Discovery of a 3-(4-Pyrimidinyl) Indazole (MLi-2), an Orally Available

Article pubs.acs.org/jmc

Discovery of a 3‑(4-Pyrimidinyl) Indazole (MLi-2), an Orally Available and Selective Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitor that Reduces Brain Kinase Activity Jack D. Scott,*,† Duane E. DeMong,*,‡ Thomas J. Greshock,*,§ Kallol Basu,∥ Xing Dai,† Joel Harris,†,⊥ Alan Hruza,† Sarah W. Li,§ Sue-Ing Lin,† Hong Liu,† Megan K. Macala,†,# Zhiyong Hu,† Hong Mei,† Honglu Zhang,† Paul Walsh,∥ Marc Poirier,† Zhi-Cai Shi,† Li Xiao,† Gautam Agnihotri,†,∇ Marco A. S. Baptista,†,○ John Columbus,‡,◆ Matthew J. Fell,‡ Lynn A. Hyde,† Reshma Kuvelkar,† Yinghui Lin,† Christian Mirescu,‡ John A. Morrow,†,¶ Zhizhang Yin,† Xiaoping Zhang,† Xiaoping Zhou,† Ronald K. Chang,§ Mark W. Embrey,§ John M. Sanders,§ Heather E. Tiscia,§ Robert E. Drolet,§ Jonathan T. Kern,§,+ Sylvie M. Sur,§ John J. Renger,§,□ Mark T. Bilodeau,§,● Matthew E. Kennedy,‡ Eric M. Parker,† Andrew W. Stamford,† Ravi Nargund,† John A. McCauley,§ and Michael W. Miller† †

Merck Merck § Merck ∥ Merck ‡

& & & &

Co., Co., Co., Co.,

Inc., Inc., Inc., Inc.,

2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United States 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States 126 East Lincoln Avenue, Rahway, New Jersey 07065, United States

S Supporting Information *

ABSTRACT: Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein which contains a kinase domain and GTPase domain among other regions. Individuals possessing gain of function mutations in the kinase domain such as the most prevalent G2019S mutation have been associated with an increased risk for the development of Parkinson’s disease (PD). Given this genetic validation for inhibition of LRRK2 kinase activity as a potential means of affecting disease progression, our team set out to develop LRRK2 inhibitors to test this hypothesis. A high throughput screen of our compound collection afforded a number of promising indazole leads which were truncated in order to identify a minimum pharmacophore. Further optimization of these indazoles led to the development of MLi-2 (1): a potent, highly selective, orally available, brain-penetrant inhibitor of LRRK2.

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INTRODUCTION Parkinson’s disease (PD) is a progressive neurodegenerative disease that afflicts over 10 million people worldwide.1 Characteristic primary symptoms of PD develop gradually, with the most common initial clinical manifestation of resting hand or foot tremor followed by bradykinesia, muscle rigidity, and postural instability. At a neuropathological level, this collection of motor symptoms is caused by the progressive loss of dopaminergic neurons in the substantia nigra, occurring over the course of many years.2 The current standard of care for Parkinsonian motor symptoms is dopamine replacement therapy. To date, there are no predictive biomarkers of clinical PD and no treatments available to slow or halt the course of PD neurodegeneration. Therefore, the discovery of the root causes of disease and targeting therapies to such mechanisms hold promise to fill the gap of high unmet medical need for PD disease modification. © 2017 American Chemical Society

Current views on PD pathoetiology presume that the vast majority of disease results from an interplay between environmental and genetic factors.3 These cases are classified as sporadic as they occur in patients with no apparent familial history of the disorder. Approximately 15% of PD follows a pattern of Mendelian transmission, with disease linked to specific genetic polymorphisms. Familial cases of PD are associated with monogenic mutations in leucine-rich repeast kinase 2 (LRRK2) among several others that have been identified to date.4−6 Among the familial PD population, mutations in the LRRK2 gene account for 4% of cases.7 More broadly, LRRK2 variants account for only approximately 1% of the sporadic cases. Although these incidences are relative low, LRRK2 is the most prevalent genetic risk factor for PD. Received: January 10, 2017 Published: February 28, 2017 2983

DOI: 10.1021/acs.jmedchem.7b00045 J. Med. Chem. 2017, 60, 2983−2992

Journal of Medicinal Chemistry

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identification of several 3,5-disubstituted indazoles, with nanomolar LRRK2 IC50s and modest to good ligand efficiencies (LE).22 Indazoles 2 and 3 are representative of the hits uncovered during the screening campaign (Figure 1). In addition to their LRRK2 potencies, these analogues were attractive due to their ability to partition into the CNS and their promising kinase selectivity profiles. CNS penetration was observed with compound 2, which was found to have a modest exposure of 36 nM in the CSF along with an unbound plasma concentration of 42 nM in rat following a 5 mg/kg ip dose. With respect to kinase selectivity, at a concentration of 1 μM, 2 was found to have an inhibition greater than 50% in only 10 out of the 305 kinases screened. Although these leads met several of the requirements we sought in the profile of a LRRK2 inhibitor, the lead ID/lead optimization process often trends toward more complicated analogues with higher molecular weight as the program progresses, leading to the rise of new issues such as reduced cellular permeability and CNS exposure. Thus, we sought a more optimal starting point that would allow for more chemical flexibility and diversity without compromising CNS penetration. To that end, we set out to identify the minimal indazole pharmacophore for LRRK2 inhibition, focusing on improvement of LE relative to the original HTS hits. Further optimization of LRRK2 potency would be progressed in parallel with the maintenance of physicochemical properties within a range of previously reported CNS drugs.23 For the lead optimization campaign, it was decided to assay compounds against G2019S LRRK2 because we wanted to ensure that any chemical matter developed could be profiled in individuals that possessed this mutation. Compounds selected for kinome screening would also be screened against wt LRRK2 to ensure that there was no divergence in enzyme inhibition between variants. As shown in Table 1, compounds 4−10 which are truncated at the 5-position of the indazole relative to the initial hits led to LRRK2 inhibitors with improved LEs compared to compounds 2 and 3. High ligand efficiencies could also be obtained by the complementary approach of reducing substitution at C-3 to a methyl group while leaving C-5 intact. These compounds, however, suffered from a significant erosion in the kinase selectivity profile (data not shown). Of the three pyridine regioisomers, the 3- and 4-pyridyl analogues (4 and 5, respectively) had similar LRRK2 IC50s while the 2-pyridyl analogue 6 was less potent. Further elaboration of the C-3 pyridine ring with amino substituents, as is present in 3, provided analogues with improved potencies. In general, analogues with cyclic secondary amines such as morpholine (e.g., 8) and piperazine were found to be much more selective LRRK2 inhibitors relative to acyclic inhibitors such as 7. As the potencies of the inhibitors increased, the analogues with a 4pyridyl at C-3 were found to be significantly more potent than the corresponding 3-pyridyl analogues. For example, 8 was 21fold more potent than the 3-pyridine regioisomer 9. It is also noteworthy that the pyrimidine analogue 10 was 11-fold less potent than the 4-pyridyl analogue 8. Introduction of heteroaromatic groups at C-5, while maintaining the pyridyl morpholine at C-3, led to the identification of a series of very potent LRRK2 inhibitors with single-digit nanomolar IC50s; however, these analogues were generally found to be P-gp efflux substrates in LLC-PK1 cells expressing MDR124 and/or possessed significantly eroded kinase selectivities. For example, in a panel of 68 kinases, the pyrazole analogue 12, at a concentration of 1 μM, was found to

LRRK2 is a large multidomain protein consisting of a kinase, a GTPase, and several protein−protein interacting regions.8 The most common LRRK2 variant in these populations results in the G2019S missense mutation, which is located in the kinase domain of LRRK2. Enzymatic studies have shown that this single nucleotide change results in a gain of kinase function.9 Unlike other familial PD variants, LRRK2-linked PD typically manifests itself as a late-onset and slowly progressing disease which may be more amenable to clinical intervention for disease modification, compared to juvenile or early onset familial forms of PD.10−15 Collectively, these fundamental observations anchored in human genetics serve as cornerstone evidence for the LRRK2 kinase hypothesis for PD and accordingly have served as the rationale for the development of LRRK2 kinase inhibitors as possible disease-modifying treatments for PD.16−18 To date, it is not yet fully understood how LRRK2 kinase activity increases the risk of PD or more specifically what downstream signal events lead to the subsequent degeneration of dopaminergic neurons. However, recent substrate mining efforts have now unambiguously identified a subset of Rab GTPases as key LRRK2 substrates.19 These observations should enable fine pathway mapping of LRRK2 substrates to identify pathogenic signaling. The key pharmacological tool which enabled the discovery of these LRRK2 substrates via phosphoproteomics was cis-2,6-dimethyl-4-(6-(5-(1-methylcyclopropoxy)-1H-indazol-3-yl)pyrimidin-4-yl)morpholine (MLi2, 1, Figure 1), a highly potent and selective LRRK2 kinase

Figure 1. Indazole lead 1, HTS hits 2 and 3, and G2019S LRRK2 enzyme inhibition.

inhibitor.20 Here, we describe our medicinal chemistry efforts and strategy within an indazole pharmacophore, leading to the development of selective LRRK2 kinase inhibitors and ultimately the discovery of 1.

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RESULTS AND DISCUSSION The identification of selective and brain-penetrant LRRK2 kinase inhibitors began with a high throughput screen (HTS) of our sample collection using recombinant wild-type (wt) LRRK2 in an enzymatic assay.21 We chose to screen with the wt LRRK2 enzyme so that we could identify compounds that would ideally be suitable for use in both individuals with idiopathic PD and carriers of the G2019S mutation. Confirmation of the activity in both enzymes was assessed in followup kinome screens. The HTS screen led to the 2984



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Table 1. LRRK2 Inhibition, Efflux Ratio, Permeability, and Hepatocycte Clearance Data

a

G2019S LRRK2 IC50 values are the average of a minimum of two independent determinations (see ref 20). bEfflux ratio A−B/B−A. Pgp, Pglycoprotein; h, human; r, rat; Hep Cl, hepatocyte clearance; NT, not tested.

addition, the increased polarity imparted by the sulfonyl moiety of 15 (PSA 101 Å2)25 likely contributed to that analogue being a P-gp substrate. Strikingly, while the methoxy analogue 16 was found to have a moderate IC50 of 15 nM, the isopropoxy compound 17 (MLi-1)20 was found to be 21-fold more potent, with an LE similar to that of 8. Equally important, 17 possessed excellent selectivity over other kinases. Across a panel of 306 kinases at a concentration of 1 μM, 17 inhibited only nine

exhibit greater than 50% inhibition of 24 in the kinases screened. In an effort to identify potent chemical matter with nonaromatic C-5 substituents, a series of analogues exemplified by compounds 13−18 was evaluated (Table 1). The cyclopropyl, cyano, and sulfonyl substituents (analogues 13−15, respectively) were found to provide no improvement in LRRK2 potency over that of the C-5 unsubstituted analogue 8. In 2985



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20−-22) did not lead to an improvement in clearance compared to the morpolino analogue 17. The triazole substituted piperidine analogue 23 exhibited improved hepatocyte stability, but it was found to be a P-gp efflux substrate and possessed reduced kinase selectivity compared to the morpholino analogues as indicated by the heat maps in Table 2. Excitingly, replacement of the 4-pyridyl in the indazole 3-position with a 2,4-pyrimidinyl group (compound 24, MLi3)20 led to improved stability in both rat and human hepatocytes, albeit with a 10-fold loss in LRRK2 potency compared to its homologue 17. This was consistent with the potency difference observed between previously discussed analogues 8 and 10. Additional improvement in the LRRK2 potency was obtained by increasing the steric bulk of the C-5 alkoxy group with methylcyclopropoxy analogue 25, showing a modest improvement in LRRK2 IC50 compared to the isopropoxy analogue 24. The addition of the methyl group on the cyclopropane ring provided a striking improvement in LRRK2 potency considering the modest potency (41 nM) of the cyclopropoxy analogue 18. In the panel of 306 kinases at a concentration of 1 μM, 24 only inhibited four kinases at greater than 50%, while 11 kinases were inhibited by greater than 50% by 25. Analogues 26, 27, and 1 with the addition of one or two methyl groups on the morpholine moiety had enhanced selectivities over the same panel of kinases compared to 25. The dimethylmorpholino analogue 1 was found to be a very potent LRRK2 inhibitor (0.76 nM) and extremely selective with at least 295-fold selectivity over the entire kinase panel.20 With a LE of 0.45 and exquisite selectivity for 1, we achieved our goal of identifying a LRRK2 inhibitor with a superior profile compared to the original HTS hits. Our LRRK2 model was able to provide insight into the improved potency and selectivity of 1. The larger methylcyclopropoxy moiety in the catalytic lysine region is able to make enhanced hydrophobic interactions with Val1893 of LRRK2 (not shown in Figure 2 for clarity). In addition, the two methyl groups on the morpholine of 1 could form van der Waals (VDW) contacts with the side chain of Leu1949,21 providing stronger binding to LRRK2, whereas, these two methyl substituents would cause negative VDW clashes with larger residues, such as Phe or Tyr, that are observed in many human kinases at this Leu1949 position (see Supporting Information). More specifically, 1 exhibited enhanced selectivity over TTK and NUAK1 compared to 24 with TTK having a Cys and NUAK1 a Tyr at that position. Because of the lack of a cell-based assay at the time that these LRRK2 inhibitors were initially evaluated, a biochemical assay performed in the presence of a physiologically relevant concentration of 5 mM ATP was used to mimic cellular conditions compared to the standard assay conditions at the ATP Km concentration of 134 μM. Compounds evaluated in this higher ATP concentration assay showed a 5- to 35-fold loss in potency compared to the IC50s obtained under the standard assay conditions. Despite this shift in potency, several analogues were still found to be very potent inhibitors of LRRK2 with IC50s less than 15 nM in the modified assay. We have since reported the development of a cell-based assay in SHSY5Y cells that expressed LRRK2 monitoring for dephosphorylated Ser935 LRRK2 using Western blot analysis, confirming enzyme occupancy of our LRRK2 inhibitors through the use of 35Slabeled 28 (MLi-A).20 In this communication, we report a set of IC50s generated via a high throughput cellular assay that has

kinases at a level greater than 50%, with TTK being inhibited the most at 91%. Without access to an X-ray crystal structure of LRRK2, we constructed a homology model of the LRRK2 kinase domain based on the X-ray structure of an indazole inhibitor bound to ERK2. This allowed us to gain further insight into the binding of our indazole inhibitors in LRRK2 and rationalize the selectivity over other kinases. As shown in Figure 2, the model

Figure 2. Overlay of X-ray structure of 17 in ERK2 (tan) (PDB code: 5U6I) with the model of 17 in LRRK2 (green). Note: the surfaces for LRRK2 residues Lys1906 and Leu1949 are included.

of 17 in LRRK2 shows that the indazole is bound to the hinge region of LRRK2 in a hydrogen bond donor/acceptor pair with the indazole NH and nitrogen. The morpholine is positioned adjacent to Leu1949 on the hinge and directed toward bulk water, and the isopropoxy resides in the region of the catalytic lysine (Lys1906). Although the model suggested that 17 would display a very similar binding mode between LRRK2 and ERK2, the biochemical inhibition of ERK2 by 17 (41% inhibition at 1 μM) is significantly reduced compared to the LRRK2 potency. While residue Leu1949 of LRRK2 is conserved in ERK2, several other amino acid residues are not conserved between the two kinase binding sites, most notably the inserted Ser1951 in LRRK2 in the hinge region, which likely influences the inhibitor binding. In addition to high LRRK2 potency and selectivity over other kinases, we set a goal of the identification of inhibitors with oral bioavailability and sufficient CNS penetration to allow for pharmacodynamic evaluation in rodents following oral dosing. Generally, the C-3 pyridyl indazoles showed high metabolic turnover in hepatocytes, which translated into low exposures and short half-lives in rat. For example the isopropoxy analogue 17 was found to have a modest AUC(0−8h) of 1.0 μM·h with a half-life of 1 h after a 2 mg/kg iv dose. Efforts to reduce the in vivo clearance of this class of inhibitors focused on the probable metabolic hotspots, the morpholino and alkyl ether substituents, along with reducing the electron density of the aminopyridine moiety. To quickly assess the likelihood of a given compound to show an improvement in in vivo clearance, we utilized in vitro metabolic stability studies with both human and rat hepatocytes. Attempts to increase the hepatocyte stability through replacement of the morpholine with electron-deficient cyclic amines (analogues 2986



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Table 2. LRRK2 Inhibition, Cellular Potency, and Kinase Selectivity for Indazole Analogues

a

G2019S LRRK2 IC50 values are the average of a minimum of two independent determinations (see ref 20). bEnzymatic assay using 5 mM ATP. Efflux ratio A−B/B−A. dHeat map indicating the amount of inhibition for a panel of kinases listed in alphabetical order (A-Z left to right) not including LRRK2. Pgp, P-glycoprotein; h, human; r, rat; Hep Cl, hepatocyte clearance; NT, not tested.

c

been used to profile the advanced inhibitors. In comparison of the enzymatic and cellular readouts, generally the high ATP enzymatic evaluation was found to be a good surrogate for a cellular assay with our most potent inhibitors, such as 27, 28, and 1, exhibiting similar potencies across the three assays.

The dimethylmorpholine analogue 1 was selected for further profiling based on both the excellent LRRK2 potency and selectivity over other kinases. The rat pharmacokinetic profile of 1 is summarized in Table 3. Overall, 1 exhibited modest plasma exposure after oral dosing with good bioavailability and 2987



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Table 3. Pharmacokinetic Parameters of 1 in Han−Wistar Rat parameter AUC(0−8h) [μM·h] Cmax [μM] Tmax [h] T1/2 [h] MRT [h] Cl [mL/min/kg] F [%]

iv administrationa (2 mg/kg)

oral administrationb (10 mg/kg)

0.66 ± 0.24 1.17 ± 0.15c

1.3 ± 0.56 0.48 ± 0.36 1.3 ± 0.75

1.8 ± 1.0 1.7 ± 0.46 153 ± 55

4.0 ± 1.0 39 ± 18

Figure 3. Oral dose response for 1 and its effect on decrease in ratio of pS935/LRRK2 in male CD rats (n = 5 rats per dose, * p ≤ 0.05; *** p ≤ 0.001).

a

Dosed as a solution of the free base (2 mg/mL) in a mixture of DMSO:PEG400:water (20:60:20). bDosed as a fine suspension of the free base (5 mg/mL) in a mixture of DMSO:PEG400:water (20:60:20). cInitial concentration. MRT, mean residence time; F, bioavailability.

treatment with acetic anhydride afforded the desired acetamide 33 in excellent yield over the three steps. Indazole ring formation was accomplished via modified Jacobson conditions, and subsequent treatment with ammonia provided the indazole 35.26 Treatment of 35 with SEM-Cl and the hindered base N,N-dicyclohexylmethylamine resulted in selective SEM protection of the N2 position of the indazole to afford 36. Regioselective lithiation of the 3-position of the 36, followed by Negishi coupling with 4,6-dichloropyrimidine, resulted in the formation of intermediate 37.27 Displacement of the chloride with cis-2,6-dimethylmorpholine, followed by removal of the SEM protecting group, led to the formation of 1. Late-stage intermediates exemplified by 37 allowed for rapid analogue syntheses and SAR expansion in the solvent front region of these indazole inhibitors via focused parallel medicinal chemistry library generation. Prior to synthesis, virtual libraries were enumerated and the in silico properties, including CNS MPO,23 were calculated with the analogues meeting our criteria being selected for synthesis. These library compounds were prepared in parallel followed by mass-triggered HPLC purification28 on sufficient scale that allowed for evaluation in our standard set of assays (i.e., LRRK2 enzyme IC50, P-gp, Hep Cl, and kinome selectivity). Analogues that were progressed for further evaluation were scaled up in singleton format as needed.

a favorable apparent half-life. The difference in oral bioavailability observed between rats is likely due to the low equilibrium solubility of 1 at 24 h in a variety of buffers ( 50 μM) nor was it a timedependent inhibitor of CYP3A4. In addition to the excellent kinase selectivity, 1 did not show any significant inhibition in a broad panel of enzymes, ion channels, or receptors.20 We have previously reported the results of the pharmacokinetic−pharmacodynamic (PK/PD) evaluation of 1 in wildtype mice. After oral dosing, 1 signficantly reduced the ratio of phosphorylated Ser935 LRRK2 (pS935) to total LRRK2 in the mouse cortex in both single dose and 11-day in-diet studies.20 In the acute study, a greater than 90% reduction of the ratio of pS935 to total LRRK2 was observed at oral doses of 10 mg/kg or higher. An additional 15-week study in MitoPark mice showed that 1 was well tolerated, with an indication that type II pneumocytes were enlarged in the lung. No additional overt changes were observed in the lung or kidney of the mice, organs in which LRRK2 is highly expressed and have been shown to have overt phenotypes in LRRK2 knockout mice.20 In addition to the aforementioned studies in mice, compound 1 was shown to significantly reduce pS935 in a dose dependent manner (1−100 mg/kg) 1 h post treatment in rats (Figure 3A). Reductions of greater than 90% were observed at oral doses of 30 mg/kg and higher. The unbound brain IC50 was found to be 0.18 nM (see Figure 3B) and the unbound plasma IC50 was 0.2 nM, which is consistent with 1 not being a Pgp substrate. These IC50 values were found to be similar to the values that we reported in mice: 0.8 and 1.1 nM in the brain and plasma, respectively.20

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CONCLUSION With the identification of a number of promising indazole screening hits, followed by a focus on the optimization of ligand efficiency, in vitro P-gp efflux, and in vitro hepatocyte stability, we were able to discover a series of potent, orally bioavailable LRRK2 inhibitors. Additional refinement led us to the orally available and brain-penetrant 1. A dose-dependent reduction in the ratio of pS935 LRRK2 to total LRRK2 in the brains of rats after oral dosing with 1 has provided confirmation of target engagement. The potent and selective LRRK2 inhibitor 1 has emerged as a key tool in the understanding of LRRK2 function and may provide additional insight into the role of LRRK2 in the pathogenesis of Parkinson’s disease.

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EXPERIMENTAL SECTION

Synthetic Materials and Methods. Reagents and solvents were obtained from commercial sources and used without further purification. 1H NMR spectra (400 and 500 MHz) were collected on Varian spectrometers. Chemical shifts are reported in ppm relative to the residual solvent peak in the indicated solvent, and for 1H NMR spectra, multiplicities, coupling constants in hertz, and numbers of protons are indicated parenthetically. Purities of all reported compounds were greater than 95% based on HPLC chromatograms obtained on an Agilent 1100 LCMS system. All active compounds

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SYNTHESIS As summarized in Scheme 1, the preparation of 1 commenced with an SNAr reaction between the commercially available 1methyl-1-cyclopropanol and aryl fluoride 29 to afford ether 30. Palladium-mediated methylation followed by reduction of the nitro group afforded the amino derivative 32, which upon 2988



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Scheme 1. Synthesis of 1a

Reagents and conditions: (a) 1-methylcyclopropan-1-ol, NaH, DMF, 0 °C then RT, 5 h, 87%; (b) trimethylboroxine, Pd(Ph3P)4, K2CO3, Cs2CO3, 1,4-dioxane, 100 °C, 16 h; (c) ammonium formate, 10% Pd/C EtOH, RT, 5 h, 96% (2 steps); (d) Ac2O, Et3N, CH2Cl2, RT, 16 h, 96%; (e) Ac2O, KOAc, 3-methylbutyl nitrite, toluene, 80 °C, 16 h, 86%; (f) 3.5 M ammonia in MeOH, RT, 2 h, 94%; (g) c-hex2NMe, SEM-Cl, THF, RT 16 h, 79%; (h) (1) n-BuLi, THF, −78 °C then −20 °C, (2) ZnCl2, −78 °C then −20 °C, (3) Pd(PPh3)4, 4,6-dichloropyrimidine, THF, RT, 5 h, 71%; (i) cis-2,6dimethylmorpholine, Et3N, DMSO, 100 °C, 2 h, 82%; (j) 4 N HCl in 1,4-dioxane, MeOH, RT then 65 °C, 30 min, 91%.

a

were analyzed for and found to be free of pan assay interference compounds (PAINS).29 2-Bromo-4-(1-methylcyclopropoxy)-1-nitrobenzene (30). To a cold (0 °C), stirred mixture of 2-bromo-4-fluoro-1-nitrobenzene (10 g, 45.5 mmol) and 1-methylcyclopropan-1-ol (3.61 g, 50.0 mmol) in DMF (200 mL) was added NaH (2.36 g, 59.1 mmol, 60% w/w in mineral oil) and the mixture was stirred at RT for 5 h. The reaction was quenched with water and extracted with EtOAc (3×). The combined organic layers were washed with water (3×) and brine (2×), dried, filtered, and concentrated to leave an oil which was purified by silica gel column (gradient elution with 100:0 to 20:1 hexane:EtOAc) to yield 30 (10.8 g, 87%) as a light-yellow oil. 1H NMR (400 MHz, chloroform-d) δ 7.95 (d, J = 9.1 Hz, 1H), 7.31 (d, J = 2.6 Hz, 1H), 7.00 (dd, J = 9.1, 2.6 Hz, 1H), 1.55 (s, 3H), 1.04−0.92 (m, 2H), 0.86− 0.74 (m, 2H). 2-Methyl-4-(1-methylcyclopropoxy)-1-nitrobenzene (31). A stirred mixture of 30 (8.2 g, 30.1 mmol), K2CO3 (8.33 g, 60.3 mmol), and Cs2CO3 (9.82 g, 30.1 mmol) in 1,4-dioxane (502 mL) was purged with argon for 15 min. Trimethylboroxine (9.27 mL, 66.3 mmol) and Pd(Ph3P)4 (3.48 g, 3.01 mmol) were added, and the mixture was heated at 100 °C overnight. The reaction was cooled to RT and concentrated under vacuum. To this residue was added 10:1 hexanes:EtOAc (500 mL), and the mixture was filtered through a pad of silica. The filter pad was washed with another 1 L of 10:1 hexanes:EtOAc solution. The filtrate was concentrated to afford crude 31 (5.3 g), which was used in the next step without further purification. 1H NMR (400 MHz, chloroform-d) δ 8.06 (d, J = 9.1 Hz, 1H), 6.92 (ddd, J = 9.1, 2.7, 0.5 Hz, 1H), 6.87−6.85 (m, 1H), 2.62 (t, J = 0.5 Hz, 3H), 1.56 (t, J = 0.7 Hz, 3H), 1.05−0.95 (m, 2H), 0.85−0.72 (m, 2H). 2-Methyl-4-(1-methylcyclopropoxy)aniline (32). To a solution of 31 (5.3 g, 30.1 mmol) in EtOH (502 mL) were added 10% Pd/C (3.21 g, 3.01 mmol) and ammonium formate (22.80 g, 362 mmol).

The resulting heterogeneous mixture was stirred at RT for 5 h. To this solution was added 5:1 hexanes:EtOAc (1000 mL), and the mixture was filtered through a pad of silica gel. The filtrate was concentrated to afford 32 (5.12 g, 96%, 2 steps), which was used directly in the next step. LCMS: (ESI MS) m/z = 178.2 [M + H]+. 1H NMR (400 MHz, chloroform-d) δ 6.76−6.68 (m, 2H), 6.63−6.55 (m, 1H), 3.36 (broad s, 2H), 2.14 (s, 3H), 1.49 (t, J = 0.8 Hz, 3H), 1.00−0.91 (m, 2H), 0.70−0.58 (m, 2H). N-(2-Methyl-4-(1-methylcyclopropoxy)phenyl)acetamide (33). To a cold (0 °C), stirred mixture of 32 (5.12 g, 28.9 mmol) and Et3N (8.05 mL, 57.8 mmol) in CH2Cl2 (50 mL) was added Ac2O (4.09 mL, 43.3 mmol). The mixture was slowly warmed to RT and stirred overnight. The reaction was quenched with a saturated solution of aqueous NaHCO3 and extracted with CH2Cl2 (3×). The combined organic layers were dried, filtered, and concentrated to leave a residue which was purified by silica gel column (gradient elution with 2:1 to 1:1 hexanes:EtOAc) to yield 33 (6.1 g, 96%) as a colorless gum. LCMS (ESI MS) m/z = 220.0 [M + H]+. 1-(5-(1-Methylcyclopropoxy)-1H-indazol-1-yl)ethan-1-one (34). To a stirred solution of 33 (6.1 g, 27.8 mmol) in toluene (150 mL) were added KOAc (4.10 g, 41.7 mmol) and Ac2O (12.07 mL, 128 mmol). The mixture was then heated to 80 °C. 3-Methylbutyl nitrite (15.57 mL, 111 mmol) was added dropwise, and the resulting mixture was heated at 80 °C overnight. The reaction was then filtered through a pad of Celite, and the filtrate was concentrated to leave a residue which was then purified by silica gel column (elution with 20:1 hexanes:EtOAc) to yield 34 (5.5 g, 86%) as a yellow solid. LCMS (ESI MS) m/z = 231.2 [M + H]+; 253.2 [M + Na]+. 1H NMR (400 MHz, chloroform-d) δ 8.28 (ddd, J = 9.0, 0.8, 0.8 Hz, 1H), 8.03 (d, J = 0.9 Hz, 1H), 7.28 (dd, J = 2.4, 0.7 Hz, 1H), 7.16 (dd, J = 9.0, 2.4 Hz, 1H), 2.74 (s, 3H), 1.56 (t, J = 0.7 Hz, 3H), 1.06−0.95 (m, 2H), 0.83−0.68 (m, 2H). 2989



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(ESI+) m/z 380.2077 [(M+ H)+ calcd for C21H25N5O2: 380.2086]. 1H NMR (500 MHz, CDCl3) δ 10.33 (s, 1H), 8.79 (d, J = 1.0 Hz, 1H), 8.31 (s, 1H), 7.41 (d, J = 9.0 Hz, 1H), 7.34 (s, 1H), 7.15 (dd, J = 9.0, 2.5 Hz, 1H), 4.35−4.29 (m, 2H), 3.72 (m, 2H), 2.68 (dd, J = 13.0, 10.5 Hz, 2H), 1.66 (s, 3H), 1.30 (d, J = 6.0 Hz, 6H), 1.13−1.07 (m, 2H), 0.84−0.77 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 161.94, 159.27, 158.45, 152.76, 143.01, 137.65, 122.39, 120.13, 110.68, 106.65, 98.08, 71.53, 56.60, 49.26, 20.16, 18.85, 13.68.

5-(1-Methylcyclopropoxy)-1H-indazole (35). To a stirred suspension of 34 (6.5 g, 28.2 mmol) in MeOH (20 mL) was added 7 M ammonia in MeOH (20.16 mL, 141 mmol), and the mixture was stirred at RT for 2 h. The reaction was concentrated, and the residue was purified by silica gel column (gradient elution with 5:1 to 1:1 hexanes:EtOAc) to yield 35 (5g, 94%) as a yellow solid. LCMS (ESI MS) m/z = 189.2 [M + H]+. 1H NMR (400 MHz, chloroform-d) δ 7.99 (d, J = 1.1 Hz, 2H), 7.41−7.30 (m, 4H), 7.24 (s, 1H), 7.04 (ddd, J = 9.0, 2.3, 0.4 Hz, 2H), 1.11−0.94 (m, 4H), 0.81−0.68 (m, 4H). 5-(1-Methylcyclopropoxy)-2-((2-(trimethylsilyl)ethoxy)methyl)-2H-indazole (36). To a stirred solution of 35 (3.36 g, 17.85 mmol) in THF (50 mL) were added N,N-dicyclohexylmethylamine (4.97 mL, 23.21 mmol) and 2-(trimethylsilyl)ethoxymethyl chloride (3.78 mL, 21.42 mmol). The mixture was stirred at RT overnight. The reaction was quenched with water and extracted with CH2Cl2 (3×). The combined organic layers were washed with 1 N HCl (aq) (2×), 1N NaOH (aq) (2×), and brine, dried, filtered, and concentrated to leave a residue which was purified by silica gel column (elution with 10:1 hexanes:EtOAc) to yield 36 (4.5 g, 79%) as a light-yellow oil. LCMS (ESI MS) m/z = 319.2 [M + H]+ 1. 1H NMR (400 MHz, chloroform-d) δ 7.96 (s, 1H), 7.60 (dddd, J = 9.3, 1.0, 1.0, 1.0 Hz, 1H), 7.14 (ddd, J = 2.1, 0.9, 0.9 Hz, 1H), 6.95 (ddd, J = 9.3, 2.3, 1.0 Hz, 1H), 5.67 (d, J = 0.9 Hz, 2H), 3.64−3.55 (m, 2H), 1.58 (s, 3H), 1.02 (m, 2H), 0.95−0.89 (m, 2H), 0.73−0.69 (m, 2H). 3-(6-Chloropyrimidin-4-yl)-5-(1-methylcyclopropoxy)-2-((2(trimethylsilyl)ethoxy)methyl)-2H-indazole (37). To a cold (−78 °C), stirred solution of 36 (2.09 g, 6.56 mmol) in THF (13 mL) was added n-BuLi (1.6 M in hexane, 5.33 mL, 8.53 mmol). The mixture was then stirred at −78 °C for 15 min and then raised to −20 °C for 5 min. The mixture was cooled back down to −78 °C, and a solution of ZnCl2 (19.69 mL, 9.84 mmol) in THF was added. The mixture was then raised to −20 °C and stirred for 10 min at −20 °C. A mixture of 4,6-dichloropyrimidine (1.075 g, 7.22 mmol) and tetrakis(triphenylphosphine) palladium (0.379 g, 0.328 mmol) was added. The cold bath was removed, and the mixture was stirred at RT for 5 h before being quenched by saturated aqueous NH4Cl. The solution was then extracted with CH2Cl2 (3×). The combined organic layers were dried, filtered, and concentrated to leave a residue which was purified by silica gel column (elution with 10:1 hexanes:EtOAc) to yield 37 (2g, 71%) as a colorless gum. LCMS (ESI+, m/z): 431 [M + H]+. 1H NMR (300 MHz, CDCl3, ppm): δ 9.12 (s,1H), 7.97 (s, 1H), 7.73 (d, J = 9.3 Hz, 1H), 7.58 (d, J = 2.1 Hz, 1H), 7.14−7.10 (dd, J = 9.3 Hz, 1H), 6.13 (s, 2H), 3.75−3.69, (m, 2H), 1.65 (s, 3H), 1.12−1.08 (m, 2H), 0.95−0.90 (m, 2H), 0.82−0.78 (m, 2H), 0.04 (s, 9H). cis-2,6-Dimethyl-4-(6-(5-(1-methylcyclopropoxy)-2-((2(trimethylsilyl)ethoxy)methyl)-2H-indazol-3-yl)pyrimidin-4-yl)morpholine (38). A solution of 37 (5 g, 11.60 mmol), cis-2,6dimethylmorpholine (4.29 mL, 34.8 mmol), and Et3N (9.70 mL, 69.6 mmol) in DMSO (50 mL) was heated at 100 °C in a sealed glass reactor for 2 h. The mixture was cooled, diluted with EtOAc, washed with water and brine, dried over anhydrous MgSO4, filtered, and evaporated. The resulting crude material was purified via silica gel chromatography (gradient elution: 15−70% EtOAc in hexanes) to afford 38 (4.85 g, 82%).. LCMS (ESI+, m/z): 510.2 [M + H]+. 1H NMR (500 MHz, CDCl3, ppm): δ 8.76 (s,1H), 7.68 (d, J = 9.4 Hz, 1H), 7.47 (d, J = 2.1 Hz, 1H), 7.14 (d, J = 0.9, 1H), 7.04 (dd, J = 9.4, 2.4 Hz, 1H), 6.10 (s, 2H), 4.27 (broad s, 2H), 3.74−3.69 (m, 4H), 2.70 (dd, J = 12.8, 12.8 Hz, 2H), 1.62 (s, 3H), 1.31 (s, 3H), 1.29 (s, 3H), 1.08 (dd, J = 6.9, 6.9 Hz, 2H), 0.91 (dd, J = 8.5, 2.8 Hz, 2H), 0.74 (m, 2H), −0.05 (s, 6H). cis-2,6-Dimethyl-4-(6-(5-(1-methylcyclopropoxy)-1H-indazol-3-yl)pyrimidin-4-yl)morpholine (1). Compound 38 (4.85 g, 9.52 mmol) was dissolved in MeOH (30 mL), and 4 M HCl in 1,4dioxane (35.7 mL, 143 mmol) was added. The resulting mixture was heated at 65 °C for 30 min. The mixture was cooled to room temperature, diluted with EtOAc and washed with satd aq NaHCO3. The EtOAc phase was dried over anhydrous MgSO4, filtered, and evaporated. The resulting crude material was purified by silica gel chromatography (80 g ISCO Redi Sep Rf; gradient elution, 30−100% EtOAc in hexanes) to afford 1 (3.3 g, 91%) as a white solid. HRMS

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00045. Homology model file (PDB) Molecular formula strings, IC50, P-gp and hepatocyte clearance values (CSV) Synthetic methods and characterization data for compounds 4−28, methods for in vitro assays, in vivo studies and molecular modeling along with crystallographic information (PDF) Accession Codes

Coordinates have been deposited in the PDB with accession code for 17: 5U6I. Authors will release the atomic coordinates and experimental data upon article publication.

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AUTHOR INFORMATION

Corresponding Authors

*For J.D.S.: phone, 908-740-4729; E-mail, jack.scott@merck. com. *For D.E.D.: phone, 617-992-3489; E-mail, duane.demong@ merck.com. *For T.J.G.: phone, 215-652-4873; E-mail, thomas_greshock@ merck.com. ORCID

Jack D. Scott: 0000-0002-7678-9699 John M. Sanders: 0000-0002-3788-4220 Present Addresses ⊥

For J. H.: H. B. Fuller, Vadnais Heights, Minnesota 55110, United States. # For M.K.M.: AECOM, Pittsburgh, Pennsylvania 15219, United States. ∇ For G.A: WuXi AppTec, Plainsboro, New Jersey 08536, United States. ○ For M.A.S.B: The Michael J. Fox Foundation, New York, New York 10018, United States. ◆ For J.C.: Leidos Biomedical Research, Inc., Frederick Maryland 21701, United States. ¶ For J.A.M: IOmet Pharm, Edinburgh EH16 4UX, United Kingdom + For J.T.K: AbbVie, North Chicago, Illinois 60064, United States. □ For J.J.R.: Purdue Pharma, Stamford, Connecticut 06901, United States. ● For M.T.B.: Tarveda Therapeutics, Watertown, Massachusetts 02472, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Corey Strickland and Michael Ellis for their valuable input toward this article. 2990



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ABBREVIATIONS USED ATP, adenosine triphosphate; AUC, area under the curve; CNS, central nervous system; CSF, cerebrospinal fluid; CYP, cytochrome P450; ERK2, extracellular signal-regulated kinase 2; HTS, high throughput screen; LE, ligand efficiency; LLCPK1, Lilly Laboratories Cell-porcine kidney 1; LRRK2, leucinerich repeast kinase 2; MDR1, multidrug resistance protein 1; Papp, apparent permeability; PD, Parkinson’s disease; P-gp, Pglycoprotein; pS935, phosphorylated LRRK2 serine 935; PSA, polar surface area; SEM, 2-(trimethyl)ethoxymethyl; VDW, van der Waals; MRT, mean residence time; F, bioavailability

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Discovery of a 3-(4-Pyrimidinyl) Indazole (MLi-2), an Orally Available

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