Discovery of 2,4-diaminopyrimidine derivatives targeting p21-activated kinase 4: Biological evaluation and docking studies

Article abstract of DOI:10.1002/ardp.202000097

In this study, novel 2,4-diaminopyrimidine derivatives targeting p21-activated kinase 4 (PAK4) were discovered and evaluated for their biological activity against PAK4. Among the derivatives studied, promising compounds A2, B6, and B8 displayed the highest inhibitory activities against PAK4 (IC₅₀ = 18.4, 5.9, and 20.4 nM, respectively). From the cellular assay, compound B6 exhibited the highest potency with an IC₅₀ value of 2.533 μM against A549 cells. Some compounds were selected for computational ADME (absorption, distribution, metabolism, and elimination) properties and molecular docking studies against PAK4. The detailed structure–activity relationship based on the biochemical activities and molecular docking studies were explored. According to the docking studies, compound B6 had the lowest docking score (docking energy: ?7.593 kcal/mol). The molecular docking simulation indicated the binding mode between compound B6 and PAK4. All these results suggest compound B6 as a useful candidate for the development of a PAK4 inhibitor.

Full text of DOI:10.1002/ardp.202000097

Received: 30 March 2020
Revised: 8 June 2020
Accepted: 11 June 2020
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DOI: 10.1002/ardp.202000097
F U L L P A P E R
Discovery of 2,4‐diaminopyrimidine derivatives targeting
p21‐activated kinase 4: Biological evaluation and docking studies
Qiaohua Qin | Tianxiao Wu | Wenbo Yin | Yixiang Sun | Xiangyu Zhang |
Ruifeng Wang | Jing Guo | Dongmei Zhao
| Maosheng Cheng
Key Laboratory of Structure‐Based Drug
Design & Discovery of Ministry of Education,
Abstract
Shenyang Pharmaceutical University,
In this study, novel 2,4‐diaminopyrimidine derivatives targeting p21‐activated kinase
4 (PAK4) were discovered and evaluated for their biological activity against PAK4.
Among the derivatives studied, promising compounds A2, B6, and B8 displayed the
highest inhibitory activities against PAK4 (IC50 = 18.4, 5.9, and 20.4 nM, respec-
tively). From the cellular assay, compound B6 exhibited the highest potency with an
IC50 value of 2.533 μM against A549 cells. Some compounds were selected for
computational ADME (absorption, distribution, metabolism, and elimination) prop-
erties and molecular docking studies against PAK4. The detailed structure–activity
relationship based on the biochemical activities and molecular docking studies were
explored. According to the docking studies, compound B6 had the lowest docking
score (docking energy: −7.593 kcal/mol). The molecular docking simulation indicated
the binding mode between compound B6 and PAK4. All these results suggest
compound B6 as a useful candidate for the development of a PAK4 inhibitor.
Shenyang, China
Correspondence
Dongmei Zhao, Key Laboratory of Structure‐
Based Drug Design & Discovery of Ministry of
Education, Shenyang Pharmaceutical
University, 110016 Shenyang, China.
Email: medchemzhao@163.com
Funding information
National Natural Science Foundation of China,
Grant/Award Numbers: 81230077, 81872729
K E Y W O R D S
anticancer, docking study, inhibitors, PAK4
in oncogenic transformation.[8] Moreover, studies have shown that
the inhibition of PAK1 may contribute to PAK2 inhibition, resulting in
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1
INTRODUCTION
A protein substrate can be phosphorylated by protein kinases,
changing the protein molecules from an inactive state to an activated
state. A portion of protein kinases is highly expressed in tumor cells,
which become excellent drug targets.[1–3] The p21‐activated kinases
(PAKs) are serine/threonine (Ser/Thr) protein kinases, which can
phosphorylate numerous proteins related to the human tissue
function.[4–8] According to their structural difference and sequence
homology, six existing mammalian isoforms of PAKs are distributed
into two groups: group I includes PAK1–3 and group II includes
PAK4–6.[9–11] In human tissue, overexpression of PAKs, particularly
PAK1 and PAK4, is often linked to cancers.[12] Hence, PAKs are de-
veloped as the potential targets of novel anticancer strategies and
the attractive subject for drug discovery. PAK4 is the most studied
group II PAK member among all of the PAKs for its capability to
facilitate cell survival and follow‐up unbounded proliferation.[8]
Overexpression or activation of PAK4 has became a crucial process
acute cardiovascular toxicity, which leaves people in doubt whether
group I PAKs can serve as potential targets.[13] It is highly necessary
to develop specific and effective PAK4 inhibitors which can decrease
the risk caused by the inhibition of group I PAKs.[14] Furthermore,
studies have shown that the combined application of a PAK4 in-
hibitor and a PD‐1 inhibitor provides a new approach to avoid the
resistance of PD‐1 blockade therapy.[15]
To date, several PAK inhibitors have been reported, exhibiting
varying selectivity to the PAKs (Figure 1). In 1972, natural medicine
chemist isolated staurosporine (1) from natural products. However,
staurosporine has a poor kinase selectivity, so it is mainly used as a
positive control drug.[16,17] PF‐3758309 (2) was developed by Pfizer,
entered Phase I clinical trails in 2009, and was subsequently sus-
pended due to insufficient oral bioavailability.[18] KPT‐9274 (3),
presenting dual inhibitory activities against NAMPT and PAK4, ac-
cessed Phase I clinical trials to treat solid malignancy.[19] In 2019,
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Arch Pharm. 2020;e2000097.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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FIGURE 1 Several representative small‐molecule p21‐activated kinase inhibitors
(a)
(b)
FIGURE 2 (a) The co‐crystal structure of 7
with PAK4 (PDB code: 5BMS).[21] (b) The
design of the target compounds

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KPT‐9274 was also planned to access Phase I clinical trials in com-
bination with PD‐1 inhibitor nivolumab.[15] GNE‐2861 (4), type I 1/2
PAK4 inhibitor, is the first selective PAK4 inhibitor reported in the
literature.[20] Czh‐226 (5), a PAK4 inhibitor obtained from structure‐
based drug design (SBDD) method, revealed high activity, strong
structural novelty, and significant selectivity.[14] Compound 6, a se-
lective PAK1 inhibitor with aminopyrazole scaffold, was discovered
by Genentech in 2015.[21]
10b–d was performed as shown in Scheme 1. The commercially
available 8b–d were treated with acetonitrile and sodium hydride
(NaH) in anhydrous tetrahydrofuran (THF), leading to the inter-
mediates 9b–d in 44.4–55.6% yield. The ketonitriles 9b–d were
condensed with hydrazine, affording the aminopyrazoles 10b–d in
35.2–60.5% yield.[22] The synthesis of the intermediates 13a,b
is shown in Scheme 2. Acylation of the commercially available
11a,b with tert‐butylpiperazine‐1‐carboxylate in the presence of
1‐(3‐dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride (EDC‐
HCl), 1‐hydroxybenzotriazole (HOBt), and the base N,N‐
diisopropylethylamine (DIPEA) afforded the intermediates 12a,b in
71.3–79.5% yield. Deprotection of the N‐Boc moiety of intermediates
12a,b with trifluoroacetic acid (TFA) provided the intermediates
13a,b in 88.2–91.3% yield.[23]
We selected compound 6 as the lead compound to carry out
structural modification. Compound 7 (Figure 1), a homolog to compound
6, makes a co‐crystal structure with PAK4.[21] According to the co‐crystal
structure of compound 7 with PAK4 (Figure 2a), the suitable substituent
of the 5′‐position on the pyrazole ring accesses the hydrophobic region of
PAK4 and makes an effective hydrophobic interaction with the gate-
keeper residue Met395. Hence, the structural modification of the
5′‐position on the pyrazole ring was implemented. In contrast, the amino
acid residues of PAK4 protein (Asp440, Asp444, Ser445, and Asp458)
located in the ribose‐binding pocket are unable to form a hydrogen bond
network and adopt a strained conformation (Figure 2a).[21] On the basis
of the spatial constraint resulting from the residues in the ribose‐binding
pocket, it was reasonable to assume that adjusting the size and length of
the substituents at the 2‐position of the pyrimidine nucleus can improve
inhibitory activity against PAK4. Finally, considering the introduction of
polar groups in the 2‐position of the pyrimidine, we designed a benzyl
group in the 6‐position of the pyrimidine to compensate the impact
of the polar groups. To test our hypothesis, a new library of 2,4‐
diaminopyrimidine derivatives was designed and synthesized (Figure 2b).
We progressed the enzymatic activities of these compounds as PAK4
inhibitors and also performed detailed structure–activity relationship
(SAR) studies. These compounds, A1–8 and B1–12, were configured
against PAK4. In addition, the activities of selected compounds against
the A549 cell line were quested. In the meantime, molecular docking
studies and the prediction of ADME (absorption, distribution, metabolism,
and elimination) properties were performed.
General procedures of synthesizing the 2,4‐diaminopyrimidine
derivatives were carried out in accordance with Scheme 3. The
synthesis was started from the substitution of the commercially
available 2,4,6‐trichloropyrimidine (14) with benzylmagnesium
bromide and a catalytic amount of (dppe)NiCl2, generating the
intermediate 15 in 75.2% yield.[24] The substitution of intermediate
15 with 1H‐pyrazol‐3‐amine, 5‐methyl‐1H‐pyrazol‐3‐amine, 5‐
cyclopropyl‐1H‐pyrazol‐3‐amine, 5‐(p‐tolyl)‐1H‐pyrazol‐3‐amine, or
10a–d in the presence of the base DIPEA delivered the intermediates
16a–g or 18 in 71.4–80.5% yield. The target compounds, A1–8 and
B1–12, were obtained by substitution of the intermediates 16a–g or
18 with amine and then deprotection with TFA to provide the de-
sirable compounds (for B1–3 and B7–12, the deprotection was not
required).[25,26]
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2.2
Biology
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2.2.1
Inhibitory activities against PAK4 and SAR
studies
Homogeneous time‐resolved fluorescence (HTRF) assay was utilized
to evaluate the inhibition of the synthesized compounds against
PAK4 (Tables 1 and 2). PF‐3758309 (2), the positive control, was
utilized to verify the screening condition. The IC50 value showed by
PF‐3758309 (2) was similar with the report.[18]
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2
RESULTS AND DISCUSSION
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2.1
Chemistry
The synthesis of intermediate 10a was conducted by following the
reported patent (Scheme 1).[22] The synthesis of the intermediates
Considering the co‐crystal structure of compound 7 with PAK4,
it is observed that the substituents on the 5′‐position of the pyrazole
SCHEME 1 The synthesis of the
intermediates 10a–d. Reagents and
conditions: (a) CH3CN, NaH, anhydrous
tetrahydrofuran, reflux, 12 hr, 44.4–55.6%; (b)
EtOH, N2H4‐H2O, reflux, 3 hr, 35.2–60.5%

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SCHEME 2 The synthesis of the intermediates 13a,b. Reagents and conditions: (a) tert‐butylpiperazine‐1‐carboxylate, 1‐(3‐
dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride, 1‐hydroxybenzotriazole, N,N‐diisopropylethylamine, dichloromethane (DCM), 0°C,
2 hr, 71.3–79.5%; (b) tetrahydrofuran, DCM, room temperature, 12 hr, 88.2–91.3%
ring can affect PAK4 affinity (Figure 2b). The analogs with methyl and
ethyl substituents (A2, A3) displayed the activities with IC50 values of
18.4 and 12.3 nM, respectively, and exhibited better inhibitory ac-
tivities than the hydrogen substituent (A1), suggesting that methyl or
ethyl moiety extends into the hydrophobic cavity of the protein and
forms a valid hydrophobic interaction with the gatekeeper Met395.
Unfortunately, compared with A2 and A3, the analogs with
cyclopropyl substituent (A4) and aromatic substituents, for instance,
phenyl, 4‐chlorophenyl, and 4‐methoxyphenyl (A5, A6, and A8, re-
spectively) on the 5′‐position of the pyrazole ring encountered a 2‐ to
40‐fold reduction of potency; also, the analog with 4‐methylphenyl
(A7) completely lost the potency. The introduction of bulkier sub-
stituents on the 5′‐position of the pyrazole ring led to the overall
decline of enzymatic activity, indicating that the hydrophobic cavity
SCHEME 3 The synthesis of compounds A1–8 and B1–12. Reagents and conditions: (a) PhCH2MgBr, (dppe)NiCl2, anhydrous
tetrahydrofuran, room temperature, 12hr, 75.2%; (b) 1H‐pyrazol‐3‐amine/5‐methyl‐1H‐pyrazol‐3‐amine/5‐cyclopropyl‐1H‐pyrazol‐3‐amine/5‐
(p‐tolyl)‐1H‐pyrazol‐3‐amine/10a–d, N,N‐diisopropylethylamine, 80°C, 12hr, 71.4–80.5%; (c) amine/13a–b, i‐, sealed tube, 135°C, 12hr,
70.2–85.9%; (d) trifluoroacetic acid, room temperature, 12hr, 95.2–97.5%

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TABLE 1 Inhibitory activities against PAK4 of compounds bearing R1 moieties
a,b
a,b
Entry
A1
R1
IC50 (nM)
Entry
A6
R1
IC50 (nM)
H
294.8
362.3
A2
A3
Me
Et
18.4
12.3
A7
2,379
280.8
24.0
A8
A4
A5
31.5
PF‐3758309c
124.3
aHomogeneous time‐resolved fluorescence assay was utilized to evaluate values of IC50
bThe IC50 values were the average of two separate experiments.
cA positive control.
.
at that location is tolerant to small hydrophobic moieties. However, it
is worth mentioning that compound A8 displayed better inhibitory
activity than compound A7, speculating that the methoxy group
makes hydrogen bond interactions with the surrounding amino acid
residues as a hydrogen bond acceptor group.
makes an electrostatic interaction with Asp444, which should be the
main factor for compound B8 showing a higher inhibitory activity
than B1. The inhibitory activities of compounds B9 and B10 were not
as satisfactory as expected, indicating that the introduction of bulkier
functional groups at the terminal position is unsuitable. Combining
above results, the 2‐position substituent of the pyrimidine nucleus
has a length of about 6 Å and contains a hydrogen bond donor group
at the tail position, which may be beneficial to the inhibitory activity.
In summary, compound B6 showed the best potency with an IC50
value of 5.9 nM, drawing a conclusion that compound B6 is the
On the basis of the spatial constraint in the ribose‐binding
pocket, further structural optimization of the substituents at the 2‐
position of the pyrimidine nucleus was adopted to increase the PAK4
inhibitory activity. Compounds B1, B2, and B3 showed a decline of
enzymatic activity as compared with that of A2, manifesting IC50
values of 351.9, 187.5, and 303.3 nM, respectively. Also, compared
with A2, compounds B4, B6, and B7 showed a similiar magnitude in
potency, and compounds B11 and B12 exhibited a three‐ to fivefold
decrease of PAK4 affinity, suggesting that hydrogen bond donor
moiety at the tail position of the 2‐position substituents played a key
role. From the specific interaction of A2 in the PAK4 ATP pocket, the
NH group of the piperazine ring of A2 forms an H‐bond interaction
with Asp458 (Figure 3a). Compared with B4, compound B5 had a
threefold decrease in activity due to the N‐methylation. Surprisingly,
compounds B1 and B8 did not contain a hydrogen bond donor group
at the tail position; however, compound B8 showed a similiar potency
as A2 and presented a near 17‐fold improvement in potency against
PAK4, compared with B1. The superimposition of compounds B1
with B8 manifested that both compounds B8 and B1 could
form critical donor–acceptor–donor H‐bonds with PAK4 kinase
(Figure 3b). However, the terminal position on the 2‐position of B8
preferred compound for
a two‐round structural modification.
Compounds B6 and A2 contain a hydrogen bond donor group at the
the tail position.
It is to be expected that the interaction between B6 and PAK4 is
similiar to that of A2. From the superimposition of compounds B6
with B8 (Figure 3c), the NH group of the piperazine ring of B6 makes
an H‐bond interaction with Asp458, whereas the terminal position on
the 2‐position of B8 forms an electrostatic interaction with Asp444.
These results verify our expectation.
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2.2.2
Antiproliferative activity
Overexpression of PAK4 was found in A549 cells. Hence, on the basis of
enzymatic activities of the synthesized compounds, we selected com-
pounds A2, B5, B6, B7, and B8 to proceed the antiproliferative efficacy

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TABLE 2 Inhibitory activities against PAK4 of compounds bearing R2 moieties
a,b
a,b
Entry
A2
R2
IC50 (nM)
Entry
B7
R2
IC50 (nM)
18.4
37.3
B1
B2
351.9
187.5
B8
B9
20.4
375.7
B3
203.3
B10
339.5
B4
B5
B6
22.6
72.7
5.9
B11
63.4
111.7
24.0
B12
PF‐3758309c
aHomogeneous time‐resolved fluorescence assay was utilized to evaluate values of IC50
bThe IC50 values were the average of two separate experiments.
cA positive control.
.
study in A549 cells by utilizing the Cell Counting Kit‐8 (CCK‐8) assay
method, and PF‐3758309 served as the positive control. The anti-
proliferative activities shown by these selected compounds are depicted
in Table 3. As shown in Table 3, these compounds displayed anti-
proliferative activities against A549 cells with IC50 values ranging from
2.533 to 7.252 μM. Among them, compound B6 suppressed the pro-
liferation in A549 cells with an IC50 value of 2.533 μM.
scores. According to the binding energy calculations against PAK4,
the most potential compound B6 showed a better docking score with
the docking energy of −7.593 kcal/mol (Table 4). The docking results
of all the compounds (A1–8 and B1–12) are also provided in Table 4.
We reported the prediction of the binding mode of B6 with PAK4
(Figure 4a). The 3‐aminopyrazole fragment binds to the hinge region
residues (Glu396 and Leu398 in PAK4) to form critical
donor–acceptor–donor H‐bonds. The hydrophobic segment at 5′‐
position of the pyrazole ring occupies the hydrophobic cavity of the
protein, and the NH group of the piperazine ring forms an H‐bond
interaction with Asp458; however, the benzyl group extends into the
solvent region (Figure 4b).
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2.3
Molecular docking
We utilized Glide standard precision (SP) mode to calculate the
docking results based on the binding energy and docking poses. Ac-
cording to the positional root mean square deviation, the docking
results showing a difference <2.0 Å were taken together and were
ranked on the basis of their free binding energy. Most of the binding
energy scores were positively correlated with the inhibitory activities
for PAK4 via the results from the native docking simulations. For
example, compounds A2, A3, B4, B6, and B8 displayed better in-
hibitory activities against PAK4 and also possessed better docking
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2.3.1
Theoretical prediction of ADME properties
Several physicochemical parameters, shown in Table 5, are related
with ADME properties, which can evaluate the druggability of the
optimized molecules. The prediction of druggability of compounds
A2, B6, and B8 was carried out through the SYBYL‐X, and

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(a)
(b)
(c)
FIGURE 3 (a) The 2D diagram of specific interaction of A2 in the PAK4 ATP pocket. (b) The superimposition of B1 (yellow) with B8 (blue) in
the PAK4 ATP pocket binding site. (c) The superimposition of B6 (yellow) with B8 (blue) in the PAK4 ATP pocket binding site
PF‐3758309 served as the positive control. The results of calculation
are shown in Table 5. The molecular weight (M.W.) in all cases con-
formed with the optimum of the bioavailability range.[27] The M.W. of
the preferred compound B6 was 363.46 g/mol, which is lower than
400. All the clogP values were close to 3, near the optimal scope
(clogP ≈ 3).[28,29] On the basis of the physicochemical characteristics
shown by 28 small‐molecule kinase inhibitors, topological polar sur-
face area (tPSA) is in the scope of 60–140 Å2, the sum of H‐bond
donor and H‐bond acceptor is less than four and nine, respectively,
and the number of rotatable bonds is in the scope of 3–9.[30] The
estimated tPSA values in all cases were lower than 140 Å2. Com-
pound B6 contains four H‐bond donors, three H‐bond receptors, as
well as three rotatable bonds, showing a clogP value of 3.45. The
ligand efficiency (LE), ligand lipophilicity efficiency (LLE), and ligand
efficiency‐dependent lipophilicity (LELP) values in all cases also
conformed with the preferred range,[31] and they were computed on
the basis of the pIC50 values from inhibitory activities of the syn-
thesized compounds against PAK4.
TABLE 3 The antiproliferative activity of selected compounds
a
Entry
PAK4 (IC50, nM)
A549 (IC50
7.252
,
μM)
A2
18.4
72.7
5.9
B5
2.725
B6
2.533
B7
37.3
20.4
24.0
6.289
B8
6.989
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3
CONCLUSION
PF‐3758309
0.463
aMTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide)
assay was utilized to evaluate the antiproliferative activity, and the values
of IC50 symbolize the concentration that inhibited 50% cell growth after
72 hr of drug exposure.
In summary, a novel series of 2,4‐diaminopyrimidine skeleton PAK4
inhibitors was designed and synthesized by using an SBDD strategy,
and their biochemical activities were determined. Among them,

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TABLE 4 The docking energies of compounds A1–8 and B1–12 binding in PAK4
Docking
energy
Docking
energy
Docking
energy
a,b
a,b
a,b
IC50
IC50
IC50
Entry
A1
(kcal/mol)
(nM)
294.8
18.4
Entry
A8
B1
(kcal/mol)
(nM)
280.8
351.9
187.5
203.3
22.6
Entry
B7
(kcal/mol)
(nM)
−6.168
−7.17
−6.232
−5.711
−6.769
−6.492
−7.599
−6.917
−7.593
−6.689
−7.049
−5.465
−5.705
−6.775
−6.898
37.3
A2
B8
20.4
A3
−7.381
−7.276
−6.323
−5.33
12.3
B2
B9
375.7
339.5
63.4
A4
31.5
B3
B10
B11
B12
A5
124.3
362.3
2,379
B4
A6
B5
72.7
111.7
A7
−5.171
B6
5.9
aHomogeneous time‐resolved fluorescence assay was utilized to evaluate values of IC50
.
bThe IC50 values were the average of two separate experiments.
compound B6 exhibited the strongest biochemical activity with an
IC50 value of 5.9 nM against PAK4 and antiproliferative potency
against A549 cell line with an IC50 value of 2.533 μM. We proposed
that the binding mode between compound B6 and PAK4, Glu396,
and Leu398 in the hinge region plays a key role. Compound B6
showed the lowest docking score from the docking results (docking
energy = −7.593 kcal/mol). Furthermore, the detailed SAR studies of
the synthesized compounds were reported in terms of biochemical
activities and molecular docking studies. Finally, druggability pre-
dictions indicate that compound B6 has a potential value as a PAK4
inhibitor for further optimization. Relevant research is going on and
will be published in due course.
solvents were dried and purified by conventional methods before use.
Column chromatography was carried out on silica gel (100–200 mesh and
200–300 mesh). All reactions were monitored by thin‐layer chromato-
graphy on silica gel plates with fluorescence F‐254 and visualized with
UV light. 1H NMR spectral data were recorded in dimethyl sulfoxide
(DMSO)‐d6 on Bruker ARX‐400 and ARX‐600 NMR spectrometers with
TMS as an internal standard. High‐resolution accurate mass spectrometry
(HRMS) determinations for all final target compounds were obtained on a
Bruker Micromass Time‐of‐Flight Mass Spectrometer equipped with an
electrospray ionization detector.
The original spectra of the investigated compounds, together
with their InChI codes as well as some biological activity data, are
provided as Supporting Information Data.
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4
EXPERIMENTAL
|
4.1.2
The general procedure for the synthesis of
|
4.1
Chemistry
intermediates 10b–d
|
4.1.1
General
An example of the synthetic procedure of 5‐phenyl‐1H‐pyrazol‐3‐
amine (10b) is given as follows: A mixture of MeCN (0.46 g,
6.45 mmol) and NaH (0.39 g, 6.45 mmol) in dry THF (3.00 ml) was
stirred for 0.5 hr at room temperature, followed by the addition of
All reagents and solvents were obtained from commercial suppliers and
used without further purification, unless otherwise indicated. Anhydrous
(a)
(b)
FIGURE 4 The binding mode of B6 with the PAK4 ATP pocket. (a) Specified interactions with residues of the PAK4 ATP pocket are shown.
Red dotted lines represent H‐bonds. (b) The surface on the rim of the binding site of compound B6 with the PAK4 ATP pocket. The PAK4 pocket
is shown as a surface that is color‐coded based on hydrophobicity

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TABLE 5 ADME properties of compounds A2, B6, and B8
THF (5 ml). The mixture was stirred below 30°C for 2 hr. A liquid with
a dark gray metallic luster was obtained, which was used directly in
the next reaction.
Entry
A2
M.W.
pIC50 clogP tPSA
LE
LLE
LELP
8.00
8.08
349.43 7.70
363.46 8.23
377.49 7.69
3.21
3.45
3.78
4.43
139.93 0.40 4.53
123.00 0.43 4.78
B6
B8
87.39 0.37 3.92 10.15
84.49 0.30 3.19 14.77
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4.1.5
Synthesis of benzyl‐2,
6‐dichloropyrimidine (15)
PF‐3758309 490.64 7.62
Abbreviations: ADME, absorption, distribution, metabolism, and
elimination; clogP, partition coefficient; LE, ligand efficiency; LELP, ligand
efficiency‐dependent lipophilicity; LLE, ligand lipophilicity efficiency;
M.W., molecular weight; pIC50, −log[IC50 (PAK4, mol/L)]; tPSA, topological
polar surface area.
(Dppe)NiCl2 (0.04 g, 0.06 mmol) and benzylmagnesium bromide were
added to a solution of 2,4,6‐trichloropyrimidine (14; 1 g, 5.46 mmol) in
anhydrous THF (5 ml). The mixture was stirred for 12 hr at room tem-
perature. A saturated ammonium chloride solution (0.5 ml) was added to
the reaction mixture to obtain a large amount of yellow solid, and then
the insoluble matter was removed by filtration. The filtrate was then
concentrated and diluted with water (100 ml) and extracted with ethyl
acetate (3 × 50 ml). The combined organic layers were were washed with
brine, dried over anhydrous Na2SO4, and concentrated under vacuum.
The concentrated residue was purified and separated by column chro-
matography, using silica with 0.2% petroleum ether in ethyl acetate as an
eluent to give 15 as a light yellow liquid.
methyl benzoate (8b; 0.59 g, 4.3 mmol). The resulting mixture was
stirred for 12 hr at 66°C. After cooling to room temperature, the
reaction mixture was treated with saturated ammonium chloride
solution (2.0 ml) and diluted with water (100.0 ml), and then ex-
tracted with ethyl acetate (3 × 50 ml). The combined organic layers
were washed with brine, dried over anhydrous Na2SO4, and con-
centrated under vacuum to give 9b, a light yellow solid. MeOH
(50 ml) and hydrazine (0.42 ml, 8.79 mmol) were added to the solid,
and the mixture was stirred for 3 hr at 65°C, cooled to room tem-
perature, and then concentrated under vacuum. The concentrated
residue was purified and separated by column chromatography, using
silica with 2% MeOH in dichloromethane as an eluent to obtain 10b
as a light yellow solid.
|
4.1.6
The general procedure for the synthesis of
intermediates 16a–g and 18
An example of the synthetic procedure of 6‐benzyl‐2‐chloro‐N‐(5‐methyl‐
1H‐pyrazol‐3‐yl)pyrimidin‐4‐amine (18) is given as follows: The commer-
cially available 5‐methyl‐1H‐pyrazol‐3‐amine (0.02 g, 0.23 mmol) and DI-
PEA (0.04 ml, 0.25 mmol) were added to a solution of 4‐benzyl‐2,6‐
dichloropyrimidine (15; 0.05 g, 0.21 mmol) in DMF (5 ml). The mixture
was stirred at 100°C for 12 hr and then cooled to room temperature. The
reaction mixture was diluted with water (100 ml) and then extracted with
ethyl acetate (3 × 50 ml). The combined organic layers were washed with
brine, dried over anhydrous Na2SO4, and concentrated under vacuum.
The concentrated residue was purified and separated by column chro-
matography, using silica with 1.25% MeOH in DCM as an eluent to
provide 18 as a light yellow solid.
Other intermediates 10c,d were prepared as described in the
procedure for synthesizing 10b.
|
4.1.3
The general procedure for the synthesis of
intermediates 13a,b
An example of the synthetic procedure of furan‐2‐yl(piperazin‐1‐yl)
methanone (13a) is given as follows: EDC‐HCl (1.39 g, 6.69 mmol),
HOBt (0.90 g, 6.69 mmol), and DIPEA (1.5 ml, 8.92 mmol) were added
to a solution of 11a (0.50 g, 4.46 mmol) in DCM (50 ml). The mixture
was stirred for 1 hr at 0°C. tert‐Butylpiperazine‐1‐carboxylate (1.0 g,
5.35 mmol) was added to the reaction mixture and stirred for 1 hr at
0°C. The reaction mixture was treated with water (500 ml), forming a
large amount of white solid. The mixture was filtered to afford the
desired solid product 12a. TFA (2.0 ml) was added to a solution of
12a in DCM (2.0 ml) and stirred for 12 hr at room temperature. Then,
the resulting solid was filtered and dried to afford the desired solid
product 13a.
Other intermediates, 16a–g, were prepared as described in the
procedure for synthesizing 18.
|
4.1.7
The general procedure for the synthesis of
intermediates 17a–g, 19a–c, 20, compounds B1–3
and B7–12
Piperazin‐1‐yl(1H‐pyrrol‐2‐yl)methanone (13b) was prepared as
described in the procedure for synthesizing 13a.
An example of the synthetic procedure for tert‐butyl 4‐{4‐[(1H‐
pyrazol‐3‐yl)amino]‐6‐benzylpyrimidin‐2‐yl}piperazine‐1‐carboxylate
(17a) is given as follows: 4‐Boc‐aminopiperazine (0.02 mg, 0.09 mmol)
and a catalytic amount of triethylamine were added to a solution of
16a (0.03 g, 0.01 mmol) in sec‐butyl alcohol (3.00 ml). The mixture
was stirred for 12 hr at 135°C in the sealed tube and then cooled to
room temperature. The reaction mixture was concentrated, and then
the concentrated residue was purified and separated by column
|
4.1.4
Preparation of benzylmagnesium bromide
Benzyl bromide (0.30 ml, 2.73 mmol) was added slowly to a solution
of a catalytic amount of I2 and Mg (0.98 g, 40.95 mmol) in anhydrous

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chromatography, using silica with 4% MeOH in DCM as an eluent to
give 17a.
(400 MHz, DMSO‐d6) δ ppm: 12.74 (s, 1H, NH‐pyrazole), 9.53 (s, 1H,
Ar‐NH‐Ar), 7.73–7.67 (m, 2H, pyrazole‐Ar‐Ho), 7.45 (t, J = 7.7 Hz, 2H,
pyrazole‐Ar‐Hm), 7.39–7.26 (m, 5H, pyrimidine‐Ph‐H), 7.21 (ddd,
J = 6.7, 5.6, 2.5 Hz, 1H, pyrazole‐Ph‐Hp), 6.70 (s, 1H, pyrazole‐H), 6.24
(brs, 1H, pyrimidine‐H), 3.82 (t, J = 5.1 Hz, 4H, piperazine‐H), 3.75
(s, 2H, Ph‐CH2‐Ar), 2.97 (t, J = 5.2 Hz, 4H, piperazine‐H). HRMS calcd.
for C24H26N7, [M+H]+, 412.2250; found 412.2243.
Other intermediates, 17b–g, 19a–c, and 20, including compounds
B1–3 and B7–12, were prepared as described in the procedure for
synthesizing 17a.
6‐Benzyl‐2‐(piperazin‐1‐yl)‐N‐(1H‐pyrazol‐3‐yl)pyrimidin
‐4‐amine (A1)
TFA (2.00 ml) was added to a solution of intermediate 17a in DCM
(2.00 ml). The mixture was stirred for 12 hr at room temperature, and
then the resulting solid was filtered and dried to afford the desired solid
product A1. Light yellow solid (0.04 g, 78.7% yield). 1H NMR (400 MHz,
DMSO‐d6) δ ppm: 12.20 (s, 1H, NH‐pyrazole), 7.60–7.24 (m, 7H, Ph‐H),
6.35 (s, 1H, Ar‐NH‐Ar), 3.87–3.59 (m, 6H, piperazine‐H, and Ph‐CH2‐Ar),
2.99–2.90 (m, 2H, piperazine‐H), 2.87 (t, J = 5.0 Hz, 2H, piperazine‐H).
HRMS calcd. for C18H22N7, [M+H]+, 336.1937; found 336.1934.
6‐Benzyl‐N‐[5‐(4‐chlorophenyl)‐1H‐pyrazol‐3‐yl]‐2‐(piperazin‐1‐yl)‐
pyrimidin‐4‐amine (A6)
Compound A6 was prepared in a similar manner as compound A1,
17e instead of 17a. Light yellow solid (0.03 g, 82.0% yield). 1H NMR
(400 MHz, DMSO‐d6) δ ppm: 12.89 (s, 1H, NH‐pyrazole), 9.99 (s, 1H,
Ar‐NH‐Ar), 8.01 (d, J = 7.7 Hz, 1H, Cl‐Ph‐H), 8.00–7.55 (m, 3H, Cl‐Ph‐
H), 7.53 (q, J = 9.5, 8.4 Hz, 3H, pyrimidine‐Ph‐H), 7.30 (d, J = 6.1 Hz,
2H, pyrimidine‐Ph‐H), 6.84 (s, 1H, pyrazole‐H), 6.71 (s, 1H,
pyrimidine‐H), 3.73 (s, 2H, Ph‐CH2‐Ar), 3.68 (t, J = 5.0 Hz, 4H,
piperazine‐H), 2.79 (t, J = 5.1 Hz, 4H, piperazine‐H). HRMS calcd. for
C24H25N7Cl, [M+H]+, 446.1860; found 446.1864.
6‐Benzyl‐N‐(5‐methyl‐1H‐pyrazol‐3‐yl)‐2‐(piperazin‐1‐yl)pyrimidin‐
4‐amine (A2)
Compound A2 was prepared in a similar manner as compound A1,
19a instead of 17a. White solid (0.03 g, 97.1% yield). 1H NMR
(400 MHz, DMSO‐d6) δ ppm: 11.03 (s, 1H, NH‐pyrazole), 9.94–9.60
(m, 2H, 2 × NH), 7.38–7.30 (m, 5H, Ph‐H), 6.30 (s, 1H, pyrazole‐H),
6.10 (s,1H, pyrimidine‐H), 4.22–4.17 (m, 6H, piperazine‐H, and Ph‐
CH2‐Ar), 3.28 (s, 4H, piperazine‐H), 2.24 (s, 3H, CH3). HRMS calcd. for
C19H24N7, [M+H]+, 350.2093; found 350.2088.
6‐Benzyl‐2‐(piperazin‐1‐yl)‐N‐[5‐(p‐tolyl)‐1H‐pyrazol‐3‐yl]pyrimidin‐
4‐amine (A7)
Compound A7 was prepared in a similar manner as compound A1,
17f instead of 17a. Light yellow solid (0.04 g, 83.6% yield). 1H NMR
(400 MHz, DMSO‐d6) δ ppm: 12.73 (s, 1H, NH‐pyrazole), 9.51 (s, 1H,
Ar‐NH‐Ar), 7.77–7.50 (m, 4H, pyrazole‐Ph‐H), 7.43–7.13 (m, 5H,
pyrimidine‐Ph‐H), 6.64 (s, 1H, pyrazole‐H), 6.49–5.67 (m, 1H,
pyrimidine‐H), 3.91–3.76 (m, 4H, piperazine‐H, and Ph‐CH2‐Ar), 3.75
(s, 2H, piperazine‐H), 2.97 (q, J = 8.7, 6.9 Hz, 4H, piperazine‐H), 2.33
(d, J = 4.4 Hz, 3H, CH3). HRMS calcd. for C25H28N7, [M+H]+,
426.2406; found 426.2400.
6‐Benzyl‐N‐(5‐ethyl‐1H‐pyrazol‐3‐yl)‐2‐(piperazin‐1‐yl)pyrimidin‐4‐
amine (A3)
Compound A3 was prepared in a similar manner as compound A1,
17b instead of 17a. Light yellow solid (0.05 g, 88.2% yield). 1H NMR
(400 MHz, DMSO‐d6) δ ppm: 11.92 (s, 1H, NH‐pyrazole), 9.33 (s, 1H,
Ar‐NH‐Ar), 7.37–7.04 (m, 5H, Ph‐H), 6.19 (s, 1H, pyrazole‐H), 6.10
(s, 1H, pyrimidine‐H), 3.97–3.67 (m, 6H, piperazine‐H, and Ph‐CH2‐
Ar), 2.97 (t, J = 5.1 Hz, 4H, piperazine‐H), 2.57 (t, J = 7.7 Hz, 2H, CH2‐
pyrazole), 1.17 (t, J = 7.6 Hz, 3H, CH3). HRMS calcd. for C20H26N7,
[M+H]+, 364.2250; found 364.2246.
6‐Benzyl‐N‐[5‐(4‐methoxyphenyl)‐1H‐pyrazol‐3‐yl]‐2‐(piperazin‐1‐
yl)pyrimidin‐4‐amine (A8)
Compound A8 was prepared in a similar manner as compound A1,
17g instead of 17a. Light yellow solid (0.03 g, 79.8% yield). 1H NMR
(400 MHz, DMSO‐d6) δ ppm: 12.57 (s, 1H, NH‐pyrazole), 9.48 (s, 1H,
Ar‐NH‐Ar), 7.65–7.60 (m, 2H, pyrazole‐Ph‐Ho), 7.30 (d, J = 6.2 Hz, 5H,
pyrimidine‐Ph‐H), 7.06–6.98 (m, 2H, pyrazole‐Ph‐Hm), 6.60 (s, 1H,
pyrazole‐H), 6.22 (s, 1H, pyrimidine‐H), 3.80 (m, 7H, OCH3, and
piperazine‐H), 3.74 (s, 2H, Ph‐CH2‐Ar), 2.97 (t, J = 5.1 Hz, 4H,
piperazine‐H). HRMS calcd. for C25H28N7O, [M+H]+, 442.2355; found
442.2348.
6‐Benzyl‐N‐(5‐cyclopropyl‐1H‐pyrazol‐3‐yl)‐2‐(piperazin‐1‐yl)‐
pyrimidin‐4‐amine (A4)
Compound A4 was prepared in a similar manner as compound A1, 17c
instead of 17a. Light yellow solid (0.03 g, 88.0% yield). 1H NMR
(600 MHz, DMSO‐d6) δ ppm 11.02 (s, 1H, NH‐pyrazole), 9.74 (s, 1H, Ar‐
NH‐Ar), 7.49–7.18 (m, 5H, Ph‐H), 6.20 (s, 1H, pyrazole‐H), 6.15 (s, 1H,
pyrimidine‐H), 4.36–4.09 (m, 6H, piperazine‐H, and Ph‐CH2‐Ar), 3.27
(t, J = 5.1 Hz, 4H, piperazine‐H), 2.01–1.86 (m, 1H, cyclopropane‐H),
0.98–0.90 (m, 2H, cyclopropane‐H), 0.74–0.66 (m, 2H, cyclopropane‐H).
HRMS calcd. for C21H26N7, [M+H]+, 376.2250; found 376.2253.
6‐Benzyl‐N‐(5‐methyl‐1H‐pyrazol‐3‐yl)‐2‐(4‐methylpiperazin‐1‐yl)‐
pyrimidin‐4‐amine (B1)
Light yellow solid (0.03 g, 77% yield). 1H NMR (400 MHz, DMSO‐d6) δ
ppm: 11.83 (s, 1H, NH‐pyrazole), 9.24 (s, 1H, Ar‐NH‐Ar), 7.47–7.09
(m, 5H, Ph‐H), 6.11 (s, 1H, pyrazole‐H), 6.09 (s, 1H, pyrimidine‐H),
3.70–3.64 (m, 6H, piperazine‐H, and Ph‐CH2‐Ar), 2.43–2.39 (m, 4H,
piperazine‐H), 2.25 (s, 3H, CH3‐pyrazole), 2.18 (s, 3H, CH3‐
piperazine). HRMS calcd. for C20H26N7, [M+H]+, 364.2250; found
364.2244.
6‐Benzyl‐N‐(5‐phenyl‐1H‐pyrazol‐3‐yl)‐2‐(piperazin‐1‐yl)pyrimidin‐
4‐amine (A5)
Compound A5 was prepared in a similar manner as compound A1,
17d instead of 17a. Light yellow solid (0.04 g, 80.1% yield). 1H NMR

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6‐Benzyl‐2‐(4‐ethylpiperazin‐1‐yl)‐N‐(5‐methyl‐1H‐pyrazol‐3‐yl)‐
pyrimidin‐4‐amine (B2)
piperidine‐H). HRMS calcd. for C20H26N7, [M+H]+, 364.2250; found
364.2241.
Light yellow solid (0.04 g, 82.2% yield). 1H NMR (400 MHz, DMSO‐
d6) δ ppm: 12.00 (s, 1H, NH‐pyrazole), 9.32 (s, 1H, Ar‐NH‐Ar),
7.37–7.10 (m, 5H, Ph‐H), 6.15 (s, 1H, pyrazole‐H), 6.03 (s, 1H,
pyrimidine‐H), 3.72 (s, 2H, Ph‐CH2‐Ar), 3.31–3.33 (m, 4H, piperazine‐
H), 2.21–2.19 (s, 6H, N‐CH2 ‐Me and piperazine‐H), 1.29–1.06 (m, 3H,
CH3‐pyrazole), 0.89–0.80 (m, 3H, CH3 in Et). HRMS calcd. for
C21H28N7, [M+H]+, 378.2406; found 378.2398.
6‐Benzyl‐2‐(1,4‐diazepan‐1‐yl)‐N‐(5‐methyl‐1H‐pyrazol‐3‐yl)‐
pyrimidin‐4‐amine (B7)
Light yellow solid (0.04 g, 80.1% yield). 1H NMR (400 MHz, DMSO‐d6) δ
ppm: 11.89 (s, 1H, NH‐pyrazole), 9.33 (s, 1H, Ar‐NH‐Ar), 7.45–7.08 (m,
5H, Ph‐H), 6.14 (s, 2H, pyrazole‐H, and pyrimidine‐H), 3.96
(d, J = 6.0 Hz, 2H, Ph‐CH2‐Ar), 3.82 (t, J = 6.0 Hz, 2H, diazepane‐H), 3.72
(s, 2H, diazepane‐H), 3.21 (s, 2H, diazepane‐H), 3.05 (d, J = 7.1 Hz, 2H,
diazepane‐H), 2.19 (s, 3H, CH3), 2.06 (s, 2H, diazepane‐H). HRMS calcd.
for C20H26N7, [M+H]+, 364.2250; found 364.2246.
6‐Benzyl‐N‐(5‐methyl‐1H‐pyrazol‐3‐yl)‐2‐morpholinopyrimidin‐4‐
amine (B3)
Light yellow solid (0.03 g, 76.5% yield). 1H NMR (400 MHz, DMSO‐
d6) δ ppm: 11.83 (s, 1H, NH‐pyrazole), 9.27 (s, 1H, Ar‐NH‐Ar),
7.39–7.10 (m, 5H, Ph‐H), 6.20 (s, 1H, pyrazole‐H), 6.11 (s, 1H,
pyrimidine‐H), 3.71 (s, 2H, Ph‐CH2‐Ar), 3.71–3.64 (m, 8H,
morpholine‐H), 2.18 (s, 3H, CH3). HRMS calcd. for C19H24N6O, [M
+H]+, 351.1933; found 351.1928.
6‐Benzyl‐2‐(4‐methyl‐1,4‐diazepan‐1‐yl)‐N‐(5‐methyl‐1H‐pyrazol‐3‐
yl)pyrimidin‐4‐amine (B8)
Light yellow solid (0.03 g, 82% yield). 1H NMR (400 MHz, DMSO‐d6) δ
ppm: 11.89 (s, 1H, NH‐pyrazole), 9.34 (s, 1H, Ar‐NH‐Ar), 7.41–7.08
(m, 5H, Ph‐H), 6.14 (s, 2H, pyrazole‐H, and pyrimidine‐H), 4.39 (s, 1H,
Ph‐CH2‐Ar), 4.00 (s, 1H, Ph‐CH2‐Ar), 3.72 (s, 2H, diazepane‐H), 3.51
(d, J = 2.7 Hz, 1H, diazepane‐H), 3.19–2.98 (m, 5H, diazepane‐H), 2.75
(s, 3H, CH3‐N), 2.20 (s, 3H, CH3). HRMS calcd. for C21H28N7, [M+H]+,
378.2406; found 378.2403.
2‐(4‐Aminopiperidin‐1‐yl)‐6‐benzyl‐N‐(5‐methyl‐1H‐pyrazol‐3‐yl)‐
pyrimidin‐4‐amine (B4)
Compound B4 was prepared in a similar manner as compound A1, 19b
instead of 17a. Light yellow solid (0.02 g, 76.2% yield). 1H NMR
(400 MHz, DMSO‐d6) δ ppm: 9.22 (s, 1H, Ar‐NH‐Ar), 7.29–7.19 (m, 5H,
Ph‐H), 6.12 (s, 1H, pyrazole‐H), 6.08 (s, 1H, pyrimidine‐H), 4.57–4.54
(m, 2H, Ph‐CH2‐Ar), 3.69 (s, 2H, piperidine‐H), 2.92–2.86 (m, 2H,
piperidine‐H), 2.18 (s, 3H, CH3), 1.84–1.78 (m, 2H, piperidine‐H),
1.23–1.21 (m, 3H, piperidine‐H). HRMS calcd. for C20H26N7, [M+H]+,
364.2250; found 364.2245.
{4‐{4‐Benzyl‐6‐[(5‐methyl‐1H‐pyrazol‐3‐yl)amino]pyrimidin‐2‐yl}‐
piperazin‐1‐yl}(1H‐pyrrol‐2‐yl)methanone (B9)
Light yellow solid (0.03 g, 76.4% yield). 1H NMR (400 MHz, DMSO‐
d6) δ ppm: 12.36 (s, 1H, NH‐pyrazole), 11.53 (s, 1H, NH‐pyrrole),
10.96 (s, 1H, Ar‐NH‐Ar), 7.51–7.19 (m, 5H, Ph‐H), 6.93 (td, J = 2.7,
1.3 Hz, 1H, pyrrole‐H), 6.69–6.55 (m, 1H, pyrrole‐H), 6.38 (s, 1H,
pyrrole‐H), 6.26–6.12 (m, 1H, pyrazole‐H), 6.05 (s, 1H, pyrimidine‐H),
4.40–4.26 (s, 2H, Ph‐CH2‐Ar), 4.26–3.85 (m, 8H, piperazine‐H), 2.24
(s, 3H, CH3). HRMS calcd. for C24H27N8O, [M+H]+, 443.2308; found
443.2308.
6‐Benzyl‐N‐(5‐methyl‐1H‐pyrazol‐3‐yl)‐2‐[4‐(methylamino)
piperidin‐1‐yl]pyrimidin‐4‐amine (B5)
Compound B5 was prepared in a similar manner as compound A1,
19c instead of 17a. Light yellow solid (0.03 g, 79.4% yield). 1H NMR
(400 MHz, DMSO‐d6) δ ppm: 12.83 (brs, 1H, NH‐pyrazole), 11.00 (s,
1H, Ar‐NH‐Ar), 7.39–7.30 (m, 5H, Ph‐H), 6.32 (s, 1H, pyrazole‐H),
6.04 (s, 1H, pyrimidine‐H), 4.74 (d, J = 12.4 Hz, 2H, Ph‐CH2‐Ar), 4.32
(s, 2H, piperidine‐H), 3.32–3.30 (m, 1H, piperidine‐H), 3.22
(t, J = 12.4 Hz, 2H, piperidine‐H), 2.54–2.52 (m, 2H, piperidine‐H),
2.24–2.20 (m, 6H, 2 × CH3), 1.69 (dd, J = 12.4 Hz, 8.1 Hz, 2H,
piperidine‐H). HRMS calcd. for C21H28N7, [M+H]+, 378.2406; found
378.2402.
{4‐{4‐Benzyl‐6‐[(5‐methyl‐1H‐pyrazol‐3‐yl)amino]pyrimidin‐2‐yl}‐
piperazin‐1‐yl}(furan‐2‐yl)methanone (B10)
Light yellow solid (0.04 g, 85.1% yield). 1H NMR (400 MHz, DMSO‐
d6) δ ppm: 11.84 (s, 1H, NH‐pyrazole), 9.31 (s, 1H, Ar‐NH‐Ar), 7.86 (d,
J = 1.7 Hz, 1H, furan‐H), 7.37–7.13 (m, 5H, Ph‐H), 7.04 (d, J = 3.4 Hz,
1H, furan‐H), 6.64 (dd, J = 3.5, 1.8 Hz, 1H, furan‐H), 6.21 (s, 1H,
pyrazole‐H), 6.14 (s, 1H, pyrimidine‐H), 3.91–3.82 (s, 2H, Ph‐CH2‐Ar),
3.91–3.63 (m, 8H, piperazine‐H), 2.19 (s, 3H, CH3). HRMS calcd. for
C
24H26N7O2, [M+H]+, 444.2148; found 444.2147.
6‐Benzyl‐N4‐(5‐methyl‐1H‐pyrazol‐3‐yl)‐N2‐(piperidin‐4‐yl)‐
pyrimidine‐2,4‐diamine (B6)
N2,6‐Dibenzyl‐N4‐(5‐methyl‐1H‐pyrazol‐3‐yl)pyrimidine‐2,4‐
diamine (B11)
Compound B6 was prepared in a similar manner as compound A1, 20
instead of 17a. Light yellow solid (0.03 g, 95.3% yield). 1H NMR
(400 MHz, DMSO‐d6) δ ppm: 11.20 (s, 1H, NH‐pyrazole), 8.72 (s, 1H,
Ar‐NH‐Ar), 7.47–7.24 (m, 5H, Ph‐H), 6.41 (s, 1H, pyrazole‐H),
6.19–5.83 (m, 1H, pyrimidine‐H), 4.00 (s, 2H, Ph‐CH2‐Ar), 3.29 (d,
J = 12.3 Hz, 2H, piperidine‐H), 3.16–2.91 (m, 3H, piperidine‐H), 2.27
(s, 3H, CH3), 2.15 (d, J = 12.3 Hz, 2H, piperidine‐H), 1.90–1.73 (m, 2H,
Light yellow solid (0.03 g, 81.2% yield). 1H NMR (400 MHz, DMSO‐
d6) δ ppm: 11.74 (s, 1H, NH‐pyrazole), 9.16 (s, 1H, Ar‐NH‐Ar),
7.43–7.02 (m, 10H, 2 × Ph‐H), 6.08 (s, 2H, pyrazole‐H, and
pyrimidine‐H), 4.47 (d, J = 6.2 Hz, 2H, Ph‐CH2‐N), 3.67 (s, 2H, Ph‐
CH2‐Ar), 2.12 (s, 3H, CH3). HRMS calcd. for C22H23N6, [M+H]+,
371.1984; found 371.1977.

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6‐Benzyl‐N4‐(5‐methyl‐1H‐pyrazol‐3‐yl)‐N2‐(pyridin‐3‐ylmethyl)‐
pyrimidine‐2,4‐diamine (B12)
force field and saved in mol2 format. Protein preparation: The X‐ray
crystal structures of PAK4 were downloaded from the RCSB Protein
Data Bank, PDB code 2X4Z (http://www.rcsb.org/pdb/).
Light yellow solid (0.03 g, 80.4% yield). 1H NMR (400 MHz, DMSO‐
d6) δ ppm: 12.28 (s, 1H, NH‐pyrazole), 10.91 (s, 1H, Ar‐NH‐Ar),
8.59–8.45 (m, 1H, pyridine‐H), 7.80 (s, 1H, pyridine‐H), 7.63–7.10 (m,
7H, pyridine‐H, and Ph‐H), 6.06–5.89 (m, 2H, pyrazole‐H, and
pyrimidine‐H), 4.65 (d, J = 17.2 Hz, 1H, pyridine‐CH2‐N), 3.95 (s, 2H,
Ph‐CH2‐Ar), 2.16 (s, 3H, CH3). HRMS calcd. for C21H22N7, [M+H]+,
372.1937; found 372.1936.
Docking: Crystal structures of PAK4 (PDB code: 2X4Z) were
imported into Glide 9.7, defined as the receptor structure and the
location of the active site with a box of size 15 × 15 × 15 Å. The OPLS
2005 force field was used for grid generation. The SP was set for
docking studies with two crucial residues, Glu396 and Leu398, in
constrained binding to get accurate results. All other parameters
were maintained as the default.
|
4.2
Biological assays
ACKNOWLEDGMENTS
The authors acknowledge the National Natural Science Foundation
of China (Grant: 81872729, 81230077) and Program of Innovative
Research Team of the Ministry of Education, and Program of Liaoning
Innovative Research Team in University for their financial support.
|
4.2.1
PAK4 HTRF assay
HTRF assays were performed in white 384‐well small volume plates
with a total working volume of 20 μl. Compounds were dispensed,
with 4 μl per well, from a concentrated stock of 20 mM in 100%
DMSO and with serial kinase reaction buffer dilutions. The IC50
measurements were performed in replicates. All HTRF reagents were
purchased from CisBio Bioassays and reconstituted according to the
supplier protocols. For each assay, 6 μl of mix 1 (kinase + ATP +
substrate S2) was added in the assay wells containing the previously
dispensed inhibitors. The assay wells were incubated at room tem-
perature for 60 min and terminated by adding 10 μl of mix 2
(Sa‐XL665 + STK Antibody‐Cryptate). After a final incubation (60 min
at room temperature), HTRF signal was obtained by reading the plate
in Infinite® F500 microplate reader (Tecan, Switzerland). The fluor-
escence was measured at 620 nM (Cryptate) and 665 nM (XL665). A
ratio was calculated (665/620) for each well. For the primary com-
pound screening, compounds were diluted in kinase reaction buffer
and tested at 100, 10, and 1.0 μM in 10‐μl kinase reaction with
DMSO below or at 0.5%. The inhibition ratio was calculated,
respectively.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interests.
ORCID
Dongmei Zhao
http://orcid.org/0000-0001-5156-6125
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Qin Q, Wu T, Yin W, et al. Discovery
of 2,4‐diaminopyrimidine derivatives targeting p21‐activated
kinase 4: Biological evaluation and docking studies. Arch
Pharm. 2020;e2000097.
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