Discovery of Highly Potent, Selective, and Brain-Penetrant

Article pubs.acs.org/jmc

Discovery of Highly Potent, Selective, and Brain-Penetrant Aminopyrazole Leucine-Rich Repeat Kinase 2 (LRRK2) Small Molecule Inhibitors Anthony A. Estrada,*,† Bryan K. Chan,*,† Charles Baker-Glenn,∇ Alan Beresford,◆ Daniel J. Burdick,† Mark Chambers,∇ Huifen Chen,† Sara L. Dominguez,‡ Jennafer Dotson,† Jason Drummond,§ Michael Flagella,⊥ Reina Fuji,⊥ Andrew Gill,○ Jason Halladay,∥ Seth F. Harris,# Timothy P. Heffron,† Tracy Kleinheinz,§ Donna W. Lee,⊥ Claire E. Le Pichon,‡ Xingrong Liu,∥ Joseph P. Lyssikatos,† Andrew D. Medhurst,○ John G. Moffat,§ Kevin Nash,○ Kimberly Scearce-Levie,‡ Zejuan Sheng,‡ Daniel G. Shore,† Susan Wong,∥ Shuo Zhang,‡ Xiaolin Zhang,∥ Haitao Zhu,‡ and Zachary K. Sweeney† Departments of †Discovery Chemistry, ‡Neurosciences, §Biochemical and Cellular Pharmacology, ∥Drug Metabolism and Pharmacokinetics, ⊥Safety Assessment, and #Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States Departments of ∇Chemistry, ○Biochemical and Cellular Pharmacology, and ◆Drug Metabolism and Pharmacokinetics, BioFocus, Chesterford Research Park, Saffron Walden, CB10 1XL, United Kingdom S Supporting Information *

ABSTRACT: Leucine-rich repeat kinase 2 (LRRK2) has drawn significant interest in the neuroscience research community because it is one of the most compelling targets for a potential disease-modifying Parkinson’s disease therapy. Herein, we disclose structurally diverse small molecule inhibitors suitable for assessing the implications of sustained in vivo LRRK2 inhibition. Using previously reported aminopyrazole 2 as a lead molecule, we were able to engineer structural modifications in the solvent-exposed region of the ATPbinding site that significantly improve human hepatocyte stability, rat free brain exposure, and CYP inhibition and induction liabilities. Disciplined application of established optimal CNS design parameters culminated in the rapid identification of GNE-0877 (11) and GNE-9605 (20) as highly potent and selective LRRK2 inhibitors. The demonstrated metabolic stability, brain penetration across multiple species, and selectivity of these inhibitors support their use in preclinical efficacy and safety studies.

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INTRODUCTION Parkinson’s disease (PD) is a degenerative disorder of the central nervous system (CNS) that affects approximately 5% of the population greater than 80 years of age.1 It is characterized pathologically by gradual loss of dopaminergic neurons and dopamine secretion in the substantia nigra, and a diseasemodifying or neuroprotective therapy remains a major unmet medical need. Currently available PD treatments only address the dopamine deficiencies characteristic of the disorder, and the most widely used symptomatic treatment, levodopa (L-dopa), often causes L-dopa-induced dyskinesias (LID) after prolonged use.2,3 The LRRK2 gene is a multidomain protein comprising 2527 amino acids and is expressed throughout the brain and other peripheral organs. The association of this protein with PD was established in 2004, and it has since been determined that mutations in the LRRK2 gene are the most common cause of familial PD.4−8 Furthermore, LRRK2 genetic polymorphisms have been linked to idiopathic PD in terms of age of onset, symptoms, and pathology, suggesting that a LRRK2-derived © XXXX American Chemical Society

therapy could benefit both sporadic and familial cases of PD.9−11 Multiple LRRK2 mutations have been reported to demonstrate increased kinase activity in vitro and in vivo, with the most common being the autosomal dominant G2019S mutation that resides in the activation loop of the kinase domain.12−18 As a result, several groups have recently reported structurally diverse small molecule LRRK2 kinase inhibitors. These molecules should facilitate efforts to unlock the normal functions of LRRK2, the role of LRRK2 in PD and other diseases,19,20 and the consequences of prolonged inhibition in preclinical species.21−35 Our group was the first to report on a series of potent, selective, and brain-penetrable aminopyrimidine LRRK2 inhibitors.24,36 The demonstrated kinase selectivity was achieved through the discovery and exploitation of a selectivity hotspot that was identified through kinase counterscreening Received: October 25, 2013

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1 μM). A docking overlay of inhibitors 1 and 2 using our LRRK2 homology model can be visualized in Figure 2.24

and a matched molecular pair activity cliff analysis.24,37−42 Lead optimization using property- and structure-based drug design provided highly efficient LRRK2 inhibitors such as GNE-7915 (1, Figure 1)25 with excellent selectivity against the broader

Figure 1. Previously reported LRRK2 diaminopyrimidine inhibitors.25,26

kinome and good free brain-to-plasma ratios across preclinical species. Experiments employing 1 and additional lead compounds demonstrated potent cellular activity and robust PK/PD responses in G2019S bacterial artificial chromosome (BAC) transgenic mice. These studies were enabled by the discovery of Ser1292 as a direct, specific autophosphorylation site suitable for use as an in vitro and in vivo biomarker of LRRK2 activity.43 In an effort to reduce size, improve aqueous solubility (thermodynamic solubility of crystalline 1 at pH 7.4 < 2 μg mL−1), and eliminate the potential for ortho-quinoneimine reactive metabolite formation, we successfully designed a series of aminopyrazoles such as 2 (LELP44,45 = 5.7; thermodynamic solubility of crystalline 2 at pH 7.4 = 20 μg mL−1) as aniline bioisosteres (Figure 1).26 In this contribution, we report our successful efforts to identify improved aminopyrazole LRRK2 inhibitors that possess profiles suitable for early development evaluation.

Figure 2. Docking model of 1 (cyan) and 2 (magenta) in LRRK2 (green). The side-chain Y931 of JAK2 is also shown (yellow). Intermolecular hydrogen-bond interactions are shown as yellow dashed lines.

Gratifyingly, JAK2 continued to serve as a reliable indicator of general kinase selectivity for aminopyrazoles with a chloro or methyl substituent ortho to the aminopyrimidine hinge binder (an unfavorable proximity of the chloro substituent of inhibitor 2 to Y931 of JAK2 can be seen in Figure 2).24,25 All of the inhibitors disclosed herein have a JAK2 biochemical selectivity index of >150-fold unless otherwise specified. Compound 2 also possesses good human liver microsomal (LM) stability and shows a favorable rat in vitro−in vivo stability correlation with excellent free-drug clearance (Table 1). Unfortunately, upon

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RESULTS AND DISCUSSION As previously reported,26 aminopyrazole 2 has LRRK2 biochemical and cellular potencies of 9 and 28 nM, respectively (Table 1), and demonstrates very good kinase selectivity (185 kinases, 116-fold over LRRK2 Ki, 1 kinase at >75% inhibition at

Table 1. Summarized Profile of Aminopyrazole 2 and Representative SAR for Fused Morpholinopyrazolesa

compd

R1

R2

R3

LRRK2 Ki (nM)b

2 3 4 5 6l

H H Mel Me

Me H (S)-Me H

Me H H H

9 0.9 3 6 3

pLRRK2 IC50 (nM)c

LM Clhep (mL min−1 kg−1)d h/re

hep Clhep (mL min−1 kg−1)f h/re

28 20 18 49 42

3/13 11/47 14/17 8/32 4/11

10/18 12/− 8/− 3/19

rat CI (Clu) (mL min−1 kg−1)g 21(84)

43 (105)

MDR1h P-gp ERi (B:A/A:B)j 0.9 0.7 1.0 1.0 1.3

rat Bu/Puk 0.37

0.2

Compounds were dosed po (1 mg kg−1) as an aqueous suspension with 1% methylcellulose, iv (0.5 mg kg−1) as either a 60% PEG solution or a 20−60% NMP solution for systemic PK, and iv (0.5 mg kg−1) as a 60% NMP solution for brain PK. bBiochemical assay. cCellular assay; all biochemical and cellular assay results represent the arithmetic mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the reported mean. dLiver microsome-predicted hepatic clearance. eh/r = human/rat. fIn vitro stability in cryopreserved hepatocytes. gClu = unbound clearance = total clearance/fup, where fup is the unbound plasma free fraction. hMDCK-MDR1 human P-gp transfected cell line. iEfflux ratio. jBasolateral-to-apical/apical-to-basolateral. kUnbound brain/unbound plasma AUC ratio. lAbsolute stereochemistry unknown. a

B

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expanded profiling, we observed higher turnover when inhibitor 2 was incubated with human hepatocytes (10 mL min−1 kg−1, Table 1), and in vitro metabolite identification studies revealed high levels of glucuronidated parent (>60%) after 3 h. Additionally, the unbound brain/unbound plasma concentration (Bu/Pu) ratio for 2 was suboptimal (Bu/Pu = 0.37, Table 1), and bioavailability in cynomolgus monkey pharmacokinetic (PK) studies was poor (oral bioavailability 5% at 1 and 20 mg kg−1), potentially because of high intestinal metabolism via glucuronidation. As a consequence, there was significant uncertainty in the human PK prediction for compound 2 and thus further progression was halted. Our initial strategy to address the issues with aminopyrazole 2 led to the fused morpholinopyrazoles shown in Table 1. We hypothesized that cyclization of the tertiary alcohol would potentially improve the Bu/Pu concentration ratio by lowering topological polar surface area (TPSA) and removing one hydrogen-bond donor (HBD). Additionally, we hoped to improve metabolic stability by reducing the number of rotatable bonds, increasing rigidity, and attenuating the observed glucuronidation with tertiary alcohol 2. Thus, morpholinopyrazoles 3, 4, and 5 (Table 1) were prepared. Although all of these fused bicycles showed similar cellular potencies as 2 and lacked P-gp-mediated efflux in vitro, desirable microsomal stability improvements were not achieved. Pleasingly, monomethyl-substituted morpholine 6 (Table 1) demonstrated low turnover in both human LM and hepatocytes and retained good cellular activity. Assessment of the rat in vivo PK profile of 6 revealed low free-drug clearance (105 mL min−1 kg−1), but a slight in vitro−in vivo disconnection reduced our confidence in the improved in vitro human stability relative to our previous lead (2). Additionally, brain cassette rat PK studies of 6 revealed an unacceptable Bu/Pu concentration ratio (0.2, Table 1).25,46 As a result, further evaluation of 6 was halted in favor of pursuing inhibitors with superior profiles. In addition to pursuing bicyclic pyrazoles, a separate parallel strategy to identify polar, metabolically stable pyrazole Nsubstituents was developed. We have previously shown that simple, linear N-alkyl-substituted pyrazoles exhibited potent reversible and time-dependent inhibition (TDI) of CYP1A2 (IC50 values typically 95 Å2) are above suggested values for brain-penetrant small molecules.49,50 The first amide synthesized (7, Table 2) possessed good LRRK2 cellular activity and LM stability and was devoid of CYP inhibition. Unlike morpholinopyrazole 6, compound 7 also showed a promising in vitro−in vivo correlation (rat iv Cl = 12 mL min−1 kg−1; Clu = 27 mL min−1 kg−1). Unfortunately, as predicted by the high TPSA (96 Å2), 7 was effluxed in the MDCK-MDR1 cell line and exhibited poor in vivo brain penetration (rat Bu/Pu = 0.08). Attempts to mask further or block P-gp recognition of the amide motif led to the preparation of ethyl- (8), isopropyl- (9), and trifluoroethylsubstituted (10) secondary amides. Although we were

Table 2. Investigation of Pyrazole Amide Substitutions

compd

R1

pLRRK2 IC50 (nM)a

7 8 9 10

Me Et iPr −CH2CF3

36 99 140 53

LM Clhep (mL min−1 kg−1)b h/rc

MDR1d P-gp ERe (B:A/A:B)f

6/8 8/27 7/36 9/27

5.5 4.0 1.3 1.5

a

Cellular assay; all cellular assay results represent the arithmetic mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the reported mean. bLiver microsome-predicted hepatic clearance. ch/r = human/rat. dMDCKMDR1 human P-gp transfected cell line. eEfflux ratio. fBasolateral-toapical/apical-to-basolateral.

successful in reducing the P-gp efflux for 9 and 10, modest cellular potencies of compounds in this series discouraged further progression.51 A series of gem-disubstituted cyano pyrazole LRRK2 inhibitors were then prepared via dehydration of the precursor amide. We were hopeful that removing the amide functionality and maintaining a low MW would enhance brain penetration. Validation of this strategy was provided with GNE-0877 (11, Table 3), which showed significantly enhanced LRRK2 cellular potency (3 nM) and low turnover in human liver microsomes and hepatocytes with no evidence of glucuronidation. We hypothesize that the significant enhancement in cellular potency is primarily due to a better fit and tighter binding of the less bulky gem-dimethyl cyano group. The flexibility afforded by the lack of intramolecular hydrogen bonding should allow the methyl groups to form better van der Waals contacts with the hydrophobic residues in the protein, whereas the cyano group can form favorable electrostatic interactions with the side chain of Arg1957. Invitrogen kinase-selectivity profiling (188 kinases) of aminopyrazole 11 at 0.1 μM (145-fold over LRRK2 Ki) resulted in only four kinases showing greater than 50% inhibition (Aurora B = 51%, RSK2 = 52%, RSK4 = 62%, and RSK3 = 68%) and suggested that 11 is a highly selective LRRK2 inhibitor. Furthermore, 11 possessed a 212-fold biochemical-selectivity index over TTK (Ki = 150 nM), which was previously highlighted as an off-target kinase of concern because of the suggested role of TTK in the maintenance of chromosomal stability.25 The in vivo rat clearance for inhibitor 11 was within 2-fold of measured in vitro stability, and good oral bioavailability (88%) was achieved at 50 mg kg−1 following administration of a methylcellulose/tween (MCT) suspension. Crystalline 11 also demonstrated good thermodynamic solubility (pH 7.4 solubility = 100 μg mL−1). Importantly, the absence of in vitro P-gp efflux translated to a good in vivo rat free brain-toplasma ratio (0.6) and corresponding CSF-to-free plasma ratio (0.9).52,53 Despite previous success in eliminating CYP1A2 inhibition with alkyl branching of the pyrazole N-substituents,26 11 was found to be a reversible CYP1A2 inhibitor (IC50 = 0.7 μM), but this compound did not exhibit TDI of CYP1A2. C

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Table 3. gem-Disubstituted Aminopyrazole SAR and DMPK Profilesa

Compounds were dosed po (1 mg kg−1) as an aqueous suspension with 1% methylcellulose, iv (0.5 mg kg−1) as either a 60% PEG solution or a 20−60% NMP solution for systemic PK, and iv (0.5 mg kg−1) as a 60% NMP solution for brain PK. bBiochemical assay. cCellular assay; all biochemical and cellular assay results represent the arithmetic mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the reported mean. dLiver microsome-predicted hepatic clearance. eh/r = human/rat. fIn vitro stability in cryopreserved hepatocytes. gClu = unbound clearance = total clearance/fup, where fup is the unbound plasma free fraction. hMDCK-MDR1 human P-gp transfected cell line. iEfflux ratio. jBasolateral-to-apical/apical-to-basolateral. kUnbound brain/unbound plasma AUC ratio. lNT = not tested. a

kg−1). Unfortunately, although 16 did not show significant reversible CYP inhibition, time-dependent inhibition of CYP1A2 was observed (IC50 = 2 μM). This result suggested that more sterically encumbered substitutions, such as a tetrahydropyranyl (THP) group, may be required for attenuation of CYP1A2 inhibition. As predicted, THP analogue 17 showed excellent LRRK2 potency and in vitro metabolic stability similar to that of 16, whereas it showed no evidence of time-dependent inhibition of CYP1A2. Because of its favorable cellular potency and in vitro metabolic stability, 17 was progressed into in vivo PK evaluation. Intravenous dosing of 17 at 0.5 mpk in rats returned a total plasma clearance of 74 mL min−1 kg−1 and a volume of distribution of 1.1 L kg−1. The resultant short halflife (0.17 h), potential for extrahepatic clearance, undesired in vitro−in vivo disconnection, and suboptimal brain penetration (Bu/Pu = 0.3) halted the progression of 17. Other minor modifications of 17 involving substitution of the THP ring and incorporation of asymmetry (data not shown) did not reduce in vivo clearance. With the uncertainties surrounding the efficacy drivers for disease modification, it was a program goal to ensure that adequate half-lives were achieved by our LRRK2 inhibitors. We envisioned that the incorporation of a weakly basic group into the solvent-exposed region of our inhibitor would potentially increase the volume of distribution and thereby extend the in

Additional profiling suggested that 11 was a pan-inducer of CYP enzymes in human hepatocytes regulated through nuclear receptors PXR (3A4), AhR (1A2), and CAR (2B6). There are several literature examples where minor structural modifications and/or stereoisomers can alleviate the induction potential of small molecule inhibitors.54 We therefore targeted close-in analogues of 11 such as pyrazole regioisomer 12 (Table 3), spiro-cyclopropyl matched pair 13, spiro-cyclopropyl chloropyrazole 14, and carbon-linked gem-dimethyl nitrile 15. Although these compounds exhibited similar in vitro and in vivo profiles as 11 and several showed reduced CYP1A2 potential liabilities, all of these inhibitors suffered from variable levels of CYP induction. It was then hypothesized that incorporating polarity more distal to the pyrazole ring while maintaining branched pyrazole N-alkyl substitutions for CYP1A2 inhibition relief55,56 may be necessary to attenuate the observed human CYP induction.57−59 To minimize the number of rotatable bonds, the compact N-oxetyl pyrazole analogue (16, Table 4) was synthesized. Because of the structural similarity of the oxetane to the N-capping group of compound 2, it was hoped that the N-oxetyl pyrazole would retain the favorable properties of 2 without suffering from glucuronidation of the related alcohol. Indeed, 16 demonstrated good LRRK2 cellular potency (pLRRK2 IC50 = 29 nM) and excellent stability upon incubation in hepatocytes (human hep Clhep = 2 mL min−1 D

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Table 4. Oxetyl, Pyranyl, and Piperidinyl Aminopyrazole SAR and DMPK Profilesa

Compounds were dosed po (1 mg kg−1) as an aqueous suspension with 1% methylcellulose, iv (0.5 mg kg−1) as either a 60% PEG solution or a 20−60% NMP solution for systemic PK, and iv (0.5 mg kg−1) as a 60% NMP solution for brain PK. bBiochemical assay. cCellular assay; all biochemical and cellular assay results represent the arithmetic mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the reported mean. dLiver microsome-predicted hepatic clearance. eh/r = human/rat. fIn vitro stability in cryopreserved hepatocytes. gClu = unbound clearance = total clearance/fup, where fup is the unbound plasma free fraction. hMDCK-MDR1 human P-gp transfected cell line. iEfflux ratio. jBasolateral-to-apical/apical-to-basolateral. kUnbound brain/unbound plasma AUC ratio. lAbsolute stereochemistry unknown. a

fluoro-piperidinyl system to give 19. Although MDCK-MDR1 results suggested an improved efflux ratio (ER = 2.2), fluoropiperidine 19 showed minimal improvement in in vivo unbound brain penetration (Bu/Pu = 0.08). One potential explanation for the impaired brain penetration could be attributed to efflux by the BCRP transporter (MDCK-BCRP ER = 4.2). During the optimization leading to tertiary alcohol 2, it was recognized that the replacement of the methylpyrazole with a chloropyrazole within the current series of LRRK2 aminopyrazole inhibitors could translate to improved brain penetration. In addition to the obvious structural difference, it was also envisioned that such a methyl-to-chloro substitution would increase the lipophilicity of the inhibitor (ΔcLogD7.4 = +0.4), thereby potentially leading to improved intrinsic permeability. Indeed, notable improvement in the MDCKMDR1 and MDCK-BCRP efflux ratios (ER = 0.80 and 0.90, respectively) was observed for chloropyrazole analogue GNE9605 (20). Despite the minor increase in lipophilicity, 20 retained excellent predicted human metabolic stability when assayed in human liver microsomes and hepatocytes. In addition, no reversible or time-dependent inhibition of any of

vivo half-life without compromising potency. Replacement of the THP group in 17 with a piperidine-N-oxetyl moiety afforded 18 (Table 4). As predicted, 18 showed good LRRK2 biochemical (LRRK2 Ki = 4 nM) and cellular potency (pLRRK2 IC50 = 20 nM) with no evidence of reversible or time-dependent CYP inhibition. The in vivo rat clearance (Clp = 22 mL min−1 kg−1) correlated well with the in vitro metabolic stability results (rLM = 16.3 mL min−1 kg−1). In addition, the volume of distribution (1.7 L kg−1) was slightly higher than 17, leading to a significantly improved half-life (2.1 h). Despite attempts to modulate the basicity of the piperidine nitrogen with the electron-withdrawing oxetyl group, inhibitor 18 (cpKa = 7.6) showed significant in vivo brain-penetration impairment (Bu/Pu = 0.06). MDCK-MDR1 results confirmed 18 to be a substrate of P-gp (ER = 5.1). Nonetheless, the low unbound clearance suggested that the inhibitor possessed excellent systemic metabolic stability, which supported further optimization of 18. To reduce further the basicity of the inhibitor without significantly altering the structure and overall physicochemical properties, the piperidinyl group in 18 was exchanged for a 3E

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the major CYP isoforms was observed. Similar to chloropyrazole 2, less than 0.2% of trapping adducts were observed when 20 was incubated in human liver microsomes in the presence of potassium cyanide, and no adducts were detected after incubation with glutathione and methoxyamine. Importantly, our strategy to attenuate the CYP induction observed with other aminopyrazole inhibitors by installing polarity distal to the pyrazole ring proved successful, as 20 was not an inducer of any CYP isoform tested (3A4, 1A2, and 2B6). Inhibitor 20 was highly potent against LRRK2 in both biochemical (Ki = 2.0 nM) and cellular (IC50 = 18.7 nM) assays. When assayed against a 178-membered Invitrogen kinase panel (0.1 μM; 50-fold over LRRK2 Ki), 20 only inhibited 1 kinase greater than 50% (TAK1-TAB1 = 54%). In addition, 20 did not exhibit any inhibitory activity against a representative panel of receptors and ion channels. In vivo rat PK studies with 20 demonstrated a total plasma clearance of 26 mL min−1 kg−1 (Table 4) with excellent oral bioavailability (90%). As predicted by the in vitro permeability data, unbound brain-to-plasma and CSF-to-unbound plasma AUC ratios were between 0.5 and 1.0. Promising aminopyrazole inhibitors 11 and 20 were evaluated for their ability to inhibit in vivo LRRK2 Ser1292 autophosphorylation using BAC transgenic mice expressing human LRRK2 protein with the G2019S Parkinson’s disease mutation (Figure 3). Tissues from the hippocampus (brain) were harvested at 1, 3, and 6 h post intraperotineal (ip) injection at 10 and 50 mg kg−1 to evaluate pharmacodynamic knockdown.43 Using free-drug concentrations, robust concentration-dependent inhibition of Ser1292 autophosphorylation was observed for both inhibitors. In vivo unbound brain IC50 values of 3 and 20 nM were calculated for 11 and 20, respectively, using a pharmacodynamic inhibition model. Cynomolgus monkey PK studies were carried out with 11 and 20 because these inhibitors possessed well-balanced profiles and satisfied our target candidate profile criteria. Both 11 and 20 demonstrated excellent in vitro−in vivo correlations and free-drug clearance values ≤100 mL min−1 kg−1. As anticipated from in vitro human MDR1 permeability data and in vivo rat brain-penetration studies, 11 and 20 exhibited excellent brain penetration in higher species (cyno CSF/Pu = 1.2 and 1.1, respectively, Table 5). For several reasons, inhibitor 11 was chosen as a representative of the pyrazole series for further evaluation in preclinical toxicity and genotoxicity studies: (a) approximately 10-fold more potent in vivo unbound brain IC50 for 11 compared to 20, (b) in vivo multiday PK studies in rat and cyno with 11 demonstrated excellent and sustained oral exposure without evidence of autoinduction, which may suggest species-dependent nuclear receptor binding,60,61 and (c) human induction potential of 11 could be derisked in early clinical trials. In assessing the genotoxicity risk of pyrazole 11, it was determined that the inhibitor did not induce increases in micronucleated polychromatic erythrocytes after administration of 11 daily for 2 days at 200 mg kg−1 (mean unbound plasma concentration = 4.8 μM). The results of preclinical multiday toxicity studies in both rats and monkeys employing 11 and previously disclosed aminopyrimidine 1 will be disclosed shortly.

Figure 3. In vivo G2019S LRRK2 transgenic mouse PK/PD results measuring brain pSer1292 autophosphorylation. The circles represent the observed data, and the lines represent the predicted data from a direct inhibition model. Percent inhibition is normalized to pSer1292 levels observed in mice dosed with vehicle alone (n = 3). Plotted data are shown for mice treated with (A) 11 [10 and 50 mg kg−1, ip, at 1, 3, and 6 h (n = 3/dose)] and (B) 20 [10 and 50 mg kg−1, ip, at 1 and 6 h (n = 3/dose)].

trimethylsilyldiazomethane followed by debenzylation afforded pyrazoles 23. Installation of the nitro group and activation of the hydroxyl group were achieved via NBS bromination and nitration. Treatment of crude 24 with sodium hydride triggered the ring-closing alkylation to give the [5,6]-fused nitropyrazoles. Reduction of the nitropyrazole to the corresponding aminopyrazole followed by a standard SNAr reaction with 2chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (26) provided 3−6. The syntheses of 7−11 were carried out as outlined in Scheme 2. Alkylation of 5-methyl-4-nitropyrazole with methyl 2-bromo-2-methylpropanoate followed by saponification of the methyl ester yielded common intermediate 28. Amide coupling, nitro-group reduction, and acid- or base-catalyzed addition to 26 yielded inhibitors 7−10. Dehydration of primary amide 34 using phosphorus oxychloride yielded 11. Close-in analogues of 11, inhibitors 12 and 13, were prepared similarly according to Scheme 3. The preparation of 5-chloropyrazole 14 began with the cyclopropanation of acetate 39 via a bis-alkylation protocol (Scheme 4). Regioselective lithiation of the resulting nitropyrazole followed by trapping with hexachloroethane installed the C5-chloro substitution. The synthesis was then completed

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CHEMISTRY Fused bicyclic inhibitors 3−6 were prepared according to Scheme 1. Propargylation of alcohols 21 yielded the cycloaddition precursors 22. Formal cycloadditions between 22 and F

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Table 5. In Vivo Cyno PK Profiles of 11 and 20a compd

hepatocytes Clhep (mL min−1 kg−1) b cyno

% cyno PPB

CI (Clu) (mL min−1 kg−1)c

iv t1/2 (h)

F(%) 1 mg kg−1

CSF/Pud

11 20

19 13

80 3 82.1

19.7 (100) 7.8 (43)

2.2 4.0

35 74

1.2 1.1

Compounds were dosed po (1 mg kg−1) with crystalline material as an aqueous suspension with 1% methylcellulose and iv (0.5 mg kg−1) as a 20− 60% NMP solution. bIn vitro stability in cryopreserved hepatocytes. cClu = unbound clearance = total clearance/fup, where fup is the unbound plasma free fraction. dCSF/unbound plasma AUC ratio. a

give aminopyrazoles 45 and 46 (4-amino-3-methylpyrazole analogues not shown). Finally, palladium-catalyzed coupling with 26 afforded a mixture of the isomeric products, which were separated by preparative HPLC. Piperidine-based inhibitors 18−20 were synthesized as outlined in Schemes 7 and 8. Installation of the N-piperidinyl moiety was achieved via alkylation or Mitsunobu reactions. NBoc deprotection by treatment with HCl in dioxane and reductive amination using oxetan-3-one provided the N-oxetylpiperidine intermediates. Installation of the 5-chloro substitution to give 53 (Scheme 8) was accomplished via lithiation and hexachloroethane trapping as described above. Standard reduction of the nitropyrazole using either iron or palladiumcatalyzed hydrogenation set the stage for the acid-catalyzed SNAr reactions to give the final inhibitors as enantiomeric mixtures. The individual enantiomers were separated by SFC purification.

Scheme 1. Synthesis of Fused Morpholinopyrazole Analoguesa

Reagents and conditions: (a) NaH, 3-bromoprop-1-yne, THF, 0 °C; (b) Me3SiCHN2, 135 °C, microwave; (c) H2, Pd(OH)2, EtOH, 100 °C; (d) NBS, CH3CN; (e) HNO3, 0 °C; (f) NaH, DMF, 0 °C; (g) hydrazine hydrate, Raney nickel, THF, MeOH, 0 °C; (h) 26, t-BuOH, 100 °C. a

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CONCLUSIONS Expanding upon the identification of aminopyrazoles as aniline bioisosteres, we identified 11 and 20 as highly efficient, LRRK2specific small molecule inhibitors with ADME profiles suitable for multiday efficacy and toxicity studies. Both inhibitors demonstrated robust, concentration-dependent knockdown of pLRRK2 in the brain of G2019S transgenic mice expressing human LRRK2 protein. Advantages of the aminopyrazole series include elimination of potential anilino-derived ortho-quinoneimine reactive metabolite formation, good overall aqueous solubility, and low free-drug clearance values demonstrated by multiple compounds. The aminopyrazole scaffold is also structurally differentiated from the majority of LRRK2 inhibitors disclosed in the literature, which should prove beneficial in assessing the risk of prolonged inhibition of LRRK2 in vivo. A detailed report of toxicity studies and associated histopathology findings with 11 and previously disclosed anilino-aminopyrimidine LRRK2 inhibitor 1 will be reported shortly.

Scheme 2. Synthesis of N-Alkyl Pyrazole Analoguesa

a

Reagents and conditions: (a) NaH, methyl 2-bromo-2-methylpropanoate, DMF, 70%; (b) LiOH, THF-H2O, 90%; (c) (i) (COCl)2, CH2Cl2, (ii) R-NH2, THF; (d) Pd/C, H2, MeOH; (e) 26, Et3N, nBuOH, 120 °C; (f) 26, TFA, 2-methoxyethanol, 70 °C; (g) POCl3, 90 °C, 42%.

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EXPERIMENTAL SECTION

All chemicals were purchased from commercial suppliers and used as received. Flash chromatography was carried out with prepacked SiO2 cartridges from either ISCO or SiliCycle on an ISCO CombiFlash chromatography system using gradient elution or with prepacked silica gel cartridges from Biotage using a Biotage SP4 or an Isolara 4 MPLC system using gradient elution. NMR spectra were recorded on a Bruker Avance 400, Bruker DPX 400M, or Bruker Avance III 400 or 500 NMR spectrometers and referenced to tetramethylsilane. The following abbreviations are used: br = broad signal, s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, and m = multiplet. Preparative HPLC was performed on a Polaris C18 5 μM column (50 × 21 mm), a Waters Sunfire OBD Phenomenex Luna Phenyl Hexyl column (150 × 19 mm), or a Waters Xbridge Phenyl column (150 × 19 mm), eluting with mixtures of water−acetonitrile or water−methanol, optionally containing a modifier (0.1% v/v formic

in a manner analogous to that which yielded compound 11. Clinked analogue 15 was prepared via a straightforward two-step sequence (Scheme 5). Cyclization between methylhydrazine and 2,2-dimethyl-3-oxopentanedinitrile (42) provided aminopyrazole 43. Acid-catalyzed coupling with 2-chloro-N-ethyl-5(trifluoromethyl)pyrimidin-4-amine (44) yielded 15. Compounds 16 and 17 were synthesized according to Scheme 6. Alkylation of 5-methyl-4-nitropyrazole yielded an isomeric mixture of the corresponding N-alkyl-5-methyl-4nitropyrazole and N-alkyl-3-methyl-4-nitropyrazole. The mixtures of regioisomers were then reduced via hydrogenation to G

dx.doi.org/10.1021/jm401654j | J. Med. Chem. XXXX, XXX, XXX−XXX

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Scheme 3. Synthesis of Analogues 12 and 13a

a

Reagents and conditions: (a) Cs2CO3, methyl 2-bromoacetate, DMF, 89%; (b) NaH, CH3I, DMF, 39%; (c) (i) LiOH, THF, H2O, (ii) (COCl)2, cat. DMF, DCM, (iii) NH4OH, THF, 86%; (d) Pd/C, H2, EtOH; (e) 26, TFA, 2-methoxyethanol, 90 °C; (f) POCl3, 90 °C; (g) NaH, ethylene dibromide, DMF, 0 °C, 40%.

Scheme 4. Synthesis of Inhibitor 14a

Reagents and conditions: (a) NaH, ethylene dibromide, DMF, 0 °C, 20%; (b) LiHMDS, Cl3CCCl3, −78 °C, THF, 54%; (c) (i) LiOH, water, THF, (ii) (COCl)2, cat. DMF, CH2Cl2, (iii) NH4OH, THF, 69%, three steps; (d) iron dust, NH4Cl, EtOH, quant.; (e) 26, TFA, 2-methoxyethanol, 70 °C; (f) POCl3, 90 °C, 30%, two steps.

a

Scheme 5. Synthesis of Inihibitor 15a

Scheme 7. Synthesis of N-Piperidinyl Inhibitorsa

a

Reagents and conditions: (a) methylhydrazine sulfate, HCl, EtOH, reflux, 28%; (b) 44, TFA, n-BuOH, 100 °C, microwave, 11%.

Scheme 6. Synthesis of Inhibitors 16 and 17a

a

Reagents and conditions: (a) Cs2CO3, N-Boc-4-bromopiperidine, DMF, 120 °C, 52%; (b) (±)-(cis)-tert-butyl 3-fluoro-4-hydroxypiperidine-1-carboxylate, PPh3, diisopropyl azodicarboxylate, THF, quant.; (c) HCl, 1,4-dioxane, 55 °C; (d) oxetan-3-one, DIPEA, NaBH(OAc)3, acetic acid, DCE; (e) Pd/C, H2 , EtOH; (f) 26, TFA, 2methoxyethanol, 95 °C. acid or 10 mM ammonium bicarbonate). Low-resolution mass spectra were recorded on a Sciex 15 mass spectrometer in ES+ mode, a Micromass ZQ single quadrapole LC−MS in ES+, ES− mode, or a Quattro Micro LC-MS-MS in ES+, ES− mode. All final compounds were purified to >95% chemical and optical purity, as assayed by either (a) HPLC (Waters Acquity UPLC column 21 × 50 mm, 1.7 μM) with gradient of 0−90% acetonitrile (containing 0.038% TFA) in 0.1% aqueous TFA, with UV detection at λ = 254 and 210 as well as CAD detection with an ESA Corona detector, (b) HPLC (Phenomenex Luna C18 (2) column 4.6 × 100 mm, 5 μM) with gradient of 5−95% acetonitrile in water (with 0.1% formic acid in each mobile phase), with UV DAD detection between λ = 210 and 400 nm, (c) HPLC

a Reagents and conditions: (a) Cs2CO3, 3-iodo-oxetane or 4-bromotetrahydropyran, 100 °C, DMF; (b) Pd/C, H2, EtOH; (c) 26, Cs2CO3, XPhos, Pd2(dba)3, 1,4-dioxane, 135 °C, microwave.

H

dx.doi.org/10.1021/jm401654j | J. Med. Chem. XXXX, XXX, XXX−XXX

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Scheme 8. Synthesis of Inhibitor 20a

propanamide (7). To the solution of N,2-dimethyl-2-(3-methyl-4nitro-1H-pyrazol-1-yl)propanamide (29, 0.4 g, 1.77 mmol) in MeOH (25 mL) was added Pd/C (80 mg, 10 wt %). The reaction was stirred under a hydrogen atmosphere (1 atm) for 1 h. The mixture was filtered through Celite and concentrated under reduced pressure to give crude 2-(4-amino-3-methyl-1H-pyrazol-1-yl)-N,2-dimethylpropanamide (0.41 g), which was used in the next step without further purification. To a solution of 2-(4-amino-3-methyl-1H-pyrazol-1-yl)-N,2-dimethylpropanamide (0.41 g, 1.83 mmol) in n-BuOH (15 mL) were added 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (26, 0.43 g, 2.01 mmol) and triethylamine (0.77 mL, 5.49 mmol), and the reaction mixture was stirred at 120 °C for 1 h before it was cooled to room temperature. Water (20 mL) was added, the aqueous phase was extracted with ethyl acetate (3×), and the combined organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a residue that was purified by silica gel chromatography (CH2Cl2/MeOH, 20:1) to give 7 (0.18 g, 23% yield) as a white solid. 1H NMR (400 MHz, DMSO) δ 9.09 (s, 1H), 8.14 (s, 1H), 8.10 (s, 1H), 7.20 (s, 1H), 2.90 (s, 3H), 2.55 (s, 3H), 2.18 (s, 3H), 1.63 (s, 6H). LCMS, m/z = 372 [M + H]+. N-Ethyl-2-methyl-2-(3-methyl-4-((4-(methylamino)-5(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)propanamide (8). The title compound was prepared in a manner analogous to 7 (37% yield). 1H NMR (500 MHz, CDCl3) δ 8.08 (s, 1H), 7.91 (s, 1H), 6.55 (brs, 1H), 5.36 (brs, 1H), 5.19 (brs, 1H), 3.23−3.28 (m, 2H), 3.03 (d, J = 4.5 Hz, 3H), 2.19 (s, 3H), 1.82 (s, 6H), 1.06 (t, 3H). LCMS, m/z = 386 [M + H]+. N-Isopropyl-2-methyl-2-(3-methyl-4-((4-(methylamino)-5(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)propanamide (9). The title compound was prepared in a manner analogous to 7. 1H NMR (500 MHz, CD3OD) δ 8.03 (s, 1H), 7.85 (s, 1H), 4.00−3.97 (m, 1H), 2.99 (s, 3H), 2.23 (s, 3H), 1.76 (s, 6H), 1.11 (d, J = 7.0 Hz, 6H). LCMS, m/z = 400 [M + H]+. 2-Methyl-2-(3-methyl-4-((4-(methylamino)-5(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-N(2,2,2-trifluoroethyl)propanamide (10). The title compound was prepared in a manner analogous to 7. 1H NMR (500 MHz, CDCl3) δ 8.13 (s, 2H), 7.02 (s, 1H), 6.79 (s, 1H), 5.24 (s, 1H), 3.87−3.80 (m, 2H), 3.05 (d, J = 4.5 Hz, 3H), 2.30 (s, 3H), 1.84 (s, 6H). LCMS, m/z = 440 [M + H]+. 2-Methyl-2-(3-methyl-4-((4-(methylamino)-5(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)propanenitrile (11). A solution of 2-methyl-2-(3-methyl-4-((4(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol1-yl)propanamide (34, 250 mg, 0.7 mmol) in POCl3 (5 mL) was stirred at 90 °C for 1 h. The POCl3 was removed by evaporation. The mixture was then slowly poured onto ice (10 mL). The pH of the solution was adjusted to 8 with saturated sodium carbonate. The aqueous phase was extracted with EtOAc (3×). The combined organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a residue that was purified by recrystallization to give 11 (100 mg, 42% yield) as a white solid. 1H NMR (300 MHz, DMSO) δ 9.18 (s, 1H), 8.29 (s, 1H), 8.14 (s, 1H), 7.10 (s, 1H), 2.91 (d, 3H), 2.22 (s, 3H), 1.94 (s, 6H). HRMS (ES) m/ z: [M + H]+ calcd for C14H16F3N7H+, 340.1492; found, 340.1484. 2-Methyl-2-(5-methyl-4-((4-(methylamino)-5(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)propanenitrile (12). The title compound was synthesized from 2-(4amino-5-methyl-1H-pyrazol-1-yl)-2-methylpropanamide (36) in a manner analogous to 11 (17% yield). 1H NMR (400 MHz, DMSO) δ 8.93 (s, 1H), 8.07 (s, 1H), 7.79 (s, 1H), 6.95 (s, 1H), 2.83 (s, 3H), 2.41 (s, 3H), 1.95 (s, 6H). LCMS, m/z = 340 [M + H]+. 1-(3-Methyl-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)cyclopropanecarbonitrile (13). A mixture of 1-(4-amino-3-methyl1H-pyrazol-1-yl)cyclopropanecarboxamide (38, 0.30 g, 1.7 mmol), 2chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (26, 0.40 g, 1.9 mmol), and trifluoroacetic acid (0.1 mL) in 2-methoxyethanol (5 mL) was stirred at 90 °C for 45 min. The reaction was cooled to room temperature and diluted with saturated sodium bicarbonate. The

a

Reagents and conditions: (a) (±)-(cis)-tert-butyl 3-fluoro-4-hydroxypiperidine-1-carboxylate, PPh3, diisopropyl azodicarboxylate, THF; (b) TFA, DCM, 58% over two steps; (c) oxetan-3-one, DIPEA, NaBH(OAc)3, acetic acid, DCE, 85%; (d) LiHMDS then C2Cl6, THF, −78 °C, 65%; (e) iron dust, NH4Cl, EtOH, 90 °C; (f) 26, TFA, 2methoxyethanol, 90 °C, 40%, two steps. (Waters Xterra MS C18 column 4.6 × 100 mm, 5 μM) with gradient of 5−95% acetonitrile in water (with 10 mM ammonium bicarbonate in the aqueous mobile phase), with UV DAD detection between λ = 210 and 400 nm, (d) HPLC (Supelco, Ascentis Express C18 or Hichrom Halo C18 column 4.6 × 150 mm, 2.7 μM) with gradient of 4−100% acetonitrile in water (with 0.1% formic acid in each mobile phase), with UV DAD detection between λ = 210 and 400 nm, or (e) HPLC (Phenomenex, Gemini NX C18 column 4.6 × 150 mm, 3 μM) with gradient of 4.5−100% acetonitrile in water (with 10 mM ammonium bicarbonate in the aqueous mobile phase), with UV DAD detection between λ = 210 and 400 nm. The synthesis of compound 2 was described previously.26 N2-(6,6-Dimethyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin3-yl)-N4-methyl-5-(trifluoromethyl)pyrimidine-2,4-diamine (3). To a solution of 6,6-dimethyl-3-nitro-6,7-dihydro-4H-pyrazolo[5,1c][1,4]oxazine (25, 35.0 mg, 0.177 mmol) in THF/MeOH (3 mL/3 mL) were added Raney Ni (100 mg) and hydrazine hydrate (1 mL) at 0 °C. After being stirred for 16 h, the insoluble material was filtered off. The filtrate was concentrated in vacuo to give 6,6-dimethyl-6,7dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-3-amine (26 mg, 89% yield). LCMS, m/z = 168 [M + H]+. A microwave vial equipped with a magnetic stirrer was charged with 6,6-dimethyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-3-amine (13 mg, 0.078 mmol), 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4amine (26, 20 mg, 0.095 mmol), and t-BuOH (0.5 mL). The mixture was heated at 100 °C under microwave irradiation for 15 min. After concentration, the residue was purified by preparative HPLC to give 3 (9.5 mg, 36% yield). 1H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H), 7.67 (s, 1H), 6.79 (s, 1H), 5.18 (s, 1H), 4.83 (s, 2H), 3.93 (s, 2H), 3.00 (d, J = 4.5 Hz, 3H), 1.63 (s, 6H). LCMS, m/z = 343 [M + H]+. N 2 -(6,7-Dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-3-yl)-N 4 methyl-5-(trifluoromethyl)pyrimidine-2,4-diamine (4). The title compound was prepared in a manner analogous to 3 (34% yield). 1H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.60 (s, 1H), 6.37 (s, 1H), 5.13 (s, 1H), 4.81 (s, 2H), 4.19 (t, J = 5.0 Hz, 2H), 4.11 (t, J = 5.0 Hz, 2H), 3.01 (d, J = 4.8 Hz, 3H). LCMS, m/z = 315 [M + H]+. N2-((6S)-4,6-Dimethyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-3-yl)-N4-methyl-5-(trifluoromethyl)pyrimidine-2,4-diamine (5). The title compound was prepared in a manner analogous to 3 (22% yield). 1H NMR (400 MHz, DMSO) δ 9.10 8.57 (m, 1H), 8.06 (s, 1H), 7.81 7.34 (m, 1H), 6.95 (s, 1H), 5.31 (s, 1H), 4.29 4.18 (m, 1H), 4.09 (dd, J = 12.4, 3.1 Hz, 1H), 3.64 (dd, J = 12.3, 9.5 Hz, 1H), 2.85 (s, 3H), 1.31 (d, J = 5.6 Hz, 3H), 1.25 (d, J = 6.2 Hz, 3H). LCMS, m/z = 343 [M + H]+. N 4 -Methyl-N 2 -(4-methyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-3-yl)-5-(trifluoromethyl)pyrimidine-2,4-diamine (6). The title compound was prepared in a manner analogous to 3 (14% yield). 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.55 (s, 1H), 5.15 (s, 1H), 4.96 (m, 1H), 4.32−3.93 (m, 4H), 2.99 (d, J = 4.7 Hz, 3H), 1.47 (d, J = 6.5 Hz, 3H). LCMS, m/z = 329 [M + H]+. N , 2 - D i m e t h y l - 2 - ( 3 -m e t h y l -4 -( (4 - ( m e t h yl a m i n o ) - 5 (trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)I

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to give the title compound (68 mg, 26% yield). 1H NMR (400 MHz, DMSO) δ 8.84 (s, 1H), 8.04 (s, 1H), 7.71 (s, 1H), 6.89 (s, 1H), 4.54 (t, J = 6.5 Hz, 2H), 4.43 (t, J = 6.1 Hz, 2H), 4.07 (s, 1H), 3.45− 3.43 (m, 1H), 2.89−2.79 (m, 5H), 2.12 (s, 3H), 2.04−1.72 (m, 6H). LCMS, m/z = 412 [M + H]+. N2-(1-((cis)-3-Fluoro-1-(oxetan-3-yl)piperidin-4-yl)-3-methyl1H-pyrazol-4-yl)-N4-methyl-5-(trifluoromethyl)pyrimidine-2,4diamine (19). The title compound was prepared in a manner analogous to 18 (11% yield). The enantiomers were separated by chiral SFC purification. 1H NMR (400 MHz, DMSO) δ 8.89 (s, 1H), 8.05 (s, 1H), 7.78 (s, 1H), 6.92 (s, 1H), 4.86 (m, 1H), 4.56 (m, 2H), 4.46 (m, 2H), 4.27 (m, 1H), 3.58 (m, 1H), 3.22−3.11 (m, 1H), 2.96− 2.71 (m, 4H), 2.29−1.98 (m, 6H), 1.90 (m, 1H). LCMS, m/z = 430 [M + H]+. N2-(5-Chloro-1-((trans)-3-fluoro-1-(oxetan-3-yl)piperidin-4yl)-1H-pyrazol-4-yl)-N4-methyl-5-(trifluoromethyl)pyrimidine2,4-diamine (20). A mixture of (±)-(trans)-4-(5-chloro-4-nitro-1Hpyrazol-1-yl)-3-fluoro-1-(oxetan-3-yl)piperidine (53, 2.2 g, 3.9 mmol), iron dust (1.6 g, 29 mmol), and ammonium chloride (1.5 g, 29 mmol) in ethanol (20 mL) was stirred at 90 °C for 30 min. The reaction was filtered and concentrated. The residue was sonicated with 100 mL of EtOAc for 5 min. The mixture was filtered to remove all insoluble solids. The filtrate was then concentrated to give crude (±)- (trans)-4(5-chloro-4-amino-pyrazol-1-yl)-3-fluoro-1-(oxetan-3-yl)piperidine (1.9 g). To a mixture of the crude (±)-(trans)-4-(5-chloro-4-amino-pyrazol1-yl)-3-fluoro-1-(oxetan-3-yl)piperidine (1.9 g) and 2-chloro-Nmethyl-5-(trifluoromethyl)pyrimidin-4-amine (26, 1.5 g, 6.9 mmol) in 2-methoxyethanol (25 mL) was added TFA (0.60 mL, 7.7 mmol). The reaction was stirred at 90 °C for 15 min. The mixture was then diluted with saturated sodium bicarbonate and extracted with EtOAc (3×). The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by preparative HPLC, chiral SFC, and recrystallized in isopropanol to give 20 (0.70 g, 40% yield). 1H NMR (400 MHz, DMSO) δ 8.91 (s, 1H), 8.08 (s, 1H), 7.87 (s, 1H), 7.00 (s, 1H), 5.03 4.79 (m, 1H), 4.56 (m, 1H), 4.46 (m, 2H), 3.68−3.51 (m, 1H), 3.26− 3.12 (m, 1H), 2.92−2.73 (m, 3H), 2.54 (s, 2H), 2.20−1.88 (m, 3H). HRMS (ES) m/z: [M + H]+ calcd for C17H20ClF4N7OH+, 450.1427; found, 450.1418. ((2-Methyl-2-(prop-2-ynyloxy)propoxy)methyl)benzene (22). To a mixture of NaH (5.0 g) in THF (50 mL) at 0 °C was added dropwise 1-(benzyloxy)-2-methylpropan-2-ol (21, 9.36 g, 51.9 mmol). After being stirred at room temperature for 30 min, 3-bromoprop-1yne (12.4 g, 104 mmol) was added dropwise at 0 °C. The mixture was then stirred overnight at reflux. The reaction was quenched by the addition of saturated ammonium chloride. The resulting mixture was extracted with ethyl acetate (3×). The organic layer was combined, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/EtOAc, 20:1) to afford the title compound (9.7 g, 86% yield) as pale-yellow oil. 1H NMR (500 MHz, CDCl3) δ 7.34−7.35 (m, 5H), 4.56 (s, 2H), 4.18 (d, J = 2.0 Hz, 2H), 3.38 (s, 2H), 2.34 (t, J = 2.0 Hz, 1H), 1.25 (s, 6H). 2-((1H-Pyrazol-5-yl)methoxy)-2-methylpropan-1-ol (23). A mixture of ((2-methyl-2-(prop-2-ynyloxy)propoxy)methyl)benzene (1.00 g, 4.58 mmol) and (diazomethyl)trimethylsilane (2.29 mL) was stirred at 135 °C under microwave irradiation for 1 h. Removal of the solvent under vacuum afforded 3-((1-(benzyloxy)-2-methylpropan-2-yloxy)methyl)-1H-pyrazole (1.19 g, 80% yield). The crude residue was carried to the next step without further purification. LCMS, m/z = 261 [M + H]+. A mixture of 3-((1-(benzyloxy)-2-methylpropan-2-yloxy)methyl)1H-pyrazole (5.5 g, 21 mmol) and 10% Pd(OH)2 on carbon (2.2 g) in EtOH (100 mL) was stirred at 100 °C under H2 (4 atm) for 12 h. The insoluble material was filtered off, and the filtrate was concentrated to afford 2-((1H-pyrazol-5-yl)methoxy)-2-methylpropan-1-ol (3.0 g, 83% yield). LCMS, m/z = 171 [M + H]+. 2-Methyl-2-((4-nitro-1H-pyrazol-5-yl)methoxy)propyl Nitrate (24). To a solution of 2-((1H-pyrazol-3-yl)methoxy)-2methylpropan-1-ol (3.00 g, 17.6 mmol) in acetonitrile (30 mL) was

product was then extracted with EtOAc (3×). The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel chromatography (0−100% EtOAc in heptane) to give 1-[3-methyl-4[[4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl]amino]pyrazol1-yl]cyclopropanecarboxamide (0.31 g, 50%). LCMS, m/z = 256 [M + H]+. A mixture of 1-[3-methyl-4-[[4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl]amino]pyrazol-1-yl]cyclopropanecarboxamide (0.30 g, 0.84 mmol) in POCl3 (3 mL) was stirred at 90 °C for 1 h. The reaction was slowly poured onto an ice-cold 1 M sodium carbonate solution. The product was extracted with EtOAc (3×). The combined extracts were washed with saturated sodium bicarbonate and then brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by preparative HPLC to give 13 (176 mg, 62% yield). 1H NMR (400 MHz, DMSO) δ 9.09 (s, 1H), 8.12 (s, 2H), 7.03 (s, 1H), 2.91 (d, J = 4.4 Hz, 3H), 2.16 (s, 3H), 1.87 1.78 (m, 2H), 1.78 1.70 (m, 2H). LCMS, m/z = 338. [M + H]+. 1-(5-Chloro-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)cyclopropanecarbonitrile (14). The title compound was prepared in a manner analogous to 11 (yield =30%). 1H NMR (400 MHz, DMSO) δ 9.12 (s, 1H), 8.11 (s, 1H), 7.94 (s, 1H), 7.06 (m, 1H), 2.84 (d, J = 2.7 Hz, 3H), 2.10−1.98 (m, 2H), 1.89 1.76 (m, 2H). LCMS, m/z = 358 [M + H]+. 2-(5-((4-(Ethylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1-methyl-1H-pyrazol-3-yl)-2-methylpropanenitrile (15). To a solution of 2-(5-amino-1-methyl-1H-pyrazol-3-yl)-2methylpropanenitrile (43, 100 mg, 0.61 mmol) in n-BuOH (1 mL) were added 2-chloro-N-ethyl-5-(trifluoromethyl) pyrimidin-4-amine (44, 137 mg, 0.61 mmol) and a drop of trifluoroacetic acid. After heating in microwave oven at 100 °C for 1 h, the resulting solution was concentrated in vacuo, and the residue was purified by preparative HPLC to afford the title compound as a white solid (23 mg, 11%). 1H NMR (500 MHz, DMSO) δ 9.54 (s, 1H), 8.17 (s, 1H), 7.24 (s, 1H), 6.36 (s, 1H), 3.67 (s, 3H), 3.44−3.38 (m, 2H), 1.62 (s, 6H), 1.10 (t, J = 7.0 Hz, 3H). LCMS, m/z = 354 [M + H]+. N4-Methyl-N2-(5-methyl-1-(oxetan-3-yl)-1H-pyrazol-4-yl)-5(trifluoromethyl)pyrimidine-2,4-diamine (16). A microwave tube was charged a mixture of 3-methyl-1-(oxetan-3-yl)-1H-pyrazol-4amine and 5-methyl-1-(oxetan-3-yl)-1H-pyrazol-4-amine (45, 0.26 g, 1.7 mmol), 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (26, 0.28 g, 1.3 mmol), cesium carbonate (0.87 g, 2.7 mmol), XPhos (64 mg, 0.13 mmol), tris(dibenzylideneacetone)dipalladium(0) (61 mg, 0.067 mmol), and 1,4-dioxane (16 mL). The reaction was sealed and heated in a microwave reactor at 135 °C for 30 min. The reaction mixture was then filtered and concentrated. The crude product was purified by preparative HPLC to give 16 (22 mg, 4.9% yield). 1H NMR (400 MHz, DMSO) δ 8.91 (s, 1H), 8.05 (s, 1H), 7.86 (s, 1H), 6.94 (s, 1H), 5.53 (m, 1H), 4.95−4.91 (m, 2H), 4.90 4.83 (m, 2H), 2.85 (s, 3H), 2.14 (s, 3H). LCMS, m/z = 329 [M + H]+. N4-Methyl-N2-(5-methyl-1-(tetrahydro-2H-pyran-4-yl)-1Hpyrazol-4-yl)-5-(trifluoromethyl)pyrimidine-2,4-diamine (17). The title compound was prepared in a manner analogous to 16 (yield =8.2%). 1H NMR (400 MHz, DMSO) δ 8.83 (s, 1H), 8.04 (s, 1H), 7.65 (s, 1H), 6.89 (s, 1H), 4.33 (m, 1H), 3.95 (m, 2H), 3.47 (m, 2H), 2.84 (s, 3H), 2.21 (s, 3H), 2.02 (m, 2H), 1.82 1.67 (m, 2H). LCMS, m/z = 357 [M + H]+. N4-Methyl-N2-(3-methyl-1-(1-(oxetan-3-yl)piperidin-4-yl)1H-pyrazol-4-yl)-5-(trifluoromethyl)pyrimidine-2,4-diamine (18). A suspension of 4-(3-methyl-4-nitro-1H-pyrazol-1-yl)-1-(oxetan3-yl)piperidine (49) and minor amount of 4-(5-methyl-4-nitropyrazol-1-yl)-1-(oxetan-3-yl)piperidine (0.158 g, 0.593 mmol) and palladium on carbon (10 wt %, 0.1 g) in ethanol (15 mL) was stirred under a hydrogen atmosphere for 18 h. The reaction mixture was then filtered through Celite and concentrated. The residue was redissolved in 2-methoxythanol (3 mL) and mixed with 2-chloro-N-methyl-5(trifluoromethyl)pyrimidin-4-amine (26, 0.13 g, 0.63 mmol) and TFA (0.1 mL, 1 mmol). The resulting mixture was stirred at 95 °C for 1 h. The reaction was allowed to cool to room temperature and concentrated. The crude product was purified by preparative HPLC J

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N-Ethyl-2-methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)propanamide (30). The title compound was prepared in a manner analogous to 29. LCMS, m/z = 241 [M + H]+. N-Isopropyl-2-methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)propanamide (31). The title compound was prepared in a manner analogous to 29. LCMS, m/z = 255 [M + H]+. 2-Methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)-N-(2,2,2trifluoroethyl)propanamide (32). The title compound was prepared in a manner analogous to 29. LCMS, m/z = 295 [M + H]+. 2-Methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)propanamide (33). To the solution of 2-methyl-2-(3-methyl-4-nitro-1H-pyrazol-1yl)propanoic acid (2.5 g, 11.7 mmol) in CH2Cl2 (50 mL) was added dropwise oxalyl chloride (2.97 g, 23.4 mmol). The reaction was stirred at room temperature for 2 h and then concentrated under reduced pressure to remove the solvent. The residual solid was dissolved in THF (30 mL), which was then added dropwise into a stirred solution of ammonium hydroxide (50 mL). The reaction was stirred at room temperature for 1 h. The solution was then concentrated under reduced pressure and partitioned between EtOAc (50 mL) and water (100 mL), and the aqueous phase was extracted with EtOAc (3×). The combined organic was washed with saturated ammonium chloride (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product 2-methyl-2-(3-methyl-4nitro-1H-pyrazol-1-yl)propanamide (2.5 g, quant.) as white solid that was used in the next step without further purification. 2-Methyl-2-(3-methyl-4-((4-(methylamino)-5(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)propanamide (34). To the solution of 2-methyl-2-(3-methyl-4-nitro1H-pyrazol-1-yl)propanamide (2.5g, 11.7 mmol) in MeOH (50 mL) was added Pd/C (1 g, 10 wt %). The reaction was stirred under a hydrogen atmosphere (1 atm) for 1 h at room temperature. The solution was filtered and concentrated under reduced pressure to give the crude 2-(4-amino-3-methyl-1H-pyrazol-1-yl)-2-methylpropanamide (2.0 g, 93% yield), which was used in the next step without further purification. To a solution of 2-(4-amino-3-methyl-1H-pyrazol-1-yl)-2-methylpropanamide (250 mg, 1.37 mmol) in 2-methoxyethanol (5 mL) were added 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (290 mg, 1.37 mmol) and trifluoroacetic acid (156 mg, 1.37 mmol). The reaction was stirred at 70 °C for 30 min. The reaction mixture was cooled to room temperature followed by the addition of water (10 mL). The pH of the aqueous solution was adjusted to 8 by adding saturated sodium carbonate. The aqueous phase was extracted with EtOAc (3×). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give a residue that was purified by silica gel chromatography (CH2Cl2/MeOH, 20:1) to give 2-methyl-2-(3-methyl-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-1H- pyrazol-1-yl)propanamide (250 mg, 51% yield) as white solid.). LCMS, m/z = 358 [M + H]+. Methyl 2-(5-Methyl-4-nitro-1H-pyrazol-1-yl)acetate and methyl 2-(3-methyl-4-nitro-1H-pyrazol-1-yl)acetate (35 and 37). A microwave vial equipped with a magnetic stirrer was charged with methyl 2-bromoacetate (7.22 g, 47.2 mmol), 5-methyl-4nitropyrazole (5 g, 39.4 mmol), cesium carbonate (19.2 g, 59.1 mmol), and DMF (40 mL). The reaction mixture was stirred at room temperature for 1 h. It was then filtered to remove all insoluble solids. The filtrate was concentrated and purified by silica gel chromatography (petroleum ether/EtOAc, 3:1) to afford the mixture of methyl 2-(5methyl-4-nitro-1H-pyrazol-1-yl)acetate and methyl 2-(3-methyl-4nitro-1H-pyrazol-1-yl)acetate as a yellow oil (7.0 g, 89% yield). LCMS, m/z = 200 [M + H]+. 2-(4-Amino-5-methyl-1H-pyrazol-1-yl)-2-methylpropanamide (36). To a mixture of methyl 2-(5-methyl-4-nitro-1H-pyrazol-1yl)acetate and methyl 2-(3-methyl-4-nitro-1H-pyrazol-1-yl)acetate (4.5 g, 23 mmol) in DMF (50 mL) were added NaH (60% dispersion, 2.3 g, 56 mmol) and iodomethane (3.5 mL, 56 mmol). The reaction was stirred at room temperature for 4 h. The mixture was diluted with saturated ammonium chloride and extracted with EtOAc (3×). The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel chromatography (0−100% EtOAc in heptane) to give a mixture of

added NBS (3.45g, 19.4 mmol) in one portion. After overnight stirring, the mixture was concentrated. The residue was purified by preparative HPLC to give 2-((4-bromo-1H-pyrazol-5-yl)methoxy)-2methylpropan-1-ol (1.8 g, 41%). LCMS, m/z = 249 [M + H]+. Fuming nitric acid (3.0 mL) was added to 2-((4-bromo-1H-pyrazol3-yl)methoxy)-2-methylpropan-1-ol (300 mg, 1.2 mmol) at 0 °C. After being stirred for 1 h at 0 °C, the reaction was quenched with ice. The mixture was extracted with ethyl acetate (3×). The organic layers were washed with water and brine, dried over sodium sulfate, and concentrated to give crude 2-methyl-2-((4-nitro-1H-pyrazol-5-yl)methoxy)propyl nitrate (260 mg, 52% yield). LCMS, m/z = 261 [M + H]+. 6,6-Dimethyl-3-nitro-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine (25). To a cooled (0 °C) solution of 2-methyl-2-((4-nitro1H-pyrazol-5-yl)methoxy)-propyl nitrate (580 mg, 1.12 mmol) in DMF (15 mL) was added NaH (89.0 mg, 2.23 mmol, 60% dispersion). The mixture was stirred overnight at room temperature. This reaction was quenched by ice, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic layers were washed with water and brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with (5−20% EtOAc in petroleum ether) to give 6,6-dimethyl-3-nitro6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine (43.5 mg, 20% yield). LCMS, m/z = 198 [M + H]+. 2-Methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)propanoic Acid (28). To a solution of 5-methyl-4-nitropyrazole (27, 10 g, 78.6 mmol) in DMF (50 mL) was added NaH (4.72 g, 118.0 mmol, 60% dispersion) in several portions. The reaction mixture was stirred at room temperature for 2 h followed by the addition of methyl 2-bromo2-methylpropanoate (21.3 g, 118.0 mmol). The resulting mixture was stirred for an additional 12 h before water (250 mL) was added to quench the reaction. The aqueous phase was extracted with EtOAc (3×), and the combined organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give a residue that was purified by silica gel chromatography to give methyl 2-methyl-2-(3-methyl-4-nitro-1Hpyrazol-1-yl)propanoate (11.5 g, 80% yield) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 3.78 (s, 3H), 2.57 (s, 3H), 1.89 (s, 6H). LCMS, m/z = 228 [M + H]+. To a solution of methyl 2-methyl-2-(3-methyl-4-nitro-1H-pyrazol-1yl)propanoate (2 g, 8.81 mmol) in THF (40 mL) and water (10 mL) was added LiOH (250 mg, 10.6 mmol), and the resulting mixture was stirred at room temperature for 5 h. THF was removed under reduced pressure, the aqueous phase was extracted with EtOAc (2×), and the organic phases were discarded. The pH value of the aqueous phase was adjusted to 1 to 2 by adding a 3 N HCl solution, and the mixture was extracted with EtOAc (3×). The combined organic phase was washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give crude 28 (1.4 g, 74% yield). 1H NMR (300 MHz, CD3OD) δ 8.71 (s, 1H), 2.53 (d, J = 1.1 Hz, 3H), 1.88 (s, 6H). N,2-Dimethyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)propanamide (29). To the solution of 2-methyl-2-(3-methyl-4-nitro1H-pyrazol-1-yl)propanoic acid (1.4 g, 6.47 mmol) in CH2Cl2 (30 mL) was added dropwise oxalyl chloride (1.64 g, 12.9 mmol), and the reaction was stirred at room temperature for 2 h. The reaction mixture was then concentrated under reduced pressure to remove the solvent. The residual solid was redissolved in THF (30 mL), and methylamine (6.5 mL, 12.94 mmol, 2 M in THF) was added dropwise. The reaction was stirred at room temperature for 12 h. The reaction solution was concentrated under reduced pressure and partitioned between EtOAc (15 mL) and water (10 mL), and the aqueous phase was extracted with EtOAc (2×). The combined organic phase was washed with saturated ammonium chloride (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give a residue that was purified by silica gel chromatography (petroleum ether/EtOAc, 2:1) to give 29 (920 mg, 63% yield) as white solid. 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 6.29 (s, 1H), 2.79 (d, J = 4.8 Hz, 3H), 2.58 (s, 3H), 1.84 (s, 6H). LCMS, m/z = 358 [M + H]+. K

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brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel chromatography (0−5% CH3OH in CH2Cl2) to give 1-(3-methyl-4-nitro-pyrazol-1-yl)cyclopropanecarboxamide (0.53 g, 86% over two steps). LCMS, m/z = 211 [M + H]+. A suspension of 1-(3-methyl-4-nitro-pyrazol-1-yl)cyclopropanecarboxamide (0.50 g, 2.4 mmol) and palladium on carbon (10 wt %, 0.2 g) in EtOH (5 mL) was stirred under a hydrogen atmosphere (1 atm) for 3 h. The reaction was then filtered through Celite and concentrated. The crude material (0.30 g, 71% yield) was used in the next step without purification. LCMS, m/z = 181 [M + H]+. Methyl 1-(4-Nitro-1H-pyrazol-1-yl)cyclopropanecarboxylate (40). To a solution of methyl 2-(4-nitropyrazol-1-yl)acetate (39, 1.5 g, 8.1 mmol) in DMF (25 mL) were added a 60% dispersion of NaH (0.81 g, 20 mmol) and ethylene dibromide (0.85 mL, 9.7 mmol) at 0 °C. The reaction was stirred at 0 °C for 10 min prior to stirring at room temperature for 2 h. The reaction was then diluted with saturated ammonium chloride and extracted with EtOAc (3×). The organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel chromatography (0−100% EtOAc in heptane) to give methyl 1-(4nitropyrazol-1-yl)cyclopropanecarboxylate (0.35 g, 20%). LCMS, m/z = 212 [M + H]+. 1-(4-Amino-5-chloro-1H-pyrazol-1-yl)cyclopropanecarboxamide (41). To as a solution of methyl 1-(4nitropyrazol-1-yl)cyclopropanecarboxylate (40, 0.35 g, 1.7 mmol) in THF (15 mL) was added a 1 M solution of LiHMDS in THF (3.3 mL, 3.3 mmol) at −78 °C. The reaction was stirred at −78 °C for 30 min before the addition of a solution of hexachloroethane (0.47 g, 2.0 mmol) in THF (5 mL). The reaction was then stirred at −78 °C for 30 min before warming to room temperature. The reaction was diluted with saturated ammonium chloride and extracted with EtOAc (3×). The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel chromatography (0−100% EtOAc in heptane) to give methyl 1-(5-chloro-4-nitro-pyrazol-1-yl)cyclopropanecarboxylate (0.22 g, 54% yield). LCMS, m/z = 246 [M + H]+. To a solution of methyl 1-(5-chloro-4-nitro-pyrazol-1-yl)cyclopropanecarboxylate (0.15 g, 0.61 mmol) in THF (8 mL) were added water (2 mL) and lithium hydroxide (22 mg, 0.92 mmol). The reaction was stirred at room temperature for 7 h. The mixture was then diluted with water and washed with diethyl ether. The aqueous layer was then acidified to pH 1 by adding concentrated hydrochloric acid followed by extraction by EtOAc (3×). The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated to give 1-(5-chloro-4-nitro-pyrazol-1-yl)cyclopropanecarboxylic acid (97 mg, 69%). The crude product was carried to the next step without purification. To a solution of the crude 1-(5-chloro-4-nitro-pyrazol-1-yl)cyclopropanecarboxylic acid (97 mg, 0.42 mmol) in CH2Cl2 (2 mL) were added oxalyl chloride (0.5 mL) and 1 drop of DMF. The reaction was stirred at room temperature for 45 min prior to concentration in vacuo. The residue was the redissolved in THF (10 mL), which was then added dropwise to a stirring mixture of ammonium hydroxide (10 mL) and THF (10 mL). The reaction was stirred at room temperature for 1 h. The mixture was then diluted with brine and extracted with EtOAc (3×). The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated to give crude 1-(5chloro-4-nitro-pyrazol-1-yl)cyclopropanecarboxamide (99 mg, quant. yield). A mixture of the crude 1-(5-chloro-4-nitro-pyrazol-1-yl)cyclopropanecarboxamide (90 mg, 0.39 mmol), ammonium chloride (62 mg, 1.1 mmol), and iron dust (66 mg, 1.17 mmol) in ethanol (10 mL) was stirred at 90 °C for 30 min. The reaction was the filtered through Celite and concentrated. To the residue was added EtOAc (50 mL), and the mixture was sonicated for 2 min. The mixture was then filtered to remove all insoluble solids. The filtrate was concentrated to give 1-(4-amino-5-chloro-pyrazol-1-yl)cyclopropanecarboxamide (0.09

methyl 2-methyl-2-(5-methyl-4-nitro-pyrazol-1-yl)propanoate and methyl 2-methyl-2-(3-methyl-4-nitro-pyrazol-1-yl)propanoate (2.0 g, 39% yield). LCMS, m/z = 228 [M + H]+. The mixture of methyl 2-methyl-2-(5-methyl-4-nitro-pyrazol-1yl)propanoate and methyl 2-methyl-2-(3-methyl-4-nitro-pyrazol-1yl)propanoate (1.56 g, 6.87 mmol) was then dissolved in THF (20 mL). To the solution were then added water (5 mL) and LiOH (0.17 g, 6.87 mmol). The resulting mixture was stirred overnight at room temperature. The reaction was then diluted with brine and washed with diethyl ether. The aqueous layer was acidified to pH 2 by adding a 2 M HCl solution. The mixture was then extracted with EtOAc (4×). The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated to give a crude mixture of 2-methyl2-(5-methyl-4-nitro-pyrazol-1-yl)propanoic acid and 2-methyl-2-(3methyl-4-nitro-pyrazol-1-yl)propanoic acid (1.3 g, 88% yield). LCMS, m/z = 214 [M + H]+. To a crude mixture of 2-methyl-2-(5-methyl-4-nitro-pyrazol-1yl)propanoic acid and 2-methyl-2-(3-methyl-4-nitro-pyrazol-1-yl)propanoic acid (0.63 g, 2.9 mmol) were added CH2Cl2 (10 mL), oxalyl chloride (0.6 mL, 7 mmol), and 1 drop of DMF. The reaction was stirred at room temperature for 2 h prior to concentrating to a solid. The solid was redissolved in THF (5 mL), which was then added dropwise to a stirred solution of ammonium hydroxide (10 mL). After stirring at room temperature for 35 min, the reaction was diluted with brine and extracted with EtOAc (4×). The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated to give a crude mixture of 2-methyl-2-(5-methyl-4nitro-pyrazol-1-yl)propanamide and 2-methyl-2-(3-methyl-4-nitro-pyrazol-1-yl)propanamide (0.60 g, 96% yield). LCMS, m/z = 213 [M + H]+. A suspension of the mixture of 2-methyl-2-(5-methyl-4-nitropyrazol-1-yl)propanamide and 2-methyl-2-(3-methyl-4-nitro-pyrazol1-yl)propanamide (0.65 g, 3.1 mmol) and palladium on carbon (10 wt %, 0.2 g) in EtOH (10 mL) was stirred under a hydrogen atmosphere (1 atm) for 1 h. The mixture was filtered through Celite and concentrated to give a mixture of 2-(4-amino-5-methyl-pyrazol-1-yl)-2methyl-propanamide and 2-(4-amino-3-methyl-pyrazol-1-yl)-2-methylpropanamide (0.58 g, quant.). The crude product was carried to the next step without purification. LCMS, m/z = 183 [M + H]+. 1-(4-Amino-3-methyl-1H-pyrazol-1-yl)cyclopropanecarboxamide (38). To a solution of a mixture of methyl 2-(5-methyl-4-nitro-pyrazol-1-yl)acetate and methyl 2-(3methyl-4-nitro-pyrazol-1-yl)acetate (0.70 g, 3.5 mmol) in DMF (10 mL) were added NaH (0.30 g, 7.5 mmol, 60% dispersion) and ethylene dibromide (0.80 mL, 9.2 mmol) at 0 °C. The reaction was stirred at room temperature for 2 h. The reaction was then diluted with saturated ammonium chloride and extracted with EtOAc. The organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel chromatography (0−100% EtOAc in heptane) to give methyl 1-(5methyl-4-nitro-pyrazol-1-yl)cyclopropanecarboxylate (0.30 g, 38% yield). LCMS, m/z = 226 [M + H]+. A mixture of methyl 1-(5-methyl-4-nitro-pyrazol-1-yl)cyclopropanecarboxylate (0.50 g, 2.2 mmol), lithium hydroxide (0.05 g, 2 mmol) in THF (10 mL), and water (5 mL) was stirred at room temperature for 24 h. The reaction was diluted with brine and washed with diethyl ether. The aqueous layer was then acidified to pH 2 by the addition of a 2 M HCl solution. The mixture was then extracted with EtOAc (4×). The combined extracts were washed with brine, dried over sodium sulfate, and concentrated to give crude 1-(3-methyl-4nitro-1H-pyrazol-1-yl)cyclopropanecarboxylic acid (0.62 g, quant.). The material was carried to the next step without purification. To a solution of 1-(3-methyl-4-nitro-1H-pyrazol-1-yl)cyclopropanecarboxylic acid (0.62 g, 2.9 mmol) in CH2Cl2 (5 mL) were added oxalyl chloride (0.78 mL, 8.8 mmol) and 1 drop of DMF. The reaction was stirred for 30 min prior to being concentrated under vacuum. The residue was dissolved in THF (2 mL) and added to a stirred mixture of ammonium hydroxide (10 mL) in THF (10 mL). After 30 min of stirring, the mixture was diluted with water and extracted with EtOAc (3×). The combined extracts were washed with L

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g, quant.). The crude material was carried onto the next step without further purification. LCMS, m/z = 201 [M + H]+. 2-(5-Amino-1-methyl-1H-pyrazol-3-yl)-2-methylpropanenitrile (43). To a solution of 2,2-dimethyl-3-oxo-pentanedinitrile (42, 900 mg, 6.62 mmol) and methylhydrazine sulfate (953 mg, 20 mmol) in EtOH (10 mL) was added concentrated HCl (1.7 mL, 198 mmol). After heating at reflux overnight, the solvent was removed in vacuo, and the residue was purified by silica gel chromatography (CH2Cl2/ MeOH, 20:1) to give the 43 as a white solid (300 mg, 28% yield). LCMS, m/z = 165 [M + H]+. 5-Methyl-1-(oxetan-3-yl)-1H-pyrazol-4-amine (45). To a mixture of 3-methyl-4-nitro-pyrazole (0.80 g, 6.3 mmol), cesium carbonate (4.1 g, 12 mmol) in DMF (10 mL) was added 3-iodooxetane (3.47 g, 19 mmol). The mixture was stirred at 100 °C for 3 h. The reaction was diluted with water and extracted with EtOAc (3×). The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel chromatography (20−100% EtOAc in heptane) to give a mixture of 5-methyl-4-nitro-1-(oxetan-3-yl)-1H-pyrazole and 3-methyl-4-nitro-1-(oxetan-3-yl)-1H-pyrazole (0.85 g, 74% yield). LCMS, m/ z = 184 [M + H]+. To a solution of a mixture of 5-methyl-4-nitro-1-(oxetan-3-yl)-1Hpyrazole and 3-methyl-4-nitro-1-(oxetan-3-yl)-1H-pyrazole (0.137 g, 0.75 mmol) in ethanol (2 mL) was added Pd/C (10 wt %, 0.10 g). The mixture was stirred under a hydrogen atmosphere for 24 h. The reaction was filtered through Celite and concentrated to give a mixture of 5-methyl-1-(oxetan-3-yl)-1H-pyrazol-4-amine and 3-methyl-1-(oxetan-3-yl)-1H-pyrazol-4-amine (83 mg, 73% yield). LCMS, m/z = 154 [M + H]+. 5-Methyl-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-amine (46). The title compound was prepared in a manner analogous to 45 (72% yield, two steps). LCMS, m/z = 182 [M + H]+. tert-Butyl 4-(3-Methyl-4-nitro-1H-pyrazol-1-yl)piperidine-1carboxylate (47). A mixture of 3-methyl-4-nitropyrazole (1.47 g, 11.6 mmoL), N-Boc-4-bromopiperidine (3.7 g, 14 mmol), and cesium carbonate (7.5 g, 23 mmol) in DMF (15 mL) was stirred in a sealed reaction tube at 120 °C for 2 days. The reaction was then diluted with water and extracted with EtOAc (3×). The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product was then purified by silica gel chromatography (0−100% EtOAc in heptane) to give a 2:1 mixture of tert-butyl 4-(3-methyl-4-nitro-1H-pyrazol-1-yl)piperidine-1-carboxylate and tert-butyl 4-(5-methyl-4-nitro-1H-pyrazol-1-yl)piperidine-1-carboxylate as an off-white solid (1.9 g, 52% combined yield). The crude product was carried to the next step without purification. LCMS, m/z = 311 [M + H]+. (±)-(trans)-tert-Butyl 3-Fluoro-4-(3-methyl-4-nitro-1H-pyrazol-1-yl)piperidine-1-carboxylate (48). To a solution of 5methyl-4-nitro-1H-pyrazole (1.2 g, 9.6 mmol), tert-butyl (cis)-3fluoro-4-hydroxypiperidine-1-carboxylate (2.1 g, 9.6 mmol), and triphenylphosphine (2.8 g, 11 mmol) in THF (8 mL) was added diisopropyl azodicarboxylate (2.4 mL, 11 mmol). The reaction was stirred at room temperature for 18 h. The mixture was then diluted with brine and extracted with EtOAc (3×). The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel chromatography (0−100% EtOAc in heptane) to give a mixture of the title compound (3.24 g, quant.) containing a trace amount of (trans)-tert-butyl 3-fluoro-4-(5-methyl-4-nitro-1H-pyrazol-1-yl)piperidine-1- carboxylate. LCMS, m/z = 329 [M + H]+. 4-(3-Methyl-4-nitro-1H-pyrazol-1-yl)-1-(oxetan-3-yl)piperidine (49). To a solution of tert-butyl 4-(3-methyl-4-nitro-1Hpyrazol-1-yl)piperidine-1-carboxylate (0.65 g, 2.1 mmol) in 1,4dioxane (5 mL) was added a 4 M HCl in 1,4-dioxane solution (5 mL, 20 mmol). The reaction was stirred at 55 °C for 3 h. The resulting precipitate was collected by filtration to give a 2:1 mixture of crude hydrochloride salts of 4-(3-methyl-4-nitro-1H-pyrazol-1-yl)piperidine and 4-(5-methyl-4-nitro-1H-pyrazol-1-yl)piperidine (0.51 g, quant.). The crude product was carried to the next step without purification.

To a suspension of 4-(3-methyl-4-nitro-pyrazol-1-yl)piperidine hydrochloride (0.25 g, 1.2 mmol) in 1,2-dichloroethane (5 mL) were added DIPEA (0.3 mL, 1.8 mmol) and oxetan-3-one (0.21 g, 3.0 mmol). The suspension was stirred at room temperature for 15 min prior to the addition of sodium triacetoxyborohydride (0.53 g, 2.4 mmol) and glacial acetic acid (0.082 mL, 1.4 mmol). The reaction was stirred at room temperature for 2 h. The reaction was diluted with saturated sodium bicarbonate and extracted with CH2Cl2 (3×). The combined extracts were washed with water, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel chromatography (0−100% EtOAc in heptane) to give a mixture of 4(3-methyl-4-nitro-pyrazol-1-yl)-1-(oxetan-3-yl)piperidine and 4-(5methyl-4-nitro-pyrazol-1-yl)-1-(oxetan-3-yl)piperidine. (0.15 g, 48% yield). LCMS, m/z = 267 [M + H]+. (±)-(trans)-3-Fluoro-4-(3-methyl-4-nitro-1H-pyrazol-1-yl)-1(oxetan-3-yl)piperidine (50). The title compound was prepared in a manner analogous to 49 (81% yield). LCMS, m/z = 285 [M + H]+. (±)-trans-3-Fluoro-4-(4-nitro-1H-pyrazol-1-yl)piperidine (52). To a mixture of (±)-cis-butyl 3-fluoro-4-hydroxypiperidine-1carboxylate (25 g, 114 mmol), 4-nitropyrazole (51, 15.48 g, 137 mmol), and PPh3 (44.8 g, 171 mmol) in THF (800 mL) was added diisopropyl azodicarboxylate (34.54 g, 171 mmol) dropwise at 0 °C under a nitrogen atmosphere. The reaction was stirred at 30 °C for 20 h and then concentrated in vacuo, and the residue was diluted with EtOAc (100 mL) and washed with water (50 mL) and brine (50 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated, and the residue was purified by silica gel chromatography (petroleum ether/EtOAc, 5:1) to give the crude product as a yellow solid. To a solution of the yellow solid in CH2Cl2 (200 mL) was added TFA (60 g, 0.53 mol), and the mixture was stirred at room temperature for 2 h. The reaction mixture was then concentrated in vacuo, the residue was diluted with DCM (100 mL) and water (50 mL), and the pH value was adjusted to 8 with a saturated K2CO3 solution. The organic layer was dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography (10% MeOH in CH2Cl2) to give the title compound as a yellow solid (14.3 g, 58% yield over two steps). LCMS, m/z = 215 [M + H]+. (±)-(trans)-4-(5-Chloro-4-nitro-1H-pyrazol-1-yl)-3-fluoro-1(oxetan-3-yl)piperidine (53). To a solution of (±)-(trans)-3-fluoro4-(4-nitro-1H-pyrazol-1-yl)piperidine (12.8 g, 60 mmol), oxetan-3-one (4.75 g, 66 mmol), and ZnCl2 (82 mg, 0.6 mmol) in MeOH (200 mL) was added NaBH3CN (5.67 g, 90 mmol) at 0 °C. The reaction was stirred at 30 °C for 20 h and then quenched with ice water, and the solvent was removed in vacuo. The residue was diluted with EtOAc (100 mL) and washed with water and brine. The organic layer was then dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography (CH2Cl2/MeOH, 15:1) to give (±)-(trans)-3-fluoro-4-(4-nitro-1H-pyrazol-1-yl)-1-(oxetan-3-yl)piperidine as a yellow solid (13.5 g, 85%). LCMS, m/z = 271 [M + H]+. To a solution of (±)-(trans)-3-fluoro-4-(4-nitro-1H-pyrazol-1-yl)-1(oxetan-3-yl)piperidine (13.5 g, 50 mmol) in THF (150 mL) was added LiHMDS (60 mL, 1 M in THF, 60 mmol) dropwise at −70 °C under a nitrogen atmosphere. The reaction was stirred at −70 °C for 2 h, and C2Cl6 (14.2 g, 60 mmol) in THF (30 mL) was then added. The mixture was allowed to warm to room temperature and stirred for 2 h. The mixture was quenched with ice water and concentrated in vacuo. The residue was diluted with EtOAc (100 mL) and washed with water and brine. The organic layer was dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography (50% EtOAc in petroleum ether) to give the (±)-(trans)-4-(5-chloro-4-nitro-1H-pyrazol-1-yl)-3-fluoro-1-(oxetan-3yl)piperidine as a white solid (11.9 g, 65% yield). 1H NMR (500 MHz, CDCl3) δ 8.26 (s, 1H), 5.15−5.01 (m, 1H), 4.74−4.71 (m, 2H), 4.66−4.63 (m, 2H), 4.51−4.43 (m, 1H), 3.73−3.67 (m, 1H), 3.28− 3.26 (m, 1H), 2.93−2.89 (m, 1H), 2.44−2.35 (m, 1H), 2.19−2.05 (m, 3H). LCMS, m/z = 305 [M + H]+. Molecular Modeling. Homology models of LRRK2 were constructed using the modeling program MOE version 2009.1062 and AMBER9963 force field. The human LRRK2 sequence was M

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retrieved from Swiss-Prot64 and aligned to template structure sequences using ClustalW65 followed by manual fine-tuning of residues adjacent to loop regions, insertions, and deletions. The models were further refined with bound ligand using the Macromodel utility implemented in Maestro and OPLS2005 force field.66 Inhibitor docking studies were carried out using docking program Glide SP with one hydrogen-bond constraint to the carbonyl oxygen of hinge residue Ala1950. The docking poses were evaluated on the basis of a combination of criteria including the Glide docking score (cutoff = −6), favorable intermolecular interactions with the hinge and other parts of the ATP-binding pocket, low strain energy of the bound ligand (Estrain < 2 kcal/mol), and so forth.

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(3) Muller, T.; Russ, H. Levadopa, motor fluctuations and dyskinesia in Parkinson’s disease. Expert Opin. Pharmacother. 2006, 7, 1715− 1730. (4) Zimprich, A.; Biskup, S.; Leitner, P.; Lichtner, P.; Farrer, M.; Lincoln, S.; Kachergus, J.; Hulihan, M.; Uitti, R. J.; Calne, D. B.; Stoessl, A. J.; Pfeiffer, R. F.; Patenge, N.; Carbajal, I. C.; Vieregge, P.; Asmus, F.; Muller-Myhsok, B.; Dickson, D. W.; Meitinger, T.; Strom, T. M.; Wszolek, Z. K.; Gasser, T. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 2004, 44, 601−607. (5) Paisan-Ruiz, C.; Jain, S.; Evans, E. W.; Gilks, W. P.; Simon, J.; van der Brug, M.; Lopez de Munain, A.; Aparicio, S.; Gil, A. M.; Khan, N.; Johnson, J.; Martinez, J. R.; Nicholl, D.; Carrera, I. M.; Pena, A. S.; de Silva, R.; Lees, A.; Marti-Masso, J. F.; Perez-Tur, J.; Wood, N. W.; Singleton, A. B. Cloning of the gene containing mutations that cause PARK8-linked Parkinson’s disease. Neuron 2004, 44, 595−600. (6) Mata, I. F.; Wedemeyer, W. J.; Farrer, M. J.; Taylor, J. P.; Gallo, K. A. LRRK2 in Parkinson’s disease: Protein domains and functional insights. Trends Neurosci. 2006, 29, 286−293. (7) Greggio, E.; Cookson, M. R. Leucine-rich repeat kinase 2 mutations and Parkinson’s disease: Three questions. ASN Neuro 2009, 1, e00002-1−e00002-12. (8) Gandhi, P. N.; Chen, S. G.; Wilson-Delfosse, A. L. Leucine-rich repeat kinase 2 (LRRK2): A key player in the pathogenesis of Parkinson’s disease. J. Neurosci. Res. 2009, 87, 1283−1295. (9) Dachsel, J. C.; Farrer, M. J. LRRK2 and Parkinson disease. Arch. Neurol. 2010, 67, 542−547. (10) Satake, W.; Nakabayashi, Y.; Mizuta, I.; Hirota, Y.; Ito, C.; Kubo, M.; Kawaguchi, T.; Tsunoda, T.; Watanabe, M.; Takeda, A.; Tomiyama, H.; Nakashima, K.; Hasegawa, K.; Obata, F.; Yoshikawa, T.; Kawakami, H.; Sakoda, S.; Yamamoto, M.; Hattori, N.; Murata, M.; Nakamura, Y.; Toda, T. Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson’s disease. Nat. Genet. 2009, 41, 1303−1307. (11) Simón-Sánchez, J.; Schulte, C.; Bras, J. M.; Sharma, M.; Gibbs, J. R.; Berg, D.; Paisan-Ruiz, C.; Lichtner, P.; Scholz, S. W.; Hernandez, D. G.; Krüger, R.; Federoff, M.; Klein, C.; Goate, A.; Perlmutter, J.; Bonin, M.; Nalls, M. A.; Illig, T.; Gieger, C.; Houlden, H.; Steffens, M.; Okun, M. S.; Racette, B. A.; Cookson, M. R.; Foote, K. D.; Fernandez, H. H.; Traynor, B. J.; Schreiber, S.; Arepalli, S.; Zonozi, R.; Gwinn, K.; van der Brug, M.; Lopez, G.; Chanock, S. J.; Schatzkin, A.; Park, Y.; Hollenbeck, A.; Gao, J.; Huang, X.; Wood, N. W.; Lorenz, D.; Deuschl, G.; Chen, H.; Riess, O.; Hardy, J. A.; Singleton, A. B.; Gasser, T. Genome-wide association study reveals genetic risk underlying Parkinson’s disease. Nat. Genet. 2009, 41, 1308−1312. (12) West, A. B.; Moore, D. J.; Biskup, S.; Bugayenko, A.; Smith, W. W.; Ross, C. A.; Dawson, V. L.; Dawson, T. M. Parkinson’s diseaseassociated mutations in leucine-rich repeat kinase 2 augment kinase activity. Proc. Natl. Acad. Sci.U.S.A. 2005, 102, 16842−16847. (13) Greggio, E.; Jain, S.; Kingsbury, A.; Bandopadhyay, R.; Lewis, P.; Kaganovich, A.; van der Brug, M. P.; Beilina, A.; Blackinton, J.; Thomas, K. J.; Ahmad, R.; Miller, D. W.; Kesavapany, S.; Singleton, A.; Lees, A.; Harvey, R. J.; Harvey, K.; Cookson, M. R. Kinase activity is required for the toxic effects of mutant LRRK2/dardarin. Neurobiol. Dis. 2006, 23, 329−341. (14) Cookson, M. R. The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson’s disease. Nat. Rev. Neurosci. 2010, 11, 791− 797. (15) Rudenko, I. N.; Chia, R.; Cookson, M. R. Is inhibition of kinase activity the only therapeutic strategy for LRRK2-associated Parkinson’s disease? BMC Med. 2012, 10, 20−27. (16) Liu, Z.; Hamamichi, S.; Lee, B. D.; Yang, D.; Ray, A.; Caldwell, G. A.; Caldwell, K. A.; Dawson, T. M.; Smith, W. W.; Dawson, V. L. Inhibitors of LRRK2 kinase attenuate neurodegeneration and Parkinson-like phenotypes in Caenorhabditis elegans and Drosophila Parkinson’s disease models. Hum. Mol. Genet. 2011, 20, 3933−3942. (17) Lee, B. D.; Shin, J.-H.; VanKampen, J.; Petrucelli, L.; West, A. B.; Ko, H. S.; Lee, Y.-I.; Maguire-Zeiss, K. A.; Bowers, W. J.; Federoff, H. J.; Dawson, V. L.; Dawson, T. M. Inhibitors of leucine-rich repeat

ASSOCIATED CONTENT

S Supporting Information *

Additional experimental procedures and references. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(A.A.E.) Phone: 650-225-4934. Fax: 650-742-4943. E-mail: [email protected]. *(B.K.C.) Phone: 650-467-3195. Fax: 650-742-4943. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank our Analytical, Bioanalytical, NMR, Pharmacokinetic, Biopharmacology, and Safety Assessment colleagues for their contributions. We also thank John Atherall, Linda Bao, Leo Berezhkovskiy, Diane Carrera, Jonathan Cheong, Po-Chang Chiang, Kang-Jye Chou, Xiao Ding, Quynh Ho, Claire Holt, Heather Kennedy, Emile Plise, Veronica Rivera, Kimberley Smith, Hilda Solanoy, and Hervé Van de Poël for their individual contributions.

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ABBREVIATIONS USED AUC, area under the curve; BAC, bacterial artificial chromosome; BCRP, breast cancer resistance protein; Bu, unbound brain concentration; CNS, central nervous system; CSF, cerebrospinal fluid; CYP, cytochrome P450; DIPEA, N,Ndiisopropylethylamine; ER, efflux ratio; h, human; ip, intraperotineal; iv, intravenous; JAK2, janus kinase 2; LELP, ligandefficiency-dependent lipophilicity; LM, liver microsome; LRRK2, leucine-rich repeat kinase 2; MCT, methylcellulose/ tween; MDCK-MDR1, Madin-Darby canine kidney cellsmultidrug resistance protein 1; NMP, N-methyl-2-pyrrolidone; PD, Parkinson’s disease; PE, petroleum ether; PEG, poly(ethylene glycol); P-gp, P-glycoprotein; PK/PD, pharmacokinetics/pharmacodynamics; pLRRK2, phospho-LRRK2; PPB, plasma protein binding; Pu, unbound plasma concentration; r, rat; SFC, supercritical fluid chromatography; TDI, timedependent inhibition; TPSA, topological surface area; Vd, volume of distribution; XPhos, 2-dicyclohexylphosphino2′,4′,6′-triisopropylbiphenyl

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REFERENCES

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