Structure-based drug design: Synthesis and biological evaluation of quinazolin-4-amine derivatives as selective Aurora A kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.08.053

Aurora kinases play critical roles in the regulation of the cell cycle and mitotic spindle assembly. Aurora A kinase, a member of the Aurora protein family, is frequently highly expressed in tumors, and selective Aurora A inhibition serves as a significant component of anticancer therapy. However, designing highly selective Aurora A inhibitors is difficult because Aurora A and B share high homology and differ only by three residues in their ATP-binding pockets. Through structure-based drug design, we designed and synthesized a series of novel quinazolin-4-amine derivatives. These derivatives act as selective Aurora A kinase inhibitors by exploiting the structural differences between Aurora A and B. The selectivities of most compounds were improved (the best up to >757-fold) when comparing with the lead compound (3-fold). In vitro biochemical and cellular assays revealed that compound 6 potently inhibited Aurora A kinase and most human tumor cells. Furthermore, compound 6 effectively suppressed carcinoma, such as triple-negative breast cancers (TNBC) in an animal model. Therefore, compound 6 might serve as a promising anticancer drug. Moreover, through molecular dynamic (MD) analysis, we have identified that a salt bridge formed in Aurora B is key contributor for the isoform selectivity of the inhibitor. This salt bridge has not been previously detected in the reported crystal structure of Aurora B. These results might provide a crucial basis for the further development of highly potent inhibitors with high selectivity for Aurora A.

Full text of DOI:10.1016/j.ejmech.2018.08.053

Accepted Manuscript
Structure-based drug design: Synthesis and biological evaluation of quinazolin-4-
amine derivatives as selective Aurora A kinase inhibitors
Liang Long, Yong-Heng Wang, Jun-Xiao Zhuo, Zheng-Chao Tu, Ruibo Wu, Min Yan,
Quentin Liu, Gui Lu
PII:
S0223-5234(18)30723-2
10.1016/j.ejmech.2018.08.053
EJMECH 10664
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 February 2018
Revised Date: 17 August 2018
Accepted Date: 18 August 2018
Please cite this article as: L. Long, Y.-H. Wang, J.-X. Zhuo, Z.-C. Tu, R. Wu, M. Yan, Q. Liu, G. Lu,
Structure-based drug design: Synthesis and biological evaluation of quinazolin-4-amine derivatives as
selective Aurora A kinase inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.08.053.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Structure-Based Drug Design: Synthesis and Biological
Evaluation of Quinazolin-4-Amine Derivatives as Selective
Aurora A Kinase Inhibitors
Liang Long,a,+ Yong-Heng Wang,a,+ Jun-Xiao Zhuo,b,+ Zheng-Chao Tu,c Ruibo Wu,a
Min Yan,b,* Quentin Liu,b,d,* Gui Lua,*
a
Institute of Medicinal Chemistry, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou
510006, P. R. China
b State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou 510060,
P. R. China
c Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, P. R. China
d Institute of Cancer Stem Cell, Dalian Medical University, Dalian 116044, P. R. China
* Corresponding author.
Keywords: Aurora A kinase inhibitors, quinazolin-4-amine derivatives, molecular dynamic
simulations, TNBC
ABSTRACT: Aurora kinases play critical roles in the regulation of the cell cycle and mitotic
spindle assembly. Aurora A kinase, a member of the Aurora protein family, is frequently highly
expressed in tumors, and selective Aurora A inhibition serves as a significant component of
anticancer therapy. However, designing highly selective Aurora A inhibitors is difficult because
Aurora A and B share high homology and differ only by three residues in their ATP-binding
pockets. Through structure-based drug design, we designed and synthesized a series of novel
quinazolin-4-amine derivatives. These derivatives act as selective Aurora A kinase inhibitors by
exploiting the structural differences between Aurora A and B. The selectivities of most compounds
were improved (the best up to >757-fold) when comparing with the lead compound (3-fold). In
vitro biochemical and cellular assays revealed that compound 6 potently inhibited Aurora A kinase
and most human tumor cells. Furthermore, compound 6 effectively suppressed carcinoma, such as
triple-negative breast cancers (TNBC) in an animal model. Therefore, compound 6 might serve as
a promising anticancer drug. Moreover, through molecular dynamic (MD) analysis, we have
identified that a salt bridge formed in Aurora B is key contributor for the isoform selectivity of the
inhibitor. This salt bridge has not been previously detected in the reported crystal structure of
Aurora B. These results might provide a crucial basis for the further development of highly potent
inhibitors with high selectivity for Aurora A.
1. Introduction
Over the past decades, the inhibition of protein kinases through ATP-binding site has
emerged as an attractive strategy for the treatment of multiple tumor types. The Aurora family of
serine–threonine protein kinases, which comprise Aurora A, B, and C, were originally identified in
Drosophila melanogaster by Dr. Glover’s group in 1995.1 Since then, a number of studies have
demonstrated that Aurora A and B play diverse roles in the regulation of various events during
mitosis. Aurora A is responsible for centrosome maturation, mitotic entry, and spindle formation,
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as evidenced by its subcellular localization in centrosomes and spindle poles. In contrast to Aurora
A, Aurora B and C are chromosome passenger proteins. Specifically, Aurora B is crucial for
precise chromosome segregation and cytokinesis, and Aurora C is mainly expressed in the testis.2-5
Small-molecule inhibitors that target Aurora A and B kinases have been fully investigated
over the past decades. Most of these compounds are dual Aurora A and B kinase inhibitors,6-11 and
only few selective Aurora A inhibitors are currently undergoing clinical trials (Fig. 1).12-14
Although pan-Aurora kinase inhibitors exhibit major phenotypes consistent with Aurora B
inhibition, they are significantly active against Aurora A and B kinases in vivo.15 A recent report
has shown that numerous toxicities are associated with Aurora B inhibition, which might produce
unstable polyploid cells to facilitate tumorigenesis.16 In addition, some antagonistic effects may
occur when both Aurora kinases are inhibited.16 Notably, a recent study indicated that the
expression of Aurora A, but not of Aurora B, is related to the survival of breast cancer patients.17
This relationship indicated that therapeutic agents targeting Aurora A kinase might be preferable
to those targeting Aurora B kinase in the treatment of breast cancer.
Figure 1. Several selective Aurora A kinase inhibitors in clinical test.
Aurora A and B kinases share high structural and sequence similarities. Their ATP-binding
sites differ only by three residues: L215, T217, and R220 in Aurora A, and R159, E161, and K164
in Aurora B.18 Given the considerable homology between Aurora A and B kinases, the selective
inhibition of one specific Aurora kinase is difficult. Nevertheless, some groups have reported that
inhibitors selective for Aurora A exploit the non-conserved residue T217 in active site of Aurora A,
whereas introducing a steric clash with the equivalent residue E161 in Aurora B/C.19-23
Nevertheless, the lack of relevant data complicates the use of residues L215 and R220 in Aurora A
as drivers of inhibitor selectivity for Aurora A over Aurora B.
We previously identified N-trisubstituted pyrimidine derivatives as pan-Aurora kinases
inhibitors. Among these derivatives, compound 1 exhibited potent anticancer activity (Fig. 2).24-25
We sought to further improve the therapeutic efficacy by improving its selectivity for Aurora A.
For this purpose, compound 2, a highly selective Aurora A inhibitor, established by Rueth et al.,19
drew our attention (Fig. 2). They inferred that T217 is the most likely contributor to specific
Aurora A kinase activity. E161 in Aurora B undergoes steric hindrance with the N-2 phenyl ring.
In addition, the 4-aminopyrazole motif enables key interactions with the hinge region of Aurora A
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(Glu211, Ala213, Pro214) essential for maintaining activity.19 However, the antiproliferative
activities of compound 2 on cell level require further development. Taking these elements into
account, we set out to design and prepare a new class of isoform selective Aurora A kinase
inhibitors on the basis of the quinazolin-4-amine scaffold. Modification on the C-2 and C-7
positions of quinazolin-4-amine can greatly affect the selectivity of inhibitors for Aurora A over
Aurora B. To our delight, the selectivity of these quinazolin-4-amine derivatives was superior to
that of our previously synthesized derivatives. In particular, compounds 6 and 3d were highly
selective for Aurora A kinase, with approximately 66-fold and >757-fold Aurora A selectivity,
respectively. Therefore, we investigated the cancer biology behaviors of these inhibitors. However,
we found that 3d exhibited unfavorable cellular activities. By contrast, compound 6 showed potent
biological behaviors that corresponded to Aurora A kinase inhibition in cultured human cancer
cells and inhibited the growth of MDA-MB-231 xenografts nude mice without producing obvious
side effects. These behaviors are essential for the effectiveness of cancer-targeted therapies.
Meanwhile, we preformed MD simulations to investigate the binding modes of the inhibitors. We
found that a salt bridge in Aurora B play key roles in the selectivity of inhibitors toward Aurora A.
Figure 2. Structure-based design of quinazoline-4-amines as selective Aurora A inhibitors.
2. Chemistry
As shown in Schemes 1-4, a variety of quinazoline-4-amine derivatives were designed and
synthesized. The key intermediate quinazolinones 2a-2g (Scheme 1) were obtained through the
treatment of anthranilic acid analogs 1a-1f with the corresponding acyl chlorides, and 2h-2k were
obtained through the conversion of the anthranilamide 1g via a HATU-mediated coupling reaction
(Scheme 2). Subsequently, quinazolin-4-amine derivatives 3a-3k were prepared through direct
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ammoniation with 3-amino-5-methylpyrazole in accordance with a previously established
method26 with some modifications. Derivatives 3a-3k could be directly isolated from the reaction
mixture as a solid product. The C-N coupling reaction realized the displacement of the 7-chloro
group with piperidine 4-piperidinecarboxylic acid tert-butyl ester. Then, the direct ammoniation of
intermediate 4 resulted in structure 5. Compounds 6 and 7 were obtained through the substitution
of the tertiary butyl of 5 by ethyl and the removal of the tertiary butyl (Scheme 1).
Scheme 1. Synthesis of target compounds 3a-3g, 6 and 7.
Reagents and conditions: (a) K2CO3, MeCN, 95 °C, 12 h; (b) (MeCO)2O, 130-160 °C, 2 h; (c) NH4Ac, 160-190 °C,
5 h; (d) Pd2dba3, Xphos, NaOtBu, 1,4-dioxane, 90 °C, 16 h; (e) PyBrop, DBU, MeCN, 70 °C, 48-72 h; (f) SOCl2,
EtOH, rt～80 °C, 12 h; (g) TFA, CH2Cl2, rt, 2 h.
Scheme 2. Synthesis of compounds 3h-3k and 10a.
Reagents and conditions: (h) HATU, DIPEA, CH2Cl2, rt, 24 h; (i) 10 N NaOH, rt, 0.5 h; (j) I2, DMSO, 110 °C, 12
h.
High yield of 2-amino-4-chlorobenzamide 12 was obtained from 2-amino-4-chlorobenzoic
acid 11 upon the treatment with EDCI, HOBT and ammonium hydroxide (Scheme 3). Access to
quinazolin-4-amine derivatives 10a-10c was accomplished through one-pot oxidation and
aromatization to provide the 2-benzoylquinazolin-4(3H)-ones 9a-c,27 which were then subjected to
ammoniation in a manner similar to that described for 3a-3k (Schemes 2 and 3). We subsequently
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attempted to replace 7-chlorine with piperidinecarboxylic acid tert-butyl ester followed by
ammoniation. However, although the 2-benzoylquinazolin-4(3H)-one derivatives 9b and 9c were
successfully linked with piperidinecarboxylic acid tert-butyl ester, attempts to obtain the final
ammoniation products failed. We inferred that the carbonyl of benzoyl disturbed the ammoniation
reaction. Therefore, we tried to increase the steric hindrance around benzoyl and bring in an
electron-donating group to decrease the reactivity of benzoyl. Finally, the desired compound 13
was obtained with 2’,6’-dimethoxyacetophenone as the starting material (Scheme 3).
Scheme 3. Synthesis of compounds 10b-10c and 13.
Reagents and conditions: (k) EDCI, HOBT, NH4OH, DMF, 0 °C~rt.
Intermediate 15 was produced through nucleophilic substitution at the C4-position of
.
4-chloro-2-nitrobenzonitrile 14 followed by in situ reduction with 80% N2H4 H2O under the
catalysis of 10% Pd/C (Scheme 4). The amino compound 15 was converted to amide through
coupling and subsequent cyclization in a strong alkaline condition to afforded structure 16.
Compounds 17a and 17b were prepared in a similar ammoniation reaction with the above final
compounds in the presence of 3-amino-5-methylpyrazole or 3-aminopyrazole (Scheme 4).
Scheme 4. Synthesis of compounds 17a and 17b.
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.
Reagents and conditions: (l) 4-methylpiperazine, 1,4-dioxane, 110 °C, 48 h; (m) Pd/C, N2H4 H2O, 80 °C, EtOH, 12
h.
3. Results and Discussion
In vitro kinases and cells assay. Biochemical potency is a key metric used in compound
optimization. The in vitro Aurora enzyme inhibition and antiproliferative activities of these new
quinazoline-4-amine derivatives were evaluated, the results were summarized in Tables 1 and 2,
respectively.
To obtain derivatives with high affinity for Aurora A, we preliminarily modified the
quinazoline-4-amine scaffold by introducing R3 with phenyl, benzyl, naphthyl, isopropyl, and
benzoyl into the C-2 position of quinazoline (compounds 3h-3k and 10a). Compound 3h with
phenyl group showed desirable Aurora A inhibition activity (IC50 = 0.028 µM) but modest
selectivity over Aurora B (30-fold), while compound 10a with benzoyl group exhibited slightly
low Aurora A potency (IC50 = 0.188 µM) but high selectivity (61-fold). Compound 3j with bulky
naphthyl group also showed high selectivity (40-fold), while 3i and 3k afforded pronounced losses
in selectivity. Furthermore, most compounds exhibited good antiproliferative activities against
seven human tumor cells, while compounds 3j and 10a displayed pronounced inferior effect.
We then tested the influence of R2 in the C-8 quinazoline moiety. The Aurora potencies of the
methoxy-substituted inhibitors 3a and 3b were comparable with unsubstituted inhibitors 3h and 3i,
but higher than those with nitro-substituted 3c. And the selectivities of these compounds for
Aurora B were at the same level.
We continued to investigate the effects of additional substituents around the
quinazoline-4-amine scaffold by focusing our modification efforts on the C-7 moiety (R1
substituent group). Surprisingly, when R1 was chlorine, fluorine, or nitro, as exemplified by 3d,
10b, 10c, 3e, and 3f, Aurora B potency sharply decreased (IC50 > 100 µM), thus the selectivity
also sharply increased (>757-fold, >134-fold, >77-fold, 88-fold, and >308-fold, respectively).
However, the Aurora A activity of these compounds decreased (IC50 values of 0.132 µM, 0.744
µM, 1.30 µM, 0.072 µM, and 0.325 µM, respectively), and their intracellular potency weakened.
These results indicated that the physical properties of this series caused poor cell penetrability.
When the R1 moiety was methoxy or N-containing heterocyclic compounds, apart from compound
13 (the relatively high molecular weight and TPSA probably indicated poor druggability according
to the extension of Lipinski's Rule of Five), the activity levels of compound 17a, 6, and 7 toward
Aurora kinases improved.
In particular, compound 17a showed remarkable Aurora A inhibition (IC50 = 0.010 µM) and
antiproliferative activities against tumor cells (IC50 values of 0.142–2.49 µM). Its specificity for
Aurora A, however, was only 30-fold. Notably, compound 6, which featured
a
piperidinecarboxylic acid ethyl ester substitution, displayed satisfactory Aurora A kinase potency
(IC50 = 0.038 µM) and isoform selectivity (66-fold over Aurora B), as well as considerable
cytotoxicities against seven human cancer cell lines (IC50 values of 0.45–2.40 µM). By contrast,
the Aurora A and B potency of compound 17b drastically declined due to the removal of methyl
group at the C-4 moiety (compared with 17a).
Table 1. Kinase inhibition activities of quinazoline-4-amine analogues.
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IC50 (µM)a
Selectivity
Compd
R1
R2
R3
TPSAb
(Aur A/B)
30
Aurora A Aurora B
H
H
H
H
Ph
0.028
0.069
0.128
0.103
0.188
0.025
0.041
0.174
0.132
0.744
1.30
0.858
1.271
5.07
66
66
66
66
84
76
76
112
66
93
84
66
112
76
3h
3i
CH2Ph
18
H
H
Nap
40
3j
H
H
iPr
1.147
11.5
11
3k
10a
3a
3b
3c
H
H
COPh
61
H
OMe
OMe
NO2
H
Ph
0.547
0.893
4.594
>100
>100
>100
6.282
>100
0.773
21
H
CH2Ph
22
H
Ph
26
Cl
Cl
Cl
F
Ph
>757
>134
>77
88
3d
10b
10c
3e
H
CO(4-OMe)Ph
H
CO(4-Me)Ph
H
Ph
Ph
Ph
0.072
0.325
0.034
NO2
OMe
H
>308
22
3f
H
3g
H
H
H
H
Ph
Ph
Ph
0.010
0.038
0.006
0.270
2.522
0.127
30
66
20
73
96
17a
6
107
7
CO(2,6-diOMe)Ph 0.217
>60
>276
2.4
132
73
13
17bc
5.951
14.36
a
b
IC50 values were reported as the mean from at least two independent experiments. The polar
surface area was calculated using the Molinspiration molecular property calculation engine
(http://www.molinspiration.com). c The structure of 17b was displayed above Table 1.
Table 2. Antiproliferative activities of quinazoline-4-amine analogues. (IC50 values in µM; NT =
not tested, NA = not active; values lower than 1 µM were highlighted in bold)
Compd
K562
MCF-7
HELA
DU145
MOLT-4
MDA-MB-231
SK-BR-3
2.99
2.29
15.0
3.92
6.16
13.3
>30
10.7
7.99
4.44
10.4
7.43
5.15
5.79
10.4
NT
2.88
3.12
11.0
4.80
2.54
9.10
7.15
11.9
3h
3i
0.883
1.52
3.75
2.81
3j
3k
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>30
2.30
1.44
6.43
NA
>30
4.17
6.28
4.53
>30
NA
5.22
0.817
1.45
2.46
>30
>30
6.36
>10
8.82
2.86
4.81
6.48
NA
17.0
1.90
2.62
3.47
>30
14.7
1.85
2.72
4.59
NA
10a
3a
3b
3c
6.62
>30
3d
10b
10c
3e
NA
>30
12.5
16.0
>30
NT
12.3
>30
NA
NA
NA
NA
NT
NA
>30
>30
>30
3.60
0.654
2.23
6.04
1.51
7.88
9.7
NA
11.2
9.83
1.32
0.142
1.19
10.5
1.38
3.98
10.7
8.86
2.03
2.49
2.40
16.3
2.75
6.12
12.0
2.12
0.191
1.09
5.52
0.373
6.75
10.1
1.31
0.179
0.450
1.86
0.526
7.85
7.36
4.73
0.409
2.05
21.5
3.61
5.27
NT
3f
1.20
0.149
0.821
2.20
NT
3g
17a
6
7
13
17b
NT
Binding mode and Aurora A kinase selectivity. To better understand the binding modes
between inhibitors and Aurora proteins and the origin of selectivity toward Aurora A, we
performed MD simulations on Aurora A and B with compound 6 at the binding site, Figure 3
shows the protein surface and the binding sites (with compound 6 inside) of Aurora A and B. The
active pocket of Aurora A is more opened with a crevice accessing to solvent; while Aurora B is
more closed with only a hole accessing to outside solvent. This is attributed to the salt bridge
between K85 and E161 in Aurora B, whereas the corresponding residues in Aurora A are K141
and T217 without salt bridge formed (also see Figure S1 in SI). The binding modes of compound
6 in Aurora A and B are similar, thus we suspect that the salt bridge is mainly responsible for the
selectivity of Aurora A against Aurora B. Structurally, the benzene ring of compound 6 is torsional
relative to the quinazolin ring in Aurora B (Fig. 3B), resulting from the steric clash between the
benzene ring and the salt bridge (Fig. 3A); while the two aromatic rings are nearly coplanar in
Aurora A binding. This will bring energy penalty to Aurora B and lead to the selectivity toward
Aurora A.
To further confirm our speculation, we calculated binding free energies of Aurora A and B
with compound 6 using MMGBSA method (without considering the contribution of entropy). The
binding free energy of compound 6 with Aurora A and B kinases is -38.3 and -36.0 kcal/mol (Fig.
3), respectively, indicating the bindings are thermodynamically favorable and compound 6 prefers
to bind with Aurora A protein comparing with Aurora B. The calculated binding free energy
difference of 2.3 kcal/mol is comparable to the experimental value of 2.5 kcal/mol (converted
from the 66-fold selectivity using Boltzmann distribution), guaranteeing the credibility of the
calculated method. Additionally, to inspect the contribution of residues around the binding site to
the binding, we decomposed the binding free energy as shown in Figure 4. The total energy
contribution of E161 in Aurora B is positive (about 1.5 kcal/mol), unfavorable to the binding;
while the corresponding contribution of T217 in Aurora A is negative (about -1.0 kcal/mol),
favorable to the binding. For E161 in Aurora B, the leading unfavorable factor comes from the
electrostatic repulsion (about 2.5 kcal/mol) with the inhibitor compound 6, in agreement with our
initial guess; with respect to T217 in Aurora A, van der Waals interaction and electrostatic
attraction contribute to the negative energy. Because of the salt bridge between E161 and K85, the
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total energy contribution of K85 in Aurora B is a little positive (about 0.5 kcal/mol), whereas the
equivalent K141 in Aurora A nearly has no contribution to the binding. These are responsible for
the selectivity toward Aurora A kinase. In addition, the R220 in Aurora A has energy contribution
of about -0.7 kcal/mol, whereas the corresponding K164 nearly has no contribution, also
responsible for the selectivity. Despite the different position of L215 (R159) in Aurora A (B), these
residues have approximately equal contributions to the binding and thus have no contribution to
the selectivity. Other residues around the binding site are similar in Aurora A and B proteins, and
have little contribution to the selectivity.
From the above analysis, we found that the salt bridge is the main contribution to the
selectivity and the other two different positions (R220 and K164) at the pocket entry has only a
little contribution. This well explained the fact that the modification of R3 group (near to the salt
bridge) greatly affected the selectivity (such as compounds 3h-3k and 10a in Table 1). However,
the further enhancement of selectivity comes from the modification of R1 group. To explain this
phenomenon, we performed further MD simulations on Aurora B in complex with the
Cl-substituent compound 3d with the highest selectivity of > 757-fold over Aurora B. In contrast
to compound 6, the salt bridge between K85 and E161 in the complex of Aurora B with compound
3d is broken after 34 ns during MD simulation (Fig. 5A), which will bring energy penalty to the
binding of Aurora B with compound 3d and lead to the high selectivity to Aurora A. Furthermore,
we monitored the dihedral between the benzene ring and the quinazolin core system of compounds
6 and 3d in the pocket of Aurora B during MD simulations as shown in Figure 5B. Compared with
compound 6, the two aromatic rings in compound 3d are more coplanar, especially after the salt
bridge was broken. This may result from different electronic effect of the two different
substituents at C-7 position (R1 group). The coplanar benzene ring in compound 3d is closer and
more clash sterically with the salt bridge (than the non-coplanar benzene ring in compound 6), and
finally leads to the breakage of salt bridge. Hence, the high selectivity of compound 3d is actually
attributed to electronic effect of -Cl atom, which changes the interaction between the benzene ring
and the salt bridge. Similarly, the selectivity of compounds 3e, 3f and 3g are subject to the
electronic effect of substituent group at C-7 position. Because the C-7 position of quinazolin core
is located at the solvent-accessible region, the charged substituent group at C-7 such as
compounds 7 and 17a are more active for both Aurora A and B (Table 1). Moreover, the methyl
group at C-4 moiety points to the inside hydrophobic region of the binding pocket and interacts
with hydrophobic residues such as A160 (A104), L194 (L154) and L210 (L154) in Aurora-A (-B)
(Fig. 3 and 4), thus removing this substituent will lead to potency loss of both Aurora A and B
proteins (comparing with compound 17a, the IC50 values of compound 17b against Aurora A and
B were 5.951 µM and 14.36 µM, respectively).
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Figure 3. Representative surface maps and binding modes of compound 6 interact with Aurora A
(A) and Aurora B (B) from molecular dynamics simulations. Compound 6 (cyan) and surrounding
residues are shown as sticks. The binding free energies (∆Gbinding) were calculated using
MMGBSA method.
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Figure 4. The individual energy contribution of residues to the binding free energy of Aurora A
and B in complex with compound 6. The total energy contribution (black) consists of van der
Waals interaction energy (red), electrostatic interaction energy (blue) and solvation free energy
(olive).
A
K85
d
E161
B
NH
N
HN
N
R1
N
ϕ
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Figure 5. (A) The time evolutions of the distances between K85 and E161 of Aurora B binding
with compounds 6 and 3d during molecular dynamics simulations. (B) The time evolutions of the
dihedral between the benzene ring and the quinazolin ring of compounds 6 and 3d in the pocket of
Aurora B during molecular dynamics simulations.
Compound 6 potently inhibits Aurora A kinase in cultured human cancer cells. Having
identified a potent and specific Aurora A inhibitor, compound 6, through structure-guided lead
compound optimization, we focused on the biochemical behaviors of this novel inhibitor on the
cellular level.
Upon activation, Aurora A is autophosphorylated on Thr288 in its activation loop.28 Histone
H3 is a direct downstream target of Aurora B kinase, and inhibiting Aurora B kinase decreases the
phosphorylation of histone H3 (Ser10).29 First, we determined if compound 6 could lower the
levels of p-Aurora A (Thr288) in 293T cells. Cells were treated with the control (0.1% DMSO) or
increasing doses of compound 6 for 24 h and subjected to Western blot analysis. As shown in
Figure 6A, p-Aurora A (Thr288) levels in 293T cells treated with compound 6 decreased to levels
comparable with those in cells treated with the specific Aurora A inhibitor MLN8237. However,
compound 6 did not decrease the phosphorylation of histone H3 (Ser10). These results indicated
that compound 6 selectively inhibits the activity of Aurora A kinase in human cells.
We then investigated if compound 6 can induce cell cycle arrest and apoptosis in breast
cancer cells. Flow cytometry analysis showed that compared with treatment with DMSO,
treatment with compound 6 induced dramatic G2/M arrest and increased the numbers of 4N/8N
cells in a dose dependent manner (Fig. 6B). These data were consistent with reports that Aurora
kinase A is crucial for cell-cycle progression. Apoptotic cell death was assessed through Annexin
V/propidium iodide (PI) staining. As shown in Figure 6C, the percentage of Annexin-V-positive
cells significantly increased after 48 h of treatment with compound 6 (2 µM, 42.33±0.07%; 5 µM,
47.10±0.01%) relative to that with treatment with DMSO (15.46±1.18%) (p<0.001). These results
indicated that compound 6 induces apoptosis in cancer cells.
Cells with impaired Aurora A activity typically form abnormal mitotic spindles.30 Therefore,
we examined mitotic spindle morphology and chromosome alignment in MDA-MB-231 cells
treated with DMSO or compound 6 (1 µM) for 24 h. The DMSO-treated control cells displayed
normal bipolar mitotic spindles with proper chromosome alignment along the metaphase plate. By
contrast, compound 6-treated cells showed monopolar or multipolar spindles with various defects
in chromosome alignment (Fig. 6D). This phenotype is consistent with those observed in cells
with Aurora A suppression by RNAi.31
Finally, we performed a colony formation assay to test whether compound 6 can inhibit
cancer cell proliferation. As shown in Figure 6E, MDA-MB-231 cells treated with compound 6 (3
µM) (16±20) formed significantly fewer colonies than cells treated with DMSO (180±30)
(p<0.01). Thus, in general, these data suggested that in breast cancer cells, compound 6 inhibits
Aurora A kinase and suppresses growth.
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Figure 6. Compound 6 selectively inhibits Aurora A over Aurora B in cultured human tumor cells.
(A) 293T cells were treated with indicated doses of compound 6 or control (0.1% DMSO) for 24 h
and subjected to Western-blot assay with p-Aurora A (Thr288), p-Histone H3 (Ser10) and GAPDH
antibodies. (B) MDA-MB-231 cells were treated with indicated doses of compound 6, MLN8237
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or control (0.1% DMSO) for 24 h and subjected to flow cytometry analysis of cell cycle. (C)
Annexin V/PI analysis of the cells described in (B). (D) Representative immunofluorescent
images of mitotic spindle structures in MDA-MB-231 cells incubated with compound 6 (1 µM) or
control (0.1% DMSO): centrosome (green), spindle (red), nuclei (blue), original magnification×
1000. (E) Colony formation assay to assess the cell proliferation of MDA-MB-231 cells treated
with compound 6 (3 µM) or control (0.1% DMSO) for 7 days.
Compound 6 inhibits the growth of xenografts in nude mice. On the basis of the above
intracellular data, we then evaluated the antitumor activity of compound 6 in a nude mice
xenograft model of triple-negative human breast cancer (TNBC). Nude mice bearing
MDA-MB-231 xenograft tumors were treated with compound 6 (30 mg/kg, per 2 days) through
intragastric administration for 18 days. As shown in Figure 7A, the tumor size in the compound
6-treated group (n=4) was smaller than that in the control vehicle group (n=4). This result
indicated that compound 6 significantly inhibits the growth of xenograft tumors (TGI: 45%,
p<0.05). Consistently, the tumor weight in the compound 6-treated group (0.85±0.45 g) was
significantly lower than that in vehicle-treated group (1.70±0.84 g; p<0.01; Fig. 7B). During the
experimental period, compound 6 was well tolerated by the mice with no significant weight loss
observed (Fig. 7C), indicating the low toxicity of intragastrically administered compound 6. In
addition, the weights of key organs, such as liver, lung, kidney, and spleen, were not significantly
altered in response to treatment with compound 6 (Fig. 7D). Accordingly, the blood cell count
assay showed that the composition of blood cell in the compound 6-treated group was comparable
with that in vehicle-treated group (Fig. 7E). These results suggested that compound 6 efficiently
suppresses tumor growth in vivo without causing evident side-effects in normal tissues and has
potential applications in targeted cancer therapy.
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Figure 7. Compound 6 causes tumor growth inhibition without showing obvious impact on mice
weight, normal organs and blood routine level in vivo. (A) Nude mice bearing MDA-MB-231
xenograft tumors were intragastrically treated with vehicle or compound 6 for 18 days (30 mg/kg,
per 2 days) after tumor formation. The estimated tumor volume is plotted versus time. (B) Weights
of dissected tumors were measured. (C) Body weights of mice were monitored and plotted versus
time. (D) Organ weights of liver, lung, kidney, and spleen among non-treatment (blank), vehicle-
and drug-treated mice were measured after treatment had finished. (E) Blood-cell count was
accessed among non-treatment (blank), vehicle- and drug-treated mice after treatment had finished.
For the statistical analysis of differences in tumor volume between treatment groups, a two-way
ANOVA with repeated measures on day factor was performed. The unpaired two-tailed t-tests of
vehicle-treated and compound 6-treated mice were performed to look for significant changes in
the data generated from blood and organs. All data are presented as the mean ±SD (n=4; *, p <
0.05, Student’s test).
4. Conclusion
The aberrant expression and localization of Aurora A have been frequently detected in various
types of cancers, such as leukemia, breast, ovarian, prostate, and colorectal cancers.32-37 TNBCs,
15

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which do not express the estrogen receptor, progesterone receptor, and human epidermal growth
factor receptor 2, account for 15–20% of all reported breast cancer cases. TNBCs are highly
aggressive and are associated with worse prognosis than other breast cancer subtypes. The
development of new effective targeted therapies for TNBCs is urgently needed.38
The active pockets of Aurora A and Aurora B/C differ only by three residues. Some groups
have designed selective Aurora A inhibitors on the basis of the non-conserved T217 residue in
Aurora A, which is smaller than the equivalent E161 residue in Aurora B, and may distinguish the
isoform-specific Aurora A inhibitor. By contrast, through MD simulations, we identified that a salt
bridge between E161 and K85 in Aurora B might cause a steric clash with compounds 6 and 3d.
This interaction is crucial for the selectivity of inhibitors for Aurora A.
In this paper, we report a series of quinazolin-4-amine derivatives, which represent a novel
class of inhibitors selective for the Aurora A kinase isoform. Compound 6, in particular, exhibits
potential anticancer effects in cell and animal models. This compound is not associated with
significant human ether-a-go-go-related gene (hERG)-related problems and has an IC50 value
of >10 µM for the hERG K+ channel. These data suggested that compound 6 is a promising drug
candidate for the treatment of TNBC. In addition, MD analysis revealed that compound 3d could
disrupt the salt bridge in Aurora B. This novel finding inspired us to design more potent and
highly isoform selective Aurora A inhibitors. Our research group is conducting additional detailed
studies in this field, and the results of these studies will be reported in due time.
5. Experimental protocols
5.1. Chemistry
All starting materials, reagents and anhydrous solvents were obtained from commercial
sources and were distilled from standard drying agents unless otherwise specified. All reactions
were monitored by thin-layer chromatography on silica gel plates (GF-254) and visualized with
UV light. Purification of reaction products were carried out by flash chromatography with silica
gel (200-300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd or silica-based packing
materials (SP-120-40/60-ODS-RPS) from Daisogel. 1H and 13C NMR spectra were recorded using
TMS as the internal standard in DMSO-d6 or CDCl3 with a Bruker Avance III spectrometer at 400
MHz and 101 MHz, respectively. HRMS spectra were recorded on an ESI-ion trap mass
spectrometer (Shimadzu LCMS-IT-TOF). Mass spectra were measured with an Agilent 6120
spectrometer using an ESI+APCI source coupled to an Agilent 1200 HPLC system operating in
reverse mode. Purity of all biologically evaluated compounds was greater than 95%.
5.1.1 General procedure for the synthesis of 2a-2g
1a-g (1.0 equiv.) and K2CO3 (3.0 equiv.) were dissolved in MeCN (200 mL), then benzoyl
chloride (1.2 equiv.) was slowly added under rt. The solution was stirred at 90 °C overnight. After
removal of the solvent under vacuum, the residue was quenched with 2 N HCl solution. The
formed precipitate was collected by filtration and washed with water, then treated with acetic
anhydride (25 mL) and heated under reflux for 2 h. The solid obtained upon concentration was
collected and refluxed with NH4OAc at 170 °C for 5 h. The mixture was poured into water,
filtered and washed with CH2Cl2 and water successively to give products 2a-g.
5.1.1.1 8-Methoxy-2-phenylquinazolin-4(3H)-one (2a)
Compound 2a was prepared from 1a (694 mg, 46% yield). 1H NMR (400 MHz, DMSO-d6) δ:
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12.55 (s, 1H), 8.19 (d, J = 1.6 Hz, 1H), 8.17 (d, J = 2.0 Hz, 1H), 7.71 (dd, J = 1.2, 1.2 Hz, 1H),
7.59-7.53 (m, 5H), 7.45 (t, J = 8.0 Hz, 1H), 7.39 (dd, J = 1.2, 1.2 Hz, 1H), 3.95 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ: 158.65, 157.68, 155.16, 142.52, 139.30, 130.48, 128.84, 128.39, 126.28,
114.81, 112.76, 98.02, 56.33 ppm. MS (ESI+APCI) m/z 253.1 [M+H]+.
5.1.1.2 2-Benzyl-8-methoxyquinazolin-4(3H)-one (2b)
Compound 2b was prepared from 1a (483 mg, 60% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.21 (s, 1H), 7.64 (d, J = 7.2 Hz, 1H), 7.40-7.35 (m, 3H), 7.32-7.28 (m, 3H), 7.24-7.21 (m, 1H),
3.96 (s, 2H), 3.89 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 162.36, 155.02, 140.05, 137.05,
129.17, 128.91, 127.22, 126.96, 122.24, 117.47, 116.16, 56.76, 41.38 ppm. MS (ESI+APCI) m/z
267.1 [M+H]+.
5.1.1.3 8-Nitro-2-phenylquinazolin-4(3H)-one (2c)
Compound 2c was prepared from 1b (475 mg, 40% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.95 (s, 1H), 8.38 (dd, J = 1.2, 1.2 Hz, 1H), 8.32 (d, J = 7.6 Hz, 1H), 8.17 (d, J = 7.2 Hz, 2H),
7.67-7.62 (m, 2H), 7.60-7.56 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 158.18, 158.00, 146.32,
141.08, 139.83, 136.34, 133.70, 132.50, 129.64, 129.56, 128.69, 124.83, 121.12, 112.94, 99.15,
11.35 ppm. MS (ESI+APCI) m/z 268.1 [M+H]+.
5.1.1.4 7-Chloro-2-phenylquinazolin-4(3H)-one (2d)
1
Compound 2d was prepared from 1c (1.2 g, 48% yield). H NMR (400 MHz, DMSO-d6) δ:
12.66 (s, 1H), 8.19-8.13 (m, 3H), 7.78 (d, J = 1.6 Hz, 1H), 7.63-7.52 (m, 4H). 13C NMR (101
MHz, DMSO-d6) δ: 161.59, 153.74, 149.85, 139.13, 132.36, 131.64, 128.57, 127.88, 126.72,
126.54, 119.79 ppm. MS (ESI+APCI) m/z 257.2 [M+H]+.
5.1.1.5 7-Fluoro-2-phenylquinazolin-4(3H)-one (2e)
Compound 2e was prepared from 1d (570 mg, 59% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.62 (s, 1H), 8.23-8.17 (m, 3H), 7.61-7.49 (m, 4H), 7.38 (t, J = 7.6 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6) δ: 167.56, 165.06, 162.08, 154.27, 151.44, 151.31, 132.91, 132.13, 129.50, 129.39,
129.09, 128.37, 118.49, 115.66, 115.43, 112.98, 112.77 ppm. MS (ESI+APCI) m/z 241.1 [M+H]+.
5.1.1.6 7-Nitro-2-phenylquinazolin-4(3H)-one (2f)
Compound 2f was prepared from 1e (595 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.89 (s, 1H), 8.39 (s, 1H), 8.33 (d, J = 8.8 Hz, 1H), 8.20 (d, J = 8.0 Hz, 3H), 7.65-7.55 (m, 3H).
13C NMR (101 MHz, DMSO-d6) δ: 161.80, 154.95, 151.78, 149.62, 132.54, 132.44, 129.15,
128.62, 128.51, 125.78, 122.84, 120.48 ppm. MS (ESI+APCI) m/z 268.1 [M+H]+.
5.1.1.7 7-Methoxy-2-phenylquinazolin-4(3H)-one (2g)
Compound 2g was prepared from 1f (715 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.38 (s, 1H), 8.19 (d, J = 6.8 Hz, 2H), 8.06 (d, J = 8.8 Hz, 1H), 7.60-7.54 (m, 3H), 7.19 (d, J =
2.4 Hz, 1H), 7.10 (dd, J = 2.4, 2.4 Hz, 1H), 3.93 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ:
164.67, 162.83, 162.23, 153.39, 133.20, 131.85, 129.04, 128.21, 127.92, 116.62, 114.90, 108.99,
56.19 ppm. MS (ESI+APCI) m/z 253.1 [M+H]+.
5.1.2 General procedure for the synthesis of 2h-2k, 16
To a mixture of substituted benzoic acid (1.0 equiv.), HATU (1.2 equiv.) and CH2Cl2 (50
mL) was added DIPEA (2.0 equiv.). The solution was stirred at rt for 1 h, then anthranilamide 1g
was added. The reaction mixture was stirred at 50 °C for 24 h, then 1 N HCl (50.0 mL) was added,
the resulting solution was extracted with CH2Cl2. The combined organic phases were dried over
17

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Na2SO4. After removal of the solvent under vacuum, the residue was dissolved in EtOH (50 mL)
at 0 °C. 10 N NaOH (4.0 equiv.) was added, and the mixture was stirred for 30 min. The solution
was neutralized with concentrated HCl before the solvent was removed under vacuum. The
residue was purified by silica gel chromatography using CH2Cl2/MeOH (V: V= 30: 1) as eluent to
afford compounds 2h-2k, 16.
5.1.2.1 2-Phenylquinazolin-4(3H)-one (2h)
Compound 2h was prepared from 1g (674 mg, 61% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.56 (s, 1H), 8.23-8.18 (m, 3H), 7.87-7.83 (m, 1H), 7.78-7.76 (m, 1H), 7.61-7.52 (m, 4H). 13C
NMR (101 MHz, DMSO-d6) δ: 162.72, 152.78, 149.15, 134.99, 133.19, 131.80, 129.02, 128.22,
127.90, 126.98, 126.30, 121.44 ppm. MS (ESI+APCI) m/z 223.1 [M+H]+.
5.1.2.2 2-Benzylquinazolin-4(3H)-one (2i)
Compound 2i was prepared from 1g (220 mg, 40% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.43 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.80-7.75 (m, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.50-7.45 (m,
13
1H), 7.40 (d, J = 7.2 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.25 (t, J = 7.2 Hz, 1H), 3.95 (s, 2H). C
NMR (101 MHz, DMSO-d6) δ: 162.34, 156.44, 149.39, 137.04, 134.85, 129.35, 128.97, 127.40,
127.27, 126.67, 126.18, 121.23, 41.26 ppm. MS (ESI+APCI) m/z 237.1 [M+H]+.
5.1.2.3 2-(Naphthalen-2-yl)quinazolin-4(3H)-one (2j)
Compound 2j was prepared from 1g (312 mg, 38% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.66 (s, 1H), 8.84 (s, 1H), 8.32 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 7.6 Hz, 1H), 8.08 (d, J = 8.0 Hz,
2H), 8.02 (d, J = 7.2 Hz, 1H), 7.87 (t, J = 7.4 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.67-7.63 (m, 1H),
7.55 (t, J = 7.4 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ: 162.69, 152.84, 149.26, 134.94,
134.71, 132.90, 130.61, 129.35, 128.57, 128.50, 128.27, 128.08, 127.28, 127.00, 126.36, 124.95,
121.61 ppm. MS (ESI+APCI) m/z 273.1 [M+H]+.
5.1.2.4 2-isoPropylquinazolin-4(3H)-one (2k)
Compound 2k was prepared from 1g (285 mg, 63% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.14 (s, 1H), 8.10 (dd, J = 1.2, 1.2 Hz, 1H), 7.81-7.76 (m, 1H), 7.63 (d, J = 8.0 Hz, 1H),
13
7.49-7.45 (m, 1H), 2.90 (dt, J = 6.8, 7.2Hz, 1H), 1.28 (s, 3H), 1.27 (s, 3H). C NMR (101 MHz,
DMSO-d6) δ: 162.46, 162.09, 149.30, 134.79, 127.40, 126.48, 126.14, 121.33, 33.77, 20.81 ppm.
MS (ESI+APCI) m/z 187.1 [M-H]-
5.1.2.5 7-(4-Methylpiperazin-1-yl)-2-phenylquinazolin-4(3H)-one (16)
Compound 16 was prepared from 15 (531 mg, 83% yield). 1H NMR (400 MHz, DMSO-d6) δ:
8.13 (d, J = 6.8 Hz, 1H), 7.97 (d, J = 9.2 Hz, 1H), 7.62-7.53 (m, 4H), 7.19 (dd, J = 2.0, 2.4 Hz,
13
1H), 7.04 (d, J = 2.4 Hz, 1H), 3.40 (t, J = 4.8 Hz, 4H), 2.50 (t, J = 4.6 Hz, 4H), 2.25 (s, 3H). C
NMR (101 MHz, DMSO-d6) δ: 161.75, 155.06, 152.53, 150.45, 132.79, 131.27, 128.59, 127.53,
126.90, 114.86, 111.25, 108.81, 54.04, 46.51, 45.38 ppm. MS (ESI+APCI) m/z 321.2 [M+H]+.
5.1.3 tert-Butyl 1-(4-oxo-2-phenyl-3,4-dihydroquinazolin-7-yl)piperidine-4-carboxylate (4)
To a solution of 2d (1 g, 4.0 mmol) in dioxane (15 mL), Pd2dba3 (73 mg, 0.08 mmol), XPhos
(152 mg, 0.32 mmol), NaOtBu (769 mg, 8.0 mmol) and 4-piperidinecarboxylic acid t-butyl ester
(1.15 g, 5.2 mmol) were added subsequently. The mixture was heated at 95 °C overnight, then the
solution was quenched with saturated NH4Cl solution. The solvent was removed under reduced
pressure and the residue was purified by flash chromatography (CH2Cl2/MeOH, V: V= 50: 1) to
afford compound 4 (737 mg, 48% yield). 1H NMR (400 MHz, DMSO-d6) δ: 12.15 (s, 1H), 8.16 (d,
18

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J = 7.2 Hz, 2H), 7.93 (d, J = 8.8 Hz, 1H), 7.54 (d, J = 7.6 Hz, 3H), 7.17 (d, J = 8.4 Hz, 1H), 7.02
(s, 1H), 3.92 (d, J = 12.8 Hz, 2H), 3.35 (s, 1H), 3.01 (t, J = 11.4 Hz, 2H), 1.88 (d, J = 11.6 Hz, 2H),
1.60 (d, J = 10.8 Hz, 2H), 1.41 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ: 173.93, 162.13, 155.23,
152.85, 151.04, 133.42, 131.66, 129.00, 128.08, 127.41, 115.47, 111.60, 109.42, 80.15, 46.97,
41.48, 28.18, 27.62 ppm. MS (ESI+APCI) m/z 406.2 [M+H]+.
5.1.4 2-Amino-4-chlorobenzamide (12)
To a solution of 2-amino-4-chlorobenzoic acid 11 (3.4 g, 20 mmol) and HOBt (3.0 g, 22
mmol) in DMF (50 mL) was added EDCI (4.6 g, 24 mmol). The mixture was stirred at rt for 2 h.
The resulting solution was cooled to 0 °C, then 25% ammonia solution (20 mL) was added. The
mixture was warmed to ambient temperature and stirred for 1 h. The reaction mixture was poured
1
into crushed ice, filtered and washed with water to give 12 (3.0 g, 88% yield) as gray solid. H
NMR (400 MHz, DMSO-d6) δ: 7.77 (s, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.14 (s, 1H), 6.82 (s, 2H),
6.74 (d, J = 2.1 Hz, 1H), 6.49 (dd, J = 8.5, 2.1 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ: 170.90,
151.98, 136.78, 131.06, 115.55, 114.45, 112.86 ppm. MS (ESI+APCI) m/z 171.0 [M+H]+.
5.1.5 General procedure for the synthesis of 9a-9d 27
DMSO (3 mL) was added to a mixture of acetophenone (1.0 equiv.) and I2 (1.1 equiv.). The
solution was stirred at 110 °C, then 2-aminobenzamide (1.0 equiv.) in 2 mL DMSO was added
dropwise to the mixture during 1-2 h. After a complete disappearance of the starting material, 50
mL water and 30 mL saturated brine were added to the mixture. The solution was extracted with
CH2Cl2 (50 mL×3). The organic layer was washed with 10% Na2S2O3 solution, dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel
chromatography (petroleum ether/EtOAc = 5:1) to yield the desired products 9a-9d.
5.1.5.1 2-Benzoylquinazolin-4(3H)-one (9a)
1
Compound 9a was prepared from 1g (275 mg, 55% yield). H NMR (400 MHz, CDCl3) δ:
10.38 (s, 1H), 8.52 (d, J = 7.6 Hz, 2H), 8.41 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.85 (t, J
= 7.4 Hz, 1H), 7.71-7.63 (m, 2H), 7.55 (t, J = 7.6 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ: 185.65,
160.97, 147.56, 145.97, 134.88, 134.33, 133.97, 131.83, 129.50, 129.43, 128.42, 126.89, 123.28
ppm. MS (ESI+APCI) m/z 251.1 [M+H]+.
5.1.5.2 7-Chloro-2-(4-methoxybenzoyl)quinazolin-4(3H)-one (9b)
Compound 9b was prepared from 12 (647 mg, 68% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.75 (s, 1H), 8.20-8.14 (m, 3H), 7.82 (d, J = 17.6 Hz, 1H), 7.67-7.61 (m, 1H), 7.12-7.06 (m, 2H),
3.88 (d, J = 17.6 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 185.55, 164.84, 161.04, 151.39,
148.90, 139.73, 133.94, 131.79, 128.85, 128.49, 127.77, 126.90, 122.06, 114.57, 114.24, 56.27
ppm. MS (ESI+APCI) m/z 315.1 [M+H]+.
5.1.5.3 7-Chloro-2-(4-methylbenzoyl)quinazolin-4(3H)-one (9c)
Compound 9c was prepared from 12 (495 mg, 41% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.81 (s, 1H), 8.20 (d, J = 8.8 Hz, 1H), 8.07 (d, J = 8.0 Hz, 2H), 7.85 (s, 1H), 7.67 (d, J = 8.4 Hz,
1H), 7.40 (d, J = 8.0 Hz, 2H), 2.43 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 186.92, 161.03,
151.17, 148.85, 145.85, 139.76, 131.75, 131.47, 129.74, 128.96, 128.52, 127.79, 122.13, 21.85
ppm. MS (ESI+APCI) m/z 299.1 [M+H]+.
5.1.5.4 7-Chloro-2-(2,6-dimethoxybenzoyl)quinazolin-4(3H)-one (9d)
Compound 9d was prepared from 12 (853 mg, 50% yield). 1H NMR (400 MHz, DMSO-d6) δ:
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12.83 (s, 1H), 8.18 (d, J = 8.8 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.65 (dd, J = 2.0, 2.0 Hz, 1H),
7.48 (t, J = 8.4 Hz, 1H), 6.79 (d, J = 8.8 Hz, 2H), 3.68 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ:
189.12, 161.00, 158.94, 150.79, 148.99, 139.93, 133.46, 129.36, 128.63, 127.87, 122.17, 115.36,
105.22, 56.60, 55.26 ppm. MS (ESI+APCI) m/z 345.1 [M+H]+.
5.1.6 tert-Butyl 1-(2-(2,6-dimethoxybenzoyl)-4-oxo-3,4-dihydroquinazolin-7-yl)piperidine-4-
carboxylate (10d)
Compound 10d was prepared from 9d and 4-piperidinecarboxylic acid t-butyl ester following
a similar procedure for the preparation of 4 (127 mg, 15% yield). 1H NMR (400 MHz, CDCl3) δ:
9.70 (s, 1H), 8.15 (d, J = 8.8 Hz, 1H), 7.42 (t, J = 8.4 Hz, 1H), 7.12 (dd, J = 2.4, 2.4 Hz, 1H), 7.01
(d, J = 2.4 Hz, 1H), 6.66 (s, 1H), 6.64 (s, 1H), 3.88-3.83 (m, 2H), 3.75 (s, 6H), 3.02-2.95 (m, 2H),
2.47-2.40 (m, 1H), 1.96 (dd, J = 3.6, 4.0 Hz, 2H), 1.75-1.71 (m, 2H), 1.45 (s, 9H). 13C NMR (101
MHz, CDCl3) δ: 190.06, 173.75, 160.51, 158.74, 155.16, 150.26, 146.85, 132.23, 127.86, 116.91,
114.91, 113.70, 111.50, 104.28, 80.59, 72.80, 56.16, 47.22, 41.74, 29.71, 29.33, 28.07, 27.57 ppm.
MS (ESI+APCI) m/z 494.1 [M+H]+.
5.1.7 2-Amino-4-(4-methylpiperazin-1-yl)benzamide (15)
To a stirred solution of 4-chloro-2-nitrobenzonitrile 14 (5.5 g, 30 mmol) in dioxane (100 mL)
was added morpholine (7.4 mL, 66 mmol). The reaction was refluxed overnight. The solution was
cooled to rt and partitioned between EtOAc (150 mL) and saturated NaHCO3 (50 mL). The
separated organic layer was dried over Na2SO4, filtered and evaporated in vacuo. After the residue
was dissolved in EtOH (100 mL), Pd/C (300 mg) and hydrazine hydrate (5.8 mL, 4 equiv.) were
added. The solution was stirred at rt for 0.5 h, then heated to 80 °C for 8 h. After cooling, the Pd/C
catalyst was filtered through a celite pad and washed with EtOH. The filtrate was concentrated
under reduced pressure and the residue was purified by chromatography on silica gel to give
compound 15 (6.4 g, 91% yield). 1H NMR (400 MHz, DMSO-d6) δ: 7.41 (d, J = 8.8 Hz, 1H), 6.57
(s, 2H), 6.13 (dd, J = 8.9, 2.2 Hz, 1H), 6.09 (d, J = 2.1 Hz, 1H), 3.24-2.96 (m, 4H), 2.45-2.27 (m,
4H), 2.20 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 171.57, 153.90, 152.31, 130.39, 105.17,
103.40, 100.55, 54.92, 47.42, 46.24 ppm. MS (ESI+APCI) m/z 235.2 [M+H]+.
5.1.8. General procedure for the synthesis of 3a-3k, 5, 10a-10c, 13, 17a-17b26
To a solution of 2 (1.0 equiv.) and PyBrop (1.3 equiv.) in MeCN (6 mL) was added DBU
(1.5 equiv.) at rt. The mixture was stirred for 30 min, then 3-amino-5-methylpyrazole (2.0 equiv.)
was added. The resulting mixture was stirred at 70 °C and monitored by TLC until a complete
disappearance of the starting material. The mixture was filtered and washed with solvent to give
the pure product. It was further purified by a flash chromatography on silica gel (CH2Cl2/MeOH,
V: V= 30: 1) to afford 3a-3k, 5, 10a-10c, 13, 17a-17b.
5.1.8.1 8-Methoxy-N-(5-methyl-1H-pyrazol-3-yl)-2-phenylquinazolin-4-amine (3a)
Compound 3a was prepared from 2a (113 mg, 80% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.23 (s, 1H), 10.24 (s, 1H), 8.48 (d, J = 7.2 Hz, 2H), 8.18 (d, J = 8.0 Hz, 1H), 7.56-7.50 (m, 3H),
7.45 (t, J = 8.4 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H), 6.77 (s, 1H), 3.99 (s, 3H), 2.35 (s, 3H). 13C
NMR (101 MHz, DMSO-d6) δ: 164.09, 158.03, 157.48, 146.25, 140.43, 139.82, 131.63, 128.32,
127.01, 115.18, 111.67, 99.03, 56.28, 11.35 ppm. HRMS (ESI-TOF): m/z calcd. for C19H18N5O
[M+H]+: 332.1506; found: 332.1487.
5.1.8.2 2-Benzyl-8-methoxy-N-(5-methyl-1H-pyrazol-3-yl)quinazolin-4-amine (3b)
Compound 3b was prepared from 2b (206 mg, 79% yield). 1H NMR (400 MHz, DMSO-d6) δ:
20

ACCEPTED MANUSCRIPT
12.38 (s, 1H), 10.87 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.40-7.38 (m, 3H),
7.35-7.3 (m, 2H), 7.26 (t, J = 7.0 Hz, 1H), 6.22(s, 1H), 4.25 (s, 2H), 3.99 (s, 3H), 2.20 (s, 3H). 13C
NMR (101 MHz, DMSO-d6) δ: 163.40, 156.74, 152.36, 152.12, 146.78, 138.69, 137.83, 129.51,
128.27, 126.46, 126.37, 114.51, 113.40, 113.27, 97.84, 56.06, 43.68, 10.88 ppm. HRMS
(ESI-TOF): m/z calcd. for C20H20N5O [M+H]+: 346.1662; found: 346.1652.
5.1.8.3 N-(5-Methyl-1H-pyrazol-3-yl)-8-nitro-2-phenylquinazolin-4-amine (3c)
Compound 3c was prepared from 2c (50 mg, 38% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.33 (s, 1H), 10.79 (s, 1H), 8.92 (d, J = 8.0 Hz, 1H), 8.40 (d, J = 4.0 Hz, 2H), 8.31 (d, J = 7.6 Hz,
1H), 7.65 (t, J = 7.6 Hz, 1H), 7.55 (s, 3H), 6.78 (s, 1H), 2.36 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) δ: 158.18, 158.00, 146.32, 141.08, 139.83, 136.34, 133.70, 132.50, 129.64, 129.56,
128.69, 124.83, 121.12, 112.94, 99.15, 11.35 ppm. HRMS (ESI-TOF): m/z calcd. for C18H15N6O2
[M+H]+: 347.1251; found: 347.1244.
5.1.8.4 7-Chloro-N-(5-methyl-1H-pyrazol-3-yl)-2-phenylquinazolin-4-amine (3d)
Compound 3d was prepared from 2d (124 mg, 43% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.28 (s, 1H), 10.55(s, 1H), 8.70 (d, J = 8.8 Hz, 1H), 8.48 (d, J = 5.6 Hz, 2H), 7.87 (s, 1H),
7.58-7.41 (m, 4H), 6.78 (s, 1H), 2.36 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 161.04, 157.64,
151.87, 147.97, 138.85, 138.64, 138.14, 131.00, 128.93, 128.56, 127.09, 126.53, 125.99, 112.90,
98.11, 11.42 ppm. HRMS (ESI-TOF): m/z calcd. for C18H15ClN5 [M+H]+: 336.0797; found:
336.1002.
5.1.8.5 7-Fluoro-N-(5-methyl-1H-pyrazol-3-yl)-2-phenylquinazolin-4-amine (3e)
Compound 3e was prepared from 2e (152 mg, 56% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.27 (s, 1H), 10.50 (s, 1H), 8.75 (dd, J = 6.0, 6.0 Hz, 1H), 8.48-8.46 (m, 2H), 7.58-7.53 (m, 4H),
7.47-7.42 (m, 1H), 6.76 (s, 1H), 2.35 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 166.42, 163.93,
161.01, 157.60, 152.96, 152.83, 138.70, 130.95, 128.93, 128.54, 127.00, 126.89, 115.67, 115.43,
112.32, 112.12, 111.35, 100.00, 98.06, 11.49 ppm. HRMS (ESI-TOF): m/z calcd. for C18H15FN5
[M+H]+: 320.1306; found: 320.1170.
5.1.8.6 N-(5-Methyl-1H-pyrazol-3-yl)-7-nitro-2-phenylquinazolin-4-amine (3f)
Compound 3f was prepared from 2f (114 mg, 44% yield). 1H NMR (400 MHz, DMSO-d6) δ:
10.78 (s, 1H), 8.88 (d, J = 8.4 Hz, 1H), 8.47 (s, 3H), 8.17 (d, J = 8.0 Hz, 1H), 7.55 (s, 3H), 6.76 (s,
1H), 2.36 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 161.78, 157.44, 150.86, 150.56, 147.46,
139.22, 138.22, 131.29, 128.98, 128.68, 126.27, 123.33, 119.20, 117.86, 98.12, 11.48 ppm. HRMS
(ESI-TOF): m/z calcd. for C18H15N6O2 [M+H]+: 347.1251; found: 347.1241.
5.1.8.7 7-Methoxy-N-(5-methyl-1H-pyrazol-3-yl)-2-phenylquinazolin-4-amine (3g)
Compound 3g was prepared from 2g (125 mg, 48% yield). 1H NMR (400 MHz, DMSO-d6) δ:
11.75 (s, 1H), 8.76 (d, J = 9.2 Hz, 1H), 8.33 (d, J = 7.2 Hz, 2H), 7.78-7.69 (m, 3H), 7.58 (d, J =
2.4 Hz, 1H), 7.38 (dd, J = 2.4, 2.4 Hz, 1H), 6.65 (s, 1H), 3.98 (s, 3H), 2.51, 2.35 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ: 165.10, 157.82, 157.55, 146.37, 139.79, 133.71, 132.29, 129.63, 129.37,
126.87, 118.85, 106.67, 101.89, 99.04, 56.72, 11.33 ppm. HRMS (ESI-TOF): m/z calcd. for
C19H17N5O [M+H]+: 332.1506; found: 332.1490.
5.1.8.8 N-(5-Methyl-1H-pyrazol-3-yl)-2-phenylquinazolin-4-amine (3h)
1
Compound 3h was prepared from 2h (125 mg, 48% yield). (58 mg, 42% yield). H NMR
(400 MHz, DMSO-d6) δ: 11.98 (s, 1H), 8.86 (d, J = 7.6 Hz, 1H), 8.36 (d, J = 7.2 Hz, 2H), 8.18 (d,
21

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J = 8.0 Hz, 1H), 8.08 (t, J = 7.2 Hz, 1H), 7.81-7.71 (m, 5H), 6.71 (s, 1H), 2.36 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ: 158.18, 158.00, 146.32, 141.08, 139.83, 136.34, 133.70, 132.50, 129.64,
129.56, 128.69, 124.83, 121.12, 112.94, 99.15, 11.35 ppm. HRMS (ESI-TOF): m/z calcd. for
C18H16N5 [M+H]+: 302.1400; found: 302.1383.
5.1.8.9 2-Benzyl-N-(5-methyl-1H-pyrazol-3-yl)quinazolin-4-amine (3i)
Compound 3i was prepared from 2i (145 mg, 39% yield). 1H NMR (400 MHz, DMSO-d6) δ:
8.13 (d, J = 8.4 Hz, 1H), 7.71-7.65 (m, 2H), 7.42 (t, J = 7.2 Hz, 1H), 7.29 (d, J = 7.2 Hz, 2H), 7.22
(t, J = 7.4 Hz, 2H), 7.13 (t, J = 7.2 Hz, 1H), 6.12 (s, 1H), 4.08 (s, 2H), 2.15 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ: 165.51, 157.34, 153.48, 150.47, 148.20, 139.56, 133.23, 129.84, 128.67,
127.63, 126.55, 125.93, 123.54, 113.61, 98.06, 46.05, 11.43 ppm. HRMS (ESI-TOF): m/z calcd.
for C19H18N5 [M+H]+: 316.1557; found: 316.1541.
5.1.8.10 N-(5-Methyl-1H-pyrazol-3-yl)-2-(naphthalen-2-yl)quinazolin-4-amine (3j)
1
Compound 3j was prepared from 2j (52 mg, 20% yield). H NMR (400 MHz, DMSO-d6) δ:
11.95 (s, 1H), 9.00 (s, 1H), 8.86 (d, J = 8.0 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 8.23-8.07 (m, 6H),
7.81-7.68 (m, 3H), 6.80 (s, 1H), 2.39 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 158.08, 157.92,
146.41, 139.86, 136.29, 135.33, 132.61, 130.70, 129.98, 129.77, 129.36, 129.32, 128.63, 128.34,
127.91, 125.19, 124.83, 121.44, 113.00, 99.25, 11.35 ppm. HRMS (ESI-TOF): m/z calcd. for
C22H17N5 [M+H]+: 352.1557; found: 352.1547.
5.1.8.11 2-isoPropyl-N-(5-methyl-1H-pyrazol-3-yl)quinazolin-4-amine (3k)
Compound 3k was prepared from 2k (252 mg, 89% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.69 (s, 1H), 12.02 (s, 1H), 8.84 (d, J = 8.0 Hz, 1H), 8.07-8.03 (m, 1H), 7.97 (d, J = 7.6 Hz, 1H),
7.76 (d, J = 6.8 Hz, 1H), 6.72 (s, 1H), 3.33-3.30 (m, 1H), 2.32 (s, 3H), 1.39 (d, J = 6.0 Hz, 6H).
13C NMR (101 MHz, DMSO-d6) δ: 168.71, 158.31, 146.39, 139.56, 136.40, 128.56, 124.97,
119.66, 112.64, 99.14, 34.67, 21.19, 11.28 ppm. HRMS (ESI-TOF): m/z calcd. for C15H18N5
[M+H]+: 268.1557; found: 268.1553.
5.1.8.12 tert-Butyl 1-(4-((5-methyl-1H-pyrazol-3-yl)amino)-2-phenylquinazolin-7-yl)piperidine-4-
carboxylate (5)
1
Compound 5 was prepared from 4 (125 mg, 60% yield). H NMR (400 MHz, DMSO-d6) δ:
13.70 (s, 1H), 11.55 (s, 1H), 8.60 (d, J = 9.2 Hz, 1H), 8.27 (d, J = 7.2 Hz, 2H), 7.78-7.69 (m, 3H),
7.49 (d, J = 7.2 Hz, 1H), 7.26 (s, 1H), 6.62 (s, 1H), 4.00 (d, J = 13.2 Hz, 2H), 3.19 (t, J = 11.8 Hz,
2H), 2.65-2.60 (m, 1H), 2.33 (s, 3H), 1.94 (d, J = 11.2 Hz, 2H), 1.60 (dd, J = 11.2, 10.4 Hz, 2H),
1.42 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ: 173.34, 158.10, 156.49, 153.86, 146.84, 138.91,
135.53, 135.48, 131.36, 128.62, 128.21, 124.87, 116.49, 104.12, 103.83, 97.78, 79.70, 46.32,
40.87, 27.68, 27.14, 10.98 ppm. HRMS (ESI-TOF): m/z calcd. for C28H32N6O2 [M+H]+: 485.2660;
found: 485.2658.
5.1.8.13 (4-((5-Methyl-1H-pyrazol-3-ylamino)quinazolin-2-yl)(phenyl)methanone (10a)
Compound 10a was prepared from 9a (107 mg, 36% yield). 1H NMR (400 MHz, DMSO-d6)
δ: 12.23 (s, 1H), 10.71 (s, 1H), 8.75 (d, J = 7.6 Hz, 1H), 7.99-7.85 (m, 4H), 7.73-7.66 (m, 2H),
7.57 (t, J = 6.8 Hz, 2H), 6.52 (s, 1H), 2.18 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 192.19,
158.71, 157.27, 148.96, 147.30, 138.15, 135.26, 133.60, 133.49, 130.26, 128.56, 128.19, 127.37,
123.38, 114.62, 97.76, 10.74 ppm. HRMS (ESI-TOF): m/z calcd. for C19H16N5O [M+H]+:
330.1092; found: 330.1341.
5.1.8.14 (7-Chloro-4-(5-methyl-1H-pyrazol-3-ylamino)quinazolin-2-yl)(4-methoxyphenyl)
22

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methanone (10b)
Compound 10b was prepared from 9b (107 mg, 45% yield). 1H NMR (400 MHz, DMSO-d6)
δ: 12.23 (s, 1H), 10.80 (s, 1H), 8.76 (d, J = 8.8 Hz, 1H), 7.96-7.90 (m, 3H), 7.70 (dd, J = 1.6, 2.0
13
Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 6.50 (s, 1H), 3.86 (s, 3H), 2.18 (s, 3H). C NMR (101 MHz,
DMSO-d6) δ: 190.82, 164.20, 160.88, 157.71, 150.58, 147.32, 139.07, 138.58, 133.22, 128.10,
127.88, 127.32, 126.12, 114.46, 113.75, 98.27, 56.11, 11.29 ppm. HRMS (ESI-TOF): m/z calcd.
for C20H17ClN5O2 [M+H]+: 394.1065; found: 394.1051.
5.1.8.15 (7-Chloro-4-(5-methyl-1H-pyrazol-3-ylamino)quinazolin-2-yl)(p-tolyl)methanone (10c)
Compound 10c was prepared from 9c (495 mg, 41% yield). 1H NMR (400 MHz, DMSO-d6)
δ: 12.25 (s, 1H), 10.83 (s, 1H), 8.78 (d, J = 8.8 Hz, 1H), 7.91-7.85 (m, 3H), 7.71 (d, J = 8.4 Hz,
1H), 7.37 (d, J = 7.6 Hz, 2H), 6.50 (s, 1H), 2.41 (s, 3H), 2.18 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) δ: 191.98, 160.64, 157.68, 153.80, 150.58, 147.60, 144.92, 138.61, 132.89, 130.90,
129.69, 127.99, 127.39, 126.15, 113.82, 98.26, 21.77, 11.28 ppm. HRMS (ESI-TOF): m/z calcd.
for C20H17ClN5O [M+H]+: 378.1116; found: 378.1118.
5.1.8.16 tert-Butyl 1-(2-(2,6-dimethoxybenzoyl)-4-(5-methyl-1H-pyrazol-3-ylamino)quinazolin-7-
yl)piperidine-4-carboxylate (13)
1
Compound 13 was prepared from 10d (8 mg, 6% yield). H NMR (400 MHz, DMSO-d6) δ:
11.95 (s, 1H), 10.22 (s, 1H), 8.47 (d, J = 8.4 Hz, 1H), 7.42 (t, J = 7.6 Hz, 2H), 7.07 (s, 1H), 6.78 (d,
J = 8.4 Hz, 2H), 6.01 (s, 1H), 3.95 (d, J = 12.4 Hz, 2H), 3.62 (s, 6H), 3.31 (s, 1H), 3.01 (t, J = 11.4
13
Hz, 2H), 2.12 (s, 3H), 1.89 (d, J = 11.6 Hz, 2H), 1.65-1.56 (m, 2H), 1.41 (s, 9H). C NMR (101
MHz, DMSO-d6) δ: 193.97, 173.44, 157.42, 157.02, 156.40, 153.49, 151.80, 133.13, 130.44,
124.09, 119.58, 117.97, 108.84, 106.46, 104.50, 99.50, 96.78, 79.64, 55.81, 46.51, 41.00, 27.69,
27.16, 10.72 ppm. HRMS (ESI-TOF): m/z calcd. for C31H37N6O5 [M+H]+: 573.2820; found:
573.2803.
5.1.8.17
N-(5-Methyl-1H-pyrazol-3-yl)-7-(4-methylpiperazin-1-yl)-2-phenylquinazolin-4-amine
(17a)
Compound 17a was prepared from 16 (56 mg, 23% yield). 1H NMR (400 MHz, DMSO-d6) δ:
11.01 (s, 1H), 8.62 (d, J = 8.8 Hz, 1H), 8.37 (d, J = 5.2 Hz, 2H), 7.65 (s, 3H), 7.49 (d, J = 8.8 Hz,
1H), 7.33 (s, 1H), 6.68 (s, 1H), 3.37 (s, 4H), 2.89 (s, 3H), 2.51 (s, 4H), 2.34 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ: 159.62, 156.68, 153.37, 152.22, 147.32, 147.27, 138.96, 129.97, 128.33,
127.86, 124.25, 116.16, 108.56, 106.24, 97.12, 53.47, 46.05, 44.43, 29.09, 11.28 ppm. HRMS
(ESI-TOF): m/z calcd. for C23H26N7 [M+H]+: 400.2244; found: 400.2239.
5.1.8.18 7-(4-Methylpiperazin-1-yl)-2-phenyl-N-(1H-pyrazol-3-yl)quinazolin-4-amine (17b)
Compound 17b was prepared from 16 (66 mg, 42% yield). 1H NMR (400 MHz, DMSO-d6) δ:
12.68 (s, 1H), 9.86 (s, 1H), 8.47 (d, J =7.2 Hz, 2H), 8.33 (d, J = 9.2 Hz, 1H), 8.12 (s, 2H), 7.53 (dd,
J = 6.0, 8.8 Hz, 3H), 7.37 (d, J = 9.2 Hz, 1H), 7.09 (s, 1H), 3.44 (s, 4H), 2.60 (s, 4H), 2.32 (s, 3H).
13C NMR (101 MHz, DMSO-d6) δ: 160.35, 156.77, 154.13, 152.40, 139.63, 130.36, 128.76,
128.29, 124.05, 122.57, 116.47, 109.08, 106.69, 54.59, 47.20, 45.77 ppm. HRMS (ESI-TOF): m/z
calcd. for C22H24N7 [M+H]+: 386.2088; found: 386.2078.
5.1.9
Ethyl
1-(4-((5-methyl-1H-pyrazol-3-yl)amino)-2-phenylquinazolin-7-yl)piperidine-4-
carboxylate (6)
SOCl2 (0.08 mL, 1.15 mmol) was added to a solution of 5 (185 mg, 0.38 mmol) in EtOH (20
23

ACCEPTED MANUSCRIPT
mL) under 0 °C. The solution was stirred at rt for 2 h, then stirred at 80 °C overnight. The mixture
was concentrated, neutralized with Na2CO3 solution, purified by chromatography to afford
compound 6 (120 mg, 69% yield). 1H NMR (400 MHz, DMSO-d6) δ: 12.15 (s, 1H), 10.06 (s, 1H),
8.46 (d, J = 6.8 Hz, 3H), 7.54-7.48 (m, 3H), 7.29 (d, J = 8.8 Hz, 1H), 7.06 (s, 1H), 6.76 (s, 1H),
4.09 (dd, J = 6.4, 6.8 Hz, 2H), 3.96 (d, J = 12.8 Hz, 2H), 3.01 (t, J = 11.4 Hz, 2H), 2.64-2.58 (m,
1H), 2.33 (s, 3H), 1.94 (d, J = 11.2 Hz, 2H), 1.71-1.62 (m, 2H), 1.19 (t, J = 7.2 Hz, 3H). 13C NMR
(101 MHz, DMSO-d6) δ: 174.57, 160.00, 157.15, 153.99, 152.77, 148.61, 139.47, 138.58, 130.36,
128.75, 128.30, 124.60, 116.79, 108.76, 106.21, 97.88, 60.41, 47.16, 27.62, 14.57, 11.40 ppm.
HRMS (ESI-TOF): m/z calcd. for C26H28N6O2 [M+H]+: 457.2347; found: 457.2347.
5.1.10 1-(4-((5-Methyl-1H-pyrazol-3-yl)amino)-2-phenylquinazolin-7-yl)piperidine-4-carboxylic
acid (7)
A solution of 5 (125 mg, 0.26 mmol) in TFA/CH2Cl2 (V: V=1: 1) was stirred for 5 h, then the
mixture was concentrated, neutralized with Na2CO3 solution, purified by reverse column
chromatography (CH2Cl2/MeOH, V: V= 20: 1, 0.5% NH4OH) to afford compound 7 (122 mg,
1
99% yield). H NMR (400 MHz, DMSO-d6) δ: 12.36 (s, 1H), 10.83 (s, 1H), 8.52 (d, J = 9.6 Hz,
1H), 8.38-8.35 (m, 2H), 7.62-7.60 (m, 4H), 7.39 (dd, J = 2.0, 2.4 Hz, 1H), 7.19 (s, 1H), 6.66 (s,
1H), 3.98 (d, J = 13.2 Hz, 2H), 3.11 (t, J = 11.2 Hz, 2H), 2.62-2.56 (m, 1H), 2.33 (s, 3H),
1.97-1.91 (m, 2H), 1.70-1.60 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 176.09, 158.59, 158.45,
158.27, 157.00, 154.43, 147.19, 139.44, 132.03, 129.18, 128.76, 125.49, 119.21, 117.03, 116.23,
98.34, 46.90, 27.62, 11.46 ppm. HRMS (ESI-TOF): m/z calcd. for C24H25N6O2 [M+H]+: 429.2034;
found: 429.2019.
METHODS
All methods and NMR spectra are found within the Supporting Information.
CONFLICTS OF INTEREST
The authors have no competing financial interests to declare.
AUTHOR INFORMATION
Corresponding Author
*E-mail address: lugui@mail.sysu.edu.cn (G. Lu)
*E-mail address: liuq9@mail.sysu.edu.cn (Q. Liu)
*E-mail address: yanmin@sysucc.org.cn (M. Yan)
AUTHOR CONTRIBUTIONS
+These authors contributed equally to this work. L.L., Q.L. and L.G. conceived the project. L.L.
performed and contributed to synthesize and design all experiments. W.Y.H. and W.R.B performed
the molecular dynamic simulations. J.X.Z., M.Y. and Z.C.T. performed all in vitro and in vivo
biochemical assays. L.L., L.G., W.Y.H., J.X.Z. and M.Y. wrote the paper. All authors approved the
final version of the manuscript.
ACKNOWLEDGMENTS
This work was financially supported by the National Science and Technology Major Project of the
Ministry of Science and Technology of China (2018ZX09735010), the National Natural Science
24

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Foundation of China (No. 81630005 to QL), the Guangdong Province Natural Science Foundation
(No. 2015A030313184) and Science and Technology Program of Guangzhou (No.
201607010118).
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