Synthesis and Quantum Chemical Studies of New 4-aminoquinazoline Derivatives as Aurora A/B Kinase Inhibitors

Article abstract of DOI:10.1111/cbdd.12089

Nine novel 4-aminoquinazoline derivatives were designed and synthesized. Biochemical and cellular analyses demonstrated that most of the derivatives exhibited a strong activity to inhibit Aurora A and B kinases and to suppress the proliferation of a panel of human tumor cell lines (U937, K562, A549, LoVo, and HT29). Quantum chemical studies were also carried out to determine the structural features of these compounds engaged in the inhibition of Aurora kinases.

Full text of DOI:10.1111/cbdd.12089

Chem Biol Drug Des 2012
Research Article
Synthesis and Quantum Chemical Studies of New
4-aminoquinazoline Derivatives as Aurora A⁄B Kinase
Inhibitors
Ming Zheng1, Youguang Zheng2, Yunsheng Xue2,
has a crucial role in the bipolar spindle formation (2). Aur-
ora B is a chromosomal passenger protein and localizes
at the centromeres during prometaphase and associates
with midzone microtubules and midbodies during ana-
Yi Liu2, Lin An2, Ling Zhang2, Min Ji1,3,*, Bai
Xue1,4, Xuan Wu2, Xuedong Gong5, Ning Gu1 and
Xi Zhan6
phase and telophase (1,3). Aurora
C is specifically
1School of Biological Science & Medical Engineering,
Southeast University, Nanjing 211189, China
expressed in the testis and may play a role in spermato-
genesis (4). The initial link between Aurora kinases and
cancer was based on the observation that Aurora A and
B were frequently overexpressed in colon cancer (5). Fur-
ther studies found that Aurora A and B were overexpres-
sed in a variety of solid tumors including prostate (6,7),
pancreatic (8), breast (9), and thyroid cancers (10). Over-
expression of Aurora A or B was mainly attributed to gene
amplification and correlated with histological malignancy
and poor prognosis (11). Recently, several small molecules
with selective anti-Aurora activities have been developed
and entered into preclinical or clinical trials for cancer
treatment (12–14).
2School of Pharmacy, Xuzhou Medical College, Xuzhou
221004, China
3School of Chemistry & Chemical Engineering, Southeast
University, Nanjing 211189, China
4JiangSu Simcere Pharmaceutical R&D Co., Ltd, Nanjing
210042, China
5Department of Chemistry, Nanjing University of Science
and Technology, Nanjing 210094, China
6The Center for Inflammatory and Vascular Diseases,
Department of Pathology, University of Maryland School of
Medicine Baltimore, 800 West Baltimore Street, MD
21201, USA
ZM447439 (AstraZeneca, Wilmington, DE), which has a 4-
aminoquinazoline skeleton, was the first reported small
inhibitor of Aurora kinases. In vitro, ZM447439 inhibits both
Aurora A and B with IC50 values of 110 and 130 nM, respec-
tively; impairs certain mitosis events, including chromosome
alignment, chromosome segregation, and cytokinesis; and
induces ultimately cell deaths (15,16). The report herein
described our effort to improve the efficacy of inhibiting
Aurora kinases by introducing an active imatinib group into
several 4-aminoquinazoline analogs. This was achieved by
intramolecular cyclization of the amide group on the
benzene ring. We found that several new compounds gen-
erated by this approach have acquired improved potency in
inhibiting both Aurora A and B kinases (Figure 1).
*Corresponding author: Min Ji, jimin@seu.edu.cn
Ming Zheng and Youguang Zheng contributed equally to
this work.
Nine novel 4-aminoquinazoline derivatives were
designed and synthesized. Biochemical and cellular
analyses demonstrated that most of the derivatives
exhibited a strong activity to inhibit Aurora A and B
kinases and to suppress the proliferation of a panel of
human tumor cell lines (U937, K562, A549, LoVo, and
HT29). Quantum chemical studies were also carried
out to determine the structural features of these com-
pounds engaged in the inhibition of Aurora kinases.
Key words: 4-aminoquinazolines, anti-proliferative activity,
Aurora A and B, quantum chemistry
Methods and Materials
Received 30 July 2012, revised 7 October 2012 and accepted
for publication 2 November 2012
Chemistry
The strategy for synthesis of 4-aminoquinazoline derivatives
is outlined in Scheme 1 (17). Interaction of cyclocondensa-
tion of 2-amino-4-fluorobenzoic acid 1 with formamidine
acetate in the solvent of 2-methoxyethanol produced a high
yield of compound 2. Compound 2 was then treated with
propylene glycol in the presence of sodium hydride to form
compound 3, which was further used to interact with thio-
nyl chloride to generate chlorinated compound 4. Using
Mammalian Aurora kinases constitute a small family of
three closely related Ser ⁄ Thr kinases, named as Aurora A,
B, and C. These kinases contain a highly conserved cata-
lytic site and are involved in the progression of mitosis or
meiosis, although the function of Aurora C is less under-
stood (1,2). Aurora A localizes to the spindle poles and
ª 2012 John Wiley & Sons A/S. doi: 10.1111/cbdd.12089
1

Zheng et al.
Figure 1: Design of the compounds.
Scheme 1: Synthesis route of the
Target Compounds. Reagents
and conditions: a) Formamidine
Acetate, 2-methoxyethanol;
b) propylene glycol and NaH;
c) SOCl2 ⁄ DMF; d) aniline deriva-
tives, isopropanol; e) secondary
amine ⁄ DMF.
isopropanol as solvent, compound 4 reacted with aniline
derivatives to yield 4-aminoquinazoline 5, which furnished
the final compound 6 upon treatment with the secondary
amine in N, N-dimethylformamide.
Aurora kinase assays
The kinase activity of Aurora A and B was measured using
HTRF-KinEASE-STK discovery kit (Cisbio, Bedford, MA,
USA). Briefly, Aurora A (65 nM) or Aurora B (316 nM) (Milli-
pore, Billerica, MA, USA) was incubated with 1 lM STK
substrate 2-biotin (Cisbio) and 15 lM ATP (Sigma-Aldrich)
in the presence of tested compounds for 1 h at room tem-
perature. The reaction was terminated by adding 200 mM
EDTA. Phosphorylated substrates were incubated with
STK antibody labeled with Eu3+-cryptate and streptavidin-
XL665. The Eu3+-cryptate-conjugated antibody was
excited at 340 nm, and cryptate and fluorescence reso-
nance energy transfer emissions were detected at 615
and 665 nm, respectively. GRAPHPAD PRISM was used to
generate IC50 values.
Cell proliferation assay
The effect of the synthesized compounds on cell prolifera-
tion was assessed by MTT (Sigma-Aldrich Shanghai Trad-
ing Co Ltd Shanghai, China). The cell lines that were
assessed include the following: U937 human acute mono-
cytic leukemia cells, K562 human chronic myeloid leuke-
mia cells, A540 human non-small cell lung cancer cells,
and LoVo and HT29 human colon cancer cells. 5 · 103
cells were seeded in each well in a 96-well culture plate.
The cell cultures were added with compounds to be
tested at various concentrations in triplicates. Seventy-two
hours later, the cell growth medium was removed, and
each well was added with 5 mg ⁄ mL of MTT followed by
incubation at 37 ꢀC. After 4-h incubation, 50 lL of 20%
SDS was added to each well, which was further incu-
bated for overnight. Absorbance was measured with a
plate reader at 570 nm with correction at 630 nm. The
results were expressed as the percentage of absorbance
of treated wells versus that of vehicle control. IC50, the
drug concentration causing 50% growth inhibition, was
calculated via sigmoid curve fitting using GRAPHPAD
PRISM 5.0 (GraphPad Software, Inc., La Jolla, CA, USA).
Quantum chemical calculation
The chemical structures of new compounds were drawn
using HYPERCHEM 7.0 and optimized with its module of
molecular mechanics (MM+ force field). The calculated
structures at low energy were subjected to conformational
analysis using HyperChem’s Conformational Search mod-
ule, and only those that have the most stable conformation
were used in the next level of quantum chemical calcula-
tions. These calculations were performed using density
functional theory (DFT) with the aid of GAUSSIAN03 software
(Gaussian, Inc., Wallingford, CT, USA). B3LPY functional
2
Chem Biol Drug Des 2012

New 4-aminoquinazoline Derivatives as Aurora A ⁄ B Kinase Inhibitors
and the 6-31G (d) basis set were used in the DFT calcula-
1334.52, 1297.88, 1224.60, 1132.03, 1002.82, 804.18,
651.83; 1H-NMR (DMSO-d6, 300 MHz) d (ppm): 1.50 (m,
2H, -CH2-CH2-CH2-O-),1.71 (s, 4H, 2 · piperidine-CH2),
2.24 (s, 5H, -CH3 and piperidine-CH2), 2.95 (s, 4H,
2 · piperidine-CH2), 3.33 (s, 2H, -CH2-CH2-CH2-O-), 4.21
(s, 2H, -CH2-CH2-CH2-O-), 7.22 (d, 3H, Ar-H), 7.42–7.56
(d, 3H, Ar-H), 8.13 (s, 1H, Ar-H), 8.43–8.51 (d, 4H, Ar-H),
8.66 (s, 1H, Ar-H), 8.98 (s, 1H, Ar-H), 9.28 (s, 1H, -NH-),
9.70 (s, 1H, -NH-); 13C-NMR (DMSO-d6, 75 MHz) d(ppm):
17.61, 22.16, 23.18, 23.91, 52.52, 53.69, 65.70, 107.42,
107.67, 109.43, 117.35, 118.74, 119.10, 123.66, 124.78,
127.58, 129.81, 132.12, 134.20, 137.26, 137.62, 148.11,
151.31, 151.91, 155.05, 157.39, 159.40, 161.16, 161.51,
161.62; ESI-MS m ⁄ z: 547.3 [M + H]+.
tionsb (18–21). The fundamental vibrations were also cal-
culated using the same method to determine whether they
were true minima. The highest occupied molecular orbital
(HOMO) and lowest unoccupied molecular orbital (LUMO)
values, which were calculated as molecular orbital coeffi-
cients in artificial unit (a.u.), were converted into energy
units in eV. The plots of HOMO and LUMO were gener-
ated using GAUSSVIEW 3.0 (Gaussian, Inc.).
Experimental Protocols
All reagents were purchased from commercial sources
and used without further purification. Melting points were
measured in open capillaries without correction. IR spectra
were determined as KBr pellets on a Thermo Nicolet 6700
spectrophotometer. 1H-NMR and 13C-NMR spectra were
recorded in DMSO-d6 on a Bruker Avance 300 or 500
spectrometer; chemical shifts (d) were reported in parts
per million (ppm) relative to tetramethylsilane and used as
an internal standard. Mass spectra (MS) were obtained
from Agilent 1100LC ⁄ MS Spectrometry Services. Elemen-
tary analyses were performed on Elementar Vario EL III
instrument. The purity of all compounds was routinely veri-
fied by TLC with silica gel GF-254 glass plates and viewed
under UV light at 254 nm.
N1-(7-(3-(diethylamino)propoxy)quinazolin-4-yl)-4-
methyl-N3-(5-(pyridin-3-yl)pyrimidin-2-yl)benzene-
1,3-diamine(6c)
Yield 46.8%, mp 157 ꢀC. IR (KBr, per cm): 3384.51,
2979.53, 2651.68, 1618.01, 1597.44, 1525.44, 1452.16,
1415.52, 1332.59, 1295.95, 1224.60, 1130.10, 1004.75,
987.39, 800.33, 707.76, 653.76; 1H-NMR (DMSO-d6,
300 MHz) d (ppm): 1.89 (s, 2H, -CH2-CH2-CH2-O-), 1.97–
2.04 (d, 5H, diethylamine-CH2-CH3), 2.24 (s, 3H, -CH3),
2.78–2.87 (t, 5H, diethylamine-CH2-CH3), 3.98–4.05 (m,
2H, -CH2-CH2-CH2-O-), 4.21 (s, 2H, -CH2-CH2-CH2-O-),
7.15–7.24 (t, J = 7.05 Hz, 3H, Ar-H), 7.40–7.46 (t,
J = 4.95 Hz, 2H, Ar-H), 7.59 (d, J = 7.74 Hz, 1H, Ar-H),
8.14 (s, 1H, Ar-H), 8.43 (s, 2H, Ar-H), 8.49–8.57 (dd,
J1 = 4.80 Hz, J2 = 9.18 Hz, 2H, Ar-H), 8.66 (s, 1H, Ar-H),
8.99 (s, 1H, Ar-H), 9.28 (s, 1H, -NH-), 9.71 (s, 1H, -NH-);
13C-NMR (DMSO-d6, 75 MHz) d(ppm): 7.78, 9.97, 14.04,
17.64, 20.71, 24.51, 46.26, 48.04, 58.56, 59.70, 65.71,
107.43, 107.58, 109.43, 117.53, 118.78, 119.14, 123.68,
124.84, 127.59, 129.82, 132.15, 134.22, 137.21, 137.64,
148.14, 151.33, 151.96, 155.07, 157.42, 159.42, 161.18,
161.53, 161.73; ESI-MS m ⁄ z: 535.34 [M + H]+.
Compounds 6a–6i were characterized as follows:
4-methyl-N1-(7-(3-morpholinopropoxy)quinazolin-4-
yl)-N3-(5-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-
diamine(6a)
Yield 52.3%, mp 143 ꢀC. IR (KBr, per cm): 3243.73,
2958.31, 2858.03, 1618.01, 1577.51, 1529.30, 1452.16,
1419.37, 1332.59, 1299.81, 1224.60, 1118.53, 1025.96,
794.54, 649.90; 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
1.90–1.97 (m, 2H, -CH2-CH2-CH2-O-), 2.24 (s, 3H, –CH3),
2.40 (m, 4H, 2 · morpholine-CH2), 2.50 (m, 4H, 2 · mor-
pholine-CH2), 3.59 (s, 2H, -CH2-CH2-CH2-O-), 4.20 (t,
J = 6.06 Hz, 2H, -CH2-CH2-CH2-O-), 7.14 (s, 1H, Ar-H),
7.25 (t, J = 8.31 Hz, 2H, Ar-H), 7.41–7.49 (m, 2H, Ar-H),
7.59 (d, J = 8.31 Hz, 1H, Ar-H), 8.13 (s, 1H, Ar-H), 8.43
(s, 2H, Ar-H), 8.52 (t, J = 2.91 Hz, 2H, Ar-H), 8.67 (d,
J = 3.51 Hz, 2H, Ar-H), 8.98 (s, 1H, Ar-H), 9.28 (s, 1H, -
NH-), 9.66 (s, 1H, -NH-); 13C-NMR (DMSO-d6, 75 MHz) d
(ppm): 17.61, 25.52, 53.20, 54.64, 66.01, 66.16, 107.41,
107.51, 109.30, 117.50, 118.71, 119.07, 123.65, 124.68,
127.54, 129.81, 132.12, 134.19, 137.28, 137.63, 148.11,
151.30, 151.94, 155.01, 157.37, 159.39, 161.16, 161.51,
161.88; ESI-MS m ⁄ z: 549.33 [M + H]+.
4-methyl-N3-(5-(pyridin-3-yl)pyrimidin-2-yl)-N1-(7-
(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-yl)benzene-
1,3-diamine(6d)
Yield 31.5%, mp 125 ꢀC. IR (KBr, per cm): 3423.08,
2960.24, 2805.96, 1618.01, 1577.51, 1529.30, 1454.09,
1419.37, 1334.52, 1297.88, 1224.60, 1130.10, 798.40,
705.83, 651.83; 1H-NMR (DMSO-d6, 500 MHz) d(ppm):
1.68 (s, 4H, 2 · pyrrolidine-CH2),1.90–1.98 (m, 2H, -CH2-
CH2-CH2-O-), 2.25 (s, 3H, -CH3), 2.46–2.50 (m, 4H,
2 · pyrrolidine-CH2), 2.59 (t, J = 7.15 Hz, 2H, -CH2-CH2-
CH2-O-), 4.17 (t, J = 6.35 Hz, 2H, -CH2-CH2-CH2-O-),
7.15 (d, J = 2.45 Hz, 1H, Ar-H), 7.20–7.24 (m, 2H, Ar-H),
7.41 (d, J = 5.15 Hz, 1H, Ar-H), 7.45–7.47 (dd,
J1 = 4.80 Hz, J2 = 4.75 Hz, 1H, Ar-H), 7.55–7.57 (dd,
J1 = 2.05 Hz, J2 = 2.00 Hz, 1H, Ar-H), 8.13 (s, 1H, Ar-H),
8.41–8.44 (m, 2H, Ar-H), 8.46–8.51 (dd, J1 = 9.25 Hz,
J2 = 5.10 Hz, 1H, Ar-H), 8.65–8.67 (dd, J1 = 1.55 Hz,
J2 = 1.50 Hz, 1H, Ar-H), 8.96 (s, 1H, Ar-H), 9.28 (s, 1H,
4-methyl-N1-(7-(3-(piperidin-1-
yl)propoxy)quinazolin-4-yl)-N3-(5-(pyridin-3-
yl)pyrimidin-2-yl)benzene-1,3-diamine(6b)
Yield 52.1%, mp 170 ꢀC. IR (KBr, per cm): 3423.08,
2944.82, 1618.01, 1577.51, 1523.51, 1452.16, 1413.59,
Chem Biol Drug Des 2012
3

Zheng et al.
-NH-), 9.59 (s, 1H, -NH-); 13C-NMR (DMSO-d6, 125 MHz)
d(ppm): 17.63, 23.06, 27.91, 52.08, 53.55, 66.25, 107.43,
107.52, 109.29, 117.54, 118.68, 119.04, 123.67, 124.58,
127.55, 129.85, 132.14, 134.20, 137.28, 137.67, 148.13,
151.32, 151.96, 155.04, 157.36, 159.41, 161.18, 161.53,
161.93; ESI-MS m ⁄ z: 533.32 [M + H]+.
1H-NMR (DMSO-d6, 300 MHz) d(ppm): 1.32–1.39 (m, 5H,
–CH2-CH3), 1.50 (d, 4H, 2 · piperidine-CH2), 1.93 (t,
J = 6.96 Hz, 2H, -CH2-CH2-CH2-O-), 2.34–2.44 (m, 6H,
3 · piperidine-CH2), 4.18 (t, J = 6.33 Hz, 2H, -CH2-CH2-
CH2-O-), 4.32–4.39 (m, 2H, -CH2-CH2-CH2-O-), 7.17 (d,
J = 2.37 Hz, 1H, Ar-H), 7.24–7.27 (dd, J1 = 2.37 Hz,
J2 = 2.37 Hz, 1H, Ar-H), 7.93–7.97 (d, J1 = 1.98 Hz,
J2 = 1.86 Hz, 1H, Ar-H), 8.03 (d, J = 8.82 Hz, 1H, Ar-H),
8.21 (s, 1H, Ar-H), 8.47–8.53 (m, 3H, Ar-H), 9.85 (s, 1H, -
NH-); 13C-NMR (DMSO-d6, 125 MHz) d(ppm): 14.11,
24.10, 25.57, 26.10, 54.07, 55.00, 61.38, 66.42, 107.53,
109.26, 117.82, 118.26, 122.69, 123.54, 124.56, 130.74,
133.65, 136.41, 138.79, 152.05. 154.91, 157.42, 161.96,
162.08; ESI-MS m ⁄ z: 491.4 [M + H]+.
Ethyl 6-(7-(3-morpholinopropoxy)quinazolin-4-
ylamino)benzo[b]thiophene-3-carboxylate(6e)
Yield 61.3%, mp 145 ꢀC. IR (KBr, per cm): 3234.09,
2942.89, 1664.29, 1567.87, 1554.37, 1403.95, 1357.66,
1274.74, 1201.45, 1114.67, 958.46, 821.54; 1H-NMR
(DMSO-d6, 500 MHz) d(ppm): 1.36 (t, J = 7.15Hz, 3H, –
CH2-CH3), 1.93–1.97 (dd, J1 = 6.80 Hz, J2 = 6.65 Hz, 2H,
–CH2-CH3), 2.38 (s, 4H, 2 · morpholine-CH2), 2.44–2.50
(dd, J1 = 7.10 Hz, J2 = 12.85 Hz, 2H, -CH2-CH2-CH2-O-),
3.58 (t, J = 4.40 Hz, 4H, 2 · morpholine-CH2), 4.19 (t,
J = 6.30 Hz, 2H, -CH2-CH2-CH2-O-), 4.34–4.38 (m, 2H, -
CH2-CH2-CH2-O-), 7.18 (d, J = 2.25 Hz, 1H, Ar-H), 7.24–
7.26 (dd, J1 = 2.00 Hz, J2 = 2.00 Hz, 1H, Ar-H), 7.94–
7.96 (dd, J1 = 1.60 Hz, J2 = 1.50 Hz, 1H, Ar-H), 8.04 (d,
J = 8.80 Hz, 1H, Ar-H), 8.20 (s, 1H, Ar-H), 8.47–8.53 (m,
3H, Ar-H), 9.83 (s, 1H, -NH-); 13C-NMR (DMSO-d6,
125 MHz) d(ppm): 14.12, 25.68, 53.33, 54.73, 61.39,
66.18, 66.26, 107.54, 109.29, 117.81, 118.25, 122.69,
123.52, 124.57, 130.74, 133.66, 136.41, 136.97, 138.80,
152.05, 154.52, 157.43, 161.97, 162.05; ESI-MS m ⁄ z:
493.22 [M + H]+.
Ethyl 6-(7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-
ylamino)benzo[b]thiophene-3-carboxylate(6h)
Yield 36.9%, mp 170 ꢀC. IR (KBr, per cm): 3426.94,
2964.10, 2875.39, 2788.61, 1618.01, 1575.58, 1519.66,
1456.02, 1432.58, 1417.45, 1338.38, 1230.38, 1130.10,
81.32, 717.40, 676.90; 1H-NMR (DMSO-d6, 300 MHz)
d(ppm): 1.85–2.00 (m, 5H, –CH2-CH3), 2.49–2.54 (m, 4H,
2 · pyrrolidine-CH2), 2.65 (t, J = 6.93 Hz, 2H, -CH2-CH2-
CH2-O-), 3.51–3.55 (t, 4H, 2 · pyrrolidine-CH2), 3.85 (d,
J = 6.15 Hz, 2H, -CH2-CH2-CH2-O-), 4.20 (t, J = 6.27 Hz,
2H, -CH2-CH2-CH2-O-), 7.17 (d, J = 2.43 Hz, 1H, Ar-H),
7.23–7.27 (dd, J1 = 2.43 Hz, J2 = 2.43 Hz, 1H, Ar-H),
7.81–7.84 (dd, J1 = 2.07 Hz, J2 = 1.92 Hz, 1H, Ar-H),
7.96–8.02 (dd, J1 = 5.10 Hz, J2 = 11.91 Hz, 2H, Ar-H),
8.49–8.62 (m, 3H, Ar-H), 10.02 (s, 1H, -NH-); 13C-NMR
(DMSO-d6, 125 MHz) d(ppm): 22.96, 23.51, 26.15, 27.19,
47.25, 48.47, 51.86, 53.45, 66.01, 107.61, 109.37, 113.64,
117.69, 117.87, 118.37, 122.12, 123.11, 124.65, 125.14,
134.80, 136.54, 139.64, 140.58, 152.01, 154.97, 155.60,
157.55, 160.79, 161.92; ESI-MS m ⁄ z: 477.2 [M + H]+.
Ethyl 6-(7-(3-(4-methylpiperazin-1-yl)propoxy)
quinazolin-4-ylamino)benzo[b]thiophene-3-
carboxylate(6f)
Yield 43.7%, mp 162 ꢀC. IR (KBr, per cm): 3274.59,
2937.10, 1712.51, 1619.94, 1577.51, 1529.30, 1450.23,
1413.59, 1292.09, 1230.38, 1149.38, 1072.24, 1014.39,
782.97, 754.04; 1H-NMR (DMSO-d6, 500 MHz) d(ppm):
1.34 (t, J = 8.30 Hz, 3H, –CH2-CH3), 1.90–1.93(t,
J = 6.20, 2H, –CH2-CH3), 2.18 (s, 4H, 2 · morpholine-
CH2), 2.40–2.49 (m, 9H, 2 · morpholine-CH2, -CH2-CH2-
CH2-O- and –CH3), 4.17 (d, J = 5.60 Hz, 2H, -CH2-CH2-
CH2-O-), 4.33–4.37 (m, 2H, -CH2-CH2-CH2-O-), 7.15 (s,
1H, Ar-H), 7.24 (d, J = 8.85 Hz, 1H, Ar-H), 7.97 (d,
J = 8.55 Hz, 1H, Ar-H), 8.03 (d, J = 8.70 Hz, 1H, Ar-H),
8.19 (s, 1H, Ar-H), 8.50–8.53 (m, 3H, Ar-H), 9.86 (s, 1H, -
NH-); 13C-NMR (DMSO-d6, 125 MHz) d(ppm): 14.10,
25.95, 45.31, 52.34, 54.15, 54.45, 61.36, 66.25, 107.50,
109.30, 117.74, 118.20, 122.63, 123.49, 124.63, 130.72,
133.62, 136.38, 137.00, 138.77, 152.03, 154.90, 157.42,
161.95, 162.02; ESI-MS m ⁄ z: 506.26 [M + H]+.
Ethyl 6-(7-(3-(diethylamino)propoxy)quinazolin-4-
ylamino)benzo[b]thiophene-3-carboxylate(6i)
Yield 35.7%, mp 160 ꢀC. IR (KBr, per cm): 3455.87,
3025.81, 2971.81, 2933.24, 2802.11, 1706.72, 1618.01,
1577.51, 1526.27, 1450.23, 1409.73, 1326.81, 1172.53,
1149.38, 1130.10, 1070.32, 1020.18, 912.18, 755.97,
674.97; 1H-NMR (DMSO-d6, 300 MHz) d(ppm): 0.89–0.97
(m, 6H, 2 · diethylamine-CH3), 1.28–1.37 (m, 3H, –CH2-
CH3), 1.85–1.91 (dd, J1 = 6.54 Hz, J2 = 5.12 Hz, 2H, -
CH2-CH2-CH2-O-), 2.42–2.57 (m, 6H, 2 · diethylamine-
CH2 and –CH2-CH3), 4.17(t, J = 6.12 Hz, 2H, -CH2-CH2-
CH2-O-), 4.32–4.39 (t, 2H, -CH2-CH2-CH2-O-), 7.16 (s,
1H, Ar-H), 7.26 (d, J = 9.03 Hz, 1H, Ar-H), 7.97 (d,
J = 8.91 Hz, 1H, Ar-H), 8.05 (d, J = 8.76 Hz, 1H, Ar-H),
8.20 (s, 1H, Ar-H), 8.47–8.53 (m, 3H, Ar-H), 9.86 (s, 1H, -
NH-); 13C-NMR (DMSO-d6, 125 MHz) d(ppm): 11.83,
14.09, 26.45, 46.42, 48.57, 61.35, 66.23, 107.46, 109.27,
117.76, 118.20, 122.63, 123.48, 124.56, 130.71, 133.63,
136.38, 137.00, 138.77, 139.36, 152.05, 154.87, 157.41,
161.94, 162.10; ESI-MS m ⁄ z: 479.4 [M + H]+.
Ethyl 6-(7-(3-(piperidin-1-yl)propoxy)quinazolin-4-
ylamino)benzo[b]thiophene-3-carboxylate(6g)
Yield 57.9%, mp 170–171 ꢀC. IR (KBr, per cm): 3390.58,
2933.73, 1709.27, 1619.05, 1570.93, 1526.82, 1454.64,
1410.53, 1228.07, 1127.82, 1069.67, 1053.63, 756.89;
4
Chem Biol Drug Des 2012

New 4-aminoquinazoline Derivatives as Aurora A ⁄ B Kinase Inhibitors
6e–6i. To facilitate structure–activity relationship (SAR)
analysis, we maintained the aniline group at R2 while
varying the side chain substituent at R1. We found that
variations of the secondary amine substituent at R1 also
Results and Discussion
Analysis of the inhibitory activity of new
compounds against Aurora A ⁄ B kinases
resulted in dramatic changes in the inhibition of Aurora kin-
ases. Compounds 6d and 6h with a pyrrole side chain
showed the strongest inhibitory activity. Compounds 6b
and 6g with a piperidine were significantly less potent than
that of 6d and 6h. Compound 6a with a morpholine at R1
had also a modest activity to inhibit Aurora kinases. How-
ever, compound 6e, which has a similar side chain, did
We have synthesized nine compounds derived from 4-am-
inoquinazoline. To explore the mechanism of action of
these new compounds, they were analyzed for the inhibi-
tion of Aurora A ⁄ B kinases. As shown in Table 1, most of
the derivatives exhibit a higher selectivity for Aurora A than
Aurora B. For example, compound 6d has a lower IC50
value (77 nM) for Aurora A than that for Aurora B (144 nM).
Introduction of the active group of imatinib at the amine of
C-4 of the quinazoline ring in compounds 6a–6d improved
the inhibitory activity as compared to aniline analogs
not show
a detectable activity against Aurora A ⁄ B.
Replacement of the pyrrole group in compound 6d with
diethylamine group did not change the anti-Aurora activity
(6c). On the other hand, the similar replacement in 6h led
to a significant reduction in the potency (6i). Compound
6f, which has an N-methyl piperazine at R1, exhibited a
similar activity as ZM447439. Thus, changes in R1 and R2
side chains of 4-aminoquinazoline can improve the
potency against Aurora kinases.
Table 1: Aurora A ⁄ B inhibitory activity of 4-aminoquinazoline
derivates
R2
HN
N
R1
O
N
Analysis of antiproliferation activity
IC50(nM)a
Aurora A Aurora B
Compound
R1
R2
All the new compounds were evaluated for the potential
to inhibit the growth of human tumor cells. For this pur-
pose, we chose five cell lines representing different tumor
types, including human acute monocytic leukemia cell line
(U937), human chronic myeloid leukemia cell line (K562),
human non-small cell lung cancer (A549), and human
colon cancer (LoVo and HT29). The result of the study
was summarized in Table 2. Most of the compounds had
evident antiproliferation effect on solid tumor cell lines
(A549, LoVo, and HT29) but poor effect on leukemia cells
(U937 and K562). In particular, compounds 6c and 6d
have IC50 values ranging from 0.99 to 4.35 lM toward
solid tumor cells, which are similar to or better than that
of ZM447439 (IC50 = 1.87–3.36 lM). In addition, com-
pounds 6c and 6d also showed increased potency
against leukemia cell lines as compared to ZM447439.
We proposed that the higher cytotoxicity of these com-
pounds could be attributed to the active group of imatinib
substituted at the R2 side chain. Indeed, compound 6b,
which has the same active group, also showed a similar
activity against solid tumor cell lines with an IC50 value of
2.01–2.85 lM. Also compound 6g, which has a R1 side
chain (piperidine) same as 6b but has an aniline group at
R2, exhibited a significant decrease in the cytotoxic activ-
ity (IC50 > 100 lM for the most tumor cells) and poor
potency against Aurora A ⁄ B kinases. On the other hand,
compounds 6d and 6h, which have a pyrrolidine ring at
the R1 side chain but with different R2 groups, inhibited
the growth of solid tumor cells with IC50 values from 1.98
to 5.44 lM. Thus, the side chain at R1 also has a signifi-
cant impact on the antiproliferation activity. Another inter-
esting observation is that compound 6f, which has an N-
methyl piperazine at R1 and a similar anti-Aurora kinase
activity as ZM447439, was more potent than ZM447439
in inhibiting tumor cell growths. This difference may be
N
N
N
N
N
179
323
98
191
489
202
145
6a
O
N
N
N
N
N
N
H
N
N
N
6b
6c
6d
N
N
H
N
N
H
77
N
N
H
COOC2H5
NT
111
822
62
NT
136
NT
6e
6f
O
N
S
COOC2H5
N
N
S
COOC2H5
6g
6h
N
S
COOC2H5
146
N
S
COOC2H5
788
138
893
156
6i
N
S
ZM447439
NT, not tested.
aThe mean of at least two experiments.
Chem Biol Drug Des 2012
5

Zheng et al.
Table 2: In vitro cytotoxic activity
of target compounds against can-
cer cells
IC50 (lM)a
Compound
U937
K562
A549
LoVo
HT29
6a
6b
6c
6d
6e
6f
6g
6h
>100
11.30
7.20
4.90
>100
4.90
>100
10.60
>100
>100
>100
>100
4.00
2.00
>100
2.00
>100
>100
>100
>100
>100
2.85
0.99
2.85
6.23
2.26
>100
2.53
8.85
3.36
6.09
2.20
1.65
1.98
5.88
1.21
6.88
5.44
7.35
1.87
9.90
2.01
4.35
1.99
2.16
1.89
>100
4.26
>100
2.23
6i
ZM447439
aCellular proliferation was determined by MTT assay.
A
B
Figure 2: (A) Binding mode of
compounds 6d with Aurora A; (B)
Binding interaction of ZM-447439
with Aurora A.
due to different permeability of these two compounds
into cells.
structure was presented using SYBYL 7.3 software (Tripos
International, St. Louis, MO, USA) and subjected to molecu-
lar modeling, which indicates that compound 6d would
dock strongly into the ATP-binding site of Aurora A (Fig-
ure 2A). It also reveals that the compound participates in a
hydrogen bond interaction with the amine group of Ala213
within the protein hinge region (Figure 2A). Interestingly, the
Molecular docking analysis
The coordinate for the Aurora A structure was obtained from
the RCSB Protein Data Bank (PDB ID: 2NP8). The protein
A
C
B
D
Figure 3: The optimized structures of the most active compounds 6d (A, B) and 6f (C, D).
6
Chem Biol Drug Des 2012

New 4-aminoquinazoline Derivatives as Aurora A ⁄ B Kinase Inhibitors
amine between benzene and pyrimidine ring also makes a
drugs (22). In SAR analysis, quantum chemical parameters
have yielded promising estimation for the correlation of the
electronic effects with the biological activity (23–25). These
parameters include the HOMO and the LUMO (26), which
reflect the frontier molecular orbitals that dominate the reac-
tivity of a molecule. Thus, we calculated the orbital energies
of both HOMO and LUMO and their gaps for all the new
compounds, the result of which is presented in Table 3. Sig-
nificantly, we observed that compounds 6d and 6f have the
lowest energy gap (DE) of 4.0115 and 3.9426 eV, respec-
tively, and both exhibit the highest cytotoxic activity.
hydrogen bond to the hinge region of the kinase, which is
apparently absent in the binding mode of ZM 447439 with
Aurora A (Figure 2B). We proposed that this extra hydrogen
bond may contribute to the apparent high activity of com-
pound 6d to inhibit Aurora kinases and cell growth.
Quantum chemical studies
To gain further insight into the structural basis for these
compounds to interact with their targets, conformational
analysis was performed using MM+ force field in HYPER-
CHEM 7.0a. The analysis demonstrated that the lowest
energy-minimized structures of compounds 6a–d take a
coplanar configuration of the benzopyrimidine, benzene,
and pyrimidine moieties. Such configuration is apparently
stabilized by intramolecular hydrogen bond between the
CH group in the benzene ring and the adjacent nitrogen
atom in the pyrimidine ring (Figure 3A, B). The compounds
6a–c have a structure similar to 6d. As for compounds
6e–i, their benzopyrimidine and benzothiophene moieties
are also configured into a structural plane (Figure 3C, D).
We also plotted the HOMO and LUMO of these com-
pounds to analyze the atomic contributions of these orbi-
tals to the sites for electronic transfer between individual
compound and its biological target. As the representatives,
the plots of the HOMO and LUMO of the most active
compounds (6d and 6f) are presented in Figure 4. The
results illustrate that the HOMO molecular orbital of 6d is
mainly located in the benzopyrimidine, benzene, and
pyrimidine moieties, indicative of the existence of possible
reactive sites that may be involved in electrophilic attacks.
On the other hand, the LUMO of 6d is primarily concen-
trated on pyridine and pyrimidine rings in which the nega-
tively charged polar residues of the receptor may be
It has been reported that molecular electronic effects play a
critical role in controlling the pharmacological activities of
Table 3: Energies of both HOMO
and LUMO and their gaps (in eV)
Compound
EHOMO (eV)
ELUMO (eV)
DE (eV)
calculated for all compounds
6a
)5.36205
)5.33647
)5.33783
)5.33538
)5.56205
)5.46627
)5.53484
)5.53321
)5.53647
)1.34126
)1.32493
)1.32520
)1.32384
)1.54507
)1.52358
)1.51024
)1.50834
)1.51052
4.020786
4.011534
4.012623
4.011534
4.016976
3.942689
4.024596
4.024868
4.025956
6b
6c
6d
6e
6f
6g
6h
6i
HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital.
Figure 4: Plots of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the most active
compounds 6d (upper) and 6f (lower).
Chem Biol Drug Des 2012
7

Zheng et al.
favorable. When compared with 6d, the HOMO and
LUMO of 6f are quite similar and delocalized in benzopyr-
imidine and benzothiophene moieties.
man G.D. (1998) A homologue of Drosophila Aurora
kinase is oncogenic and amplified in human colorectal
cancers. EMBO J;17:3052–3065.
6. Chieffi P., Cozzolino L., Kisslinger A., Libertini S., Staib-
ano S., Mansueto G., De Rosa G., Villacci A., Vitale M.,
Linardopoulos S., Portella G., Tramontano D. (2006)
Aurora B expression directly correlates with prostate
cancer malignancy and influence prostate cell prolifera-
tion. Prostate;66:326–333.
7. Ewart-Toland A., Dai Q., Gao Y.T., Nagase H., Dunlop
M.G., Farrington S.M., Barnetson R.A. et al. (2005) Aur-
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Conclusion
We have designed a series of 4-aminoquinazoline deriva-
tives and analyzed their anti-Aurora kinase activities. Most
of the compounds showed a significant antiproliferation
activity against three solid tumor cell lines, but no or poor
activity against two leukemia cell lines. Introduction of the
active group of imatinib at the R2 side chain or a pyrroli-
dine ring at the R1 side chain impacts significantly the
activity for both anti-Aurora kinases and antigrowth of
tumor cells. Molecular modeling suggested that the strong
inhibitory activity of compound 6d may be attributed to an
extra hydrogen bond between the amine at the pyrimidine
ring and the hinge region of targeted kinases. Quantum
chemical calculation of electronic parameters including
HOMO and LUMO indicates an association of molecular
orbitals at low energy with antiproliferation activities of
these compounds. These findings may be useful in
improving the efficacy of Aurora A ⁄ B kinase inhibitors by
incorporating some structural groups that are able to mod-
ify the electronic property of the existing anti-Aurora com-
pounds.
10. Ulisse S., Delcros J.G., Baldini E., Toller M., Curcio F.,
Giacomelli L., Prigent C., Ambesi-Impiombato F.S.,
D’Armiento M., Arlot-Bonnemains Y. (2006) Expression
of Aurora kinases in human thyroid carcinoma cell lines
and tissues. Int J Cancer;119:275–282.
11. McLaughlin J., Markovtsov V., Li H., Wong S., Gelman
M., Zhu Y., Franci C. et al. (2010) Preclinical character-
ization of Aurora kinase inhibitor R763 ⁄ AS703569 iden-
tified through an image-based phenotypic screen. J
Cancer Res Clin Oncol;136:99–113.
Acknowledgments
This work was supported by National Basic Research Program of China
(No. 2011CB933503), ’Qing-Lan’ Project of Jiangsu Province; The National
Natural Science Foundation of China (No. 81202490); Natural Science
Foundation of Jiangsu Province (BK2009253); The Industrialization of
Scientific Research Promotion Projects of Colleges and Universities in Ji-
angsu Province (JHZD09-23); Natural Science Fund for Colleges and Uni-
versities in Jiangsu Province (09KJB350003; 11KJB350005); and Science
and Technology Project of Xuzhou, China (XZZD1224); The authors are
grateful for these financial supports.
12. Harrington E.A., Bebbington D., Moore J., Rasmussen
R.K., Ajose-Adeogun A.O., Nakayama T., Graham J.A.,
Demur C., Hercend T., Diu-Hercend A., Su M., Golec
J.M.C., Miller K.M. (2004) VX-680, a potent and selec-
tive small-molecule inhibitor of the Aurora kinases, sup-
presses tumor growth in vivo. Nat Med;10:262–267.
13. Manfredi M., Ecsedy J., Meetze K., Balani S., Burenk-
ova O., Chen W., Hoar K. et al. (2006) 97th Annual
AACR Meeting, Washington, DC, Abstract 4724.
14. Wilkinson R.W., Keen N., Odedra R., Heaton S.P.,
Wedge S.R., Foote K.M., Mortlock A., Jung F., Heron
N.M., Brady M.C., Walker M., Khatri L., Barrass N.C.,
Foster J.J., Green S. (2006) 97th Annual AACR Meet-
ing, Washington, DC, Abstract 5673.
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