Discovery of 3,6-disubstutited-imidazo[1,2-a]pyridine derivatives as a new class of CLK1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2021.127881

Inhibition of cdc2-like kinase1 (CLK1) could efficiently induce autophagy and it has been thought as a potential target for treatment of autophagy-related diseases. Herein we report the discovery of a series of 3,6-disubstutited-imidazo[1,2-a]pyridine derivatives as a new class of CLK1 inhibitors. Among them, compound 9e is the most potent one, which exhibits an IC₅₀ value of 4 nM against CLK1 kinase. In vitro, this compound reduces the phosphorylation level of the typical downstream substrates of CLK1 and affects their subcellular redistribution. Further study indicates that 9e is efficient to induce autophagy. Overall, this study provides a promising lead compound for drug discovery targeting CLK1 kinase.

Full text of DOI:10.1016/j.bmcl.2021.127881

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Bioorg. Med. Chem. Lett. 41 (2021) 127881
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 3,6-disubstutited-imidazo[1,2-a]pyridine derivatives as a new
class of CLK1 inhibitors
,
c
,
,
,
Yun Zhanga 1, Anjie Xiaa 1, Shiyu Zhangb 1, Guifeng Lina, Jingming Liua, Pei Chenb,
,
,*
Bo Mua d, Yan Jiaoa, Wenwen Xub, Mingxin Chena, Linli Lib
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China
b Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education, West China School of Pharmacy, Sichuan University, Sichuan 610041, China
c Macular Disease Research Laboratory, Department of Ophthalmology, West China Hospital, Sichuan University, Sichuan 610041, China
d Basic Medical College of North Sichuan Medical College, Nanchong 637000, Sichuan, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Inhibition of cdc2-like kinase1 (CLK1) could efficiently induce autophagy and it has been thought as a potential
target for treatment of autophagy-related diseases. Herein we report the discovery of a series of 3,6-disubstutited-
imidazo[1,2-a]pyridine derivatives as a new class of CLK1 inhibitors. Among them, compound 9e is the most
potent one, which exhibits an IC50 value of 4 nM against CLK1 kinase. In vitro, this compound reduces the
phosphorylation level of the typical downstream substrates of CLK1 and affects their subcellular redistribution.
Further study indicates that 9e is efficient to induce autophagy. Overall, this study provides a promising lead
compound for drug discovery targeting CLK1 kinase.
CLK1
Small molecule inhibitor
Structure-activity relationship
Autophagy
Introduction
molecular docking based virtual screening (VS) against SPECS and our
in-house compound database (for detail, see Fig. S1), which gave a hit
Autophagy is a mechanism conversed among eukaryotes by which
the cells engulf cellular material in vesicles formed autophagosomes
which fuse with lysosomes into autolysosomes for degradation to
maintain homeostasis and protect cells from death1–3. Autophagy can
degrade long-lived proteins, misfolded proteins, and damaged organ-
elles4. Dysfunction of autophagy has been shown to be associated with
many diseases, such as cancer, infectious, neurodegenerative, and car-
diovascular disease5–9. Many protein kinases have been reported to
participate in autophagy process regulation, one of them namely cdc2-
like kinase1 (CLK1), which plays an important role in alternative
splicing regulation of pre-mRNA by phosphorylation of the serine-
arginine-rich (SR) protein10,11. Previous studies revealed the inhibition
of CLK1 could efficiently induce autophagy through the mTOR/PI3K
pathway12,13. Currently, several CLK1 small molecular inhibitors have
been reported14–18, but few of them were related to autophagy in-
ducers17. Thus, discovering more CLK1 inhibitors with new scaffolds for
additional treatment options for autophagy-related diseases has attrac-
ted much attention.
compound, (S)-6-(1-methyl-1H-pyrazol-4-yl)-N-(1-phenylethyl)- imi-
dazo[1,2–a]pyridine-3-carboxamide (E242, Fig. 2). This compound
showed moderate inhibitory activity against CLK1 (IC50 = 296 nM) and
required further optimization to improve its potency. The subsequent
structural optimization was focused on two regions of E242 (region A
and region B, Fig. 1). Various substituents were used to replace these two
regions, and a total of 25 new 3,6-disubstutited-imidazo[1,2-a]pyridine
derivatives were synthesized.
To examine the influence of chiral benzylamine substituents (region
A, R1) on the bioactivity, we first fixed region B as the original 1-methyl-
1H-pyrazol-4-yl group and varied the R1 group substituents. The syn-
thetic routes for compounds 6a-l are depicted in Scheme 1. Briefly,
treatment of commercially available 1 with DMF-DMA resulted in the
formation of 2, which was cyclized with ethyl 2-bromoacetate to ob-
tained 3. Suzuki coupling between 3 and 1-methyl-4-pyrazole boronic
acid pinacol ester delivered intermediate 4a, which was then hydrolyzed
to offer the corresponding acid 5a. Target compounds 6a-l were finally
generated by a condensation reaction of 5a and various amines.
The chemical structures and bioactivities of compounds 6a-l are
In the effort to seek potent CLK1 inhibitors, we performed a
* Corresponding author.
E-mail address: lilinli@scu.edu.cn (L. Li).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2021.127881
Received 25 November 2020; Received in revised form 4 February 2021; Accepted 13 February 2021
Available online 2 March 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

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Y. Zhang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127881
Next, we introduced methoxyl or chlorine in the C4 position at
phenyl group, delivered potency enhanced R configuration com-
pounds 6c and 6e, and potency decreased S configuration com-
pounds 6b and 6d, further confirmed that the R configuration 1-
phenethyl groups are preferred at the R1 position. Then, we
removed the chiral methyl on 1-phenethyl group and synthesized 5
non-chiral compounds (6f-j). Among them, 6f, which with a para-
methoxybenzyl group, further increased the activity against CLK1
kinase. Finally, we replaced the benzyl group with smaller substi-
tution groups (6k and 6l), and the two generated compounds showed
reduced bioactivity. Therefore, it seems that para-methoxybenzyl is
Fig. 1. (A) Chemical structure of E242. (B) Focuses of the structural
modification.
the best choice for R1
.
In the second step, to explore the effect of the substituent at region B
(R2), we installed different subgroups, including pyridyl, substituted
five-membered heterocyclic rings, and bicyclic aromatic groups in this
position (R2) and fixed region A as the optimal para-methoxybenzyl
group. Another 5 new compounds (9a-e) were then synthesized.
Scheme 2 illustrates the synthetic routes of these compounds. In-
termediates 3 was readily prepared as depicted above. Hydrolyzation of
3 delivered acid 7, which then reacted with 4-methoxybenzylamine
gave intermediate 8. Suzuki coupling of 8 with commercially available
aromatic boric acid (or ester) yielded products 9a-e.
The chemical structures and bioactivities of compounds 9a-e are
shown in Table 2, revealing that substitutions at region B substantially
impacted the activity. Replacement of the 1-methyl-1H-pyrazol-4-yl
group with pyridin-4-yl group maintained the potency (9a vs 6f), but
introduction of 3,5-dimethylisoxazol-4-yl (9b), benzo[d]oxazol-5-yl
(9c) and naphthalen-2-yl (9d) at R2 position resulted in significantly
decreased or completely absent CLK1 activities. Compound 9e, which
has a quinolin-6-yl moiety at the R2 position displayed the best perfor-
mance, with an IC50 value of 4 nM against CLK1. Collectively, quinolin-
6-yl group may be the best choice for region B.
In the last step, to examine whether para-methoxybenzyl group is
still optimal when region B is fixed as quinolin-6-yl a moiety, we pre-
pared eight compounds with different substituents at region A (10a-h).
The synthetic routes for these compounds are given in Scheme 3, which
is similar to that in Scheme 1.
Fig. 2. Predicted binding mode of 9e with CLK1 (the CLK1 structure was taken
from PDB entry 5X8I).
Table 3 shows the CLK1 inhibitory activities of compounds 10a-h.
It can be seen that the introduction of chiral 1-phenethyl group at R3
position resulted in no improvement of the potency, and again S
configuration compounds 10a and 10c exhibited weaker potency
compared with R configuration enantiomers 10b and 10d. Replace-
ment of the para-methoxyl of 9e with para-ethoxyl or para-
isopropoxyl led to the obvious loss in CLK1 inhibitory activity.
given in Table 1. Because E242 contains an S configuration 1-phe-
nethyl group at the R1 position, we first examined the bioactivity
of its enantiomer 6a with an R configuration 1-phenethyl group. 6a
displayed an obvious improvement in CLK1 inhibitory potency,
indicating that the R configuration 1-phenethyl group is more
favorable than the corresponding S configuration at the R1 position.
Scheme 1. Synthetic routes for compounds 6a-l, and E242. Reagents and conditions: (i) DMF-DMA, DMF 65 ℃; (ii) NaHCO3, ethyl 2-bromoacetate, DMF, 85 ℃; (iii)
1-methyl-4-pyrazole boronic acid pinacol ester, PdCl2(dppf), K2CO3, 1,4-dioxane/H2O, 100 ◦C; (iv) LiOH⋅H2O, THF/MeOH/H2O, 60 ◦C; (v) substituted amine, EDCI,
1-Hydroxybenzotriazole, triethylamine, DCM, rt.
2

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Y. Zhang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127881
Table 1
Bioactivities of compounds 6a-l and E242a.
Compound
R1
IC50 (nM)
296
%Control @10 µM
ꢀ 7
E242
6a
6b
14
ꢀ 14
809
0
6c
8
ꢀ 3
6d
6e
6f
399
11
6
12
5
ꢀ 12
6g
8
0
6h
6i
14
11
1
1
6j
12
7
1
3
6k
6l
Methyl
23
1
a IC50 values were determined from Kinase Profiler of Eurofins. [ATP] = 10 µM.
3

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Y. Zhang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127881
Scheme 2. Synthetic routes for compounds 9a-e. Reagents and conditions: (i) DMF-DMA, DMF 65 ℃; (ii) NaHCO3, ethyl 2-bromoacetate, DMF, 85 ℃; (iii)
LiOH⋅H2O, THF/MeOH/H2O, 60 ◦C; (iv) 4-methoxybenzylamine, EDCI, 1-Hydroxybenzotriazole, triethylamine, DCM, rt; (v) commercially available aromatic boric
acid (or ester), PdCl2(dppf), K2CO3, 1,4-dioxane/H2O, 100 ◦C.
Table 2
Bioactivities of compounds 9a-ea.
Compound
R2
IC50 (nM)
13
%Control @10 µM
ꢀ 9
9a
9b
9c
>10000
59
122
ꢀ 5
9d
9e
>10000
88
2
4
a IC50 values were determined from Kinase Profiler of Eurofins. [ATP] = 10 µM.
(10e-f). The shift of methoxyl from para-position to meta-position also
reduced the potency (10g). At last, Hydrazide 10h was synthesized,
which showed high bioactivity but still did not exceed 9e in terms of
potency.
hydrogen bond with Lys191. Another hydrogen bond is formed between
the imidazo[1,2-a]pyridine nitrogen atom and Leu244 on the hinge. The
oxygen atom of methoxyl also forms one hydrogen bond with Ser247,
which might be the reason why para-methoxybenzyl is the best choice
on 3-position of the scaffold. In addition, it is also obvious that the
Then we used molecular docking to predict the binding model of 9e
and CLK1. As shown in Fig. 2, 9e suitably occupies the ATP binding
pocket of CLK1. The nitrogen atom of quinoline of 9e forms one
quinoline of 9e forms a
π–π stacking interaction with the benzene ring of
Phe172, and the imidazo[1,2-a]pyridine scaffold formed a T-shape
π–π
4