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

Article abstract of DOI:10.1021/acs.jmedchem.7b00045

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.

Full text of DOI:10.1021/acs.jmedchem.7b00045

Subscriber access provided by University of Newcastle, Australia
Article
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 P Nargund, John A McCauley, and Michael W Miller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00045 • Publication Date (Web): 28 Feb 2017
Downloaded from http://pubs.acs.org on February 28, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Bilodeau, Mark; Tarveda Therapeutics,
Kennedy, Matthew; Merck and Co Inc
Parker, Eric; NA
Stamford, Andrew; NA
Nargund, Ravi; Merck Research Laboratories, Dept. of Medicinal Chemistry
McCauley, John; Merck Research Laboratories, Department of Medicinal
Chemistry
Miller, Michael; NA
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 30
1
2
3
4
5
6
7
8
9
Discovery of a 3ꢀ(4ꢀPyrimidinyl) Indazole (MLiꢀ2), an
Orally Available and Selective LeucineꢀRich Repeat
Kinase 2 (LRRK2) Inhibitor that Reduces Brain Kinase
Activity
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Jack D. Scott*,1, Duane E. DeMong*,2, Thomas J. Greshock*,3, Kallol Basu4, Xing Dai1, Joel
Harris1,±,Alan Hruza1, Sarah W. Li3, SueꢀIng Lin1, Hong Liu1, Megan K. Macala1,∆, Zhiyong Hu1, Hong
Mei1, Honglu Zhang1, Paul Walsh4, Marc Poirier1, ZhiꢀCai Shi1, Li Xiao1, Gautam Agnihotri1,◊, Marco
A. S. Baptista1,▲, John Columbus2,┴, Matthew J. Fell2, Lynn A. Hyde1, Reshma Kuvelkar1, Yinghui Lin1,
Christian Mirescu2, John A. Morrow1,○, Zhizhang Yin1, Xiaoping Zhang1, Xiaoping Zhou1, Ronald K.
Chang3, Mark W. Embrey3, John M. Sanders3, Heather E. Tiscia3, Robert E. Drolet3, Jonathan T.
Kern3,#, Sylvie M. Sur3, John J. Renger3,‡, Mark T. Bilodeau3,=, Matthew E. Kennedy2, Eric M. Parker1,
Andrew W. Stamford1, Ravi Nargund1, John A. McCauley3, and Michael W. Miller1.
1 Merck & Co., Inc., 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States; 2 Merck & Co.,
Inc., 33 Avenue Louis Pasteur, Boston, MA 02115, United States; 3 Merck & Co., Inc., 770
Sumneytown Pike, West Point, PA 19486, United States; 4 Merck & Co., Inc., 126 E. Lincoln Avenue,
Rahway, NJ 07065, United States.
KEYWORDS: MLiꢀ2, LeucineꢀRich Repeat Kinase 2, LRRK2, kinase inhibitor, kinase selectivity,
Parkinson’s disease.
ACS Paragon Plus Environment

Page 3 of 30
Journal of Medicinal Chemistry
ABSTRACT: LeucineꢀRich 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 effecting disesase 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 MLiꢀ2 (1):
a potent, highly selective, orally available, brain penetrant inhibitor of LRRK2.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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.
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 leucineꢀrich repeast kinase 2 (LRRK2) among
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 30
several others that have been identified to date.4ꢀ6 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 prevelant
genetic risk factor for PD.
1
2
3
4
5
6
7
8
9
LRRK2 is a large multidomain protein consisting of a kinase, a GTPase and several proteinꢀprotein
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,
LRRK2ꢀlinked PD typically manifests itself as a lateꢀonset and slowly progressing disease which may
be more amenable to clinical intervention for disease modification, compared to juvenile or earlyꢀonset
familial forms of PD.10ꢀ15 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 diseaseꢀmodifying treatments
for PD.16ꢀ18
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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 cisꢀ2,6ꢀdimethylꢀ4ꢀ(6ꢀ(5ꢀ(1ꢀ
methylcyclopropoxy)ꢀ1Hꢀindazolꢀ3ꢀyl)pyrimidinꢀ4ꢀyl)morpholine (MLiꢀ2, 1, Fig. 1), a highly potent
and selective LRRK2 kinase 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.
RESULTS AND DISCUSSION
ACS Paragon Plus Environment

Page 5 of 30
Journal of Medicinal Chemistry
The identification of selective and brainꢀpenetrant LRRK2 kinase inhibitors began with a high
1
2
throughput screen (HTS) of our sample collection using recombinant wildꢀtype (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 follow up kinome
screens. The HTS screen led to the identification of several 3,5ꢀdisubstituted indazoles, with nanomolar
LRRK2 IC50’s and modest to good ligand efficiencies (LE).22 Indazoles 2 and 3 are representative of the
hits uncovered during the screening campaign (Fig. 1). In addition to their LRRK2 potencies, these
analogs 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 i.p. 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.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
N
N
O
N
N
1 (MLi-2)
O
N
H
N
H
N
N
N
N
HN
NH2
N
N
MeO2S
2
3
LRRK2 IC50 = 44 nM
LE = 0.37
LRRK2 IC50 = 728 nM
LE = 0.37
Figure 1. Indazole lead 1, HTS hits 2 and 3 and G2019S LRRK2 Enzyme Inhibition
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 towards more complicated analogs 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
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 30
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
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For the lead optimization campaign, it was decided to assay compounds against G2019S LRRK2
since 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 4ꢀ10 which are truncated at the 5ꢀposition 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 Cꢀ3 to a methyl group
while leaving Cꢀ5 intact. These compounds, however suffered from a significant erosion in the kinase
selectivity profile (data not shown). Of the three pyridine regioisomers, the 3ꢀ and 4ꢀpyridyl analogs (4
and 5 respectively) had similar LRRK2 IC50s while the 2ꢀpyridyl analog 7 was less potent. Further
elaboration of the Cꢀ3 pyridine ring with amino substituents, as is present in 3, provided analogs with
improved potencies. In general, analogs 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 analogs with a 4ꢀpyridyl at Cꢀ3 were found to be
significantly more potent than the corresponding 3ꢀpyridyl analogs. For example, 8 was 21ꢀfold more
potent than the 3ꢀpyridine regioisomer 9. It is also noteworthy that the pyrimidine analog 10 was 11ꢀ
fold less potent than the 4ꢀpyridyl analog 8.
Introduction of heteroaromatic groups at Cꢀ5, while maintaining the pyridyl morpholine at Cꢀ3, led to
the identification of a series of very potent LRRK2 inhibitors with single digit nanomolar IC50s;
however, these analogs were generally found to be Pꢀgp efflux substrates in LLCꢀPK1 cells expressing
MDR124 and/or possessed significantly eroded kinase selectivities. For example, in a panel of 68
ACS Paragon Plus Environment

Page 7 of 30
Journal of Medicinal Chemistry
kinases, the pyrazole analog 12, at a concentration of 1 µM, was found to exhibit greater than 50%
inhibition of 24 of the kinases screened.
1
2
3
4
5
6
7
8
In an effort to identify potent analogs with nonꢀaromatic Cꢀ5 substituents, a series of analogs with
alternative substituents exemplified by compounds 13ꢀ18 was evaluated (Table 1). The cyclopropyl,
cyano and sulfonyl substituents (analogs 13ꢀ15 respectively) were found to provide no improvement in
LRRK2 potency over that of the Cꢀ5 unsubstituted analog 8. In addition, the increased polarity imparted
by the sulfonyl moiety of 15 (PSA 101 Å2)25 likely attributed to that analog being a Pꢀgp substrate.
Strikingly, while the methoxy analog 16 was found to have a moderate IC50 of 15 nM, the isopropoxy
compound 17 (MLiꢀ1)20 was found to be 14ꢀfold 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 kinases at a level greater than 50%, with TTK being
inhibited the most at 91%.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. LRRK2 inhibition, efflux ratio, permeability and hepatocycte clearance data.
Pgp
h/rb
Hep Cl
LRRK2 IC50
(nM)a
Cmpd
R1
R2
h/r
LE
(uL/min/
[hPapp]
106cells)
(10ꢀ6 cm/s)
H
H
H
H
904
1231
6186
98
0.55
0.54
0.48
0.48
NT
NT
NT
NT
NT
NT
NT
NT
4
5
6
7
0.3/0.2
[19]
H
H
H
12
0.51
0.43
0.45
NT
NT
8
9
0.4/0.5
[38]
256
134
NT
9/30
10
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 30
Pgp
h/rb
Hep Cl
1
2
3
4
R1
R2
LRRK2 IC50
LE
Cmpd
h/r
(nM)a
(uL/min/
[hPapp]
106cells)
(10ꢀ6 cm/s)
5
6
7
8
16/38
[32]
6.4
<0.6
13
0.42
>0.47
0.45
0.43
0.39
0.48
0.50
0.41
10/6
7/31
11
12
13
14
15
16
17
18
1.5/6.3
[38]
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.2/0.1
[25]
31/52
NT
0.7/1.3
[34]
CN
64
20/44
[35]
18
7.3/15
23/28
22/49
37/32
0.2/0.4
[33]
OMe
15
0.5/0.7
[30]
0.7
41
0.3/0.3
[31]
a G2019S LRRK2 IC50 values are the average of a minimum of two independent determinations (see
ref 20). bEfflux ratio AꢀB/BꢀA. Pgp: Pꢀglycoprotein, h: human, r: rat, Hep Cl: hepatocyte clearance, NT:
Not Tested.
Without access to an Xꢀray crystal structure of LRRK2, we constructed a homology model of the
LRRK2 kinase domain based on the Xꢀray 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 Fig. 2 the model 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).
ACS Paragon Plus Environment

Page 9 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Figure 2. Overlay of Xꢀray structure of 17 in ERK2 (tan)[PDB code: 5U6I] with the model of 17 in
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
LRRK2 (green). Note: the surfaces for LRRK2 residues Lys1906 and Leu1949 are included.
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 Cꢀ3 pyridyl indazoles
showed high metabolic turnover in hepatocytes which translated into low exposures and short halfꢀlives
in rat. For example the isopropoxy analog 17 was found to have a modest AUC(0ꢀ8h) of 1.0 µM·h with a
halfꢀlife of 1 hour after a 2 mg/kg i.v. 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. In order 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
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 30
with electronꢀdeficient cyclic amines (analogs 20ꢀ22) did not lead to an improvement in clearance
compared to the morpolino analog 17. The triazole substituted piperidine analog 23 exhibited improved
hepatocyte stability, but it was found to be a Pꢀgp efflux substrate and possessed reduced kinase
selectivity compared to the morpholino analogs as indicated with the heat maps in Table 2. Excitingly,
replacement of the 4ꢀpyridyl in the indazole 3ꢀposition with a 2,4ꢀpyrimidinyl group (compound 24,
MLiꢀ3)20 led to improved stability in both rat and human hepatocytes, albeit with a 10ꢀfold loss in
LRRK2 potency compared to its homologue 17. This was consistent with the potency difference
observed between previously discussed analogs 8 and 10. Additional improvement in the LRRK2
potency was obtained by increasing the steric bulk of the Cꢀ5 alkoxy group with methylcyclopropoxy
analog 25 showing a modest improvement in LRRK2 IC50 compared to the isopropoxy analog 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 analog 18.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. LRRK2 inhibition, Cellular potency and kinase selectivity for indazole analogs.
Kinome Selectivity
5mM ATP
LRRK2
IC50
Pgp
Hep Cl
LRRK2
IC50
Cell
IC50
% inhibition
h/rc
R1
R2
h/r
Cmpd
(uL/min/
[hPapp]
(nM) a
(nM) a
106cells)
(nM) a,b
(10ꢀ6cm/s)
# kinases (conc.)d
ACS Paragon Plus Environment

Page 11 of 30
Journal of Medicinal Chemistry
0.5/0.7
[30]
1
2
3
4
5
6
7
8
17
0.7
0.8
2.7
2.6
14
22
14
91
43
52
83
59
22/49
7/36
306 (1 µM)
54 (0.1 µM)
304 (1 µM)
306 (1 µM)
0.6/1.7
[27]
19
0.4/0.6
[29]
20
21
7/32
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3.2/15
[38]
16/38
0.4/0.7
[31]
22
23
6.6
4.4
128
69
341
194
25/39
2/19
73 (1 µM)
73 (1 µM)
40/41
[46]
0.6/0.4
[38]
24
25
26
27
7.2
1.9
5.8
2.0
96
10
76
13
41
NT
3.8
5.0
6/13
8/18
7/26
7/15
306 (1 µM)
305 (1 µM)
306 (1 µM)
306 (1 µM)
0.3/0.4
[35]
0.5/0.5
[27]
0.3/0.4
[28]
0.8/0.6
[24]
1
0.76
6.4
4.8
8/15
306 (1 µM)
2.2/12
[30]
28
2.2
11
7.6
ꢀꢀ/10
305 (1 µM)
a G2019S LRRK2 IC50 values are the average of a minimum of two independent determinations (see
ref 20). bEnzymatic assay using 5 mM ATP. cEfflux ratio AꢀB/BꢀA. dHeat map indicating the amount of
inhibition for a panel of kinases listed in alphabetical order (AꢀZ left to right) not including LRRK2.
Pgp: Pꢀglycoprotein, h: human, r: rat, Hep Cl: hepatocyte clearance, NT: Not Tested.
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. Analogs 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 analog 1 was found to
be a very potent LRRK2 inhibitor (0.76 nM) and extremely selective with at least 295ꢀfold
selectivity over the entire kinase panel.20 With a LE of 0.45 and exquisite selectivity for 1, we
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 30
1
2
3
4
5
6
achieved our goal of identifying a LRRK2 inhibitor with a superior profile compared to the
original HTS hits.
7
8
9
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 Fig.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.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Due to 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 5ꢀ to 35ꢀfold loss in potency compared to the IC50s obtained
under the standard assay conditions. Despite this shift in potency, several analogs 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 35Sꢀlabelled 28 (MLiꢀ
A)20. In this communication we report a set of IC50s generated via a high throughput cellular
assay that has been used to profile the advanced inhibitors. In comparison of the enzymatic and
11
ACS Paragon Plus Environment

Page 13 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
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.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Pharmacokinetic parameters of 1 in HanꢀWistar rat.
IV administrationa Oral administrationb
Parameter
(2 mg/kg)
0.66 ± 0.24
1.17 ± 0.15c
ꢀ
(10 mg/kg)
1.3 ± 0.56
0.48 ± 0.36
1.3 ± 0.75
AUC(0ꢀ8h)
[ꢁM·h]
Cmax [ꢁM]
Tmax
[h]
T1/2
[h]
1.8 ± 1.0
1.7 ± 0.46
153 ± 55
ꢀ
ꢀ
MRT
[h]
4.0 ± 1.0
ꢀ
Cl
[mL/min/kg]
F
[%]
39 ± 18
a
Dosed as a solution of the free base (2 mg/mL) in a mixture of DMSO:PEG400:water
(20:60:20). Dosed as a fine suspension of the free base (5 mg/mL) in a mixture of
DMSO:PEG400:water (20:60:20). initial concentration. MRT: mean residence time, F:
b
c
bioavailability.
The dimethylmorpholine analog 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 a favorable apparent halfꢀlife. The difference in oral bioavailability
observed between rats is likely due to the low equilibrium solubility of 1 at 24h in a variety of
buffers (<0.014 mg/mL to 0.038 mg/mL, see supporting information). The high and variable IV
clearance may also be attributed to the poor solubility of 1 in media covering a wide pH range.
12
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 30
1
2
3
4
5
6
Given the acceptable oral exposure observed in the rat pharmacokinetic study, the decision was
made to progress 1 into an in vivo target engagement study in rats.
7
8
9
Additionally, the free fraction of 1 in rat plasma was found to be 1.2%. Compound 1 does not
inhibit human CYP isoforms 2D6, 2C9 or 3A4 (all IC50s > 50 µM) nor was it a timeꢀdependent
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
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We have previously reported the results of the pharmacokineticꢀpharmacodynamic (PK/PD)
evaluation of 1 in wildꢀtype 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 11ꢀday inꢀdiet 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 15ꢀweek
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 observations 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 (1ꢀ100 mg/kg) one hour post treatment in rats (Fig.
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 Fig 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 nM and 1.1 nM in the brain and plasma
respectively.20
13
ACS Paragon Plus Environment

Page 15 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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).
SYNTHESIS
As summarized in Scheme 1, the preparation of 1 commenced with an SNAr reaction between
the commercially available 1ꢀmethylꢀ1ꢀcyclopropanol and aryl fluoride 29 to afford ether 30.
Palladiumꢀmediated methylation followed by reduction of the nitro group afforded the amino
derivative 32 which upon 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 SEMꢀCl and the hindered base N,Nꢀdicyclohexylmethylamine, resulted in
selective SEM protection of the N2 position of the indazole to afford 36. Regioselective lithiation
of the 3ꢀposition of the 36, followed by Negishi coupling with 4,6ꢀdichloropyrimidine resulted in
14
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 30
1
2
the formation of intermediate 37.27 Displacement of the chloride with cisꢀ2,6ꢀ
dimethylmorpholine, followed by removal of the SEM protecting group led to the formation of 1.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthesis of 1.
Reagents and conditions: (a) NaH, DMF, 0 °C then RT, 5h, 87%. (b) trimethylboroxine,
Pd(Ph3P)4, K2CO3, Cs2CO3, 1,4ꢀdioxane, 100 °C, 16h. (c) ammonium formate, 10% Pd/C EtOH,
RT, 5h, 96% (2 steps). (d) Ac2O, Et3N, CH2Cl2, RT, 16h, 96%. (e) Ac2O, KOAc, 3ꢀmethylbutyl
nitrite, toluene, 80 °C, 16h, 86%. (f) 3.5 M ammonia in MeOH, RT, 2h, 94%. (g) cꢀHex2NMe,
SEMꢀCl, THF, RT 16h, 79%. (h) 1) nꢀBuLi, THF, ꢀ78 °C then ꢀ20 °C. 2) ZnCl2, ꢀ78 °C then ꢀ20
°C. 3) Pd(PPh3)4, 4,6ꢀdichloropyrimidine, THF, RT, 5h, 71%. (i) cisꢀ2,6ꢀdimethylmorpholine,
Et3N, DMSO, 100 °C, 2h, 82%. (j) 4N HCl in 1,4ꢀdioxane, MeOH, RT then 65 °C, 30 min, 91%.
Lateꢀstage intermediates exemplified by 37 allowed for rapid analog 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
15
ACS Paragon Plus Environment

Page 17 of 30
Journal of Medicinal Chemistry
1
2
silico properties, including CNS MPO,23 were calculated with the analogs meeting our criteria
being selected for synthesis. These library compounds were prepared in parallel followed by
massꢀtriggered HPLC purification28 on sufficient scale that allowed for evaluation in our
standard set of assays (e.g. LRRK2 enzyme IC50, Pꢀgp, Hep Cl and kinome selectivity). Analogs
that were progressed for further evaluation were scaled up in singleton format as needed.
CONCLUSION
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
With the identification of a number of promising indazole screening hits, followed by a focus
on the optimization of ligand efficiency, in vitro Pꢀgp 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 brainꢀpenetrant 1. A doseꢀdependent 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.
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
1
solvent peak in the indicated solvent, and for H 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
16
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 30
1
2
3
4
5
6
LCMS system. All active compounds were analyzed for and found to be free of pan assay
interference compounds (PAINS).29
7
8
9
2ꢀbromoꢀ4ꢀ(1ꢀmethylcyclopropoxy)ꢀ1ꢀnitrobenzene (30). To a cold (0 °C), stirred mixture of
2ꢀbromoꢀ4ꢀfluoroꢀ1ꢀnitrobenzene (10 g, 45.5 mmol) and 1ꢀmethylcyclopropanꢀ1ꢀol (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(3x). The combined organic layers were washed with water (3x), brine (2x), dried, filtered
and concentrated to leave an oil which was purified by silica gel column (gradient elution with
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
100:0 to 20:1 hexane:EtOAc) to yield 30 (10.8g, 87%) as a light yellow oil. H NMR (400 MHz,
Chloroformꢀd) 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).
2ꢀmethylꢀ4ꢀ(1ꢀmethylcyclopropoxy)ꢀ1ꢀnitrobenzene (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 1,4ꢀdioxane (502 ml)
was purged 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 hex: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 hex:EtOAc solution. The filtrate was concentrated to afford crude 31 (5.3 g), which
1
was used in the next step without further purification. H NMR (400 MHz, Chloroformꢀd) δ 8.06
(d, J = 9.1 Hz, 1H), 6.92 (ddd, J = 9.1, 2.7, 0.5 Hz, 1H), 6.87ꢀ6.85 (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).
17
ACS Paragon Plus Environment

Page 19 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2ꢀmethylꢀ4ꢀ(1ꢀmethylcyclopropoxy)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 heterogenous mixture was stirred at RT for 5 h. To this solution was
added 5:1 hexane: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, Chloroformꢀd) 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).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Nꢀ(2ꢀmethylꢀ4ꢀ(1ꢀmethylcyclopropoxy)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
(3x). 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
(6.1g, 96%) as a colorless gum. LCMS (ESI MS) m/z = 220.0 [M + H]+
1ꢀ(5ꢀ(1ꢀmethylcyclopropoxy)ꢀ1Hꢀindazolꢀ1ꢀyl)ethanꢀ1ꢀone (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. 3ꢀMethylbutyl 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 hexane:EtOAc) to yield
34 (5.5g, 86%) as a yellow solid. LCMS (ESI MS) m/z = 231.2 [M + H]+; 253.2 [M + Na]+
18
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 30
1
2
3
4
5
6
7
8
9
1H NMR (400 MHz, Chloroformꢀd) 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).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5ꢀ(1ꢀmethylcyclopropoxy)ꢀ1Hꢀindazole (35). To a stirred suspension of 34 (6.5 g, 28.2 mmol)
in MeOH (20 mL) was added 7M ammonia in MeOH (20.16 ml, 141 mmol) and the mixture was
stirred at RT for 2h. The reaction was concentrated and the residue was purified by silica gel
column (gradient elution with 5:1 to 1:1 hexane:EtOAc) to yield 35 (5g, 94%) as a yellow solid.
LCMS (ESI MS) m/z = 189.2 [M + H]+ 1H NMR (400 MHz, Chloroformꢀd) 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).
5ꢀ(1ꢀmethylcyclopropoxy)ꢀ2ꢀ((2ꢀ(trimethylsilyl)ethoxy)methyl)ꢀ2Hꢀindazole (36).
To a
stirred solution of 35 (3.36 g, 17.85 mmol) in THF (50 ml) were added N,Nꢀ
dicyclohexylmethylamine (4.97 ml, 23.21 mmol) and 2ꢀ(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 (x3). The combined organic layers were washed with 1N HCl
(aq.) (2x), 1N NaOH (aq.) (2x) and brine, dried, filtered and concentrated to leave a residue
which was purified by silica gel column (elution with 10:1 hexane:EtOAc ) to yield 36 (4.5g,
79%) as a light yellow oil. LCMS (ESI MS) m/z = 319.2 [M + H]+ 1H NMR (400 MHz,
Chloroformꢀd) 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).
3ꢀ(6ꢀchloropyrimidinꢀ4ꢀyl)ꢀ5ꢀ(1ꢀmethylcyclopropoxy)ꢀ2ꢀ((2ꢀ(trimethylsilyl)ethoxy)methyl)ꢀ
2Hꢀindazole (37). To a cold (ꢀ78 °C), stirred solution of 36 (2.09 g, 6.56 mmol) in THF (13 ml)
19
ACS Paragon Plus Environment

Page 21 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
was added nꢀBuLi (1.6M 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 4,6ꢀdichloropyrimidine (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 sat. aq. NH4Cl. The
solution was then extracted with CH2Cl2 (3x). The combined organic layers were dried, filtered
and concentrated to leave a residue which was purified by silica gel column (elution with 10:1
hexane:EtOAc) to yield 37 (2g, 71%) as a colorless gum. LCMS (ESI+, m/z): 431 [M+H]+
1HꢀNMR (300MHz, CDCl3, ppm): δ 9.12 (s,1H), 7.97 (s, 1H), 7.73 (d, J = 9.3Hz, 1H), 7.58 (d, J
= 2.1Hz, 1H), 7.14ꢀ7.10 (dd, J = 9.3Hz, 1H), 6.13 (s, 2H), 3.75ꢀ3.69, (m, 2H), 1.65 (s, 3H), 1.12ꢀ
1.08 (m, 2H), 0.95ꢀ0.90 (m, 2H), 0.82ꢀ0.78 (m, 2H), 0.04 (s, 9H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
cisꢀ2,6ꢀdimethylꢀ4ꢀ(6ꢀ(5ꢀ(1ꢀmethylcyclopropoxy)ꢀ2ꢀ((2ꢀ(trimethylsilyl)ethoxy)methyl)ꢀ2Hꢀ
indazolꢀ3ꢀyl)pyrimidinꢀ4ꢀyl)morpholine (38). A solution of 37 (5 g, 11.60 mmol), cisꢀ2,6ꢀ
dimethylmorpholine (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 2h. 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% to
70% EtOAc in hexanes) to afford 38 (4.85 g, 82%).. LCMS (ESI+, m/z): 510.2 [M+H]+ 1Hꢀ
NMR (500MHz, 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), 3.74ꢀ3.69
(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).
20
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 30
1
2
3
4
cisꢀ2,6ꢀdimethylꢀ4ꢀ(6ꢀ(5ꢀ(1ꢀmethylcyclopropoxy)ꢀ1Hꢀindazolꢀ3ꢀyl)pyrimidinꢀ4ꢀ
5
6
7
8
yl)morpholine (1). Compound 38 (4.85 g, 9.52 mmol) was dissolved in MeOH (30 ml) and 4M
HCl in 1,4ꢀdioxane (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
sat. 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 to 100% EtOAc in hexanes) to afford 1 (3.3 g, 91%) as a white
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
solid. HRMS (ESI+) m/z 380.2077 [(M+H)+ calcd for C21H25N5O2: 380.2086 H 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.
ASSOCIATED CONTENT
The following files are available free of charge.
Supporting Information
Synthetic methods and characterization data for compounds 4ꢀ28, methods for in vitro assays,
in vivo studies and molecular modeling along with crystallographic information (PDF)
Molecular formula strings, IC50, Pꢀgp and hepatocyte clearance values (CSV) Homology model
file (PDB).
Accession Code
21
ACS Paragon Plus Environment

Page 23 of 30
Journal of Medicinal Chemistry
1
2
3
4
Coordinates have been deposited in the PDB with accession code for 17: 5U6I
5
6
7
Authors will release the atomic coordinates and experimental data upon article publication.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
AUTHOR INFORMATION
Corresponding Authors
* Phone: 908ꢀ740ꢀ4729. Eꢀmail: jack.scott@merck.com
* Phone: 617ꢀ992ꢀ3489. Eꢀmail: duane.demong@merck.com
* Phone: 215ꢀ652ꢀ4873. Eꢀmail: thomas_greshock@merck.com
Notes
The authors declare no competing financial interest.
Present Addresses
± (J. H.): H. B. Fuller, Vadnais Heights, Minnesota, USA
∆(M.K.M.): AECOM, Pittsburgh, Pennsylvania, USA
◊(G.A): WuXi AppTec, Plainsboro, New Jersey, USA
▲(M.A.S.B): The Michael J. Fox Foundation, New York, New York, USA
┴(J.C.): Leidos Biomedical Research, Inc., Frederick Maryland, USA
○(J.A.M): IOmet Pharm, Edinburgh, United Kingdom
#(J.T.K): AbbVie, North Chicago, Illinois, USA
‡(J.J.R): Purdue Pharma, Stamford, Connecticut, USA
=(M.T.B): Tarveda Therapeutics, Watertown, Massachusetts, USA
22
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 30
1
2
3
4
ACKNOWLEDGMENT
5
6
7
Corey Strickland and Michael Ellis their valuable input towards this mansuscript.
8
9
ABBREVIATIONS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ATP, adenosine triphosphate; AUC, area under the curve; CNS, central nervous system; CSF,
cerebrospinal fluid; CYP, Cytochrome P450; ERK2, extracellular signalꢀregulated kinase 2;
HTS, high throughput screen; LE, ligand efficiency; LLCꢀPK1, Lilly Laboratories Cellꢀporcine
kidney 1; LRRK2, leucineꢀrich repeast kinase 2; MDR1, multidrug resistance protein 1; Papp,
apparent permeability; PD, Parkinson’s disease; Pꢀgp, Pꢀglycoprotein; pS935, phosphorylated
LRRK2 serine 935; PSA, polar surface area; SEM, 2ꢀ(trimethyl)ethoxymethyl; VDW, Van der
Waals; MRT, Mean Residence Time; F, Bioavailability.,
REFERENCES
(1)
Parkinson’s
Disease
Foundation
(online).
Available
at:
www.pdf.org/en/parkinson_statistics. Accessed November 17, 2016.
(2) Goedert, M.; Spillantini, M. G.; Del Tredici, K.; Braak, H. 100 years of Lewy pathology.
Nature Rev. Neurol. 2013, 9, 13–24.
(3) Lin, M. K.; Farrer, M. J. Genetics and genomics of Parkinson’s disease. Genome Med.
2014, 6, 48.
(4) PaisanꢀRuiz, 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.; MartiꢀMasso, J. F.; PerezꢀTur, J.; Wood, N. W.;
23
ACS Paragon Plus Environment

Page 25 of 30
Journal of Medicinal Chemistry
1
2
3
4
Singleton, A. B. Cloning of the gene containing mutations that cause PARK8ꢀlinked Parkinson's
5
6
disease. Neuron, 2004, 44, 595ꢀ600.
7
8
9
(5) 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.; MullerꢀMyhsok, B.; Dickson, D. W.; Meitinger, T.; Strom, T. M.;
Wszolek, Z. K.; Gasser, T. Mutations in LRRK2 cause autosomalꢀdominant parkinsonism with
pleomorphic pathology. Neuron, 2004, 44, 601ꢀ607.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(6) Kachergus, J.; Mata, I. F.; Hulihan, M.; Taylor, J. P.; Lincoln, S.; Aasly, J.; Gibson, J. M.;
Ross, O. A.; Lynch, T.; Wiley, J.; Payami, H.; Nutt, J.; Maraganore, D. M.; Czyzewski, K.;
Styczynska, M.; Wszolek, Z. K.; Farrer, M. J.; Toft, M. Identification of a novel LRRK2
mutation linked to autosomal dominant parkinsonism: evidence of a common founder across
European populations. Am. J. Hum. Genet., 2005, 76, 672ꢀ680.
(7) Healy, D. G.; Falchi, M.; O’Sullivan, S. S.; Bonifati, V.; Durr, A.; Bressman, S.; Brice, A.;
Aasly, J.; Zabetian, C. P.; Goldwurm, S.; Ferreira, J. J.; Tolosa, E.; Kay, D. M.; Klein, C.;
Williams, D. R.; Marras, C.; Lang, A. E.; Wszolek, Z. K.; Berciano, J.; Schapira, A. H. V.;
Lynch, T.; Bhatia, K. P.; Gasser, T.; Lees, A. J.; Wood, N. W., and International LRRK2
consortium. Phenotype, genotype, and worldwide genetic penetrance of LRRK2ꢀassociated
Parkinson’s disease: a caseꢀcontrol study. Lancet Neurol. 2008, 7, 583–590.
(8) Cookson, M. R. The role of leucineꢀrich repeat kinase 2 (LRRK2) in Parkinson’s disease.
Nat. Rev. Neurosci. 2010, 11, 791–797.
24
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 30
1
2
3
4
5
6
7
8
9
(9) West, A. B.; Moore, D. J.; Biskup S.; Bugayenko, A.; Smith, W. W. Ross, C. A.; Dawson,
V. L.; Dawson, T. M. Parkinson’s diseaseꢀassociated mutations in leucineꢀrich repeat kinase 2
augment kinase activity. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16842–16847.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(10) Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A., Pike,
B., Root, H., Rubenstein, J., Boyer, R., Stenroos, E. S., Chandrasekharappa, S., Athanassiadou,
A., Papepetropoulos, T., Johnson, W. G., Lazzarini, A. M., Duvoisin, R. C., Di Iorio, G., Golbe,
L. I., Nussbaum, R. L. Mutation in the αꢀsynuclein gene identified in families with Parkinson's
disease. Science 2007, 276, 2045ꢀ2047.
(11) Lucking, C. B.; Durr, A.; Bonifati, V.; Vaughan, J.; De Michele, G.; Gasser, T.;
Harhangi, B. S.; Meco, G.; Denefle, P.; Wood, N. W.; Agid, Y.; Brice, A. Association between
earlyꢀonset Parkinson's disease and mutations in the parkin gene. N. Engl. J. Med. 2000, 342,
1560–1567.
(12) Periquet, M.; Latouche, M.; Lohmann, E.; Rawal, N.; De Michele, G.; Ricard, S.; Teive,
H.; Fraix, V.; Vidailhet, M.; Nicholl, D.; Barone, P.; Wood, N. W.; Raskin, S.; Deleuze, J. F.;
Agid, Y.; Durr, A.; Brice, A. Parkin mutations are frequent in patients with isolated earlyꢀonset
parkinsonism. Brain, 2003, 126, 1271–1278.
(13) Valente, E. M.; AbouꢀSleiman, P. M.; Caputo V.;, Muqit, M. M.; Harvey, K.; Gispert, S.;
Ali, Z.; Del Turco, D.; Bentivoglio, A. R.; Healy, D. G.; Albanese, A.; Nussbaum, R.; Gonzalezꢀ
Maldonado, R.; Deller, T.; Salvi, S.; Cortelli, P.; Gilks, W. P.; Latchman, D. S.; Harvey, R. J.;
Dallapiccola, B.; Auburger, G.; Wood, N. W. Hereditary earlyꢀonset Parkinson's disease caused
by mutations in PINK1. Science, 2004, 304, 1158–1160.
25
ACS Paragon Plus Environment

Page 27 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(14) Valente, E. M.; Bentivoglio, A. R.; Dixon, P. H.; Ferraris, A.; Ialongo, T.; Frontali, M.;
Albanese, A.; Wood, N. W. Localization of a novel locus for autosomal recessive earlyꢀonset
parkinsonism, PARK6, on human chromosome 1p35ꢀp36. Am. J. Hum. Genet. 2001, 68, 895–
900.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(15) Hague, S.; Rogaeva, E.; Hernandez, D.; Gulick, C.; Singleton, A.; Hanson, M.; Johnson,
J.; Weiser, R.; Gallardo, M.; Ravina, B.; GwinnꢀHardy, K.; Crawley, A.; St. GeorgeꢀHyslop, P.
H.; Lang, A. E.; Heutink, P.; Bonifati, V.; Hardy, J.; Singleton, A. Earlyꢀonset Parkinson's
disease caused by a compound heterozygous DJꢀ1 mutation. Ann. Neurol.2003, 54, 271–274.
(16) Henderson, J. L.; Kormos, B. L.; Hayward, M. M.; Coffman, K. J.; Jasti, J.; Kurumbail,
R. G.; Wager, T. T.; Verhoest, P. R.; Noell, G. S.; Chen, Y.; Needle, E.; Berger, Z.; Steyn, S. J.;
Houle, C.; Hirst, W. D.; Galatsis, P. Discovery and preclinical profiling of 3ꢀ[4ꢀ(morpholinꢀ4ꢀ
yl),7Hꢀpyrrolo[2,3ꢀd]pyrimidinꢀ5ꢀyl]benzonitrile (PFꢀ06447475), a highly potent, selective,
brain penetrant, and in vivo active LRRK2 kinase inhibitor. J. Med. Chem. 2015, 58, 419ꢀ432.
(17) Estrada, A. A.; Chan, B. K.; BakerꢀGlenn, C.; Beresford, A.; Burdick, D. J.; Chambers,
M.; Chen, H.; Dominguez, S. L.; Dotson, J.; Drummond, J.; Michael Flagella, M.; Reina Fuji,
R.; Andrew Gill, A.; Halladay, J.; Harris, S. F.; Timothy P. Heffron, T. P.; Kleinheinz, T.; Lee,
D. W.; Le Pichon, C. E.; Liu, X.; Lyssikatos, J. P.; Medhurst, A. D.; Moffat, J. G.; Nash, K.;
ScearceꢀLevie, K.; Sheng, Z.; Shore, D. G.; Wong, S.; Zhang, S.; Zhang, X.; Zhu, H.; Sweeney,
Z. K. Discovery of highly potent, selective, and brainꢀpenetrant aminopyrazole leucineꢀrich
repeat kinase 2 (LRRK2) Small Molecule Inhibitors. J. Med. Chem.2015, 57, 921ꢀ936.
26
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 30
1
2
3
4
5
6
7
8
9
(18) Hatcher, J. M.; Zhang, J.; Choi, H. G.; Ito, G.; Alessi, D. R.; Gray, N. S. Discovery of a
Pyrrolopyrimidine (JHꢀIIꢀ127), a highly potent, selective, and brain penetrant LRRK2 inhibitor.
ACS Med. Chem. Lett. 2015, 6, 584ꢀ589.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(19) Steger, M.; Tonelli, F.; Ito, G.; Davies, P.; Trost, M.; Vetter, M.; Wachter, S.; Lorentzen,
E.; Duddy, G.; Wilson, S.; Baptista, M. A. S.; Fiske, B. K.; Fell, M. J.; Morrow, J. A.; Reith, A.
D.; Alessi, D. R.; Mann, M. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2
regulates a subset of Rab GTPases. eLife, 2016, 10.7554/eLife.12813.
(20) Fell, M. J.; Mirescu, C.; Basu, K.; Cheewatrakoolpong, B.; DeMong, D. E.; Ellis, J. M.;
Hyde, L. A.; Lin, Y.; Markgraf, C. G.; Mei, H.; Miller, M.; Poulet, F. M.; Scott, J. D.; Smith, M.
D.; Yin, Z.; Zhou, Z.; Parker, E. M.; Kennedy, M. E.; Morrow, J. A. MLiꢀ2, a potent, selective,
and centrally active compound for exploring the therapeutic potential and safety of LRRK2
kinase inhibition. J. Pharmacol. Exp. Ther. 2015, 355, 397ꢀ409.
(21) A series of dihydrobenzothiophene based LRRK2 inhibitors was also identifed from our
HTS screening, see: Greshock, T. J.; Sanders, J. M.; Drolet, R. E.; Rajapakse, H. A.; Chang, R.
K.; Kim, B.; Rada, V. L.; Tiscia, H. E.; Su, H.; Lai, M.ꢀT.; Sur, S. M.; Sanchez, R. I.; Bilodeau,
M. T.; Renger, J. J.; Kern, J. T.; McCauley, J. A.; Potent, selective and orally bioavailable
leucineꢀrich repeat kinase 2 (LRRK2) inhibitors. Bio. Org. Med. Chem. Lett. 2016, 26, 2631ꢀ
2635.
(22) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a useful metric for lead
selection. Drug Discovery Today 2004, 9, 430–431.
27
ACS Paragon Plus Environment

Page 29 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(23) Wager, T. T.; Chandrasekaran, R.; Y.; Hou, X.; Troutman, M. D.; Verhoest, P. R.;
Villalobos, A.; Will, Y. Defining desirable central nervous system drug space through the
alignment of molecular properites, in vitro ADME, and safety attributes. ACS Chem. Neurosci.
2010, 1, 420ꢀ434.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(24) He, H.; Lyons, K. A.; Shen, X.; Yao, Z.; Bleasby, K.; Chan, G.; Hafey, M.; Li, X.; Xu, S.;
Salituro, G. M.; Cohen, L. H.; Tang, W. Utility of unbound plasma drug levels and Pꢀ
glycoprotein transport data in prediction of central nervous system exposure. Xenobiotica, 2009,
39, 687ꢀ693.
(25) The PSA value was calculated using the method described in Ertl, P.; Rohde, B.; Selzer,
P. Fast calculation of molecular polar surface area as a sum of fragmentꢀbased contributions and
its application to the prediction of drug transport properties. J. Med. Chem. 2000, 43, 3714ꢀ
3717, excluding the contribution from the sulfur atom.
(26) Ruchardt, C.; Hassmann, V. Eine Vereinfachung der Jacobsonschen indazolꢀsynthese.
Synthesis 1972, 7, 375ꢀ376.
(27) Basu, K.; Poirier, M.; Ruck, R. T. Solution to the C3ꢀarylation of indazoles: development
of a scalable method. Org. Lett. 2016, 18, 3218.
(28) Liu, M.; Chen, K.; Christian, D.; Fatima, T.; Pissarnitski, N.; Streckfuss, E.; Zhang, C.;
Xia, L.; Borges, S.; Shi, Z.; Vachal, P.; Tata, J.; Athanasopoulos, J. Highꢀthroughput purification
platform in support of drug discovery. ACS Comb. Sci. 2012, 14, 51ꢀ59.
28
ACS Paragon Plus Environment