deuterated colony stimulating factor-1 receptor (csf-1r) inhibitors

Abstract

Disclosed herein are deuterated compounds of the formulae below, which are useful as colony stimulating factor 1 receptor inhibitors ("CSF-1R inhibitors").

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 129,939, filed December 23, 2020, and U.S. Provisional Application No. 63 / 226,549, filed July 28, 2021, which are incorporated herein by reference in their entirety for any purpose. Background Technology

[0002] Drugs often exhibit poor absorption, distribution, metabolism, and / or excretion (ADME) properties, hindering their broader use or limiting their application in certain indications. Poor ADME properties can also be a major cause of drug candidate failure in clinical trials. While formulation and prodrug strategies can be employed in some cases to improve certain ADME properties, these approaches typically fail to address the fundamental ADME problems present in many drugs and drug candidates.

[0003] One such problem is rapid metabolism, which causes many drugs to be rapidly eliminated from the body—drugs that would otherwise be highly effective in treating the disease. A possible solution to rapid drug clearance is frequent dosing or high-dose administration to achieve sufficiently high plasma drug levels. However, this approach has potential drawbacks, including poorer patient adherence to dosing regimens, more severe side effects at higher doses, and increased treatment costs. Rapidly metabolized drugs may also expose patients to unwanted toxic or reactive metabolites.

[0004] The degradation of toxic or bioreactive metabolites can also be a problem, causing some patients receiving the drug to experience toxicity, or limiting safe dosing, resulting in patients receiving suboptimal doses of the active agent. Sometimes, changing the dosing interval or formulation can help reduce adverse clinical reactions, but often the formation of such undesirable metabolites is inherent to the compound's metabolism.

[0005] Enzymes in living organisms, such as aldehyde oxidases, can lead to unwanted metabolic degradation. Aldehyde oxidases (AOs) are molybdenum-containing enzymes involved in the biotransformation of many drugs. The challenges represented by AO-mediated metabolism are driven by several overlapping factors, including the complex biology of the enzymes and the widespread use of structural motifs that serve as substrates for AOs (e.g., nitrogen-containing heterocyclic compounds and amides). See, for example, Manevski, N. et al., Metabolism by Aldehyde Oxidase: Drug Design and Complementary Approaches to Challenges in Drug Discovery, J. Med. Chem. 2019, 62, 10955-10994. Furthermore, variability in AO-mediated metabolism (not only between species but also between individuals) contributes to the variability in exposure and complicates human dose selection.

[0006] While the strategy of simply avoiding substrates susceptible to AO metabolism may seem appealing, this would impractically eliminate a vast potential pharmacore. Therefore, various strategies have been proposed to modulate the potential AO metabolism of drug compounds. These strategies include attempting to block the AO response (e.g., by conjugating the compound with an AO inhibitor); attempting to reduce the rate of the AO response; and using AO metabolites as novel scaffolds or prodrugs. See, for example, Manevski et al. Furthermore, to halt or mitigate AO metabolism of the pharmacore, the reaction site between the AO and the pharmacore must be identified. Manevski et al. provide a table of suggested strategies for mitigating AO metabolism, such as blocking the AO reaction site, replacing carbon with heteroatoms, replacing nitrogen with carbon, removing aromaticity, reducing ring size, kinetic deuterium isotope effect (“KDIE”), and reducing logD; however, knowledge of the AO degradation site is crucial in each instance. See Table 4 of Manevski et al. These strategies all include supplementary measures based on laboratory testing to predict human clearance. In other words, for the average person skilled in the art, there is no predictable way to know whether any proposed strategy will be able to develop a drug that targets a specific target, maintains the intended effect of the drug for its intended purpose (e.g., high efficacy, target binding, or bioavailability), and also mitigates AO degradation without extensive testing using appropriate biological samples. Summary of the Invention

[0007] Surprisingly, it has been found that deuterium-substituted CSF-1R inhibitor compounds, such as those shown in WO 2017 / 015267, can improve ADME properties. In some aspects of this disclosure, deuterium-substituted CSF-1R inhibitor compounds exhibit improved ADME properties, particularly significant resistance to AO degradation, thus potentially improving drug efficacy and in vivo drug exposure. This document discloses deuterated colony-stimulating factor-1 receptor inhibitors (“CSF-1R inhibitors”) resistant to enzymatic degradation in vivo. The CSF-1R inhibitors of this disclosure are small molecule compounds capable of crossing the blood-brain barrier to reach the central nervous system (CNS). Because these compounds are capable of advantageously crossing the blood-brain barrier (a highly desirable property in neurological indications), the compounds need to exhibit sufficient absorption, metabolism, distribution, and excretion (ADME) properties to ensure appropriate dosing. Metabolic issues can include rapid metabolism and metabolic degradation, both of which can lead to toxicity and / or suboptimal dosing of the active agent.

[0008] This disclosure relates to deuterated CSF-1R inhibitors and to the use of deuterated CSF-1R inhibitors and pharmaceutical compositions comprising CSF-1R inhibitors for the treatment of diseases, said inhibitors and said pharmaceutical compositions having surprisingly reduced AO degradation and high efficacy as CSF-1R inhibitors for the treatment of diseases.

[0009] Such compounds include those of formula (I):

[0010]

[0011] and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts, wherein:

[0012] The dashed lines represent optional double bonds;

[0013] X 1 Is it C, N, or CR? 7 ;

[0014] X 2 X 3 X 4 X 5 X 6 and X 7 Each is independently selected from N and NR. 7 or CR 7 ;

[0015] X 8 and X 9 Each is independently selected from N or C;

[0016] Each R 7Independently selected from H, D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C2-C 10 )alkylynyl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, (C2-C 10 ) alkynylamine, C(O)-, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl-,COOH-(C3-C 10 )cycloalkyl-, (C1-C 10 )alkoxy-, R 8 -(C1-C 10 )alkyl-, R 8 -(C3-C 10 )cycloalkyl, R 8 -(C2-C9) heterocyclic alkyl, R 8 -(C6-C 14 )Aryl, R 8 -(C2-C9) heteroaryl, R 8 -(C2-C 10 )alkylynyl, R 8 -(C1-C 10 )alkylamine, R 8 -((C1-C 10 )alkyl)2amine, R 8 -(C2-C 10 ) acetylinylamine, R 8 -C(O)-、R 8 -(C1-C 10 )alkyl-C(O)O-, R 8 -(C1-C 10 )alkoxy-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 )aryl-O-, (C2-C9)heteroaryl-O-, R 8 -(C3-C 10 )cycloalkyl-O-, R 8 -(C2-C9)heterocyclic alkyl-O-, R 8 -(C6-C 14 )Aryl-O-, R 8 -(C2-C9)heteroaryl-O-, HO-, halogen, cyano, H2N-, (CH3)HN-, (CH3)2N-, R8 R 9 N-, R 8 R 9 N(O)C-, R 8 (R 9 C(O))N-、R 8 R 9 NC(O)O-、R 8 C(O)-, R 8 R 9 NC(O)R 8 N-, (C1-C 10 )alkyl-OC(O)R 8 N-, (C3-C 10 )cycloalkyl-OC(O)R 8 N-, (C2-C9) heterocyclic alkyl-OC(O)R 8 N-, (C6-C 14 )Aryl-OC(O)R 8 N-,(C2-C9)heteroaryl-OC(O)R 8 N-, F3C-, F2HC-, CH3F2C-, FH2C-, CH3FHC-, (CH3)2FC-; NC-, (C1-C 10 )alkyl(O)P-,(C1-C 10 )alkyl-S-, (C1-C 10 )alkyl-S-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-S-, (C6-C 14 aryl-S-, (C2-C9)heteroalkyl-S-, (C2-C9)heterocycloalkyl-S-, (C2-C9)heteroaryl-S-, (C1-C 10 )alkyl-S(O)-, (C3-C 10 )cycloalkyl-S(O)-, (C6-C 14 aryl-S(O)-, (C2-C9)heterocyclic alkyl-S(O)-, (C2-C9)heteroaryl-S(O)-, (C3-C 10 )alkyl-S(O)2-, (C3-C 10 )cycloalkyl-S(O)2-, (C6-C 14 aryl-S(O)2-, (C2-C9)heterocyclic alkyl-S(O)2-, (C2-C9)heteroaryl-S(O)2-, R 8 R 9 NS(O)2-、(C1-C 10 )alkyl-S(O)2R 8 N-, (C3-C 10 )cycloalkyl-S(O)2R8 N-, (C6-C 14 )Aryl-S(O)2R 8 N-, (C2-C9) heterocyclic alkyl-SO2R 8 N- and (C2-C9)heteroaryl-S(O)2R 8 N-;

[0017] Where R 8 and R 9 Each is independently selected from H, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl, COOH-(C3-C 10 )cycloalkyl, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 )aryl-O-, (C2-C9)heteroaryl-O-, HO-, halogen, (CH3)2N- and H2N-;

[0018] Or R 8 and R 9 Together they form 3 to 10-membered cycloalkyl, heterocycloalkyl, aryl, or heteroaryl rings;

[0019] Each of (C1-C) 10 )alkyl, (C6-C 14 ) aryl, (C2-C9) heteroaryl, (C3-C 10 The cycloalkyl or (C2-C9) heterocycloalkyl group is further optionally substituted by one to four groups selected from the following: (C1-C9) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10)alkyl-C(O)O-,COOH-(C1-C 10 )alkyl, COOH-(C3-C 10 )cycloalkyl, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 )aryl-O-, (C2-C9)heteroaryl-O-, HO-, halogen or H2N-;

[0020] T 1 T 2 and T 3 Each is independently selected from N or CR. 10 ,

[0021] Each R 10 Independently selected from H, D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C2-C 10 )alkylynyl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, (C2-C 10 ) alkynylamine, C(O)-, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl-,COOH-(C3-C 10 )cycloalkyl-, (C1-C 10 )alkoxy-, R 10A -(C1-C 10 )alkyl-, R 10A -(C3-C 10 )cycloalkyl, R 10A -(C2-C9) heterocyclic alkyl, R 10A -(C6-C 14 )Aryl, R 10A -(C2-C9) heteroaryl, R 10A -(C2-C 10 )alkylynyl, R 10A -(C1-C 10 )alkylamine, R 10A -((C1-C 10 )alkyl)2amine, R 10A -(C2-C 10) acetylinylamine, R 10A -C(O)-、R 10A -(C1-C 10 )alkyl-C(O)O-, R 10A -(C1-C 10 )alkoxy-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 )aryl-O-, (C2-C9)heteroaryl-O-, R 10A -(C3-C 10 )cycloalkyl-O-, R 10A -(C2-C9)heterocyclic alkyl-O-, R 10A -(C6-C 14 )Aryl-O-, R 10A -(C2-C9)heteroaryl-O-, HO-, halogen, cyano, H2N-, (CH3)HN-, (CH3)2N-, R 10A R 11 N-, R 10A R 11 N(O)C-, R 10A (R 11 C(O))N-、R 10A R 11 NC(O)O-、R 10A C(O)-, R 10A R 11 NC(O)R 10A N-, (C1-C 10 )alkyl-OC(O)R 10A N-, (C3-C 10 )cycloalkyl-OC(O)R 10A N-, (C2-C9) heterocyclic alkyl-OC(O)R 10A N-, (C6-C 14 )Aryl-OC(O)R 10A N-,(C2-C9)heteroaryl-OC(O)R 10A N-, F3C-, F2HC-, CH3F2C-, FH2C-, CH3FHC-, (CH3)2FC-; NC-, (C1-C 10 )alkyl(O)P-,(C1-C 10 )alkyl-S-, (C1-C 10 )alkyl-S-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-S-, (C6-C 14aryl-S-, (C2-C9)heteroalkyl-S-, (C2-C9)heterocycloalkyl-S-, (C2-C9)heteroaryl-S-, (C1-C 10 )alkyl-S(O)-, (C3-C 10 )cycloalkyl-S(O)-, (C6-C 14 aryl-S(O)-, (C2-C9)heterocyclic alkyl-S(O)-, (C2-C9)heteroaryl-S(O)-, (C3-C 10 )alkyl-S(O)2-, (C3-C 10 )cycloalkyl-S(O)2-, (C6-C 14 aryl-S(O)2-, (C2-C9)heterocyclic alkyl-S(O)2-, (C2-C9)heteroaryl-S(O)2-, R 10A R 11 NS(O)2-、(C1-C 10 )alkyl-S(O)2R 10A N-, (C3-C 10 )cycloalkyl-S(O)2R 10A N-, (C6-C 14 )Aryl-S(O)2R 10A N-, (C2-C9) heterocyclic alkyl-SO2R 10A N- and (C2-C9)heteroaryl-S(O)2R 10A N-;

[0022] Where R 10A and R 11 Each is independently selected from H, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl, COOH-(C3-C 10 )cycloalkyl, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 )aryl-O-, (C2-C9)heteroaryl-O-, HO-, halogen, (CH3)2N- and H2N-;

[0023] Or R 10A and R 11 Together they form 3 to 10-membered cycloalkyl, heterocycloalkyl, aryl, or heteroaryl rings;

[0024] Each of (C1-C) 10 )alkyl, (C6-C 14 ) aryl, (C2-C9) heteroaryl, (C3-C 10 The cycloalkyl or (C2-C9) heterocycloalkyl group is further optionally substituted by one to four groups selected from the following: D, (C1-C9) cycloalkyl groups. 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl, COOH-(C3-C 10 )cycloalkyl, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 aryl-O-, (C2-C9)heteroaryl-O-, HO-, halogen or H2N-

[0025] Y 1 It is O, NR 12 or CR 12 R 13 ,

[0026] Where R 12 Does not exist or R 12 and R 13 Each is independently selected from H, D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10)alkyl, COOH-(C3-C 10 )cycloalkyl, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 )aryl-O-, (C2-C9)heteroaryl-O-, HO-, halogen and H2N-;

[0027] R 1 Together with the attached carbon, it forms a carbonyl group and R 2 Does not exist or R 1 and R 2 Each is independently selected from H, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 )aryl-O-, (C2-C9)heteroaryl-O-, HO-, halogen and H2N-, or R 1 and R 2 Together with the attached carbon, they form 3 to 10-membered rings;

[0028] R 5 It does not exist or is selected from H, D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14)aryl-O-, (C2-C9)heteroaryl-O-, HO-, halogen and H2N-;

[0029] R 6 Selected from H, D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C2-C 10 )alkylynyl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, (C2-C 10 ) alkynylamine, C(O)-, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl-,COOH-(C3-C 10 )cycloalkyl-, (C1-C 10 )alkoxy-, R 14 -(C1-C 10 )alkyl-, R 14 -(C3-C 10 )cycloalkyl, R 14 -(C2-C9) heterocyclic alkyl, R 14 -(C6-C 14 )Aryl, R 14 -(C2-C9) heteroaryl, R 14 -(C2-C 10 )alkylynyl, R 14 -(C1-C 10 )alkylamine, R 14 -((C1-C 10 )alkyl)2amine, R 14 -(C2-C 10 ) acetylinylamine, R 14 -C(O)-、R 14 -(C1-C 10 )alkyl-C(O)O-, R 14 -(C1-C 10 )alkoxy-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 )aryl-O-, (C2-C9)heteroaryl-O-, R 14 -(C3-C 10 )cycloalkyl-O-, R 14 -(C2-C9)heterocyclic alkyl-O-, R 14 -(C6-C 14 )Aryl-O-, R14 -(C2-C9)heteroaryl-O-, HO-, halogen, cyano, H2N-, (CH3)HN-, (CH3)2N-, R 14 R 15 N-, R 14 R 15 N(O)C-, R 14 (R 15 C(O))N-、R 14 R 15 NC(O)O-、R 14 C(O)-, R 14 R 15 NC(O)R 14 N-, (C1-C 10 )alkyl-OC(O)R 14 N-, (C3-C 10 )cycloalkyl-OC(O)R 14 N-, (C2-C9) heterocyclic alkyl-OC(O)R 14 N-, (C6-C 14 )Aryl-OC(O)R 14 N-,(C2-C9)heteroaryl-OC(O)R 14 N-, F3C-, F2HC-, CH3F2C-, FH2C-, CH3FHC-, (CH3)2FC-; NC-, (C1-C 10 )alkyl(O)P-,(C1-C 10 )alkyl-S-, (C1-C 10 )alkyl-S-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-S-, (C6-C 14 aryl-S-, (C2-C9)heteroalkyl-S-, (C2-C9)heterocycloalkyl-S-, (C2-C9)heteroaryl-S-, (C1-C 10 )alkyl-S(O)-, (C3-C 10 )cycloalkyl-S(O)-, (C6-C 14 aryl-S(O)-, (C2-C9)heterocyclic alkyl-S(O)-, (C2-C9)heteroaryl-S(O)-, (C3-C 10 )alkyl-S(O)2-, (C3-C 10 )cycloalkyl-S(O)2-, (C6-C 14 aryl-S(O)2-, (C2-C9)heterocyclic alkyl-S(O)2-, (C2-C9)heteroaryl-S(O)2-, R 14 R 15 NS(O)2-、(C1-C10 )alkyl-S(O)2R 14 N-, (C3-C 10 )cycloalkyl-S(O)2R 14 N-, (C6-C 14 )Aryl-S(O)2R 14 N-, (C2-C9) heterocyclic alkyl-SO2R 14 N- and (C2-C9)heteroaryl-S(O)2R 14 N-;

[0030] Where R 14 and R 15 Each is independently selected from H, D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl, COOH-(C3-C 10 )cycloalkyl, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 )aryl-O-, (C2-C9)heteroaryl-O-, HO-, F2HC-O-, halogen, (CH3)2N-, H2N-, F3C-C(O)-, F3C- and F2HC-;

[0031] Or R 14 and R 15 Together they form 3 to 10-membered cycloalkyl, heterocycloalkyl, aryl, or heteroaryl rings;

[0032] Each of (C1-C) 10 )alkyl, (C6-C 14 ) aryl, (C2-C9) heteroaryl, (C3-C 10 The cycloalkyl or (C2-C9) heterocycloalkyl group is further optionally substituted by one to four groups selected from the following: (C1-C9) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14)aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl, COOH-(C3-C 10 )cycloalkyl, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, (C3-C 10 )cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 )aryl-O-, (C2-C9)heteroaryl-O-, HO-, halogen or H2N-;

[0033] Z 1 Selected from H, halogen, (C1-C) 10 alkyl, (C2-C9)heteroalkyl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, (C2-C 10 ) alkynylamine, (C1-C 10 )alkoxy- or H2N-;

[0034] Y 2 It is O, S, NR 17 or CR 17 R 18 ,and

[0035] Where R 17 Does not exist or R 17 and R 18 Each is independently selected from H, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl, COOH-(C3-C 10 )cycloalkyl, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, (C3-C 10)cycloalkyl-O-, (C2-C9)heterocyclic alkyl-O-, (C6-C 14 )aryl-O-, (C2-C9)heteroaryl-O-, HO-, halogen or H2N-;

[0036] Where R 7 R 1 Or R 2 At least one of them is D.

[0037] In at least one aspect, this disclosure relates to compounds of formula (I):

[0038]

[0039] and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts, wherein:

[0040] The dashed lines represent optional double bonds;

[0041] X 1 Is it C, N, or CR? 7 ;

[0042] X 2 X 3 X 4 X 5 X 6 X 7 Each is independently selected from N and NR. 7 or CR 7 ;

[0043] X 8 and X 9 Each is independently selected from N or C;

[0044] Each R 7 Independently selected from H, D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C2-C9)heteroaryl, (C2-C 10 ) alkynylamine, (C1-C 10 )alkyl-C(O)O-, (C1-C 10 )alkoxy-, R 8 -(C2-C9) heterocyclic alkyl, R 8 -(C2-C9) heteroaryl, R 8 -(C2-C 10 )alkylynyl, R 8 -(C2-C 10 ) acetylinylamine, R 8 -(C1-C 10)alkoxy-, R 8 -(C2-C9)heterocyclic alkyl-O-, halogen, cyano, H2N-, (CH3)HN-, (CH3)2N-, R 8 C(O)-, F3C-, F2HC-, CH3F2C-, FH2C-, CH3FHC- and (CH3)2FC;

[0045] Where R 8 Each is independently selected from H, (C1-C) 10 )alkyl, (C2-C9)heterocyclic alkyl, (C1-C 10 )alkylamines, (C1-C 10 )alkyl-C(O)O-, (C1-C 10 )alkoxy-, HO-, halogen, (CH3)2N- and H2N-;

[0046] Each of (C1-C) 10 alkyl, (C2-C9)heteroaryl, or (C2-C9)heterocyclic alkyl groups are further optionally substituted by one to four groups selected from the following: deuterium, (C1-C9)heteroaryl, (C2-C9)heterocyclic alkyl groups. 10 )alkyl or (C1-C 10 Alkylamines;

[0047] T 1 T 2 and T 3 Each is independently selected from N or CR 10 ;

[0048] Each R 10 Independently selected from H, D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, (C2-C 10 ) alkynylamine, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl-,COOH-(C3-C 10 )cycloalkyl-, (C1-C 10 )alkoxy-, R 10A -(C1-C 10 )alkyl-, R 10A -(C1-C 10 )alkylamine, R 10A -((C1-C 10 )alkyl)2amine, R 10A -(C2-C 10 ) acetylinylamine, R 10A-C(O)-、R 10A -(C1-C 10 )alkyl-C(O)O-, R 10A -(C1-C 10 )alkoxy-, HO- and halogen, cyano, H2N-, (CH3)HN-, (CH3)2N-, R 10A R 11 N-, R 10A R 11 N(O)C-, R 10A (R 11 C(O))N-、R 10A R 11 NC(O)O-、R 10A C(O)-, R 10A R 11 NC(O)R 10A N-, (C1-C 10 )alkyl-OC(O)R 10A N-, F3C-, F2HC-, CH3F2C-, FH2C-, CH3FHC-, (CH3)2FC-;

[0049] Where R 10A and R 11 Each is independently selected from H, D, (C1-C) 10 )alkyl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, HO-, halogenated, (CH3)2N- and H2N-;

[0050] Each of (C1-C) 10 The alkyl group is further optionally substituted by one to four groups selected from the following: D, (C1-C2) 10 )alkyl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, HO-, halogen or H2N-

[0051] Y 1 It is O, NR 12or CR 12 R 13 ;

[0052] Where R 12 Does not exist or R 12 and R 13 Each is independently selected from H, D, (C1-C) 10 )alkyl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 Alkyl-, HO-, halogenated and H2N-;

[0053] R 1 and R 2 Each is independently selected from H, D, (C1-C) 10 Alkyl, HO-, halogenated and H2N;

[0054] R 5 It does not exist or is selected from H, D, (C1-C) 10 Alkyl, HO-, halogen, and H2N-; and

[0055] R 6 Selected from D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, R 14 -(C3-C 10 )cycloalkyl, R 14 -(C6-C 14 )Aryl, R 14 -(C2-C9) heteroaryl and R 14 -(C1-C 10 Alkylamines;

[0056] Where R 14 Each is independently selected from H, D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, (C1-C 10)alkoxy-, HO-, F2HC-O-, halogen, (CH3)2N-, F3C-C(O)-, F3C- and F2HC-;

[0057] Each of (C1-C) 10 )alkyl, (C6-C 14 ) aryl, (C2-C9) heteroaryl, (C3-C 10 The cycloalkyl or (C2-C9) heterocycloalkyl group is further optionally substituted by one to four groups selected from the following: (C1-C9) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, HO-, halogen or H2N-; and

[0058] Z 1 Selected from H, halogen groups and (C1-C) 10 )alkyl;

[0059] Y 2 It is O, NR 17 or CR 17 R 18 ;

[0060] Where R 17 Does not exist or R 17 and R 18 Each is independently selected from H, (C1-C) 10 Alkyl, HO-, halogenated and H2N-;

[0061] Where R 7 R 1 Or R 2 At least one of them is D.

[0062] In at least one aspect, this disclosure relates to compounds of formula (I'):

[0063]

[0064] and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts, wherein:

[0065] The dashed lines represent optional double bonds;

[0066] A is selected from H and D;

[0067] X 3’ It is CR 3’ , where R 3’ Selected from H and D;

[0068] X 4’It is CR 4’ , where R 4’ Selected from H, D and R 7 ;and

[0069] X 5’ It is CR 5’ , where R 5’ Selected from H and D,

[0070] Among them, A and R 3’ R 4’ and R 5’ At least one of them is D.

[0071] This disclosure also relates to pharmaceutical formulations comprising deuterated CSF-1R inhibitors and to the use of deuterated CSF-1R inhibitors and pharmaceutical compositions comprising CSF-1R inhibitors for the treatment of diseases. Further, this document discloses the use of deuterated CSF-1R inhibitors and pharmaceutical compositions comprising deuterated CSF-1R inhibitors for the treatment of immune-mediated diseases and neurological disorders, wherein the inhibitors are resistant to aldehyde oxidase degradation. The immune-mediated diseases include multiple sclerosis, lupus nephritis, and rheumatoid arthritis, and the neurological disorders include amyotrophic lateral sclerosis (ALS), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and Huntington's disease.

[0072] Regarding deuterium modification, researchers have sought to slow down CYP-mediated metabolism of drugs or reduce the formation of undesirable metabolites by replacing one or more hydrogen atoms with deuterium atoms. Deuterium is a safe, stable, and non-radioactive isotope of hydrogen. Compared to hydrogen, deuterium forms stronger bonds with carbon. In selected cases, the increased bond strength conferred by deuterium can positively influence the ADME properties of drugs, potentially leading to improved drug efficacy, safety, and / or tolerability. Simultaneously, since deuterium is substantially the same size and shape as hydrogen, deuterium substitution for deuterium is not expected to substantially affect the biochemical potency and selectivity of drugs compared to the original chemical entity containing only hydrogen.

[0073] It should be noted that the effects of deuterium substitution on metabolic rates have been reported for a very small percentage of approved drugs (see, for example, Blake, MI et al., J Pharm Sci, 1975, 64:367-91; Foster, AB, Adv Drug Res 1985, 14:1-40 (“Foster”); Kushner, DJ et al., Can J Physiol Pharmacol 1999, 79-88; Fisher, MB et al., Curr Opin Drug Discov Devel, 2006, 9:101-09 (“Fisher”)). However, the results are variable and unpredictable. For some compounds, deuteration causes a decrease in metabolic clearance in vivo. For others, metabolism remains unchanged. Still others exhibit increased metabolic clearance. The variability of the deuterium effect has also led experts to question or disregard deuterium modification as a viable drug design strategy for inhibiting undesirable metabolism (see Foster's page 35 and Fisher's page 101).

[0074] The compounds disclosed herein are deuterium-substituted CSF-1R inhibitor compounds as shown in WO 2017 / 015267, and possess improved ADME properties and, in particular, high resistance to AO degradation, thus potentially improving drug efficacy and in vivo drug exposure. Given the interlocking and contradictory challenges described by Manevski et al. in overcoming AO-mediated metabolism (e.g., the need to assess and balance multiple conflicting factors such as compound structural motifs), experimentally determining in vitro and in vivo properties (e.g., in liver microsomes or hepatocytes); and the aforementioned uncertainties regarding the improved ADME properties, particularly the reduction in AO degradation, obtained through deuterium substitution, this result is quite surprising and unexpected.

[0075] In one embodiment, this disclosure relates to a method of treating a subject requiring such treatment for a disease or disorder mediated by colony-stimulating factor-1 receptor (CSF-1R) or involving CSF-1R, the method comprising administering to the subject an effective amount of a compound according to formula (I) or (I') and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts. In another embodiment, the disease or disorder is a neurological or immune-mediated disease requiring such treatment in a subject, including multiple sclerosis, ALS, MSA, PSP, Huntington's disease, lupus, lupus nephritis, and rheumatoid arthritis, such treatment comprising administering to the subject an effective amount of a compound according to formula (I) or (I') and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts.

[0076] This disclosure also relates to pharmaceutical compositions comprising compounds according to formula (I) or formula (I'). Attached Figure Description

[0077] Figures 1A and 1B illustrate the effect of the exemplary CSF-1R inhibitory compound (compound 24) on the production of MCP-1 chemokines after CSF-1 stimulation. Mean values ​​(Figure 1A) and standard deviations (Figure 1B) are shown.

[0078] Figures 2A and 2B illustrate the effect of the exemplary deuterated CSF-1R inhibitory compound (compound 6) on the production of MCP-1 chemokines after CSF-1. Mean values ​​(Figure 2A) and standard deviations (Figure 2B) are shown.

[0079] Figures 3A and 3B show the IC values ​​of the experiment in Figure 1. 50 curve.

[0080] Figures 4A and 4B show the IC values ​​of the experiment in Figure 2. 50 curve.

[0081] Figures 5A-5C show that the CSF-1R inhibitor compound 6 (Figure 5A) and the PLX3397 control (Figure 5C) significantly reduced MCP-1 production in a concentration-dependent manner. The results for compound 49 are shown in Figure 5B.

[0082] Figures 6A-6C show that CSF-1 stimulation significantly increased Iba1 levels. + The area was significantly reduced, and treatment with the CSF-1R inhibitor compound 6 (Figure 6A) and the control PLX3397 (Figure 6C) significantly eliminated this effect in a concentration-dependent manner. The results for compound 49 are shown in Figure 6B.

[0083] Figures 7A-7C show that CSF-1 stimulation increased the number of cells in the culture (e.g., via DAPI). + (Cellular evidence), and CSF-1R inhibitor compound 6 (Fig. 7A) and control PLX3397 (Fig. 7C) reduced this amount in a concentration-dependent manner. Results for compound 49 are shown in Fig. 7B.

[0084] Figures 8A-8B show cell viability after pretreatment with DMSO (control) or compound 6 and CSF-1 in wild-type (Figure 8A) or SOD1 (Figure 8B) cells.

[0085] Figures 9A-9B show cell viability after treatment with DMSO (control) or compound 6 and LPS in wild-type (Figure 9A) or SOD1 (Figure 9B) cells.

[0086] Figures 10A-10B show the production of MCP-1 in wild-type (Figure 10A) or SOD1 (Figure 10B) cells after stimulation with DMSO (control) or compound 6 and CSF-1.

[0087] Figures 11A-11B show the production of IL-12p40 in wild-type (Figure 11A) or SOD1 (Figure 11B) cells after stimulation with DMSO (control) or compound 6 and LPS.

[0088] Figure 12A shows the in vitro metabolic curve of compound 24 after incubation in human cryopreserved hepatocytes in the absence of the AO inhibitor hydralazine.

[0089] Figure 12B shows the in vitro metabolic curve of compound 24 after incubation in human cryopreserved hepatocytes in the presence of the AO inhibitor hydralazine.

[0090] Figure 13 illustrates the proposed metabolic pathway of compound 24 in cryopreserved human hepatocytes in the absence and presence of the AO inhibitor hydralazine.

[0091] Figure 14A shows the in vitro metabolic curve of compound 6 after incubation in human cryopreserved hepatocytes in the absence of the AO inhibitor hydralazine.

[0092] Figure 14B shows the in vitro metabolic curve of compound 6 after incubation in human cryopreserved hepatocytes in the presence of the AO inhibitor hydralazine.

[0093] Figure 15 illustrates the proposed metabolic pathway of compound 6 in cryopreserved human hepatocytes in the absence and presence of the AO inhibitor hydralazine.

[0094] Figure 16 shows cell viability after CSF1R inhibitor treatment and CSF1 stimulation.

[0095] Figure 17 shows the blocking effect of compound 6 on CSF1-induced MCP-1 production in this experiment.

[0096] Figures 18A-18B compare the MCP1 production of compound 6 (Figure 18A) and compound 24 (Figure 18B), showing that they have similar effects on MCP1.

[0097] Figures 19A-19B show the significant average disease scores of the two CSF1R inhibitors (compound 6 and compound 24). Compared with the non-deuterated compound 24, the deuterated CSF1R inhibitor compound 6 improved paralysis symptoms to a surprisingly greater extent.

[0098] Figure 20 shows the XRPD diagram of compound 6, form A.

[0099] Figure 21 shows a PLM image of compound 6, form A.

[0100] Figure 22 shows the TGA (top) / DSC (bottom) overlay of compound 6 form A.

[0101] Figure 23 shows the HPLC of compound 6, form A. Detailed Implementation

[0102] This disclosure relates to colony-stimulating factor-1 receptor inhibitors (“CSF-1R inhibitors”), which are small molecules capable of crossing the blood-brain barrier to reach the central nervous system (CNS). This disclosure also relates to pharmaceutical formulations comprising CSF-1R inhibitors and to the use of CSF-1R inhibitors and pharmaceutical compositions comprising CSF-1R inhibitors for the treatment of diseases. Such diseases include immune-mediated diseases (including multiple sclerosis, lupus nephritis, and rheumatoid arthritis) and neurological diseases (including amyotrophic lateral sclerosis (ALS) and Huntington's disease).

[0103] Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) that causes intermittent relapses and progressive neurological deterioration. In MS, activated microglia and macrophages contribute to CNS damage and play a significant role in disease progression and neurodegeneration. These activated innate immune cells can participate in antigen presentation and produce inflammatory and neurotoxic mediators that are destructive to neurons and oligodendrocytes. CSF-1R is a receptor-tyrosine kinase expressed on macrophages, monocytes, and microglia and represents a potential target for therapeutic modulation of effector function.

[0104] The CSF-1R inhibitors described in this article are particularly useful in the treatment of multiple sclerosis (MS), and have demonstrated the following in preclinical in vitro and in vivo studies: reduction of inflammatory cytokines / chemokines, inhibition of macrophage / microglia expansion and activation without negatively impacting their phagocytic activity, inhibition of CNS infiltration in multiple in vivo disease models, and therapeutic benefit in mouse disease models. These data suggest that inhibiting CNS macrophage / microglia effector function through CSF-1R antagonism provides neuroprotection in MS by reducing inflammation, demyelination, and axonal loss. CSF-1R signaling has also been found to be upregulated in ALS, and it may also be upregulated in, for example, PSP and MSA, and CSF-1R inhibition has been noted in the literature as appearing effective in preclinical models of ALS, MSA, and PSP. See, e.g., Gowing, G. et al., Macrophage colony stimulating factor (M-CSF) exacerbates ALS disease in a mouse model through altered responses of microglia expressing mutant superoxide dismutase, Exp Neurol. 2009 Dec;220(2):267-75; Martínez-Muriana, A. et al., CSF1R blockade slows the progression of amyotrophic lateral sclerosis by reducing lateral sclerosis. microgliosis and invasion of macrophages into peripheral nerves, Sci Rep. 2016 May 13;6:25663; Neal, ML et al., Pharmacological inhibition of CSF1R by GW2580 reduces microglialproliferation and is protective against neuroinflammation and dopaminergicneurodegeneration. FASEB J. 2020 Jan;34(1):1679-1694; Oh, SJMancuso, R. et al., CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice. Brain. 2019 Oct 1;142(10):3243-3264; Lodder, C. et al., CSF1R inhibition rescues tau pathology and neurodegeneration in an A / T / N model with combined AD pathologies, while preserving plaque associated microglia. Acta Neuropathol Commun. 2021 Jun 8;9(1):108. .

[0105] In one embodiment, this disclosure relates to compounds of formula (I):

[0106]

[0107] and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts, wherein:

[0108] The dashed lines represent optional double bonds;

[0109] X 1 Is it C, N, or CR? 7 ;

[0110] X 2 X 3 X 4 X 5 X 6 X 7 Each is independently selected from N and NR. 7 or CR 7 ;

[0111] X 8 and X 9 Each is independently selected from N or C

[0112] Each R 7 Independently selected from H, D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C2-C9)heteroaryl, (C2-C 10 ) alkynylamine, (C1-C 10 )alkyl-C(O)O-, (C1-C 10 )alkoxy-, R 8 -(C2-C9) heterocyclic alkyl, R 8 -(C2-C9) heteroaryl, R 8 -(C2-C 10 )alkylynyl, R 8 -(C2-C 10 ) acetylinylamine, R 8 -(C1-C 10 )alkoxy-, R 8 -(C2-C9)heterocyclic alkyl-O-, halogen, cyano, H2N-, (CH3)HN-, (CH3)2N-, R 8 C(O)-, F3C-, F2HC-, CH3F2C-, FH2C-, CH3FHC- and (CH3)2FC;

[0113] Where R 8 Each is independently selected from H, (C1-C) 10 )alkyl, (C2-C9)heterocyclic alkyl, (C1-C 10 )alkylamines, (C1-C 10 )alkyl-C(O)O-, (C1-C 10 )alkoxy-, HO-, halogen, (CH3)2N- and H2N-;

[0114] Each of (C1-C) 10 alkyl, (C2-C9)heteroaryl, or (C2-C9)heterocyclic alkyl groups are further optionally substituted by one to four groups selected from the following: deuterium, (C1-C9)heteroaryl, (C2-C9)heterocyclic alkyl groups. 10 )alkyl or (C1-C 10 Alkylamines;

[0115] T 1 T 2 and T 3 Each is independently selected from N or CR 10 ,

[0116] Each R 10 Independently selected from H, D, (C1-C) 10 )alkyl, (C3-C 10)cycloalkyl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, (C2-C 10 ) alkynylamine, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl-,COOH-(C3-C 10 )cycloalkyl-, (C1-C 10 )alkoxy-, R 10A -(C1-C 10 )alkyl-, R 10A -(C1-C 10 )alkylamine, R 10A -((C1-C 10 )alkyl)2amine, R 10A -(C2-C 10 ) acetylinylamine, R 10A -C(O)-、R 10A -(C1-C 10 )alkyl-C(O)O-, R 10A -(C1-C 10 )alkoxy-, HO- and halogen, cyano, H2N-, (CH3)HN-, (CH3)2N-, R 10A R 11 N-, R 10A R 11 N(O)C-, R 10A (R 11 C(O))N-、R 10A R 11 NC(O)O-、R 10A C(O)-, R 10A R 11 NC(O)R 10A N-, (C1-C 10 )alkyl-OC(O)R 10A N-, F3C-, F2HC-, CH3F2C-, FH2C-, CH3FHC-, (CH3)2FC-;

[0117] Where R 10A and R 11 Each is independently selected from H, D, (C1-C) 10 )alkyl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkyl-C(O)O-,COOH-(C1-C 10 )alkyl, (C1-C 10)alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, HO-, halogenated, (CH3)2N- and H2N-;

[0118] Each of (C1-C) 10 The alkyl group is further optionally substituted by one to four groups selected from the following: D, (C1-C2) 10 )alkyl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 )alkyl-, HO-, halogen or H2N-

[0119] Y 1 It is O, NR 12 or CR 12 R 13 ;

[0120] Where R 12 Does not exist or R 12 and R 13 Each is independently selected from H, D, (C1-C) 10 )alkyl, (C1-C 10 )alkylamines, ((C1-C 10 alkyl)2-amine, (C1-C3)alkynylamine, (C1-C 10 )alkoxy-, (C1-C 10 )alkoxy-(C1-C 10 Alkyl-, HO-, halogenated and H2N-;

[0121] R 1 and R 2 Each is independently selected from H, D, (C1-C) 10 Alkyl, HO-, halogenated and H2N;

[0122] R 5 It does not exist or is selected from H, D, (C1-C) 10 Alkyl, HO-, halogen, and H2N-; and

[0123] R 6 Selected from D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, R 14 -(C3-C10 )cycloalkyl, R 14 -(C6-C 14 )Aryl, R 14 -(C2-C9) heteroaryl and R 14 -(C1-C 10 Alkylamines;

[0124] Where R 14 Each is independently selected from H, D, (C1-C) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, (C1-C 10 )alkylamines, ((C1-C 10 )alkyl)2amine, (C1-C 10 )alkoxy-, HO-, F2HC-O-, halogen, (CH3)2N-, F3C-C(O)-, F3C- and F2HC-;

[0125] Each of (C1-C) 10 )alkyl, (C6-C 14 ) aryl, (C2-C9) heteroaryl, (C3-C 10 The cycloalkyl or (C2-C9) heterocycloalkyl group is further optionally substituted by one to four groups selected from the following: (C1-C9) 10 )alkyl, (C3-C 10 )cycloalkyl, (C2-C9)heterocycloalkyl, (C6-C 14 )aryl, (C2-C9)heteroaryl, HO-, halogen, and H2N-; and

[0126] Z 1 Selected from H, halogen groups and (C1-C) 10 )alkyl;

[0127] Y 2 It is O, NR 17 or CR 17 R 18 ;

[0128] Where R 17 Does not exist or R 17 and R 18 Each is independently selected from H, (C1-C) 10 Alkyl, HO-, halogenated, or H2N-;

[0129] Where R 7 R 1 Or R 2 At least one of them is D.

[0130] In at least one embodiment of this disclosure, the compound according to formula (I) causes X to... 1 It is N. In at least one embodiment of this disclosure, the compound according to formula (I) makes X 2 It is N. In at least one embodiment of this disclosure, the compound according to formula (I) makes X 3 It is CR 7 In at least one embodiment of this disclosure, the compound according to formula (I) causes X to... 4 It is CR 7 In at least one embodiment of this disclosure, the compound according to formula (I) causes X to... 5 It is CR 7 In at least one embodiment of this disclosure, the compound according to formula (I) causes X to... 6 It is N. In at least one embodiment of this disclosure, the compound according to formula (I) makes X 7 It is CR 7 In at least one embodiment of this disclosure, the compound according to formula (I) causes X to... 8 It is C. In at least one embodiment of this disclosure, the compound according to formula (I) causes X to... 9 It is C. In at least one embodiment of this disclosure, the compound according to formula (I) causes X to... 1 It is N; x 2 It is N; X 3 It is CR 7 ;X 4 It is CR 7 ;X 5 It is CR 7 ;x 6 It is N; X 7 It is CR 7 ;X 8 It is C; and X 9 It's C.

[0131] In at least one embodiment of this disclosure, the compound according to formula (I) causes T 1 It is CR 10 In at least one embodiment of this disclosure, the compound according to formula (I) causes T 2 It is CR 10 In at least one embodiment of this disclosure, the compound according to formula (I) causes T 3 It is CR 10 In at least one embodiment of this disclosure, the compound according to formula (I) causes T 1 T 2 and T 3At least two of them are CR independently. 10 In at least one embodiment of this disclosure, the compound according to formula (I) causes T 1 T 2 and T 3 Each is CR independently 10 .

[0132] In at least one embodiment of this disclosure, the compound according to formula (I) makes each R 10 Independently selected from H, (C1-C 10 )alkyl, (C3-C 10 )cycloalkyl, (C1-C 10 Alkyl and halogen groups. In at least one embodiment of this disclosure, the compound according to formula (I) makes each R 10 Independently selected from H, (C1-C 10 Alkyl and halogen groups. In at least one embodiment of this disclosure, the compound according to formula (I) makes each R 10 Independently selected from H, (C1-C 10 )alkyl, (C1-C 10 Alkyl and halogen groups. In at least one embodiment of this disclosure, the compound according to formula (I) makes each R 10 Independently selected from H, (C1-C 10 )alkyl, (C3-C 10 ) Cycloalkyl and halogen groups. In at least one embodiment of this disclosure, the compound according to formula (I) makes each R 10 It is independently selected from H and halogen groups.

[0133] In at least one embodiment of this disclosure, the compound according to formula (I) causes Y to... 1 It is O. In at least one embodiment of this disclosure, the compound according to formula (I) causes Y to... 2 It is O. In at least one embodiment of this disclosure, the compound according to formula (I) makes Y1 and Y2 each O.

[0134] In at least one embodiment of this disclosure, the compound according to formula (I) causes Z to... 1 Selected from H, halogen groups and (C1-C) 10 )alkyl. In at least one embodiment of this disclosure, the compound according to formula (I) makes Z 1 It is (C1-C) 10 )alkyl. In at least one embodiment of this disclosure, the compound according to formula (I) makes Z 1It is a halogen group. In at least one embodiment of this disclosure, the compound according to formula (I) makes Z 1 It is H.

[0135] In at least one embodiment of this disclosure, the compound according to formula (I) causes R to... 1 and R 2 Each is independently selected from H and D. In at least one embodiment of this disclosure, the compound according to formula (I) causes R to... 1 and R 2 All are H. In at least one embodiment of this disclosure, the compound according to formula (I) causes R to... 1 and R 2 All are D. In at least one embodiment of this disclosure, the compound according to formula (I) causes R to... 1 and R 2 One of them is H and the other is D.

[0136] In at least one embodiment of this disclosure, the compound according to formula (I) causes R to... 6 Selected from (C3-C) 10 )cycloalkyl, (C2-C9)heteroaryl, R 14 -(C6-C 14 )Aryl, R 14 -(C2-C9) heteroaryl and R 14 -(C1-C 10 )alkylamines; wherein R 14 Each is independently selected from H, (C1-C) 10 )alkyl, (C1-C 10 )alkylamines, (C1-C 10 )alkoxy-, HO-, F2HC-O-, F3C-C(O)-, F3C- and F2HC-; and each of which (C3-C 10 The cycloalkyl or (C2-C9) heterocycloalkyl group is further optionally substituted by one to four groups selected from the following: (C1-C9) 10 Alkyl, HO-, halogen, or H2N-. In at least one embodiment of this disclosure, the compound according to formula (I) causes R to... 6 Selected from (C3-C) 10 )cycloalkyl and (C2-C9)heteroaryl; and wherein each (C3-C 10 The cycloalkyl or (C2-C9) heterocycloalkyl group is further optionally substituted by one or two groups selected from the following: (C1-C9) 10 Alkyl, HO-, halogen or H2N-.

[0137] On the other hand, this disclosure relates to compounds of formula (I'):

[0138]

[0139] and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts, wherein:

[0140] The dashed lines represent optional double bonds;

[0141] A is selected from H and D;

[0142] X 3’ It is CR 3’ , where R 3’ Selected from H and D;

[0143] X 4’ It is CR 4’ , where R 4’ Selected from H, D and R 7 ;and

[0144] X 5’ It is CR 5’ , where R 5’ Selected from H and D,

[0145] Among them, A and R 3’ R 4 'and R 5’ At least one of them is D.

[0146] In at least one embodiment of this disclosure, the compound according to formula (I') causes R to... 1 and R 2 Each is independently selected from H and D. In at least one embodiment of this disclosure, the compound according to formula (I') causes R to... 6 Selected from (C3-C) 10 )cycloalkyl, (C2-C9)heteroaryl, R 14 -(C6-C 14 )Aryl, R 14 -(C2-C9) heteroaryl and R 14 -(C1-C 10 )alkylamines; wherein R 14 Each is independently selected from H, (C1-C) 10 )alkyl, (C1-C 10 )alkylamines, (C1-C 10 )alkoxy-, HO-, F2HC-O-, F3C-C(O)-, F3C- and F2HC-; and each of which (C1-C 10 )alkyl, (C6-C 14 ) aryl, (C2-C9) heteroaryl, (C3-C 10The cycloalkyl or (C2-C9) heterocycloalkyl group is further optionally substituted by one to four groups selected from the following: (C1-C9) 10 Alkyl, HO-, halogen or H2N-.

[0147] On the other hand, this disclosure relates to the compounds of Table A, and / or their stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts:

[0148] Table A

[0149]

[0150] In at least one embodiment of this disclosure, the compound is selected from 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts. In at least one embodiment of this disclosure, the compound is 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d.

[0151] Another aspect of this disclosure is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of formula (I) and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts. In at least one embodiment, the pharmaceutical composition comprises a compound of formula (I) and / or its pharmaceutically acceptable salts.

[0152] Another aspect of this disclosure is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and at least one compound of formula (I') and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts. In at least one embodiment, the pharmaceutical composition comprises a compound of formula (I') and / or its pharmaceutically acceptable salts.

[0153] Another aspect of this disclosure is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of Table A and / or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts. In at least one embodiment, the pharmaceutical composition comprises a compound of Table A and / or its pharmaceutically acceptable salts. In one aspect of this disclosure, the pharmaceutical composition comprises 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d and / or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts. In one aspect of this disclosure, the pharmaceutical composition comprises 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d form A.

[0154] Another aspect of this disclosure is a method for treating a disease or disorder (such as a neurological disorder and an immune-mediated disorder) in a subject of need, the method comprising administering a therapeutically effective amount of a compound of formula (I) as described herein and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts. In at least one embodiment, the method comprises administering a therapeutically effective amount of a compound of formula (I') as described herein and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts. In at least one embodiment, the method comprises administering a therapeutically effective amount of a compound of Table A as described herein and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts. In one aspect of this disclosure, the pharmaceutical composition comprises 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts. In another aspect of this disclosure, the pharmaceutical composition comprises form A of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d.

[0155] Another aspect of this disclosure is a method for treating a disease or disorder (such as a neurological disorder and an immune-mediated disorder) in a subject of need, the method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) as described herein and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts. In at least one embodiment, the method comprises administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I') as described herein and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts. In at least one embodiment, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of Table A as described herein and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts. In one aspect of this disclosure, the method includes administering a therapeutically effective amount of a pharmaceutical composition comprising 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts and / or other pharmaceutically acceptable salts as described herein. In one aspect of this disclosure, the method includes administering a therapeutically effective amount of a pharmaceutical composition comprising 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d form A and / or a pharmaceutically acceptable salt thereof as described herein.

[0156] On the other hand, this disclosure provides compounds of formula (I) and / or stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts thereof used as pharmaceutical agents as described herein. In at least one embodiment, the compound of formula (I) and / or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts thereof used as pharmaceutical agents as described herein are compounds of formula (I') and / or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts thereof. In at least one embodiment, the compound of formula (I) and / or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts thereof used as pharmaceutical agents as described herein are compounds of Table A herein and / or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts thereof. In at least one embodiment, the compound of formula (I) and / or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts used as pharmaceutical agents as described herein are selected from compounds of the following: 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d and / or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts. In one aspect of this disclosure, the compound comprises 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d as described herein and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts and / or other pharmaceutically acceptable salts. In another aspect of this disclosure, the compound comprises 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d form A as described herein and / or other pharmaceutically acceptable salts.

[0157] On the other hand, this disclosure provides compounds of formula (I) and / or stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts thereof for treating diseases or disorders (such as neurological diseases and immune-mediated diseases) in subjects of need, as described herein. In at least one embodiment, a compound of formula (I) and / or stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts thereof for treating diseases or disorders (such as neurological diseases and immune-mediated diseases) in subjects of need, as described herein, is a compound of formula (I') and / or stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts thereof. In at least one embodiment, the compound of formula (I) and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts thereof for treating a disease or disorder (such as a neurological disease and an immune-mediated disease) of a subject in need, as described herein, are compounds of Table A as described herein and / or their stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts thereof. In at least one embodiment, the compound of formula (I) and / or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts thereof for treating a disease or disorder (such as a neurological disease and an immune-mediated disease) of a subject in need, as described herein, is selected from the group consisting of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d and / or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and / or pharmaceutically acceptable salts thereof. In one aspect of this disclosure, the compound comprises 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d as described herein and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts and / or other pharmaceutically acceptable salts. In another aspect of this disclosure, the compound comprises 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d form A as described herein and / or other pharmaceutically acceptable salts.

[0158] In some aspects of this disclosure, the neurological and immune-mediated diseases include multiple sclerosis, ALS, MSA, PSP, Huntington's disease, lupus, lupus nephritis, and rheumatoid arthritis.

[0159] The examples describe the in vitro and in vivo effects of deuterated CSF-1R inhibitors, their ability to withstand metabolic degradation, and methods for preparing the selected deuterated CSF-1R inhibitors of this disclosure.

[0160] Although specific embodiments of this disclosure will now be described with reference to preparations and schemes, it should be understood that such embodiments are by way of example only and only a small number of the many possible specific embodiments that may represent the application of the principles of this disclosure. Various variations and modifications will be apparent to those skilled in the art in view of the benefits of this disclosure, and as further defined in the appended claims, such variations and modifications are considered to be within the spirit and scope of this disclosure.

[0161] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although other compounds or methods may be used in practice or testing, certain preferred methods are now described in the context of the following preparation and schemes.

[0162] As used herein, the term "amino" means a functional group having a nitrogen atom and one to two hydrogen atoms. "Amino" can generally be used herein to describe primary, secondary, or tertiary amines, and the identity of said amino group will be readily determined by those skilled in the art in the context of its use in this disclosure. The terms "amine" or "aminegroup" or "ammonia group" mean a functional group containing a nitrogen atom derived from ammonia (NH3). An amine group is preferably a primary amine, meaning that nitrogen is bonded to two hydrogen atoms and a substituent comprising a substituted or unsubstituted alkyl, aryl, aliphatic, or aromatic group. An amine group can be a secondary amine, meaning that nitrogen is bonded to one hydrogen atom and two substituents comprising a substituted or unsubstituted alkyl, aryl, aliphatic, or aromatic group, as defined below. An amine group can be a tertiary amine, meaning that nitrogen is bonded to three substituents comprising a substituted or unsubstituted alkyl, aryl, aliphatic, or aromatic group. The amino group can also be a quaternary ammonium, which means that the specified amino group is bonded to a fourth group, resulting in a positively charged ammonium group.

[0163] It should be understood that any or all of the amines in this disclosure may be in the form of a free amine (i.e., -NH2 as a primary amine) or in a protonated form having a pharmaceutically acceptable anion (i.e., -NH3 as a primary amine). + Y - , where Y - It is a pharmaceutically acceptable anion.

[0164] As used herein, the term "amide group" refers to a functional group that contains a carbonyl group linked to nitrogen.

[0165] As used in this article, "carbonyl" refers to a functional group containing a carbon atom bonded to an oxygen atom by a double bond represented by (C=O).

[0166] As used herein, the term "alkane" refers to a saturated hydrocarbon bonded by a single bond. Alkanes can be straight-chain or branched. "Cycloalkanes" are saturated hydrocarbon rings bonded by single bonds.

[0167] As used in this article, the term "(C1-C" is used in conjunction with the terminology used in this article. 10 "Alkyl" refers to a saturated straight-chain, branched, or cyclic hydrocarbon consisting essentially of 1 to 10 carbon atoms and a corresponding number of hydrogen atoms. Typically, the straight-chain or branched group has one to ten carbon atoms, or more typically one to five. Example (C1-C1) 10 Alkyl groups include methyl (represented by -CH3), ethyl (represented by -CH2-CH3), n-propyl, isopropyl, n-butyl, isobutyl, etc. In view of the benefits of this disclosure, other (C1-C-) 10 The alkyl group will be obvious to those skilled in the art.

[0168] As used herein, the term "(C2-C9)heteroalkyl" means a saturated straight-chain, branched, or cyclic hydrocarbon consisting of essentially 2 to 10 atoms, wherein 2 to 9 atoms are carbon and the remaining one or more atoms are selected from nitrogen, sulfur, and oxygen. Exemplary (C2-C9)heteroalkyls will be apparent to those skilled in the art in light of the benefits of this disclosure.

[0169] As used in this article, the term "(C3-C" 10 "Cycloalkyl" refers to a non-aromatic saturated hydrocarbon group that forms at least one ring consisting of approximately 3 to 10 carbon atoms and a corresponding number of hydrogen atoms. (C3-C) 10 Cycloalkyl groups can be monocyclic or polycyclic. Besides covalent substitution, the individual rings of polycyclic cycloalkyl groups can have different linkages, such as fusion, bridging, spirosynthesis, etc. Example (C3-C 10Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo-octyl, octahydropentaenoyl, spiro-decyl, cyclopropyl substituted with cyclobutyl, cyclobutyl substituted with cyclopentyl, cyclohexyl substituted with cyclopropyl, etc. For the benefit of this disclosure, other (C3-C) 10 The cycloalkyl group will be obvious to those skilled in the art.

[0170] As used herein, the term "(C2-C9)heterocyclic alkyl" means a non-aromatic group having 3 to 10 atoms forming at least one ring, wherein 2 to 9 ring atoms are carbon and the remaining one or more ring atoms are selected from nitrogen, sulfur, and oxygen. (C2-C9)heterocyclic alkyl groups can be monocyclic or polycyclic. In addition to covalent substitution, the individual rings of such polycyclic heterocyclic alkyl groups can have different linkages, such as fused, bridged, spiro, etc. Exemplary (C2-C9) heterocyclic alkyl groups include pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydropyranyl, pyranyl, thiopyranyl, aziridinyl, aziridine, epoxyethyl, methylenedioxy, chromenyl, barbituryl, isoxazolyl, 1,3-oxazolidine-3-yl, isothiazolyl, 1,3-thiazolyl-3-yl, 1,2-pyrazolidine-2-yl, 1,3-pyrazolidine-1-yl, piperidinyl, thiomorpholinyl, 1,2-tetrahydrothiazin-2-yl, 1,3-tetrahydrothiazin-3-yl, tetrahydrothiadiazinyl, morpholinyl, 1,2-tetrahydrodiazin-2-yl, 1,3-tetrahydrodiazin-1-yl, tetrahydroachenginyl, piperazinyl, piperazin-2-one, and piperazinyl. Zine-3-keto, chromanyl, 2-pyrrolinyl, 3-pyrrolinyl, imidazoalkyl, 2-imidazoalkyl, 1,4-dioxane, 8-azabicyclo[3.2.1]octyl, 3-azabicyclo[3.2.1]octyl, 3,8-diazabicyclo[3.2.1]octyl, 2,5-diazabicyclo[2.2.1]heptyl, 2,5-diazabicyclo[2.2.2]octyl, octahydro-2H-pyrido[1,2-a]pyrazinyl, 3-azabicyclo[4.1.0]heptyl, 3-azabicyclo[3.1.0]hexyl, 2-azaspiro[4.4]nonyl, 7-oxa-1-aza-spiro[4.4]nonyl, 7-azabicyclo[2.2.2]heptyl, octahydro-1H-indoleyl, etc. The (C2-C9) heterocyclic alkyl groups are typically attached to the main structure via carbon or nitrogen atoms. Other (C2-C9) heterocyclic alkyl groups will be apparent to those skilled in the art given the benefits of this disclosure.

[0171] The term "aliphatic group" or "aliphatic" refers to a non-aromatic group consisting of carbon and hydrogen, and may optionally include one or more double and / or triple bonds. In other words, an aliphatic group is any group consisting of carbon and hydrogen containing no aromatic functional groups. Aliphatic groups can be straight-chain, branched, or cyclic, and typically contain between about 1 and about 24 carbon atoms.

[0172] The term "aryl group" is used interchangeably with "aryl," "aryl ring," "aromatic," "aromatic group," and "aromatic ring." Aryl groups include carbocyclic aromatic groups, typically having six to fourteen ring carbon atoms. Aryl groups also include heteroaryl groups, which typically have five to fourteen ring atoms and one or more heteroatoms selected from nitrogen, oxygen, and sulfur.

[0173] As used in this article, the term "(C6-C" 14 "Aryl" refers to an aromatic functional group having 6 to 14 carbon atoms that form at least one ring.

[0174] As used herein, the term "(C2-C9) heteroaryl" refers to an aromatic functional group having 5 to 10 atoms forming at least one ring, wherein 2 to 9 ring atoms are carbon and the remaining one or more ring atoms are selected from nitrogen, sulfur, and oxygen. (C2-C9) heteroaryl groups can be monocyclic or polycyclic. In addition to covalent substitution, the individual rings of such polycyclic heteroaryl groups can have different degrees of connectivity, such as fusion. Exemplary (C2-C9) heteroaryl groups include furanyl, thiophene, thiazolyl, pyrazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrroleyl, triazolyl, tetrazolyl, imidazolyl, 1,3,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-oxadiazolyl, 1,3,5-thiadiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, 1,2,4-triazinyl, 1,2,3-triazinyl, and 1,3,5-triazinyl. Pyrazolo[3,4-b]pyridinyl, cyclolinyl, pteridinyl, purinyl, 6,7-dihydro-5H-[1]pyridinyl, benzo[b]phenylthio, 5,6,7,8-tetrahydro-quinoline-3-yl, benzoxazolyl, benzothiazolyl, benzoisothiazolyl, benzoisoxazolyl, benzoimidazolyl, thioindenyl, isothioindenyl, benzofuranyl, isobenzofuranyl, isoindolyl, indolyl, indoleazinyl, indoleazinyl, indazole, isoquinolinyl, quinolinyl, phthalazinyl, quinoxolinyl, quinazolinyl, and benzoxazinyl, etc. (C2-C9) heteroaryl groups are typically attached to the main structure via carbon atoms; however, those skilled in the art will recognize that certain other atoms (e.g., heterocyclic atoms) may be attached to the main structure at some point. In view of the benefits of this disclosure, other (C2-C9) heteroaryl groups will be apparent to those skilled in the art.

[0175] The term "alkynyl" refers to a functional group containing a triple-bonded carbon atom, consisting of (C2-C) groups. 10 ) yyn group - indicates.

[0176] As used herein, the term "alkylamine" refers to a (C1-C2) amine containing a primary, secondary, or tertiary amine group that substitutes for a hydrogen atom. 10 )alkyl, composed of (C1-C 10 )alkylamines and ((C1-C 10 It is represented by alkyl)2amine.

[0177] The term "alkynylamine" refers to an amino group containing a triple-bonded carbon atom and an amine group (C2-C). 10 ) group, composed of (C2-C 10 ) alkynylamine is indicated.

[0178] The term "alkoxy" refers to an oxygen-bonded (C1-C2) group. 10 )alkyl, composed of (C1-C 10 )alkyl-O- or (C1-C 10)Alkoxy - represented. The term "alkoxyalkyl" means (C1 - C 10 )alkyl bonded to another oxygen atom bonded to (C1 - C 10 )alkyl, represented by (C1 - C 10 )alkyl - O - (C1 - C 10 )alkyl - or (C1 - C 10 )alkoxy - (C1 - C 10 )alkyl -.

[0179] The term "alkyl ester" means (C1 - C 10 )alkyl containing an ester group in place of one hydrogen atom, represented by - O(O)C - (C1 - C 10 )alkyl.

[0180] The term "alkyl acid" means (C1 - C 10 )alkyl containing a carboxylic acid group in place of one hydrogen atom, represented by (C1 - C 10 )alkyl - COOH.

[0181] The term "aliphatic acid" means an acid of non - aromatic hydrocarbons, represented by (C1 - C 10 )alkyl - COOH and (C3 - C 10 )cycloalkyl - COOH.

[0182] As used herein, both "D" and "d" refer to deuterium.

[0183] The term "dicarbonyl" refers to an organic molecule containing two or more adjacent carbonyl groups. The carbonyl group represented by C = O can be, for example, an aldehyde, a ketone, and other groups having an oxygen atom double - bonded to a carbon atom. Examples include glyoxal, methylglyoxal, dimethylglyoxal, and 3 - deoxyglucosone.

[0184] The term "halo" or "Hal" means a fluorine (F), chlorine (Cl), bromine (Br), iodine (I), or astatine (At) ion.

[0185] As used herein, "i -" refers to iso.

[0186] The term "methoxy" means (C1)alkyl containing an oxygen in place of one hydrogen atom, represented by -(O)CH3.

[0187] As used herein, "n -" refers to normal.

[0188] The term "polyol" means an alcohol containing multiple hydroxyl (-OH) groups.

[0189] As used herein, "Sec" or "s -" each refer to secondary.

[0190] As used in this article, the term "stereoisomer" refers to both the enantiomer and the diastereomer.

[0191] "Substituted" means that the carbon in an alkyl, heterocyclic, or aryl group is replaced by one or more non-carbon substituents. Non-carbon substituents are selected from nitrogen, oxygen, and sulfur.

[0192] As used in this article, "Tert" and "t-" each refer to an uncle.

[0193] "Unsubstituted" means a group consisting only of hydrogen and carbon.

[0194] 3- to 10-membered rings refer to closed rings; 3- to 10-membered rings can be acyclic, aromatic, or heterocyclic.

[0195] The term "pharmaceutically acceptable anion" refers to anion suitable for pharmaceutical use. Pharmaceutically acceptable anions include halide ions, carbonate ions, bicarbonate ions, sulfate ions, hydrogen sulfate ions, hydroxide ions, nitrate ions, persulfate ions, phosphate ions, sulfite ions, acetate ions, ascorbate ions, benzoate ions, citrate ions, dihydrogen citrate ions, hydrogen citrate ions, oxalate ions, succinate ions, tartrate ions, taurocholate ions, glycocholate ions, and cholate ions.

[0196] "Replaced by deuterium" means replacing one or more hydrogen atoms with a corresponding number of deuterium atoms.

[0197] All pharmaceutically acceptable salts, prodrugs, tautomers, hydrates, and solvates of the compounds disclosed in this invention are also within the scope of this disclosure.

[0198] This invention discloses compounds that are inherently basic and are generally capable of forming a variety of different salts having various inorganic and / or organic acids. While such salts are generally pharmaceutically acceptable for administration to animals and humans, in practice it is often desirable to first isolate the compound from the reaction mixture as a pharmaceutically unacceptable salt, and then simply convert the latter back to a free basic compound by treatment with a basic reagent, and subsequently convert the free base into a pharmaceutically acceptable acid addition salt. Acid addition salts of basic compounds can be readily prepared using conventional techniques, for example, by treating the basic compound in an aqueous solvent medium or in a suitable organic solvent (e.g., methanol or ethanol) with substantially equal amounts of a selected inorganic or organic acid. After careful evaporation of the solvent, the desired solid salt is obtained.

[0199] The acids that can be used to prepare pharmaceutically acceptable acid addition salts of basic compounds are those that can form non-toxic acid addition salts, namely salts containing pharmacologically acceptable anions, such as chlorides, bromides, iodides, nitrates, sulfates or bisulfates, phosphates or acidic phosphates, acetates, lactates, citrates or acidic citrates, tartrates or hydrotartrates, succinates, maleates, fumarates, gluconates, glycosides, benzoates, methanesulfonates, and bis(hydroxynaphthyl) salts [i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthyl)].

[0200] This invention discloses compounds that are inherently acidic (e.g., containing a COOH or tetrazolium moiety) and are generally capable of forming various different salts having a variety of inorganic and / or organic bases. While such salts are generally pharmaceutically acceptable for administration to animals and humans, in practice it is often desirable to first isolate the compound from the reaction mixture as a pharmaceutically unacceptable salt, and then simply convert the latter back to a free acid compound by treatment with an acidic reagent, and subsequently convert the free acid to a pharmaceutically acceptable base addition salt. These base addition salts can be readily prepared using conventional techniques, for example, by treating the corresponding acidic compound with an aqueous solution containing the desired pharmaceutically acceptable cation, and then evaporating the resulting solution to dryness (preferably under reduced pressure). Alternatively, they can also be prepared by mixing a lower alkane solution of the acidic compound with the desired alkali metal alkoxide, and then evaporating the resulting solution to dryness in the same manner as before. In either case, stoichiometric amounts of reagents are preferred to ensure the integrity of the reaction and the maximum product yield of the desired solid salt.

[0201] The bases that can be used to prepare pharmaceutically acceptable base addition salts are those that can form non-toxic base addition salts, namely, salts containing pharmacologically acceptable cations, such as alkali metal cations (e.g., potassium and sodium), alkaline earth metal cations (e.g., calcium and magnesium), ammonium or other water-soluble amine addition salts such as N-methylglucosamine (glucosamine), lower alkanol ammonium and other organic amines.

[0202] The stereoisomers (e.g., cis and trans isomers) and all optical isomers (e.g., R and S enantiomers) of the compounds disclosed in this invention, as well as racemic isomers, diastereomers and other mixtures of such isomers, are within the scope of this disclosure.

[0203] The compounds, salts, prodrugs, hydrates, and solvates disclosed in this invention can exist in several tautomeric forms (including enol and imine forms and ketone and enamine forms) and geometric isomers and mixtures thereof. Tautomers exist as mixtures of tautomer sets in solution. In solid form, typically one tautomer is dominant. Even if one tautomer can be described, all tautomers are within the scope of this disclosure.

[0204] Transisomers are also within the scope of this disclosure. A transisomer is a compound that can be isolated into a rotation-restricted isomer.

[0205] This disclosure also provides pharmaceutical compositions comprising at least one compound disclosed herein and at least one pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be any such carrier known in the art, including those described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., (ARGennaro, editor 1985). Pharmaceutical compositions of the compounds disclosed herein may be prepared by conventional methods known in the art, including, for example, mixing at least one compound disclosed herein with a pharmaceutically acceptable carrier.

[0206] The pharmaceutical compositions disclosed in this invention can be used in animals or humans. Therefore, the compounds disclosed in this invention can be formulated into pharmaceutical compositions for oral, oral, parenteral (e.g., intravenous, intramuscular, or subcutaneous), topical, rectal, or nasal administration, or in a form suitable for inhalation or inhalation.

[0207] The compounds disclosed in this invention can also be formulated for continuous delivery using methods well known to those skilled in the art. Examples of such formulations can be found in U.S. Patents 3,119,742; 3,492,397; 3,538,214; 4,060,598; and 4,173,626.

[0208] For oral administration, the pharmaceutical composition may be in the form of tablets or capsules prepared by conventional means with one or more pharmaceutically acceptable excipients such as: binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium glycolate); and / or wetting agents (e.g., sodium dodecyl sulfate). Tablets may be coated using methods well known in the art. Liquid formulations for oral administration may be in the form of, for example, solutions, syrups, or suspensions, or they may be presented as dry products for reconstitution with water or other suitable media prior to use. Such liquid formulations can be prepared by conventional means with one or more pharmaceutically acceptable additives such as: suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous mediators (e.g., almond oil, oily esters, or ethanol); and / or preservatives (e.g., methylparaben, propylparaben, or sorbic acid).

[0209] The recommended dose of the compounds disclosed in this invention for oral, parenteral, or buccal administration to a general adult for the treatment or prevention of CSF-1R-related disease states is from about 0.1 mg to about 2000 mg. In some embodiments, the recommended dose is from about 0.1 mg to about 200 mg of active ingredient per unit dose. Regardless of the recommended dose, administration of the compounds can occur, for example, 1 to 4 times per day.

[0210] Pharmaceutical compositions and methods of treatment or prevention including the administration of at least one of the compounds disclosed herein are also within the scope of this disclosure.

[0211] Non-limiting examples of suitable CSF-1R inhibitors according to formulas (I) and (I') are presented in the following examples. It should be understood that any or all amines present in the structures of the inhibitors according to formulas (I) and (I') presented in the following examples may be in the form of a free amine or in a protonated form having a pharmaceutically acceptable anion. Preferred pharmaceutically acceptable anions include halide ions, carbonate ions, bicarbonate ions, sulfate ions, hydrogen sulfate ions, hydroxide ions, nitrate ions, persulfate ions, phosphate ions, sulfite ions, acetate ions, ascorbate ions, benzoate ions, citrate ions, dihydrocitrate ions, hydrogen citrate ions, oxalate ions, succinate ions, tartrate ions, taurocholate ions, glycocholate ions, and cholate ions. Most preferred pharmaceutically acceptable anions include chloride ions, carbonate ions, and bicarbonate ions. It should also be understood that any or all CSF-1R inhibitors according to formulas (I) and (I') may be racemic or enantiomers of racemic mixtures.

[0212] Example

[0213] Example 1: Synthesis Method

[0214] By referring to the preparations, schemes, and examples described herein, those skilled in synthetic chemistry can readily synthesize compounds of Formula I. Similar preparations, schemes, and procedures for preparing compounds of Formula I and their intermediates are disclosed in the general schemes and synthetic examples of WO2017 / 015267. Specific embodiments of this disclosure are described with reference to the synthetic preparations and schemes presented below; it should be understood that such embodiments are by way of example only and only a few of the many possible specific embodiments that may represent the application of the principles of this disclosure are illustrated. Various variations and modifications to the preparations, schemes, and examples will be apparent to those skilled in the art in view of the benefits of this disclosure.

[0215] Synthesis Examples (Examples 1-9)

[0216] Example 1: Synthesis of (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-5-d (compound 1)

[0217]

[0218] Example 1-1: Preparation of (+ / -)-(trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid methyl ester

[0219]

[0220] Carbon tetrachloride (1.9 mL, 19.40 mmol) was added to a stirred solution of methyl 4-hydroxy-3-((1-hydroxy-1-(6-methoxypyridin-3-yl)prop-2-yl)oxy)-5-methoxybenzoate (1.41 g, 3.88 mmol, see WO 2017015267 for preparation), triphenylphosphine (1.23 g, 4.66 mmol), and N,N-diisopropylethylamine (1.0 mL, 5.82 mmol) in acetonitrile (30 mL). The resulting colorless solution was heated to reflux and stirred under an inert atmosphere. After 45 min, LC / MS analysis showed that the reaction was complete. The mixture was cooled to room temperature and concentrated to provide a brown solid. Chromatographic purification (CombiFlash, 80 g SiO2 gold column, elution with 10%-30% ethyl acetate / heptane) yielded (+ / -)-(trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid methyl ester (0.75 g, 2.18 mmol, 56% yield) as a white solid: 1 H NMR (400MHz, CDCl3) δ8.19(d,J=2.4Hz,1H),7.59(dd,J=8.6,2.4Hz,1H),7.34(d,J=1.9Hz,1H),7.23(d,J=1.9Hz,1H),6.80(d,J =8.6Hz, 1H), 4.70 (d, J = 7.8Hz, 1H), 4.15 (dq, J = 7.8, 6.4Hz, 1H), 3.96 (s, 3H), 3.90 (s, 6H), 1.22 (d, J = 6.4Hz, 3H) ppm; (M+1) = 346.

[0221] Examples 1-2: Preparation of (+ / -)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methanol

[0222]

[0223] Lithium aluminum hydride (0.12 g, 3.27 mmol) was added in a single addition to a solution of (+ / -)-trans-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (0.75 g, 2.18 mmol) stirred in tetrahydrofuran (30 mL) at 0 °C (note a small amount of gas release). The resulting gray mixture was stirred at 0 °C under an inert atmosphere. After 10 min, LC / MS analysis showed that the reaction was complete. The mixture was quenched by adding water (0.12 mL), 1 N sodium hydroxide solution (0.12 mL), and water (0.38 mL). The resulting mixture was stirred at 0 °C for 10 min, and then magnesium sulfate (approximately 5 g) was added. The mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (50 mL). The filtrate was concentrated to provide (+ / -)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methanol (0.69 g, 2.18 mmol, 100% yield) as a viscous white foam: 1 HNMR (400MHz, DMSO-d6) δ8.26(d,J=2.3Hz,1H),7.77(dd,J=8.5,2.3Hz,1H),6.89(d,J=8.5Hz,1H),6.55(d,J=1.9Hz,1H),6.49(d,J=1.9Hz,1H),5.09(br s,1H),4.74(d,J=7.7Hz,1H),4.38(s,2H),4.31(dq,J=7.7,6.3Hz,1H),3.88(s,3H),3.72(s,3H),1.09(d,J=6.3Hz,3H); (M+1)=318.

[0224] Examples 1-3: Preparation of (+ / -)-5-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine

[0225]

[0226] To a stirred solution of 5-bromo-1H-imidazo[4,5-b]pyridine (0.21 g, 0.99 mmol) and (+ / -)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methanol (0.35 g, 1.10 mmol) in toluene (10 mL), (tributylphosphine)acetonitrile (0.43 g, 1.74 mmol) was added. The resulting mixture was heated to 75 °C in a sealed container with stirring allowed. After 18 h, LC / MS analysis showed that the reaction was complete. The mixture was cooled to room temperature and concentrated to provide a brown oil. Chromatographic purification (CombiFlash, 40g SiO2 gold column, 20%-60% ethyl acetate:ethanol / heptane elution) yielded (+ / -)-5-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine as a brownish-brown solid (0.33g, 0.67mmol, 67% yield): 1 H NMR (400MHz, DMSO-d6) δ8.61 (s, 1H), 8.23 ​​(d, J = 2.3Hz, 1H), 8.08 (d, J = 8.3Hz, 1H), 7.73(dd,J=8.6,2.3Hz,1H),7.49(d,J=8.3Hz,1H),6.88(d,J=8.6Hz,1H),6.79(d, J=1.9Hz,1H),6.46(d,J=1.9Hz,1H),5.36(s,2H),4.73(d,J=7.8Hz,1H),4.29(dq, J=7.8,6.3Hz,1H),3.87(s,3H),3.73(s,3H),1.05(d,J=6.3Hz,3H)ppm; (M+1)=497. Confirmation of regional chemistry: 1 H- 13 C HSQC NMR data identified the imidazole C-2 proton and carbon at 8.61 and 145.8 ppm, respectively. Next, 1 H- 13 HMBC NMR data showed a multibond correlation between this proton at 8.61 ppm and the quaternary ring carbons at 134.1 and 146.4 ppm, with the carbon at 146.4 ppm adjacent to pyridine nitrogen. Finally, the correlation between the adjacent methylene proton at 5.36 ppm and the quaternary carbons at 145.8 ppm and 146.4 ppm was demonstrated in the HMBC NMR data. 1 H- 13 C-multi-bond correlation is used to confirm the connection.

[0227]

[0228] Examples 1-4: Preparation of (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-5-d

[0229] Add tris(dibenzylacetone)dipalladium(0) (0.19 g, 0.21 mmol), 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (0.27 g, 0.63 mmol), and potassium carbonate (0.29 g, 2.12 mmol) to a stirred solution of (+ / -)-5-bromo-trans-3-((-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (0.53 mg, 1.06 mmol) in 2-propanol-d8 (5 mL) (Note: carried out in a microwave-safe reaction vessel dried in an oven). Seal the vessel and degas the contents under vacuum / backfill with N2 (x 3). Heat the mixture to 100 °C and allow stirring. After 2 hours, LC / MS analysis showed that the reaction was complete. The mixture was cooled to room temperature and concentrated to provide a brown oil. Chromatographic purification (CombiFlash, 40 g SiO2 gold column, 20%-70% ethyl acetate:ethanol / heptane elution) gave (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-5-d as a brown solid (0.30 g, 0.72 mmol, 68% yield): 1 H NMR(400MHz, DMSO-d6)δ8.60(s,1H),8.22(d,J=2.3Hz,1H),8.10(d,J=8.1Hz,1H), 7.73(dd,J=8.6,2.3Hz,1H),7.30(d,J=8.1Hz,1H),6.88(d,J=8.6Hz,1H),6.76(d, J=1.9Hz,1H),6.49(d,J=1.9Hz,1H),5.39(s,2H),4.72(d,J=7.8Hz,1H),4.27(dq, J=7.8,6.4Hz,1H),3.86(s,3H),3.70(s,3H),1.04(d,J=6.4Hz,3H)ppm; (M+1)=420.

[0230] Example 2: Synthesis of (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2,5-d2 (compound 2)

[0231]

[0232] Example 2-1: Preparation of 2-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-5-d

[0233]

[0234] To a stirred solution of 2-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-5-d (0.25 g, 0.59 mmol) in N,N-dimethylformamide (3 mL), carbon tetrabromide (0.32 g, 0.96 mmol) and sodium tert-butoxide (0.23 g, 2.35 mmol) were added. The resulting dark brown mixture was stirred at room temperature. After 30 min, LC / MS analysis showed the formation of a new product and the presence of the starting material. An additional portion of carbon tetrabromide (0.32 g, 0.96 mmol) and sodium tert-butoxide (0.23 g, 2.35 mmol) was added to the mixture. After 1 h, LC / MS analysis showed the reaction was still incomplete. The mixture was quenched in a saturated ammonium chloride solution (50 mL). The mixture was extracted with ethyl acetate (40 mL). The organic phase was washed with brine (30 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to provide a brown oil. Chromatographic purification (CombiFlash, 40 g SiO2 gold column, 10%-50% 3:1 ethyl acetate:ethanol / heptane elution) gave (+ / -)-2-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-5-d as a grayish-white solid (0.089 g, 0.18 mmol, 31% yield): 1H NMR (400MHz, DMSO-d6) δ8.22(d,J=2.3Hz,1H),8.11(d,J=8.0Hz,1H),7.73(dd, J=8.6,2.3Hz,1H),7.35(d,J=8.0Hz,1H),6.88(d,J=8.6Hz,1H),6.69(d,J=1.9H z,1H),6.27(d,J=1.9Hz,1H),5.41(s,2H),4.72(d,J=7.8Hz,1H),4.28(dq,J=7 .8, 6.3Hz, 1H), 3.87 (s, 3H), 3.69 (s, 3H), 1.02 (d, J = 6.3Hz, 3H) ppm; (M+1) = 498.

[0235] Example 2-2: Preparation of (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2,5-d2

[0236] Add tris(dibenzylacetone)dipalladium(0) (0.031 g, 0.034 mmol), 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (0.043 g, 0.10 mmol), and potassium carbonate (0.047 g, 0.34 mmol) to a stirred solution of (+ / -)-2-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-5-d (0.085 mg, 0.17 mmol) in 2-propanol-d8 (3 mL) (Note: carried out in a microwave-safe reaction vessel dried in a 20 mL oven) to a container. Seal the container and degas the contents under vacuum / backfill with N2 (x3). The mixture was heated to 100°C and stirred. After 2 h, LC / MS analysis showed that the reaction was complete. The mixture was cooled to room temperature and concentrated to provide a brown oil. Chromatographic purification (CombiFlash, 24 g SiO2 gold column, 20%-70% ethyl acetate:ethanol / heptane elution) gave (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2,5-d2 as a pale yellow solid (0.047 g, 0.11 mmol, 66% yield): 1H NMR (400MHz, DMSO-d6) δ8.22(d,J=2.3Hz,1H),8.10(d,J=8.0Hz,1H),7.73(dd, J=8.7,2.3Hz,1H),7.30(d,J=8.0Hz,1H),6.88(d,J=8.7Hz,1H),6.76(d,J=2.0H z,1H),6.49(d,J=2.0Hz,1H),5.39(s,2H),4.72(d,J=7.8Hz,1H),4.27(dq,J=7 .8, 6.3Hz, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 1.04 (d, J = 6.3Hz, 3H) ppm; (M+1) = 421.

[0237] Example 3: Synthesis of (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3H-imidazo[4,5-b]pyridine (compound 3)

[0238]

[0239] Example 3-1: Preparation of (+ / -)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2-ol

[0240]

[0241] Lithium aluminum deuteride (0.34 g, 8.13 mmol) was added in a single addition to a stirred solution of (+ / -)-(trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (2.08 g, 6.02 mmol) in tetrahydrofuran (60 mL) at 0 °C (note a small amount of gas release). The resulting gray mixture was stirred at 0 °C. After 15 min, LC / MS analysis showed that the reaction was complete. The mixture was quenched by adding water (0.50 mL), 1 N sodium hydroxide solution (0.50 mL), and water (1.5 mL). The resulting mixture was stirred at 0 °C for 15 min, and then magnesium sulfate (approximately 10 g) was added. The mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (100 mL). The filtrate was concentrated to provide (+ / -)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2-ol (1.92 g, 6.01 mmol, 100% yield) as a viscous white foam: 1 HNMR (400MHz, CDCl3) δ8.15(d,J=2.5Hz,1H),7.59(dd,J=8.6,2.5Hz,1H),6.79(d,J=8.6Hz,1H),6.59(d,J=1.9Hz,1H),6.57(d ,J=1.9Hz,1H),4.63(d,J=7.8Hz,1H),4.14(dq,J=7.8,6.4Hz,1H),3.95(s,3H),3.86(s,3H),1.19(d,J=6.4Hz,3H); (M+1)=320.

[0242] Example 3-2: Preparation of (+ / -)-5-((trans)-6-(azidomethyl-d2)-8-methoxy-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-2-yl)-2-methoxypyridine

[0243]

[0244] 1,8-diazabicyclo[5.4.0]undec-7-ene (1.4 mL, 9.62 mmol) was added to a stirred solution of (+ / -)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methane-d2-ol (1.92 g, 6.01 mmol) and diphenylphosphoazide (2.07 mL, 9.62 mmol) in tetrahydrofuran (50 mL). The resulting mixture was heated to reflux and stirred under an inert atmosphere. After 1 h, LC / MS analysis showed that the reaction was complete. The colorless solution was cooled to room temperature and concentrated to provide a yellow oil. Chromatographic purification (CombiFlash, 40 g SiO2 gold column, elution with 10%-30% ethyl acetate / heptane) yielded (+ / -)-5-((trans)-6-(azidomethyl-d2)-8-methoxy-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-2-yl)-2-methoxypyridine as a white solid (1.78 g, 5.17 mmol, 86% yield): 1 H NMR (400MHz, CDCl3) δ8.19(d,J=2.4Hz,1H),7.60(dd,J=8.7,2.4Hz,1H),6.80(d,J=8.7Hz,1H),6.57(d,J=2.0Hz,1H),6.47(d,J =2.0Hz, 1H), 4.65 (d, J = 7.9Hz, 1H), 4.15 (dq, J = 7.9, 6.4Hz, 1H), 3.96 (s, 3H), 3.86 (s, 3H), 1.20 (d, J = 6.4Hz, 3H) ppm; (M+1) = 345.

[0245] Example 3-3: Preparation of (+ / -)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2-amine

[0246]

[0247] Polymer-bonded triphenylphosphine (3.50 g, approximately 10.50 mmol) was added to a stirred solution of (+ / -)-5-((trans)-6-(azidomethyl-d2)-8-methoxy-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-2-yl)-2-methoxypyridine (1.78 g, 5.17 mmol) in tetrahydrofuran (50 mL) and water (5 mL). The orange suspension was heated to reflux and stirred under an inert atmosphere. After 2 h, LC / MS analysis showed that the reaction was complete. The mixture was cooled to room temperature and filtered through diatomaceous earth using ethyl acetate (50 mL). The filtrate was dried over magnesium sulfate, filtered, and concentrated to provide (+ / -)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2-amine (1.61 g, 5.06 mmol, 98% yield) as a colorless oil: 1 H NMR (400MHz, CDCl3) δ8.17(d,J=2.4Hz,1H),7.59(dd,J=8.6,2.4Hz,1H),6.78(d,J=8.6Hz,1H),6.54(d,J=1.9Hz,1H),6.50(d,J=1.9Hz ,1H),4.63(d,J=7.8Hz,1H),4.13(dq,J=7.8,6.4Hz,1H),3.95(s,3H),3.85(s,3H),2.04(s,2H),1.19(d,J=6.4Hz,3H)ppm; (M-16)=302.

[0248] Examples 3-4: Preparation of (+ / -)-N-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3-nitropyridine-2-amine

[0249]

[0250] 2-Chloro-3-nitropyridine (0.84 g, 5.31 mmol) was added to a stirred solution of (+ / -)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2-amine (1.61 g, 5.06 mmol) and N,N-diisopropylethylamine (1.3 mL, 7.59 mmol) in acetonitrile (30 mL). The resulting mixture was heated to reflux and stirred under an inert atmosphere. After 16 h, LC / MS analysis of the yellow mixture indicated that the reaction was complete. The mixture was cooled to room temperature and diluted with water (50 mL). The resulting mixture was extracted with ethyl acetate (2 x 50 mL). The combined organic phases were dried over magnesium sulfate, filtered, and concentrated to provide (+ / -)-N-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3-nitropyridin-2-amine as a yellow solid (2.05 g, 4.65 mmol, 92% yield): 1 H NMR (400MHz, CDCl3) δ8.51–8.40(m,3H),8.17(d,J=2.4Hz,1H),7.58(dd,J=8.6,2.4Hz,1H),6.79(d,J=8.6Hz,1H),6.70–6.66(m,1H),6.61(d, J=1.9Hz,1H),6.55(d,J=1.9Hz,1H),4.63(d,J=7.8Hz,1H),4.18–4.09(m,1H),3.95(s,3H),3.84(s,3H),1.19(d,J=6.4Hz,3H)ppm; (M+1)=441.

[0251] Examples 3-5: (+ / -)-N 2 Preparation of -(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)pyridine-2,3-diamine

[0252]

[0253] Zinc powder (2.43 g, 37.23 mmol) was added to a stirred solution of (+ / -)-N-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3-nitropyridin-2-amine (2.05 g, 4.65 mmol) and ammonium chloride (1.99 g, 37.23 mmol) in a mixture of tetrahydrofuran (50 mL) / methanol (20 mL) / water (10 mL). The resulting mixture was stirred at room temperature. After 45 min, LC / MS analysis showed that the reaction was complete. The gray suspension was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (75 mL). The filtrate was washed with 5N ammonium hydroxide solution (50 mL). The organic phase was dried over magnesium sulfate, filtered, and concentrated to provide (+ / -)-N-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3-nitropyridin-2-amine as a dark brown solid. 2 -(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)pyridine-2,3-diamine (1.72 g, 4.19 mmol, 90% yield): 1 H NMR (400MHz, CDCl3) δ8.17(d,J=2.4Hz,1H),7.78(dd,J=5.1,1.5Hz,1H),7.58(dd,J=8.6,2.4Hz,1H),6.88(dd,J=7.4,1.5Hz,1H) ,6.78(d,J=8.6Hz,1H),6.65(d,J=1.9Hz,1H),6.59(d,J=1.9Hz,1H),6.56(dd,J=7.4,5.1Hz,1H),4.63(d,J=7.7Hz,1H),4.40(br s,1H),4.18–4.09(m,1H),3.95(s,3H),3.83(s,3H),3.22(br s, 2H), 1.19 (d, J = 6.3Hz, 3H) ppm; (M+1) = 411.

[0254] Examples 3-6: Preparation of (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3H-imidazo[4,5-b]pyridine

[0255] To (+ / -)-N 2-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)pyridine-2,3-diamine (1.72 g, 4.19 mmol) and triethyl orthoformate (2.0 mL, 11.78 mmol) in a stirred suspension in ethanol (75 mL) were supplemented with p-toluenesulfonic acid monohydrate (approximately 0.050 g). The resulting mixture was heated to reflux and stirred under an inert atmosphere. After 16 h, LC / MS analysis showed that the reaction was complete. The mixture was cooled to room temperature and concentrated to provide a brown oil. Chromatographic purification (CombiFlash, 120 g SiO2 gold column, 20%–50% ethyl acetate:ethanol / heptane elution) provided a light brown solid. The solid was suspended in a mixture of methyl tert-butyl ether (12 mL) / ethyl acetate (0.50 mL). The mixture was heated to 55°C. After 3 h, the warm mixture was filtered, and the filter cake was washed with methyl tert-butyl ether (10 mL) and dried to give (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3H-imidazo[4,5-b]pyridine (0.99 g, 2.37 mmol, 57% yield) as a brown solid. 1 H NMR (400MHz, CDCl3) δ8.45 (dd, J=4.8, 1.4Hz, 1H), 8.16 (d, J=2.4Hz, 1H), 8.10 (d d,J=8.0,1.4Hz,1H),8.06(s,1H),7.56(dd,J=8.6,2.4Hz,1H),7.27(dd,J=8.0, 4.8Hz,1H),6.78(d,J=8.6,1H),6.54(s,2H),4.62(d,J=7.8Hz,1H),4.12(dq,J= 7.8, 6.3Hz, 1H), 3.94 (s, 3H), 3.79 (s, 3H), 1.17 (d, J = 6.3Hz, 3H) ppm; (M+1) = 421.

[0256] Example 4: Synthesis of (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3H-imidazo[4,5-b]pyridine-2-d (compound 4)

[0257]

[0258] Example 4-1: Synthesis of (+ / -)-2-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3H-imidazo[4,5-b]pyridine

[0259] Adding carbon tetrabromide (0.54 g, 1.62 mmol) and sodium tert-butoxide (0.41 g, 4.31 mmol) to a stirred solution of (+ / -)3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3H-imidazo[4,5-b]pyridine (0.45 g, 1.08 mmol) in N,N-dimethylformamide (5 mL) resulted in the formation of a dark brown mixture. The mixture was stirred at room temperature. After 20 min, LC / MS analysis showed the formation of a new product and the presence of the starting material (approximately 1:1). Additional carbon tetrabromide (0.54 g, 1.62 mmol) and sodium tert-butoxide (0.41 g, 4.31 mmol) were added to the mixture (repeated at t = 40 min, t = 60 min, and t = 80 min). After a total of 100 min, LC / MS analysis showed that the reaction was nearing completion. The mixture was quenched in a saturated ammonium chloride solution (50 mL). The mixture was extracted with ethyl acetate (40 mL). The organic phase was washed with brine (30 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to provide a brown oil. Chromatographic purification (CombiFlash, 40g SiO2 gold column, elution with 10%-50% ethyl acetate:ethanol / heptane) yielded (+ / -)-2-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3H-imidazo[4,5-b]pyridine as a grayish-white solid (0.39g, 0.79mmol, 73% yield): 1H NMR (400MHz, DMSO-d6) δ8.42(dd,J=4.9,1.5Hz,1H),8.22(d,J=2.3Hz,1H),8.10(dd,J =8.1,1.5Hz,1H),7.73(dd,J=8.6,2.3Hz,1H),7.35(dd,J=8.1,4.9Hz,1H),6.88(d,J= 8.6Hz,1H),6.70(d,J=1.9Hz,1H),6.28(d,J=1.9Hz,1H),4.72(d,J=7.9Hz,1H),4.28( dq,J=7.9,6.3Hz,1H),3.86(s,3H),3.69(s,3H),1.03(d,J=6.3Hz,3H)ppm; (M+1)=499.

[0260] Example 4-2: Preparation of (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3H-imidazo[4,5-b]pyridine-2-d

[0261] Add tris(dibenzylacetone)dipalladium (0) (0.12 g, 0.13 mmol), 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (0.16 g, 0.39 mmol), and potassium carbonate (0.18 g, 1.29 mmol) to a stirred solution of (+ / -)-2-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3H-imidazo[4,5-b]pyridine (0.32 g, 0.65 mmol) in propanol-d8 (3 mL) (Note: the reaction was carried out in a microwave-safe reaction vessel dried in a 20 mL oven). Seal the container and degas the contents under vacuum / backfill with N2 (x3). Heat the mixture to 100 °C in a heating block. After 2 hours, LC / MS analysis showed the reaction was complete. The mixture was cooled to room temperature and concentrated to provide a brown oil. Chromatographic purification (CombiFlash, 80 g SiO2 gold column, 20%–70% ethyl acetate:ethanol / heptane elution) yielded an impure brown oil. Preparative HPLC purification (Interchim column: F0040-51 g–51.0 g (20 bar) column, 20% acetonitrile / water / 0.1% formic acid to 100% acetonitrile / 0.1% formic acid elution) provided two pure fractions. The fractions were combined and diluted with saturated sodium bicarbonate solution (30 mL). The mixture was extracted with ethyl acetate (30 mL). The organic phase was dried over magnesium sulfate, filtered, and concentrated to provide (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl-d2)-3H-imidazo[4,5-b]pyridine-2-d as a white solid (0.16 g, 0.37 mmol, 57% yield): 1 H NMR (400MHz, DMSO-d6) δ8.40(dd,J=4.8,1.5Hz,1H),8.22(d,J=2.5Hz,1H),8.10(dd,J =8.0,1.5Hz,1H),7.73(dd,J=8.6,2.5Hz,1H),7.30(dd,J=8.0,4.8Hz,1H),6.88(d,J= 8.6Hz,1H),6.76(d,J=2.0Hz,1H),6.50(d,J=2.0Hz,1H),4.72(d,J=7.8Hz,1H),4.27( dq,J=7.8,6.3Hz,1H),3.86(s,3H),3.70(s,3H),1.04(d,J=6.3Hz,3H)ppm; (M+1)=421.

[0262] Example 5: Synthesis of 3-(((2R,3R)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d (compound 5)

[0263]

[0264] Example 5-1: Preparation and isolation of 3-(((2R,3R)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine and 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine

[0265]

[0266] The preparation of (+ / -)-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine was carried out in five steps from (+ / -)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine according to the procedure described in Examples 3-2 to 3-6. The racemic product (approximately 95:5 trans:cis) was subjected to chiral SFC separation (Whelk-01 21 x 250 mm column, flow rate 70 mL / min, elution in 50% ethanol in CO2 / 0.1% diethylamine, with the compound (2.24 g) dissolved in 60 mL methanol / 15 mL dichloromethane, 1.8 mL solution injected each time) to provide three fractions. The first fraction contained a small amount of one of the trans enantiomers. The second fraction contains 3-(((2R,3R)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (contaminated with a small amount of one of the trans enantiomers), and the third fraction contains 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine: 1H NMR (400MHz, CDCl3) δ8.45 (dd, J=4.8, 1.5Hz, 1H), 8.16 (d, J=2.5Hz, 1H), 8.09 (d d,J=8.0,1.5Hz,1H),8.04(s,1H),7.56(dd,J=8.6,2.5Hz,1H),7.29–7.26(m,1H ),6.78(d,J=8.6Hz,1H),6.54–6.52(m,2H),5.38(s,2H),4.62(d,J=7.8Hz,1H), 4.11(dq,J=7.8,6.3Hz,1H),3.94(s,3H),3.79(s,3H),1.17(d,J=6.3Hz,3H)ppm.

[0267] Example 5-2: Preparation of 2-bromo-3-(((2R,3R)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine

[0268]

[0269] Carbon tetrabromide (0.38 g, 1.16 mmol) and sodium tert-butoxide (0.40 g, 4.21 mmol) were added to a stirred solution of 3-(((2R,3R)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (0.44 g, 1.05 mmol) in N,N-dimethylformamide (5 mL). The resulting dark brown mixture was stirred at room temperature. After 30 min, LC / MS analysis showed the formation of a new product and the presence of the starting material (approximately 1:1). The mixture was quenched in a saturated ammonium chloride solution (50 mL). The mixture was extracted with ethyl acetate (40 mL). The organic phase was washed with brine (30 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to provide a brown oil. Chromatographic purification (CombiFlash, 24 g SiO2 gold column, elution with 10%-50% ethyl acetate:ethanol / heptane) yielded a brownish-red solid of 2-bromo-3-(((2R,3R)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (0.18 g, 0.37 mmol, 35% yield): 1HNMR(400MHz,DMSO-d6)δ8.42(dd,J=4.9,1.5Hz,1H),8.22(d,J=2.4Hz,1H),8.11(dd,J=8 .1,1.5Hz,1H),7.73(dd,J=8.6,2.4Hz,1H),7.36(dd,J=8.1,4.9Hz,1H),6.88(d,J=8.6Hz ,1H),6.69(d,J=1.9Hz,1H),6.27(d,J=1.9Hz,1H),5.41(s,2H),4.72(d,J=7.8Hz,1H),4. 28(dq,J=7.8,6.3Hz,1H),3.86(s,3H),3.69(s,3H),1.02(d,J=6.3Hz,3H)ppm; (M+1)=497.

[0270] Example 5-3: Preparation of 3-(((2R,3R)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d

[0271] Add tris(dibenzylacetone)dipalladium(0) (0.059 g, 0.064 mol), 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (0.080 g, 0.19 mmol), and potassium carbonate (0.089 g, 0.64 mmol) to a stirred solution of 2-bromo-3-(((2R,3R)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (0.16 g, 0.32 mmol) in 2-propanol-d8 (5 mL) (Note: the reaction was carried out in a microwave-safe reaction vessel dried in a 20 mL oven). Seal the container and degas the contents under vacuum / backfill with N2 (x 3). Heat the mixture to 100 °C in a heating block. After 2 hours, LC / MS analysis showed that the reaction was complete. The mixture was cooled to room temperature and concentrated to provide a brown oil. Chromatographic purification (CombiFlash, 40 g SiO2 gold column, 20%-70% ethyl acetate:ethanol / heptane elution) gave a yellow solid of 3-(((2R,3R)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d (0.075 g, 0.18 mmol, 56% yield): 1H NMR (400MHz, DMSO-d6) δ8.40 (dd, J=4.7, 1.5Hz, 1H), 8.22 (d, J=2.4Hz, 1H), 8.10 (dd, J=8. 0,1.5Hz,1H),7.73(dd,J=8.6,2.4Hz,1H),7.30(dd,J=8.0,4.7Hz,1H),6.88(d,J=8.6Hz, 1H),6.76(d,J=1.9Hz,1H),6.49(d,J=1.9Hz,1H),5.39(s,2H),4.72(d,J=7.8Hz,1H),4.2 8(dq,J=7.8,6.3Hz,1H),3.86(s,3H),3.70(s,3H),1.04(d,J=6.3Hz,3H)ppm; (M+1)=420.

[0272] Example 6: Synthesis of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d (compound 6)

[0273] Method A:

[0274]

[0275] Example 6-1: Synthesis of 2-bromo-3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine

[0276]

[0277] Carbon tetrabromide (0.53 g, 1.58 mmol) and sodium tert-butoxide (0.50 g, 5.22 mmol) were added to a stirred solution of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (0.51 g, 1.22 mmol) in N,N-dimethylformamide (5 mL). The resulting dark brown mixture was stirred at room temperature. After 1 h, LC / MS analysis showed the formation of a new product and the presence of the starting material (approximately 1:1). The mixture was quenched in a saturated ammonium chloride solution (50 mL). The mixture was extracted with ethyl acetate (40 mL). The organic phase was washed with brine (30 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to provide a brown oil. Chromatographic purification (CombiFlash, 40g SiO2 gold column, elution with 10%-60% ethyl acetate:ethanol / heptane) yielded a grayish-white solid of 2-bromo-3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (0.20 mg, 0.39 mmol, 32% yield): 1 H NMR (400MHz, DMSO-d6) δ8.42(dd,J=4.8,1.5Hz,1H),8.22(d,J=2.4Hz,1H),8.11(dd,J=8. 1,1.5Hz,1H),7.73(dd,J=8.6,2.4Hz,1H),7.36(dd,J=8.1,4.8Hz,1H),6.88(d,J=8.6Hz, 1H),6.69(d,J=2.0Hz,1H),6.27(d,J=2.0Hz,1H),5.41(s,2H),4.72(d,J=7.9Hz,1H),4.2 8(dq,J=7.9,6.3Hz,1H),3.86(s,3H),3.69(s,3H),1.02(d,J=6.3Hz,3H)ppm; (M+1)=497.

[0278] Example 6-2: Preparation of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d

[0279] Add tris(dibenzylacetone)dipalladium(0) (0.054 g, 0.059 mmol), 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (0.074 g, 0.18 mmol), and potassium carbonate (82.52 mg, 591.13 μmol) to a stirred solution of 2-bromo-3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (0.15 mg, 0.30 mmol) in 2-propanol-d8 (5 mL) (Note: the reaction was carried out in a microwave-safe reaction vessel dried in an oven). Seal the container and degas the contents under vacuum / backfill with N2 (x 3). The mixture was heated to 100°C in a heating block. After 1 h, LC / MS analysis showed that the reaction was complete. The mixture was cooled to room temperature and concentrated to provide a brown oil. Chromatographic purification (CombiFlash, 40 g SiO2 gold column, 20%-70% ethyl acetate:ethanol / heptane elution) gave a yellow solid of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d (0.088 g, 0.21 mmol, 71% yield): 1 H NMR (400MHz, DMSO-d6) δ8.40 (dd, J=4.8, 1.5Hz, 1H), 8.22 (d, J=2.4Hz, 1H), 8.10 (dd, J=8. 0,1.5Hz,1H),7.73(dd,J=8.7,2.4Hz,1H),7.30(dd,J=8.0,4.8Hz,1H),6.88(d,J=8.7Hz, 1H),6.76(d,J=1.9Hz,1H),6.49(d,J=1.9Hz,1H),5.39(s,2H),4.72(d,J=7.8Hz,1H),4.2 8(dq,J=7.8,6.3Hz,1H),3.86(s,3H),3.70(s,3H),1.04(d,J=6.3Hz,3H)ppm; (M+1)=420.

[0280] Example 7: Synthesis of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d (compound 6)

[0281] Method B:

[0282] 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (8.00 g, 19.12 mmol) was dissolved in 2-methyltetrahydrofuran (220 mL) under heating. The solution was distilled to remove 20 mL of solvent to dry the mixture. After cooling to room temperature, the mixture was treated with potassium tert-butoxide (3.2 g, 28.7 mmol) and methanol-d1 (24.00 mL, 646 mmol). The solution was heated to 58-61 °C. After 4 h, the solution was cooled to room temperature and washed with 10% w / w ammonium chloride aqueous solution (150 mL). The organic layer was washed twice with brine, dried over magnesium sulfate, filtered, and concentrated to provide 7.60 g of solid. The material was dissolved in 76 mL of 2-methyltetrahydrofuran by heating to 60 °C. The solution was inoculated and stirred at 45 °C for 1 h. The mixture was stirred at room temperature for 1 h, and then stirred at 0 °C–5 °C for 1 h. The resulting solid was filtered, washed with a small amount of 2-methyltetrahydrofuran, and dried under vacuum to give 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d (6.3 g, 79% yield, LCMS: 94.5% D, 1H NMR: 94% D) as a grayish-white crystalline solid.

[0283] Example 8: Large-scale synthesis of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d (compound 6)

[0284] 50 g of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine was added to 500 mL of toluene. The resulting slurry was heated to 120-130 °C and refluxed for 2-3 h, and water was removed using Dean Stark solvent. After most of the solvent was removed, the mixture was added to a volume of 3.0 to 4.0 with heptane (2 × 250 mL) at a temperature below 60 °C. The resulting slurry was then exchanged with 2-methylTHF (2 × 250 mL) at a temperature below 60 °C to a volume of 3.0 to 4.0. After adding 800 mL (16 volumes) of 2-methylTHF, the mixture was heated to 60-65°C and 50 mL of MeOD and 67 mL (1.0 equivalent) of a 20% potassium tert-butoxide solution in THF were added at 60-65°C. The reaction mixture was maintained at 60-65°C for 3 h. The reaction was cooled to 20-30°C and quenched with 1000 mL (20 volumes) of a 10% ammonium chloride aqueous solution. The organic layer was diluted with ethyl acetate and washed with water (3 × 250 mL) and a 25% salt solution (250 mL). The organic layer was distilled under vacuum at 60°C to 3.0-4.0 volumes. The mixture was then added to toluene (2 × 250 mL) to 3.0-4.0 volumes below 60°C, and then added to heptane (2 × 250 mL) to 3.0-4.0 volumes below 60°C. The resulting slurry was solvent-exchanged with 2-methylTHF (2 × 250 mL) at below 60 °C to a volume of 3.0 to 4.0. 1050 mL (21 volumes) of 2-methylTHF was added to the mixture, and the reactants were heated to 60-65 °C to obtain a clear solution. The obtained pale yellow clear solution, obtained by 1H NMR, contained 80%-85% of compound D6. 100 mL of MeOD and 13.4 mL (0.2 equivalents) of a 20% potassium tert-butoxide solution in THF were added to the solution at 60-65 °C. The reaction was maintained at 60-65 °C for 3 h. The reaction was cooled to 20-30 °C and quenched with 500 mL (10 volumes) of a 10% ammonium chloride solution. The organic layer was further washed with water (3 × 250 mL). The organic layer was distilled under vacuum at 60 °C to a volume of 7.5 to 8.0. The resulting slurry was refluxed at 65-70°C to obtain a clear solution. The mixture was cooled to 60-65°C over a period of 20 minutes and inoculated with 0.025 g of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d.The mixture was slowly cooled to 25°C to 30°C over a period of 2–3 h and further cooled to 0°C to 5°C over a period of 2–3 h, while stirring for 1–2 h. The solids were filtered and washed with 50 mL (1.0 volume) of pre-cooled 2-methylTHF. The wet material (39.5 g) was placed under high vacuum at 45°C to 50°C for 16 h to obtain 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d (39.2 g; 78% yield; by LCMS 95% D).

[0285] Example 9: Further large-scale synthesis of compound 6 with advantageous stereoselectivity

[0286] Example 9-1: Preparation of 2-(5-((3H-imidazo[4,5-b]pyridin-3-yl)methyl)-2-(benzyloxy)-3-methoxyphenoxy)-1-(6-methoxypyridin-3-yl)prop-1-one

[0287]

[0288] A mixture of 2-bromo-1-(6-methoxypyridin-3-yl)prop-1-one (21.2 g, 87 mmol, 1 equivalent, CAS 1391089-35-2), 5-((3H-imidazo[4,5-b]pyridin-3-yl)methyl)-2-(benzyloxy)-3-methoxyphenol (32.9 g, 91.3 mmol, 1.05 equivalent) (WO 2017015267 Example 1-193) and potassium carbonate (30 g, 218 mmol, 2.5 equivalent) in acetonitrile (330 mL) was stirred at room temperature for 4 h. HPLC analysis showed complete consumption of 2-bromo-1-(6-methoxypyridin-3-yl)prop-1-one. Methyl tert-butyl ether (330 mL) was added to the slurry, and the mixture was filtered and the solids were washed with methyl tert-butyl ether. The filtrate was washed with diluted sodium hydroxide solution (350 mL) and saturated sodium chloride solution (300 mL). The solvent was exchanged with methanol. The methanol solution was stirred with seed crystals (20 mg) at room temperature. After stirring at room temperature for 16 h, the crystallized product was separated by filtration, washed with methanol and dried to give 2-(5-((3H-imidazo[4,5-b]pyridin-3-yl)methyl)-2-(benzyloxy)-3-methoxyphenoxy)-1-(6-methoxypyridin-3-yl)prop-1-one (35.9 g, 85% yield) as a grayish-white crystalline solid with a melting point of 72 °C. 1H NMR (400MHz, CDCl3) δ8.83 (dd, J=2.4, 0.7Hz, 1H), 8.39 (dd, J=4.8, 1.4Hz, 1H), 8.13 –8.03(m,2H),7.96(s,1H),7.48–7.41(m,2H),7.36–7.21(m,4H),6.69(dd,J=8.8,0 .8Hz,1H),6.55(d,J=1.9Hz,1H),6.44(d,J=2.0Hz,1H),5.32(s,2H),5.29(q,J=6.8 ,1H),4.99(s,2H),3.98(s,3H),3.74(s,3H),1.60(d,J=6.8Hz,3H)ppm; (M+1)=525.

[0289] Example 9-2: Preparation of 4-((3H-imidazo[4,5-b]pyridin-3-yl)methyl)-2-(((1S,2S)-1-hydroxy-1-(6-methoxypyridin-3-yl)prop-2-yl)oxy)-6-methoxyphenol

[0290]

[0291] 2-(5-((3H-imidazo[4,5-b]pyridin-3-yl)methyl)-2-(benzyloxy)-3-methoxyphenoxy)-1-(6-methoxypyridin-3-yl)prop-1-one (14 g, 25.2 mmol), potassium tert-butoxide (1.35 g, 12.01 mmol, 0.48 equivalents), and RuCl2[(S)-(DM-BINAP)][(S)-DAIPEN](CAS 220114-01-2, 0.33 g, 0.27 mmol, 0.01 equivalents) were dissolved in isopropanol (230 mL) and loaded into a hydrogenation reactor. The reactor was purged with nitrogen and filled with hydrogen to 70 psi. After stirring at 22 °C and 70 psi hydrogen pressure for 5 h, HPLC analysis showed that the starting material was completely consumed. Hydrogenolysis was carried out by charging a reactor with Pd / C (4.8 g, 34 wt%, 5% activated carbon-supported Pd, 50% humidity). The Parr reactor was purged with nitrogen and charged with hydrogen to 70 psi. After stirring at 22 °C and 70 psi hydrogen pressure for 48 h, HPLC analysis showed that the reaction was essentially complete. The reaction mixture was filtered through a diatomaceous earth mat and washed with isopropanol and methanol. The filtrate was concentrated to a clear yellow oil. The oil was dissolved in ethyl acetate (250 mL) and washed with an aqueous solution of ammonium chloride (130 mL). The aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with a saturated sodium chloride solution, dried with sodium sulfate, filtered, and concentrated to obtain 4-((3H-imidazo[4,5-b]pyridin-3-yl)methyl)-2-(((1S,2S)-1-hydroxy-1-(6-methoxypyridin-3-yl)prop-2-yl)oxy)-6-methoxyphenol (10.1 g, 23.1 mmol, 92% yield), which was a pale yellow, hard foam. The product was a diastereomer of 1S,2S and 1R,2S in a ratio of approximately 84:16 (via...). 1 HNMR); >98% ee (by chiral HPLC) 1 H NMR (400MHz, CDCl3) δ8.43 (d, J=4.6Hz, 1H), 8.13–8.05 (m, 2H), 8.02 (d, J=1.7Hz, 1H), 7.68 and 7.61 (2br d,J=8.7Hz,1H),7.31–7.23(m,2H),6.77–6.67(m,2H),6.66(d,J=2.7Hz,1H),5.36(s,2H),4.82 and 4.71(br s and d,J=8.3Hz,1H),4.13(m,1H),3.94(br s,3H),3.83(br s,3H),1.18–1.07(d,J=6.4Hz,3H)ppm; (M+1)=437.

[0292] Example 9-3: Preparation of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine

[0293]

[0294] A solution of 4-((3H-imidazo[4,5-b]pyridin-3-yl)methyl)-2-(((1S,2S)-1-hydroxy-1-(6-methoxypyridin-3-yl)propyl-2-yl)oxy)-6-methoxyphenol (10.10 g; 23.14 mmol; 84:16 diastereomer mixture; 1.00 equivalent) in ethyl acetate (90 mL) was stirred with diisopropylethylamine (16.02 mL; 92.56 mmol; 4.00 equivalent) and CCl4 (5.58 mL; 57.85 mmol; 2.50 equivalent) at 45-50 °C. Tri-n-butylphosphine (11.99 mL; 48.60 mmol; 2.10 equivalent) was added dropwise over 10 min, accompanied by slight exothermic reaction. The resulting brown solution was stirred at 45-50 °C for 1.5 h. Add sodium hydroxide solution (15 wt%, 40 mL, 6.5 equivalents) to the reaction mixture and stir at 45 °C for 0.5–1 h. Cool the reaction mixture to room temperature. Separate the layers. Extract the aqueous layer with ethyl acetate (40 mL). Wash the combined organic layers with saturated sodium chloride solution (50 mL), dry over sodium sulfate, filter, and concentrate to a wet solid. Stir the solid in methyl tert-butyl ether (60 mL) for 2 h, filter, and dry under vacuum. Dissolve the off-white solid in ethanol (55 mL) at elevated temperature. Stir the solution with the seed crystals at room temperature and cool to 0 °C–5 °C. The resulting solid was filtered and dried to give 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (6.16 g, 63.6%) 99A% (by HPLC), 98% ee, Pd: 1 ppm; Ru: 225 ppm; Form A. Melting point 164.9 °C. 1H NMR(400MHz, CDCl3)δ8.45(dd,J=4.8,1.5Hz,1H),8.16(d,J=2.4Hz,1H),8.13–8.03(m,2H ),7.56(dd,J=8.6,2.5Hz,1H),7.27(dd,J=8.0,4.8Hz,1H),6.78(d,J=8.6Hz,1H),6.53(br s,2H),5.38(s,2H),4.62(d,J=7.8Hz,1H),4.13(m,1H)3.94(s,3H),3.79(s,3H),1.17(d,J=6.4Hz,3H)ppm. (M+1)=419.

[0295] Examples 9-4: Preparation of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d

[0296]

[0297] 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (20 g; 47.80 mmol) was dissolved in 2-methyltetrahydrofuran (400 mL) at 60 °C. Methanol-d (20 mL; 1 V, 99% D) was added, followed by potassium tert-butoxide (5.36 g; 47.80 mmol; 1 equivalent). The solution was heated for 2 h. LCMS showed 87% D. The reaction solution was cooled to 22 °C. The suspension was washed with 10% w / w ammonium chloride aqueous solution (400 mL). The organic layer was separated, diluted with ethyl acetate (200 mL), washed with water (3 x 100 mL), and then washed with 1 / 2 saturated sodium chloride solution (100 mL). The organic layer was dried (sodium sulfate), filtered, and concentrated to a solid. The solid was azeotropically dried with toluene (2 x 100 mL). The resulting brownish solid was dissolved in 2-methyltetrahydrofuran (500 mL) at 60 °C, and methanol-d (40 mL; 2 V) was added, followed by potassium tert-butoxide (1.1 g; 9.80 mmol; 0.2 equivalents). The solution was heated at 60 °C for 3 h. After 3 h, LCMS showed 96%–97% D. The reaction solution was cooled to room temperature and washed with 10% w / w ammonium chloride aqueous solution (200 mL; 10 V). The organic layer was separated and washed three times with water (200 mL each time). The organic solution was filtered, concentrated, and azeotropically dried with toluene. The solid was dissolved in 2-methyltetrahydrofuran (560 mL) at 80 °C. The reaction solution was cooled to 75 °C and inoculated with form A (200 mg). The mixture was stirred while the temperature was cooled to 22 °C and maintained for 1 h. The mixture was stirred at 0-5°C for 1 h. The resulting solid was filtered, washed with cold 2-methyltetrahydrofuran, and dried in a vacuum oven to obtain 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d form A (100% (by HPLC), chiral purity: 99.5%; D (by LCMS); Pd 1 ppm; Ru 20 ppm, 84% yield) as a grayish-white powder. 1¹H NMR (400MHz, DMSO-d⁶) δ 8.57 (s, undeuterated, 0.02H), 8.40 (dd, J = 4.8, 1.5Hz, 1H), 8.22 (d, J = 2.3Hz, 1H), 8.10 (dd, J = 8.0, 1.5, 1H), 7.72 (dd, J = 8.6, 2.4Hz, 1H), 7.36 (dd, J = 8.0, 4.8) ,1H),6.88(d,J=8.6Hz,1H),6.76(d,J=1.9,1H),6.50(d,J=1.9,1H),5.40(s,2H),4.72( d,J=7.8Hz,1H),4.26-4.29(m,1H),3.87(s,3H),3.70(s,3H),1.03(d,J=6.3Hz,3H)ppm. (M+1)=420

[0298] The 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d form A was further characterized by XRPD, PLM, DSC, TGA and HPLC. The results are summarized in Table 2-1, indicating that the starting material was a crystalline anhydrous hydrate.

[0299]

[0300] Analysis method:

[0301] X-ray powder diffraction (XRPD)

[0302] Instrument: Panalytical Empyrean powder diffractometer

[0303] Parameters: X-ray tube Cu (Kα); tube voltage 45kV; tube current 40mA

[0304] 2-θ scan from 2 to 40 degrees; 0.013 degrees / step; scan rate 6 degrees / min

[0305] Thermogravimetric analysis (TGA)

[0306] Instrument: TA Instruments Discovery TGA Q5500

[0307] Parameters: ramp up 10°C / min, ambient temperature up to 250°C / 300°C, N2 flushing at 50 mL / min

[0308] Differential scanning calorimetry (DSC)

[0309] Instrument: TA Instruments Discovery DSC

[0310] Parameters: Inclined rise 10℃ / min, from ambient temperature to 250℃ / 300℃, N2 flushing 50mL / min

[0311] Polarized light microscopy (PLM)

[0312] Instrument: Nikon Eclipse Ci Pol

[0313] Camera: Nikon

[0314] Software: NIS-Elements image software

[0315] The sample is dispersed in slurry form on a microscope slide, or if dry, dispersed with silicone oil and examined under transmitted polarized light.

[0316] Phosphorylation cFMS activity

[0317] Reagents and consumables were purchased from Sigma Aldrich, Gibco Life Technologies, BD Biosciences, Perkin Elmer, R&D Systems, Cell Signaling, Thermo Scientific (Pierce), and Santa Cruz Biotechnology. HEK293 cells overexpressing human cFMS (HEK293 / hFMS) were cultured in RPMI medium in T225 flasks and divided twice weekly. For experiments, cells were trypsinized, counted, and diluted to 600,000 cells / ml (30,000 cells / well) with serum-free Megacell medium (Sigma catalog number M3817). Serial dilutions of test compounds were prepared using Echo LDV plates (catalog number LP-0200) via Echo 555 (LABCYTE); and 500 nmol of each compound concentration was added to DMSO (0.5% final) in 96-well BD Biocoat poly-d-lysine plates (BD catalog number 356640). Then add 50 μL / well MegaCell serum-free medium to cover the compound, followed by cell addition at 50 μL / well (30,000 / well). Rotate the plate at 1000 rpm for 1 minute and incubate on a plate for 15–30 minutes; transfer the plate to a CO2 incubator at 37°C for overnight incubation. Pre-coat white 96-well Perkin Elmer OptiPlates (catalog number 6005509) with 50 ng / well (100 μL / well) of anti-cFMS / CSF-1R (C-20) (Santa Cruz catalog number sc-692) in PBS, seal with foil sealant, rotate at 1000 rpm for 1 minute and incubate overnight at 4°C.

[0318] On the second day, pre-coated OptiPlates plates were blocked at room temperature with 200 μL / well of 1% BSA in 1x PBST (PBS with 0.1% Triton-X) for 2–3 hours. In parallel, 100 μL / well of 2x hCSF1 (final 150 ng / ml) (R&D Systems, catalog number 216-MC-025 / CF) (or culture medium, as a negative control) was added to HEK293 / hFMS cells (BD culture plates) incubated overnight with the compound. On each plate, 100% reaction (treated with CSF1) and 0% reaction (without CSF1) control columns were used to calculate the percentage of inhibition and Z-prime value of the test compound. The plates were incubated at 37°C for 10 minutes. Aspirate the culture medium / hCSF1 and lyse the cells with 100 μL / well of pre-chilled lysis buffer, which is prepared with lysis buffer (Cell Signaling catalog 9803), protease / phosphatase inhibitor (Pierce catalog 78444), and PMSF (Sigma catalog 93482). Shake the plate for 60 seconds; then, incubate at 3200 rpm for 5 minutes at 4°C on ice. Transfer 90 μL of the lysate to pre-coated / blocked OptiPlates. Then, incubate the plate overnight at 1000 rpm in a sealed container at 4°C.

[0319] The next day, the lysate was removed from the plate; and the plate was washed 6 times with 300 μL / well of 1xPBS using a Biotek washer. The remaining PBS on the plate was tapped off. 90 μL / well of 1:10,000 antiphosphorylated Eu (Tyr 100) in 1% BSA in PBST (Perkin Elmer catalog number AD0159) was added to the plate; and the plate was incubated sealed at room temperature for one hour. After one hour, the antibody was removed, and the plate was washed 6 times with 300 μL / well of PBST using a Biotek washer. Next, 90 μL / well of enhancement solution (Perkin Elmer catalog number 4001-0010) was added, and the plate was sealed and shaken for 5 minutes. The signal was immediately read on a Perkin Elmer Envision to obtain time-resolved fluorescence with excitation at 320 nm and emission at 615 nm.

[0320] Analyze the data via Pipeline Pilot to calculate IC. 50 Value; for selected CSF-1R inhibitors, the IC50 value of phosphor c-FMS is... 50 The values ​​are provided in Table B below.

[0321] Table B

[0322]

[0323]

[0324] Biological Examples (Examples 10-17xx): In vitro studies

[0325] Example 10

[0326] To compare the effects of CSF-1R inhibitory compounds and the deuterated CSF-1R inhibitory compounds of this disclosure on the production of cytokines / chemokines after CSF-1 stimulation, the following experiments were performed in BV2 mouse microglia.

[0327] Two different generations of BV2 mouse microglia were plated in separate 96-well plates to provide biological quadruplicates.

[0328] Group number simulation group Hole / Group deal with 1 Non-irritating 4 holes / generation DMSO 2 CSF-1 stimulation 4 holes / generation DMSO, compound 24 or compound 6

[0329] Test items:

[0330] DMSO

[0331] Compound 24:

[0332]

[0333] Compound 6

[0334]

[0335] • Recombinant mouse M-CSF (R&D Systems, catalog number 416-ML / CF, batch number ME4518091) - Prepare a 100 μg / mL stock solution by dissolving 50 μg in 500 μl PBS and then treating with 100 ng / mL.

[0336] Compound 24 was prepared according to the procedure outlined in Examples 1-92 of WO 2017 / 015267.

[0337] Two test compounds were prepared in diluted stock solutions (10 mM) with culture medium to obtain 100 μM working solutions, and were treated at 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM or 400 nM.

[0338] method

[0339] Treatment and stimulation of BV2 microglia

[0340] BV2 mouse microglia were divided into 5 x 10⁻⁶ cells. 5Cells were suspended at a concentration of [number] cells / mL, and 100 μL of this cell suspension was added to each well of a 96-well plate. Microglia were allowed to stand overnight at 37°C and 5% CO2. The next day, the culture medium was removed, and the cells were treated with dimethyl sulfoxide (DMSO), compound 24, or compound 6 at 37°C and 5% CO2 for 30 minutes. The cells were then stimulated with 100 ng / mL recombinant mouse M-CSF for 24 hours. After stimulation, the culture supernatant was removed from each well, and the cells were aliquoted into two different 96-well plates for subsequent ELISA assays.

[0341] Mouse MCP-1 ELISA

[0342] The culture supernatant was assayed using the Quantikine Mouse MCP-1 ELISA kit from R&D Systems. Samples were diluted 1:10 with calibrator diluent. First, 50 μL of assay diluent was added to each well. Then, 50 μL of standard, assay control, and diluted sample were added to the wells. The plate was mixed by gently tapping the frame and sealed with tape. The plate was incubated at room temperature for 2 hours. After incubation, the plate was washed 5 times with approximately 400 μL of wash buffer using a spray bottle. After the final wash, the plate was gently tapped on a paper towel to remove excess moisture. 100 μL of mouse MCP-1 conjugate was added to each well, covered with new tape, and incubated at room temperature for 2 hours. After incubation, the plate was washed as described above. Then, substrate solution was added to each well and incubated in the dark at room temperature for 30 minutes. After incubation, acidic stop solution was added to each well, and the plate was read at 450 nm using an ELISA plate reader.

[0343] result

[0344] BV2 mouse microglia were seeded at 50,000 cells / well and incubated overnight. Cells were pretreated with DMSO, Compound 24, or Compound 6 for 30 min and then subjected to CSF-1 stimulation. Cell culture supernatant from this experiment was processed in an MCP1 ELISA to determine whether stimulation / treatment affected chemokine production. As shown in Figures 1A-1B and 2A-2B, CSF-1 stimulation significantly increased the release of MCP-1 (CCL2-chemokine), and both small molecule CSF-1R inhibitors significantly reduced MCP-1 production in a concentration-dependent manner. The percentage of inhibition was calculated based on the unstimulated and stimulated controls, and IC50 was generated. 50 The curves, as shown in Figures 3A-3B and 4A-4B, indicate that for this determination, the two compounds exhibited similar IC50 values ​​between 28.8 nM and 36.5 nM. 50 value.

[0345] The bars in the graph represent the mean and standard deviation. Statistical significance was determined using a one-way ANOVA with multiple comparisons, and p-values ​​are expressed as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0346] Example 11

[0347] To compare the effects of two CSF-1R inhibitory compounds and one deuterated CSF-1R inhibitory compound of this disclosure on the production of cytokines / chemokines in microglia after CSF-1 stimulation, the following experiments were performed in primary mouse microglia.

[0348] Primary mouse microglia

[0349]

[0350]

[0351] Test items:

[0352] DMSO

[0353] Compound 49

[0354]

[0355] ·PLX3397 (Pedatitinib)

[0356]

[0357] Compound 6

[0358] • Recombinant mouse CSF-1 (R&D Systems, catalog number 416-ML / CF, batch number ME4518091) - prepared as a 100 μg / mL stock solution by dissolving 50 μg in 500 μL PBS and treating microglia with 100 ng / mL.

[0359] Compound 49 was prepared according to the procedure outlined in Examples 1-5 of WO 2017 / 015267.

[0360] All test compounds were prepared in diluted stock solutions (10 mM) with culture medium to obtain 100 μM working solutions, and treated at 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM or 400 nM.

[0361] method

[0362] Treatment and stimulation of primary microglia

[0363] Primary mouse microglia were cultured at 5 x 10⁻⁶ cells per cell line. 5 Cells were suspended at a concentration of 100 μL / mL and 100 μL of this cell suspension was added to the inner 60 wells of three 96-well plates. Microglial cells were allowed to stand overnight at 37°C and 5% CO2. The next day, the culture medium was removed, and the cells were treated with dimethyl sulfoxide (DMSO), compound 49, PLX3397, or compound 6 at 37°C and 5% CO2 for 30 minutes. The cells were then stimulated with 100 ng / mL recombinant mouse CSF-1 for 24 hours. After stimulation, the culture supernatant was removed from each well and aliquoted into two different 96-well plates for subsequent ELISA assays. The plates were fixed with 4% PFA for future immunocytochemical analysis.

[0364] Mouse MCP-1 ELISA

[0365] The culture supernatant was analyzed using the Quantikine Mouse MCP-1 ELISA Kit (R&D Systems, catalog number SMJE00B). Samples were diluted 1:10 with calibrator diluent. First, 50 μL of assay diluent was added to each well. Then, 50 μL of standard, assay control, and diluted sample were added to the wells. The plate was mixed by gently tapping the frame and sealed with tape. The plate was incubated at room temperature for 2 hours. After incubation, the plate was washed 5 times with approximately 400 μL of wash buffer using a spray bottle. After the final wash, the plate was gently tapped on a paper towel to remove excess moisture. 100 μL of mouse MCP-1 conjugate was added to each well, covered with new tape, and incubated at room temperature for 2 hours. After incubation, the plate was washed as described above. Then, substrate solution was added to each well and incubated in the dark at room temperature for 30 minutes. After incubation, an acid-terminating solution was added to each well, and the plate was read at 450 nm using a FlexStation3 multimode microplate reader (Molecular Devices, catalog number Flex3) with SoftMax Pro software.

[0366] Immunostaining of primary microglia

[0367] Following stimulation, cells were fixed with 4% PFA for 20 minutes at room temperature. Cells were then washed in PBS at room temperature, washed 3 times for 5 minutes in 0.2% PBT (0.2% Triton X-100 in PBS), and blocked with 10% donkey serum / 0.2% PBT for 1 hour. Cells were then incubated overnight at 4°C with primary antibody diluted in 10% donkey serum / 0.2% PBT (rabbit anti-Iba1, 1:500; Wako, catalog number 019-19741 or rabbit anti-Ki67, 1:500; Abcam, catalog number ab15580). The next day, cells were washed 3 times for 5 minutes in 0.2% PBT and incubated for 1 hour at room temperature with secondary antibody diluted in 1% donkey serum / 0.2% PBT (donkey anti-rabbit Alexa Fluoro 488, 1:500; Life Technologies, catalog number A21206). The cells were then washed 3 times for 5 minutes in 0.2% PBT, incubated for 5 minutes in DAPI (1:10,000 in PBS) at room temperature, and rinsed in PBS.

[0368] Microscopy

[0369] After staining, the plate was imaged on an IN Cell Analyzer 2200, obtaining 9 fields of view per well. Quantification was performed using IN Cell Imaging Analysis Software, calculating the total area of ​​IBA1 staining in the 9 fields (in μm²). 2 The number of cells (either counted or Ki67+) was calculated. For each technique, the mean of each field of view in each well was calculated in triplicate (excluding some fields due to staining artifacts, typically 6-9 fields per well), and normalized relative to the mean of the DMSO control wells. One-way ANOVA was used to determine the statistical significance of differences between samples. Statistical analysis was performed using Prism 6 (GraphPad software), and p-values ​​were indicated as *≤0.05, **≤0.01, ***≤0.001, and ****≤0.0001.

[0370] result

[0371] Primary mouse microglia were seeded at 50,000 cells / well and incubated overnight. Cells were pretreated with DMSO, compound 49, PLX3397, or compound 6 for 30 min and then subjected to CSF-1 stimulation. Cell culture supernatant from this experiment was processed in an MCP1 ELISA to determine whether stimulation / treatment affected chemokine production. As shown in Figures 5A-5C, CSF-1 stimulation induced a significant increase in the release of MCP-1 (CCL2-chemokine). The CSF-1R inhibitors PLX3397 and compound 6 significantly reduced MCP-1 production in a concentration-dependent manner (ordinary one-way ANOVA, p < 0.0001). PLX3397 (IC50) was calculated. 50 =17.4nM) and compound 6 (IC) 50 =23.2nM) IC of the two 50 Value. CSF-1-induced MCP-1 production is not robust in plates treated with compound 49 (Fig. 5B), therefore IC50 of this compound cannot be generated. 50 Values. MCP-1 secretion was assessed 24 hours later using the R&D MCP-1 ELISA kit. Each data point represents a single well, while the graph bars represent the mean and standard deviation of six wells.

[0372] Following microglial cell stimulation, immunocytochemistry was performed using Iba1, Ki67, and DAPI to determine microglial cell morphology, proliferation status, and number. InCell imaging microscopy and analysis software were used to quantify Iba1 levels within the culture. + Area and DAPI + The number of cell nuclei. Ki67 could not be quantified because aggregation during ICC cross-transfers the Iba1 antibody into the Ki67 wells. Quantitative results (Figs. 6A-6C and 7A-7C) demonstrate the effect of CSF-1 stimulation on Iba1. + Area and DAPI + Significant effect on cell number. It can be seen that CSF-1R inhibition blocks these CSF-1-induced cellular changes in a dose-dependent manner. The IC50 of PLX3397 (Iba1) was calculated. 50 =50.43 nM, and DAPI is 68.2 nM) and compound 6 (IC50 of Iba1) 50 =84.6nM, and DAPI is 248nM) IC 50 value.

[0373] In Figures 6A-6C, Iba1 was quantified after microglial cell stimulation assay. + Area. CSF-1 stimulation significantly increased Iba1. +The area was measured, and treatment with a CSF-1R inhibitor significantly eliminated this effect in a concentration-dependent manner. Nine images were taken from three different wells for each condition to quantify the microglial cell area. Data points represent the mean Iba1 area per well. + Area, and error bars represent standard deviations (n=3). Statistical significance was determined by one-way ANOVA, with p-values ​​indicating *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0374] In Figures 7A-7C, DAPI was quantified using the following microglial cell stimulation assay. + Labeled cell nuclei. CSF-1 stimulation increased the number of cells in the culture, and CSF-1R inhibitors reduced this number in a concentration-dependent manner. Nine images taken from three different wells for each condition to quantify DAPI. + Cell nucleus. Data points represent the average Iba1 in each well. + Area (from 9 images), and error bars represent standard deviations (n=3). Statistical significance was determined by one-way ANOVA, and p-values ​​are indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0375] Example 12

[0376] The effects of the deuterated CSF-1R inhibitor of this disclosure on cytokine / chemokine production after stimulation with CSF-1 or LPS were examined in wild-type primary microglia and SOD1-mutant primary microglia in the following experiments.

[0377] Primary mouse microglia - isolated in ELN 20200624-062

[0378] Group number simulation group Hole / Group deal with 1 Non-irritating 6 wells / genotype DMSO 2 CSF-1 stimulation 6 wells / genotype DMSO, compound 6 3 LPS stimulation 6 wells / genotype DMSO, compound 6

[0379] Test items:

[0380] DMSO

[0381] Compound 6 - The stock solution (10 mM) was diluted with culture medium to obtain a 100 μM working solution and microglia were treated at 50 nM, 100 nM or 200 nM.

[0382] • Recombinant mouse M-CSF (R&D Systems, catalog number 416-ML / CF, batch number ME4518091) - prepared by dissolving 50 μg in 500 μl PBS to prepare a 100 μg / mL stock solution and treating microglia with 100 ng / mL.

[0383] • Lipopolysaccharide, derived from Escherichia coli O55:B5 (Sigma, catalog number L6529-1mg, batch number 059M4103V) ​​- was prepared as a 0.2mg LPS / mL PBS stock solution and microglia were treated with 10ng / mL.

[0384] method

[0385] Treatment and stimulation of primary microglia

[0386] Primary mouse microglia were cultured at 5 x 10⁻⁶ cells per cell line. 5 Cells were suspended at a concentration of [number] cells / mL, and 100 μL of this cell suspension was added to each well of a 96-well plate. Microglial cells were allowed to stand overnight at 37°C and 5% CO2. The next day, the culture medium was removed, and the cells were treated with dimethyl sulfoxide (DMSO) or Compound 6 for 30 min or 24 h at 37°C and 5% CO2. The cells were then stimulated with 100 ng / mL recombinant mouse M-CSF for 30 min or with 10 ng / mL lipopolysaccharide for 24 h. After stimulation, the culture supernatant was removed from each well and aliquoted into two different 96-well plates for subsequent ELISA assays.

[0387] CellTiter Glo 2.0 Viability Assay

[0388] Cell viability was determined using a Promega Cell Titer Glo chemiluminescence cell viability assay. The assay reagents were first allowed to equilibrate to room temperature for 30 minutes. After removing the culture supernatant, 100 μL of fresh room temperature culture medium was added to each well. Subsequently, 100 μL of the assay reagent was added to each well. The assay plate was then shaken for two minutes and then allowed to stand for 10 minutes. 100 μL was transferred from each well to a white plate, and the luminescence was immediately read on a FlexStation3 plate reader.

[0389] Mouse MCP-1 ELISA

[0390] The culture supernatant was assayed using the Quantikine Mouse MCP-1 ELISA Kit (catalog number SMJE00B) from R&D Systems. Samples were diluted 1:10 with calibrator diluent. First, 50 μL of assay diluent was added to each well. Then, 50 μL of standard, assay control, and diluted sample were added to the wells. The plate was mixed by gently tapping the frame and then sealed with tape. The plate was incubated at room temperature for 2 hours. After incubation, the plate was washed 5 times with approximately 400 μL of wash buffer using a spray bottle. After the final wash, the plate was gently tapped on a paper towel to remove excess moisture. 100 μL of mouse MCP-1 conjugate was added to each well, covered with new tape, and incubated at room temperature for 2 hours. After incubation, the plate was washed as described above. Then, substrate solution was added to each well and incubated in the dark at room temperature for 30 minutes. After incubation, acidic stop solution was added to each well, and the plate was read at 450 nm using an ELISA plate reader.

[0391] Mouse IL-12p40 ELISA

[0392] Cell culture supernatant was measured using the Quantikine Mouse IL-12p40 ELISA Kit (catalog number MP400) from R&D Systems. Samples were diluted 1:10 with calibrator diluent. First, 50 μL of assay diluent was added to each well. Then, 50 μL of standard, assay control, and diluted sample were added to each well as a single sample. The plate was mixed by gently tapping the frame and then sealed with tape. The plate was incubated at room temperature for 2 hours. After incubation, the plate was washed 5 times with approximately 400 μL of wash buffer using a spray bottle. After the final wash, the plate was gently tapped on a paper towel to remove excess moisture. 100 μL of mouse IL-12p40 conjugate was added to each well, covered with new tape, and incubated at room temperature for 2 hours. After incubation, the plate was washed as described above. Then, substrate solution was added to each well and incubated in the dark at room temperature for 30 minutes. After incubation, an acidic stop solution was added to each well, and the plate was read at 450 nm using an ELISA plate reader.

[0393] result

[0394] Primary mouse microglia were seeded at 50,000 cells / well and incubated overnight. Cells were pretreated with DMSO or Compound 6 for 30 min or 24 h, and then stimulated with CSF-1 or LPS, respectively. Cell viability was assessed using Promega's Cell Titer Glo assay kit after 24 h. Both CSF-1 and LPS stimulation induced a slight increase in cell viability readings compared to unstimulated cells (Figs. 8A-8B and 9A-9B).

[0395] As shown in Figures 8A and 8B, CSF-1R inhibitor treatment at the assessed concentrations had no toxic effect on microglia. The deuterated CSF-1R inhibitor, compound 6, slightly reduced the CSF-1-induced increase in cell viability. The graph bars represent the mean and standard deviation of the six wells. As shown in Figures 9A-9B, CSF-1R inhibition had no detrimental effect on cell viability. The graph bars represent the mean and standard deviation of the six wells. No significant difference in cell viability was observed between wild-type and SOD1 microglia in response to CSF-1 or LPS stimulation.

[0396] Cell culture supernatant from this experiment was treated with two separate ELISAs (MCP-1 and IL12p40) to determine whether stimulation / treatment affected chemokine / cytokine production.

[0397] As shown in Figures 10A-10B, CSF-1 stimulation significantly increased the release of MCP-1 (CCL2-chemokine), and compound 6 significantly reduced MCP-1 production in a concentration-dependent manner. The bars represent the mean and standard deviation of the six wells. Ordinary one-way ANOVA was performed to determine statistical differences between groups, and p-values ​​are expressed as ***p<0.001 and ****p<0.0001.

[0398] As shown in Figures 11A-11B, LPS stimulation induced a significant increase in IL12-p40 production in mouse microglia culture. CSF-1R inhibition of compound 6 significantly reduced IL12-p40 production in a concentration-dependent manner. The bars represent the mean and standard deviation of the six wells. Ordinary one-way ANOVA was performed to determine statistical differences between groups, and p-values ​​are expressed as **p<0.01, ***p<0.001, and ****p<0.0001.

[0399] Example 13: Caco-2 permeability and efflux determination to compare compound 6 with compound 24.

[0400] Caco-2 permeability and efflux assays were performed using Caco-2 / TC7 cells in a cell-based permeability model. For permeability assays, Caco-2 / TC7 cells were seeded on Millipore Millicell 96 plates, and for efflux assays, 24-well plates were used. Plates with cells were prepared for use between 21 and 25 days of culture. Both permeability and efflux assays were performed using the TECAN automated liquid handling platform. For permeability assays, the test compound was prepared at a 20 μM test concentration in a permeability assay buffer (10 mM HEPES in HBSS buffer, pH 6.5) containing 0.5% BSA. The basolateral permeability buffer (pH 7.4) contained 5% BSA. For efflux assays, the test compound was prepared at a 1 μM test concentration in a permeability assay buffer (10 mM HEPES in HBSS buffer, pH 7.4) containing 0.5% BSA. For efflux assays, the basolateral permeability buffer (pH 7.4) contained 0.5% BSA. Permeability assays were initiated by adding 20 μM test solution to the top side of a plate containing Caco-2 / TC7 cells. In efflux assays, 1 μM test compound was added to the top compartment for top-to-base side (A to B) permeability determination. For base-to-top (B to A) permeability determination, the test compound was added to the base side. The plates were incubated at 37°C with constant shaking for 90 min. At the end of the incubation period, the obtained samples were analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). For each assay, apparent permeability (P0) was calculated from the mass spectrometry data. app ) and recovery rate. For permeability determination, P app The value is reported as a number x 10 -07 cm / s. For efflux determination, in addition to the recovery value, P is used. app (from basal side to apex) and P app Calculate the outflow rate (from the top to the base).

[0401] Permeability data:

[0402] compound <![CDATA[P app (x 10 -07 cm / s)20μM]]> Recovery (%) 20 μM 24 377.00 80％ 6 377.00 73％

[0403] External discharge data:

[0404]

[0405] CYP inhibition

[0406] The purpose of this assay is to determine the in vitro inhibitory potential of the test compound against a specific cytochrome P450 (CYP) enzyme using human liver microsomes (HLM). The test compound was diluted from pure DMSO stock solution to a final test concentration range of 10 μM–0.07 μM in 0.5% DMSO solution. The compound was co-incubated at 37°C with 0.22 mg / mL human liver microsomes (HLM), 50 mM phosphate buffer, 1.33 mM NADPH, 3.33 mM glucose-6-phosphate, 3.33 mM magnesium hexahydrate, 0.4 units / mL glucose-6-phosphate dehydrogenase, and an appropriate concentration of a single chemical probe for 10–30 minutes. After incubation, the sample was extracted, and the protein was precipitated in acetonitrile containing 0.1% formic acid. The sample was centrifuged to remove excess protein, and the IC50 was determined by LDTD / MS / MS analysis. 50 Values. Key substrate concentrations were as follows: CYP2D6 substrate - 10 μM dextromethorphan; CYP3A4 substrate - 60 μM testosterone and 2 μM midazolam. Dextromethorphan and testosterone were incubated with the test compounds individually for 30 minutes. Midazolam was incubated with the test compounds for 10 minutes. As shown in the data table below, compounds 6 and 24 showed no CYP inhibition at concentrations up to 10 μM.

[0407] data:

[0408]

[0409] Human, rat, dog, mouse and monkey hepatocytes

[0410] Two replicates of the test compound at a concentration of 1 μM were incubated with rat, human, dog, monkey, or mouse hepatocytes at a concentration of 500,000 hepatocytes / mL. Samples were removed from the incubation at the assay time point, and then analyzed by liquid chromatography and tandem mass spectrometry to determine the concentration-time process at which the test compound was depleted. Incubation was terminated using an organic solvent.

[0411]

[0412] Human cytosol and S-9 fraction

[0413] Two replicates of the test compound were incubated at a concentration of 1 μM in either 1 mg / mL human liver cytosol (with high aldehyde oxidase (AO) / xanthine oxidase (XO) activity) or 2.5 mg / mL human liver S-9 fraction (with high AO / XO activity). The concentration-time process at which the test compound was depleted was determined by removing the sample from the incubation at the assay time point and performing liquid chromatography-tandem mass spectrometry analysis. Incubation was terminated using an organic solvent.

[0414] Calibration method for aldehyde oxidase substrates

[0415] Zientek M, Jiang Y, Youdim K, Obach RS. In vitro-in vivo correlation for intrinsic clearance for drugs metabolized by human aldehyde oxidase. DrugMetabDispos. 2010; 38(8):1322-1327. doi:10.1124 / dmd.110.033555 describes a basic calibration method for AO substrates.

[0416] This method provides a benchmark tool for in vitro-in vivo correlations using the inherent clearance rates of commercially available drugs known to be metabolized by AO.

[0417] It is known that preclinical species (mice, rats, and dogs) cannot accurately predict AO metabolism due to differential expression of human AOX1 isoforms of the enzyme. Mice and rats contain all four active isoforms (AOX1, AOX2, AOX3, and AOX4), dogs lack active AOX1 enzymes, and only monkeys contain active AOX1 isoforms. Traditional analogical methods for predicting human pharmacokinetics are difficult due to the lack of preclinical species available for accurate prediction of AO substrates. Therefore, in vitro-in vivo calibration methods are used with known AO substrates that have human pharmacokinetics in clinical settings. Several of these drugs have failed in clinical trials due to poor PK properties. A ranking calibration method can be developed by using zaleplon (an AO substrate with lower clearance) as a benchmark compound, which has acceptable human pharmacokinetic properties.

[0418] These available known AO substrates were analyzed using three in vitro systems (combined human liver cytosol, liver S-9 fraction, and human hepatocytes isolated from livers perfused with HTK medium). The amplified unbound intrinsic clearance of the test compound / novel chemical entity was calculated and compared to the in vivo unbound intrinsic clearance of known AO substrates. Compounds with predicted AO-mediated amplified in vitro unbound intrinsic clearance lower than that of zaleplon showed acceptable in vivo AO clearance.

[0419] Raw data

[0420]

[0421] Enlarged data

[0422]

[0423] Example 14: In the absence and presence of the aldehyde oxidase inhibitor hydralazine, in human cryopreserved liver Metabolic characteristics of compound 24 in cells

[0424] The metabolic characterization of compound 24 was investigated in vitro in human cryopreserved hepatocytes in both the absence and presence of the aldehyde oxidase (AO) inhibitor hydralazine. After incubating compound 24 in human cryopreserved hepatocytes for 2 hours, a total of nine metabolites were identified and quantified by LC-MS.

[0425] After incubation for 2 hours in human cryopreserved hepatocytes, 72.2% of the parental compounds remained unchanged, a figure calculated based on the total integrated MS peak area of ​​compound 24 and its identified metabolites. H10 was the most abundant metabolite detected, accounting for 20.3% of the total integrated MS peak area of ​​compound 24 and its identified metabolites. Metabolite H4a accounted for 4.5% of the total integrated MS peak area of ​​compound 24 and its identified metabolites. Each other identified metabolite accounted for <2% of the total integrated MS peak area of ​​compound 24 and its identified metabolites.

[0426] After incubation for 2 hours in human cryopreserved hepatocytes in the presence of hydralazine, compound 24 remained unchanged, accounting for 90.9% of the total integrated MS peak area of ​​compound 24 and its identified metabolites. The formation of the hydroxylated metabolite H10 was significantly inhibited, accounting for 1.6% of the total integrated MS peak area of ​​compound 24 and its identified metabolites. H4a was the major metabolite, accounting for 5.4% of the total integrated MS peak area of ​​compound 24 and its identified metabolites. Each of the other identified metabolites accounted for <1% of the total integrated MS peak area of ​​compound 24 and its identified metabolites.

[0427] H10 is proposed to originate from the hydroxylation at the 2C position of the 3H-imidazolium[4,5-b]pyridine moiety of compound 24. H4a is proposed to originate from the hydration of the 3H-imidazolium[4,5-b]pyridine moiety of compound 24 and the glucuronidation at the 2C position of the 3H-imidazolium[4,5-b]pyridine moiety. H11a is proposed to originate from the glucuronidation of the 3H-imidazolium[4,5-b]pyridine moiety of H10. H7 is proposed to originate from the O-demethylation of the 2-methoxy-pyridine moiety of compound 24. H6 is proposed to be a glucuronidated conjugate of compound 24.

[0428] The proposed major metabolic pathways include AO-mediated hydroxylation followed by glucuronidation and combinations of hydration and glucuronidation. Other observed metabolic pathways include non-AO-mediated hydroxylation followed by glucuronidation, direct glucuronidation, demethylation followed by glucuronidation, and combinations of oxidative deamination followed by oxidation.

[0429] Compound 24 was prepared according to the procedure outlined in Examples 1-92 of WO 2017 / 015267.

[0430] Incubation conditions

[0431] The following is a general experimental design containing hydralazine:

[0432] Reagents or parameters Final concentration / conditions Compound 24 1μM Cryopreserved monkey liver cells <![CDATA[0.5x 10 6 [cells / mL]]> Cryopreserved human liver cells <![CDATA[1x 10 6 [cells / mL]]> Incubation time 0, 15, 30, 60, 90 and 120 min Hydralazine HCl 10μM Incubation <![CDATA[At 37°C in a CO2 incubator]]> Incubation medium KHB buffer Total incubation volume 0.5mL

[0433] After sampling, the remaining samples from the triplicate incubation were combined and processed for metabolite identification studies.

[0434] Sample preparation

[0435] Add an equal volume of ice-cold acetonitrile (v / v) to each sample and then vortex to mix. After centrifugation at approximately 13,000 rpm for 10 min, concentrate the supernatant at 35 °C under a nitrogen stream until approximately 0.1–0.2 mL of extract remains. Before analysis, centrifuge the remaining extract at approximately 13,000 rpm for 15 min. Inject the supernatant into LC / UV / MS for analysis.

[0436] Instrument conditions

[0437] Metabolite identification was performed on UPLC (Thermo Vanquish) coupled with UV (Thermo Vanquish) and mass spectrometry (MS) detection (Thermo Orbitrap ID-X).

[0438]

[0439] Data Evaluation

[0440] Due to the low sample concentration, the mass peak area was used for metabolite characterization. Assuming that the mass spectrometric reactions of metabolites or parental compounds at equivalent molar concentrations are equal, the percentage of metabolites or unchanged parental compounds was calculated based on the total integrated MS peak area of ​​compound 24 and its identified metabolites. Table 1 below reports metabolites whose peak area represents 0.1% or greater of the total integrated MS peak area.

[0441] Based on its accurate quality (tolerance ≤ 5 ppm), mass fragmentation pattern, and comparison with other in vitro studies, metabolites are characterized.

[0442] Table 1: Metabolite characteristics of compound 24 after incubation for 2 hours in frozen human hepatocytes, with or without hydralazine.

[0443]

[0444] The metabolic characterization of compound 24 was investigated in vitro in human cryopreserved hepatocytes in both the absence and presence of the AO inhibitor hydralazine. After incubating compound 24 in human cryopreserved hepatocytes for 2 hours, a total of nine metabolites were identified and quantified by LC-MS (see Figures 12A-12B).

[0445] After incubation for 2 hours in human cryopreserved hepatocytes, 72.2% of the parental compounds remained unchanged, a figure calculated based on the total integrated MS peak area of ​​compound 24 and its identified metabolites. H10 was the most abundant metabolite detected, accounting for 20.3% of the total integrated MS peak area of ​​compound 24 and its identified metabolites. Metabolite H4a accounted for 4.5% of the total integrated MS peak area of ​​compound 24 and its identified metabolites. Each other identified metabolite accounted for <2% of the total integrated MS peak area of ​​compound 24 and its identified metabolites.

[0446] After incubation for 2 hours in human cryopreserved hepatocytes in the presence of the AO inhibitor hydralazine, compound 24 remained unchanged, accounting for 90.9% of the total integrated MS peak area of ​​compound 24 and its identified metabolites. The formation of the hydroxylated metabolite H10 was significantly inhibited, accounting for 1.6% of the total integrated MS peak area of ​​compound 24 and its identified metabolites. H4a was the major metabolite, accounting for 5.4% of the total integrated MS peak area of ​​compound 24 and its identified metabolites. Each of the other identified metabolites accounted for <1% of the total integrated MS peak area of ​​compound 24 and its identified metabolites.

[0447] H10 is proposed to originate from the hydroxylation at the 2C position of the 3H-imidazolium[4,5-b]pyridine moiety of compound 24. H4a is proposed to originate from the hydration of the 3H-imidazolium[4,5-b]pyridine moiety of compound 24 and the glucuronidation at the 2C position of the 3H-imidazolium[4,5-b]pyridine moiety. H11a is proposed to originate from the glucuronidation of the 3H-imidazolium[4,5-b]pyridine moiety of H10. H7 is proposed to originate from the O-demethylation of the 2-methoxy-pyridine moiety of compound 24. H6 is proposed to be a glucuronidated conjugate of compound 24.

[0448] The proposed major metabolic pathways (Figure 13) include AO-mediated hydroxylation followed by glucuronidation and a combination of hydration and glucuronidation. Other observed metabolic pathways include non-AO-mediated hydroxylation followed by glucuronidation, direct glucuronidation, demethylation followed by glucuronidation, and a combination of oxidative deamination followed by oxidation.

[0449] Example 15: In the absence and presence of the aldehyde oxidase inhibitor hydralazine, in human cryopreserved liver Metabolic characteristics of compound 6 in cells

[0450] The metabolic characterization of compound 6 was investigated in vitro in human cryopreserved hepatocytes with and without the aldehyde oxidase (AO) inhibitor hydralazine. After incubating compound 6 in human cryopreserved hepatocytes for 2 hours, a total of nine metabolites were identified and quantified by LC-MS.

[0451] After incubation for 2 hours in human cryopreserved hepatocytes, 87.1% of the parental compound remained unchanged, a figure calculated based on the total integrated MS peak area of ​​compound 6 and its identified metabolites. H10 and H4a were the major metabolites detected, accounting for 6.6% and 4.6% of the total integrated MS peak area of ​​compound 6 and its identified metabolites, respectively. Each other identified metabolite accounted for <1% of the total integrated MS peak area of ​​compound 6 and its identified metabolites.

[0452] After incubation for 2 hours in human cryopreserved hepatocytes in the presence of the AO inhibitor hydralazine, compound 6 remained unchanged, accounting for 92.0% of the total integrated MS peak area of ​​compound 6 and its identified metabolites. The formation of the hydroxylated metabolite H10 was significantly inhibited, accounting for 0.9% of the total integrated MS peak area of ​​compound 6 and its identified metabolites. H4a was the major metabolite, accounting for 5.7% of the total integrated MS peak area of ​​compound 6 and its identified metabolites. Each of the other identified metabolites accounted for <1% of the total integrated MS peak area of ​​compound 6 and its identified metabolites.

[0453] H10 is proposed to originate from the hydroxylation at the 2C position of the 3H-imidazolium[4,5-b]pyridine moiety of compound 6. H4a is proposed to originate from the hydration of the 3H-imidazolium[4,5-b]pyridine moiety of compound 6 and the glucuronidation at the 2C position of the 3H-imidazolium[4,5-b]pyridine moiety. H11a is proposed to originate from the glucuronidation of the 3H-imidazolium[4,5-b]pyridine moiety of H10. H7 is proposed to originate from the O-demethylation of the 2-methoxy-pyridine moiety of compound 6. H6 is proposed to be a glucuronidated conjugate of compound 6.

[0454] The proposed major metabolic pathways include AO-mediated hydroxylation followed by glucuronidation and combinations of hydration and glucuronidation. Other observed metabolic pathways include non-AO-mediated hydroxylation followed by glucuronidation, direct glucuronidation, demethylation followed by glucuronidation, and combinations of oxidative deamination followed by oxidation.

[0455] Incubation conditions

[0456] The following is a general experimental design containing hydralazine:

[0457] Reagents or parameters Final concentration / conditions Compound 6 1μM Cryopreserved human liver cells <![CDATA[1x 10 6 [cells / mL]]> Incubation time 0, 15, 30, 60, 90 and 120 min Hydralazine HCl 10μM Incubation <![CDATA[in a CO2 incubator at 37°C]]> Incubation medium KHB buffer Total incubation volume 0.5mL

[0458] After sampling, the remaining samples from the triplicate incubation were combined and processed for metabolite identification studies.

[0459] Sample preparation

[0460] Add an equal volume of ice-cold acetonitrile (v / v) to each sample and vortex to mix. After centrifugation at approximately 13,000 rpm for 10 min, concentrate the supernatant at 35 °C under a nitrogen stream until approximately 0.1–0.2 mL of extract remains. Before analysis, centrifuge the remaining extract at approximately 13,000 rpm for 15 min. Inject the supernatant into LC / UV / MS for analysis.

[0461] Instrument conditions

[0462] Metabolite identification was performed on UPLC (Thermo Vanquish) coupled with UV (Thermo Vanquish) and mass spectrometry (MS) detection (Thermo Orbitrap ID-X).

[0463]

[0464] Data Evaluation

[0465] Due to the low sample concentration, the mass peak area was used for metabolite characterization. Assuming that the mass spectrometric reactions of metabolites or parental compounds at equivalent molar concentrations are equal, the percentage of metabolites or unchanged parental compounds was calculated based on the total integrated MS peak area of ​​compound 6 and its identified metabolites. Table 2 below reports metabolites whose peak area represents 0.1% or greater of the total integrated MS peak area.

[0466] Based on its accurate quality (tolerance ≤ 5 ppm), mass fragmentation pattern, and comparison with other in vitro studies, metabolites are characterized.

[0467] result

[0468] Table 2: Metabolite characteristics of compound 6 after 2 hours of incubation in frozen human hepatocytes, with or without hydralazine.

[0469]

[0470]

[0471] The metabolic characterization of compound 6 was investigated in vitro in human cryopreserved hepatocytes in both the absence and presence of the AO inhibitor hydralazine. After incubating compound 6 in human cryopreserved hepatocytes for 2 hours, a total of nine metabolites were identified and quantified by LC-MS (see Figures 14A-14B).

[0472] After incubation for 2 hours in human cryopreserved hepatocytes, 87.1% of the parental compound remained unchanged, a figure calculated based on the total integrated MS peak area of ​​compound 6 and its identified metabolites. H10 and H4a were the major metabolites detected, accounting for 6.6% and 4.6% of the total integrated MS peak area of ​​compound 6 and its identified metabolites, respectively. Each other identified metabolite accounted for <1% of the total integrated MS peak area of ​​compound 6 and its identified metabolites.

[0473] After incubation for 2 hours in human cryopreserved hepatocytes in the presence of the AO inhibitor hydralazine, compound 6 remained unchanged, accounting for 92.0% of the total integrated MS peak area of ​​compound 6 and its identified metabolites. The formation of the hydroxylated metabolite H10 was significantly inhibited, accounting for 0.9% of the total integrated MS peak area of ​​compound 6 and its identified metabolites. H4a was the major metabolite, accounting for 5.7% of the total integrated MS peak area of ​​compound 6 and its identified metabolites. Each of the other identified metabolites accounted for <1% of the total integrated MS peak area of ​​compound 6 and its identified metabolites.

[0474] H10 is proposed to originate from hydroxylation at the 2C position of the 3H-imidazolium[4,5-b]pyridine moiety of compound 6. H4a is proposed to originate from hydration at the 3H-imidazolium[4,5-b]pyridine moiety of compound 6 and glucuronidation at the 2C position of the 3H-imidazolium[4,5-b]pyridine moiety. H11a is proposed to originate from glucuronidation of the 3H-imidazolium[4,5-b]pyridine moiety of H10. H7 is proposed to originate from O-demethylation of the 2-methoxy-pyridine moiety of compound 6. H6 is proposed to be a glucuronidated conjugate of compound 6. The proposed major metabolic pathways (Figure 15) include AO-mediated hydroxylation followed by glucuronidation and combinations of hydration and glucuronidation. Other observed metabolic pathways include non-AO-mediated hydroxylation followed by glucuronidation, direct glucuronidation, demethylation followed by glucuronidation, and combinations of oxidative deamination followed by oxidation.

[0475] Example 16: in vitro Microglial cell stimulation study

[0476] To determine the effect of CSF1 stimulation on the production of cytokines / chemokines in human microglia, the following experiments were conducted.

[0477] Test items:

[0478] DMSO

[0479] Compound 6 - Dilute the stock solution (10 mM) with culture medium to obtain a 100 μM working solution and treat microglia at 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM or 200 nM.

[0480] Compound 24 - Dilute the stock solution (10 mM) with culture medium to obtain a 100 μM working solution and treat microglia at 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM or 200 nM.

[0481] • Recombinant human CSF1 (R&D Systems, catalog number 216-ML / CF, batch number MVN1218101) - Prepare a 100 μg / mL stock solution by dissolving 50 μg in 500 μl PBS and treating microglia with 100 ng / mL.

[0482] method

[0483] iCell microglia processing and stimulation

[0484] iCell microglia (microglia derived from human iPSCs) were obtained from FujiFilm (catalog number C1110, batch number 105458). These cells were thawed and maintained in complete iCell microglia culture medium according to the manufacturer's instructions. For this assay, iCell microglia were cultured at 5 x 10⁻⁶ cells / mL. 5 The cells were suspended at a concentration of 100 μL / mL, and 100 μL of this cell suspension was added to the inner 60 wells of a 96-well plate. The iCell microglia were allowed to stand overnight at 37°C and 5% CO2. The following evening, the culture medium was removed and replaced with Neurobasal medium (Gibco, catalog 21103049) containing B27 additive (Gibco, catalog 17504044). Again, the microglia were allowed to stand overnight at 37°C and 5% CO2. The following evening, the cells were treated with dimethyl sulfoxide (DMSO), compound 6, or compound 24 for 30 minutes at 37°C and 5% CO2. The cells were then stimulated with 100 ng / mL recombinant human CSF1 for 24 hours. After stimulation, the culture supernatant was removed from each well and aliquoted into two different 96-well plates for subsequent ELISA assays.

[0485] CellTiter Glo 2.0 Viability Assay

[0486] Cell viability was determined using the CellTiter Glo 2.0 chemiluminescence cell viability assay (Promega, catalog number G9242). The assay reagents were first allowed to equilibrate to room temperature for 30 minutes. After removing the culture supernatant, 100 μL of fresh room temperature culture medium was added to each well. Then, 100 μL of the assay reagent was added to each well. The assay plate was then shaken for two minutes and then allowed to stand for 10 minutes. 100 μL was transferred from each well to a white plate, and the chemiluminescence was immediately read on a FlexStation3 multimode microplate reader (Molecular Devices, catalog number Flex3) with SoftMax Pro software.

[0487] Mouse MCP-1 ELISA

[0488] The culture supernatant was analyzed using the Quantikine Human MCP-1 ELISA Kit (R&D Systems, catalog SCP00). The sample was diluted 1:10 with calibrator diluent. Then, 200 μL of standard and diluted sample were added to each well. The plate was mixed by gently tapping the frame and sealed with tape. The plate was incubated at room temperature for 2 hours. After incubation, the plate was washed 5 times with approximately 400 μL of wash buffer using a spray bottle. After the final wash, the plate was gently tapped on a paper towel to remove excess moisture. 200 μL of human MCP-1 conjugate was added to each well, covered with a new strip of tape, and incubated at room temperature for 2 hours. After incubation, the plate was washed as described above. Then, 200 μL of substrate solution was added to each well and incubated in the dark at room temperature for 30 minutes. After incubation, 50 μL of acid-terminating solution was added to each well, and the plate was read at 450 nm using a FlexStation3 multimode microplate reader (MolecularDevices, catalog number Flex3) with SoftMax Pro software.

[0489] result

[0490] iCell microglia (microglia derived from human iPSCs) were seeded at 50,000 cells / well and incubated overnight. The culture medium containing growth factors was removed, and the cells were then allowed to incubate again overnight. Next, the cells were pretreated with DMSO or Compound 6 or Compound 24 for 30 minutes and then stimulated with CSF1. Cell viability was assessed using Promega's Cell Titer Glo assay kit. The cell culture supernatant from this experiment was processed in an MCP1 ELISA to determine whether the stimulation / treatment affected chemokine production.

[0491] In this experiment, compound 6 did not affect the viability of human microglia at the evaluated concentration (Figure 16). Figure 16 depicts cell viability after treatment with the CSF1R inhibitor and CSF1 stimulation as described above. iCell human microglia were seeded at 50,000 cells / well and incubated overnight after growth factor starvation. Cells were pretreated with DMSO or RA16100017 for 30 min and then subjected to CSF1 stimulation. Cell viability was assessed using Promega's Cell Titer Glo 2.0 assay kit 24 hours later. CSF1 stimulation induced an increase in cell viability, while the CSF1R inhibitor had no effect on this effect. Each data point represents a single well, and the graph bars represent the mean and standard deviation of six wells.

[0492] As shown in Figure 17, CSF1 stimulation significantly increased the release of MCP-1 (CCL2-chemokine). Figure 17 illustrates the blocking effect of compound 6 on CSF1-induced MCP-1 production in this experiment. iCell human microglia were seeded at 50,000 cells / well and incubated overnight after growth factor starvation. Cells were pretreated with DMSO or RA16100017 for 30 min and then subjected to CSF1 stimulation. MCP-1 secretion was assessed 24 hours later using the R&D MCP1 Elisa kit. CSF1R inhibitor treatment significantly reduced MCP1 production in a concentration-dependent manner (ordinary one-way ANOVA). Each data point represents a single well, and the graph bars represent the mean and standard deviation of six wells.

[0493] Compound 6 significantly reduced MCP1 production in a concentration-dependent manner (ordinary one-way ANOVA, p < 0.0001). CSF1 stimulation induced a significant increase in MCP1 production, and CSF1R inhibition of compound 6 eliminated this effect in a concentration-dependent manner. Figure 18 compares the MCP1 production of compound 6 with that of compound 24, showing similar effects on MCP1.

[0494] Example 17: MOG-EAE

[0495] Experimental autoimmune encephalomyelitis (EAE) is primarily used as a nonclinical model of autoimmune inflammatory diseases of the central nervous system and is similar to many aspects of human multiple sclerosis. Myelin oligodendrocyte glycoprotein (MOG)-induced EAE models are ideal for exploring this immune-mediated mechanism of neuroinflammation and demyelination. In the following experiments, compounds 24 and 6 were tested in a mouse c57BL / 6 EAE model to evaluate their potential efficacy in reducing disease scores.

[0496] Species: 8-9 week old females C57BL / 6J

[0497]

[0498] Test items:

[0499] MOG 35-55 Peptides (New England Peptides, batch number BU01787) - 250 μg / mouse in 4 mg / mL complete Freund's adjuvant (CFA; Chondrex Inc, catalog number 7009, batch number 190446).

[0500] Bordetella pertussis toxin (PTX, Sigma, catalog number P7208-50UG, batch number MKCL 1350) - 280 ng / mouse in 200 μL PBS

[0501] CSF-1R inhibitor - 15 mg / kg

[0502] Medium - 0.5% methylcellulose / 0.2% Tween-80

[0503] deliver:

[0504] MOG peptide and CFA: Subcutaneous injection into two sites on the posterior ventral side (100 μL / site).

[0505] Bordetella pertussis toxin: 280 ng / mouse was administered intravenously in 200 μL PBS on days 0 and 2.

[0506] CSF1R inhibitors: administered orally via gavage every 12 hours.

[0507] Mediator: Oral gavage every 12 hours

[0508] Time point:

[0509] Day 0 - MOG peptides in CFA and PTX will be administered to groups 1-3.

[0510] Day 2 - PTX was administered intravenously to groups 1-3.

[0511] Day 9 - Begin scoring the mice daily.

[0512] Days 11-14 - When the score reaches 1, mice are randomly assigned to groups and treatment begins.

[0513] Approximately 7 days after treatment (days 18-21), the mice were perfused and euthanized.

[0514] Mice were clinically assessed daily to assess signs of paralysis, and were weighed intermittently to record weight loss. At the end of the study, half of the brain was fixed for histological examination, and brain / liver / plasma were collected for exposure. Spinal cord and whole blood were collected for flow cytometry. Additional plasma aliquots were preserved for subsequent analysis.

[0515] method:

[0516] EAE induction and scoring

[0517] Female C57BL / 6J mice were used in MOG with complete Freund's adjuvant (CFA). 35-55 Immunization was performed using a peptide emulsion (250 μg / mouse). The emulsion was delivered subcutaneously at a volume of 100 μL per injection site to the posterior ventral region twice. Pertussis botulinum toxin (PTX) was administered via tail vein injection on days 0 and 2 at a dose of 280 ng / animal in 200 μL of PBS. Following EAE induction, paralysis symptoms in mice were monitored daily, and their clinical presentation was scored using a progression scoring system (score 0: no disease; score 1: drooping tail; score 2: hind limb weakness; score 3: hind limb paralysis; score 4: forelimb weakness or partial paralysis; score 5: death).

[0518] Animals were included in the study once they achieved a disease score of 1. Each day, animals achieving a score of 1 for the first time were evenly distributed among the treatment groups, and treatment began that evening. The mediators and compounds were color-coded so that those scoring the study were unaware of the treatment groups. Animals were treated for seven days. One hour after the last dose (out of 14 doses over 7 days), the animals were anesthetized, and blood was collected via retroorbital sampling into EDTA tubes. The animals were then perfused with ice-cold PBS, and appropriate tissue was collected for the study endpoint.

[0519] result

[0520] The proposed scheme aims to use higher concentrations of MOG. 35-55 The EAE model was induced by CFA emulsion. EAE mice with a score of 1 or higher were randomly assigned to three different treatment groups: the mediator, compound 24 (15 mg / kg), or compound 6 (15 mg / kg). The mean disease score in this study (Figure 19) demonstrates the standard disease course of the MOG35-55-induced C57BL / 6 EAE model of multiple sclerosis. Data points and error bars represent the group mean and the standard error of the mean, respectively. As shown in Figure 19, both CSF1R inhibitors significantly improved the mean disease score. However, compared to the non-deuterated compound 24, the deuterated CSF1R inhibitor compound 6 surprisingly improved paralysis symptoms to a greater extent.

Claims

1. A compound of formula (I'): (I’) and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts, wherein: The dashed lines represent optional double bonds; A is selected from H and D; X 3’ It is CR 3’ , where R 3’ Selected from H and D; X 4’ It is CR 4’ , where R 4’ It is H; X 5’ It is CR 5’ , where R 5’ It is H; T 1 and T 3 It is CH; T 2 It is CR 10 ;where R 10 It is (C1-C) 10 )alkoxy-; Y 1 It is O; R 1 and R 2 Each is independently selected from H and D; R 5 It is H; and R 6 It is R 14 -(C2-C9) heteroaryl; wherein R 14 It is (C1-C) 10 )alkoxy-; Z 1 It is (C1-C) 10 )alkyl; and Y 2 It is O; Among them, A and R 3’ At least one of them is D.

2. A compound, said compound being selected from: And / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts.

3. A compound selected from 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts.

4. The compound according to any one of claims 1 to 3, wherein the compound is 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d.

5. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound according to any one of claims 1 to 4 and / or a stereoisomer, optical isomer, racemic and diastereomer mixture and / or a pharmaceutically acceptable salt thereof.

6. Use of the compound and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts, or the pharmaceutical composition according to any one of claims 1 to 4, in the preparation of a medicament for treating an immune-mediated disease in a subject of need.

7. Use of the compound and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts, or the pharmaceutical composition of claim 5, in the preparation of a medicament for treating multiple sclerosis in a subject of need.

8. Use of the compound and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts, or the pharmaceutical composition according to any one of claims 1 to 4, in the preparation of a medicament for treating lupus nephritis in a subject of need.

9. Use of the compound according to any one of claims 1 to 4 and / or its stereoisomers, optical isomers, racemic and diastereomer mixtures and / or pharmaceutically acceptable salts, or the pharmaceutical composition according to claim 5, in the preparation of a medicament for treating a neurological disease in a subject of need.

10. The use according to claim 9, wherein the neurological disease is ALS.

11. The use according to claim 9, wherein the neurological disease is PSP.

12. The use according to claim 9, wherein the neurological disease is MSA.

Images