Identification of 5-(2,3-Dihydro-1 H-indol-5-yl)-7 H-pyrrolo[2,3- d]pyrimidin-4-amine Derivatives as a New Class of Receptor-Interacting Protein Kinase 1 (RIPK1) Inhibitors, Which Showed Potent Activity in a Tumor Metastasis Model

Article abstract of DOI:10.1021/acs.jmedchem.8b01652

We herein report the structural optimization and structure-activity relationship studies of 5-(2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine derivatives as a new class of receptor-interacting protein kinase 1 (RIPK1) inhibitors. Among all obtained RIPK1 inhibitors, 1-(5-{4-amino-7-ethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl}-2,3-dihydro-1H-indol-1-yl)-2-[3-(trifluoromethoxy)phenyl]ethan-1-one (22b) is the most active one. This compound potently inhibited RIPK1 with a binding affinity (K_D) of 0.004 μM and an enzymatic IC₅₀ value of 0.011 μM and also showed good kinase selectivity. It could efficiently protect cells from necroptosis and attenuate the necrotic cell death of vascular endothelial cells induced by tumor cells both in vitro and in vivo. Importantly, compound 22b exhibited excellent antimetastasis activity in the experimental B16 melanoma lung metastasis model. It also displayed favorable pharmacokinetic properties. Collectively, 22b could be a promising agent for preventing tumor metastasis.

Full text of DOI:10.1021/acs.jmedchem.8b01652

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Article
Identification of 5-(2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-
amine Derivatives as a New Class of Receptor-Interacting Protein Kinase 1
RIPK1) Inhibitors, Which Showed Potent Activity in a Tumor Metastasis Model
(
Yueshan Li, Yu Xiong, Guo Zhang, Liting Zhang, Wei Yang, Jiao Yang, Lu-Yi Huang, Zeen Qiao,
Zhuang Miao, Guifeng Lin, Qiu Sun, Ting Niu, LiJuan Chen, Dawen Niu, Linli Li, and Shengyong Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01652 • Publication Date (Web): 27 Nov 2018
Downloaded from http://pubs.acs.org on November 28, 2018
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Li, Linli; Key Laboratory of Drug Targeting and Drug Delivery System of
Ministry of Education, West China School of Pharmacy, Sichuan
University
Yang, Shengyong; Sichuan University, State Key Laboratory of
Biotherapy, West China Hospital
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Identification of 5-(2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
Derivatives as a New Class of Receptor-Interacting Protein Kinase 1 (RIPK1)
Inhibitors, Which Showed Potent Activity in a Tumor Metastasis Model
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†
,#
†,#
‡
†
‡
†
Yueshan Li, Yu Xiong, Guo Zhang, Liting Zhang, Wei Yang, Jiao Yang, Luyi
†
†
†
†
†
§
†
Huang, Zeen Qiao, Zhuang Miao, Guifeng Lin, Qiu Sun, Ting Niu, Lijuan Chen,
†
‡,
†,
Dawen Niu, Linli Li, * Shengyong Yang *
†State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan
University / Collaborative Innovation Center of Biotherapy, Chengdu, Sichuan 610041,
China.
‡Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education,
West China School of Pharmacy, Sichuan University, Sichuan 610041, China.
§Department of Hematology and Research laboratory of Hematology, West China
Hospital, Sichuan University, Sichuan 610041, China.
#These authors contributed equally to this work.
*
To whom correspondence should be addressed. Shengyong Yang, State Key Laboratory
of Biotherapy and Cancer Center, West China Hospital, Sichuan University / Collaborative
Innovation Center of Biotherapy, Chengdu, Sichuan 610041, China. E-mail:
yangsy@scu.edu.cn. Linli Li, Key Laboratory of Drug Targeting and Drug Delivery
System of Ministry of Education, West China School of Pharmacy, Sichuan University,
Sichuan 610041, China. E-mail: ysylilinli@sina.com.cn.
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Abstract
We herein report the structural optimization and structure-activity relationship (SAR)
studies of 5-(2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
derivatives as a new class of receptor-interacting protein kinase 1 (RIPK1) inhibitors.
Among all the obtained RIPK1 inhibitors,
-(5-{4-amino-7-ethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl}-2,3-dihydro-1H-indol-1-yl)-2-[
-(trifluoromethoxy)phenyl]ethan-1-one (22b) is the most active one. This compound
) of 0.004 μM and an enzymatic IC50
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potently inhibited RIPK1 with a binding affinity (K
D
value of 0.011 μM, and also showed good kinase selectivity. It could efficiently protect
cells from necroptosis and attenuate the necrotic cell death of vascular endothelial cells
induced by tumor cells both in vitro and in vivo. Importantly, Compound 22b exhibited
excellent anti-metastasis activity in the experimental B16 melanoma lung metastasis
model. It also displayed favorable pharmacokinetic properties. Collectively, 22b could be
a promising agent for preventing tumor metastasis.
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1
. INTRODUCTION
Tumor metastasis is one of the principal events leading to death in individuals with
1
,2
cancer. Medical treatments that can prevent the tumor metastasis are expected to be able
to help reduce mortality.3-5 Nevertheless, the progress in this field is unsatisfactory,4
possibly due to the fact that the tumor metastasis is an extremely complex process and its
molecular mechanism is poorly understood.6-10 Recently Strilic et al revealed for the first
time that tumor cells could induce programmed necrosis (necroptosis) of vascular
endothelial cells, which results in tumor cell extravasation, and hence promoting tumor
metastasis. The receptor-interacting protein kinase 1 (RIPK1) is a key regulator of
necroptosis,12-15 and RIPK1 inhibitors have been shown to be able to efficiently inhibit cell
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necroptosis. Therefore, RIPK1 inhibitors could be potential agents for preventing tumor
metastasis17,18
.
Up to now, a few RIPK1 inhibitors have been publicly reported. Figure 1 shows
1
9
20
21
chemical structures of typical RIPK1 inhibitors, including Nec-1, Nec-1s, PN10,
2
2
23
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25
26
RIPA-56, Cpd 8, GSK’963, GSK’2982772, and Cpd 22 . Despite a number of
RIPK1 inhibitors reported, there is only one (GSK’2982772) in clinical trials, which is for
treating psoriasis, rheumatoid arthritis, and ulcerative colitis.25,27 Many of the known
RIPK1 inhibitors are not suitable for clinical studies due to low potency or poor
pharmacokinetic properties.27,28 Therefore, discovery of more new RIPK1 inhibitors,
particularly with novel scaffolds and drug-likeness, is still necessary at present.
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Figure 1. Chemical structures of previously reported RIPK1 inhibitors.
In an effort to discover new RIPK1 inhibitors, we adopted the classical
TNFα/Smac-mimetic/z-VAD-FMK (TSZ)-induced HT29 cell necroptosis model22 to
screen an in-house small molecular library (see Supplementary data), which contains more
than 20,000 agents. The mechanism of the TSZ-induced necroptosis model and the
screening details are given in the experimental section 5.4. From this screening, 6
compounds were found to be able to protect HT29 cells from TSZ-induced necroptosis (see
Supplementary
data,
Table
S1).
Among
these
compounds,
N-(4-{4-amino-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl}phenyl)-2-[3-(trifluoromethy
l)phenyl]acetamide (1, Figure 2) is the most active one, which showed an EC50 (half
maximal effective concentration) value of 1.45 µM. Importantly, compound 1 contains a
new scaffold, 3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (no RIPK1 inhibitors
publicly reported possess this kind of scaffold). The enzymatic activity of compound 1
against RIPK1 was then measured, which gave an IC50 (half maximal inhibitory
concentration) value of 0.077 µM. Because RIPK3 is also involved in the regulation of
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necroptosis, we next tested the activity of compound 1 against RIPK3. In this assay,
compound 1 did not show activity against RIPK3 (IC50 > 10 µM ). Obviously, we
obtained a new RIPK1 inhibitor, but the potency of this compound is poor. The purpose
of this investigation was to further improve its potency and explore its effect on
preventing tumor metastasis.
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Figure 2. The chemical structure of hit compound 1, together with regions that are the
focus of structural modifications.
2
. CHEMISTRY
To optimize the potency of compound 1 and explore its structure-activity relationship
(SAR), a series of derivatives of compound 1 were synthesized according to the synthetic
routes outlined in Schemes 1−4. Synthetic routes of 3-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4-amine derivatives 8a-g are outlined in Scheme 1. Direct iodination was
carried out on commercially available 1H-pyrazolo[3,4-d]pyrimidin-4-amine (2) under N
2
atmosphere to give intermediate 3, which was subsequently coupled with iodomethane to
afford intermediate 4. Suzuki-Miyaura coupling of 4 with various substituted
phenylamines produced intermediates 5a-g. These intermediates then reacted with
2-(3-(trifluoromethyl)phenyl)acetyl chloride (7), which was prepared by refluxing
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reaction of 2-(3-trifluoromethylphenyl)acetic acid (6) in SOCl , to generate final products
2
1
or 8a-g with 53-72% yields.
Scheme 1. Synthetic Routes of Compounds 1, 8a-g a.
1
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a
Reagents and conditions: (i) DMF, NIS, 85 °C, N
2
, 18 h, 90%; (ii) DMF, K
2
CO ,
3
Iodomethane, rt, 1 h, 80 °C, 12 h, 55%; (iii) substituted 4-aminobenzoic boricacid ester,
,4-dioxane/H O (5/1), PdCl (dppf). CH Cl adduct, tricyclohexylphospine, Cs CO , N
95 °C, 18 h, 50-65%; (iv) SOCl , 85 °C, 10 h; (v) Pyridine, rt, 12 h, 53-72%.
1
2
2
2
2
2
3
2
,
2
Scheme 2 depicts the synthetic routes of compound 13a-r. Firstly, indoline boronate
derivative 11 was obtained by two steps from commercially available 5-bromoindoline 9.
Then Suzuki-Miyaura coupling of 11 with intermediate 4, which was catalyzed by
+
-, gave intermediate 12 in a 60% yield. Finally
Pd
2
(dba)
3
cooperated with (t-Bu)
3
PH BF
4
the deprotection product of intermediate 12 reacted with substituted phenyl acetic acids to
offer compounds 13a-r with 50-62% yields.
Scheme 2. Synthetic Routes of Compounds 13a-r a.
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a
Reagents and conditions: (i) CH
,4-dioxane, bis(pinacolato)diboron, PdCl
O (5/1), Pd (dba) , (t-Bu) PH BF
3
CN, DIEA, DMAP, Boc
2
O, 80 °C, 3 h, 90%; (ii) dry
1
2
(dppf), KOAc, N
2
, 95 °C, 24 h, 60%; (iii)
, 95 °C, 18 h, 58%; (iv)
+
-, K
1,4-dioxane/H
2
2
3
3
4
3
PO
4
, N
2
DCM, TFA, rt, 12 h; (v) DMF, DIEA, HATU, 12 h, 65 °C, 50-62%.
Synthetic routes of compounds 17a-b are shown in Scheme 3. Commercially
available 14a-b reacted with iodomethane by slightly different conditions to generate
intermediates 15a-b. Suzuki-Miyaura coupling of 15a-b with intermediate 11 produced
compounds 16a-b. Deprotection of the Boc group (TFA, DCM) of 16a-b and followed by
reaction with 2-(3-(trifluoromethoxy)phenyl)acetic acid provided final compounds 17a-b
with 51-54% yields.
Scheme 3. Synthetic Routes of Compounds 17a-b a.
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a
Reagents and conditions: (i) iodomethane, sodium hydroxide, DMF, 50 °C, 12 h, 70%. (ii)
DMF, Cs
PdCl (dppf).CH
Pd(OAc) , KOAc, N
2
CO3, Iodomethane, 80 °C, 8 h, 45%; (iii)1,4-dioxane/H
Cl adduct, Cs CO , N , 95 °C, 18 h, 53%; (iv) 1,4-dioxane/H
, 95 °C, 18 h, 48%; (v) DCM, TFA, rt, 12 h; (vi) DMF,
2
O (5/1),
2
2
2
2
3
2
2
O (10/1),
2
2
2-[3-(trifluoromethoxy)phenyl]acetic acid, DIEA, HATU, 65°C, 12 h, 51-54%.
Compounds 22a-f were generated according to the reaction routes displayed in
Scheme 4. Commercially available starting material 18 was iodinated with NIS to give
3
-iodine derivative 19 in a perfect yield. Alkylation reactions of 19 with various
haloalkanes generated 20a-f under 80 °C calefaction. Suzuki-Miyaura coupling of 20a-f
with intermediate 11 provided compounds 21a-f. Similar as before, deprotection of the
Boc group of compounds 21a-f under HCl in dioxane solution and subsequent reaction
with 2-(3-(trifluoromethoxy)phenyl)acetic acid gave final products 22a-f in 51-67%
yields.
Scheme 4. Synthetic Routes of Compounds 22a-f a.
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Reagents and conditions: (i) DCM, KOH, NIS, rt, 12 h, 90%; (ii) DMF, Cs
2
CO
adduct,
, 95 °C, 18 h, 58-66%; (iv) 1,4-dioxane, HCl in dioxane ,
3
,
Haloalkane, 80 °C, 12 h, 40-55%; (iii) 1,4-dioxane/H
tricyclohexylphospine, Cs CO , N
2
O (5/1), PdCl
2
(dppf).CH
2
Cl
2
2
3
2
rt, 4 h; (v) DMF, 2-[3-(trifluoromethoxy)phenyl]acetic acid, DIEA, HATU, 65°C, 12 h,
51-67%.
3
3
. RESULTS AND DISCUSSION
.1. SAR Analyses of the Synthesized Compounds.
In the SAR study, kinase inhibition assays were utilized to evaluate the bioactivities
of the synthesized compounds. The SAR analyses below will mainly focus on discussing
effects of the following three regions on the bioactivities (Figure 2), namely the linker
phenylamine group (Region Ⅰ), the terminal phenyl group (Region Ⅱ), and the head
heterocyclic rings (Region Ⅲ).
3
.1.1 Impact of the Linker Phenylamine Group (Region Ⅰ).
In this section, we shall investigate the effect of the linker phenylamine group
(Region Ⅰ, Figure 2) on the bioactivity. For this purpose, all the other parts of compound 1
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were fixed as their original structures. A total of eight compounds (8a-g, 13e) were
synthesized. Structures and bioactivities of these compounds are shown in Table 1.
Substitution on the phenyl ring of the aniline (8a-g) did not bring an obvious change in the
RIPK1 kinase inhibition activity. Of special note is that replacement of aniline by indoline
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(13e) led to a significant improvement in the enzymatic inhibition activity. Therefore, in
the subsequent structural modifications, region I was fixed as indoline.
Table 1. RIPK1 Kinase Inhibition Activities of Compounds 1, 8a-g, and 13e.
compd
Region Ⅰ
RIPK1 kinase (IC50, μM)a
1
0.077 ± 0.004
8
a
0.079 ± 0.013
0.094 ± 0.010
0.049 ± 0.023
8
b
8c
8
d
0.134±0.030
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
e
0.424±0 .015
8
f
0.107±0.009
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8g
0.531±0.026
0.014 ± 0.002
1
3e
a
The data are averages of two independent experiments and reported as the mean value ±
SD. IC50 values were determined from Kinase Profiler of Eurofins.
3
.1.2 Influence of the Terminal Phenyl Group (Region Ⅱ).
To examine the possible influence of the terminal phenyl group of compound 1
(
Region II, Figure 2), various substituted phenyl or benzoyl groups were used to replace
the original 3-(trifluoromethyl)benzoyl group with region I fixed as the optimal group
indoline and region III fixed as its original group. Seventeen compounds (13a-r) were
synthesized and bioactivities of these compounds are listed in Table 2. From Table 2, we
can see that the RIPK1 inhibition activities of compounds 13a-b, which contain a chiral
center, were remarkably reduced compared with compound 13e. Compounds 13c and 13d,
which contain 3-(trifluoromethyl)aniline and 1-ethyl-3-(trifluoromethyl)benzene at region
II, respectively, showed considerable RIPK1 inhibition activities. Compounds 13f-r,
bearing a monosubstituted or disubstituted benzyl group, all displayed considerable
RIPK1 inhibition potency. Among these compounds, 13i showed the highest activity
(
IC50 = 0.010 µM). This compound contains a 2-(3-(trifluoromethoxy)phenyl)acetyl group
at the region II position. Therefore, we fixed region II as
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2
-(3-(trifluoromethoxy)phenyl)acetyl group in the following structural optimization.
Table 2. RIPK1 Kinase Inhibition Activities of Compounds 13a-r.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compd
Region Ⅱ
RIPK1 kinase (IC50, μM)a
1
3e
0.014 ± 0.002
1
3a
3b
3c
3.457 ± 0.035
2.994 ± 0.040
1
1
0.049 ± 0.016
0.017 ± 0.002
1
3d
13f
3g
3h
0.061 ± 0.025
0.058 ± 0.009
0.026 ± 0.008
1
1
1
3i
0.010 ± 0.003
0.056 ± 0.029
1
3j
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3k
0.116 ± 0.021
1
3l
0.190 ± 0.044
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3m
3n
13o
0.128 ± 0.012
0.704 ± 0.061
0.392 ± 0.015
1
1
1
3p
3q
0.018 ± 0.004
0.264 ± 0.045
0.028 ± 0.008
1
3r
a
The data are averages of two independent experiments and reported as the mean value ±
SD. IC50 values were determined from KinaseProfiler of Eurofins.
3
.1.3 Effect of the Modification of the Head Heterocyclic Rings (Region III).
To examine the influence of the modification of the head heterocyclic rings on the
bioactivity (Region III, Figure 2), we fixed the region I and II as their optimal fragments,
namely, indoline for region I and 3-(trifluoromethoxy)benzyl for region II. Eight
compounds (17a-b, 22a-f) were synthesized, and bioactivities of these compounds are
shown
in
Table
3.
Firstly,
the
4-amine
group
of
1
-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was removed, which led to a significant
decrease in the RIPK1 activity (17a vs 13i, Table 3), implying the importance of 4-amine.
We then tested the impact of the N atom at the 2-position of pyrazolo[3,4-d]pyrimidin.
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As
shown
in
Table
3,
compound
22a,
which
contains
7
-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine, showed a comparable activity in RIPK1
inhibition. We further tested the bioactivity of compound 22a and 13i at the cellular level,
namely the ability to protect HT29 cells from TSZ-induced necroptosis. In this assay, 22a
exhibited a relatively higher potency (IC50 = 3 nM) than 13i (IC50 = 8 nM), indicating that
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2a probably has a better cell membrane permeability than 13i (Table 4). We thus
replaced the nitrogen atom at the 2-position of pyrazolo[3,4-d]pyrimidin by a carbon
atom in the subsequent structural modification. 17b, a compound obtained by removing
the 4-amine group of 7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine of 22a, displayed a
significantly decreased potency in the RIPK1 kinase activity assay (17b vs 22a, Table 3),
demonstrating again the importance of 4-amine. Finally, we examined the possible
influences of substituents at the N-7 position of 7H-pyrrolo[2,3-d]pyrimidin-4-amine
moiety. Various alkyl groups with different sizes were used to replace the original methyl
group. The generated compounds all displayed potent activity against RIPK1 except 22f
containing an alkyl ether group, which exhibited a decreased activity compared with 13i.
Table 3. RIPK1 Inhibition Activity of Compounds 13i, 17a-b, and 22a-f.
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
RIPK1 kinase (IC50, μM)a
compd
Region III
1
3i
0.010±0.003
0.634 ±0.031
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
17a
17b
22a
0.468±0.089
0.013±0.004
2
2b
0.011±0.001
0.010±0.005
2
2c
22d
0.031±0.012
0.010±0.006
0.105±0.025
2
2e
2
2f
a
The data are averages of two independent experiments and reported as the mean value ±
SD. IC50 values were determined from KinaseProfiler of Eurofins.
The above structural optimization and SAR studies led to the discovery of a series of
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new RIPK1 inhibitors. Because one of the main objectives of this investigation is to
obtain potent necroptosis inhibitors, we then selected compounds with high RIPK1 kinase
inhibition activity (IC50 < 0.015 μM) to further test their cell protection effect in the
TSZ-induced HT29 cell necroptosis model. As shown in Table 4, compound 22b
displayed the most potent activity (EC50 = 0.002 μM) in the cellular assays. Therefore,
further investigations, including kinase selectivity, in vitro and in vivo cell necroptosis
protection bioactivities, anti-tumor metastasis effect, mechanism of action, and
preliminary pharmacokinetic characterization were carried out on compound 22b.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Cell Protection Effect of Selected Potent RIPK1 Inhibitors in the
TSZ-induced HT29 Cell Necroptosis Model.
compd
RIPK1 kinase (IC50, μM)
HT29 EC50 (μM) a
13e
0.014 ± 0.002
0.010 ± 0.003
0.013 ± 0.004
0.011 ± 0.001
0.010 ± 0.005
0.010 ± 0.006
0.035 ± 0.0044
0.008 ± 0.0007
0.003 ± 0.0002
0.002 ± 0.0002
0.003 ± 0.0003
0.004 ± 0.0003
1
3i
2a
2b
22c
2e
2
2
2
a
The data are averages of three independent experiments and reported as the mean value
SD.
±
3
.2 Binding Mode of 22b with RIPK1
Molecular docking was used to investigate the possible binding mode of compound
2b with RIPK1. In the molecular docking study, the crystal structure of RIPK1 kinase
2
domain was taken from the RCSB Protein Data Bank (http://www.rcsb.org, pdb entry:
4
NEU, 2.57 Å resolution). The predicted binding mode of 22b with RIPK1 is displayed
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1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
in Figure 3. Compound 22b is a typical type II kinase inhibitor 29 and interacts with the
inactive
conformation
(DFG-out)
of
RIPK1
kinase
domain.
The
1H-pyrazolo[3,4-d]pyrimidin-4-amine group forms two hydrogen bonds with residues
Glu93 and Met95 in the hinge region and the carbonyl of 22b forms a hydrogen bond
with Asp156. The 3-trifluoromethoxyphenyl group locates in the allosteric pocket and
forms hydrophobic interactions with residues including Val75, Leu129 and Ile154.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. The predicted binding mode of 22b with the kinase domain of RIPK1. Hydrogen
bonding interactions are indicated by dashed yellow lines. The crystal structure of RIPK1
kinase domain was taken from the RCSB Protein Data Bank (PDB entry: 4NEU).
3
.3 Kinase Selectivity of Compound 22b.
To examine the kinome-wide selectivity of 22b, we tested the kinase inhibition
profiles of this compound against a panel of 360 recombinant human protein kinases
through the Eurofins Kinase Profiling Service. The inhibitory activities of 22b at a fixed
concentration of 10 µM were measured first, the results of which are shown in
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Supplementary Table S2. For kinases that had a high inhibitory rate at 10 μM, further
IC50 values were examined. As shown in Table 5 and Supplementary Figure S2, 22b
potently inhibited RIPK1 with an IC50 value of 11 nM. In addition to RIPK1, it also
displayed considerable activity against several other kinases, including Flt4, TrkA, TrkB,
TrkC, Axl, HRI, Mer, and MAP4K5. Importantly, 22b had very weak or no inhibitory
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
0
activity against another 312 protein kinases (Table S2). The calculated selectivity scores ,
S(1), S(5), and S(20) were 0.014, 0.077, and 0.213, respectively. Furthermore, we tested
the kinase binding affinities of 22b against RIPK sub-family members. As shown in
Table 6, 22b displayed at least 1500-fold selectivity toward RIPK1 over other sub-family
members (RIPK2, RIPK3 and RIPK4).
Table 5. IC50 Values of Compound 22b against Selected Kinases
kinase
Abl
IC50 (nM) a
335
kinase
Flt1
IC50 (nM) a
kinase
Tie2
IC50 (nM) a
76
20
75
26
ACK1
Arg
995
Flt4
TrkA
TrkB
TrkC
WNK3
Yes
306
Fyn
187
387
142
26
8
Aurora-B
Axl
210
GCK
Hck
HRI
Itk
7
35
97
BTK
862
384
457
138
111
11
cKit
182
60
ZAK
Blk
cSRC
DDR1
EphA2
173
Lck
43
35
Met
92
BRK
RIPK1
341
MLK1
52
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EphA7
EphA8
FAK
190
233
89
NEK2
PKR
268
210
29
DDR2
Lyn
116
85
Mer
MST2
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
FGFR1
MAP4K5
MEKK2
127
37
MLK2
Ret
50
MAP4K3
Src(1-530)
Fgr
106
207
470
66
255
SAPK4
347
a
IC50 values were determined from KinaseProfiler of Eurofins. The ATP concentration
used was 10 μM.
Table 6. Binding Affinities of 22b against RIPK Subfamily Members
(nM) a
K
D
compd
RIPK1 RIPK2 RIPK3 RIPK4
4.4 >10000 7200 >10000
values were determined from DiscoverX (DiscoverX, USA)
2
2b
a
K
D
3
.4. 22b Could Potently Protect Cells from TSZ-induced Necroptosis in a Cell
Line-independent Manner.
In the screening stage, we have identified that 22b could protect HT29 cells from
TSZ-induced necroptosis (Table 4 and Figure 4A). To examine whether 22b could
protect other cell types from necroptosis, the same experiments were performed on U937,
L929 and J774A.1 cell lines. The results showed that 22b could also potently protect
these cells from necroptosis with EC50 values of 11.55 ± 0.92 nM, 0.999 ± 0.008 nM and
0
.517 ± 0.088 nM for U937, L929 and J774A.1, respectively, suggesting a cell
line-independent effect. Moreover, dual-staining with CytoCalcein™ Violet 450 (for
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living cells) and 7-AAD (for necrotic cells) in HT29 and L929 cells visually showed that
2
2b inhibited TSZ-induced necroptosis and rescued cell survival in
a
concentration-dependent manner (Figure 4B).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. 22b protected cells from TSZ-induced necroptosis. (A) Dose response curves
of 22b in TSZ-induced HT29, U937, L929, and J774A.1 cell necroptosis models.
Necroptosis of HT29, U937 and J774A.1 cells was induced by TNFα (10 ng/mL),
Smac-mimetic (100 nM), and Z-VAD-FMK (20 M). Necroptosis of L929 cells was
induced by TNFα (10 ng/mL) and Z-VAD-FMK (20 M). Cell viability was measured
with CCK8 staining after 24h inducing. (B) Dual-staining with CytoCalcein™ Violet 450
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2
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2
2
2
2
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(
blue, live cell) 7-AAD in HT29 and L929 cell lines with/without 22b treatment after
TSZ-induced for 24h.
3
.5. 22b Inhibited Phosphorylation of RIPK1 and Its Downstream Kinases RIPK3
0
1
2
3
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and MLKL in Intact Cells.
Western blot assays were used to examine the activity of 22b in inhibiting the
phosphorylation of RIPK1 and its downstream signaling proteins in intact cells. As
shown in Figure 5, 22b significantly and dose-dependently inhibited the phosphorylation
of RIPK1 and its downstream signaling proteins RIPK3 and MLKL. It had no effect on
the expressions of RIPK1, RIPK3 and MLKL proteins.
Figure 5. 22b inhibited the phosphorylation of RIPK1, RIPK3 and MLKL. RIPK1,
RIPK3, and MLKL and their phosphorylated states were measured 8h with or without
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2b treatment after TSZ inducing.
3.6. 22b Attenuated the Necrotic Cell Death of Human Umbilical Vein Endothelial
Cells (HUVEC) Induced by Tumor Cells in vitro.
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An in vitro assay was conducted to examine the effect of 22b on the necrotic cell
death of HUVEC induced by tumor cells. In this assay, MDA-MB-231-GFP cells were
co-cultured with HUVEC for 3 days, and spontaneous necrotic cell death of HUVEC was
detected by positive 7-AAD labeling. As shown in Figure 6A, the MDA-MB-231 tumor
cells indeed induced the necrotic cell death of HUVEC. Treatment by 22b significantly
attenuated the HUVEC cell necrosis (Figure 6A).
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.7. In Vivo Effects of 22b on the Necroptosis of Vascular Endothelial Cells (VEC)
and Tumor Metastasis.
The mouse B16 melanoma lung metastasis model was used to examine the ability of
22b to inhibit the necroptosis of VEC induced by tumor cells and prevent the tumor
metastasis in vivo. As displayed in Figure 6B, intravenous injection of B16 cells into
C57BL/6 mice induced necrotic cell death of a part of VECs, as measured by 7-AAD
positive cells in lungs of animals sacrificed 6h after inoculation. Treatment with 22b
(20mg/kg/d) by i.p. injection just before inoculation significantly reduced 7-AAD positive
cells in the lungs analyzed, implying that necroptosis of VECs was inhibited.
Anti-metastatic efficacy of 22b was tested in a 12-day model by treating mice
inoculated i.v. with B16 cells with daily i.p. injections of vehicle or 22b, sacrificing the
animals, and examining the lungs for metastatic foci both visually and by H&E staining. As
shown in Figure 6C and 6D, the 22b treated group showed fewer and smaller metastasis
loci, and exhibited a relatively normal morphology.
Moreover, the same in vivo experiments as above were performed, but
intraperitoneal injection was replaced by oral administration with multiple doses used (1,
3, 10, and 30 mg/kg/d). The results showed that 22b also markedly reduced metastasis
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loci in a concentration dependent manner (see Figure S3A in Supplementary data).
Immunohistochemical analyses of the mouse lung tissue indicated that the vehicle group
was positive with phosphorylated RIPK1 (p-RIPK1) staining, which was reduced by 22b
treatment dose-dependently (see Figure S3B in Supplementary data). These results
demonstrated that 22b reduced metastasis via inhibiting RIPK1 phosphorylation and
necroptotic signaling.
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Figure 6. 22b exhibited a good ability to inhibit the necroptosis of vascular endothelial
cells induced by tumor cells both in vitro and vivo, and prevent the tumor metastasis in
vivo. (A) HUVEC and MDA-MB-231-GFP cells were co-cultured for 4 days and necrotic
HUVECs were detected by CytoCalcein™ Violet 450 and 7-AAD staining. 22b was added
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on day 2 after cells were seeded. (B) 5x10 B16 cells were injected to C57BL/6 mice
intravenously. 22b (20mg/kg) or vehicle was pre-administrated i.p. before injection.
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-AAD was injected intravenously 30 min before sacrifice. Lungs were obtained to
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conduct immunofluorescence with anti-CD31 and DAPI. (C, D) 5x10 B16 cells were
injected to C57BL/6 mice intravenously. 22b (20mg/kg/d) or vehicle was administrated
24h after the inoculation. Mice were sacrificed on day 12 and their lungs were obtained to
evaluate.
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.7. A Preliminary Assessment for the Pharmacokinetic Properties of 22b.
The pharmacokinetic characteristics of 22b were assessed on rats. A dose of 10
mg/kg was administrated through intravenous infusion or oral administration. The plasma
concentration versus time profile is presented in Figure S4, and the key oral
administration pharmacokinetic parameters calculated are summarized in Table 7. The
area under the concentration-time curve (AUC0-∞) is 14855.22 mg/L*h. The half-life
(T1/2), the clearance rate (CL), and apparent distribution volume (Vss) are 2.254 h, 0.673
L/h/kg, and 2.19 L/kg, respectively. The maximum plasma concentration (Cmax) is 2307
mg/L within about 2 h. Importantly, 22b had an excellent bioavailability of 58%. All of
these indicate that 22b possesses favorable pharmacokinetic properties.
Table 7. Key Pharmacokinetic Parameters of 22b (p.o.).
Pharmacokinetic parameter
Value
2.254
2307
2
T
1/2
C
max(mg/L)
T
max
CL(L/h/kg)
0.673
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V
ss(L/kg)
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14855.21
58
AUC(0-∞)mg/L*h
Bioavailability (%)
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. CONCLUSION
In this investigation, structural optimization and SAR analyses of the hit compound
led to the discovery of a series of new RIPK1 inhibitors. Among these compounds,
compound 22b showed a high enzymatic inhibition activity against RIPK1 with an IC50
value of 0.011µM, and the most potent cell protection effect in the TSZ-induced HT29
cell necroptosis model with an EC50 value of 0.002 µM. Further in-depth studies were
carried out on this compound, including kinase selectivity, in vitro and in vivo
bioactivities, mechanism of action, and preliminary pharmacokinetic characterization.
The results showed that 22b had a good kinase selectivity with the calculated selectivity
scores, S(1), S(5), and S(20) being 0.014, 0.077, and 0.213, respectively. It could
efficiently inhibit phosphorylation of RIPK1 and its downstream signaling proteins in
intact cells. This compound was able to attenuate the necrotic cell death of vascular
endothelial cell induced by tumor cells both in vitro and in vivo. In the experimental B16
melanoma lung metastatic model, 22b exhibited excellent anti-tumor metastasis activity.
This compound also displayed favorable pharmacokinetic properties. All of the data
presented here indicate that 22b has a potential in the treatment of tumor metastasis, and
deserves further research and development.
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5
. EXPERIMENTAL SECTION
.1. Molecular Docking.
Molecular docking studies were carried out by GOLD (version 5.2). GOLD adopts
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the genetic algorithm to dock flexible ligands into protein binding site. The crystal
structure of the RIPK1 kinase domain in complex with isoquinolin-1-amine analog (PDB
code 4NEU) was used as the receptor structure in the molecular docking studies. The
ligand (isoquinolin-1-amine analog) in the complex and water molecules were removed,
and hydrogen atoms were added to the protein by using Discovery Studio 3.1 (Accelrys
Inc., San Diego, CA, USA). The Charmm force field was assigned. Compound 22b was
also prepared by using Discovery Studio 3.1. The binding site was defined as a sphere
containing residues that stay within 10 Å from its original ligand, which is large enough
to cover the ATP binding region at the active site. GoldScore was selected as the scoring
function, and other parameters were set as default. Images were created using PyMOL.
5
.2. In Vitro Kinase Binding Affinity and Kinase Enzymatic Inhibition Assays.
The in vitro kinase binding assays were conducted by the Kinase Binding Constants
Services provided by DiscoverX (DiscoverX, USA). The in vitro kinase enzymatic
inhibition assays were carried out by the Kinase Profiling Services provided by Eurofins
(Eurofins, UK).
5
.3. Cell Lines and Cell Culture Conditions.
All the cell lines used in this investigation were purchased from the American Type
Culture Collection (ATCC). These cell lines were cultured in DMEM (Dulbecco's
Modified Eagle Medium, Gibco) culture medium supplemented with 10% FBS, 100
U/mL penicillin, and 100 U/mL streptomycin. All cellular incubations were performed at
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7 °C and 5% CO
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unless stated otherwise.
5
.4. Mechanism of the TSZ-induced Necroptosis Model and Cell Necroptosis
Protection Assay.
The detailed mechanism of the TSZ-induced necroptosis model could be found in
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reference . Here a brief introduction is given as follows. TNFα binds to its receptor
TNFR1, which signals to its downstream kinase RIPK1. Smac-mimetic can promote
RIPK1-caspase-8 complex formation and then induce apoptosis. Z-VAD-FMK, a
pan-caspase inhibitor, can shift apoptosis to necroptosis by making RIPK1 to associate
with RIPK3 to form an intracellular complex, called necrosome, which acts as a
transducer of the necroptotic signaling. Obviously, the combined use of
TNFα/Smac-mimetic/z-VAD-FMK (TSZ) can induce cell necroptosis, and RIPK1
inhibition is able to protect cells from necroptosis in this model.
Cell necroptosis protection assays were performed in 96-well cell culture plate. 8000
cells were plated in each well and cultured at 37 °C overnight. HT-29，U937, or J774A.1
cells were treated with 10 ng/mL TNFα, 100 nM Smac mimetic, and 20 μM z-VAD-FMK
for 24 h. L929 cells were treated with 10 ng/mL TNFα, and 20 μM z-VAD-FMK for 24 h.
The cell survival rate was determined using the CCK8 Cell Viability Assay kit. All
experiments were performed in triplicate, and each EC50 value was expressed as mean ±
SD.
5
.5. Western Blot Analysis.
Cells were seeded in 60×15mm dishes overnight prior to treatment and incubated
with the indicated doses of 22b for 24 h at 37 °C. TNFα (Peprotech), Smac mimetic
(Selleck), and Z-VAD-FMK (Selleck) were added in the meanwhile with 22b. Then cell
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pellets were collected and resuspended with NP40 lysis buffer (Beyotime), which was
added with extra proteasome inhibitor PMSF and phosphatase inhibitor Cocktail (Sigma).
Whole-cell protein lysates were incubated on ice for 30 min and centrifuged at 12000
r/min and 4 °C for 20 min. The supernatants were determined using the BCA method.
Proteins were separated by SDS−PAGE and transferred to PVDF membranes (Millipore).
The membranes were incubated at 4 °C overnight with the primary antibodies. After
washing, the membranes were incubated for 1 h at room temperature with the secondary
antibody (anti-IgG-HRP 1:5000, CST) and washed again. Changes in protein expression
and phosphorylation were detected using an enhanced chemiluminescence detection
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reagent . RIPK1 antibody was purchased from R&D. RIPK3 antibody was purchased
from Santa Cruz. Antibodies for MLKL, p-MLKL, p-MLKL(m) and p-RIPK3 were
purchased from ABcam. P-RIPK1 antibody was purchased from Cell Signaling
Technology (CST).
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.6. Anti-tumor Metastasis Model.
Animal studies were conducted under the approval of the Experimental Animal
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Management Committee of Sichuan University. 5×10 B16 cells were injected to C57BL/6
intravenously. Vehicle or 22b (20mg/kg) was administrated once a day. Lung vascular
endothelial cells and extravascular TCs were marked by DAPI and CD31. Mice were
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sacrificed 12 day and their lungs were harvested to evaluate by visual inspection and HE
staining.
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.7. Immunohistochemical Analysis.
Paraffin-embedded lungs were subjected to immunostaining with p-RIPK1 (SAB
Signaling Antibody, 13224). The representative images were taken on a LEICA DM 2500
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