Aminopyrimidinyl derivatives for treatment of Parkinson's disease

Abstract

Heterobifunctional compounds capable of inducing degradation of leucine rich repeat kinase 2 (LRRK2) and PDE6D are disclosed. These compounds are capable of binding to LRRK2 at one end and binding to an ubiquitin E3 ligase (e.g., cerebron, Von Hippel-Lindau or an apoptosis inhibitor (cIAP)) at the other end, and thus bring LRRK2 very close to the E3 ligase and induce ubiquitination and degradation of the LRRK2 protein. Also disclosed are pharmaceutical compositions comprising the heterobifunctional compounds and methods of using the compounds in the treatment of diseases and disorders associated with LRRK2 and PDE6D.

Description

Technical Field

[0001] This article describes a novel small-molecule protein hydrolysis targeting chimera (PROTAC) that targets and induces rapid and selective degradation of leucine-rich repeat kinase 2 (LRRK2) at nanomolar concentrations in multiple cells. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease, currently affecting approximately 10 million people worldwide. This number is projected 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 due to a rapidly aging population (Feigin et al., Lancet Neurol. 2019 May; 18(5):459-480.). To date, all attempts to slow the progression of PD have failed, and successful treatment remains an extremely important unmet medical need.

[0003] The leucine-rich repeat kinase 2 (LRRK2), encoded by the LRRK2 gene, is a large (286 kDa) multidomain protein. The armadillo repeat motif (ARM), ankyrin repeat domain (ANK), leucine-rich repeat domain, and WD40 repeat domain are involved in protein-protein interactions. The Ras (ras of complex, ROC) and C-terminus (COR) tandem domains of the ROC complex function as GTPases. The kinase domain is the most studied domain of LRRK2, responsible for autophosphorylation at Ser-1292 (Sheng et al., Sci Transl Med. 2012 Dec 12; 4(164):164ra161.) and phosphorylation of a group of Rab GTPases involved in vesicle transport (Stegeret et al. eLife 2016; 5:e12813). LRRK2 was found to play a role in both sporadic and familial PD (Tolosa et al., Nat. Rev. Neurol. 2020; 16(2):97-107.). Pathological mutations in the RocCOR GTPase domain (R1441C / G / H, N1437H, Y1699C) and kinase domain (G2019S, I2020T) lead to alterations in GTPase and / or kinase activity, resulting in changes in vesicle transport, cytoskeleton dynamics, autophagy, lysosomal degradation, neurotransmission, mitochondrial function, and many other PD-related pathogenic markers (Tolosa et al., Nat. Rev. Neurol. 2020; 16(2):97-107.).Therefore, targeting the GTPase and kinase activity of LRRK2 is considered a potential PD treatment to alleviate the disease (Tolosa et al., Nat. Rev. Neurol. 2020; 16(2):97-107.), among which many LRRK2 kinase inhibitors have been described in the past, 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.). (al., J. Med. Chem. 2015; 58(1): 419-32.), and DNL201 and DNL151, which are the two earliest LRRK2 kinase inhibitors in clinical trials (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, which enhance LRRK2 mislocalization and microtubule association by preferentially 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.), and have been observed to have potential undesirable effects on vesicle transport and on-target side effects in the lungs and kidneys (Andersen et al.). al. Toxicology.2018;395:15-22; Baptista et al.Sci.Transl.Med.2020Apr 22;12(540):eaav0820.).

[0004] Therefore, alternative LRRK2 targeting strategies have been proposed and explored, such as the G2019SLRRK2 selective inhibitor (Garofalo et al., J.Med.Chem.2020; 63(23):14821-14839; LRRK2 dimerization inhibitors (Helton et al., ACSChem. 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 LRRK2 proteolytic targeting chimeras (PROTAC) (Konstantinidou et al., ChemMedChem 2021; 16(6): 959-965.) (WO2020081682A1, WO2021194878A and WO2021194879A1). However, to date, no effective, rapid and selective LRRK2 degraders have been reported.

[0005] Induced target protein degradation is a paradigm-shifting approach to drug discovery. Heterofunctional degraders (also known as PROTACs) induce target protein degradation by recruiting E3 ubiquitin ligases near the target protein, leading to ubiquitination and subsequent degradation of the target protein (Schapira et al. Nat. Rev. Drug Discov. 2019; 18(12):949-963.). Previous efforts to develop LRRK2 PROTACs have resulted in 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 based on cereblon, which at most shows moderate and / or slow degrading of LRRK2.

[0006] There is a need to identify GTPase and kinase activities targeting LRRK2, particularly faster, more effective, orally bioavailable, and / or blood-brain barrier-penetrating LRRK2 PROTAC degraders for the treatment of Parkinson's disease and related conditions. Summary of the Invention

[0007] In the first aspect, compounds of formula (I) are provided:

[0008] X-L1-L2-Y

[0009] Formula (I)

[0010] Where X is:

[0011]

[0012] R1 is selected from: (i)F, (ii)Cl, (iii)Br, (iv)CF3, (v)CN, (vi)NO2, (vii)CHF2, (viii)CH2F;

[0013] Wherein R2 is selected from: (i)-Me, (ii)-CD3, (iii)-OMe, (iv)-OCD3, (v)-OCF3, (vi)-OCHF2, (vii) ethyl, (viii)-OCH2CH3, (ix) isopropyl, (x) cyclopropyl and the following groups:

[0014]

[0015] L1 is selected from:

[0016]

[0017] L2 is selected from:

[0018]

[0019] Where m is independently selected from 2, 3, 4, 5, 6, 7 and 8; and where n is independently selected from 1, 2, 3, 4, 5 and 6;

[0020] Where Y is selected from:

[0021]

[0022] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0023] In the compound of formula (I), X can be selected from:

[0024]

[0025] In the compound of formula (I), L1 can be selected from:

[0026]

[0027] In some preferred embodiments, L1 can be selected from:

[0028] Preferred

[0029] In the compound of formula (I), L2 can be selected from:

[0030]

[0031] Where m is independently selected from 3, 4, and 5; Where n is independently selected from 1, 2, 3, and 4;

[0032] In some preferred embodiments, L2 can be selected from:

[0033] Where m is independently selected from 3, 4, and 5 (especially where m is 3). Where n is independently selected from 1, 2, 3, and 4 (especially where n is 1 or 2), In some other preferred embodiments, L2 can be selected from:

[0034]

[0035] In embodiments of the compound of formula (I), Y may be selected from:

[0036]

[0037] In some preferred embodiments of the compound of formula (I), Y may be selected from:

[0038]

[0039] In some preferred embodiments of the compound of formula (I), Y may be selected from:

[0040]

[0041] In some preferred embodiments of the compound of formula (I), Y may be selected from:

[0042]

[0043] In some preferred embodiments, Y can be selected from:

[0044]

[0045] In a particularly preferred embodiment, Y can be selected from:

[0046]

[0047] In the most preferred implementation, Y can be:

[0048]

[0049] The preferred compound of formula (I) may be selected from:

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0059] The preferred compound of formula (I) may be selected from:

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0069] Some preferred compounds of formula (I) may be selected from:

[0070]

[0071] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0072] In addition, some preferred compounds of formula (I) may be selected from:

[0073]

[0074] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0075] Furthermore, compounds of formula (I) can be selected from:

[0076]

[0077] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0078] Furthermore, the preferred compound of formula (I) can be selected from: (i)

[0079]

[0080] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0081] Compounds of formula (I) may also be selected from:

[0082]

[0083] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0084] Some other preferred (I) compounds may be selected from:

[0085]

[0086] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0087] In addition, another particularly preferred compound of formula (I) may be:

[0088]

[0089] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0090] Some other preferred (I) compounds may be selected from:

[0091]

[0092] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0093] Some other preferred compounds of formula (I) may be selected from:

[0094]

[0095] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0096] The particularly preferred compound of formula (I) may be:

[0097]

[0098] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0099] In other words, the preferred compound of formula (I) can be selected from:

[0100] (2S,4R)-N-(2-(3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propoxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (SD13);

[0101] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (SD12);

[0102] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (SD79);

[0103] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (SD82);

[0104] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (SD75);

[0105] (2S,4R)-1-((R)-1-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-17-(1-fluorocyclopropane-1-amido)-16,16-dimethyl-3,6,9,12-tetraoxa-15-thiaoctadecane-18-acyl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (SD74);

[0106] (2S,4R)-1-((R)-3-((4-((4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (SD100);

[0107] 5-(4-(3-(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);

[0108] 5-(4-(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);

[0109] (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)thiazolyl-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethyl)-2-(methylamino)acrylamide HCl salt (SD113);

[0110] (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)thiazolyl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethyl)-2-(methylamino)acrylamide (SD112);

[0111] (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)thiazolyl-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethyl)-2-(methylamino)acrylamide (SD114);

[0112] (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-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-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01078B);

[0113] (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-((3-(4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazin-1-yl)propyl)thio)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01072);

[0114] (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-((4-((4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazin-1-yl)methyl)benzyl)thio)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01070B);

[0115] (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-amido)-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL01119);

[0116] (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-amido)-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL01118);

[0117] (2S,4R)-1-((R)-3-((4-((4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL01120);

[0118] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01126);

[0119] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01134);

[0120] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01076);

[0121] (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-amido)-3-methylbutyryl)-N-(3-fluoro-4-(4-methylthiazolyl-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL01123);

[0122] (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-amido)-3-methylbutyryl)-N-(3-fluoro-4-(4-methylthiazolyl-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL01122);

[0123] (2S,4R)-1-((R)-3-((4-((4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-N-(3-fluoro-4-(4-methylthiazolyl-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL01121);

[0124] (2S,4R)-1-((R)-3-((2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01131);

[0125] (2S,4R)-1-((R)-3-(((S)-1-(4-(4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl-2-yl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01140);

[0126] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01111);

[0127] (2S,4R)-1-((S)-1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenoxy)-14-(1-fluorocyclopropane-1-amido)-13,13-dimethyl-3,6,9-trioxa-12-thiapentadecanoyl-15-acyl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01145);

[0128] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01149);

[0129] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01168);

[0130] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02017);

[0131] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02031);

[0132] (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]undecyl-3-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02038);

[0133] (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-amido)-3-methylbutyryl)-N-(2-fluoro-4-(4-methylthiazolyl-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL02047);

[0134] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02048);

[0135] (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-amido)-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL02049);

[0136] (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-amido)-3-methylbutyryl)-N-(2-fluoro-4-(4-methylthiazolyl-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL02058);

[0137] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02059);

[0138] (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-amido)-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL02060);

[0139] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02063);

[0140] (2S,4R)-1-((R)-3-((3-(9-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)-3,9-diazaspiro[5.5]undecyl-3-yl)propyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02067B);

[0141] (2S,4R)-1-((R)-3-((((1R,4R)-4-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazine-1-carbonyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02068);

[0142] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02093);

[0143] (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-di Alkyl)methyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02098);

[0144] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02133);

[0145] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02134);

[0146] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02127);

[0147] (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-amido)-3-methylbutyryl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02152);

[0148] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)-4,5-dihydro-1H-pyrrole-2-carboxamide (XL02146);

[0149] (2S,4R)-N-(2-(3-(1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperidin-4-yl)propoxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02117);

[0150] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02126);

[0151] (2S,4R)-N-(2-((5-(4-(4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)pentyl)oxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02115);

[0152] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02135);

[0153] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02120);

[0154] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02119);

[0155] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02137);

[0156] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02138);

[0157] (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-amido)-3-methylbutyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01077);

[0158] (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-amido)-3-methylbutyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01083);

[0159] (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-amido)-3-methylbutyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01088);

[0160] (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-amido)-3-methylbutyryl)-N-(4-cyanobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01089);

[0161] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0162] Preferred compounds may also be selected from:

[0163] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (SD75);

[0164] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (SD82);

[0165] (2S,4R)-1-((R)-3-((4-((4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (SD100); or its pharmaceutically acceptable salts, enantiomers, stereoisomers, hydrates, solvates or polymorphs.

[0166] The preferred compounds may also be selected from:

[0167] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01126);

[0168] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (cis-XL01126);

[0169] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01134);

[0170] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0171] Some preferred compounds may also be selected from:

[0172] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02017);

[0173] (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-amido)-3-methylbutyryl)-N-(2-fluoro-4-(4-methylthiazolyl-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (XL02047);

[0174] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02048);

[0175] (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-amido)-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (XL02049);

[0176] (2S,4R)-1-((R)-3-((((1R,4R)-4-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazine-1-carbonyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02068);

[0177] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02093);

[0178] (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-di Alkyl)methyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02098);

[0179] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02133);

[0180] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02134);

[0181] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02127);

[0182] (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-amido)-3-methylbutyryl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02152);

[0183] (2S,4R)-N-(2-(3-(1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperidin-4-yl)propoxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02117);

[0184] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02126);

[0185] (2S,4R)-N-(2-((5-(4-(4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)pentyl)oxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02115);

[0186] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02135);

[0187] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02120);

[0188] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02119);

[0189] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02137);

[0190] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (XL02138);

[0191] (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-amido)-3-methylbutyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01077);

[0192] (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-amido)-3-methylbutyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01083);

[0193] (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-amido)-3-methylbutyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01088);

[0194] (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-amido)-3-methylbutyryl)-N-(4-cyanobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01089);

[0195] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0196] The particularly preferred compound of formula (I) may be selected from XL01126, cis-XL01126 and XL01134, especially XL01126, or its pharmaceutical salt, enantiomer, stereoisomer, hydrate, solvate or polymorph.

[0197] The particularly preferred compounds of formula (I) may also be selected from:

[0198] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01126);

[0199] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01134);

[0200] (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-amido)-3-methylbutyryl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL02152);

[0201] (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-amido)-3-methylbutyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01088);

[0202] Alternatively, it can be a medicinal salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph.

[0203] In some embodiments, the preferred compound of formula (I) may be (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01126) or its pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph.

[0204] Furthermore, the preferred compound of formula (I) may be selected from XL02047, XL02048, XL02049, XL02017 and XL02068, or its pharmaceutical salt, enantiomer, stereoisomer, hydrate, solvate or polymorph.

[0205] In addition, the preferred compound of formula (I) may be selected from SD82, SD75 and DS100, or its pharmaceutical salt, enantiomer, stereoisomer, hydrate, solvate or polymorph.

[0206] In addition, the preferred compound of formula (I) may be selected from SD12, SD13 and XL01131, or its pharmaceutical salt, enantiomer, stereoisomer, hydrate, solvate or polymorph.

[0207] In addition, the preferred compound of formula (I) may be selected from XL02152, XL02098 and MP01088, or its pharmaceutical salt, enantiomer, stereoisomer, hydrate, solvate or polymorph.

[0208] In a second aspect, a pharmaceutical composition is provided comprising the compound as defined above and its pharmaceutically acceptable carrier or diluent.

[0209] The pharmaceutical composition may comprise an effective amount of the compound as defined above, in combination with a pharmaceutically acceptable carrier, additive or excipient, and optionally in combination with another bioactive agent.

[0210] In the third aspect, compounds as defined herein are provided for medical use. The medical uses of the compounds as defined herein are also provided herein.

[0211] In a fourth aspect, compounds as defined herein are provided for the prevention or treatment of diseases or conditions associated with altered GTPase and / or kinase activity of leucine repeat kinase 2 (LRRK2) protein, comprising administering a compound of formula (I)PROTAC to a subject who has or may be exposed to said disease or condition. The use of compounds as defined herein for the prevention or treatment of diseases or conditions associated with altered GTPase and / or kinase activity of leucine repeat kinase 2 (LRRK2) protein is also provided herein, comprising administering a compound of formula (I)PROTAC to a subject who has or may be exposed to said disease or condition.

[0212] In the fifth aspect, compounds as defined herein are provided for the prevention or treatment of diseases or conditions associated with altered PDE6D activity, comprising administering a PROTAC compound of formula (I) as defined herein to a subject suffering from or potentially exposed to a PDE6D-related disease or condition. The use of compounds as defined herein for the prevention or treatment of diseases or conditions associated with altered PDE6D activity is also provided herein, comprising administering a PROTAC compound of formula (I) as defined herein to a subject suffering from or potentially exposed to a PDE6D-related disease or condition.

[0213] In the sixth aspect, compounds as defined herein are provided for the prevention or treatment of diseases or conditions 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 tau protein disorders, or inflammation-related diseases such as leprosy, neuroinflammation, and Crohn's disease. The use of compounds as defined herein for the prevention or treatment of diseases or conditions independently selected from the following is also provided herein.

[0214] In a seventh aspect, a method is provided for modulating the activity of a protein rich in leucine repeat kinase 2 (LRRK2) protein in a patient in need, comprising administering to the patient an amount of a compound as described herein.

[0215] In the eighth aspect, a method for degrading leucine-rich repeat kinase 2 (LRRK2) in patients in need is provided, comprising administering an effective amount of a compound as described herein to the patient.

[0216] In another aspect, a method for modulating the protein activity of protein PDE6D in patients in need is provided, comprising administering to the patient an amount of a compound as described herein.

[0217] In another aspect, a method for degrading the protein PDE6D in a patient in need is provided, comprising administering an effective amount of a compound as described herein to the patient.

[0218] In another aspect, a method is provided for treating patients with a disease or condition associated with altered activity of GTPases and / or kinases rich in leucine repeat kinase 2 (LRRK2) protein, comprising administering a compound of formula (I)PROTAC to a subject who has or may be exposed to said disease or condition.

[0219] In another aspect, a method is provided for treating a patient with a disease or condition associated with altered PDE6D activity, comprising administering to a subject who has or may be exposed to a PDE6D-related disease or condition a compound of formula (I) PROTAC as defined herein, the method comprising administering a therapeutically effective dose of the compound or pharmaceutical composition as described herein.

[0220] In another aspect, a method is provided for treating patients suffering from diseases or conditions independently selected from: Parkinson's disease, idiopathic Parkinson's disease, idiopathic late-onset Parkinson's disease, familial Parkinson's disease, LRRK2 mutation-associated Parkinson's disease, Lewy body dementia, primary tau protein disease, or inflammation-related diseases such as leprosy, neuroinflammation, and Crohn's disease, the method comprising administering a therapeutically effective dose of a compound or pharmaceutical composition as described herein. Attached Figure Description

[0221] Figure 1 This is an image of a Western blot, showing the effects of a first-generation degrading agent on WT mouse embryonic fibroblasts (MEF) and LRRK2 cells. G2019S MEF treatment with 33 nM / 4 h and 1 μM / 4 h resulted in total LRRK2 degradation, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation at Thr73.

[0222] Figure 2 This is an image of a Western blot, showing the effects of the first-generation degradative agents on WT MEF and LRRK2. G2019S MEF treatment with 33 nM / 24 h and 1 μM / 24 h resulted in total LRRK2 degradation, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation at Thr73.

[0223] Figure 3 This is an image of a Western blot, showing the effects of second-generation (Group 1) degraders on WT MEF and LRRK2. G2019S After MEF treatment with 33 nM / 4 h and 1 μM / 4 h, total LRRK2 degradation, phosphorylation inhibition of LRRK2 at Ser935, and phosphorylation inhibition of Rab10 at Thr73 were observed.

[0224] Figure 4 This is an image of a Western blot, showing the effects of second-generation (Group 1) degraders on WT MEF and LRRK2. G2019S MEF treatment with 33 nM / 24 h and 1 μM / 24 h resulted in total LRRK2 degradation, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation at Thr73.

[0225] Figure 5 This is an image of a Western blot, showing the effects of second-generation (Group 2) degraders on WT MEF and LRRK2. G2019SMEF treatment with 33 nM / 4 h and 1 μM / 4 h resulted in total LRRK2 degradation, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation at Thr73.

[0226] Figure 6 This is an image of a Western blot, showing the effects of second-generation (Group 2) degraders on WT MEF and LRRK2. G2019S MEF treatment with 33 nM / 24 h and 1 μM / 24 h resulted in total LRRK2 degradation, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation at Thr73.

[0227] Figure 7 This is an image of a Western blot, showing the effects of third-generation (Group 1) degradative agents on WT MEF and LRRK2. G2019S MEF treatment with 33 nM / 4 h and 1 μM / 4 h resulted in total LRRK2 degradation, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation at Thr73.

[0228] Figure 8 This is an image of a Western blot, showing the effects of third-generation (Group 1) degradative agents on WT MEF and LRRK2. G2019S MEF treatment with 33 nM / 24 h and 1 μM / 24 h resulted in total LRRK2 degradation, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation at Thr73.

[0229] Figure 9 This is an image of a Western blot, showing the effects of third-generation (Group 2) degradative agents on WT MEF and LRRK2. G2019S MEF treatment with 33 nM / 4 h and 1 μM / 4 h resulted in total LRRK2 degradation, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation at Thr73.

[0230] Figure 10 This is an image of a Western blot, showing the effects of third-generation (Group 2) degradative agents on WT MEF and LRRK2. G2019S MEF treatment with 33 nM / 24 h and 1 μM / 24 h resulted in total LRRK2 degradation, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation at Thr73.

[0231] Figure 11AThis is an image of a Western blot, showing the results of comparing WT MEF and LRRK2. G2019S After 24 hours of MEF treatment, SD75 showed dose-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0232] Figure 11B This shows the results for WT MEF and LRRK2 respectively. G2019S 24 hours after MEF treatment, SD75 showed total LRRK2 in a dose-dependent manner. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0233] Figure 11C This shows the results for WT MEF and LRRK2 respectively. G2019S A plot showing the inhibition of Rab10 phosphorylation at Thr73 by SD75 in a dose-dependent manner 24 hours after MEF treatment.

[0234] Figure 12A This is an image of a Western blot, showing the results of comparing WT MEF and LRRK2. G2019S After 4 hours of MEF treatment, XL01134 showed dose-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0235] Figure 12B This shows the results for WT MEF and LRRK2 respectively. G2019S Four hours after MEF treatment, XL01134 showed a dose-dependent total LRRK2. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0236] Figure 12C This shows the results for WT MEF and LRRK2 respectively. G2019S A plot showing the inhibition of Rab10 phosphorylation at Thr73 in a dose-dependent manner by XL01134 after 4 hours of MEF treatment.

[0237] Figure 13A This is an image of a Western blot, showing the results of comparing WT MEF and LRRK2. G2019S After 4 hours of MEF treatment, XL01126 showed dose-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0238] Figure 13BThis shows the results for WT MEF and LRRK2 respectively. G2019S Four hours after MEF treatment, XL01126 showed total LRRK2 in a dose-dependent manner. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0239] Figure 13C This shows the results for WT MEF and LRRK2 respectively. G2019S A plot showing the inhibition of Rab10 phosphorylation at Thr73 in a dose-dependent manner by XL01126 after 4 hours of MEF treatment.

[0240] Figure 14A This is an image of a Western blot, showing the results after processing WT MEF and LRRK2 with 1000 nm SD75. G2019S Following MEF, SD75 exhibited time-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0241] Figure 14B This demonstrates the processing of WT MEF and LRRK2 using a 1000nm SD75. G2019S Following MEF, SD75's total LRRK2 in a time-dependent manner WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0242] Figure 14C This demonstrates the processing of WT MEF and LRRK2 using a 1000nm SD75. G2019S A graph showing the time-dependent inhibition of Rab10 phosphorylation at Thr73 by SD75 after MEF.

[0243] Figure 15A This is an image of a Western blot, showing the results after processing WT MEF and LRRK2 with XL01134 at 300 nm. G2019S Following MEF, XL01134 exhibited time-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0244] Figure 15B This demonstrates the processing of WT MEF and LRRK2 using a 300nm XL01134. G2019S Following MEF, XL01134 exhibits a time-dependent total LRRK2. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0245] Figure 15C This demonstrates the processing of WT MEF and LRRK2 using a 300nm XL01134. G2019S Following MEF, a graph showing the time-dependent inhibition of Rab10 phosphorylation at Thr73 by XL01134.

[0246] Figure 16A This is an image of a Western blot, showing the results after processing WT MEF and LRRK2 with 300 nm XL01126. G2019S Following MEF, XL01126 exhibited time-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0247] Figure 16B This demonstrates the processing of WT MEF and LRRK2 using XL011126 at 300 nM. G2019S Following MEF, XL01126 exhibits a time-dependent total LRRK2. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0248] Figure 16C This demonstrates the processing of WT MEF and LRRK2 using XL01126 at 300 nM. G2019S Following MEF, a graph showing the time-dependent inhibition of Rab10 phosphorylation at Thr73 by XL01126.

[0249] Figure 17A This is an image of a Western blot, showing LRRK2 in the presence of VHL ligand (VH101), MLN4924, and MG132. WT The degradation of LRRK2 and the dephosphorylation of Rab10.

[0250] Figure 17B This shows the total LRRK2 induced by XL01126. WT The degradation can be rescued by pretreatment with VHL ligand (VH101), MLN4924 and MG132.

[0251] Figure 17C The figure shows that the dephosphorylation of Rab10 induced by XL01126 at Thr73 on WT MEF can be partially rescued by pretreatment with VHL ligand (VH101), MLN4924 and MG132.

[0252] Figure 17D This shows XL01126-induced LRRK2 WTDephosphorylation at Ser935 can be partially salvaged by pretreatment with VHL ligand (VH101), MLN4924, and MG132.

[0253] Figure 18A This is an image of a Western blot, showing LRRK2 in the presence of VHL ligand (VH101), MLN4924, and MG132. G2019S LRRK2 on MEF G2019S The degradation of LRRK2 G2019S And dephosphorylation of Rab10.

[0254] Figure 18B This shows the total LRRK2 induced by XL01126. G2019S The degradation can be rescued by pretreatment with VHL ligand (VH101), MLN4924 and MG132.

[0255] Figure 18C This is shown in LRRK2 G2019S The dephosphorylation of Rab10 at Thr73 induced by XL01126 on MEF can be partially rescued by pretreatment with VHL ligand (VH101), MLN4924 and MG132 (Figure).

[0256] Figure 18D This shows XL01126-induced LRRK2 G2019S Dephosphorylation at Ser935 can be partially salvaged by pretreatment with VHL ligand (VH101), MLN4924, and MG132.

[0257] Figure 19A This is an image of a Western blot, showing the effect on LRRK2. R1441C After 4 hours of MEF treatment, XL01126 was able to degrade LRRK2 in a dose-dependent manner. R1441C And inhibit LRRK2 R1441C Phosphorylation at Ser935 and phosphorylation at Thr73 of Rab10, while cis-XL01126 can only inhibit LRRK2 in a dose-dependent manner. R1441C Phosphorylation at Ser935 and phosphorylation of Rab10 at Thr73.

[0258] Figure 19B This shows the results for LRRK2 R1441C After 4 hours of MEF treatment, XL01126 was able to degrade LRRK2 in a dose-dependent manner. R1441C cis-XL01126 cannot degrade LRRK2 R1441C The image.

[0259] Figure 19C The figure shows that XL01126 is more effective than cisXL01126 in inhibiting the phosphorylation of Rab10 at Thr73.

[0260] Figure 20A The image is a Western blot image showing that XL01126 is able to degrade total LRRK2 in macrophages in a dose-dependent manner, while cis-XL01126 cannot degrade LRRK2 in macrophages.

[0261] Figure 20B The figure shows that XL01126 can degrade total LRRK2 in macrophages in a dose-dependent manner, while cis-XL01126 cannot degrade LRRK2 in macrophages.

[0262] Figure 21A The image is a Western blot image showing that after 4 hours of treatment with human peripheral blood mononuclear cells (PBMCs), XL01126 degrades LRRK2 in a dose-dependent manner and inhibits LRRK2 phosphorylation at Ser935 and Rab10 phosphorylation at Thr73, while cis-XL01126 is only able to inhibit LRRK2 phosphorylation at Ser935 and Rab10 phosphorylation at Thr73 in a dose-dependent manner.

[0263] Figure 21B The figure shows that after 4 hours of treatment with human peripheral blood mononuclear cells (PBMCs), XL01126 degraded LRRK2 in a dose-dependent manner, while cis-XL01126 did not degrade LRRK2 at the tested concentration.

[0264] Figure 21C The figure shows that both XL01126 and cisXL01126 inhibited Rab10 phosphorylation on human peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner after 4 hours of treatment, but XL01126 was more effective.

[0265] Figure 22A The image is a Western blot image showing that after 24 hours of treatment with human peripheral blood mononuclear cells (PBMCs), XL01126 degrades LRRK2 in a dose-dependent manner and inhibits phosphorylation of LRRK2 at Ser935 and Rab10 at Thr73, while cis-XL01126 is only able to inhibit phosphorylation of LRRK2 at Ser935 and Rab10 at Thr73 in a dose-dependent manner.

[0266] Figure 22B The figure shows that after 24 hours of treatment with human peripheral blood mononuclear cells (PBMCs), XL01126 degraded LRRK2 in a dose-dependent manner, while cis-XL01126 hardly degraded LRRK2 at the tested concentration.

[0267] Figure 22C The figure shows that both XL01126 and cisXL01126 inhibited Rab10 phosphorylation on human peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner after 24 hours of treatment, but XL01126 was more effective.

[0268] Figure 23A The images are Western blot images showing that after treatment of human peripheral blood mononuclear cells (PBMCs) with 300 nM XL01126, XL01126 degrades LRRK2 in a time-dependent manner and inhibits LRRK2 phosphorylation at Ser935 and Rab10 phosphorylation at Thr73. 300 nM cis-XL01126 does not degrade LRRK2 and barely inhibits LRRK2 phosphorylation at Ser935 and Rab10 phosphorylation at Thr73.

[0269] Figure 23B The figure shows that after treatment of human peripheral blood mononuclear cells (PBMCs) with 300 nM XL01126, XL01126 degraded LRRK2 in a time-dependent manner and inhibited phosphorylation of LRRK2 at Ser935 and phosphorylation of Rab10 at Thr73.

[0270] Figure 23C The graph shows that 300 nM cis-XL01126 cannot degrade LRRK2 and hardly inhibits the phosphorylation of LRRK2 at Ser935 and the phosphorylation of Rab10 at Thr73.

[0271] Figure 24 This is a graph of quantitative proteomics analysis, showing the ratio of intracellular individual protein levels in cells treated with XL01126 to those treated with cis-XL01126 (WT MEF).

[0272] Figure 25A This is an image of a Western blot, showing the results of comparing WT MEF and LRRK2. G2019S After 4 hours of MEF treatment, XL02047 showed dose-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0273] Figure 25BThis shows the results for WT MEF and LRRK2 respectively. G2019S Four hours after MEF treatment, XL02047 showed total LRRK2 in a dose-dependent manner. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0274] Figure 25C This shows the results for WT MEF and LRRK2 respectively. G2019S A plot showing the inhibition of Rab10 phosphorylation at Thr73 in a dose-dependent manner by XL02047 after 4 hours of MEF treatment.

[0275] Figure 26A This is an image of a Western blot, showing the results of comparing WT MEF and LRRK2. G2019S After 4 hours of MEF treatment, XL02048 showed dose-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0276] Figure 26B This shows the results for WT MEF and LRRK2 respectively. G2019S Four hours after MEF treatment, XL02048 showed a dose-dependent total LRRK2 concentration. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0277] Figure 26C This shows the results for WT MEF and LRRK2 respectively. G2019S A plot showing the inhibition of Rab10 phosphorylation at Thr73 in a dose-dependent manner by XL02048 after 4 hours of MEF treatment.

[0278] Figure 27A This is an image of a Western blot, showing the results of comparing WT MEF and LRRK2. G2019S After 4 hours of MEF treatment, XL02049 showed dose-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0279] Figure 27B This shows the results for WT MEF and LRRK2 respectively. G2019S Four hours after MEF treatment, XL02049 showed a dose-dependent total LRRK2. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0280] Figure 27CThis shows the results for WT MEF and LRRK2 respectively. G2019S A plot showing the inhibition of Rab10 phosphorylation at Thr73 in a dose-dependent manner by XL02049 after 4 hours of MEF treatment.

[0281] Figure 28A This is an image of a Western blot, showing the results of comparing WT MEF and LRRK2. G2019S After 4 hours of MEF treatment, XL02017 showed dose-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0282] Figure 28B This shows the results for WT MEF and LRRK2 respectively. G2019S Four hours after MEF treatment, XL02017 showed total LRRK2 in a dose-dependent manner. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0283] Figure 28C This shows the results for WT MEF and LRRK2 respectively. G2019S A plot showing the inhibition of Rab10 phosphorylation at Thr73 in a dose-dependent manner by XL02017 after 4 hours of MEF treatment.

[0284] Figure 29A This is an image of a Western blot, showing the results of comparing WT MEF and LRRK2. G2019S After 4 hours of MEF treatment, XL02038 showed dose-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0285] Figure 29B This shows the results for WT MEF and LRRK2 respectively. G2019S Four hours after MEF treatment, XL02038 showed total LRRK2 in a dose-dependent manner. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0286] Figure 29C This shows the results for WT MEF and LRRK2 respectively. G2019S A plot showing the inhibition of Rab10 phosphorylation at Thr73 in a dose-dependent manner by XL02038 after 4 hours of MEF treatment.

[0287] Figure 30A This is an image of a Western blot, showing the results of comparing WT MEF and LRRK2.G2019S After 4 hours of MEF treatment, XL02068 showed dose-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0288] Figure 30B This shows the results for WT MEF and LRRK2 respectively. G2019S Four hours after MEF treatment, XL02068 showed total LRRK2 in a dose-dependent manner. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0289] Figure 30C This shows the results for WT MEF and LRRK2 respectively. G2019S A plot showing the inhibition of Rab10 phosphorylation at Thr73 in a dose-dependent manner by XL02068 after 4 hours of MEF treatment.

[0290] Figure 31A This is an image of a Western blot, showing the results of comparing WT MEF and LRRK2. G2019S After 4 hours of MEF treatment, XL02059 showed dose-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0291] Figure 31B This shows the results for WT MEF and LRRK2 respectively. G2019S Four hours after MEF treatment, XL02059 showed total LRRK2 in a dose-dependent manner. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0292] Figure 31C This shows the results for WT MEF and LRRK2 respectively. G2019S A plot showing the inhibition of Rab10 phosphorylation at Thr73 in a dose-dependent manner by XL02059 after 4 hours of MEF treatment.

[0293] Figure 32A This is an image of a Western blot, showing the results of comparing WT MEF and LRRK2. G2019S After 4 hours of MEF treatment, XL02060 showed dose-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0294] Figure 32B This shows the results for WT MEF and LRRK2 respectively. G2019SFour hours after MEF treatment, XL02060 showed total LRRK2 in a dose-dependent manner. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0295] Figure 32C This shows the results for WT MEF and LRRK2 respectively. G2019S A plot showing the inhibition of Rab10 phosphorylation at Thr73 in a dose-dependent manner by XL02060 after 4 hours of MEF treatment.

[0296] Figure 33 The figure shows the plasma concentrations of XL01126 in mice after administration of XL01126 via intravenous (5 mg / Kg), intraperitoneal (30 mg / Kg), and oral (30 mg / Kg), respectively.

[0297] Figure 34 The figure shows the brain concentrations of XL01126 in mice after administration of XL01126 via intravenous (5 mg / Kg), intraperitoneal (30 mg / Kg), and oral (30 mg / Kg), respectively.

[0298] Figure 35 The figure shows the cerebrospinal fluid concentration of XL01126 in mice after administration of XL01126 via intravenous (5 mg / Kg), intraperitoneal (30 mg / Kg), and oral (30 mg / Kg), respectively.

[0299] Figure 36 The figure shows the binary binding of VH101, XL01126, SD75, XL01134 and cis-XL01126 with VHL as measured by fluorescence polarization assay.

[0300] Figure 37A and Figure 37B This is a graph showing the VHL binding assays of VH101, XL01126, SD75, and XL01134 in permeabilized and live HEK293 cells.

[0301] Figure 38A and Figure 38B This is a graph of LRRK2 binding assays, which shows that HG-10-102-01, XL01126, SD75, and XL01134 can bind to LRRK2 in permeabilized and live HEK293 cells.

[0302] Figure 39A , Figure 39B and Figure 39CThis is a graph showing the LRRK2 binding assays of XL01126, SD75, and XL01134 in the presence and absence of VCB protein, using HG-10-102-01 as a reference compound.

[0303] Figure 40 This diagram illustrates the formation of ternary complexes with VHL and LRRK2 induced by cis-XL01126, XL01126, SD75, and XL01134.

[0304] Figure 41 , 42 Images 43 and 44 are Western blot images showing the effects of a fourth-generation degrader at specified concentrations on WT mouse embryonic fibroblasts (MEF) and LRRK2 cells. G2019S After 4 hours of MEF treatment, total LRRK2 degradation, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation at Thr73 were observed.

[0305] Figure 44A This is an image of a Western blot, showing the effects of processing WT MEF and LRRK2 with a specified concentration of XL02098. G2019S MEF, after 4 hours, showed that XL02098 exhibited time-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0306] Figure 44B This demonstrates the effects of treating WT MEF and LRRK2 with various concentrations of XL02098. G2019S Following MEF, XL02098 showed total LRRK2 in a dose-dependent manner. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0307] Figure 44C This demonstrates the effects of treating WT MEF and LRRK2 with various concentrations of XL02098. G2019S Following MEF, a plot showing the inhibition of Rab10 phosphorylation at Thr73 by XL02098 in a dose-dependent manner.

[0308] Figure 45A This is an image of a Western blot, showing the effects of treating WT MEF and LRRK2 with a specified concentration of XL02152. G2019S MEF, after 4 hours, showed that XL02152 exhibited time-dependent degradation of total LRRK2, inhibition of LRRK2 phosphorylation at Ser935, and inhibition of Rab10 phosphorylation.

[0309] Figure 45B This demonstrates the effects of treating WT MEF and LRRK2 with various concentrations of XL02098. G2019S Following MEF, XL02152 showed total LRRK2 in a dose-dependent manner. WT Degradation and total LRRK2 G2019S A diagram of the degradation process.

[0310] Figure 45C This demonstrates the effects of treating WT MEF and LRRK2 with various concentrations of XL02098. G2019S Following MEF, a plot showing the inhibition of Rab10 phosphorylation at Thr73 by XL02152 in a dose-dependent manner. Detailed Implementation

[0311] The present invention aims to identify faster and more effective, and potentially orally bioavailable and / or blood-brain barrier-penetrating LRRK2 PROTAC degraders, and to qualify them as chemical probes for studying LRRK2 biology, further validating the target as a therapeutic mechanism in PD, and providing potential treatment or prevention for PD and related diseases.

[0312] Using HG-10-102-01 as the warhead and recruiting a variety of different E3 ligase ligands, the inventors developed the first-generation LRRK2 PROTAC.

[0313]

[0314] Among the first-generation compounds, SD75, SD82, and SD100 exhibited moderate LRRK2 degradation ( Figure 1 and Figure 2 ):

[0315]

[0316] Then, based on the most effective first-generation LRRK2 PROTAC SD75, SD82 and SD100 groups, the second-generation LRRK2 PROTAC was designed and synthesized.

[0317] Two sets of second-generation LRRK2 PROTAC were designed and synthesized:

[0318] Group 1

[0319]

[0320] Group 2

[0321]

[0322] Western blot analysis of LRRK2 protac degradation was performed using WT and PD-associated LRRK2 [G2019S] mouse embryonic fibroblasts (MEFs). Second-generation protacs were tested in parallel with first-generation protacs SD75, SD82, and SD100. Compounds were tested at 33 nM or 1 mM concentrations for 4 or 24 hours, with two biological replicates run under each condition, and quantification representing both replicates was achieved. Quantitative immunoblotting analysis was performed using readouts of total LRRK2 (the C-terminal epitope of the antibody-recognized protein), Rab10 pThr73 (a physiological substrate), and LRRK2 pSer935 (a biomarker phosphorylation site).

[0323] From the selection of second-generation LRRK2 PROTAC ( Figure 3 , 4 Of the compounds identified in (5 and 6), XL01126 and XL01134 are the two best-identified compounds.

[0324]

[0325] Therefore, in dose-dependent assays, head-to-head comparisons of SD75 vs XL01126 vs XL01134 showed that SD75 induced partial LRRK2 degradation ( Figure 11A (C), XL01134 induces LRRK2 degradation, accompanied by a "hook effect" (Douglass, et al., J Am Chem Soc. 2013; 135(16):6092–6099). Figure 12A (to C) and XL01126 induce almost complete LRRK2 degradation ( Figure 13A To C).

[0326] Table 1 - Head-to-head degradation comparison of LRRK2 degraders XL01126, XL01134 and SD75

[0327]

[0328] The results in Table 1 also show that XL01126 and XL01134 are more effective degraders than SD75.

[0329] Table 2 - Head-to-head comparison of pRab10 dephosphorylation by XL01126, XL01134 and SD75

[0330]

[0331] The results in Table 2 show that XL01126 and XL01134 are more effective than SD75 in dephosphorylating pRab10.

[0332] Table 3 - Head-to-head comparison of pSer935-LRRK2 dephosphorylation by XL01126, XL01134 and SD75.

[0333]

[0334] Table 3 shows that XL01126 and XL01134 are more effective than SD75 in dephosphorylating pSer935-LRRK2.

[0335] In the time-dependent determination, side-by-side comparisons of SD75, XL01134, and XL01126 showed that SD75 ( Figure 14A To C), XL01134 ( Figure 15A To C) and XL01126 ( Figure 16A All of these can induce time-dependent degradation of LRRK2, as well as time-dependent dephosphorylation of pRab10 and LRRK2.

[0336] Table 4 - Summary of Dose Response and Time Process Experiments

[0337]

[0338] The results showed that

[0339] 1) XL01126 is the most effective LRRK2 degrader and induces the fastest LRRK2 degradation among SD75 and XL01134.

[0340] 2) Compared with SD75 and XL01134, XL01126 induces the most efficient and fastest LRRK2 dephosphorylation and pRab10 dephosphorylation.

[0341] Further screening of compounds based on XL01126 was then conducted, and cis-XL01126 was synthesized as a non-degradation control to analyze inhibition from degradation. Cis-XL01126 does not bind to VHL E3 ligase.

[0342]

[0343] Table 5 - Binding affinities of VH101, cis-XL01126, and XL01126 as determined by fluorescence polarization assay

[0344]

[0345] XL01126-induced LRRK2 degradation depends on the proteasome pathway. Figure 17A To D and Figure 18A To D).

[0346] XL01126 dose-dependently degrades LRRK2 R1441C MEF( Figure 19A To C) and macrophages ( Figure 20A LRRK2 in B) and also dose- and time-dependently degrade LRRK2 in human PBMC cells. Figure 21A To C, Figure 22A To C, Figure 23A To C).

[0347] As shown in TMT-labeled proteomics studies, XL01126 induces selective LRRK2 degradation in WT MEF. Figure 24 ).

[0348] Table 6 shows that the solubility of XL01126 in PBS and Fed-State Simulated Intestinal Fluid (FeSSIP) is 0.55 μM and 26.05 μM, respectively. XL01126 can penetrate Caco-2 monolayers. XL01126 is stable in mouse plasma and mouse hepatocytes, with half-lives of 108.29 min and 314.33 min, respectively. XL01126 is metabolized in mouse liver microsomes, with a half-life of 3.65 min.

[0349] Table 6. Physicochemical properties and in vitro ADME properties of XL01126

[0350]

[0351] XL01126 exhibits a remarkable in vivo pharmacokinetic profile, with an oral bioavailability of 15% and the ability to cross the blood-brain barrier. Figure 33 , 34 and 35).

[0352] Despite XL01126 being VHL ( Figure 36 , 37A and 37B) and LRRK2 ( Figure 38A and 38B The binary binding affinity of XL01126 is poor compared to XL01134 and SD75 in the permeation mode of VHL and LRRK2 target binding assays, and XL01126 is worse in binding to VHL and LRRK2 than XL01134 and SD75. However, XL01126 is the most permeable in PROTAC testing, as evidenced by its highest IC50 value between permeation and live mode target binding assays. 50As demonstrated, XL01126 also forms a higher and more synergistic ternary complex with VHL and LRRK2 than with XL01134 and SD75 (Figure 39).

[0353] Considering the favorable degradation profiles of XL01126 and XL01134, and the excellent in vivo PK profile of XL01126, further modifications (third generation) of LRRK2PROTAC are based on XL01126 and XL01134:

[0354] XL02031, XL02038, and XL02017 are based on XL01126, with modifications to the piperidine moiety:

[0355]

[0356] XL02058, XL02059, XL02060, and XL02063 are based on XL01134 and modify the VHL ligands and connectors accordingly.

[0357]

[0358] XL02047, XL02048, and XL02049 are based on XL01126 and have modified VHL ligands; XL02067B, XL02068, and XL02077 have modified connectors.

[0359]

[0360] Most compounds from the third generation showed degradation efficiencies comparable to XL01126 and XL01134.

[0361] Results and discussion

[0362] Figure 1 The results showed that after treatment at 33 nM for 4 hours, SD12, SD13, SD79, SD75, SD82, SD100, SD10, SD11, SD112, SD113, and SD114 exhibited a degradation of 0% to 25% of LRRK2. WT and 0 to 60% LRRK2 G2019S It also inhibits 0 to 25% of pRab10 and LRRK2 in WT MEF. G2019S 0 to 60% of pRab10 in MEF Figure 1 After treatment with 1 μM for 4 hours, SD12, SD13, SD79, SD75, SD82, SD100, SD10, SD11, SD112, SD113, and SD114 degraded LRRK2 by 0% to 45%. WT and 0 to 75% LRRK2G2019S It also inhibits 18% to 90% of pRab10 and LRRK2 in WT MEF. G2019S pRab10 in MEF accounts for 30% to 95% ( Figure 1 ).

[0363] As in Figure 2 As observed, after treatment at 33 nM / 24 hours, SD12, SD13, SD79, SD75, SD82, SD100, SD10, SD11, SD112, SD113, and SD114 showed a degradation of 0% to 40% of LRRK2. WT and 0 to 70% LRRK2 G2019S It also inhibits 10% to 40% of pRab10 and LRRK2 in WTMEF. G2019S 0% to 70% of pRab10 in MEF Figure 2 After treatment with 1 μM for 24 hours, SD12, SD13, SD79, SD75, SD82, SD100, SD10, SD11, SD112, SD113, and SD114 degraded LRRK2 by 0% to 60%. WT and 0 to 85% LRRK2 G2019S It also inhibits 30% to 90% of pRab10 and LRRK2 in WT MEF. G2019S 30% to 100% of pRab10 in MEF ( Figure 2 ).

[0364] Figure 3 and Figure 5 The results showed that after treatment at 33 nM / 4 hours, SD75, XL01078B, XL0119, XL01126, SD82, XL01072, XL01118, XL01134, SD100, XL01070B, XL01120, XL01076, XL01123, XL01131, XL01145, XL01122, XL01140, XL01149, XL01121, XL01111, and XL01168 exhibited a degradation of 0% to 25% of LRRK2. WT and 0 to 60% LRRK2 G2019S It also inhibits 0% to 40% of pRab10 and LRRK2 in WT MEF. G2019S 0% to 70% of pRab10 in MEF Figure 3 and Figure 5After 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, and XL01168 degraded LRRK2 by 0% to 60%. WT and 10% to 80% of LRRK2 G2019S It also inhibits 10% to 95% of pRab10 and LRRK2 in WT MEF. G2019S 20% to 100% of pRab10 in MEF ( Figure 3 and Figure 5 ).

[0365] Figure 4 and Figure 6 The results showed that after treatment at 33 nM / 24 hours, SD75, XL01078B, XL01119, XL01126, SD82, XL01072, XL01118, XL01134, SD100, XL01070B, XL01120, XL01076, XL01123, XL01131, XL01145, XL01122, XL01140, XL01149, XL01121, XL01111, and XL01168 exhibited a degradation of 0% to 65% of LRRK2. WT and 0 to 80% LRRK2 G2019S It also inhibits 0 to 60% of pRab10 and LRRK2 in WT MEF. G2019S pRab10 in MEF (0 to 90%) Figure 4 and Figure 6 After treatment with 1 μM for 24 hours, SD75, XL01078B, XL01119, XL01126, SD82, XL01072, XL01118, XL01134, SD100, XL01070B, XL01120, XL01076, XL01123, XL01131, XL01145, XL01122, XL01140, XL01149, XL01121, XL01111, and XL01168 degraded LRRK2 by 10% to 90%. WT and 20% to 90% of LRRK2 G2019S It also inhibits 30% to 100% of pRab10 and LRRK2 in WT MEF. G2019S 10% to 100% of pRab10 in MEF ( Figure 4 and Figure 6 ).

[0366] Figure 7 and Figure 9 The results showed that after treatment at 33 nM / 4 hours, XL01126, XL02017, XL02031, XL02038, XL02047, XL02048, XL02049, XL02058, XL02059, XL02060, XL02063, XL02067B, and XL02068 degraded LRRK2 by 0% to 60%. WT and 0 to 70% LRRK2 G2019S It also inhibits 0 to 70% of pRab10 and LRRK2 in WT MEF. G2019S pRab10 in MEF (0 to 90%) Figure 7 and Figure 9 After treatment with 1 μM for 4 hours, XL01126, XL02017, XL02031, XL02038, XL02047, XL02048, XL02049, XL02058, XL02059, XL02060, XL02063, XL02067B, and XL02068 degraded 0% to 90% of LRRK2. WT and 40% to 90% of LRRK2 G2019S It also inhibits 80% to 100% of pRab10 and LRRK2 in WT MEF. G2019S 10% to 100% of pRab10 in MEF ( Figure 7 and Figure 9 ).

[0367] Figure 8 and Figure 10 The results showed that after treatment with 33 nM / 24 hours, XL01126, XL02017, XL02031, XL02038, XL002047, XL02048, XL02049, XL02058, XL02059, XL02060, XL02063, XL02067B, and XL02068 degraded LRRK2 by 0% to 80%. WT and 20% to 90% of LRRK2 G2019S It also inhibits 0 to 80% of pRab10 and LRRK2 in WT MEF. G2019S pRab10 in MEF accounts for 30% to 90% ( Figure 8 and Figure 10After treatment with 1 μM for 24 hours, XL01126, XL02017, XL02031, XL02038, XL02047, XL02048, XL02049, XL02058, XL02059, XL02060, XL02063, XL02067B, and XL02068 degraded 60% to 90% of LRRK2. WT and 60% to 95% of LRRK2 G2019S It also inhibits 80% to 100% of pRab10 and LRRK2 in WT MEF. G2019S 80% to 100% of pRab10 in MEF ( Figure 8 and Figure 10 ).

[0368] Figure 11A , 11B 11C shows the SD75 in WT MEF and LRRK2 respectively. G2019S LRRK2 induction in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT) = 51% and D max (G2019S) = 58% Figure 11A and Figure 11B ). SD75 in WT MEF and LRRK2 respectively. G2019S MEF induces dose-dependent dephosphorylation of Rab10, in which EC 50 (WT) = 2270 nM and EC 50 (G2019S)=379nM( Figure 11A and Figure 11C ).

[0369] Figure 12A , 12B 12C shows XL01134 in WT MEF and LRRK2 respectively. G2019S LRRK2 induction in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT) = 59% DC 50 (WT) = 32nM, D max (G2019S) = 81% and DC 50 (G2019S)=7nM( Figure 12A and Figure 12B XL01134 was tested in WT MEF and LRRK2 respectively. G2019S MEF induces dose-dependent dephosphorylation of Rab10, in which EC 50(WT) = 74 nM, and EC 50 (G2019S)=12nM( Figure 12A and Figure 12C ).

[0370] from Figure 13A , 13B As can be seen from 13C, XL01126 is in WT MEF and LRRK2 respectively. G2019S LRRK2 induction in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT)=82%, DC50(WT)=32nM, D max (G2019S) = 90% and DC 50 (G2019S)=14nM( Figure 13A and Figure 13B XL01126 was tested in WT MEF and LRRK2 respectively. G2019S MEF induces dose-dependent dephosphorylation of Rab10, in which EC 50 (WT) = 54nM and EC 50 (G2019S)=15nM( Figure 13A and Figure 13C ).

[0371] Figure 14A , 14B The 14C display shows that at 1000nm, the SD75 performs well at WT MEF and LRRK2 respectively. G2019S LRRK2 induction in MEF WT and LRRK2 G2019S Time-dependent degradation, where T 1 / 2 (WT) = 5.1 hours, D max (WT) = 52%, T 1 / 2 (G2019S) = 1.4 hours and D max (G2019S) = 81% Figure 14A and Figure 14B At 1000 nM, SD75 showed performance at WTMEF and LRRK2, respectively. G2019S Time-dependent dephosphorylation of Rab10 induced in MEF, where T 1 / 2 (WT) = 6.7 hours, D max (WT) = 41%, T 1 / 2 (G2019S) = 1.1 hours and D max (G2019S) = 74% Figure 14A and Figure 14C ).

[0372] As in Figure 15A , 15B As observed in 15C, at 300nM, XL01134 exhibits performance at WT MEF and LRRK2. G2019S LRRK2 induction in MEF WT and LRRK2 G2019S Time-dependent degradation, where T 1 / 2 (WT) = 2.7 hours, D max (WT) = 75%, T 1 / 2 (G2019S) = 1.4 hours and D max (G2019S) = 82% Figure 15A and Figure 15B At 300 nM, XL01134 exhibits performance at WT MEF and LRRK2, respectively. G2019S Time-dependent dephosphorylation of Rab10 induced in MEF, where T 1 / 2 (WT) = 2.1 hours, D max (WT) = 68%, T 1 / 2 (G2019S) = 0.3 hours and D max (G2019S) = 87% Figure 15A and Figure 15C ).

[0373] Figure 16A , 16B The 16C display shows that at 300nm, XL01126 is at WT MEF and LRRK2 respectively. G2019S LRRK2 induction in MEF WT and LRRK2 G2019S Time-dependent degradation, where T 1 / 2 (WT) = 1.2 hours, D max (WT) = 82%, T 1 / 2 (G2019S) = 0.6 hours and D max (G2019S) = 92% Figure 16A and Figure 16B At 300 nM, XL01126 was observed at WT MEF and LRRK2, respectively. G2019S Time-dependent dephosphorylation of Rab10 induced in MEF, where T 1 / 2 (WT) = 2.1 hours, D max (WT) = 84%, T 1 / 2 (G2019S) = 0.3 hours and D max (G2019S) = 92% Figure 16A and Figure 16C ).

[0374] Figure 17A , Figure 17B , Figure 17C and Figure 17D The total LRRK2 induced by XL01126 was shown. WT The degradation of LRRK2 can be rescued by pretreatment with VHL ligand (VH101), MLN4924, and MG132. WT Inhibition of phosphorylation at Ser935 and Rab10 at Thr73 can be partially salvaged by pretreatment with VHL ligand (VH101), MLN4924, and MG132. Figure 17A , Figure 17B , Figure 17C and Figure 17D ).

[0375] Figure 18A , Figure 18B , Figure 18C and Figure 18D This indicates that XL01126 induces total LRRK2 G2019S Degradation can be rescued by pretreatment with VHL ligand (VH101), MLN4924, and MG132. LRRK2 G2019S Inhibition of phosphorylation at Ser935 and Rab10 at Thr73 can be partially salvaged by pretreatment with VHL ligand (VH101), MLN4924, and MG132. Figure 18A , Figure 18B , Figure 18C and Figure 18D ).

[0376] Figure 19A , 19B 19C shows XL01126 induces LRRK2 R1441C Dose-dependent degradation, of which DC 50 =15nM and D max =89% ( Figure 19A and Figure 19B XL01126 induces LRRK2 R1441C Dose-dependent dephosphorylation of Rab10 in MEF, including EC 50 =30nM( Figure 19A and Figure 19C ). cis-XL01126 induces LRRK2 R1441C Dose-dependent dephosphorylation of Rab10 in MEF, including EC 50 =158nM( Figure 19A and Figure 19C ).

[0377] Figure 20A , 20BThis indicates that XL01126 induces dose-dependent degradation of LRRK2 in macrophages, with DC... 50 =55 nM. cis-XL01126 cannot degrade LRRK2 in macrophages. Figure 20A and 20B ).

[0378] Figure 21A , 21B 21C showed that after 4 hours of treatment, XL01126 induced dose-dependent degradation of LRRK2 in PBMCs, with DC... 50 =72nM and D max =88%. cis-XL01126 cannot degrade LRRK2 in PBMCs. Figure 21A and 21B Following 4 hours of treatment, XL01126 induced dose-dependent dephosphorylation of Rab10 in PBMC cells, with EC... 50 =69 nM. cis-XL01126 also dephosphorylates Rab10 in PBMC cells, including EC... 50 >3μM ( Figure 21A and Figure 21C ).

[0379] Figure 22A , 22B 22C analysis showed that after 24 hours of treatment, XL01126 induced dose-dependent degradation of LRRK2 in PBMCs, with DC... 50 =17nM and D max =93%. Cis-XL01126 cannot degrade LRRK2 in PBMCs. Figure 22A and 22B Following 24-hour treatment, XL01126 induced dose-dependent dephosphorylation of Rab10 in PBMC cells, with EC... 50 =20 nM. cis-XL01126 also dephosphorylates Rab10 in PBMC cells, including EC... 50 =705nM( Figure 22A and Figure 22C ).

[0380] Figure 23A , 23B 23C assays showed that XL01126 induced time-dependent degradation of LRRK2 in PBMCs at 300 nM, where T 1 / 2 = 2.4 hours and D max =92%. At 300 nM, XL01126 induces time-dependent dephosphorylation of LRRK2 and Rab10 in PBMC cells, of which T 1 / 2The times were 1 hour and 0.38 hours respectively. Figure 23A and Figure 23B At 300 nM, cis-XL01126 cannot degrade LRRK2 and induces minor dephosphorylation of LRRK2 and Rab10. Figure 23A and Figure 23C ).

[0381] Figure 24 The results showed that XL01126 selectively reduced intracellular LRRK2 protein levels in TMT-labeled proteomics studies.

[0382] The data presented in Table 1 show that the solubility of XL01126 in PBS and fed-state simulated intestinal fluid (FeSSIP) is 0.55 μM and 26.05 μM, respectively (Table 6). XL01126 can penetrate Caco-2 monolayers (Table 6). XL01126 is stable in mouse plasma and mouse hepatocytes, with half-lives of 108.29 min and 314.33 min, respectively (Table 6). XL01126 is metabolized in mouse liver microsomes, with a half-life of 3.65 min (Table 6).

[0383] Figure 25A , 25B 25C showed that XL02047 induced WT MEF and LRRK2, respectively. G2019S LRRK2 in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT) = 78%, DC 50 (WT) = 45nM, D max (G2019S) = 79%, and DC 50 (G2019S)=60nM( Figure 25A and Figure 25B XL02047 induced WT MEF and LRRK2, respectively. G2019S Dose-dependent dephosphorylation of Rab10 on MEF, including EC 50 (WT) = 228 nM, and EC 50 (G2019S)=73nM( Figure 25A and Figure 25C ).

[0384] Figure 26A , 26B 26C indicates that XL02048 induces WT MEF and LRRK2, respectively. G2019S LRRK2 in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max(WT) = 81.5% DC 50 (WT) = 46nM, D max (G2019S) = 83.5%, and DC 50 (G2019S)=23nM( Figure 26A and Figure 26B XL02048 induced WT MEF and LRRK2, respectively. G2019S Dose-dependent dephosphorylation of Rab10 on MEF, including EC 50 (WT) = 72nM and EC 50 (G2019S)=53nM( Figure 26A and Figure 26C ).

[0385] Figure 27A , 27B 27C showed that XL02049 induced WT MEF and LRRK2, respectively. G2019S LRRK2 in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT) = 76.5% DC 50 (WT) = 40nM, D max (G2019S) = 90%, and DC 50 (G2019S)=27nM( Figure 27A and Figure 27B XL02049 induced WT MEF and LRRK2, respectively. G2019S Dose-dependent dephosphorylation of Rab10 on MEF, including EC 50 (WT) = 65nM and EC 50 (G2019S)=31nM( Figure 27A and Figure 27C ).

[0386] Figure 28A , 28B 28C indicates that XL02017 induces WT MEF and LRRK2, respectively. G2019S LRRK2 in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT) = 68%, DC 50 (WT) = 44nM, D max (G2019S) = 79%, and DC 50 (G2019S)=22nM( Figure 28A and Figure 28B XL02017 induced WT MEF and LRRK2, respectively.G2019S Dose-dependent dephosphorylation of Rab10 on MEF, including EC 50 (WT) = 83nM and EC 50 (G2019S)=25nM( Figure 28A and Figure 28C ).

[0387] like Figure 29A , 29B As shown in Figure 29C, XL02038 induces WT MEF and LRRK2, respectively. G2019S LRRK2 in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT) = 65% and D max (G2019S) = 79% Figure 29A and Figure 29B XL02038 induced WT MEF and LRRK2, respectively. G2019S Dose-dependent dephosphorylation of Rab10 on MEF, including EC 50 (WT) = 14nM and EC 50 (G2019S)=10nM( Figure 29A and Figure 29C ).

[0388] like Figure 30A , 30B As shown in Figure 30C, XL02068 induces WT MEF and LRRK2, respectively. G2019S LRRK2 in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT) = 89% DC 50 (WT) = 93nM, D max (G2019S) = 87% and DC 50 (G2019S)=103nM( Figure 30A and Figure 30B XL02068 induced WT MEF and LRRK2, respectively. G2019S Dose-dependent dephosphorylation of Rab10 on MEF, including EC 50 (WT) = 272nM and EC 50 (G2019S)=150nM( Figure 30A and Figure 30C ).

[0389] like Figure 31A , 31BAs shown in Figure 31C, XL02059 induces WT MEF and LRRK2, respectively. G2019S LRRK2 in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT) = 77.5% and D max (G2019S) = 76% Figure 31A and Figure 31B XL02059 induced WT MEF and LRRK2, respectively. G2019S Dose-dependent dephosphorylation of Rab10 on MEF, including EC 50 (WT) = 227 nM and EC 50 (G2019S)=55nM( Figure 31A and Figure 31C ).

[0390] like Figure 32A , 32B As shown in Figure 32C, XL02060 induces WT MEF and LRRK2, respectively. G2019S LRRK2 in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT) = 71% and D max (G2019S) = 78.5% Figure 32A and Figure 32B XL02060 induced WT MEF and LRRK2, respectively. G2019S Dose-dependent dephosphorylation of Rab10 on MEF, including EC 50 (WT) = 32nM and EC 50 (G2019S)=27nM( Figure 32A and Figure 32C ).

[0391] As in Figure 33 As observed, after intravenous administration of 5 mg / kg XL01126 (single dose) to mice, the plasma clearance of XL01126 was 0.208 L / h / kg, the volume of distribution was 0.511 L / kg, the metabolic half-life was 1.52 hours, and the AUC was [data missing]. last and AUC INF The areas under the curve (AUC) were 23663 h*ng / mL and 23981 h*ng / mL, respectively. Figure 33Following a single intraperitoneal injection of 30 mg / kg, XL01126 plasma concentrations reached their maximum (7700 ng / mL) at 0.25 hours, with a metabolic half-life of 5.2 hours and an AUC of [missing value]. last and AUC INF The areas under the curve (AUC) were 41434 h*ng / mL and 64068 h*ng / mL, respectively. The bioavailability of XL01126 after intraperitoneal injection was 29.2% (…). Figure 33 Following administration of a single dose of 30 mg / kg via tube feeding, the plasma concentration of XL01126 reached its maximum (3620 ng / mL) at 2 hours, with a metabolic half-life of 21.9 hours and an AUC of [missing value]. last and AUC INF The areas under the curve (AUC) were 21337 h*ng / mL and 109271 h*ng / mL, respectively. Following intraperitoneal injection, the bioavailability of XL01126 was 15% (…). Figure 33 ).

[0392] As in Figure 34 As observed, after intravenous administration of 5 mg / kg (single dose) of XL01126 to mice, the concentration of XL01126 in brain tissue reached its maximum level (155 ng / mL) at 0.083 hours, with a metabolic half-life of 3.67 hours and an AUC of [missing information]. last and AUC INF The areas under the curve (AUC) were 258 h*ng / mL and 349 h*ng / mL, respectively. Figure 34 Following intraperitoneal injection of 30 mg / kg XL01126 (single dose) into mice, the concentration of XL01126 in brain tissue reached its maximum level (218 ng / mL) at 0.5 hours, with a metabolic half-life of 2.79 hours and an AUC of [missing value]. last and AUC INF The areas under the curve (AUC) were 752 h*ng / mL and 884 h*ng / mL, respectively. Figure 34 Following administration of a single dose of 30 mg / kg XL01126 to mice via tube feeding, the brain tissue concentration of XL01126 reached its maximum (27 ng / mL) at 2 hours, with a metabolic half-life of 10.7 hours and an AUC of [missing value]. last and AUC INF The areas under the curve (AUC) were 142 h*ng / mL and 402 h*ng / mL, respectively.

[0393] Figure 35The results showed that after intravenous administration of 5 mg / kg XL01126 (single dose) to mice, the concentration of XL01126 in cerebrospinal fluid (CSF) reached its maximum concentration (27.4 ng / mL) at 0.083 hours, with a metabolic half-life of 8.59 hours and an AUC of [missing value]. last and AUC INF The areas under the curve (AUC) were 107 h*ng / mL and 240 h*ng / mL, respectively. Figure 35 XL01126 also achieved CSF after intraperitoneal (30 mg / Kg) and oral (30 mg / Kg) administration.

[0394] Leucine-rich repeat kinase 2 (LRRK2) is one of the most promising disease-remission targets for Parkinson's disease, but current LRRK2 targeting strategies have focused on kinase inhibitors with potentially undesirable side effects. Here, we disclose the discovery of an LRRK2 proteolytic targeting chimeric (PROTAC) degrader, exemplified by compound XL01126 as an alternative LRRK2 targeting strategy. XL01126 induces rapid and efficient degradation of LRRK2 protein in multiple cell lines in a dose- and time-dependent manner. Specifically, XL01126 is able to induce degradation of LRRK2 protein in DC cells in a dose- and time-dependent manner. 50 At 32nM, 14nM, 15nM, 55nM and 17nM, WT MEF, LRRK2 G2019S MEF, LRRK2 R1441C LRRK2 protein levels were reduced by 80% to 90% in MEF, macrophages, and PBMCs. XL01126 showed reduced LRRK2 levels in WT MEF. G2019S The degradation half-lives on MEF and PBMC are 1.2 h, 0.6 h, and 2.4 h, respectively. XL01126 is orally bioavailable (F = 15%) and crosses the blood-brain barrier in mice following a single dose administered intravenously (5 mg / kg), intraperitoneally (30 mg / kg), or orally (30 mg / kg). All these properties qualify XL01126 as a chemical probe for studying the in vitro and in vivo biology of LRRK2 and provide a starting point and potential therapeutic approaches for future PD drug development.

[0395] Figure 36 The results showed that VH101, XL01126, SD75, XL01134, and cis-XL01126 replaced the fluorescent tracer from the VHL protein in a dose-dependent manner, with XL01134 (K) being the most effective. i=0.16 μM) showed the most potent substitution and therefore the highest binding affinity for VHL, followed by VH101(K i =0.44μM) and SD75(K i =0.82μM), while XL01126(K i >2.33M) showed poor substitution (binding affinity with VHL) and cis-XL01126 did not substitute the fluorescent tracer.

[0396] Figure 37A The results showed that in permeabilized HEK293 cells, VH101, XL01126, SD75, and XL01134 could displace VHL tracers derived from VHL proteins in a dose-dependent manner, with XL01134 (IC) being the most effective. 50 =0.78μM) exhibited the most potent substitution and therefore the highest binding affinity for VHL, followed by VH101 (IC). 50 =1.0μM) and SD75 (IC 50 =1.9 μM), while XL01126 exhibits poor substitution (binding affinity with VHL).

[0397] Figure 37B The results showed that in live HEK293 cells, VH101, XL01126, SD75, and XL01134 could replace VHL tracers derived from VHL proteins in a dose-dependent manner, with XL01134 exhibiting the most potent substitution (IC50). 50 =0.88μM), and therefore has the highest binding affinity for VHL, followed by VH101 (IC). 50 =1.8μM) and SD75 (IC 50 =2.5μM), while XL01126 (IC 50 =10.1 μM) exhibits poor substitution (binding affinity with VHL). XL01126 has the highest IC50 between permeation and active-mode VHL binding. 50 Ratio (IC) 50，per / IC 50，活 >0.99), and therefore had the highest permeability in the PROTAC test, followed by XL01134 (IC). 50，per / IC 50，活 =0.89) and SD75 (IC 50，per / IC 50，活 =0.76).

[0398] Figure 38AThe results showed that in permeabilized HEK293 cells, HG-10-102-01, XL01126, SD75, and XL01134 could replace LRRK2 tracers derived from the LRRK2 protein in a dose-dependent manner, among which HG-10-102-01 (IC50) was the most effective. 50 =0.23μM) exhibited the most potent substitution and therefore the highest binding affinity for LRRK2, followed by SD75 (IC). 50 =0.78μM) and XL01134 (IC 50 =2.0 μM), while XL01126 showed poor substitution.

[0399] Figure 38B The results showed that in live HEK293 cells, HG-10-102-01, XL01126, SD75, and XL01134 were able to replace LRRK2 tracers derived from the LRRK2 protein in a dose-dependent manner, with HG-10-102-01 exhibiting the most potent substitution (IC50). 50 =0.7 μM), and therefore has the highest binding affinity for LRRK2, followed by XL01126 (IC). 50 =1.4μM), SD75 (IC) 50 =1.8μM) and XL01134 (IC 50 =2.2μM). XL01126 has the highest IC50 between permeation mode and active mode LRRK2 binding. 50 The ratio (1.89) was the highest, and therefore the highest permeability was observed in the PROTAC test, followed by XL01134 (IC). 50，per / IC 50，活 =0.91) and SD75 (IC 50，per / IC 50，活 =0.43).

[0400] Figure 39A The results showed that XL01126 binds to LRRK2 better in the presence of VCB protein than in the absence of VCB (IC). 50 =0.35μM vs 2μM), resulting in a positive cooperativity (α) of 5.7.

[0401] Figure 39B The results showed that XL01134 binds to LRRK2 better in the presence of VCB protein than in the absence of VCB (IC). 50 =0.79μM vs 1.1μM), resulting in a positive cooperativity (α) of 1.4.

[0402] Figure 39CThe results showed that SD75 binding to LRRK2 was worse in the presence of VCB protein than in the absence of VCB (IC). 50 =1.4μM vs 1.1μM), resulting in a negative cooperativity (α) of 0.79.

[0403] Figure 40 The results show 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.

[0404] Figure 41 , 42 XL01126, XL02152, XL02098, XL02093, and MP01088 showed that after 4 hours / 1 μM treatment, they degraded 60% to 80% of LRRK2 in WT MEF. WT Degradation of LRRK2 G2019S 75% to 90% of LRRK2 in MEF G2019S It also inhibits 75% to 90% of pRab10 and LRRK2 in WT MEF. G2019S 80% to 100% of pRab10 in MEF. After 4 hours / 33 nM treatment, XL01126, XL02152, XL02093, and MP01088 degraded 25% to 40% of LRRK2 in WT MEF. WT and LRRK2 G2019S 25% to 50% of LRRK2 in MEF G2019S It inhibits 20% to 40% of pRab10 and LRRK2 in WT MEF. G2019S pRab10 accounts for 20% to 50% of MEF.

[0405] Figure 44A , 44B 44C showed that XL02098 induced WT MEF and LRRK2, respectively. G2019S LRRK2 in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT) = 59% DC 50 (WT) = 50nM, D max (G2019S) = 81% and DC 50 (G2019S)=45nM( Figure 44A and Figure 44B XL02098 induced WT MEF and LRRK2, respectively. G2019S Dose-dependent dephosphorylation of Rab10 on MEF, including EC50 (WT) = 282nM and EC 50 (G2019S)=157.2nM( Figure 44A and Figure 44C ).

[0406] Figure 45A , 45B 45C showed that XL02152 induced WT MEF and LRRK2, respectively. G2019S LRRK2 in MEF WT and LRRK2 G2019S The dose-dependent degradation of D, where D max (WT) = 68% DC 50 (WT) = 62nM, D max (G2019S) = 80% and DC 50 (G2019S)=44nM( Figure 45A and Figure 45B XL02152 induced WT MEF and LRRK2, respectively. G2019S Dose-dependent dephosphorylation of Rab10 on MEF, including EC 50 (WT) = 144 nM and EC 50 (G2019S)=63nM( Figure 45A and Figure 45C ).

[0407] experiment

[0408] Production of mouse embryonic fibroblasts (MEF)

[0409] Primary MEFs were generated as described in a previous study (Wiggin et al., Mol. Cell Biol. 2002 Apr; 22(8):2871-81.). Briefly, uterine horns were collected from adult female mice on day E12.5 and transferred to 10 cm tissue culture dishes containing cold PBS. The yolk sac was torn open using two forceps to separate each embryo. The forceps were thoroughly cleaned with 70% ethanol between each embryo separation. Embryos were selected, and tissue fragments were collected in PCR tubes for genotyping. Red tissue from embryos that developed into hearts and livers was removed. The remaining embryos were minced with a scalpel and incubated for 10 minutes in a 37°C CO2 tissue culture incubator with 7.5 ml trypsin-EDTA solution. The dishes were removed from the incubator, and individual cells were examined under an optical microscope. 7.5 ml of complete culture medium was added to the trypsin-digested cells, and the cell suspension was transferred to 15 ml Falcon tubes and centrifuged at 1200 rpm for 5 minutes at room temperature. Trypsin was aspirated, and the cell pellet was resuspended in 5 ml of fresh complete culture medium. The cell suspension was then plated in 60 mm tissue culture dishes and incubated at 37°C in a 5% CO2 tissue culture incubator. MEFs at this stage were considered generation 0, and once the genotype was confirmed by allele sequencing and Western blotting, they were passaged and amplified for experimental use. MEFs 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 1X non-essential amino acids and 1 mM sodium pyruvate.

[0410] Production of bone marrow-derived macrophages (BMDM)

[0411] Macrophages were cultured in complete medium containing DMEM, 10% (v / v) heat-inactivated FBS, 20% (v / v) L929 pretreated medium, 2.5% (v / v) HEPES, 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin, 2% sterile filtered β-mercaptoethanol, 1X non-essential amino acids, and 1 mM sodium pyruvate. Bone marrow isolation and macrophage differentiation were modified from (Wells et al., 2004) using L929 pretreated medium as the source of M-CSF for differentiation. Briefly, the femur and tibia were dissected from adult mice using scissors and forceps, and muscle tissue was carefully removed from the bone. The cleaned femur and tibia were placed in tissue culture dishes containing complete medium. The ends of each bone were cut open with scissors to expose the bone marrow. The bone marrow was flushed with a 25G needle attached to a 10 ml syringe containing complete medium. Bone marrow-containing culture medium was passed through a 70 μm cell filter, and precursor cell plates were seeded onto 10 cm non-tissue culture-treated bacteriological plates containing 10 ml of complete culture medium. This was marked as day 0 of isolation. On day 3 post-isolation, macrophages were flooded with 5 ml of fresh complete culture medium. On day 7 post-isolation, macrophages were washed once with PBS and incubated with Versene for 5 minutes in a 37°C, 5% CO2 tissue culture incubator. Macrophages were separated using a cell scraper and centrifuged at 1200 rpm for 5 minutes at room temperature. Versene was removed, and the remaining cell pellet was resuspended in complete culture medium. The cell suspension was counted, and cells were seeded into 6-well tissue culture dishes for experimental analysis, resulting in a final cell density of one million cells / 6 wells.

[0412] PBMC cell isolation and processing

[0413] According to the existing plan ( dx.doi.org / 10.17504 / protocols.io.bnhxmb7nPBMCs were isolated from human blood and precipitated by centrifugation at 1000g for 2 minutes. The supernatant was discarded, and the PBMC pellet was resuspended in PBS containing 2% FBS and washed. The suspension was centrifuged again at 1000g for 2 minutes, and the PBMC pellet was resuspended in RPMI-1640 (Gibco) medium supplemented with 10% FBS. The cells were then seeded into 6-well plates and treated with the test compound at the specified concentration and time period. After treatment, the cells were collected into 2 ml Eppendorf tubes and centrifuged at 500g for 2 minutes to precipitate the cells. The supernatant was discarded, and the pellet was resuspended in 1 ml PBS and centrifuged again at 500g for 2 minutes. The PBMC precipitate was lysed with 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 / ester, 5 mM sodium pyrophosphate, 0.1 μg / ml mycrocystin-LR (Enzo Life Sciences), 270 mM sucrose, and 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. The lysed cells were then centrifuged at 1500 g for 15 min at 0 °C. Collect the supernatant for quantitative immunoblotting analysis. For long-term storage, rapidly freeze the supernatant and store at -80°C. Use Pierce... TM The BCA Protein Assay Kit (ThermoFisher) determines the protein concentration of cell lysates.

[0414] Cell culture, processing and lysis

[0415] Adherent cell lines were cultured and passaged using aseptic techniques in a CL1 or CL2 (for PBMC isolation) biosafety cabinet. All cells were incubated in a 37°C incubator containing 5% CO2. Mycoplasma contamination tests were performed on the cell lines periodically. For Western blot assays, cells were seeded in 6-well plates. After treatment with the test compound at the specified concentration and time period, the culture medium was removed, and the cells were washed with PBS and 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 / ester, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 270 mM sucrose, supplemented with 1 μg / ml microcystin-LR, 1 mM sodium orthovanadate, a completely EDTA-free protease inhibitor mixture (Roche), and 1% (v / v) Triton X-100. Cells were immediately placed on ice and scraped off into 1.5 ml Eppendorf tubes. The cell lysates were incubated on ice for 10 minutes, then centrifuged at 15,000 g for 15 minutes at 4°C. The cell pellet was discarded, and the supernatant was collected for quantitative immunoblotting analysis. For long-term storage, the supernatant was rapidly frozen and stored at -80°C. Protein concentrations in the cell lysates were determined using the Bradford assay.

[0416] Quantitative immunoblotting

[0417] Cell lysates containing one-quarter volume of 4×NuPAGE LDS sample buffer (NP0007) supplemented with 5% β-mercaptoethanol were heated at 95°C for 5 minutes. 15–20 μg of sample was loaded into pre-cast 4%–12% Bis-Tris midi 20W or 26W gels (Thermo Fisher Scientific). On Cat#WG1402BOX or WG1403BOX, use NuPAGE MOPSSDS running buffer (Thermofisher) The protein (Cat#NP0001-02) was resolved at 130V for 2 hours. The protein was then electrophoretically transferred to a 0.45μm nitrocellulose membrane (GE Healthcare, Amersham). On a supported 0.45 mm NC membrane, run on ice at 90 V for 90 min in transfer buffer (48 mM Tris base supplemented with 20% methanol and 39 mM glycine). At room temperature, block the transferred membrane with 5% (w / v) skim milk dissolved in Tween-buffered saline (TBS-T) (50 mM Tris base, 150 mM sodium chloride (NaCl), 0.1% (v / v) Tween-20) for 1 h. Wash the membrane three times with TBS-T and incubate overnight with primary antibody at 4 °C. Before secondary antibody incubation, wash the membrane three times with TBS-T for 15 min each time. Incubate the membrane with secondary antibody at room temperature in the dark for 1 h. Following this, the membrane was washed three times with TBS-T, with each wash followed by incubation for 15 minutes. Protein bands were then obtained using an Odyssey CLx imaging system via near-infrared fluorescence detection, and quantified using Image Studio software. Graphs were generated using Graphpad Prism version 8 software.

[0418] Antibody

[0419] Monoclonal rabbit LRRK2 Ser935 (Cat#UDD2) was purified by MRC PPU Reagents and Services at the University of Dundee, UK, and used at a final concentration of 1 μg / ml. Total LRRK2 (C-terminus) was obtained from Antibodies Inc. / Neuromab (Cat#75-253) and diluted 1:1000. Previously characterized MJFF monoclonal rabbit Rab10 pThr73 (Lis et al., Biochem J. 2018 Jan 2; 475(1):1-22.) was purchased from Abcam Inc. (ab230261) and diluted 1:1000. Mouse monoclonal α-tubulin (#3873) was purchased from Cell Signaling Technology and used at a 1:1000 concentration. 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 Bioloicals and used at a final concentration of 1:500. All rabbit and mouse primary antibodies were diluted in 5% (w / v) bovine serum albumin (BSA) dissolved in TBS-T (50 mM Tris base, 150 mM sodium chloride (NaCl), 0.1% (v / v) Tween 20). 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 derived from LI-COR and diluted 1:10,000 in 5% (w / v) milk in TBS-.

[0420] Total proteome sample preparation and MS analysis

[0421] Wild-type MEFs were seeded at a density of two million cells / dish in 10 cm tissue culture dishes. Cells were treated with 0.1% DMSO, 300 nM XL01126 or 300 nM cis-XL01126 for 4 h, and then harvested in 400 μl of complete lysis buffer supplemented with 1 μg / ml microcystin-LR, 1 mM sodium orthovanadate, a completely EDTA-free protease inhibitor mixture (Roche), and 1% (v / v) Triton X-100. Cell lysates were incubated on ice for 10 min, followed by three rounds of high-energy sonication using a Diagenode Bioruptor for 15 cycles (30 sec on, 30 sec off). Cell lysates were centrifuged at 15,000 g for 15 min at 4 °C. The cell pellet was discarded, and the supernatant was collected for protein quantification using a BCA protein assay kit (Pierce #23225). Total proteomic analysis was performed using 100 μg of cell lysate. Proteins in cell lysates were reduced to a final concentration of 10 mM with 0.1 M tris(2-carboxyethyl)phosphine (TCEP) diluted in 300 mM triethylammonium bicarbonate (TEABC). The samples were incubated on a Thermomixer at 60 °C and 800 rpm for 30 min. The samples were then incubated to room temperature and alkylated with 0.4 M iodoacetamide (IAA) freshly dissolved in water. The samples were then incubated on a Thermomixer at room temperature in the dark at 800 rpm for 30 min. Alkylation was quenched by adding 0.1 M TCEP dissolved in 300 mM TEABC (final concentration 5 mM). The samples were incubated on a Thermomixer at room temperature at 800 rpm for 20 min. Sodium dodecyl sulfate (SDS) was added from a 20% (w / v) stock solution to a final concentration of 5% (w / v). Then add 12% (v / v) phosphate to a final concentration of 1.2% (v / v). Dilute the sample in 6 times the sample volume of S-trap wash buffer containing 90% (v / v) methanol diluted in 100 mM (v / v) TEAB pH 7.1.

[0422] S trap clears and digests

[0423] The sample was S-trap cleaned using a 2 ml Eppendorf S-trap microcolumn (PROTIFI Cat#MSPPC02-MINI-80) to remove detergent and other impurities. The protein mixture was added to the column and briefly centrifuged (1000g / 1 min / room temperature). The column was then cleaned with... Wash the column four times with S-trap buffer, centrifuging at 1000g / 1min / room temperature after each wash. Place the column in a fresh 2ml Eppendorf container and add 100μl of 5μg trypsin / Lys-C dissolved in 50mM TEAB, pH 8.5. Briefly centrifuge the column (200g / 1min / room temperature) and transfer the trypsin / Lys-C mixture back onto the column. Add 100μl of 50mM TEAB (pH 8.5) directly to the 2ml Eppendorf container to cover any remaining digested peptides in the tube. Incubate the S-trap column in the 2ml Eppendorf container at 47°C without shaking for 1.5 hours, then overnight at room temperature.

[0424] Add 80 μl of 50 mM TEAB to an S-trap column, centrifuge the column, and collect the eluent in a new 1.5 mL Eppendorf tube. Add 80 μl of 0.2% (v / v) formic acid to the column, centrifuge the column, and combine the second eluent with the first eluent. Add 80 μl of 50% (v / v) acetonitrile diluted in 0.2% (v / v) formic acid to the column, centrifuge the column, and combine the third eluent with the previous eluent. Set aside 500 ng of the digested peptide for separate vacuum drying to verify digestion efficiency >90%. Divide the remaining peptide in half (50 μg peptide / tube), vacuum dry, and store at -80 °C, then proceed with tandem mass tag (TMT) labeling.

[0425] TMT Marking

[0426] Dissolve 800 μg of TMT quality tag reagent in 80 μl of 100% (v / v) anhydrous acetonitrile to obtain a final concentration of 10 μg / μl. Incubate the resuspended TMT reagent at room temperature for 10 min, then vortex and briefly centrifuge (2000 g / 2 min / room temperature). Resuspend 50 μg of lyophilized peptide in 50 μl of a mixture containing 38 μl of 50 mM TEAB and 8 μl of 100% (v / v) anhydrous acetonitrile. Sonicate the resuspended peptide for 10 min, then centrifuge at 17,000 g for 10 min at room temperature. Transfer the peptide to a fresh protein lo-bind 1.5 ml Eppendorf tube. Add 20 μl of 10 μg / μl TMT reagent to the dissolved peptide, vortex, briefly centrifuge (2,000 g / 1 min / room temperature), and incubate on a Thermomixer at 800 rpm for 2 h at room temperature. Add 50 μl of 50 mM TEAB to each reaction, followed by vortexing, brief centrifugation (2,000 g / 1 min / room temperature), and incubation for an additional 10 min at 800 rpm on a Thermomixer at room temperature. Set aside 5 μl of each TMT-labeled sample, vacuum dry, and inject into an MS sample to confirm labeling efficiency >98%. Store the remaining reactants at -80 °C until labeling efficiency is verified. Quench the thawed TMT samples and labeling reactions to room temperature by adding 5 μl of 5% (v / v) hydroxylamine (dissolved in water from a 50% (v / v) stock solution). Incubate the samples on a Thermomixer at 800 rpm for 20 min at room temperature. Combine the quenched TMT-labeled samples and desalt using a C18 stage-tip.

[0427] Pharmacokinetic (PK) studies

[0428] PK spectroscopy analysis was outsourced and undertaken by Shanghai ChemPartner Co., Ltd. All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) and the Office of Laboratory Animal Welfare (OLAW). Six- to eight-week-old male C57BL / 6 mice purchased from Jihui Laboratory Animal Co., Ltd. were used in this study. XL01126 was prepared in 10% HP-β-CD in 50 mM citrate buffer (pH 3.0) and administered at 1 mg / mL for intravenous injection and 3 mg / mL for intraperitoneal (IP) and peritoneal (PO) injections. For IV injection, 5 mg / kg of XL01126 was administered via the tail vein of nine mice. For IP injection, 30 mg / kg of XL01126 was administered via intraperitoneal injection in mice. Animals were manually restrained at specified time points (0.083, 0.25, 0.5, 1, 2, 4, and 8 hours); approximately 110 μL of blood sample was collected into K2EDTA tubes via the facial vein. Three mice were used at each time point, resulting in a total of 21 mice for each administration route. Blood samples were placed on ice and centrifuged at 2000 g for 5 min to obtain plasma samples within 15 min. Plasma, brain, and CSF samples were stored at approximately -70°C until analysis. 200 μL of internal standard (Glipozode, 40 ng / mL) in MeCN containing 5% citri was added to 30 μL of plasma aliquots. The mixture was then vortexed for 1 min and then centrifuged at 5800 rpm for 10 min. The supernatant (100 μL) was transferred to a new plate. Solvent (1 μL) was injected into LC–MS / MS. LC–MS / MS instrument used: SCIEX LC–MS / MS-49 (Triple Quad 6500+). The data was analyzed using WinNonLin and Microsoft Excel.

[0429] Fluorescence polarization measurement

[0430] FP competitive binding assays were performed according to the previously described method (Bond et al, J. Med. Chem. 2021, 64(20), 15477–15502.). All measurements were performed on a PHERstar (BMG LABTECH) plate reader equipped with an FP filter that set the excitation and emission wavelengths at 485 nm and 520 nM, respectively. Each well of a 384-well plate (Coring 3575) contained 10 nM VCB protein, 5 nM FAM-labeled HIF-1α peptide (FAM-DEALAHypYIPMDDDFQLRSF, “JC9”), and PROTAC (14 concentrations with 2-fold sequential dilutions starting at 250 μM) in FP assay buffer (100 mM Bis-Tris propane, 100 mM NaCl, 1 mM TCEP, pH 7) at decreasing concentrations, with a final DMSO concentration of 5%. Control wells containing VCB and JC9 but without the compound were set to the maximum signal (zero substitution). Control wells containing JC9 but without the protein were set to the minimum signal. The substitution percentage relative to the Log[compound] was obtained using the control values. The average IC50 for each titration was determined using nonlinear regression analysis with Prism (v.9.3.1). 50 Value. As mentioned before, by JC9's K d (1.5 nM to 3.4 nM) and fitted IC 50 Value inverse calculation K i value.

[0431] NanoBRET target binding assay

[0432] For VHL and LRRK2 target conjugation assays in live and permeable cells, HEK293 cells were transfected with either the VHL-NanoLuc fusion vector (Promega, N275A) or the LRRK2-NanoLuc fusion vector (Promega, NV3401) according to the Promega protocol and seeded at a density of 6000 cells / well in white 384-well plates (Corning 3570). To measure NanoBRET in permeable cells, cells were treated with 50 μg / mL digitalis saponin (Sigma, D141), 125 nM VHL tracer / 125 nM LRRK2 tracer, decreasing concentrations (12 concentrations with 2-fold serial dilutions starting from 33 μM) of the test compound, and NanoBRET NanoGlo substrate (Promega) at the manufacturer's recommended concentration. DMSO was added instead of the test compound in the maximum signal control sample (DMSO control). In the minimum signal control sample (no tracer control), DMSO and tracer dilution buffer were used instead of the test compound and tracer, respectively. Filtered luminescence was measured 10 minutes after substrate addition on a GloMax Discover microplate reader (Promega) or a PHERAstar (BMG LABTECH) plate reader equipped with a 450 nm bandpass filter (donor) and a 600 nm longpass filter (recipient). To measure NanoBRET in live cells, cells were treated with 250 nM VHL tracer / 500 nM LRRK2 tracer at decreasing concentrations (12 concentrations with 2-fold sequential dilutions starting at 33 μM) of the test compound and incubated at 37 °C for 2 hours. The plates were then cooled and the NanoBRET NanoGlo substrate and the extracellular NanoLuc inhibitor (Promega, N2160) were added, followed by NanoBRET readings on the plate reader in the same permeation mode. The NanoBRET ratio per well is expressed in milliBRET according to the following equation: mBRET = [(signal at 610 nM / signal at 450 nM) - (signal at 610 nM without tracer control / signal at 450 nM without tracer control)] × 1000. Fractional occupancy is calculated according to the following equation: Fractional occupancy = (mBRET test compound - mBRET without tracer control) / (mBRET DMSO control - mBRET without tracer control).

[0433] Ternary binding and synergistic determination based on NanoBRET

[0434] HEK293 cells were transfected with the LRRK2-NanoLuc fusion vector (Promega, NV3401) according to the Promega protocol and seeded at a density of 6000 cells / well in white 384-well plates (Corning 3570). Cells were then treated with: 50 μg / mL digitalis saponin, 125 nM LRRK2 tracer, decreasing concentrations (11 consecutive 2-fold dilutions starting at 10 μM) of the test compound or a mixture of the test compound and VCB (for the compound, 11 consecutive 2-fold dilutions starting at 10 μM; the first 6 VCB concentrations starting at 32 μM with 2-fold dilutions, and the last 5 VCB concentrations maintained at 1 μM), and the NanoBRET NanoGlo substrate (Promega) at the manufacturer's recommended concentration. In the maximum signal control sample (DMSO control), DMSO was added instead of the test compound. In the minimum signal control sample (tracer-free control), DMSO and LRRK2 tracer dilution buffer were used instead of the test compound and LRRK2 tracer, respectively. Filtered luminescence was measured 10 minutes after substrate addition on a PHERAstar (BMGLABTECH) plate reader equipped with a 450 nm bandpass filter (donor) and a 600 nm longpass filter (acceptor). Fractional occupancy was calculated using the following equation: Fractional occupancy = (mBRET test compound - mBRET tracer-free control) / (mBRET DMSO control - mBRET tracer-free control).

[0435] VCB's Bodipy 576 / 589 markings

[0436] VCB was labeled with Bodipy 576 / 589 according to previously reported protocol 76. In short, the VCB complex was mixed with Bodipy 576 / 589 NHS ester at a molar ratio of 20:1 and incubated in reaction buffer (0.1 M sodium phosphate, 75 mM KOAc, 2 mM DTT, pH 7.4) at room temperature (protected from light) for 2 hours. The reaction was quenched by dilution 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, and 1 mM DTT pH 7. The eluted labeled protein solution was collected and concentrated using a Pierce concentrator, 3K MWCO (Thermo Scientific).

[0437] NanoBRET ternary complex formation assay

[0438] Hek293 cells were transfected with the LRRK2-NanoLuc vector (Promega, NV3401) for 24 hours, harvested, and resuspended in phenol red-free OptiMEM medium (Life Technologies). Cells were then seeded at a density of 6000 cells / well in white 384-well plates (Corning3570). Digitalis saponin solution (final concentration 50 μg / mL), DMSO, or a decreasing concentration (11 sequentially diluted concentrations starting at 33 μM) of the test PROTAC, and Bodipy576 / 589-labeled VCB protein (final concentration 0.5 μM) were added to each well. NanoBRET NanoGlo substrate (Promega, N2160) was added to each well, and NanoBRET readings were performed on a PHERAstar (BMG LABTECH) plate reader equipped with a 450 nm bandpass filter (donor) and a 600 nm longpass filter (acceptor). The NanoBRET ratio for each well is expressed in milliBRET according to the following equation: mBRET = [(signal at 610 nM / signal at 450 nM) - (signal at 610 nM without tracer control / signal at 450 nM without tracer control)] × 1000. The background signal shown in the DMSO control sample is subtracted from each sample.

[0439] Chemistry - Materials and Methods

[0440] Commercially available chemicals were purchased from Apollo Scientific, Sigma-Aldrich, Fluorochem, and Enamine and were ready for use without further purification. All solvents used in the reactions were anhydrous. LC-MS was performed on a Shimadzu HPLC / MS2020 equipped with a Hypersil gold column (1.9 μm 50 × 2.1 mm), a photodiode array detector, and an ESI detector. Samples were eluted for 3 minutes with a gradient of 5% to 95% acetonitrile in water containing 0.1% formic acid at a flow rate of 0.8 mL / min. Rapid column chromatography was performed on a Teledyne ISCO Combiflash Companion equipped with disposable normal-phase RediSep Rf columns (230 to 400 mesh, 40 to 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% to 95% acetonitrile in water containing 0.1% formic acid over 10 minutes at a flow rate of 25 mL / min. Compound characterization was performed using NMR on a Bruker 500 Ultrashield or a Bruker Ascend 400 spectrometer. 1 H NMR and 13 The reference solvents for C NMR were d1-CDCl3 (δH = 7.26 ppm / δC = 77.16 ppm), d4-CD3OD (δH = 3.31 ppm / δC = 49.00 ppm), or d 6- DMSO (δH = 2.50 ppm / δC = 39.52 ppm). Signal modes were described as singlets (s), doublets (D), triplets (t), quartets (q), quintets, multiplets (m), broad peaks (br), or combinations of the listed split modes. Coupling constants (J) were measured in Hertz (Hz). HRMS was performed on a Bruker MicroTOF II focus ESI mass spectrometer connected in parallel with a Dionex Ultimate 3000 RSLC system, featuring a diode array detector and a Waters XBridge C18 column (50 mm × 2.1, 3.5 μm particle size). Purity of all final compounds was >95% by HPLC.

[0441] synthesis

[0442] General Scheme 1 (Boc Protection): At 0°C, Boc₂O (1.1 equivalents) is added to a solution of benzylamine compound (1 equivalent) and TEA (2 equivalents) in DCM. The resulting mixture is stirred at room temperature for 4 hours or overnight. DCM is added, and the mixture is washed with 1N aqueous HCl, saturated NaHCO₃ aqueous solution, water, and brine, respectively. The collected organic phase is then dried over sodium sulfate, filtered, and concentrated under vacuum to obtain the Boc-protected compound.

[0443] Universal Scheme 2 (Pd(OAc)2-mediated coupling): Under nitrogen protection, Pd(OAc)2 (0.1 equivalents) was added to a mixture of a bromide compound (1 equivalent), KOAc (2 equivalents), and 4-methylthiazole (2 equivalents) in DMA. The resulting mixture was heated to 110°C and stirred overnight at this temperature. The reaction mixture was then diluted with EtOAc and filtered through a diatomaceous earth pad. The collected filtrate was washed with water and brine, dried over sodium sulfate, filtered, and concentrated under vacuum to give a residue, which was purified by rapid column chromatography on silica gel (0% to 100% EtOAc in heptane) to give the desired compound.

[0444] General Scheme 3 (Boc Deprotection and Condensation with Hydroxyproline): A solution of the Boc-protected (1 equivalent) compound in DCM (the same volume as 4N HCl in dioxane) was added to 4N HCl in dioxane (8 equivalents). The resulting mixture was stirred at room temperature for 1 hour and then concentrated to give a solid, which was washed with diethyl ether and dried under vacuum to give an HCl salt. The salt was suspended in DCM, and then TEA (4 equivalents), (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (1 equivalent) or (2S,4S)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (1 equivalent), and HATU (1.05 equivalents) were added. After stirring at room temperature for 2 hours, the resulting mixture was diluted with DCM, washed with water and brine, dried with sodium sulfate, filtered, and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel (0% to 100% EtOAc in heptane) to obtain the desired compound.

[0445] General Scheme 4 (Boc Deprotection and Condensation with Fmoc-S-Triphenylmethyl-L-Penicillamine): A stirred solution of the Boc-protected substrate (1 equivalent) in DCM was added to 4N HCl (10 equivalents, same volume as DCM). After stirring at room temperature for 1 hour, the mixture was concentrated under vacuum to obtain a solid, which was washed with diethylethyl to quantitatively obtain a pale yellow solid as an HCl salt. The solid (1 equivalent) was dissolved in DMF, and TEA (1.5 equivalents) was added to neutralize the HCl, followed by dropwise addition of a mixed solution of Fmoc-S-Triphenylmethyl-L-Penicillamine (1 equivalent), HATU (1.1 equivalent), and TEA (2 equivalents) in DMF at 0°C. The resulting mixture was stirred at 0°C for 2 hours, and then EtOAc and water were added. The aqueous phase was extracted with EtOAc (3×), and the combined organic phases were washed with water and brine, dried with sodium sulfate, filtered and concentrated to obtain the residue, which was purified by rapid column chromatography on silica gel (0% to 100% EtOAc in heptane) to obtain the desired compound.

[0446] General Procedure 5 (Fmoc Deprotection): Piperidine (5 equivalents) is added dropwise to a solution of the Fmoc-protected compound (1 equivalent) in DCM. The resulting mixture is stirred at room temperature for 2 hours, then concentrated under vacuum to obtain a residue, which is purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM containing 0.7 M ammonia) to obtain the desired product.

[0447] General Procedure 6 (Condensation with 1-fluorocyclopropane-1-carboxylic acid): 1-fluorocyclopropane-1-carboxylic acid (1 equivalent) was added to a solution of an amine compound (1 equivalent), TEA (2 equivalents), and HATU (1.05 equivalents) in DMF. After stirring overnight at room temperature, water was added to the mixture, and extraction was performed with EtOAc (3×). The combined organic phases were washed with water and brine, respectively, dried over sodium sulfate, filtered, and concentrated to obtain a residue. This residue was purified by rapid column chromatography on silica gel (0% to 100% EtOAc in PE) to obtain the desired product.

[0448] General Procedure 7 (Triphenylmethyl Deprotection): At 0°C, triisopropylsilane (TIPS, 10 equivalents) and trifluoroacetic acid (10 equivalents) are added to a solution of a triphenylmethyl-protected VHL ligand (1 equivalent) in DCM. After stirring at 0°C for 10 minutes, the mixture is concentrated under vacuum to obtain a residue, which is purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to obtain the desired thiol compound.

[0449] General Procedure 8 (Substitution Reaction): A solution of a toluenesulfonate compound or bromide compound (1 equivalent) in THF is added to a solution of a thiol compound (1 equivalent) and DBU (6 equivalents). After stirring overnight at room temperature, the reaction mixture is concentrated to obtain a residue, which is purified under acidic conditions by preparative HPLC (5% to 95% CH3CN in 0.1% aqueous HCO2H) to obtain the desired compound.

[0450] Exemplary synthesis of exemplary compound 1 (SD13)

[0451]

[0452] 2,5-Dichloro-N-methylpyrimidin-4-amine

[0453] At 0 °C, DIPEA (6.05 mL, 34.78 mmol) was added to a stirred solution of 2,4,5-trichloropyrimidine (5.8 g, 31.62 mmol) in anhydrous THF (100 mL), followed by dropwise addition of methylamine (33% solution in ethanol, 4.33 mL, 34.78 mmol). The reaction mixture was stirred at 0 °C for 1 hour, then warmed to room temperature (RT) and concentrated under reduced pressure. The crude residue was purified by rapid column chromatography on silica gel (0% to 50% EtOAc in petroleum spirit) to give 2,5-dichloro-N-methylpyrimidine-4-amine (5.26 g, 29.54 mmol, 93% yield) as a white solid.

[0454]

[0455]

[0456] 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid

[0457] At room temperature, 4-amino-3-methoxybenzoic acid (4.13 g, 24.70 mmol) was added to a solution of 2,5-dichloro-N-methylpyrimidin-4-amine (4.39 g, 24.65 mmol) in a mixture of dioxane and water (70 mL:70 mL), followed by a solution of 4N HCl in dioxane (6.18 mL, 24.72 mmol). The reaction mixture was refluxed overnight at 100 °C, and then cooled to precipitate a white solid. The solid was filtered, washed with water, and dried under vacuum to give 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (5.95 g, 19.32 mmol, 78% yield) as a white solid.

[0458]

[0459]

[0460] 3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl 4-methylbenzenesulfonate

[0461] A mixture of 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (220 mg, 0.7 mmol) and SOCl2 (2 ml) was heated at 100 °C for 1 hour, then cooled and concentrated to obtain a residue, which was dissolved in 2 ml of DMF. 1.26 ml of the DMF solution was mixed with 3-(piperazin-1-yl)propyl 4-methylbenzenesulfonate (125 mg, 0.42 mmol) and DIPEA (0.22 ml, 1.26 mmol). The resulting mixture was stirred overnight at room temperature. After standard treatment, 3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl 4-methylbenzenesulfonate was obtained and could be used for the next step without further purification.

[0462]

[0463] (2S,4R)-N-(2-(3-(4-(4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propoxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (1,SD13)

[0464] To 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-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (6.65 mg, 0.0125 mmol) ((Farnaby et al. (al., 2019) K₂CO₃ (5 mg, 0.036 mmol) and KI (0.2 mg, 0.0125 mmol) were added to a solution of DMF (0.2 mL). After stirring overnight at 100 °C, the resulting mixture was cooled, filtered through a 0.45 μm PTFE filter, and purified by preparative HPLC (5% to 95% CH₃CN in 0.1% HCO₂H aqueous solution) under acidic conditions to give SD₁₃ (2.95 mg, 25% yield).

[0465]

[0466] Exemplary synthesis of exemplary compound 2 (SD12)

[0467]

[0468]

[0469] 2-(2-(2-(4-(4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate

[0470] A mixture of 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (220 mg, 0.7 mmol) and SOCl2 (2 ml) was heated at 100 °C for 1 hour, then cooled and concentrated to obtain a residue, which was dissolved in 2 ml of DMF. 0.96 ml of the DMF solution was mixed with 119 mg (0.32 mmol) of 2-(2-(2-(piperazin-1-yl)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate and DIPEA (0.18 ml, 1.0 mmol). The resulting mixture was stirred overnight at room temperature. After standard treatment, 2-(2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (167 mg, two-step yield 36%) was obtained.

[0471]

[0472]

[0473] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (2,SD12)

[0474] Add K2CO3 (5 mg, 0.036 mmol) and KI (0.2 mg, 0.0125 mmol) 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) and (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (6.65 mg, 0.0125 mmol) in DMF (0.2 ml). After stirring overnight at 100°C, the resulting mixture was cooled, filtered through a 0.45 μm PTFE filter, and purified by preparative HPLC (5% to 95% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to give SD12 (3.54 mg, 28% yield).

[0475]

[0476] Exemplary synthesis of exemplary compound 3 (SD79)

[0477]

[0478]

[0479] 4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazine-1-carboxylic acid tert-butyl ester

[0480] At room temperature, HOBt (0.98 g, 7.29 mmol), EDCl (1.39 g, 7.29 mmol), 1-Boc-piperazine (1.19 g, 6.38 mmol), and DIPEA (4.23 mL, 24.33 mmol) were added to a solution of 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (2.1 g, 6.08 mmol) in DMF (25 mL). The mixture was stirred at room temperature for 16 hours, then diluted with water (50 mL) and extracted with EtOAc (200 mL). The organic layer was washed with brine (25 mL) and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel (0% to 100% EtOAc in DCM) to give 4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazine-1-carboxylic acid tert-butyl ester (2.52 g, 5.28 mmol, 87%) as a white solid.

[0481]

[0482]

[0483] (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methyl ketone HCl salt

[0484] At room temperature, a solution of tert-butyl 4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazine-1-carboxylic acid in HCl (2.52 g, 5.28 mmol) in a mixture of DCM and MeOH 9:1 (30 ml) was added to a solution of 4 M HCl in dioxane (5.28 ml, 21.12 mmol). The reaction mixture was stirred overnight, diluted with Et2O (200 ml), the precipitate was collected on a glass filter, washed with Et2O (100 ml), and dried overnight to give (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazine-1-yl) methyl ketone (2.13 g, 5.17 mmol, 98%) as an HCl salt.

[0485]

[0486]

[0487] 14-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonate

[0488] Add pentylene glycol di(p-toluenesulfonate) (159.3 mg, 0.292 mmol) and K2CO3 (67 mg, 0.49 mmol) to a solution of (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl) methyl ketone (40 mg, 0.097 mmol) in acetone (3 mL). After stirring overnight at 50°C, the mixture was diluted with DCM, washed with water and brine, dried with sodium sulfate, filtered, and concentrated to give a crude product. The crude product was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give 14-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonate (30 mg, 41% yield) as a white solid.

[0489]

[0490]

[0491] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (3,SD79)

[0492] Add K2CO3 (5 mg, 0.036 mmol) and KI (0.2 mg, 0.0125 mmol) to a solution of 14-(4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonate (9.39 mg, 0.0125 mmol) and (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (6.65 mg, 0.0125 mmol) in DMF (0.2 ml). After stirring overnight at 100°C, the resulting mixture was cooled, filtered through a 0.45 μm PTFE filter, and purified by preparative HPLC (5% to 95% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to give SD79 (2.24 mg, 16% yield).

[0493]

[0494] Exemplary synthesis of exemplary compound 4 (SD82)

[0495]

[0496] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (4,SD82)

[0497] Add K2CO3 (5 mg, 0.036 mmol) and KI (0.2 mg, 0.0125 mmol) 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-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-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). After stirring overnight at 80°C, the resulting mixture was cooled and purified by preparative HPLC (10% to 80% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to give SD82 (3.01 mg, 25% yield).

[0498]

[0499] Exemplary synthesis of exemplary compound 5 (SD75)

[0500]

[0501] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (5,SD75)

[0502] Add K2CO3 (5 mg, 0.036 mmol) and KI (0.2 mg, 0.0125 mmol) 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) and (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (6.68 mg, 0.0125 mmol) in DMF (0.2 ml). After stirring overnight at 80°C, the resulting mixture was cooled and purified by preparative HPLC (10% to 95% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to give SD75 (2.12 mg, 17% yield).

[0503]

[0504] Exemplary synthesis of exemplary compound 6 (SD74)

[0505]

[0506] (2S,4R)-1-((R)-1-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-17-(1-fluorocyclopropane-1-amido)-16,16-dimethyl-3,6,9,12-tetraoxa-15-thiaoctadecane-18-acyl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (6, SD74)

[0507] SD74 was prepared according to general scheme 8. 7.55 mg of SD74 was obtained from 7.0 mg of the thiol compound, in 51% yield.

[0508]

[0509] Exemplary synthesis of exemplary compound 7 (SD100)

[0510]

[0511] (4-(4-(bromomethyl)benzyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[0512] To a suspension of (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl) methyl ketone HCl salt (80 mg, 0.19 mmol) and 4-(bromomethyl)benzaldehyde (38.5 mg, 0.19 mmol) in DCM (3 ml), Na(OAc)3BH (123 mg, 0.58 mmol) was added. After stirring overnight at room temperature, the mixture was diluted with DCM, washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give (4-(4-(bromomethyl)benzyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone (70 mg, 65% yield) as a white solid.

[0513]

[0514]

[0515] (2S,4R)-1-((R)-3-((4-((4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (7, SD100)

[0516] DBU (9.6 μl, 0.064 mmol) was added to a solution of (4-(4-(bromomethyl)benzyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) ketone (6 mg, 0.011 mmol) and (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (6.0 mg, 0.011 mmol) in THF (1 mL). After stirring overnight at room temperature, the mixture was concentrated under reduced pressure to obtain a residue, which was purified by rapid column chromatography on silica gel to give SD100 (4.2 mg, 39% yield) as a white solid.

[0517]

[0518] Exemplary synthesis of exemplary compound 8 (SD11)

[0519]

[0520] 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazine-1-carboxylic acid tert-butyl ester

[0521] DIPEA (0.324 mL, 1.86 mmol) was added to a solution of 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (644 mg, 2.33 mmol) and Boc-piperidine (868 mg, 4.66 mmol) in NMP (4.5 mL). After stirring overnight at 90 °C, the mixture was cooled to room temperature, water was added, and it was extracted with EtOAc (2 × 20 mL). The combined organic phases were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give a residue, which was purified by rapid column chromatography on silica gel (0 to 50% EtOAc in heptane) to give 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazine-1-carboxylic acid tert-butyl ester (815 mg, 80% yield) as a powder.

[0522]

[0523]

[0524] 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione, HCl salt

[0525] Add 1 ml of 4N HCl to a solution of 50 mg (0.11 mmol) of 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazine-1-carboxylic acid tert-butyl ester (50 mg, 0.11 mmol) in DCM (1 ml). Stir the mixture at room temperature for 1 hour and then concentrate it under reduced pressure to give the title compound as a residue, which can be used for the next step without further purification.

[0526]

[0527] 5-(4-(3-(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 (8,SD11)

[0528] K₂CO₃ (15 mg, 0.11 mmol) and KI (0.6 mg, 0.00375 mmol) were added to a solution of 3-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl 4-methylbenzenesulfonate (22 mg, 0.0375 mmol) in DMF (0.4 mL). After stirring overnight at 100 °C, the resulting mixture was cooled and purified by preparative HPLC (10% to 50% CH₃CN in 0.1% HCO₂H aqueous solution) under acidic conditions to give SD₁₁ (5.4 mg, 19% yield).

[0529]

[0530] Exemplary synthesis of exemplary compound 9 (SD10)

[0531]

[0532] 5-(4-(2-(2-(2-(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 (9, SD10)

[0533] Add 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione HCl salt (14.2 mg, 0.0375 mmol), K₂CO₃ (15 mg, 0.109 mmol), and KI (0.6 mg, 0.00375 mmol) 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 (24.9 mg, 0.0375 mmol) in DMF (0.5 mL) to the solution. Heat the resulting mixture to 100 °C and stir overnight at 100 °C. The mixture was filtered through a 0.45 μM filter and purified by preparative HPLC (10% to 85% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to give SD10 (2.82 mg, 9% yield).

[0534]

[0535] Exemplary synthesis of exemplary compound 10 (SD113)

[0536]

[0537] (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)thiazolyl-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethyl)-2-(methylamino)acrylamide HCl salt (10, SD113)

[0538] Add ((S)-1-(((S)-1-cyclohexyl-2-((S)-2-(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) to a solution of 2-(2-(2-(4-(3-hydroxybenzoyl)thiazolyl-2-yl)pyrrolidine-1-yl)-2-oxoethyl)amino)-1-oxopropyl-2-yl)(methyl)carbamate tert-butyl ester (7.5 mg, 0.0125 mmol) (Shibata et al., 2018), K2CO3 (5 mg, 0.036 mmol) and KI (0.6 mg, 0.00375 mmol) 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 (7.5 mg, 0.0125 mmol) in DMF (0.2 ml). The resulting mixture was heated to 100 °C and stirred overnight at 100 °C. The mixture was filtered through a 0.45 μM filter and purified by preparative HPLC (10% to 85% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to give ((S)-1-(((S)-2-((S)-2-(4-(3-(2-(2-(2-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)benzoyl)thiazolyl-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropyl-2-yl)(methyl)carbamate (3 mg, 22% yield); HRMS (ESI) + )m / z, for C 54 H 74 ClN 10 O 10 The calculated value of S is: 1089.4993[M+H] +The measured value was 1089.5198. A solution of ((S)-1-(((S)-2-((S)-2-(4-(3-(2-(2-(2-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)benzoyl)thiazo-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropyl-2-yl)(methyl)carbamate tert-butyl ester (3 mg, 0.0028 mmol) in 0.2 mL of DCM was added to a 4N HCl solution (0.2 mL) in 1,4-dioxane. The resulting mixture was stirred at room temperature for 2 hours and then concentrated to give SD113 as an HCl salt.

[0539]

[0540] Exemplary synthesis of exemplary compound 11 (SD112)

[0541]

[0542] (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)thiazolyl-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethyl)-2-(methylamino)acrylamide (11, SD112)

[0543] To a solution of 14-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonate (9.4 mg, 0.0125 mmol) in DMF (0.2 mL), tert-butyl (methyl)carbamate (7.5 mg, 0.0125 mmol), 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 overnight at 100 °C. The mixture was filtered through a 0.45 μM filter and purified by preparative HPLC (10% to 85% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to give ((S)-1-(((S)-2-((S)-2-(4-(3-(2-(2-(2-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)benzoyl)thiazolyl-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropyl-2-yl)(methyl)carbamate (3.8 mg, 26% yield); HRMS (ESI) + )m / z, for C 58 H 82 ClN 10 O 12 The calculated value of S is: 1177.5517[M+H] + The measured value was 1177.5747. A solution of 4N HCl in 1,4-dioxane was added to a solution of ((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)thiazolyl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropyl-2-yl)(methyl)carbamate tert-butyl ester (3.8 mg, 0.0028 mmol) in 0.2 ml of DCM. The resulting mixture was stirred at room temperature for 2 hours and then concentrated to give SD112 as an HCl salt.

[0544]

[0545] Example synthesis of compound 12 (SD114)

[0546]

[0547] (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)thiazolyl-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethyl)-2-(methylamino)acrylamide, HCl salt (SD114)

[0548] 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) in DMF (0.2 mL), tert-butyl (methyl)carbamate (7.5 mg, 0.0125 mmol), 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 overnight at 100 °C. The mixture was filtered through a 0.45 μM filter and purified by preparative HPLC (10% to 85% of aqueous solution in 0.1% HCO2H) under acidic conditions to give ((S)-1-(((S)-2-((S)-2-(4-(3-(3-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propoxy)benzoyl)thiazolyl)pyrrolidone-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropyl-2-yl)(methyl)carbamate (4 mg, 32% yield); HRMS (ESI) + )m / z, for C 51 H 67 ClN 10 O8S, calculated value: 1015.462534 [M+H] +The measured value was 1015.4770. A solution of ((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)thiazolyl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropyl-2-yl)(methyl)carbamate tert-butyl ester (4 mg, 0.0039 mmol) in 0.2 ml of DCM was added to a 4N HCl solution (0.2 ml) in 1,4-dioxane. The resulting mixture was stirred at room temperature for 2 hours and then concentrated to give SD114 as an HCl salt.

[0549]

[0550] Exemplary synthesis of exemplary compound 13 (XL01078B)

[0551]

[0552] 2-Chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine

[0553] At 0 °C, DIPEA (2.3 mL, 13.18 mmol) was added to a stirred solution of 2,4-dichloro-5-(trifluoromethyl)pyrimidine (2.6 g, 11.99 mmol) in anhydrous THF (50 mL), followed by dropwise addition of methylamine (33% solution in ethanol, 1.64 mL, 13.18 mmol). The reaction mixture was stirred at 0 °C for 1 hour, then warmed to room temperature and concentrated under vacuum. The crude residue was purified by rapid column chromatography on silica gel with a gradient of 0% to 50% EtOAc in petroleum solvent to give 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidine-4-amine (1.36 g, 6.42 mmol, 53% yield).

[0554]

[0555]

[0556] 3-Methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoic acid

[0557] 4-Amino-3-methoxybenzoic acid (0.87 g, 5.20 mmol) was added 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), followed by a 4 M HCl solution (1.3 mL, 5.20 mmol) in dioxane. After reflux at 100 °C for 2 hours, the mixture was cooled to room temperature to precipitate a white solid. The solid was filtered, washed with water, and dried under vacuum to give 3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoic acid (1.25 g, 77% yield) as a white solid.

[0558]

[0559]

[0560] 4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazine-1-carboxylic acid tert-butyl ester

[0561] At room temperature, 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) were added 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). After stirring at room temperature for 16 hours, the mixture was diluted with water (30 mL) and extracted with EtOAc (100 mL). The organic layer was washed with brine (15 mL) and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel (0% to 100% EtOAc in DCM) to give 4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazine-1-carboxylic acid tert-butyl ester (1.12 g, 2.19 mmol, 85% yield).

[0562]

[0563]

[0564] (3-Methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)(piperazin-1-yl)methyl ketone HCl salt

[0565] At room temperature, a solution of tert-butyl 4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazine-1-carboxylic acid (1.12 g, 2.19 mmol) in a mixture of DCM and MeOH 9:1 (20 ml) was added to a 4N HCl solution (2.2 ml, 8.76 mmol) in dioxane. After stirring overnight at room temperature, the reaction mixture was diluted with Et2O (100 ml) to precipitate a white solid. The white solid 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)(piperazine-1-yl)methyl ketone HCl salt (0.97 g, 2.17 mmol, 99%) as a white powder.

[0566]

[0567]

[0568] 2-(2-(2-(4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate

[0569] Triethylene glycol dimethylbenzenesulfonate (205 mg, 0.448 mmol) and K2CO3 (124 mg, 0.896 mmol) were added to a solution of (3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)(piperazin-1-yl) methyl ketone HCl salt (50 mg, 0.111 mmol) in acetone (3 ml). After stirring overnight at 50°C, the mixture was cooled, diluted with DCM (10 ml), washed with water and brine, dried over sodium sulfate, filtered, and concentrated to obtain a residue. The residue was purified by rapid column chromatography on silica gel to give 2-(2-(2-(4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazin-1-yl)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (36 mg, 46% yield) as a white solid.

[0570]

[0571]

[0572] (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-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-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (13, XL01078B)

[0573] XL01078B was prepared according to general scheme 8. 3.6 mg of XL01078B was obtained from 5 mg of the thiol compound, in a 36% yield.

[0574]

[0575] Exemplary synthesis of exemplary compound 14 (XL01072)

[0576]

[0577] (4-(3-bromopropyl)piperazin-1-yl)(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)methyl ketone

[0578] To a solution of (3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)(piperazin-1-yl)methyl ketone HCl salt (100 mg, 0.22 mmol) in acetone (2 mL), 1,3-dibromopropane (181 mg, 0.90 mmol) and K₂CO₃ (93 mg, 0.67 mmol) were added. After stirring overnight at room temperature, the resulting mixture was diluted with DCM, washed with water and brine respectively, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give (4-(3-bromopropyl)piperazin-1-yl)(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)methyl ketone (51 mg, 42% yield) as a white solid.

[0579]

[0580]

[0581] (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-((3-(4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazin-1-yl)propyl)thio)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (14, XL01072)

[0582] XL01072 was prepared according to general scheme 8. 6.1 mg of XL01072 was obtained from 7 mg of the thiol compound, in a 47% yield.

[0583]

[0584] Exemplary synthesis of exemplary compound 15 (XL01070B)

[0585]

[0586] (4-(4-(bromomethyl)benzyl)piperazin-1-yl)(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)methyl ketone

[0587] Na(OAc)3BH (114 mg, 0.54 mmol) was added to a mixture of 4-bromomethylbenzaldehyde (36 mg, 0.18 mmol) and (3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)(piperazin-1-yl)methyl ketone HCl salt (80 mg, 0.18 mmol) in DCM (5 ml). After stirring at room temperature for 3 hours, the resulting mixture was diluted with DCM, washed with water and brine respectively, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give (4-(4-(bromomethyl)benzyl)piperazin-1-yl)(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)methyl ketone (70 mg, 66% yield).

[0588]

[0589]

[0590] (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-((4-((4-(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl)piperazin-1-yl)methyl)benzyl)thio)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (15, XL01070B)

[0591] XL01070B was prepared according to general scheme 8. 7.8 mg of XL01070B was obtained from thiols, in 57% yield.

[0592]

[0593] Exemplary synthesis of exemplary compound 16 (XL01119)

[0594]

[0595] (S)-(1-(4-bromophenyl)ethyl)carbamate tert-butyl ester was prepared according to general scheme 1. 3.6 g of (S)-(1-(4-bromophenyl)ethyl)carbamate tert-butyl ester was obtained from 4.17 g of the amine compound, in 58% yield.

[0596]

[0597]

[0598] (S)-(1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamate tert-butyl ester was prepared according to general scheme 2. 600 mg of (S)-(1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamate tert-butyl ester was obtained from 1.03 g of the bromide compound, in 55% yield.

[0599]

[0600]

[0601] (2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared according to general scheme 3. 380 mg of (2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was obtained from 337 mg of (S)-(1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester, in 83% yield.

[0602]

[0603]

[0604] (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate was prepared according to general scheme 4. 190 mg of (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was obtained from 118 mg of (2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate, in 75% yield.

[0605]

[0606]

[0607] (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide was prepared according to General Scheme 5. 80 mg of (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide was obtained from 150 mg of Fmoc-protected starting material in 70% yield.

[0608]

[0609]

[0610] (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide was prepared according to General Scheme 6. 80 mg of (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-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.

[0611]

[0612]

[0613]

[0614] (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide was prepared according to General Scheme 7. 25 mg of (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide was obtained from 40 mg of triphenylmethyl-protected starting material in 90% yield.

[0615]

[0616]

[0617] (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-amido)-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (16, XL01119)

[0618] XL01119 was prepared according to general scheme 8. 6.7 mg of XL01119 was obtained from 8 mg of thiol starting material, in a 40% yield.

[0619]

[0620] Exemplary synthesis of exemplary compound 17 (XL01118)

[0621]

[0622] (4-(3-bromopropyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[0623] K₂CO₃ (50 mg, 0.36 mmol) and 1,3-dibromopropane (98 mg, 0.48 mmol) were added to a solution of (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl) methyl ketone HCl salt (50 mg, 0.12 mmol) in DMF (1 mL). 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) methyl ketone (30 mg, 50% yield) as a white solid.

[0624]

[0625]

[0626] (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-amido)-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (17, XL01118)

[0627] XL01118 was prepared according to general scheme 8. 3.1 mg of XL01118 was obtained from 8 mg of the thiol compound, in a 20% yield.

[0628]

[0629] Exemplary synthesis of exemplary compound 18 (XL01120)

[0630]

[0631] (2S,4R)-1-((R)-3-((4-((4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (18, XL01120)

[0632] XL01120 was prepared according to general scheme 8. 9.5 mg of XL01120 was obtained from 8 mg of the thiol compound, in 65% yield.

[0633]

[0634] Exemplary synthesis of exemplary compound 19 (XL01126)

[0635]

[0636] trans-1,4-bis(p-toluenesulfonyloxymethyl)cyclohexane

[0637] At 0 °C, TEA (1.93 mL, 13.9 mmol), DMAP (42 mg, 0.34 mmol), and TsCl (1.655 g, 8.68 mmol) were added to a solution of trans-1,4-cyclohexanediethanol (500 mg, 3.47 mmol) in DCM (10 mL). The resulting mixture was then warmed to room temperature and stirred overnight at room temperature. DCM was added, and the mixture was washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. This residue was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give trans-1,4-bis(p-toluenesulfonyloxymethyl)cyclohexane (1.29 g, 82% yield) as a white solid.

[0638]

[0639]

[0640] trans-1,4-dibromomethylcyclohexane

[0641] LiBr (95 mg, 1.1 mmol) was added to a solution of trans-1,4-bis(p-toluenesulfonyloxymethyl)cyclohexane (125 mg, 0.28 mmol) in acetone (3 mL). After stirring under reflux overnight, the mixture was cooled, ethyl acetate was added, and the mixture was washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. This residue was purified by rapid column chromatography on silica gel (0% to 20% ethyl acetate in heptane) to give trans-1,4-dibromomethylcyclohexane (61 mg, 82% yield) as a white solid.

[0642]

[0643]

[0644] (4-(((1r,4r)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[0645] Add trans-1,4-dibromomethylcyclohexane (50 mg, 0.18 mmol) and K2CO3 (42 mg, 0.30 mmol) to a solution of (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methyl ketone HCl salt (25 mg, 0.06 mmol) in acetone (2 ml). After stirring overnight at 50°C, the mixture was cooled to room temperature, diluted with DCM, washed with water and brine respectively, dried with sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to produce (4-(((1r,4r)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone (10 mg, 29% yield) as a white solid.

[0646]

[0647]

[0648] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (19, XL01126)

[0649] XL01126 was prepared according to general scheme 8. 7.7 mg of XL01126 was obtained from 7.6 mg of the thiol compound, in 53% yield.

[0650]

[0651] Exemplary synthesis of exemplary compound 20 (XL01134)

[0652]

[0653] cis-1,4-cyclohexanediethanol

[0654] Me2S-BH3 (2M in THF, 5.8 mL, 11.6 mmol) was added to a solution of cis-cyclohexane-1,4-dicarboxylic acid (500 mg, 2.9 mmol) in THF (5 mL) at 0 °C. After stirring at 0 °C for 2 h, the reaction mixture was quenched with methanol and then concentrated under reduced pressure to obtain a residue, which was purified by rapid column chromatography on silica gel to give cis-1,4-cyclohexanediethanol (418 mg, 100% yield) as a colorless oil.

[0655]

[0656]

[0657] cis-1,4-bis(p-toluenesulfonyloxymethyl)cyclohexane

[0658] At 0 °C, TsCl (437 mg, 2.3 mmol) was added to a mixture of cis-1,4-cyclohexanediethanol (150 mg, 1.04 mmol), TEA (0.58 mL, 4.17 mmol), and DMAP (13 mg, 0.1 mmol) in DCM (20 mL). The mixture was then warmed to room temperature and stirred overnight. DCM was added, and the reaction mixture was washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel (0% to 30% ethyl acetate in heptane) to give cis-1,4-bis(p-toluenesulfonyloxymethyl)cyclohexane (208 mg, 44% yield) as a white solid.

[0659]

[0660]

[0661] cis-1,4-dibromomethylcyclohexane

[0662] LiBr (358 mg, 1.32 mmol) was added to a solution of cis-1,4-bis(p-toluenesulfonyloxymethyl)cyclohexane (150 mg, 0.33 mmol) in acetone (5 mL). After refluxing overnight, the reaction mixture was cooled to room temperature, diluted with ethyl acetate, washed with water and brine respectively, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel (0% to 30% ethyl acetate in heptane) to give cis-1,4-dibromomethylcyclohexane (75 mg, 83% yield) as a white solid.

[0663]

[0664]

[0665] (4-(((1s,4s)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[0666] Add cis-1,4-dibromomethylcyclohexane (50 mg, 0.18 mmol) and K2CO3 (50 mg, 0.36 mmol) to a solution of (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methyl ketone HCl salt (25 mg, 0.06 mmol) in acetone (2 ml). After stirring overnight at 50°C, the mixture was cooled to room temperature, diluted with DCM, washed with water and brine respectively, dried with sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to produce (4-(((1s,4s)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone (10 mg, 29% yield) as a white solid.

[0667]

[0668]

[0669] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (20, XL01134)

[0670] XL01134 was prepared according to general scheme 8. 9.3 mg of XL01134 was obtained from 8.0 mg of the thiol compound, in 61% yield.

[0671]

[0672] Exemplary synthesis of exemplary compound 21 (XL01076)

[0673]

[0674] (4-(4-bromobut-2-yn-1-yl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[0675] To a solution of (60 mg, 0.134 mmol) HCl salt of (4-(5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl) methyl ketone in acetone (2 mL), 1,4-dibromo-2-butyne (85.4 mg, 0.403 mmol) and K₂CO₃ (46.4 mg, 0.336 mmol) were added. After stirring at room temperature for 3 hours, the resulting mixture was diluted with DCM, washed with water and brine respectively, dried over sodium sulfate, filtered, and concentrated to obtain a residue. This residue was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give (4-(4-bromobut-2-yn-1-yl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone. This product was unstable and was used as an impure mixture for the next step.

[0676]

[0677] (2S,4R)-1-((R)-3-((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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (21, XL01076)

[0678] XL01076 was prepared according to general protocol 8. 3.1 mg of XL01076 was obtained from 5 mg of thiol starting material, in a 34% yield.

[0679]

[0680] Exemplary synthesis of exemplary compound 22 (XL01123)

[0681]

[0682] (4-Bromo-3-fluorobenzyl) tert-butyl carbamate

[0683] Trifluoroacetic acid (0.754 mL, 9.9 mmol) was added to a solution of 4-bromo-3-fluorobenzaldehyde (1 g, 4.9 mmol), tert-butyl carbamate (1.73 g, 14.8 mmol), and triethylsilane (1.72 g, 14.8 mmol) in DCM and acetonitrile (10 mL DCM mixed with 30 mL acetonitrile). After stirring overnight at room temperature, the mixture was diluted with DCM, washed with water and brine respectively, dried over sodium sulfate, filtered, and concentrated to give a residue. This residue was purified by rapid column chromatography on silica gel (0% to 10% ethyl acetate in heptane) to give tert-butyl (4-bromo-3-fluorobenzyl) carbamate as a white solid (710 mg, 59% yield).

[0684]

[0685]

[0686] (3-fluoro-4-(4-methylthiazol-5-yl)benzyl)tert-butyl carbamate was prepared according to general scheme 2. 550 mg of (3-fluoro-4-(4-methylthiazol-5-yl)benzyl)tert-butyl carbamate was obtained from 865 mg of (4-bromo-3-fluorobenzyl)carbamate, in 60% yield.

[0687]

[0688]

[0689] (2S,4R)-2-((3-fluoro-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester was prepared according to general scheme 3. Starting with 502 mg of (3-fluoro-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)carbamoyl ester, 550 mg of (2S,4R)-2-((3-fluoro-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester was obtained in 81% yield.

[0690]

[0691]

[0692] (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-2-((3-fluoro-4-(4-methylthiazo-5-yl)benzyl)carbamoyl)-4-hydroxypyrrolidone-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate was prepared according to general scheme 4. 104 mg of (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-2-((3-fluoro-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester was obtained from 119 mg of (2S,4R)-2-((3-fluoro-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)-4-hydroxypyrrolidine-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate, in 41% yield.

[0693]

[0694]

[0695] (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide was prepared according to General Scheme 5. 86 mg of (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide was obtained from 150 mg of the compound protected by Fmoc, in 75% yield.

[0696]

[0697]

[0698] (2S,4R)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide was prepared according to General Scheme 6. 77 mg of (2S,4R)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide was obtained from 85 mg of the amine compound in 81% yield.

[0699]

[0700]

[0701] (2S,4R)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide was prepared according to General Scheme 7. 27 mg of (2S,4R)-N-(3-fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide, 97%, was obtained from 40 mg of the triphenylmethyl-protected compound.

[0702]

[0703]

[0704] (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-amido)-3-methylbutyryl)-N-(3-fluoro-4-(4-methylthiazolyl-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (22, XL01123)

[0705] XL01123 was prepared according to general scheme 8. 5 mg of XL01123 was obtained from 8 mg of the thiol compound, in a 40% yield.

[0706]

[0707] Exemplary synthesis of exemplary compound 23 (XL01122)

[0708]

[0709] (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-amido)-3-methylbutyryl)-N-(3-fluoro-4-(4-methylthiazolyl-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (23, XL01122)

[0710] XL01122 was prepared according to general scheme 8. 1.4 mg of XL01122 (10%) was obtained from 8 mg of the thiol compound.

[0711]

[0712] Exemplary synthesis of exemplary compound 24 (XL01121)

[0713]

[0714] (2S,4R)-1-((R)-3-((4-((4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)methyl)benzyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-N-(3-fluoro-4-(4-methylthiazolyl-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (24, XL01121)

[0715] XL01121 was prepared according to general scheme 8. 12.2 mg of XL01121 (83%) was obtained starting with 8 mg of the thiol compound.

[0716]

[0717] Exemplary synthesis of exemplary compound 25 (XL01131)

[0718]

[0719]

[0720] 2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethyl 4-methylbenzenesulfonate

[0721] Add Dess-Martin periodinane (195 mg, 0.46 mmol) to a solution of 2-(2-hydroxyethoxy)ethyl 4-methylbenzenesulfonate (100 mg, 0.38 mmol) in DCM (3 ml). After stirring at room temperature for 1 hour, dilute the reaction mixture with DCM, wash with water and brine, dry with sodium sulfate, filter, concentrate, and give the residue, which is used for the next step without further purification. Dissolve half of the residue from the previous step in DCM (4 ml), then add (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methyl ketone 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 with sodium sulfate, filtered, and concentrated to obtain the residue. The residue was purified by rapid 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).

[0722]

[0723]

[0724] (2S,4R)-1-((R)-3-((2-(2-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)ethoxy)ethyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (25, XL01131)

[0725] XL01131 was prepared according to general scheme 8. 5.9 mg of XL01131 was obtained from 8 mg of the thiol compound, in a 40% yield.

[0726]

[0727] Exemplary synthesis of exemplary compound 26 (XL01140)

[0728]

[0729]

[0730] A mixture of piperazine-1-carboxylic acid tert-butyl ester (500 mg, 2.69 mmol), propylene oxide (234 mg, 4.0 mmol), and ethanol (3 mL) was stirred overnight in a sealed microwave-safe tube at room temperature. The mixture was concentrated under reduced pressure to obtain a residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give (S)-4-(2-hydroxypropyl)piperazine-1-carboxylic acid tert-butyl ester (390 mg, 60% yield).

[0731]

[0732]

[0733] Triphenylphosphine (298 mg, 1.1 mmol) was added to a solution of (S)-4-(2-hydroxypropyl)piperazine-1-carboxylic acid tert-butyl ester (150 mg, 0.61 mmol) and CBr4 (367 mg, 1.1 mmol) in DCM (6 mL) at 0 °C. After stirring at 0 °C for 1 h, an aqueous solution of NH4Cl was added and the mixture was extracted with DCM (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 rapid column chromatography on silica gel (0% to 50% ethyl acetate in heptane) to give (R)-4-(2-bromopropyl)piperazine-1-carboxylic acid tert-butyl ester (64 mg, 34% yield).

[0734]

[0735]

[0736] 4-((S)-2-(((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)propyl)piperazine-1-carboxylic acid tert-butyl ester was prepared according to general scheme 8. 16 mg of 4-((S)-2-(((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)propyl)piperazine-1-carboxylic acid tert-butyl ester was obtained from 20 mg of the thiol compound in 57% yield.

[0737]

[0738]

[0739] (2S,4R)-1-((R)-3-(((S)-1-(4-(4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)propyl-2-yl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01140)

[0740] HCl (1,4-dioxane, 4N, 1 mL) was added to a stirred solution of 4-((S)-2-(((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)propyl)piperazine-1-carboxylic acid tert-butyl ester (16 mg, 0.021 mmol) in DCM (1 mL). After stirring overnight at room temperature, the mixture was concentrated to obtain a solid, which was washed with diethyl ether and used in the next step. The solid was dissolved in DMF (1 ml), and 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (7 mg, 0.023 mmol), DIPEA (16 μL, 0.084 mmol), and PyOxim (12 mg, 0.023 mmol) were added, respectively. After stirring at room temperature for 2 hours, the reaction mixture was diluted with DCM (5 ml), washed with water and brine, dried over sodium sulfate, filtered, and concentrated to obtain a residue. This residue was purified by preparative HPLC (5% to 95% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to give XL01140 (3.6 mg, 17% yield).

[0741]

[0742] Exemplary synthesis of exemplary compound 27 (XL01111)

[0743]

[0744] (4-(3-(bromomethyl)benzyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[0745] A suspension of (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl) methyl ketone HCl salt (80 mg, 0.19 mmol) and 3-bromomethylbenzaldehyde (38.5 mg, 0.19 mmol) in DCM (4 mL) was added with sodium triacetoxyborohydride (123 mg, 0.58 mmol). After stirring overnight at room temperature, the mixture was diluted with DCM, washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give (4-(3-(bromomethyl)benzyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone (51 mg, 46% yield).

[0746]

[0747]

[0748] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (XL01111)

[0749] XL01111 was prepared according to general scheme 8. 7.5 mg of XL01111 (42%) was obtained from 10 mg of the thiol compound.

[0750]

[0751] Exemplary synthesis of exemplary compound 28 (XL01145)

[0752]

[0753] 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate

[0754] At 0 °C, TEA (2.92 mL, 20.98 mmol), DMAP (64 mg, 0.52 mmol), and TsCl (2 g, 10.5 mmol) were added to a solution of tetraethylene glycol (6.1 g, 31.5 mmol) in DCM (100 mL). After stirring overnight at room temperature, the 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 rapid column chromatography on silica gel (0% to 100% ethyl acetate in heptane) to give 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (2.4 g, 66% yield) as a white solid.

[0755]

[0756]

[0757] 2-(2-(2-(2-(3-methoxy-4-nitrophenoxy)ethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate

[0758] Under nitrogen protection, triphenylphosphine (275 mg, 1.05 mmol) was added to a solution of 3-methoxy-4-nitrophenol (111 mg, 0.658 mmol), 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (229 mg, 0.658 mmol), and diisopropyl azodicarboxylate (199 mg, 0.987 mmol) in THF (5 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl acetate, washed with aqueous NaOH, aqueous 1N HCl, and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. This residue was purified by rapid column chromatography on silica gel (0% to 100% ethyl acetate in heptane) to give 2-(2-(2-(2-(3-methoxy-4-nitrophenoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (85 mg, 24%).

[0759]

[0760]

[0761] 2-(2-(2-(2-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenoxy)ethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate

[0762] Under nitrogen protection, 10% palladium on carbon (10 mg) was added to a solution of 2-(2-(2-(2-(3-methoxy-4-nitrophenoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (85 mg, 0.17 mmol) in ethyl acetate (3 ml). The nitrogen was then replaced with hydrogen, and the resulting mixture was stirred overnight under hydrogen pressure (hydrogen balloon). The mixture was filtered through a diatomaceous earth pad and concentrated to obtain a residue, which was dissolved in DCM, and then TEA (0.047 ml, 0.46 mmol) and di-tert-butyl decarbonate (37 mg, 0.17 mmol) were added. After stirring at room temperature for 2 hours, the resulting mixture was concentrated and purified by rapid column chromatography on silica gel (0% to 100% ethyl acetate in heptane) to give 2-(2-(2-(2-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenoxy)ethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (58 mg for both steps, 60% yield).

[0763]

[0764]

[0765] (4-(((R)-1-(1-fluorocyclopropyl)-3-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-4,4-dimethyl-1-oxo-8,11,14-trioxa-5-thia-2-azahexadecyl-16-yl)oxy)-2-methoxyphenyl)tert-butyl carbamate

[0766] Add 1,8-diazabicyclo[5.4.0]undecyl-7-ene (0.072 mL, 0.5 mmol) to a solution of 2-(2-(2-(2-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (29 mg, 0.05 mmol) and (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (27 mg, 0.05 mmol) in THF (2 mL). After stirring overnight at room temperature, the mixture was concentrated and purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give tert-butyl (4-(((R)-1-(1-fluorocyclopropyl)-3-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-4,4-dimethyl-1-oxo-8,11,14-trioxa-5-thia-2-azahexadecyl-16-yl)oxy)-2-methoxyphenyl)carbamate (36 mg, 77% yield).

[0767]

[0768]

[0769] (2S,4R)-1-((S)-1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenoxy)-14-(1-fluorocyclopropane-1-amido)-13,13-dimethyl-3,6,9-trioxa-12-thiapentadecano-15-acyl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (28, XL01145)

[0770] Add HCl (4N in 1,4-dioxane, 1 mL) to a solution of tert-butyl carbamate (36 mg, 0.039 mmol) in DCM (1 mL). After stirring overnight at room temperature, concentrate and dissolve in isopropanol (1.5 mL). 2,5-Dichloro-N-methylpyrimidin-4-amine (8 mg, 0.045 mmol) and HCl (4N in 1,4-dioxane, one drop) were added, and the resulting mixture was heated in a microwave at 100 °C for 8 hours. The solvent was removed under reduced pressure to obtain a residue, which was purified by preparative HPLC (5% to 95% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to give XL01145 (22% yield for 8.3 mg in both steps).

[0771]

[0772] Exemplary synthesis of exemplary compound 29 (XL01149)

[0773]

[0774] 3-(benzyloxy)propyl 4-methylbenzenesulfonate

[0775] At 0 °C, TsCl (867 mg, 4.54 mmol) was added to a solution of 3-(benzyloxy)prop-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). 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 rapid column chromatography on silica gel (0% to 50% ethyl acetate in heptane) to give 3-(benzyloxy)propyl 4-methylbenzenesulfonate (750 mg, 54% yield).

[0776]

[0777]

[0778] 2-(3-(benzyloxy)propoxy)ethanol-1-ol

[0779] At 0 °C, 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). The resulting mixture was heated to reflux and stirred overnight under reflux. The reaction mixture was then cooled in an ice bath, and an aqueous solution of NH4Cl 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, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give 2-(3-(benzyloxy)propoxy)ethanol-1-ol (534 mg, 54% yield).

[0780]

[0781]

[0782] 4-(2-(3-(benzyloxy)propoxy)ethoxy)-2-methoxy-1-nitrobenzene

[0783] At 0 °C, diisopropyl azodicarboxylate (192 mg, 0.95 mmol) was added to a solution of 3-methoxy-4-nitrophenol (81 mg, 0.48 mmol), 2-(3-(benzyloxy)propoxy)eth-1-ol (100 mg, 0.48 mmol), and triphenylphosphine (312 mg, 1.19 mmol) in THF. The resulting reaction mixture was stirred overnight at room temperature under nitrogen protection. Ethyl acetate was added. The resulting mixture was washed with 1N NaOH aqueous solution, water, and brine, respectively, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel (0% to 100% ethyl acetate in heptane) to give 4-(2-(3-(benzyloxy)propoxy)ethoxy)-2-methoxy-1-nitrobenzene (80 mg, 38% yield).

[0784]

[0785]

[0786] (4-(2-(3-hydroxypropoxy)ethoxy)-2-methoxyphenyl)tert-butyl carbamate

[0787] Under nitrogen protection, 10% palladium on carbon (8 mg) was added to a solution of 4-(2-(3-(benzyloxy)propoxy)ethoxy)-2-methoxy-1-nitrobenzene (80 mg, 0.22 mmol) in methanol (3 ml). The nitrogen was evacuated and backfilled with hydrogen, and the resulting mixture was stirred overnight under positive hydrogen pressure (hydrogen balloon). The mixture was then filtered through a diatomaceous earth pad, and the filtrate was concentrated to obtain a residue, which was dissolved in DCM (3 ml), followed by the addition of TEA (0.092 ml, 0.66 mmol) and di-tert-butyl decarbonate / salt (48 mg, 0.22 mmol). After stirring overnight at room temperature, the mixture was diluted with DCM, washed with 1M HCl aqueous solution, water and brine respectively, dried with sodium sulfate, filtered and concentrated under reduced pressure to obtain the residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give tert-butyl (4-(2-(3-hydroxypropoxy)ethoxy)-2-methoxyphenyl)carbamate (14.3 mg, 19% yield).

[0788]

[0789]

[0790] 3-(2-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenoxy)ethoxy)propyl4-methylbenzenesulfonate

[0791] TsCl (20 mg, 0.1 mmol) was added to a solution of (4-(2-(3-hydroxypropoxy)ethoxy)-2-methoxyphenyl)carbamate tert-butyl ester (33 mg, 0.10 mmol), TEA (0.027 mL, 0.19 mmol), and DMAP (1 mg, 0.008 mmol) in DCM (2 mL). After stirring overnight at room temperature, the mixture was diluted with DCM, washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give 3-(2-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenoxy)ethoxy)propyl 4-methylbenzenesulfonate (30 mg, 62% yield).

[0792]

[0793]

[0794] (4-(2-(3-(((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)propoxy)ethoxy)-2-methoxyphenyl)tert-butyl carbamate was prepared according to general scheme 8. 30 mg of tert-butyl carbamate (4-(2-(3-(((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)propoxy)ethoxy)-2-methoxyphenyl)carbamate was obtained from 27 mg of thiol compound in 70% yield.

[0795]

[0796]

[0797] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (29, XL01149)

[0798] HCl (1,4-dioxane, 4N, 1 mL) was added to a solution of tert-butyl carbamate (30 mg, 0.035 mmol) in DCM (1 mL). After stirring overnight at room temperature, the mixture was concentrated under reduced pressure to obtain a solid, which was dissolved in isopropanol, followed by the addition of 2,5-dichloro-N-methylpyrimidin-4-amine (7 mg, 3.9 mmol). After heating at 100°C for 8 hours in a microwave reactor, the mixture was diluted with DCM, washed with water and brine, dried with sodium sulfate, filtered and concentrated to obtain a residue, which was purified by preparative HPLC (5% to 95% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to obtain XL01149 (7.5 mg for both steps, 24%).

[0799]

[0800] Exemplary synthesis of exemplary compound 30 (XL01168)

[0801]

[0802] (4-Hydroxy-2-methoxyphenyl) tert-butyl carbamate

[0803] Under nitrogen protection, 10% palladium on carbon (100 mg) was added to a solution of 3-methoxy-4-nitrophenol (1 g, 5.9 mmol) in methanol (10 mL). The resulting mixture was evacuated and backfilled with hydrogen via a connected hydrogen balloon. After stirring overnight at room temperature, the mixture was filtered through a diatomaceous earth pad, and the filtrate was concentrated to obtain a residue, which was dissolved in DCM (20 mL). TEA (1.65 mL, 11.8 mmol) and BoC2O (1.29 g, 5.9 mmol) were then added, and the mixture was stirred overnight at room temperature. DCM (30 mL) was added, and the resulting mixture was washed with 1 N HCl aqueous solution, water, and brine, respectively, then dried over sodium sulfate, filtered, and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give tert-butyl (4-hydroxy-2-methoxyphenyl)carbamate (14% yield for 200 mg in both steps).

[0804]

[0805]

[0806] (4-(((1r,4r)-4-(bromomethyl)cyclohexyl)methoxy)-2-methoxyphenyl)tert-butyl carbamate

[0807] Add tert-butyl (4-hydroxy-2-methoxyphenyl)carbamate (20 mg, 0.084 mmol), trans-1,4-bis(bromomethyl)cyclohexane (68 mg, 0.25 mmol), DMF (1 ml), and Cs₂CO₃ (82 mg, 0.25 mmol) to a microwave-safe tube. After heating in the microwave at 60 °C for 30 minutes, the mixture was cooled and water was added. The resulting mixture was then extracted with ethyl acetate (3×), the combined organic phases were washed with water and brine, dried over sodium sulfate, filtered, and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel to give tert-butyl (4-(((1r,4r)-4-(bromomethyl)cyclohexyl)methoxy)-2-methoxyphenyl)carbamate (15 mg, 42% yield).

[0808]

[0809]

[0810] (4-(((1S,4R)-4-((((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methoxy)-2-methoxyphenyl)tert-butyl carbamate was prepared according to general scheme 8. 25 mg of tert-butyl carbamate (4-(((1S,4R)-4-((((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methoxy)-2-methoxyphenyl)carbamate was obtained from 18 mg of thiol compound in 81% yield.

[0811]

[0812]

[0813] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (30, XL01168)

[0814] (25 mg, 0.028 mmol) of tert-butyl carbamate (4-(((1S,4R)-4-((((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methoxy)-2-methoxyphenyl)carbamate (1 mL)) was dissolved in DCM (1 mL), followed by the addition of HCl (4N HCl in 1,4-dioxane, 1 mL). After stirring overnight at room temperature, the mixture was concentrated under reduced pressure to obtain a solid, which was used directly in the next step without further purification. The solid from the previous step was dissolved in isopropanol (1 mL), followed by the addition of 2,5-dichloro-N-methylpyrimidin-4-amine (4.8 mg, 0.027 mmol) and HCl (4N in 1,4-dioxane, 0.006 mL). The resulting mixture was heated in a microwave at 100 °C for 9 hours, cooled, diluted with water, and extracted with DCM (3×). The combined organic phases were washed with water and brine, dried over sodium sulfate, filtered, and concentrated to obtain a residue, which was purified by preparative HPLC (5% to 95% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to give XL01168 (26% yield for 10 mg of both steps).

[0815]

[0816] Exemplary synthesis of exemplary compound 31 (XL02017)

[0817]

[0818] 4-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenoxy)piperidine-1-carboxylic acid benzyl ester

[0819] 1,1'-(azodicarbonyl)dipiperidine (633 mg, 2.5 mmol) was added 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). 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, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel (0% to 50% ethyl acetate in heptane) to give 4-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenoxy)piperidine-1-carboxylate (450 mg, 79% yield).

[0820]

[0821]

[0822] (2-Methoxy-4-(piperidin-4-yloxy)phenyl)tert-butyl carbamate

[0823] Under nitrogen protection, 10% palladium on carbon (50 mg) was added to a solution of 4-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenoxy)piperidine-1-carboxylic acid benzyl ester (450 mg, 0.99 mmol) in methanol (15 ml). The resulting mixture was stirred overnight at room temperature under positive hydrogen pressure generated by a hydrogen balloon, then filtered through a diatomaceous earth pad, and the filtrate was concentrated and purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM containing 0.7 M ammonia) to give tert-butyl (2-methoxy-4-(piperidine-4-yloxy)phenyl)carbamate (210 mg, 66%).

[0824]

[0825]

[0826] (4-((1-(((1R,4R)-4-(bromomethyl)cyclohexyl)methyl)piperidin-4-yl)oxy)-2-methoxyphenyl)tert-butyl carbamate

[0827] K₂CO₃ (126 mg, 0.91 mmol) was added to a solution of (2-methoxy-4-(piperidin-4-yloxy)phenyl)carbamate (56 mg, 0.17 mmol) and trans-1,4-bis(bromomethyl)cyclohexane (147 mg, 0.54 mmol) in acetone (3 mL). After stirring overnight at 60 °C, the reaction mixture was cooled, diluted with water, and extracted with ethyl acetate (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 rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give (4-((1-(((1R,4R)-4-(bromomethyl)cyclohexyl)methyl)piperidin-4-yl)oxy)-2-methoxyphenyl)carbamate (41 mg, 45% yield).

[0828]

[0829]

[0830] (4-((1-(((1S,4r)-4-((((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidone-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methyl)piperidin-4-yl)oxy)-2-methoxyphenyl)tert-butyl carbamate)

[0831] (4-((1-(((1S,4r)-4-((((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methyl)piperidin-4-yl)oxy)-2-methoxyphenyl)tert-butyl carbamate was prepared according to general scheme 8. 27 mg of tert-butyl carbamate (4-((1-(((1S,4r)-4-((((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methyl)piperidin-4-yl)oxy)-2-methoxyphenyl)carbamate was obtained from 21 mg of the thiol compound in 72% yield.

[0832]

[0833]

[0834] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (31, XL02017)

[0835] Add HCl (4N in 1,4-dioxane, 1 mL) to a solution of (4-((1-(((1S,4R)-4-((((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methyl)piperidin-4-yl)oxy)-2-methoxyphenyl)tert-butyl carbamate (27 mg, 0.028 mmol) in DCM (1 mL). After stirring overnight at room temperature, concentrate the mixture and use it for the next step without further purification. The concentrated residue from the previous step (18.6 mg, 0.02 mmol) was dissolved in isopropanol (1 mL) in a microwave tube, followed by the addition of 2,5-dichloro-N-methylpyrimidin-4-amine (3.7 mg, 0.02 mmol) and HCl (1,4-dioxane, 4N HCl, one drop). After heating in a microwave reactor at 100 °C for 8 hours, the reaction mixture was concentrated and purified by preparative HPLC (5% to 95% CH3CN in 0.1% HCO2H aqueous solution) under acidic conditions to give XL02017 (2.8 mg, 13% yield in two steps).

[0836]

[0837] Exemplary synthesis of exemplary compound 32 (XL02031)

[0838]

[0839] (3S,5R)-4-(((1R,4R)-4-(bromomethyl)cyclohexyl)methyl)-3,5-dimethylpiperazine-1-carboxylic acid tert-butyl ester

[0840] N,N-diisopropylethylamine (0.083 mL, 0.48 mmol) was added to a solution of (3S,5R)-3,5-dimethylpiperazine-1-carboxylic acid tert-butyl ester (34 mg, 0.16 mmol) and trans-1,4-bis(bromomethyl)cyclohexane (130 mg, 0.48 mmol) in isopropanol (1 mL). After stirring in a microwave reactor at 120 °C for 6 h, the mixture was cooled and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel (0% to 100% ethyl acetate in heptane) to give (3S,5R)-4-(((1R,4R)-4-(bromomethyl)cyclohexyl)methyl)-3,5-dimethylpiperazine-1-carboxylic acid tert-butyl ester (11 mg, 17% yield).

[0841]

[0842]

[0843] (3R,5S)-4-(((1S,4R)-4-((((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methyl)-3,5-dimethylpiperazine-1-carboxylic acid tert-butyl ester was prepared according to general scheme 8. 15 mg of (3R,5S)-4-(((1S,4R)-4-((((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methyl)-3,5-dimethylpiperazine-1-carboxylic acid tert-butyl ester was obtained from 15 mg of the thiol compound in 62.5% yield.

[0844]

[0845]

[0846] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (32, XL02031)

[0847] Add HCl (1,4-dioxane-1-amido) to a solution of (3R,5S)-4-(((1S,4R)-4-((((S)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methyl)-3,5-dimethylpiperazine-1-carboxylic acid tert-butyl ester (15 mg, 0.018 mmol) in DCM (1 mL). After stirring overnight at room temperature, concentrate the mixture and use it for the next step without further purification. The residue from the previous step was dissolved in DMF (1 mL), followed by the addition of 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (6 mg, 0.019 mmol), N,N-diisopropylethylamine (0.012 mL, 0.070 mmol), and Pyoxim (11 mg, 0.02 mmol). The resulting mixture was stirred overnight at room temperature, filtered through a 0.45 μM filter, and injected into a preparative HPLC (5% to 95% CH3CN in 0.1% HCO2H aqueous solution) for purification under acidic conditions to give XL02031 (11% yield for 2 mg of both steps).

[0848]

[0849] Exemplary synthesis of exemplary compound 33 (XL02038)

[0850]

[0851] 9-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester

[0852] Add Pyoxim (171 mg, 0.32 mmol) to a solution of 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (100 mg, 0.32 mmol), 3,9-diazaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester (83 mg, 0.33 mmol) and N,N-diisopropylethylamine (0.113 mL, 0.65 mmol) in DMF (5 mL). After stirring at room temperature for 2 hours, the reaction mixture was diluted with ethyl acetate, washed with water and brine, dried with sodium sulfate, filtered and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give 9-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester (80 mg, 45% yield).

[0853]

[0854]

[0855] (9-(((1r,4r)-4-(bromomethyl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undec-3-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[0856] HCl (1,4-dioxane-4N HCl, 1 mL) was added to a solution of 9-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester (70 mg, 0.128 mmol) in DCM (1 mL). After stirring overnight at room temperature, the mixture was concentrated and used for the next step. The concentrated residue was dissolved in acetone, followed by the addition of trans-1,4-bis(bromomethyl)cyclohexane (44 mg, 0.163 mmol) and K2CO3 (37 mg, 0.27 mmol). The resulting mixture was stirred overnight at 50 °C and then cooled to room temperature. Water was added, and the mixture was extracted with ethyl acetate (3×). The combined organic phases were washed with water and brine, dried with sodium sulfate, filtered and concentrated to give a crude product as a residue. The crude product was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give (9-(((1r,4r)-4-(bromomethyl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undec-3-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone (12 mg, 35% yield).

[0857]

[0858]

[0859] (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]undecyl-3-yl)methyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide(33, XL02038)

[0860] XL02038 was prepared according to general scheme 8. 6.7 mg of XL02038 was obtained from 10 mg of the thiol compound, in a 33% yield.

[0861]

[0862] Exemplary synthesis of exemplary compound 34 (XL02047)

[0863]

[0864] (4-Bromo-2-fluorobenzyl) tert-butyl carbamate was prepared according to general scheme 1. 6.1 g of (4-bromo-2-fluorobenzyl) tert-butyl carbamate was obtained from 5 g of the amine compound, in 83% yield.

[0865]

[0866]

[0867] (2-fluoro-4-(4-methylthiazol-5-yl)benzyl)tert-butyl carbamate was prepared according to general scheme 2. 3.4 g of (2-fluoro-4-(4-methylthiazol-5-yl)benzyl)tert-butyl carbamate was obtained from 4 g of (4-bromo-2-fluorobenzyl)carbamate, in 80% yield.

[0868]

[0869]

[0870] (2S,4R)-2-((2-fluoro-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester was prepared according to general scheme 3. 1.8 g of (2S,4R)-2-((2-fluoro-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester was obtained from 1.48 g of (2-fluoro-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)carbamoyl ester, in 90% yield.

[0871]

[0872]

[0873] (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-2-((2-fluoro-4-(4-methylthiazo-5-yl)benzyl)carbamoyl)-4-hydroxypyrrolidone-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate was prepared according to general scheme 4. 170 mg of (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-2-((2-fluoro-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester was obtained from 119 mg of (2S,4R)-2-((2-fluoro-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)-4-hydroxypyrrolidine-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate, in 67% yield.

[0874]

[0875]

[0876] (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-N-(2-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide was prepared according to General Scheme 5. 105 mg of (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-N-(2-fluoro-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide was obtained from 150 mg of the compound protected by Fmoc, in 92% yield.

[0877]

[0878]

[0879] (2S,4R)-N-(2-fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide was prepared according to General Scheme 6. 71 mg of (2S,4R)-N-(2-fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide was obtained from 105 mg of the amine compound in 60% yield.

[0880]

[0881]

[0882] (2S,4R)-N-(2-fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide was prepared according to General Scheme 7. 39 mg of (2S,4R)-N-(2-fluoro-4-(4-methylthiazol-5-yl)benzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide was obtained from 71 mg of the triphenylmethyl-protected compound in 80% yield.

[0883]

[0884]

[0885] (2S,4R)-1-( -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-amido)-3-methylbutyryl)-N-(2-fluoro-4-(4-methylthiazolyl-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (34, XL02047)

[0886] XL01047 was prepared according to general scheme 8. 12 mg of XL02047 was obtained from 10 mg of the thiol compound, in 65% yield.

[0887]

[0888] Exemplary synthesis of exemplary compound 35 (XL02048)

[0889]

[0890] (4-Bromo-2-methylbenzyl) tert-butyl carbamate was prepared according to general scheme 1. 5.7 g of (4-bromo-2-methylbenzyl) tert-butyl carbamate was obtained from 4 g of the amine compound, in 95% yield.

[0891]

[0892]

[0893] (2-Methyl-4-(4-methylthiazol-5-yl)benzyl)tert-butyl carbamate was prepared according to general scheme 2. 2.35 g of (2-methyl-4-(4-methylthiazol-5-yl)benzyl)tert-butyl carbamate was obtained from 3 g of the bromide compound, in 70% yield.

[0894]

[0895]

[0896] (2S,4R)-4-hydroxy-2-((2-methyl-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared according to general scheme 3. 1.2 g of (2S,4R)-4-hydroxy-2-((2-methyl-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was obtained from 1.18 g of (2-methyl-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)carbamoyl ester, in 75% yield.

[0897]

[0898]

[0899] (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-4-hydroxy-2-((2-methyl-4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate was prepared according to general scheme 4. 310 mg of (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-4-hydroxy-2-((2-methyl-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was obtained from 176 mg of (2S,4R)-4-hydroxy-2-((2-methyl-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate, in 82% yield.

[0900]

[0901]

[0902] (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide was prepared according to General Scheme 5. 135 mg of (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide was obtained from 250 mg of the compound protected by Fmoc, in 71% yield.

[0903]

[0904]

[0905] (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazo-5-yl)benzyl)pyrrolidine-2-carboxamide was prepared according to General Scheme 6. 79.5 mg of (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazo-5-yl)benzyl)pyrrolidine-2-carboxamide was obtained from 102 mg of the amine compound in 70% yield.

[0906]

[0907]

[0908] (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide was prepared according to General Scheme 7. 40 mg of (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide was obtained from 79.5 mg of the triphenylmethyl-protected compound in 73% yield.

[0909]

[0910]

[0911] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (35, XL02048)

[0912] XL02048 was prepared according to general scheme 8. 12.6 mg of XL02048 was obtained from 10 mg of the thiol compound, in 69% yield.

[0913]

[0914] Exemplary synthesis of exemplary compound 36 (XL02049)

[0915]

[0916] (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-amido)-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (36, XL02049)

[0917] XL02049 was prepared according to general scheme 8. 8.95 mg of XL02049 was obtained from 10 mg of the thiol compound, in a 49% yield.

[0918]

[0919] Exemplary synthesis of exemplary compound 37 (XL02058)

[0920]

[0921] (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-amido)-3-methylbutyryl)-N-(2-fluoro-4-(4-methylthiazolyl-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (37, XL02058)

[0922] XL02058 was prepared according to general scheme 8. 16.99 mg of XL02058 was obtained from 10 mg of the thiol compound, in 71% yield.

[0923]

[0924] Exemplary synthesis of exemplary compound 38 (XL02059)

[0925]

[0926] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(2-methyl-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (38, XL02059)

[0927] XL02059 was prepared according to general scheme 8. 14.29 mg of XL02059 was obtained from 10 mg of the thiol compound, in 59% yield.

[0928]

[0929] Exemplary synthesis of exemplary compound 39 (XL02060)

[0930]

[0931] (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-amido)-3-methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (39, XL02060)

[0932] XL02060 was prepared according to general scheme 8. 17.22 mg of XL02060 was obtained from 10 mg of the thiol compound, in 71% yield.

[0933]

[0934] Exemplary synthesis of exemplary compound 40 (XL02063)

[0935]

[0936] (cis-cyclohexane-1,3-diyl)diethanol

[0937] Me2S-BH3 (3.2 mL, 6.38 mmol) was added dropwise to a solution of cis-cyclohexane-1,3-dicarboxylic acid (500 mg, 2.9 mmol) in THF (10 mL) at 0 °C. After stirring at 0 °C for 2 to 3 hours, the reaction was slowly quenched with methanol and then water was added. The mixture was extracted with DCM (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 rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give (cis-cyclohexane-1,3-diyl)diethanol (250 mg, 60% yield).

[0938]

[0939]

[0940] cis-1,3-bis(bromomethyl)cyclohexane

[0941] CBr4 (690 mg, 2.08 mmol) was dissolved in DCM (5 mL) and added dropwise to a solution of (cis-cyclohexane-1,3-diyl)diethanol (100 mg, 0.69 mmol) and triphenylphosphine (636 mg, 2.43 mmol) in DCM (6 mL) (at 0 °C). The resulting mixture was slowly warmed to room temperature and stirred overnight at room temperature. Water was added and the mixture was extracted with DCM (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 rapid column chromatography on silica gel (0% to 50% ethyl acetate in heptane) to give cis-1,3-bis(bromomethyl)cyclohexane (140 mg, 75% yield).

[0942]

[0943]

[0944] (4-(((1S,3R)-3-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[0945] K₂CO₃ (42 mg, 0.3 mmol) was added to a solution of (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl) methyl ketone (25 mg, 0.06 mmol) and cis-1,3-bis(bromomethyl)cyclohexane (50 mg, 0.185 mmol) in acetone. After stirring overnight at 50 °C, the mixture was cooled to room temperature and diluted with water. The mixture was then extracted with DCM (3×), and the combined organic phases were washed with water and brine, dried with sodium sulfate, filtered and concentrated to give a residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give (4-(((1S,3R)-3-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone (6 mg, 17% yield).

[0946]

[0947]

[0948] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (40, XL02063)

[0949] XL02063 was prepared according to general scheme 8. 5.56 mg of XL02063 was obtained from 5.5 mg of the thiol compound, in 53% yield.

[0950]

[0951] Exemplary synthesis of exemplary compound 41 (XL02067B)

[0952]

[0953] (2S,4R)-1-((R)-3-((3-(9-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)-3,9-diazaspiro[5.5]undecyl-3-yl)propyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (41, XL02067B)

[0954] HCl (4N in 1,4-dioxane, 1 mL) was added to a solution of 9-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester (200 mg, 0.367 mmol) in DCM (1 mL). The mixture was stirred overnight at room temperature and then concentrated to give a white solid (180 mg, 91% yield). The solid (60 mg, 0.125 mmol) was suspended in 2 mL of acetone, followed by the addition of 1,3-dibromopropane (75.5 mg, 0.374 mmol) and K₂CO₃ (86 mg, 0.623 mmol). After stirring overnight at room temperature, water was added to the mixture, and the mixture was extracted with DCM (3×). The combined organic phases were washed with water and brine, dried with sodium sulfate, filtered, and concentrated to obtain a residue. The residue was purified by rapid chromatography on silica gel (0% to 10% methanol in DCM) to give a bromide compound as an impure product, which can be used for the next step without further purification. (9-(3-bromopropyl)-3,9-diazaspiro[5.5]undecyl-3-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone (10 mg, 0.018 mmol) and (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (10 mg, 0.019 mmol) were dissolved in THF (1.5), followed by the addition of DBU (16 μL, 0.112 mmol). After stirring overnight at room temperature, the mixture was concentrated and purified by rapid column chromatography on silica gel (0 to 10% methanol in DCM) to give XL02067B as a white solid (13.6 mg, 71% yield).

[0955]

[0956] Exemplary synthesis of exemplary compound 42 (XL02068)

[0957]

[0958] 4-(trans-4-(methoxycarbonyl)cyclohexane-1-carbonyl)piperazine-1-carboxylic acid tert-butyl ester

[0959] HATU (2.145 g, 5.65 mmol) was added to a solution of 1-Boc-piperazine (1 g, 5.38 mmol), trans-4-(methoxycarbonyl)cyclohexane-1-carboxylic acid (1 g, 5.38 mmol), and TEA (1.5 mL, 10.8 mmol) in DCM (25 mL). After stirring overnight at room temperature, the mixture was diluted with DCM (25 mL), washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel (0% to 100% ethyl acetate in heptane) to give tert-butyl 4-(trans-4-(methoxycarbonyl)cyclohexane-1-carbonyl)piperazine-1-carboxylic acid (1.2 g, 63% yield).

[0960]

[0961]

[0962] 4-(trans-4-(hydroxymethyl)cyclohexane-1-carbonyl)piperazine-1-carboxylic acid tert-butyl ester

[0963] At 0 °C, LiBH4 (0.846 mL, 1.69 mmol) was added dropwise to a solution of 200 mg (0.564 mmol) of 4-(trans-4-(methoxycarbonyl)cyclohexane-1-carbonyl)piperazine-1-carboxylic acid tert-butyl ester (5 mL) in THF. The reaction mixture was stirred at 0 °C for 2 to 3 hours, then quenched with methanol and 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, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give 4-(trans-4-(hydroxymethyl)cyclohexane-1-carbonyl)piperazine-1-carboxylic acid tert-butyl ester (180 mg, 98% yield).

[0964]

[0965]

[0966] 4-((1R,4R)-4-(bromomethyl)cyclohexane-1-carbonyl)piperazine-1-carboxylic acid tert-butyl ester

[0967] CBr4 (152 mg, 0.46 mmol) was dissolved in DCM (5 mL) and added dropwise to a solution of 4-(trans-4-(hydroxymethyl)cyclohexane-1-carbonyl)piperazine-1-carboxylic acid tert-butyl ester (100 mg, 0.306 mmol) and triphenylphosphine (161 mg, 0.61 mmol) in DCM (6 mL) (at 0 °C). After stirring at 0 °C for 2 to 3 hours, the reaction was quenched with water, extracted with DCM (3×), and the combined organic phases were washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a crude product. The crude product was purified by rapid column chromatography on silica gel (0% to 100% ethyl acetate in heptane) to give 4-((1R,4R)-4-(bromomethyl)cyclohexane-1-carbonyl)piperazine-1-carboxylic acid tert-butyl ester (93 mg, 78% yield).

[0968]

[0969]

[0970] 4-((1R,4R)-4-((((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexane-1-carbonyl)piperazine-1-carboxylic acid tert-butyl ester was prepared according to general scheme 8. 22 mg of 4-((1R,4R)-4-((((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexane-1-carbonyl)piperazine-1-carboxylic acid tert-butyl ester was obtained from 15 mg of the thiol compound in 100% yield.

[0971]

[0972]

[0973]

[0974] (2S,4R)-1-((R)-3-((((1R,4R)-4-(4-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazine-1-carbonyl)cyclohexyl)methyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (42, XL02068)

[0975] Add HCl (4N HCl in 1,4-dioxane, 0.5 mL) to a solution of 4-((1R,4R)-4-((((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexane-1-carbonyl)piperazine-1-carboxylic acid tert-butyl ester (22 mg, 0.026 mmol) in DCM (0.5 mL). After stirring overnight at room temperature, concentrate the mixture and wash with diethyl ether to obtain a residue, which is used for the next step. The residue was dissolved in DMF (2 mL), followed by the addition of 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (9 mg, 0.029 mmol), N,N-diisopropaneethylamine (0.021 mL, 0.117 mmol), and Pyoxim (17.2 mg, 0.033 mmol). The resulting mixture was stirred at room temperature for 2 hours. Water was added, and the mixture was extracted with DCM (3×). The combined organic phases were washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a crude product, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give XL02068 (18.36 mg, 60% yield).

[0976]

[0977] Exemplary synthesis of exemplary compound 43 (XL02093)

[0978]

[0979] (cis-cyclopentane-1,3-diyl)diethanol

[0980] Mes2S-BH3 (2M in THF, 3.48 mL, 6.96 mmol) was added to a solution of (1R,3S)-cyclopentane-1,3-dicarboxylic acid (500 mg, 3.125 mmol) in 10 mL of THF at 0 °C. After stirring at 0 °C for 2 to 3 hours, the reaction was quenched with methanol and concentrated under reduced pressure to give a crude product, which was purified by rapid column chromatography on silica gel to give (cis-cyclopentane-1,3-diyl)diethanol (400 mg, 97% yield).

[0981]

[0982]

[0983] cis-1,3-bis(bromomethyl)cyclopentane

[0984] CBr4 (955 mg, 2.88 mmol) was dissolved in DCM (5 mL) and added to a solution of (cis-cyclopentane-1,3-diyl)diethanol (150 mg, 1.15 mmol) and triphenylphosphine (906 mg, 3.46 mmol) in DCM (10 mL) (at -15 °C). The mixture was slowly warmed to room temperature and stirred overnight at room temperature. Water was added and the mixture was extracted with DCM. The organic phase was then washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid chromatography on silica gel (0 to 20% ethyl acetate in heptane) to give cis-1,3-bis(bromomethyl)cyclopentane (51 mg, 17% yield).

[0985]

[0986]

[0987] (4-(((1S,3R)-3-(bromomethyl)cyclopentyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[0988] A solution of (30 mg, 0.067 mmol) methyl ketone HCl salt in acetone (1 mL) was treated with cis-1,3-bis(bromomethyl)cyclopentane (56 mg, 0.217 mmol) and K₂CO₃ (60 mg, 0.434 mmol). After stirring at 50 °C for two days, the mixture was cooled, diluted with water, and extracted with DCM (3×). The combined organic phases were washed with water and brine, dried with sodium sulfate, filtered and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel (0% to 10% methanol in DCM) to give (4-(((1S,3R)-3-(bromomethyl)cyclopentyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone (8 mg, 20% yield).

[0989]

[0990]

[0991] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (43, XL02093)

[0992] XL02093 was prepared according to general scheme 8. 10.6 mg of XL02093 was obtained from 10 mg of the thiol compound, in 58% yield.

[0993]

[0994] Exemplary synthesis of exemplary compound 44 (cis-XL01126)

[0995]

[0996] (2S,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared according to general scheme 3. 560 mg of (2S,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was obtained from 500 mg of the amine compound, in 65% yield.

[0997]

[0998]

[0999] (2S,4S)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(4-(4-methylthiazo-5-yl)benzyl)pyrrolidine-2-carboxamide

[1000] A solution of (2S,4S)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester (568 mg, 1.36 mmol) in DCM (6.8 mL) was added with 4N HCl (6.8 mL). The resulting mixture was stirred overnight at room temperature and concentrated to give a solid (530 mg, 100% yield). A mixture of Fmoc-S-triphenylmethyl-L-penicillamine (329 mg, 0.54 mmol), HATU (215 mg, 0.57 mmol), and TEA (79 μL) in DMF (5 mL) was added dropwise to a solution of the resulting solid (200 mg, 0.57 mmol) and TEA (236 μL). After stirring overnight at room temperature, the mixture was diluted with DCM, water was added, and the organic layer was collected. The organic layer was then washed with water and brine, dried with sodium sulfate, filtered, and concentrated to obtain a residue, which was purified by rapid column chromatography to give the residue as an amine compound: (2S,4S)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (120 mg, 32% yield, LC-MS, ESI). - 689.4 [MH] - The amine compound (60 mg, 0.087 mmol) was added to a solution of DMF (1.5 mL) with TEA (24 μL, 0.174 mmol), HATU (35 mg, 0.092 mmol), and 1-fluorocyclopropane-1-carboxylic acid (9 mg, 0.087 mmol). After stirring at room temperature for 4 hours, the resulting mixture was diluted with ethyl acetate, washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel to give (2S,4S)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(4-(4-methylthiazo-5-yl)benzyl)pyrrolidine-2-carboxamide (57 mg, 85% yield).

[1001]

[1002]

[1003] (2S,4S)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide was prepared according to General Scheme 7. 36 mg of (2S,4S)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide was obtained from 57 mg of the triphenylmethyl-protected compound in 92% yield.

[1004]

[1005]

[1006]

[1007] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide(44,cis-XL01126)

[1008] Compound 44 (cis-XL01126) was prepared according to general scheme 8. 11.92 mg of 44 (cis-XL01126) was obtained from 10 mg of the thiol compound, in 65% yield.

[1009]

[1010] Exemplary synthesis of exemplary compound 45 (XL02093)

[1011]

[1012] (1S,3S)-1,3-bis(bromomethyl)cyclopentane

[1013] At -15 °C, CBr4 (955 mg, 2.88 mmol) and PPh3 (906 mg, 3.46 mmol) were added to a solution of ((1S,3S)-cyclopentane-1,3-diyl)diethanol (150 mg, 1.15 mmol) in DCM (3 mL). The resulting mixture was slowly heated to room temperature and stirred overnight at room temperature. The mixture was concentrated and purified by rapid chromatography on silica gel to obtain the dibromide compound (35 mg, 12% yield).

[1014]

[1015] (4-(((1S,3S)-3-(bromomethyl)cyclopentyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[1016] Cs₂CO₃ (138 mg, 0.42 mmol) was added to a solution of (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl) methyl ketone hydrochloride (35 mg, 0.085 mmol) and (1S,3S)-1,3-bis(bromomethyl)cyclopentane (60 mg, 0.23 mmol) in 1.5 mL of acetone. The mixture was stirred overnight at 50 °C, then cooled, filtered, and concentrated to obtain a residue, which was purified by rapid chromatography on silica gel to give (4-(((1S,3S)-3-(bromomethyl)cyclopentyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone (10 mg, 21% yield).

[1017]

[1018]

[1019] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (45, XL02093)

[1020] To a solution of (1S,3S)-3-(bromomethyl)cyclopentyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone (10 mg, 0.018 mmol) in THF (1.5 mL), (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (10 mg, 0.019 mmol) and DBU (26 μL, 0.18 mmol) were added. The resulting mixture was stirred overnight at room temperature, then concentrated and purified by rapid chromatography on silica gel to give 45(XL02093) (10.6 mg, 58% yield).

[1021]

[1022]

[1023] Exemplary synthesis of exemplary compound 46 (XL02098)

[1024]

[1025] (2R,5S)-2,5-bis(bromomethyl)-1,4-di alkyl

[1026] At -15℃, towards ((2S,5R)-1,4-di The solution of alkyl-2,5-diyl)diethanol (45 mg, 0.3 mmol) in DCM (3 mL) was supplemented with CBr4 (252 mg, 0.76 mmol) and PPh3 (239 mg, 0.91 mmol). The resulting mixture was slowly heated to room temperature and stirred overnight. The reaction was then quenched with water, diluted with DCM, washed with water and brine, dried over sodium sulfate, and concentrated to give a residue, which was purified by rapid chromatography on silica gel to give (2R,5S)-2,5-bis(bromomethyl)-1,4-diol. Alkane (44 mg, 53% yield).

[1027]

[1028] (4-(((2S,5S)-5-(bromomethyl)-1,4-di) (Alkyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[1029] (2R,5S)-2,5-bis(bromomethyl)-1,4-di A solution of alkyl (44 mg, 0.16 mmol) and (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methyl ketone hydrochloride (66 mg, 0.16 mmol) in acetone (4 mL) was supplemented with K₂CO₃ (110.8 mg, 0.8 mmol). The resulting mixture was heated to 50 °C and stirred for 2 to 3 days, then diluted with DCM and washed with water and brine. The organic phase was collected, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid chromatography on silica gel to give (4-(((2S,5S)-5-(bromomethyl)-1,4-di) Alk-2-yl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone (13 mg, 14% yield).

[1030]

[1031]

[1032] (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-di Alkyl)methyl)thio)-2-(1-fluorocyclopropane-1-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (46, XL02098)

[1033] To (4-(((2S,5S)-5-(bromomethyl)-1,4-di) A solution of (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (10 mg, 0.019 mmol) and DBU (26 μL, 0.18 mmol) was added to THF (1.5 mL). The resulting mixture was stirred overnight at room temperature, then concentrated and purified by rapid chromatography on silica gel to give compound 46, (XL02098) (6.1 mg, 26.5% yield).

[1034]

[1035] Exemplary synthesis of exemplary compound 47 (XL02133)

[1036]

[1037] ((1R,3R)-cyclobutane-1,3-diyl)diethanol

[1038] Me2S-THF (2M, 1.6ml, 3.2mmol) was added to a solution of (1R,3R)-cyclobutane-1,3-dicarboxylic acid (150 mg, 1.04 mmol) in THF (3.2 ml) at 0 °C. The resulting mixture was slowly warmed to room temperature and then quenched with methanol. The mixture was then concentrated and purified by rapid chromatography on silica gel to give ((1R,3R)-cyclobutane-1,3-diyl)diethanol (110 mg, 92%).

[1039] ((1R,3R)-cyclobutane-1,3-diyl)bis(methylene)bis(4-methylbenzenesulfonate)

[1040] At 0 °C, triethylamine (0.396 μL, 2.84 mmol) and TsCl (453 mg, 2.37 mmol) were added to a solution of ((1R,3R)-cyclobutane-1,3-diyl)diethanol (110 mg, 0.95 mmol) in DCM (5 mL). The resulting mixture was stirred overnight at room temperature and then diluted with DCM. The mixture was then washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel to give ((1R,3R)-cyclobutane-1,3-diyl)bis(methylene)bis(4-methylbenzenesulfonate) (330 mg, 82% yield).

[1041]

[1042] (1R,3R)-1,3-bis(bromomethyl)cyclobutene

[1043] LiBr (1.05 g, 3.88 mmol) was added to a solution of ((1R,3R)-cyclobutane-1,3-diyl)bis(methylene)bis(4-methylbenzenesulfonate) (330 mg, 0.78 mmol) in acetone (5 mL). The resulting mixture was heated to 60 °C and stirred overnight at this temperature. The mixture was then diluted with ethyl acetate, washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid chromatography on silica gel to give (1R,3R)-1,3-bis(bromomethyl)cyclobutene (95 mg, 51% yield).

[1044]

[1045] (4-(((1R,3R)-3-(bromomethyl)cyclobutyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone

[1046] K₂CO₃ (67.5 mg, 0.49 mmol) was added to a solution of (1R,3R)-1,3-bis(bromomethyl)cyclobutene (47.5 mg, 0.20 mmol) and (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methyl ketone hydrochloride (40.5 mg, 0.098 mmol) in acetone (4 ml). The resulting mixture was heated to 50 °C and stirred overnight, then diluted with DCM and washed with water and brine. The organic phase was collected, dried with sodium sulfate, filtered, and concentrated to obtain a residue, which was purified by rapid chromatography on silica gel to give (4-(((1R,3R)-3-(bromomethyl)cyclobutyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) ketone (13 mg, 38.5% yield).

[1047]

[1048]

[1049] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (47, XL02133)

[1050] To a solution of (1R,3R)-3-(bromomethyl)cyclobutyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)methyl ketone (10 mg, 0.019 mmol) in THF (1.5 mL), (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (10 mg, 0.019 mmol) and DBU (26.2 μL, 0.18 mmol) were added. The resulting mixture was stirred overnight at room temperature, then concentrated and purified by rapid chromatography on silica gel to give 47(XL02133) (11 mg, 60% yield).

[1051]

[1052] Exemplary synthesis of exemplary compound 48 (XL02134)

[1053]

[1054] (R)-2-((((1R,4R)-4-(bromomethyl)cyclohexyl)methoxy)methyl)morpholine-4-carboxylic acid tert-butyl ester

[1055] At 0 °C, (R)-2-(hydroxymethyl)morpholine-4-carboxylic acid tert-butyl ester (135 mg, 0.622 mmol) and (1R,4R)-1,4-bis(bromomethyl)cyclohexane (500 mg, 1.87 mmol) were added to a suspension of NaH (50 mg, 1.25 mmol) in THF (6 mL). The resulting mixture was stirred overnight at 60 °C, then cooled to 0 °C and quenched with an aqueous solution of NH4Cl. The mixture was diluted with ethyl acetate, washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel to give the title compound (83 mg, 33% yield).

[1056]

[1057] (R)-2-((((1R,4R)-4-((((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolid-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methoxy)methyl)morpholine-4-carboxylic acid tert-butyl ester

[1058] Add DBU (15 μL, 0.1 mmol) to a solution of (R)-2-((((1R,4R)-4-(bromomethyl)cyclohexyl)methoxy)methyl)morpholine-4-carboxylic acid tert-butyl ester (7 mg, 0.017 mmol) and (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (9 mg, 0.017 mmol) in THF (1.5 mL). The mixture was stirred overnight at room temperature and then concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel to give (R)-2-((((1R,4R)-4-((((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methoxy)methyl)morpholine-4-carboxylic acid tert-butyl ester (10 mg, 0.012 mmol).

[1059]

[1060]

[1061] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (48, XL02134)

[1062] Add 0.6 ml of HCl to a solution of (R)-2-((((1R,4R)-4-((((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)methyl)cyclohexyl)methoxy)methyl)morpholine-4-carboxylic acid tert-butyl ester (10 mg, 0.012 mmol) in DCM (0.6 ml). The mixture was stirred at room temperature for 1 hour, then concentrated to obtain a solid. This solid was dissolved in DMF (1 ml), and DIPEA (6 μL, 0.034 mmol), 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (3.6 mg, 0.011 mmol), and pyoxim (6 mg, 0.011 mmol) were added (at 0 °C). After stirring at 0 °C for 2 hours, the mixture was diluted with ethyl acetate, washed with water and brine, dried over sodium sulfate, filtered, and concentrated to obtain a crude product. This crude product was purified by rapid column chromatography on silica gel to give 48 (XL02134) (5.7 mg, 47% yield).

[1063]

[1064] Exemplary synthesis of exemplary compound 49 (XL02127)

[1065]

[1066] 4-(2-(toluenesulfonyloxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester

[1067] At 0 °C, TEA (572 μl, 4.1 mmol) and TsCl (432 mg, 2.26 mmol) were added to a solution of 4-(2-hydroxyethyl)piperidine-1-carboxylic acid tert-butyl ester (471 mg, 2.05 mmol) in DCM (10 mL). The resulting mixture was stirred overnight at room temperature and then diluted with DCM. The mixture was then washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel to give 4-(2-(toluenesulfonyloxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester (700 mg, 89% yield).

[1068]

[1069] 4-(2-Iodoethyl)piperidine-1-carboxylic acid tert-butyl ester

[1070] KI (260 mg, 1.57 mmol) was added to a solution of 4-(2-(toluenesulfonyloxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester (285 mg, 0.744 mmol) in acetone (7 mL). The resulting mixture was stirred overnight at 50 °C, then cooled to room temperature and diluted with ethyl acetate. The mixture was then washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel to give 4-(2-iodoethyl)piperidine-1-carboxylic acid tert-butyl ester (177 mg, 70% yield).

[1071]

[1072] 4-(2-(2-hydroxyethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester

[1073] At 0 °C, ethylene glycol (82 μL, 1.5 mmol) and tert-butyl 4-(2-iodoethyl)piperidine-1-carboxylic acid (100 mg, 0.29 mmol) were added to a suspension of NaH (24 mg, 0.6 mmol) in THF (5 mL). The resulting mixture was heated to 50 °C and stirred at 50 °C for 2 days. The mixture was then cooled to 0 °C and quenched with aqueous NH4Cl solution, followed by extraction with ethyl acetate. The collected organic phase was washed with water and brine, dried over sodium sulfate, filtered, and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel to give tert-butyl 4-(2-(2-hydroxyethoxy)ethyl)piperidine-1-carboxylic acid (41 mg, 51% yield).

[1074]

[1075] 4-(2-(2-(toluenesulfonyloxy)ethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester

[1076] TEA (41.7 μl, 0.3 mmol) and TsCl (31.5 mg, 0.16 mmol) were added to a solution of 4-(2-(2-hydroxyethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester (41 mg, 0.15 mmol) in DCM (3 mL). The resulting mixture was stirred overnight at room temperature and then diluted with DCM. The mixture was then washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel to give 4-(2-(2-(toluenesulfonyloxy)ethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester (43 mg, 67% yield).

[1077]

[1078] 4-(2-(2-iodoethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester

[1079] KI (67 mg, 0.14 mmol) was added to a solution of 4-(2-(2-(toluenesulfonyloxy)ethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester (43 mg, 0.1 mmol) in acetone (3 mL). The mixture was stirred overnight at 50 °C, then cooled to room temperature and diluted with ethyl acetate. The resulting mixture was then washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel to give 4-(2-(2-iodoethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester (38 mg, 91% yield).

[1080]

[1081] 4-(2-(2-(((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)ethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester

[1082] Add TEA (10.4 μl, 0.075 mmol) to a solution of (2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (20 mg, 0.037 mmol) and 4-(2-(2-iodoethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester (14 mg, 0.038 mmol) in THF (1 mL). Stir the mixture overnight at room temperature and then dilute with ethyl acetate. The resulting mixture was then washed with water and brine, dried with sodium sulfate, filtered, and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel to give 4-(2-(2-(((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)ethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester (21 mg, 71% yield).

[1083]

[1084]

[1085] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (49, XL02127)

[1086] 1 ml of 4N HCl was added to a solution of 4-(2-(2-(((R)-3-(1-fluorocyclopropane-1-amido)-4-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-2-methyl-4-oxobut-2-yl)thio)ethoxy)ethyl)piperidin-1-carboxylic acid tert-butyl ester (8 mg, 0.01 mmol) in DCM (1 ml). The mixture was stirred at room temperature for 1 hour, then concentrated to obtain a residue, which was dissolved in DMF (1 ml) and DIPEA (8.8 μl, 0.05 mmol), 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (3.11 mg, 0.01 mmol) and pyoxim (5.34 mg, 0.01 mmol) were added. The mixture was stirred at room temperature for 2 hours, then filtered through a 0.45 μm filter and injected into a preparative HPLC (5% to 95% ACN in water containing 0.5% formic acid) for purification to give 49 (XL02127) (6.2 mg for both steps, 61% yield).

[1087]

[1088] Exemplary synthesis of exemplary compounds 50 (XL02146) and 51 (XL02152)

[1089]

[1090] (2R,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[1091] According to General Scheme 3, (2R,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester (90 mg, 52% yield) was obtained from (2R,3R,4S)-1-(tert-butoxycarbonyl)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester (90 mg, 52% yield) by (2R,3R,4S)-1-(tert-butoxycarbonyl)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester (90 mg, 52% yield) from (2R,3R,4S)-1-(tert-butoxycarbonyl)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester (90 mg, 52% yield) and (4-(4-methylthiazol-5-yl)benzyl)carbamoyl)methylamine hydrogen chloride (96.6 mg, 0.401 mmol).

[1093] (9H-fluorene-9-yl)methyl((R)-1-((2S,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolid-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate

[1094] According to General Scheme 4, (9H-fluorene-9-yl)methyl((R)-1-((2S,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester (52.7 mg, 0.121 mmol) was prepared from (2R,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate (64 mg, 57% yield) from (2R,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate.

[1095]

[1096] (2S,3R,4S)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazo-5-yl)benzyl)pyrrolidine-2-carboxamide

[1097] According to General Scheme 5, (2S,3R,4S)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)butyryl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (45 mg, 92% yield) was obtained from (9H-fluorene-9-yl)methyl((R)-1-((2S,3R,4S)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (64 mg, 0.069 mmol).

[1098]

[1099] (2S,3R,4S)-3-fluoro-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(4-(4-methylthiazo-5-yl)benzyl)pyrrolidine-2-carboxamide

[1100] According to General Scheme 6, (2S,3R,4S)-3-fluoro-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (45 mg, 0.063 mmol) was obtained from (2S,3R,4S)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (30 mg, 60% yield) from (2S,3R,4S)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (30 mg, 60% yield).

[1101]

[1102] (2S,3R,4S)-3-fluoro-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazo-5-yl)benzyl)pyrrolidine-2-carboxamide

[1103] According to General Scheme 7, (2S,3R,4S)-3-fluoro-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (30 mg, 0.038 mmol) was prepared from (2S,3R,4S)-3-fluoro-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (15 mg, 75% yield).

[1104]

[1105]

[1106] (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-amido)-3-methylbutyryl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide(51, XL02152)

[1107] Add TEA (12 μl, 0.087 mmol) to a solution of (2S,3R,4S)-3-fluoro-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (8 mg, 0.0145 mmol) and (4-(((1R,4R)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone (8.2 mg, 0.0145 mmol) in THF (1 ml). The resulting mixture was concentrated and purified by preparative HPLC (5% to 95% ACN in water containing 5% formic acid) to give 51 (XL02152) (10 mg, 67% yield).

[1108]

[1109]

[1110] (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-amido)-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)-4,5-dihydro-1H-pyrrole-2-carboxamide (50, XL02146)

[1111] According to general scheme 8, 50 (XL02146) (8 mg, 30% yield) was obtained from (2S,3R,4S)-3-fluoro-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (15 mg, 0.027 mmol) and (4-(((1R,4R)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone (17 mg, 0.03 mmol)

[1112]

[1113] Exemplary synthesis of exemplary compound 52 (XL02117)

[1114]

[1115] 4-(3-(toluenesulfonyloxy)propyl)piperidine-1-carboxylic acid tert-butyl ester

[1116] At 0 °C, TEA (572 μl, 4.1 mmol) and TsCl (432 mg, 2.26 mmol) were added to a solution of 4-(3-hydroxypropyl)piperidine-1-carboxylic acid tert-butyl ester (500 mg, 2.05 mmol) in DCM (10 mL). The mixture was stirred overnight at room temperature and then diluted with DCM. The mixture was then washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel to give 4-(3-(toluenesulfonyloxy)propyl)piperidine-1-carboxylic acid tert-butyl ester (615 mg, 75% yield).

[1117]

[1118] 4-(3-Iodopropyl)piperidine-1-carboxylic acid tert-butyl ester

[1119] KI (250.6 mg, 1.5 mmol) was added to a solution of 300 mg (0.75 mmol) of 4-(3-(toluenesulfonyloxy)propyl)piperidine-1-carboxylic acid tert-butyl ester in acetone (15 mL). The mixture was stirred overnight at 50 °C and then cooled to room temperature. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by rapid column chromatography on silica gel to give 200 mg (75% yield) of 4-(3-iodopropyl)piperidine-1-carboxylic acid tert-butyl ester.

[1120]

[1121] 4-(3-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-amido)methyl)-5-(4-methylthiazolyl-5-yl)phenoxy)propyl)piperidine-1-carboxylic acid tert-butyl ester

[1122] Cs₂CO₃ (38 mg, 0.12 mmol) was added to a solution of tert-butyl 4-(3-iodopropyl)piperidin-1-carboxylate (20 mg, 0.056 mmol) and (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (25 mg, 0.047 mmol) in DMF (1 mL). After stirring at room temperature for 1 hour, water was added to the mixture and the mixture was extracted with ethyl acetate. The organic phase was collected, washed with water and brine, dried with sodium sulfate, filtered and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel to give 4-(3-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-amido)methyl)-5-(4-methylthiazolyl-5-yl)phenoxy)propyl)piperidine-1-carboxylic acid tert-butyl ester (25 mg, 69% yield).

[1123]

[1124]

[1125] (2S,4R)-N-(2-(3-(1-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperidin-4-yl)propoxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (52, XL02117)

[1126] Add 4N HCl (1 ml) to a solution of tert-butyl piperidine-1-carboxylic acid (25 mg, 0.033 mmol) in DCM (1 ml). Stir the mixture at room temperature for 1 hour, then concentrate under reduced pressure to obtain the residue. Dissolve the residue in DMF (1 ml) and add DIPEA (17 μL, 0.098 mmol), 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (19 mg, 0.036 mmol) and pyoxim (19 mg, 0.036 mmol) (at 0 °C). After stirring at room temperature for 2 hours, the mixture was filtered through a 0.45 μm filter and purified by preparative HPLC (5% to 95% ACN in water containing 0.5% formic acid) to give 52 (XL02117) (16.5 mg, 53.1% yield).

[1127]

[1128] Exemplary synthesis of exemplary compound 53 (XL02126)

[1129]

[1130] 4-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-amido)methyl)-5-(4-methylthiazolyl-5-yl)phenoxy)ethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester

[1131] Cs₂CO₃ (36.7 mg, 0.112 mmol) was added to a solution of tert-butyl piperidine-1-carboxylic acid (14.4 mg, 0.038 mmol) and (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (20 mg, 0.038 mmol) in DMF (1 ml). The mixture was stirred overnight at room temperature and then diluted with ethyl acetate. The mixture was then washed with water and brine, dried with sodium sulfate, filtered, and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel to give 4-(2-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-amido)methyl)-5-(4-methylthiazolyl-5-yl)phenoxy)ethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester (31 mg, 100% yield).

[1132]

[1133]

[1134] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (53, XL02126)

[1135] Add 4N HCl (1 ml) to a solution of 4-(2-(2-((((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-amido)methyl)-5-(4-methylthiazolyl-5-yl)phenoxy)ethoxy)ethyl)piperidine-1-carboxylic acid tert-butyl ester (15 mg, 0.019 mmol) in DCM (1 ml). Stir the mixture overnight at room temperature, then concentrate to obtain a residue, which is used directly in the next step. The residue was dissolved in DMF (1 ml), and DIPEA (16.5 μL, 0.07 mmol), 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (5.86 mg, 0.019 mmol), and Pyoxim (10 mg, 0.019 mmol) were added. After stirring overnight at room temperature, the mixture was filtered through a 0.45 μm filter and purified by preparative HPLC to give 53(XL02126) (9 mg, 48% yield).

[1136]

[1137] Exemplary synthesis of exemplary compound 54 (XL02115)

[1138]

[1139] (2S,4R)-N-(2-((5-bromopentyl)oxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide

[1140] Add 1,5-dibromopentane (25.9 mg, 0.113 mmol) and Cs2CO3 (36.7 mg, 0.112 mmol) to a solution of (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (20 mg, 0.037 mmol) in DMF (1 ml). After stirring at room temperature for 3 hours, the mixture was diluted with ethyl acetate, washed with water and brine, dried with sodium sulfate, filtered and concentrated to obtain the residue, which was purified by rapid column chromatography on silica gel to give (2S,4R)-N-(2-((5-bromopentyl)oxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (20 mg, 77% yield).

[1141]

[1142]

[1143] (2S,4R)-N-(2-((5-(4-(4-(((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoyl)piperazin-1-yl)pentyl)oxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (54, XL02115)

[1144] Add (4-((5-bromopentyl)oxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (10 mg, 0.0147 mmol) to a solution of (2S,4R)-N-(2-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(piperazin-1-yl)methyl ketone hydrogen chloride (6.67 mg, 0.0161 mmol) and Cs2CO3 (20 mg, 0.061 mmol) to a solution of (2S,4R)-N-(2-((5-bromopentyl)oxy)-4-(4-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (10 mg, 0.0147 mmol) in acetone (1 ml ... After stirring overnight at room temperature, the mixture was diluted with ethyl acetate, washed with water and brine, dried over sodium sulfate, filtered and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel to give 54 (XL02115) (6 mg, 42% yield).

[1145]

[1146] Exemplary synthesis of exemplary compound 55 (XL02135)

[1147]

[1148] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (55, XL02135)

[1149] Add (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (10 mg, 0.0186 mmol) and Cs₂CO₃ (30 mg, 0.092 mmol) to a solution of (4-(((1R,3R)-3-(bromomethyl)cyclobutyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) ketone (10 mg, 0.0186 mmol) in DMF (1 ml). Stir the mixture overnight at 60 °C, then cool and dilute with ethyl acetate. The resulting mixture was then washed with water and brine, dried with sodium sulfate, filtered and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel to give 55 (XL02135) (8 mg, 42% yield).

[1150]

[1151] Exemplary synthesis of exemplary compound 56 (XL02120)

[1152]

[1153] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (56, XL02120)

[1154] Cs₂CO₃ (24.5 mg, 0.075 mmol) was added to a solution of (4-(((1R,4R)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) ketone (10.6 mg, 0.0187 mmol) and (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (10 mg, 0.0187 mmol) in DMF (1 ml). After stirring overnight at 60 °C, the mixture was cooled and diluted with DCM. The resulting mixture was then washed with water and brine, dried with sodium sulfate, filtered and concentrated to obtain a residue, which was dissolved in 1 ml of methanol and purified by preparative HPLC (5% to 95% ACN in water containing 0.5% formic acid) to give 56 (XL02120) (7 mg, 37% yield).

[1155]

[1156] Exemplary synthesis of exemplary compound 57 (XL02119)

[1157]

[1158] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (57, XL02119)

[1159] To ((4-(((1s,4s)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl) methyl ketone (10.6 mg, 0.0187 mmol) and (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (10 mg, 0.01 mmol) A solution of 87 mmol Cs₂CO₃ (24.5 mg, 0.075 mmol) in DMF (1 ml) was added. After stirring overnight at 60 °C, the mixture was cooled and diluted with DCM. The resulting mixture was then washed with water and brine, dried over sodium sulfate, filtered, and concentrated to obtain a residue, which was dissolved in 1 ml of methanol and purified by preparative HPLC (5% to 95% ACN in water containing 0.5% formic acid) to give 57 (XL02119) (7.8 mg, 41% yield).

[1160]

[1161] Exemplary synthesis of exemplary compound 58 (XL02137)

[1162]

[1163] Cs₂CO₃ (37 mg, 0.11 mmol) was added to a solution of (19 mg, 0.037 mmol) of tert-butyl carbamate (19 mg, 0.037 mmol) and (2S, 4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (20 mg, 0.037 mmol) in DMF (1 ml). After stirring overnight at 50 °C, the mixture was cooled and diluted with DCM. The resulting mixture was then washed with water and brine, dried with sodium sulfate, filtered, and concentrated to obtain a residue, which was purified by rapid column chromatography on silica gel to give tert-butyl carbamate (4-((1-(((1S,4r)-4-((2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-amido)methyl)-5-(4-methylthiazolyl-5-yl)phenoxy)methyl)cyclohexyl)methyl)piperidin-4-yl)oxy)-2-methoxyphenyl)carbamate (10 mg, 29% yield).

[1164]

[1165]

[1166] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (58, XL02137)

[1167] Add 0.5 ml of 4N HCl to a solution of (10 mg, 0.01 mmol) of tert-butyl carbamate (4-((1-(((1S,4R)-4-((2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-amido)methyl)-5-(4-methylthiazolyl-5-yl)phenoxy)methyl)cyclohexyl)methyl)piperidin-4-yl)oxy)-2-methoxyphenyl)carbamate (10 mg, 0.01 mmol) in DCM (0.5 ml). Stir the mixture overnight at room temperature, then concentrate under reduced pressure to obtain a residue, which is dissolved in 1 ml of isopropanol and 2,5-dichloro-N-methylpyrimidin-4-amine (4 mg, 0.022 mmol) and 5 drops of 4N HCl are added. The resulting mixture was heated in a microwave tube at 100°C for 12 hours, then concentrated under reduced pressure and purified by preparative HPLC (5% to 95% CAN in water containing 0.5% formic acid) to give 58 (XL02137) (48% yield for 5 mg of both steps).

[1168]

[1169] Exemplary synthesis of exemplary compound 59 (XL02138)

[1170]

[1171] (R)-2-((((1S,4R)-4-((2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-amido)methyl)-5-(4-methylthiazolyl-5-yl)phenoxy)methyl)cyclohexyl)methoxy)methyl)morpholine-4-carboxylic acid tert-butyl ester

[1172] Add Cs₂CO₃ (14.1 mg, 0.043 mmol) to a solution of (R)-2-((((1R,4R)-4-(bromomethyl)cyclohexyl)methoxy)methyl)morpholine-4-carboxylic acid tert-butyl ester (7 mg, 0.017 mmol) and (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (9.2 mg, 0.017 mmol) in DMF (0.5 ml). After stirring overnight at 50°C, the mixture was cooled to room temperature, diluted with ethyl acetate, washed with water and brine, dried over sodium sulfate, filtered, and concentrated to obtain a residue. This residue was purified by rapid column chromatography on silica gel to give (R)-2-((((1S,4R)-4-((2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-amido)methyl)-5-(4-methylthiazolyl-5-yl)phenoxy)methyl)cyclohexyl)methoxy)methyl)morpholine-4-carboxylic acid tert-butyl ester (12 mg, 86% yield).

[1173]

[1174]

[1175] (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-methylthiazolyl-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (59, XL02138)

[1176] Add 0.5 ml of 4NHCl to a solution of (R)-2-((((1S,4R)-4-((2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-amido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-amido)methyl)-5-(4-methylthiazolyl-5-yl)phenoxy)methyl)cyclohexyl)methoxy)methyl)morpholine-4-carboxylic acid tert-butyl ester (12 mg, 0.014 mol) in 0.5 ml of DCM. After stirring at room temperature for 1 hour, the mixture was concentrated to obtain a residue, which was dissolved in DMF (1 ml) and treated with DIPEA (12 μL, 0.069 mmol), 4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxybenzoic acid (4.3 mg, 0.014 mmol), and Pyoxim (7.4 mg, 0.014 mmol) (at 0 °C). After stirring at 0 °C for 2 hours, the mixture was filtered through a 0.45 μm filter and injected into a preparative HPLC system (5% to 95% ACN in water containing 0.5% formic acid) for purification to give 59 (XL02138) (9 mg, 61% yield).

[1177]

[1178] Exemplary synthesis of exemplary compound 60 (MP01077)

[1179]

[1180] (2S,4R)-2-((4-chlorobenzyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester

[1181] TEA (1.18 mL, 8.47 mmol), (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (1.63 g, 7.06 mmol), and HATU (2.82 g, 7.42 mmol) were added to a solution of 4-chlorobenzylamine (0.86 mL, 7.06 mmol) in a DCM / methanol mixture (38.0 / 2.0 mL). The reaction mixture was stirred at room temperature for 1.5 h, then DCM was added, and the organic phase was washed with water and brine, dried over sodium sulfate, filtered, and concentrated to give a crude residue. This crude residue was purified by rapid column chromatography on silica gel (0 to 10% methanol in DCM) to give the compound (2S,4R)-2-((4-chlorobenzyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester (1.79 g, 71% yield).

[1182]

[1183] (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-2-((4-chlorobenzyl)carbamoyl)-4-hydroxypyrrolidone-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate

[1184] Following general protocol 4, (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-2-((4-chlorobenzyl)carbamoyl)-4-hydroxypyrrolidone-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate was obtained (356 mg for both steps, 81% yield).

[1185]

[1186] (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide

[1187] According to General Scheme 5, (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate (214 mg, 0.25 mmol) was used to obtain (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (142 mg, 90% yield) from (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-2-((4-chlorobenzyl)carbamoyl)-4-hydroxypyrrolidine-2-carboxamide (142 mg, 90% yield).

[1188]

[1189] (2S,4R)-N-(4-chlorobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide

[1190] According to General Scheme 6, (2S,4R)-N-(4-chlorobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide (142 mg, 65% yield) was obtained from (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide (192 mg, 0.31 mmol)

[1191]

[1192] (2S,4R)-N-(4-chlorobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide

[1193] Following General Scheme 7, (2S,4R)-N-(4-chlorobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide (91 mg, 97% yield) was obtained from (2S,4R)-N-(4-chlorobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (142 mg, 0.20 mmol)

[1194]

[1195]

[1196] (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-amido)-3-methylbutyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (60, MP01077)

[1197] According to general scheme 8, 60 (MP01077) (24 mg, 92% yield) was obtained from (2S,4R)-N-(4-chlorobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (13 mg, 0.027 mmol) and (4-(((1R,4R)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(3-methoxy-4-((4-(methylamino)pyrimidin-2-yl)amino)phenyl)methyl ketone (15 mg, 0.027 mmol).

[1198]

[1199] Exemplary synthesis of exemplary compound 61 (MP01083)

[1200]

[1201] (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-amido)-3-methylbutyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (MP01083)

[1202] According to general scheme 8, 61 (MP01083) (22 mg, 85% yield) was obtained from (4-(((1s,4s)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(3-methoxy-4-((4-(methylamino)pyrimidin-2-yl)amino)phenyl) ketone (15 mg, 0.027 mmol) and (2S,4R)-N-(4-chlorobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (13 mg, 0.027 mmol).

[1203]

[1204] Exemplary synthesis of exemplary compound 62 (MP01088)

[1205]

[1206] (2S,4R)-2-((4-cyanobenzyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester

[1207] Add (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (1.75 g, 7.57 mmol), TEA (1.266 mL, 9.079 mmol), and HATU (3.021 g, 7.944 mmol) to a solution of 4-(aminomethyl)benzylnitrile (1 g, 7.57 mmol) in DCM (40 mL). Stir the resulting mixture overnight at room temperature, then dilute with DCM. Wash the mixture with water and brine, dry with sodium sulfate, filter, and concentrate to obtain a residue, which was purified by rapid column chromatography on silica gel to give (2S,4R)-2-((4-cyanobenzyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester (933 mg, 36% yield).

[1208]

[1209] (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-2-((4-cyanobenzyl)carbamoyl)-4-hydroxypyrrolidone-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate

[1210] According to general scheme 4, (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-2-((4-cyanobenzyl)carbamoyl)-4-hydroxypyrrolidone-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)but-2-yl)carbamate (329 mg, 74% yield) was obtained.

[1211]

[1212] (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-N-(4-cyanobenzyl)-4-hydroxypyrrolidine-2-carboxamide

[1213] According to General Scheme 5, (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-N-(4-cyanobenzyl)-4-hydroxypyrrolidine-1-yl)-3-methyl-1-oxo-3-(triphenylmethylthio)butyryl)carbamate (329 mg, 82% yield) was obtained from (9H-fluorene-9-yl)methyl((R)-1-((2S,4R)-2-((4-cyanobenzyl)carbamoyl)-4-hydroxypyrrolidine-2-carboxamide (199 mg, 82% yield)

[1214]

[1215] (2S,4R)-N-(4-cyanobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide

[1216] According to General Scheme 6, (2S,4R)-N-(4-cyanobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide (167 mg, 74% yield) was obtained from (2S,4R)-1-((R)-2-amino-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide (199 mg, 0.322 mmol)

[1217]

[1218] (2S,4R)-N-(4-cyanobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide

[1219] According to General Scheme 7, (2S,4R)-N-(4-cyanobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-methyl-3-(triphenylmethylthio)butyryl)-4-hydroxypyrrolidine-2-carboxamide (167 mg, 0.237 mmol) was obtained from (2S,4R)-N-(4-cyanobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (89 mg, 81% yield) from (2S,4R)-N-(4-cyanobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (89 mg, 81% yield).

[1220]

[1221]

[1222] (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-amido)-3-methylbutyryl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide (62, MP01088)

[1223] According to General Scheme 8, MP01088 (22 mg, 85% yield) was obtained from (4-(((1R,4R)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(3-methoxy-4-((4-(methylamino)pyrimidin-2-yl)amino)phenyl) ketone (15 mg, 0.027 mmol) and (2S,4R)-N-(4-cyanobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (13 mg, 0.027 mmol).

[1224]

[1225] Exemplary synthesis of exemplary compound 63 (MP01089)

[1226]

[1227] (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-amido)-3-methylbutyryl)-N-(4-cyanobenzyl)-4-hydroxypyrrolidine-2-carboxamide (63, MP01089)

[1228] According to General Scheme 8, MP01089 (9 mg, 42% yield) was obtained from (2S,4R)-N-(4-cyanobenzyl)-1-((R)-2-(1-fluorocyclopropane-1-amido)-3-mercapto-3-methylbutyryl)-4-hydroxypyrrolidine-2-carboxamide (12 mg, 0.027 mmol) and (4-(((1s,4s)-4-(bromomethyl)cyclohexyl)methyl)piperazin-1-yl)(3-methoxy-4-((4-(methylamino)pyrimidin-2-yl)amino)phenyl)methyl ketone (15 mg, 0.027 mmol).

[1229]

Claims

1. Compound of formula (I): X-L1-L2-Y Formula (I) Where X is: wherein R1 is selected from: (i) F, (ii) Cl, (iii) Br, (iv) CF3, (v) CN, (vi) NO2, (vii) CHF2, (viii) CH2F; Wherein R2 is selected from: (i) -Me, (ii) -CD3, (iii) -OMe, (iv) -OCD3, (v) -OCF3, (vi) -OCHF2, (vii) ethyl, (viii) -OCH2CH3, (ix) isopropyl, (x) cyclopropyl and the following groups: Wherein L1 is selected from: Wherein L2 is selected from: wherein m is independently selected from 2, 3, 4, 5, 6, 7 and 8; and wherein n is independently selected from 1, 2, 3, 4, 5 and 6; Wherein Y is selected from: or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof.

2. The compound of formula (I) according to claim 1, wherein X is selected from (i) 3. The compound of formula (I) according to claim 1 or 2, wherein L1 is selected from: (i) 4. A compound of formula (I) according to any one of claims 1 to 3, wherein L2 is selected from: wherein m is independently selected from 3, 4 and 5; wherein n is independently selected from 1, 2, 3 and 4; 5. A compound of formula (I) according to any one of claims 1 to 4, wherein Y is selected from:

6. A compound of formula (I) according to any one of claims 1 to 5, wherein Y is selected from:

7. A compound of formula (I) according to any one of claims 1 to 6, wherein the compound is selected from: or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof.

8. A compound of formula (I) according to any one of claims 1 to 7, wherein: a) one of the compounds is selected from: or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof; or b) one of the compounds is selected from: (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-amido)-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-amido)-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-amido)-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-amido)-3-methylbutanoyl)-N-(4-chlorobenzyl)-4-hydroxypyrrolidine-2-carboxamide; or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof; or c) One of the compounds 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-amido)-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. A compound of formula (I) according to any one of claims 1 to 7, wherein: One of the compounds is selected from: or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof; or, One of the compounds is selected from: or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof.

10. A compound of formula (I) according to any one of claims 1 to 7, wherein the compound is selected from: or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof.

11. A compound of formula (I) according to any one of claims 1 to 7, wherein the compound is selected from: or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof.

12. A compound of formula (I) according to any one of claims 1 to 7, wherein the compound is selected from: or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof.

13. A compound of formula (I) according to any one of claims 1 to 7 or 9, wherein the compound is selected from: or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof.

14. A compound of formula (I) according to any one of claims 1 to 7 or 10, wherein: At least one of the compounds is selected from: or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof; or, At least one of the compounds is: or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof.

15. A pharmaceutical composition comprising a compound as defined in any preceding claim and a pharmaceutically acceptable carrier or diluent thereof, optionally wherein the pharmaceutical composition comprises an effective amount of a compound as defined in any one of claims 1 to 14 in combination with a pharmaceutically acceptable carrier, additive or excipient.

16. A compound as defined in any one of claims 1 to 14 for use in medicine.

17. A compound as defined in any one of claims 1 to 14 for use in preventing or treating a disease or condition associated with altered GTPase and / or kinase activity of a leucine-rich repeat kinase 2 (LRRK2) protein, comprising administering a PROTAC compound of formula (I) to a subject suffering from or likely to be exposed to the disease or condition.

18. A compound as defined in any one of claims 1 to 14 for use in preventing or treating a disease or condition associated with altered PDE6D activity, comprising administering a PROTAC compound according to formula (I) as defined herein to a subject suffering from or potentially exposed to a disease or condition associated with PDE6D.

19. A compound as defined in any one of claims 1 to 14 for use in the prevention or treatment of a disease or condition independently selected from the group consisting of Parkinson's disease, idiopathic Parkinson's disease, idiopathic late-onset Parkinson's disease, familial Parkinson's disease, LRRK2 mutation-associated Parkinson's disease, Lewy body dementia, primary tauopathy, or inflammation-related diseases such as leprosy, neuroinflammation, and Crohn's disease.

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