BOOTSMA ANDREA NICOLE (US)
CHOI CHULHO (US)
DOW ROBERT LEE (US)
EDMONDS DAVID JAMES (CH)
GARCIA-IRIZARRY CARMEN NOEMI (US)
GERSTENBERGER BRIAN STEPHEN (US)
INGLE GAJENDRA (US)
O’BRIEN JESSICA GLORIA KATHERINE (US)
PARIKH MIHIR DINESHKUMAR (US)
RESCOURIO GWENAELLA CHRISTINE (US)
SCHMITT DANIEL COPLEY (US)
WO2009144554A1 | 2009-12-03 | |||
WO2009144554A1 | 2009-12-03 | |||
WO2000035298A1 | 2000-06-22 | |||
WO1991011172A1 | 1991-08-08 | |||
WO1994002518A1 | 1994-02-03 | |||
WO1998055148A1 | 1998-12-10 | |||
WO2013185103A1 | 2013-12-12 | |||
WO2021028806A1 | 2021-02-18 | |||
WO2013185103A1 | 2013-12-12 |
US20160220557A1 | 2016-08-04 | |||
US20100113418A1 | 2010-05-06 | |||
US6106864A | 2000-08-22 |
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Claims 1. A compound of formula (I) having the structure: or (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein: R is selected from the group consisting of H, C1-C6 alkoxy, C1-C6 alkyl and -(CH2)m-W, where W is C3-C8 cycloalkyl, bicyclo alkyl, bridged bicycloalkyl, , phenyl, , 5- or 6-membered heteroaryl or heterocyclic containing one, two or three heteroatoms selected from the group consisting of N, S and O atoms; wherein each of said alkyl, cycloalkyl, heterocyclic, phenyl, naphthyl or heteroaryl may be unsubstituted or substituted by phenyl, halo, cyano, deuterium, hydroxy, C1-C6 alkyl, C1- C6 alkoxy, -SO2-R’, -CONR’R”, NR’COR”, -NR’CONR’R”, -NR’CO2R”, -(CH2)n-SO2-R’, -NHSO2- R’, -NR”SO2-R’, -SO2NR’R”, NR’R” or SR’ where R’ and R’’ are independently H, C1-C6 alkyl or C3-C8 cycloalkyl; R1 is selected from the group consisting of phenyl, naphthyl, 5- or 6-membered heteroaryl or heterocyclic containing one, two, three or four heteroatoms selected from the group consisting of N, S and O atoms; and, a 9- or 10-membered bicyclic aryl, heteroaryl or heterocyclic containing one, two or three heteroatoms selected from the group consisting of N, S and O atoms; wherein each of said phenyl, naphthyl, aryl, heterocyclic, or heteroaryl may be unsubstituted or substituted by halo, cyano, deuterium, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, phenyl, -SO2-R’, -CONR’R”, NR’COR”, -NR’CONR’R”, -NR’CO2R”, -(CH2)n-SO2-R’, -NHSO2-R’, -NR”SO2-R’, -SO2NR’R”, NR’R”, -P(O)R’R”, or SR’ where R’ and R’’ are independently H, C1-C alkyl or C-C 6 3 8 cycloalkyl; and, m and n are independently 0, 1, 2 or 3. 2. The compound of claim 1 wherein R is selected from the group consisting of H, C1-C6 alkyl and -(CH2)m-W, where W is C3-C8 cycloalkyl, wherein each of said alkyl, cycloalkyl, bicycloalkyl, and bridged bicycloalkyl may be unsubstituted or substituted by halo, cyano, deuterium, hydroxy, C1-C6 alkyl and C1-C6 alkoxy; and, m and n are independently 0, 1, 2 or 3. 3. The compound of claim 1 wherein R is t-butyl. 4. A compound according to claim 1 wherein R1 is phenyl, pyridyl, indolyl, indazolyl, pyrrolopyridinyl, quinolinyl, isoquinolinyl or naphthyl; wherein each of said phenyl, pyridyl, indolyl, indazolyl, pyrrolopyridinyl, quinolinyl, isoquinolinyl or naphthyl may be unsubstituted or substituted by halo, cyano, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, phenyl, -CONR’R”, NR’R” or SR’ where R’ and R’’ are independently H, C1-C6 alkyl or C3-C8 cycloalkyl; and, m and n are independently 0, 1, 2 or 3. 5. The compound of claim 1 selected from the group consisting of: 2-(tert-butyl)-1'-(7-methoxy-1,3-dimethyl-1H-indazole-5-carbonyl)-5H-spiro[benzo[d]thiazole- 6,4'-piperidin]-4(7H)-one; 2-(tert-butyl)-1'-(7-methyl-1H-indole-5-carbonyl)-5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)- one; 2-(tert-butyl)-1'-(8-methyl-3-(methylamino)quinoline-6-carbonyl)-5H-spiro[benzo[d]thiazole-6,4'- piperidin]-4(7H)-one; 2-(tert-butyl)-1'-(7-ethoxy-1,3-dimethyl-1H-indazole-5-carbonyl)-5H-spiro[benzo[d]thiazole-6,4'- piperidin]-4(7H)-one; and, 2-(tert-butyl)-1'-(4-methyl-2-naphthoyl)-5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one; or, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt. 6. The compound of claim 1 wherein the compound is 2-(tert-butyl)-1'-(7-methoxy-1,3-dimethyl- 1H-indazole-5-carbonyl)-5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one; or, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt. 7. The compound of claim 1 wherein the compound is 2-(tert-butyl)-1'-(7-methyl-1H-indole-5- carbonyl)-5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one; or, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt. 8. The compound of claim 1 wherein the compound is 2-(tert-butyl)-1'-(8-methyl-3- (methylamino)quinoline-6-carbonyl)-5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one; or, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt. 9. The compound of claim 1 wherein the compound is 2-(tert-butyl)-1'-(7-ethoxy-1,3-dimethyl- 1H-indazole-5-carbonyl)-5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one; or, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt. 10. The compound of claim 1 wherein the compound is 2-(tert-butyl)-1'-(4-methyl-2-naphthoyl)- 5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one; or, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt. 11. A pharmaceutical composition comprising a compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, and a pharmaceutically acceptable excipient. 12. A method of treating a disease or condition selected from inflammation, autoimmune disease, neuroinflammation, arthritis, rheumatoid arthritis, spondyloarthropathies, systemic lupus erythematous, lupus nephritis, osteoarthritis, gouty arthritis, pain, fever, pulmonary sarcoidosis, silicosis, cardiovascular disease, atherosclerosis, myocardial infarction, thrombosis, congestive heart failure and cardiac reperfusion injury, cardiomyopathy, stroke, ischemia, reperfusion injury, brain edema, brain trauma, neurodegeneration, liver disease, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, nephritis, retinitis, retinopathy, macular degeneration, glaucoma, diabetes (type 1 and type 2), diabetic neuropathy, viral and bacterial infection, myalgia, endotoxic shock, toxic shock syndrome, osteoporosis, multiple sclerosis, endometriosis, menstrual cramps, vaginitis, candidiasis, cancer, fibrosis, obesity, muscular dystrophy, polymyositis, dermatomyositis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, vitiligo, Alzheimer’s disease, skin flushing, eczema, psoriasis, atopic dermatitis, sunburn, keloid, hypertrophic scar, rheumatic diseases, urticaria, discoid lupus, cutaneous lupus, central nervous system lupus, psoriatic arthritis, asthma, allergic asthma, type I interferonopathies including Aicardi–Goutières syndrome and other mendelian diseases of overexpression of type I interferon, primary progressive multiple sclerosis, relapsing remitting multiple sclerosis, non- alcoholic fatty liver disease, non-alcoholic steatohepatitis, scleroderma, alopecia areata, scarring alopecia, prurigo, prurigo nodularis, CPUO, lichen diseases, lichen planus, Steven’s Johnson’s syndrome, spondylopathy, myositis, vasculitis, pemphigus, lupus, major depression disorder, allergy, dry eye syndrome, transplant rejection, cancer, septic shock, cardiopulmonary dysfunction, acute respiratory disease, ankylosing spondylitis, cachexia, chronic graft-versus-host disease, acute graft-versus-host disease, Celiac Sprue, idiopathic thrombocytopenic thrombotic purpura, thrombotic thrombocytopenic purpura, myasthenia gravis, Sjogren's syndrome, epidermal hyperplasia, cartilage inflammation, bone degradation, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile Reter's Syndrome, SEA Syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, enteropathic arthritis, reactive arthritis, Reter's Syndrome, myolitis, polymyolitis, dermatomyolitis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, dermatitis, Still's disease, chronic obstructive pulmonary disease, Guillain-Barre disease, Graves' disease, Addison's disease, Raynaud's phenomenon, psoriatic epidermal hyperplasia, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, an immune disorder associated with or arising from activity of pathogenic lymphocytes, noninfectious uveitis, Behcet’s disease and Vogt–Koyanagi–Harada syndrome, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of claim 1 to 10, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt. 13. The method of claim 12 wherein the compound is administered topically. 14. The method of claim 12, wherein the compound is administered as a cream, ointment, lotion, gel, solution, suspension, foam, aerosol, spray, shampoo, patch or tape. 15. A method of treating acne, comprising administering to the subject a therapeutically effective amount of a compound of claim 1 to 10 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt. 16. The method of claim 15 wherein the compound is administered topically. 17. The method of claim 15, wherein the compound is administered as a cream, ointment, lotion, gel, solution, suspension, foam, aerosol, spray, shampoo, patch or tape. 18. A method of treating inflammatory skin disease, seborrheic dermatitis, rosacea, steroid acne, papulopustular drug eruption, and hidradenitis suppurativa, comprising administering to the subject a therapeutically effective amount of a compound of claim 1 to 10 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt. 19. The method of claim 18 wherein the compound is administered topically. 20. The method of claim 18, wherein the compound is administered as a cream, ointment, lotion, gel, solution, suspension, foam, aerosol, spray, shampoo, patch or tape. 21. Use of a compound according to any of claims 1 to 10 for the manufacture of a medicament for the treatment of a disorder for which an ACC inhibitor is indicated. 22. Use of a compound according to any of claims 1 to 10 for the manufacture of a medicament for the treatment of acne. 23. A compound according to any of claims 1 to 10 for use in the treatment of a disorder for which an ACC inhibitor is indicated. |
Scheme 1 Compounds prepared via Scheme 1 Compounds of formula B may be prepared from compounds of formula A according to process step (a), a bromination under suitable conditions including treatment with TMS triflate in the presence of an organic base such as triethyl amine, followed by reaction with a brominating agent such as N-bromosuccinimide. Alternative conditions for step (b) include use of trimethyl- phenylammonium tribromide in THF at 25°C. Compounds of formula C may be prepared from compounds of formula B according to process step (b), an annellation with 2,2-dimethylpropanethioamide under suitable basic conditions. Preferred conditions comprise pyridine in ethanol at about 80°C. Compounds of formula D may be prepared from compounds of formula C according to process step (c) bromo- alkoxylation, which may be effected using a brominating agent such as N-bromosuccinimide.in the presence of an alcohol. The reaction typically proceeds under ambient conditions. Compounds of formula E may be prepared from compounds of formula D according to process step (d), an elimination reaction which is carried out using a nonnucleophilic base such as potassium tert-butoxide under ambient conditions in an inert solvent such as tetrahydrofuran. Compounds of formula F may be prepared from compounds of formula E according to process step (e), a deprotection/hydrolysis with a suitable acid such as hydrochloric acid in a mixed aqueous/organic solvent such as dioxane. Compounds of formula G may be prepared from compounds of formula F according to process step (f), an acylation with a heteroaryl carboxylic acid under suitable basic conditions. Preferred conditions comprise N-ethyl-N-(propan-2-yl)propan-2-amine (DIPEA) in the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyri dinium 3-oxide hexafluorophosphate (HATU). In alternative conditions, reagents may include propanephosphonic acid anhydride in the presence of triethylamine in a dipolar solvent such as DMF at 25°C. Other effective conditions include a water-soluble carbodiimide such as 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDCI) in the presence of hydroxybenzotriazole (HOBt) and triethylamine in DMF at 25°C Scheme 2 Compounds prepared via Scheme 2 Compounds of formula I may be prepared from compounds of formula H via Minisci coupling with a carboxylic acid using 9-mesityl-10-methyl acridinium perchlorate and irradiation Steps (e) and (f) follow the same processes as the ones described in Scheme 1 Accordingly, the derivatives of the formula I can be prepared by the procedures described in the general methods presented below or by routine modifications thereof. The present invention also encompasses any one or more of these processes for preparing the derivatives of formula I, in addition to any novel intermediates used therein. The person skilled in the art will appreciate that the following reactions may be heated thermally or under microwave irradiation. In the non-limiting Examples and Preparation set out below that illustrate the invention, and in the aforementioned Scheme, the following the abbreviations, definitions and analytical procedures may be referred to: AcOH: acetic acid atm: atmosphere aq: aqueous BOC 2 O: BOC anhydride, di-tert-butyl dicarbonate br: broad °C: degrees Celsius CBZ: carboxybenzyl; benzyloxycarbonyl conc. or c.: concentrated δ: chemical shift d: doublet dd: doublet of doublets ddd: doublet of doublet of doublets dt: doublet of triplets DCM: dichloromethane DHP: dihydropyran DMAC: N,N-dimethylacetamide DMAP: 4-dimethylaminopyridine DMF: dimethylformamide DMSO: dimethyl sulfoxide EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ESI-MS: electrospray ionization mass spectrometry EtOAc: ethyl acetate Et 3 N: triethylamine equiv.: equivalent g: gram h: hour(s) HATU: (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyr idinium 3-oxide hexafluorophosphate HOPO: 2-hydroxypyridine 1-oxide HPLC: high pressure liquid chromatography iPr 2 NEt: N,N-diisopropylethylamine amine, Hunig's base iPrOH : isopropanol, 2-propanol Kg: Kilogram KOtBu: potassium tert-butoxide L: liter LAH: Lithium aluminum hydride, LiAlH 4 LCMS: liquid chromatography mass spectrometry LDA: Lithium diisopropylamide LiHMDS: Lithium bis(trimethylsilyl)amide M: multiplet M: molar MeCN: acetonitrile MHz: mega Hertz min: minutes mL: milliliter mm: millimeter mmol: millimole µmol: micromole mol: mole MS m/z: mass spectrum peak MTBE: methyl tert-butyl ether N: normal n-BuLi: n-butyl lithium NBS: N-bromosuccinimide NCS: N-chlorosuccinimide NH 4 OH: 33 aq. ammonia NMP: N-methyl pyrrolidine NMR: nuclear magnetic resonance Pd 2 (dba) 3 : Tris(dibenzylideneacetone)dipalladium(0) Pd/C: palladium on carbon PE: petroleum ether Prep: preparatory pTSA·H 2 O: p-toluenesulfonic acid monohydrate q: quartet quint: quintet RT: room temperature s: singlet sat.: saturated SFC: supercritical fluid chromatography t: triplet t-BuOH: tert-butanol TFA: trifluoroacetic acid THF: tetrahydrofuran TMSOTf: trimethylsilyl trifluoromethanesulfonate TTBP . HBF 4 : tri-tert-butylphosphonium tetrafluoroborate T3P: propylphosphonic anhydride X-Phos: 2-dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl µm: micrometer µL: microliter General experimental section: Unless otherwise stated all reactions are run under a nitrogen atmosphere. The abbreviation RT refers to “room temperature” and is generally taken to mean approximately 22 °C (± 5 °C). Unless otherwise stated, the term “concentrated” refers to the process of removal of volatile compounds such as solvents by use of a rotary evaporator under reduced pressure. The term “chromatography” refers to silica gel chromatography with mobile phase consisting of mixtures or gradients of either EtOAc/heptane or methanol/DCM or some combination thereof. 1H NMR spectra were in all cases consistent with the proposed structures. Characteristic δ for 1 H NMR are reported relative to residual solvent signals (for CDCl 3 , δH = 7.27 ppm; for DMSO-d 6 , δH = 2.50 ppm, for CD 3 OD, δH = 3.30 ppm) using conventional abbreviations for designation of major peaks. The skilled person will appreciate that tautomers may be recorded within the NMR data and some exchangeable protons may not be visible. Likewise, the skilled person will appreciate that a mixture of rotamers may be recorded within the NMR data. Mass spectra were recorded using either ESI-MS. Where relevant and unless otherwise stated the m/z data provided are for isotopes 19 F, 35 Cl, 79 Br and/or 81 Br. Where silica gel chromatography, preparative HPLC or SFC chromatography have been used, the skilled person will appreciate that any suitable solvent or solvent combination may be employed to purify the desired compound. Nomenclature for the compounds of the Preparations and Examples that follow was generated using ChemDraw Professional 19.0, Perkin Elmer, in accordance with the IUPAC (International Union of Pure and Applied Chemistry). Amidation methods A) To a mixture of carboxylic acid (1.0 equiv.) was added DIPEA (4.0 equiv.) in DMF (c = 0.14 M), followed by HATU (1.5 equiv.). The resulting mixture was stirred at about 15 ºC for about 10 min, then amine (1.0 equiv.) was added to the mixture. The reaction was stirred for about 16 h at about 15 °C. The reaction was filtered, and the filtrate was purified by prep HPLC. B) To the carboxylic acid (1.0 equiv.) was added TPTU stock solution (1.5 equiv., 0.30 M in DMF), followed by amine (1.0 equiv., 0.20 M stock solution in DMF) with 2 equiv. DIPEA (2.20 mmol, 383 µl). The mixture was stirred at about 65 ºC for about 14 h. The solvent was evaporated under a stream of N 2 gas. The resulting residue was dissolved in DMSO, filtered, and purified by prep HPLC. C) A mixture of amine (1.0 equiv.), acid (1.0 equiv.), and EDCI (1.5 equiv.) in pyridine (final concentration = 65 mM) were heated to about 110 ºC using a microwave reactor for about 30 min. The solvent was evaporated, and it was further purified by prep HPLC. D) To a mixture of carboxylic acid (1.0 equiv.) under N 2 , a solution of amine (1.0 equiv.) in DMF (c = 0.25 M) and triethylamine (6.0 equiv.) were added. The resulting mixture was stirred at about 23 ºC for about 5 min, then cooled in an ice/water bath for another 5 min. T3P (50% in DMF) was slowly added (2.0 equiv.) dropwise to the reaction mixture over 10 min. The reaction was slowly warmed to about 15 ºC over about 1.5 h, after which time H 2 O (3x volume of the reaction mixture) was added. The workup mixture was filtered, and the precipitate was collected, which was dried and purified by prep HPLC. Preparation 1: 2-(tert-butyl)-5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7 H)-one hydrochloride salt (P1) Step 1: Synthesis of tert-butyl 2-(tert-butyl)-7H-spiro[benzo[d]thiazole-6,4'-piperidine]-1' - carboxylate (C1) To a solution of tert-butyl 9-oxo-3-azaspiro[5.5]undec-7-ene-3-carboxylate (CAS: 873924- 07-3; 160 g, 603 mmol) in DCM (2.4 L) was added Et 3 N (251 mL, 1.81 mol). The solution was cooled to about -78 °C and a solution of TMSOTf (164 mL, 904 mmol) in DCM (800 mL) was added dropwise. The solution was stirred at about -78 °C for about 30 min, then warmed to about 0 °C and stirred for about 2 h at about 0 °C. The solution was cooled to about -78 °C and a suspension of NBS (118 g, 663 mmol) in DCM (800 mL) was added. The mixture was stirred at about -78 °C for about 2 h, then warmed to about 0 °C. Boc 2 O (52.6 g, 241 mmol) was added, and the mixture was stirred at about 0 °C for about 2 h. Saturated NaHCO 3 (aq) (1.6 L) was added, and the organic phase was separated and concentrated under reduced pressure. To the residue was added 2,2-dimethylpropanethioamide (84.8 g, 723 mmol), pyridine (436 mL, 542 mmol), and EtOH (2.1 L). The solution was heated to about 80 °C for about 16 h and then cooled to about 23 °C. The mixture was washed with saturated NaHCO 3 (aq) (2.0 L) and the organic phase was concentrated under reduced pressure. The residue was purified by column chromatography (silica, petroleum ether: EtOAc = 1: 0 to 3: 1) to provide the title compound (149 g, 68%). 1 H NMR (400 MHz, CDCl 3 ) δ = 6.65 (d, 1H), 5.87 (d, 1H), 3.57-3.51 (m, 2H), 3.40-3.33 (m, 2H), 2.82 (s, 2H), 1.71-1.67 (m, 2H), 1.53-1.48 (m, 2H), 1.46 (s, 9H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 363.1. Step 2: Synthesis of 2-(tert-butyl)-5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7 H)-one hydrochloride (P1) To compound C1 (188 g, 519 mmol) was added MeOH (1.88 L). The solution was cooled to about 15 °C and NBS (96.9 g, 545 mmol) was added in batches. The resulting mixture was stirred at about 15 °C for about 2 h and concentrated under reduced pressure. To the residue was added THF (2.5 L) and the resulting solution was cooled to about 15 °C. KOtBu (1 M in THF, 2.1 L) was added and the solution was stirred at about 15 °C for 16 h. Water (4.9 L) was added and the mixture was extracted with isopropyl acetate (3 X 2.8 L). The combined organic phases were concentrated under reduced pressure to provide crude tert-butyl 2-(tert-butyl)-4-methoxy- 7H-spiro[benzo[d]thiazole-6,4'-piperidine]-1'-carboxylate (intermediate C1b). To the resulting residue was added additional intermediate C1b (21.6 g, 55 mmol) from a prior batch and dioxane (970 mL), followed by dropwise addition of HCl (4 M in dioxane, 2.26 L). The solution was stirred at about 25 °C for about 16 h and then concentrated under reduced pressure. MeOH (3.5 L) was added and the solution was heated to about 60 °C and cooled to about 25 °C. The solution was concentrated under reduced pressure to remove 2.8 L of MeOH. The resulting mixture was stirred at about 25 °C for about 16 h. EtOAc (2.0 L) was added, and the resulting mixture was stirred at about 25 °C for about 16 h. The resulting slurry was filtered, and the cake was dried under reduced pressure at about 40 °C to afford the title compound (157 g, 87%). 1 H NMR (400 MHz, DMSO-d6) δ = 9.01 (br s, 1H), 8.93 (br s, 1H), 3.22 (s, 2H), 3.06 (br s, 4H), 2.62 (s, 2H), 1.76-1.65 (m, 4H), 1.37 (s, 9H); LC/MS m/z (M+H) + = 279.2. Preparation 2: 5-methyl-2-(methylamino)quinoline-7-carboxylic acid (P2) Step 1: 7-bromo-N,5-dimethylquinolin-2-amine (C2) To a solution of 7-bromo-2-chloro-5-methylquinoline (8.96 g, 34.9 mmol, prepared by the method of Aciro, C. et al. PCT Int. Appl. (2013), WO 2013185103) in methylamine (175 mL of 2 M solution in THF, 349 mmol) at about 25 °C was added cesium fluoride (10.6 g, 69.9 mmol). The mixture was heated to about 100 °C for about 48 h. The mixture was diluted with saturated NH 4 Cl (30 mL) and extracted with EtOAc. The combined organic phases were washed with brine (30 mL), dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by SFC (Column: Chiral Tech OX-H, 30x250 mm, 5 µm; Mobile Phase A: carbon dioxide; Mobile phase B: MeOH / 1% NH 3 ; 95%-70% B gradient, 3.5 min; flow rate 80 mL/min) to provide the title compound (2.81 g, 32%). LC/MS m/z (M+H) + = 252.1. Step 2: methyl 5-methyl-2-(methylamino)quinoline-7-carboxylate (C3) To a solution of compound C2 (1.15 g, 4.59 mmol) in MeOH (50 mL) was added triethylamine (2.0 mL, 14 mmol) and Pd(dppf)Cl 2 . CH 2 Cl 2 (613 mg, 0.751 mmol). The solution was added to a Parr reactor and flushed with nitrogen three times. The reactor was flushed with CO three times, then sealed at about 75 PSI CO. The reactor was heated to about 80 °C for about 24 h. The solution was cooled to about 25 °C and diluted with EtOAc. The solution was washed with water and brine. The organic phase was dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by column chromatography to provide the title compound (853 mg, 81%). LC/MS m/z (M+H) + = 231.3. Step 3: 5-methyl-2-(methylamino)quinoline-7-carboxylic acid (P2) To a solution of compound C3 (840 mg, 3.65 mmol) in THF (18 mL) was added 1 M sodium hydroxide (22 mL, 22 mmol). The solution was stirred at about 25 °C for about 16 h. HCl (1 M) was added dropwise until a precipitate formed (pH ~5). The precipitate was filtered to provide the title compound (789 mg, 88%). 1 H NMR (400 MHz, DMSO-d6) δ 8.01 (d, 1H), 7.94 (s, 1H), 7.49 (s, 1H), 7.11 (d, 1H), 6.86 (d, 1H), 2.91 (d, 3H), 2.55 (s, 3H); LC/MS m/z (M+H) + = 217.2. Preparation 3: 2-(tert-butyl)-5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7 H)-one hydrochloride salt (P3) Step 1: Synthesis of 4-(1,3-dimethyl-1H-pyrazol-4-yl)-3-(ethoxycarbonyl)but-3-eno ic acid (C4) To a solution of solid KOtBu (36.2 g, 322 mmol) in ethanol (200 mL) at about 25 °C was added 1,3-dimethyl-1H-pyrazole-4-carbaldehyde (CAS: 25016-21-0; 20 g, 160 mmol) and diethyl succinate (42.1 g, 40 mL, 242 mmol). The reaction was heated at about 80 °C for about 18 h, then cooled to RT and quenched using AcOH (23 mL, 403 mmol). The reaction mixture was concentrated to about (40 mL) and heptane (2 x 100 mL) was added, and reaction mixture concentrated. The reaction was cooled to about 0-10 °C, charged with water (200 mL) and the pH of the aqueous layer adjusted to 4-5 with aqueous HCl. The resulting suspension was filtered and rinsed with water (60 mL). The solids were dried to provide the title compound (33.4 g, 82.1%). 1 H NMR (400 MHz, Methanol-d4) δ 7.81 (s, 1H), 7.63 (s, 1H), 4.25 (q, 2H), 3.85 (s, 3H), 3.53 (s, 2H), 2.28 (s, 3H), 1.32 (t, 2H); LC/MS m/z (M+H) + = 253.1. Step 2: Synthesis of ethyl 7-acetoxy-1,3-dimethyl-1H-indazole-5-carboxylate (C5) To a solution of compound C4 (32.0 g, 126.9 mmol) in DMAC (128 mL) was added sodium acetate (26.0 g, 317 mmol) and acetic anhydride (32 mL, 2.67 mmol). The reaction was heated at about 95-105 °C for about 18 h, then cooled to about 0-10 °C, and water (512 mL) was added dropwise. The resulting suspension was filtered and rinsed with water (100 mL). The precipitate was recrystallized using water and dried to provide the title compound (28.8 g, 82.9%). 1 H NMR (400 MHz, Methanol-d4) δ 8.33 (s, 1H), 7.76 (s, 1H), 4.42 (q, 2H), 4.11 (s, 3H), 2.59 (s, 3H), 2.47 (s, 3H), 1.44 (t, 3H); %); LC/MS m/z (M+H) + = 277.1. Step 3: Synthesis of ethyl 7-hydroxy-1,3-dimethyl-1H-indazole-5-carboxylate (C6) To a solution of compound C5 (1.11 g, 4.0 mmol) in ethanol (16 mL) was added potassium carbonate (2.76 g, 5.0 mmol). The reaction was heated at reflux for about 1.5 h and then concentrated. The residue was diluted with EtOAc (25 mL) and water (25 mL) and pH of the aqueous layer was adjusted to 4-5 with aqueous citric acid. The organic layer was separated, washed with brine (25 mL), dried over MgSO 4 , filtered, and concentrated, residue was diluted with hexanes (25 mL) and the resulting mixture concentrated under reduced pressure, dried using high vacuum around 50 °C to provide the title compound (0.89 g, 95% yield) as a light pink solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 7.81 (d, 1H), 7.26 (s, 1H), 4.29 (q, 2H), 4.15 (s, 3H), 2.45 (s, 3H), 1.33 (t, 3H); LC/MS m/z (M+H) + = 235.1. Step 4: Synthesis of ethyl 7-methoxy-1,3-dimethyl-1H-indazole-5-carboxylate (C7) To a solution of compound C6 (0.7 g, 3.0 mmol) in acetone (20 mL) was added potassium carbonate (0.83 g, 6.0 mmol) and methyl iodide (0.64 g, 0.28 mL, 4.5 mmol). The reaction was heated at reflux for about 24 h and then concentrated. The residue was diluted with water (25 mL) and extracted with EtOAc (2 x 25 mL). The combined EtOAc extracts were washed with brine (25 mL), dried over MgSO 4 , filtered, and concentrated. The residue was purified by chromatography (silica, 0-25% EtOAc in hexanes) to provide the title compound (0.71 g, 95.3% yield) as a white solid. 1 H NMR (400 MHz, Chloroform-d) δ 8.04 (s, 1H), 7.37 (s, 1H), 4.44 (q, 2H), 4.27 (s, 3H), 4.03 (s, 3H), 2.59 (s, 3H), 1.46 (t, 3H); LC/MS m/z (M+H) + = 249.1. Step 5: Synthesis of 7-methoxy-1,3-dimethyl-1H-indazole-5-carboxylic acid (P3) To compound C7 (0.3 g, 1.21 mmol) in EtOH (4.8 mL), THF (4.3 mL), was added 1N NaOH (4.83 mL, 4.83 mmol). The mixture was heated to about 45 °C for about 2 h, then cooled to about 25 °C and concentrated. The residue was diluted with water (2 mL), the pH of aqueous layer adjusted to 4-5 using 1.5 M citric acid. The resulting suspension was filtered and rinsed with water (5 mL). The precipitate was dried at 50 °C to provide the title compound (0.23 g, 86.4%). 1 H NMR (400 MHz, Methanol-d4) δ 7.99 (d, J = 1.2 Hz, 1H, 4), 7.38 (d, J = 1.2 Hz, 1H, 2), 4.19 (s, 3H, 16), 4.00 (s, 3H, 10), 2.51 (s, 3H, 11); LC/MS m/z (M+H) + = 221.1. Examples Example 1: 1'-(3,7-dimethyl-2H-indazole-5-carbonyl)-2-(1-methylcyclopro pyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one A solution of 2-(1-methylcyclopropyl)-5H-spiro[benzo[d]thiazole-6,4'-piper idin]-4(7H)-one, prepared like compound P1, (0.06 g, 0.22 mmol), 3,7-dimethyl-1H-indazole-5-carboxylic acid (0.05 g, 0.26 mmol), EDCI . HCl (0.08 g, 0.43 mmol), and pyridine (2 mL) was microwaved at about 110 °C for about 30 min. The resulting mixture was filtered, and the residue was purified by prep- HPLC (Column: YMC Actus Triart C18, 150x30 mm, 5 µm); Mobile Phase A: water (0.225% v/v formic acid); Mobile Phase B: MeCN; 30-50% B gradient, 11 min, hold at 100% B for 2 min; flow rate 35 mL/min to provide the title compound (15 mg, 16%). 1 H NMR (400 MHz, Methanol-d4) δ 7.64 (d, 1H), 7.20 (t, 1H), 3.79 (m, 2H), 3.55 (m, 2H), 3.21 (s, 2H), 2.70 (s, 2H), 2.56 (s, 3H), 2.55 (s, 3H), 1.64 (s, 4H), 1.55 (s, 3H), 1.29 (q, 2H), 1.04 – 0.95 (m, 2H); LC/MS m/z (M+H) + = 449.4. Example 2: 1'-(3,7-dimethyl-1H-indazole-5-carbonyl)-2-(1-methylcyclobut yl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one A solution of 2-(1-methylcyclobutyl)-5H-spiro[benzo[d]thiazole-6,4'-piperi din]-4(7H)-one, prepared like compound P1, (0.022 g, 0.077 mmol), 3,7-dimethyl-1H-indazole-5-carboxylic acid (0.017 g, 0.092 mmol), EDCI . HCl (0.029 g, 0.154 mmol), and pyridine (1 mL) was microwaved at about 110 °C for about 30 min. The resulting mixture was filtered, and the residue was purified by prep-HPLC using water/acetonitrile (Column: Sunfire C18, 19x100 mm, 5 µm) to provide the title compound (6.9 mg, 19%). LC/MS m/z (M+H) + = 463.3. Example 3: 1'-(3,7-dimethyl-1H-indazole-5-carbonyl)-2-(2,3-dimethylbuta n-2-yl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: Synthesis of tert-butyl 2-(2,3-dimethylbutan-2-yl)-4-oxo-4,7-dihydro-5H- spiro[benzo[d]thiazole-6,4'-piperidine]-1'-carboxylate (C8) To a solution of tert-butyl 4-oxo-4,7-dihydro-5H-spiro[benzo[d]thiazole-6,4'-piperidine] -1'- carboxylate, prepared like compound P1, (0.1 g, 0.31 mmol) in acetonitrile (1.5 mL) and water (1.5 mL) was added 2,2,3-trimethylbutanoic acid (0.121 g, 0.93 mmol), Na 2 HPO 4 (0.132 g, 0.93 mmol) and 9-mesityl-10-methyl acridinium perchlorate (3.83 g, 0.0093 mmol). The reaction mixture was irradiated with a 72 W blue LED strip for about 60 h. The resulting mixture was filtered, and the residue was purified by prep-HPLC (Column: Phenomenex Gemini-NX C18, 50x250 mm, 10 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile Phase B: MeCN; 63-83% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min to provide the title compound (30 mg, 23.8%). 1 H NMR (400 MHz, Methanol-d4) δ 3.52 – 3.44 (m, 4H), 3.20 (s, 2H), 2.68 (s, 2H), 2.12 (h, 1H), 1.60 (m, 4H), 1.47 (s, 9H), 1.38 (s, 6H), 0.88 (s, 3H), 0.87 (s, 3H); LC/MS m/z (M+H) + = 407.4. Step 2: Synthesis of 2-(2,3-dimethylbutan-2-yl)-5H-spiro[benzo[d]thiazole-6,4'-pi peridin]- 4(7H)-one (C9) To a solution of compound C8 (0.03 g, 0.074 mmol) in MeOH (2 mL) was added 4 N HCl in dioxane (1 mL) and stirred at about 20 °C for about 2 h. The reaction mixture was concentrated to give yellow oil, acetonitrile (2 x 5 mL) was added, and reaction was concentrated under reduced pressure, dried in vacuum to provide title compound (25 mg, 99%). LC/MS m/z (M+H) + = 307.1. Step 3: Synthesis of 1'-(3,7-dimethyl-1H-indazole-5-carbonyl)-2-(2,3-dimethylbuta n-2-yl)- 5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one A solution of 2-(2,3-dimethylbutan-2-yl)-5H-spiro[benzo[d]thiazole-6,4'-pi peridin]-4(7H)- one, (0.015 g, 0.08 mmol), 3,7-dimethyl-1H-indazole-5-carboxylic acid (0.025 g, 0.07 mmol), EDCI . HCl (0.027 g, 0.146 mmol), and pyridine (2 mL) was microwaved at about 110 °C for about 30 min. The resulting mixture was concentrated, and the residue was purified by prep-HPLC (Column: Phenomenex Gemini-NX C18, 40x80 mm, 3 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile Phase B: MeCN; 31-71% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min to provide the title compound (5.56 mg, 16%). 1 H NMR (400 MHz, Methanol-d 4 ) δ 7.67 (dd, 1H), 7.23 (t, 1H), 3.82 (m, 2H), 3.58 (m, 2H), 3.27 (s, 2H), 2.76 (s, 2H), 2.55 (s, 3H), 2.54 (s, 3H), 2.12 (h, 1H), 1.53 (m, 4H), 1.37 (s, 6H), 0.88 (s, 3H), 0.87 (s, 3H); LC/MS m/z (M+H) + = 479.4. Example 4: 1'-(3,7-dimethyl-1H-indazole-5-carbonyl)-2-(1-methylcyclopen tyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one A solution of 2-(1-methylcyclopentyl)-5H-spiro[benzo[d]thiazole-6,4'-piper idin]-4(7H)-one, prepared like compound C9, (0.024 g, 0.07 mmol), 3,7-dimethyl-1H-indazole-5-carboxylic acid (0.015 g, 0.07 mmol), EDCI . HCl (0.027 g, 0.141 mmol), and pyridine (2 mL) was microwaved at about 110 °C for about 30 min. The resulting mixture was filtered, and the residue was purified by prep-HPLC (Column: Phenomenex Gemini-NX C18, 40x80 mm, 3 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile Phase B: MeCN; 29-69% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min to provide the title compound (7.2 mg, 21%). 1 H NMR (400 MHz, Methanol-d4) δ 7.66 (s, 1H), 7.22 (s, 1H), 3.82 (m, 2H), 3.59 (m, 2H), 3.27 (s, 2H), 2.75 (s, 2H), 2.58 (m, 6H), 2.19 (m, 2H), 1.86 – 1.75 (m, 7H), 1.68 (s, 3H), 1.46 (s, 3H); LC/MS m/z (M+H) + = 477.3. Example 5: 1'-(3,7-dimethyl-1H-indazole-5-carbonyl)-2-(tert-pentyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 3, substituting 2,2-dimethylbutanoic acid for 2,2,3-trimethylbutanoic acid in Step 1, to provide the title compound (8.3 mg). LC/MS m/z (M+H) + = 465.3. Example 6: 1'-(3,7-dimethyl-1H-indazole-5-carbonyl)-2-ethyl-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one According to amidation method A, 2-ethyl-5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)- one, prepared like compound P1, (0.08 g, 0.28 mmol) was coupled with 3,7-dimethyl-1H- indazole-5-carboxylic acid (0.056 g, 0.28 mmol). The residue was purified by prep-HPLC (Column: Phenomenex Gemini-NX C18, 40x80 mm, 3 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile Phase B: MeCN; 9-60% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min to provide the title compound (45 mg, 38%). 1 H NMR (400 MHz, Methanol-d 4 ) δ 7.67 (d, 1H), 7.22 (d, 1H), 3.83 (m, 2H), 3.55 (m, 2H), 3.26 (s, 2H), 3.03 (q, 2H), 2.75 (s, 2H), 2.58 (s, 3H), 2.57 (s, 3H), 1.75 (m, 2H), 1.65 (m, 2H), 1.38 (t, 3H); LC/MS m/z (M+H) + = 423.1. Example 7: 2-(bicyclo[1.1.1]pentan-1-yl)-1'-(3,7-dimethyl-1H-indazole-5 -carbonyl)- 5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one According to amidation method A, 2-(bicyclo[1.1.1]pentan-1-yl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one, prepared like compound P1, (0.08 g, 0.28 mmol) was coupled with 3,7-dimethyl-1H-indazole-5-carboxylic acid (0.056 g, 0.28 mmol). The residue was purified by prep-HPLC (Column: Phenomenex Gemini-NX C18, 40x80 mm, 3 µm); Mobile Phase A: water (0.225% v/v formic acid); Mobile Phase B: MeCN; 19-59% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min to provide the title compound (28.5 mg, 40%). 1 H NMR (400 MHz, Methanol-d 4 ) δ 7.66 (dd, 1H), 7.22 (t, 1H), 3.81 (s, 2H), 3.57 (s, 2H), 3.26 (s, 2H), 2.75 (s, 2H), 2.57 (s, 3H) 2.56 (s, 3H), 2.27 (s, 7H), 1.74 (s, 2H), 1.65 (s, 2H); LC/MS m/z (M+H) + = 461.4. Example 8: 2-isopropyl-1'-(5-methyl-2-(methylamino)quinoline-7-carbonyl )-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one According to amidation method A, 2-isopropyl-5H-spiro[benzo[d]thiazole-6,4'-piperidin]- 4(7H)-one, prepared like compound P1, (0.034 g, 0.116 mmol) was coupled with 5-methyl-2- (methylamino)quinoline-7-carboxylic acid (0.03 g, 0.14 mmol). The residue was purified by prep- HPLC (Column: Phenomenex Gemini-NX C18, 40x80 mm, 3 µm); Mobile Phase A: water (0.225% v/v formic acid); Mobile phase B: MeCN; 20-50% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min to provide the title compound (23.2 mg, 36%). 1 H NMR (400 MHz, Methanol-d 4 ) δ 8.06 (dd, 1H), 7.57 – 7.50 (m, 1H), 7.05 (dd, 1H), 6.83 (d, 1H), 3.85 (m, 2H), 3.55 (m, 2H), 3.31 (h, 1H), 3.27 (s, 2H), 3.03 (s, 3H), 2.75 (d, 2H), 2.61 (s, 3H), 1.78 (m, 2H), 1.66 (m, 2H), 1.40 (d, 6H); LC/MS m/z (M+H) + = 463.4. Example 9: 1'-(5-methyl-2-(methylamino)quinoline-7-carbonyl)-2-(1- methylcyclopropyl)-5H-spiro[benzo[d]thiazole-6,4'-piperidin] -4(7H)-one According to amidation method A, 2-(1-methylcyclopropyl)-5H-spiro[benzo[d]thiazole- 6,4'-piperidin]-4(7H)-one, prepared like example P1, (0.036 g, 0.116 mmol) was coupled with 5- methyl-2-(methylamino)quinoline-7-carboxylic acid (0.03 g, 0.14 mmol). The residue was purified by prep-HPLC (Column: Boston Prime C18, 150x30 mm, 5 µm); mobile phase A: water (0.05% v/v conc. NH 4 OH); mobile phase B: MeCN; 26-56% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min to provide the title compound (24.7 mg, 38%). 1 H NMR (400 MHz, Methanol- d 4 ) δ 8.06 (dd, 1H), 7.57 – 7.50 (m, 1H), 7.05 (dd, 1H), 6.83 (d, 1H), 3.91 – 3.78 (m, 2H), 3.57 – 3.51 (m, 2H), 3.25 (s, 2H), 3.03 (s, 3H), 2.73 (d, 2H), 2.61 (s, 3H), 1.80 – 1.74 (m, 2H), 1.68 – 1.62 (m, 2H), 1.58 (s, 3H), 1.36 – 1.27 (m, 2H), 1.06 – 0.93 (m, 2H); LC/MS m/z (M+H) + = 475.4. Example 10: 2-isopropyl-1'-(7-methoxy-1,3-dimethyl-1H-indazole-5-carbony l)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one According to amidation method A, 2-isopropyl-5H-spiro[benzo[d]thiazole-6,4'-piperidin]- 4(7H)-one, prepared like example P1, (0.047 g, 0.159 mmol) was coupled with 7-methoxy-1,3- dimethyl-1H-indazole-5-carboxylic acid (0.035 g, 0.16 mmol). The residue was purified by prep- HPLC (Column: Boston Prime C18, 150x30 mm, 5 µm); mobile phase A: water (0.05% v/v formic acid); mobile phase B: MeCN; 14-54% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min to provide the title compound (17.7 mg, 24%). 1 H NMR (400 MHz, Methanol-d 4 ) δ 7.35 (d, 1H), 6.87 (d, 1H), 4.21 (s, 3H), 4.02 (s, 3H), 3.80 (m, 2H), 3.61 (m, 2H), 3.29 (h, 1H), 3.27 (s, 2H), 2.75 (s, 2H), 2.51 (s, 3H), 1.71 (m, 4H), 1.40 (d, 6H); LC/MS m/z (M+H) + = 467.3. Example 11: 1'-(7-methoxy-1,3-dimethyl-1H-indazole-5-carbonyl)-2-(1- methylcyclopropyl)-5H-spiro[benzo[d]thiazole-6,4'-piperidin] -4(7H)-one According to amidation method A, 2-(1-methylcyclopropyl)-5H-spiro[benzo[d]thiazole- 6,4'-piperidin]-4(7H)-one, prepared like example P1, (0.05 g, 0.16 mmol) was coupled with 7- methoxy-1,3-dimethyl-1H-indazole-5-carboxylic acid (0.035 g, 0.16 mmol). The residue was purified by prep-HPLC (Column: YMC Triart C18, 150x25 mm, 5 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile phase B: MeCN; 26-56% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min to provide the title compound (19 mg, 25%). 1 H NMR (400 MHz, Methanol-d 4 ) δ 7.35 (d, 1H), 6.86 (d, 1H), 4.21 (s, 3H), 4.02 (s, 3H), 3.83 – 3.76 (m, 2H), 3.63 – 3.57 (m, 2H), 3.24 (s, 2H), 2.73 (s, 2H), 2.51 (s, 3H), 1.72 – 1.66 (m, 4H), 1.58 (s, 3H), 1.36 – 1.27 (m, 2H), 1.07 – 0.97 (m, 2H); LC/MS m/z (M+H) + = 479.4. Example 12: 1'-(7-methoxy-1,3-dimethyl-1H-indazole-5-carbonyl)-2-(2-(met hyl- d3)propan-2-yl-1,1,1,3,3,3-d6)-5H-spiro[benzo[d]thiazole-6,4 '-piperidin]-4(7H)-one To a solution of 2-(2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6)-5H-spiro[benzo[d ]thiazole- 6,4'-piperidin]-4(7H)-one, prepared like preparation P1 (0.105 g, 0.366 mmol), 7-methoxy-1,3- dimethyl-1H-indazole-5-carboxylic acid (0.1 g, 0.454 mmol), EDCI . HCl (0.126 g, 1.8 mmol), and HOBt (0.79 g, 0.586 mmol) in DMF (15 mL) at about 25 °C was added Et 3 N (0.51 mL, 3.66 mmol). The mixture was stirred at about 25 °C for about 16 h, then diluted with EtOAc (50 mL). The mixture was washed with saturated aqueous Na 2 CO 3 (2 x 25 mL), and brine (2 x 25 mL). The organic phase was dried over MgSO 4 , filtered, and concentrated. The residue was purified by chromatography (silica, EtOAc: Heptane’s, 0-100%) to provide the title compound (0.15 g, 67.5%) as a white solid. 1 H NMR (400 MHz, Methanol-d4) δ 7.35 (s, 1H), 6.87 (s, 1H), 4.22 (s, 3H), 4.03 (s, 3H), 3.64 (m, 4H, 11), 3.28 (s, 2H), 2.76 (s, 2H), 2.52 (s, 3H), 1.72 (m, 4H); LC/MS m/z (M+H) + = 490.2. Example 13: 2-(tert-butyl)-1'-(7-methoxy-1,3-dimethyl-1H-indazole-5-carb onyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one To a solution of preparation P1 (0.97 g, 3.1 mmol), preparation P3 (0.68 g, 3.1 mmol), EDCI . HCl (0.89 g, 4.65 mmol), and HOBt (0.73 g, 4.65 mmol) in DMF (25.8 mL) at about 25 °C was added i Pr 2 NEt (2.97 mL, 17.0 mmol). The mixture was stirred at about 25 °C for about 16 h, then diluted with EtOAc (50 mL). The mixture was washed sequentially with 5% aqueous LiCl (25 mL), 0.5 N HCl (25 mL), saturated aqueous NaHCO 3 (20 mL), and 1:1 brine-water (30 mL). The organic phase was dried over MgSO 4 , filtered, concentrated, heptane (2 x 25 mL) was added and resulting mixture was concentrated under reduced pressure. The residue product was purified by chromatography (silica, EtOAc/Hexanes, 0-100% then MeOH: EtOAc, 0-10%) to provide the title compound (1.05 g, 70.4% yield) as a white solid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.23 (s, 1H), 6.74 (s, 1H), 4.22 (s, 3H), 3.96 (s, 3H), 3.73 (m, 2H), 3.62 (m, 2H), 3.10 (s, 2H), 2.71 (s, 2H), 2.50 (s, 3H), 1.68 (m, 4H), 1.45 (s, 9H); LC/MS m/z (M+H) + = 481.3. Example 14: 2-(tert-butyl)-1'-(7-ethoxy-1,3-dimethyl-1H-indazole-5-carbo nyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: Synthesis of ethyl 7-ethoxy-1,3-dimethyl-1H-indazole-5-carboxylate (C10) To a solution of compound C6 (0.328 g, 1.4 mmol) in acetone (9.3 mL) was added potassium carbonate (0.33 g, 1.7 mmol) and ethyl iodide (0.30 g, 0.16 mL, 1.96 mmol). The reaction was heated at reflux for about 24 h and then concentrated. The residue was diluted with water (25 mL) and extracted with EtOAc (2 x 25 mL). The combined EtOAc extracts, washed with brine (25 mL), dried over MgSO 4 , filtered, and concentrated. The residue was purified by chromatography (silica, EtOAc/Hexanes, 0-25%) to provide the title compound (0.27 g, 74% yield) as a white solid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.99 (s, 1H), 7.32 (s, 1H), 4.40 (q, 2H), 4.23 (s, 3H), 4.22 (q, 2H), 2.56 (s, 3H), 1.52 (t, 3H), 1.42 (t, 3H); LC/MS m/z (M+H) + = 263.1. Step 2: Synthesis of 7-ethoxy-1,3-dimethyl-1H-indazole-5-carboxylic acid (C11) To compound C10 (268 mg, 1.02 mmol) in EtOH (5.1 mL) and THF (5.1 mL) was added 1 M aqueous NaOH (5.1 mL, 5.1 mmol). The mixture was stirred at about 25 °C for about 17 h, then concentrated under reduced pressure. To the mixture was added 1.5 M aqueous citric acid (2.5 mL) and water (2 mL). After stirring for about 3 minutes, the precipitate was isolated via filtration to provide the title compound (230 mg, 96.3%). 1 H NMR (400 MHz, DMSO-d6) δ 12.78 (s, 1H), 7.91 (d, 1H), 7.27 (s, 1H), 4.22 (q, 2H), 4.16 (s, 3H), 2.46 (s, 3H), 1.45 (t, 3H). Step 3: 2-(tert-butyl)-1'-(7-ethoxy-1,3-dimethyl-1H-indazole-5-carbo nyl)-5H-spiro[ben- zo[d]thiazole-6,4'-piperidin]-4(7H)-one (Example 14) To a flask containing compound C11 (1.23 g, 5.0 mmol), EDCI.HCl (1.2 g, 6.25 mmol) and HOPO (0.694 mg, 6.25 mmol) was added acetonitrile (25.0 mL) followed by i Pr 2 NEt (2.18 mL, 2.5 mmol). The mixture was heated to about 50 o C for about 2 h and then cooled to room temperature. Preparation P1 (2.23 g, 5.5 mmol), i Pr 2 NEt (3.48 mL, 4.00 mmol) and water (5.0 mL) was added, and mixture heated again to about 50 o C for about 2 h, followed by stirring at room temperature for about 16 h. The reaction mixture was diluted with EtOAc (150 mL) and washed with water (2 x 100 mL). The aqueous layer was back extracted using EtOAc (50 mL). The combined EtOAc extracts were then washed with 0.25 N HCl (2 x 50 mL) and the aqueous layer back extracted using EtOAc (50 mL). The combined EtOAc extracts were then washed with saturated aqueous NaHCO 3 (2 x 50 mL) and aqueous layer back extracted using EtOAc (50 mL). The combined EtOAc extracts were washed with brine (50 mL), dried over MgSO 4 , filtered over celite and concentrated to give light yellow solid (2.47 g, 99%). The solid was dissolved in EtOAc (20 ml) and heated to about 50 o C for about 10 min followed by addition of heptanes (200 mL) and further heating to about 70 o C for about 2 h. After cooling to room temperature, the mixture was stirred for about 72 h. The solid was filtered, washed with chilled premixed 25% EtOAc- Heptanes (100 mL), dried on high vacuum to provide the title compound (2.1 g, 84.9%). 1 H NMR (400 MHz, CD 3 OD) δ 7.31 (s, 1H), 6.82 (s, 1H), 4.23 (q, 2H), 4.21 (s, 3H), 3.80-3.60 (m, 4H), 3.25 (s, 2H), 2.73 (s, 2H), 2.49 (s, 3H), 1.68 (m, 4H), 1.52 (t, 3H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 495.1 Example 15: 2-(tert-butyl)-1'-(8-methyl-3-(methylamino)quinoline-6-carbo nyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: Synthesis of ethyl 8-methylquinoline-6-carboxylate (C12) To a solution of 6-bromo-8-methylquinoline (900 mg, 4.05 mmol) in EtOH (25 mL) was added Pd(OAc) 2 (91 mg, 0.405 mmol), DBU (0.91 mL, 6.08 mmol), Mo(CO) 6 (588 mg, 2.23 mmol), and TTBP . HBF 4 (118 mg, 0.405 mmol). The mixture was sparged with N 2 for about 1 min, sealed in a microwave tube, and irradiated in the microwave (Biotage Smith Synthesizer) at about 120 °C for about 70 min. The mixture was concentrated under reduced pressure to provide the crude residue. Purification by chromatography (0 to 25% EtOAc in petroleum ether) provided the title compound (800 mg, 92%). LC/MS m/z (M+H) + = 215.9. Step 2: Synthesis of ethyl 3-bromo-8-methylquinoline-6-carboxylate (C13) The following reaction was carried out in 2 batches in parallel. To a solution of compound C12 (350 mg, 1.63 mmol) in CCl 4 (15 mL) was added pyridine (0.26 mL, 3.25 mmol) and Br 2 (0.10 mL, 1.95 mmol). The resulting solution was stirred at about 70 °C for about 4 h. The mixture was cooled to about 25 °C and poured into H 2 O (20 mL). The mixture was extracted with EtOAc (2 X 15 mL), washed with sat. aq. NaHCO 3 solution, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The 2 batches were combined, and the residue was purified by chromatography (0% to 20% EtOAc in petroleum ether) to provide the title compound (500 mg, 26% per reaction). 1 H NMR (400 MHz, DMSO-d6) δ = 9.08 (d, 1H), 8.91 (d, 1H), 8.49 (s, 1H), 8.10 (s, 1H), 4.39 (q, 2H), 2.74 (s, 3H), 1.38 (t, 3H); LC/MS m/z (M+H) + = 295.8. Step 3: ethyl 3-((tert-butoxycarbonyl)(methyl)amino)-8-methylquinoline-6-c arboxylate (C14) To compound C13 (500 mg, 1.70 mmol) was added tert-butyl methylcarbamate (334 mg, 2.55 mmol), Pd 2 (dba) 3 (78 mg, 0.085 mmol), X-Phos (81 mg, 0.17 mmol), Cs 2 CO 3 (1.66 g, 5.1 mmol) and toluene (17 mL). The mixture was sparged with N 2 . The mixture was stirred at about 120 °C for about 16 h. The mixture was cooled to about 25 °C and filtered. The filtrate was concentrated under reduced pressure and diluted with EtOAc (20 mL). The mixture was washed with H 2 O (3 X 5 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by chromatography (0% to 30% EtOAc in petroleum ether) to provide the title compound (450 mg, 77%). LC/MS m/z (M+H) + = 345.2. Step 4: 3-((tert-butoxycarbonyl)(methyl)amino)-8-methylquinoline-6-c arboxylic acid (C15) To compound C14 (450 mg, 1.31 mmol) in MeOH (12 mL) and H 2 O (4.0 mL) was added LiOH . H2O (164 mg, 3.92 mmol). The mixture was stirred at about 25 °C for about 4 h. The reaction mixture was concentrated under reduced pressure then diluted with H 2 O (10 mL). 1 N HCl was added until the pH was 3-4 and the mixture was extracted with EtOAc (2 X 20 mL). The combined organic phases were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to provide the title compound (400 mg, 97%). 1 H NMR (400 MHz, DMSO-d6) δ = 9.01 (d, 1H), 8.46 (s, 1H), 8.39 (d, 1H), 8.03 (s, 1H), 3.33 (s, 3H), 2.74 (s, 3H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 317.3. Step 5: 8-methyl-3-(methylamino)quinoline-6-carboxylic acid hydrochloride salt (C16) To compound C15 (500 mg, 1.58 mmol) in dioxane (6.0 mL) was added 4 N HCl in dioxane (6.0 mL). The solution was stirred at about 25 °C for about 1.5 h, then concentrated under reduced pressure to provide the title compound (399 mg, 100%). LC/MS m/z (M+H) + = 217.0. Step 6: Synthesis of 2-(tert-butyl)-1'-(8-methyl-3-(methylamino)quinoline-6-carbo nyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one (Example 15) To a solution of compound C16 (200 mg, 0.79 mmol) in DMF (4.0 mL) was added HATU (301 mg, 0.79 mmol) and iPr 2 NEt (0.40 mL, 2.31 mmol). The mixture was stirred at about 25 °C for about 10 min. Then was added Preparation P1 (208 mg, 0.66 mmol). The mixture was stirred at about 25 °C for about 1 h. The residue was concentrated and purified by prep-HPLC (Column: Boston Prime C18, 30x150 mm, 5 µm; Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile phase B: MeCN; 28-58% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min) to provide the title compound (229 mg, 61%). 1 H NMR (400 MHz, CD 3 OD) δ = 8.45 (d, 1H), 7.58 (d, 1H), 7.21 (dd, 1H), 7.10 (d, 1H), 3.83 (m, 2H), 3.52 (s, 2H), 3.25 (s, 2H), 2.89 (s, 3H), 2.73 (d, 2H), 2.69 (s, 3H), 1.70 (m, 4H), 1.43 (s, 9H). LC/MS m/z (M+H) + = 477.3. Example 16: 2-(tert-butyl)-1'-(4-methyl-2-naphthoyl)-5H-spiro[benzo[d]th iazole-6,4'- piperidin]-4(7H)-one Step 1: Synthesis of methyl 4-hydroxy-2-naphthoate (C17) To a solution of 4-hydroxy-2-naphthoic acid (18.8 g, 100 mmol) in MeOH (500 mL) was added conc. H 2 SO 4 (8.33 mL, 150 mmol) slowly than the reaction was refluxed for about 48 h. The solvent was concentrated under reduced pressure and the residue was diluted with EtOAc (250 mL), washed sequentially with water (150 mL), and brine (150 mL), dried over MgSO 4 , filtered, and concentrated to provide dark brown solid (19.6 g, 96.9%) used directly in the next step without further purification; 1 H NMR (400 MHz, Chloroform-d) δ 8.28 (d, 1H), 8.23 (s, 1H), 7.94 (d, 1H), 7.67 – 7.54 (m, 4H), 4.02 (s, 3H); LC/MS m/z (M-H) + = 201.1. Step 2: Synthesis of methyl 4-(((trifluoromethyl)sulfonyl)oxy)-2-naphthoate (C18) To a solution of methyl 4-hydroxy-2-naphthoate C17 (19.4 g, 96 mmol) in CH 2 Cl 2 (480 mL) at about 0 °C was added iPr 2 NEt (83.6 mL, 480 mmol) followed by slow addition of N-Phenyl- bis(trifluoromethanesulfonamide) (45.3 g, 127 mmol) portion-wise over about 10 min. The mixture was allowed to stir at about 25 °C for about 16 h. The reaction was quenched using 1 N HCl (350 mL), then the organic phase was separated. The aqueous phase was extracted with additional CH 2 Cl 2 . The combined organic phases were washed sequentially with saturated aqueous Na 2 CO 3 (200 mL) and brine (100 mL), dried over MgSO 4 , filtered, and concentrated. The residue was purified by chromatography (silica, EtOAc/Hexanes, 0-10%) to provide the title compound (30 g, 93% yield) as a white solid; 1 H NMR (400 MHz, Chloroform-d) δ 8.65 (s, 1H), 8.12 (d, 1H), 8.08 – 8.01 (m, 2H), 7.78 (t, 1H), 7.69 (t, 1H), 7.56 (dt, 1H), 7.41 (d, 1H), 4.01 (s, 3H); LC/MS m/z (M+H) + = 335.1. Step 3: Synthesis of methyl 4-methyl-2-naphthoate (C19) To a solution of methyl 4-(((trifluoromethyl)sulfonyl)oxy)-2-naphthoate C18 (22.4 g, 67 mmol) in dioxane (134 mL) was added potassium carbonate (30.6 g, 221 mmol), trimethylboroxine (25.2 g, 28.1 mL, 210 mmol), and water (3.36 mL, 2.78 mmol). The mixture was degassed and purged with N 2 /vacuum cycle (3 times) then Pd(dppf)Cl 2 (1.37 g, 1.68 mmol) was added. The reaction was heated to about 60 °C for about 2 h. The mixture was cooled to about 25 °C and diluted with EtOAc (250 mL). The EtOAc layer washed with water (100 mL), brine (100 ml), dried over MgSO 4 , filtered, and concentrated. The residue was purified by chromatography (silica, EtOAc/Hexanes, 0-5%) to provide the title compound (10.2 g, 75% yield) as a white solid; 1 H NMR (400 MHz, Chloroform-d) δ 8.38 (s, 1H), 7.93 (d, 1H), 7.87 (d, 1H), 7.83 (d, 1H), 7.59 – 7.39 (m, 2H), 3.92 – 3.87 (m, 3H), 2.64 (s, 3H); LC/MS m/z (M+H) + = 201.1. Step 4: Synthesis of 4-methyl-2-naphthoic acid (C20) To a solution of methyl 4-methyl-2-naphthoate C19 (13.8 g, 68.9 mmol) in THF (230 mL) and methanol (230 mL) was added a premixed solution of LiOH (6.6 g, 276 mmol) in water (138 mL). After about 96 h, the mixture was concentrated, and the residue diluted with 1 N NaOH (50 mL). The aqueous residue was extracted with EtOAc (2 x 50 mL), and the organic layer was discarded. The pH of the aqueous layer was adjusted to 4-5 with conc. HCl. The resulting suspension was filtered. The solids were dried to provide the title compound (9.6 g, 74.8%). 1 H NMR (400 MHz, Methanol-d4) δ 8.48 (s, 1H), 8.10 (d, 1H), 8.01 (d, 1H), 7.91 (s, 1H), 7.72 – 7.64 (m, 1H), 7.59 (t, 1H), 2.75 (s, 3H); LC/MS m/z (M-H) + = 185.2. Step 5: Synthesis of 2-(tert-butyl)-1'-(4-methyl-2-naphthoyl)-5H-spiro[benzo[d]th iazole- 6,4'-piperidin]-4(7H)-one (Example 16) To a solution of preparation P1 (15.7 g, 50 mmol), 4-methyl-2-naphthoic acid C20 (9.31 g, 50 mmol), EDCI . HCl (14.4 g, 75 mmol), and HOBt (11.9 g, 75 mmol) in DMF (333 mL) at about 25 °C was added i Pr 2 NEt (61 mL, 350 mmol). The mixture was stirred at about 25 °C for about 16 h, then diluted with EtOAc (300 mL). The mixture was washed sequentially with 1 N HCl (300 mL), saturated aqueous NaHCO 3 (200 mL), and brine (200 mL). The organic phase was dried over MgSO4, filtered, and concentrated. The residue was redissolved in DCM (300 mL) and washed with 5% aqueous LiCl (100 mL). The DCM layer was separated and dried over MgSO 4 to provide the solid. The residue was purified by chromatography (silica, EtOAc/Hexanes, 10- 86%) to provide the title compound (16.9 g, 76% yield) as a white solid; 1 H NMR (400 MHz, Methanol-d4) δ 8.08 (d, 1H), 7.95 (d, 1H), 7.80 (s, 1H), 7.60 (m, 2H), 7.37 (s, 1H), 3.87 (m, 2H), 3.55 (m, 2H), 3.27 (s, 2H), 2.75 (s, 2H), 2.74 (s, 3H), 1.79 (m, 2H), 1.65 (m, 2H), 1.45 (s, 9H); LC/MS m/z (M+H) + = 447.2. Example 17: 2-(tert-butyl)-1'-(7-methyl-1H-indole-5-carbonyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one To a solution of preparation P1 (6.3 g, 20 mmol), 7-methyl-1H-indole-5-carboxylic acid (4.2 g, 24 mmol, commercial vendor PharmaBlock), EDCI . HCl (4.79 g, 25 mmol), and HOBt (3.97 g, 25 mmol) in DMF (100 mL) at about 25 °C was added i Pr 2 NEt (20.9 mL, 120 mmol). The mixture was stirred at about 25 °C for about 16 h, then diluted with EtOAc (200 mL). The resulting mixture was washed sequentially with 5% aqueous LiCl (100 mL), saturated aqueous NaHCO 3 (50 mL), and brine (50 mL). The organic phase was dried over MgSO 4 , filtered, and concentrated. The residue was purified by chromatography (silica, EtOAc/Hexanes 5-100% then MeOH-EtOAc, 0-10%) to provide the title compound (8.0 g, 84% yield) as a white solid; 1 H NMR (400 MHz, Methanol-d4) δ 7.52 (s, 1H), 7.32 (d, 1H), 7.00 (s, 1H), 6.53 (d, 1H), 3.65 (m, 4H), 3.23 (s, 2H), 2.72 (s, 2H), 2.54 (s, 3H), 1.67 (m, 4H), 1.45 (s, 9H); LC/MS m/z (M+H) + = 436.1. Example 18: 2-(tert-butyl)-1'-(3-(ethylamino)-8-methylquinoline-6-carbon yl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 15, substituting tert-butyl ethylcarbamate for tert-butyl methylcarbamate in Step 3, to provide the title compound (353 mg). 1 H NMR (400 MHz, CD 3 OD) δ 8.46 (s, 1H), 7.56 (s, 1H), 7.20 (s, 1H), 7.11 (s, 1H), 3.86-3.79 (m, 4H), 3.52 (m, 2H), 3.25-3.20 (m, 2H), 2.72 (s, 2H), 2.69 (s, 3H), 1.76-1.63 (m, 4H), 1.43 (s, 9H), 1.32 (t, 3H); LC/MS m/z (M+H) + = 491.2. Example 19: 2-(tert-butyl)-1'-(3-methoxy-8-methylquinoline-6-carbonyl)-5 H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 15, substituting methanol for tert-butyl methylcarbamate in Step 3, to provide the title compound (56 mg). 1 H NMR (400 MHz, CD 3 OD) δ 8.63 (s, 1H), 7.75 (s, 1H), 7.72 (s, 1H), 7.43 (s, 1H), 3.98 (s, 3H), 3.88-3.82 (m, 2H), 3.51 (m, 2H), 3.26 (s, 2H), 2.76 (s, 3H), 2.73 (s, 2H), 1.77-1.64 (m, 4H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 478.3. Example 20: 2-(tert-butyl)-1'-(3-ethoxy-8-methylquinoline-6-carbonyl)-5H - spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 15, substituting ethanol for tert-butyl methylcarbamate in Step 3, to provide the title compound (2.5 mg). 1 H NMR (400 MHz, CD 3 OD) δ 8.64 (s, 1H), 7.75 (s, 1H), 7.71 (s, 1H), 7.44 (s, 1H), 4.24 (q, 2H), 3.80-3.60 (m, 2H), 3.53 (m, 2H), 3.28 (s, 2H), 2.77 (s, 3H), 2.76 (s, 2H), 1.79-1.63 (m, 4H), 1.52 (t, 3H), 1.45 (s, 9H); LC/MS m/z (M+H) + = 492.3. Example 21: 2-(tert-butyl)-1'-(8-methyl-3-(methylthio)quinoline-6-carbon yl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 15, substituting 2-methyl-2-thiopseudourea hemisulfate for tert-butyl methylcarbamate and PdCl 2 dppf in Step 3, to provide the title compound (28 mg). 1 H NMR (400 MHz, CD 3 OD) δ 8.78 (d, 1H), 8.13 (d, 1H), 7.77 (s, 1H), 7.54 (s, 1H), 3.90-3.84 (m, 2H), 3.53 (m, 2H), 3.28 (s, 2H), 2.78 (s, 3H), 2.76 (s, 2H), 2.66 (s, 3H), 1.79-1.60 (m, 4H), 1.45 (s, 9H); LC/MS m/z (M+H) + = 494.3. Example 22: 4-(4-(2-(tert-butyl)-4-oxo-4,7-dihydro-5H-spiro[benzo[d]thia zole-6,4'- piperidine]-1'-carbonyl)-6-(dimethylamino)pyridin-2-yl)benza mide Step 1: Synthesis of 2-(4-carbamoylphenyl)-6-(dimethylamino)isonicotinic acid (C21) To methyl 2-chloro-6-(dimethylamino)isonicotinate (104 mg, 0.486 mmol) in dioxane (3.0 mL) and water (1.0 mL) in a microwave vial was added Na 2 CO 3 (64 mg, 0.61 mmol), Pd(PPh 3 ) 4 (14 mg, 0.012 mmol), and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (100 mg, 0.41 mmol). The mixture was sparged with nitrogen, placed in the microwave, and irradiated at about 100 °C for about 45 min. The mixture was cooled to about 25 °C and concentrated under reduced pressure. The aqueous residue was acidified to approximately pH 5 with 2 N HCl and the resulting precipitate was filtered to provide the title compound (78 mg, 68% yield). 1 H NMR (400 MHz, CD 3 OD) δ 8.20 (d, 1H), 7.99 (d, 1H), 7.65 (s, 1H), 7.19 (s, 1H), 3.24 (s, 6H); LC/MS m/z (M+H) + = 285.9. Step 2: 4-(4-(2-(tert-butyl)-4-oxo-4,7-dihydro-5H-spiro[benzo[d]thia zole-6,4'-piperidine]- 1'-carbonyl)-6-(dimethylamino)pyridin-2-yl)benzamide (Example 22) To a solution of preparation P1 (50 mg, 0.16 mmol), compound C21 (68 mg, 0.24 mmol), and EDCI (61 mg, 0.32 mmol) in a microwave vial was added pyridine (3.0 mL). The mixture was irradiated in the microwave at about 110 °C for about 30 min. The mixture was concentrated under reduced pressure and purified by prep-HPLC (Column: C18-1, 30x150 mm, 5 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile phase B: MeCN; 30-70% B gradient, 9 min, hold at 100% B for 2 min; flow rate 30 mL/min) to provide the title compound (21 mg, 24%). 1 H NMR (400 MHz, CD 3 OD) δ 8.16 (d, 2H), 7.96 (d, 2H), 7.13 (s, 1H), 6.58 (s, 1H), 3.90-3.75 (m, 2H), 3.54 (m, 2H), 3.26 (s, 2H), 3.21 (s, 6H), 2.76 (s, 2H), 1.80-1.62 (m, 4H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 546.4. Example 23: 4-(4-(2-(tert-butyl)-4-oxo-4,7-dihydro-5H-spiro[benzo[d]thia zole-6,4'- piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzamide Prepared in the same manner as Example 22, substituting 2-chloro-6-methoxyisonicotinic acid for methyl 2-chloro-6-(dimethylamino)isonicotinate in Step 1, to provide the title compound (18 mg). 1 H NMR (400 MHz, CD 3 OD) δ 8.23 (d, 2H), 8.01 (d, 2H), 7.54 (s, 1H), 6.77 (s, 1H), 4.06 (s, 3H), 3.49 (m, 2H), 3.30-3.25 (m, 4H), 2.73 (s, 2H), 1.80-1.63 (m, 4H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 533.4. Example 24: 2-(tert-butyl)-1'-(5-methyl-1-(methylamino)isoquinoline-7-ca rbonyl)- 5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: 3-bromo-N-(2,2-dimethoxyethyl)-5-methylbenzamide (C22) To a solution of 2,2-dimethoxyethan-1-amine (1.34 g, 12.8 mmol) in DCM (50 mL) was added triethylamine (1.76 g, 17.4 mmol). The reaction was cooled to about 0 °C, then 3-bromo- 5-methylbenzoyl chloride (2.71 g, 11.6 mmol) in DCM (10 mL) was added dropwise. The reaction was allowed to warm to about 30 °C over about 16 h. Water (10 mL) was added and the mixture was extracted with DCM (2 X 20 mL). The combined organic phases were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to provide the title compound (3.0 g, 85%). Step 2: 7-bromo-5-methylisoquinolin-1(2H)-one (C23) To compound C22 (3.50 g, 12.3 mmol) at about 0 °C was added concentrated H 2 SO 4 (30 mL). The mixture was stirred at about 25 °C for about 16 h, then heated to about 50 °C for about 5 h. The mixture was slowly poured into ice water (200 mL), then extracted with EtOAc (3 X 300 mL). The combined organic phases were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was suspended in petroleum ether / EtOAc (50 mL / 20 mL) and stirred at about 25 °C for about 10 min. The solids were collected by filtration to provide a mixture of the title compound and regioisomer, 5-bromo-7-methylisoquinolin-1(2H)-one (1.8 g, 76%). LC/MS m/z (M+H) + = 238.0. Step 3: methyl 5-methyl-1-oxo-1,2-dihydroisoquinoline-7-carboxylate (C24) To compound C23 (mix of regioisomers, 1.00 g, 4.2 mmol) in MeOH (50 mL) was added Pd(dppf)Cl 2 (461 mg, 0.63 mmol) and triethylamine (1.28 g, 12.6 mmol). The mixture was heated to about 80 °C under CO atmosphere (50 psi) for about 48 h. The mixture was cooled to about 25 °C, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography (0% to 30% EtOAc in petroleum ether) to provide the title compound and regioisomer, methyl 7-methyl-1-oxo-1,2-dihydroisoquinoline-5-carboxylate (600 mg, 66%). The isomers were separated via prep-SFC (Column: Daicel Chiralpak AD, 30x250 mm, 10 µm; Mobile Phase A: water (0.1% v/v NH 4 OH); Mobile phase B: EtOH; 35% B, flow rate 80 mL/min) to provide the title compound (100 mg, 11%). LC/MS m/z (M+H) + = 218.0. Step 4: methyl 1-chloro-5-methylisoquinoline-7-carboxylate (C25) To compound C24 (100 mg, 0.46 mmol) was added POCl 3 (1.41 g, 9.2 mmol). The mixture was heated to about 100 °C for about 2 h. The reaction was concentrated under reduced pressure and the pH was adjusted to about 8 with NaHCO 3 (aq). The organic phase was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by chromatography (0% to 50% EtOAc in petroleum ether) to provide the title compound (105 mg, 97%). LC/MS m/z (M+H) + = 236.0. Step 5: methyl 5-methyl-1-(methylamino)isoquinoline-7-carboxylate (C26) To compound C25 (105 mg, 0.45 mmol) in NMP (5 mL) was added i-Pr 2 NEt (0.39 mL, 2.23 mmol) and methylamine hydrochloride (90 mg, 1.3 mmol). The mixture was stirred at about 110 °C for about 16 h. The solution was cooled to about 30 °C and diluted with water (10 mL). The mixture was extracted with EtOAc (2 X 10 mL) and the combined organic phases were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by chromatography (0% to 50% EtOAc in petroleum ether) to provide the title compound (50 mg, 49%). LC/MS m/z (M+H) + = 231.0. Step 6: 5-methyl-1-(methylamino)isoquinoline-7-carboxylic acid (C27) To compound C26 (50 mg, 0.22 mmol) in MeOH (5 mL) and water (2 mL) was added LiOH monohydrate (36 mg, 0.87 mmol). The mixture was stirred at about 30 °C for about 16 h. The mixture was concentrated under reduced pressure and cooled to about 0 °C. 1 M HCl (aq) was added to adjust the solution to pH 5. A precipitate formed and was collected by filtration to provide the title compound (28 mg, 60%). LC/MS m/z (M+H) + = 217.0. Step 7: 2-(tert-butyl)-1'-(5-methyl-1-(methylamino)isoquinoline-7-ca rbonyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one According to amidation method A, compound C27 (28 mg, 0.13 mmol) was coupled to preparation P1. The residue was purified by prep-HPLC (Column: Boston Prime C18, 30x150 mm, 5 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile phase B: MeCN; 29-69% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min) to provide the title compound (40 mg, 64%). 1 H NMR (400 MHz, CD 3 OD) δ 8.02 (s, 1H), 7.93 (d, 1H), 7.51 (s, 1H), 7.04 (d, 1H), 3.88-3.83 (m, 2H), 3.53 (m, 1H), 3.27 (s, 2H), 3.06 (s, 3H), 2.75 (s, 2H), 2.62 (s, 3H), 1.78-1.66 (m, 4H), 1.45 (s, 9H). LC/MS m/z (M+H) + = 477.3. Example 25: 2-(tert-butyl)-1'-(1-cyclopropyl-5-methylisoquinoline-7-carb onyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 24, substituting cyclopropylboronic acid for methylamine hydrochloride in Step 5, to provide the title compound (32 mg). 1 H NMR (400 MHz, CD 3 OD) δ 8.37 (s, 1H), 8.27 (d, 1H), 7.65 (d, 1H), 7.53 (s, 1H), 3.81-3.77 (m, 2H), 3.46 (m, 2H), 3.18 (s, 2H), 2.76 (m, 1H), 2.66 (s, 2H), 2.63 (s, 3H), 1.71 (m, 2H), 1.57 (m, 2H), 1.35 (s, 9H), 1.08 (d, 4H); LC/MS m/z (M+H) + = 488.4. Example 26: 2-(tert-butyl)-1'-(4-methyl-1-(methylamino)isoquinoline-7-ca rbonyl)- 5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: methyl 1-((tert-butoxycarbonyl)(methyl)amino)-4-methylisoquinoline- 7- carboxylate (C28) To 1-chloro-4-methylisoquinoline-7-carboxylate (140 mg, 0.594 mmol), tert-butyl-N- methylcarbamate (100 mg, 0.594 mmol), Pd 2 (dba) 3 (27 mg, 0.030 mmol), Cs 2 CO 3 (581 mg, 1.78 mmol), and X-Phos (28 mg, 0.059 mmol) was added toluene (5 mL). The mixture was sparged with nitrogen. The mixture was heated to about 110 °C for about 16 h. The solution was concentrated under reduced pressure and the residue was purified by chromatography (0% to 30% EtOAc in petroleum ether) to provide the title compound (15 mg, 8%). LC/MS m/z (M+H - Boc) + = 231.1. Step 2: methyl 4-methyl-1-(methylamino)isoquinoline-7-carboxylate (C29) To compound C28 (15 mg, 0.045 mmol) in MeOH (1.0 mL) was added HCl (4 M in dioxane, 1.0 mL). The reaction was stirred at about 50 °C for about 2 h. The mixture was concentrated under reduced pressure to provide the title compound (10 mg, 96%). LC/MS m/z (M+H) + = 231.0. Step 3: 4-methyl-1-(methylamino)isoquinoline-7-carboxylic acid (C30) To compound C29 (10 mg, 0.043 mmol) in EtOH (4.0 mL) was added NaOH (8.7 mg, 0.217 mmol) and water (4.0 mL). The reaction was stirred at about 50 °C for about 1 h. The EtOH was removed under reduced pressure and the mixture was acidified to pH 7 with 2 N HCl. The precipitate was filtered and dried in vacuo to provide the title compound (9.4 mg, 100%). LC/MS m/z (M+H) + = 217.0. Step 3: 2-(tert-butyl)-1'-(4-methyl-1-(methylamino)isoquinoline-7-ca rbonyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one According to amidation method C, compound C30 (9 mg, 0.044 mmol) was coupled to preparation P1. The residue was purified by prep-HPLC (Column: Boston Prime C18, 30x150 mm, 5 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile phase B: MeCN; 32-62% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min) to provide the title compound (2.9 mg, 14%). 1 H NMR (400 MHz, CD 3 OD) δ 8.17 (s, 1H), 7.89 (d, 1H), 7.72-7.70 (m, 2H), 3.87-3.81 (m, 2H), 3.50 (m, 2H), 3.25 (s, 2H), 3.03 (s, 3H), 2.73 (s, 2H), 2.40 (s, 3H), 1.77-1.58 (m, 4H), 1.42 (s, 9H); LC/MS m/z (M+H) + = 477.3. Example 27: 2-(tert-butyl)-1'-(1,4-dimethylisoquinoline-7-carbonyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 26, substituting trimethylboroxine for tert-butyl- N-methylcarbamate in Step 1, to provide the title compound (42 mg). 1 H NMR (400 MHz, CD 3 OD) δ 8.31 (s, 1H), 8.20 (s, 1H), 8.15 (d, 1H), 7.84 (d, 1H), 3.91-3.86 (m, 2H), 3.53 (m, 2H), 3.27 (s, 2H), 2.93 (s, 3H), 2.75 (s, 2H), 2.63 (s, 3H), 1.85-1.65 (m, 4H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 462.3. Example 28: 2-(tert-butyl)-1'-(5-methoxy-1-methylisoquinoline-7-carbonyl )-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: methyl 5-methoxyisoquinoline-7-carboxylate (C31) To methyl 5-bromoisoquinoline-7-carboxylate (1.00 g, 3.76 mmol; prepared as described by World patent application WO 2021/028806), RockPhos-Pd-G3 (95 mg, 0.113 mmol), and Cs 2 CO 3 (1.22 g, 3.76 mmol) was added MeOH (1.2 g, 37.6 mmol) and dioxane (20 mL). The mixture was heated to about 70 °C for about 16 h. The mixture was cooled to about 25 °C, filtered, and concentrated under reduced pressure. The residue was purified by chromatography (0% to 40% EtOAc in petroleum ether) to provide the title compound (450 mg, 55%). LC/MS m/z (M+H) + = 217.9. Step 2: 5-methoxy-7-(methoxycarbonyl)isoquinoline 2-oxide (C32) To a solution of compound C31 (450 mg, 2.07 mmol) in DCM (10 mL) at about 0 °C was added m-CPBA (429 mg, 2.49 mmol). The mixture was stirred at about 45 °C for about 16 h. Saturated Na 2 S 2 O 3 (aq) was added, followed by saturated Na 2 CO 3 (aq) until the pH was >8. The mixture was stirred for about 30 min, then extracted with DCM (2 X 10 mL) and H 2 O (2 X 10 mL). The DCM extracts were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to provide the title compound (450 mg, 93%). LC/MS m/z (M+H) + = 234.0. Step 3: methyl 1-chloro-5-methoxyisoquinoline-7-carboxylate (C33) To a solution of compound C32 (450 mg, 1.93 mmol) in DCM (10 mL) was added POCl 3 (0.90 mL, 9.65 mmol). The mixture was stirred at about 50 °C for about 2 h, then cooled to about 25 °C. The mixture was concentrated under reduced pressure and NaHCO 3 (aq) was added until the pH was about 8. The organic phase was separated, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by chromatography (0% to 40% EtOAc in petroleum ether) to provide the title compound (200 mg, 41%). LC/MS m/z (M+H) + = 252.0. Step 4: methyl 5-methoxy-1-methylisoquinoline-7-carboxylate (C34) To a solution of compound C33 (60 mg, 0.24 mmol) and trimethylboroxine (60 mg, 0.24 mmol) in THF (2 mL) was added Pd(dtbpf)Cl 2 (7.8 mg, 0.012 mmol) and Cs 2 CO 3 (155 mg, 0.477 mmol). The mixture was heated to about 70 °C for about 2 h, then concentrated under reduced pressure. The residue was purified by chromatography (0% to 30% EtOAc in petroleum ether) to provide the title compound (40 mg, 73%). LC/MS m/z (M+H) + = 232.0. Step 5: 5-methoxy-1-methylisoquinoline-7-carboxylic acid (C35) To a solution of compound C34 (40 mg, 0.17 mmol) in MeOH (2 mL) and water (1 mL) was added LiOH . H2O (15 mg, 0.35 mmol). The mixture was stirred at about 25 °C for about 2 h, then heated to about 50 °C for about 2 h. The solution was concentrated under reduced pressure and the pH was adjusted to about 5 with 1 M HCl. The resulting precipitate was filtered to provide the title compound (38 mg, 100%). LC/MS m/z (M+H) + = 217.9. Step 6: 2-(tert-butyl)-1'-(5-methoxy-1-methylisoquinoline-7-carbonyl )-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one (Example 28) According to amidation method A, compound C35 (38 mg, 0.17 mmol) was coupled to preparation P1. The residue was purified by prep-HPLC (Column: Boston Prime C18, 30x150 mm, 5 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile phase B: MeCN; 29-59% B gradient, 9 min, hold at 100% B for 2 min; flow rate 30 mL/min) to provide the title compound (24 mg, 28%). 1 H NMR (400 MHz, CD 3 OD) δ 8.33 (d, 1H), 7.97 (d, 1H), 7.81 (s, 1H), 7.18 (s, 1H), 4.07 (s, 3H), 3.89-3.84 (m, 2H), 3.54 (m, 2H), 3.26 (s, 2H), 2.93 (s, 3H), 2.75 (s, 2H), 1.79-1.65 (m, 4H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 478.4. Example 29: 2-(tert-butyl)-1'-(1-ethyl-5-methoxyisoquinoline-7-carbonyl) -5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 28, substituting triethylborane for trimethylboroxine in Step 1, to provide the title compound (5.0 mg). 1 H NMR (400 MHz, CD 3 OD) δ 8.37 (d, 1H), 7.98 (d, 1H), 7.85 (s, 1H), 7.18 (s, 1H), 4.07 (s, 3H), 3.91-3.83 (m, 2H), 3.55 (m, 2H), 3.35-3.31 (m, 2H), 3.27 (s, 2H), 2.75 (s, 2H), 1.80-1.65 (m, 4H), 1.43 (s, 9H), 1.37 (t, 3H); LC/MS m/z (M+H) + = 492.3. Example 30: 2-(tert-butyl)-1'-(2-(isopropylamino)quinoline-7-carbonyl)-5 H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: methyl 2-(isopropylamino)quinoline-7-carboxylate (C36) To a solution of 7-(methoxycarbonyl)quinoline 1-oxide (200 mg, 0.98 mmol) in DCM (15 mL) at about -70 °C was added trifluoromethanesulfonic anhydride (304 mg, 1.08 mmol) dropwise. The mixture was stirred for about 5 min at about -70 °C, then a 2 M solution of isopropylamine in THF was added (2.94 mL, 5.88 mmol) dropwise. The mixture was stirred for about 5 min at about -70 °C, then water (15 mL) was added. The layers were separated, and the aqueous phase was extracted with DCM (20 mL). The combined DCM extracts were washed with brine (2 X 10 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by chromatography (0% to 40% EtOAc in petroleum ether) to provide the title compound (150 mg, 63%). LC/MS m/z (M+H) + = 245.1. Step 2: 2-(isopropylamino)quinoline-7-carboxylic acid (C37) To a solution of compound C36 (150 mg, 0.614 mmol) in EtOH (4 mL) and water (4 mL) was added NaOH (123 mg, 3.07 mmol). The mixture was heated to about 50 °C for about 16 h. The mixture was concentrated under reduced pressure and treated with 2 M HCl until the pH was about 5. The resulting precipitate was filtered to provide the title compound (135 mg, 96%). LC/MS m/z (M+H) + = 231.1. Step 3: 2-(tert-butyl)-1'-(2-(isopropylamino)quinoline-7-carbonyl)-5 H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one (Example 30) According to amidation method A, compound C37 (50 mg, 0.22 mmol) was coupled to preparation P1. The residue was purified by prep-HPLC (Column: Boston Prime C18, 30x150 mm, 5 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile phase B: MeCN; 32-62% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min) to provide the title compound (36 mg, 34%). 1 H NMR (400 MHz, CD 3 OD) δ 7.83 (d, 1H), 7.68-7.63 (m, 2H), 7.19 (dd, 1H), 6.78 (d, 1H), 4.30 (sept, 1H), 3.88-3.83 (m, 2H), 3.55 (m, 2H), 3.27 (s, 2H), 2.75 (d, 2H), 1.78-1.66 (m, 4H), 1.45 (s, 9H), 1.28 (d, 6H); LC/MS m/z (M+H) + = 491.4. Example 31: 2-(tert-butyl)-1'-(2-(cyclobutylamino)quinoline-7-carbonyl)- 5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 30, substituting cyclobutylamine for isopropylamine in Step 1, to provide the title compound (46 mg). 1 H NMR (400 MHz, CD 3 OD) δ 7.83 (d, 1H), 7.65 (d, 1H), 7.60 (s, 1H), 7.17 (d, 1H), 6.76 (d, 1H), 4.54 (m, 1H), 3.87-3.79 (m, 2H), 3.52 (m, 1H), 3.26 (s, 2H), 2.73 (s, 2H), 2.46 (m, 2H), 2.04 (m, 2H), 1.82-1.64 (m, 6H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 503.4. Example 32: 2-(tert-butyl)-1'-(2-(ethylamino)quinoline-7-carbonyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 30, substituting ethylamine for isopropylamine in Step 1, to provide the title compound (27 mg). 1 H NMR (400 MHz, CD 3 OD) δ 7.82 (d, 1H), 7.66 (d, 1H), 7.63 (s, 1H), 7.17 (d, 1H), 6.78 (d, 1H), 3.87-3.81 (m, 2H), 3.52-3.46 (m, 4H), 3.26 (s, 2H), 2.73 (s, 2H), 1.76-1.64 (m, 4H), 1.43 (s, 9H), 1.28 (t, 3H); LC/MS m/z (M+H) + = 477.4. Example 33: 2-(tert-butyl)-1'-(5-methoxy-4-methyl-1H-indazole-7-carbonyl )-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: Synthesis of 6-bromo-4-methoxy-2,3-dimethylaniline (C38) To 4-methoxy-2,3-dimethylaniline (500 mg, 3.31 mmol) in MeCN (10 mL) was added NBS (706 mg, 3.97 mmol). The mixture was stirred at about 25 °C for about 2 h. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by chromatography (17% EtOAc in petroleum ether, then 20% MeOH in EtOAc) to provide the title compound (600 mg, 79%). 1 H NMR (400 MHz, CDCl 3 ) δ = 6.88 (s, 1H), 3.77 (s, 3H), 2.18 (s, 3H), 2.15 (s, 3H); LC/MS m/z (M+H) + = 231.9. Step 2: Synthesis of 7-bromo-5-methoxy-4-methyl-1H-indazole (C39) To compound C38 (600 mg, 2.61 mmol) in H 2 O (4 mL) was added conc. HCl (4 mL). The mixture was heated to about 60 °C for about 30 min, then cooled to about 0 °C. A solution of NaNO 2 (198 mg, 2.87 mmol) in H 2 O (1 mL) was added dropwise and the mixture was stirred at about 0 °C for about 1 h. To the mixture was added sat. aq. NaOAc until the pH was 4-5. A solution of 2-methylpropane-2-thiol (259 mg, 2.87 mmol) in EtOH (7 mL) was added. The mixture was slowly warmed to about 25 °C and stirred about 16 h. The mixture was diluted with EtOAc (20 mL), washed with water (20 mL), and washed with brine (20 mL). The organic phase was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The resulting residue was dissolved in DMSO (5 mL) and a solution of KOtBu (1.69 g, 15 mmol) in DMSO (10 mL) was added dropwise. The mixture was stirred at about 25 °C for about 2 h. The mixture was diluted with EtOAc (50 mL), washed with water (50 mL), and washed with brine (50 mL). The organic phase was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by chromatography (17% EtOAc in petroleum ether) to provide the title compound (170 mg, 27%). 1 H NMR (400 MHz, CDCl 3 ) δ = 8.12 (s, 1H), 7.29 (s, 1H), 3.90 (s, 3H), 2.45 (s, 3H); LC/MS m/z (M+H) + = 242.8. Step 3: Synthesis of ethyl 5-methoxy-4-methyl-1H-indazole-7-carboxylate (C40) To compound C39 in EtOH (5.0 mL) was added Mo(CO) 6 (93 mg, 0.353 mmol), TTBP . HBF 4 (21 mg, 0.0705 mmol), DBU (0.16 mL, 1.06 mmol), and Pd(OAc) 2 (15.8 mg, 0.0705 mmol). The mixture was irradiated at about 100 °C for about 1 h in a microwave reactor, then cooled to about 25 °C. The mixture was concentrated under reduced pressure and purified by chromatography (17% EtOAc in petroleum ether) to provide the title compound (70 mg, 42%). 1 H NMR (400 MHz, CDCl 3 ) δ = 8.11 (s, 1H), 7.78 (s, 1H), 4.51 (q, 2H), 3.96 (s, 3H), 2.57 (s, 3H), 1.51 (t, 3H); LC/MS m/z (M+H) + = 235.0. Step 4: Synthesis of 5-methoxy-4-methyl-1H-indazole-7-carboxylic acid (C41) To compound C40 (70 mg, 0.30 mmol) in MeOH (1.0 mL) and THF (2.0 mL) was added a solution of LiOH . H 2 O (38 mg, 0.90 mmol) in H 2 O (1.0 mL). The mixture was stirred at about 25 °C for about 16 h, then H 2 O (10 mL) was added. The mixture was washed with EtOAc (10 mL), then the aqueous phase was acidified with sat. aq. citric acid to pH ~6. The resulting suspension was filtered and the filter cake was collected to provide the title compound (62 mg, 100%). LC/MS m/z (M+H) + = 207.0. Step 5: Synthesis of 2-(tert-butyl)-1'-(5-methoxy-4-methyl-1H-indazole-7-carbonyl )-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one (Example 33) To a solution of compound C41 (62 mg, 0.30 mmol) in DMF (2.0 mL) at about 0 °C was added Et 3 N (0.17 mL, 1.2 mmol) and T3P (0.36 mL of a 50% w/w solution in EtOAc, 0.60 mmol). Preparation P1 (95 mg, 0.30 mmol) was added. The resulting solution was stirred at about 25 °C for about 16 h. The mixture was diluted with EtOAc (15 mL), washed with water (15 mL) and brine (15 mL). The organic phase was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC (Column: Boston Green ODS, 30x150 mm, 5 µm; Mobile Phase A: water (0.2% v/v conc. HCl); Mobile phase B: MeCN; 23-63% B gradient, 9 min, hold at 100% B for 2 min; flow rate 30 mL/min) to provide the title compound (27 mg, 19%). 1 H NMR (400 MHz, CD3OD) δ = 8.19 (d, 1H), 7.29 (t, 1H), 3.90 (s, 3H), 3.65 (m, 4H), 3.24 (s, 2H), 2.73 (s, 2H), 2.48 (s, 3H), 1.69 (m, 4H), 1.43 (s, 9H). LC/MS m/z (M+Na) + = 489.1. Example 34: 2-(tert-butyl)-1'-(4-chloro-5-methyl-1H-indazole-7-carbonyl) -5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 33, substituting 3-chloro-2,4-dimethylaniline for 4-methoxy-2,3-dimethylaniline in Step 1, to provide the title compound (15 mg). 1 H NMR (400 MHz, CD 3 OD) δ 8.11 (s, 1H), 7.37 (s, 1H), 3.87-3.48 (m, 4H), 3.24 (s, 2H), 2.73 (s, 2H), 2.49 (s, 3H), 1.77-1.58 (m, 4H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 471.3. Example 35: 2-(tert-butyl)-1'-(4,5-dimethyl-1H-indazole-7-carbonyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Prepared in the same manner as Example 33, substituting 2,3,4-trimethylaniline in place of 4-methoxy-2,3-dimethylaniline in Step 1, to provide the title compound (26 mg). 1 H NMR (400 MHz, CD 3 OD) δ 8.13 (s, 1H), 7.26 (s, 1H), 3.70-3.59 (m, 4H), 3.23 (s 2H), 2.73 (s, 2H), 2.55 (s, 3H), 2.39 (s, 3H), 1.76-1.67 (m, 4H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 451.4. Example 36: 2-(tert-butyl)-1'-(4-methoxy-5-methyl-1H-indazole-7-carbonyl )-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: Synthesis of 4-methoxy-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (C42) To 5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-ol (2.5 g, 10.76 mmol) and K 2 CO 3 (2.98 g, 21.5 mmol) was added DMF (30 mL). The mixture was cooled to about 0-5 °C and stirred for about 10 min. Iodomethane (1.01 mL, 16.1 mmol) was added dropwise, and the mixture was stirred at about 25 °C for about 20 h. Water (30 mL) was added and the mixture was extracted with EtOAc (3 X 30 mL). The combined EtOAc extracts were washed with saturated brine, dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0-20% EtOAc in petroleum ether) to provide the title compound (2.40 g, 91%). LC/MS m/z (M+H) + = 247.0. Step 2: Synthesis of 7-bromo-4-methoxy-5-methyl-1H-indazole (C43) To a solution of compound C42 (2.40 g, 9.74 mmol) in DCM (50 mL) was added pyridinium tribromide (3.43 g, 10.7 mmol). The mixture was stirred at about 25 °C for about 2 h. Water (30 mL) was added, and the mixture was extracted with EtOAc (3 X 20 mL). The combined EtOAc extracts were washed with saturated brine, dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0-20% EtOAc in petroleum ether) to provide the title compound (1.90 g, 81%). LC/MS m/z (M+H) + = 242.8. Step 3: Synthesis of ethyl 4-methoxy-5-methyl-1H-indazole-7-carboxylate (C44) To a solution of compound C43 (1.20 g, 3.0 mmol) in EtOH (20 mL) was added Pd(OAc) 2 (54 mg, 0.239 mmol), tBu 3 P . BF 4 (87 mg, 0.30 mmol), DBU (682 mg, 4.48 mmol), and Mo(CO) 6 (237 mg, 0.90 mmol). The mixture was irradiated in the microwave at 100 °C for 30 min. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0-30% EtOAc in petroleum ether) to provide the title compound (450 mg, 64%). LC/MS m/z (M+H) + = 235.0. Step 4: Synthesis of 4-methoxy-5-methyl-1H-indazole-7-carboxylic acid (C45) To a solution of compound C44 (700 mg, 2.99 mmol) in EtOH (5 mL) was added NaOH (598 mg, 14.9 mmol) and water (5 mL). The mixture was stirred at about 50 °C for about 16 h. The mixture was concentrated under reduced pressure and the residue was acidified to pH 5 with 2 N HCl. The precipitate was filtered to provide the title compound (580 mg, 94%). LC/MS m/z (M+H) + = 206.9. Step 5: Synthesis of 2-(tert-butyl)-1'-(4-methoxy-5-methyl-1H-indazole-7-carbonyl )-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one According to amidation method A, compound C45 (300 mg, 1.45 mmol) was coupled to preparation P1. The residue was purified by prep-HPLC (Column: Boston Prime C18, 30x150 mm, 5 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile phase B: MeCN; 30-60% B gradient, 9 min, hold at 100% B for 2 min; flow rate 30 mL/min) to provide the title compound (345 mg, 51%). 1 H NMR (400 MHz, CD 3 OD) δ 8.32 (s, 1H), 7.28 (s, 1H), 4.25 (s, 3H), 3.69 (m, 4H), 3.24 (s, 2H), 2.73 (s, 2H), 2.29 (s, 3H), 1.68 (m, 4H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 467.4. Example 37: 2-(tert-butyl)-1'-(4-chloro-5-methoxy-1H-indole-7-carbonyl)- 5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: Synthesis of 1-bromo-4-chloro-5-methoxy-2-nitrobenzene (C46) To 4-bromo-1-chloro-2-methoxybenzene (1.00 g, 4.52 mmol) was added H2SO4 (3 mL) and the mixture was cooled to about 0 °C and stirred for about 10 min. HNO 3 (0.43 mL, 11.3 mmol) was added portion-wise. The mixture was allowed to slowly warm to about 20 °C over about 16 h. The mixture was poured onto ice water and adjusted to pH 7-9 with Na 2 CO 3 . The mixture was extracted with EtOAc (3 X 200 mL) and the combined EtOAc extracts were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0-2% EtOAc in DCM) to provide the title compound and the dinitrated byproduct, 2-bromo-5-chloro-4-methoxy-1,3-dinitrobenzene (1.10 g, <91% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 8.10 (s, 1H), 7.22 (s, 1H), 4.01 (s, 3H). Step 2: Synthesis of 7-bromo-4-chloro-5-methoxy-1H-indole (C47) To compound C46 (1.10 g, about 20% purity, 0.83 mmol) in THF (10 mL) at about -50 °C was added vinylmagnesium bromide (1 M, 15 mL, 15 mmol). The mixture was allowed to warm to about -20 °C and stirred for about 16 h. The mixture was warmed to 0 °C and NH 4 Cl (30 mL) and EtOAc (50 mL) were added. The phases were separated, and the aqueous phase was extracted with EtOAc (3 X 20 mL). The combined EtOAc extracts were washed with brine (20 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (5% EtOAc in petroleum ether) to provide the title compound (30 mg, 14% yield). LC/MS m/z (M+H) + = 261.8. Step 3: Synthesis of ethyl 4-chloro-5-methoxy-1H-indole-7-carboxylate (C48) To a solution of compound C47 (50 mg, 0.19 mmol) in EtOH (10 mL) was added Pd(OAc) 2 (3.5 mg, 0.0154 mmol), tBu 3 P . BF 4 (5.6 mg, 0.0192 mmol), DBU (44 mg, 0.288 mmol), and Mo(CO) 6 (51 mg, 0.192 mmol). The mixture was irradiated in the microwave at about 100 °C for about 1 h. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0-2% EtOAc in petroleum ether) to provide the title compound (20 mg, 41%). LC/MS m/z (M+H) + = 254.0. Step 4: Synthesis of 4-chloro-5-methoxy-1H-indole-7-carboxylic acid (C49) To a solution of compound C48 (20 mg, 0.079 mmol) in water (1 mL) and THF (2 mL) was added LiOH . H 2 O (6.6 mg, 0.16 mmol) at about 15 °C. The mixture was stirred at about 50 °C for about 16 h. The mixture was concentrated under reduced pressure and the residue was acidified to pH 5 with 2 N HCl. The solution was concentrated to provide the title compound (15 mg, 84%). LC/MS m/z (M+H) + = 225.9. Step 5: Synthesis of 2-(tert-butyl)-1'-(4-chloro-5-methoxy-1H-indole-7-carbonyl)- 5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one According to amidation method A, compound C49 (15 mg, 1.45 mmol) was coupled to preparation P1. The residue was purified by prep-HPLC (Column: Boston Prime C18, 30x150 mm, 5 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile phase B: MeCN; 40-70% B gradient, 9 min, hold at 100% B for 2 min; flow rate 30 mL/min) to provide the title compound (4.5 mg, 14%). 1 H NMR (400 MHz, CD 3 OD) δ 7.35 (d, 1H), 7.00 (s, 1H), 6.52 (d, 1H), 3.90 (s, 3H), 3.89-3.48 (m, 4H), 3.23 (s, 2H), 2.73 (s, 2H), 1.79-1.64 (m, 4H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 486.3. Example 38: 2-(tert-butyl)-1'-(5-methyl-2-(methylamino)quinoline-7-carbo nyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one To a solution of preparation P1 (1.26 g, 4.01 mmol), preparation P2 (581 mg, 2.69 mmol), EDCI (773 mg, 4.04 mmol), and HOBt (582 mg, 4.31 mmol) in DMF (35 mL) at about 25 °C was added i Pr 2 NEt (5 mL, 26.9 mmol). The mixture was stirred at about 25 °C for about 16 h, then diluted with EtOAc. The mixture was washed sequentially with 5% aqueous LiCl, saturated aqueous NaHCO 3 , and 1:1 brine:water (30 mL). The organic phase was dried over MgSO4, filtered, and concentrated. The residue was purified by column chromatography (0 to 20% MeOH in DCM) to provide the title compound (1.28 g, 86%). 1 H NMR (400 MHz, DMSO-d6) δ 7.99 (d, 1H), 7.31 (s, 1H), 7.07 (s, 1H), 6.96 (s, 1H), 6.81 (d, 1H), 3.75-3.35 (m, 4H), 3.23 (s, 2H), 2.91 (d, 3H), 2.66 (s, 2H), 2.54 (s, 3H), 1.60-1.53 (m, 4H), 1.38 (s, 9H); LC/MS m/z (M+H) + = 477.5. Example 39: 2-(tert-butyl)-1'-(2-(ethylamino)-5-methylquinoline-7-carbon yl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: Synthesis of 7-bromo-N-ethyl-5-methylquinolin-2-amine (C50) To 7-bromo-2-chloro-5-methylquinoline (prepared as described in patent application WO 2013185103 A1, 50 mg, 0.19 mmol) was added iPr 2 NEt (67 μL, 0.39 mmol), ethylamine (70% solution in water, 0.234 mmol), and NMP (0.2 mL). The mixture was heated to about 110 °C for about 18 h, then heated to about 140 °C for about 24 h. The mixture was cooled to about 25 °C and diluted with water. The mixture was extracted with diethyl ether (3X). The combined ether extracts were dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0% to 60% EtOAc in heptanes) to provide the title compound (14 mg, 27%). LC/MS m/z (M+H) + = 265.2. Step 2: Synthesis of 2-(tert-butyl)-1'-(2-(ethylamino)-5-methylquinoline-7-carbon yl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one To compound C50 (80 mg, 0.30 mmol) was added 2,4,6-trichlorophenyl formate (136 mg, 0.60 mmol) and Xantphos (11 mg, 0.018 mmol). A degassed solution of triethylamine (85 μL, 0.60 mmol) and toluene (0.30 mL) was added. The mixture was heated to about 80 °C for about 18 h. The mixture was diluted with ether and filtered through celite. The mixture was concentrated under reduced pressure, redissolved in DCM, and filtered through silica. The mixture was concentrated under reduced pressure. To the residue was added THF (0.2 mL), triethylamine (21 μL, 0.15 mmol), preparation P1 (25 mg, 0.087 mmol), and DMAP (0.5 mg, 0.004 mmol). The mixture was stirred at about 50 °C for about 16 h. Water was added and the mixture was extracted with EtOAc (2x). The combined EtOAc extracts were dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC (Column: XBridge C18, 19x100 mm, 5 µm) to provide the title compound (27 mg, 18%). LC/MS m/z (M+H) + = 491.5. Example 40: 2-(tert-butyl)-1'-(5-methoxy-2-(methylamino)quinoline-7-carb onyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: Synthesis of 5-methoxy-7-(methoxycarbonyl)quinoline 1-oxide (C51) The following reaction was carried out in 2 batches in parallel. To methyl 5- methoxyquinoline-7-carboxylate (1.20 g, 4.70 mmol) in DCM (25 mL) was added m-CPBA (972 mg, 5.63 mmol). The mixture was stirred at about 20 °C for about 16 h. The two batches were combined and saturated aqueous Na 2 S 2 O 3 was added. Saturated aqueous Na2CO3 was added until the pH was >8, then the mixture was stirred for about 30 min. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was diluted with DCM (50 mL) and washed with water (3 X 20 mL). The DCM extract was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified via column chromatography (0% to 15% MeOH in EtOAc) to provide the title compound (1.90 g, 95% average yield). LC/MS m/z (M+H) + = 233.9. Step 2: Synthesis of methyl 5-methoxy-2-(methylamino)quinoline-7-carboxylate (C52) To compound C51 (1.20 g, 5.15 mmol) in DCM (30 mL) at about -70 °C was added Tf 2 O (1.60 g, 5.66 mmol) dropwise. The mixture was stirred for about 15 min at -70 °C, then MeNH 2 (2 M in THF, 20.6 mL, 41.2 mmol) was added dropwise. The mixture was stirred at about -70 °C for about 15 min, then water (30 mL) was added. The aqueous phase was extracted with DCM (30 mL). The combined DCM extracts were washed with brine (2 X 10 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified via column chromatography (0% to 60% EtOAc in petroleum ether) to provide the title compound (800 mg, 63% yield). LC/MS m/z (M+H) + = 247.0. Step 3: Synthesis of 5-methoxy-2-(methylamino)quinoline-7-carboxylic acid (C53) To compound C52 (800 mg, 3.25 mmol) in EtOH (6 mL) was added NaOH (650 mg, 16.2 mmol) and water (6 mL). The reaction was stirred at about 50 °C for about 2 h. Ethanol was removed under reduced pressure and the residue was acidified to pH 5 with 2 N HCl. The precipitate was filtered to provide the title compound (730 mg, 97% yield). LC/MS m/z (M+H) + = 232.9. Step 4: Synthesis of 2-(tert-butyl)-1'-(5-methoxy-2-(methylamino)quinoline-7-carb onyl)- 5H-spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Compound C53 (300 mg, 1.29 mmol) was coupled according to general amide coupling procedure A. The residue was purified by prep-HPLC (Column: Boston Prime C18, 30x150 mm, 5 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile phase B: MeCN; 28-58% B gradient, 9 min, hold at 100% B for 2 min; flow rate 30 mL/min) to provide the title compound (259 mg, 41%). 1 H NMR (400 MHz, CD3OD) δ 8.14 (d, 1H), 7.25 (s, 1H), 6.72 (d, 1H), 6.66 (s, 1H), 3.97 (s, 3H), 3.88-3.79 (m, 2H), 3.55 (m, 2H), 3.26 (s, 2H), 2.99 (s, 3H), 2.74 (s, 2H), 1.77-1.65 (m, 4H), 1.43 (s, 9H); LC/MS m/z (M+H) + = 493.4. Example 41: 2-(tert-butyl)-1'-(4-methoxy-8-methylquinoline-6-carbonyl)-5 H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: Synthesis of 6-bromo-4-methoxy-8-methylquinoline (C54) To 6-bromo-4-chloro-8-methylquinoline (2.67 g, 9.9 mmol) in MeOH (30 mL) was added sodium methoxide (2.61 g, 48.3 mmol). The mixture was stirred at about 80 °C for about 48 h. The mixture was cooled to about 25 °C and diluted with EtOAc. The mixture was washed with water (2x) and brine. The combined EtOAc extracts were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to provide a 1:1 mixture of 6-bromo-4-chloro-8- methylquinoline the title compound (2.50 g). LC/MS m/z (M+H) + = 254.2. Step 2: Synthesis of methyl 4-methoxy-8-methylquinoline-6-carboxylate (C55) To a solution of compound C54 (3.06 g, 12.2 mmol) in MeOH (100 mL) was added triethylamine (5.5 mL, 39 mmol) and Pd(dppf)Cl 2 (720 mg, 0.882 mmol). The mixture was added to a Parr reactor, flushed with nitrogen (3X), and flushed with CO (3X). The mixture was stirred at about 80 °C and 75 PSI of CO for about 16 h. The mixture was cooled to about 25 °C and filtered through celite. The filtrate was concentrated under reduced pressure, then purified via column chromatography (0% to 100% EtOAc in heptanes) to provide the title compound (1.96 g, 70%). LC/MS m/z (M+H) + = 232.5. Step 3: Synthesis of 4-methoxy-8-methylquinoline-6-carboxylic acid (C56) To a solution of compound C55 (1.96 g, 8.48 mmol) in THF (65 mL) was added 2 M NaOH (25 mL, 50 mmol). The mixture was stirred at about 25 °C for about 18 h. The mixture was acidified with conc. HCl to pH 6. The mixture was extracted with EtOAc (2X) and the combined EtOAc extracts were washed with brine, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to provide the title compound (1.46 g, 79%). LC/MS m/z (M+H) + = 218.0. Step 4: Synthesis of 2-(tert-butyl)-1'-(4-methoxy-8-methylquinoline-6-carbonyl)-5 H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one To a solution of preparation P1 (325 mg, 1.03 mmol), compound C56 (264 mg, 1.22 mmol), EDCI (243 mg, 1.27 mmol), and HOBt (263 mg, 1.80 mmol) in DMF (5 mL) at about 25 °C was added i Pr 2 NEt (1.0 mL, 5.6 mmol). The mixture was stirred at about 25 °C for about 16 h. To the mixture was added additional EDCI (225 mg, 1.17 mmol), HOBt (198 mg, 1.40 mmol), and Et 3 N (0.72 mL, 5.2 mmol). The mixture was stirred at about 25 °C for about 2 h, then diluted with EtOAc. The mixture was washed sequentially with 5% aqueous LiCl, saturated aqueous NaHCO 3 , and 1:1 brine:water. The organic phase was dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0 to 8% MeOH in DCM) to provide the title compound (435 mg, 88%). 1 H NMR (400 MHz, DMSO- d6) δ 8.82 (d, 1H), 7.98 (s, 1H), 7.60 (s, 1H), 7.11 (d, 1H), 4.06 (s, 3H), 3.66-3.41 (m, 4H), 3.23 (s, 2H), 2.72 (s, 3H), 2.66 (s, 2H), 1.62-1.51 (m, 4H), 1.38 (s, 9H); LC/MS m/z (M+H) + = 478.5. Example 42: 2-(tert-butyl)-1'-(3-chloro-7-methyl-1H-indole-5-carbonyl)-5 H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: Synthesis of ethyl 3-chloro-7-methyl-1H-indole-5-carboxylate (C61) To a solution of ethyl 7-methyl-1H-indole-5-carboxylate (commercial; 0.45 g, 2.21 mmol) in THF (22 mL) was added NCS (0.5 g, 3.76 mmol). The reaction was stirred at about 25 °C for about 3 h and diluted with EtOAc (20 mL) and water (20 mL). The organic layer was separated, and the aqueous layer was extracted using EtOAc (2 x 20 mL). The combined EtOAc extracts were dried over MgSO 4 , filtered, and concentrated under reduced pressure to provide the title compound (380 mg, 72%) as a yellow solid. 1 H NMR (400 MHz, Chloroform-d) δ 8.36 (s, 0.5H), 8.31 – 8.26 (m, 0.5H), 8.24 (s, 0.5H), 8.18 (d, 0.5H), 7.79 (d, 1H), 7.24 (d, 1H), 4.43 (q, 2H), 2.53 (s, 3H), 1.45 (t, 3H). Step 2: Synthesis of 3-chloro-7-methyl-1H-indole-5-carboxylic acid (C62) To compound ethyl 3-chloro-7-methyl-1H-indole-5-carboxylate C61 (0.38 g, 1.6 mmol) in MeOH (9 mL), and H 2 O (3 mL), was added LiOH . H 2 O (0.2 g, 4.80 mmol). The mixture was heated to about 25 °C for about 4 h and then concentrated. The mixture was diluted with water (6 mL) and extracted with EtOAc (2 x 20 mL); the EtOAc extract was discarded. The pH of the aqueous layer was adjusted to 3-4 with 1 N HCl and extracted using EtOAc (2 x 20 mL). The combined EtOAc extracts were dried over MgSO 4 , filtered, and concentrated under reduced pressure to provide the title compound (210 mg, 63%) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 11.77 (s, 1H), 8.04 (s, 1H), 7.74 – 7.67 (m, 1H), 7.66 (s, 1H), 2.56 (s, 3H, overlap with d-DMSO). Step 3: Synthesis of 2-(tert-butyl)-1'-(3-chloro-7-methyl-1H-indole-5-carbonyl)-5 H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one According to amidation method C, (EDCI, Pyridine, MW) preparation P1, (0.3 g, 0.95 mmol) was coupled with 3-chloro-7-methyl-1H-indole-5-carboxylic acid (0.2 g, 0.95 mmol). The residue was purified by prep-HPLC (Column: Boston Prime C18, 150x30 mm, 3 µm); Mobile Phase A: water (0.05% v/v conc. NH 4 OH); Mobile phase B: MeCN; 9-60% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min to provide the title compound (170 mg, 34.5%). 1 H NMR (400 MHz, DMSO-d6) δ 11.56 (d, 1H), 7.60 (d, 1H), 7.36 – 7.30 (m, 1H), 7.03 (d, 1H), 3.52 (m, 4H), 3.22 (s, 2H), 2.64 (s, 2H), 2.48 (s, 3H), 1.54 (m, 4H), 1.37 (s, 9H); LC/MS m/z (M+H) + = 470.3. Example 43: 2-(tert-butyl)-1'-(4,8-dimethoxyisoquinoline-6-carbonyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one Step 1: Synthesis of ethyl 8-bromoisoquinoline-6-carboxylate (C63) To a solution of ethyl isoquinoline-6-carboxylate (commercial; 22.6 g, 112.3 mmol) in conc. H 2 SO 4 (200 mL) was added NBS (22 g, 124 mmol) in portions. The reaction was stirred at about 10 °C for about 16 h. Additional NBS (3 g, 16.9 mmol) was added, and reaction was stirred at about 10 °C for an additional 16 h. The reaction was quenched by pouring into ice (500 mL), cooled to 0 °C and the pH was adjusted to ~8 using 3 N NaOH (500 mL). The reaction mixture was extracted using DCM (2 x 800 mL), washed with brine (100 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by chromatography using silica gel [(Petroleum ether/DCM = 1:1): EtOAc = 100:0 to 90:10] to provide the title compound (22.2 g, 70.6%) as a white solid. 1 H NMR (400 MHz, Chloroform-d) δ 9.69 (s, 1H), 8.72 (d, 1H), 8.54 (t, 1H), 8.45 (d, 1H), 7.79 – 7.73 (m, 1H), 4.49 (q, 2H), 1.48 (t, 3H); LC/MS m/z (M+H) + = 279.9. Step 2: Synthesis of ethyl 8-bromo-4-chloroisoquinoline-6-carboxylate (C64) To a solution of ethyl 8-bromoisoquinoline-6-carboxylate C63 (0.25 g, 0.89 mmol) in AcOH (5 mL) was added NCS (0.143 g, 1.07 mmol). The reaction mixture was stirred at about 50 °C for about 16 h and then the temperature was increased to about 60 °C for an additional 16 h. The reaction was concentrated, then diluted using EtOAc (50 mL) and sat. aqueous NaHCO3 (20 mL). The layers were separated, and the aqueous layer extracted using EtOAc (3 x 50 mL). The combined EtOAc layers was washed with brine (2 X 30 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by chromatography (0% to 20% EtOAc in petroleum ether) to provide the title compound (0.2 g, 71.2%). 1 H NMR (400 MHz, Chloroform-d) δ 9.51 (d, 1H), 8.82 (t, 1H), 8.67 (s, 1H), 8.42 (d, 1H), 4.43 (q, 2H), 1.41 (t, 3H); LC/MS m/z (M+H) + = 315.9. Step 3: Synthesis of methyl 4,8-dimethoxyisoquinoline-6-carboxylate (C65) To a solution of ethyl 8-bromo-4-chloroisoquinoline-6-carboxylate (0.2 g, 0.636 mmol) in dioxane (5 mL) was added Rockphos-Pd-G3 (0.053 g, 0.0636 mmol), Cs 2 CO 3 (0.414 g, 1.27 mmol) and MeOH (0.102 g, 3.18 mmol). The reaction mixture was stirred at about 80 °C for about 16 h, cooled to room temperature, filtered, and concentrated. The residue was purified by chromatography (0% to 40% EtOAc in petroleum ether) to provide the title compound (0.09 g, 57%) as a yellow solid. 1 H NMR (400 MHz, Chloroform-d) δ 9.22 (d, 1H), 8.42 (dt, 1H), 8.11 (s, 1H), 7.43 (d, 1H), 4.10 – 3.99 (m, 6H), 3.93 (s, 3H); LC/MS m/z (M+H) + = 248.1. Step 4: Synthesis of 4,8-dimethoxyisoquinoline-6-carboxylic acid (C66) To compound methyl 4,8-dimethoxyisoquinoline-6-carboxylate C65 (0.09 g, 0.36 mmol) in MeOH (3 mL), and H 2 O (1 mL), was added LiOH . H 2 O (0.076 g, 1.82 mmol). The reaction mixture was stirred at about 20 °C for about 16 h. The solvent was concentrated, and pH of residue was adjusted to 3-4 using 1N HCl. The resulting solid was filtered and dried under vacuum to provide the title compound (40 mg, 47%) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 13.45 (s, 1H), 9.16 (s, 1H), 8.32 (s, 2H), 7.51 (s, 1H), 4.08 (d, 6H); LC/MS m/z (M+H) + = 233.9. Step 5: Synthesis of 2-(tert-butyl)-1'-(4,8-dimethoxyisoquinoline-6-carbonyl)-5H- spiro[benzo[d]thiazole-6,4'-piperidin]-4(7H)-one (Example 43) According to amidation method A, preparation P1 (0.06 g, 0.17 mmol) was coupled with 4,8-dimethoxyisoquinoline-6-carboxylic acid (0.04 g, 0.17 mmol). The residue was purified by prep-HPLC (Column: Boston Prime C18, 150x30 mm, 5 µm); Mobile Phase A: water (0.05% v/v conc. NH4OH); Mobile Phase B: MeCN; 25-55% B gradient, 9 min, hold at 100% B for 2 min; flow rate 25 mL/min to provide the title compound (57.4 mg, 68%). 1 H NMR (400 MHz, Methanol-d4) δ 9.03 (s, 1H), 8.03 (s, 1H), 7.65 (t, 1H), 7.00 (d, 1H), 3.99 (s, 6H), 3.76 (d, 2H), 3.39 (s, 2H), 3.16 (s, 2H), 2.63 (d, 2H), 1.68 (s, 2H), 1.52 (s, 2H), 1.32 (s, 9H); LC/MS m/z (M+H) + = 494.4. The following compounds of the invention were prepared similarly using the amidation methods described above. For those examples characterized by HPLC retention time, the following HPLC conditions were used: Method 1 Column: ACQUITY UPLC BEH C1850x2.1mm,1.7 µm. Mobile phase A: 10 mM Ammonium Acetate in Water / acetonitrile-95/5 v/v. Mobile phase B: 10 mM Ammonium Acetate in acetonitrile / Water-95/5 v/v. Gradient: 5% D increase to 100% D within 1 min; hold at 100% D for 0.2 min; then back to 0% D at 1.21 min and hold for 0.29 min. Flow: 1.0 mL/min. Method 2 Column: Atlantis dC184.6x50mm 5 µm. Mobile phase A: 0.05% TFA in water (v/v). Mobile phase B: 0.05% TFA in Acetonitrile (v/v). Gradient: 95% water / 5% acetonitrile linear to 5% water / 95% acetonitrile in 4.0 min, HOLD at 5% water / 95% acetonitrile for 5 min. Flow: 2mL/min. Method 3 Column: Xbridge C182.1×50mm 5μm Mobile phase A: 0.0375% TFA in water Mobile phase B: 0.01875% TFA in acetonitrile Gradient: Hold 10%B for 0.5 min then linear to 100% B at 4 min, drop to 10%B at 4.30 min until 4.70 min. Flow: 0.8 mL/min. Method 4 Column: Xbridge C182.1×50mm 5μm Mobile phase A: 0.0375% TFA in water. Mobile phase B: 0.01875% TFA in acetonitrile. Gradient: Hold 1%B for 0.6 min then linear to 100% B at 4 min, drop to 1%B at 4.30 min until 4.70 min. Flow: 0.8 mL/min. Method 5 Column: Waters Acquity HSS T3, 2.1mmx50mm, 1.7µm. Mobile phase A: 0.1% formic acid in water (v/v). Mobile phase B: 0.1% formic acid in acetonitrile (v/v). Gradient: Initial conditions A-95%:B-5%; hold at initial from 0.0-0.1min; Linear Ramp to A-5%:B- 95% over 0.1-1.0min; hold at A-5%:B-95% from 1.0-1.1min; return to initial conditions 1.1-1.5 min. Flow: 1.25mL/min.
Example 109: rac-(R)-(2-(tert-butyl)-4-hydroxy-4,7-dihydro-5H-spiro[benzo [d]thiazole- 6,4'-piperidin]-1'-yl)(7-ethoxy-1,3-dimethyl-1H-indazol-5-yl )methanone To a solution of Example 14 (100 mg, 0.2 mmol) in MeOH (3.5 mL) was added NaBH 4 (23 mg, 0.6 mmol) and stirred at about 25 °C for about 17 h. The reaction was diluted with 10% MeOH-EtOAc (20 mL) and water (20 mL) and organic layer was separated. The aqueous layer was extracted using EtOAc (2 x 20 mL). The combined EtOAc extracts were dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by HPLC (Column: Xbridge C18, 19x100 mm, 5 µm) Mobile phase A: 0.05% TFA in water (v/v); Mobile phase B: 0.05% TFA in acetonitrile (v/v), gradient time of 10 min to provide the title compound (67 mg, 68%); 1 H NMR (600 MHz, DMSO-d6) δ 7.27 (s, 1H), 6.77 (s, 1H), 5.35 – 5.02 (m, 1H), 4.62 (t, 1H), 4.19 (q, 2H), 4.14 (s, 3H), 3.45 – 3.39 (m, 4H, overlap with d-DMSO), 2.75 (d, 1H), 2.68 (d, 1H), 2.43 (s, 3H), 1.99 (dd, 1H), 1.72 (dd, 1H), 1.69 – 1.59 (m, 2H), 1.53 (dd, 2H), 1.44 (t, 3H), 1.35 (s, 9H); LC/MS m/z (M+H) + = 497.3. Deuterated Analogues of the Compound of Example 14 The metabolite profile of compound of Example 14 was evaluated in liver microsomes and hepatocytes (mouse, rat, rabbit, dog, monkey, and human), recombinant human cytochrome P450 enzymes, recombinant human UGT enzymes, and plasma from animals (mouse, rat, and dog). The metabolite profile of Compound XXCAN is comprised of oxidation and glucuronidation. General methods / reviews of obtaining metabolite profile and identifying metabolites of a compound are described in: King, R., “Biotransformations in Drug Metabolism,” Ch.3, Drug Metabolism Handbook Introduction, https://doi.org/10.1002/9781119851042.ch3; Wu, Y., et al, “Metabolite Identification in the Preclinical and Clinical Phase of Drug Development,” Current Drug Metabolish, 2021, 22, 11, 838-857, 10.2174/1389200222666211006104502; Godzien, J., et al, “Chapter Fifteen - Metabolite Annotation and Identification,” Comprehensive Analytical Chemistry, 2018, 82, 415-445, https://doi.org/10.1016/bs.coac.2018.07.004; Zhang, Z., et al, “Drug metabolism in drug discovery and development,” Acta Pharmaceutica Sinica B, 2018, 8(5), 721-732, https://doi.org/10.1016/j.apsb.2018.04.003. The metabolite profile of a compound can also be obtained from publicly available and commercially available software tools. Examples of such tools include, BioTransofrmer 3.0 (biotransformer.ca/new) which predicts the metabolic biotransformations of small molecules using a database of known metabolic reactions; Lhasa Meteor Nexus (www.lhasalimited.org/products/meteor-nexus.htm) offers prediction of metabolic pathways and metabolite structures using a range of machine learning models, which covers phase I and phase II biotransformations of small molecules. Prophetic deuterate69d analogs 110-122 set forth hereinbelow may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life, reduced dosage requirements, reduced CYP450 inhibition (competitive or time dependent), or an improvement in therapeutic index or tolerability. A person with ordinary skill may make additional deuterated analogs of the compound of Example 14. Such additional deuterated analogs may provide similar therapeutic advantages than may be achieved by nondeuterated analogs. Example 110 2-tert-butyl-1'-{7-[(1,1-dideuterio)ethyloxy]-1,3-dimethyl-1 H-indazole-5-carbonyl}-5H- spiro[[1,3]benzothiazole-6,4'-piperidin]-4(7H)-one Example 111 2-tert-butyl-1'-{7-[(pentadeuterio)ethyloxy]-1,3-dimethyl-1H -indazole-5-carbonyl}-5H- spiro[[1,3]benzothiazole-6,4'-piperidin]-4(7H)-one Example 112 1'-{7-[(pentadeuterioethyloxy]-1,3-bis[(trideuterio)methyl]( dideuterio)-1H-indazole- 5-carbonyl}-2-[2-(trideuterio)methyl(hexadeuterio)propan-2-y l](dodecadeuterio)-5H- spiro[[1,3]benzothiazole-6,4'-piperidin]-4(7H)-one Example 113 2-tert-butyl-1'-{7-[(pentadeuterio)ethyloxy]-1,3-bis[(trideu terio)methyl](dideuterio)- 1H-indazole-5-carbonyl}-5H-spiro[[1,3]benzothiazole-6,4'-pip eridin]-4(7H)-one Example 114 2-tert-butyl-1'-{7-[(1,1-dideuterio)ethyloxy]-1,3-bis[(tride uterio)methyl]-1H-indazole- 5-carbonyl}-5H-spiro[[1,3]benzothiazole-6,4'-piperidin]-4(7H )-one Example 115 2-tert-butyl-1'-{7-ethoxy-1,3-bis[(trideuterio)methyl]-1H-in dazole-5-carbonyl}-5H- spiro[[1,3]benzothiazole-6,4'-piperidin]-4(7H)-one Example 116 2-tert-butyl-1'-[7-ethoxy-1-methyl-3-(trideuterio)methyl-1H- indazole-5-carbonyl]-5H- spiro[[1,3]benzothiazole-6,4'-piperidin]-4(7H)-one Example 117 2-tert-butyl-1'-[7-ethoxy-3-methyl-1-(trideuterio)methyl-1H- indazole-5-carbonyl]-5H- spiro[[1,3]benzothiazole-6,4'-piperidin]-4(7H)-one Example 118 2-tert-butyl-1'-{7-[(pentadeuterio)ethyloxy]-1,3-bis[(trideu terio)methyl]-1H-indazole- 5-carbonyl}-5H-spiro[[1,3]benzothiazole-6,4'-piperidin]-4(7H )-one
Example 119 1'-{7-[(pentadeuterio)ethyloxy]-1,3-bis[(trideuterio)methyl] -1H-indazole-5-carbonyl}-2-[2- (trideuterio)methyl(hexadeuterio)propan-2-yl]-5H-spiro[[1,3] benzothiazole-6,4'-piperidin]- 4(7H)-one Example 120 1'-{7-[(pentadeuterio)ethyloxy]-1,3-bis[(trideuterio)methyl] -1H-indazole-5-carbonyl}-2-[2- (trideuterio)methyl(hexadeuterio)propan-2-yl](5,5-dideuterio )-5H-spiro[[1,3]benzothiazole- 6,4'-piperidin]-4(7H)-one Example 121 1'-{7-[(pentadeuterio)ethyloxy]-1,3-bis[(trideuterio)methyl] -1H-indazole-5-carbonyl}-2-[2- (trideuterio)methyl(hexadeuterio)propan-2-yl]-5H-spiro[[1,3] benzothiazole-6,4'-piperidin]- 4(7H)-one
Example 122 2-tert-butyl-1'-(7-ethoxy-1,3-dimethyl-1H-indazole-5-carbony l)(5,5-dideuterio)-5H- spiro[[1,3]benzothiazole-6,4'-piperidin]-4(7H)-one Biological Protocols The utility of the compounds of present invention in the treatment and/or prevention of acne vulgaris in patients may be demonstrated by the activity in the in vitro assays described below. Such assays also provide a means whereby the activities of the compounds of the present invention can be compared with the activities of other known compounds. Direct Inhibition of the Activity of ACC1 The ACC inhibitory activity of the compounds of the present invention was demonstrated by methods based on standard procedures. The direct inhibition of ACC1 for the compounds of the present invention was determined using preparations of recombinant human ACC1 (rhACC1) (SEQ ID NO.1) Preparation of rhACC1 Two liters of SF9 cells, infected with recombinant baculovirus containing full length human ACC1 cDNA, were suspended in ice-cold lysis buffer (25 mM Tris, pH 7.5; 150 mM NaCI; 10% glycerol; 5 mM imidazole (EMD Bioscience; Gibbstown, NJ); 2mM TCEP (BioVectra; Charlottetown, Canada); Benzonase nuclease (10000U/100 g cell paste; Novagen; Madison, Wl); EDTA-free protease inhibitor cocktail (1 tab/50 ml; Roche Diagnostics; Mannheim, Germany). Cells were Iysed by 3 cycles of freeze-thaw and centrifuged at 40,000 X g for 40 minutes (4°C). Supernatant was directly loaded onto a HisTrap FF crude column (GE Healthcare; Piscataway, NJ) and eluted with an imidazole gradient up to 0.5 M over 20 column volumes (CV). ACC1-containing fractions were pooled and diluted 1:5 with 25 mM Tris, pH 7.5, 2mM TCEP, 10% glycerol and direct loaded onto a CaptoQ (GE Healthcare) column and eluted with an NaCI gradient up to 1 M over 20 CV’s. Phosphate groups were removed from purified ACC1 by incubation with lambda phosphatase (100U/10 μM target protein; New England Biolabs; Beverly, MA) for 14 hours at 4°C; okadaic acid was added (1 μM final concentration; Roche Diagnostics) to inhibit the phosphatase. Purified ACC1 was exchanged into 25 mM Tris, pH 7.5, 2 mM TCEP, 10% glycerol, 0.5 M NaCI by 6 hour dialysis at 4°C. Aliquots were prepared and frozen at -80°C. Measurement of rhACC1 inhibition rhACC1 was assayed in a Corning #3820 (Corning, Tewksbury, MA) 384-well plate using the Transcreener ADP detection FP assay kit (Bellbrook Labs, Madison, Wisconsin) using the manufacturer’s recommended conditions for a 50 μM ATP reaction. The final conditions for the assay were 50 mM HEPES, pH 7.2, 10 mM MgCl 2 , 7.5 mM tripotassium citrate, 2 mM DTT, 0.1 mg/mL BSA, 30 μM acetyl-CoA, 50 μΜ ATP, and 10 mM KHCO3. Typically, a 10 μΜ reaction was run for 60 min at room temp, and 10 μl of Transcreener stop and detect buffer was added and the combination incubated at room temp for an overnight (18 hours). The data was acquired on an Envision Fluorescence reader (PerkinEImer) using a 620 excitation Cy5 FP general dual mirror, 620 excitation Cy5 FP filter, 688 emission (S) and a 688 (P) emission filter. Table 1
Geomean IC 50 given, number of repeats n≥2 unless symbol “*” then n=1; NA = not available High content imaging assay to quantify lipid droplets in a human sebocyte cell line One week prior to cell dosing, SZ95 human sebocytes were thawed and grown in a T175 tissue culture flask containing 50 mL media. Media was prepared as follows: Sebomed basal medium with stable glutamine and without phenol red (Sigma; Catalog No. F8205), 10% heat inactivated fetal bovine serum (Invitrogen; Cat. No.10082), 5 ng/mL recombinant human epidermal growth factor (Gibco; Catalog No. PHG0311), 1 mM calcium chloride (Fisher Scientific; Catalog No. BP9742), and 1X penicillin/streptomycin (Thermo Fisher; Catalog No.15140-122). Cells were cultured at 37 o C and the media was replaced every 48-72 hours until start of assay. Compounds were delivered as a 75 nL spot by the Echo 550 (Labcyte) into 384-well assay plates (PerkinElmer; Catalog No. 6057308) with final compound concentrations of 10, 3.162, 1.000, 0.316, 0.100, 0.032, 0.010, 0.003, 0.001, 0.0003, and 0.0001 μM. The final DMSO concentration was 0.1%. SZ-95 cells were washed with Dulbecco’s phosphate buffered saline (DPBS, Lonza; Catalog No. 17-512Q) and then detached with 0.25% Trypsin-EDTA (Gibco; Catalog No.25200056). Growth media (25 mL) was added to the flask and the cells were further diluted to 1.33 x 10^5 cells/mL. SZ-95 cells were plated at a density of 10,000 cells/well in 75 μL and incubated for 48 hours at 37 o C. Using the Biomek FX (Beckman), 25 μL of media was removed and the cells were fixed by adding 18.7 μL of 16% paraformaldehyde (Electron Microscopy Sciences; Cat. No.50980488). After a 30 minute incubation at room temperature, the plates were washed twice with 75μl DPBS. Following the second wash, all remaining DPBS was removed. Staining solution was prepared with 2 μM Bodipy (Invitrogen; Cat. No D3922, diluted 1:1000) and Hoechst (Life Technologies; Cat. No. H3570, diluted 1:2000) in DPBS. Using the Biomek FX, 30 μL of the staining solution was added to each well. The cells were incubated for 20 minutes at room temperature, then washed once with 75 μL DPBS. Finally, 30 μL of DPBS was added to each well and the plates were sealed with light blocking film. The plates were read on an Opera Phenix (PerkinElmer) for high content imaging. Nuclei were detected by Hoechst staining and lipid droplets by Bodipy, which stains neutral lipids. Active compounds caused a reduction in the number and area of lipid droplets. The percent (%) effect at each concentration of compound is calculated by Genedata Screener analysis program using a four-parameter logistic dose response equation and is based on and relative to the amount of lipid droplets in the positive and negative control wells contained within each assay plate to determine the 50% inhibition concentration (IC50). Table 2
Geomean IC 50 given, number of repeats n≥2 unless symbol “*” then n=1; NA = not available Radiometric measurement of de novo lipogenesis in cultured human sebocytes SZ95 sebocytes were grown in Human Sebocyte Growth Medium (HSGM) containing Sebomed® basal medium (Sigma-Aldrich, F8205) supplemented with 10% heat- inactivated fetal bovine serum (Gibco, 10100-147), 1% penicillin/streptomycin (Gibco, 15070-063), 1 mM calcium chloride (Fisher, BP9742-10X5) and 5 ng/mL recombinant human epidermal growth factor (Gibco, PHG0311). At 90% confluence, cells were washed with PBS and then detached with 0.05% Trypsin-EDTA (Gibco, 25300054). Prior to starting the assay, cells were centrifuged and resuspended in HSGM containing 5% charcoal-stripped serum (Life Technologies, 12676-029) instead of 10% heat-inactivated fetal bovine serum. Cells were added to 24-well plates at a density of 0.25x10 6 cells/well and incubated overnight at 37°C to allow the cells to adhere to the culture plate. Cells were then treated with a dose response of compound (30, 1, 0.03, 0.006, 0.0009, 0.0002, and 0.00003 μM) with each concentration tested in duplicate. Briefly, compounds were dissolved in DMSO stocks and diluted 1:1000 into HSGM with charcoal-stripped media. The vehicle control wells were treated with 0.1% DMSO. After a 1-hour preincubation with compound or vehicle at 37°C, 0.25 μCi 14 C sodium acetate (American Radiolabeled Chemicals: ARC, 0173A) was added to each well. Plates were incubated for an additional two hours at 37°C. At the end of the incubation period, cells were removed from incubator, placed on ice, and then washed twice with ice-cold PBS to remove free 14 C-sodium acetate. Plates were sealed and stored at -20°C until analysis. To induce lysis, 125 μL of mammalian protein extraction reagent (MPER; Fisher, 78501) was added to each well. Plates were shaken for 1 hour at room temperature and lysates transferred to individual 2 mL polypropylene tubes. Wells were then washed with 175 μL PBS, which was added to lysates. A chloroform:methanol solution (1:1 v/v, 450 μL) was added to each tube. All tubes were vortexed for 10 seconds and centrifuged at 14,000 x g for 5 minutes at room temperature to separate the aqueous and organic phases. A 25 μL aliquot was removed from the bottom organic layer of each sample and added to 6 mL Optiphase Supermix scintillation fluid (PerkinElmer, 1200-439). Counts of 14 C were assessed by scintillation counting. DNL (counts of 14 C incorporated into lipids) was expressed as a percentage for compound treated cells relative to the vehicle control. IC50 values were determined using a non-linear regression (four parameter with variable slope) in GraphPad Prism. Table 3. Geo mean IC 50 given, number of repeats n≥2 unless symbol “*” then n=1
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