Discovery of 5-substituent-N-arylbenzamide derivatives as potent, selective and orally bioavailable LRRK2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.07.052

Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic target for Parkinson's disease. Herein we report the discovery of 5-substituent-N-arylbenzamide derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds 8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome, good brain exposure, and high oral bioavailability.

Full text of DOI:10.1016/j.bmcl.2017.07.052

Accepted Manuscript
Discovery of 5-substituent-N-arylbenzamide derivatives as potent, selective and
orally bioavailable LRRK2 inhibitors
Xiao Ding, Xuedong Dai, Kai Long, Cheng Peng, Daniele Andreotti, Paul
Bamborough, Andrew J. Eatherton, Colin Edge, Karamjit S. Jandu, Paula L.
Nichols, Oliver J. Philps, Luigi Piero Stasi, Zehong Wan, Jia-Ning Xiang, Kelly
Dong, Pamela Dossang, Ming-Hsun Ho, Yi Li, Lucy Mensah, Xiaoming Guan,
Alastair D. Reith, Feng Ren
PII:
S0960-894X(17)30759-X
http://dx.doi.org/10.1016/j.bmcl.2017.07.052
BMCL 25165
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 June 2017
10 July 2017
20 July 2017
Please cite this article as: Ding, X., Dai, X., Long, K., Peng, C., Andreotti, D., Bamborough, P., Eatherton, A.J.,
Edge, C., Jandu, K.S., Nichols, P.L., Philps, O.J., Piero Stasi, L., Wan, Z., Xiang, J-N., Dong, K., Dossang, P., Ho,
M-H., Li, Y., Mensah, L., Guan, X., Reith, A.D., Ren, F., Discovery of 5-substituent-N-arylbenzamide derivatives
as potent, selective and orally bioavailable LRRK2 inhibitors, Bioorganic & Medicinal Chemistry Letters (2017),
doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.052
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Discovery of 5-substituent-N-arylbenzamide
derivatives as potent, selective and orally
bioavailable LRRK2 inhibitors
Leave this area blank for abstract info.
Xiao Dinga, Xuedong Daia, Kai Longa, Cheng Penga, Daniele Andreottib, Paul Bamboroughc, Andrew
J. Eathertond, Colin Edgec, Karamjit S. Jandue, f, Paula L. Nicholsd, Oliver J. Philpse, Luigi Piero Stasia,
Zehong Wana, Jia-Ning Xianga, Kelly Dongg, Pamela Dossangc, Ming-Hsun Hog, Yi Lig, Lucy
Mensahc,†, Xiaoming Guana, Alastair D. Reithd, e, and Feng Rena,*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery of 5-substituent-N-arylbenzamide derivatives as potent, selective and
orally bioavailable LRRK2 inhibitors
Xiao Dinga, Xuedong Daia, Kai Longa, Cheng Penga, Daniele Andreottib, Paul Bamboroughc, Andrew J.
Eathertond, Colin Edgec, Karamjit S. Jandue, f, Paula L. Nicholsd, Oliver J. Philpse, Luigi Piero Stasia,
Zehong Wana, Jia-Ning Xianga, Kelly Dongg, Pamela Dossangc, Ming-Hsun Hog, Yi Lig, Lucy Mensahc,†,
Xiaoming Guana, Alastair D. Reithd, e, and Feng Rena,*
a Neurodegeneration DPU, Neurosciences Therapeutic Area Unit, GSK Pharmaceuticals R&D, 898 Halei Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai
201203, PR China.
b Neurosciences CEDD, GSK Pharmaceuticals R&D, Verona, Italy.
c Platform Technology Sciences, GSK Pharmaceuticals R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts UK.
d Neurosciences CEDD, GSK Pharmaceuticals R&D, New Frontiers Science Park, Harlow, Essex, UK.
e Neurodegeneration DPU, Neurosciences Therapeutic Area Unit, GSK Pharmaceuticals R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage,
Herts, UK.
f Current address: Discovery, Charles River, Chesterford Research Park, Saffron Walden, UK.
g Platform Technology Sciences, GSK Pharmaceuticals R&D, 898 Halei Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, PR China.
† Deceased.
ARTICLE INFO
ABSTRACT
Abstract: Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic
target for Parkinson’s disease. Herein we report the discovery of 5-substituent-N-arylbenzamide
derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds
8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome,
good brain exposure, and high oral bioavailability.
Article history:
Received
Revised
Accepted
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved
.
Parkinson’s disease
LRRK2 inhibitor
Arylbenzamide
CNS penetration
Kinase selectivity
acceptable CNS penetration, and the ability to inhibit LRRK2
activity both in vitro and in vivo. However, no LRRK2 inhibitor
has yet been reported to be progressed to clinical development.
We have previously reported GSK2578215A,15 a novel 5-
substituent-N-arylbenzamide analogue, as a potent LRRK2
inhibitor with high selectivity across the kinome and good CNS
penetration (brain to plasma exposure ratio = 1.4) in mice. The
compound was observed to substantially inhibit the
phosphorylation of S910 and S935 of LRRK2 in spleen and
kidney after intraperitoneal injection (100 mg/kg) to normal mice.
The compound’s potent LRRK2 activity, high kinase selectivity,
and good PK profile have made it a versatile tool for the
exploration of the biological roles of LRRK2 including roles in
autophagy,16 receptor trafficking,17 protein degradation,18 and
inflammation,19 as well as definition of physiologic substrates of
LRRK220 and pharmacodynamic markers assays21. The 5-
substituent-N-arylbenzamide pharmacophore is a distinct
structure scaffold for LRRK2 small molecule inhibitors. In this
report, we describe the detailed SAR studies of 5-substituent-N-
arylbenzamide series leading to the discovery of the potent,
selective and orally bioavailable LRRK2 inhibitor 8e.
Parkinson’s disease (PD), a slowly progressive
neurodegenerative disorder mainly affecting the motor function,
has been recognized as the second most prevalent
neurodegenerative diseases.1 It was reported that PD affected ~4
million people over age 50 worldwide in 2005 and the number
was estimated to be doubled in 2030.2 Current therapies for PD
are limited to alleviating the symptoms of the disease in early
stage,3 and development of disease-modifying therapies to slow
down disease progression is therefore profoundly needed.4
Leucine-rich repeat kinase 2 (LRRK2) gene mutations have been
considered as the most common genetic cause of familial and
sporadic PD.5 Among the several mutations presented in PD
patients carrying LRRK2 mutations, G2019S is the most frequent
one identified which results in aberrant kinase activity,
suggesting small molecules inhibiting LRRK2 activity could be
potential therapeutic treatments for PD.6
A number of structurally diverse small molecule LRRK2
inhibitors have been disclosed in the past several years (Figure
1),7 including LRRK2-IN-1 (1), 8 GNE-7915 (2),9 PF-06447475
(3),10 Nov-LRRK2-11 (4),11 MLi-2 (5),12 and compounds with
quinoline13 andtriazolopyridazine14 core structures. Some of
these compounds demonstrated good pharmacokinetic profile,

O
N
O
F
O
CN
potency (pIC50 =8.1). Changing the position of fluorine from
ortho (6b) to meta (6c) or para (6d) resulted in similar potency.
Because para-substituted benzyl alcohols are readily available,
we thus focused our SAR exploration on para-substitutions.
Either electron-donating group (methoxy, 6e) or electron-
withdrawing group (cyanide, 6f) led to reduced LRRK2 potency
by ~10 folds. On the other hand, the un-substituted analogue (6g)
demonstrated good potency comparable to that of 6d. These data
suggested only small substitutions such as hydrogen and fluorine
were well tolerated at the para-position of the phenyl ring, while
bulkier substitutions resulted in decreased potency. The 3,4-
difluoro analogue (6h) showed slightly decreased potency which,
again, might due to steric effect. Furthermore, introducing a
methyl group to the benzylic position provided compound (6i)
with lower potency.
O
CF3
NH
N
N
N
N
N
N
O
N
N
N
N
N
H
N
H
O
N
N
O
N
H
3, PF-06447475
2, GNE-7915
1, LRRK2-IN-1
N
F
O
N
O
N
N
N
O
N
H
O
O
O
N
N
H
NH
O
N
H
N
5, MLi-2
4, Nov-LRRK2-11
GSK2578215A
Figure 1. Structures of reported LRRK2 inhibitors
Table 2. LRRK2 HTRF potency for compounds 7a‒7l.
Br
O
NH
O
R2
7a 7l
−
a
Compd
R2
HTRF pIC50
7.9
Figure 2. The predicted docking pose of KCS Hit (6a, cyan)
in the LRRK2 homology model.
6g
7a
7b
7.7
7.3
A KCS (kinase-focused set of compounds for lead discovery)
screening of the GSK in-house compounds using a homogeneous
time-resolved fluorescence (HTRF) assay resulted in the
identification of several LRRK2 inhibitor hits including the 5-
substituent-N-arylbenzamide compound 6a with a pIC50 of 7.0.
Given its structural novelty and low molecule weight, this hit
series was selected for further optimization. Docking of
compound 6a to a LRRK2 homology model22 suggested that 6a
bound to the LRRK2 ATP pocket (Figure 2). The amide carbonyl
group of 6a formed a hydrogen bond interaction with A1950 in
the hinge region and was proposed as the key interaction with
LRRK2. The bromide group of 6a pointed toward the solvent-
exposed area of the kinase. 2-Cl-phenyl ring and the pyridine
ring were both buried in the binding pocket: the former was
closer to D2017 and the latter to the gatekeeper residue, M1947.
7c
5.7
7d
7e
<4.6
<4.6
7f
7.2
6.6
7g
7h
7i
7.6
6.9
4.8
Table 1. LRRK2 HTRF potency for compounds 6a‒6i.
Br
O
NH
O
R1
N
7j
6a 6i
−
a
Compd
6a
R1
HTRF pIC50
7.0
7k
7l
5.6
2-Cl-C6H4CH2-
2-F-C6H4CH2-
3-F-C6H4CH2-
4-F-C6H4CH2-
4-OCH3-C6H4CH2-
4-CN-C6H4CH2-
C6H5CH2-
6b
6c
8.1b
<4.6
7.8
a HTRF assay data is the average of at least two determinations.
6d
6e
8.1
7.4
Having identified the un-substituted benzyl group as one of the
best R1 moieties, attention was then turned to optimizing the
heteroaryl (R2) group (Table 2). Several different heteroaromatic
analogues were synthesized and their LRRK2 HTRF potency
evaluated. Among them, the 3-pyridinyl (6g) and the 4-
pyridazinyl (7a) groups proved to be the most optimal fragments.
Changing the position of the nitrogen atom of 7a or adding
substitutions such as chlorine to 6g resulted in decreased LRRK2
activities to different extents (7b‒7e). The predicted binding
model showed the pyridine group of R2 bounds in a shallow
pocket closer to M1947 and E1948 residues of LRRK2.
6f
7.2
6g
7.9
7.7b
6h
3,4-di-F-C6H3CH2-
6i
C6H5-CH(CH3)-
7.4
a HTRF assay data is the average of at least two determinations. bOne
determination.
SAR exploration of the 5-substituent-N-arylbenzamide series
started with different substitutions on the benzyl ether moiety (R1)
in order to improve potency of the hit compound 6a (Table 1).
Replacing the chloro group of 6a with a smaller fluoro
Introduction of a N atom or a Cl substitution to the 5-position of
the pyridine introduces an electrostatic or steric repulsion, which
substitution (6b) provided more than 10 fold improvement in

could result in inactive compounds (7d and 7e). Replacing 3-
pyridinyl with 3-methyl phenyl (7f) or 3-fluoro phenyl (7g)
groups resulted in decreased potency. However, the
corresponding chloro analog (7h) demonstrated comparable
potency (pIC50 = 7.6) to that of the pyridine analogue (6g). When
electron-donating groups such as methoxy (7i) or dimethylamino
(7j) groups were introduced, the potency decreased dramatically.
Changing the six-membered heteroaryl ring to the five-membered
ring (7k) yielded a much-decreased activity. Furthermore, the
saturated ring such as cyclohexyl led to totally inactive
Heteroaromatic rings were also explored including pyridines (8h
and 8i) and pyrazoles (8j‒8m), and all compounds demonstrated
high potency and improved metabolic stability (except for 8h and
8j).
Table 4. Mouse PK profile of 8e, 8i, and 8l.a
DNAUC0~t
((ng·h/mL)/ F%
(mg/kg))
T1/2
(h)
CLb
(mL/min/kg)
Vss
(L/kg)
Br/Bl
ratio
8eb
8ic
8lb
1.2
1.1
0.4
27.7
30
2.7
2.3
1.6
597
520
253
91.6
12.2
10
1.3
compound (7l), which might be caused by the twisted dihedral
angle between the amide group and the cyclohexyl ring.
1.4
69.9
NDd
aExperiments were done in male Swiss Albino Mice. DNAUC = dose
normalized area under the curve (measure of exposure) after i.v., T1/2
half-life, CLb = blood clearance, Vss = volume of distribution, F = oral
Table 3 LRRK2 HTRF potency for compounds 8a‒8m.
=
bioavailability. b1 mg/kg i.v. and 2 mg/kg p.o. 1 mg/kg i.v. and 10 mg/kg
c
p.o. dNot determined.
Representative compounds 8e, 8i and 8l were selected for
further progression. They all demonstrated good passive
permeability (> 100 nm/s) and proved not to be Pgp efflux
transporter substrates in the Polarized Madin-Darby canine
kidney (MDCKII) cells heterologously expressing human Pgp
(MDCKII-MDR1 cell line)24. They were then progressed to in
vivo to evaluate their brain penetration and pharmacokinetic
properties in the mouse crossover PK study25 (i.v. and p.o.). As
shown in Table 4, compound 8e was observed to be brain
penetrable in mice with high brain to blood ratio (Br/Bl = 1.3) in
terms of total drug exposure. In addition, compound 8e also
demonstrated excellent oral bioavailability (F = 91.6%), and it
was the first compound in the 5-substituent-N-arylbenzamide
series observed to be orally bioavailable. In comparison, the
previously reported compound 8i (GSK2578215A) had very low
oral bioavailability (F = 12.2%) even though its brain penetration
was good (Br/Bl = 1.4).15 Both compounds 8e and 8i showed low
clearance in mice (27.7 and 30 mL/min/kg, respectively). On the
other hand, compound 8l had much higher clearance (69.9
mL/min/kg) thus a short half-life (T1/2 = 0.43 h), and its oral
bioavailability was low (F = 10%). Compound 8e was further
evaluated for its selectivity over 300 other kinases using the
HotSpot assay platform.26 The results indicated 8e is a highly
selective inhibitor of LRRK2. Only LRRK2 and one other kinase
(MSSK1-STK23) had inhibition levels of over 40% at 1µM
compound concentration. Subsequent dose-response titrations for
LRRK2 and MSSK1-STK23 gave pIC50 of 8.1 and 6.8,
respectively.
HTRF
Cli
(mL/min/g)b
Compd
R3
R4
a
pIC50
7.9
7.4
7.8
7.9
7.8
7.7
6.7
5.9c
6g
8a
8b
8c
8d
8e
8f
-Br
H
H
H
H
H
F
2.8
-F
18.3
NDd
16.2
22.6
0.8
-Cl
-CH3
-OCH3
-CF3
-CON(CH3)2
-SO2N(CH3)2
H
H
0.7
NDd
8g
8h
8i
H
H
H
8
5.1
1.2
2.3
8
8j
7.9
8k
H
8.1
1.2
8l
F
F
8
8
1.5
0.6
8m
aHTRF assay data is the average of at least two determinations. b In vitro
human liver microsome clearance. cOne determination. dNot determined.
From the preliminary SAR exploration, compound 6g was
discovered with high potency and good “drug likeness” profile
(low MW, cLogP, etc.) based on Lipinski’s rule.23 However, the
in vitro clearance profile of 6g in human liver microsome24 (2.8
mL/min/g) was observed to be sub-optimal which precluded its
further progression. We then focused our efforts on SAR
exploration of R3 to improve this series’ metabolic stability and
physicochemical properties at the same time to maintain
potencies. A variety of functional groups was well tolerated at the
R3 position, including electron-withdrawing groups such as
fluorine and chlorine (8a and 8b) as well as electron-donating
groups such as methyl and methoxyl (8c and 8d). Even though
these compounds demonstrated similar potency as 6g, their
clearance in human liver microsome was still high. To our
delight, introduction of a trifluoromethyl group to the R3 position
provided the compound (8e) with good potency (pIC50 = 7.7) and
metabolic stability in human liver microsome (Cli = 0.8
mL/min/g). In addition, the solubility of 8e (11 µM) was also
improved compared to that of 6g (<1 µM). We further evaluated
more polar groups such as amide (8f) and sulfonamide (8g).
However, both compounds turned out to be less potent.
As reported previously, compound 8i (GSK2578215A)15b
showed strong inhibition against phosphorylation of S910 and
S935 of LRRK2 in mouse spleen and kidney, whereas no
significant inhibition in mouse brain despite its high brain
exposure25. Mouse brain tissue binding was measured for both
compound 8e and 8i. Not surprisingly, both compounds showed
very high tissue binding in mouse brain (99.6% and 99.7% for 8e
and 8i, respectively). The low free unbound drug concentration of
8i in mouse brain might account for its lack of efficacy in CNS.
Efficient syntheses of 5-substituent-N-arylbenzamide
derivatives 6‒8 were developed (Scheme 1). Treatment of
commercial benzoic acids 9 with various alkylation reagents in
the presence of a base (K2CO3) in acetone provided benzoic
esters 10. Hydrolysis of intermediates 10 using LiOH in the
mixed solvent of THF and water resulted in benzoic acids 11.
Treatment of 11 with different amines in the presence of coupling
reagents such as EDCI/HOBT or HATU in DMF yielded target
compounds 6a‒6h, 7, 8a‒8g. Compounds 6i could be obtained
by alkylation of phenols 12 under basic conditions (KOH) in
methanol. Intermediates 12 were prepared from commercial

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reactions of 6d or 6g with various boronic acids in the presence
of Pd catalysts at elevated temperatures provided compounds
8h‒8m.
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Dominguez, S. L.; Dotson, J.; Drummond, J.; Flagella, M.; Flynn, S.; Fuji,
R.; Gill, A.; Gunzner-Toste, J.; Harris, S. F.; Heffron, T. P.; Kleinheinz,
T.; Lee, D. W.; Le Pichon, C. E.; Lyssikatos, J. P.; Medhurst, A. D.;
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Scheme 1. Synthesis of 5-substituent-N-arylbenzamide
derivatives 6‒8. Reagents and conditions: (a) R1Br, K2CO3,
acetone; (b) LiOH, THF/water; (c) R2NH2, EDCI/HOBT or
HATU, DMF; (d) R1Br, KOH, MeOH; (e) R3B(OH)2, Pd(PPh3)4
or Pd(PPh3)2Cl2, DME/water.
12. 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.;
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In summary, we discovered a novel series of 5-substituent-N-
arylbenzamide derivatives as potent LRRK2 inhibitors. Extensive
SAR studies led to the identification of compounds 8e and 8i
with high potencies of LRRK2 inhibition and good selectivity
over hundreds of kinases. Compound 8e demonstrated good in
vitro and in vivo pharmacokinetic profile with high exposure in
both brain and blood (Br/Bl ratio = 1.3). 8e was orally
bioavailable (F = 91.6%) in comparison with the previously
reported compound 8i (GSK2578215A, F = 12.2%). Both
compound 8e and 8i were observed to be highly tissue bound
(99.7% and 99.6%, respectively) in mouse brain. The low free
unbound drug concentration of 8i in brain was proposed to be
accountable for its lack of efficacy in CNS (no significant
inhibition against phosphorylation of S910 or S935 in mouse
brain). Further efforts will be focused on improving the series’
free unbound fraction in brain.
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Acknowledgments
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Authors thank Dr. Qian Liu for useful discussions on binding
mode of 5-substituent-N-arylbenzamide analogues with LRRK2
protein, Mr. Morris Sui and Mrs. Yun Liu for NMR and LCMS
analysis. This work was supported in part by a Therapeutics
Development Initiative Award from the Michael J. Fox
Foundation for Parkinson’s Research (to A.D.R.).
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Elife 2016, 5, e12813; (b) Ito, G.; Katsemonova, K.; Tonelli, F.; Lis, P.;
Baptista, M. A.; Shpiro, N.; Duddy, G.; Wilson, S.; Ho, P. W.; Ho, S. L.;
Reith, A. D.; Alessi, D. R., Biochem. J. 2016, 473, 2671.
21. Perera, G.; Ranola, M.; Rowe, D. B.; Halliday, G. M.; Dzamko, N., Sci.
Rep. 2016, 6, 31391.
Supplementary Material
22. The LRRK2 homology model was initially built on a ROCK1 template as
described in reference 15b; the depiction in Figure 2 is taken from a
more recent homology model based on the MLK1 kinase domain (PDB:
4UY9): Marusiak, A. A.; Stephenson, N. L.; Baik, H.; Trotter, E. W.; Li,
Y.; Blyth, K.; Mason, S.; Chapman, P.; Puto, L. A.; Read, J. A.;
Brassington, C.; Pollard, H. K.; Phillips, C.; Green, I.; Overman, R.;
Collier, M.; Testoni, E.; Miller, C. J.; Hunter, T.; Sansom, O. J.; Brognard,
J., Cancer Res. 2016, 76, 724.
Supplementary data associated with this article can be found in
the online version.
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