Potent, selective and orally bioavailable leucine-rich repeat kinase 2 (LRRK2) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.04.021

Familial Parkinson's disease cases have recently been associated with the leucine rich repeat kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent, selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of the internal Merck sample collection. Initial SAR studies around the core established the series as a tractable small molecule lead series of LRRK2 inhibitors for potential treatment of Parkinson's disease. It was also found that incorporation of a lactam into the core drastically improved the CNS and DMPK properties of these small molecules.

Full text of DOI:10.1016/j.bmcl.2016.04.021

Accepted Manuscript
Potent, selective and orally bioavailable Leucine-Rich Repeat Kinase 2
(LRRK2) Inhibitors
Thomas J. Greshock, John M. Sanders, Robert E. Drolet, Hemaka A. Rajapakse,
Ronald K. Chang, Boyoung Kim, Vanessa L. Rada, Heather E. Tiscia, Hua Su,
Ming-Tain Lai, Sylvie M. Sur, Rosa I. Sanchez, Mark T. Bilodeau, John J.
Renger, Jonathan T. Kern, John A. McCauley
PII:
S0960-894X(16)30382-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.04.021
BMCL 23783
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 February 2016
7 April 2016
8 April 2016
Please cite this article as: 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 Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.04.021
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Potent, Selective and Orally Bioavailable
Leucine-Rich Repeat Kinase 2 (LRRK2)
Inhibitors
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Thomas J. Greshock,a,* John M. Sanders,b,* Robert E. Drolet,c Hemaka A. Rajapakse,a Ronald K.
Chang,a Boyoung Kim,a Vanessa L. Rada,a Heather E. Tiscia,a Hua Su,d Ming-Tain Lai,e Sylvie M.
Sur,e Rosa I. Sanchez,f Mark T. Bilodeau,a John J. Renger,c Jonathan T. Kernc and John A. McCauleya

Bioorganic & Medicinal Chemistry Letters
jo u r n a l h o m e p a g e : w w w . e ls e v ie r . c o m
Potent, selective and orally bioavailable Leucine-Rich Repeat Kinase 2 (LRRK2)
Inhibitors
Thomas J. Greshock,a,* John M. Sanders,b,* Robert E. Drolet,c Hemaka A. Rajapakse,a Ronald K. Chang,a
Boyoung Kim,a Vanessa L. Rada,a Heather E. Tiscia,a Hua Su,d Ming-Tain Lai,e Sylvie M. Sur,e Rosa I.
Sanchez,f Mark T. Bilodeau,a John J. Renger,c Jonathan T. Kern,c and John A. McCauleya
Departments of aMedicinal Chemistry, bChemistry Modeling and Informatics, cNeuroscience Research,dStructural Chemistry, eIn Vitro Pharmacology, and
fDrug Metabolism, Merck Research Laboratories, West Point, PA 19486
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Familial Parkinson’s disease cases have recently been associated with the leucine rich repeat
kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may
have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent,
selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of
the internal Merck sample collection. Initial SAR studies around the core established the series
as a tractable small molecule lead series of LRRK2 inhibitors for potential treatment of
Parkinson’s disease. It was also found that incorporation of a lactam into the core drastically
improved the CNS and DMPK properties of these small molecules.
Keywords:
LRRK2
Parkinson’s disease
Leucine rich repeat kinase
CNS
2009 Elsevier Ltd. All rights reserved.
Kinase
Parkinson’s disease (PD) is
a
chronic, progressive
broad role in disease pathogenesis, and therapeutic intervention is
likely to impact both familial and sporadic disease.
neurodegenerative disorder characterized by the onset and
gradual worsening of cardinal motor symptoms. Motor symptoms
are attributed to the primary pathological hallmark of the disease,
the loss of nigrostriatal dopamine neurons. There are several non-
motor symptoms associated with the disease, as well as extra-
nigral pathology, however, diagnosis is still made based on
presence of motor symptoms and responsiveness to dopamine
replacement therapies. There are no effective therapies that
prevent neuron cell death and symptomatic treatments only
partially ameliorate motor symptoms. Disease risk increases
substantially with advancing age1 and, with the aging of Western
populations, incidence and prevalence rates are projected to
increase.2 These circumstances highlight the need to better
understand disease pathogenesis and identify effective
therapeutic strategies to lessen the impact of this disease.
LRRK2 is a large (268 kDa) multidomain protein consisting
of: N-terminal ankyrin repeats, a leucine-rich repeat region, the
catalytic core comprised of a GTPase ROC (Ras of Complex)
and COR (C-terminus Of ROC) domains, the serine/threonine
kinase domain, and a C-terminal WD-40 domain. Over the last
decade, several mutations have been identified throughout the
LRRK2 gene. Six of these mutations (R1441G, R1441C,
R1441H, Y1699C, G2019S, and I2020T) were deemed
pathogenic,5 and 5 of the 6 pathogenic mutations occur within the
enzymatic cores of the protein. Additionally, the LRRK2 triple
mutation N551K-R1398H-K1423K was identified as decreasing
disease risk.6 Together, these data support the hypothesis that
pharmacological mainpulation of LRRK2 enzymatic activity can
be of therapeutic value in treating PD.
Though most PD cases are idiopathic, meta-analyses of
genome-wide association studies of Parkinson's patients and
controls in the United States and Europe identified 11 genetic
loci associated with increasing PD risk. Mapping of these loci
identified variants in several genes including α-synuclein,
microtubule-associated protein Tau, and leucine rich repeat
kinase 2 (LRRK2) that increased disease risk.3 LRRK2 mutations
are associated with both familial and sporadic disease and are the
most common known cause of inheritable PD.4 LRRK2 PD
pathology is nearly indistinguishable from sporadic disease with
respect to age of onset, motor symptoms and responsiveness to
dopamine replacement therapies.5 LRRK2 may therefore play a
The kinase domain of LRRK2 is of particular interest, as it
represents a classically druggable target. There has been
considerable success in developing relatively selective LRRK2
kinase inhibitors, and recently some of these have been publically
disclosed.7-17 Continued discovery of selective LRRK2 kinase
inhibitors with “drug-like” properties is critical to enabling the
long-term safety and efficacy studies needed to fully interrogate
the therapeutic potential of this important target. To this end, we
carried out a screen of the Merck sample collection using
recombinant wt LRRK2 in a binding assay.18 This led to
identification of the potent LRRK2 inhibitor thiophene 3a (Table

1) which showed inhibition of both wt LRRK2 and the G2019S
mutant with Ki 377 and 234 nM, respectively. The
ethyl substituent (5g). Aromatic residues including benzyl (5e),
phenethyl (5f) and thiadiazole (5h) led to a reduction of LRRK2
activity. The gem-dimethyl group seemed to have little effect, see
compound 3a vs. 3b and 5c vs. 5i. We then explored the
requirement of a sulfur atom at R1. Interestingly, a nitrogen
substituent at R1 (5j and 5k) gave a large difference in activity
between wt and mutant LRRK2 enzymes and were in fact
equipotent to 5c against the G2019S mutant, while significantly
less potent against wt. An O linker (5l) was moderately potent,
whereas the all carbon case led to poor activity (5m). The
unsubstituted pyrazole substituent was found to be important for
maintaining LRRK2 binding affinity (Table 2); N-methylation of
the pyrazole led to an inactive compound (6) and replacement
with a thiazole (7) also lowered activity compared to 5g. The
SAR around the pyrazole group, together with induced fit
docking results based on a homology model of LRRK2, led us to
conjecture that the pyrazole was involved in two hydrogen bonds
with the backbone atoms of hinge residues Glu-1948 and Ala-
1950 (Figure 2a).
=
dihydrobenzothiophene series initially drew our attention as a
result of its limited activity in other high-throughput screening
(HTS) campaigns. For example, compound 3a had been assayed
in 274 HTS campaigns and had not been confirmed to be active
in any of these screens.19 Recognizing that other HTS campaigns
had been conducted for kinases prior to the LRRK2 screen, we
were attracted to the dihydrobenzothiophenes for their lack of
activity against other kinases. In order to enhance our
understanding of the general kinase activity properties of this
series, 5c was screened against a panel of 192 kinases. As seen
in the heat map in Figure 1, minimal off-target activity was
observed, further solidifying the selectivity profile of this series.
Table 1.
Table 2.
LRRK2 wt
Ki (nM)
LRRK2 G2019S
Compound
Structure
Ki (nM)
LRRK2 wt
Ki (nM)
LRRK2 G2019S
Compound
R1
R2
Ki (nM)
SMe
SO2Me
S(2-propyl)
SEt
Me
Me
Me
Me
Me
377
7130
256
156
389
234
2793
137
177
94
133
863
454
240
3a
4a
5a
5b
5c
5d
5e
5f
5583
>10,000
6
S(cyclopentyl)
SCH2(cyclopropyl) Me
SBn
SCH2CH2Ph
SCH2CH2OH
172
Me
Me
Me
>10 uM
1892
515
5g
nd
5464
7
Me
993
3994
5h
SMe
H
H
H
Me
Me
Me
312
295
>10 uM
2794
475
189
432
160
97
301
2242
3b
5i
5j
5k
5l
5m
S(cyclopentyl)
NH(cyclopentyl)
NH(cyclopentyl)
O(cyclopentyl)
Propyl
The synthesis of keto-thiophenes 5a-m commenced with
thiocarbonylation of 1,3-cyclohexanedione with carbon
1
disulfide, followed by alkylation of the resultant dithioic acid
with chloroacetone to produce a dithioester intermediate (Scheme
1).21,22 Trapping of the dithioester in situ with MeI effected a
condensation/aromatization sequence that smoothly provided the
desired thiophenes 2 in good yields. Subsequent treatment of
ketone 2 with DMF-DMA followed by hydrazine-mediated
cyclization of the resultant vinylogous amide afforded pyrazoles
3. The thiomethyl ether of 3 can be readily displaced to introduce
alternative functionality by straighforward oxidation to the
sulfone 4 with mCPBA followed by SnAr reaction with a
corresponding nucleophile to give rise to the desired keto-
thiophenes 5a-m.
5293
Compound 5c was shown to have very high clearance in rats
(Cl = 96 mL/min/kg) and did not achieve good CNS penetration
with a measured CSF level of 10 nM at 1 h after systemic
injection (5 mpk IP), demonstrating the need for optimization of
its DMPK properties. This result is not entirely unexpected, as 5c
has a rather high cLogP value of 5.5 which is a key contributing
factor to a poor CNS MPO score20 of 3.8. High plasma protein
binding (99.3%) is likely responsible for the low observed CNS
exposure, as 5c was not a substrate for P-glycoprotein, one of the
major efflux pumps in the CNS (BA/AB Ratio = 0.6).
Figure 1. Heat map of kinase inhibition (reported as percent inhibition)
across a panel of 192 kinases at a testing concentration of 10 μM. Red =
100% inhibition, yellow = 50% inhibition, green = 0% inhibition, gray = not
available. Note: LRRK2 wt and G2019S on far right.
Figure 2. (a) Induced fit docking pose for compound 5c in a LRRK2
homology model based on the TAK1 crystal structure (PDB ID 2EVA).
Schrödinger’s induced fit docking protocol was used to generate this pose. (b)
Crystal structure of compound 13e in TrkA (PDB code: 5I8A).23
Initial SAR studies around 3a showed that the oxidation state
of the sulfur was important for activity as evidenced by the
reduction in potency of sulfone 4a (Table 1). Alkyl substituents
on the sulfur were well-tolerated (5a–5d), as was an hydroxy

Scheme 1. Synthesis. Reagents and conditions: (a) K2CO3, CS2, DMF;
ClCH2COCH3 (b) MeI; (c) DMF-DMA; (d) N2H4, EtOH; (e) mCPBA,
CH2Cl2; (f) i. R1SH, NaH, THF, 0 oC – rt; ii. R1NH2, DMSO, 100 oC; iii.
R1OH, NaH, THF, 60 oC; iv. R1MgCl, CuBr-DMS, TMSCl, THF, -78 oC.
We then explored the possibility of replacing the
cyclohexanone with a lactam as shown in Table 3. Compound
13a, the direct lactam analog of 3c, did not show very good
activity, but larger alkyl substituents (15a–15f) were tolerated in
this series. As in the cyclohexanone case, aromatic groups at R1
led to a loss of potency (15g). Furthermore, substituents on the
amide nitrogen were not tolerated (14a and 14b).
Scheme 2. Synthesis. Reagents and conditions: (a) Meldrum’s Acid, EDC,
DMAP, CH2Cl2; (b) EtOAc, reflux; (c) ClCH2COCH3, Cs2CO3, DMF; CS2;
(d) MeI; (e) DMF-DMA; (f) N2H4, EtOH; (g) TFA, CH2Cl2; (h) SEMCl,
NaH, CH2Cl2; (i) TFA; (j) R2I, NaH, DMF; (k) HCl, MeOH; (l) mCPBA,
CH2Cl2; (m) R1SH, NaH, THF, 0 oC – rt.
Thiophene 12 proved to be a useful intermediate to probe the
SAR of the lactam series. Final compounds were prepared from
12 via the following three synthetic methods (Scheme 2): 1)
Simple deprotection of the Boc group with TFA afforded lactams
13a-s; 2) Protecting group manipulations of 12, which included
SEM protection of the pyrazole (NaH, SEMCl), followed by
deprotection of the Boc group with TFA, allowed for selective
functionalization of the lactam nitrogen. Alkylation of the lactam
with alkyl halides, followed by subsequent deprotection of the
SEM with HCl/MeOH afforded the desired tertiary lactams 14a-
b; and 3) Oxidation of the thiomethyl ether to the sulfone with
mCPBA and deprotection of the Boc group under standard
conditions produced the SnAr substrate, which was readily
displaced with thiol nucleophiles to provide the lactams 15a-k in
excellent yields (NaH, RSH).22
Table 3.
LRRK2 wt
Ki (nM)
LRRK2 G2019S
Compound
R1
R2
Ki (nM)
Me
Et
H
H
H
H
H
H
H
H
Me
Ph
3407
589
572
988
459
919
614
4458
5566
4706
776
348
355
557
286
372
305
1598
2535
2219
13a
15a
15b
15c
15d
15e
15f
15g
14a
14b
1-propyl
2-propyl
cyclopentyl
cyclohexyl
CH2(cyclopropyl)
Ph
Table 4.
Me
Me
The requisite dioxopiperidines 10 for the preparation of
lactam thiophenes 13a-s, 14a-b, 15a-k (Scheme 2) were prepared
as follows in a two-step sequence from various N-Boc-protected
b-amino acids 8.22 EDC coupling of Meldrum’s acid to b-amino
acid 8 afforded tricarbonyl 9 which, upon subjection to
thermolysis in ethyl acetate, the intermediate ketene produced
was intramolecularly trapped by the Boc-amine to smoothly
provide lactams 10 in good yields. Similarly to Scheme 1,
dicarbonyls 10 were transformed to amido-thiophenes 11 via
cyclization of the corresponding dithioester intermediate upon
treatment with MeI. Subsequently, the pyrazole was introduced
via standard vinylogous amide formation of the acetyl group of
11, followed by cyclization with hydrazine to provide lactam 12.
LRRK2
wt
Ki (nM)
LRRK2
G2019S
Ki (nM)
Compound
R3
R4
Me (rac)
Bn (rac)
CF3 (rac)
Ph (R)
Ph (S)
H
H
H
H
H
H
H
H
H
H
H
H
H
Me (R)
Me (S)
1238
928
501
174
120
3293
271
>10 uM
561
3564
198
64
53
1640
92
4226
341
293
1650
143
225
186
367
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
3-F-Ph (rac)
4-F-Ph (rac)
3-pyridyl (S)
CH2OMe (rac)
cyclobutyl (rac)
spiro-cyclopropyl
spiro-cyclobutyl
spiro-cyclopentyl
H
1045
>10 uM
233
411
319
H
603

H
H
H
Et (R)
Et (S)
Bn (S)
82
1443
342
46
700
159
13q
13r
13s
effort as a surrogate for LRRK-2. To enable this, we submitted
several dihydrobenzothiophenes to our in-house TrkA Caliper
assay despite our selectivity panel data which indicated low
activity against the Trk kinases. Our rationale for doing this was
that the TrkA crystal system had already demonstrated its ability
to structurally characterize weakly active compounds, and some
inhibition observed in the Caliper assay in a full dose response
might enable the use of TrkA as a surrogate for LRRK-2. Indeed,
compound 13e was shown to be ~8 uM vs. TrkA in the Caliper
assay, so we leveraged our in-house TrkA crystal system to solve
the structure of 13e with TrkA (Figure 2b, PDB code: 5I8A).23
This structure confirmed that our hypothesis about the binding
mode of this series in LRRK-2 was reasonable, although it does
not provide definitive proof that this mode is in fact correct for
the LRRK-2 protein.
These synthetic routes to the lactam compounds gave us the
opportunity to access both the 6- and 7- positions of the bicyclic
ring system for substitution by using readily available b-amino
acid starting materials. Substitution at R3 (Table 4) generally
resulted in improvements in potency over the parent compound
13a (see 13b – 13n). Specifically, introduction of phenyl and 4-
fluorophenyl provided a 10-fold boost (13e, 13f, 13h). Smaller
substitution, such as methyl and trifluoromethyl did not have as
drastic effect, but were tolerated. Interestingly, spirocyclic
substitution at C-6 resulted in potency increases in the
spirocyclobutyl (13m) and spirocyclopentyl (13n) cases, but not
with the spirocyclopropyl analogue 13l.
Nevertheless, what is immediately obvious from the TrkA
structure, which is in good agreement with our structural
hypothesis derived from induced fit docking to a homology
model of LRRK-2, is that the gatekeeper+2 residue (G+2) in
TrkA (Tyr-591) is in close proximity to the SMe group in 13e
(Figure 2a). The G+2 residue in LRRK-2 is Leu-1949 whereas
most kinases have a larger residue at this position (Figure 3), so
LRRK-2 can be expected to accommodate larger substitutions in
the region of the SMe of 13e, for instance the cyclopentyl of 5c,
with minimal loss of activity.
In addition, it was found that substitution at the C-7 position
of the lactam resulted in improvements in potency as seen with
the methyl and ethyl analogues 13o, 13p, and 13q. We next
looked to see if SAR was additive in the lactam series by
exploring thioether substituents as we had in the cyclohexanone
series. Combining the optimal R4 substituents R-Me and R-Et
with two of our known potency enhancing S-alkyl substituents
led to compounds 15h–15k (Table 5), each of which showed
<100 nM activity against both the wt and G2019S mutant
LRRK2 variants.
Table 5.
LRRK2 wt
Ki (nM)
LRRK2 G2019S
Compound
R1
R4
Ki (nM)
1-propyl
CH2(cyclopropyl) Me
1-propyl
CH2(cyclopropyl)
Me
84
83
68
58
39
34
30
28
15h
15i
15j
15k
Et
Et
Figure 3. Pie chart showing the frequency of residue identity at the G+2
position. The kinase sequence alignment used was taken from
www.kinase.com.
The lactam series generally showed improved cLogP values
and CNS MPO scores, with clogP being ~1 unit lower and the
CNS MPO score being ~0.5 higher for paired molecules.
Experimentally, these compounds also demonstrated improved in
vivo intrinsic clearance values (Clint) versus the cyclohexanones.
While the rat in vivo Clint values of the cyclohexanones were high
(undefined for 5c, 6,500 mL/min/kg for 3b), the lactam series
compounds 13a, 13e, and 15h demonstrated improved values
(1,300, 3,300, and 1,200 mL/min/kg, respectively).24 The
improved Clint values for the lactam series seems to correlate well
with the higher observed bioavailabilities (%F) with PO dosing
and improved CSF levels following IP dosing (Table 6).
Compounds in the cyclohexanone series (5c and 3b) showed
relatively poor rat PK with high clearance and low
bioavailability. The lactam series, on the other hand, gave
improved rat PK with compound 15h showing 98%
bioavailability and low to moderate clearance.
Indeed, of the compounds submitted for selectivity data 5c
showed the least inhibition of off-target kinases (Figure 1: top to
bottom: 5c, 3b, 13a, 13e), consistent with the idea that larger
substitutions in this region of the protein create destabilizing
interactions with most kinases due to unfavorable interactions
with larger residues that are homologous to Leu-1949 in LRRK-2
(e.g. Tyr-591 in TrkA). For instance, at a testing concentration
of 1 uM, compounds 3b, 13a, and 13e all inhibit Aurora A by
~40-65%, Aurora B by ~55%, and CHK2 by ~45-70%, whereas
5c inhibits each of these proteins by <~10%. Aurora A and
Aurora B both have Tyr at the G+2 position, supporting the
hypothesis that the size of the G+2 residue and the substitution
pattern of the inhibitor play an important role in the selectivity
profile of our compounds, although CHK2 has Leu at this
position so clearly other factors are at work as well.
In order to improve our structural understanding of this series,
we considered employing our in-house TrkA crystallography

Table 6. Rat pharmacokinetic parameters
Compound
Cla (mL/min/kg)
95.5
T1/2a (h)
1.2
%F
0.2
POb Cmax (mM)
POb AUC (mM*h)
IPc [Plasma] (mM)
IPc [CSF] (mM)
0.12
0.01
0.97 @ 1 h
0.01 @ 1 h
5c
56.0
47.5
22.0
11.9
6.5
1.4
4.3
3.6
33
70
84
98
1.6
4.8
3.8
2.33 @ 1 h
28.3 @ 1 h
5.65 @ 1 h
24.4 @ 1 h
0.05 @ 1 h
0.50 @ 1 h
0.07 @ 1 h
0.15 @ 1 h
3b
14.0
19.7
46.1
13a
13e
15h
5.1
16.0
aIV: 2 mg/kg, n = 2, 50%PEG400/50%DMSO or 60%PEG200/30%DMA/10%water
bPO: 10 mg/kg, n = 2, PEG400; cIP: 5 mg/kg, n = 3, PEG400
In conclusion, a dihydrobenzothiophene series of potent,
selective, orally bioavailable LRRK2 inhibitors were identified
from a high throughput screen of the Merck sample collection.
Initial SAR studies around the core established the series as a
tractable small molecule lead series of LRRK2 inhibitors for
potential treatment of Parkinson’s disease. It was also found that
incorporation of a lactam into the core drastically improved the
CNS and DMPK properties of these small molecules. Further
efforts to optimize the overall drug-like profile are ongoing and
will be reported in due course.
Brighina, L.; Van Broeckhoven, C.; Carr, J.; Chartier-Harlin, M. C.;
Dardiotis, E.; Dickson, D. W.; Diehl, N. N.; Elbaz, A.; Ferrarese, C.;
Ferraris, A.; Fiske, B.; Gibson, J. M.; Gibson, R.; Hadjigeorgiou, G. M.;
Hattori, N.; Ioannidis, J. P.; Jasinska-Myga, B.; Jeon, B. S.; Kim, Y. J.;
Klein, C.; Kruger, R.; Kyratzi, E.; Lesage, S.; Lin, C. H.; Lynch, T.;
Maraganore, D. M.; Mellick, G. D.; Mutez, E.; Nilsson, C.; Opala, G.;
Park, S. S.; Puschmann, A.; Quattrone, A.; Sharma, M.; Silburn, P. A.;
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