Design, Synthesis, and Biological Evaluation of 1-Benzyl-1H-pyrazole Derivatives as Receptor Interacting Protein 1 Kinase Inhibitors

Article abstract of DOI:10.1111/cbdd.12689

Receptor interacting protein 1 (RIP1) kinase plays an important role in necroptosis, and inhibitors of the RIP1 kinase are thought to have a potential therapeutic value in the treatment of diseases related to necrosis. Herein, we report the structural optimization of a RIP1 kinase inhibitor, 1-(2,4-dichlorobenzyl)-3-nitro-1H-pyrazole (1a). A number of 1-benzyl-1H-pyrazole derivatives were synthesized and structure-activity relationship (SAR) analysis led to the discovery of a potent compound, 4b, which showed a K_d value of 0.078 μm against the RIP1 kinase and an EC₅₀ value of 0.160 μm in a cell necroptosis inhibitory assay. Compound 4b also displayed considerable ability to protect the pancreas in an l-arginine-induced pancreatitis mouse model.

Full text of DOI:10.1111/cbdd.12689

Chem Biol Drug Des 2016; 87: 569–574
Research Article
Design, Synthesis, and Biological Evaluation of
1-Benzyl-1H-pyrazole Derivatives as Receptor
Interacting Protein 1 Kinase Inhibitors
1
,2,†
1,2,†
1,2,†
Chan Zou
, Yu Xiong
, Lu-Yi Huang
,
understood (7,8), and receptor interacting protein 1 (RIP1)
kinase is thought to be one of the key regulators of
necroptosis (9,10). RIP1 is a serine–threonine protein
kinase and has been demonstrated to directly regulate
RIP3-MLKL-dependent necroptosis (11); the RIP3-MLKL
cascade is a driving signaling pathway leading to necrop-
tosis recently discovered, which is triggered by death
receptor such as tumor necrosis factor receptor (TNFR).
Discovery of RIP1 kinase inhibitors could help in elucidat-
ing the regulation mechanism of necroptosis and might
provide potential therapeutic agents for diseases related to
necrosis.
3
1,2
3,
Chun-Li Song , Xiao-Ai Wu , Lin-Li Li * and
Sheng-Yong Yang
1
,2,
*
1
State Key Laboratory of Biotherapy and Cancer Center,
West China Hospital, Sichuan University, Chengdu,
Sichuan 610041, China
Collaborative Innovation Center of Biotherapy, Chengdu,
Sichuan 610041, China
West China School of Pharmacy, Sichuan University,
Chengdu, Sichuan 610041, China
Corresponding authors: Lin-Li Li, ysylilinli@sina.com;
2
3
*
Sheng-Yong Yang, yangsy@scu.edu.cn
These authors contributed equally.
†
Up to now, a number of RIP1 kinase inhibitors have been
reported. Representative ones including Nec1 (12), Nec-3a
(13), Nec-4 (14), Nec-5 (15), Nec-21 (16), and Cpd27 (17)
are shown in Figure 1, and Nec1 is often used as a
molecular tool in necroptosis-related studies (10). Even so,
most of these compounds just showed moderate activity
against the RIP1 kinase. Therefore, discovering more
potent RIP1 kinase inhibitors, particularly those containing
new chemical scaffolds, is still necessary at present.
Receptor interacting protein 1 (RIP1) kinase plays an
important role in necroptosis, and inhibitors of the
RIP1 kinase are thought to have a potential therapeutic
value in the treatment of diseases related to necrosis.
Herein, we report the structural optimization of a RIP1
kinase inhibitor, 1-(2,4-dichlorobenzyl)-3-nitro-1H-pyra-
zole (1a). A number of 1-benzyl-1H-pyrazole derivatives
were synthesized and structure-activity relationship
(SAR) analysis led to the discovery of a potent com-
pound, 4b, which showed a K value of 0.078 lM
d
We recently identified a new necroptosis inhibitor, namely
against the RIP1 kinase and an EC50 value of 0.160 lM
in a cell necroptosis inhibitory assay. Compound 4b
also displayed considerable ability to protect the pan-
creas in an L-arginine-induced pancreatitis mouse
model.
1
-(2,4-dichlorobenzyl)- 3-nitro-1H-pyrazole (1a, Fig-
ure 2); the chemical scaffold contained in this compound,
1
1
-Benzyl-1H-pyrazole, is a novel one, which means that
-Benzyl-1H-pyrazole derivatives have never been
reported as RIP1 kinase inhibitors. Compound 1a was
obtained through screening our in-house database by a
cell necroptosis inhibitory assay, in which human colon cell
line HT29 was treated with TNF-a, Smac mimetic, and Z-
VAD-FMK (TSZ). Here, TNF-a and Smac mimetic can
cause cells to undergo apoptosis by triggering the forma-
tion of a caspase-8 activating complex containing RIP1,
and Z-VAD-FMK is a pan-caspase inhibitor and can switch
apoptosis to necroptosis in RIP3-expressing cells upon
caspase-8 inhibition (18). In this assay, Nec1 was used as
a positive control, which displayed an EC50 (concentration
for 50% of maximal effect) value of 0.860 lM. Compound
1a exhibited an EC50 value of 1.048 lM. A further kinase
inhibition assay showed that compound 1a was a RIP1
Key words: 1-benzyl-1H-pyrazoles, kinase, necroptosis,
pancreatitis, receptor interacting protein 1
Received 14 June 2015, revised 4 November 2015 and
accepted for publication 5 November 2015
Necrosis, an alternative form of programmed cell death,
plays a prominent role in many pathological conditions,
including ischemic brain injury (1), neurodegenerative dis-
eases (2), heart ischemia/reperfusion injury (3), and viral
infection (4,5). It has conventionally been viewed as an
accidental and unwanted cell death, carried out in a non-
regulated manner. However, recent studies have shown
that necrosis can also be highly regulated in certain condi-
tion, hence termed necroptosis (6). The signaling pathway
responsible for carrying out necroptosis has just been
d
kinase inhibitor with a K value of 0.130 lM and exhibited
no inhibitory activity against RIP3. The purpose here is to
further optimize its potency and carry out a structure-activ-
ity relationship (SAR) analysis.
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12689
569

Zou et al.
Figure 1: Structures of representative RIP1
kinase inhibitors together with their
bioactivities (*EC50 obtained in this study).
Biological experiments
Cell culture and necroptosis induction
HT29 was cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal calf serum (FCS), L-glutamine
(200 mM), and sodium pyruvate (400 mM). Necroptosis was
induced by 20 ng/mL TNF-a (PEPROTECH, Rocky Hill, NJ,
USA), 100 nM Smac mimetic (SELLECK), and 20 lM Z-
VAD-FMK (APExBIO, Houston, TX, USA).
Figure 2: The structure of hit compound 1a.
Methods and Materials
Kinase binding assay
General procedure for the synthesis of compounds
The kinase binding affinity of compounds was measured
TM
3
a–3c (exemplified by 3a)
through the KINOMEscan kinase binding assay (Dis-
A solution of 3-nitro-1H-pyrazole (2.0 g, 17.7 mmol) and
N-chloro-succinimide (3.54 g, 26.6 mmol) in DMF (25 mL)
was stirred at 55 °C for 12 h under an N2 atmosphere.
After completion of the reaction as monitored by TLC,
coveRX). In this assay, the binding affinity of a compound
to a kinase is calculated by quantitatively measuring the
competition binding ability of this compound and an
immobilized, active-site-directed ligand with the kinase.
2
0 mL of water and 10 mL of saturated sodium thiosulfate
solution were added to the reaction mixture. After stirring
at room temperature for 10 min, the precipitate formed
was collected by filtration, washed with water, and dried
Immunoprecipitation and Western Blot
HT29 cells at 90% confluence grown were washed once with
PBS and lysed for 20 min on ice in the RIPA lysis buffer (Bey-
otime, Jiangsu, China). Cell lysates were centrifuged at
160609 g. The solution fraction was collected, and protein
concentration was determined by BCA Protein Assay kit
under vacuum to afford a yellow powder. (1.1 g, 42.0%
1
yield). H-NMR (400 MHz, DMSO-d ) d: 14.32 (s, 1H),
8
6
+
.37 (s, 1H); m/z: 148.1 [M + H] .
(Beyotime). Then, 1 mg of the extracted protein was immuno-
General procedure for the synthesis of compounds
a–u, 1w–z, 4a–c (exemplified by 1a)
precipitated overnight with antiphosphoserine (Invitrogen,
Camarillo, CA, USA) at 4 °C and then captured by protein A
agarose (Roche, Mannheim, Germany) for another 6 h. Next,
theagarosewaswashedthreetimeswith PBSandthenboiled
in1%SDSloadingbuffer, andthesupernatantwasconducted
Western blot with RIP1 (BD) and RIP3 (ABcam, Cambridge,
UK). The other antibodies, p-MLKL and MLKL, were
purchasedfromABcamandb-actinwasfromZSGB-BIO.
1
A mixture of 3-Nitro-1H-pyrazole (1.0 g, 8.9 mmol), 2, 4-
dichlorobenzyl chloride (1.9 g, 9.7 mmol), potassium car-
bonate (3.7 g, 26.7 mmol), and tetrabutylammonium bro-
mide (145 mg, 0.89 mmol) in N, N-dimethylformamide was
stirred at room temperature for 1 h. After completion of the
reaction as monitored by TLC, 10 mL of water was added to
the reaction mixture. The resulting precipitate was collected
by filtration, washed successfully with water, saturated brine,
and then dried under vacuum to afford a white solid. Yield:
Animal experiments
1
9
6
4%, white solid; H-NMR (400 MHz, DMSO-d ) d: 8.16 (d,
Specific pathogen-free male C57BL/6 mice (weight 20–
24 g) were supplied from experimental animal center of
Sichuan University. Mice after overnight fasting were
injected with 8% L-arginine (dissolved in saline, PH = 7.3,
J = 2.4 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 7.49 (dd, J = 8.4,
2
1
.0 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.11 (d, J = 2.4 Hz,
H), 5.59 (s, 2H); MS (ESI), m/z: 272.1 [M + H] .
+
5
0 lL/g) by i.p. twice in an hour. For drug administration,
Additional data for characterization of all target com-
compound 4b was dissolved in drinking water with 12.5%
pounds 1b–z, 4a–c can be found in Appendix S1.
alcohol and 12.5% castor oil. The mice were killed after
5
70
Chem Biol Drug Des 2016; 87: 569–574

Pyrazole Derivatives as RIP1 Kinase Inhibitors
5
days drug administration. To evaluate the changes in
which is due to the following reasons. A SAR analysis
based on cellular assays may permit the evaluation of both
the intrinsic activity of compounds against the protein tar-
get and the ability of compounds to permeate the cell wall.
In addition, relative to the enzymatic assay, the cell-based
assay is much cheaper. For the most potent compound in
the cell-based assay, its enzymatic activity will be mea-
sured.
pancreas histology, sections of paraformaldehyde-fixed
pancreas tissues were stained with hematoxylin and eosin
(HE) and photographed by an optical microscope.
Results and Discussion
Chemistry
A total of 28 1-benzyl-1H-pyrazole derivatives were syn-
thesized and synthetic routes of these compounds are
outlined in Schemes 1 and 2. As shown in Scheme 1,
compounds 1a–u and 1w–z were obtained through reac-
tions of commercial available reagents containing chloro-
methyl or bromomethyl with pyrazoles in the presence of
alkali in excellent yields. 1s was stirred with N-methylpiper-
azine in an alkaline condition to give compound 1v.
Scheme 2 depicts the synthetic routes for compounds
Substitution effect of benzyl
Table 1 shows the chemical structures of compounds 1a–v
and their bioactivities. Replacement of the benzene ring
with benzoheterocycle or biphenyl abolished the bioactivity
(EC50 > 10 lM, 1b–d). A pure benzyl (without any sub-
stituent) resulted in a diminished activity (1e). Compound
1f–h bearing a monosubstituted ortho-, meta- or para-
chloride displayed one to three times lower activity than 1a.
When the para-chloride atom was replaced by more polar
methoxyl, amino, or trifluoromethyl group, the bioactivity
was further decreased (1i, 1j, and 1k). Compounds 1l and
1m, which contain a monosubstituted para-bromide and
ortho-methyl, respectively, also showed decreased bioac-
tivity. However, compound 1n, containing a bromide sub-
stituted at ortho position, exhibited about twofold higher
potency than compound 1a. Among compounds 1o–v,
which contain disubstituted benzyl, three compounds (1p,
1q and 1s) showed an increased bioactivity compared with
4a–c. 3-nitropyrazole underwent halogenation reactions
with halogenated reagents to produce intermediates 3a–c,
which then reacted with 2, 5-difluorobenzyl bromide in the
presence of alkali to afford final products 4a–c.
Necroptosis inhibitory activities and structure-
activity relationships
The SAR analysis below will be based on the cell necrop-
tosis inhibitory assay rather than a direct enzymatic assay,
1
a. It is obvious that different substitution number and sites
of halide (F, Cl, Br) on the benzene ring have some influ-
ence on the bioactivity of compounds. Compound 1q,
which bears two fluoride substituents at C-2 and C-5 posi-
tions of the benzene ring, corresponds to the most active
compound (EC50: 0.290 lM).
Influence of substituents of pyrazole
In this section, we shall discuss the influence of sub-
stituents of pyrazole with the benzyl moiety fixed as its
optimal substituents, namely 2,5-difluorobenzyl. For this
purpose, a total of seven compounds were synthesized
(see Table 2). Compound 1w with no any substituent on
the pyrazole ring showed a significantly decreased bioac-
tivity compared with compound 1q. Replacement of nitro
by carboxylate or amino also led to reduced bioactivity
(1x, 1y). Translocation of nitro from the C-3 position to the
C-4 position (1z) resulted in a significantly decreased
bioactivity. Introduction of a chlorine or bromine atom at
the C-4 position of pyrazole ring slightly increased the
bioactivity (4a and 4b), and introduction of an iodine atom
led to a weakly decreased bioactivity (4c).
Scheme 1: Reagents and conditions: (a) reagents containing
+
À
chloromethyl or bromomethyl, K CO , Bu N Br , DMF, rt, 2 h; (b)
2 3 4
N-methylpiperazine, ethyldiisopropylamine, rt, overnight.
The above SAR studies led to the discovery of compound
4
b, which displayed the most potent activity in the cell
necroptosis inhibitory assay. Then, the enzymatic activity
TM
of 4b was measured by the KINOMEscan kinase binding
Scheme 2: Reagents and conditions: (a) NCS (or NBS/NIS), ethyl-
d
assay. In this assay, compound 4b showed a K value of
+
À
diisopropylamine, rt, overnight; (b) K
2
CO
3
, Bu
4
N Br , DMF, rt, 1 h.
0.078 lM. For comparison, the enzymatic activity of Nec1
Chem Biol Drug Des 2016; 87: 569–574
571

Zou et al.
was also measured at the same experimental condition,
Protein-ligand docking studies
which gave a K
pound 4b is a little more potent than Nec1.
d
value of 0.130 lM, indicating that com-
To understand the interaction mode of 4b with RIP1,
molecular docking was performed based on the crystal
structure of RIP1 (PDB code: 4ITJ). Here, GOLD v5.0 was
used for the docking studies (19,20). The predicted bind-
ing mode is shown in Figure 3. 4b suitably resides in the
allosteric pocket of RIP1 kinase. Two hydrogen bonds are
formed between the 4-bromo-3-nitro-1H-pyrazole moiety
of 4b and residue Asp156 of the RIP1 kinase. The 2,5-
difluorobenzyl moiety of 4b locates in a hydrophobic
pocket formed by a number of hydrophobic residues
including Leu157, Ala155, Val76, Ile154, and Met92.
Table 1: Structures of compounds 1a–v and their bioactivities
R1
R2
R3
N
N
NO2
R4
a
Compound R1
R2 R3
R4 EC50 (lM)
1
a
Cl
H
Cl
H
H
H
1.048 Æ 0.117
Inhibitory effect on necroptotic signaling
1
b
-CH=CH-
CH=CH-
-O-CH2-
CH2-O-
H
H
Cl
H
H
H
H
H
H
CH3
Br
H
F
F
H
>10
Western blot assays were performed to examine the sig-
naling inhibition ability of 4b in intact cells. In these
assays, HT29 cells were treated with TSZ by 8 h. The
results showed that the phosphorylation of MLKL (p-
MLKL) was inhibited by 4b in a concentration-dependent
manner (Figure 4A). Because there is no commercially
available phosphorylated RIP1 (p-RIP1) and RIP3 (p-RIP3)
antibodies, immunoprecipitation was performed to detect
the levels of phosphorylated RIP1 and RIP3, in which
antiphosphoserine antibody and protein A agarose were
used to pull down p-RIP1 and p-RIP3(21). The results
1
c
H
H
>10
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
d
e
f
H
H
H
Cl
H
H
H
H
H
H
H
F
H
H
F
H
H
H
H
4-cyanophenyl
H
H
H
Cl
OCH3
NH2
CF3
Br
H
H
F
F
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
F
>10
2.463 Æ 0.072
2.543 Æ 0.026
3.240 Æ 0.277
1.449 Æ 0.129
5.940 Æ 0.181
5.603 Æ 0.432
7.371 Æ 0.522
1.955 Æ 0.092
1.158 Æ 0.052
0.550 Æ 0.073
3.012 Æ 0.288
0.881 Æ 0.007
0.290 Æ 0.003
1.872 Æ 0.134
0.827 Æ 0.014
5.880 Æ 0.354
3.671 Æ 0.218
>10
g
h
i
j
k
l
(see Figure 4B) indicated that 4b inhibited the phosphory-
m
n
o
p
q
r
lation of RIP1 and RIP3 in a concentration-dependent
manner. Overall, our results demonstrated that 4b could
dose dependently inhibit the activation of RIP1/RIP3/
MLKL signaling in intact cells.
F
s
t
Cl
Cl
F
H
Cl
Cl
H
H
H
The protective effect on pancreas in an L-arginine-
induced pancreatitis mouse model
u
v
Cl
Methylpiperazine
Finally, an L-arginine-induced pancreatitis mouse model
was used to examine the in vivo effect of 4b. Excessive
doses of L-arginine could reduce polyamine biosynthesis
a
All assays were conducted in duplicate.
Table 2: Structures of compounds 1q, 1w–z, 4a–c and their
bioactivities
F
N
N
R1
R2
F
a
Compound
R1
R2
EC50 (lM)
1
1
1
1
1
4
4
4
q
w
x
y
z
NO2
H
COOH
NH2
H
NO2
NO2
NO2
H
H
H
H
NO2
Cl
0.290 Æ 0.003
>10
0.421 Æ 0.019
>10
>10
a
b
c
0.179 Æ 0.006
0.160 Æ 0.002
0.413 Æ 0.015
0.860
Br
I
Nec1
Figure 3: The predicted binding mode of 4b with the RIP1
kinase.
a
All assays were conducted in duplicate.
72
5
Chem Biol Drug Des 2016; 87: 569–574

Pyrazole Derivatives as RIP1 Kinase Inhibitors
Figure 4: (A) Western Blot conducted in HT29 after TSZ inducing to measure RIP1, RIP3, MLKL, and p-MLKL with 4b or Nec1 treated.
(B) Immunoprecipitation performed with antiphosphoserine and protein A agarose to capture p-RIP1 and p-RIP3, blotted with RIP1 and
RIP3 antibody in HT29 cells. (C) HE staining of paraffin section of pancreas with or without L-arginine injection and 4b or Nec1
administration.
and thus inhibit the synthesis of protein. The pancreatic
acinar cells have the most active protein synthesis and
would be injured by administration of large doses of L-argi-
nine. In this model, C57BL/6 mice after overnight fasting
were injected with 8% L-arginine (dissolved in saline and
the dosage is 50 lL/g) two times interval of an hour. Then
1-Benzyl-1H-pyrazole. This compound showed an EC50
value of 0.160 lM in the cell necroptosis inhibitory assay
and a K value of 0.078 lM against the RIP1 kinase. It also
d
exhibited considerable ability to inhibit the RIP1/RIP3/
MLKL signaling in intact cells and showed a good protec-
tive effect on pancreas in the L-arginine-induced pancreati-
tis mouse model. Overall, compound 4b could be taken
as a good lead compound for further studies.
4b or Nec1 was administrated by i.p. 10 mg/kg/day for
5
days. Mice were killed on the sixth day, and pancreas
damage was evaluated by HE staining. As shown in Fig-
ure 4C, 4b displayed a considerable pancreas-protecting
effect in the L-arginine-induced pancreatitis mouse model
Acknowledgments
(22).
This work was supported by the 973 Program
(2013CB967204), and the National Natural Science Foun-
Conclusion
dation of China (81172987, 81325021, 81473140), and
partly by the National Science and Technology Major Pro-
ject (2012ZX09501001-003, 2012ZX09102-101-002), and
PCSIRT (IRT13031).
In summary, SAR analysis led to the discovery of a potent
RIP1 kinase inhibitor, 4b, which contains a new scaffold of
Chem Biol Drug Des 2016; 87: 569–574
573

Zou et al.
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Supporting Information
1
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Additional Supporting Information may be found in the
online version of this article:
1
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Denu R., Yuan J.Y., Cuny G.D. (2005) Structure-activ-
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Appendix S1. Chemical characterizations of compounds
1
b-1z, 3b-3c, 4a-4c.
5
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