield) as crystalline yellow solid after evaporation of the desired chromatographic fractions. .sup.1H nmr ((CD.sub.3).sub.2SO): δ 3.906 (s, 3H), 8.592 (s, 1H), 8.862 (s, 1H).

Preparation 9

Methyl 5-bromo-4-(2-pyridyl)pyridine-3-carboxylate

(22) ##STR00011##
References: Bioorg. Med. Chem. Lett., 2017, 27(16), 3817; Bioorg. Med. Chem. Lett., 2016, 26(1), 160.

(23) A 25 mL microwave vessel containing, methyl 5-bromo-4-iodo-pyridine-3-carboxylate (710 mg, 2.1 mmol), CsF (630 mg, 4.1 mmol), LiCl (176 mg, 4.1 mmol), tetrakis(triphenyl-phosphine)palladium(0) (120 mg, 0.1 mmol) is evacuated and purged with nitrogen (two cycles of vacuum/nitrogen). 1,4-Dioxane (18 mL, 210.8 mmol) is added, nitrogen bubbled through the mixture, and tributyl(2-pyridyl)stannane (540 μL, 1.7 mmol) is added. The sealed reaction mixture is heated to 125° C. in a heating block and heated overnight. The resulting mixture is stirred over 48 h at RT. The crude mixture is filtered through diatomaceous earth and rinsed with EtOAc, and the filtrate is concentrated under reduced pressure. The resulting residue is purified by flash chromatography over silica gel, eluting with 0 to 70% EtOAc in isohexanes, to obtain the title compound (140 mg, 23% yield) as a golden oil after evaporation of the desired chromatographic fractions. ESMS (m/z): (.sup.79Br/.sup.81Br) 292/294 (M+H).

Preparation 10

2-fluoro-5-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzamide

(24) ##STR00012##

(25) 2-Fluoro-5-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzoic acid (33.1 g, 115 mmol), NH.sub.4Cl (8.0 g, 150 mmol), HATU (57 g, 142.4 mmol), DIPEA (44 mL, 252 mmol) and DMF (300 mL) are combined in a round-bottomed flask and stirred at RT for 1 h. The reaction mixture is diluted with water and the resulting precipitate is collected by filtration, washed with water, and dried under vacuum at 50° C. to obtain the title compound (24 g, 72% yield) as an off-white, crystalline solid. The filtrate is concentrated under reduced pressure and diluted with water. The resulting mixture is extracted with EtOAc, and the organic layer is washed with saturated aqueous NaCl, dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting residue is triturated with EtOAc, collected by filtration and air-dried, to obtain additional title compound (5.4 g, 16% yield) as a white, crystalline solid. The title compound is combined with previously recovered title material (29.0 g, 92% total yield). ESMS (m/z): 288 (M+H).

Preparation 11

Methyl 5-[[2-fluoro-5-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzoyl]amino]-4-(2-pyridyl)pyridine-3-carboxylate

(26) ##STR00013##

(27) A 25 mL microwave vial is charged with, 2-fluoro-5-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzamide (282 mg, 1 mmol), CsCO.sub.3 (800 mg, 2.5 mmol), tris(dibenzylideneacetone)dipalladium(0) (150 mg, 0.2 mmol) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (190 mg, 0.3 mmol). The vial is sealed, subjected to two cycles of vacuum/nitrogen, and a solution of methyl 5-bromo-4-(2-pyridyl)pyridine-3-carboxylate (240 mg, 0.8 mmol) in 1,4-dioxane (8 mL) is added. Nitrogen is bubbled through the mixture for 5 min and mixture is heated in a heating block at 130° C. overnight. The mixture is filtered through diatomaceous earth and rinsed sequentially with EtOAc and MeOH, and the filtrate is concentrated to give a brown oil. The resulting residue is diluted in MeOH (to a total volume of 8.5 ml), filtered over a bed of diatomaceous earth, and purified by preparative HPLC over C18 silica gel (PHENOMENEX® Gemini, 5μ, 30×100 mm, 60 mL/min, 210 nm), eluting with a gradient of 5-100% ACN in water containing 10 mM NH.sub.4CO.sub.3, adjusted to pH ˜9 with aqueous NH.sub.4OH, over 9 minutes (1 total injection), to obtain the title compound (40 mg, 8.5% yield) after evaporation of the desired chromatographic fractions. ESMS (m/z): 500 (M+H).

Preparation 12

5-[[2-fluoro-5-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzoyl]amino]-4-(2-pyridyl)pyridine-3-carboxylic acid

(28) ##STR00014##

(29) Methyl 5-[[2-fluoro-5-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzoyl]amino]-4-(2-pyridyl)pyridine-3-carboxylate (40 mg, 0.07 mmol) is dissolved in MeOH (5 mL) and treated with a 2M aqueous solution of NaOH (5 mL, 10 mmol), and the resulting mixture is stirred at for 30 min. The reaction is acidified with an aqueous solution of 2M HCl and extracted with DCM. The organic layer is separated, dried through a hydrophobic frit, and concentrated under reduced pressure to give a pale yellow solid. The aqueous phase is additionally extracted with 20% MeOH/DCM, the layers are separated, and the organic extract is dried over MgSO.sub.4, filtered, combined with previous organic phase, and concentrated to give the title compound (40 mg, 118% crude yield) as a pale yellow solid suitable for use without additional purification. ESMS (m/z): 486 (M+H).

Example 1

5-[[2-fluoro-5-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzoyl]amino]-4-(2-pyridyl)pyridine-3-carboxamide

(30) ##STR00015##

(31) 5-[[2-fluoro-5-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzoyl]amino]-4-(2-pyridyl)pyridine-3-carboxylic acid (40 mg, 0.08 mmol) is dissolved in anhydrous DMF (2 mL). HATU (58 mg, 0.2 mmol) and NH.sub.4Cl (55 mg, 1.0 mmol), and DIPEA (270 μL, 1.6 mmol) are added. The reaction mixture is stirred at RT for 60 min, diluted in MeOH (to a total volume of 6 mL), filtered over diatomaceous earth, and purified by preparative HPLC (PHENOMENEX® Gemini 5μ, 30×100 mm, 60 mL/min, 220 nm) over C18 silica gel, eluting with a gradient of 15-100% ACN in water containing 10 mM NH.sub.4CO.sub.3, adjusted to pH ˜9 with aqueous NH.sub.4OH over 9 minutes, to obtain the title compound (25 mg, 56% yield) after evaporation of the desired chromatographic fractions. ESMS (m/z): 485 (M+H).

Assays

(32) Human Tropomyosin Receptor Kinase (hTrkA, hTrkB, hTrkC) Assays to Examine hTrk Kinase Inhibitors

(33) The reaction of non-phosphorylated hTrk kinase domains (hTrkA, aa: 441-796, hTrkB, aa: 526-838 or hTrkC, aa: 510-825) with a fluorescein-labeled poly-GT substrate (poly-GT, Invitrogen) generates a fluorescein-labeled phosphorylated product. Binding of the terbium-labeled antibody (Invitrogen) to the phosphorylated tyrosine product brings the terbium and fluorescein into proximity, resulting in an increase in time-resolved fluorescence resonance energy transfer (TR-FRET). In the presence of an inhibitor, formation of phosphorylated product is reduced, and the TR-FRET value is decreased.

(34) Inhibition of hTrk kinase activity: The activity of human tropomyosin receptor hTrk kinase is quantitated using TR-FRET technology (Invitrogen) as per vendor instructions. Briefly, compounds for dose-response studies are serially diluted in dimethyl sulfoxide (1:2 for 20 concentrations) using an acoustic dispensing instrument Echo Access (Labcyte) and dispensed into ProxiPlate-384 plus plates (PerkinElmer). Kinase domain for hTrkA, hTrkB or hTrkC is added and the plates are incubated at room temperature (RT) for one hour. ATP and poly-GT are next added and plates are incubated at RT for one hour. EDTA and terbium-labeled antibody are added and plates are incubated at RT for one hour after which the TR-FRET signal is detected using an ENVISION® plate reader (Perkin-Elmer). The ratio of fluorescence at 520 to 495 nm is calculated.

(35) Data Analysis: The data are converted into % Inhibition and relative IC50 values are calculated from the top-bottom range of the concentration response curve using a four-parameter logistic curve fitting program (GENEDATA SCREENER® v13.0.5) with the equation Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log IC50−X)*HillSlope)). The top of the curve is 100% inhibition and defined by the kinase wells treated with an internal standard, which is a non-selective hTrk kinase inhibitor ([final]=7.9 uM), whereas the bottom of the curve is 0% inhibition and defined by the kinase wells in the absence of compound.

(36) The internal standard used in this procedure is N-[5-[7-[(1S)-2-hydroxy-1-methyl-ethyl]pyrrolo[2,3-d]pyrimidine-5-carbonyl]-3-pyridyl]-2-[4-(trifluoromethyl)phenyl]acetamide:

(37) ##STR00016##
This internal standard is commercially available as CAS Registry Number 1402438-37-2. This internal standard may also be made using the teachings of U.S. Pat. No. 8,846,698.

(38) In Vitro Inhibition of SH2 Domain Protein Recruitment in Human Bone Osteosarcoma Epithelial Cells by Human Tropomyosin Receptor Kinase (hTrk) Inhibitors

(39) Activation of hTrkA, TrkB or TrkC is examined using PathHunter® (DiscoverX) enzyme fragment complementation technology. In this approach, one fragment of β-galactosidase (β-gal) ProLink (PK) is fused to the C-terminal of the Trk receptor and co-expressed with a phosphotyrosine SH2 domain protein fused with the remaining fragment of β-gal, Enzyme Acceptor (EA), in human Bone Osteosarcoma Epithelial (hU2OS) cells also expressing the p75 neurotrophic receptor (DiscoverX). Activation of hTrk receptors by agonists results in receptor phosphorylation. The SH2-EA fusion protein binds the phosphorylated hTrk receptor resulting in complementation of PK and EA to form an active β-gal enzyme. β-gal enzymatic activity is quantitatively measured using a chemiluminescent substrate in the PathHunter® detection kit (DiscoverX). Human nerve growth factor (hNGF, PeproTech), human brain-derived neurotrophic factor (hBDNF, PeproTech) and recombinant human neurotrophin-3 protein (hNT-3, PeproTech) are used as agonists for hTrkA, hTrkB or hTrkC receptors respectively.

(40) Cell Culture: Cultured hU2OS cells that express hTrk receptors and p75 (PathHunter® system, DiscoverX) are grown in AssayComplete™ U2OS Cell Culture Kit 11 (DiscoverX) to about 80% confluence. On the day before the assay, cells are detached using AssayComplete™ Cell Detachment Reagent (DiscoverX), harvested in AssayComplete™ Cell Plating 16 Reagent (DiscoverX) to the correct cell concentration (250,000/ml) and seeded at 5K/well into 384-well poly-D-lysine coated white plates (BD Biosciences). Cell plates are incubated at 37° C. overnight.

(41) Inhibition of hTrk kinase activity: The activity of hTrkA and hTrkC kinase is quantitated using Pathhunter® technology (DiscoverX) as per vendor instructions.

(42) Briefly, on the assay day, compounds for dose-response studies are serially diluted in dimethyl sulfoxide (1:3 for 10 concentrations) using an acoustic dispensing instrument Echo Access (Labcyte) and dispensed into cell plates that are subsequently incubated at room temperature (RT) for one hour. For studies in hTrKA-p75 cells, recombinant human NGF-β (PeproTech) is added to the cells at an ECK) concentration ([final]=27 ng/ml) and plates are incubated at RT for three hours. Pathhunter® detection reagents (DiscoverX) are added and plates are read in an ENVISION® plate reader (Perkin-Elmer) at 700 nm after 1 h incubation at RT in the dark. Human brain-derived neurotrophic factor (hBDNF, PeproTech, [final]=20 ng/ml) or recombinant human neurotrophin-3 protein (hNT-3, PeproTech, [final]=29 ng/ml) are used as agonist for studies in hTrkB-p75 or hTrkC-p75 cells, respectively.

(43) Data Analysis: The data are converted into % Inhibition and relative IC.sub.50 values are calculated from the top-bottom range of the concentration response curve using a four-parameter logistic curve fitting program (GENEDATA SCREENER® v13.0.5) with the equation Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((Log IC.sub.50−X)*HillSlope)). The top of the curve is 100% inhibition and defined by the wells without agonist stimulation. The bottom of the curve is 0% inhibition and defined by the agonist-stimulated (hNGF, hBDNF or hNT) wells in the absence of inhibitor.

(44) TABLE-US-00001 TABLE 1 Relative IC.sub.50 in nM of Example 1 against hTrkA/B/C ± SEM (N = number of times tested) PathHunter.sup. ® cell-based assay Binding Assays hTrkA hTrkB hTrkC hTrkA hTrkB hTrkC (nM) (nM) (nM) (nM) (nM) (nM) 1.24 6,700 9,010 1.09 >4,290 8,360 (N = 32) (N = 25) (N = 24) (N = 4) (N = 1) (N = 1)
These data indicate that the compound of Example 1 is a potent inhibitor of and selective for hTrkA in vitro.

Preparation of Hemi Succinic Acid Cocrystal Formation of Example 1

(45) To a flask, add 16 g of the compound of Example 1 (33.0 mmol, 1 eq). Add THF (288 mL) and water (32 mL). Heat this mixture to 55° C. and obtain a solution. Polish filter the solution and rinse with 9:1 v:v THF:water (2×16 mL). In a separate flask, add succinic acid (3.9 g, 33.0 mmol, 1 eq) and ethanol (80 mL). Mix until a solution results.

(46) Transfer the solution of the compound of Example 1 to a flask with overhead stirring and a distillation head and rinse with 9:1 v:v THF:water (16 mL). Add the succinic acid solution in EtOH. Heat the solution to reflux and begin distillation. After 300 mL distillate are collected, solids crystallize from the mixture. Add back EtOH (160 mL) and stir. Turn off the heat. Cool to room temperature. Filter and rinse with EtOH (4×16 mL). Dry the wetcake under vacuum at 50° C. Isolate white solids, the hemi succinic acid salt of the compound of Example 1 (16.58 g, 30.5 mmol, 92.5% yield).

(47) Co-Crystal Formation

(48) The following is a description of a crystallization process for the hemi succinic acid cocrystal. Those skilled in the art would appreciate that a similar process could be used to crystallize the free base (with modifications as needed, as would be appreciated by a skilled artisan).

(49) To a flask, add the hemi succinic acid cocrystal of Example 1 (40.1 g, 72.2 mmol, 1 eq). Add DMSO (120 mL), and heat to 50° C. to obtain a solution. Polish filter the solution and rinse with DMSO (2ט2 mL).

(50) To another flask, add succinic acid (14.5 g, 123 mmol, 1.7 eq) and EtOH (980 mL). Mix until a solution results.

(51) To the crystallization vessel, add DMSO (20 mL) and a portion of the succinic acid solution in EtOH (140 mL). Heat to 50° C. Add seed crystals of the hemi succinic acid salt of the compound of Example 1 (1.4 g) and stir.

(52) Add the solution of compound of Example 1 in DMSO and the solution of succinic acid in EtOH to the crystallization vessel in separate feed streams over 4 hours, maintaining the temperature of the crystallization vessel at 50° C. After the co-addition is complete, cool the mixture slowly to 20° C. Stir at 20° C., then filter and rinse with a solution of succinic acid in EtOH (2 mg/mL succinic acid in EtOH, 4×70 mL rinses). Dry the wetcake under vacuum at 50° C. Isolate white solids, the hemi succinic acid cocrystal of the compound of Example 1 (38.2 g, 70.3 mmol, 92% yield corrected for the seed amount).

(53) NMR: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.14 (s, 1H), 10.66 (d, J=3.1 Hz, 1H), 9.08 (s, 1H), 8.70-8.63 (m, 1H), 8.61 (s, 1H), 8.06 (s, 1H), 7.94-7.85 (m, 2H), 7.83 (d, J=10.6 Hz, 1H), 7.80 (d, J=2.2 Hz, 1H), 7.60-7.52 (m, 2H), 7.46-7.38 (m, 1H), 6.46-6.41 (m, 1H), 3.93 (s, 3H), 2.43 (s, 2H).

(54) Mass spec: found 485.0 m/z, theory 485.1

(55) X-Ray Powder Diffraction (XRPD) of Crystalline Forms

(56) The XRPD patterns of crystalline solids are obtained on a Bruker D8 Endeavor X-ray powder diffractometer, equipped with a CuKα (1.5418 Å) source and a Linxeye detector, operating at 40 kV and 40 mA. The sample is scanned between 4 and 42 2θ°, with a step size of 0.009 2θ° and a scan rate of 0.5 seconds/step, and using 0.3° primary slit opening, and 3.9° PSD opening. The dry powder is packed on a quartz sample holder and a smooth surface is obtained using a glass slide. The crystal form diffraction patterns are collected at ambient temperature and relative humidity. Crystal peak positions are determined in MDI-Jade after whole pattern shifting based on an internal NIST 675 standard with peaks at 8.853 and 26.774 2θ°. It is well known in the crystallographic art that, for any given crystal form, the relative intensities of the diffraction peaks may vary due to preferred orientation resulting from factors such as crystal morphology and habit. Where the effects of preferred orientation are present, peak intensities are altered, but the characteristic peak positions of the polymorph are unchanged. See, e.g. The United States Pharmacopeia #23, National Formulary #18, pages 1843-1844, 1995. Furthermore, it is also well known in the crystallography art that for any given crystal form the angular peak positions may vary slightly. For example, peak positions can shift due to a variation in the temperature at which a sample is analyzed, sample displacement, or the presence or absence of an internal standard. In the present case, a peak position variability of ±0.2 2θ° is presumed to take into account these potential variations without hindering the unequivocal identification of the indicated crystal form. Confirmation of a crystal form may be made based on any unique combination of distinguishing peaks.

(57) XRPD of Hemisuccinic Acid Form of Example 1 (3848608) RS7-H81933-069A

(58) A prepared sample of the hemisuccinic acid of Example 1 is characterized by an XRPD pattern using CuKα radiation as having diffraction peaks (2-theta values) as described in Table A below, and in particular having a peak at 10.5 in combination with one or more of the peaks selected from the group consisting of 12.6, and 22.2; with a tolerance for the diffraction angles of 0.2 degrees.

(59) TABLE-US-00002 TABLE A XRPD peaks of crystalline hemisuccinic acid of Example 1 hemisuccinic acid of Example 1 Angle (°2- Relative Intensity Peak Theta) +/− 0.2° (% of most intense peak)  1 10.5 100.0%  2 11.8  33.6%  3 12.6  37.2%  4 14.1  34.4%  5 16.0  32.2%  6 18.6  36.5%  7 20.6  42.0%  8 21.3  54.3%  9 22.2  67.1% 10 24.7  69.5%

COMPARATIVE EXAMPLES

(60) The following two molecules were made from U.S. Pat. No. 9,815,846 using the methods and procedures described in that patent. Specifically, Examples 89 and 141 of U.S. Pat. No. 9,815,846 were prepared and have the following structures:

Example 89 of U.S. Pat. No. 9,815,846

(61) ##STR00017##

Example 141 of U.S. Pat. No. 9,815,846

(62) ##STR00018##

(63) These two compounds from U.S. Pat. No. 9,815,846 were run in the Assays (which procedures were outlined above) in order to determine their binding of Trk A, Trk B and Trk C. The data is shown below in Table 2 (and is compared to the data for Example 1 found in Table 1 above)).

(64) TABLE-US-00003 TABLE 2 Comparative IC50 data for binding to Trk A, Trk B, and Trk C. Example 1 Example 89 Example of the present of US 141 of US disclosure Patent No. Patent No. (data taken from 9815846 9815846 Table 1 above) hTrkA IC50 1.55 nM 11.1 nM 1.24 nM hTrkB IC50 713 nM >47,900 nM 6,700 nM hTrkC IC50 452 nM 37,800 nM 9,010 nM

(65) As can see from the data in Table 2, the compound of Example 1 is a potent binder of Trk A, and is selective for Trk A. Example 89 of U.S. Pat. No. 9,815,846 is potent with respect to Trk A, but is not as selective for Trk B and Trk C as in Example 1. Example 141 of U.S. Pat. No. 9,815,846 has similar selectivity for Trk A over Trk B and Trk C, but is less potent at the target Trk A. However, it should be noted that the compound of Example 141 of U.S. Pat. No. 9,815,846 in which there is a two pyridine rings is difficult to achieve synthetically, and it would likely not be possible to make this molecule on a kilogram scale using the techniques outlined in U.S. Pat. No. 9,815,846. In fact, the following article shows the difficulty of creating this type of two-pyridine system:

(66) Xinlan A. F. Cook, Antoine de Gombert, Janette McKnight, Loïc R. E. Pantaine and Michael C. Willis, “The 2-Pyridyl Problem: Challenging Nucleophiles in Cross-Coupling Arylations”, Angew. Chem. Int. Ed. 2020, 59, 2-26.

(67) Further, in examining U.S. Pat. No. 9,815,846, Example 152 has the following structure:

(68) ##STR00019##
The difference between Example 152 and Example 141 of U.S. Pat. No. 9,815,846 is that Example 141 has a pyridine ring whereas Example 152 has a phenyl ring. If you compare the listed Trk A IC50 values for these two molecules that are found in Table 2 of U.S. Pat. No. 9,815,846, Example 141 (with the pyridine ring) has a listed value of 14 nM whereas Example 152 (with the phenyl ring) has a listed value of 0.069 nM. Thus, by using a phenyl ring in Example 152 (rather than a pyridine ring of Example 141), there is a dramatic effect on the potency at TrkA—over 200 fold (e.g., 14 divided by 0.069. is 202.9). However, when compared with the molecule of present Example 1 (which also has a pyridine ring like Example 141 of U.S. Pat. No. 9,815,846), the present Example 1 has good the potency and selectivity.