Indolinone based LRRK2 kinase inhibitors with a key hydrogen bond

Article abstract of DOI:10.1016/j.bmcl.2014.08.049

The most prevalent leucine-rich repeat kinase 2 (LRRK2) mutation G2019S is associated with Parkinson's disease (PD). It enhances kinase activity and has been identified in both familial and sporadic cases. Kinase activity was reported to be required for LRRK2 mutants to exert their toxic effects. Hence LRRK2 kinase inhibition may be a promising therapeutic target for PD. Here we report on the discovery and characterization of indolinone based LRRK2 inhibitors. Indolinone 15b, the most potent and selective inhibitor of the present series, is characterized by an IC₅₀of 15 nM against wild-type LRRK2 and 10 nM against the LRRK2 G2019S mutant, respectively. Compound 15b was further evaluated in a kinase panel including 46 human protein kinases and in a zebrafish embryo phenotype assay, which enabled toxicity determination in whole organisms.

Full text of DOI:10.1016/j.bmcl.2014.08.049

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4630–4637
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Indolinone based LRRK2 kinase inhibitors with a key hydrogen bond
Stefan Göring a, , Jean-Marc Taymans b, Veerle Baekelandt b, Boris Schmidt a,
⇑
a Clemens Schöpf—Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, 64287 Darmstadt, Hessen, Germany
b KU Leuven, Laboratory for Neurobiology and Gene Therapy, Leuven B-3000, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The most prevalent leucine-rich repeat kinase 2 (LRRK2) mutation G2019S is associated with Parkinson’s
disease (PD). It enhances kinase activity and has been identified in both familial and sporadic cases.
Kinase activity was reported to be required for LRRK2 mutants to exert their toxic effects. Hence LRRK2
kinase inhibition may be a promising therapeutic target for PD. Here we report on the discovery and
characterization of indolinone based LRRK2 inhibitors. Indolinone 15b, the most potent and selective
inhibitor of the present series, is characterized by an IC50 of 15 nM against wild-type LRRK2 and
10 nM against the LRRK2 G2019S mutant, respectively. Compound 15b was further evaluated in a kinase
panel including 46 human protein kinases and in a zebrafish embryo phenotype assay, which enabled
toxicity determination in whole organisms.
Received 11 June 2014
Revised 20 August 2014
Accepted 21 August 2014
Available online 29 August 2014
Keywords:
Parkinson’s disease
Leucine-rich repeat kinase 2 (LRRK2)
LRRK2-inhibitors
Ó 2014 Elsevier Ltd. All rights reserved.
Zebrafish phenotype
Parkinson’s disease (PD) is after Alzheimer’s disease the second
most common, age related, neurodegenerative disorder, affecting
about 2% of the population older than 60 years.1 It is characterized
clinically by a number of symptoms, for example, tremor at rest,
bradykinesia, rigidity and postural instability. Specific neuropatho-
logical hallmarks for PD are the neuronal cell loss in dopaminergic
neurons of the substantia nigra pars compacta and the formation of
intracellular fibrils and Lewy bodies in the surviving neurons. A
compared to its wild type counterpart.7–9 This effect may guide
the pharmacological development of kinase inhibitors for
a
potential PD therapy. Second, the cellular toxicity of acute overex-
pression of LRRK2 clinical mutants is ablated when these muta-
tions are combined with mutations inactivating the kinase
activity, both in cell culture and primary neurons,7,10 as well as
in viral vector mediated brain overexpression in rodents.11,12
Although pharmacological studies have begun to indicate that
LRRK2 kinase inhibition may be beneficial in reversing LRRK2 med-
iated toxicity,12 further confirmation studies with more potent and
specific LRRK2 inhibitors are required.13,14
major component of Lewy bodies is
underlying molecular mechanism of PD remains poorly under-
stood and there is no cure for this disease.
a
-synuclein.2 To date the
The leucine-rich repeat kinase 2 (LRRK2) is a multi-domain
protein and consists of 2527 amino acids. Several independent
domains are known for the LRRK2 protein including both a ser-
ine/threonine kinase and a GTPase domain. Several mutations have
been identified inside the protein, at least five of them (R1441C,
R1441G, Y1699C, G2019S and I2020T) are currently assumed to
be pathogenic and are localized in the catalytic core.3–5 The most
prevalent mutation is the glycine to serine amino acid substitution,
which is present in more than 85% of familial PD patients carrying
LRRK2 mutations.6
At present different chemotypes of LRRK2 inhibitors are known
(Fig. 1). Compounds of several structural families as indolinone 1
(GW5074),12 the diaminopyridines 2 (LRRK2-IN-1),15 3 (TAE684)16
and 4 (HG-10-102-01),17 the cinnoline derivative 518 or the triazol-
pyridine 619 are potent LRRK2 kinase inhibitors. Especially the
LRRK2 inhibitor HG-10-102-01 4 exhibited good selectivity and
pharmacokinetic properties regarding brain penetration and
dephosphorylated Ser910 and Ser935 in tissues including kidney,
spleen and brain.17
Nevertheless, the ideal LRRK2 inhibitor has not yet been
reported. Thus the interest to explore activities of multiple classes
of compounds that provide desirable characteristics such as
potency, selectivity, cellular activity and brain penetrance, to name
a few, remains in focus.3
Converging studies point to the inhibition of LRRK2 kinase
activity as a therapeutic concept. First, the G2019S mutation in
the kinase domain increases the kinase activity 2- to 3-fold
In a previous study we have reported the inhibition of the FMS-
like tyrosine kinase-3 (FLT-3) by substituted indolinones.20 FLT-3 is
involved and aberrantly active in acute myeloid leukemia
(AML).21,22 Working on FLT-3 inhibitors we have identified a highly
⇑
Corresponding author. Tel.: +49 6151 163075, +49 6151 164531; fax: +49 6151
163278.
E-mail address: schmidt_boris@t-online.de (B. Schmidt).
Tel.: +49 6151 164531; fax: +49 6151 163278.
http://dx.doi.org/10.1016/j.bmcl.2014.08.049
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

S. Göring et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4630–4637
4631
Cl
N
O
H
N
N
HN
N
N
NH
O
S
O
Br
O
O
N
N
OH
Br
O
N
N
I
O
N
H
N
N
N
N
Indolinone
Diaminopyrimidine
Diaminopyrimidine
1
(
2
3
(
GW5074)
(
LRRK2-IN-1)
TAE684)
Cl
NH
N
N
NH
O
HN
N
NH
N
N
NH2
O
N
N
HO
N
O
N
O
Triazolpyridine
O
Cinnoline
(6 Compound 8)
(5)
Diaminopyrimidine
(4 HG-10-102-01)
Figure 1. Selected chemotypes as lead structures for the development of new LRRK2 inhibitors.12,15–19
active and selective compound based on the indolinone scaffold
(Fig. 2). The indolinone 7 exhibited an IC50 value for FLT-3 in the
low nanomolar range. Additionally, the selectivity of indolinone
This model revealed that the indolinone moiety favorably inter-
acts with the backbone of LRRK2 ATP pocket. Additionally an
important interaction was observed with the diethylaminoethoxy
anchor. The nitrogen of the diethylamine group interacts with
the rear of the ATP-binding pocket and forms a strong hydrogen
bond to K1906 in addition to several hydrophobic interactions with
amino acids in the close surroundings of the phenol moiety. To
confirm the docking results of indolinone derivative 7 in the LRRK2
homology model the potency of this compound was determined
against LRRK2 in an in vitro peptide substrate based kinase activity
derivative 7 was determined at a concentration of 1 lM against
50 human protein kinases.20 Beside FLT-3 only two other kinases
(MAP4K4 and JAK3) were also inhibited by this compound. Unfor-
tunately, LRRK2 was not part of this kinase screening. Due to the
structural analogy of compound 7 to the known LRRK2 inhibitors
1 (GW5074) and Sunitinib, a well-known multi-kinase inhibitor,
we refocused our interest on the inhibition of LRRK2. A recent
publication by Novartis confirmed the efficiency of indolinones
as selective and brain penetrant LRRK2 inhibitors.23
assay at concentrations of 10 and 1 lM (assay information is avail-
able in the Supplementary data and referenced in 27 and 26).
Those results agreed with our hypothesis and previous docking
studies. Thus, indolinone 7 reduces in vitro LRRK2 activity by more
than 50% relative to control for both concentrations (Table 1). At 10
and 1 lM of compound, residual LRRK2 kinase activities of 12% and
11% were observed, respectively. To assess the activity of com-
In the absence of a full-length LRRK2 crystal structure analysis,
the use and construction of LRRK2 homology models is the key for
structure based design. In order to gain insight in how the indoli-
none derivative 7 interacts with the ATP-binding pocket of LRRK2
we performed docking studies of 7 in a LRRK2 homology model.
Based on the sequence identity with the LRRK2 kinase domain,
the tyrosine-kinase like kinase B-Raf (PDB 4DBN) was selected as
template to model LRRK2 kinase. The alignment between the
LRRK2 kinase domain and B-Raf kinase was performed using the
automatic mode of the Swiss Model server.24,25 The final homology
model was visualized using Molegro Virtual Docker 6. The validity
of docking compounds onto a modeled structure of LRRK2 kinase
was recently demonstrated using a kinome-wide kinase inhibitor
panel correlating docking scores to compound.26
pound 7 more precisely we determined the IC50 value additionally.
An IC50 value of 0.265 lM clearly demonstrated the submicromolar
potency of compound 7 and consequently indolinones as LRRK2
inhibitors. Based on these encouraging results indolinone 7 was
an attractive lead structure for further structure–activity-
relationship (SAR) optimization.
Additional indolinone derivatives were synthesized by known
conditions using a Knoevenagel condensation between indolinone
and the corresponding aldehydes in methanol and a catalytic
amount of piperidine. The Knoevenagel condensation is a reliable
and quick method to establish chemical diversity for the variation
of the SAR study based on compound 7. The synthesis is outlined
in Scheme 1. The first step providesthe aldehydes for the subsequent
Knoevenagel condensation. Hence, the alkyl and aryl benzyl halides
were connected with derivatives of 4-hydroxybenzaldehydes 8a–c
N
O
Cl
Table 1
O
LRRK2 activity of compound 7
N
H
7
Compound
LRRK2 activity
M of compd
IC50
10
l
1
l
M of compd
IC50 (FLT3) = 4.1 nM
7
12%
11%
0.265 lM
Figure 2. Highly potent and selective indolinone inhibitor for FLT3.20

4632
S. Göring et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4630–4637
R3
O
OH
R2
R1
R2
R1
alkyl-/benzylbromide or- chloride
K2CO3
DMF or aceton
70°C, 12h
H
O
H
O
8a R1 = R2 = H
1
2
8b
R = H, R = OMe
9a-m
8c R1 = OMe, R2 = H
F
X
R4
O
O
14
5-chloroindolin-2-one
piperidine
K2CO3
Cl
O
DMSO or DMF
120°C, 3d
methanol
100°C, 30 min
microwave
N
H
H
O
H
O
15a-t
11a
X = morpholine
10
11b X = 1-methylpiperazin
11c X = methylimidazole
11d
X = sulfonylmethane
alkyl chloride
Cs2CO3
H
H
N
H
N
DMSO
RT, 20h
N
O
O
12
13
Scheme 1. Synthesis of aldehydes derivatives for the following Knoevenagel condensation.
Table 2
Synthesized indolinone derivatives and their in vitro LRRK2 inhibitory activity
N
R3
N
R2
R1
R4
Cl
Cl
O
O
N
H
N
H
15a-s
15t
Compound
R1
R2
R3
R4
LRRK2 activity
IC50
—
0.0035
0.261
0.015
12
0.163
0.466
10
0.205
—
(lM)
10
l
M of compd (%)
1
5
lM of compd (%)
Staurosporine*
—
—
F
—
—
OH
OH
—
—
H
H
H
H
H
H
H
H
H
2
5
2
4
8
9
3
16
15
24
28
LRRK2-IN-1*
15a
13
5
3
12
8
22
27
37
26
26
F
Br
H
OMe
H
H
H
OMe
H
15b
15c
15d
15e
15f
15g
15h
15i
Br
H
H
H
H
H
H
H
OH
2-Morpholinoethoxy
2-(Dimethylamino)ethoxy
H
2-Morpholinoethoxy
2-(Diethylamino)ethoxy
Acetamide
10
O
15j
H
OMe
H
24
26
—
3O
15k
15l
H
H
H
H
H
H
H
H
H
—
4-Methylpiperazin-1-yl
4-Morpholino
OMe
2-(Dimethylamino)ethoxy
3-(1H-tetrazol-5-yl)propoxy
4-Methyl-1H-imidazol-1-yl
Tosyl
4-Methoxybenzonitrile
Methyl 4-(methoxy)benzoate
—
OMe
OMe
H
H
H
OMe
OMe
H
H
—
H
H
H
OMe
H
H
H
H
H
26
33
42
29
38
41
48
74
85
26
32
22
47
62
85
52
50
52
89
56
—
—
—
—
—
—
—
—
—
—
15m
15n
15o
15p
15q
15r
15s
15t
—
*
Control.

S. Göring et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4630–4637
4633
Scheme 2. Synthesis of compounds 20a and 20b.
under basic conditions to obtain a number of structurally diverse
compounds 9a–m in good yields (see Supporting information).20
Furthermore 4-fluoro-3-methoxybenzaldehyde 10 was connected
to the corresponding cyclic amines 11a–c and to its corresponding
sulfonyl derivative 11d. An aminoalkylation of 1H-indole-6-carbal-
dehyde 12 resulted in aldehyde 13. The final condensation was
carried out in a microwave reactor. The reaction of 5-chloroindo-
lin-2-one 14 with the commercial and synthesized aldehydes
yielded the indolinone derivatives 15a–t in good yields up to 97%
(Table 2).
of derivative 15b have substitutions in the 3rd and 5th position of
the phenol ring. Compounds 15c and 15f, which lack substitutions
in this position, displayed a dramatic decrease in LRRK2 inhibitory
activity (IC50 = 12 lM and IC50 = 10 lM, respectively) in compari-
son to the compounds 15a–b and 15d.
The chlorine of lead compound 15b was further substituted
(Scheme 2). Two residues with different sizes were chosen to
analyze the impact regarding LRRK2 activity on this position.
Intermediate 17 was generated via Suzuki coupling between
5-bromoindolin-2-one 16 and benzeneboronic acid. Furthermore
isatin 18 was reduced using hydrazine monohydrate to yield
indolinone derivative 19. The final compounds 20a and 20b were
coupled via Knoevenagel condensation. The LRRK2 activities of
compounds 20a and 20b are given in Table 3. Derivative 20a,
carrying the phenyl residue, exhibited a 6-fold decrease in LRRK2
The obtained indolinone derivatives 15a–t were tested for their
ability to inhibit LRRK2 kinase activity (assay conditions available
in Supplementary data).26,27 Indeed most of the synthesized indoli-
none derivatives 15a–t exhibited good in vitro activity reducing
LRRK2 activity below 50% at 10 lM (Table 2). Two exceptions are
the derivatives 15r and 15s, which were unable to significantly
reduce the in vitro LRRK2 activity (reduction to 74% and 85%,
respectively). This may be caused by the length and the size of
the second phenyl ring. On the contrary, indolinone derivatives
which have no or a short substitution at the 4-hydroxyl of the phe-
nol exhibited high potency against LRRK2 at a concentration of
activity (0.204
derivative 20b with 0.031
l
M) compared to its corresponding methoxy
l
M LRRK2 inhibitory activity.
Next, docking studies were performed to analyze the potential
binding mode of the most active LRRK2 inhibitor 15b. Models of
compound 15b docked into the ATP-binding site of LRRK2 are
shown in Figures 3A and B. The indolinone core favorably interacts
with the backbone of LRRK2. Especially glutamic acid 1948 is close
to the N–H motif of the indole and forms a strong hydrogen bond,
whereas the chlorine of the indolinone core binds towards the sol-
vent exposed region of the ATP-binding site (Fig. 3A and B). More
important are the interactions of the 3,5-dibromo-4-phenol moiety
with the rear of the mostly hydrophobic binding pocket (Fig. 3A
and B). Hydrophobic interactions were observed between the phe-
nol ring and the gatekeeper residue M1947 in addition to several
hydrophobic interactions with the 3,5-dibromo substitutions
(e.g., L1924 and G1888). The 4-hydroxy group of the phenol turns
10
lM. Three of them, the derivatives 15a–b and 15d, also reduced
LRRK2 activity below 10% in the presence of 1
lM test compound.
Additionally we determined the IC50’s of selected indolinone deriv-
atives (Table 2). Compound 15b is highly potent against LRRK2
with an IC50 value of 15 nM. Interestingly, indolinone derivative
15b is closely related to the known brain-penetrant, non-selective
LRRK2 inhibitor GW5074 1, only the substitution to chlorine in the
5th position of the indol ring is different (Fig. 1). Remarkably this
compound 15b exhibited a 53-fold higher activity against LRRK2
than GW5074 1 (IC50 of 880 nM).12 Further impacts on the activity
Figure 3. Suggested binding mode of compound 15b. The homology model of LRRK2 was made using the Swiss Model server.24,25 The molecular docking was performed
using the software Molegro Virtual Docker 6. (A) Compound 15b (brown) docked in the ATP-binding site of LRRK2. Important interactions are shown as green dashed lines. (B)
Surface illustration of the ATP-binding site of LRRK2 and the ligand 15b (white).

4634
S. Göring et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4630–4637
round of derivatization was performed. A replacement of the
OH
O
A
MeI
hydroxyl should result in loss of LRRK2 inhibitory activity. There-
fore additional indolinone derivatives were synthesized (outlined
in Scheme 3 and Table 4). The easiest way to establish the role of
a hydroxyl in this position is replacement of the hydrogen atom
by a methyl group. Therefore, in the presence of methyl iodide
and potassium carbonate compound 21 was converted to its corre-
sponding aldehyde 23. Substitutions in the 3rd and 5th position of
the phenol ring also influence LRRK2 inhibitory activity. The com-
mercial 4-hydroxy-3,5-dimethylbenzaldehyde 22, its correspond-
ing methylated counterpart 24 and the commercial aldehyde 25
which carries only one bromine in the 3rd position were also cou-
pled under the same Knoevenagel conditions with indolinone 14 in
good yields between 77% and 89% (Scheme 3, Table 3).
R
R
R
R
K2CO3
DMF
RT or 55°C
H
O
H
O
21
22
23
24
R = Br
R = Me
R = Br
R = Me
R3
R2
R1
R2
B
R1
R3
5-chloroindolin-2-one14
piperidine
Cl
O
methanol
100°C, 30 min
microwave
N
H
O
H
22 R1 = Me, R2 = OH, R3 = Me
23 R1 = Br, R2 = OMe, R3 = Br
26a R1 = Me, R2 = OH, R3 = Me
26b R1 = Br, R2 = OMe, R3 = Br
26c
26d
The obtained indolinone derivatives 26a–d were also tested for
their ability to inhibit LRRK2 kinase activity in vitro (Table 4). As
expected, the methylation of the phenol oxygen resulted in a dra-
matic loss of in vitro LRRK2 activity. The comparison of compounds
15b and 26b indicates a multiple drop of LRRK2 inhibitory activity
24
25
R
R
1 = Me, R2 = OMe, R3 = Me
1 = Br, R2 = OH, R3 = H
R
R
1 = Me, R2 = OMe, R3 = Me
1 = Br, R2 = OH, R3 = H
Scheme 3. (A) Methylation of aldehydes 21 and 22 to its corresponding counter-
parts 23 and 24. (B) Connecting the aldehydes with indolinone 14 via Knoevenagel
condensation.
from 0.015 lM to >10 lM. This was also confirmed by the indoli-
none derivatives 26a and 26c, respectively. The 3,5-dimethyl-4-
hydroxy moiety of compound 26a exhibited a reduced activity
with an IC50 of 0.194
but showed a 7 times higher activity concerning its corresponding
lM relative to compound 15b (IC50 = 15 nM),
Table 3
Indolinone derivatives 20a and 20b and their in vitro LRRK2 inhibitory activity
methylated form 26c with an IC50 of 1.5 lM (Table 3). This con-
firms the importance of a hydroxyl in position R2 (Table 1 and
Table 3) for the inactivating activity of indolinone based com-
pounds. Remarkably compound 26d, which carries one bromine
only, exhibited also high potency against LRRK2 with an IC50 value
of 0.046 lM.
G2019S, the most common disease-causing mutation in LRRK2
dramatically increased kinase activity. Additionally to the wild-
type LRRK2 activity we determined the LRRK2 activity against
the G2019S mutant for compounds 15b and 26d (Table 4). Both
compounds inhibited the LRRK2 G2019S mutant in a similar man-
Compound
R1
IC50 (lM)
20a
20b
Ph
OMe
0.204
0.031
ner as obtained for wild type LRRK2 with 0.010
lM for compound
15b and 0.064 M for compound 26d, respectively.
l
Table 4
The indolinone scaffold is well known of its activity in inhibiting
several different classes of kinases. Therefore we next examined
the kinase selectivity of lead compound 15b. The kinase selectivity
of compound 15b was assessed against a panel (ExpresS Diversity
kinase profile) of 46 human protein kinases (Fig. 4). At a concentra-
Synthesized indolinone derivatives and their in vitro LRRK2 inhibitory activity
R3
R2
R1
tion of 1 lM compound 15b merely inhibited the serine/threonine
Cl
kinases of HGK (MAPAK4) and Pimp2 with a residual activity
below 30%. These results suggest that 15b is a selective LRRK2
inhibitor, however further profiling against additional kinases are
needed.
O
N
H
Compound R1
R2
R3
Br
IC50 (wt LRRK2) (
l
M) IC50 (LRRK2
G2019S)
After the evaluation of the in vitro activity of this set of indoli-
none derivatives the most potent and promising compounds 15b
and 26d were additionally tested for their in vivo activity on zebra-
fish embryos. The zebrafish (Danio rerio) has become a useful ver-
tebrate model for assessing toxicological effects of chemicals and
drugs and is standardized international.28,29 Rapid development
of the embryos, their small size, high fecundity and the ease of hus-
bandry are advantages of zebrafish for a pharmacological and tox-
icological research.28,30,31 Treatment of the zebrafish embryo with
chemicals or drugs a phenotypic description of the zebrafish devel-
opment provides data for compound permeability and safety.
The zebrafish embryos were collected and maintained in E3
medium at 28 °C. The indolinone derivatives 15b and 26d were
added at 24 hpf (hours post fertilization) and the phenotypes were
15b
26a
26b
26c
26d
Br
OH
0.015
0.010
—
l
M
Me OH
Br
Me OMe Me 1.5
Br OH 0.046
Me 0.194
>10
OMe Br
—
—
H
0.064
lM
out to be particularly important. As already speculated by Chen
et al.,19 the 4-hydroxyl of the triazolpyridine 6 (Fig. 1) may act as
a donor and as an acceptor, but this has not been proven. This
interaction is also clearly represented in Figure 3A. On the one
hand the 4-hydroxyl group of compound 15b is donating two
hydrogen bonds to the conserved residues E1920 and D2017,
whereas it also acts as an acceptor by forming a strong H-bond
with K1906.
compared at 96 hpf (Fig. 5). At 20
reduced growth compared to concentrations of 1, 5, 10
the control (Fig. 5A–D and I). The zebrafish displayed stunted
and crooked tails at a higher concentration of 30 M. The lethality
of the zebrafish was observed every 24 h up to 120 hpf. Above a
l
M compound 15b caused a
lM and
To confirm the docking results and to confirm the importance of
the hydroxyl group in the 4th position of the phenol ring a second
l

S. Göring et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4630–4637
4635
Figure 4. Screening of compound 15b against 46 human protein kinases at 1 lM. Each bar represents the activity of one individual protein kinase (see Supplementary data
for more details).
Figure 5. In vivo cytotoxicity with zebrafish embryos. The zebrafish embryos were collected and maintained in E3 medium at 28 °C. Compounds 15b and 26d were added
24 hpf (hours post fertilization) and the phenotypes were compared after 96 hpf. (A–E) Zebrafish embryos treated with compound 15b in different concentrations of 1 M (A),
M (B), 10 M (C), 20 M (D) and 30 M (E). (F–H) Exposure of the zebrafish embryos to 1 M, 5 M and 10 M of 26d. 26d caused stunted and crooked tail at 5 M, which
is more pronounced at 10 M. (I) Control embryo in 1% DMSO.
l
5
l
l
l
l
l
l
l
l
l

4636
S. Göring et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4630–4637
Figure 6. Lethality representation of the zebrafish embryos at different time points for indolinone derivative 15b and 26d. The observed data are expressed as averages of
duplicates. (A) Lethality caused by compound 15b. (B) Lethality caused by compound 26d.
concentration of 30
over time (Fig. 6A). After 120 hpf the lethality for concentrations
l
M 15b we observed an increased lethality
4-hydroxy group of the phenol and the conserved residues E1920
and D2017 is essential for LRRK2 inhibition. During the develop-
ment of drugs, safety is one of the most important factors. In our
in vivo zebrafish embryo phenotype assay we have evaluated the
toxicity of both compounds. We observed that indolinone deriva-
of 50
at 30
l
l
M and 100
lM of compound 15b exhibited 100%, whereas
M 40% of the zebrafish embryos died. The zebrafish assay
did not indicate any abnormalities and lethality at a concentration
below 10 M (Fig. 5A–D and 6). Related, but more distinct pheno-
l
tive 15b displayed no significant toxicity below 10 lM, whereas
types were observed for compound 26d. Reduced growth and
stunted and crooked tails were already observed at a concentration
compound 26d displayed an increased toxicity over time.
Further improvements are required to pursue with more detail
phenotypic testing of analogues compounds in vivo and enable
development in preclinical models.
of 5
Indolinone derivative 26d also displayed a higher in vivo toxicity
compared to 15b (Fig. 6B). At a concentration above 10 M the
lM, and this is more pronounced at 10 lM (Fig. 5G and H).
l
lethality increased dramatically over time. After 48 hpf the lethal-
Acknowledgment
ity of the zebrafish embryos increased over 80% for concentrations
of 20 and 30
l
M, respectively. Therefore, there is a window of safe
M and
lM which may be used in evaluating neuroprotective
The authors thank the Hans und Ilse Breuer Stiftung for finan-
cial support.
treatment doses of these compounds situated below 5
above 15
effects.
l
Supplementary data
The observed deformities of the zebrafish indicated evidences of
exposure and cell penetration of the indolinone derivatives 15b
and 26d. Nevertheless, it cannot be ruled out that the defects on
the axis is an off-target effect via glycogen synthase kinase-3
(GSK-3) inhibition. It is known that Wnt signaling, and thus GSK-
3 activity, plays a crucial role in the development of metazoan
and was confirmed for known GSK-3 inhibitors, which interrupt
zebrafish development.32–34 Interestingly, the eyeless phenotype
caused by GSK-3 inhibitors,35,36 was not observed for both indoli-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.08.
049.
References and notes
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