Aminopyrimidinyl derivatives for the treatment of Parkinson's disease

Abstract

Disclosed are heterobifunctional compounds that induce the degradation of leucine-rich repeat kinase 2 (LRRK2) and PDE6D. These compounds fit into one end of LRRK2 and bind to a ubiquitin E3 ligase (e.g., cereblon, Von Hippel-Lindau, or Cellular Inhibitor of Apoptosis (cIAP)) at the other end, thereby bringing LRRK2 very close to the E3 ligase and enabling ubiquitination and degradation of the LRRK2 protein. Also disclosed are pharmaceutical compositions containing the heterobifunctional compounds and methods of using the compounds for treating diseases and disorders associated with LRRK2 and PDE6D.

Description

Technical Field

[0001] Disclosed herein is a novel small molecule chimeric proteolysis targeting chimera (PROTAC) that targets and induces rapid and selective degradation of leucine-rich repeat kinase 2 (LRRK2) in the nanomolar concentration range in polyploid cells.

Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disorder, currently affecting approximately 10 million people worldwide. Due to the rapidly increasing elderly population, this number is predicted to reach 17.5 million by 2040 (Dorsey et al., J Parkinsons Dis. 2018;8(s1):S3-S8.), posing a significant public health problem and economic burden (Feigin et al., Lancet Neurol. 2019 May;18(5):459-480.). So far, all attempts to slow the progression of PD have failed, and effective treatment remains a very important unmet medical need.

[0003] Leucine-rich repeat kinase 2 (LRRK2), encoded by the LRRK2 gene, is a large (286 kDa) multi-domain protein. Armadillo repeat motifs (ARM), ankyrin repeat domains (ANK), leucine-rich repeat domains and WD40 repeat domains are involved in protein-protein interactions; the Ras of complex proteins as a GTPase (ROC) and C-terminal ROC (COR) tandem domain function; the kinase domain (the most studied domain of LRRK2) is involved in autophosphorylation at Ser-1292 (Sheng et al., Sci Transl Med. 2012 Dec 12;4(164):164ra161.) and phosphorylation of the base of Rab GTPases involved in vesicle transport (Steger et al. eLife 2016; 5:e12813). LRRK2 has been found to play a role in both sporadic PD and familial PD (Tolosa et al., Nat. Rev. Neurol. 2020; 16(2):97-107.). Pathogenic mutations in the RocCOR GTPase domain (R1441C / G / H, N1437H, Y1699C) and kinase domain (G2019S, I2020T) result in changes in GTPase and / or kinase activity, and lead to functional changes in vesicle transport, cytoskeletal dynamics, autophagy, lysosomal degradation, neurotransmission, mitochondrial function and many other pathogenic features associated with PD (Tolosa et al., Nat. Rev. Neurol. 2020; 16(2):97-107.).Therefore, targeting the GTPase and kinase activities of LRRK2 is considered a PD treatment method that modifies the underlying pathology by a number of previously described LRRK2 kinase inhibitors, including LRRK2-IN-1 (Deng et al., Nat. Chem. Biol. 2011; 7(4):203-5.), HG-10-102-01 (Choi et al., ACS Med Chem Lett. 2012; 3(8):658-662.), MLi-2 (Fell et al., J. Pharmacol. Exp. Ther. 2015; 355(3):397-409.), PF-06447475 (Henderson et al., J. Med. Chem. 2015; 58(1):419-32.), and the first two LRRK2 kinase inhibitors in clinical trials, DNL201 and DNL151 (Tolosa et al., Nat. Rev. Neurol. 2020; 16(2):97-107.). However, all of these LRRK2 kinase inhibitors are ATP-competitive type 1 kinase inhibitors that preferably promote LRRK2 mislocalization and microtubule binding by binding to the closed conformation of LRRK2 (Deniston et al., Nature 2020; 588(7837):344-349; Watanabe et al. Cell 2020; 182(6):1508-1518.e16.), causing unwanted effects on vesicular transport and on-target side effects observed in the lung and kidney (Andersen et al. Toxicology. 2018; 395:15-22; Baptista et al. Sci. Transl. Med. 2020 Apr 22;12(540):eaav0820.).

[0004] Therefore, alternative LRRK2 targeting strategies, such as, for example, G2019S LRRK2 selective inhibitors (Garofalo et al., J. Med. Chem. 2020; 63(23):14821-14839; Leśniak et al., Eur. J. Med. Chem. 2022; 229:114080.), LRRK2 dimerization inhibitors (Helton et al., ACS Chem. Biol. 2021; 16(11):2326-2338.), GTPase inhibitors, antisense oligonucleotides, type 2 LRRK2 kinase inhibitors (Tasegian et al. Biochem J. 2021; 478(19):3555-3573.), and chimeric LRRK2 target proteolysis compounds (PROTACs) (Konstantinidou et al., ChemMedChem 2021; 16(6):959-965.) (WO2020081682A1, WO2021194878A1, and WO2021194879A1), have been proposed and investigated. However, to date, no potent, rapid, and selective LRRK2 degrading compounds have been reported.

[0005] Induced target proteolysis is a paradigm-shifting drug discovery approach. Heterobifunctional degrading compounds (also known as PROTACs) induce the degradation of target proteins by recruiting an E3 ubiquitin ligase near the target protein, and result in ubiquitination and subsequent degradation of the target protein (Schapira et al. Nat. Rev. Drug Discov. 2019; 18(12):949-963.). Previous efforts towards the development of LRRK2 PROTACs have led to three patents (WO2020081682A1, WO2021194878A1, and WO2021194879A1) and one publication (Konstantinidou et al., ChemMedChem 2021; 16(6):959-965.). However, all of these reported compounds are cereblon-based and show not-so-large LRRK2 degradation and / or are slow degrading compounds.

[0006] In particular, for the treatment of Parkinson's disease and related disorders, there is a need to identify faster, more potent, orally bioavailable, and / or blood-brain barrier penetrating LRRK2 PROTAC degrader compounds that target the GTPase and kinase activities of LRRK2. SUMMARY OF THE INVENTION

[0007] In a first aspect, the following formula (I): X-L1-L2-Y Formula (I) {wherein, X is the following:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0008] In the compound of formula (I), X is one of the following:

Chemical formula

[0009] In the compound of formula (I), L1 is one of the following:

Chemical formula

[0010] In some preferred embodiments, L1 is

Chemical formula

Chemical formula

[0011] In the compound of formula (I), L2 is one of the following:

Chemical formula

Chemical formula

Chemical formula

[0012] In some preferred embodiments, L2 is one of the following: [Chemical formula] (wherein m is 3, 4, and 5 (in particular, wherein m is 3)); [Chemical formula] (wherein n is independently selected from 1, 2, 3, and 4 (in particular, wherein n is 1 or 2)); [Chemical formula] It may be selected from.

[0013] In another preferred embodiment, L2 is the following: [Chemical formula] It may be selected from.

[0014] In an embodiment of the compound of formula (I), Y is the following: [Chemical formula] It may be selected from.

[0015] In some preferred embodiments of the compound of formula (I), Y is the following: [Chemical formula] It may be selected from.

[0016] In some preferred embodiments of the compound of formula (I), Y is the following: [Chemical formula] It may be selected from.

[0017] In some preferred embodiments of the compound of formula (I), Y is the following: [Chemical formula] may be selected from

[0018] In some preferred embodiments, Y is one of the following:

Chemical formula

[0019] In particularly preferred embodiments, Y is one of the following:

Chemical formula

[0020] In the most preferred embodiments, Y is one of the following:

Chemical formula

[0021] Preferred compounds of formula (I) are the following:

Chemical formula

[0022]

Chemical formula

[0023]

Chemical formula

[0024]

Chemical formula

[0025]

Chemical formula

[0026]

Chemical formula

[0027]

Chem.

[0028]

Chem.

[0029] Preferred compounds of formula (I) are the following:

Chem.

[0030]

Chem.

[0031]

Chem.

[0032]

Chem.

[0033]

Chem.

[0034]

Chem.

[0035]

Chem.

[0036] [Chemistry] Alternatively, it may be selected from its pharmaceutically acceptable salts, enantiomers, stereoisomers, hydrates, solvates, or polymorphs.

[0037] Some preferred compounds of formula (I) are the following: [Chemistry] Alternatively, it may also be selected from its pharmaceutically acceptable salts, enantiomers, stereoisomers, hydrates, solvates, or polymorphs.

[0038] Also, some preferred compounds of formula (I) are the following: [Chemistry] Alternatively, it may also be selected from its pharmaceutically acceptable salts, enantiomers, stereoisomers, hydrates, solvates, or polymorphs.

[0039] Also, the compounds of formula (I) are the following: [Chemistry] Alternatively, it may also be selected from its pharmaceutically acceptable salts, enantiomers, stereoisomers, hydrates, solvates, or polymorphs.

[0040] Also, the preferred compounds of formula (I) are the following: [Chemistry] Alternatively, it may also be selected from its pharmaceutically acceptable salts, enantiomers, stereoisomers, hydrates, solvates, or polymorphs.

[0041] Also, the compounds of formula (I) are the following: [Chemistry] Alternatively, it may be selected from its pharmaceutically acceptable salts, enantiomers, stereoisomers, hydrates, solvates, or polymorphs.

[0042] Some other preferred compounds of formula (I) are the following:

Chem.

[0043] Also, another particularly preferred compound of formula (I) is the following:

Chem.

[0044] Some other preferred compounds of formula (I) are the following:

Chem.

[0045] Some other preferred compounds of formula (I) are the following:

Chem.

[0046] Particularly preferred compounds of formula (I) are the following:

Chem.

[0047] In other words, the compounds of the preferred formula (I) can be selected from the following: (2S,4R)-N-(2-(3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamide)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (SD13); (2S,4R)-N-(2-(2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamide)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (SD12); (2S,4R)-N-(2-((14-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamide)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (SD79);

[0048] (2S,4R)-1-((R)-3-((3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (SD82); (2S,4R)-1-((R)-3-((2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (SD75); (2S,4R)-1-((R)-1-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-17-(1-fluorocyclopropane-1-carboxamido)-16,16-dimethyl-3,6,9,12-tetraoxa-15-thiaoctadecane-18-yl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (SD74); (2S,4R)-1-((R)-3-((4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (SD100);

[0049] 5-(4-(3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (SD11); 5-(4-(2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (SD10); (S)-N-((S)-2-((S)-2-(4-(3-(2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)benzoyl)thiazol-2-yl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)-2-(methylamino)propenamide HCl salt (SD113); (S)-N-((S)-2-((S)-2-(4-(3-((14-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl)oxy)benzoyl)thiazol-2-yl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)-2-(methylamino)propenamide (SD112); (S)-N-((S)-2-((S)-2-(4-(3-(3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propoxy)benzoyl)thiazol-2-yl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)-2-(methylamino)propenamide (SD114);

[0050] (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamide)-3-((2-(2-(2-(4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl)thio)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01078B); (2S,4R)-1-((R)-2-(1-Fluorocyclopropane-1-carboxamide)-3-((3-(4-(3-Methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazin-1-yl)propyl)thio)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01072); (2S,4R)-1-((R)-2-(1-Fluorocyclopropane-1-carboxamide)-3-((4-((4-(3-Methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazin-1-yl)methyl)benzyl)thio)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01070B); (2S,4R)-1-((R)-3-((2-(2-(2-(4-(4-((5-Chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL01119);

[0051] (2S,4R)-1-((R)-3-((3-(4-(4-((5-Chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL01118); (2S,4R)-1-((R)-3-((4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL01120); (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01126); (2S,4R)-1-((R)-3-((((1S,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01134); (2S,4R)-1-((R)-3-((4-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)but-2-yn-1-yl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01076);

[0052] (2S,4R)-1-((R)-3-((2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL01123); (2S,4R)-1-((R)-3-((3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL01122); (2S,4R)-1-((R)-3-((4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL01121); (2S,4R)-1-((R)-3-((2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01131);

[0053] (2S,4R)-1-((R)-3-(((S)-1-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propan-2-yl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01140); (2S,4R)-1-((R)-3-((3-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01111); (2S,4R)-1-((S)-1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenoxy)-14-(1-fluorocyclopropane-1-carboxamide)-13,13-dimethyl-3,6,9-trioxa-12-thiapentadecan-15-oil)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01145); (2S,4R)-1-((S)-3-((3-(2-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenoxy)ethoxy)propyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01149); (2S,4R)-1-((S)-3-((((1R,4S)-4-((4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenoxy)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01168);

[0054] (2S,4R)-1-((S)-3-((((1R,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenoxy)piperidin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02017); (2S,4R)-1-((S)-3-((((1R,4S)-4-(((2R,6S)-4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)-2,6-dimethylpiperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02031); (2S,4R)-1-((S)-3-((((1R,4S)-4-((9-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02038); (2S,4R)-1-((R)-3-((((1r,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(2-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL02047);

[0055] (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02048); (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL02049); (2S,4R)-1-((R)-3-((((1s,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(2-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL02058); (2S,4R)-1-((R)-3-((((1s,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02059); (2S,4R)-1-((R)-3-((((1s,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL02060);

[0056] (2S,4R)-1-((R)-3-((((1S,3R)-3-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02063); (2S,4R)-1-((R)-3-((3-(9-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02067B); (2S,4R)-1-((R)-3-((((1R,4R)-4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazine-1-carbonyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02068); (2S,4R)-1-((S)-3-((((1S,3S)-3-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclopentyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02093); (2S,4R)-1-((S)-3-((((2S,5S)-5-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)-1,4-dioxan-2-yl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02098);

[0057] (2S,4R)-1-((S)-3-((((1r,3S)-3-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclobutyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02133); (2S,4R)-1-((R)-3-((((1R,4R)-4-((((R)-4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)morpholin-2-yl)methoxy)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02134); (2S,4R)-1-((R)-3-((2-(2-(1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperidin-4-yl)ethoxy)ethyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02127); (2S,3R,4S)-1-((R)-3-((((1r,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02152);

[0058] (R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-4,5-dihydro-1H-pyrrole-2-carboxamide (XL02146); (2S,4R)-N-(2-(3-(1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperidin-4-yl)propoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02117); (2S,4R)-N-(2-(2-(2-(1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperidin-4-yl)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02126); (2S,4R)-N-(2-((5-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)pentyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02115);

[0059] (2S,4R)-N-(2-(((1r,3S)-3-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclobutyl)methoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02135); (2S,4R)-N-(2-(((1r,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02120); (2S,4R)-N-(2-(((1s,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02119); (2S,4R)-N-(2-(((1r,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenoxy)piperidin-1-yl)methyl)cyclohexyl)methoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02137); (2S,4R)-N-(2-(((1R,4S)-4-((((R)-4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)morpholin-2-yl)methoxy)methyl)cyclohexyl)methoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02138);

[0060] (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01077); (2S,4R)-1-((R)-3-((((1r,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01083); (2S,4R)-1-((R)-3-((((1s,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01088); (2S,4R)-1-((R)-3-((((1s,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(4-cyanobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01089); Or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

[0061] Preferred compounds may also be selected from the following: (2S,4R)-1-((R)-3-((2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (SD75); (2S,4R)-1-((R)-3-((3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (SD82); (2S,4R)-1-((R)-3-((4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (SD100); Or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

[0062] Further preferred compounds may also be selected from the following: (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01126); (2S,4S)-1-((R)-3-((((1r,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (cis-XL01126); (2S,4R)-1-((R)-3-((((1S,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01134); or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

[0063] Some preferred compounds may also be selected from the following: (2S,4R)-1-((S)-3-((((1R,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenoxy)piperidin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02017); (2S,4R)-1-((R)-3-((((1r,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(2-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL02047); (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02048);

[0064] (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL02049); (2S,4R)-1-((R)-3-((((1R,4R)-4-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-carbonyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02068); (2S,4R)-1-((S)-3-((((1S,3S)-3-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclopentyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02093); (2S,4R)-1-((S)-3-((((2S,5S)-5-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)-1,4-dioxan-2-yl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02098);

[0065] (2S,4R)-1-((S)-3-((((1r,3S)-3-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclobutyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02133); (2S,4R)-1-((R)-3-((((1R,4R)-4-((((R)-4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)morpholin-2-yl)methoxy)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02134); (2S,4R)-1-((R)-3-((2-(2-(1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperidin-4-yl)ethoxy)ethyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02127); (2S,3R,4S)-1-((R)-3-((((1r,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02152);

[0066] (2S,4R)-N-(2-(3-(1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperidin-4-yl)propoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamide)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02117); (2S,4R)-N-(2-(2-(2-(1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperidin-4-yl)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamide)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02126); (2S,4R)-N-(2-((5-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)pentyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02115); (2S,4R)-N-(2-(((1r,3S)-3-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclobutyl)methoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02135);

[0067] (2S,4R)-N-(2-(((1r,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02120); (2S,4R)-N-(2-(((1s,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02119); (2S,4R)-N-(2-(((1r,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenoxy)piperidin-1-yl)methyl)cyclohexyl)methoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamide)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02137); (2S,4R)-N-(2-(((1R,4S)-4-((((R)-4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)morpholin-2-yl)methoxy)methyl)cyclohexyl)methoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamide)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (XL02138);

[0068] (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01077); (2S,4R)-1-((R)-3-((((1r,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01083); (2S,4R)-1-((R)-3-((((1s,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01088); (2S,4R)-1-((R)-3-((((1s,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(4-cyanobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01089); or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

[0069] Particularly preferred compounds of formula (I) can be selected from XL01126, Cis-XL01126, and XL01134, particularly XL01126, and pharmaceutically acceptable salts, enantiomers, stereoisomers, hydrates, solvates, or polymorphs thereof.

[0070] Particularly preferred compounds of formula (I) can also be selected from the following: (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01126); (2S,4R)-1-((R)-3-((((1S,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01134); (2S,3R,4S)-1-((R)-3-((((1r,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02152); (2S,4R)-1-((R)-3-((((1s,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01088); Or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

[0071] In some embodiments, a preferred compound of formula (I) is (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01126), or may be a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

[0072] Also, a preferred compound of formula (I) may also be selected from XL02047, XL02048, XL02049, XL02017, and XL02068, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof. Also, a preferred compound of formula (I) may also be selected from SD82, SD75, and DS100, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

[0073] Also, a preferred compound of formula (I) may also be selected from SD12, SD13, and XL01131, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof. Also, a preferred compound of formula (I) may also be selected from XL02152, XL02098, and MP01088, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

[0074] In a second aspect, there is provided a pharmaceutical composition comprising a compound as previously defined herein and a pharmaceutically acceptable vehicle or diluent therefor. The pharmaceutical composition may comprise an effective amount of a compound as previously defined herein, in combination with a pharmaceutically acceptable carrier, additive, or excipient, and optionally further in combination with an additional bioactive substance.

[0075] In a third aspect, a compound as defined herein is provided for use in a medicament. Use of the compound as defined herein in a medicament is also provided herein.

[0076] In a fourth aspect, use in the prevention or treatment of a disease or condition associated with a change in the GTPase and / or kinase activity of protein leucine-rich repeat kinase 2 (LRRK2), said use comprising administration of a PROTAC compound of formula (I) to a subject suffering from or potentially exposed to said disease or condition, a compound as defined herein is provided. Also provided is the use of a compound as defined herein, including administration of a PROTAC compound of formula (I) to a subject suffering from or potentially exposed to a disease or condition associated with a change in the GTPase and / or kinase activity of protein leucine-rich repeat kinase 2 (LRRK2) for the prevention or treatment of said disease or condition.

[0077] In a fifth aspect, use in the prevention or treatment of a disease or condition associated with a change in PDE6D activity, said use comprising administration of a PROTAC compound of formula (I) as defined herein to a subject suffering from or potentially exposed to a PDE6D-related disease or condition, a compound as defined herein is provided. Also provided is the use of a compound as defined herein, including administration of a PROTAC compound of formula (I) as defined herein to a subject suffering from or potentially exposed to a PDE6D-related disease or condition in the prevention or treatment of a disease or condition associated with a change in PDE6D activity.

[0078] In a sixth aspect, there is provided a compound as defined herein for use in the prevention or treatment of a disease or condition independently selected from the following: Parkinson's disease, idiopathic Parkinson's disease, idiopathic late-onset Parkinson's disease, familial Parkinson's disease, LRRK2 mutation-related Parkinson's disease, dementia with Lewy bodies, primary tauopathy, or an inflammation-related disease such as Hansen's disease, neuroinflammation, and Crohn's disease. Also provided is the use of a compound as defined herein in the prevention or treatment of a disease or condition independently selected from the following: Parkinson's disease, idiopathic Parkinson's disease, idiopathic late-onset Parkinson's disease, familial Parkinson's disease, LRRK2 mutation-related Parkinson's disease, dementia with Lewy bodies, primary tauopathy, or an inflammation-related disease such as Hansen's disease, neuroinflammation, and Crohn's disease.

[0079] In a seventh aspect, there is provided a method of modulating the protein activity of a patient's protein leucine-rich repeat kinase 2 (LRRK2), the method comprising administering to the patient an amount of a compound as described herein. In an eighth aspect, there is provided a method of degrading a patient's leucine-rich repeat kinase 2 (LRRK2), the method comprising administering to the patient an effective amount of a compound as described herein.

[0080] In a further aspect, there is provided a method of modulating the protein activity of a patient's protein PDE6D, the method comprising administering to the patient an amount of a compound as described herein. In a further aspect, there is provided a method of degrading a patient's protein PDE6D, the method comprising administering to the patient an effective amount of a compound as described herein.

[0081] In a further aspect, there is provided a method of treating a patient suffering from a disease or condition associated with a change in the GTPase and / or kinase activity of protein leucine-rich repeat kinase 2 (LRRK2), the method comprising administering to a subject suffering from or at risk of being exposed to said disease or condition a PROTAC compound of formula (I). In a further aspect, there is provided a method of treating a patient suffering from a disease or condition associated with a change in PDE6D activity, the method comprising administering to a subject suffering from or at risk of being exposed to a PDE6D-related disease or condition a PROTAC compound of formula (I) as defined herein, and the method comprises administering a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein.

[0082] In a further aspect, there is provided a method of treating a patient suffering from a disease or condition independently selected from the following: Parkinson's disease, idiopathic Parkinson's disease, idiopathic late-onset Parkinson's disease, familial Parkinson's disease, LRRK2 mutation-related Parkinson's disease, Lewy body dementia, primary tauopathy, or an inflammation-related disease, such as leprosy, neuroinflammation, and Crohn's disease, etc., and the method comprises administering a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0083]

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Mode for Carrying Out the Invention

[0084] Detailed Description of the Invention The present invention is directed to identifying more rapid, more potent, and potentially orally bioavailable and / or blood-brain barrier permeable LRRK2 PROTAC degrader compounds, qualifying them as chemical probes for studying the biology of LRRK2, further confirming the target validity as a therapeutic concept for PD, and providing potential treatments or preventive methods for PD and related diseases.

[0085] By using HG-10-102-01 as a warhead and engaging multiple different E3 ligase ligands, the inventors developed a first-generation LRRK2 PROTAC. [Chemistry]

[0086] Among the first-generation compounds, SD75, SD82, and SD100 showed moderate LRRK2 degradation (Figures 1 and 2): [Chemistry]

[0087] Next, the second-generation LRRK2 PROTACs were designed and synthesized based on the set of SD75, SD82, and SD100, which are the most potent first-generation LRRK2 PROTACs. Two sets of second-generation LRRK2 PROTACs were designed and synthesized: [Chemistry]

[0088] The Western blot degradation assay of LRRK2 protac was performed using WT and PD-related LRRK2[G2019S] mouse embryonic fibroblasts (MEFs). The second-generation PROTACs were tested in parallel with the first-generation PROTACs SD75, SD82, and SD100. The compounds were tested at concentrations of 33 nM or 1 mM for 4 hours or 24 hours, and two biological replicates per condition were performed to achieve quantification representative of both replicates. Analysis by quantitative immunoblotting was performed using readouts of total LRRK2 (antibody recognizes an epitope at the C-terminus of the protein), Rab10 pThr73 (physiological substrate), and LRRK2 pSer935 (phosphorylation site, a biomarker).

[0089] From the screening of the second-generation LRRK2 PROTACs (Figures 3, 4, 5, and 6), XL01126 and XL01134 were the top two identified compounds: [Chemistry]

[0090] Therefore, in the direct comparison of SD75 vs. XL01126 vs. XL01134 in a dose-dependent assay, SD75 induced partial LRRK2 degradation (Figures 11A - C), XL01134 induced LRRK2 degradation with a “hook effect” (Douglass, et al., J Am Chem Soc. 2013; 135(16): 6092 - 6099.) (Figures 12A - C), and XL01126 induced almost complete LRRK2 degradation (Figures 13A - C).

[0091] Table 1 - Direct degradation comparison of XL01126, XL01134, and SD75, which are LRRK2 - degrading compounds

Table 1

[0092] The results in Table 1 also show that XL01126 and XL01134 are more potent degrading compounds than SD75.

[0093] Table 2 - Direct comparison of pRab10 dephosphorylation by XL01126, XL01134, and SD75

Table 2

[0094] The results in Table 2 show that XL01126 and XL01134 are more potent than SD75 in dephosphorylating pRab10.

[0095] Table 3 - Direct comparison of pSer935 - LRRK2 dephosphorylation by XL01126, XL01134, and SD75

Table 3

[0096] Table 3 shows that XL01126 and XL01134 are more potent than SD75 in the dephosphorylation of pSer935-LRRK2. Parallel comparison of SD75, XL01134, and XL01126 in the time-dependent assay shows that all of SD75 (Figs. 14A - C), XL01134 (Figs. 15A - C), and XL01126 (Figs. 16A - C) can induce the time-dependent degradation of LRRK2, as well as the time-dependent dephosphorylation of pRab10 and LRRK2.

[0097] Table 4 - Summary of dose - response and time - course experiments [Table 4]

[0098] The results show the following: 1) XL01126 is the most potent LRRK2 - degrading compound and induced the fastest LRRK2 degradation among SD75 and XL01134. 2) XL01126 induced the most potent and fastest LRRK2 dephosphorylation and pRab10 dephosphorylation compared to SD75 and XL01134.

[0099] Next, further screening of XL01126 - based compounds was carried out, and Cis - XL01126 was synthesized as a non - degrading control for separating degradation from inhibition. Cis - XL01126 does not bind to the VHL E3 ligase. [Chemical Structure]

[0100] Table 5 - Binding affinities of VH101, Cis - XL01126, and XL01126 tested by fluorescence polarization assay [Table 5]

[0101] XL01126-induced LRRK2 degradation is dependent on the proteasome pathway (Figures 17A-D and 18A-D). XL01126 is LRRK2 R1441C It degraded LRRK2 in a dose-dependent manner in MEF (Figures 19A-C) and macrophage cells (Figures 20A-B), and also degraded LRRK2 in a dose- and time-dependent manner in human PBMC cells (Figures 21A-C, 22A-C, 23A-C). XL01126 induced selective LRRK2 degradation in WT MEFs, as shown by TMT labeling proteomic analysis (Figure 24).

[0102] Table 6 shows that the solubility of XL01126 in PBS and FeSSIP is 0.55 μM and 26.05 μM, respectively. XL01126 can permeate Caco-2 monolayers. XL01126 is stable in mouse plasma and mouse hepatocytes with half-lives of 108.29 and 314.33 minutes, respectively. XL01126 is metabolized in mouse liver microsomes with a half-life of 3.65 minutes.

[0103] Table 6. Physicochemical and in vitro ADME properties of XL01126 [Table 6]

[0104] XL01126 exhibits an attractive in vivo pharmacokinetic profile with an oral bioavailability of 15% and is capable of penetrating the blood-brain barrier (Figures 33, 34, and 35). XL01126 has only a slight binary binding affinity for VHL (Figures 36, 37A, and 37B) and LRRK2 (Figures 38A and 38B), and is inferior to XL01134 and SD75 in binding to VHL and LRRK2 in the permeabilized mode of the VHL and LRRK2 target engagement assays. However, XL01126 has the highest permeability among the PROTACs tested, as demonstrated by the highest IC50 ratio between the permeabilized mode and the live mode engagement assays. XL01126 also forms a higher and more cooperative ternary complex with VHL and LRRK2 compared to XL01134 and SD75 (Figure 39).

[0105] Considering the favorable degradation profiles of XL01126 and XL01134, and the excellent in vivo PK profile of XL01126, further modifications (third generation) of the LRRK2 PROTACs are based on XL01126 and XL01134:

[0106] XL02031, XL02038, and XL02017 are based on XL01126 with modifications to the piperidine moiety:

Chemical Structure

[0107] XL02058, XL02059, XL02060, and XL02063 are based on XL01134 with modifications to the VHL ligand and linker:

Chemical Structure

[0108] XL02047, XL02048, and XL02049 are based on XL01126 with modifications to the VHL ligand; XL02067B, XL02068, and XL02077 have modifications in the linker:

Chemical Structure

[0109] Most of the compounds from the third generation showed comparable resolution that exceeded XL01126 and XL01134.

[0110] Results and Discussion Figure 1 shows that after treatment with 33 nM / 4 h, SD12, SD13, SD79, SD75, SD82, SD100, SD10, SD11, SD112, SD113, SD114 degraded 0 - 25% of LRRK2 WT and 0 - 60% of LRRK2 G2019S and inhibited 0 - 25% of pRab10 and LRRK2 in WT MEF G2019S and 0 - 60% of pRab10 in MEF (Figure 1). After treatment with 1 μM / 4 h, SD12, SD13, SD79, SD75, SD82, SD100, SD10, SD11, SD112, SD113, SD114 degraded 0 - 45% of LRRK2 WT and 0 - 75% of LRRK2 G2019S and inhibited 18 - 90% of pRab10 and LRRK2 in WT MEF G2019S and 30 - 95% of pRab10 in MEF (Figure 1).

[0111] As is evident from Figure 2, after treatment with 33 nM / 24 h, SD12, SD13, SD79, SD75, SD82, SD100, SD10, SD11, SD112, SD113, SD114 degraded 0 - 40% of LRRK2 WT and 0 - 70% of LRRK2 G2019S and inhibited 10 - 40% of pRab10 and LRRK2 in WT MEF G2019S and 0 - 70% of pRab10 in MEF (Figure 2). After treatment with 1 μM / 24 h, SD12, SD13, SD79, SD75, SD82, SD100, SD10, SD11, SD112, SD113, SD114 degraded 0 - 60% of LRRK2 WT and 0 - 85% of LRRK2 G2019S and inhibited 30 - 90% of pRab10 and LRRK2 in WT MEF G2019SIn MEF, 30 - 100% of pRab10 was inhibited (Figure 2).

[0112] Figures 3 and 5 show that after treatment with 33 nM for 4 hours, SD75, XL01078B, XL01119, XL01126, SD82, XL01072, XL01118, XL01134, SD100, XL01070B, XL01120, XL01076, XL01123, XL01131, XL01145, XL01122, XL01140, XL01149, XL01121, XL01111, XL01168 degraded 0 - 25% of LRRK2 WT and 0 - 60% of LRRK2 G2019S and inhibited 0 - 40% of pRab10 and LRRK2 in WT MEF G2019S Figures 3 and 5 show that after treatment with 1 μM for 4 hours, SD75, XL01078B, XL01119, XL01126, SD82, XL01072, XL01118, XL01134, SD100, XL01070B, XL01120, XL01076, XL01123, XL01131, XL01145, XL01122, XL01140, XL01149, XL01121, XL01111, XL01168 degraded 0 - 60% of LRRK2 WT and 10 - 80% of LRRK2 G2019S and inhibited 10 - 95% of pRab10 and LRRK2 in WT MEF G2019S Figures 3 and 5 show that after treatment with 20 - 100% of pRab10 was inhibited in MEF (Figures 3 and 5).

[0113] Figures 4 and 6 show that after treatment with 33 nM for 24 hours, SD75, XL01078B, XL01119, XL01126, SD82, XL01072, XL01118, XL01134, SD100, XL01070B, XL01120, XL01076, XL01123, XL01131, XL01145, XL01122, XL01140, XL01149, XL01121, XL01111, XL01168 degraded 0 - 65% of LRRK2 WT and 0 - 80% of LRRK2 G2019Swere degraded, and 0 - 60% of pRab10 and LRRK2 in WT MEF G2019S showed inhibition of 0 - 90% of pRab10 in MEF (Figures 4 and 6). After treatment with 1 μM / 24 h, SD75, XL01078B, XL01119, XL01126, SD82, XL01072, XL01118, XL01134, SD100, XL01070B, XL01120, XL01076, XL01123, XL01131, XL01145, XL01122, XL01140, XL01149, XL01121, XL01111, XL01168 were 10 - 90% of LRRK2 WT and 20 - 90% of LRRK2 G2019S were degraded, and 30 - 100% of pRab10 and LRRK2 in WT MEF G2019S showed inhibition of 10 - 100% of pRab10 in MEF (Figures 4 and 6).

[0114] Figures 7 and 9 show that after treatment with 33 nM / 4 h, XL01126, XL02017, XL02031, XL02038, XL02047, XL02048, XL02049, XL02058, XL02059, XL02060, XL02063, XL02067B, XL02068 were 0 - 60% of LRRK2 WT and 0 - 70% of LRRK2 G2019S were degraded, and 0 - 70% of pRab10 and LRRK2 in WT MEF G2019S showed inhibition of 0 - 90% of pRab10 in MEF (Figures 7 and 9). After treatment with 1 μM / 4 h, XL01126, XL02017, XL02031, XL02038, XL02047, XL02048, XL02049, XL02058, XL02059, XL02060, XL02063, XL02067B, XL02068 were 0 - 90% of LRRK2 WT and 40 - 90% of LRRK2 G2019S were degraded, and 80 - 100% of pRab10 and LRRK2 in WT MEF G2019S showed inhibition of 10 - 100% of pRab10 in MEF (Figures 7 and 9).

[0115] Figures 8 and 10 show that after treatment with 33 nM / 24 h, XL01126, XL02017, XL02031, XL02038, XL02047, XL02048, XL02049, XL02058, XL02059, XL02060, XL02063, XL02067B, XL02068 decomposed 0 - 80% of LRRK2 WT and 20 - 90% of LRRK2 G2019S and inhibited 0 - 80% of pRab10 and LRRK2 in WT MEF G2019S and 30 - 90% of pRab10 in LRRK2 MEF (Figures 8 and 10). After treatment with 1 μM / 24 h, XL01126, XL02017, XL02031, XL02038, XL02047, XL02048, XL02049, XL02058, XL02059, XL02060, XL02063, XL02067B, XL02068 decomposed 60 - 90% of LRRK2 WT and 60 - 95% of LRRK2 G2019S and inhibited 80 - 100% of pRab10 and LRRK2 in WT MEF G2019S and 80 - 100% of pRab10 in LRRK2 MEF (Figures 8 and 10).

[0116] Figures 11A, 11B, 11C show that SD75 induced dose - dependent degradation of LRRK2 max in D max (WT)=51% and D G2019S in LRRK2 WT MEF at D G2019S (G2019S)=58% respectively (Figures 11A and 11B). SD75 induced dose - dependent dephosphorylation of Rab10 in WT MEF and LRRK2 50 MEF at EC 50 (WT)=2270 nM and EC G2019S (G2019S)=379 nM respectively (Figures 11A and 11C).

[0117] Figures 12A, 12B, 12C show that XL01134 has D max (WT)=59%, DC 50(WT) = 32 nM, D max (G2019S) = 81% and DC 50 (G2019S) = 7 nM, respectively, in WT MEF and LRRK2 G2019S LRRK2 in MEF WT and LRRK2 G2019S induced dose-dependent degradation (Figures 12A and 12B). XL01134 is EC 50 (WT) = 74 nM and EC 50 (G2019S) = 12 nM, respectively, in WT MEF and LRRK2 G2019S induced dose-dependent dephosphorylation of Rab10 in MEF (Figures 12A and 12C).

[0118] From Figures 13A, 13B, 13C, XL01126 is D max (WT) = 82%, DC 50 (WT) = 32 nM, D max (G2019S) = 90% and DC 50 (G2019S) = 14 nM, respectively, in WT MEF and LRRK2 G2019S LRRK2 in MEF WT and LRRK2 G2019S clearly induced dose-dependent degradation (Figures 13A and 13B). XL01126 is EC 50 (WT) = 54 nM and EC 50 (G2019S) = 15 nM, respectively, in WT MEF and LRRK2 G2019S induced dose-dependent dephosphorylation of Rab10 in MEF (Figures 13A and 13C).

[0119] Figures 14A, 14B, 14C show that at 1000 nM, SD75 is T 1 / 2 (WT) = 5.1 hours, D max (WT) = 52%, T 1 / 2 (G2019S) = 1.4 hours, and D max (G2019S) = 81%, respectively, in WT MEF and LRRK2 G2019S LRRK2 in MEF WT and LRRK2 G2019Sshowed the induction of the time-dependence decomposition (FIGS. 14A and 14B). At 1000 nM, SD75 had T 1 / 2 (WT)=6.7 hours, D max (WT)=41%, T 1 / 2 (G2019S)=1.1 hours, and D max (G2019S)=74% respectively, and induced the time-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 G2019S MEF (FIGS. 14A and 14C).

[0120] As shown in FIGS. 15A, 15B, and 15C, at 300 nM, XL01134 had T 1 / 2 (WT)=2.7 hours, D max (WT)=75%, T 1 / 2 (G2019S)=1.4 hours, and D max (G2019S)=82% respectively, and induced the time-dependent decomposition of LRRK2 G2019S and LRRK2 WT in WT MEF and LRRK2 G2019S MEF (FIGS. 15A and 15B). At 300 nM, XL01134 had T 1 / 2 (WT)=2.1 hours, D max (WT)=68%, T 1 / 2 (G2019S)=0.3 hours, and D max (G2019S)=87% respectively, and induced the time-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 G2019S MEF (FIGS. 15A and 15C).

[0121] FIGS. 16A, 16B, and 16C show that at 300 nM, XL01126 had T 1 / 2 (WT)=1.2 hours, D max (WT)=82%, T 1 / 2 (G2019S)=0.6 hours, and D max (G2019S)=92% respectively, and induced the time-dependent decomposition of LRRK2 G2019S and LRRK2 WT in WT MEF and LRRK2 G2019SFigures 16A and 16B show that at 300 nM, XL01126 induced a time-dependent degradation of T 1 / 2 (WT) = 2.1 hours, D max (WT)=84%, T 1 / 2 (G2019S) = 0.3 hours, and D max (G2019S)=92%, respectively, in WT MEFs and LRRK2 G2019S It induced a time-dependent dephosphorylation of Rab10 in MEFs (Figures 16A and 16C).

[0122] FIG. 17A, FIG. 17B, FIG. 17C, and FIG. 17D show LRRK2 WT We show that XL01126 induces global degradation of LRRK2 at Ser935, but this can be rescued by pretreatment with VHL Ligand (VH101), MLN4924, and MG132. WT Inhibition of phosphorylation and of Rab10 phosphorylation at Thr73 could be partially rescued by pretreatment with VHL Ligand (VH101), MLN4924, and MG132 (Figures 17A, 17B, 17C, and 17D).

[0123] FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D show LRRK2 G2019S We demonstrate that XL01126 induces global degradation of VHL, which can be rescued by pretreatment with VHL Ligand (VH101), MLN4924, and MG132. G2019S Inhibition of phosphorylation and of Rab10 phosphorylation at Thr73 could be partially rescued by pretreatment with VHL Ligand (VH101), MLN4924, and MG132 (Figures 18A, 18B, 18C, and 18D).

[0124] Figures 19A, 19B, and 19C show that XL01126 is 50 = 15 nM, and D max =89%, LRRK2 R1441C The results show that XL01126 induced dose-dependent degradation of EC 50 At =30nM, LRRK2R1441C Induced dose-dependent dephosphorylation of Rab10 in MEFs (Figs. 19A and 19C). CisXL01126 has an EC 50 = 158 nM and induces dose-dependent degradation of LRRK2 in R1441C MEFs (Figs. 19A and 19C).

[0125] Figs. 20A and 20B demonstrate that XL01126 induces dose-dependent degradation of LRRK2 in macrophages at a DC 50 = 55 nM. Cis-XL01126 is unable to degrade LRRK2 in macrophage cells (Figs. 20A and 20B). Figs. 21A, 21B, and 21C show that XL01126 induces dose-dependent degradation of LRRK2 in PBMCs at a DC 50 = 72 nM and a D max = 88% after 4 hours of treatment. Cis-XL01126 is unable to degrade LRRK2 in PBMCs (Figs. 21A and 21B). XL01126 induces dose-dependent dephosphorylation of Rab10 in PBMC cells at an EC 50 = 69 nM after 4 hours of treatment. Cis-XL01126 also dephosphorylates Rab10 in PBMC cells at an EC 50 > 3 μM (Figs. 21A and 21C).

[0126] Figs. 22A, 22B, and 22C show that XL01126 induces dose-dependent degradation of LRRK2 in PBMCs at a DC 50 = 17 nM and a D max = 93% after 24 hours of treatment. Cis-XL01126 is unable to degrade LRRK2 in PBMCs (Figs. 22A and 22B). XL01126 induces dose-dependent dephosphorylation of Rab10 in PBMC cells at an EC 50 = 20 nM after 24 hours of treatment. Cis-XL01126 also dephosphorylates Rab10 in PBMC cells at an EC 50Rab10 was dephosphorylated in PBMC cells at = 705 nM (Figures 22A and 22C).

[0127] Figures 23A, 23B, and 23C show that at 300 nM, XL01126 induced time-dependent degradation of LRRK2 in PBMCs at 1 / 2 T = 2.4 hours and max D = 92%. At 300 nM, XL01126 induced time-dependent dephosphorylation of LRRK2 and Rab10 in PBMCs at 1 / 2 T = 1 hour and 0.38 hour, respectively (Figures 23A and 23B). At 300 nM, Cis-XL01126 was unable to degrade LRRK2 and induced little dephosphorylation of LRRK2 and Rab10 (Figures 23A and 23C). Figure 24 shows that XL01126 selectively reduced intracellular LRRK2 protein levels in a TMT-labeled proteomics survey.

[0128] The data presented in Table 1 show that the solubility of XL01126 in PBS and fed simulated intestinal fluid (FeSSIP) is 0.55 μM and 26.05 μM, respectively (Table 6). XL01126 can penetrate the Caco-2 monolayer (Table 6). XL01126 is stable in mouse plasma and mouse hepatocytes with half-lives of 108.29 minutes and 314.33 minutes, respectively (Table 6). XL01126 can be metabolized in mouse liver microsomes with a half-life of 3.65 minutes (Table 6).

[0129] Figures 25A, 25B, and 25C show that XL02047 at max (WT) = 78%, DC 50 (WT) = 45 nM, D max (G2019S) = 79% and DC 50 (G2019S) = 60 nM induced LRRK2 in WT MEF and LRRK2 G2019S in MEF and LRRK2 WT and LRRK2 G2019Sshowed induction of dose-dependent dissociation (FIGS. 25A and 25B). XL02047 induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 50 (WT) = 228 nM and EC 50 (G2019S) = 73 nM, respectively, in WT MEF and LRRK2 G2019S MEF (FIGS. 25A and 25C).

[0130] FIGS. 26A, 26B, and 26C demonstrate that XL02048 induced dose-dependent dissociation of LRRK2 max (WT) = 81.5%, DC 50 (WT) = 46 nM, D max (G2019S) = 83.5% and DC 50 (G2019S) = 23 nM, respectively, in WT MEF and LRRK2 G2019S MEF (FIGS. 26A and 26B). XL02048 induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 WT and LRRK2 G2019S at EC 50 (WT) = 72 nM and EC 50 (G2019S) = 53 nM, respectively, in WT MEF and LRRK2 G2019S MEF (FIGS. 26A and 26C).

[0131] FIGS. 27A, 27B, and 27C show that XL02049 induced dose-dependent dissociation of LRRK2 max (WT) = 76.5%, DC 50 (WT) = 40 nM, D max (G2019S) = 90% and DC 50 (G2019S) = 27 nM, respectively, in WT MEF and LRRK2 G2019S MEF (FIGS. 27A and 27B). XL02049 induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 WT and LRRK2 G2019S at EC 50 (WT) = 65 nM and EC 50 (G2019S) = 31 nM, respectively, in WT MEF and LRRK2 G2019SDephosphorylation of Rab10 in MEF was induced in a dose-dependent manner (Figures 27A and 27C).

[0132] Figures 28A, 28B, and 28C show that XL02017 at D max (WT) = 68%, DC 50 (WT) = 44 nM, D max (G2019S) = 79% and DC 50 (G2019S) = 22 nM induced dose-dependent disassembly of WT MEF and LRRK2 G2019S LRRK2 in MEF WT and LRRK2 G2019S respectively (Figures 28A and 28B). XL02017 induced dose-dependent dephosphorylation of Rab10 in MEF at EC 50 (WT) = 83 nM and EC 50 (G2019S) = 25 nM for WT MEF and LRRK2 G2019S in MEF (Figures 28A and 28C).

[0133] As shown in Figures 29A, 29B, and 29C, XL02038 at D max (WT) = 65% and D max (G2019S) = 79% induced dose-dependent disassembly of WT MEF and LRRK2 G2019S LRRK2 in MEF WT and LRRK2 G2019S respectively (Figures 29A and 29B). XL02038 induced dose-dependent dephosphorylation of Rab10 in MEF at EC 50 (WT) = 14 nM and EC 50 (G2019S) = 10 nM for WT MEF and LRRK2 G2019S in MEF (Figures 29A and 29C).

[0134] As shown in Figures 30A, 30B, and 30C, XL02068 at D max (WT) = 89%, DC 50 (WT) = 93 nM, D max (G2019S) = 87% and DC 50(G2019S) induced dose-dependent degradation of LRRK2 in WT MEF and LRRK2 MEF at 103 nM, respectively (Figures 30A and 30B). XL02068 induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at EC(WT) = 272 nM and EC(G2019S) = 150 nM, respectively (Figures 30A and 30C). G2019S LRRK2 in MEF WT and LRRK2 G2019S as shown in Figures 30A and 30B. XL02068 induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at EC(WT) = 272 nM and EC(G2019S) = 150 nM, respectively (Figures 30A and 30C). 50 (WT)=272nM and EC 50 (G2019S)=150nM induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at EC(WT) = 272 nM and EC(G2019S) = 150 nM, respectively (Figures 30A and 30C). G2019S as shown in Figures 30A and 30C).

[0135] As shown in Figures 31A, 31B, and 31C, XL02059 induced dose-dependent degradation of LRRK2 in WT MEF and LRRK2 MEF at D(WT) = 77.5% and D(G2019S) = 76%, respectively (Figures 31A and 31B). XL02059 induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at EC(WT) = 227 nM and EC(G2019S) = 55 nM, respectively (Figures 31A and 31C). max (WT)=77.5% and D max (G2019S)=76% induced dose-dependent degradation of LRRK2 in WT MEF and LRRK2 MEF at D(WT) = 77.5% and D(G2019S) = 76%, respectively (Figures 31A and 31B). XL02059 induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at EC(WT) = 227 nM and EC(G2019S) = 55 nM, respectively (Figures 31A and 31C). G2019S LRRK2 in MEF WT and LRRK2 G2019S as shown in Figures 31A and 31B). XL02059 induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at EC(WT) = 227 nM and EC(G2019S) = 55 nM, respectively (Figures 31A and 31C). 50 (WT)=227nM and EC 50 (G2019S)=55nM induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at EC(WT) = 227 nM and EC(G2019S) = 55 nM, respectively (Figures 31A and 31C). G2019S as shown in Figures 31A and 31C).

[0136] As shown in Figures 32A, 32B, and 32C, XL02060 induced dose-dependent degradation of LRRK2 in WT MEF and LRRK2 MEF at D(WT) = 71% and D(G2019S) = 78.5%, respectively (Figures 32A and 32B). XL02060 induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at EC(WT) = 32 nM and EC(G2019S) = 27 nM, respectively (Figures 32A and 32C). max (WT)=71% and D max (G2019S)=78.5% induced dose-dependent degradation of LRRK2 in WT MEF and LRRK2 MEF at D(WT) = 71% and D(G2019S) = 78.5%, respectively (Figures 32A and 32B). XL02060 induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at EC(WT) = 32 nM and EC(G2019S) = 27 nM, respectively (Figures 32A and 32C). G2019S LRRK2 in MEF WT and LRRK2 G2019S as shown in Figures 32A and 32B). XL02060 induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at EC(WT) = 32 nM and EC(G2019S) = 27 nM, respectively (Figures 32A and 32C). 50 (WT)=32nM and EC 50 (G2019S)=27nM induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at EC(WT) = 32 nM and EC(G2019S) = 27 nM, respectively (Figures 32A and 32C). G2019SDose-dependent dephosphorylation of Rab10 in MEF was induced (Figures 32A and 32C).

[0137] As shown in Figure 33, after intravenous injection of 5 mg / kg of XL01126 (single dose) into mice, the plasma XL01126 had a clearance of 0.208 L / hour / kg, a volume of distribution of 0.511 L / kg, a metabolic half-life of 1.52 hours, and, respectively, AUC last and AUC INF with areas under the curve (AUC) of 23663 hour*ng / mL and 23981 hour*ng / mL (Figure 33). After intraperitoneal injection of 30 mg / kg (single dose), the plasma concentration of XL01126 had a metabolic half-life of 5.2 hours and, respectively, AUC last and AUC INF with areas under the curve (AUC) of 41434 hour*ng / mL and 64068 hour*ng / mL and reached a maximum value (7700 ng / mL) at 0.25 hour. The bioavailability of XL01126 was 29.2% after intraperitoneal injection (Figure 33). After oral gavage of 30 mg / kg (single dose), the plasma concentration of XL01126 had a metabolic half-life of 5.2 hours and, respectively, AUC last and AUC INF with areas under the curve (AUC) of 21337 hour*ng / mL and 109271 hour*ng / mL and reached a maximum value (3620 ng / mL) at 2 hours. The bioavailability of XL01126 was 15% after intraperitoneal injection (Figure 33).

[0138] As shown in Figure 34, after intravenous injection of 5 mg / kg of XL01126 (single dose) into mice, the concentration of XL01126 in the brain tissue had a metabolic half-life of 3.67 hours and, respectively, AUC last and AUC INFFor [the relevant one], the area under the curve (AUC) was 258 h*ng / mL and 349 h*ng / mL, and it reached its maximum level (155 ng / mL) at 0.083 h (Figure 34). After administering 30 mg / kg of XL01126 (single dose) to mice by intraperitoneal injection, the concentration of XL01126 in the brain tissue had a metabolic half-life of 2.79 h, and respectively, AUC last and AUC INF For the area under the curve (AUC) of 752 h*ng / mL and 884 h*ng / mL, it reached its maximum level (218 ng / mL) at 0.5 h (Figure 34). After administering 30 mg / kg of XL01126 (single dose) to mice by forced feeding, the concentration of XL01126 in the brain tissue had a metabolic half-life of 10.7 h, and respectively, AUC last and AUC INF For the area under the curve (AUC) of 142 h*ng / mL and 402 h*ng / mL, it reached the maximum (27 ng / mL) at 2 h.

[0139] Figure 35 shows that after administering 5 mg / kg of XL01126 (single dose) to mice by intravenous injection, the concentration of XL01126 in the cerebrospinal fluid (CSF) had a metabolic half-life of 8.59 h, and respectively, AUC last and AUC INF For the area under the curve (AUC) of 107 h*ng / mL and 240 h*ng / mL, it reached its highest concentration (27.4 ng / mL) at 0.083 h (Figure 35). XL01126 also reaches the CSF after being administered intraperitoneally (30 mg / kg) and orally (30 mg / kg).

[0140] Leucine-rich repeat kinase 2 (LRRK2) is, so far, one of the most promising disease-modifying targets for Parkinson's disease, although current LRRK2 targeting strategies have focused on kinase inhibitors with potential unwanted side effects. Herein, we disclose the discovery of a chimeric protein degradation compound (PROTAC) of LRRK2, exemplified by the compound XL01126, as an alternative LRRK2 targeting strategy. XL01126 induces rapid and potent degradation of LRRK2 protein in multiple cell lines in a dose- and time-dependent manner. Specifically, XL01126 reduces the LRRK2 protein level by 80 - 90% in WT MEF, LRRK2 50 MEF, LRRK2 G2019S MEF, macrophages, and PBMC cells at DC R1441C of 32 nM, 14 nM, 15 nM, 55 nM, and 17 nM, respectively. XL01126 has degradation half-lives of 1.2 hours, 0.6 hours, and 2.4 hours in WT MEF, LRRK2 G2019S MEF, and PBMC, respectively. XL01126 has oral bioavailability (F = 15%) and can penetrate the blood-brain barrier after a single dose of XL01126 by intravenous (5 mg / kg), intraperitoneal (30 mg / kg), or oral administration (30 mg / kg) in mice. All of these properties qualify XL01126 as a chemical probe for studying the biology of LRRK2 in vitro and in vivo, leading to a starting point for future PD drug development and potential therapies.

[0141] Figure 36 shows that VH101, XL01126, SD75, XL01134, and Cis-XL01126 replaced the fluorescent tracer from the VHL protein in a dose-dependent manner, and XL01134 (Ki = 0.16 μM), which showed the most potent replacement and thus the highest binding affinity for VHL, was followed by VH101 (Ki = 0.44 μM) and SD75 (Ki = 0.82 μM). In contrast, XL01126 (Ki > 2.33 μM) showed only slight replacement (binding affinity for VHL), and Cis-XL01126 did not replace the fluorescent tracer.

[0142] Figure 37A shows that in permeabilized HEK293 cells, VH101, XL01126, SD75, and XL01134 can replace the VHL fluorescent tracer from the VHL protein in a dose-dependent manner, and XL01134 (IC 50 = 0.78 μM), which showed the most potent replacement and thus the highest binding affinity for VHL, was followed by VH101 (IC 50 = 1.0 μM) and SD75 (IC 50 = 1.9 μM). In contrast, XL01126 showed only slight replacement (binding affinity for VHL).

[0143] Figure 37B shows that in live HEK293 cells, VH101, XL01126, SD75, and XL01134 can replace the VHL fluorescent tracer from the VHL protein in a dose-dependent manner, and XL01134, which showed the most potent replacement (IC 50 = 0.88 μM) and thus the highest binding affinity for VHL, was followed by VH101 (IC 50 = 1.8 μM) and SD75 (IC 50 = 2.5 μM). In contrast, XL01126 (IC 50=10.1 μM) showed only minor replacement (binding affinity for VHL). XL01126 had the highest IC 50 ratio between the permeabilized mode and live cell mode VHL engagement (IC 50、透過化per / IC 50、生細胞 > 0.99), and thus had the highest permeability among the PROTACs tested, followed by XL01134 (IC 50、透過化per / IC 50、生細胞 = 0.89), and SD75 (IC 50、透過化per / IC 50、生細胞 = 0.76).

[0144] Figure 38A shows that in permeabilized HEK293 cells, HG-10-102-01, XL01126, SD75, and XL01134 can replace the LRRK2 fluorescent tracer from the LRRK2 protein in a dose-dependent manner, and HG-10-102-01 (IC 50 = 0.23 μM), which showed the most potent replacement and thus the highest binding affinity for LRRK2, was followed by SD75 (IC 50 = 0.78 μM), and XL01134 (IC 50 = 2.0 μM), while XL01126 showed only minor replacement.

[0145] Figure 38B shows that in live HEK293 cells, HG-10-102-01, XL01126, SD75, and XL01134 can replace the LRRK2 fluorescent tracer from the LRRK2 protein in a dose-dependent manner, and HG-10-102-01, which showed the most potent replacement (IC 50 = 0.7 μM) and thus the highest binding affinity for LRRK2, was followed by XL01126 (IC 50 = 1.4 μM), SD75 (IC 50 = 1.8 μM), and XL01134 (IC 50 = 2.2 μM). XL01126 had the highest IC 50has a ratio (1.89) and thus has the highest permeability among the PROTACs tested, followed by XL01134 (IC 50、透過化per / IC 50、生細胞 = 0.91), and SD75 (IC 50、透過化per / IC 50、生細胞 = 0.43).

[0146] Figure 39A shows that XL01126 binds to LRRK2 better in the presence of the VCB protein than in its absence (IC 50 = 0.35 μM vs 2 μM), leading to a positive cooperativity (α) of 5.7. Figure 39B shows that XL01134 binds to LRRK2 better in the presence of the VCB protein than in its absence (IC 50 = 0.79 μM vs 1.1), leading to a positive cooperativity (α) of 1.4. Figure 39C shows that SD75 binds to LRRK2 worse in the presence of the VCB protein than in its absence (IC 50 = 1.4 μM vs 1.1), leading to a negative cooperativity (α) of 0.79.

[0147] Figure 40 shows that XL01126 can induce the highest ternary complex with VHL and LRRK2, followed by SD75 and XL01134. Cis-XL01126 cannot form a ternary complex with VHL and LRRK2.

[0148] Figures 41, 42, and 43 show that after 4-hour / 1 μM treatment, XL01126, XL02152, XL02098, XL02093, and MP01088 degrade 60 - 80% of LRRK2 in WT MEF, WT degrade 75 - 90% of LRRK2 in LRRK2 G2019S MEF, and degrade 80 - 100% of pRab10 and LRRK2 in WT MEF G2019S and G2019SAfter 4 h / 33 nM treatment, XL01126, XL02152, XL02093, and MP01088 inhibited LRRK2 by 25%-40% in WT MEFs. WT , and LRRK2 G2019S 25% to 50% of LRRK2 in MEFs G2019S and 20-40% of pRab10 and LRRK2 in WT MEFs. G2019S It inhibited pRab10 by 20-50% in MEFs.

[0149] Figures 44A, 44B, and 44C show that XL02098 is D max (WT)=59%, DC 50 (WT) = 50 nM, D max (G2019S)=81% and DC 50 (G2019S)=45 nM in WT MEFs and LRRK2, respectively G2019S LRRK2 in MEFs WT and LRRK2 G2019S The results show that XL02098 induced dose-dependent degradation of EC 50 (WT) = 282 nM and EC 50 (G2019S) = 157.2 nM in WT MEFs and LRRK2, respectively. G2019S It induced a dose-dependent dephosphorylation of Rab10 in MEFs (Figures 44A and 44C).

[0150] Figures 45A, 45B, and 45C show that XL02152 is D max (WT)=68%, DC 50 (WT) = 62 nM, D max (G2019S)=80% and DC 50 (G2019S)=44 nM in WT MEFs and LRRK2, respectively G2019S LRRK2 in MEFs WT and LRRK2 G2019S The results show that XL02152 induced dose-dependent degradation of EC 50 (WT) = 144 nM and EC 50(G2019S) induced dose-dependent dephosphorylation of Rab10 in WT MEF and LRRK2 MEF at 63 nM (Figures 45A and 45C). G2019S EXAMPLE

[0151] Generation of Mouse Embryonic Fibroblasts (MEF) Primary MEF were generated as described in previous studies (Wiggin et al., Mol. Cell Biol. 2002 Apr;22(8):2871-81.). Briefly, uterine horns were harvested from mature female mice at 12.5 E days and transferred to a 10 cm tissue culture dish containing cold PBS. Two forceps were used to tear the yolk sac to isolate each fetus. During each fetus isolation, the forceps were thoroughly washed with 70% ethanol. Fetuses were sorted and tissue pieces were collected into PCR tubes for genotyping. The red tissues of the fetuses (which grow into the heart and liver) were removed. The remaining parts of the fetuses were minced with a surgical scalpel blade and incubated with 7.5 ml of trypsin-EDTA solution in a 37 °C tissue culture CO2 incubator for 10 minutes. The dish was removed from the incubator and single cells were confirmed under an optical microscope. 7.5 ml of complete medium was added to the trypsinized cells, and the cell suspension was transferred to a 15 ml Falcon tube and centrifuged at 1200 rpm for 5 minutes at room temperature. The trypsin was aspirated, the cell pellet was resuspended in 5 ml of fresh complete medium, and the cell suspension was plated in a 60 mm tissue culture dish and incubated at 37 °C in a 5% CO2 tissue culture incubator. MEF at this stage were considered passage 0 and were passaged and expanded for experimental use to confirm genotype by allele sequencing and immunoblotting. MEF were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin supplemented with 1× non-essential amino acids and 1 mM sodium pyruvate.​

[0152] Generation of bone marrow-derived macrophages (BMDMs) Macrophages were cultured in complete medium containing DMEM, 10% (v / v) heat-inactivated FBS, 20% (v / v) L929 conditioned medium, 2.5% (v / v) HEPES, 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin, 2% sterile-filtered β-mercaptoethanol, 1× non-essential amino acids, and 1 mM sodium pyruvate. Bone marrow isolation and macrophage differentiation were modified from (Wells et al., 2004), and L929 conditioned medium was utilized as the source of M-CSF for differentiation. Briefly, using scissors and forceps, femurs and tibias were dissected from adult mice, and muscle tissue was carefully removed from the bones. The clean femurs and tibias were placed into a tissue culture dish containing complete medium. The ends of each bone were cut with scissors to expose the bone marrow. The bone marrow was flushed using a 10 ml syringe fitted with a 25-gauge needle containing complete medium. The medium containing the bone marrow was passed through a 70 μm cell strainer, and the progenitor cells were plated onto an uncoated 10 cm bacteriological plate containing 10 ml of complete medium. This was designated as day 0 of isolation. On day 3 after isolation, the macrophages were replenished with 5 ml of fresh complete medium. On day 7 after isolation, the macrophages were rinsed once with PBS and incubated with Versene for 5 minutes in a 37 °C, 5% CO2 tissue culture incubator. The macrophages were detached using a cell scraper and centrifuged at 1200 rpm for 5 minutes at room temperature. The Versene was aspirated, and the remaining cell pellet was resuspended in complete medium. The cell suspension was counted, and the cells were seeded in a 6-well format into tissue culture-treated dishes for experimental analysis at a final cell density of 1 million cells per well.

[0153] PBMC cell isolation and treatment PBMC cells were isolated from human blood according to an existing protocol (dx.doi.org / 10.17504 / protocols.io.bnhxmb7n) and pelleted by centrifugation at 1000 g for 2 minutes. The supernatant was discarded, and for washing, the PBMC pellet was resuspended in PBS containing 2% FBS. The suspension was centrifuged again at 1000 g for 2 minutes, and the PBMC pellet was resuspended in RPMI-1640 (Gibco) medium supplemented with 10% FBS. Next, the cells were seeded in 6-well plates and treated with the test compound at the indicated concentrations and for the indicated periods. After treatment, the cells were harvested into 2 ml Eppendorf tubes, centrifuged at 500 g for 2 minutes to pellet the cells, the supernatant was discarded, and the pellet was resuspended in 1 ml of PBS and centrifuged again at 500 g for 2 minutes. The PBMC pellet was added to a complete EDTA-free protease inhibitor cocktail (Sigma-Aldrich Cat#11836170001) and lysed using 60 μL of lysis buffer containing 50 mM Tris-HCl, pH 7.5, 1% (v / v) Triton X-100, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM NaF, 0.1% (v / v) 2-mercaptoethanol, 10 mM 2-glycerophosphate, 5 mM sodium pyrophosphate, 0.1 μg / ml microcystin-LR (Enzo Life Sciences), 270 mM sucrose, 0.5 mM DIFP (Sigma, Cat#D0879). DIFP is highly toxic and must be prepared as a 0.5 M stock solution in isopropanol in a fume hood. Next, the lysed cells were centrifuged at 1500 g for 15 minutes at 0 °C. The supernatant was recovered for analysis by quantitative immunoblotting. For long-term storage, the supernatant was snap-frozen and stored at -80 °C. The protein concentration of the cell lysate was measured using the Pierce™ BCA Protein Assay Kit (ThermoFisher).

[0154] Cell Culture, Treatment, and Lysis Subsequent culturing and passage of the cell line were performed using aseptic techniques within a CL1 or CL2 (for PBMC isolation) safety cabinet. All cells were incubated in a 37 °C incubator with 5% CO2. The cell line was periodically tested for mycoplasma contamination. For Western blot assays, cells were seeded in 6-well plates. After treatment with the test compound at the indicated concentrations and time periods, the medium was removed, the cells were washed with PBS, and then lysed in 100 μl of ice-cold complete lysis buffer containing 50 mM Tris HCl, pH 7.4, 1 mM EGTA, 10 mM 2-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 270 mM sucrose, supplemented with 1 μg / ml microcystin-LR, 1 mM sodium orthovanadate, a complete EDTA-free protease inhibitor cocktail (Roche), and 1% (v / v) Triton X-100. The cells were immediately placed on ice, scraped, and collected into a 1.5 ml Eppendorf tube. The cell lysate was incubated on ice for 10 minutes and then centrifuged at 15,000 g for 15 minutes at 4 °C. The cell pellet was discarded and the supernatant was collected for analysis by quantitative immunoblotting. For long-term storage, the supernatant was snap-frozen and stored at -80 °C. The protein concentration of the cell lysate was measured using the Bradford assay.

[0155] Quantitative immunoblotting Cell lysates containing 4×NuPAGE LDS sample buffer (NP0007) supplemented with 5% β-mercaptoethanol in a quarter volume were heated at 95°C for 5 minutes. Samples of 15 - 20 μg were loaded onto precast 4 - 12% Bis-Tris midi 20W or 26W gels (Thermofisher Scientific®, Cat# WG1402BOX or WG1403BOX) and resolved at 130V for 2 hours using NuPAGE MOPS SDS running buffer (Thermofisher Scientific®, Cat#NP0001-02). Proteins were transferred electrophoretically onto a 0.45 μm nitrocellulose membrane (GE Healthcare, Amersham Protran® Supported 0.45mm NC) at 90V for 90 minutes on ice in transfer buffer (48 mM Tris base and 39 mM glycine supplemented with 20% methanol). The transferred membrane was blocked at room temperature for 1 hour with 5% (w / v) non-fat dry milk dissolved in Tris-buffered saline with tween (TBS-T) (50 mM Tris base, 150 mM sodium chloride (NaCl), 0.1% (v / v) Tween-20). The membrane was washed 3 times with TBS-T and incubated overnight at 4°C in the primary antibody. Prior to secondary antibody incubation, the membrane was washed 3 times with TBS-T for 15 minutes each. The membrane was incubated with the secondary antibody for 1 hour at room temperature in the dark. Subsequently, the membrane was washed 3 times with TBS-T with 15-minute incubations for each wash, and protein bands were acquired by near-infrared fluorescence detection using an Odyssey CLx imaging system and quantified using Image Studio software. Graphs were created using Graphpad Prism version 8 software.

[0156] antibody Monoclonal rabbit LRRK2 Ser935 (Cat# UDD2) was purified by MRC PPU Reagents and Services at the University of Dundee and used at a final concentration of 1 μg / ml. Full LRRK2 (C-terminal) was from Antibodies Inc. / Neuromab (Cat# 75-253) and diluted 1:1000. The previously characterized (Lis et al., Biochem J. 2018 Jan 2;475(1):1-22.), MJFF monoclonal rabbit Rab10 pThr73 was purchased from Abcam Inc. (ab230261) and diluted 1:1000. Mouse monoclonal α-tubulin (#3873) was purchased from Cell Signaling Technology and used at 1:1000. Mouse monoclonal anti-Rab10 total antibody was purchased from Nanotools (#0680-100 / Rab10-605B11) and used at a final concentration of 1 μg / ml. Mouse monoclonal Hif-1α was purchased from R&D Systems (Cat# MAB1536) and diluted 1:1000. Mouse monoclonal Ubiquitin was purchased from Biolegend (Cat# 646302) and diluted 1:1000. Rabbit polyclonal PDE6D antibody was purchased from Novus Biologicals and used at a final concentration of 1:500. All rabbit and mouse primary antibodies were diluted with 5% (w / v) bovine serum albumin (BSA) dissolved in TBS-T (50 mM Tris base, 150 mM sodium chloride (NaCl), 0.1% (v / v) Tween20). Goat anti-mouse IRDye 800CW (#926-32210), goat anti-mouse IRDye 680LT (#926-68020), goat anti-rabbit IRDye 800CW (#926-32211), and goat anti-rabbit IRDye 680LT (#926-68021) IgG(H+L) secondary antibodies were from LI-COR and diluted 1:10,000 with 5% (w / v) milk in TBS-T.

[0157] Whole proteome sample preparation and MS analysis Wild-type MEFs were seeded in 10-cm tissue culture dishes at a density of 2 million cells per dish. Cells were treated 4 hours prior to harvest with 0.1% DMSO, 300 nM XL01126, or 300 nM cisXL01126 and harvested into 400 μl of complete lysis buffer supplemented with 1 μg / ml microcystin-LR, 1 mM sodium orthovanadate, a protease inhibitor cocktail without EDTA (Roche), and 1% (v / v) Triton X-100. Cell lysates were incubated on ice for 10 minutes and then subjected to three rounds of high-energy sonication for 15 cycles (30 seconds on, 30 seconds off) using a Diagenode Bioruptor. Cell lysates were centrifuged at 15,000 g for 15 minutes at 4 °C. The cell pellet was discarded and the supernatant was recovered for protein quantification using a BCA protein assay kit (Pierce #23225). 100 μg of cell lysate was utilized for global proteome analysis. Proteins in the cell lysate were reduced with 0.1 M Tris(2-carboxyethyl)phosphine (TCEP) (TEABC) diluted with 300 mM triethylammonium bicarbonate to a final concentration of 10 mM. Samples were incubated at 60 °C, 800 rpm for 30 minutes in a thermomixer. Samples were incubated to room temperature and subjected to alkylation with 0.4 M iodoacetamide (IAA) freshly dissolved in water. Samples were then incubated in the dark at room temperature, 800 rpm for 30 minutes in a thermomixer. Alkylation was quenched by the addition of 0.1 M TCEP dissolved in 300 mM TEABC to a final concentration of 5 mM. Samples were incubated at room temperature, 800 rpm for 20 minutes in a thermomixer. Sodium dodecyl sulfate (SDS) was added from a 20% (w / v) stock to a final concentration of 5% (w / v). Next, 12% (v / v) phosphoric acid was added to a final concentration of 1.2% (v / v). Samples were diluted 6-fold with S-trap wash buffer containing 90% (v / v) methanol diluted with 100 mM (v / v) TEAB pH 7.1 to the sample volume.

[0158] S-trap Purification and Digestion The sample was subjected to S-trap purification and the washing agent and other impurities were removed using an S-trap mini column (PROTIFI Cat# MSPPC02-MINI-80) placed in a 2 ml Eppendorf. The protein mixture was added to the column and centrifuged gently (1000 g / 1 minute / RT). The column was washed 4 times with 400 μl of S-trap buffer and centrifuged after each wash (1000 g / 1 minute / RT). The column was placed in a new 2 ml Eppendorf and 100 μl of 5 μg of trypsin / Lys-C newly dissolved in 50 mM TEAB, pH 8.5 was added. The column was centrifuged gently (200 g / 1 minute / RT), and the trypsin / Lys-C mixture was pipetted back onto the column. 100 μl of 50 mM TEAB, pH 8.5 was added directly to the 2 ml Eppendorf to cover all the digested peptides remaining in the tube. The S-trap column in the 2 ml Eppendorf was incubated at 47 °C for 1.5 hours without shaking and then incubated overnight at RT.

[0159] 80 μl of 50 mM TEAB was added to the S-trap column, and it was centrifuged, and the eluate was collected in a new 1.5 ml Eppendorf tube. 80 μl of 0.2% (v / v) formic acid was added to the column, and it was centrifuged, and the second eluate was stored together with the first eluate. 80 μl of 50% (v / v) acetonitrile diluted with 0.2% (v / v) formic acid was added to the column, and it was centrifuged, and the third eluate was stored together with the previous eluates. 500 ng of the digested peptides were taken for vacuum drying respectively to confirm that the digestion efficiency was >90%. The remaining peptides were divided in half (50 μg of peptides in each tube), vacuum dried, and stored at -80 °C before the continuation of tandem mass tag (TMT) labeling.

[0160] TMT Labeling 800 μg of the TMT mass tag reagent was dissolved in 80 μl of 100% (v / v) anhydrous acetonitrile to obtain a final concentration of 10 μg / μl. The resuspended TMT reagent was incubated at RT for 10 minutes, then vortexed and gently centrifuged (2000 g / 2 minutes / RT). 50 μg of lyophilized peptide was resuspended in a 50 μl mixture containing 38 μl of 50 mM TEAB and 8 μl of 100% (v / v) anhydrous acetonitrile. The resuspended peptide was sonicated for 10 minutes and then centrifuged at 17,000 g, RT for 10 minutes. The peptide was transferred to a new protein low-binding 1.5 ml Eppendorf tube. 20 μl of 10 μg / μl TMT reagent was added to the solubilized peptide, vortexed, gently centrifuged (2,000 g / 1 minute / RT), and incubated in a thermomixer at 800 rpm, RT for 2 hours. 50 μl of 50 mM TEAB was added to each reaction, followed by vortexing, gentle centrifugation (2,000 g / 1 minute / RT), and incubation in a thermomixer at 800 rpm, RT for an additional 10 minutes. 5 μl of each TMT-labeled sample was taken, vacuum dried, and injected into the MS to confirm that the labeling efficiency was >98%. The remaining reactions were stored at -80 °C until the labeling efficiency was confirmed. The TMT samples were thawed to RT, and the labeled reactions were quenched using the addition of 5 μl of 5% (v / v) hydroxylamine (dissolved in water from a 50% (v / v) stock solution). The samples were incubated in a thermomixer at 800 rpm, RT for 20 minutes. The quenched TMT-labeled samples were pooled and processed using C18 stage-tip desalting.

[0161] Pharmacokinetic (PK) investigation PK profiling was outsourced and commissioned by Shanghai ChemPartner Co., Ltd. All animal experiments conducted were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) and Office of Laboratory Animal Welfare (OLAW) guidelines. Six- to eight-week-old male C57BL / 6 mice purchased from Jihui Laboratory Animal Co., LTD were used in the study. XL01126 was formulated in 10% HP-β-CD in 50 mM citrate buffer pH = 3.0 at 1 mg / mL for intravenous injection and 3 mg / mL for IP and PO injection. For intravenous injection, 5 mg / kg of XL01126 was administered via the tail vein to 9 mice. For IP injection, 30 mg / kg of XL01126 was administered by intraperitoneal injection in mice. Animals were manually restrained at the indicated time points (0.083, 0.25, 0.5, 1, 2, 4, and 8 hours); approximately 110 μL of blood samples were collected into K2EDTA tubes via the facial vein. Three mice were used per time point, and as a result, a total of 21 mice were used per administration route. Blood samples were placed on ice and centrifuged at 2000 g for 5 minutes to obtain plasma samples within 15 minutes. Plasma, brain, and CSF samples were stored at approximately -70 °C until analysis. To 30 μL aliquots of plasma, 200 μL of internal standard (Glipozode, 40 ng / mL) in MeCN containing 5% citric acid was added. The mixture was then vortexed for 1 minute and then centrifuged at 5800 rpm for 10 minutes. The supernatant (100 μL) was transferred to a new plate. Solvent (1 μL) was injected into the LC-MS / MS. LC-MS / MS instrument used: SCIEX LC-MS / MS-49 (Triple Quad 6500+). Data were analyzed by WinNonLin and Microsoft Excel.

[0162] Fluorescence polarization assay The FP competition binding assay was carried out according to the previously described method (Bond et al, J. Med. Chem. 2021, 64 (20), 15477-15502.). All measurements were obtained using a PHERstar (BMG LABTECH) plate reader incorporating an FP filter with excitation and emission wavelengths set at 485 nm and 520 nm (nM), respectively. Each well of a 384-well plate (Coring 3575) contained 10 nM of VCB protein, 5 nM of FAM-labeled HIF-1α peptide (FAM-DEALAHypYIPMDDDFQLRSF, "JC9"), and the concentration of PROTAC was lowered (14 concentrations by 2-fold serial dilution starting from 250 μM) with FP assay buffer (100 mM Bis-Tris propane, 100 mM NaCl, 1 mM TCEP, pH 7) having a final DMSO concentration of 5%. Control wells containing VCB and JC9 without compound were set to the maximum signal (replaced with zero). Additionally, JC9-containing control wells without protein were set to the minimum signal. The control values were used to obtain the percentage of replacement graphed against Log[compound]. For each titer measurement, mean IC 50 values were measured using non-linear regression analysis with Prism (v.9.3.1). K i values were calculated inversely from the K d (1.5 nM - 3.4 nM) of JC9 and the fitted IC 50 values as previously described.

[0163] NanoBRET Target Engagement Assay For VHL and LRRK2 engagement experiments in live and permeabilized cells, HEK293 cells were transfected with a VHL-NanoLuc fusion vector (Promega, N275A) or an LRRK2-NanoLuc fusion vector (Promega, NV3401) according to the Promega protocol and seeded into white 384-well plates (Corning 3570) at a density of 6000 cells / well. To measure NanoBRET in permeabilized cells, the cells were treated with 50 μg / mL digitonin (Sigma, D141), 125 nM VHL tracer / 125 nM LRRK2 tracer, serially diluted concentrations of the test compound (12 concentrations by 2-fold serial dilution starting from 33 μM), and NanoBRET NanoGlo Substrate (Promega) at a concentration recommended by the manufacturer's protocol. In the maximum signal control sample (DMSO control), DMSO was added instead of the test compound. In the minimum signal control sample (no-tracer control), DMSO and tracer dilution buffer were used to replace the test compound and tracer, respectively. The filtered luminescence was measured within 10 minutes after substrate addition using a GloMax Discover microplate reader (Promega) equipped with a 450 nm bandpass filter (donor) and a 600 nm longpass filter (acceptor) or a PHERAstar (BMG LABTECH) plate reader. To measure NanoBRET in live cells, the cells were treated with 250 nM VHL tracer / 500 nM LRRK2 tracer, serially diluted concentrations of the test compound (12 concentrations by 2-fold serial dilution starting from 33 μM), and incubated for 2 hours at 37 °C in an incubator. The plates were then cooled and NanoBRET NanoGlo Substrate and Extracellular NanoLuc Inhibitor (Promega, N2160) were added before NanoBRET readings in the same mode as for permeabilized cells using a plate reader.The NanoBRET ratio for each well was expressed in milliBRET according to the following equation: mBRET = [(signal at 610 nM / signal at 450 nM) - (signal at 610 nM of the no-tracer control / signal at 450 nM of the no-tracer control)] × 1000. The fractional occupancy was calculated according to the following equation: fractional occupancy = (mBRET of the tested compound - mBRET of the no-tracer control) / (mBRET of the DMSO control - mBRET of the no-tracer control).

[0164] NanoBRET-based ternary complex and cooperativity assay HEK293 cells were transfected with the LRRK2-NanoLuc fusion vector (Promega, NV3401) according to the Promega protocol and seeded into white 384-well plates (Corning 3570) at a density of 6000 cells / well. Next, the cells were treated with 50 μg / mL digitonin, 125 nM LRRK2 tracer, decreasing concentrations of the test compound (11 concentrations by 2-fold serial dilution starting from 10 μM), a mixture of the test compound and VCB (11 concentrations by 2-fold serial dilution starting from 10 μM for the compound. The first six concentrations of VCB were by 2-fold dilution starting from 32 μM, and the last five concentrations of VCB were maintained at 1 μM), and NanoBRET NanoGlo Substrate (Promega) at the concentration recommended by the manufacturer's protocol. For the maximum signal control sample (DMSO control), DMSO was added in place of the test compound. For the minimum signal control sample (no-tracer control), DMSO and LRRK2 tracer dilution buffer were used to replace the test compound and LRRK2 tracer, respectively. The filtered luminescence was measured within 10 minutes after substrate addition using a PHERAstar (BMG LABTECH) plate reader equipped with a 450 nm bandpass filter (donor) and a 600 nm longpass filter (acceptor). The fractional occupancy was calculated according to the following equation: fractional occupancy = (mBRET of the tested compound - mBRET of the no-tracer control) / (mBRET of the DMSO control - mBRET of the no-tracer control).

[0165] Bodipy576 / 589 labeling of VCB VCB was labeled with Bodipy576 / 589 according to the previously reported protocol, 76. Briefly, the VCB complex was mixed with Bodipy576 / 589 NHS ester at a molar ratio of 20:1 and incubated for 2 h at room temperature (in the dark) in reaction buffer (0.1 M sodium phosphate, 75 mM KOAc, 2 mM DTT, pH 7.4). The reaction was quenched by diluting 10-fold with reaction buffer and unreacted dye was removed using a PD-10 MiniTrap desalting column (GE Healthcare) equilibrated with 100 mM BisTris pH 7.0, 100 mM NaCl, 1 mM DTT, pH 7. The eluted labeled protein solution was collected and concentrated using a Pierce Concentrator, 3K MWCO (Thermo scientific).

[0166] NanoBRET ternary complex formation assay Hek293 cells were transfected with the LRRK2-NanoLuc vector (Promega, NV3401) for 24 hours and then resuspended in OptiMEM medium without phenol red (Life Technologies). Next, the cells were seeded into white 384-well plates (Corning 3570) at a density of 6000 cells / well. Digitonin solution (final concentration 50 μg / mL), serial dilutions of the test PROTAC (11 concentrations by 2-fold dilution starting from 33 μM), or DMSO, and the VCB protein labeled with Bodipy576 / 589 (final concentration 0.5 μM) were added respectively. NanoBRET NanoGlo Substrate (Promega, N2160) was added to each well, and then NanoBRET readings were performed using a PHERAstar (BMG LABTECH) plate reader equipped with a 50 nm bandpass filter (donor) and a 600 nm long pass filter (acceptor). The NanoBRET ratio of each well was expressed in milliBRET according to the following equation: mBRET = [ (signal at 610 nM / signal at 450 nM) - (signal at 610 nM of the no-tracer control / signal at 450 nM of the no-tracer control) ] × 1000. The background signal shown by the DMSO control samples was subtracted from each sample.

[0167] Chemistry-Materials and Methods Commercially available chemicals were purchased from Apollo Scientific, Sigma-Aldrich, Fluorochem, and Enamine and used without further purification. All solvents used in the reactions were anhydrous. LC-MS was performed on a Shimadzu HPLC / MS 2020 equipped with a Hypersil Gold column (1.9 μm 50×2.1 mm), a photodiode array detector, and an ESI detector. Samples were eluted at a flow rate of 0.8 mL / min using a 3-minute gradient of 5-95% acetonitrile in water containing 0.1% formic acid. Flash column chromatography was performed on a Teledyne ISCO Combiflash Companion incorporating a disposable normal-phase RediSep Rf column (230-400 mesh, 40-63 mm, SiliCycle). Preparative HPLC purification was performed on a Gilson Preparative HPLC system equipped with a Waters X-Bridge C18 column (100 mm×19 mm and 5 μm particle size) using a gradient of 5-95% acetonitrile in water containing 0.1% formic acid over 10 minutes at a flow rate of 25 mL / min. Characterization of the compounds using NMR was performed on either a Bruker 500 Ultrashield or a Bruker Ascend 400 spectrometer. The 1 1H NMR and 13The 13C NMR reference solvents are d1-CDCl3 (δH = 7.26 ppm / δC = 77.16 ppm), d4-CD3OD (δH = 3.31 ppm / δC = 49.00 ppm), or d6-DMSO (δH = 2.50 ppm / δC = 39.52 ppm). Signal patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), quintet (quint.), multiplet (m), broad (br), or combinations of the listed splitting patterns. Coupling constants (J) are measured in Hertz (Hz). HRMS was performed using a Bruker MicroTOF II focus ESI Mass Spectrometer connected in parallel with a Dionex Ultimate 3000 RSLC system equipped with a diode array detector and a Waters XBridge C18 column (50 mm × 2.1, 3.5 μm particle size). All final compounds are >95% pure according to HPLC.

[0168] Synthesis Basic Procedure 1 (Boc Protection) To a solution of benzylamine compound (1 eq) and TEA (2 eq) in DCM, Boc2O (1.1 eq) was added at 0 °C, and the resulting mixture was stirred at room temperature for 4 h or overnight. DCM was added, and the mixture was washed with 1 N aqueous HCl, saturated aqueous NaHCO3, water, and brine, respectively. Next, the recovered organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain the Boc-protected compound.

[0169] Basic Procedure 2 (Pd(OAc)2-Mediated Coupling) Under nitrogen protection, Pd(OAc)2 (0.1 eq) was added to a mixture of the bromine compound (1 eq), KOAc (2 eq), and 4-methylthiazole (2 eq) in DMA. The resulting mixture was heated to 110 °C and stirred at this temperature overnight. Next, the reaction mixture was diluted with EtOAc and filtered through a pad of celite. The collected filtrate was washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, which was purified by flash column chromatography on silica (0% to 100% EtOAc in heptane) to obtain the desired compound.

[0170] Basic Procedure 3 (Condensation using Boc deprotecting group and hydroxyl proline) To a solution of the Boc-protected (1 eq) compound in DCM (same volume as 4N HCl in dioxane), 4N HCl in dioxane (8 eq) was added. The resulting mixture was stirred at room temperature for 1 hour and then concentrated to obtain a solid, which was washed with diethyl ether and dried under reduced pressure to obtain the HCl salt. The salt was suspended in DCM, followed by the addition of TEA (4 eq), (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (1 eq) or (2S,4S)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (1 eq), and HATU (1.05 eq). After stirring at room temperature for 2 hours, the resulting mixture was diluted with DCM, washed with water and brine, dried over sodium sulfate, filtered, and concentrated to obtain a residue, which was purified by flash column chromatography on silica (0% to 100% EtOAc in heptane) to obtain the desired compound.

[0171] Basic Procedure 4 (Condensation of Boc deprotecting group and Fmoc-S-trityl-L-penicillamine) To a stirred solution of the Boc-protected substrate (1 eq) in DCM was added 4N HCl in dioxane (same volume as DCM, 10 eq). After stirring at room temperature for 1 h, the mixture was concentrated under reduced pressure to give a solid, which was washed with diethyl ether to afford the HCl salt quantitatively as a pale yellow solid. The solid (1 eq) was dissolved in DMF, TEA (1.5 eq) was added to neutralize HCl, and subsequently, a mixed solution of Fmoc-S-trityl-L-penicillamine (1 eq), HATU (1.1 eq), and TEA (2 eq) in DMF was added dropwise at 0 °C. The resulting mixture was stirred at 0 °C for 2 h, then EtOAc and water were added. The aqueous phase was extracted with EtOAc (3×), the combined organic phases were washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by flash column chromatography on silica (0% to 100% EtOAc in heptane) to afford the desired compound.

[0172] Basic Procedure 5 (Fmoc Deprotection) To a solution of the Fmoc-protected compound (1 eq) in DCM was added piperidine (5 eq) dropwise. The resulting mixture was stirred at room temperature for 2 h, then concentrated under reduced pressure to give a residue, which was purified by flash column chromatography on silica (0% to 10% methanol in DCM containing 0.7M ammonia) to afford the desired product.

[0173] Basic Procedure 6 (Condensation with 1-Fluorocyclopropane-1-carboxylic Acid) To a solution of the amine compound (1 eq), TEA (2 eq), and HATU (1.05 eq) in DMF was added 1-fluorocyclopropane-1-carboxylic acid (1 eq). After stirring at room temperature overnight, water was added to the mixture, which was extracted with EtOAc (3×), and the combined organic phases were washed with water and brine respectively, dried over sodium sulfate, filtered, concentrated to give a residue, which was purified by flash column chromatography on silica (0% to 100% EtOAc in PE) to afford the desired product.

[0174] Basic Procedure 7 (Trityl Group Deprotection) To a solution of trityl-protected VHL ligand (1 eq) in DCM, triisopropylsilane (TIPS, 10 eq) and trifluoroacetic acid (10 eq) were added at 0 °C. After stirring at 0 °C for 10 minutes, the mixture was concentrated under reduced pressure to obtain a residue, which was purified by flash column chromatography on silica (0% to 10% methanol in DCM) to give the desired thiol compound.

[0175] Basic Procedure 8 (Substitution Reaction) To a solution of tosylated or brominated compound (1 eq) in THF, thiol compound (1 eq) and DBU (6 eq) were added. After stirring overnight at room temperature, the reaction mixture was concentrated to obtain a residue, which was purified by preparative HPLC under acidic conditions (5 to 95% CH3CN in 0.1% aqueous HCO2H) to give the desired compound.

[0176] Exemplary Synthesis of Exemplified Compound 1 (SD13) [Chemical Structure]

[0177] [Chemical Structure] 2,5-Dichloro-N-methylpyrimidin-4-amine To a stirred solution of 2,4,5-trichloropyrimidine (5.8 g, 31.62 mmol) in anhydrous THF (100 mL) was added DIPEA (6.05 ml, 34.78 mmol), followed by dropwise addition of methylamine (33% solution in ethanol, 4.33 mL, 34.78 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h, then warmed to room temperature (RT) and concentrated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (0% to 50% EtOAc in petroleum spirit) to afford 2,5-dichloro-N-methylpyrimidin-4-amine (5.26 g, 29.54 mmol, 93% yield) as a white solid. 1 H NMR (500 MHz, CDCl3) δ 8.00 (s, 1H), 5.55 (s, 1H), 3.10 (d, J = 5.0 Hz, 3H); 13 C NMR (126 MHz, CDCl3) δ 159.58, 158.77, 153.37, 113.49, 28.28; and LC-MS, ESI + , m / z 178 [M+H] + 。

[0178]

Chemical formula

[0179]

Chem.

[0180]

Chem.

[0181] Exemplary Synthesis of Exemplified Compound 2 (SD12)

Chem.

[0182]

Chem.

[0183]

Chem.

[0184] Exemplary synthesis of exemplary compound 3 (SD79)

Chemical Structure

[0185]

Chemical Structure

[0186] [Chemical formula] (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methanone HCl salt To a solution of tert-butyl 4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazine-1-carboxylate (2.52 g, 5.28 mmol) in a mixture of DCM and MeOH 9:1 (30 ml) was added a 4 M solution of HCl in dioxane (5.28 ml, 21.12 mmol) at RT. The reaction mixture was stirred overnight, diluted with Et2O (200 ml), The precipitate was collected by a glass filter, washed with Et2O (100 ml), and dried overnight to give (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methanone as the HCl salt (2.13 g, 5.17 mmol, 98%). 1 H NMR (500 MHz, DMSO-d6) δ 9.65 (s, 2H), 8.72 (s, 1H), 8.28 (s, 1H), 8.13 (d, J = 8.20 Hz, 1H), 7.20 (d, J = 1.70 Hz, 1H), 7.11 (dd, J1 = 1.70 Hz, J2 = 8.20 Hz, 1H), 3.91 (s, 3H), 3.75 (br s, 4H), 3.15 (br s, 4H), 2.99 (d, J = 4.6 Hz, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 169.28, 159.06, 152.3, 150.3, 127.80, 122.0, 120.02, 111.07, 105.06, 65.34, 56.67, 43.0, 29.0. LC-MS, ESI + , m / z 377.15 [M+H] + .

[0187] [Chemical formula] 14-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonate To a solution of (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methanone (40 mg, 0.097 mmol) in acetone (3 mL) were added pentaethylene glycol di(p-toluenesulfonate) (159.3 mg, 0.292 mmol) and K2CO3 (67 mg, 0.49 mmol). After stirring overnight at 50 °C, the mixture was diluted with DCM, washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give the crude product, which was purified by flash column chromatography on silica gel (0% to 10% methanol in DCM) to afford 14-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonate as a white solid (30 mg, 41% yield). 1 H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 8.7 Hz, 1H), 7.90 (s, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.61 (s, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.02 - 6.96 (d, J = 7.0 Hz, 2H), 5.32 (q, J = 4.3 Hz, 1H), 4.19 - 4.11 (m, 2H), 3.91 (s, 3H), 3.77 - 3.50 (m, 20H), 3.09 (d, J = 4.8 Hz, 3H), 2.61 (t, J = 5.5 Hz, 2H), 2.52 (s, 4H), 2.43 (s, 3H); 1313C NMR (101 MHz, CDCl3) δ 170.53, 158.67, 157.92, 152.75, 147.50, 144.86, 133.24, 131.43, 129.92, 128.07, 127.99, 120.26, 116.85, 109.68, 105.61, 70.87, 70.76, 70.71, 70.65, 70.52, 69.32, 69.03, 68.81, 57.84, 55.98, 53.77, 28.18, 21.72. LC-MS, ESI - , m / z 749 [M-H] - 。

[0188]

Chem.

[0189] Exemplary synthesis of Exemplary Compound 4 (SD82) [Chemical formula]

[0190] (2S,4R)-1-((R)-3-((3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (4, SD82) To a solution of 3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl 4-methylbenzenesulfonate (7.36 mg, 0.0125 mmol) and (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-carboxamide)-3-mercapto-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Girardini et al., Bioorg. Med. Chem. 2019; 27(12):2466 - 2479.) (6.675 mg, 0.0125 mmol) in DMF (0.2 ml), K2CO3 (5 mg, 0.036 mmol) and KI (0.2 mg, 0.0125 mmol) were added. After stirring at 80 °C overnight, the resulting mixture was cooled and purified by preparative HPLC under acidic conditions (in 0.1% aqueous HCO2H, 10 to 80% CH3CN) to obtain SD82 (3.01 mg, 25% yield). 11H NMR (500 MHz, MeOD-d4) δ 8.87 (s, 1H), 8.60 (d, J = 8.3 Hz, 1H), 7.88 (s, 1H), 7.51 - 7.39 (m, 4H), 7.10 - 7.00 (m, 2H), 4.94 (s, 1H), 4.63 (t, J = 8.4 Hz, 1H), 4.58 (d, J = 15.6 Hz, 1H), 4.53 (s, 1H), 4.37 (d, J = 15.7 Hz, 1H), 3.98 (s, 3H), 3.94 - 3.86 (m, 2H), 3.78 - 3.62 (m, 4H), 3.06 (s, 3H), 2.76 - 2.55 (m, 8H), 2.49 (s, 3H), 2.29 (dd, J = 12.9, 7.8 Hz, 1H), 2.12 (ddd, J = 13.3, 9.2, 4.4 Hz, 1H), 1.81 - 1.72 (m, 2H), 1.46 (d, J = 10.8 Hz, 6H), 1.40 - 1.28 (m, 4H). HRMS (ESI + ) m / z, C 45 H 56 ClFN 10 O6S2: Calculated for 951.3571 [M + H] + , found 951.3565.

[0191] Exemplary Synthesis of Exemplified Compound 5 (SD75)

Chemical Structure

[0192] Exemplary synthesis of Exemplary Compound 6 (SD74) [Chemical Structure] (2S,4R)-1-((R)-1-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-17-(1-fluorocyclopropane-1-carboxamide)-16,16-dimethyl-3,6,9,12-tetraoxa-15-thiaoctadecane-18-yl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6, SD74) SD74 was prepared according to Basic Procedure 8. 7.55 mg of SD74 was obtained from 7.0 mg of the thiol compound, with a 51% yield. 1 H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.54 (d, J = 8.7 Hz, 1H), 7.92 (s, 1H), 7.65 (s, 1H), 7.42 - 7.30 (m, 5H), 7.19 (d, J = 7.8 Hz, 1H), 7.03 - 6.96 (m, 2H), 5.39 - 5.27 (m, 1H), 4.85 (d, J = 8.2 Hz, 1H), 4.76 (t, J = 7.7 Hz, 1H), 4.59 - 4.33 (m, 3H), 3.92 (s, 3H), 3.91 - 3.82 (m, 2H), 3.82 - 3.64 (m, 5H), 3.65 - 3.44 (m, 15H), 3.10 (d, J = 4.8 Hz, 3H), 2.85 - 2.05 (m, 8H), 2.51 (s, 3H), 2.46 - 2.39 (m, 1H), 2.25 - 2.15 (m, 1H), 1.39 - 1.20 (m, 10H); 1313C NMR (101 MHz, CDCl3) δ 170.98, 170.66, 170.25 (d, J = 20.48 Hz), 169.96, 158.74, 157.91, 152.74, 150.39, 148.65, 147.61, 138.31, 131.69, 131.10, 129.62, 129.08, 128.33, 126.03, 120.40, 116.97, 109.76, 105.72, 79.43, 70.72, 70.68, 70.63, 70.55, 70.25, 69.88, 68.36, 59.35, 57.69, 56.51, 56.06, 55.94, 53.61, 48.10, 43.19, 36.91, 28.72, 28.25, 25.97, 25.19, 21.48, 16.22, 14.00, 13.91; HRMS (ESI + ) m / z, C 52 H 70 ClFN 10 O 10 S2: Calculated for 1113.4463 [M + H] + , found 1113.4220.

[0193] Exemplary Synthesis of Exemplary Compound 7 (SD100)

Chem.

[0194]

Chem.

[0195]

Chem.

[0196] lFN O6S2 1013.3727 [M + H] , found 1013.3849.

[0196] Exemplary Synthesis of Exemplary Compound 8 (SD11)

Chem.

[0197]

Chem.

[0198]

Chem.

[0199]

Chemical formula

[0200] Exemplary synthesis of Exemplified Compound 9 (SD10)

Chemical Structure

[0201] Exemplary Synthesis of Exemplary Compound 10 (SD113) [Chemical Formula] (S)-N-((S)-2-((S)-2-(4-(3-(2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)benzoyl)thiazol-2-yl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)-2-(methylamino)propenamide HCl salt (10, SD113) To a solution of 2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (8.29 mg, 0.0125 mmol) in DMF (0.2 ml), tert-butyl ((S)-1-(((S)-1-cyclohexyl-2-((S)-2-(3-hydroxybenzoyl)thiazol-2-yl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (7.5 mg, 0.0125 mmol) (Shibata et al., 2018), K2CO3 (5 mg, 0.036 mmol), and KI (0.6 mg, 0.00375 mmol) were added. The resulting mixture was heated to 100 °C and stirred at 100 °C overnight. The mixture was filtered through a 0.45 μm filter and purified by preparative HPLC under acidic conditions (10 to 85% CH3CN in 0.1% aqueous HCO2H) to give tert-butyl ((S)-1-(((S)-2-((S)-2-(4-(3-(2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)benzoyl)thiazol-2-yl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (3 mg, 22% yield); HRMS (ESI +) m / z, C 54 H 74 ClN 10 O 10 Calculated value for S 1089.4993 [M + H] + , found value 1089.5198.

[0202] To a solution of tert-butyl ((S)-1-(((S)-2-((S)-2-(4-(3-(2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)benzoyl)thiazol-2-yl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (3 mg, 0.0028 mmol) in 0.2 ml of DCM was added an aqueous solution of 4N HCl in 1,4-dioxane (0.2 ml). The resulting mixture was stirred at room temperature for 2 hours and then concentrated to give SD113 as the HCl salt. 11H NMR (500 MHz, DMSO-d6) δ 8.48 (s, 1H), 8.39 (d, J = 8.3 Hz, 1H), 8.25 - 8.19 (m, 3H), 7.96 (s, 1H), 7.69 - 7.60 (m, 3H), 7.45 (t, J = 8.0 Hz, 1H), 7.34 (q, J = 4.3 Hz, 1H), 7.27 - 7.21 (m, 1H), 7.01 (d, J = 1.6 Hz, 1H), 6.96 (dd, J = 8.3, 1.6 Hz, 1H), 5.39 (dd, J = 8.0, 3.0 Hz, 1H), 4.55 - 4.44 (m, 1H), 4.18 - 4.15 (m, 2H), 3.89 (s, 3H), 3.82 - 3.76 (m, 4H), 3.62 - 3.58 (m, 4H), 3.55 - 3.51 (m, J = 7.6, 4.5 Hz, 6H), 3.30 (dd, J = 13.7, 6.8 Hz, 1H), 2.90 (d, J = 4.6 Hz, 3H), 2.49 (d, J = 5.9 Hz, 1H), 2.42 (s, 4H), 2.29 (s, 3H), 2.27 - 2.16 (m, 2H), 2.08 - 1.99 (m, 2H), 1.72 - 1.59 (m, 4H), 1.55 (d, J = 9.7 Hz, 2H), 1.19 (d, J = 6.9 Hz, 3H), 1.15 - 0.93 (m, 6H). HRMS (ESI + ) m / z, C 49 H 65 ClN 10 O8S: Calculated for 989.4468 [M + H] + , found 989.4537.

[0203] Exemplary synthesis of Exemplified Compound 11 (SD112)

Chemical Structure

[0204] To a solution of tert-butyl ((S)-1-(((S)-2-((S)-2-(4-(3-((14-(4-((4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl)oxy)benzoyl)thiazol-2-yl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (3.8 mg, 0.0028 mmol) in 0.2 ml of DCM was added an aqueous solution of 4N HCl in 1,4-dioxane (0.2 ml). The resulting mixture was stirred at room temperature for 2 hours and then concentrated to give SD112 as the HCl salt. 11H NMR (500 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.40 (d, J = 8.3 Hz, 1H), 8.27 (s, 2H), 8.06 (d, J = 8.7 Hz, 1H), 7.96 (s, 1H), 7.70 - 7.60 (m, 3H), 7.46 (t, J = 8.0 Hz, 1H), 7.34 (q, J = 4.4 Hz, 1H), 7.29 - 7.21 (m, 1H), 7.02 (d, J = 1.6 Hz, 1H), 6.97 (dd, J = 8.3, 1.6 Hz, 1H), 5.39 (dd, J = 8.0, 2.9 Hz, 1H), 4.49 (dd, J = 8.5, 7.1 Hz, 1H), 4.18 - 4.13 (m, 2H), 3.89 (s, 3H), 3.82 - 3.78 (m, 2H), 3.77 - 3.74 (m, 2H), 3.60 - 3.58 (m, 2H), 3.55 - 3.52 (m, 2H), 3.52 - 3.46 (m, 15H), 3.12 (q, J = 6.8 Hz, 1H), 2.90 (d, J = 4.6 Hz, 3H), 2.48 (d, J = 5.8 Hz, 1H), 2.42 (s, 4H), 2.30 - 2.24 (m, 1H), 2.22 (s, 3H), 2.20 - 2.15 (m, 1H), 2.07 - 1.99 (m, 2H), 1.74 - 1.61 (m, 4H), 1.55 (d, J = 10.2 Hz, 2H), 1.14 (d, J = 6.9 Hz, 3H), 1.12 - 0.92 (m, 6H); HRMS (ESI + ) m / z, C 53 H 73 ClN 10 O 10 Calculated for 1077.4993 [M + H] + , found 1077.5042.

[0205] Exemplary Synthesis of Exemplified Compound 12 (SD114)

Chem.

[0206] To a solution of tert-butyl ((S)-1-(((S)-2-((S)-2-(4-(3-(3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propoxy)benzoyl)thiazol-2-yl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (4 mg, 0.0039 mmol) in 0.2 ml of DCM, an aqueous 4N HCl solution in 1,4-dioxane (0.2 ml) was added. The resulting mixture was stirred at room temperature for 2 hours and then concentrated to obtain SD114 as the HCl salt. 1 H NMR (500 MHz, DMSO-d6) δ 8.50 (s, 1H), 8.41 (d, J = 8.3 Hz, 1H), 8.06 (br, s, 1H), 7.97 (s, 1H), 7.73 - 7.63 (m, 3H), 7.51 - 7.41 (m, 1H), 7.35 (q, J = 4.3 Hz, 1H), 7.27 - 7.20 (m, 1H), 7.03 (d, J = 1.5 Hz, 1H), 6.98 (dd, J = 8.3, 1.6 Hz, 1H), 5.40 (dd, J = 7.9, 2.7 Hz, 1H), 4.52 - 4.44 (m, 1H), 4.10 (t, J = 6.2 Hz, 2H), 3.90 (s, 3H), 3.85 - 3.77 (m, 4H), 3.52 - 3.47 (m, 4H), 2.91 (d, J = 4.6 Hz, 3H), 2.48 (d, J = 7.2 Hz, 1H), 2.46 - 2.35 (m, 4H), 2.31 - 2.15 (m, 4H), 2.14 - 2.08 (m, 1H), 2.08 - 2.00 (m, 2H), 1.98 - 1.88 (m, 2H), 1.72 - 1.56 (m, 4H), 1.56 - 1.49 (m, 2H), 1.19 - 0.86 (m, 9H). HRMS (ESI + ) m / z, C 46 H 59 ClN 10Calculated value for O6S: 915.4101 [M + H] + , measured value 915.4190.

[0207] Exemplary synthesis of Exemplary Compound 13 (XL01078B)

Chemical Structure

[0208]

Chemical Structure

[0209] [Chemical formula] 3-Methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoic acid To a solution of 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (1.0 g, 4.72 mmol) in a mixture of dioxane and water (20 ml:20 ml), 4-amino-3-methoxybenzoic acid (0.87 g, 5.20 mmol) was added, followed by a solution of 4M HCl in dioxane (1.3 ml, 5.20 mmol). After refluxing at 100 °C for 2 hours, the mixture was cooled to room temperature and a white solid was precipitated. The solid was filtered, washed with water and dried under reduced pressure to give 3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoic acid (1.25 g, 77% yield) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 12.71 (br, 1H), 9.19 (s, 1H), 8.44 (s, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.07 (br, 1H), 7.63 (dd, J = 8.4, 1.7 Hz, 1H), 7.57 (d, J = 1.7 Hz, 1H), 3.95 (s, 3H), 2.99 (d, J = 4.4 Hz, 3H). 19 F NMR (471 MHz, DMSO-d6) δ -61.32. 13 C NMR (126 MHz, DMSO-d6) δ 167.31, 158.41, 156.47, 149.50, 149.21, 131.41, 126.51, 124.00 (q, J = 270.37 Hz), 122.85, 120.39, 111.79, 99.33, 56.55, 29.19. LC-MS, ESI + , m / z 343 [M+H] + .

[0210] [Chemical formula] tert-Butyl 4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazine-1-carboxylate To a solution of 3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoic acid (0.98 g, 2.58 mmol) in DMF (15 mL) were added HOBt (0.42 g, 3.09 mmol), EDCl (0.59 g, 3.09 mmol), 1-Boc-piperazine (0.51 g, 2.73 mmol), and DIPEA (1.8 mL, 10.32 mmol) at room temperature. After stirring at room temperature for 16 h, the mixture was diluted with water (30 mL) and extracted with EtOAc (100 mL). The organic layer was washed with brine (15 mL), concentrated to give a residue, which was purified by flash column chromatography on silica (0% to 100% EtOAc in DCM) to afford tert-butyl 4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazine-1-carboxylate (1.12 g, 2.19 mmol, 85% yield). 1 H NMR (500 MHz, CDCl3) δ 8.60 (d, J=8.2 Hz, 1H), 8.21 (s, 1H), 7.85 (s, 1H), 7.04 (m, 2H), 5.26 (m, 1H), 3.97 (s, 3H), 3.65 (br s, 4H), 3.49 (br s, 4H), 3.14 (d, J = 4.7 Hz, 3H), 1.50 (s, 9H). 19 F NMR (471 MHz, CDCl3) δ - 61.52. 1313C NMR (101 MHz, CDCl3) δ 170.73, 162.62, 160.80, 159.70, 154.67 (q, J = 5.0 Hz), 147.93, 130.93, 128.54, 125.05 (q, J = 267 Hz), 120.13, 117.89, 109.73, 99.56, 80.43, 56.03, 43.93, 28.50, 28.45; LC-MS, ESI-, m / z 509.2 [M-H]-.

[0211] [Chem.] (3-Methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)(piperazin-1-yl)methanone HCl salt A solution of tert-butyl 4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazine-1-carboxylate (1.12 g, 2.19 mmol) in a mixture of DCM and MeOH 9:1 (20 ml) was treated with a solution of 4N HCl in dioxane (2.2 ml, 8.76 mmol) at room temperature. After stirring overnight at room temperature, the reaction mixture was diluted with Et2O (100 ml) to precipitate a white solid, which was filtered, washed with Et2O (50 ml), and air-dried overnight to give (3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)(piperazin-1-yl)methanone HCl salt (0.97 g, 2.17 mmol, 99%) as a white powder. 11H NMR (500 MHz, DMSO-d6) δ 9.81 (s, 1H), 9.74 (s, 2H), 8.51 (s, 1H), 8.44 (s, 1H), 8.14 (d, J = 6.0 Hz, 1H), 7.21 (d, J = 1.6 Hz, 1H), 7.11 (dd, J =1.6 Hz, 8.20 Hz,1H), 3.91 (s, 3H), 3.76 (br s, 4H), 3.14 (br s, 4H), 2.99 (d, J = 4.4 Hz, 3H). 19 19F NMR (471 MHz, DMSO-d6) δ - 61.54. 13 13C NMR (126 MHz, DMSO-d6) δ 169.5, 158.2, 154.6, 150.4, 132.16, 127.53, 123.3 (q, J = 269 Hz), 119.97, 111.09, 99.4, 65.5, 56.7, 42.9, 29.7. LC-MS, ESI + , m / z 411 [M+H] + 。

[0212]

Chem.

[0213]

Chem.

[0214] Exemplary Synthesis of Exemplified Compound 14 (XL01072)

Chemical Structure

[0215]

Chemical Structure

[0216]

Chem.

[0217] Exemplary Synthesis of Exemplified Compound 15 (XL01070B)

Chemical Structure

[0218]

Chemical Structure

[0219]

Chem.

[0220] Exemplary synthesis of Exemplary Compound 16 (XL01119) [Chemical Structure]

[0221] [Chemical Structure] tert-Butyl (S)-(1-(4-bromophenyl)ethyl)carbamate was prepared according to Basic Procedure 1. 3.6 g of tert-Butyl (S)-(1-(4-bromophenyl)ethyl)carbamate was obtained from 4.17 g of the amine compound, with a yield of 58%. 1 H NMR (400 MHz, CDCl3) δ 7.51 - 7.44 (m, 2H), 7.20 (d, J = 8.4 Hz, 2H), 4.76 (s, 2H), 1.48 - 1.38 (m, 12H).

[0222] [Chemical Structure] tert-Butyl (S)-(1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamate was prepared according to Basic Procedure 2. 600 mg of tert-Butyl (S)-(1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamate was obtained from 1.03 g of the bromine compound, with a yield of 55%. 1 H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.47 - 7.41 (m, 2H), 7.38 (d, J = 8.3 Hz, 2H), 4.83 (s, 2H), 2.56 (s, 3H), 1.50 (d, J = 6.6 Hz, 3H), 1.46 (s, 9H).

[0223] [Chemical Structure] tert-Butyl (2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-carboxylate was prepared according to Basic Procedure 3. 380 mg of tert-butyl (2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-carboxylate was obtained from 337 mg of tert-butyl (S)-(1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamate in 83% yield. 1 H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.45 - 7.37 (m, 4H), 5.13 (s, 1H), 4.49 (d, J = 35.0 Hz, 2H), 3.75 - 3.35 (m, 2H), 2.55 (s, 3H), 1.94 (d, J = 3.8 Hz, 1H), 1.62 (s, 1H), 1.56 - 1.33 (m, 12H).

[0224] [Chemical formula] (9H-Fluoren-9-yl)methyl ((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3-methyl-1-oxo-3-(tritylthio)butan-2-yl)carbamate was prepared according to Basic Procedure 4. 190 mg of (9H-fluoren-9-yl)methyl ((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3-methyl-1-oxo-3-(tritylthio)butan-2-yl)carbamate was obtained from 118 mg of tert-butyl (2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-carboxylate in 75% yield.1 1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 7.78 - 7.72 (m, 2H), 7.62 - 7.52 (m, 8H), 7.41 - 7.17 (m, 18H), 5.73 (d, J = 5.3 Hz, 1H), 5.00 (p, J = 7.0 Hz, 1H), 4.65 (t, J = 8.0 Hz, 1H), 4.42 - 4.15 (m, 4H), 3.56 (d, J = 5.3 Hz, 1H), 3.45 (d, J = 11.6 Hz, 1H), 3.17 (dd, J = 11.6, 3.5 Hz, 1H), 2.83 (br, s, 1H), 2.51 (s, 3H), 2.44 - 2.32 (m, 1H), 2.08 - 1.96 (m, 1H), 1.39 - 1.31 (m, 6H), 1.17 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 170.91, 169.57, 156.61, 150.35, 148.68, 144.60, 144.02, 143.58, 143.26, 141.41, 130.99, 130.04, 129.68, 128.03, 127.94, 127.28, 127.08, 126.58, 125.29, 125.13, 120.18, 70.25, 67.79, 58.61, 58.38, 56.62, 48.92, 47.14, 35.86, 26.12, 26.05, 22.34, 16.23. LC-MS, ESI + , m / z 927 [M+H] + 。

[0225]

Chem.

[0226] [Chemical Structure] (2S,4R)-1-((R)-2-(1-Fluorocyclopropane-1-carboxamido)-3-methyl-3-(tritylthio)butanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide was prepared according to the basic procedure 6. 80 mg of (2S,4R)-1-((R)-2-(1-Fluorocyclopropane-1-carboxamido)-3-methyl-3-(tritylthio)butanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide was obtained from 80 mg of the amine compound in 89% yield. 1 H NMR (500 MHz, CDCl3) δ 8.69 (s, 1H), 7.67 - 7.60 (m, 6H), 7.44 - 7.33 (m, 6H), 7.31 - 7.27 (m, 6H), 7.26 - 7.21 (m, 3H), 5.03 (p, J = 7.0 Hz, 1H), 4.65 (t, J = 8.1 Hz, 1H), 4.35 (s, 1H), 3.59 (d, J = 4.8 Hz, 1H), 3.49 (d, J = 11.5 Hz, 1H), 3.26 (d, J = 5.9 Hz, 1H), 3.19 (dd, J = 11.5, 3.7 Hz, 1H), 2.53 (s, 3H), 2.33 (ddd, J = 13.0, 8.1, 4.5 Hz, 1H), 2.11 - 1.97 (m, 1H), 1.41 - 1.32 (m, 8H), 1.29 - 1.20 (m, 5H). 19 F NMR (471 MHz, CDCl3) δ -197.26. 1313C NMR (126 MHz, CDCl3) δ 170.38 (J = 16.7 Hz), 170.11, 169.64, 150.34, 148.65, 144.60, 143.30, 131.77, 130.95, 129.94, 129.65, 128.02, 127.04, 126.60, 79.22, 70.25, 68.52, 58.64, 57.30, 56.78, 53.67, 48.90, 36.04, 26.16, 22.39, 16.21, 13.88 (d, J = 10.3 Hz), 13.71 (d, J = 10.3 Hz). LC-MS, ESI-, m / z 789 [M-H]-.

[0227] [Chem.] (2S,4R)-1-((R)-2-(1-Fluorocyclopropane-1-carboxamido)-3-mercapto-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide was prepared according to the general procedure 7. 25 mg of (2S,4R)-1-((R)-2-(1-Fluorocyclopropane-1-carboxamido)-3-mercapto-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide was obtained from 40 mg of the trityl protected starting material in 90% yield. 11H NMR (500 MHz, CDCl3) δ 8.73 (s, 1H), 7.49 - 7.43 (m, 1H), 7.42 - 7.36 (m, 4H), 7.28 (d, J = 8.4 Hz, 1H), 5.10 (p, J = 7.0 Hz, 1H), 4.72 - 4.66 (m, 2H), 4.55 - 4.49 (m, 1H), 4.25 (s, 1H), 4.09 (d, J = 11.2 Hz, 1H), 3.76 (dd, J = 11.2, 3.9 Hz, 1H), 2.71 (s, 1H), 2.53 (s, 3H), 2.44 - 2.37 (m, 1H), 2.13 - 2.05 (m, 1H), 1.54 - 1.50 (m, 6H), 1.43 (s, 3H), 1.38 - 1.27 (m, 4H). 13 13C NMR (126 MHz, CDCl3) δ 170.72 (d, J = 21.0 Hz), 170.43, 169.73, 150.70, 148.27, 143.35, 132.01, 130.85, 129.75, 126.66, 77.24 (d, J = 232.2 Hz), 70.17, 59.14, 57.85, 56.70, 49.10, 46.45, 36.51, 30.78, 28.82, 22.34, 15.97, 14.06 (d, J = 10.4 Hz), 14.05 (d, J = 10.9 Hz). 19 19F NMR (471 MHz, CDCl3) δ -197.71. LC-MS, 549.5 [M+H] + 。

[0228]

Chem.

[0229] Exemplary Synthesis of Exemplified Compound 17 (XL01118) [Chemical Structure]

[0230] [Chemical Structure] (4-(3-Bromopropyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methanone To a solution of (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methanone HCl salt (50 mg, 0.12 mmol) in DMF (1 ml), K2CO3 (50 mg, 0.36 mmol) and 1,3-dibromopropane (98 mg, 0.48 mmol) were added. After stirring overnight at room temperature, water was added and the resulting mixture was extracted with EtOAc (3×). The combined organic phases were washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give (4-(3-bromopropyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methanone (30 mg, 50% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 8.59 - 8.47 (m, 1H), 7.91 (s, 1H), 7.62 (s, 1H), 7.03 - 6.97 (m, 2H), 5.36 - 5.26 (m, 1H), 3.91 (s, 3H), 3.77 - 3.53 (m, 4H), 3.47 (t, J = 6.6 Hz, 2H), 3.09 (d, J = 4.9 Hz, 3H), 2.56 - 2.36 (m, 6H), 2.02 (p, J = 6.7 Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ 170.56, 158.68, 157.93, 152.77, 147.52, 131.49, 127.94, 120.28, 116.87, 109.69, 105.64, 56.38, 55.98, 53.45, 31.64, 30.00, 28.18. LC-MS, ESI + , m / z, 497, [M+H] + , 499 [M+H+2] + 。

[0231]

Chemical Structure

[0232] Exemplary Synthesis of Exemplified Compound 18 (XL01120)

Chemical Structure

[0233] Exemplary Synthesis of Exemplified Compound 19 (XL01126)

Chem.

[0234]

Chem.

[0235]

Chem.

[0236]

Chem.

[0237]

Chem.

[0238] Exemplary Synthesis of Exemplified Compound 20 (XL01134)

Chem.

[0239]

Chem.

[0240]

Chem.

[0241]

Chemical Structure

[0242]

Chemical formula

[0243]

Chem.

[0244] Exemplary Synthesis of Exemplary Compound 21 (XL01076)

Chem.

[0245]

Chem.

[0246] [Chemical formula] (2S,4R)-1-((R)-3-((4-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)but-2-yn-1-yl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (21, XL01076) XL01076 was prepared according to the basic procedure 8. 3.1 mg of XL01076 was obtained starting from 5 mg of the thiol starting material, with a 34% yield. 11H NMR (500 MHz, CDCl3) δ 8.68 (s, 1H), 8.53 (d, J = 8.7 Hz, 1H), 7.91 (s, 1H), 7.70 (s, 1H), 7.36 (dd, J = 20.1, 8.2 Hz, 4H), 7.25 - 7.17 (m, 2H), 7.03 - 6.97 (m, 2H), 5.36 - 5.27 (m, 1H), 4.91 (d, J = 8.0 Hz, 1H), 4.76 - 4.70 (m, 1H), 4.57 - 4.53 (m, 1H), 4.46 (ddd, J = 37.5, 15.0, 5.9 Hz, 2H), 4.00 (d, J = 11.1 Hz, 1H), 3.92 (s, 3H), 3.85 (dd, J = 11.0, 4.3 Hz, 1H), 3.69 (s, 4H), 3.43 - 3.36 (m, 1H), 3.34 - 3.28 (m, 3H), 3.10 (d, J = 4.9 Hz, 3H), 2.55 (s, 2H), 2.52 (s, 3H), 2.51 - 2.48 (m, 1H), 2.42 (s, 2H), 2.21 - 2.14 (m, 1H), 1.40 (s, 3H), 1.35 (s, 3H), 1.34 - 1.27 (m, 4H); 13 13C NMR (126 MHz, CDCl3) δ 170.74, 170.71, 170.52 (d, J = 20.7 Hz), 169.92, 162.81, 158.76, 157.85, 152.56, 150.53, 148.63, 147.70, 138.15, 131.75, 131.65, 131.20, 129.72, 128.31, 127.66, 120.38, 117.08, 109.82, 105.73, 81.99, 79.20, 77.99, 70.05, 59.17, 56.62, 56.26, 56.08, 52.20, 48.99, 47.36, 43.38, 36.48, 28.26, 25.64, 25.46, 17.53, 16.17, 14.08 (d, J = 11.06 Hz), 13.99 (d, J = 12.26 Hz);19 19F NMR (471 MHz, CDCl3) δ -197.70; HRMS (ESI + ) m / z, C 46 H 54 ClFN 10 O6S2: calculated value for [M + H] 961.3414 + , found 961.3672.

[0247] Exemplary Synthesis of Exemplary Compound 22 (XL01123)

Chem.

[0248]

Chem.

[0249]

Chem.

[0250]

Chem.

[0251]

Chem.

[0252]

Chem.

[0253]

Chem.

[0254] [Chemical formula] (2S,4R)-N-(3-Fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-carboxamido)-3-mercapto-3-methylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide was prepared according to the basic procedure 7. 27 mg of (2S,4R)-N-(3-Fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-carboxamido)-3-mercapto-3-methylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide was obtained from 40 mg of the trityl-protected compound, 97%. 11H NMR (500 MHz, CDCl3) δ 9.04 (s, 1H), 7.53 (t, J = 5.7 Hz, 1H), 7.43 (dd, J = 8.5, 3.0 Hz, 1H), 7.29 - 7.24 (m, 1H), 7.20 - 7.13 (m, 2H), 5.67 (br, s, 1H), 4.76 - 4.66 (m, 2H), 4.60 (dd, J = 15.5, 6.8 Hz, 1H), 4.56 - 4.52 (m, 1H), 4.32 (dd, J = 15.5, 5.3 Hz, 1H), 4.08 (d, J = 11.1 Hz, 1H), 3.81 (dd, J = 11.1, 3.7 Hz, 1H), 2.62 (s, 1H), 2.42 (s, 3H), 2.35 (ddd, J = 13.0, 8.4, 4.4 Hz, 1H), 2.24 - 2.16 (m, 1H), 1.43 (s, 3H), 1.38 (s, 3H), 1.36 - 1.25 (m, 4H). 13 13C NMR (126 MHz, CDCl3) δ 171.32, 170.6 (d, J = 20.7 Hz), 170.22, 159.8 (d, J = 251.0 Hz), 152.76, 149.28, 142.32 (d, J = 7.7 Hz), 132.14 (d, J = 2.3 Hz), 125.65, 123.55 (d, J = 4.4 Hz), 117.44 (d, J = 15.2 Hz), 115.38 (d, J = 22.9 Hz), 77.27 (d, J = 232.7 Hz), 70.26, 59.37, 57.81, 56.88, 46.55, 42.90, 37.12, 30.47, 28.83, 15.40, 14.05 (d, J = 10.5 Hz), 13.99 (d, J = 9.7 Hz). 19 19F NMR (471 MHz, CDCl3) δ -111.74, -197.61. LC-MS, ESI + , m / z 553.4 [M+H] + 。

[0255] [Chemical formula] (2S,4R)-1-((R)-3-((2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (22, XL01123) XL01123 was prepared according to the basic procedure 8. A 40% yield of 5 mg of XL01123 was obtained from 8 mg of the thiol compound. 1 H NMR (500 MHz, CDCl3) δ 8.76 (s, 1H), 8.54 (d, J = 8.7 Hz, 1H), 7.92 (s, 1H), 7.63 (s, 1H), 7.44 (t, J = 5.9 Hz, 1H), 7.32 - 7.27 (m, 1H), 7.18 (dd, J = 8.3, 3.1 Hz, 1H), 7.15 - 7.11 (m, 2H), 7.02 - 6.98 (m, 2H), 5.32 - 5.27 (m, 1H), 4.87 (d, J = 8.1 Hz, 1H), 4.76 (dd, J = 8.4, 6.2 Hz, 1H), 4.58 - 4.52 (m, 1H), 4.50 - 4.40 (m, 2H), 3.92 (s, 3H), 3.91 - 3.88 (m, 2H), 3.80 - 3.45 (m, 13H), 3.10 (d, J = 4.9 Hz, 3H), 2.85 - 2.72 (m, 2H), 2.61 (s, 2H), 2.57 - 2.43 (m, 5H), 2.42 (d, J = 1.1 Hz, 3H), 2.21 - 2.14 (m, 1H), 1.36 (s, 3H), 1.34 (s, 3H), 1.33 - 1.27 (m, 4H); 1313C NMR (126 MHz, CDCl3) δ 171.05, 170.65, 170.21, 170.14, 163.36, 158.75, 157.93, 152.75, 151.85, 151.07, 147.62, 141.50, 132.40 (d, J = 2.5 Hz), 131.61, 127.76, 124.51, 123.63 (J = 3.3 Hz), 120.37, 118.59 (d, J = 15.7 Hz), 116.99, 115.5 (d, J = 22.9 Hz), 109.76, 105.71, 79.22, 70.56, 70.45, 70.22, 69.67, 68.65, 59.26, 57.77, 56.19, 56.06, 55.96, 53.63, 47.98, 42.88, 36.60, 28.89, 28.25, 26.00, 25.20, 16.13, 14.04 (d, J = 9.85 Hz), 13.96 (d, J = 9.84 Hz); HRMS (ESI + ) m / z, C 48 H 61 ClF2N 10 O8S2: Calculated for 1043.3845 [M + H] + , found 1043.3733.

[0256] Exemplary Synthesis of Exemplified Compound 23 (XL01122) [Chemical Structure] (2S,4R)-1-((R)-3-((3-(4-(4-((5-Chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (23, XL01122) XL01122 was prepared according to the general procedure 8. 1.4 mg of XL01122 was obtained from 8 mg of the thiol compound, 10%.1 1H NMR (500 MHz, CDCl3) δ 8.76 (s, 1H), 8.54 (d, J = 8.1 Hz, 1H), 7.93 (s, 1H), 7.65 (s, 1H), 7.36 - 7.28 (m, 2H), 7.23 (dd, J = 7.8, 3.2 Hz, 1H), 7.16 - 7.10 (m, 2H), 7.02 - 6.97 (m, 2H), 5.32 - 5.27 (m, 1H), 4.81 - 4.70 (m, 2H), 4.59 - 4.47 (m, 2H), 4.41 (dd, J = 15.3, 5.7 Hz, 1H), 4.08 (d, J = 11.4 Hz, 1H), 3.93 (s, 3H), 3.76 - 3.52 (m, 5H), 3.11 (d, J = 4.9 Hz, 3H), 2.62 - 2.47 (m, 4H), 2.47 - 2.36 (m, 9H), 2.26 - 2.20 (m, 1H), 1.74 - 1.64 (m, 2H), 1.37 (s, 3H), 1.36 (s, 3H), 1.34 - 1.26 (m, 4H); 13 13C NMR (126 MHz, CDCl3) δ 170.81, 170.64, 170.61, 170.26, 158.75, 157.95, 152.78, 151.88, 151.12, 147.62, 141.46, 141.41, 132.45, 132.43, 131.61, 124.41, 123.55 (d, J = 3.0 Hz), 120.35, 116.95, 115.42 (d, J = 22.4 Hz), 109.77, 105.73, 79.24, 70.31, 59.00, 57.24, 56.69, 56.45, 56.05, 53.27, 47.98, 42.95, 36.62, 28.24, 26.17, 25.73, 25.53, 16.15, 14.15 (d, J = 10.6 Hz), 14.00 (d, J = 10.5 Hz); HRMS (ESI + ) m / z, C 45 H 55 ClF2N 10Calculated value for O6S2: 969.3477 [M + H] + , measured value 969.3772.

[0257] Exemplary synthesis of Exemplary Compound 24 (XL01121) [Chemical Structure] (2S,4R)-1-((R)-3-((4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (24, XL01121) XL01121 was prepared according to Basic Procedure 8. 12.2 mg of XL01121 was obtained starting from 8 mg of the thiol compound, 83%. 11H NMR (500 MHz, CDCl3) δ 8.79 (s, 1H), 8.57 (d, J = 8.8 Hz, 1H), 7.95 (s, 1H), 7.66 (s, 1H), 7.41 (t, J = 6.0 Hz, 1H), 7.32 - 7.26 (m, 6H), 7.12 (d, J = 9.1 Hz, 2H), 7.06 - 7.00 (m, 2H), 5.34 (q, J = 4.7 Hz, 1H), 4.76 (t, J = 7.9 Hz, 1H), 4.66 (d, J = 7.5 Hz, 1H), 4.51 - 4.48 (m, 1H), 4.49 - 4.43 (m, 1H), 4.38 (dd, J = 15.4, 5.8 Hz, 1H), 3.95 (s, 3H), 3.90 (d, J = 11.2 Hz, 1H), 3.80 (dd, J = 12.5 Hz, 2H), 3.75 - 3.57 (m, 5H), 3.51 (s, 2H), 3.14 (d, J = 4.9 Hz, 3H), 2.56 - 2.38 (m, 8H), 2.24 - 2.16 (m, 1H), 1.47 (s, 3H), 1.43 (s, 3H), 1.39 - 1.29 (m, 4H); 1313C NMR (126 MHz, CDCl3) δ 171.04, 170.62, 170.46 (d, J = 20.9 Hz), 170.13, 159.86 (d, J = 250.1 Hz), 158.71, 157.95, 152.79, 151.82, 151.05, 147.54, 141.49 (d, J = 7.0 Hz), 137.04, 136.41, 132.33 (d, J = 2.4 Hz), 131.49, 129.60, 129.03, 127.99, 124.45, 123.39 (d, J = 3.5 Hz), 120.32, 118.51 (d, J = 15.7 Hz), 116.92, 115.30 (d, J = 23.3 Hz), 109.74, 105.67, 78.28 (d, J = 229.1 Hz), 70.17, 62.69, 59.15, 56.66, 56.47, 56.02, 53.30, 48.94, 42.84, 36.86, 33.39, 28.21, 25.75, 25.36, 16.11 (d, J = 2.8 Hz), 13.96 (d, J = 10.8 Hz), 13.94 (d, J = 10.9 Hz). HRMS (ESI + ) m / z, for C50H57ClF2N10O6S2: calculated 1031.3633 [M + H] + , found 1031.3403.

[0258] Exemplary Synthesis of Exemplified Compound 25 (XL01131) [Chemical Structure]

[0259] [Chemical Structure] 2-(2-(4-(4-((5-Chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethyl 4-methylbenzenesulfonate To a solution of 2-(2-hydroxyethoxy)ethyl 4-methylbenzenesulfonate (100 mg, 0.38 mmol) in DCM (3 ml) was added Dess-Martin Periodinane (195 mg, 0.46 mmol). After stirring at room temperature for 1 hour, the reaction mixture was diluted with DCM, washed with water and brine, dried over sodium sulfate, filtered, concentrated to give a residue, which was used in the next step without further purification. Half of the residue from the final step was dissolved in DCM (4 ml), followed by the addition of (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methanone HCl salt (63 mg, 0.15 mmol), sodium triacetoxyborohydride (72 mg, 0.34 mmol), and HOAc (1 drop). After stirring at room temperature for 2 hours, the mixture was diluted with DCM, washed with water and brine, dried over sodium sulfate, filtered, concentrated to give a residue, which was purified by flash column chromatography on silica gel (0% to 10% methanol in DCM) to give 2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethyl 4-methylbenzenesulfonate (46 mg, 49% yield). 1 H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 8.9 Hz, 1H), 7.92 (s, 1H), 7.81 - 7.75 (m, 2H), 7.68 (s, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.03 - 6.95 (m, 2H), 5.34 - 5.29 (m, 1H), 4.17 - 4.13 (m, 2H), 3.92 (s, 3H), 3.74 - 3.58 (m, 8H), 3.14 - 3.07 (m, 3H), 2.64 (t, J = 5.4 Hz, 2H), 2.60 - 2.51 (m, 4H), 2.43 (s, 3H). 1313C NMR (101 MHz, CDCl3) δ 170.63, 158.74, 157.90, 152.68, 147.64, 145.03, 133.26, 131.59, 129.99, 128.07, 127.79, 120.34, 117.01, 109.76, 105.65, 69.23, 68.95, 68.73, 57.61, 56.04, 53.60, 28.24, 21.76. LC-MS, ESI + , m / z 619.35 [M+H] + 。

[0260]

Chem.

[0261] Exemplary Synthesis of Exemplified Compound 26 (XL01140) [Chemical Structure]

[0262] [Chemical Structure] A mixture of tert-butyl piperazine-1-carboxylate (500 mg, 2.69 mmol), propylene oxide (234 mg, 4.0 mmol), and ethanol (3 ml) was stirred overnight at room temperature in a sealed microwave tube. The mixture was concentrated under reduced pressure to give a residue, which was purified by flash column chromatography on silica gel (0% to 10% methanol in DCM) to give tert-butyl (S)-4-(2-hydroxypropyl)piperazine-1-carboxylate (390 mg, 60% yield). 11H NMR (400 MHz, CDCl3) δ 3.93 - 3.73 (m, 1H), 3.48 - 3.34 (m, 4H), 3.30 (s, 1H), 2.66 - 2.53 (m, 2H), 2.36 - 2.17 (m, 4H), 1.43 (s, 9H), 1.11 (d, J = 6.1 Hz, 3H). 13 13C NMR (101 MHz, CDCl3) δ 154.80, 79.80, 65.91, 62.45, 53.17, 43.81, 28.53, 20.09. LC-MS, ESI + , m / z 245.1 [M+H] + 。

[0263]

Chem.

[0264]

Chem.

[0265]

Chem.

[0266] Exemplary Synthesis of Exemplified Compound 27 (XL01111)

Chem.

[0267]

Chem.

[0268] [Chemical formula] (2S,4R)-1-((R)-3-((3-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01111) XL01111 was prepared according to the basic procedure 8. 7.5 mg of XL01111 was obtained from 10 mg of the thiol compound, 42%. 1 H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.53 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.62 (s, 1H), 7.34 - 7.26 (m, 5H), 7.26 - 7.15 (m, 5H), 7.02 - 6.96 (m, 2H), 5.37 - 5.23 (m, 1H), 4.84 - 4.68 (m, 2H), 4.50 - 4.32 (m, 3H), 3.96 - 3.88 (m, 4H), 3.82 - 3.71 (m, 3H), 3.64 (s, 4H), 3.51 (d, J = 13.0 Hz, 1H), 3.44 (d, J = 13.0 Hz, 1H), 3.10 (d, J = 4.9 Hz, 3H), 2.55 - 2.36 (m, 8H), 2.23 - 2.11 (m, 1H), 1.41 - 1.25 (m, 10H). 1313C NMR (101 MHz, CDCl3) δ 170.70, 170.63, 170.50 (d, J = 20.8 Hz), 170.04, 158.72, 157.95, 152.79, 150.38, 148.68, 147.57, 138.21, 138.10, 137.31, 131.67, 131.53, 131.16, 129.81, 129.65, 128.86, 128.41, 128.20, 127.92, 120.33, 116.91, 109.75, 105.70, 78.40 (d, J = 209.1 Hz), 69.99, 62.89, 59.09, 56.43, 56.25, 56.03, 48.98, 43.25, 36.67, 33.60, 28.22, 25.73, 25.63, 16.22, 14.07 (d, J = 9.3 Hz), 13.95 (d, J = 8.9 Hz). 19 19F NMR (471 MHz, CDCl3) δ -197.75. HRMS (ESI + ) m / z, C 50 H 58 ClFN 10 O6S2: calculated value for [M + H] 1013.3727 + , found 1013.3893.

[0269] Exemplary Synthesis of Exemplified Compound 28 (XL01145) [Chemical Structure]

[0270] [Chemical Structure] 2-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate To a solution of tetraethylene glycol (6.1 g, 31.5 mmol) in DCM (100 mL) were added TEA (2.92 mL, 20.98 mmol), DMAP (64 mg, 0.52 mmol), and TsCl (2 g, 10.5 mmol) at 0 °C. After stirring overnight at room temperature, the mixture was diluted with DCM, washed with water and brine, dried over sodium sulfate, filtered, concentrated to give a residue, which was purified by flash column chromatography on silica gel (0% to 100% ethyl acetate in heptane) to afford 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (2.4 g, 66% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 4.18 -4.14 (m, 2H), 3.74 - 3.53 (m, 14H), 2.44 (s, 3H).

[0271]

Chemical formula

[0272] [Chemical formula] 2-(2-(2-(2-(4-((tert-Butoxycarbonyl)amino)-3-methoxyphenoxy)ethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate To a solution of 2-(2-(2-(2-(3-methoxy-4-nitrophenoxy)ethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (85 mg, 0.17 mmol) in ethyl acetate (3 ml) was added 10% palladium on carbon (10 mg) under nitrogen protection. Next, nitrogen was replaced with hydrogen, and the resulting mixture was stirred overnight under hydrogen pressure (hydrogen balloon). The mixture was filtered through a pad of celite, concentrated to give a residue, which was dissolved in DCM, and then TEA (0.047 ml, 0.46 mmol) and di-tert-butyl dicarbonate (37 mg, 0.17 mmol) were added. After stirring at room temperature for 2 hours, the resulting mixture was concentrated and purified by flash column chromatography on silica gel (ethyl acetate from 0% to 100% in heptane) to give 2-(2-(2-(2-(4-((tert-Butoxycarbonyl)amino)-3-methoxyphenoxy)ethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (58 mg, 60% yield in 2 steps). 1 H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 7.8 Hz, 1H), 7.83 - 7.71 (m, 2H), 7.32 (d, J = 8.0 Hz, 2H), 6.81 (s, 1H), 6.50 (d, J = 2.6 Hz, 1H), 6.44 (dd, J = 8.8, 2.6 Hz, 1H), 4.17 - 4.12 (m, 2H), 4.11 - 4.02 (m, 2H), 3.86 - 3.77 (m, 5H), 3.71 - 3.66 (m, 4H), 3.65 - 3.61 (m, 2H), 3.60 - 3.55 (m, 4H), 2.43 (s, 3H), 1.51 (s, 9H).13 13C NMR (101 MHz, CDCl3) δ 154.81, 153.12, 148.97, 144.87, 133.26, 129.93, 128.08, 121.94, 119.07, 104.95, 99.64, 80.13, 70.89, 70.81, 70.70, 69.94, 69.35, 68.83, 67.94, 55.79, 28.51, 21.72. LC-MS, ESI + , m / z 470.15 [M+H] + 。

[0273]

Chem.

[0274] [Chemical formula] (2S,4R)-1-((S)-1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenoxy)-14-(1-fluorocyclopropanecarboxamido)-13,13-dimethyl-3,6,9-trioxa-12-thiapentadecan-15-yl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (28, XL01145) To a solution of tert-butyl (4-(((R)-1-(1-fluorocyclopropyl)-3-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-4,4-dimethyl-1-oxo-8,11,14-trioxa-5-thia-2-azahexadecan-16-yl)oxy)-2-methoxyphenyl)carbamate (36 mg, 0.039 mmol) in DCM (1 mL) was added HCl (4 N in 1,4-dioxane, 1 mL). After stirring overnight at room temperature, the mixture was concentrated and then dissolved in isopropanol (1.5 mL). 2,5-Dichloro-N-methylpyrimidin-4-amine (8 mg, 0.045 mmol) and HCl (4 N in 1,4-dioxane, 1 drop) were added, and the resulting mixture was heated at 100 °C for 8 h by microwave. The residue was obtained by removal of the solvent under reduced pressure and was purified by preparative HPLC under acidic conditions (5 to 95% CH3CN in 0.1% aqueous HCO2H) to give XL01145 (8.3 mg, 22% yield over 2 steps). 11H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.25 (d, J = 8.8 Hz, 1H), 7.85 (s, 1H), 7.41 - 7.29 (m, 6H), 7.19 (dd, J = 8.0, 3.2 Hz, 1H), 6.54 (d, J = 2.6 Hz, 1H), 6.49 (dd, J = 8.9, 2.7 Hz, 1H), 5.27 - 5.20 (m, 1H), 4.81 (d, J = 7.8 Hz, 1H), 4.78 - 4.73 (m, 1H), 4.55 - 4.50 (m, 1H), 4.50 - 4.38 (m, 2H), 4.14 - 4.08 (m, 2H), 3.95 (d, J = 11.2 Hz, 1H), 3.87 - 3.78 (m, 6H), 3.74 - 3.67 (m, 2H), 3.67 - 3.62 (m, 2H), 3.62 - 3.46 (m, 6H), 3.07 (d, J = 4.9 Hz, 3H), 2.83 - 2.67 (m, 2H), 2.51 (s, 3H), 2.48 - 2.40 (m, 1H), 2.26 - 2.14 (m, 1H), 1.38 - 1.23 (m, 10H); 13 13C NMR (101 MHz, CDCl3) δ 170.83, 170.43 (d, J = 20.6 Hz), 170.06, 158.69, 158.37, 154.30, 152.65, 150.41, 149.52, 148.65, 138.26, 131.76, 131.14, 129.64, 128.33, 123.53, 119.70, 104.83, 104.62, 99.76, 78.40 (d, J = 209.1 Hz), 70.97, 70.77, 70.70, 70.58, 70.35, 70.03, 68.03, 59.19, 56.46, 56.17, 55.92, 47.94, 43.23, 36.80, 28.64, 28.13, 25.96, 25.27, 16.22, 14.05 (d, J = 9.6 Hz), 13.95 (d, J = 9.5 Hz); HRMS (ESI +) m / z, C 45 H 58 Calculated value for ClFN8O9S2: 973.3513 [M + H] + , found value 973.3903.

[0275] Exemplary synthesis of Exemplified Compound 29 (XL01149) [Chemical formula]

[0276] [Chemical formula] 3-(Benzyloxy)propyl 4-methylbenzenesulfonate TsCl (867 mg, 4.54 mmol) was added to a solution of 3-(benzyloxy)propan-1-ol (720 mg, 4.33 mmol), TEA (0.904 mL, 6.5 mmol), and DMAP (26 mg, 0.21 mmol) in DCM (10 mL) at 0 °C. After stirring overnight at room temperature, the mixture was diluted with DCM (20 mL), washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue, which was purified by flash column chromatography on silica gel (0% to 50% ethyl acetate in heptane) to give 3-(benzyloxy)propyl 4-methylbenzenesulfonate (750 mg, 54% yield). 1 H NMR (400 MHz, CDCl3) δ 7.85 - 7.77 (m, 2H), 7.39 - 7.24 (m, 7H), 4.43 (s, 2H), 4.20 (t, J = 6.1 Hz, 2H), 3.53 (t, J = 6.1 Hz, 2H), 2.45 (s, 3H), 1.97 (p, J = 6.1 Hz, 2H).

[0277] [Chemical formula] 2-(3-(Benzyloxy)propoxy)ethan-1-ol NaH (60% in mineral oil, 206 mg, 5.16 mmol) was added to a solution of ethylene glycol (1.453 g, 23.4 mmol) in THF (20 mL) at 0 °C. The resulting mixture was heated to reflux temperature and stirred overnight at reflux temperature. Next, the reaction mixture was cooled in an ice bath and NH4Cl aqueous solution was added dropwise to quench the reaction. The resulting mixture was extracted with ethyl acetate (3 × 30 mL), the combined organic phases were washed with water and brine, dried over sodium sulfate, filtered, concentrated to give a residue, and it was purified by flash column chromatography on silica gel (0% to 10% methanol in DCM) to give 2-(3-(benzyloxy)propoxy)ethan-1-ol (534 mg, 54% yield). 1 H NMR (400 MHz, CDCl3) δ 7.43 - 7.18 (m, 5H), 4.53 (s, 2H), 3.75 - 3.69 (m, 2H), 3.68 - 3.49 (m, 6H), 2.26 (t, J = 5.8 Hz, 1H), 1.93 (p, J = 6.3 Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ 138.57, 128.46, 127.74, 127.67, 73.08, 71.99, 68.34, 67.32, 61.90, 30.12. LC-MS, ESI + , m / z 233 [M+Na] + 。

[0278]

Chemical Structure

[0279]

Chemical Structure

[0280]

Chem.

[0281]

Chem.

[0282]

Chem.

[0283] Exemplary Synthesis of Exemplified Compound 30 (XL01168)

Chem.

[0284]

Chem.

[0285]

Chem.

[0286]

Chem.

[0287]

Chem.

[0288] Exemplary synthesis of Exemplary Compound 31 (XL02017) [Chemical formula]

[0289] [Chemical formula] Benzyl 4-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenoxy)piperidine-1-carboxylate To a solution of tert-butyl (4-hydroxy-2-methoxyphenyl)carbamate (300 mg, 1.26 mmol), benzyl 4-hydroxypiperidine-1-carboxylate (354 mg, 1.5 mmol), and triphenylphosphine (724 mg, 2.76 mmol) in toluene (10 ml) was added 1,1‘-(azodicarbonyl)dipiperidine (633 mg, 2.5 mmol). After stirring overnight at room temperature under nitrogen protection, the reaction mixture was diluted with ethyl acetate, washed with water and brine, dried over sodium sulfate, filtered, concentrated to give a residue, which was purified by flash column chromatography on silica gel (0% to 50% ethyl acetate in heptane) to give benzyl 4-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenoxy)piperidine-1-carboxylate (450 mg, 79% yield). 11H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 8.3 Hz, 1H), 7.39 - 7.28 (m, 5H), 6.84 (s, 1H), 6.55 - 6.37 (m, 2H), 5.15 (s, 2H), 4.46 - 4.36 (m, 1H), 3.82 (s, 3H), 3.80 - 3.68 (m, 2H), 3.49 - 3.40 (m, 2H), 1.99 - 1.83 (m, 2H), 1.83 - 1.71 (m, 2H), 1.52 (s, 9H). 13 13C NMR (101 MHz, CDCl3) δ 155.40, 153.09, 152.91, 149.10, 136.97, 128.59, 128.08, 127.96, 122.23, 119.16, 106.95, 101.14, 80.17, 72.56, 67.21, 55.77, 40.89, 30.59, 28.49. LC-MS, ESI + , m / z 479.2 [M+Na] + 。

[0290]

Chem.

[0291]

Chem.

[0292]

Chem.

[0293]

Chem.

[0294] Exemplary Synthesis of Exemplified Compound 32 (XL02031)

Chemical Structure

[0295]

Chemical Structure

[0296]

Chemical Structure

[0297]

Chem.

[0298] Exemplary Synthesis of Exemplified Compound 33 (XL02038)

Chem.

[0299]

Chem.

[0300]

Chem.

[0301]

Chem.

[0302] Exemplary Synthesis of Exemplified Compound 34 (XL02047)

Chemical Structure

[0303]

Chemical Structure

[0304]

Chem.

[0305]

Chem.

[0306]

Chemical formula

[0307]

Chem.

[0308]

Chem.

[0309] [Chemical formula] (2S,4R)-N-(2-Fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-carboxamido)-3-mercapto-3-methylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide was prepared according to the basic procedure 7. 39 mg of (2S,4R)-N-(2-fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-carboxamido)-3-mercapto-3-methylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide was obtained from 71 mg of the trityl-protected compound in 80% yield. 11H NMR (500 MHz, CDCl3) δ 8.71 (s, 1H), 7.47 - 7.40 (m, 2H), 7.30 (t, J = 5.9 Hz, 1H), 7.15 (dd, J = 7.9, 1.7 Hz, 1H), 7.11 (dd, J = 10.6, 1.6 Hz, 1H), 4.71 - 4.64 (m, 2H), 4.58 - 4.49 (m, 2H), 4.42 (dd, J = 15.2, 5.5 Hz, 1H), 4.06 (d, J = 11.3 Hz, 1H), 3.75 (dd, J = 11.2, 3.9 Hz, 1H), 3.31 (s, 1H), 2.59 (d, J = 0.7 Hz, 1H), 2.52 (s, 3H), 2.40 (ddd, J = 12.9, 7.9, 4.6 Hz, 1H), 2.18 - 2.09 (m, 1H), 1.39 - 1.25 (m, 10H). 13 13C NMR (126 MHz, CDCl3) δ 170.92, 170.69 (d, J = 21.2 Hz), 170.23, 160.74 (d, J = 248.1 Hz), 150.99, 149.04, 133.26 (d, J = 8.0 Hz), 130.66, 130.55 (d, J = 4.9 Hz), 125.35 (d, J = 3.2 Hz), 125.01 (d, J = 15.1 Hz), 116.27 (d, J = 22.8 Hz), 78.20 (d, J = 232.5 Hz), 70.20, 59.10, 57.75, 56.71, 46.39, 37.46 (d, J = 3.8 Hz), 36.69, 30.46, 28.71, 16.15, 14.07, 13.99. 19 19F NMR (471 MHz, CDCl3) δ -117.66, -197.71. LC-MS, ESI + , 553.2 [M+H] + 。

[0310]

Chem.

[0311] Exemplary Synthesis of Exemplified Compound 35 (XL02048) [Chemical Structure]

[0312] [Chemical Structure] tert-Butyl (4-bromo-2-methylbenzyl)carbamate was prepared according to general procedure 1. 5.7 g of tert-butyl (4-bromo-2-methylbenzyl)carbamate was obtained from 4 g of the amine compound, 95% yield. 1 H NMR (400 MHz, CDCl3) δ 7.43 - 7.27 (m, 2H), 7.10 (d, J = 7.9 Hz, 1H), 4.71 (s, 1H), 4.25 (d, J = 5.4 Hz, 2H), 2.29 (s, 3H), 1.45 (s, 9H). 13 C NMR (101 MHz, CDCl3) δ 155.79, 138.49, 135.83, 133.30, 129.63, 129.25, 121.31, 42.35, 28.54, 27.58, 18.88. LC-MS, ESI + , m / z 246 [M-56+H] + .

[0313] [ka] tert-Butyl (2-methyl-4-(4-methylthiazol-5-yl)benzyl)carbamate was prepared according to general procedure 2. 2.35 g of tert-butyl (2-methyl-4-(4-methylthiazol-5-yl)benzyl)carbamate was obtained from 3 g of the bromine compound, 70% yield. 1 H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.24 - 7.20 (m, 2H), 4.92 (s, 1H), 4.32 (d, J = 5.3 Hz, 2H), 2.50 (s, 3H), 2.34 (s, 3H), 1.45 (s, 9H); 13 C NMR (101 MHz, CDCl3) δ 155.85, 150.19, 148.50, 136.66, 131.69, 131.30, 131.10, 128.21, 127.03, 79.62, 42.47, 28.49, 19.03, 16.14. LC-MS, ESI+ , m / z 319.2 [M+H] + 。

[0314]

Chem.

[0315]

Chem.

[0316]

Chem.

[0317]

Chem.

[0318] [Chemical formula] (2S,4R)-1-((R)-2-(1-Fluorocyclopropane-1-carboxamido)-3-mercapto-3-methylbutanoyl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide was prepared according to the basic procedure 7. 40 mg of (2S,4R)-1-((R)-2-(1-Fluorocyclopropane-1-carboxamido)-3-mercapto-3-methylbutanoyl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide was obtained from 79.5 mg of the trityl-protected compound, with a yield of 73%. 11H NMR (500 MHz, CDCl3) δ 8.69 (s, 1H), 7.42 (d, J = 6.1 Hz, 1H), 7.36 - 7.30 (m, 1H), 7.24 - 7.19 (m, 2H), 7.14 (t, J = 4.8 Hz, 1H), 4.70 - 4.64 (m, 2H), 4.52 (s, 1H), 4.47 (dd, J = 15.0, 5.9 Hz, 1H), 4.39 (dd, J = 15.0, 5.4 Hz, 1H), 4.06 (d, J = 11.0 Hz, 1H), 3.77 (dd, J = 11.1, 3.6 Hz, 1H), 3.45 (s, 1H), 2.52 (s, 1H), 2.50 (s, 3H), 2.39 (ddd, J = 12.9, 7.9, 4.6 Hz, 1H), 2.34 (s, 3H), 2.19 - 2.11 (m, 1H), 1.40 - 1.26 (m, 10H); 13 13C NMR (126 MHz, CDCl3) δ 170.72, 170.57, 170.14, 150.56, 148.29, 136.89, 135.90, 131.94, 131.43, 131.20, 128.82, 127.20, 78.21 (d, J = 232.6 Hz), 70.20, 59.23, 57.77, 56.75, 46.40, 41.50, 36.93, 30.50, 28.62, 19.21, 16.07, 14.02 (d, J = 10.8 Hz), 14.00 (d, J = 10.4 Hz); 19 19F NMR (471 MHz, CDCl3) δ -197.64; LC-MS, ESI + , 549.3 [M+H] + 。

[0319]

Chem.

[0320] Exemplary Synthesis of Exemplified Compound 36 (XL02049) [Chemical Structure Diagram] (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (36, XL02049) XL02049 was prepared according to Basic Procedure 8. A 49% yield of 8.95 mg of XL02049 was obtained from 10 mg of the thiol compound. 1 H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.53 (d, J = 8.8 Hz, 1H), 7.92 (s, 1H), 7.63 (s, 1H), 7.43 - 7.34 (m, 5H), 7.31 - 7.27 (m, 1H), 7.03 - 6.97 (m, 2H), 5.30 (q, J = 4.6 Hz, 1H), 5.10 (p, J = 6.9 Hz, 1H), 4.82 - 4.71 (m, 2H), 4.51 (s, 1H), 4.07 (d, J = 11.3 Hz, 1H), 3.92 (s, 3H), 3.78 - 3.52 (m, 5H), 3.10 (d, J = 4.9 Hz, 3H), 2.79 (s, 1H), 2.53 (s, 3H), 2.51 - 2.30 (m, 7H), 2.20 - 2.10 (m, 3H), 1.92 - 1.78 (m, 4H), 1.49 (d, J = 6.9 Hz, 3H), 1.42 (d, J = 4.8 Hz, 6H), 1.39 - 1.23 (m, 6H), 1.03 - 0.83 (m, 4H); 1313C NMR (101 MHz, CDCl3) δ 170.70 (d, J = 20.9 Hz), 170.55, 170.35, 169.74, 158.73, 157.97, 152.81, 150.37, 148.68, 147.56, 143.17, 131.77, 131.45, 131.04, 129.68, 128.13, 126.65, 120.30, 116.92, 109.74, 105.68, 79.44, 70.25, 65.43, 58.98, 56.66, 56.47, 56.02, 53.97, 48.88, 47.62, 38.61, 36.41, 35.50, 35.12, 32.84, 31.53, 28.22, 25.82, 25.74, 22.33, 16.24, 14.11 (d, J = 10.3 Hz), 13.96 (d, J = 10.2 Hz). 19 19F NMR (471 MHz, CDCl3) δ -197.73. HRMS (ESI + ) m / z, C 51 H 66 ClFN 10 O6S2: Calcd for 1033.4353 [M + H] + , found 1033.4120.

[0321] Exemplary Synthesis of Exemplary Compound 37 (XL02058) [Chemical Structure] (2S,4R)-1-((R)-3-((((1s,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-N-(2-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (37, XL02058) XL02058 was prepared according to the basic procedure 8. A 71% yield of XL02058 was obtained from 10 mg of the thiol compound, which is 16.99 mg. 1 H NMR (500 MHz, CDCl3) δ 8.68 (s, 1H), 8.53 (d, J = 8.7 Hz, 1H), 7.92 (s, 1H), 7.62 (s, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.37 (t, J = 5.8 Hz, 1H), 7.24 (dd, J = 7.7, 3.2 Hz, 1H), 7.16 (d, J = 7.9 Hz, 1H), 7.10 (d, J = 10.6 Hz, 1H), 7.02 - 6.98 (m, 2H), 5.30 (q, J = 4.1 Hz, 1H), 4.87 - 4.66 (m, 2H), 4.57 - 4.46 (m, 3H), 4.04 (d, J = 11.2 Hz, 1H), 3.92 (s, 3H), 3.77 - 3.49 (m, 5H), 3.10 (d, J = 4.9 Hz, 3H), 2.94 (s, 1H), 2.52 (s, 3H), 2.50 - 2.31 (m, 7H), 2.25 - 2.17 (m, 2H), 1.85 - 1.65 (m, 3H), 1.56 - 1.45 (m, 4H), 1.38 - 1.28 (m, 13H), 0.97 - 0.78 (m, 1H); 1313C NMR (126 MHz, CDCl3) δ 170.97, 170.59 (d, J = 20.7 Hz), 170.55, 170.14, 160.69 (d, J = 248.1 Hz), 158.72, 157.96, 152.80, 150.79, 149.22, 147.56, 133.6 (d, J = 8.7 Hz), 131.45, 130.55, 130.44 (d, J = 4.5 Hz), 128.11, 125.34 (d, J = 2.9 Hz), 125.04 (d, J = 15.1 Hz), 120.30, 116.92, 116.13 (d, J = 22.8 Hz), 109.73, 105.67, 79.20, 70.24, 62.32, 59.03, 56.67, 56.36, 56.02, 53.86, 47.75, 37.16 (d, J = 3.7 Hz), 36.80, 35.96, 33.00, 32.09, 28.68, 28.47, 28.21, 27.13 (d, J = 3.7 Hz), 25.69, 25.47, 16.29, 14.05 (d, J = 11.1 Hz), 13.95 (d, J = 10.6 Hz). 19 19F NMR (471 MHz, CDCl3) δ -117.88, -197.71. HRMS (ESI + ) m / z, C 50 H 63 ClF2N 10 O6S2: Calculated for 1037.4102 [M + H] + , found 1037.3870.

[0322] Exemplary Synthesis of Exemplified Compound 38 (XL02059)

Chemical Structure

[0323] Exemplary Synthesis of Exemplified Compound 39 (XL02060) [Chemical Structure Diagram] (2S,4R)-1-((R)-3-((((1s,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (39, XL02060) XL02060 was prepared according to Basic Procedure 8. A 71% yield was obtained from 10 mg of the thiol compound to give 17.22 mg of XL02060. 1 H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.53 (d, J = 8.8 Hz, 1H), 7.92 (s, 1H), 7.62 (s, 1H), 7.44 - 7.34 (m, 5H), 7.29 (dd, J = 7.6, 3.2 Hz, 1H), 7.02 - 6.97 (m, 2H), 5.30 (q, J = 4.7 Hz, 1H), 5.10 (p, J = 7.0 Hz, 1H), 4.80 - 4.70 (m, 2H), 4.50 (s, 1H), 4.09 - 4.00 (m, 1H), 3.92 (s, 3H), 3.78 - 3.49 (m, 5H), 3.10 (d, J = 4.9 Hz, 3H), 2.95 (s, 1H), 2.56 - 2.49 (m, 5H), 2.47 - 2.33 (m, 5H), 2.23 (d, J = 7.4 Hz, 2H), 2.20 - 2.11 (m, 1H), 1.90 - 1.70 (m, 2H), 1.65 - 1.50 (m, 4H), 1.48 (d, J = 6.9 Hz, 3H), 1.45 - 1.30 (m, 13H), 1.03 - 0.79 (m, 1H); 1313C NMR (101 MHz, CDCl3) δ 170.74, 170.55, 170.27, 169.79, 158.72, 157.96, 152.80, 150.36, 148.66, 147.55, 143.20, 131.77, 131.45, 131.01, 129.67, 128.11, 126.64, 120.30, 116.91, 109.73, 105.67, 79.43, 70.22, 62.37, 59.04, 56.66, 56.49, 56.01, 53.88, 48.86, 47.70, 36.51, 35.99, 33.05, 32.14, 28.70, 28.56, 28.21, 27.16, 25.81, 22.35, 16.23, 14.06 (d, J = 10.8 Hz), 13.96 (d, J = 10.7 Hz); 19 19F NMR (471 MHz, CDCl3) δ -197.68; HRMS (ESI + ) m / z, C 51 H 66 ClFN 10 O6S2: calculated value for [M + H] 1033.4353 + , found 1033.4323.

[0324] Exemplary synthesis of Exemplified Compound 40 (XL02063) [Chemical Structure Diagram]

[0325] [Chemical Structure Diagram] (cis-Cyclohexane-1,3-diyl)dimethanol A solution of Cis-cyclohexane-1,3-dicarboxylic acid (500 mg, 2.9 mmol) in THF (10 mL) was added dropwise with Me2S-BH3 (3.2 ml, 6.38 mmol) at 0 °C. After stirring at 0 °C for 2 - 3 hours, the reaction was quenched slowly with methanol and then water was added. The mixture was extracted with DCM (3×), and the combined organic phases were washed with water and brine, dried over sodium sulfate, filtered, concentrated to give a residue, which was purified by flash column chromatography on silica gel (0% to 10% methanol in DCM) to give (cis-cyclohexane-1,3-diyl)dimethanol (250 mg, 60% yield). 1 H NMR (400 MHz, CDCl3) δ 3.54 - 3.37 (m, 4H), 2.04 (s, 2H), 1.93 - 1.69 (m, 4H), 1.62 - 1.42 (m, 2H), 1.30 (qt, J = 12.2, 3.2 Hz, 1H), 0.87 (qd, J = 12.6, 3.4 Hz, 2H), 0.63 (q, J = 12.1 Hz, 1H). 13 C NMR (101 MHz, CDCl3) δ 77.48, 77.16, 76.84, 68.74, 40.20, 32.70, 29.59, 25.39。

[0326]

Chemical Structure

[0327]

Chemical Structure

[0328]

Chem.

Claims

1. The following formula (I): X-L1-L2-Y Formula (I) {During the ceremony, X is one of the following: 【Chemistry 1】 (In the formula, R 1 are: (i) F, (ii) Cl, (iii) Br, (iv) CF 3 , (v) CN, (vi) NO 2 , (vii) CHF 2 , (viii) CH 2 Selected from F; R 2 are: (i)-Me, (ii)-CD 3 , (iii)-OMe, (iv)-OCD 3 , (v)-OCF 3 , (vi)-OCHF 2 , (vii) ethyl, (viii) -OCH 2 CH 3 , (ix) isopropyl, (x) cyclopropyl, and the following groups: 【Chemistry 2】 is selected from: L1 is the following: 【Transformation 3】 Selected from: L2 is the following: 【Chemistry 4】 (In the formula, m is independently selected from 2, 3, 4, 5, 6, 7, and 8; and n is independently selected from 1, 2, 3, 4, 5, and 6; Y is one of the following: 【Transformation 5】 a compound selected from or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

2. wherein X is one of the following: 【Transformation 6】 2. The compound of formula (I) according to claim 1, selected from:

3. The L1 is one of the following: 【Transformation 7】 3. A compound of formula (I) according to claim 1 or 2, selected from:

4. The L2 is one of the following: 【Transformation 8】 wherein m is independently selected from 3, 4, and 5; 【Chemistry 9】 wherein n is independently selected from 1, 2, 3, and 4; 【Chemistry 10】 2. The compound of formula (I) according to claim 1, selected from:

5. The Y is one of the following: 【Chemistry 11】 2. The compound of formula (I) according to claim 1, selected from:

6. The Y is one of the following: 【Chemistry 12】 2. The compound of formula (I) according to claim 1, selected from:

7. The compound is selected from the group consisting of: 【Chemistry 13-1】 【Chemistry 13-2】 【Chemistry 13-3】 【Chemistry 13-4】 【Chemistry 13-5】 【Chemistry 13-6】 【Chemistry 13-7】 【Chemistry 13-8】 【Chemistry 13-9】 Or a compound of formula (I) according to claim 1 selected from a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

8. a) the compound is: 【Chemistry 14】 or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof; or b) the compound is: (i) (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (ii) (2S,4R)-1-((R)-3-((((1S,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (iii) (2S,3R,4S)-1-((R)-3-((((1r,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (iv) (2S,4R)-1-((R)-3-((((1s,4S)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide; or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof; or c) The compound of formula (I) according to claim 1, wherein the compound is (2S,4R)-1-((R)-3-((((1R,4R)-4-((4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-carboxamide)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof.

9. The compound is selected from the group consisting of: 【Chemistry 15】 or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof; or The compound is selected from the group consisting of: 【Chemistry 16】 or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

10. The compound is selected from the group consisting of: 【Chemistry 17】 Or a compound of formula (I) according to claim 1 selected from a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

11. The compound is selected from the group consisting of: [Chemistry 18] Or a compound of formula (I) according to claim 1 selected from a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

12. The compound is selected from the group consisting of: 【Chemistry 19】 Or a compound of formula (I) according to claim 1 selected from a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

13. The compound is selected from the group consisting of: 【Chemistry 20】 Or a compound of formula (I) according to claim 1 selected from a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

14. The compound is selected from the group consisting of: 【Chemistry 21】 or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof; or The compound is selected from the group consisting of: 【Chemistry 22】 or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

15. 1. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable vehicle or diluent therefor, optionally comprising an effective amount of a compound of claim 1 in combination with a pharmaceutically acceptable carrier, excipient or excipient.

16. 10. The compound of claim 1 for use in medicine.

17. 10. The compound of claim 1 for use in the prevention or treatment of a disease or condition associated with altered GTPase and / or kinase activity of the protein leucine-rich repeat kinase 2 (LRRK2), said use comprising administration of a PROTAC compound of formula (I) to a subject suffering from or likely to be exposed to said disease or condition.

18. 10. The compound of claim 1 for use in the prevention or treatment of a disease or condition associated with altered PDE6D activity, said use comprising administration of a PROTAC compound of formula (I) as defined herein to a subject suffering from or likely to be exposed to a PDE6D-related disease or condition.

19. 10. The compound of claim 1 for use in the prevention or treatment of a disease or condition independently selected from the following: Parkinson's disease, idiopathic Parkinson's disease, idiopathic late-onset Parkinson's disease, familial Parkinson's disease, LRRK2 mutation-associated Parkinson's disease, dementia with Lewy bodies, primary tauopathy, or inflammation-related diseases such as leprosy, neuroinflammation, and Crohn's disease.

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