Discovery of Potent and Selective Inhibitors of Cdc2-Like Kinase 1 (CLK1) as a New Class of Autophagy Inducers

Article abstract of DOI:10.1021/acs.jmedchem.7b00665

Autophagy inducers represent new promising agents for the treatment of a wide range of medical illnesses. However, safe autophagy inducers for clinical applications are lacking. Inhibition of cdc2-like kinase 1 (CLK1) was recently found to efficiently induce autophagy. Unfortunately, most of the known CLK1 inhibitors have unsatisfactory selectivity. Herein, we report the discovery of a series of new CLK1 inhibitors containing the 1H-[1,2,3]triazolo[4,5-c]quinoline scaffold. Among them, compound 25 was the most potent and selective, with an IC₅₀ value of 2 nM against CLK1. The crystal structure of CLK1 complexed with compound 25 was solved, and the potency and kinase selectivity of compound 25 were interpreted. Compound 25 was able to induce autophagy in in vitro assays and displayed significant hepatoprotective effects in the acetaminophen (APAP)-induced liver injury mouse model. Collectively, due to its potency and selectivity, compound 25 could be used as a chemical probe or agent in future mechanism-of-action or autophagy-related disease therapy studies.

Full text of DOI:10.1021/acs.jmedchem.7b00665

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Article
Discovery of Potent and Selective Inhibitors of Cdc2-like
Kinase 1 (CLK1) as a New Class of Autophagy Inducers
Qi-Zheng Sun, Gui-Feng Lin, Lin-Li Li, Xi-Ting Jin, Lu-Yi Huang, Guo Zhang, Wei Yang, Kai
Chen, Rong Xiang, Chong Chen, Yu-Quan Wei, Guang-Wen Lu, and Sheng-Yong Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00665 • Publication Date (Web): 10 Jul 2017
Downloaded from http://pubs.acs.org on July 10, 2017
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Discovery of Potent and Selective Inhibitors of Cdc2-like Kinase 1 (CLK1) as a
New Class of Autophagy Inducers
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QiꢀZheng Sun,†,§ GuiꢀFeng Lin,†,§ LinꢀLi Li,‡,§ XiꢀTing Jin,‡ LuꢀYi Huang,†,|| Guo
Zhang,‡ Wei Yang,‡ Kai Chen,† Rong Xiang,# Chong Chen,† YuꢀQuan Wei,†
GuangꢀWen Lu,†,* and ShengꢀYong Yang†,||,*
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†State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,
Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu,
610041, PR China.
‡Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of
Education, West China School of Pharmacy, Sichuan University, Sichuan 610041,
China.
||School of Chemical Engineering, Sichuan University, and Collaborative Innovation
Center for Biotherapy, Chengdu, 610041, PR China.
#Department of Clinical Medicine, School of Medicine, Nankai University, Tianjin
300071, China.
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Abstracts
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Autophagy inducers represent new promising agents for the treatment of a wide range
of medical illnesses. However, safe autophagy inducers for clinical applications are
lacking. Inhibition of cdc2ꢀlike kinase 1 (CLK1) was recently found to efficiently
induce autophagy. Unfortunately, most of the known CLK1 inhibitors have
unsatisfactory selectivity. Herein, we report the discovery of a series of new CLK1
inhibitors containing the 1Hꢀ[1,2,3]triazolo[4,5ꢀc]quinoline scaffold. Among them,
compound 25 was the most potent and selective, with an IC50 value of 2 nM against
CLK1. The crystal structure of CLK1 complexed with compound 25 was solved, and
the potency and kinase selectivity of compound 25 were interpreted. Compound 25
was able to induce autophagy in in vitro assays and displayed significant
hepatoprotective effects in the acetaminophen (APAP)ꢀinduced liver injury mouse
model. Collectively, due to its potency and selectivity, compound 25 could be used as
a chemical probe or agent in future mechanismꢀofꢀaction or autophagyꢀrelated disease
therapy studies.
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1. INTRODUCTION
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Autophagy is a type of cell “selfꢀeating” phenomenon that can selectively remove
damaged or useless organelles and proteins, thus protecting cells from death. Impaired
autophagic activity has been linked to a wide range of diseases, including cancer,1
diabetes,2,3 neurodegenerative diseases,4,5 and drugꢀinduced organ injury.6,7
Autophagy inducers are thus thought of as promising agents for the treatment of these
diseases.8 Currently, only a few autophagy inducers are known.9 Nevertheless, most
of these agents have a wide range of pharmacological activity, which implies a
potential risk of toxicity, thus restricting their clinical application.10 For example,
rapamycin, a typical autophagy inducer, could depress the immune system11,12 and
induce insulin resistance.13,14 Therefore, there is an urgent need to discover more
efficient and specific autophagy inducers.
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In a recent study, Fant et al.15 demonstrated that inhibition of cdc2ꢀlike kinase 1
(CLK1) could efficiently induce autophagy. The authors also indicated that CLK1
could be a target for the treatment of autophagyꢀrelated diseases. To date, a number of
CLK1 inhibitors have been reported.16ꢀ21 Despite few inhibitors with definite
selectivity, most of the reported CLK1 inhibitors belong to the miscellaneous type,
which are associated with unsatisfactory kinase selectivity and are thus not suitable
for clinical use due to possible side effects. In particular, these inhibitors, without
exception, displayed identical or similar potency against CLK1 and dualꢀspecificity
tyrosineꢀphosphorylationꢀregulated kinase 1A (DYRK1A).18,22 DYRK1A is highly
homologous in sequence to CLK1,18 and inhibition of DYRK1A has been shown to
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pose a potential toxicological risk.23,24 Therefore, the identification of potent and
selective CLK1 inhibitors may have potential clinical application value in the
treatment of autophagyꢀrelated diseases.
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In the effort to seek potent and selective CLK1 inhibitors, we recently obtained a
hit compound, 1ꢀmethylꢀ8ꢀ(2ꢀmethylpyridinꢀ4ꢀyl)ꢀ1Hꢀ[1,2,3]triazolo[4,5ꢀc]quinoline
(1, Figure 1) (for details, see Supporting Information). Compound 1 showed IC50
values of 139 nM and 420 nM against CLK1 and DYRK1A, respectively. Both the
activity and selectivity of this compound were poor and required further optimization.
The main purpose of this investigation was to carry out structural optimization and
structureꢀactivity relationship (SAR) analyses of compound 1. We further explored
the ability of the most active and selective compound to induce autophagy and
explored possible hepatoprotective effects in the acetaminophen (APAP)ꢀinduced
acute liver injury mouse model.
Figure 1. (A) The chemical structure of compound 1. (B) Regions of focus for
structural optimization.
2. RESULTS AND DISCUSSION
2.1 Chemical Syntheses and SAR Analyses
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The subsequent structural optimization was focused on the R1 and R2 regions of
1 (Figure 1B). In the first step, we fixed R2 as the original 2ꢀmethylpyridinꢀ4ꢀyl group
and varied the R1 group substituents. The synthetic routes are outlined in Scheme 1.
Briefly, commercially available 2 was reacted with selfꢀprepared 2ꢀnitroacetaldehyde
oxime to obtain 3, which was dehydrated to yield 4. Chlorination of 4 afforded the
key intermediate 5. A nucleophilic substitution reaction between 5 and various
substituted amines yielded intermediates 6a–6k. The nitro groups of 6a–6k were
reduced to produce the corresponding amines 7a–7k, which then underwent a
diazotization and condensation reaction to produce the triazole compounds 8a–8k.
Final product compounds 1 and 9b–k were readily obtained through the classic
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palladiumꢀcatalyzed
Suzuki
coupling
between
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and
(2ꢀmethylpyridinꢀ4ꢀyl)boronic acid (29).
Scheme 1. Synthetic routes of 1 and 9b–9ka
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aReagents and conditions: (i) 1). HCl, H2O, room temperature (rt); 2). NaOH, H2O, 0
°C; 3). HCl, H2O, 0 °C; (ii) AcOK, Ac2O, 120 °C; (iii) POCl3, reflux; (iv) substituted
amine, triethylamine, EtOH, reflux; (v) Fe, HAc, 60 °C; (vi) NaNO2, AcOH/H2O;
(vii) 29, Pd(dppf)Cl2, K2CO3, 1,4ꢀdioxane/H2O, 100 °C, 24 h.
The chemical structures and bioactivities of 9b–9k and 1 are shown in Table 1.
Here,
a
typical
CLK1
inhibitor,
(Z)ꢀ1ꢀ(3ꢀethylꢀ5ꢀmethoxybenzo[d]thiazolꢀ2(3H)ꢀylidene)propanꢀ2ꢀone (30, called
TG003 in literature),25 was used as a positive control. In addition, we used the ratio of
activities against DYRK1A and CLK1 as a selectivity index (SI) to represent the
selectivity of compounds, which was primarily based on the following considerations.
First, the systematic evaluation of the kinase selectivity must assess the activity of
compounds against the entire kinome, which is extremely costly. Second, the amino
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acid sequences of DYRK1A and CLK1 are very similar, with an identity of 70.4%,
and the majority of CLK1 inhibitors have comparable potency against DYRK1A.
Table 1 shows that the introduction of a benzyl group at the R1 position benefits the
CLK1 activity (9d vs 1, 9b, and 9c). Methylation at the benzylic carbon atom leads to
chiral derivatives, and the Sꢀconfiguration is more favorable for selectivity (9e vs 9f,
9g vs 9h). In addition, the C4 substitution on the phenyl group is sensitive to both
potency and selectivity. Compared with the unsubstituted 9g, compound 9e with a
fluorine (ꢀF) substitution showed the most selectivity, although its potency was
marginally decreased. Other substituents, including –methyl (9i) and ꢀCl (9k) groups,
decreased both the activity and selectivity. To summarize, 9e, which contains a
(S)ꢀ1ꢀ(4ꢀfluorophenyl)ethyl group at the R1 position, had the highest selectivity and
also considerable potency against CLK1.
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Table 1. Structures and bioactivities (IC50) of compounds 9b–k and 1a
Cpd.
R1
CLK1 [nM]
DYRK1A [nM]
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aIC50 values were determined using the KinaseProfiler from Eurofins. The data
b
represent the mean values of two independent experiments; SI denotes the ratio of
IC50 (DYRK1A) to IC50 (CLK1); cpositive control; [ATP] = 10 ꢁM.
In the second step, we fixed R1 as the optimal (S)ꢀ1ꢀ(4ꢀfluorophenyl)ethyl group
and altered the R2 group using different subgroups, including phenyl, pyridyl,
substituted phenyl and pyridyl, morpholinyl, fiveꢀmembered heterocyclic rings, and
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benzoꢀfused fiveꢀmembered heterocyclic rings. Another 16 new compounds (10–25)
were then synthesized.
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The synthetic routes to these compounds are depicted in Scheme 2. Briefly,
Suzuki coupling of 8e with commercially available aromatic boric acid (or ester)
yielded products 10–15 and 17–24, and palladiumꢀcatalyzed Buchwald coupling
between morpholine and 8e produced product 16. By refluxing commercially
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available
2ꢀaminoꢀ4ꢀbromophenol
(26)
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orthoformate,
5ꢀbromobenzo[d]oxazole (27) could be readily obtained, which subsequently
underwent a Miyaura borylation reaction to yield a benzooxazoleꢀ5ꢀboronic acid
pinacol ester (28). Then, Suzuki coupling of 28 with 8e yielded the final product 25.
Scheme 2. Synthetic routes to 10–25a
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aReagents and conditions: (i) commercially available aromatic boronic acid or boronic
acid ester, Pd(dppf)Cl2, K2CO3, 1,4ꢀdioxane/H2O, 100 °C, 24 h; (ii) morpholine,
Pd2(dba)3, XꢀPhos, K2CO3, tꢀBuOH, 100 °C, overnight; (iii) triethyl orthoformate,
reflux; (iv) bis(pinacolato)diboron, Pd(dppf)Cl2, AcOK, 1,4ꢀdioxane, 100 °C; (v) 8e,
Pd(dppf)Cl2, K2CO3, 1,4ꢀdioxane/H2O, 100 °C, 24 h.
The chemical structures and bioactivities of compounds 10–25 are shown in
Table 2, revealing that substitutions at the R2 position substantially impacted the
activity and selectivity. Compared with 9e, compounds with bromo (8e), phenyl (10),
substituted phenyl (14, 15, 17, and 18), pyridyl (12, 19), substituted pyridyl (11, 13),
morpholinyl (16), or fiveꢀmembered heterocyclic (20–22) groups at the R2 position
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had significantly decreased or completely absent CLK1 activities. Compounds (23–25)
containing a benzoꢀfused fiveꢀmembered heterocyclic group at the R2 position seemed
to have overall improved activity and selectivity. Among them, compound 25 with a
benzo[d]oxazolꢀ5ꢀyl moiety at the R2 position exhibited the best performance, with an
IC50 value of 2 nM against CLK1 and 69ꢀfold increased potent activity compared with
that of the closely related kinase DYRK1A.
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Collectively, compound 25, which contained (S)ꢀ1ꢀ(4ꢀfluorophenyl)ethyl and
benzo[d]oxazolꢀ5ꢀyl groups at the R1 and R2 positions, respectively, showed the
highest potency and selectivity in enzymatic assays. Further inꢀdepth studies were
then performed on this compound, including measurement of kinomeꢀwide selectivity,
coꢀcrystal structure determination, in vitro autophagyꢀinducing ability examination,
and assessment of the in vivo effect of liver protection in an acute liver injury model.
Table 2. Structures and bioactivities (IC50) of compounds 10–25a
Cpd.
R2
Br
CLK1 [nM]
29
DYRK1A [nM]
1303
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9e
>10000
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>10000
>10000
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>10000
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>10000
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aIC50 values were determined using the KinaseProfiler of Eurofins. The data represent
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the mean values of two independent experiments; SI denotes the ratio of IC50
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(DYRK1A) to IC50 (CLK1); cpositive control; [ATP] = 10 ꢁM.
2.2 Kinome-wide Selectivity of 25
The selectivity of the compounds described above were only for DYRK1A. To
examine the kinomeꢀwide selectivity of 25, we tested the kinase inhibition profiles of
this compound against a panel of 357 recombinant human protein kinases. The
inhibitory activities of 25 at a fixed concentration of 10 µM were measured first. For
kinases with inhibitory rates higher than 50%, further IC50 values were determined.
The results are presented in Supporting Information Table S1. A dendrogram
representation of the kinase inhibition by 25 is also shown in Supporting Information
Figure S2. As expected, CLK1 was the most potently inhibited kinase (IC50: 2 nM). In
addition to CLK1, only two kinases had an IC50 value less than 100 nM, namely,
CLK2 (IC50: 31 nM) and CLK4 (IC50: 8 nM), which are isoforms of CLK1. Except for
the CLKs, DYRK1A was the strongest offꢀtarget. Furthermore, Supporting
Information Table S1 shows that 25 displayed at least 110ꢀfold selectivity toward
CLK1 over 351 of the 357 kinases tested (98.32%). Of note is that compound 25 did
not show obvious activity against 292 kinases (IC50 > 10 ꢁM). To the best of our
knowledge, 25 is the most potent and selective CLK1 inhibitor identified to date.
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2.3 Co-crystal Structure of CLK1 Complexed with 25 and the Rationale for its
Potency and Selectivity
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To elucidate the interactions between 25 and CLK1 and the molecular basis of
the observed kinase selectivity, we solved the coꢀcrystal structure of the CLK1 kinase
domain with 25 at a resolution of 1.9 Å (PDB ID 5X8I). The related data processing
and structure refinement statistics are provided in Supporting Information Table S2.
Figure 2A displays the key interactions of 25 with CLK1, illustrating that 25 suitably
resides in the ATP binding pocket of CLK1, with the benzo[d]oxazolꢀ5ꢀyl group
pointing toward the hinge region and the triazole moiety of the
1Hꢀ[1,2,3]triazolo[4,5ꢀc]quinoline scaffold pointing toward the solvent area. The
quinoline nitrogen and the kinase Lys191 forms an important hydrogen bond. Another
important hydrogen bond is formed between the oxazole oxygen atom and Leu244 on
the hinge, with the oxazole oxygen atom acting as a hydrogen bond acceptor. The
oxazole nitrogen might form some interactions with the hinge region through
waterꢀmediated hydrogenꢀbondꢀnetwork, which might partially explain the potency
increase of benzoxazole 25 compared to benzofuran 24. In addition, it was also
obvious that the tricyclic scaffold of 25 formed a Tꢀshape πꢀπ interaction with the
benzene ring of CLK1 Phe241 and a πꢀπ stacking interaction with the benzene ring of
Phe172. Here, it is worth mentioning that quinolineꢀcontaining kinase inhibitors
usually adopt a canonical hinge binding mode with quinoline making the hinge
contacts in the ATPꢀbinding pocket of kinase.26ꢀ28 However, in this case, 25, which
also contains quinoline, adopts an unusual binding mode. Thus, some important
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interactions indicated above are formed, which could be one of the reasons why the
inhibitor shows such a good selectivity.
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To understand the preference of 25 in the inhibition of CLK1 over other kinases,
the active amino acids involved in CLK1/25 interactions were compared between
different kinases via sequence alignment. Figure 2B shows the alignment of the key
residues within 4.5 Å of 25 and CLK1 with the corresponding residues of other
selected kinases (note that numbering is according to CLK1) that include CLK1–4,
DYRK isoforms, and other representative kinases inhibited by 25, with IC50 values of
different orders of magnitude. Obviously, the potencies of 25 against different kinases
were strongly dependent on the residues in their active pockets. CLK2/4 had the same
residues as CLK1 in the alignment, explaining why they were potently inhibited by
25. The main difference between CLK1/2/4 and CLK3 is the residue 324 (CLK1:
valine; CLK3: alanine). We noted that alanine had a smaller hydrophobic side chain
compared with valine and thus likely weakened the hydrophobic interactions with the
ligand. We hypothesize that this may be the reason why 25 had reduced activity
against CLK3. Similarly, DRYK1A and DYRK1B displayed moderate activity. In
sequence, these two kinases had one residue that differed from CLK1 (CLK1:
LEU167 vs DYRK1A/1B: ILE167). Notably, the substitution of LEU167 (as in
CLK1) with ILE167 (as in DRYK1A/1B) created small amounts of steric hindrance
with 25, thus requiring a possible conformational adjustment in the residue to properly
accommodate the bound ligand. Accordingly, 25 exhibited decreased activity against
DYRK1A/B compared with CLK1. Finally, other selected kinases all exhibit more
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profound residue differences in the ligand binding site, explaining the poor activities
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Figure 2. The Xꢀray crystal structure of CLK1 complexed with 25 (PDB ID 5X8I).
(A) Key interactions of 25 with the kinase active site. Yellow and cyan dashed lines
indicate hydrogen bonds and πꢀπ interactions, respectively. Gray mesh: 2FoꢀFc
omitted electron density map contoured at 2.0 σ. The images were produced using
PyMOL. (B) Multipleꢀsequence alignment of the selected key residues within the
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ATPꢀbinding pocket. The residues differing from the corresponding residues of CLK1
are highlighted in blue. Numbering is according to CLK1.
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2.4 Effects of 25 on the Subcellular Redistribution of Downstream
Serine/Arginine-rich (SR) Proteins in Intact Cells
SR proteins are typical downstream substrates of CLK1. It has been
demonstrated that activation of CLKs could induce redistribution of SR proteins from
nuclear speckles (stored form) to the nucleoplasm (active form).29,30 Therefore, to
examine whether 25 can inhibit CLK1 activation in intact cells, we measured the
distribution of SR proteins in cells by immunofluorescence before and after 25
treatment. Here, BNL CL.2 (mouse embryonic liver cell line) was selected, and three
concentrations (20 nM, 100 nM, and 10 ꢁM) of 25 were used. As shown in Figure 3
and Supporting Information Figure S4, 25 treatment indeed led to the redistribution of
SR proteins from the nucleoplasm to nuclear speckles, and the potency showed
significant dose dependency. The same effect was observed for the positive control 30.
From here, we concluded that 25 could efficiently inhibit the activation of CLK1 in
intact cells.
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Figure 3. Compound 25 altered the location and redistribution of SR proteins. BNL
CL.2 cells treated with 25 (10 ꢁM), 30 (10 ꢁM) or DMSO (0.1%) for 24 h were fixed
and probed with antiꢀSR proteins antibody (mAb1H4G7). Diffuse staining and typical
speckles demonstrated by mAb1H4G7 represent active and stored forms of SR
proteins respectively. DAPI was used to dye the nucleus. Scale bar: 10 ꢁm.
2.5 Ability of 25 to Induce Autophagy In Vitro
We next examined the ability of 25 to induce autophagy in BNL CL.2 and
SKOVꢀ3 (human ovarian cancer cell line) cells. Compound 30 and the classic
autophagy inducer rapamycin were used as positive controls. Western blot assays
showed that 25 elevated the expression level of LC3II protein (a marker of
autophagosomes31,32) as well as the ratio of LC3II to LC3I (a sensitive index of
autophagy33,34) in a doseꢀdependent (Figure 4A and 5A) and timeꢀdependent manner
(Figure 4B). In accordance with the results of immunoblot assays, confocal
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microscopy examination showed that 25 increased the number of yellow LC3 puncta
(coꢀlocalization of GFP and mRFP signals35) in SKOVꢀ3 cells infected with
AdꢀmRFPꢀGFPꢀLC3 (adenovirusꢀcoding tandem GFPꢀmRFPꢀLC3), further
confirming the formation of autophagosomes. The effects of 25 on yellow LC3 puncta
also displayed obvious dose dependency, and a dose of 10 ꢁM showed the best
performance (Figure 5B and Supporting Information Figure S5). In addition, in
25ꢀtreated cells, the number of red LC3 puncta (mRFP signals only35) increased
compared with that of DMSOꢀtreated cells, indicating the formation of
autolysosomes. Importantly, 25 stimulated the degradation of SQSTM1/p62 (a marker
of autolysosomes,36 Figure 4A and Figure 5A) and increased the ratio of red LC3
puncta to yellow LC3 puncta (Figure 5B and Supporting Information Figure S5), both
of which indicated an induction of autophagic flux by 25. All of these data
demonstrated that 25 could increase the synthesis and clearance of autophagosomes,
and induce autophagy and autophagic flux in vitro to levels higher than those resulting
from 30 and rapamycinꢀinduced autophagy, especially in BNL CL.2 cells. In contrast,
compound 18 (negative control), which is inactive against CLK1 (IC50 > 10 ꢁM),
could not trigger autophagy even at a concentration of 10 ꢁM (Supporting
Information Figure S6).
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Figure 4. Detection of autophagy and autophagic flux induced by 25 in BNL CL.2
cells. (A) BNL CL.2 cells were treated with rapamycin (positive control), 30 (positive
control) and various concentrations of 25 (0.2, 1, 5, and 10 ꢁM) for 24 h. Then, whole
cell lysates were subjected to immunoblot assay to detect LC3 and SQSTM1/p62.
Quantification of immunoblots is presented in the right panel. (B) BNL CL.2 cells
were treated with 25 or rapamycin, and the level of LC3II protein was detected after
6, 12 and 24 h. Quantification of immunoblots is presented in the right panel.
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Figure 5. Detection of autophagy and autophagic flux induced by 25 in SKOVꢀ3 cells.
(A) SKOVꢀ3 cells were treated with rapamycin (positive control), 30 (positive control)
and various concentrations of 25 (0.05, 0.2, 1, 5, and 10 ꢁM) for 24 h. Then, whole
cell lysates were subjected to an immunoblotting assay to detect LC3 and
SQSTM1/p62. Immunoblots were quantified as shown below. (B)
AdꢀmRFPꢀGFPꢀLC3ꢀinfected SKOVꢀ3 cells were treated with 25 (10 ꢁM) for 24 h
and fixed before examination by confocal microscopy. Representative photographs
are presented. Scale bar: 10 ꢁm. Alignment of green and red signals appears yellow.
The number of LC3ꢀpuncta (mean ± SEM) in overlays was quantified and is shown
below. More than 80 cells were counted in each individual experiment (n = 3). ** P <
0.01, *** P < 0.001.
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2.6 Protective Effect of 25 on APAP-induced Acute Liver Injury
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As mentioned above, autophagy inducers may have potential applications in the
treatment of relevant diseases. Drugꢀinduced liver injury (DILI) is a major public
health concern37 and accounts for more than 50% of acute liver failure cases. APAP is
a widely used antipyretic analgesic drug. However, an overdose of APAP can cause
liver injury and even liver failure in humans38 and is the most frequent cause of acute
liver failure, especially in many western countries.39 Ni et al.6 found that induction of
autophagy by rapamycin inhibited APAPꢀinduced hepatotoxicity, which restricted the
necrotic areas and promoted liver regeneration and recovery.39,40 Herein, the
APAPꢀinduced hepatotoxicity mouse model was used to examine the therapeutic
effect of 25 in vivo. As depicted in Figure 6A, APAP exposure resulted in severe liver
injury, and treatment with 25 (ip, 30 mg/kg) imparted a significant hepatoprotective
effect. We further detected the levels of the liver enzymes alanine aminotransferase
(ALT) and aspartate aminotransferase (AST), high levels of which are considered to
be indicators of liver damage. The results showed that treatment with 25 decreased
serum ALT and AST levels significantly, such that both marker enzymes returned to
normal levels (Figure 6B). Rapamycin and 30 also showed similar hepatoprotective
effects in the same model.
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Figure 6. Protective effect of 25 on APAPꢀinduced liver injury in mice. Male
C57BL/6 mice were injected (ip) with 25 (30 mg/kg), 30 (30 mg/kg, positive control),
rapamycin (30 mg/kg, positive control), or solvent (12.5% ethanol and 12.5% castor
oil, 10 ml/kg), immediately followed by APAP (500 mg/kg) injection (ip) for 6 h. (A)
Representative photographs of H&E staining are presented. (B) Serum ALT and AST
levels were quantified (n = 3 mice). ** P < 0.01.
3. CONCLUSIONS
In summary, a structural optimization of hit compound
1 led to the
discovery of a series of new CLK1 inhibitors containing the scaffold
1H
ꢀ[1,2,3]triazolo[4,5ꢀ
c
]quinolone. Among them, compound 25 is the most
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potent and selective. The coꢀcrystal structure of 25 with CLK1 explained its
potent activity and kinase selectivity. This compound also demonstrated an
ability to induce autophagy in vitro and had significant hepatoprotective effects
in the APAPꢀinduced liver injury mouse model. A preliminary pharmacokinetic
study in rats showed that 25 had good bioavailability (F = 52.86%, for more
information see Supporting Information Figure S7). All of these results imply
that 25 could be a promising lead compound for the treatment of
autophagyꢀrelated diseases. However, the mechanism by which CLK1
inhibition induces autophagy is currently unknown. Further studies, including
an investigation of the mechanism of autophagy induction and preclinical
evaluation in the treatment of autophagyꢀrelated diseases, are still underway in
our laboratory.
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4. EXPERIMENTAL SECTION
4.1. Chemistry Methods. All reagents and solvents were obtained from
commercial suppliers and used without further purification unless otherwise indicated.
Anhydrous solvents were dried and purified by conventional methods prior to use.
Column chromatography was carried out on silica gel (300−400 mesh). All reactions
were monitored by thinꢀlayer chromatography (TLC), and silica gel plates with
fluorescence Fꢀ254 were used and visualized with UV light. All of the final
compounds were purified to >95% purity, as determined by highꢀperformance liquid
chromatography (HPLC). HPLC purity analysis was performed on a Waters 2695
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HPLC system with the use of a Gemini C18 reversedꢀphase column (4.6 mm Φ × 150
mm, particle size: 5 ꢁm). The enantiomeric excess (ee) of the products was
determined by HPLC analysis on Waters 2695 system using a Daicel CHIRALPAK®
IE chiral column (Lot No. IE00CEꢀTG015; Part No. 85325; particle size: 5 ꢁm;
dimensions: 4.6 mm Φ × 150 mm). Optical rotations were measured on a Rudolph
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25
Autopol VI automatic polarimeter, with [α]D values reported in degrees and
concentration (c) reported in g/100 mL. 1H NMR and 13C NMR spectra were recorded
on a Bruker AVꢀ400 spectrometer at 400 and 100 MHz, respectively. Coupling
constants (J) are expressed in hertz (Hz). Spin multiplicities are described as s
(singlet), d (doublet), t (triplet), dd (doublet of doublet), dt (doublet of triplet), q
(quartet), quint (quintet), brs (broad singlet) and m (multiplet). Chemical shifts (δ) are
listed in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal
standard. For 19F NMR, chemical shifts are reported on a scale relative to CF3COOH
(δ ꢀ76.55 ppm in DMSO) as an external reference. Lowꢀresolution and highꢀresolution
ESIꢀMS readings were recorded on an Agilent 1200ꢀG6410A mass spectrometer.
4.1.1. 1-Methyl-8-(2-methylpyridin-4-yl)-1H-[1,2,3]triazolo[4,5-c]quinolone
(1). Compound 8a (55 mg, 209.05 µmol, 1 equiv), 29 (28.63 mg, 209.05 µmol, 1
equiv), Pd(dppf)Cl2 (15.3 mg, 20.9 µmol, 0.1 equiv) and K2CO3 (86.67 mg, 627.14
µmol, 3 equiv) were suspended in a mixed solution of 1,4ꢀdioxane and water (4:1).
Under a nitrogen atmosphere, the mixture was stirred at 100 °C for 24 h. The solvent
was evaporated to dryness under reduced pressure to obtain the crude product, which
was purified by flash column chromatography to yield the desired product as a white
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powder (37.4 mg, 43% yield). 1H NMR (400 MHz, DMSOꢀd6) δ 9.58 (s, 1H), 8.81 (d,
J = 1.8 Hz, 1H), 8.61 (d, J = 5.2 Hz, 1H), 8.37 (d, J = 8.7 Hz, 1H), 8.29 (dd, J = 8.7,
1.9 Hz, 1H), 7.86 (s, 1H), 7.79 (dd, J = 5.2, 1.5 Hz, 1H), 4.84 (s, 3H), 2.61 (s, 3H).
13C NMR (101 MHz, DMSOꢀd6) δ 159.28, 150.17, 146.79, 145.77, 145.36, 141.16,
136.93, 133.70, 131.21, 128.72, 121.61, 121.58, 119.59, 116.32, 38.10, 24.70.
ESIꢀMS m/z 276.2 [M + H]+.
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4.1.2. (E)-5-Bromo-2-((2-nitrovinyl)amino)benzoic acid (3). A suspension of
2ꢀaminoꢀ5ꢀbromoꢀbenzoic acid (25 g, 115.72 mmol, 1 equiv) in 10:1 H2O/HCl was
stirred for 8 h and then filtered (solution A). Nitromethane (8.17 g, 133.85 mmol, 1.15
equiv) was added over 10 min to an ice bathꢀcooled mixture of ice (35 g) and NaOH
(15.3 g, 382.53 mmol, 3.30 equiv). After stirring for 1 h at 0 °C and 1 h at rt, the
solution was added to an iceꢀbath cooled mixture of ice (28 g) and HCl (37%, 42 mL)
(solution B). Solutions A and B were combined, and the reaction mixture was stirred
overnight at rt. The yellow precipitate was filtered off, washed with H2O and dried in
vacuo to yield the crude product, which was used directly in the next step.
4.1.3. 6-Bromo-3-nitroquinolin-4-ol (4). Compound 3 (5 g, 17.42 mmol, 1
equiv) and potassium acetate (2.05 g, 20.90 mmol, 1.2 equiv) in acetic anhydride were
stirred for 2 h at 120 °C. The precipitate was filtered and washed with acetic acid until
the filtrate was colorless. The precipitate was further washed with water and then
dried to obtain the title compound (2.32 g, twoꢀstep yield: 14%). 1H NMR (400 MHz,
DMSOꢀd6) δ 13.21 (s, 1H), 9.24 (s, 1H), 8.32 (d, J = 2.3 Hz, 1H), 7.96 (dd, J = 8.8,
2.3 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H). ESIꢀMS m/z: 268.9 [M + H]+.
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4.1.4. 6-Bromo-4-chloro-3-nitroquinoline (5). Compound 4 (2 g, 7.43 mmol)
and POCl3 were stirred for 4 h at 100 °C. The mixture was cooled to rt and slowly
quenched with ice water. After neutralization with NaHCO3 (aq.), the aqueous phase
was extracted with ethyl acetate and dried over Na2SO4. The product was obtained by
evaporation of solution to dryness as a brown solid (2.1 g, 98% yield), which was
used directly in the next step.
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4.1.5. 6-Bromo-N-methyl-3-nitroquinolin-4-amine (6a). To a solution of 5 (1 g,
3.48 mmol, 1 equiv) and triethylamine (703.96 mg, 6.96 mmol, 2 equiv) in EtOH,
methanamine hydrochloride (234.84 mg, 3.48 mmol, 1 equiv) was added slowly at rt.
The resulting mixture was stirred at 60 °C for 4 h. The solution was concentrated in
vacuo and then poured into water. The precipitate was filtered off and dried to obtain
1
the desired product as a paleꢀyellow powder in 89% yield. H NMR (400 MHz,
DMSOꢀd6) δ 8.94 (s, 1H), 8.89 (s, 1H), 8.67 (d, J = 1.4 Hz, 1H), 7.91 (dd, J = 8.8, 1.7
Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 3.02 (d, J = 5.0 Hz, 3H). 13C NMR (101 MHz,
DMSOꢀd6) δ 147.89, 147.33, 147.29, 135.10, 131.84, 126.97, 126.88, 121.73, 119.33,
34.20. ESIꢀMS m/z 281.9 [M + H]+.
4.1.6. 6-Bromo-N-methyl-3-nitroquinolin-4-amine (6b). The title compound
was prepared from 5 (1 g, 3.48 mmol) and commercial available ethanamine
hydrochloride (284 mg, 3.48 mmol) using the procedure described for compound 6a
1
in 95% yield as an offꢀwhite powder. H NMR (400 MHz, DMSOꢀd6) δ 8.97 (d, J =
2.6 Hz, 1H), 8.74 (s, 1H), 8.61 (s, 1H), 7.97 – 7.89 (m, 1H), 7.84 – 7.76 (m, 1H), 3.44
– 3.35 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H). ESIꢀMS m/z 296.0 [M + H]+.
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4.1.7. 6-Bromo-
N
-isopropyl-3-nitroquinolin-4-amine (6c). The title compound
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was prepared from 5 (1 g, 3.48 mmol) and commercial available propanꢀ2ꢀamine (298
ꢁL, 3.48 mmol) using the procedure described for compound 6a in 93% yield as an
offꢀwhite powder. 1H NMR (400 MHz, DMSOꢀd6) δ 8.99 (s, 1H), 8.71 (s, 1H), 8.31 (d,
J = 7.5 Hz, 1H), 7.96 (d, J = 8.3 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 4.03 ꢀ 3.80 (m, 1H),
1.32 (d, J = 6.0 Hz, 6H). ESIꢀMS m/z 310.0 [M + H]+.
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4.1.8. 6-Bromo-N-(4-fluorobenzyl)-3-nitroquinolin-4-amine (6d). The title
compound was prepared from 5 (750 mg, 2.61 mmol) and commercial available
(4ꢀfluorophenyl)methanamine (327 mg, 2.61 mmol) using the procedure described for
compound 6a in 91% yield as an offꢀwhite powder. 1H NMR (400 MHz, DMSOꢀd6) δ
9.20 (t, J = 5.4 Hz, 1H), 8.94 (s, 1H), 8.75 (d, J = 1.9 Hz, 1H), 7.96 (dd, J = 8.9, 1.9
Hz, 1H), 7.83 (d, J = 8.9 Hz, 1H), 7.35 (dd, J = 8.7, 5.5 Hz, 2H), 7.23 – 7.10 (m, 2H),
4.70 (d, J = 5.7 Hz, 2H). ESIꢀMS m/z 376.0 [M + H]+.
4.1.9. (S)-6-Bromo-N-(1-(4-fluorophenyl)ethyl)-3-nitroquinolin-4-amine (6e).
The title compound was prepared from 5 (5 g, 17.39 mmol) and commercial available
(S)ꢀ1ꢀ(4ꢀfluorophenyl)ethanꢀ1ꢀamine (2.42 g, 17.39 mmol) using the procedure
described for compound 6a in 93% yield as an offꢀwhite powder. 1H NMR (400 MHz,
DMSOꢀd6) δ 8.96 (s, 1H), 8.86 (d, J = 6.5 Hz, 1H), 8.70 (d, J = 1.9 Hz, 1H), 7.94 (dd,
J = 8.8, 2.0 Hz, 1H), 7.81 (d, J = 8.8 Hz, 1H), 7.42 – 7.36 (m, 2H), 7.19 – 7.11 (m,
2H), 5.10 (quint, J = 6.6 Hz, 1H), 1.66 (d, J = 6.6 Hz, 3H). 13C NMR (101 MHz,
DMSOꢀd6) δ 163.02, 160.60, 148.01, 147.58, 146.42, 139.45, 139.42, 135.60, 131.93,
128.55, 128.46, 128.34, 128.13, 121.57, 119.58, 116.12, 115.91, 56.60, 25.85.
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