Discovery of 3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole derivatives as a novel selective inhibitor scaffold of JNK3

Article abstract of DOI:10.1080/14756366.2019.1705294

3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole derivatives were designed and synthesised as selective inhibitors of JNK3, a target for the treatment of neurodegenerative diseases. Following previous studies, we have designed JNK3 inhibitors to reduce the molecular weight and successfully identified a lead compound that exhibits equipotent activity towards JNK3. Kinase profiling results also showed high selectivity for JNK3 among 38 kinases. Among the derivatives, the IC₅₀ value of 8a, (R)-2-(1-(2-((1-(cyclopropanecarbonyl)pyrrolidin-3-yl)amino)pyrimidin-4-yl)-5-(3,4-dichlorophenyl)-1H-pyrazol-3-yl)acetonitrile exhibited 227 nM, showing the highest inhibitory activity against JNK3.

Full text of DOI:10.1080/14756366.2019.1705294

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Discovery of 3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole
derivatives as a novel selective inhibitor scaffold of
JNK3
Youri Oh, Miyoung Jang, Hyunwook Cho, Songyi Yang, Daseul Im, Hyungwoo
Moon & Jung-Mi Hah
To cite this article: Youri Oh, Miyoung Jang, Hyunwook Cho, Songyi Yang, Daseul Im, Hyungwoo
Moon & Jung-Mi Hah (2020) Discovery of 3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole derivatives as a
novel selective inhibitor scaffold of JNK3, Journal of Enzyme Inhibition and Medicinal Chemistry,
35:1, 372-376, DOI: 10.1080/14756366.2019.1705294
To link to this article: https://doi.org/10.1080/14756366.2019.1705294
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 19 Dec 2019.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 372–376
https://doi.org/10.1080/14756366.2019.1705294
SHORT COMMUNICATION
Discovery of 3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole derivatives as a novel selective
inhibitor scaffold of JNK3
Youri Oh, Miyoung Jang, Hyunwook Cho, Songyi Yang, Daseul Im, Hyungwoo Moon and Jung-Mi Hah
College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Korea
ABSTRACT
ARTICLE HISTORY
Received 11 November 2019
Revised 3 December 2019
Accepted 5 December 2019
3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole derivatives were designed and synthesised as selective inhibitors of
JNK3, a target for the treatment of neurodegenerative diseases. Following previous studies, we have
designed JNK3 inhibitors to reduce the molecular weight and successfully identified a lead compound
that exhibits equipotent activity towards JNK3. Kinase profiling results also showed high selectivity for
JNK3 among 38 kinases. Among the derivatives, the IC50 value of 8a, (R)-2-(1-(2-((1-(cyclopropanecarbo-
nyl)pyrrolidin-3-yl)amino)pyrimidin-4-yl)-5-(3,4-dichlorophenyl)-1H-pyrazol-3-yl)acetonitrile exhibited 227 nM,
showing the highest inhibitory activity against JNK3.
KEYWORDS
JNK; pyrazole;
neurodegenerative
diseases; SAR
Introduction
JNK isoforms have an ATP binding pocket with a highly conserved
sequence; thus far very few drugs that exhibit only high selectivity
for JNK3 have been discovered1. Due to the side effects that
appear in response to these selectivity issues, there is increasing
interest in research to find a JNK3-selective inhibitor.
c-Jun N-terminal Kinase (JNK) is a serine/threonine kinase that is
one of the members of the mitogen-activated protein kinase
(MAPK) family1. Activation occurs as a result of stimulation by fac-
tors such as oxidative stress, cytokines, and ultraviolet rays, thus
inducing the apoptosis pathway of cells2–6. These JNKs have three
isoforms. Among them, JNK1 and two are widely distributed in
cells and tissues, but JNK3 is known to be distributed specifically
in the brain7. These facts suggested that JNK3 may be a target for
therapeutic agents for neurodegenerative diseases such as
Alzheimer’s and Parkinson’s diseases8–10. Through many studies, it
has been shown that inhibiting JNK3 suppresses the formation of
We have found 1-heteroaryl-2-aryl-1H-benzimidazole derivatives
that have selectivity for JNK3 through optimisation of a hit com-
pound exhibiting JNK activity from our library in previous stud-
ies12. Based on the SAR results, we continued our efforts to
design a new chemical scaffold of JNK3 inhibitors with reduced
molecular weights for better Brain-Blood Barrier permeability13,14
.
During the development of new JNK3-selective inhibitors, we
beta amyloid, one of the causes of Alzheimer’s disease, and has sought to maintain three interactions of the previous scaffold;
proven its potential as a therapeutic target11. However, all three hydrogen bonds in the hinge region, hydrophobic interaction of the
Figure 1. Docking structures of the previous JNK3 inhibitor (PDB: 3OY1) and design of the present 1-pyrimidyl-3-alkyl-5-aryl-1H-pyrazole scaffold.
CONTACT Jung-Mi Hah
jhah@hanyang.ac.kr
College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan
04763, Korea
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
373
Scheme 1. Synthesis of 3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole derivatives.
aromatic ring, and the hydrogen bond of phenol in the benzimida- containing various aryl groups substituted (1) and formed enolate
with sodium methoxide to react with dimethyl oxalate to produce
beta ketone (2)15. The Knorr pyrazole synthesis was employed to
form the pyrazole cores (3) using hydrazinyl pyrimidine and beta
ketone16. After that, pyrazoyl ester was converted to alcohol (4)
using lithium aluminium hydride17. The nitrile was introduced by
the SN2 reaction with sodium cyanide following mesylation (5).
Through the oxidation with potassium peroxymonosulfate of
methyl sulphide to methylsulfone, a variety of amino groups were
introduced to the pyrimidyl moiety (SNAr)18,19; then the terminal
amino group was deprotected and acylated to give the final prod-
zole scaffold, thus attempting to reduce the molecular weight.
Therefore, we discarded benzene from the benzimidazole scaffold,
retaining the hydrogen bond-possible moiety on 5-membered
ring with final design and synthesis of the 3-alkyl-5-aryl-1-pyri-
midyl-1H-pyrazole derivatives (Figure 1).
Results and discussion
The synthetic process of 3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole
derivatives is shown in Scheme 1. We started with methyl ketones ucts (7a–d, 10a–f). For compounds 7e–f, 8a–f, and 9a–f,

374
Y. OH ET AL.
Scheme 2. Synthesis of compound 11a and 12a.
Table 1. Enzymatic activities of 1-heteroaryl-3-alkyl-5-aryl-1H-pyrazole derivatives.
Ã
Ã
(R/S)
No
Ar
n
(R/S)
R
JNK3 IC50 (lM)
No
Ar
n
R
JNK3 IC50 (lM)
7a
8a
9a
10a
11a
12a
7c
8c
9c
10c
7e
8e
9e
10e
3
2
2
1
3
3
3
2
2
1
3
2
2
1
S
R
S
-
S
S
S
R
S
-
CN
CN
CN
0.635
0.227
3.11
2.84
1.46
0.903
4.60
2.18
8.57
5.58
NA
7b
8b
9b
10b
3
2
2
1
S
R
S
-
CN
CN
CN
CN
0.824
0.361
2.90
CN
2.07
CONH2
CO2Me
CN
CN
CN
CN
CN
CN
CN
7d
8d
9d
10d
7f
8f
9f
3
2
2
1
3
2
2
1
S
R
S
-
S
R
S
-
CN
CN
CN
CN
CN
CN
CN
CN
7.90
4.42
3.25
NA
>10
7.89
NA
S
R
S
-
>10
NA
NA
CN
10f
NA
0.005
Control compound
JNKI VIII20,21
cyclopropylcarboxylated amine were directly incorporated. The nitrile was the best in terms of potency, but not a noticeable dif-
terminal nitrile group was changed to an ester (11a) and carboxa- ference. In an effort to reduce the molecular weight, the piperi-
mide (12a) through Scheme 2. They were synthesised through dine ring was diversified into pyrrolidine and azetidine with less
hydrolysis of the 10a performed at different conditions.
carbon atoms. Surprisingly, when (R)-aminopyrrolidine was
coupled to the pyrimidyl group instead of the (S)-aminopiperidine,
the activities were increased approximately two to three fold (7a
vs 8a, 7b vs 8b, 7c vs 8c, 7d vs 8d). This also suggested that the
configuration of the amino group in the ring should be consid-
ered important for binding, even in the solvent exposure part for
optimal extra-hydrogen bonding. The extra hydrogen bonding
seemed more plausible in (R)-pyrrolidine (8) than in both cases of
(S)-piperidine (7) and (S)-pyrrolidine (9) in the docking structures
(Figure 2).
All of the synthesised compounds, 7a–7f, 8a–8f, 9a–9f, and
10a–10f were evaluated for their inhibitory activity against JNK3
(Table 1). First, we investigated the effect of the aryl group on
their activity. The larger aryl groups such as the naphthyl and
dichlorophenyl bound at position 5, elicited more potent activity
towards JNK3 (a, b vs. e, f). This seems to be related to the elec-
tron density of the aromatic ring due to the sulfur-p interaction in
the active site of JNK3. Compared to the mono-substituted phenyl
groups, the relatively electron-rich dichlorophenyl and naphthyl
groups could have formed a stronger p–p interaction, which may
Next, we used kinase panel screening in duplicate for com-
affect the activity. Next, to investigate the effect of the substituent pound 7a over 38 different kinases at a single-dose concentration
at position 3, the compound 7a was hydrolysed to convert it to of 10 lM (Table 2). The compound was indeed a selective JNK3
an amide and a methyl ester (11a, 12a). As a result, the existing inhibitor with an excellent selectivity profile. This compound has

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
375
Figure 2. Comparison of docking structures of 7a and 8a at JNK3 (PDB: 3OY1).
Table 2. Percentages of enzymatic inhibition exerted by 7a (10 lM) on 38 selected protein kinases and enzymatic activities on selected protein kinases.
Ã
Compound IC50 (M):
IC50 (nM)
Control
Cmpd
Control
Cmpd ID
Kinase:
7a
GSK3b
JNK3
3.96
0.635
2.30
5.13
Staurosporine
JNKi VIII
We used Reaction Biology Corp. Kinase HotSpotSM service (www.reactionbiology.com) for screening of 7a.
Figure 3. Docking structures of 8a at JNK3 (PDB: 3OY1) and 2 D-interaction map.
an inhibitory activity of 90% on JNK3 at a concentration of 10 lM; the docking experiment of 8a with a known JNK3 structure
the inhibition activity was less than 20% for most other kinases (3OY1), it was shown that many of the interactions could contrib-
except GSK3b, but for which, was also six fold selective in terms ute to complex stabilisation. First, the amino pyrimidine used as
of IC50.
the hinge binder was found to form two hydrogen bonds with
A docking study was conducted to understand the binding Met149 of JNK3 and another hydrogen bond is plausible between
mode of the novel JNK3 inhibitors (Figure 3). When we performed the oxygen of the cyclopropyl carboxamide group in 8a and

376
Y. OH ET AL.
Gln155 in the extended hinge region. The third hydrogen bond
seems possible between the nitrile located in the three position
of pyrazole, which forms two hydrogen bonds between the back-
bone and the side chain of Asn152. Lastly, the aryl group at pos-
ition 1 of pyrazole also fits into the hydrophobic pocket formed
by residues such as Met148, Val79, Val145, Leu144, Ala91, Ile92,
Ile124 and Leu128, especially noting that the dichlorophenyl ring
could form a halogen bond with Lys93.
6. Weston CR, Davis RJ. The JNK signal transduction pathway.
Curr Opin Cell Biol 2007;19:142–9.
7. Gupta S, Barrett T, Whitmarsh AJ, et al. Selective interaction
of JNK protein kinase isoforms with transcription factors.
Embo J 1996;15:2760–70.
8. Antoniou X, Falconi M, Di Marino D, Borsello T. JNK3 as a
therapeutic target for neurodegenerative diseases.
J
Alzheimer Dis 2011;24:633–42.
9. Dou X, Huang H, Li Y, et al. Multistage screening reveals 3-
substituted indolin-2-one derivatives as novel and isoform-
selective c-Jun N-terminal Kinase 3 (JNK3) inhibitors: implica-
tions to drug discovery for potential treatment of neurode-
generative diseases. J Med Chem 2019;62:6645–64.
10. Koch P, Gehringer M, Laufer SA. Inhibitors of c-Jun N-ter-
minal kinases: an update. J Med Chem 2015;58:72–95.
11. Flemming A. JNK3 as new target in AD? Nat Rev Drug
Discov 2012;11:829.
12. Kim M, Lee J, Hah JM, et al. Syntheses and biological evalu-
ation of 1-heteroaryl-2-aryl-1 H -benzimidazole derivatives as
c-Jun N-terminal kinase inhibitors with neuroprotective
effects. Bioorg Med Chem 2013;21:2271–85.
13. Fong CW. Permeability of the blood–brain barrier: molecular
mechanism of transport of drugs and physiologically import-
ant compounds. J Membr Biol 2015;248:651–69.
14. Geldenhuys WJ, Mohammad AS, Adkins CE, Lockman PR.
Molecular determinants of blood–brain barrier permeation.
Ther Deliv 2015;6:961–71.
15. Tian W, Han G, Zhu J, et al. Synthesis and acrosin inhibitory
activities of 5-phenyl-1H-pyrazole-3-carboxylic acid amide
derivatives. Bioorg Med Chem Lett 2013;23:4177–84.
16. Gosselin F, O’Shea P, Webster R, et al. Highly regioselective
synthesis of 1-Aryl-3,4,5-substituted pyrazoles. Synlett 2006;
2006:3267–70.
17. Ren SZ, Wang ZC, Zhu XH, et al. Design and biological
evaluation of novel hybrids of 1, 5-diarylpyrazole and
Chrysin for selective COX-2 inhibition. Bioorg Med Chem
2018;26:4264–75.
Conclusions
We have successfully synthesised 3-alkyl-5-aryl-1-pyrimidyl-1H-pyr-
azole derivatives that were designed as novel JNK3 selective inhib-
itors in an effort to reduce the molecular weight from previous
lead. Twenty-six compounds were synthesised and measured for
their enzyme activity against JNK3. Particularly, compounds 7a,
7b, 8a, and 8b showed competitive activities against JNK3 with
IC50 values of 0.635 lM, 0.824 lM, 0.227 lM, and 0.361 lM, respect-
ively. Compound 7a was, indeed, a selective JNK3 inhibitor with
an excellent selectivity profile, especially compared to the activity
towards similar protein kinases such as p38a, GSKb, Erk, JNK1, and
JNK2. We believe that this novel scaffold, 3-alkyl-5-aryl-1-pyri-
midyl-1H-pyrazole will be highly useful in the development of
JNK3 selective inhibitors, as therapeutic agents for neurodegenera-
tive diseases.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by a National Research
Foundation of Korea grant [NRF-2017R1A2B4006447 and NRF-
2019M3A9A8066500; J.-M. Hah].
18. Odell LR, Abdel-Hamid MK, Hill TA, et al. Pyrimidine-based
inhibitors of dynamin I GTPase activity: competitive inhib-
ition at the Pleckstrin Homology domain. J Med Chem 2017;
60:349–61.
References
1. Davis RJ. Signal transduction by the JNK Group of MAP kin-
19. Beaulieu PL, Bousquet Y, Gauthier J, et al. Non-nucleoside
benzimidazole-based allosteric inhibitors of the hepatitis C
virus NS5B polymerase: inhibition of subgenomic hepatitis C
virus RNA replicons in Huh-7 Cells. J Med Chem 2004;47:
6884–92.
20. Vivanco I, Palaskas N, Tran C, et al. Identification of the JNK
signaling pathway as a functional target of the tumor sup-
pressor PTEN. Cancer Cell 2007;11:555–69.
21. Szczepankiewicz BG, Kosogof C, Nelson LTJ, et al.
Aminopyridine-based c-Jun N-terminal kinase inhibitors with
cellular activity and minimal cross-kinase activity. J Med
Chem 2006;49:3563–80.
ases. Cell 2000;103:239–52.
€
2. Mishra P, Gunther S. New insights into the structural dynam-
ics of the kinase JNK3. Sci Rep 2018;8:9435.
3. Kyriakis JM, Banerjee P, Nikolakaki E, et al. The stress-acti-
vated protein kinase subfamily of c-Jun kinases. Nature
1994;369:156–60.
4. Bogoyevitch MA, Ngoei KR, Zhao TT, et al. c-Jun N-terminal
kinase (JNK) signaling: recent advances and challenges.
Biochim Biophys Acta 2010;1804:463–75.
5. Haeusgen W, Boehm R, Zhao Y, et al. Specific activities of
individual c-Jun N-terminal kinases in the brain.
Neuroscience 2009;161:951–9.