Design, synthesis and biological evaluation of pyridine derivatives as selective SHP2 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.103875

SHP2 is a non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene, which affects the transduction of multiple signaling pathways, including RAS-ERK, PI3K-AKT and JAK-STAT. SHP2 also plays an important role in the programmed cell death pathway (PD-1/PD-L1). Studies have shown that SHP2 is associated with a variety of cancers, including breast, liver and gastric cancers. Therefore, the development of SHP2 inhibitors has attracted extensive attention. In this study, based on the known inhibitor 1 (SHP099), novel SHP2 inhibitors were designed by means of scaffold hopping, and 35 pyridine derivatives as SHP2 inhibitors were found. The in vitro enzyme activity assay was performed on these compounds, and multiple selective SHP2 inhibitors with activity potency similar to that of SHP099 were obtained. Among them, compound (2-(4-(aminomethyl)piperidin-1-yl)-5-(2,3-dichlorophenyl)pyridin-3-yl)methanol (11a) was the most potent and highly selective SHP2 inhibitor with an in vitro enzyme activity IC₅₀ value of 1.36 μM. Fluorescence titration assay verified that 11a bound directly to SHP2 protein. Subsequently, cell assay of representative compounds showed that these compounds could effectively inhibit the proliferation of Ba/F₃ cells. In addition, the pharmacokinetic characteristics of the designed compounds were analyzed by the in silico ADMET prediction. Molecular docking study provided more detailed information on the binding mode of compounds and SHP2 protein. In brief, this study reported for the first time that pyridine derivatives as novel SHP2 inhibitors had good inhibitory activity and selectivity, providing new clues for the development of small molecule SHP2 inhibitors.

Full text of DOI:10.1016/j.bioorg.2020.103875

Journal Pre-proofs
Design, synthesis and biological evaluation of pyridine derivatives as selec-
tive SHP2 inhibitors
Wen-Shan Liu, Bing Yang, Rui-Rui Wang, Wei-Ya Li, Yang-Chun Ma,
Liang Zhou, Shan Du, Ying Ma, Run-Ling Wang
PII:
S0045-2068(19)32039-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103875
YBIOO 103875
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
28 November 2019
31 March 2020
21 April 2020
Please cite this article as: W-S. Liu, B. Yang, R-R. Wang, W-Y. Li, Y-C. Ma, L. Zhou, S. Du, Y. Ma, R-L.
Wang, Design, synthesis and biological evaluation of pyridine derivatives as selective SHP2 inhibitors,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103875
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Design, synthesis and biological evaluation of pyridine derivatives as
selective SHP2 inhibitors
Wen-Shan Liua, b, 1, Bing Yangc,1, Rui-Rui Wanga, Wei-Ya Lia, Yang-Chun Maa, Liang Zhoua, Shan
Dua, Ying Maa, *, Run-Ling Wanga, *
aTianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and
Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070,
China.
bDepartment of pharmacy, Affiliated Hospital of Weifang Medical University, Weifang,
Shandong 261000, China.
cDepartment of Cell Biology, School of Basic Medical Science, Tianjin Medical University,
Tianjin 300070, China.
1These authors contributed equally to this work.
*Correspondence to:
Ying Ma
Email: maying@tmu.edu.cn
Run-Ling Wang
Email: wangrunling@tmu.edu.cn
Funding information
Contract grant sponsor: National Natural Science Foundation of China; Contract grant number:
81773569.
Contract grant sponsor: Natural Science Foundation of Tianjin; Contract grant number:
16JCZDJC32500, 18JCQNJC13700.
Contract grant sponsor: Cooperation and Exchange Project of the National Natural Science
Foundation of China; Contract grant number: 81611130090.
Contract grant sponsor: Science & Technology Development Fund of Tianjin Education
Commission for Higher Education; Contract grant number: 2017KJ229.

Abstract
SHP2 is a non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene, which affects
the transduction of multiple signaling pathways, including RAS-ERK, PI3K-AKT and JAK-STAT.
SHP2 also plays an important role in the programmed cell death pathway (PD-1 / PD-L1). Studies
have shown that SHP2 is associated with a variety of cancers, including breast, liver and gastric
cancers. Therefore, the development of SHP2 inhibitors has attracted extensive attention. In this
study, based on the known inhibitor 1 (SHP099), novel SHP2 inhibitors were designed by means of
scaffold hopping, and 35 pyridine derivatives as SHP2 inhibitors were found. The in vitro enzyme
activity assay was performed on these compounds, and multiple selective SHP2 inhibitors with
activity potency similar to that of SHP099 were obtained. Among them, compound (2-(4-
(aminomethyl)piperidin-1-yl)-5-(2,3-dichlorophenyl)pyridin-3-yl)methanol (11a) was the most
potent and highly selective SHP2 inhibitor with an in vitro enzyme activity IC50 value of 1.36 μM.
Fluorescence titration assay verified that 11a bound directly to SHP2 protein. Subsequently, cell
assay of representative compounds showed that these compounds could effectively inhibit the
proliferation of Ba/F3 cells. In addition, the pharmacokinetic characteristics of the designed
compounds were analyzed by the in silico ADMET prediction. Molecular docking study provided
more detailed information on the binding mode of compounds and SHP2 protein. In brief, this study
reported for the first time that pyridine derivatives as novel SHP2 inhibitors had good inhibitory
activity and selectivity, providing new clues for the development of small molecule SHP2 inhibitors.
Keywords: SHP2 inhibitor, scaffold hopping, activity, ADMET, molecular docking
1. Introduction
SHP2 is a non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene, which composes
of two SH2 domains (N-SH2 and C-SH2), a catalytic PTP domain and a C-terminal tail that has at
least two phosphorylation sites[1] [2]. X-ray crystallography studies have demonstrated that in the
basal state, SHP2 adopts an auto-inhibited conformation, which the SH2 domain associates with the
PTP domain and prevents substrates from accessing the active site[3]. Activation of SHP2 occurs
through binding of bis-phosphotyrosyl peptides (e.g., IRS-1) to the SH2 domain, disrupting the
interaction between the SH2 and PTP domains. The resulting conformational opening exposes the
active site and increases phosphatase activity, which promotes cancer dependence[4].
Gain-of-function mutations in PTPN11 that lead to hyperactivation of SHP2 have been identified in
Noonan syndrome (50%)[5], juvenile myelomonocytic leukemia (JMML, 35%), myelodysplastic
syndrome (10%), B-cell accute lymphoblastic leukemia (7%), acute myeloid leukemia (AML, 4%)
[6]. In cell, SHP2 is located in the cytoplasm and transduces cell signaling from a variety of
receptor–tyrosine kinases, and is therefore involved in numerous oncogenic cell signaling cascades,
including RAS-ERK, PI3K-AKT and JAK-STAT [8, 9]. In addition, SHP2 is also involved in the
programmed cell death pathway (PD-1/PD-L1) and contribute to immune evasion[10]. After PD-
L1 is stimulated, SHP2 binds to PD-1 and inhibits T cell activation, making it a new target for cancer
immunotherapy[11]. In view of the importance of SHP2 for human diseases, the development
of small molecule SHP2 inhibitors has attracted widespread attention.
Due to the high homology between the SHP2 catalytic site and other tyrosine phosphatases,

targeting SHP2 small molecule inhibitors pose great challenges in achieving the selectivity between
phosphatases. Moreover, the highly conserved and charged environment of SHP2 catalytic site
greatly complicates drug development, resulting in the slow progress in the clinical study of SHP2
inhibitors[12, 13] [14]. The previously reported SHP2 inhibitors did not achieve the desired potency
and selectivity, such as the first three compounds in Figure 1[15-17]. Until recently, a potent,
selective, orally bioavailable inhibitor 1 (SHP099, IC50 = 70 nM) had been shown to inhibit cancer
cell growth in vitro and in mouse tumor xenograft models and had entered clinical study,
demonstrating that inhibition of SHP2 was an effective treatment for cancer. Interestingly, X-ray
analysis of SHP099 and SHP2 protein revealed a new allosteric binding mode that inhibited SHP2
phosphorylation by stabilizing the self-inhibited conformation of SHP2[18, 19]. Based on this, the
development of SHP2 allosteric inhibitors had been triggered, and many small-molecule compounds
with high inhibitory activity and high selectivity have been developed, such as RMC-4550 (IC50
=
0.583 nM), which had been shown to inhibit RAS/MAPK signaling and slow down the growth of
various cancer[20]. Currently, the inhibitor RMC-4550 had entered clinical trials. Other SHP2
allosteric inhibitors, such as SHP394 and SHP389, had good inhibitory potency and selectivity on
SHP2 protein[21, 22]. Therefore, preventing the phosphorylation of SHP2 protein by stabilizing the
autoinhibited conformation of SHP2 protein has become a new direction for the development of
SHP2 inhibitors.
Figure 1. Representative SHP2 inhibitors that have been previously reported. Among them, the first three compounds
are inhibitors against the catalytically active region of SHP2PTP, and the latter four compounds are inhibitors against
the allosteric region of SHP2 protein.
Scaffold hopping is a very effective strategy that has been widely used to design potential bioactive
molecules[23-25]. In this study, based on the known inhibitor SHP099, the SHP2 inhibitors with 3-
substituted pyridine as the core structure was designed by scaffold hopping, as shown in Figure 2.
In scaffold A, in addition to retaining the amino substituent, hydroxymethyl substituent was also
introduced into the pyridine ring to increase structural diversity. Subsequently, 35 compounds were
designed by fragment replacement of scaffolds B and C. Through the in vitro enzyme activity and
cell proliferation assays on 35 designed compounds, it was identified that multiple compounds
exhibited good inhibitory activity and selectivity for SHP2 protein. Fluorescence quenching assay

was performed on the compound 11a with the highest activity, demonstrating that the compound
11a would bind directly to SHP2 protein. Then, cell proliferation assay was performed on the
representative compounds. Furthermore, these 35 compounds were confirmed to be drug-like by
ADMET prediction. Molecular docking study provided more detailed information on the binding
modes of compounds and SHP2 protein. In this paper, the pyridine derivatives as SHP2 inhibitors
were studied for the first time, providing a new idea for the further development of small molecule
SHP2 inhibitors.
Figure 2. Design strategy of target compounds based on scaffold hopping.
2. Results and discussion
2.1. chemistry
These 35 target compounds were prepared by the synthetic route in schemes 1 and 2. Scheme 1
showed the synthetic route of compounds 6a1-g2, which used 5-bromo-2-chloro-3-nitropyridine as
the starting material. First, in the presence of N, N-diisopropylethylamine (DIEA), the 2-position
chlorine on the 5-bromo-2-chloro-3-nitropyridine was substituted by the nucleophilic substitution
reaction to gain the intermediates 3a-j. The Suzuki-Miyaura coupling was carried out to obtain the
intermediates 4ai-g2. Subsequently, the nitro group was reduced to amino group by zinc powder to
obtain the intermediates 5a1-g2. Finally, in the presence of trifluoroacetic acid (TFA), Boc
protecting groups were removed, and the target compounds 6a1-g2 were obtained.
Scheme 2 showed the synthetic route for compound 11a-b using methyl 5-bromo-2-chloronicotinate
as starting material. First, the compound methyl 5-bromo-2-chloronicotinate (7) was subjected to
the Suzuki-Miyaura coupling reaction with (2,3-dichlorophenyl)boronic acid to obtain Intermediate
8. Then, through nucleophilic substitution reaction, the intermediates 9a-b were obtained by
replacing the 2-position chlorine on intermediate 8. Subsequently, the Boc protecting group on
intermediates 9a-b were removed in the presence of trifluoroacetic acid to gain intermediates 10a-
b. Finally, the ester group on the intermediates 10a-b were reduced to the hydroxyl group by the
reducing agent lithium aluminum hydride to gain the target compounds 11a-b.

Scheme 1. Synthesis of the target compounds 6a1-g2. Reagents and condition: (a) NHR1R2Boc,
DIEA, 1,4-Dioxane, 90oC, 87−91%; (b) ArR3B(OH)2, Pd(PPh3)4, K2CO3, 1,4-Dioxane/H2O, 100oC,
82−89%; (c) zinc powder, NH4Cl, EtOH, 80 oC, 85−93%; (d) TFA, DCM, rt, 87−92%.
Scheme 2. Synthesis of the target compounds 11a-b. Reagents and condition: (a) Pd(PPh3)4, Na2CO3,
1,4-Dioxane/H2O, 90oC, 68%; (b) NHR1R2Boc, K2CO3, DMF, 100oC, 91-92%; (c) TFA, DCM, rt,
91%; (d) LiAlH4, Et2O, 0oC, 81-82%.
2.2. Biological evaluation
2.2.1. Study on the in vitro activity assay of SHP2 and SHP1and structure-activity relationship
(SAR)
All the 35 target compounds were used to evaluate the activity of inhibiting phosphatase by using
full length wild-type SHP2 and SHP1. The activity results of the target compounds 6a1-6g2 and
11a-b were shown in Table 1. The structure-activity relationship (SAR) was explored below.
Firstly, the SAR on scaffold B was explored. As shown in Table 1, the different substituents on the
phenyl ring on the scaffold B had great influence on the activity. When the phenyl ring was a single
substituent, the activity at the 2-position substitution was significantly higher than at the 3-position
and 4-position substitutions (6a1, 6b1 and 6c1). By comparing the different 2-position substituents,
it was found that the activities of Cl, F and CF3 substituents were significantly higher than that of
other substituents (6a1, 6d1-6g1), probably due to the stronger interaction of these substituents with
surrounding residues. Subsequently, comparing the activity of two substituents on the phenyl ring,
it was found that the activity of the 2,3-position substitution was significantly higher than that of

the 2,4-position and 3,4-position substitutions (6k1-6p1). It is noteworthy that, from the results of
the activities of the compounds 6h1 and 6i1, it was found that the substitution of the phenyl ring on
the scaffold B for the pyridine ring was advantageous for enhancing the inhibitory activity, and the
activity of 6j1 was increased by about 1.7-fold compared with the 6a1.Next, we turned our attention
to scaffold C and analyzed the effect of different fragments on activity. When the scaffold C was
the aromatic amine group, from the results of the activity of 6c2 and 6f2, the activity of 3-position
amino substitution was higher than that of 4-position amino substitution, with an increase of about
1.9-fold. When the scaffold C was the aliphatic amine group, different structures cause significant
differences in activity. The difference in activity between compounds 6v1 and 6w1 indicated that
the chain length of the extended terminal NH2 had a significant improvement in activity, resulting
in an about 4.3-fold increase in potency of compound 6w1. By comparing the activity results of the
compounds 6v1 and 6z1, it was found that the activity of compound 6z1 was increased by about
4.5-fold after the introduction of the methyl group at the terminal of the piperidine, due to the
introduction of the methyl group to stabilize the pseudo-equatorial amine conformation. Finally, we
turned our attention to skeleton A. The difference in activity was investigated by changing the amino
group on the pyridine ring to the hydroxymethyl group. As could be seen from the activity results
of compounds 11a and 6w1, after the amino group was changed to the hydroxymethyl group, the
compound 11a also had a higher inhibitory activity, and the activity was improved by about 1.2-
fold. One reason for the increased activity of compound 11a was that the hydroxymethyl group itself
had the function of H bond donor, and another important reason was that the hydroxymethyl group
might better match the protein cavity than the amino group. According to the activity results in
Table 1, compound 11a (IC50 = 1.36 μM) was found to have the best inhibitory activity against
SHP2 protein.
Since SHP1 and SHP2 had 60% overall primary sequence identity and about 75% similarity in the
catalytic PTP domain, SHP1 and SHP2 proteins exhibited high homology of the protein
sequence[26]. Therefore, inhibitors tended to have low selectivity for these two phosphatases. All
compounds were tested for their inhibitory activity against SHP1 to assess their selectivity. It could
be seen from the table 1 that all compounds exhibited the IC50 values of more than 50 μM for SHP1
protein, indicating that these compounds had high selectivity for the SHP2 protein.
Table 1. The SAR studies of the target compounds 6a1-6g2 and 11a-b.
X
R1
N
Y
Z
R2
N
R3
Compd.
6a1
X
Y
C
C
Z
NR1R2
R3
IC50a (µM)
SHP2 SHP1
HN
HN
NH
NH
NH2
NH2
C
C
2-Cl
3-Cl
52.12
>100
>100
>100
6b1

HN
HN
HN
HN
HN
HN
HN
HN
HN
HN
HN
HN
HN
HN
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
6c1
6d1
6e1
6f1
6g1
6h1
6i1
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
C
C
C
C
C
N
C
N
C
C
C
C
C
C
C
C
C
C
C
C
N
C
C
C
C
C
C
C
4-Cl
2-F
>100
55.73
>100
>100
30.18
8.95
>100
>100
>100
>100
>100
72.54
75.43
87.61
>100
>100
>100
>100
>100
>100
2-OCH3
2-CH3
2-CF3
H
H
34.61
30.14
28.93
85.88
>100
>100
45.66
80.39
6j1
2-Cl
6k1
6l1
2,3-Cl
2,4-Cl
3,4-Cl
2-Cl-4-CH3
2,3-F
2,4-F
6m1
6n1
6o1
6p1

HN
HN
HN
HN
HN
N
N
N
N
N
6q1
6r1
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
C
C
C
C
C
C
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
2,3-Cl
2,3-F
2-Cl
33.92
35.74
49.36
34.33
>100
7.12
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6s1
6t1
2-CF3
3,4-Cl
2,3-Cl
2,3-Cl
H
6u1
6v1
6w1
6x1
6y1
6z1
6a2
H2N
N
N
1.64
H2N
N
20.73
27.68
1.58
H2N
HN
HN
2,3-Cl
2,3-Cl
2,3-Cl
N
H2N
H2N
16.13
N
HN
HN
6b2
6c2
6d2
NH2
NH2
NH2
C
C
C
C
C
C
2,3-Cl
2,3-Cl
2-Cl
24.18
18.24
30.60
>100
87.24
71.62
H2N
NH
NH
H2N

H2N
NH
6e2
6f2
NH2
NH2
C
C
C
C
3,4-Cl
2,3-Cl
>100
9.47
62.85
71.28
H2N
NH
H2N
6g2
11a
NH2
C
C
C
C
2,3-F
12.31
1.36
72.75
>100
NH
N
CH2OH
2,3-Cl
H2N
N
11b
CH2OH
—
C
C
2,3-Cl
—
1.41
1.48
>100
>100
H2N
SHP099b
—
—
—
aThe experiment was repeated three times and data presented is the mean ±SEM of triplicates from one representative
experiment.
bPositive control
2.2.2. fluorescence quenching assay
To further verify whether the compound bound directly to SHP2, we performed fluorescence
quenching assays on compounds 11a with the best inhibitory activity. As shown in Figure 3,
compound 11a showed strong fluorescence quenching of SHP2 protein, respectively, in a dose-
dependent manner, indicating that compound 11a would bind directly to SHP2 protein.
Figure 3. The fluorescence titration of SHP2 protein was carried out by increasing the concentration of compound
11a without changing the concentration of SHP2. The experiment was repeated three times and data presented is the
mean ±SEM of triplicates from one representative experiment.
2.2.3. Cell proliferation assay
Cell assay was performed on representative compounds to assess their antiproliferative activity
against the Ba/F3 cell line. First, the in vitro concentration of the cytotoxicity of the tested
compounds was set at 20 μM. Subsequently, these compounds were further assessed to determine
their IC50 values at concentrations of 0.1, 1.0, 5.0, 10 and 20 μM. Finally, IC50 values were calculated.

Table 2 showed the cellular activities of these compounds against the Ba/F3 cell line. It was found
that the tested compounds showed better potency in inhibiting cell proliferation compared to the
positive control SHP099. Among them, the compound 11a showed the high activity for inhibiting
cell proliferation with an IC50 value of 2.35 μM, which was 8.45-fold stronger than SHP099.
Table 2. The antiproliferation IC50 values of representative compounds against Ba/F3 cell.
Compd.
antiproliferation
IC50a (µM)
6h1
6v1
6.62
6.92
2.49
2.32
7.57
2.35
2.42
19.86
6w1
6z1
6f2
11a
11b
SHP099b
aThe experiment was repeated three times and data presented is the mean ±SEM of triplicates from one representative
experiment.
bPositive control
2.3. ADMET Prediction
The in silico ADMET prediction was used to evaluate the pharmacokinetic properties of these 35
compounds by using DS v3.5 software[27-29]. Discovery Studio 3.5 (DS 3.5) was used to define
the prediction level for all ADMET properties. ADMET-Plot was a 2D plot obtained by calculating
the PSA_2D and A logp98 features for predicting human intestinal absorption (HIA) and blood-
brain barrier (BBB) of all compounds, including 95% and 99% confidence ellipse. As shown in
Figure 4, all 35 compounds were within the 99% confidence ellipse, indicating that the target
compounds had good human intestinal absorption and could penetrate the blood brain barrier. Based
on the structure of the compounds, the parameters of ADMET were calculated in Tables S1 and S2.
According to the calculated results, these 35 compounds all were confirmed to be drug-like, and had
low side effects.

Figure 4. Human intestinal absorption (HIA) and Blood Brain Barrier (BBB) plot of compounds 6a1-g2 and 11a-b.
All the compounds in the figure are located in the innermost ellipse, showing better parameter values.
2.4. Molecular docking study
Molecular docking, as an effective and reliable method, can be used to simulate the binding mode
between protein and ligand and to understand the protein-inhibitor interaction[30, 31]. As shown in
Figures 5A and 5C, with the help of DS v3.5 software, the compounds 6w1 and 11a with higher
inhibition potency were docked well in the allosteric binding pocket of SHP2 protein. Here, the
docking scores of compounds 6w1 and 11a were 56.24 and 58.62, respectively. To further explore
the interaction of compounds 6w1 and 11a with SHP2, the docking result was visualized as 2D
diagram, as shown in Figures 5B and 5D, which clearly showed the interaction of compounds 6w1
and 11a with SHP2 protein. It was found that compound 6w1 formed H bond interactions with
residues Thr108, Glu110, Arg111, Phe113 and His114 between the C-SH2 and N-SH2 domains and
residues Leu216, Thr218 and Thr253 of the PTP domain. And compound 11a formed H bond
interactions with residues Thr108, Glu110, Arg111 and Phe113 between the C-SH2 and N-SH2
domains and residues Thr218, Thr219 and Thr253 of the PTP domain. And the dichlorophenyl
groups in both compounds formed hydrophobic interactions with the surrounding residues, and both
formed cation-Pi interactions with the key residue Arg111. Therefore, both compounds formed
stable interactions with SHP2 protein, which revealed at the molecular level why both compounds
had High inhibitory activity.
The structural differences of compounds 6w1, 11a and SHP099 were mainly in the mother nucleus.
It had been reported that on the aminopyrazine ring of SHP099, the amino group mainly formed an
H bond interaction with the residue Glu250. On the aminopyridine ring of compound 6w1, the
amino group formed three H bond interactions with residues His114, Leu216, and Thr218. On the
hydroxymethylpyridine ring of compound 11a, the hydroxymethyl group formed three H bond
interactions with the residues Thr218 and Thr219. Therefore, pyridine derivatives could effectively
match with protein pocket and formed stable interactions, thus exhibiting higher inhibitory activity.
In addition, we further analyzed the difference in H bond distance and found that the H bond formed
by compound 11a was more stable than that formed by compound 6w1, which also explained why

the inhibitory activity of compound 11a was slightly higher than that of compound 6w1.
Figure 5. The binding mode of compounds 6w1 and11a with SHP2 protein. (A) The docking pocket of compound
6w1 and SHP2. (B) 2D diagram of the interaction of compound 6w1 and SHP2 protein. (C) The docking pocket of
compound 11a and SHP2. (D) 2D diagram of the interaction of compound 11a and SHP2 protein. Among them, the
H bond interactions with residues are denoted by green dotted arrow pointing to the electron donor; the H bond
interactions with residues are denote d by blue dashed arrow pointing to the electron donor; the cation-Pi interactions
are represented by the orange line; the pink rectangles represent residues with charge or H bond interactions; the
green rectangles represent residues with VDW interactions.
3. Conclusion
SHP2 has attracted extensive attention as an anticancer target due to its direct correlation with cancer
and its influence on the transduction of various signaling pathways. Therefore, the development of
SHP2 small molecule inhibitors is particularly urgent. In this study, based on the inhibitor SHP099,
35 novel SHP2 inhibitors with 3-substituted pyridine as the core structure were designed by means
of scaffold hopping. Subsequently, analysis of the in vitro enzyme activity assay gained a number
of inhibitors with the exploitable value. Among them, the compound 11a was the most potent SHP2
inhibitor and its in vitro enzyme activity IC50 value reached 1.36 μM, and the activity against SHP1
exceeded 100 μM, showing high selectivity for SHP2 protein. Fluorescence quenching assay

verified that compound 11a bound directly to the SHP2 protein. The cell activity studies of
representative compounds found that the tested compounds all showed effective antiproliferative
activity on Ba / F3 cells, of which the IC50 value of 11a was 2.35 μM. Furthermore, the in silico
ADMET prediction validated that these compounds were potential to be drug-like. The binding
modes of the compound 11a and 6w1 with SHP2 protein were explored by the molecular docking,
and the reason for the difference in activity between the two compounds was explored at the
molecular level. It was found that the H bond interaction formed between the hydroxymethyl group
and protein was more stable, revealing that the inhibitory activity of compound 11a was slightly
higher than that of the compound 6w1. In summary, through our series of studies, we sincerely hope
that this research can provide more new ideas for the development of more effective SHP2 inhibitors.
4. Experimental section
4.1. Chemistry
Reagents and solvents were obtained from commercial suppliers and could be used directly without
further purification. Analytical thin layer chromatography (TLC) was performed on the TLC plate
(silica gel 60 F254 and aluminum foil). All compounds were detected by using UV light
(wavelength: 254 nm or 365 nm). The Isolation and purification of compound was carried out by
flash column chromatography on silica gel 60 (300-400 mesh). 1H (400 MHz) and 13C NMR (100
MHz) spectra were recorded in CDCl3 or DMSO-d6 by the spectrometer at room temperature.
Chemical shifts were reported in parts per million (ppm) and coupling constants (J) were expressed
in Hz. The characteristics of the signal were s (singlet), d (doublet), dd (doublet of doublets), t
(triplet) and m (multiplet). Mass spectra were measured on an Agilent 1100 series.
4.1.1. General procedure for the synthesis of 3a-j, 4a1-g2, 5a1-g2, 6a1-g2, 8, 9a-b, 10a-b and 11a-
b.
A mixture of 5-Bromo-2chloro-3-nitropyridine (2) (10 mmol), organicamine compound (14 mmol),
and N,N-diisopropylethylamine (30 mmol) in 1,4-dioxane (100 mL) was stirred at room temperature
for 4 h. After the reaction was completed by TLC analysis, the volatiles were removed under reduced
pressure. The mixture was extracted with CH2Cl2 (3 × 60 mL) and washed with H2O (3 × 60 mL).
The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The
resulting residue was purified by column chromatography on silica gel (PE: EA = 9:1) to obtain
the intermediates 3a-j.
A suspension of intermediate 3a-j (2 mmol), boric acid compound (3 mmol), Pd(PPh3)4 (0.16 mmol)
o
and K2CO3 (6 mmol) in 1,4-dioxane/H2O (5:1, 60 mL) was heated to 120 C and stirred for 6 h.
After completion of the reaction by TLC analysis, the solvent was removed in vacuo. The mixture
was extracted with CH2Cl2 (3 × 40 mL) and washed with H2O (3 × 40 mL). The organic phases
were combined and dried over anhydrous Na2SO4. The organic phase after drying was evaporated
under reduced pressure, and the resulting residue obtained was purified by silica gel
chromatography on silica gel (PE: EA = 6:1) to gain the intermediates 4a1-g2.
The mixture of the intermediate 4a1-g2 (1.3mmol), zinc powder (26 mmol) and ammonium chloride
(6.5 mmol) in EtOH/H2O (10:1, 55mL) was heated to 80 oC and stirred for 6 h. After the reaction
was completed, the iron powder was removed by suction filtration, and then the volatiles were
removed in vacuo. PH of the mixture was basified to 9 with saturated NaHCO3 solution, then

extracted with EtOAc (3 × 30 mL) and washed with saturated brine (3 × 30 mL). The organic phases
were combined and dried over anhydrous Na2SO4. The dried organic phase was decompressed to
remove the solvent, and the obtained residue was purified by silica gel chromatography on silica gel
(PE: EA = 3:1) to obtain the intermediates 5a1-g2.
The mixture of intermediates 5a1-g2 (0.9mmol) and trifluoroacetic acid (18mmol) in
dichloromethane (15mL) was stirred at room temperature for 20 minutes, and the reaction was
completed by TLC analysis. The volatiles were removed under reduced pressure and the residue
obtained was diluted with CH2Cl2 (10mL) and H2O (10mL). The phases were separated and the
aqueous phase was further extracted with CH2Cl2 (2 × 5 mL). The combined organic phases were
discarded and the aqueous phase was basified to pH 10 with saturated Na2CO3 and extracted with
EtOAc (3 × 10 mL). The combined organic phases were dried over anhydrous Na2SO4, and the
solvent was removed under reduced pressure to obtain the final products 6a1-g2.
A suspension of methyl 5-bromo-2-chloronicotinate (7) (8 mmol), (2,3-dichlorophenyl)boronic acid
(12 mmol), Pd(PPh3)4 (0.64 mmol) and Na2CO3 (24 mmol) in 1,4-dioxane/H2O (9:1, 80 mL) was
heated to 90 oC and stirred for 3 h. After the reaction was completed, the solvent was removed in
vacuo. The mixture was extracted with CH2Cl2 (3 × 50 mL) and washed with H2O (3 × 50 mL). The
organic phases were combined and dried over anhydrous Na2SO4. The organic phase after drying
was evaporated under reduced pressure, and the resulting residue obtained was purified by silica gel
chromatography on silica gel (PE: EA = 8:1) to gain the intermediate 8.
A mixture of intermediate 8 (4 mmol), organicamine compound (6 mmol), and K2CO3 (12 mmol)
in DMF (60 mL) was heated to 100 oC for 4 h. After the reaction was completed by TLC analysis,
the volatiles were removed under reduced pressure. The mixture was extracted with CH2Cl2 (3 × 40
mL) and washed with H2O (3 × 40 mL). The combined organic layers were dried over anhydrous
Na2SO4 and concentrated in vacuo. The resulting residue was purified by column chromatography
on silica gel (PE: EA = 5:1) to obtain the intermediates 9a-b.
The mixture of intermediates 9a-b (2 mmol) and trifluoroacetic acid (40 mmol) in dichloromethane
(40 mL) was stirred at room temperature for 20 minutes, and the reaction was completed by TLC
analysis. The volatiles were removed under reduced pressure and the residue obtained was diluted
with CH2Cl2 (20 mL) and H2O (20 mL). The phases were separated and the aqueous phase was
further extracted with CH2Cl2 (2 × 10 mL). The combined organic phases were discarded and the
aqueous phase was basified to pH 10 with saturated Na2CO3 and extracted with EtOAc (3 × 20 mL).
The combined organic phases were dried over anhydrous Na2SO4, and the solvent was removed
under reduced pressure to obtain the intermediates 10a-b.
A suspension of 10a-b (0.8 mmol) in Et2O (25 mL) was stirred in ice bath for 5 minutes. Then,
lithium aluminum hydride (8 mmol) was added to the reaction flask, and the reaction was continued
for 2 hours. After the reaction was completed, it was quenched by adding 15% sodium hydroxide
solution. The insoluble matter was then removed by filtration. The mixture was extracted with
CH2Cl2 (3 × 30 mL) and washed with H2O (3 × 20 mL). Organic phases were combined and dried
with anhydrous Na2SO4. The dried organic phase was evaporated under reduced pressure and the
residue obtained was purified by silica gel chromatography (CH2Cl2: MeOH = 10:1, 0.6%
ammonium hydroxide) to obtain the final products 11a-b.
4.1.1.1. 5-(2-chlorophenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6a1)
White solid, yield 89%, mp 167-169 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.49 (d, J = 8.0 Hz, 1H),

7.41 (d, J = 2.0 Hz, 1H), 7.28-7.37 (m, 3H), 6.80 (d, J = 2.0 Hz, 1H), 5.54 (d, J = 7.2 Hz, 1H), 4.90
(s, 2H), 3.90-4.02 (m, 1H), 2.98 (d, 2H), 2.84 (s, 1H), 2.56 (t, 2H), 1.92 (d, 2H), 1.28-1.39 (m, 2H);
13C NMR (100 MHz, DMSO-d6) δ 146.3, 138.2, 134.6, 131.6, 131.2, 129.8, 129.2, 128.2, 127.3,
123.0, 118.2, 47.6, 45.1 (2C), 33.1 (2C); MS (m/z): 303.1 [M + H]+.
4.1.1.2. 5-(3-chlorophenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6b1)
White solid, yield 88%, mp 161-163 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.73 (d, J = 2.0 Hz, 1H),
7.51 (s, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.40 (t, 1H), 7.29 (d, J = 8.0 Hz, 1H), 6.99 (d, J = 2.0 Hz,
1H), 5.61 (d, J = 7.2 Hz, 1H), 4.91 (s, 2H), 3.90-4.02 (m, 1H), 2.97 (d, 2H), 2.55 (t, 3H), 1.89 (d,
2H), 1.25-1.38 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 146.9, 141.2, 133.5, 133.0, 130.6, 130.1,
125.6, 124.8, 123.8, 122.6, 115.2, 48.0, 45.3 (2C), 33.5 (2C); MS (m/z): 303.1 [M + H]+.
4.1.1.3. 5-(4-chlorophenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6c1)
White solid, yield 87%, mp 164-166 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.71 (d, J = 2.0 Hz, 1H),
7.51 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 2.0 Hz, 1H), 5.56 (d, J = 7.2 Hz, 1H),
4.93 (s, 2H), 3.90-4.02 (m, 1H), 2.98 (d, 2H), 2.52-2.70 (m, 3H), 1.91 (d, 2H), 1.24-1.41 (m, 2H);
13C NMR (100 MHz, DMSO-d6) δ 146.7, 137.8, 132.8, 130.5, 130.1, 128.7 (2C), 126.9 (2C), 122.9,
115.1, 48.0, 45.4 (2C), 33.6 (2C); MS (m/z): 303.1 [M + H]+.
4.1.1.4. 5-(2-fluorophenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6d1)
o
1
White solid, yield 91%, mp 167-169 C, H NMR (400 MHz, DMSO-d6): δ 7.56 (s, 1H), 7.41 (t,
1H), 7.26-7.33 (m, 1H), 7.19-7.26 (m, 2H), 6.90 (s, 1H), 5.58 (d, J = 7.2 Hz, 1H), 4.92 (s, 2H), 3.90-
4.02 (m, 1H), 2.99 (d, 3H), 2.57 (t, 2H), 1.92 (d, 2H), 1.28-1.41 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 159.1 (d, J = 243.1 Hz), 146.5, 134.5 (d, J = 3.6 Hz), 129.8 (d, J = 3.8 Hz), 129.7,
128.1 (d, J = 8.1 Hz), 126.8 (d, J = 13.3 Hz), 124.8 (d, J = 3.3 Hz), 119.2 (J = 1.2 Hz), 117.4 (J =
3.0 Hz), 115.9 (d, J = 22.6 Hz), 47.7, 45.1 (2C), 33.2 (2C); MS (m/z): 287.2 [M + H]+.
4.1.1.5. 5-(2-methoxyphenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6e1)
White solid, yield 90%, mp 165-167 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.47 (d, J = 2.0 Hz, 1H),
7.24 (t, 1H), 7.19 ((dd, J = 1.6 Hz, J = 7.2 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.96 (t, 1H), 6.87 (d, J
= 2.0 Hz, 1H), 5.38 (d, J = 7.2 Hz, 1H), 4.77 (s, 2H), 3.88-4.02 (m, 1H), 3.74 (s, 3H), 2.98 (d, 2H),
2.90 (s, 1H), 2.56 (t, 2H), 1.92 (d, 2H), 1.26-1.39 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 156.2,
146.0, 134.8, 129.6, 129.0, 128.3, 127.7, 122.3, 120.6, 118.9, 111.5, 55.3, 47.8, 45.3 (2C), 33.5
(2C); MS (m/z): 299.2 [M + H]+.
4.1.1.6. N2-(piperidin-4-yl)-5-(o-tolyl)pyridine-2,3-diamine (6f1)
White solid, yield 89%, mp 161-163 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.33 (s, 1H), 7.09-7.27
(m, 4H), 6.70 (s, 1H), 5.43 (d, J = 7.2 Hz, 1H), 4.84 (s, 2H), 3.88-4.02 (m, 1H), 2.98 (d, 2H), 2.67
(s, 1H), 2.56 (t, 2H), 2.25 (s, 3H), 1.93 (d, 2H), 1.26-1.40 (m, 2H); 13C NMR (100 MHz, DMSO-
d6) δ 145.9, 139.5, 134.9, 134.4, 130.2, 129.4, 129.3, 126.5, 125.8, 125.2, 118.4, 47.9, 45.4 (2C),
33.6 (2C), 20.4; MS (m/z): 283.2 [M + H]+.
4.1.1.7. N2-(piperidin-4-yl)-5-(2-(trifluoromethyl)phenyl)pyridine-2,3-diamine (6g1)
White solid, yield 91%, mp 169-171 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.77 (d, J = 7.6 Hz, 1H),

7.66 (t, 1H), 7.53 (t, 1H), 7.37 (d, J = 7.6 Hz, 1H), 7.29 (d, J = 1.6 Hz, 1H), 6.67 (d, J = 1.6 Hz,
1H), 5.50 (d, J = 7.2 Hz, 1H), 4.91 (s, 2H), 3.86-4.03 (m, 3H), 2.97 (d, 2H), 2.56 (t, 2H), 1.91 (d,
2H), 1.26-1.40 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 146.4, 139.3 (d, J = 1.9 Hz), 133.9,
132.5, 132.1, 129.0, 127.2, 126.9, 125.9, 124.3 (d, J = 272.3 Hz), 123.5, 117.9, 47.9, 45.3 (2C), 33.5
(2C); MS (m/z): 337.2 [M + H]+.
4.1.1.8. N6-(piperidin-4-yl)-[3,3'-bipyridine]-5,6-diamine (6h1)
Pale yellow solid, yield 91%, mp 171-173 oC, 1H NMR (400 MHz, DMSO-d6): δ 8.75 (d, J = 2.0
Hz, 1H), 8.44-8.48 (m, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.37-8.44 (m, 1H),
7.02 (d, J = 2.0 Hz, 1H), 5.65 (d, J = 7.2 Hz, 1H), 4.99 (d, 2H), 3.89-4.06 (m, 1H), 2.95-3.05 (m,
2H), 2.80 (s, 1H), 2.53-2.63 (m, 2H), 1.88-2.00 (m, 2H), 1.27-1.43 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 147.0, 146.9, 146.4, 134.4, 133.0, 132.5, 130.3, 123.7, 121.0, 115.0, 48.0, 45.4 (2C),
33.5 (2C); MS (m/z): 270.2 [M + H]+.
4.1.1.9. N6-(piperidin-4-yl)-[3,4'-bipyridine]-5,6-diamine (6i1)
Pale yellow solid, yield 89%, mp 163-165 oC, 1H NMR (400 MHz, DMSO-d6): δ 8.49-8.54 (m, 2H),
7.91 (d, J = 2.0 Hz, 1H), 7.51-7.55 (m, 2H), 7.10 (d, J = 2.0 Hz, 1H), 5.85 (d, J = 7.2 Hz, 1H), 5.04
(s, 2H), 3.99-4.09 (m, 1H), 3.79-3.98 (s, 3H), 3.02-3.12 (m, 2H), 2.61-2.73 (m, 2H), 1.92-2.01 (m,
2H), 1.35-1.48 (m, 2H); 13C NMR (101 MHz, DMSO) δ 149.9 (2C), 147.6, 145.8, 133.5, 130.3,
120.8, 119.5 (2C), 114.4, 47.9, 44.7 (2C), 32.4 (2C); MS (m/z): 270.2 [M + H]+.
4.1.1.10. 2'-chloro-N6-(piperidin-4-yl)-[3,3'-bipyridine]-5,6-diamine (6j1)
White solid, yield 92%, mp 166-168 oC, 1H NMR (400 MHz, DMSO-d6): δ 8.34 (dd, J = 2.0 Hz, J
= 7.6 Hz, 1H), 7.79 (dd, J = 2.0 Hz, J = 7.6 Hz, 1H), 7.42-7.48 (m, 2H), 6.83 (d, J = 2.0 Hz, 1H),
5.63 (d, J = 7.2 Hz, 1H), 4.96 (s, 2H), 3.91-4.03 (m, 1H), 2.98 (d, 2H), 2.56 (t, 3H), 1.82 (d, 2H),
1.26-1.40 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 148.66, 147.6, 146.7, 139.8, 134.8, 134.8,
129.3, 123.4, 121.1, 117.6, 48.0, 45.4 (2C), 33.5 (2C); MS (m/z): 305.2 [M + H]+.
4.1.1.11. 5-(2,3-dichlorophenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6k1)
White solid, yield 91%, mp 170-172 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.57 (d, J = 7.6 Hz, 1H),
7.40 (d, J = 1.6 Hz, 1H), 7.37 (t, 1H), 7.30 (d, J = 7.6 Hz, 1H), 6.77 (d, J = 1.6 Hz, 1H), 5.59 (d, J
= 7.2 Hz, 1H), 4.92 (s, 2H), 3.98-4.03 (m, 1H), 2.97 (d, 2H), 2.53 (d, 2H), 2.27 (s, 1H), 1.89 (d, 2H),
1.25-1.39 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ 146.6, 140.9, 134.6, 132.2, 129.8 (2C), 129.2,
128.8, 128.1, 122.8, 117.8, 47.9, 45.3, (2C) 33.5 (2C); MS (m/z): 337.1 [M + H]+.
4.1.1.12. 5-(2,4-dichlorophenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6l1)
White solid, yield 87%, mp 172-174 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.65 (d, J = 2.4 Hz, 1H),
7.44 (dd, J = 2.0 Hz, J = 8.0 Hz, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 6.77 (d, J
= 2.4 Hz, 1H), 5.58 (d, J = 7.2 Hz, 1H), 4.92 (s, 2H), 3.89-4.01 (m, 1H), 2.96 (d, 2H), 2.54 (d, 2H),
2.28 (s, 1H), 1.90 (d, 2H), 1.25-1.37 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 146.5, 137.3,
134.7, 132.5, 132.3, 131.8, 129.2, 129.1, 127.5, 121.8, 117.8, 48.0, 45.4 (2C), 33.6 (2C); MS (m/z):
337.1 [M + H]+.
4.1.1.13. 5-(3,4-dichlorophenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6ml)

o
1
White solid, yield 89%, mp 160-162 C, H NMR (400 MHz, DMSO-d6): δ 7.75 (s, 1H), 7.71 (s,
1H), 7.61 (d, J = 7.6 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 6.99 (s, 1H), 5.65 (d, J = 7.2 Hz, 1H), 4.93
(s, 2H), 3.90-4.02 (m, 1H), 2.98 (d, 2H), 2.56 (d, 2H), 1.90 (d, 2H), 1.25-1.40 (m, 2H), 1.22 (s, 1H);
13C NMR (100 MHz, DMSO-d6) δ 147.0, 139.7, 133.1, 131.5, 130.8, 130.1, 128.2, 126.7, 125.3,
121.5, 114.9, 48.0, 45.3 (2C), 33.4 (2C); MS (m/z): 337.1 [M + H]+.
4.1.1.14. 5-(2-chloro-4-methylphenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6nl)
White solid, yield 90%, mp 161-163 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.39 (d, J = 2.0 Hz, 1H),
7.33 (s, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.16 (d, J = 7.6 Hz, 1H), 6.78 (d, J = 2.0 Hz, 1H), 5.51 (d, J
= 7.2 Hz, 1H), 4.88 (s, 2H), 3.88-4.02 (m, 1H), 2.98 (d, 2H), 2.74 (s, 1H), 2.55 (t, 2H), 2.31 (s, 3H),
1.90 (d, 2H), 1.26-1.40 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 146.2, 137.9, 135.3, 134.6,
131.2, 130.9, 130.0, 129.1, 128.0, 122.9, 118.3, 47.9, 45.4 (2C), 33.6 (2C), 20.2; MS (m/z): 317.2
[M + H]+.
4.1.1.15. 5-(2,3-difluorophenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6o1)
White solid, yield 89%, mp 166-168 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.60 (s, 1H), 7.28-7.35
(m, 1H), 7.19 -7.27 (m, 2H), 6.92 (s, 1H), 5.68 (d, J = 7.2 Hz, 1H), 4.98 (s, 2H), 3.92-4.06 (m, 1H),
3.52 (s, 1H), 3.02 (d, 2H), 2.61 (t, 2H), 1.94 (d, 2H), 1.28-1.45 (m, 2H); 13C NMR (100 MHz,
DMSO-d6): δ 150.3 (dd, J = 13.4 Hz, J = 243.3 Hz), 146.9 (dd, J = 12.8 Hz, J = 244.5 Hz), 146.8,
134.6 (d, J = 3.8 Hz), 129.8, 129.2 (d, J = 10.0 Hz), 124.8, 124.7, 118.1, 117.0 (d, J = 2.8 Hz), 114.9
(d, J = 17.1 Hz), 47.7, 45.1 (2C), 33.0 (2C); MS (m/z): 305.2 [M + H]+.
4.1.1.16. 5-(2,4-difluorophenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6p1)
White solid, yield 91%, mp 164-166 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.52 (s, 1H), 7.40-7.48
(s, 1H), 7.28 (t, 1H), 7.12 (t, 1H), 6.86 (s, 1H), 5.59 (d, J = 7.2 Hz, 1H), 4.93 (s, 2H), 3.90-4.02 (m,
1H), 3.07 (s, 1H), 2.99 (d, 2H), 2.57 (t, 2H), 1.91 (d, 2H), 1.27-1.40 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 160.8 (dd, J = 11.7 Hz, J = 243.3 Hz), 159.0 (dd, J = 12.0 Hz, J = 245.7 Hz), 146.5,
134.3 (d, J = 3.1 Hz), 130.8, 129.7, 123.5 (dd, J = 3.7 Hz, J = 13.7 Hz), 118.4 (d, J = 1.4 Hz), 117.3
(J = 2.8 Hz), 111.9 (dd, J = 3.5 Hz, J = 20.8 Hz), 104.3, 47.7, 45.2 (2C), 33.2 (2C); MS (m/z): 305.2
[M + H]+.
4.1.1.17. 5-(2,3-dichlorophenyl)-2-(piperazin-1-yl)pyridin-3-amine (6q1)
White solid, yield 90%, mp 162-164 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.65 (d, J = 8.0 Hz, 1H),
7.60 (d, J = 1.6 Hz, 1H), 7.42 (t, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 1.6 Hz, 1H), 4.93 (s,
2H), 2.85-3.01 (m, 9H); 13C NMR (100 MHz, DMSO-d6) δ 149.9, 140.0, 135.6, 134.7, 132.3, 130.0,
129.9, 129.6, 129.6, 128.3, 120.8, 49.3 (2C), 45.6 (2C); MS (m/z): 323.1 [M + H]+.
4.1.1.18. 5-(2,3-difluorophenyl)-N2-(piperidin-4-yl)pyridine-2,3-diamine (6r1)
White solid, yield 88%, mp 168-170 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.76 (s, 1H), 7.37-7.46
(m, 1H), 7.25-7.35 (m, 2H), 7.16 (s, 1H), 4.97 (s, 2H), 2.84-3.05 (m, 9H); 13C NMR (100 MHz,
DMSO-d6) δ 150.2 (dd, J = 13.3 Hz, J = 243.8 Hz), 150.2, 147.0 (dd, J = 12.9 Hz, J = 245.4 Hz),
136.0, 134.6, 128.3 (d, J = 1.6 Hz), 125.3, 125.1, 124.9, 120.2 (d, J = 2.6 Hz), 116.2 (d, J = 17.0
Hz), 49.2 (2C), 45.5 (2C); MS (m/z): 291.1 [M + H]+.

4.3.1.19. 5-(2-chlorophenyl)-2-(piperazin-1-yl)pyridin-3-amine (6s1)
White solid, yield 92%, mp 167-169 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.60 (d, J = 2.0 Hz, 1H),
7.52-7.57 (m, 1H), 7.36-7.42 (m, 3H), 7.05 (d, J = 2.0 Hz, 1H), 4.93 (s, 2H), 3.24 (s, 1H), 2.95-3.02
(m, 4H), 2.86-2.94 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 149.6, 137.4, 135.6, 134.8, 131.6,
131.4, 129.8 (2C), 129.1, 127.5, 121.1, 49.2 (2C), 45.5 (2C); MS (m/z): 289.1 [M + H]+.
4.1.1.20. 2-(piperazin-1-yl)-5-(2-(trifluoromethyl)phenyl)pyridin-3-amine (6t1)
White solid, yield 90%, mp 162-164 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.82 (d, J = 7.6 Hz, 1H),
7.71 (t, 1H), 7.60 (t, 1H), 7.50 (d, J = 2.0 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 6.94 (d, J = 2.0 Hz,
1H), 4.94 (s, 2H), 2.85-3.06 (m, 9H); 13C NMR (101 MHz, DMSO) δ 149.7, 138.2 (d, J = 1.9 Hz),
135.4, 134.3, 132.3, 132.2, 130.3, 127.9, 127.1 (d, J = 28.9 Hz), 126.0, 124.2 (d, J = 272.3 Hz),
120.7, 49.2 (2C), 45.6 (2C); MS (m/z): 323.1 [M + H]+.
4.1.1.21. 5-(3,4-dichlorophenyl)-2-(piperazin-1-yl)pyridin-3-amine (6u1)
o
1
White solid, yield 87%, mp 165-167 C, H NMR (400 MHz, DMSO-d6): δ 7.91 (s, 1H), 7.82 (s,
1H), 7.69 (d, d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.24 (s, 1H), 4.91 (s, 2H), 2.94-3.02 (m,
4H), 2.84-2.92 (m, 4H), 2.68 (s, 1H); 13C NMR (101 MHz, DMSO) δ 150.4, 138.9, 136.3, 133.1,
131.6, 131.0, 129.7, 128.4, 127.8, 126.3, 118.2, 49.2 (2C), 45.5 (2C); MS (m/z): 323.1 [M + H]+.
4.1.1.22. 2-(4-aminopiperidin-1-yl)-5-(2,3-dichlorophenyl)pyridin-3-amine (6v1)
White solid, yield 92%, mp 164-166 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.65 (dd, J = 1.2 Hz, J
= 8.0 Hz, 1H), 7.58 (d, J = 2.0 Hz, 1H), 7.42 (t, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.02 (d, J = 2.0 Hz,
1H), 4.90 (s, 2H), 3.39 (d, 2H), 2.62-2.75 (m, 3H), 1.80 (d, 2H), 1.69 (s, 2H), 1.37-1.50 (m, 2H);
13C NMR (100 MHz, DMSO-d6) δ 150.1, 140.0, 135.6, 134.7, 132.3, 130.0, 129.9, 129.6, 129.5,
128.3, 120.7, 48.5, 47.2 (2C), 35.5 (2C); MS (m/z): 337.1 [M + H]+.
4.1.1.23. 2-(4-(aminomethyl)piperidin-1-yl)-5-(2,3-dichlorophenyl)pyridin-3-amine (6w1)
White solid, yield 90%, mp 167-169 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.65 (dd, J = 1.2 Hz, J
= 8.0 Hz, 1H), 7.58 (d, J = 2.0 Hz, 1H), 7.42 (t, 1H), 7.35 (dd, J = 1.6 Hz, J = 7.6 Hz, 1H), 7.02 (d,
J = 2.0 Hz, 1H), 4.90 (s, 2H), 3.45 (d, 2H), 2.61 (t, 2H), 2.49 (d, 2H), 1.80 (d, 2H), 1.61 (s, 2H),
1.27-1.41 (m, 3H); 13C NMR (100 MHz, DMSO-d6) δ 150.3, 140.0, 135.7, 134.7, 132.3, 130.0,
129.9, 129.5 (2C), 128.3, 120.7, 48.5 (2C), 47.8, 29.9 (3C); MS (m/z): 351.1 [M + H]+.
4.1.1.24. 6-(4-(aminomethyl)piperidin-1-yl)-[3,3'-bipyridin]-5-amine (6x1)
Pale yellow solid, yield 90%, mp 170-172 oC, 1H NMR (400 MHz, DMSO-d6): δ 8.82 (d, J = 2.0
Hz, 1H), 8.55 (dd, J = 0.8 Hz, J = 4.8 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 2.0 Hz, 1H),
7.44-7.50 (m, 1H), 7.27 (d, J = 2.0 Hz, 1H), 4.94 (s, 2H), 3.46 (d, 2H), 2.63 (t, 2H), 2.52 (d, 2H),
2.26 (s, 2H), 1.80 (d, 2H), 1.26-1.44 (m, 3H); 13C NMR (100 MHz, DMSO-d6) δ 150.7, 148.1,
147.1, 136.5, 133.7, 133.6, 133.1, 127.9, 123.8, 118.1, 48.6 (2C), 47.9, 39.2, 29.8 (2C); MS (m/z):
284.2 [M + H]+.
4.1.1.25. 5-(2,3-dichlorophenyl)-N2-(piperidin-4-ylmethyl)pyridine-2,3-diamine (6y1)
White solid, yield 89%, mp 171-173 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.57 (d, J = 7.6 Hz, 1H),
7.40 (s, 1H), 7.36 (t, 1H), 7.30 (d, J = 7.6 Hz, 1H), 6.77 (s, 1H), 5.84 (t, 1H), 4.91 (s, 2H), 3.18-3.33

(m, 3H), 2.97 (d, 2H), 2.46 (t, 2H), 1.70 (d, 3H), 1.01-1.15 (m, 2H); 13C NMR (100 MHz, DMSO-
d6) δ 147.5, 140.9, 134.7, 132.2, 129.8, 129.2, 128.7, 128.1, 122.8, 117.7, 47.2, 45.6 (2C), 35.6, 30.8
(2C); MS (m/z): 351.1 [M + H]+.
4.1.1.26. 2-(4-amino-4-methylpiperidin-1-yl)-5-(2,3-dichlorophenyl)pyridin-3-amine (6z1)
White solid, yield 87%, mp 164-166 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.64 (d, J = 7.6 Hz, 1H),
7.59 (s, 1H), 7.42 (t, 1H), 7.37 (d, J = 7.6 Hz, 1H), 7.02 (s, 1H), 4.87 (s, 2H), 2.99-3.14 (m, 4H),
1.44-1.68 (m, 6H), 1.11 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 150.2, 140.1, 135.5, 134.7, 132.3,
130.0, 129.9, 129.6, 129.4, 128.3, 120.7, 54.9, 46.8, 44.7 (2C), 39.6 (3C); MS (m/z): 351.1 [M +
H]+.
4.1.1.27. 2-(3-aminopiperidin-1-yl)-5-(2,3-dichlorophenyl)pyridin-3-amine (6a2)
White solid, yield 88%, mp 167-169 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.65 (dd, J = 1.6 Hz, J
= 7.6 Hz, 1H), 7.58 (d, J = 2.0 Hz, 1H), 7.42 (t, 1H), 7.36 (dd, J = 1.6 Hz, J = 7.6 Hz, 1H), 7.01 (d,
J = 2.0 Hz, 1H), 4.98 (s, 2H), 3.28 (d, 1H), 3.17 (d, 1H), 2.89 (t, 1H), 2.71 (t, 1H), 2.51 (t, 1H), 2.33
(s, 2H), 1.80 (t, 2H), 1.56-1.70 (m, 1H), 1.14-1.30 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ
150.0, 140.0, 135.7, 134.6, 132.3, 130.0, 129.9, 129.6, 129.5, 128.3, 120.7, 57.2, 48.5, 47.5, 33.6,
23.3; MS (m/z): 337.1 [M + H]+.
4.1.1.28. 6-(pyrrolidin-3-ylmethyl)-[3,3'-bipyridin]-5-amine (6b2)
White solid, yield 90%, mp 162-164 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.58 (dd, J = 1.6 Hz, J
= 8.0 Hz, 1H), 7.42 (d, J = 2.0 Hz, 1H), 7.37 (t, 1H), 7.31 (dd, J = 1.6 Hz, J = 8.0 Hz, 1H), 6.79 (d,
J = 2.0 Hz, 1H), 5.97 (t, 1H), 4.95 (s, 2H), 3.93 (s, 1H), 3.26-3.42 (m, 2H), 2.87-2.98 (m, 2H), 2.75-
2.84 (m, 1H), 2.59-2.67 (m, 1H), 2.39-2.48 (m, 1H), 1.80-1.91 (m, 1H), 1.39-1.51 (m, 1H); 13C
NMR (100 MHz, DMSO-d6) δ 147.4, 140.8, 134.6, 132.2, 129.8 (2C), 129.3, 128.8, 128.1, 123.0,
117.7, 50.3, 45.8, 44.6, 38.4, 29.8; MS (m/z): 337.1 [M + H]+.
4.1.1.29. N2-(4-aminophenyl)-5-(2,3-dichlorophenyl)pyridine-2,3-diamine (6c2)
Pale yellow solid, yield 88%, mp 191-193 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.59 (dd, J = 1.6
Hz, J = 7.6 Hz, 1H), 7.50 (s, 1H), 7.43 (t, 1H), 7.39 (t, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.30 (d, J =
8.4 Hz, 2H), 6.90 (s, 1H), 6.53 (d, J = 8.4 Hz, 2H), 5.12 (s, 2H), 4.73 (s, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 144.8, 143.2, 140.5, 134.3, 132.3, 130.8, 129.9 (2C), 129.8, 129.0, 128.2, 124.6, 121.7
(2C), 118.9, 114.0 (2C); MS (m/z): 345.1 [M + H]+.
4.1.1.30. N2-(4-aminophenyl)-5-(2-chlorophenyl)pyridine-2,3-diamine (6d2)
Pale yellow solid, yield 87%, mp 195-197 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.52 (d, J = 7.6
Hz, 1H), 7.42-7.47 (m, 2H), 7.33-7.39 (m, 3H), 7.31 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 1.6 Hz, 2H),
6.55 (d, J = 8.4 Hz, 2H), 5.10 (s, 2H), 4.72 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 144.6, 143.1,
137.9, 134.3, 131.6, 131.3, 131.0, 129.9, 129.8, 128.5, 127.4, 124.7, 121.5 (2C), 119.3, 114.0 (2C);
MS (m/z): 311.1 [M + H]+.
4.1.1.31. N2-(4-aminophenyl)-5-(3,4-dichlorophenyl)pyridine-2,3-diamine (6e2)
Pale yellow solid, yield 89%, mp 193-195 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.78 (dd, J = 2.0
Hz, J = 8.0 Hz, 2H), 7.63 (dd, J = 2.0 Hz, J = 8.4 Hz, 1H), 7.57 (s, 1H), 7.54 (dd, J = 2.0 Hz, J =

8.4 Hz, 1H), 7.32 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 2.0 Hz, 2H), 6.54 (d, J = 8.4 Hz, 2H), 5.13 (s,
2H), 4.75 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 145.2, 143.2, 139.3, 132.7, 131.5, 130.9, 130.8,
130.8, 128.6, 126.9, 125.5, 123.3, 121.6 (2C), 116.0, 114.0 (2C); MS (m/z): 345.1 [M + H]+.
4.1.1.31. N2-(3-aminophenyl)-5-(2,3-dichlorophenyl)pyridine-2,3-diamine (6f2)
Pale yellow solid, yield 90%, mp 197-199 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.61 (d, 2H), 7.52
(d, J = 2.0 Hz, 1H), 7.41 (t, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.00 (s, 1H), 6.96 (d, J = 2.0 Hz, 1H),
6.89 (t, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.16 (d, J = 7.6 Hz, 1H), 5.25 (s, 2H), 4.95 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 148.7, 143.7, 142.3, 140.3, 134.1, 132.3, 130.8, 129.9 (2C), 129.9, 129.2,
128.6, 128.3, 125.8, 119.6, 107.2, 107.1, 104.5; MS (m/z): 345.1 [M + H]+.
4.1.1.32. N2-(3-aminophenyl)-5-(2,3-difluorophenyl)pyridine-2,3-diamine (6g2)
Pale yellow solid, yield 92%, mp 195-197 oC, 1H NMR (400 MHz, DMSO-d6): δ 7.58-7.65 (m, 2H),
7.52 (d, J = 2.0 Hz, 1H), 7.41 (t, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.00 (s, 1H), 6.96 (d, J = 2.0 Hz,
1H), 6.89 (t, 1H), 6.84 (d, J = 7.6 Hz, 1H), 6.16 (d, J = 8.0 Hz, 1H), 5.25 (s, 2H), 4.95 (s, 2H); 13
C
NMR (100 MHz, DMSO-d6) δ 150.4 (dd, J = 13.3 Hz, J = 243.6 Hz), 148.7, 147.1 (dd, J = 12.8 Hz,
J = 244.8 Hz), 144.0, 142.2, 134.0 (d, J = 3.8 Hz), 132.7 (d, J = 101.9 Hz), 132.0 (d, J = 2.7 Hz),
131.4, 128.7, 124.9, 120.0, 118.8 (d, J = 2.7 Hz), 115.5 (d, J = 17.0 Hz), 107.3, 107.2, 104.6; MS
(m/z): 313.1 [M + H]+.
4.1.1.34. (2-(4-amino-4-methylpiperidin-1-yl)-5-(2,3-dichlorophenyl)pyridin-3-yl)methanol (11a)
White solid, yield 81%, mp 159-161 oC, 1H NMR (400 MHz, DMSO-d6): δ 8.19 (d, J = 2.0 Hz, 1H),
7.84 (d, J = 2.0 Hz, 1H), 7.67 (dd, J = 1.6 Hz, J = 7.6 Hz, 1H), 7.45 (t, 1H), 5.40 (dd, J = 1.6 Hz, J
= 7.6 Hz, 1H), 5.40 (s, 1H), 4.50 (s, 2H), 3.07-3.26 (m, 4H), 1.45-1.62 (m, 4H), 1.10 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) δ 159.4, 145.2, 139.6, 137.1, 132.4, 130.0, 129.9, 129.7, 128.4, 127.3
(2C), 58.5, 46.8 (2C), 46.4 (3C), 30.3; MS (m/z): 366.1 [M + H]+.
4.1.1.35. (2-(4-(aminomethyl)piperidin-1-yl)-5-(2,3-dichlorophenyl)pyridin-3-yl)methanol (11b)
White solid, yield 82%, mp 160-162 oC, 1H NMR (400 MHz, DMSO-d6): δ 8.21 (d, J = 2.0 Hz, 1H),
7.85 (s, 1H), 7.61-7.72 (m, 1H), 7.37-7.49 (m, 2H), 4.51 (s, 2H), 3.49 (d, 2H), 2.75 (t, 2H), 2.47 (d,
2H), 1.79 (d, 2H), 1.33-1.45 (m, 1H), 1.19-1.31 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 159.6,
145.2, 139.5, 137.1, 132.4, 130.0, 129.9, 129.7, 128.4, 127.6, 127.6, 58.4, 50.2 (2C), 47.8, 30.0
(2C); MS (m/z): 366.1 [M + H]+.
4.2. Biological evaluation
4.2.1 In vitro phosphatase activity assay
The purified SHP2 and SHP1 were used as the enzymes, and the peptide sequence
EFpYAEVGRSPPDPAK (H-Glu-Phe-pTyr-Ala-Glu-Val-Gly-Arg-Ser-Pro-Pro-Asp-Pro-Ala-Lys)
was used as the substrate[32]. The assay determined free phosphate generated by dephosphorylation
of the substrate using the Malachite Green Phosphate Assay Kit. First, 0.4 mg/mLSHP2 or SHP1
protein, 0.06 mM substrate and assay buffer (25 mM Tris-HCl, 50 mM NaCl, 5 mM DTT, and 2.5
mM EDTA, pH 7.4) with the tested compounds at various concentrations were added in 96well
plates at room temperature for 0.5 h. Subsequently, 25 μL NaOH (4 M) was added to the sample,
and heated at 100 °C for 30 minutes and cooled to room temperature. Then, after the sample was

cooled to room temperature, 25 μL HCl (4 M) was added. Finally, 20 μL malachite green working
reagent was added, and OD620 was measured after 0.5 h at room temperature. The IC50 value against
SHP2 or SHP1 protein was gained. The result was analyzed by Graph-Pad Prism 7 software.
4.2.2. Fluorescence titrations
To verify whether the compound bound directly to the SHP2 protein, fluorescence quenching assay
was used to examine change in the fluorescence of the SHP2 protein. The purified SHP2 his-fusion
protein was diluted into reaction buffer with pH 7.4. The titration was carried out by increasing the
concentration of the compounds while maintaining the SHP2 protein concentration at 0.4 mg/mL.
The excitation wavelength was 295 nm, and fluorescence of 360 to 500 nm was monitored. All
reported fluorescence intensities were relative values and were not corrected for wavelength
variations in detector response.
4.2.3. Cell proliferation assay
Mouse B lymphocyte Ba/F3 from the American Type Culture Collection (ATCC; Manassas, VA,
USA) was incubated in RPMI 1640 (Gibco) medium supplemented with 10% fetal bovine serum
(FBS; Origin South America sterile filtered), 1% penicillin-streptomycin, and 2 ng/ml murine IL-3
at 37°C in a humidified atmosphere of 5% CO2. Cell growth was measured by MTT assay.
Specifically, cells were seeded in 96-well plates at a density of 1.2×105 cells/mL and were untreated
(control) or treated with test compounds solutions in 96-well plates. After incubation for 48 h, 20
μL/well MTT reagent was added and incubated for 4 h at 37°C. The reaction was stopped by adding
150 μL dimethyl sulfoxide. The absorbance density was obtained at wavelength 490 nm on a 96-
well plate reader (NEST, Nest Biotechnology). IC50 value was calculated as the concentration of the
drug required to obtain 50% of maximal inhibition in cell viability.
4.8. In silico ADMET prediction
The ADMET (absorption, distribution, metabolism, excretion, and toxicity) studies were performed
using DS v3.5 software. Some important ADMET descriptors include human intestinal absorption
(HIA)[33] (Prediction of drug absorption using multivariate statistics), plasma protein binding
(PPB)[34] (Atomic solvation parameters applied to molecular dynamics of proteins in solution),
aqueous solubility[35] (Prediction of aqueous solubility of a diverse set of compounds using
quantitative structure-property relationships.), Cytochrome P450 2D6 binding (CYP2D6)[36] (Use
of robust classification techniques for the prediction of human cytochrome P450 2D6 inhibition.),
and hepatotoxicity[37] (In silico models for the prediction of dose-dependent human hepatotoxicity).
4.7. Molecular docking
Molecular docking study was performed by using the Discovery Studio 3.5 software. The X-ray
crystal structure of SHP2 was derived from the RCSB protein database, identifier PDB ID: 5EHR.
First, the preparation of proteins and the energy of the ligands was minimized by the CHARMm
force field (). Subsequently, the receptor binding pocket was defined by the coordinate space of the
extracted co-crystallized ligand[38] (Scaffold-based novel SHP2 allosteric inhibitors design using
Receptor-Ligand pharmacophore model, virtual screening and molecular dynamics. Comput Biol
Chem). The binding pocket of a protein was generally defined by a residue having at least one heavy
atom, and the distance between this heavy atom and the heavy atom of the ligand was 5 Å[39]

(Design novel dual agonists for treating type-2 diabetes by targeting peroxisome proliferator-
activated receptors with core hopping approach.). Finally, the docking module in the DS v3.5
program was performed to dock the ligands into the protein. The conformation with the highest
docking score was used to analyze the interaction between the protein and the ligand.
CONFLICT OF INTEREST
The authors report no conflicts of interest in this work.
References
1.
Hof, P., et al., Crystal structure of the tyrosine phosphatase SHP-2. Cell, 1998. 92(4):
p. 441-50.
2.
LaRochelle, J.R., et al., Structural reorganization of SHP2 by oncogenic mutations
and implications for oncoprotein resistance to allosteric inhibition. Nat Commun,
2018. 9(1): p. 4508.
3.
4.
5.
6.
Barford, D. and B.G. Neel, Revealing mechanisms for SH2 domain mediated
regulation of the protein tyrosine phosphatase SHP-2. Structure, 1998. 6(3): p. 249-
54.
Pluskey, S., et al., Potent stimulation of SH-PTP2 phosphatase activity by
simultaneous occupancy of both SH2 domains. J Biol Chem, 1995. 270(7): p. 2897-
900.
Tartaglia, M., et al., Mutations in PTPN11, encoding the protein tyrosine phosphatase
SHP-2, cause Noonan syndrome (vol 29, pg 465, 2001). Nature Genetics, 2002.
30(1): p. 123-123.
Tartaglia, M., et al., Somatic mutations in PTPN11 in juvenile myelomonocytic
leukemia, myelodysplastic syndromes and acute myeloid leykemia (vol 34, pg 148,

2003). Nature Genetics, 2003. 34(4): p. 464-464.
7.
Bentires-Alj, M., et al., Activating mutations of the Noonan syndrome-associated
SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia.
Cancer Research, 2004. 64(24): p. 8816-8820.
8.
9.
Chan, R.J. and G.S. Feng, PTPN11 is the first identified proto-oncogene that
encodes a tyrosine phosphatase. Blood, 2007. 109(3): p. 862-867.
Neel, B.G., H.H. Gu, and L. Pao, The 'Shp'ing news: SH2 domain-containing tyrosine
phosphatases in cell signaling. Trends in Biochemical Sciences, 2003. 28(6): p. 284-
293.
10.
Pan, J.H. and X. Liu, [Assessment of landscape ecological security and optimization
of landscape pattern based on spatial principal component analysis and resistance
model in arid inland area: A case study of Ganzhou District, Zhangye City, Northwest
China]. Ying Yong Sheng Tai Xue Bao, 2015. 26(10): p. 3126-36.
Gavrieli, M., et al., Characterization of phosphotyrosine binding motifs in the
cytoplasmic domain of B and T lymphocyte attenuator required for association with
protein tyrosine phosphatases SHP-1 and SHP-2. Biochem Biophys Res Commun,
2003. 312(4): p. 1236-43.
11.
12.
13.
14.
Scott, L.M., et al., Targeting protein tyrosine phosphatases for anticancer drug
discovery. Curr Pharm Des, 2010. 16(16): p. 1843-62.
Zeng, L.F., et al., Therapeutic potential of targeting the oncogenic SHP2
phosphatase. J Med Chem, 2014. 57(15): p. 6594-609.
Zhang, X., et al., Salicylic acid based small molecule inhibitor for the oncogenic Src

homology-2 domain containing protein tyrosine phosphatase-2 (SHP2). J Med Chem,
2010. 53(6): p. 2482-93.
15.
16.
Zeng, L.F., et al., Therapeutic Potential of Targeting the Oncogenic SHP2
Phosphatase. Journal of Medicinal Chemistry, 2014. 57(15): p. 6594-6609.
Lawrence, H.R., et al., Inhibitors of Src homology-2 domain containing protein
tyrosine phosphatase-2 (Shp2) based on oxindole scaffolds. Journal of Medicinal
Chemistry, 2008. 51(16): p. 4948-4956.
17.
18.
Noren-Muller, A., et al., Discovery of protein phosphatase inhibitor classes by biology-
oriented synthesis. Proceedings of the National Academy of Sciences of the United
States of America, 2006. 103(28): p. 10606-10611.
Fortanet, J.G., et al., Allosteric Inhibition of SHP2: Identification of a Potent, Selective,
and Orally Efficacious Phosphatase Inhibitor. Journal of Medicinal Chemistry, 2016.
59(17): p. 7773-7782.
19.
20.
Chen, Y.N.P., et al., Allosteric inhibition of SHP2 phosphatase inhibits cancers driven
by receptor tyrosine kinases. Nature, 2016. 535(7610): p. 148-+.
Nichols, R.J., et al., RAS nucleotide cycling underlies the SHP2 phosphatase
dependence of mutant BRAF-, NF1-and RAS-driven cancers. Nature Cell Biology,
2018. 20(9): p. 1064-+.
21.
22.
Sarver, P., et al., 6-Amino-3-methylpyrimidinones as Potent, Selective, and Orally
Efficacious SHP2 Inhibitors (vol 62, pg 1793, 2019). Journal of Medicinal Chemistry,
2019. 62(7): p. 3781-3781.
Bagdanoff, J.T., et al., Optimization of Fused Bicyclic Allosteric SHP2 Inhibitors.

Journal of Medicinal Chemistry, 2019. 62(4): p. 1781-1792.
Krueger, B.A., et al., Scaffold-Hopping Potential of Fragment-Based De Novo Design:
The Chances and Limits of Variation. Combinatorial Chemistry & High Throughput
Screening, 2009. 12(4): p. 383-396.
23.
24.
25.
26.
27.
28.
29.
30.
The scaffold hopping potential of pharmacophores. Drug Discov Today Technol,
2010. 7(4): p. e203-70.
Hu, Y., D. Stumpfe, and J. Bajorath, Recent Advances in Scaffold Hopping. J Med
Chem, 2017. 60(4): p. 1238-1246.
Yang, J., et al., Crystal structure of human protein-tyrosine phosphatase SHP-1. J
Biol Chem, 2003. 278(8): p. 6516-20.
Norinder, U. and C.A. Bergstrom, Prediction of ADMET Properties. ChemMedChem,
2006. 1(9): p. 920-37.
Gleeson, M.P., Generation of a set of simple, interpretable ADMET rules of thumb. J
Med Chem, 2008. 51(4): p. 817-34.
Egan, W.J. and G. Lauri, Prediction of intestinal permeability. Adv Drug Deliv Rev,
2002. 54(3): p. 273-89.
Liu, J.L., et al., Structural Determination of Three Different Series of Compounds as
Hsp90 Inhibitors Using 3D-QSAR Modeling, Molecular Docking and Molecular
Dynamics Methods. International Journal of Molecular Sciences, 2011. 12(2): p. 946-
970.
31.
Wang, M.Y., et al., Synthesis, biological evaluation and 3D-QSAR studies of
imidazolidine-2,4-dione derivatives as novel protein tyrosine phosphatase 1B

inhibitors. European Journal of Medicinal Chemistry, 2015. 103: p. 91-104.
Wu, X.Q., et al., Small Molecule Inhibitor that Stabilizes the Autoinhibited
Conformation of the Oncogenic Tyrosine Phosphatase SHP2. Journal of Medicinal
Chemistry, 2019. 62(3): p. 1125-1137.
32.
33.
34.
35.
Egan, W.J., K.M. Merz, and J.J. Baldwin, Prediction of drug absorption using
multivariate statistics. Journal of Medicinal Chemistry, 2000. 43(21): p. 3867-3877.
Wesson, L. and D. Eisenberg, Atomic solvation parameters applied to molecular
dynamics of proteins in solution. Protein Sci, 1992. 1(2): p. 227-35.
Cheng, A.L. and K.M. Merz, Prediction of aqueous solubility of a diverse set of
compounds using quantitative structure-property relationships. Journal of Medicinal
Chemistry, 2003. 46(17): p. 3572-3580.
36.
37.
38.
39.
Susnow, R.G. and S.L. Dixon, Use of robust classification techniques for the
prediction of human cytochrome P450 2D6 inhibition. Journal of Chemical Information
and Computer Sciences, 2003. 43(4): p. 1308-1315.
Cheng, A. and S.L. Dixon, In silico models for the prediction of dose-dependent
human hepatotoxicity. Journal of Computer-Aided Molecular Design, 2003. 17(12): p.
811-823.
Jin, W.Y., et al., Scaffold-based novel SHP2 allosteric inhibitors design using
Receptor-Ligand pharmacophore model, virtual screening and molecular dynamics.
Computational Biology and Chemistry, 2018. 73: p. 179-188.
Ma, Y., et al., Design Novel Dual Agonists for Treating Type-2 Diabetes by Targeting
Peroxisome Proliferator-Activated Receptors with Core Hopping Approach. Plos One,

2012. 7(6).

GA

Highlights
1. This manuscript was the first reported to design SHP2 inhibitors with 3-substituted pyridine as
the core structure, and 35 new compounds were obtained.
2. In this manuscript, a series of biological evaluation were carried out on these 35 compounds,
including in vitro enzyme activity, fluorescence titration and cell proliferation assays, and multiple
inhibitors with good activity and development value were obtained.
3. In this manuscript, in vitro ADMET prediction investigated pharmacokinetic characteristics of
these compound, and molecular docking study investigated the binding pocket of protein and ligand.