Discovery of 4-aminoquinazoline - Urea derivatives as Aurora kinase inhibitors with antiproliferative activity

Article abstract of DOI:10.1016/j.bmc.2014.09.029

Two series of 20 novel 4-aminoquinazoline - urea derivatives have been designed and synthesized. The entire target compounds were investigated for their in vitro antiproliferative activity against six human cancer cell lines (K562, U937, A549, NCI-H661, HT29 and LoVo) using the MTT-based assay. Most compounds showed significant antiproliferative activities against four solid tumor cell lines, but no or poor activities against two leukemia cell lines. Furthermore, the target compounds were screened for Aurora A/B kinases inhibitory activity. Among them, 7c, 7d, 8c, and 8d are more potent against Aurora A kinase than ZM447439. Docking study of compounds 7d and ZM447439 revealed that they bound strongly to the ATP-binding sites of Aurora A and B. Thus, they may be promising lead compounds for the development of novel anti-tumor drug potentially via inhibiting Aurora kinases.

Full text of DOI:10.1016/j.bmc.2014.09.029

Bioorganic & Medicinal Chemistry 22 (2014) 5813–5823
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of 4-aminoquinazoline—urea derivatives as Aurora kinase
inhibitors with antiproliferative activity
a
a
b
c
a
d
d
a
Jin Cai , Lili Li , Kwon Ho Hong , Xiaoqing Wu , Junqing Chen , Peng Wang , Meng Cao , Xi Zong ,
Min Ji
a
a,d,
⇑
School of Chemistry and Chemical Engineering, Institute of Pharmaceutical Engineering, Southeast University, 87 Dingjiaqiao, Nanjing, Jiangsu 210096, PR China
Department of Medicinal Chemistry and The Institute for Therapeutics Discovery and Development, University of Minnesota, 717 Delaware Street SE, Minneapolis, MN 55414, USA
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA
b
c
d
School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of 20 novel 4-aminoquinazoline—urea derivatives have been designed and synthesized. The
entire target compounds were investigated for their in vitro antiproliferative activity against six human
cancer cell lines (K562, U937, A549, NCI-H661, HT29 and LoVo) using the MTT-based assay. Most com-
pounds showed significant antiproliferative activities against four solid tumor cell lines, but no or poor
activities against two leukemia cell lines. Furthermore, the target compounds were screened for Aurora
A/B kinases inhibitory activity. Among them, 7c, 7d, 8c, and 8d are more potent against Aurora A kinase
than ZM447439. Docking study of compounds 7d and ZM447439 revealed that they bound strongly to
the ATP-binding sites of Aurora A and B. Thus, they may be promising lead compounds for the develop-
ment of novel anti-tumor drug potentially via inhibiting Aurora kinases.
Received 2 August 2014
Revised 10 September 2014
Accepted 11 September 2014
Available online 19 September 2014
Keywords:
ZM447439
Aurora kinase
Antiproliferative activity
Cancer cell
Ó 2014 Elsevier Ltd. All rights reserved.
Quinazolines
Docking simulation
1
. Introduction
reports of small molecule Aurora kinase inhibitors.12–14 VX-680
(Fig. 1), the first Aurora inhibitor to enter clinical trials, is a potent
Aurora kinases are a family of three highly homologous serine/
inhibitor of all three Aurora kinases and generally has low affinities
1
5,16
threonine kinases which play critical roles during the mitotic stage
to other kinases.
AZD1152 (Fig. 1) is the first Aurora-B selective
1
–3
of the cell cycle.
They are overexpressed in various cancer types
inhibitor to enter clinical trials. It is a dihydrogen phosphate pro-
drug of a pyrazoloquinazoline Aurora kinase inhibitor, and is con-
and implicated in many aspects of tumor development. They are
expressed during the G2/M phase of the cell cycle and are critical
for the proper regulation of mitosis.4
1
7
verted rapidly to the active form in plasma.
–6
Researchers from Sunesis Pharmaceuticals reported the discov-
ery of potent Aurora kinase inhibitor SNS-314 (Fig. 1), which exhib-
ited a compelling preclinical profile and entered clinical trials in
Aurora A and B have received a lot of attention to date as anti-
7
cancer targets. In mitosis, Aurora A associates with the spindle
1
8–20
poles and is involved in both centrosomal and acentrosomal spin-
dle assembly. Aurora A is highly expressed in many tumors; its
patients with advanced solid tumors.
SNS-314 is a pan-Aurora
inhibitor with good affinity against all three isoforms.
8
upregulation may play an important role in tumor progression.
ZM447439 (Fig. 1) was the first small molecule Aurora kinase
inhibitor disclosed by AstraZeneca in 2003. It has a 4-aminoqui-
nazoline scaffold and inhibits both Aurora A and B (IC50 of 110
Aurora B regulates chromatin remodeling and phosphorylates his-
9
tone H3 at Ser-10. Aurora C has similar functions as Aurora B; it is
highly expressed in the testis but is also present at a low level in
other tissues.
and 130 nM, respectively) with good selectivity over other unre-
1
0
21,5
lated kinases (affinity for Aurora C is not reported).
In addition,
In recent years, the Aurora kinases have been actively pursued
as anticancer targets for the discovery of new cancer chemothera-
peutic drugs. Considerable research efforts from both the phar-
maceutical industry and academic laboratories have led to many
ZM447439 impairs certain mitosis events, including chromosome
alignment, chromosome segregation, and cytokinesis.5
,22
1
1
The crystal structure of SNS-314 demonstrated the importance
of the urea moiety, which extends deep into the selectivity pocket
2
3
of the protein. The urea oxygen is within close proximity of the
catalytic lysine (Lys175), while the two NH groups form hydrogen
bonding interactions with the catalytic glutamic acid residue
⇑
Corresponding author. Tel.: +86 025 83272078.
E-mail address: jimin@seu.edu.cn (M. Ji).
http://dx.doi.org/10.1016/j.bmc.2014.09.029
968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
0

5
814
J. Cai et al. / Bioorg. Med. Chem. 22 (2014) 5813–5823
N
NH
HN
N
N
N
H
N
OH
HN
N
HN
N
P
O
F
H
N
HO
O
O
O
N
S
N
O
N
VX-680
AZD1152
H
N
N
S
O
O
HN
N
N
H
N
Cl
O
HN
N
H
N
S
N
N
O
O
SNS-314
ZM447439
Figure 1. Chemical structures of VX-680, AZD1152, SNS-314 and ZM447439.
(
Glu194). The SAR of SNS-314 and its analogues also explain the
whether these compounds would manifest antiproliferative activ-
ities against cancer cells and be highly potent in inhibiting both
Aurora A and B kinases.
24
importance of the urea moiety. Disruption of these hydrogen
bonding interactions by replacing the urea moiety of SNS-314 with
an amide linkage results in significant reduction of the inhibitory
activity at enzyme as well as cellular level.18 This indicated that
the urea moiety of SNS-314 (in a specific location) is essential for
its potent Aurora kinase activity.
2
2
. Results and discussion
.1. Chemistry
We used ZM447439 as a lead compound for our work to
develop Aurora kinase inhibitors as anticancer agents according
to the SAR analysis of ZM447439 and SNS-314. On the basis of
As summarized in Table 1, up to twenty compounds (7a–7j and
a–8j) were synthesized. The synthetic route was illustrated in
Scheme 1.
Cyclization of 2-amino-4-fluorobenzoic acid 1 with formami-
8
2
5,26
our previous work,
we have devised and synthesized novel
4
-aminoquinazoline—urea derivatives (Fig. 2): (1) replacement of
the amide linkage with urea moiety as an essential binding ele-
ment in the DFG pocket. (2) Various secondary amino-substituted
propoxy side chains at position 7 of the quinazoline nucleus. (3)
Both benzyl group substituted series and cyclohexyl group substi-
tuted series were investigated. Our objective was to determine
dine acetate in 2-methoxyethanol produced 2 in satisfactory yield.
Compound 2 was then treated with propylene glycol in the pres-
ence of sodium hydride to afford 3 in 91% yield, which was chlori-
nated with thionyl chloride to give chlorinated compound 4 in 77%
yield. Aminolysis of 4 was performed using p-phenylenediamine to
generate 4-aminoquinazoline 5, which was condensed with vari-
ous isocyanates to yield the corresponding compounds 6a–6b.
Nucleophilic substitution reaction of compounds 6a–6b with vari-
ous secondary amines yielded the target compounds 7a–7j and 8a–
8j.
H
N
H
N
O
2.2. In vitro antiproliferative activity of the target compounds
Replacement
Replacement
Twenty newly synthesized ZM447439 derivatives (7a–7j and
8
a–8j) were investigated for their antiproliferative activity
H
N
R2
O
employing the MTT-based assay using ZM447439 as a positive
control against six human cancer cell lines, representing different
tumor types: human chronic myeloid leukemia cell line (K562),
human acute monocytic myeloid leukemia cell line (U937), human
lung cancer cell lines (A549 and NCI-H661) and human colon can-
cer cell lines (HT29 and LoVo). Antiproliferative activities of the
compounds indicated by IC50 values were calculated by linear
regression analysis of the concentration–response curves obtained
for each compound.
O
Replacement
O
HN
N
ZM447439
N
O
N
O
H
N
H
N
The results from the antiproliferation assay are summarized in
Table 2. Most compounds manifest evident antiproliferative activ-
ities against solid tumor cell lines (A549, NCI-H661, HT29 and
LoVo), but poor antiproliferative activities against leukemia cell
lines (K562 and U937) except for 8c and 8d. These results accord
with our previous expectation, because Aurora kinases are known
to be overexpressed in solid tumors and also may be inappropri-
R1
O
HN
N
7
a-7j and 8a-8j
R2
O
N
1
,27–34
ately activated in certain cell types.
Figure 2. A design for ZM447439 derivatives.

J. Cai et al. / Bioorg. Med. Chem. 22 (2014) 5813–5823
5815
Table 1
Structure of the target compounds 7a–7j and 8a–8j
H
N
H
N
R1
O
HN
N
N
R2
O
Compd
R
1
R
2
Mp (°C)
Yieldsa (%)
7
a
O
N
219–220
79
64
73
N
N
7
b
104–105
210–211
7
c
N
N
7
d
218–219
69
N
7
e
215–216
209–210
99–100
78
61
72
7
f
N
7
g
N
N
7
h
Oil
67
HO
HO
N
N
7
i
95–96
63
70
N
7
j
119–120
HO
O
8
a
N
276–277
115–116
172–173
78
60
72
8
b
N
N
8
c
N
8
d
179–180
66
N
8
e
N
174–175
131–132
115–116
75
67
8
f
N
8
g
N
68
(
continued on next page)

5
816
J. Cai et al. / Bioorg. Med. Chem. 22 (2014) 5813–5823
Table 1 (continued)
Compd
R
1
R
2
Mp (°C)
Yieldsa (%)
63
N
N
8
h
102–103
HO
HO
HO
N
8
i
j
100–101
120–121
68
65
N
8
a
Yield for the last step.
O
N
O
N
Cl
N
COOH
NH2
a
NH
b
NH
c
N
F
F
HO
O
Cl
O
3
1
2
4
H
N
H
H N
H
N
H
N
2
N
R1
R
1
NH
O
O
d
e
NH
f
NH
N
N
N
Cl
O
N
Cl
O
N
R
2
O
N
7a-7j and 8a-8j
5
6a-6b
Scheme 1. Synthetic route for the preparation of the target compounds 7a–7j and 8a–8j. Reagents and conditions: (a) formamidine acetate, 2-methoxyethanol, 10 h, reflux,
9%; (b) 1,3-propanediol, NaH, DMF, 24 h, 110 °C, 91%; (c) SOCl , DMF, 5 h, reflux, 77%; (d) p-phenylenediamine, isopropanol, 4 h, reflux, 82%; (e) isocyanatomethyl-benzene
or isocyanatocyclohexane, 5 h, reflux, 87%/81%; (f) HNR , KI, 6 h, 60 °C, 60–79%.
6
2
2
Compared with N-benzyl (7), N-cyclohexyl series (8) com-
pounds displayed slightly lower inhibitory activities. The terminal
phenyl ring of the urea functional group enters the region com-
monly referred to as the ‘back pocket’ in kinase literature, where
it forms hydrophobic interactions with the surrounding amino acid
residues. Removal of the terminal benzyl group away from the
urea function and replacement with cyclohexyl group (8) led to
decreased activity. These results demonstrate the importance of
aromatic hydrophobic interactions in the back pocket for their
inhibitory activities against cancer cells.
decrease in the antiproliferative activities (IC50 values ranging from
13.5 to 53.6 M toward solid tumor cells), which might be induced
l
by the electro-negativity of the oxygen atoms by our conjecture. As
the antiproliferation assay was carried out in vitro, the inhibitory
effect variance among these compounds could be attributed to
the hydrophobicity and permeability changes resulting from dif-
ferent basic side chains. Besides these factors, the structural differ-
ence among these compounds in each series was the basic side
chain at position 7 of quinazoline scaffold, which was reported to
affect the pharmacokinetic properties and solubility of compound
3
5
3
6,37
It is worth pointing out that the most significant inhibition was
achieved for compounds 7c, 7d, 8c and 8d with IC50 values ranging
in vivo.
The introduction of the basic side chains was to
increase the volume of distribution at steady state (Vdss), and thus
increased the observed half-life and a long terminal half-life was
desirable in terms of increasing drug exposure and ultimately
from 0.9 to 3.4
better than that of ZM447439 (IC50 = 1.9–3.3
l
M toward solid tumor cells, which are similar to or
M). In addition,
l
3
8
compounds 7d, 8c and 8d also showed increased potency against
leukemia cell lines as compared to ZM447439. It is noticeable that
compounds 7d and 8d with 2-methylpiperidine substitution dis-
played the relatively higher antiproliferative activities than other
compounds in their respective series, while the piperidine substi-
tuted 7c and 8c showed the lower antiproliferative activities. We
speculated that conformational restriction of methyl group in
piperidine moiety would help to form appropriate angle by amino
quinazolines ring, this was more conducive for aniline ring to
reside in the ligand binding site, meanwhile the nitrogen of 2-
methylpiperidine form a hydrogen bond to the hinge region, all
of which would help to improve the inhibitory activity. Meanwhile,
compounds 7i, 7j and 8i, 8j, which have hydroxyethyl-piperazine
and hydroxymethyl-piperidine group at R2, exhibited significant
efficacy.
2.3. Kinase inhibitory activity of the target compounds
ZM447439 was initially designed and synthesized for Aurora A/
B kinases inhibition. In order to study the mechanism of action of
the new synthesized compounds, all compounds were assayed for
their inhibitory activities against Aurora A/B kinases using
ZM447439 as a reference compound. As shown in Table 2, the com-
pounds manifest Aurora kinase inhibitory activities at submicrom-
olar concentrations, and most derivatives exhibit
a higher
selectivity for Aurora A over Aurora B. For example, compound
7d has a lower IC50 value (61 nM) for Aurora A than that for Aurora
B (172 nM). In addition, the kinase inhibitory activities are

J. Cai et al. / Bioorg. Med. Chem. 22 (2014) 5813–5823
5817
Table 2
In vitro antiproliferative activity in different cell lines and enzyme inhibition activity of target compounds 7a–7j and 8a–8j
Compd
Antiproliferation in different cell linesa,b (IC50
l
M)
Enzyme inhibition
Aurora B
K562
U937
A549
NCI-H661
HT29
LoVo
Aurora A
EGFR
c
c,d
(
IC50 nM)
(%)
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
8
a
b
c
d
e
f
g
h
i
>100
40.3
>100
42.8
>100
>100
37.8
>100
>100
>100
>100
33.8
58.9
23.8
>100
18.1
55.7
>100
>100
>100
>100
>100
>100
>100
15.6
>100
61.3
>100
>100
>100
>100
>100
>100
4.3
18.3
7.4
2.9
3.1
6.8
12.5
6.8
2.1
1.5
5.2
6.3
7.1
1.4
1.2
2.5
3.7
2.8
19.8
26.7
13.5
7.6
5.9
2.6
1.8
4.1
2.6
6.3
9.1
6.9
1.5
0.9
3.8
1.8
3.7
12.1
16.3
26.1
8.2
7.1
2.7
1.5
1.8
5.0
4.8
212
195
78
367
289
193
172
292
278
196
573
621
783
264
318
221
197
232
181
176
387
589
845
156
NT
45.3
35.8
38.1
55.6
35.8
46.7
29.6
33.5
43.6
21.9
37.1
38.3
29.4
36.3
50.2
61.8
31.9
36.5
32.7
27.5
39.8
98.5
61
219
143
128
468
496
564
193
202
103
82
157
115
123
312
436
796
138
NT
4.3
5.6
4.2
4.1
29.5
36.9
47.8
32.5
6.3
3.4
2.9
5.5
3.9
18.2
28.2
32.9
24.3
8.2
2.5
3.3
3.9
1.8
j
a
b
c
d
e
f
g
h
i
3.9
10.6
>100
>100
>100
>100
>100
>100
NT
4.1
2.9
29.6
53.6
45.3
3.3
16.6
28.9
51.6
2.9
21.5
31.1
29.8
2.2
17.8
26.8
31.6
1.9
j
ZM447439
Gefitinib
e
NT
NT
NT
NT
NT
a
b
c
Values are averages of three independent experiments, SD <10%.
IC50 is the concentration of compound required to inhibit the cell growth by 50% compared to an untreated control.
Values are averages of at least two independent experiments, SD <10%.
d
e
Compounds tested at a concentration of 10
Not tested.
lM.
Figure 3. Docked binding modes of compounds in the ATP binding site of Aurora A and B. Interactions between the protein and the ligand are shown as yellow dotted lines.
The ligands are shown in stick model, while the proteins are shown in surface model for better visualization. (A) Binding mode of ZM447439 with Aurora A (PDB ID: 3D15).
(
B) Binding mode of compounds 7d with Aurora A. (C) X-ray co-crystal structure of Aurora B in complex with ZM447439 (PDB ID: 2VRX). (D) Binding mode of compounds 7d
with Aurora B.

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J. Cai et al. / Bioorg. Med. Chem. 22 (2014) 5813–5823
consistent with their antiproliferative activities. Compounds 7c,
d, 8c and 8d with stronger antiproliferative activity also show
designed and synthesized. The entire target compounds were
investigated for their in vitro antiproliferative activity using the
MTT-based assay against six human cancer cell lines (K562,
U937, A549, NCI-H661, HT29 and LoVo). Most compounds showed
significant antiproliferative activities against four solid tumor cell
lines, but no or poor activities against two leukemia cell lines. Fur-
thermore, the target compounds were screened for Aurora A/B
kinases inhibitory activity. Among them, 7c, 7d, 8c, and 8d are
more potent against Aurora A kinase than ZM447439. Docking
study performed with selected compounds 7d and ZM447439
revealed that the compounds bound strongly to the ATP-binding
sites of Aurora A and B. The results suggest that they may be prom-
ising lead compounds for the development of novel anti-tumor
drug potentially via inhibiting Aurora kinases.
7
better Aurora A inhibitory activity than ZM447439. Since Gefitinib,
a selective EGFR inhibitor, also contains quinazoline moiety, it was
thought that the quinazoline moiety might play a role in inhibiting
EGFR and it would be valuable to evaluate the EGFR inhibitory
activity of our compounds. As shown in Table 2, the new com-
pounds exhibit much lower EGFR inhibitory activities than Gefiti-
nib, which indicates that these compounds are not specific EGFR
tyrosine kinase inhibitors. Taken the biological data together, we
could preliminarily arrive at the conclusion that some target com-
pounds are potent Aurora kinase inhibitors. Thus they may be
promising lead compounds for the development of novel anti-
tumor drug potentially via inhibiting Aurora kinases.
2
.4. Docking simulation study
4
4
. Experimental protocols
To understand the structural basis of compounds 7d and
.1. Synthesis
ZM447439 for the differential selectivity profiles, we performed
docking simulations to model the possible binding modes using
Glide in Schrodinger Suite from Schrodinger LLC. Following ligand
preparation, the compounds were docked to the ATP binding
pocket of Aurora A and Aurora B as shown in Figure 3. The docking
simulation suggested that 7d and ZM447439 might have a similar
binding mode in Aurora A (Fig. 3A and B) (PDB ID: 3D15).1 Com-
pound 7d binds to the Aurora A ATP binding pocket with the bi-
aryl urea core forming several essential hydrogen bonds. The urea
carbonyl oxygen forms a hydrogen bonding interaction with
Lys175 and the two NH groups form a bidentate hydrogen bonding
interaction with Glu194. In addition to these hydrogen bonds, the
nitrogen of the piperidine moiety is within close proximity to
Gly229 for a coulombic interaction. The terminal benzyl group of
the urea functional group occupies back pocket, where it forms
hydrophobic interactions with Phe157, Leu182, Val187, Leu191,
Gly289, and Trp290.
All reagents were purchased from commercial sources and used
without further purification. Melting points were measured on an
RY-1 hot-stage microscope, and the thermometer was uncorrected.
1
13
3
H NMR and C NMR spectra were recorded in CDCl on a Bruker-
ACF 300/500 spectrometer; chemical shifts (d) are reported in parts
per million (ppm) relative to tetramethylsilane (TMS), used as an
internal standard. Mass spectra (MS) were obtained from Agilent
8
1
100 LC/MS Spectrometry Services. IR spectra were obtained using
on FT-IR Spectrometer (Perkin–Elmer). Elementary analyses were
performed on Elementar Vario EL III instrument. All compounds
were routinely checked by TLC with silica gel GF-254 glass plates
and viewed under UV light at 254 nm.
4
.1.1. 7-Fluoroquinazolin-4(3H)-one (2)
A solution of 2-amino-4-fluorobenzoic acid 1 (31.5 g, 0.20 mol)
and formamidine acetate (42.5 g, 0.36 mol) in 2-methoxyethanol
150 mL) was heated and refluxed for 10 h. The solvent was then
removed under reduced pressure and the residue washed with
The X-ray crystal structure of the complex of Aurora B with
ZM447439 was shown in Figure 3C (PDB ID: 2VRX).39 4-Aminoan-
iline-quinazoline moiety occupies the pocket made up of Leu99,
Val107, Ala120, Lys122, Gln145, Leu154, Leu170, Glu171, Phe172,
Ala173, Gly176, Glu177, Leu223, and Ala233 and a hydrogen bond-
ing interaction is formed between Ala173 and N1 of this moiety
(
ammonia water (0.01 M), and then dried to afford 2 (23.1 g, 69%)
4
0
as beige solid, mp: 237–239 °C (lit. mp: 234 °C).
4
.1.2. 7-(3-Hydroxypropoxy)quinazolin-4(3H)-one (3)
,3-Propanediol (55.6 g, 0.73 mol) was added dropwise at 0–
°C to a solution of NaH (29.2 g, 1.22 mol) in DMF (100 mL), the
(
underlined amino acid residues interact with the 4-aminoaniline
1
moiety). Benzoylamide forms a hydrogen bonding interaction with
Lys122, a coulombic interaction with Gln145, and hydrophobic
interaction with Leu124, Leu138, Ile142, Glu141, Met156,
Leu168, and Leu170. Methoxy of the quinazoline moiety forms a
coulombic interaction with Glu177. The mopholinopropoxy moiety
is exposed to the solvent and Ala173, Pro174, Arg175, and Gly176
are in proximity (4 Å) to this moiety. When compared to the crystal
structure of ZM447439 with Aurora B, 7d forms a slightly different
interaction with the binding site in the docking simulation
5
solution was stirred and heated at 55 °C for 1 h. Then to this solu-
tion 2 (20 g, 0.122 mol) was added and the mixture was heated at
10 °C for 24 h. After DMF was evaporated under reduced pressure,
1
ice water (300 mL) was added and the pH adjusted to 6 with acetic
acid. The precipitate obtained was filtered, washed with water and
1
dried to yield 3 (24.4 g, 91%) as beige solid, mp: 190–191 °C.
NMR (CDCl -d
CH –CH –O–), 3.61 (m, 2H, OH–CH
OH), 4.47 (t, J = 6.0 Hz, 2H, OH–CH
J = 2.5 Hz, 1H, Ar-H), 7.51 (dd, J = 9.0 Hz, J
.82 (br, 1H, –NH), 8.38 (d, J = 9.0 Hz, 1H, Ar-H), 8.45 (s, 1H, Ar-
H
3
6 2
, 300 MHz) d (ppm): 2.33–2.40 (m, 2H, OH–CH –
2
2
2 2
–CH –CH
2
–O–), 3.68 (br, 1H,
(Fig. 3D). Compound 7d binds to the Aurora B in the catalytic cleft
–
2
–CH
2
2
–CH –O–), 7.29 (d,
and forms a hydrogen bonding interaction between the quinazo-
line moiety N1 and the NH group of Ala173 in the hinge region.
The bi-aryl urea moiety extends into the pocket and the oxygen
of the urea moiety forms a hydrogen bond with Gln145. An addi-
tional hydrogen bonding interaction is found for the nitrogen of
piperidine with Arg175. Significantly, besides hydrophobic interac-
tion, 7d forms a pi–cation interaction between Lys122 and the aro-
matic moiety of the benzyl group.
1
2
= 2.5 Hz, 1H, Ar-H),
7
+
H); ESI-MS m/z: 221.1 [M+H] .
4
.1.3. 4-Chloro-7-(3-chloropropoxy)quinazoline (4)
Compound 3 (20 g, 0.091 mol) was added to thionyl chloride
150 mL) with magnetic stirring. DMF (15 mL) was then added
(
dropwise and the mixture was heated to reflux for 5 h. Excess thio-
nyl chloride was then removed under reduced pressure and the
residue was dissolved in dichloromethane (200 mL), washed with
a saturated solution of sodium carbonate (3 Â 100 mL) and water
3
. Conclusion
In our study, using ZM447439 as a lead compound, two series of
(2 Â 100 mL), and dried with Na
2 4
SO . Dichloromethane was then
removed under reduced pressure to give a crude product, which
2
0
novel 4-aminoquinazoline—urea derivatives have been

J. Cai et al. / Bioorg. Med. Chem. 22 (2014) 5813–5823
5819
was washed with ethyl ether and dried to give 4 (17.9 g, 77%) as
127.15, 128.26, 128.38, 130.22, 136.45, 139.40, 151.47, 153.65,
155.80, 157.19, 163.79; ESI-MS m/z: 513.2 [M+H] ; Anal. Calcd
for C29H N O (%): C, 67.95; H, 6.29; N, 16.39. Found: C, 67.69;
32 6 3
H, 6.32; N, 16.18.
1
+
light yellow powder, mp: 74–76 °C. H NMR (CDCl
(ppm): 2.31–2.39 (m, 2H, Cl–CH –CH –CH –O–), 3.79 (t,
J = 6.3 Hz, 2H, Cl–CH –CH –CH –O–), 4.34 (t, J = 5.8 Hz, 2H, Cl–
CH –CH –CH –O–), 7.35 (dd, J = 9.1 Hz, J = 2.4 Hz, 1H, Ar-H),
3
-d
6
, 300 MHz)
d
2
2
2
2
2
2
2
2
2
1
2
7
1
.45 (d, J = 2.4 Hz, 1H, Ar-H), 8.18 (d, J = 9.2 Hz, 1H, Ar-H), 8.97 (s,
H, Ar-H); ESI-MS m/z: 257.0 [M+H] .
4.1.8. 1-Benzyl-3-(4-((7-(3-(4-methylpiperazin-1-
yl)propoxy)quinazolin-4-yl)amino)phenyl)urea (7b)
Preparation of 7b is followed the procedure for 7a. Beige solid,
+
1
À1
4
.1.4. N -(7-(3-Chloropropoxy)quinazolin-4-yl)benzene-1,4-
yield: 64%, mp: 104–105 °C. IR (KBr, cm ): 3331, 2943, 2808,
diamine (5)
A mixture of compound 4 (18 g, 0.07 mol) and p-phenylenedi-
1681, 1620, 1587, 1558, 1513, 1457, 1419, 1336, 1313, 1224,
1
1133, 1003, 843; H NMR (DMSO-d
6
, 500 MHz) d (ppm): 1.90–
amine (7.65 g, 0.07 mol) in isopropanol (200 mL) was stirred at
reflux for 4 h. The mixture was cooled to room temperature, the
1.95 (m, 2H, N–CH
2.33–2.50 (m, 10H, N–CH
CH –O–), 4.31 (d, J = 5.5 Hz, 2H, Ar–CH
2
–CH
2
–CH
2
–O–), 2.16 (s, 3H, N–CH
3
),
–
2
–), 4.17 (t, J = 6.5 Hz, 2H, N–CH
2
–CH
2
precipitate obtained was filtered and dried to yield 5 (18.9 g,
2
2
–), 6.58 (t, J = 6.0 Hz, 1H,
1
8
3
2%) as orange-yellow solid, mp: 232–234 °C. H NMR (DMSO-d
6
,
–NH–), 7.12–7.41 (m, 9H, Ar-H), 7.64 (d, J = 8.5 Hz, 2H, Ar-H),
8.41 (d, J = 9.0 Hz, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 8.52 (s, 1H, –NH–
00 MHz) d (ppm): 2.25–2.29 (m, 2H, Cl–CH
–CH –CH –O–), 4.30 (t, J = 5.9 Hz, 2H, Cl–
–O–), 6.76 (d, J = 8.6 Hz, 2H, Ar-H), 7.31–7.46 (m,
2 2 2
–CH –CH –O–), 3.85
1
3
(
t, J = 6.4 Hz, 2H, Cl–CH
2
2
2
6
), 9.53 (s, 1H, –NH–); C NMR (DMSO-d ) d (ppm): 26.29, 43.71,
CH
2
–CH
2
–CH
2
46.63, 55.98, 57.23, 58.21, 69.10, 105.08, 110.19, 117.38, 118.71,
120.32, 123.18, 126.82, 127.02, 127.09, 128.25, 128.34, 130.26,
4
H, Ar-H), 8.69 (d, J = 9.2 Hz, 1H, Ar-H), 8.74 (s, 1H, Ar-H), 11.10
+
(
s, 1H, –NH–); ESI-MS m/z: 329.2 [M+H] .
136.44, 139.38, 151.50, 153.63, 155.82, 157.21, 163.80; ESI-MS
+
m/z: 526.3 [M+H] ; Anal. Calcd for C30
H
35
N
7
O
2
(%): C, 68.55; H,
4
.1.5. 1-Benzyl-3-(4-((7-(3-chloropropoxy)quinazolin-4-
6.71; N, 18.65. Found: C, 68.63; H, 6.49; N, 18.48.
yl)amino)phenyl)urea (6a)
Compound 5 (9 g, 0.0274 mol) was added to toluene (150 mL)
at room temperature. The solution of isocyanatomethyl-benzene
4.1.9. 1-Benzyl-3-(4-((7-(3-(piperidin-1-yl)propoxy)quinazolin-
4-yl)amino)phenyl)urea (7c)
(
3.6 g, 0.0274 mol) in toluene (40 mL) was the added dropwise
Preparation of 7c is followed the procedure for 7a. Beige solid,
yield: 73%, mp: 210–211 °C. IR (KBr, cm ): 3331, 2943, 2808,
À1
and the mixture was heated at reflux for 5 h. When cooled to room
temperature, the precipitate obtained was filtered and dried to
1682, 1620, 1558, 1511, 1458, 1419, 1335, 1227, 1132, 1047,
1
1
yield 6a (11 g, 87%) as bright yellow solid, mp: 246–248 °C.
NMR (DMSO-d
CH –CH –O–), 3.86 (t, J = 6.3 Hz, 2H, Cl–CH
.33 (m, 4H, Cl–CH –CH –CH –O– and Ar–CH
H
918, 841; H NMR (DMSO-d
6
, 300 MHz) d (ppm): 1.38–1.40 (m,
6
, 300 MHz) d (ppm): 2.26–2.30 (m, 2H, Cl–CH
–CH –CH –O–), 4.30–
–), 6.76 (t,
2
–
2H, piperidine–H), 1.49–1.51 (m, 4H, piperidine–H), 1.90–1.95
(m, 2H, N–CH –CH –CH –O–), 2.35–2.50 (m, 6H, N–CH –), 4.17
(t, J = 6.4 Hz, 2H, N–CH –CH –CH –O–), 4.31 (d, J = 5.9 Hz, 2H, Ar–
CH –), 6.56 (t, J = 6.0 Hz, 1H, –NH–), 7.12–7.42 (m, 9H, Ar-H),
2
2
2
2
2
2
2
2
2
4
2
2
2
2
2
2
2
J = 6.0 Hz, 1H, –NH–), 7.23–7.53 (m, 11H, Ar-H), 8.73 (d,
2
J = 9.3 Hz, 1H, Ar-H), 8.83 (s, 1H, Ar-H), 9.06 (s, 1H, –NH), 11.32
7.64 (d, J = 8.8 Hz, 2H, Ar-H), 8.41 (d, J = 9.3 Hz, 1H, Ar-H), 8.45 (s,
1H, Ar-H), 8.52 (s, 1H, –NH–), 9.53 (s, 1H, –NH–); C NMR
+
13
(
s, 1H, –NH–); ESI-MS m/z: 462.1 [M+H] .
(
6
DMSO-d ) d (ppm): 24.19, 25.53, 26.20, 43.73, 54.07, 54.15,
4
.1.6. 1-(4-((7-(3-Chloropropoxy)quinazolin-4-yl)amino)-
57.18, 69.12, 105.06, 110.42, 117.48, 118.76, 120.33, 123.29,
126.76, 126.93, 127.18, 128.25, 128.40, 130.28, 136.51, 139.42,
phenyl)-3-cyclohexylurea (6b)
Preparation of 6b is followed the procedure for 6a started from
151.46, 153.68, 155.81, 157.23, 163.83; ESI-MS m/z: 511.3
+
isocyanatocyclohexane. Bright yellow solid, yield: 81%, mp: 232–
[M+H] ; Anal. Calcd for C30
H
34
N
6
O
2
(%): C, 70.56; H, 6.71; N,
1
2
1
3
CH
(
34 °C. H NMR (DMSO-d
6
, 300 MHz) d (ppm): 1.23–1.82 (m,
–CH –CH –O–),
.47–3.48 (m, 1H, cyclohexane–H), 3.85 (t, J = 6.4 Hz, 2H, Cl–CH
–CH –O–), 4.32 (t, J = 5.8 Hz, 2H, Cl–CH –CH –CH –O–), 6.21
d, J = 7.8 Hz, 1H, –NH–), 7.15 (s, 1H, Ar-H), 7.26 (d, J = 9.3 Hz, 1H,
16.46. Found: C, 70.58; H, 6.67; N, 16.35.
0H, cyclohexane–H), 2.25–2.30 (m, 2H, Cl–CH
2
2
2
2
–
4.1.10. 1-Benzyl-3-(4-((7-(3-(2-methylpiperidin-1-yl)propoxy)-
quinazolin-4-yl)amino)phenyl)urea (7d)
2
2
2
2
2
Preparation of 7d is followed the procedure for 7a. Beige solid,
À1
Ar-H), 7.46–7.52 (m, 4H, Ar-H), 8.60 (s, 1H, Ar-H), 8.66 (d,
yield: 69%, mp: 218–219 °C. IR (KBr, cm ): 3329, 2938, 2807,
J = 9.3 Hz, 1H, Ar-H), 8.80 (s, 1H, –NH–), 11.14 (s, 1H, –NH–); ESI-
MS m/z: 454.3 [M+H] .
1685, 1621, 1559, 1456, 1423, 1336, 1228, 1130, 1047, 915, 836;
+
1
H NMR (DMSO-d
), 1.19–1.25 (m, 2H, piperidine–H), 1.43–1.58 (m,
4H, piperidine–H), 1.91–1.96 (m, 2H, N–CH –CH –CH –O–), 2.29–
2.51 (m, 5H, N–CH – and N–CH–), 4.19 (t, J = 6.5 Hz, 2H, N–CH
CH –CH –O–), 4.30 (d, J = 6.0 Hz, 2H, Ar–CH –), 6.55 (t, J = 6.0 Hz,
6
, 500 MHz) d (ppm): 1.01 (d, J = 6.1 Hz, 3H,
piperidine–CH
3
4
.1.7. 1-Benzyl-3-(4-((7-(3-morpholinopropoxy)quinazolin-4-
2
2
2
yl)amino)phenyl)urea (7a)
2
2
–
Compound 6a (2 g, 4.34 mmol) and KI (0.1 g, 0.6 mmol) were
added to morpholine (30 mL). The solution was stirred at 60 °C
for 6 h; then slowly poured into ice water, the precipitate was col-
lected by filtration. Flash chromatography was performed with
2
2
2
1H, –NH–), 7.11–7.43 (m, 9H, Ar-H), 7.65 (d, J = 8.7 Hz, 2H, Ar-H),
8.42 (d, J = 9.2 Hz, 1H, Ar-H), 8.46 (s, 1H, Ar-H), 8.51 (s, 1H, –NH–
1
3
6
), 9.55 (s, 1H, –NH–); C NMR (DMSO-d ) d (ppm): 17.87, 23.25,
CH
2
Cl
2
/CH
3
OH (40:1, v/v) to afford 7a (1.75 g, 79%) as deep yellow
25.69, 26.31, 34.36, 43.68, 50.92, 56.16, 58.15, 69.01, 105.23,
110.34, 117.65, 118.93, 120.26, 123.12, 126.78, 127.04, 127.10,
128.36, 128.43, 130.23, 136.47, 139.34, 151.49, 153.65, 155.78,
157.18, 163.82; ESI-MS m/z: 525.2 [M+H] ; Anal. Calcd for
31 36 6 2
C H N O (%): C, 70.97; H, 6.92; N, 16.02. Found: C, 70.61; H,
À1
solid, mp: 219–220 °C. IR (KBr, cm ): 3311, 2954, 1620, 1578,
1
1
(
CH
514, 1458, 1417, 1333, 1309, 1228, 1117, 862, 700; H NMR
DMSO-d , 300 MHz) d (ppm): 1.91–1.97 (m, 2H, N–CH –CH
–O–), 2.38–2.50 (m, 6H, N–CH –), 3.57–3.61 (m, 4H, O–CH –),
–CH –CH –O–), 4.31 (d, J = 5.8 Hz,
–), 6.57–6.59 (m, 1H, –NH–), 7.14–7.42 (m, 9H, Ar-H),
.63 (d, J = 8.9 Hz, 2H, Ar-H), 8.41 (d, J = 9.3 Hz, 1H, Ar-H), 8.45 (s,
+
6
2
2
–
2
2
2
4
2
7
1
.18 (t, J = 6.5 Hz, 2H, N–CH
H, Ar–CH
2
2
2
6.82; N, 16.08.
2
4.1.11. 1-Benzyl-3-(4-((7-(3-(4-methylpiperidin-1-yl)propoxy)-
quinazolin-4-yl)amino)phenyl)urea (7e)
1
3
H, Ar-H), 8.54 (s, 1H, –NH–), 9.55 (s, 1H, –NH–); C NMR
) d (ppm): 26.32, 43.73, 56.02, 58.15, 68.38, 69.05,
05.14, 110.25, 117.39, 118.86, 120.35, 123.21, 126.84, 126.98,
(
DMSO-d
6
Preparation of 7e is followed the procedure for 7a. Beige solid,
À1
1
yield: 78%, mp: 215–216 °C. IR (KBr, cm ): 3335, 2928, 2809,

5
820
J. Cai et al. / Bioorg. Med. Chem. 22 (2014) 5813–5823
1
688, 1625, 1561, 1457, 1420, 1334, 1226, 1128, 1045, 916, 832;
9.54 (s, 1H, –NH–); 13C NMR (DMSO-d
) d (ppm): 11.98, 27.06,
6
43.85, 47.66, 49.73, 57.28, 59.30, 69.28, 105.12, 110.35, 117.50,
118.73, 120.35, 123.29, 126.71, 126.95, 127.10, 128.26, 128.39,
1
H NMR (DMSO-d
piperidine–CH ), 1.13–1.22 (m, 2H, piperidine–H), 1.27–1.35 (m,
H, piperidine–H), 1.52–1.63 (m, 2H, piperidine–H), 1.91–1.95
m, 2H, N–CH –CH –CH –O–), 2.19–2.63 (m, 6H, N–CH –), 4.25
t, J = 6.4 Hz, 2H, N–CH –CH –CH –O–), 4.31 (d, J = 6.0 Hz, 2H, Ar–
–), 6.55 (t, J = 6.0 Hz, 1H, –NH–), 7.11–7.43 (m, 9H, Ar-H),
6
, 500 MHz) d (ppm): 0.90 (d, J = 6.3 Hz, 3H,
3
1
(
(
130.28, 136.57, 139.43, 151.55, 153.76, 155.81, 157.27, 163.92;
+
2
2
2
2
ESI-MS m/z: 515.3 [M+H] ; Anal. Calcd for C29
H
34
N
6
O
3
(%): C,
2
2
2
67.68; H, 6.66; N, 16.33. Found: C, 67.55; H, 6.78; N, 16.59.
CH
2
7
1
.65 (d, J = 8.8 Hz, 2H, Ar-H), 8.43 (d, J = 9.1 Hz, 1H, Ar-H), 8.45 (s,
4
.1.15. 1-Benzyl-3-(4-((7-(3-(4-(2-hydroxyethyl)piperazin-1-
H, Ar-H), 8.52 (s, 1H, –NH–), 9.56 (s, 1H, –NH–); 13C NMR
yl)propoxy)quinazolin-4-yl)amino)phenyl)urea (7i)
(
6
DMSO-d ) d (ppm): 18.18, 25.02, 30.33, 33.96, 43.76, 53.47,
Preparation of 7i is followed the procedure for 7a. Beige solid,
5
1
1
5
4.51, 56.12, 68.92, 105.32, 110.29, 117.58, 118.87, 120.19,
23.08, 126.65, 127.03, 127.21, 128.35, 128.49, 130.19, 136.44,
39.41, 151.52, 153.67, 155.74, 157.16, 163.86; ESI-MS m/z:
25.2 [M+H] ; Anal. Calcd for C31
À1
yield: 63%, mp: 95–96 °C. IR (KBr, cm ): 3329, 2945, 2807, 1683,
1
1
2
618, 1585, 1559, 1512, 1456, 1420, 1332, 1314, 1225, 1136,
1
001, 841; H NMR (DMSO-d
H, N–CH –CH –CH –O–), 2.33–2.56 (m, 12H, N–CH
OH), 4.18 (t, J = 6.4 Hz, 2H, N–CH
O–), 4.32 (d, J = 5.6 Hz, 2H, Ar–CH –), 4.40 (br, 1H, –OH), 6.57 (t,
6
, 500 MHz) d (ppm): 1.90–1.95 (m,
–), 3.52 (t,
–CH –CH
+
H
36
N
6
O
2
(%): C, 70.97; H, 6.92;
2
2
2
2
N, 16.02. Found: C, 70.86; H, 6.85; N, 15.91.
J = 6.4 Hz, 2H, –CH
2
2
2
2
–
2
4
.1.12. 1-Benzyl-3-(4-((7-(3-(pyrrolidin-1-yl)propoxy)-
J = 6.1 Hz, 1H, –NH–), 7.11–7.42 (m, 9H, Ar-H), 7.63 (d, J = 8.5 Hz,
2H, Ar-H), 8.42 (d, J = 9.1 Hz, 1H, Ar-H), 8.44 (s, 1H, Ar-H), 8.53
(s, 1H, –NH–), 9.52 (s, 1H, –NH–); C NMR (DMSO-d ) d (ppm):
6
26.24, 43.59, 43.75, 52.78, 58.49, 60.12, 70.03, 105.02, 110.21,
117.39, 118.74, 120.35, 123.19, 126.83, 127.05, 127.11, 128.26,
quinazolin-4-yl)amino)phenyl)urea (7f)
1
3
Preparation of 7f is followed the procedure for 7a. Gray solid,
À1
yield: 61%, mp: 209–210 °C. IR (KBr, cm ): 3377, 2956, 2818,
1
676, 1616, 1558, 1513, 1456, 1419, 1335, 1301, 1226, 1132,
1
8
45;
H
NMR (DMSO-d
6
,
500 MHz)
d
(ppm): 1.69 (quint,
–CH
–), 2.58 (t, J = 7.0 Hz, 2H, N–
–O–), 4.18 (t, J = 6.5 Hz, 2H, N–CH –CH –CH –O–),
.31 (d, J = 6.0 Hz, 2H, Ar–CH –), 6.57 (t, J = 6.0 Hz, 1H, –NH–),
.12–7.42 (m, 9H, Ar-H), 7.63 (d, J = 8.6 Hz, 2H, Ar-H), 8.41 (d,
128.35, 130.23, 136.47, 139.40, 151.52, 153.63, 155.81, 157.23,
+
J = 3.0 Hz, 4H, pyrrolidine–H), 1.96 (q, J = 6.5 Hz, 2H, N–CH
CH –O–), 2.47–2.51 (m, 4H, N–CH
CH –CH –CH
2
4
7
2
2
–
163.85; ESI-MS m/z: 556.3 [M+H] ; Anal. Calcd for C31
H
37
N
7
O
3
2
2
(%): C, 67.01; H, 6.71; N, 17.64. Found: C, 67.23; H, 6.59; N, 17.39.
2
2
2
2
2
2
4
.1.16. 1-Benzyl-3-(4-((7-(3-(4-(hydroxymethyl)piperidin-1-
yl)propoxy)quinazolin-4-yl)amino)phenyl)urea (7j)
J = 9.0 Hz, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 8.52 (s, 1H, –NH–), 9.52
Preparation of 7j is followed the procedure for 7a. Beige solid,
1
3
(
s, 1H, –NH–); C NMR (DMSO-d
6
) d (ppm): 19.02, 23.56, 27.10,
À1
yield: 70%, mp: 119–120 °C. IR (KBr, cm ): 3381, 2924, 2787,
4
1
1
3.83, 54.05, 54.16, 57.29, 69.18, 105.07, 110.43, 117.50, 118.73,
20.32, 123.26, 126.74, 126.91, 127.15, 128.26, 128.41, 130.26,
36.53, 139.44, 151.43, 153.69, 155.82, 157.25, 163.85; ESI-MS
1
1
1
1
CH
CH
679, 1618, 1579, 1554, 1514, 1458, 1416, 1336, 1309, 1230,
1
128, 1041, 829; H NMR (DMSO-d
.18 (m, 3H, piperidine–H), 1.62–1.64 (m, 2H, piperidine–H),
.84–1.98 (m, 4H, –CH –), 2.44 (t, J = 7.2 Hz, 2H, N–CH –CH
–O–), 2.86–2.89 (m, 2H, N–CH –), 3.24 (t, J = 5.7 Hz, 2H, –
–OH), 4.16 (t, J = 6.4 Hz, 2H, N–CH –CH –CH –O–), 4.31–4.37
(m, 3H, Ar–CH – and –OH), 6.55–6.59 (m, 1H, –NH–), 7.12–7.41
6
, 500 MHz) d (ppm): 1.10–
+
m/z: 497.3 [M+H] ; Anal. Calcd for C29
32
H N
6
O
2
(%): C, 70.14; H,
2
2
2
–
6
.49; N, 16.92. Found: C, 70.16; H, 6.35; N, 16.81.
2
2
2
2
2
2
4
4
.1.13. 1-Benzyl-3-(4-((7-(3-(diethylamino)propoxy)quinazolin-
-yl)amino)phenyl)urea (7g)
2
(m, 9H, Ar-H), 7.63 (d, J = 9.0 Hz, 2H, Ar-H), 8.41 (d, J = 9.3 Hz, 1H,
Preparation of 7g is followed the procedure for 7a. Beige solid,
yield: 72%, mp: 99–100 °C. IR (KBr, cm ): 3313, 2968, 1659,
Ar-H), 8.45 (s, 1H, Ar-H), 8.51 (s, 1H, –NH–), 9.52 (s, 1H, –NH–);
C NMR (DMSO-d ) d (ppm): 25.05, 26.18, 30.31, 33.92, 43.78,
6
À1
13
1
9
6
2
618, 1579, 1560, 1513, 1456, 1417, 1335, 1227, 1130, 1076,
53.42, 54.53, 56.15, 68.93, 105.34, 110.28, 117.57, 118.88, 120.16,
123.09, 126.68, 127.01, 127.22, 128.35, 128.50, 130.18, 136.39,
1
84; H NMR (DMSO-d
H, N–CH CH ), 1.87 (q, J = 6.3 Hz, 2H, N–CH
.44–2.57 (m, 6H, N–CH –), 4.18 (t, J = 6.3 Hz, 2H, N–CH
–O–), 4.31 (d, J = 5.9 Hz, 2H, Ar–CH –), 6.62–6.66 (m, 1H, –
NH–), 7.11–7.42 (m, 9H, Ar-H), 7.63 (d, J = 9.0 Hz, 2H, Ar-H), 8.41
6
, 300 MHz) d (ppm): 0.96 (t, J = 7.2 Hz,
–CH –CH –O–),
–CH
2
3
2
2
2
139.40, 151.53, 153.67, 155.75, 157.19, 163.83; ESI-MS m/z:
+
2
2
2
–
541.49 [M+H] ; Anal. Calcd for C31
H
36
6
N O
3
(%): C, 68.87; H, 6.71;
CH
2
2
N, 15.54. Found: C, 68.66; H, 6.83; N, 15.61.
(
d, J = 9.2 Hz, 1H, Ar-H), 8.44 (s, 1H, Ar-H), 8.58 (s, 1H, –NH–),
.53 (s, 1H, –NH–); C NMR (DMSO-d ) d (ppm): 11.78, 17.95,
6
7.12, 43.87, 46.38, 48.25, 57.25, 69.20, 105.06, 110.39, 117.47,
18.78, 120.33, 123.26, 126.75, 126.93, 127.12, 128.21, 128.43,
4
.1.17. 1-Cyclohexyl-3-(4-((7-(3-morpholinopropoxy)-
1
3
9
2
1
1
quinazolin-4-yl)amino)phenyl)urea (8a)
Preparation of 8a is followed the procedure for 7a. Beige solid,
À1
yield: 78%, mp: 276–277 °C. IR (KBr, cm ): 3338, 2929, 2854,
30.28, 136.55, 139.41, 151.49, 153.72, 155.83, 157.26, 163.89;
1
1
1
2
649, 1620, 1578, 1556, 1514, 1458, 1417, 1333, 1225, 1117,
+
ESI-MS m/z: 499.2 [M+H] ; Anal. Calcd for C29
6
H
34
N
O
6 2
(%): C,
1
041, 862; H NMR (DMSO-d
0H, cyclohexane–H), 1.93–1.96 (m, 2H, N–CH
.39–2.51 (m, 6H, N–CH –), 3.47 (t, J = 4.0 Hz, 1H, cyclohexane–
–), 4.18 (t, J = 6.5 Hz, 2H, N–CH
–O–), 6.01 (d, J = 8.0 Hz, 1H, –NH–), 7.13 (d, J = 2.5 Hz,
1H, Ar-H), 7.20 (dd, J = 9.0 Hz, J = 2.5 Hz, 1H, Ar-H), 7.36 (dd,
= 7.0 Hz, J = 2.0 Hz, 2H, Ar-H), 7.61 (dd, J = 7.0 Hz, J = 2.0 Hz,
2H, Ar-H), 8.25 (s, 1H, –NH–), 8.41 (d, J = 9.5 Hz, 1H, Ar-H), 8.44
6
, 500 MHz) d (ppm): 1.16–1.83 (m,
9.85; H, 6.87; N, 16.85. Found: C, 70.01; H, 6.52; N, 16.73.
2
–CH –CH –O–),
2
2
2
4
.1.14. 1-Benzyl-3-(4-((7-(3-(ethyl(2-hydroxyethyl)amino)-
H), 3.59 (t, J = 4.5 Hz, 4H, O–CH
CH –CH
2
2
–
propoxy)quinazolin-4-yl)amino)phenyl)urea (7h)
2
2
Preparation of 7h is followed the procedure for 7a. Beige solid,
1
2
À1
yield: 67%, oil. IR (KBr, cm ): 3311, 2963, 1655, 1617, 1573, 1561,
1
J
1
2
1
2
1
515, 1454, 1412, 1336, 1227, 1133, 1074, 985; H NMR (DMSO-d
6
,
1
3
3
00 MHz) d (ppm): 0.97 (t, J = 7.1 Hz, 3H, N–CH
–CH –CH –O–), 2.45–2.61 (m, 6H, N–CH
.46 (t, J = 6.5 Hz, 2H, –CH OH), 4.17 (t, J = 6.3 Hz, 2H, N–CH
CH –CH –O–), 4.32 (d, J = 6.0 Hz, 2H, Ar–CH –), 6.63–6.67 (m, 1H,
NH–), 7.12–7.44 (m, 9H, Ar-H), 7.63 (d, J = 8.9 Hz, 2H, Ar-H), 8.42
d, J = 9.1 Hz, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 8.59 (s, 1H, –NH–),
2 3
CH ), 1.89 (q,
6
(s, 1H, Ar-H), 9.51 (s, 1H, –NH–); C NMR (DMSO-d ) d (ppm):
22.36, 24.63, 26.32, 30.15, 51.36, 56.08, 58.19, 68.35, 69.13,
105.20, 110.27, 117.35, 118.56, 120.15, 123.06, 124.56, 124.63,
130.21, 136.50, 151.45, 153.65, 155.63, 157.23, 163.81; ESI-MS
m/z: 505.3 [M+H] ; Anal. Calcd for C28
J = 6.3 Hz, 2H, N–CH
2
2
2
2
–),
3
2
2
–
2
2
2
+
–
(
H
36
N
6
O
3
(%): C, 66.64; H,
7.19; N, 16.65. Found: C, 66.69; H, 6.98; N, 16.38.

J. Cai et al. / Bioorg. Med. Chem. 22 (2014) 5813–5823
5821
4
.1.18. 1-Cyclohexyl-3-(4-((7-(3-(4-methylpiperazin-1-
piperidine–CH
dine–H), 1.91–1.95 (m, 2H, N–CH
6H, N–CH –), 3.43–3.47 (m, 1H, cyclohexane–H), 4.20 (t,
J = 6.3 Hz, 2H, N–CH –CH –CH –O–), 6.03 (d, J = 7.8 Hz, 1H, –NH–),
3
), 1.12–1.75 (m, 15H, cyclohexane–H and piperi-
yl)propoxy)quinazolin-4-yl)amino)phenyl)urea (8b)
2
–CH –CH –O–), 2.21–2.60 (m,
2
2
Preparation of 8b is followed the procedure for 7a. Beige solid,
2
À1
yield: 60%, mp: 115–116 °C. IR (KBr, cm ): 3334, 2931, 2852,
2
2
2
2
1
808, 1672, 1623, 1579, 1552, 1514, 1419, 1417, 1335, 1296,
225, 1132, 839, 789, 679; H NMR (DMSO-d , 300 MHz) d
6
7.13 (d, J = 2.3 Hz, 1H, Ar-H), 7.20 (d, J = 9.0 Hz, 1H, Ar-H), 7.38
(d, J = 8.9 Hz, 2H, Ar-H), 7.61 (d, J = 8.9 Hz, 2H, Ar-H), 8.26 (s, 1H,
–NH–), 8.42 (d, J = 9.1 Hz, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 9.53 (s,
1H, –NH–); C NMR (DMSO-d ) d (ppm): 18.16, 22.35, 24.65,
6
26.37, 30.17, 30.35, 33.92, 51.37, 53.49, 54.52, 56.15, 68.99,
105.22, 110.26, 117.37, 118.55, 120.13, 123.16, 124.61, 124.65,
1
(
ppm): 1.18–1.83 (m, 10H, cyclohexane–H), 1.93–1.95 (m, 2H, N–
1
3
CH
CH
N–CH
2
–CH
–), 3.49–3.53 (m, 1H, cyclohexane–H), 4.17 (t, J = 6.2 Hz, 2H,
–CH –CH –O–), 6.01 (d, J = 7.7 Hz, 1H, –NH–), 7.12 (s, 1H,
Ar-H), 7.19 (d, J = 9.3 Hz, 1H, Ar-H), 7.37 (d, J = 8.9 Hz, 2H, Ar-H),
2
–CH
2
3
–O–), 2.15 (s, 3H, N–CH ), 2.33–2.51 (m, 10H, N–
2
2
2
2
130.19, 136.57, 151.46, 153.63, 155.69, 157.28, 163.85; ESI-MS
+
7
.62 (d, J = 8.8 Hz, 2H, Ar-H), 8.27 (s, 1H, –NH–), 8.41 (d,
m/z: 517.2 [M+H] ; Anal. Calcd for C30
40
H N
6
O
2
(%): C, 69.74; H,
1
3
J = 9.3 Hz, 1H, Ar-H), 8.44 (s, 1H, Ar-H), 9.52 (s, 1H, –NH–);
C
7.80; N, 16.27. Found: C, 69.55; H, 7.76; N, 16.23.
6
NMR (DMSO-d ) d (ppm): 22.35, 24.64, 26.33, 30.18, 46.67,
5
1
1
1.32, 56.01, 57.24, 58.23, 69.10, 105.22, 110.26, 117.38, 118.57,
20.12, 123.09, 124.55, 124.67, 130.19, 136.51, 151.43, 153.66,
55.63, 157.25, 163.83; ESI-MS m/z: 518.3 [M+H] ; Anal. Calcd
4.1.22. 1-Cyclohexyl-3-(4-((7-(3-(pyrrolidin-1-
yl)propoxy)quinazolin-4-yl)amino)phenyl)urea (8f)
+
Preparation of 8f is followed the procedure for 7a. Gray solid,
À1
for C29
H
39
N
7
O
2
(%): C, 67.28; H, 7.59; N, 18.94. Found: C, 67.33;
yield: 67%, mp: 131–132 °C. IR (KBr, cm ): 3315, 2931, 2854,
H, 7.43; N, 18.59.
2812, 1672, 1618, 1578, 1552, 1514, 1458, 1415, 1336, 1223,
1
1
132, 1041, 1018, 916, 841; H NMR (DMSO-d
6
, 500 MHz) d
4
.1.19. 1-Cyclohexyl-3-(4-((7-(3-(piperidin-1-
(ppm): 1.16–1.83 (m, 14H, cyclohexane–H and pyrrolidine–H),
1.93–1.96 (m, 2H, N–CH –CH –CH –O–), 2.47–2.51 (m, 4H, N–
CH –), 2.57 (t, J = 7.1 Hz, 2H, N–CH –CH –CH –O–), 3.45–3.49 (m,
1H, cyclohexane–H), 4.18 (t, J = 6.4 Hz, 2H, N-CH –CH –CH –O–),
6.01 (d, J = 7.8 Hz, 1H, –NH–), 7.12 (d, J = 2.6 Hz, 1H, Ar-H), 7.20
(dd, = 9.1 Hz, = 2.6 Hz, 1H, Ar-H), 7.37 (dd, = 6.9 Hz,
= 2.0 Hz, 2H, Ar-H), 7.61 (dd, J = 6.9 Hz, J = 2.0 Hz, 2H, Ar-H),
8.24 (s, 1H, –NH–), 8.41 (d, J = 9.3 Hz, 1H, Ar-H), 8.44 (s, 1H, Ar-
yl)propoxy)quinazolin-4-yl)amino)phenyl)urea (8c)
2
2
2
Preparation of 8c is followed the procedure for 7a. Beige solid,
2
2
2
2
À1
yield: 72%, mp: 172–173 °C. IR (KBr, cm ): 3319, 2931, 2854,
2
2
2
1
9
1
CH
1
6
676, 1620, 1554, 1513, 1456, 1417, 1335, 1223, 1132, 1039,
1
87, 841; H NMR (DMSO-d
6H, cyclohexane–H and piperidine–H), 1.90–1.94 (m, 2H, N–
–CH –CH –O–), 2.35–2.51 (m, 6H, N–CH –), 3.44–3.48 (m,
H, cyclohexane–H), 4.16 (t, J = 6.3 Hz, 2H, N–CH –CH –CH –O–),
.01 (d, J = 7.8 Hz, 1H, –NH–), 7.12 (d, J = 2.4 Hz, 1H, Ar-H), 7.19
6
, 300 MHz) d (ppm): 1.18–1.79 (m,
J
1
J
2
J
1
J
2
1
2
2
2
2
2
1
3
2
2
2
6
H), 9.51 (s, 1H, –NH–); C NMR (DMSO-d ) d (ppm): 19.05,
22.34, 23.58, 24.69, 27.03, 30.18, 51.35, 54.08, 54.11, 57.28,
69.18, 105.23, 110.29, 117.32, 118.56, 120.15, 123.18, 124.56,
(
d, J = 9.1 Hz, 1H, Ar-H), 7.37 (d, J = 8.9 Hz, 2H, Ar-H), 7.62 (d,
J = 8.9 Hz, 2H, Ar-H), 8.27 (s, 1H, –NH–), 8.41 (d, J = 9.2 Hz, 1H,
Ar-H), 8.44 (s, 1H, Ar-H), 9.53 (s, 1H, –NH–); 13C NMR (DMSO-d
d (ppm): 22.33, 24.20, 24.65, 25.57, 26.31, 30.19, 51.34, 54.08,
124.69, 130.14, 136.56, 151.42, 153.68, 155.72, 157.25, 163.91;
+
6
)
ESI-MS m/z: 487.2 [MÀH] ; Anal. Calcd for C28
36 6 2
H N O (%): C,
68.83; H, 7.43; N, 17.20. Found: C, 68.56; H, 7.32; N, 16.98.
5
1
1
C
7
4.16, 57.20, 69.16, 105.25, 110.23, 117.35, 118.59, 120.10,
23.13, 124.58, 124.65, 130.16, 136.54, 151.47, 153.67, 155.63,
4.1.23. 1-Cyclohexyl-3-(4-((7-(3-(diethylamino)propoxy)-
quinazolin-4-yl)amino)phenyl)urea (8g)
+
57.28, 163.89; ESI-MS m/z: 503.3 [M+H] ; Anal. Calcd for
29
H
38
N
6
O
2
(%): C, 69.29; H, 7.62; N, 16.72. Found: C, 69.53; H,
Preparation of 8g is followed the procedure for 7a. Beige solid,
À1
.68; N, 16.45.
yield: 68%, mp: 115–116 °C. IR (KBr, cm ): 3423, 3296, 2968,
2
931, 2852, 1618, 1578, 1514, 1456, 1417, 1335, 1225, 1130,
1
4
.1.20. 1-Cyclohexyl-3-(4-((7-(3-(2-methylpiperidin-1-
1042, 847; H NMR (DMSO-d
J = 7.1 Hz, 6H, N–CH CH ), 1.12–1.83 (m, 10H, cyclohexane–H),
1.85–1.89 (m, 2H, N–CH –CH –CH –O–), 2.43–2.62 (m, 6H, N–
CH –), 3.45–3.51 (m, 1H, cyclohexane–H), 4.17 (t, J = 6.3 Hz, 2H,
N–CH –CH –CH –O–), 6.47 (d, J = 7.0 Hz, 1H, –NH–), 7.11 (d,
J = 2.6 Hz, 1H, Ar-H), 7.18 (dd, J = 9.1 Hz, J = 2.6 Hz, 1H, Ar-H),
6
, 300 MHz) d (ppm): 0.95 (t,
yl)propoxy)quinazolin-4-yl)amino)phenyl)urea (8d)
2
3
Preparation of 8d is followed the procedure for 7a. Beige solid,
2
2
2
À1
yield: 66%, mp: 179–180 °C. IR (KBr, cm ): 3328, 2936, 2805,
2
1
688, 1619, 1560, 1458, 1425, 1339, 1229, 1131, 1049, 916, 833;
2
2
2
1
H NMR (DMSO-d
piperidine–CH ), 1.17–1.78 (m, 16H, cyclohexane–H and piperi-
dine–H), 1.91–1.95 (m, 2H, N–CH –CH –CH –O–), 2.30–2.52 (m,
H, N–CH – and N–CH–), 3.45–3.49 (m, 1H, cyclohexane–H), 4.17
t, J = 6.3 Hz, 2H, N–CH –CH –CH –O–), 6.03 (d, J = 7.8 Hz, 1H, –
NH–), 7.11 (d, J = 2.3 Hz, 1H, Ar-H), 7.20 (d, J = 9.0 Hz, 1H, Ar-H),
6
, 500 MHz) d (ppm): 1.02 (d, J = 6.0 Hz, 3H,
1
2
3
7.38 (d, J = 9.0 Hz, 2H, Ar-H), 7.61 (d, J = 9.0 Hz, 2H, Ar-H), 8.41
(d, J = 6.3 Hz, 1H, Ar-H), 8.44 (s, 1H, Ar-H), 8.76 (s, 1H, –NH–),
2
2
2
1
3
5
(
2
6
9.51 (s, 1H, –NH–); C NMR (DMSO-d ) d (ppm): 11.76, 17.98,
2
2
2
22.36, 24.63, 27.05, 30.19, 46.33, 48.23, 51.36, 57.22, 69.21,
105.25, 110.31, 117.31, 118.54, 120.16, 123.11, 124.53, 124.70,
7
1
.36 (d, J = 8.9 Hz, 2H, Ar-H), 7.61 (d, J = 8.9 Hz, 2H, Ar-H), 8.28 (s,
H, –NH–), 8.42 (d, J = 9.1 Hz, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 9.52
130.12, 136.52, 151.44, 153.65, 155.78, 157.23, 163.87; ESI-MS
+
m/z: 491.3 [M+H] ; Anal. Calcd for C28
H
38
N
6
O
2
(%): C, 68.54; H,
1
3
(
s, 1H, –NH–); C NMR (DMSO-d
6
) d (ppm): 17.53, 22.36, 23.23,
7.81; N, 17.13. Found: C, 68.31; H, 7.55; N, 16.93.
2
6
1
4.65, 25.71, 26.35, 30.16, 34.35, 50.93, 51.34, 56.18, 58.19,
9.15, 105.23, 110.27, 117.36, 118.59, 120.12, 123.15, 124.59,
4.1.24. 1-Cyclohexyl-3-(4-((7-(3-(ethyl(2-
hydroxyethyl)amino)propoxy)quinazolin-4-
yl)amino)phenyl)urea (8h)
24.63, 130.18, 136.57, 151.43, 153.65, 155.63, 157.29, 163.82;
+
ESI-MS m/z: 517.2 [M+H] ; Anal. Calcd for C30
H
40
6
N O
2
(%): C,
6
9.74; H, 7.80; N, 16.27. Found: C, 69.65; H, 7.72; N, 16.05.
Preparation of 7r is followed the procedure for 7a. Beige solid,
yield: 63%, mp: 102–103 °C. IR (KBr, cm ): 3315, 2961, 1656,
À1
4
.1.21. 1-Cyclohexyl-3-(4-((7-(3-(4-methylpiperidin-1-
1618, 1574, 1567, 1518, 1452, 1413, 1337, 1226, 1135, 1079,
1
yl)propoxy)quinazolin-4-yl)amino)phenyl)urea (8e)
983; H NMR (DMSO-d
6
, 300 MHz) d (ppm): 0.96 (t, J = 7.1 Hz,
), 1.11–1.82 (m, 10H, cyclohexane–H), 1.87–1.91
–CH –CH –O–), 2.45–2.63 (m, 6H, N–CH –), 3.45–
3.52 (m, 3H, cyclohexane–H and –CH OH), 4.18 (t, J = 6.3 Hz, 2H,
N–CH –CH –CH –O–), 6.46 (d, J = 7.0 Hz, 1H, –NH–), 7.12 (d,
Preparation of 7o is followed the procedure for 7a. Beige solid,
2 3
3H, N–CH CH
À1
yield: 75%, mp: 174–175 °C. IR (KBr, cm ): 3336, 2927, 2810,
(m, 2H, N–CH
2
2
2
2
1
689, 1627, 1563, 1458, 1422, 1335, 1227, 1126, 1043, 917, 836;
2
1
H NMR (DMSO-d
6
, 500 MHz) d (ppm): 0.92 (d, J = 6.3 Hz, 3H,
2
2
2

5
822
J. Cai et al. / Bioorg. Med. Chem. 22 (2014) 5813–5823
J = 2.6 Hz, 1H, Ar-H), 7.19 (dd, J
.39 (d, J = 9.0 Hz, 2H, Ar-H), 7.61 (d, J = 9.0 Hz, 2H, Ar-H), 8.42
d, J = 6.3 Hz, 1H, Ar-H), 8.43 (s, 1H, Ar-H), 8.78 (s, 1H, –NH–),
1
= 9.0 Hz, J
2
= 2.6 Hz, 1H, Ar-H),
was then added, and the cells were incubated at 37 °C with 5% CO
for 72 h later. Subsequently, cell growth medium was removed,
2
7
(
and WST-8 was added to each well for another incubation of
1.5 h at 37 °C. Absorbance was finally measured with a plate reader
at 650 nm. The results were expressed as the percentage of absor-
bance of treated wells versus that of the vehicle control. IC50, the
drug concentration causing 50% growth inhibition, was calculated
via sigmoid curve fitting using GraphPad Prism 5.0.
1
3
9
2
1
1
6
.52 (s, 1H, –NH–); C NMR (DMSO-d ) d (ppm): 11.97, 22.33,
4.65, 27.01, 30.13, 47.65, 49.78, 51.33, 57.22, 59.31, 69.29,
05.23, 110.32, 117.31, 118.55, 120.14, 123.15, 124.54, 124.73,
30.19, 136.50, 151.43, 153.66, 155.79, 157.28, 163.90; ESI-MS
+
m/z: 507.3 [M+H] ; Anal. Calcd for C28
H
38
N
O
6 3
(%): C, 66.38; H,
7
.56; N, 16.59. Found: C, 66.52; H, 7.68; N, 16.53.
4
.2.2. In vitro kinase assays
4
1
.1.25. 1-Cyclohexyl-3-(4-((7-(3-(4-(2-hydroxyethyl)piperazin-
-yl)propoxy)quinazolin-4-yl)amino)phenyl)urea (8i)
The kinase activities of Aurora A and B were measured using
HTRF-KinEASE-STK discovery kit (Cisbio, Bedford, MA, USA).
Briefly, Aurora A (65 nM) or Aurora B (316 nM) (Millipore, Billerica,
Preparation of 8i is followed the procedure for 7a. Beige solid,
À1
yield: 68%, mp: 100–101 °C. IR (KBr, cm ): 3332, 2947, 2803,
MA, USA) was incubated with 1
lM STK substrate 2-biotin (Cisbio)
1
1
1
683, 1615, 1585, 1554, 1517, 1458, 1421, 1336, 1314, 1227,
135, 1001, 849; 1H NMR (DMSO-d
, 500 MHz) d (ppm): 1.18–
.83 (m, 10H, cyclohexane–H), 1.92–1.95 (m, 2H, N–CH –CH
–O–), 2.32–2.57 (m, 12H, N–CH –), 3.50–3.55 (m, 3H, cyclohex-
ane–H and –CH OH), 4.17 (t, J = 6.3 Hz, 2H, N–CH –CH –CH –O–),
.39 (br, 1H, –OH), 6.01 (d, J = 7.6 Hz, 1H, –NH–), 7.13 (s, 1H, Ar-
H), 7.20 (d, J = 9.2 Hz, 1H, Ar-H), 7.38 (d, J = 9.0 Hz, 2H, Ar-H),
and 15 M ATP (Sigma–Aldrich) in the presence of tested com-
pounds for 1 h at room temperature. The reaction was terminated
l
6
2
2
–
by adding 200 mM EDTA. Phosphorylated substrates were incu-
3
+
CH
2
2
bated with STK antibody labeled with Eu -cryptate and streptavi-
3
+
2
2
2
2
din-XL665. The Eu -cryptate-conjugated antibody was excited at
340 nm, and cryptate and fluorescence resonance energy transfer
emissions were detected at 615 and 665 nm, respectively. GRAPH-
PAD PRISM was used to generate IC50 values.
4
7
.63 (d, J = 8.9 Hz, 2H, Ar-H), 8.27 (s, 1H, –NH–), 8.42 (d,
1
3
J = 9.2 Hz, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 9.53 (s, 1H, –NH–);
NMR (DMSO-d ) d (ppm): 22.38, 24.69, 26.85, 30.16, 43.78,
6
5
1
1
C
In vitro kinase inhibitory ability of EGFR was determined using
the HTScan EGFR Kinase Assay Kit (Cell Signaling Technology), fol-
lowing the manufacturer’s instructions.
1.36, 52.75, 58.53, 60.08, 69.93, 105.25, 110.28, 117.36, 118.59,
20.15, 123.10, 124.58, 124.68, 130.23, 136.55, 151.46, 153.68,
+
55.65, 157.28, 163.89; ESI-MS m/z: 548.3 [M+H] ; Anal. Calcd
4.2.3. Procedure of docking simulation
1
8
for C30
H, 7.51; N, 17.59.
H
41
N
7
O
3
(%): C, 65.79; H, 7.55; N, 17.90. Found: C, 65.83;
Crystal structures of Aurora kinase A (PDB ID: 3D15) and Aur-
3
9
42
ora kinase B (PDB ID: 2VRX) were imported to Maestro and pre-
pared for docking simulations using Protein Preparation Wizard in
4
3
4
1
.1.26. 1-Cyclohexyl-3-(4-((7-(3-(4-(hydroxymethyl)piperidin-
-yl)propoxy)quinazolin-4-yl)amino)phenyl)urea (8j)
Schrodinger Suite. In the protein preparation step, water mole-
cules were removed, bond orders assigned, hydrogen atoms added,
H-bonds assigned, and the structure relaxed with the OPLS2005
Preparation of 7t is followed the procedure for 8j. Beige solid,
À1
44
yield: 65%, mp: 120–121 °C. IR (KBr, cm ): 3311, 2927, 2852,
force field. The receptor grid was generated in Glide: grid center
1
1
1
CH
2
676, 1620, 1579, 1555, 1514, 1458, 1417, 1335, 1225, 1130,
was defined by selecting the ligand of the structure in the work-
space, the limit of the ligand length was 620 Å, and the dimension
of the grid box was 10 Â 10 Â 10. Ligands in the SDF format were
1
039, 839; H NMR (DMSO-d
5H, cyclohexane–H and piperidine–H), 1.91–1.96 (m, 2H, N–
–CH –CH –O–), 2.49–2.56 (m, 6H, N–CH –), 3.29 (t, J = 5.8 Hz,
H, –CH –OH), 3.44–3.50 (m, 1H, cyclohexane–H), 4.17 (t,
J = 6.4 Hz, 2H, N–CH –CH –CH –O–), 4.35–4.37 (m, 1H, –OH),
.02 (d, J = 7.8 Hz, 1H, –NH–), 7.12 (d, J = 2.6 Hz, 1H, Ar-H), 7.19
dd, = 9.1 Hz, = 2.6 Hz, 1H, Ar-H), 7.36 (dd, = 7.0 Hz,
= 2.0 Hz, 2H, Ar-H), 7.61 (dd, J = 7.0 Hz, J = 2.0 Hz, 2H, Ar-H),
.26 (s, 1H, –NH–), 8.41 (d, J = 9.3 Hz, 1H, Ar-H), 8.44 (s, 1H, Ar-
6
, 500 MHz) d (ppm): 1.11–1.89 (m,
4
5
2
2
2
2
imported to Maestro and prepared using LigPrep: possible ion-
ized states of the compounds were generated at target pH 7 ± 2.
Docking simulations of the compounds prepared with LigPrep
were performed in Glide.
2
2
2
2
6
(
J
1
J
2
J
1
Acknowledgments
J
2
1
2
8
13
This work is supported by The Fundamental Research Funds for
the Central Universities (2242014R30019), Technology Supporting
Program of Jiangsu province (BE2012657), and National Basin
Research Program of China (No. 2011CB933503). We thank the
Minnesota Supercomputing Institute for their support for our
molecular modeling study.
H), 9.51 (s, 1H, –NH–); C NMR (DMSO-d
6
) d (ppm): 22.33,
2
5
1
1
4.71, 25.06, 26.89, 30.15, 30.35, 33.98, 51.38, 53.44, 54.52,
6.15, 68.95, 105.26, 110.23, 117.31, 118.52, 120.11, 123.16,
24.61, 124.65, 130.29, 136.58, 151.47, 153.68, 155.63, 157.29,
63.91; ESI-MS m/z: 533.37 [M+H] ; Anal. Calcd for C30
+
H
40
N
6
O
3
(
%): C, 67.64; H, 7.57; N, 15.78. Found: C, 67.66; H, 7.69; N, 15.55.
References and notes
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.2. Biological evaluation
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