Design, synthesis, and biological evaluation of polyphenols with 4,6-diphenylpyrimidin-2-amine derivatives for inhibition of Aurora kinase A

Article abstract of DOI:10.1007/s40199-019-00272-5

Background: Several 4,6-diarylpyrimidin-2-amine derivatives show anticancer properties. However, their mode of action is not fully characterized. To develop potent anticancer chemotherapeutic agents, we designed and synthesized 25 4,6-diphenylpyrimidin-2-amine derivatives containing a guanidine moiety. Methods: Clonogenic long-term survival assays were performed to screen anticancer compounds. To derive the structural conditions showing good cytotoxicities against cancer cells, quantitative structure-activity relationships (QSAR) were calculated. Biological activities were determined by flow cytometry for cell cycle analysis and by immunoblot analysis for the detection of Aurora kinase A (AURKA) activity. Because 2-(2-Amino-6-(2,4-dimethoxyphenyl)pyrimidin-4-yl) phenol (derivative 12) selectively inhibited AURKA activity from the kinome assay, in silico docking experiments were performed to elucidate the molecular binding mode between derivative 12 and AURKA. Results: The pharmacophores were derived based on the QSAR calculations. Derivative 12 inhibited AURKA activity and reduced phosphorylation of AURKA at Thr283 in HCT116 human colon cancer cells. Derivative 12 caused the accumulation of the G2/M phase of the cell cycle and triggered the cleavages of caspase-3, caspase ?7, and poly(ADP-ribose) polymerase. The binding energies of 30 apo-AURKA – derivative 12 complexes obtained from in silico docking ranged from ?16.72 to ?11.63 kcal/mol. Conclusions: Derivative 12 is an AURKA inhibitor, which reduces clonogenicity, arrests the cell cycle at the G2/M phase, and induces caspase-mediated apoptotic cell death in HCT116 human colon cancer cells. In silico docking demonstrated that derivative 12 binds to AURKA well. The structure-activity relationship calculations showed hydrophobic substituents and 1-naphthalenyl group at the R2 position increased the activity. The existence of an H-bond acceptor at C-2 of the R1 position increased the activity, too.

Full text of DOI:10.1007/s40199-019-00272-5

DARU Journal of Pharmaceutical Sciences (2019) 27:265–281
https://doi.org/10.1007/s40199-019-00272-5
RESEARCH ARTICLE
Design, synthesis, and biological evaluation of polyphenols
with 4,6-diphenylpyrimidin-2-amine derivatives for inhibition
of Aurora kinase A
Young Han Lee1,2 & Jihyun Park3 & Seunghyun Ahn3 & Youngshim Lee3 & Junho Lee3 & Soon Young Shin1,2
Dongsoo Koh4 & Yoongho Lim3
&
Received: 3 January 2019 /Accepted: 8 May 2019 /Published online: 1 June 2019
#
Springer Nature Switzerland AG 2019
Abstract
Background Several 4,6-diarylpyrimidin-2-amine derivatives show anticancer properties. However, their mode of action is not
fully characterized. To develop potent anticancer chemotherapeutic agents, we designed and synthesized 25 4,6-
diphenylpyrimidin-2-amine derivatives containing a guanidine moiety.
Methods Clonogenic long-term survival assays were performed to screen anticancer compounds. To derive the structural
conditions showing good cytotoxicities against cancer cells, quantitative structure-activity relationships (QSAR) were calculated.
Biological activities were determined by flow cytometry for cell cycle analysis and by immunoblot analysis for the detection of
Aurora kinase A (AURKA) activity. Because 2-(2-Amino-6-(2,4-dimethoxyphenyl)pyrimidin-4-yl) phenol (derivative 12) se-
lectively inhibited AURKA activity from the kinome assay, in silico docking experiments were performed to elucidate the
molecular binding mode between derivative 12 and AURKA.
Results The pharmacophores were derived based on the QSAR calculations. Derivative 12 inhibited AURKA activity and
reduced phosphorylation of AURKA at Thr283 in HCT116 human colon cancer cells. Derivative 12 caused the accumulation
of the G2/M phase of the cell cycle and triggered the cleavages of caspase-3, caspase −7, and poly(ADP-ribose) polymerase. The
binding energies of 30 apo-AURKA – derivative 12 complexes obtained from in silico docking ranged from −16.72 to
−11.63 kcal/mol.
Conclusions Derivative 12 is an AURKA inhibitor, which reduces clonogenicity, arrests the cell cycle at the G2/M phase, and
induces caspase-mediated apoptotic cell death in HCT116 human colon cancer cells. In silico docking demonstrated that
derivative 12 binds to AURKA well. The structure-activity relationship calculations showed hydrophobic substituents and 1-
naphthalenyl group at the R2 position increased the activity. The existence of an H-bond acceptor at C-2 of the R1 position
increased the activity, too.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s40199-019-00272-5) contains supplementary
material, which is available to authorized users.
.
.
.
Keywords Apoptosis Aurora kinase A inhibitor Cell cycle
.
.
Clonogenicity 4,6-Diphenylpyrimidin-2-amine In silico
.
docking QSAR
* Dongsoo Koh
dskoh@dongduk.ac.kr
* Yoongho Lim
Background
yoongho@konkuk.ac.kr
1
2-(2-Arylmethylthio-4-chloro-5-methylbenzenesulfonyl)-
1-(1,3,5-triazin-2-ylamino)guanidine derivatives (Fig. 1a)
show anticancer activity via targeting transmembrane tumor-
associated isozymes, such as human carbonic anhydrase IX
and XII [1]. Guanidinated anthrathiophenediones, such as
1-(2-((11-amino-5,10-dioxo-5,10-dihydroanthra[2,3-b-
]thiophen-4-yl)amino)ethyl)guanidine (Fig. 1b) show antitu-
mor characteristics [2]. 3-Amino-2-(4-chloro-2-
Department of Biological Sciences, Sanghuh College of Life
Sciences, Konkuk University, Seoul 05029, Republic of Korea
2
Cancer and Metabolism Institute, Konkuk University, Seoul 05029,
Republic of Korea
3
Division of Bioscience and Biotechnology, BMIC, Konkuk
University, Seoul 05029, Republic of Korea
4
Department of Applied Chemistry, Dongduk Women’s University,
Seoul 02748, Republic of Korea

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activities of some compounds were related with antibac-
terial, antifungal, and anticonvulsant activities, and the
synthetic methods of other compounds were reported
[4–7]. Only derivative 16 was reported to show anticancer
activity [8]. The structures of the derivatives synthesized
here were identified using NMR spectroscopy and high-
resolution mass spectrometry (HRMS). To evaluate
whether the synthetic compounds exhibited anticancer ef-
fects, we selected HCT116 human colon cancer cell line.
Colon cancer is the third most commonly diagnosed can-
cer in South Korea, as well as in the United States.
Additionally, the causative factors are not clear, although
one of the causes is the western dietary life [9]. However,
the relationship between dietary patterns and colon cancer
incidence remains unclear. If colon cancer does not
spread, the first treatment is surgery. After stage II or
surgery, chemotherapy is required. Furthermore, there
are many drugs for chemotherapy of colon cancer, but
because of side-effects or drug resistance, new chemother-
apeutic agents are needed.
In this study, we used a cell-based clonogenic long-
term survival assay (CLSA) to determine anticancer ac-
tivity. To determine the mechanism by which these com-
pounds exhibit cytotoxicities, a kinome assay was per-
formed with the title compound exhibiting the best GI50
value in the test. Because the title compound selectively
inhibited Aurora kinase A (AURKA) activity from the
kinome assay, we performed in silico docking experi-
ments to elucidate the molecular binding mode between
the title compound and AURKA. To derive the structur-
al properties showing good cell growth inhibitory ef-
fects, quantitative structure-activity relationship (QSAR)
calculations were conducted using comparative molecu-
lar field analysis (CoMFA) and comparative molecular
similarity index analysis (CoMSIA). The structural prop-
erties derived from the structure-activity relationships
could help us design new compounds that exhibited bet-
ter cancer cell growth inhibitory effects.
Fig. 1 The structures of a 2-(2-Arylmethylthio-4-chloro-5-
methylbenzenesulfonyl)-1-(1,3,5-triazin-2-ylamino)guanidine, b 1-(2-
((11-amino-5,10-dioxo-5,10-dihydroanthra[2,3-b]thiophen-4-
yl)amino)ethyl)guanidine, c 3-Amino-2-(4-chloro-2-
mercaptobenzenesulfonyl)-guanidine, and d 4,6-diphenylpyrimidin-2-
amine. The boxes denote the guanidine moiety
Materials and methods
Preparation of the synthetic compounds
mercaptobenzenesulfonyl)-guanidine derivatives (Fig. 1c)
show anticancer properties in three cancer cell lines, including
MCF-7 breast cancer,NCI-H460 lung cancer, and SF-268
CNS cancer cells [3]. They all contain a guanidine moiety.
Therefore, based on these starting points, to develop po-
tent chemotherapeutic agents, we designed and synthe-
sized 25 4,6-diphenylpyrimidin-2-amine derivatives con-
taining a guanidine moiety (Fig. 1d). Of them, several
compounds were reported previously, but the biological
Beginning with an ethanol solution of variously substituted
acetophenone (I) and substituted benzaldehydes or
naphthylaldehydes (II), an excess amount of 50% aqueous
KOH was added, and the mixtures were stirred at room
temperature for 20 h. After completion of the reaction, the
reaction mixture was poured into 6 N HCl under an ice-bath
to produce precipitates of chalcones (III). The solids were
filtered and washed with ethanol and were used for the next
reaction without further purification. The chalcone (III,

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267
1 eq) and guanidine HCl salt (1.5 eq) was dissolved in DMF
solution and was added to a solid of K2CO3 (3 eq) to make a
solution. The reaction mixture was refluxed for 2 h and
cooled to room temperature. The reaction mixture was
poured into 3 N HCl under an ice-bath to produce precipi-
tates of 2-aminopyrimidine products (1–25). When the
solids were impure, the 2-aminopyrimidines were purified
by recrystallization from ethanol. The synthetic procedure
of 4,6-diphenylpyrimidin-2-amine derivatives containing a
guanidine moiety is summarized in Scheme 1. The struc-
tures and names of 25 derivatives are listed in Table 1. All
NMR experiments were carried out on a Bruker Avance 400
NMR spectrometer (9.4 T, Karlsruhe, Germany). The deu-
terated solvents and NMR tubes were purchased from
Sigma-Aldrich (St. Louis, MO, USA). The high-resolution
mass spectroscopic (HRMS) data were obtained using an
ultraperformance liquid chromatography-hybrid quadru-
pole time-of-flight mass spectrometer with a Waters
Acquity UPLC system (Waters Corp., Milford, MA).
4-([1,1′-biphenyl]-4-yl)-6-(4-chlorophenyl)pyrimidin-2-amine
(2)
1H NMR (400 MHz, DMSO-d6) δ 8.33 (ddd, 2H, H-3, H-6,
J = 8.4, 1.9, 1.7 Hz), 8.28 (d, 2H, H-2″, H-6″, J = 8.6 Hz), 7.82
(ddd, 2H, H-2, H-5, J = 8.4, 1.9, 1.7 Hz), 7.79 (s, 1H, py-5H),
7.76 (ddd, 2H, H-2′, H-6′, J = 7.7, 1.9, 1.5 Hz), 7.59 (d, 2H,
H-3″, H-5″, J = 8.6 Hz), 7.50 (ddd, 2H, H-3′, H-5′, J = 7.7, 7.4,
1.5 Hz), 7.40 (dddd, 1H, H-4′, J = 7.4, 7.4, 1.5, 1.5 Hz), 6.80 (s,
1H, NH2); 13C NMR (400 MHz, DMSO-d6) δ 164.6 (py-4),
164.0 (py-2), 163.6 (py-6), 142.1 (C-1), 139.3 (C-1′), 136.2
(C-4, C-1″), 135.2 (C-4″), 129.0 (C-3′, C-5′), 128.8 (C-2″,
C-6″), 128.6 (C-3″, C-5″), 127.9 (C-4′), 127.6 (C-3, C-5),
126.80 (C-2, C-6), 126.75 (C-2′, C-6′), 101.7 (py-5); HRMS
(m/z): Calcd. for (M + H)+: 358.1111; Found: 358.1125.
4-([1,1′-biphenyl]-4-yl)-6-(4-nitrophenyl)pyrimidin-2-amine
(3)
1H NMR (400 MHz, DMSO-d6) δ 8.50 (d, 2H, H-2″, H-6″, J =
8.7 Hz), 8.36 (d, 2H, H-3″, H-5″, J = 8.7 Hz), 8.34 (ddd, 2H,
H-3, H-6, J = 8.4, 2.0, 1.9 Hz), 7.90 (s, 1H, py-5H), 7.83 (ddd,
2H, H-2, H-5, J = 8.4, 2.0, 1.9 Hz), 7.76 (ddd, 2H, H-2′, H-6′,
J = 7.9, 1.8, 1.3 Hz), 7.50 (ddd, 2H, H-3′, H-5′, J = 7.9, 7.3,
1.4 Hz), 7.40 (dddd, 1H, H-4′, J = 7.3, 7.3, 1.3, 1.3 Hz), 6.93
(s, 1H, NH2); 13C NMR (400 MHz, DMSO-d6) δ 165.1 (py-4),
164.1 (py-2), 162.6 (py-6), 148.5 (C-4″), 143.5 (C-1″), 142.3
(C-1), 139.3 (C-1′), 135.9 (C-4), 129.0 (C-3′, C-5′), 128.2
(C-2″, C-6″), 127.9 (C-4′), 127.7 (C-3, C-5), 126.84 (C-2,
C-6), 126.76 (C-2′, C-6′), 123.7 (C-3″, C-5″), 102.7 (py-5);
HRMS (m/z): Calcd. for (M + H)+: 369.1352; Found: 369.1377.
4-([1,1′-biphenyl]-4-yl)-6-(2,4,6-trimethoxyphenyl)
pyrimidin-2-amine (1)
1H NMR (400 MHz, DMSO-d6) δ 8.17 (ddd, 2H, H-3, H-6,
J = 8.4, 2.1, 1.7 Hz), 7.78 (ddd, 2H, H-2, H-5, J = 8.4, 2.1,
1.7 Hz), 7.74 (ddd, 2H, H-2′, H-6′, J = 7.8, 1.9, 1.4 Hz), 7.49
(ddd, 2H, H-3′, H-5′, J = 7.8, 7.4, 1.5 Hz), 7.39 (dddd, 1H,
H-4′, J = 7.4, 7.4, 1.4, 1.4 Hz), 7.02 (s, 1H, py-5H), 6.60 (s,
1H, NH2), 6.32 (s, 2H, H-3″, H-5″), 3.83 (s, 1H, 4^-OCH3),
3.69 (s, 2H, 2^-OCH3, 6^-OCH3); 13C NMR (400 MHz,
DMSO-d6) δ 164.4 (py-6), 163.9 (py-2), 162.5 (py-4), 161.1
(C-4″), 158.0 (C-2″, C-6″), 141.7 (C-1), 139.4 (C-1′), 136.3
(C-4), 129.0 (C-3′, C-5′), 127.8 (C-4′), 127.3 (C-3, C-5), 126.8
(C-2, C-6), 126.7 (C-2′, C-6′), 110.7 (C-1″), 108.3 (py-5), 90.8
(C-3″, C-5″), 55.6 (2^-OCH3, 6^-OCH3), 55.4 (4^-
OCH3);HRMS (m/z): Calcd. for (M + H)+: 414.1818;
Found: 414.1824.
4-([1,1′-biphenyl]-4-yl)-6-(4-fluorophenyl)pyrimidin-2-amine
(4)
1H NMR (400 MHz, DMSO-d6) δ 8.33 (ddd, 2H, H-3,
H-6, J = 8.5, 2.1, 1.7 Hz), 8.31 (dd, 2H, H-2″, H-6″,
Scheme 1 The synthetic procedures to prepare 25 4,6-diphenylpyrimidin-2-amine derivatives containing guanidine moiety.

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Table 1 The structures and names of 25 4,6-diphenylpyrimidin-2-amine derivatives, and their half-maximal cell growth inhibitory concentrations
(GI50), where * denotes the test set used for the QSAR calculations
Molecular GI50/µM
Derivative
R1
R2
Name
Weight
413.47
18.55
40.82
21.60
15.55
33.82
16.50
4-([1,1'-biphenyl]-4-yl)-6-
(2,4,6-trimethoxyphenyl)
pyrimidin-2-amine
1
357.84
368.39
341.38
337.42
353.42
4-([1,1'-biphenyl]-4-yl)-6-(4-
chlorophenyl)pyrimidin-2-
amine
2
3
4
5
6
4-([1,1'-biphenyl]-4-yl)-6-(4-
nitrophenyl)pyrimidin-2-
amine
4-([1,1'-biphenyl]-4-yl)-6-(4-
fluorophenyl)pyrimidin-2-
amine
4-([1,1'-biphenyl]-4-yl)-6-(p-
tolyl)pyrimidin-2-amine
4-([1,1'-biphenyl]-4-yl)-6-(3-
methoxyphenyl)
pyrimidin-2-amine

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Table 1 (continued)
269
353.42
383.44
383.44
383.44
413.47
17.08
18.17
6.75
4-([1,1'-biphenyl]-4-yl)-6-(4-
methoxyphenyl)
7
pyrimidin-2-amine
4-([1,1'-biphenyl]-4-yl)-6-
(2,3-dimethoxyphenyl)
pyrimidin-2-amine
8*
9*
10
4-([1,1'-biphenyl]-4-yl)-6-
(3,4-dimethoxyphenyl)
pyrimidin-2-amine
15.25
11.19
4-([1,1'-biphenyl]-4-yl)-6-
(3,5-dimethoxyphenyl)
pyrimidin-2-amine
4-([1,1'-biphenyl]-4-yl)-6-
(2,4,5-trimethoxyphenyl)
pyrimidin-2-amine
11
12
323.35
1.45
2-(2-amino-6-(2,4-
dimethoxyphenyl)pyrimidin-
4-yl)phenol
4-(4-chlorophenyl)-6-(4-
methoxyphenyl)pyrimidin-2-
amine
311.77
385.04
355.03
341.79
291.35
34.54
29.21
15.63
15.93
22.70
13*
14
4-(4-bromophenyl)-6-(3,4-
dimethoxyphenyl)pyrimidin-
2-amine
4-(4-bromophenyl)-6-(4-
methoxyphenyl)pyrimidin-2-
amine
15
4-(4-chlorophenyl)-6-(3,4-
dimethoxyphenyl)pyrimidin-
2-amine
16
4-(4-methoxyphenyl)-6-(p-
17*
tolyl)pyrimidin-2-amine

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Table 1 (continued)
337.37
325.34
4.21
4-(3,5-dimethoxyphenyl)-6-
(2-methoxyphenyl)pyrimidin-
2-amine
18
4-(3,4-dimethoxyphenyl)-6-
(4-fluorophenyl)pyrimidin-2-
amine
15.88
19
20
321.37
337.37
12.00
5.32
4-(3,4-dimethoxyphenyl)-6-
(p-tolyl)pyrimidin-2-amine
4-(3,5-dimethoxyphenyl)-6-
(4-methoxyphenyl)pyrimidin-
2-amine
21*
4,6-bis(3,4-
dimethoxyphenyl)pyrimidin-
2-amine
367.40
352.34
357.41
19.52
9.89
1.92
22
23
4-(3,4-dimethoxyphenyl)-6-
(4-nitrophenyl)pyrimidin-2-
amine
4-(3,4-dimethoxyphenyl)-6-
(naphthalen-1-yl)pyrimidin-2-
amine
24
25
4-(3,4-dimethoxyphenyl)-6-
(naphthalen-2-yl)pyrimidin-2-
amine
357.41
10.51
J = 8.9, 5.7 Hz), 7.82 (ddd, 2H, H-2, H-5, J = 8.5, 2.1,
1.7 Hz), 7.77 (s, 1H, py-5H), 7.76 (ddd, 2H, H-2′, H-6′,
J = 7.8, 1.6, 1.2 Hz), 7.50 (ddd, 2H, H-3′, H-5′, J = 7.8,
7.4, 1.4 Hz), 7.40 (dddd, 1H, H-4′, J = 7.4, 7.4, 1.2,
1.2 Hz), 7.35 (dd, 2H, H-3″, H-5″, J = 8.9, 8.9 Hz),
6.77 (s, 1H, NH2); 13C NMR (400 MHz, DMSO-d6) δ
164.5 (py-4), 164.0 (py-6), 163.8 (py-2), 163.6 (d, 1C,
C-4″, J = 247.8 Hz), 142.0 (C-1), 139.4 (C-1′), 136.2
(C-4), 133.8 (d, 1C, C-1″, J = 2.7 Hz), 129.3 (d, 2C,
C-2″, C-6″, J = 8.5 Hz), 129.0 (C-3′, C-5′), 127.9
(C-4′), 127.6 (C-3, C-5), 126.79 (C-2, C-6), 126.75
(C-2′, C-6′), 115.5 (d, 2C, C-3″, C-5″, J = 21.4 Hz),
101.6 (py-5); HRMS (m/z): Calcd. for (M + H)+:
342.1407; Found: 342.1433.
4-([1,1′-biphenyl]-4-yl)-6-(p-tolyl)pyrimidin-2-amine (5)
1H NMR (400 MHz, DMSO-d6) δ 8.32 (ddd, 2H, H-3,
H-6, J = 8.4, 1.7, 1.3 Hz), 8.15 (d, 2H, H-2″, H-6″, J =
8.2 Hz), 7.82 (ddd, 2H, H-2, H-5, J = 8.4, 1.7, 1.3 Hz),
7.76 (ddd, 2H, H-2′, H-6′, J = 7.2, 2.1, 1.2 Hz), 7.73 (s,
1H, py-5H), 7.50 (ddd, 2H, H-3′, H-5′, J = 7.6, 7.2,
1.2 Hz), 7.40 (dddd, 1H, H-4′, J = 7.6, 7.6, 1.2,
1.2 Hz), 7.33 (d, 2H, H-3″, H-5″, J = 8.2 Hz), 6.71 (s,
1H, NH2), 2.38 (s, 1H, 4^-CH3); 13C NMR (400 MHz,
DMSO-d6) δ 164.8 (py-6), 164.2 (py-4), 164.0 (py-2),
141.9 (C-1), 140.2 (C-4″), 139.4 (C-1′), 136.4 (C-4),
134.6 (C-1″), 129.2 (C-3″, C-5″), 129.0 (C-3′, C-5′),
127.8 (C-4′), 127.6 (C-3, C-5), 126.9 (C-2″, C-6″),

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271
126.8 (C-2, C-6), 126.7 (C-2′, C-6′), 101.4 (py-5), 20.9
(4^-CH3); HRMS (m/z): Calcd. for (M + H)+: 338.1657;
Found: 338.1671.
(C-4′), 127.4 (C-3, C-5), 127.0 (C-2, C-6), 126.8 (C-2′,
C-6′), 124.0 (C-5″), 121.6 (C-6″), 114.1 (C-4″), 106.0 (py-
5), 60.8 (2^-OCH3), 55.9 (3^-OCH3); HRMS (m/z): Calcd.
for (M + H)+: 384.1712; Found: 384.1731.
4-([1,1′-biphenyl]-4-yl)-6-(3-methoxyphenyl)
pyrimidin-2-amine (6)
4-([1,1′-biphenyl]-4-yl)-6-(3,4-dimethoxyphenyl)
pyrimidin-2-amine (9)
1H NMR (400 MHz, DMSO-d6) δ 8.31 (ddd, 2H, H-3, H-6,
J = 8.4, 2.0, 1.8 Hz), 7.81 (ddd, 3H, H-2, H-5, H-6″, J = 8.4,
2.0, 1.8 Hz), 7.74 (ddd, 2H, H-2′, H-6′, J = 7.9, 2.1, 1.6 Hz),
7.729 (s, 1H, py-5H), 7.728 (dd, 1H, H-2″, J = 1.0, 1.0 Hz),
7.49 (ddd, 2H, H-3′, H-5′, J = 7.9, 7.4, 1.3 Hz), 7.44 (dd, 1H,
H-5″, J = 8.2, 8.2 Hz), 7.39 (dddd, 1H, H-4′, J = 7.4, 7.4, 1.6,
1.6 Hz), 7.09 (ddd, 1H, H-4″, J = 8.2, 2.6, 1.0 Hz), 6.71 (s, 1H,
NH2), 3.85 (s, 1H, 3^-OCH3); 13C NMR (400 MHz, DMSO-
d6) δ 164.7 (py-6), 164.4 (py-4), 164.0 (py-2), 159.6 (C-3″),
142.0 (C-1), 139.4 (C-1′), 138.9 (C-1″), 136.3 (C-4), 129.7
(C-5″), 129.0 (C-3′, C-5′), 127.9 (C-4′), 127.6 (C-3, C-5),
126.80 (C-2, C-6), 126.75 (C-2′, C-6′), 119.4 (C-6″), 116.2
(C-4″), 112.2 (C-2″), 102.0 (py-5), 55.3 (3^-OCH3); HRMS
(m/z): Calcd. for (M + H)+: 354.1606; Found: 354.1623.
1H NMR (400 MHz, pyridine-d5) δ 8.53 (ddd, 2H, H-3, H-6,
J = 8.4, 2.1, 1.7 Hz), 8.23 (d, 1H, H-2″, J = 2.0 Hz), 8.10 (dd,
1H, H-6″, J = 8.4, 2.0 Hz), 7.97 (s, 1H, py-5H), 7.86 (ddd, 2H,
H-2, H-5, J = 8.4, 2.1, 1.7 Hz), 7.77 (ddd, 2H, H-2′, H-6′, J =
7.1, 2.1, 1.4 Hz), 7.51 (ddd, 2H, H-3′, H-5′, J = 7.5, 7.1,
1.4 Hz), 7.41 (dddd, 1H, H-4′, J = 7.5, 7.5, 1.4, 1.4 Hz),
7.15 (d, 1H, H-5″, J = 8.4 Hz), 6.06 (s, 1H, NH2), 3.87 (s,
1H, 3^-OCH3), 3.83 (s, 1H, 4^-OCH3); 13C NMR
(400 MHz, pyridine-d5) δ 166.2 (py-6), 165.9 (py-4), 165.6
(py-2), 152.8 (C-4″), 150.6 (C-3″), 143.7 (C-1), 141.1 (C-1′),
138.0 (C-4), 131.5 (C-1″), 129.9 (C-3′, C-5′), 128.8 (C-3,
C-5), 128.7 (C-4′), 128.04 (C-2, C-6), 127.97 (C-2′, C-6′),
121.5 (C-6″), 112.6 (C-5″), 111.9 (C-2″), 102.9 (py-5), 56.5
(3^-OCH3), 56.4 (4^-OCH3); HRMS (m/z): Calcd. for (M +
H)+: 384.1712; Found: 384.1732.
4-([1,1′-biphenyl]-4-yl)-6-(4-methoxyphenyl)
pyrimidin-2-amine (7)
4-([1,1′-biphenyl]-4-yl)-6-(3,5-dimethoxyphenyl)
pyrimidin-2-amine (10)
1H NMR (400 MHz, DMSO-d6) δ 8.32 (ddd, 2H, H-3, H-6,
J = 8.4, 2.1, 1.8 Hz), 8.23 (d, 2H, H-2″, H-6″, J = 8.8 Hz), 7.82
(ddd, 2H, H-2, H-5, J = 8.4, 2.1, 1.8 Hz), 7.76 (ddd, 2H, H-2′,
H-6′, J = 7.8, 1.7, 1.2 Hz), 7.71 (s, 1H, py-5H), 7.50 (ddd, 2H,
H-3′, H-5′, J = 7.8, 7.4, 1.6 Hz), 7.40 (dddd, 1H, H-4′, J = 7.4,
7.4, 1.2, 1.2 Hz), 7.07 (d, 2H, H-3″, H-5″, J = 8.8 Hz), 6.67 (s,
1H, NH2), 3.84 (s, 1H, 4^-OCH3); 13C NMR (400 MHz,
DMSO-d6) δ 164.5 (py-6), 164.1 (py-4), 163.9 (py-2), 161.3
(C-4″), 141.9 (C-1), 139.4 (C-1′), 136.5 (C-4), 129.6 (C-1″),
129.0 (C-3′, C-5′), 128.6 (C-2″, C-6″), 127.9 (C-4′), 127.5
(C-3, C-5), 126.8 (C-2, C-6), 126.7 (C-2′, C-6′), 113.9
(C-3″, C-5″), 101.0 (py-5), 55.3 (4^-OCH3); HRMS (m/z):
Calcd. for (M + H)+: 354.1606; Found: 354.1621.
1H NMR (400 MHz, DMSO-d6) δ 8.35 (ddd, 2H, H-3, H-6,
J = 8.4, 2.1, 1.8 Hz), 7.82 (ddd, 2H, H-2, H-5, J = 8.4, 2.1,
1.8 Hz), 7.76 (ddd, 2H, H-2′, H-6′, J = 7.8, 2.0, 1.3 Hz), 7.75
(s, 1H, py-5H), 7.50 (ddd, 2H, H-3′, H-5′, J = 7.8, 7.4, 1.5 Hz),
7.41 (d, 2H, H-2″, H-6″, J = 2.3 Hz), 7.40 (dddd, 1H, H-4′,
J = 7.4, 7.4, 1.3, 1.3 Hz), 6.76 (s, 1H, NH2), 6.67 (dd, 1H,
H-4″, J = 2.3, 2.3 Hz), 3.85 (s, 2H, 3^-OCH3, 5^-OCH3);
13C NMR (400 MHz, DMSO-d6) δ 164.7 (py-6), 164.4 (py-
54), 163.9 (py-2), 160.7 (C-3″, C-5″), 142.0 (C-1), 139.6
(C-1″), 139.4 (C-1′), 136.3 (C-4), 129.0 (C-3′, C-5′), 127.9
(C-4′), 127.7 (C-3, C-5), 126.8 (C-2, C-6, C-2′, C-6′), 105.0
(C-2″, C-6″), 102.4 (C-4″), 102.1 (py-5), 55.4 (3^-OCH3, 5^-
OCH3); HRMS (m/z): Calcd. for (M + H)+: 384.1712; Found:
384.1724.
4-([1,1′-biphenyl]-4-yl)-6-(2,3-dimethoxyphenyl)
pyrimidin-2-amine (8)
1H NMR (400 MHz, DMSO-d6) δ 8.18 (ddd, 2H, H-3, H-6,
J = 8.5, 2.1, 1.8 Hz), 7.82 (ddd, 2H, H-2, H-5, J = 8.5, 2.1,
1.8 Hz), 7.74 (ddd, 2H, H-2′, H-6′, J = 7.9, 1.8, 1.5 Hz), 7.51
(s, 1H, py-5H), 7.49 (ddd, 2H, H-3′, H-5′, J = 7.9, 7.4, 1.5 Hz),
7.40 (dddd, 1H, H-4′, J = 7.4, 7.4, 1.5, 1.5 Hz), 7.32 (dd, 1H,
H-6″, J = 6.2, 2.9 Hz), 7.179 (dd, 1H, H-4″, J = 8.1, 2.9 Hz),
7.177 (dd, 1H, H-5″, J = 8.1, 6.2 Hz), 6.72 (s, 1H, NH2), 3.86
(s, 1H, 3^-OCH3), 3.76 (s, 1H, 2^-OCH3); 13C NMR
(400 MHz, DMSO-d6) δ 164.8 (py-6), 163.9 (py-2), 163.5
(py-4), 152.9 (C-3″), 147.2 (C-2″), 142.0 (C-1), 139.4
(C-1′), 136.4 (C-4), 132.7 (C-1″), 129.1 (C-3′, C-5′), 127.9
4-([1,1′-biphenyl]-4-yl)-6-(2,4,5-trimethoxyphenyl)
pyrimidin-2-amine (11)
1H NMR (400 MHz, DMSO-d6) δ 8.15 (ddd, 2H, H-3, H-6,
J = 8.4, 1.9, 1.8 Hz), 7.82 (ddd, 2H, H-2, H-5, J = 8.4, 1.9,
1.8 Hz), 7.74 (ddd, 2H, H-2′, H-6′, J = 7.8, 2.0, 1.3 Hz), 7.71
(s, 1H, py-5H), 7.61 (s, 1H, H-6″), 7.50 (ddd, 2H, H-3′, H-5′,
J = 7.8, 7.3, 1.6 Hz), 7.40 (dddd, 1H, H-4′, J = 7.3, 7.3, 1.3,
1.3 Hz), 6.82 (s, 1H, H-3″), 6.61 (s, 1H, NH2), 3.92 (s, 1H, 2^-
OCH3), 3.88 (s, 1H, 4^-OCH3), 3.77 (s, 1H, 5^-OCH3); 13
NMR (400 MHz, DMSO-d6) δ 163.8 (py-2), 163.5 (py-6),
C

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163.1 (py-4), 153.2 (C-2″), 151.4 (C-4″), 142.7 (C-5″), 141.7
(C-1), 139.4 (C-1′), 136.7 (C-4), 129.0 (C-3′, C-5′), 127.8
(C-4′), 127.3 (C-3, C-5), 126.9 (C-2, C-6), 126.7 (C-2′,
C-6′), 117.7 (C-1″), 113.7 (C-6″), 106.1 (py-5), 98.5 (C-3″),
56.6 (2^-OCH3), 56.2 (5^-OCH3), 55.8 (4^-OCH3); HRMS
(m/z): Calcd. for (M + H)+: 414.1818; Found: 414.1826.
4-(4-bromophenyl)-6-(4-methoxyphenyl)pyrimidin-2-amine
(15)
1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, 2H, H-2, H-6, J =
8.9 Hz), 8.17 (d, 2H, H-2″, H-6″, J = 8.6 Hz), 7.71 (d, 2H,
H-3″, H-5″, J = 8.6 Hz), 7.67 (s, 1H, py-5H), 7.06 (d, 2H, H-3,
H-5, J = 8.9 Hz), 6.70 (s, 1H, NH2), 3.83 (s, 1H, 4-OCH3); 13
C
NMR (400 MHz, DMSO-d6) δ 164.7 (py-4), 163.9 (py-2),
163.3 (py-6), 161.3 (C-4), 136.7 (C-1″), 131.6 (C-3″, C-5″),
129.5 (C-1), 129.0 (C-2″, C-6″), 128.6 (C-2, C-6), 124.0
(C-4″), 114.0 (C-3, C-5), 101.0 (py-5), 55.3 (4-OCH3);
HRMS (m/z): Calcd. for (M + H)+: 356.0398; Found:
356.0431.
2-(2-amino-6-(2,4-dimethoxyphenyl)pyrimidin-4-yl)phenol
(12)
1H NMR (400 MHz, DMSO-d6) δ 7.90 (d, 1H, H-6″, J =
8.6 Hz), 7.88 (dd, 1H, H-6, J = 8.7, 1.6 Hz), 7.71 (s, 1H, py-
5H), 7.34 (ddd, 1H, H-4, J = 8.0, 7.1, 1.6 Hz), 7.04 (s, 1H,
NH2), 6.92 (dd, 1H, H-5, J = 8.7, 7.1 Hz), 6.91 (d, 1H, H-3,
J = 8.0 Hz), 6.70 (d, 1H, H-3″, J = 2.3 Hz), 6.67 (dd, 1H,
H-5″, J = 8.6, 2.3 Hz), 3.91 (s, 1H, 4^-OCH3), 3.84 (s, 1H,
2^-OCH3); 13C NMR (400 MHz, DMSO-d6) δ 164.3 (py-4),
163.7 (py-6), 162.3 (C-2″), 161.1 (py-2), 160.3 (C-2), 159.3
(C-4″), 132.4 (C-4), 131.5 (C-6″), 127.2 (C-6), 118.9 (C-5),
118.8 (C-1″), 118.1 (C-3), 117.7 (C-1), 105.6 (C-5″), 103.9
(py-5), 98.0 (C-3″), 55.9 (4^-OCH3), 55.5 (2^-OCH3);
HRMS (m/z): Calcd. for (M + H)+: 324.1348; Found:
324.1357.
4-(4-chlorophenyl)-6-(3,4-dimethoxyphenyl)
pyrimidin-2-amine (16)
1H NMR (400 MHz, DMSO-d6) δ 8.24 (d, 2H, H-2″, H-6″,
J = 8.7 Hz), 7.84 (dd, 1H, H-6, J = 8.4, 1.9 Hz), 7.78 (d, 1H,
H-2, J = 1.9 Hz), 7.67 (s, 1H, py-5H), 7.57 (d, 2H, H-3″, H-5″,
J = 8.7 Hz), 7.07 (d, 1H, H-5, J = 8.4 Hz), 6.67 (s, 1H, NH2),
3.87 (s, 1H, 3-OCH3), 3.83 (s, 1H, 4-OCH3); 13C NMR
(400 MHz, DMSO-d6) δ 164.9 (py-4), 163.9 (py-2), 163.3
(py-6), 151.1 (C-4), 148.8 (C-3), 136.3 (C-1″), 135.2 (C-4″),
129.7 (C-1), 128.8 (C-2″, C-6″), 128.7 (C-3″, C-5″), 120.4
(C-6), 111.5 (C-5), 110.3 (C-2), 101.3 (py-5), 55.7 (3-
OCH3), 55.6 (4-OCH3); HRMS (m/z): Calcd. for (M + H)+:
342.1009; Found: 342.1026.
4-(4-chlorophenyl)-6-(4-methoxyphenyl)pyrimidin-2-amine
(13)
1H NMR (400 MHz, DMSO-d6) δ 8.24 (d, 2H, H-2″, H-6″,
J = 8.6 Hz), 8.20 (d, 2H, H-2, H-6, J = 8.9 Hz), 7.67 (s, 1H,
py-5H), 7.57 (d, 2H, H-3″, H-5″, J = 8.6 Hz), 7.06 (d, 2H,
H-3, H-5, J = 8.9 Hz), 6.69 (s, 1H, NH2), 3.83 (s, 1H, 4-
OCH3); 13C NMR (400 MHz, DMSO-d6) δ 164.7 (py-4),
163.9 (py-2), 163.2 (py-6), 161.3 (C-4), 136.3 (C-1″), 135.1
(C-4″), 129.5 (C-1), 128.7 (C-2″, C-6″), 128.6 (C-2, C-6,
C-3″, C-5″), 113.9 (C-3, C-5), 101.0 (py-5), 55.3 (4-OCH3);
HRMS (m/z): Calcd. for (M + H)+: 312.0904; Found:
312.0911.
4-(4-methoxyphenyl)-6-(p-tolyl)pyrimidin-2-amine (17)
1H NMR (400 MHz, DMSO-d6) δ 8.19 (d, 2H, H-2, H-6, J =
8.9 Hz), 8.11 (d, 2H, H-2″, H-6″, J = 8.2 Hz), 7.61 (s, 1H, py-
5H), 7.31 (d, 2H, H-3″, H-5″, J = 8.2 Hz), 7.05 (d, 2H, H-3,
H-5, J = 8.9 Hz), 6.59 (s, 1H, NH2), 3.83 (s, 1H, 4-OCH3),
2.37 (s, 1H, 4^-CH3); 13C NMR (400 MHz, DMSO-d6) δ
164.5 (py-6), 164.3 (py-4), 163.9 (py-2), 161.2 (C-4), 140.1
(C-4″), 134.7 (C-1″), 129.7 (C-1), 129.2 (C-3″, C-5″), 128.5
(C-2, C-6), 126.9 (C-2″, C-6″), 114.0 (C-3, C-5), 100.8 (py-5),
55.3 (4-OCH3), 21.0 (4^-CH3); HRMS (m/z): Calcd. for (M +
H)+: 292.1450; Found: 292.1440.
4-(4-bromophenyl)-6-(3,4-dimethoxyphenyl)
pyrimidin-2-amine (14)
1H NMR (400 MHz, DMSO-d6) δ 8.18 (d, 2H, H-2″, H-6″,
J = 8.6 Hz), 7.87 (dd, 1H, H-6, J = 8.6, 2.0 Hz), 7.80 (d, 1H,
H-2, J = 2.0 Hz), 7.72 (s, 1H, py-5H), 7.72 (d, 2H, H-3″, H-5″,
J = 8.6 Hz), 7.08 (d, 1H, H-5, J = 8.6 Hz), 7.04 (s, 1H, NH2),
3.88 (s, 1H, 3-OCH3), 3.84 (s, 1H, 4-OCH3); 13C NMR
(400 MHz, DMSO-d6) δ 164.7 (py-4), 163.6 (py-6), 163.2
(py-2), 151.5 (C-4), 148.9 (C-3), 136.3 (C-1″), 131.8 (C-3″,
C-5″), 129.3 (C-2″, C-6″), 129.2 (C-1), 124.5 (C-4″), 120.7
(C-6), 111.6 (C-5), 110.4 (C-2), 101.5 (py-5), 55.9 (3-OCH3),
55.8 (4-OCH3); HRMS (m/z): Calcd. for (M + H)+: 386.0504;
Found: 386.0515.
4-(3,5-dimethoxyphenyl)-6-(2-methoxyphenyl)
pyrimidin-2-amine (18)
1H NMR (400 MHz, DMSO-d6) δ 7.76 (dd, 1H, H-6, J = 7.5,
1.7 Hz), 7.49 (s, 1H, py-5H), 7.45 (ddd, 1H, H-4, J = 8.4, 7.5,
1.7 Hz), 7.18 (d, 2H, H-2″, H-6″, J = 2.2 Hz), 7.16 (d, 1H,
H-3, J = 8.4 Hz), 7.06 (dd, 1H, H-5, J = 7.5, 7.5 Hz), 6.65 (s,
1H, NH2), 6.64 (dd, 1H, H-4″, J = 2.2, 2.2 Hz), 3.85 (s, 1H, 2-
OCH3), 3.82 (s, 2H, 3^-OCH3, 5^-OCH3); 13C NMR
(400 MHz, DMSO-d6) δ 164.5 (py-4), 163.8 (py-2), 163.4
(py-6), 160.7 (C-3″, C-5″), 157.4 (C-2), 139.8 (C-1″), 131.0

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(C-4), 130.2 (C-6), 127.0 (C-1), 120.4 (C-5), 112.0 (C-3),
106.9 (py-5), 104.7 (C-2″, C-6″), 102.1 (C-4″), 55.7 (2-
OCH3), 55.3 (3^-OCH3, 5^-OCH3); HRMS (m/z): Calcd.
for (M + H)+: 338.1505; Found: 338.1519.
4,6-bis(3,4-dimethoxyphenyl)pyrimidin-2-amine (22)
1H NMR (400 MHz, DMSO-d6) δ 7.81 (dd, 2H, H-6, H-6″,
J = 8.6, 2.0 Hz), 7.74 (d, 2H, H-2, H-2″, J = 2.0 Hz), 7.61 (s,
1H, py-5H), 7.07 (d, 2H, H-5, H-5″, J = 8.6 Hz), 6.99 (s, 1H,
NH2), 3.85 (s, 2H, 3-OCH3, 3^-OCH3), 3.82 (s, 2H, 4-OCH3,
4^-OCH3); 13C NMR (400 MHz, DMSO-d6) δ 163.8 (py-4,
py-6), 162.2 (py-2), 151.4 (C-4, C-4″), 148.8 (C-3, C-3″),
128.8 (C-1, C-1″), 120.8 (C-6, C-6″), 111.5 (C-5, C-5″),
110.4 (C-2, C-2″), 101.0 (py-5), 55.8 (3-OCH3, 3^-OCH3),
55.7 (4-OCH3, 4^-OCH3); HRMS (m/z): Calcd. for (M +
H)+: 368.1610; Found: 368.1622.
4-(3,4-dimethoxyphenyl)-6-(4-fluorophenyl)
pyrimidin-2-amine (19)
1H NMR (400 MHz, DMSO-d6) δ 8.38 (dd, 2H, H-2″, H-6″,
J = 8.9, 5.4 Hz), 7.98 (dd, 1H, H-6, J = 8.6, 2.2 Hz), 7.90 (s,
1H, py-5H), 7.87 (d, 1H, H-2, J = 2.2 Hz), 7.45 (dd, 2H, H-3″,
H-5″, J = 8.9, 8.9 Hz), 7.16 (d, 1H, H-5, J = 8.6 Hz), 3.91 (s,
1H, 3-OCH3), 3.87 (s, 1H, 4-OCH3); 13C NMR (400 MHz,
DMSO-d6) δ 165.1 (d, 1C, C-4″, J = 250.7 Hz), 163.8 (py-2),
163.5 (py-6), 159.4 (py-4), 153.0 (C-4), 149.5 (C-3), 131.3 (d,
1C, C-1″, J = 2.9 Hz), 131.0 (d, 2C, C-2″, C-6″, J = 9.1 Hz),
126.3 (C-1), 122.4 (C-6), 116.6 (d, 2C, C-3″, C-5″, J =
21.7 Hz), 111.7 (C-5), 110.8 (C-2), 102.1 (py-5), 56.4 (3-
OCH3), 56.3 (4-OCH3); HRMS (m/z): Calcd. for (M + H)+:
326.1305; Found: 326.1320.
4-(3,4-dimethoxyphenyl)-6-(4-nitrophenyl)pyrimidin-2-amine
(23)
1H NMR (400 MHz, DMSO-d6) δ 8.49 (d, 2H, H-2″, H-6″,
J = 9.0 Hz), 8.37 (d, 2H, H-3″, H-5″, J = 9.0 Hz), 7.94 (dd,
1H, H-6, J = 8.5, 2.0 Hz), 7.91 (s, 1H, py-5H), 7.85 (d, 1H,
H-2, J = 2.0 Hz), 7.13 (d, 1H, H-5, J = 8.5 Hz), 3.90 (s, 1H, 3-
OCH3), 3.86 (s, 1H, 4-OCH3); 13C NMR (400 MHz, DMSO-
d6) δ 164.8 (py-4), 163.0 (py-2), 162.4 (py-6), 152.3 (C-4),
149.3 (C-4″), 149.2 (C-3), 142.7 (C-1″), 129.1 (C-2″, C-6″),
128.2 (C-1), 124.3 (C-3″, C-5″), 121.6 (C-6), 112.0 (C-5),
110.6 (C-2), 103.3 (py-5), 56.24 (3-OCH3), 56.18 (4-
OCH3); HRMS (m/z): Calcd. for (M + H)+: 353.1250;
Found: 353.1272.
4-(3,4-dimethoxyphenyl)-6-(p-tolyl)pyrimidin-2-amine (20)
1H NMR (400 MHz, DMSO-d6) δ 8.12 (d, 2H, H-2″, H-6″,
J = 8.2 Hz), 7.83 (dd, 1H, H-6, J = 8.3, 2.0 Hz), 7.78 (d, 1H,
H-2, J = 2.0 Hz), 7.63 (s, 1H, py-5H), 7.32 (d, 2H, H-3″,
H-5″, J = 8.2 Hz), 7.07 (d, 1H, H-5, J = 8.3 Hz), 6.60 (s, 1H,
NH2), 3.88 (s, 1H, 3-OCH3), 3.83 (s, 1H, 4-OCH3), 2.37 (s,
1H, 4^-CH3); 13C NMR (400 MHz, DMSO-d6) δ 164.5 (py-
6), 164.4 (py-4), 163.8 (py-2), 150.9 (C-4), 148.8 (C-3),
140.1 (C-4″), 134.7 (C-1″), 129.9 (C-1), 129.2 (C-3″,
C-5″), 126.9 (C-2″, C-6″), 120.2 (C-6), 111.4 (C-5), 110.2
(C-2), 101.0 (py-5), 55.7 (3-OCH3), 55.6 (4-OCH3), 21.0
(4^-CH3); HRMS (m/z): Calcd. for (M + H)+: 322.1556;
Found: 322.1562.
4-(3,4-dimethoxyphenyl)-6-(naphthalen-1-yl)
pyrimidin-2-amine (24)
1H NMR (400 MHz, DMSO-d6) δ 8.21 (dd, 1H, H-8″, J = 7.2,
2.3 Hz), 8.02 (dd, 1H, H-4″, J = 7.4, 1.5 Hz), 8.01 (dd, 1H,
H-5″, J = 7.2, 2.2 Hz), 7.78 (dd, 1H, H-6, J = 8.4, 1.9 Hz),
7.76 (d, 1H, H-2, J = 1.9 Hz), 7.70 (dd, 1H, H-2″, J = 7.2,
1.5 Hz), 7.62 (dd, 1H, H-3″, J = 7.4, 7.2 Hz), 7.55 (ddd, 1H,
H-7″, J = 7.2, 7.2, 2.2 Hz), 7.54 (ddd, 1H, H-6″, J = 7.2, 7.2,
2.3 Hz), 7.37 (s, 1H, py-5H), 7.06 (d, 1H, H-5, J = 8.4 Hz),
6.75 (s, 1H, NH2), 3.85 (s, 1H, 3-OCH3), 3.83 (s, 1H, 4-
OCH3); 13C NMR (400 MHz, DMSO-d6) δ 167.5 (py-6),
164.1 (py-4), 163.6 (py-2), 151.0 (C-4), 148.8 (C-3), 137.2
(C-1″), 133.3 (C-10″), 130.2 (C-9″), 129.7 (C-1), 129.1
(C-4″), 128.3 (C-5″), 126.9 (C-2″), 126.5 (C-7″), 126.0
(C-6″), 125.7 (C-3″), 125.6 (C-8″), 120.1 (C-6), 111.5 (C-5),
110.1 (C-2), 106.1 (py-5), 55.61 (4-OCH3), 55.57 (3-OCH3);
HRMS (m/z): Calcd. for (M + H)+: 358.1556; Found:
358.1567.
4-(3,5-dimethoxyphenyl)-6-(4-methoxyphenyl)
pyrimidin-2-amine (21)
1H NMR (400 MHz, DMSO-d6) δ 8.22 (d, 2H, H-2, H-6, J =
8.9 Hz), 7.63 (s, 1H, py-5H), 7.36 (d, 2H, H-2″, H-6″, J =
2.3 Hz), 7.06 (d, 2H, H-3, H-5, J = 8.9 Hz), 6.64 (s, 1H, NH2),
6.64 (dd, 1H, H-4″, J = 2.3, 2.3 Hz), 3.841 (s, 2H, 3^-OCH3,
5^-OCH3), 3.835 (s, 1H, 4-OCH3); 13C NMR (400 MHz,
DMSO-d6) δ 164.4 (py-4), 164.3 (py-6), 163.8 (py-2), 161.2
(C-4), 160.7 (C-3″, C-5″), 139.7 (C-1″), 129.6 (C-1), 128.6
(C-2, C-6), 113.9 (C-3, C-5), 104.9 (C-2″, C-6″), 102.3
(C-4″), 101.4 (py-5), 55.4 (3^-OCH3, 5^-OCH3), 55.3 (4-
OCH3); HRMS (m/z): Calcd. for (M + H)+: 338.1505;
Found: 338.1517.
4-(3,4-dimethoxyphenyl)-6-(naphthalen-2-yl)
pyrimidin-2-amine (25)
1H NMR (400 MHz, pyridine-d5) δ 9.06 (d, 1H, H-1″, J =
1.4 Hz), 8.57 (dd, 1H, H-3″, J = 8.6, 1.4 Hz), 8.25 (d, 1H, H-2,

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DARU J Pharm Sci (2019) 27:265–281
J = 2.0 Hz), 8.13 (dd, 1H, H-6, J = 8.4, 2.0 Hz), 8.10 (s, 1H,
py-5H), 8.06 (dd, 1H, H-8″, J = 6.3, 1.0 Hz), 8.05 (d, 1H,
H-4″, J = 8.6 Hz), 7.96 (dd, 1H, H-5″, J = 6.3, 1.0 Hz), 7.57
(ddd, 1H, H-6″, J = 6.3, 6.3, 1.0 Hz), 7.54 (ddd, 1H, H-7″, J =
6.3, 6.3, 1.0 Hz), 7.15 (d, 1H, H-5, J = 8.4 Hz), 6.56 (s, 1H,
NH2), 3.88 (s, 1H, 3-OCH3), 3.83 (s, 1H, 4-OCH3); 13C NMR
(400 MHz, pyridine-d5) δ 166.2 (py-6), 166.1 (py-4), 165.5
(py-2), 152.9 (C-4), 150.6 (C-3), 135.5 (C-10″), 134.6 (C-9″),
131.4 (C-1), 129.9 (C-8″), 129.6 (C-2″), 129.2 (C-4″), 128.7
(C-5″), 128.4 (C-1″), 128.1 (C-6″), 127.5 (C-7″), 125.7
(C-3″), 121.7 (C-6), 112.6 (C-5), 112.0 (C-2), 103.4 (py-5),
56.6 (3-OCH3), 56.5 (4-OCH3); HRMS (m/z): Calcd. for
(M + H)+: 358.1556; Found: 358.1577.
rapamycin, cyclin-dependent kinase 2/cyclin E, cyclin-
dependent kinase 5/p25, cyclin-dependent kinase 6/cyclin
D3, insulin-like growth factor 1 receptor, protein kinase (A,
Bβ, Cγ), phosphatidylinositol-4,5-bisphosphate 3-kinase,
Abelson murine leukemia viral oncogene homolog 1, and ap-
optosis signal-regulating kinase 1. Aurora A kinase was sup-
plied by EMD Millipore Corp. The substrate for phosphory-
lation, LeuArgArgAlaSerLeuGly, was 200 μM and the con-
centration of ATP was 10 μM [13]. All experiments were
repeated three times at 10 μM.
Immunoblot analysis
HCT116 cells were treated with derivative 12 for the indicated
times. The cells were lysed in an extraction buffer containing
20 mM HEPES (pH 7.2), 150 mM NaCl, 1% Triton X-100,
10% glycerol, 10 μg/mL leupeptin, and 1 mM
phenylmethylsulfonyl fluoride. Lysates (20 μg) were electro-
phoresed on a 10% SDS-polyacrylamide gel, transferred onto
nitrocellulose membrane (Bio-Ras, Richmond, CA, USA).
After blocking in TBST buffer (10 mM Tris-HCl, pH 8.0,
150 mM NaCl, and 0.1% Tween 20) containing 10% non-fat
milk for 1 h at room temperature, primary antibodies were
incubated for 4 h, followed by washing in TBST and incuba-
tion with horseradish peroxidase-conjugated secondary anti-
bodies for 4 h as described previously [14]. Antibody-reactive
protein bands were visualized using an enhanced chemilumi-
nescence detection system.
Materials
HCT116 human colon cancer cells were obtained from the
American Type Culture Collection (Rockville, MD, USA).
Antibodies against phospho-pan Aurora kinase A (T288)/
Aurora kinase B (T232)/Aurora kinase C (T198), caspase-3,
cleaved caspase-7 (Asp198), and poly(ADP-ribose) polymer-
ase (PARP) were obtained from Cell Signaling Technology
(Beverly, MA, USA). An antibody specific to GAPDH was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). Other chemicals were from Sigma-Aldrich (St. Louis,
MO, USA). Enhanced chemiluminescence detection system
was purchased from GE Healthcare (Piscataway, NJ, USA).
Clonogenic long-term survival assay
Cell cycle analysis
The clonogenic long-term survival assay (CLSA) of HCT116
human colon cancer cells followed the methods reported pre-
viously [10]. HCT116 cell lines were treated with 4,6-
diphenylpyrimidin-2-amine derivatives containing a guani-
dine moiety at different concentrations (0, 1, 5, 10, and
20 μM). After 7 days, cell colonies were fixed with 6% glu-
taraldehyde and stained with 0.1% crystal violet. Quantitation
of the viable cell colonies was determined using densitometry
(MultiGuage, Fujifilm, Japan). Their half-maximal cell
growth inhibitory concentrations (GI50) were determined
using SigmaPlot software (SYSTAT, Chicago, IL) [11].
HCT116 cells were treated with either a vehicle (0.1% DMSO)
or 20 μM derivative 12 for 24 h, fixed in 70% ethanol, washed
twice with phosphate-buffered saline, and then stained with
50 μg/ml propidium iodide, as previously described [15].
Analysis of the cell cyle phase was measured using a
NucleoCounter NC-3000 (ChemoMetec, Allerød, Denmark).
Quantitative structure-activity relationships
Quantitative structure-activity relationships (QSAR) calcula-
tions were conducted on an Intel Core 2 Quad Q6600
(2.4 GHz) Linux PC with Sybyl 7.3 (Tripos, St. Louis, MO,
USA) [16]. As the three-dimensional (3D) QSAR calcula-
tions, CoMFA and CoMSIA were adopted. Because the 3D
X-ray crystallographic structure of derivative 18, 4-(3,5-
dimethoxyphenyl)-6-(2-methoxyphenyl)pyrimidin-2-amine
was determined by the authors [17], the 3D structures of all
derivatives were determined based on the modification of de-
rivative 18 using the Sybyl program. For all derivatives, a
conformational search was performed using the grid search
method with a rotation of the selected bond in 15° increments.
The energy minimization process was followed using the
In vitro kinases assay
The kinase assay for the title compound was performed using
the EMD Millipore KinaseProfiler service assay protocol
(MilliporeSigma, Burlington, MA, USA) [12]. Cancer-
related kinases were selected as follows: Aurora kinase (A,
B), 5’-AMP-activated protein kinase (α1,β1,γ1), RAF
proto-oncogene serine/threonine-protein kinase, epidermal
growth factor receptor, glycogen synthase kinase 3β, c-Jun
N-terminal kinase (1a, 2a, 3), kinase insert domain receptor,
mitogen-activated protein kinase (1, 2), mammalian target of

DARU J Pharm Sci (2019) 27:265–281
275
Tripos force field and Gasteiger–Huckel charges, and ceased
at the convergence criteria of the total energy (0.05 kcal/
mol Å). The most stable structures were used for the QSAR
calculations. The detailed experimental procedures followed
to the methods previously reported [18].
and 17 were known, but their biological activities were not
reported [22]. Two derivatives, 16 and 20, have been reported
to show anticancer activity [8]. Several derivatives were
known to show other biological activities: 2, 3, 4, 7 (antibac-
terial), 14 (nucleoside binding affinity), 19 (anticonvulsant),
23 (antifungal), and 25 (antimicrobial) [4–7].
In silico docking
Clonogenic long-term survival assay
In silico docking to elucidate the molecular binding mode
between the title compound, 2-(2-amino-6-(2,4-
dimethoxyphenyl)pyrimidin-4-yl)phenol, and aurora kinase A
(AURKA) was performed on an Intel Core 2 Quad Q6600
(2.4 GHz) Linux PC with Sybyl 7.3 (Tripos). The 3D structure
of AURKA was adopted from the X-ray crystallographic
structure deposited in the protein data bank as 3uod.pdb
[19]. Although it is not the 3D structure containing the greatest
number of residues (2j4z.pdb) [20], because the ligand
contained in this 3D structure is similar to 2-(2-amino-
6-(2,4-dimethoxyphenyl)pyrimidin-4-yl)phenol, 3uod.pdb
was selected. The detailed experimental procedures followed
to the methods previously reported [21].
Of 25 4,6-diphenylpyrimidin-2-amine derivatives containing
a guanidine moiety, two (16 and 20) exhibited anticancer ac-
tivity [8]; however, other derivatives remained unknown for
their anticancer activity. We thus tested the effects of 25 de-
rivatives on the inhibition of clonogenicity using a CLSA,
which allowed a sensitive cell-based assay for distinguishing
the growth inhibitory effect of compounds with similar struc-
tural features. HCT116 human colon cancer cells were treated
with increasing concentrations of each derivative (0, 1, 5, 10,
and 20 μM) for 7 days, and live cell colonies were stained
with crystal violet (Fig. 2). The half-maximal cell growth in-
hibitory concentrations (GI50) of 25 derivatives were calculat-
ed and are listed in Table 1. They ranged between 1.45 μM
(derivative 12) and 40.82 μM (derivative 2). A graph of GI50
values with error bars is plotted in Suppl. Fig. 1.
Results and discussion
Synthesis
In vitro kinases assay
Of 25 4,6-diphenylpyrimidin-2-amine derivatives containing
We selected derivative 12 as the best inhibitor for the
a guanidine moiety, the synthetic methods of derivatives 13
clonogenicity of HCT116 cells and further evaluated whether
Fig. 2 Clonogenic long-term survival assays performed at five different concentrations (0, 1, 5, 10, and 20 μM). 0.1% DMSO was used a positive control

276
DARU J Pharm Sci (2019) 27:265–281
it could inhibit protein kinase activity. In vitro kinase assay
shows that derivative 12 was highly specific to AURKA
among the 25 cancer-associated kinases used in this test.
The 95% inhibition of the AURKA kinase activity was com-
pared to control without any compound.
Cell cycle analysis
AURKA functions during prophase of mitosis and regu-
lates centrosome maturation and the formation of the mi-
totic spindle [23]. Accumulating evidence has demonstrat-
ed that inhibition of AURKA induces an abnormal mitotic
spindle, cell cycle arrest at the G2/M phase, and apoptotic
cell death [24]. To determine whether derivative 12 affects
cell cycle progression, we carried out a flow cytometric
analysis. At 24 h after derivative 12 treatment, G0/G1
phase cells decreased from 54.4% in the vehicle
(DMSO)-treated control to 28.3% with a concomitant in-
crease in the G2/M phase cell population from 22.7 to
50.1% (Fig. 3b). In general, sub-G1 phase cells appeared
at the early stage of apoptosis. Derivative 12 also trig-
gered the accumulation of sub-G1 cells from 1.3 to
5.4%. These results suggest that derivative 12 inhibition
of AURKA is linked functionally to the inhibition of the
cell cycle progression at the G2/M phase and the induc-
tion of apoptosis.
Immunoblot analysis
There are three subtypes of Aurora kinases: Aurora kinase A
(AURKA), Aurora kinase B (AURKB), and Aurora kinase C
(AURKC). To determine the specificity of derivative 12 against
other Aurora kinase subtypes, we performed immunoblot anal-
ysis with a phospho-specific pan-Aurora kinase antibody.
HCT116 cells were treated with 50 μM derivative 12 for differ-
ent periods (0, 15, 30, 60, and 120 min), and the phosphorylation
status of the three subtypes of Aurora kinases was examined. We
found that only AURKA phosphorylation at Thr288 was sub-
stantially decreased, but not AURKB at Thr232 and AURKC at
Thr198, in response to derivative 12 treatment (Fig. 3a).
Fig. 3 Effect of derivative 12 on
the inhibition of Aurora kinase A
(AURKA) and cell cycle pro-
gression. a HCT116 cells were
treated with derivative 12 for var-
ious periods (0, 15, 30, 60, and
120 min) and immunoblot analy-
sis was performed using a
phospho-specific pan-Aurora ki-
nase antibody. GAPDH was used
as an internal control. Bars, mo-
lecular weight markers. b
HCT116 cells were treated with
derivative 12 for 24 h, fixed with
ethanol, and stained with
propidium iodide. Cell cycle pro-
files were analyzed using a
NucleoCounter NC-3000.
Vehicle, 0.1% DMSO; M1, sub-
G1 phase; M2, G1 phase; M3, S
phase; M4, G2/M phase

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277
Activation of the caspase cascade
using the Sybyl/DATABASE Alignment module. As shown
in Suppl. Fig. 3, because all derivatives included 4,6-
diphenylpyrimidin-2-amine containing a guanidine moiety
(Fig. 1d), they superimposed well. All derivatives were placed
in a 2 Å spacing lattice, and their steric energy based on the
Lennard-Jones potential and electrostatic energy were deter-
mined by the sp3-hybridized carbon atom with +1 charge at
grid points. The detailed procedures for CoMFA followed the
manufacturer’s manual. To generate the CoMFA model, a
cross-validated partial least-squares (PLS) analysis was per-
formed. Until finding a value over 0.5 for the cross-validated
correlation coefficient q2, indicating the quality of the predic-
tion, PLS analysis was iterated. To enhance the contribution of
the lattice points in the CoMFA region, region focusing was
performed at the same time. When the weight by discriminant
power and grid spacing were 1 and 1, respectively, q2 was
0.679, which was the best cross-validation correlation coeffi-
cient generated in these CoMFA calculations. This CoMFA
model provided non-cross-validated correlation coefficient
(r2) of 0.922, standard error of prediction of 0.112, the number
of components used in the regression was 3, and the F-test
value was 62.797. Here, steric and electrostatic field descrip-
tors contributed 75.4% and 24.6%, respectively. The pGI50
values were predicted using this CoMFA model and compared
with those obtained from the clonogenic long-term survival
assays performed in this research (Suppl. Table 1). A graph
showing this comparison was built as shown in Suppl. Fig. 4.
The differences between the pGI50 values predicted by the
CoMFA model and the experimental pGI50 values ranged
from 0.30 to 10.77%. To validate whether this CoMFA model
wassss reliable, the pGI50 values contained in the test set were
predicted using this model. As listed in Suppl. Table 1, the
Caspases are a family of proteases that play essential roles in
apoptosis progression [25]. There are two types of caspases
involved in apoptosis; initiators (caspase-2, −8, −9, and − 10)
and effectors (caspase-3 and -7). Both types of caspases are
activated by proteolytic cleavages. To determine whether
caspases were activated by derivative 12, HCT116 cells were
treated with derivative 12 for various periods, and the cleav-
ages of effector caspases were examined by immunoblot anal-
ysis. Both caspase-3 and caspase-7 cleavages began to be
detected at 12 h after derivative 12 treatment (Fig. 4a). Also,
poly(ADP-ribose) polymerase (PARP), a substrate protein of
caspase-3 and caspase-7, was also proteolytically cleaved.
The quantitative densitometric analysis showed that derivative
12-induced cleavages of caspase-3, caspase-7, and PARP after
12 h of treatment were all significant (Fig. 4b). These results
suggested that derivative 12 could induce apoptosis through
the activation of caspase cascade.
QSAR
The negative logarithmic scales of GI50 values (pGI50) were
adopted as the biological data for the 3D-QSAR calculations.
Twenty percent of 25 derivatives was chosen arbitrarily for the
test set to validate the QSAR model: 8, 9, 13, 17, and 21. They
were analyzed using hierarchical cluster analysis. As shown in
Suppl. Fig. 2, they belonged to separate clusters. For QSAR
calculations, first, CoMFAwas performed. To achieve the goal
of deriving the relationships between biological activities and
the 3D structures, all molecules participating in the calcula-
tions should be superimposed. All derivatives were aligned
Fig. 4 Effects of derivative 12 on
the activation of caspases. a
HCT116 cells were treated with
derivative 12 for various periods
(0, 6, 12, and 24 h) and
immunoblot analysis was
performed using caspase-3,
caspase-7, and PARP antibodies.
GAPDH was used as an internal
control. Bars, molecular weight
markers. b The band intensities of
cleaved caspase-3, caspase-7, and
PARP relative to GAPDH level
were measured using ImageJ
software. The data are presented as
means SD (n = 3). NS, not
significant. Statistical analysis was
carried out using one-way analysis
of variance (ANOVA) followed by
Dunnet’s multiple comparisons
tests using GraphPad Prism ver-
sion 7.04 software

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DARU J Pharm Sci (2019) 27:265–281
residuals between the experimental data and the predicted
values ranged from 7.20 to 22.01%. Therefore, this CoMFA
model was considered to be reliable. To visualize the steric
and electrostatic field descriptors, the contour maps were gen-
erated using Sybyl software. The steric field descriptors favor-
ing and disfavoring bulky groups were 83% and 17%, respec-
tively (Suppl. Fig. 5). While the average GI50 value of deriv-
atives 1–11 with the biphenyl group was 19.57 μM, that of
derivatives 12–25 was 14.19 μM. The bulky group near the
guanidyl group favored the activity. Derivative 24 with the 1-
naphthyl group at the R2 position of the figure contained in
Table 1 and derivative 12 with the 2,4-dimethoxy group
showed good GI50 values of 1.92 and 1.46 μM, respectively.
The contribution of the electrostatic field descriptors was not
large. In addition, as mentioned later, CoMSIA did not include
the electrostatic field descriptors. The visualization of the elec-
trostatic field descriptors was ignored.
CoMSIA provided steric and electrostatic field descriptors,
as well as hydrophobic, hydrogen-bond (H-bond) acceptor,
and donor filed descriptors. Of several CoMSIA models gen-
erated based on the same procedures as CoMFA, a model
showing an q2 of 0.575 was chosen, which included steric,
hydrophobic, and H-bond donor filed descriptors. Their con-
tributions were 21.6, 55.0, and 23.4%, respectively. In this
CoMSIA model, the non-cross-validated correlation coeffi-
cient (r2), standard error of prediction, the number of compo-
nents used in the regression, and F-test value were 0.962,
0.087, 6, and 54.157, respectively. The pGI50 values predicted
using this CoMSIA model were compared with those obtained
from the clonogenic long-term survival assay as listed in
Suppl. Table 2. The experimental data was plotted against
the predicted values as shown in Suppl. Fig. 6. The residuals
between pGI50 values of the training set predicted by the
CoMSIA model and the experimental pGI50 values ranged
from 0.06 to 10.05%. For the test set, the residuals ranged
between 6.98 and 27.20%. Therefore, this CoMSIA model
was considered to be reliable. To visualize the results predict-
ed using CoMSIA, contour maps were generated (Suppl.
Fig. 7). The steric field descriptor of CoMSIA showed a
similar result as CoMFA. Hydrophobic substituents at the
R2 position of the figure contained in Table 1 increased the
activity. The 1-naphthalenyl group at the R2 position showed
good activity. The existence of an H-bond acceptor at C-2 of
the R1 position, such as the hydroxyl or methoxy group, in-
creased the activity: derivatives 12 and 18. The structural con-
ditions derived from CoMFA and CoMSIA analysis can be
summarized in Fig. 5.
In silico docking
As the X-ray crystallographic structure of AURKA, 3uod.pdb
was selected from the protein databank based on the reasons
mentioned above. Its apo-protein was prepared for in silico
docking experiments by removing its ligand, which co-
c r y s t a l i z e d
w i t h ,
4-[(4-{[2-(trifluoromethyl)phenyl]amino}pyrimidin-2-
yl)amino]benzoic acid (named as 0c3) (Suppl. Fig. 8). All
hydrogen atoms were added and the structure was subsequent-
ly submitted to energy minimization using the conjugate gra-
dient algorithm. Tripos force field and Gasteiger-Hückell
charges were used for the energy minimization. A 3D struc-
ture of derivative 12 was obtained using the X-ray crystallo-
graphic structure of derivative 18 as mentioned above. The
same procedures as apo-protein were carried out for the 3D
structure of derivative 12. The binding pocket of AURKAwas
analyzed using the LigPlot software as previously reported
[26], which consists of 13 residues; Arg137, Leu139,
Gly140, Val147, Ala160, Glu211, Tyr212, Ala213, Gly216,
Thr217, Arg220, Glu 260, and Leu263. These residues were
used for the flexible docking experiments between AURKA
and derivative 12. To confirm parameters for the docking ex-
periments, the original ligand, 0c3 in the 3uod.pdb structure
was docked into the apo-protein of AURKA (named as apo-
AURKA). Because the flexible docking procedure was iterat-
ed 30 times, 30 protein-ligand complexes were generated.
Their binding energies varied from −32.39 to −23.53 kcal/
mol. The most poses with low binding energy were very sim-
ilar to the pose of the original structure. Likewise, flexible
Fig. 5 Structural conditions to
show good cancer cell growth
inhibitory effects

DARU J Pharm Sci (2019) 27:265–281
279
docking was performed between apo-AURKA and derivative
12. The binding energies of 30 apo-AURKA – derivative 12
complexes ranged from −16.72 to −11.63 kcal/mol. The
fourth complex was selected based on the pose of the ligand
and binding energy. Its binding mode was analyzed using
LigPlot. As shown in Fig. 6, there were seven hydrophobic
interactions (Arg137, Leu139, Glu211, Tyr212, Gly216,
Arg220, and Leu263) and two hydrogen bonds (Ala213 and
Pro214) for the binding of derivative 12. Based on the 3D
structure analysis using the PyMol software (PyMOL
Molecular Graphics System, version 1.0r1, Schrödinger,
LLC, Portland, OR, USA) [27], an additional hydrogen bond
between Leu139 and 1-methoxy phenyl group was observed
(Fig. 7). It was reported that only three residues (Leu215,
Thr217, and Arg220) in the active site of AURKA were dif-
ferent from AURKB or AURKC [28]. Because the 3D struc-
ture of AURKB was known, we tried to perform in silico
docking between AURKB and derivative 12. However, we
could not get the protein-ligand complex, even if structures
of Aurora kinases were highly conserved. Based on the cur-
rent docking results, Arg220 may participate in the binding of
derivative 12 through hydrophobic interactions. This phenom-
enon can explain the selectivity of derivative 12 for AURKA.
Fig. 7 The image of the binding pocket of the apo-AURKA – derivative
12 complex visualized using the PyMol program
pyrimidinamine contains guanidine moiety [29]. Another in-
hibitor of AURKA, cyclopropanecarboxylic acid-(3-(4-(3-
trifluoromethyl-phenylamino)-pyrimidin-2-ylamino)-phe-
nyl)-amide includes guanidine moiety, too [30]. Therefore, it
w a s c o n s i d e r e d t h a t 2 - ( 2 - A m i n o - 6 - ( 2 , 4 -
dimethoxyphenyl)pyrimidin-4-yl)phenol (derivative 12) with
guanidine moiety may show the inhibitory effect on
AURKA. The current results demonstrated that it acts as an
AURKA inhibitor, and arrests the cell cycle at the G2/M
phase, and induces caspase-mediated apoptotic cell death
against HCT116 human colon cancer cells. At the present
time, we could not get the evidence for the title compound
to bind to AURKA directly, we carried out in silico docking to
elucidate the binding mode between derivative 12 and
AURKA, which demonstrated that derivative 12 binds to
Conclusion
In this research, 4,6-diphenylpyrimidin-2-amine derivatives
containing a guanidine moiety were designed and synthesized.
As expected, they showed inhibitory effects on the cancer cell
growth. An inhibitor of AURKA and AURKB, 4-(2-amino-4-
methyl-5-thiazolyl)-N-[4-(4-morpholinyl)phenyl]-2-
Fig. 6 Residues residing in the
binding site of the apo-AURKA –
derivative 12 complex obtained
by the LigPlot analysis

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Acknowledgments This work was supported by the Konkuk University
Research Support Program (YHL).
Authors’ contribution YHL: designed the experiments and wrote the
manuscript. JP, YLee, JL, SYS: conducted the experiments. SA: synthe-
sized chemicals. DK and YLim: supervised the study, analyzed the data,
and edited the manuscript. All authors read and approved the final
manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Abbreviations AURKA, Aurora kinase A; AURKB, Aurora kinase B;
AURKC, Aurora kinase C; CLSA, Clonogenic long-term survival assay;
CoMFA, Comparative molecular field analysis; CoMSIA, Comparative
molecular similarity indices analysis; GI50, Half-maximal growth inhibi-
tory concentrations; HR/MS, High-resolution mass spectrometry; PARP,
Poly(ADP-ribose) polymerase; QSAR, Quantitative structure-activity
relationships
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