Optimization of ligand and lipophilic efficiency to identify an in vivo active furano-pyrimidine aurora kinase inhibitor

Article abstract of DOI:10.1021/jm4006059

Ligand efficiency (LE) and lipophilic efficiency (LipE) are two important indicators of "drug-likeness", which are dependent on the molecule's activity and physicochemical properties. We recently reported a furano-pyrimidine Aurora kinase inhibitor 4 (LE = 0.25; LipE = 1.75), with potent activity in vitro; however, 4 was inactive in vivo. On the basis of insights obtained from the X-ray co-crystal structure of the lead 4, various solubilizing functional groups were introduced to optimize both the activity and physicochemical properties. Emphasis was placed on identifying potential leads with improved activity as well as better LE and LipE by exercising tight control over the molecular weight and lipophilicity of the molecules. Rational optimization has led to the identification of Aurora kinase inhibitor 27 (IBPR001; LE = 0.26; LipE = 4.78), with improved in vitro potency and physicochemical properties, resulting in an in vivo active (HCT-116 colon cancer xenograft mouse model) anticancer agent.

Full text of DOI:10.1021/jm4006059

Article
pubs.acs.org/jmc
Optimization of Ligand and Lipophilic Efficiency To Identify an in
Vivo Active Furano-Pyrimidine Aurora Kinase Inhibitor
Hui-Yi Shiao,†,∥ Mohane Selvaraj Coumar,‡,∥ Chun-Wei Chang,†,∥ Yi-Yu Ke,† Ya-Hui Chi,§
Chang-Ying Chu,† Hsu-Yi Sun,† Chun-Hwa Chen,† Wen-Hsing Lin,† Ka-Shu Fung,† Po-Chu Kuo,†
Chin-Ting Huang,† Kai-Yen Chang,† Cheng-Tai Lu,† John T. A. Hsu,† Chiung-Tong Chen,†
Weir-Torn Jiaang,† Yu-Sheng Chao,† and Hsing-Pang Hsieh*,†
†Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli
County 350, Taiwan, ROC
‡Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605014, India
§Institute of Cellular and System Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 350,
Taiwan, ROC
S
* Supporting Information
ABSTRACT: Ligand efficiency (LE) and lipophilic efficiency (LipE) are two important indicators of “drug-likeness”, which are
dependent on the molecule’s activity and physicochemical properties. We recently reported a furano-pyrimidine Aurora kinase
inhibitor 4 (LE = 0.25; LipE = 1.75), with potent activity in vitro; however, 4 was inactive in vivo. On the basis of insights
obtained from the X-ray co-crystal structure of the lead 4, various solubilizing functional groups were introduced to optimize
both the activity and physicochemical properties. Emphasis was placed on identifying potential leads with improved activity as
well as better LE and LipE by exercising tight control over the molecular weight and lipophilicity of the molecules. Rational
optimization has led to the identification of Aurora kinase inhibitor 27 (IBPR001; LE = 0.26; LipE = 4.78), with improved in
vitro potency and physicochemical properties, resulting in an in vivo active (HCT-116 colon cancer xenograft mouse model)
anticancer agent.
therapy” for the treatment of cancer.1 In particular, the 2001
approval of imatinib, which inhibits the activity of Bcr-Abl
INTRODUCTION
■
Cancer is caused by abnormalities in the genetic material of the
transformed cells. Cancer-promoting oncogenes are activated
and tumor suppressor genes are often inactivated in cancer
kinase, for the treatment of chronic myelogenous leukemia
(CML), has opened a new field of “kinase targeted therapy”.2
Currently a total of 17 small molecule kinase inhibitors are
cells, leading to abnormal protein expression which result in an
approved by U.S. Food and Drug Administration (FDA) for the
aberrant cell cycle. Traditional approaches to cancer chemo-
therapy using cytotoxic drugs such as antimitotic agents are
treatment of various cancers.3
A subclass of protein kinases is known as Aurora kinases,
hindered by a narrow therapeutic window, severe toxicity, and
which phosphorylate serine/threonine amino acid residues and
development of drug resistance. These limitations have fueled
interest in developing “targeted therapies” to minimize toxicity
to healthy tissues. Targeted therapy constitutes the use of
agents specific for the deregulated proteins of cancer cells, with
are involved in the regulation of various stages of mitosis.4,5
Because the mitotic phase of the cell cycle is a key step in
ensuring the genetic integrity of daughter cells, any aberration
in the regulation of the mitotic phase could lead to genetic
an aim to avoid unwanted toxicity to normal tissues.
instability and ultimately cancer. Therefore, Aurora kinases are
Protein kinases, the enzymes that catalyze phosphorylation of
an important regulator in maintaining normal cell cycle process.
serine, threonine, or tyrosine residues of predetermined target
Three Aurora kinase isoforms, A, B, and C, have a conserved
proteins, are deregulated in a number of diseases, including
cancer. Because protein phosphorylation is an important step in
ATP binding pocket but different amino acid sequences at the
the cellular signal transduction pathway regulating cell
differentiation, growth, and migration, targeting abnormal
protein kinase activity has become an attractive “targeted
Received: November 3, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Aurora kinase inhibitors. Compound 17 was developed by Vertex Pharmaceuticals and has undergone testing in phase II clinical trials.
Compound 2 was developed by Sunesis Pharmaceuticals and has undergone testing in phase I clinical trials. Compounds 3,12 4,13 and 2714 were
developed in our laboratory.
a
Scheme 1
a
(a) R-NH2, ethanol, 8 h, reflux, 86−50%; (b) method A, R-NCO, CH2Cl2, rt, 8 h, 86−73%; (c) method B, CDI, R-NH2, CH2Cl2, rt, 8 h, 78−70%.
N-terminal domain.6 Aurora A is expressed from the late S
phase, peaking at G2/M phase and declining at the G1 phase
and is involved in centrosome maturation and separation,
bipolar spindle assembly, and mitotic entry. Aurora B, known as
the chromosomal passenger protein, is essential for accurate
chromosome segregation and cytokinesis, and Aurora C
complements the function of Aurora B. It is expressed in the
testes.
Aurora kinases are known to be overexpressed in solid
tumors and also may be inappropriately activated in certain cell
types.4 Inhibition of Aurora activity by inhibitors such as 17 and
28 (Figure 1) is known to slow cell growth and division and
encouraged us to initiate a kinase-based drug discovery
program,9−11 which has led to the identification of novel
furano-pyrimidine Aurora kinase inhibitor 3 from our in-house
compound library.12 Further modification of hit 3 through
construction of a small molecule library and structure-based
drug design concepts has led to the development of potent
Aurora kinase inhibitor lead 4.13 Importantly, lead 4 exhibited
antiproliferative activity when tested against the HCT-116
colon cancer cell line and showed pan Aurora kinase inhibition
(Aurora A, IC50 = 43 nM; Aurora B, IC50 = 97 nM). However,
4 did not demonstrate significant in vivo antitumor activity up
to 100 mg/kg, perhaps due to poor drug physicochemical
properties. Hence we started a lead optimization program to
modify lead 4, with an aim to identify potent compounds with
an improved in vitro activity profile as well as good in vivo
efficacy in a HCT-116 tumor xenograft mouse model.
Structure-based lead optimization of 4 by introduction of
various amino functionality, along with tight control over drug
properties such as molecular weight and lipophilicity, resulted
in the identification of 27 (IBPR001)14 as a potent Aurora
kinase inhibitor with improved physicochemical properties
(Figure 1). Compound 27 showed superior tumor growth
inhibition in an in vivo HCT-116 xenograft mouse model
compared to the known Aurora kinase inhibitor 17 and has
potential for further development as an anticancer agent. The
results leading to the identification of 27 are reported here.
CHEMISTRY
■
Synthesis of compounds 15−24 bearing modifications on the
urea side chain were carried out starting from the previously
reported chloropyrimidine 512 (Scheme 1). Substitution
reactions of 5 with appropriate amines/anilines (6a−e) in
ethanol gave the desired intermediate anilines 7a−e, which
were converted to the corresponding urea compounds 15−24,
either by reacting with appropriate isocyanates (method A) or
by first reacting with CDI and then treating with desired
substituted anilines (method B).
Compound 25 was synthesized from the chloro compound
9a12 by reacting first with 4-(2-amino-ethyl)-phenylamine in
ethanol, followed by reaction with phenyl isocyanate.
Compounds 25a,b were prepared from 9b,c in a manner
similar to 25. The chloro compounds 9b,c were synthesized
from substituted 2-bromo-1-phenyl-ethanones 8b,c in a manner
similar to 9a (Scheme 2).
B
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 2
a
(a) (i) Malononitrile, DMF, Et2NH, rt, 16 h, (ii) HCO2H, Ac2O, 0 °C → reflux, 8 h, (iii) POCl3, neat, 70 °C, 3 h, 40−55% for three steps; (b) (i)
4-(2-amino-ethyl)-phenylamine, Et3N, EtOH, reflux, 5 h, (ii) PhNCO, CH2Cl2, rt, 8 h, 88−79% for two steps.
a
Scheme 3
a
(a) BBr3, CH2Cl2, 0 °C, 96−89%; (b) method C, R2N-(CH2)n-CH2Cl, K2CO3, KI, acetone/DMF, reflux 6 h, 52−29%; (c) method D, R2N-
(CH2)n-CH2OMs, K2CO3, DMF, reflux overnight, 41−27%; (d) method E, Br-(CH2)n-Cl, K2CO3, KI, acetone/DMF, reflux 6 h, then R2-NH, DMF,
reflux, 8 h, 40−35%.
a
Scheme 4
a
(a) (i) Malononitrile, DMF, Et2NH, rt, 16 h, 71%, (ii) HCO2H, Ac2O, 0 °C → reflux, 8 h, (iii) POCl3, neat, 70 °C, 3 h, 37% for two steps; (b) (i)
4-(2-amino-ethyl)-phenylamine, Et3N, EtOH, reflux, 5 h, (ii) PhNCO, CH2Cl2, rt, 8 h, 92% for two steps.
Figure 2. X-ray co-crystal structure of Aurora kinase A in complex with inhibitor 4 (PDB ID: 3M11). Hydrogen bonding interactions between the
protein and the ligand are shown as yellow lines. Interacting amino acid residues shown in stick form. The 6-position phenyl ring (attached to the
furano-pyrimidine core) projecting toward the solvent exposed section of the Aurora A protein could be an appropriate place for further chemical
modification. Figure was created using UCSF Chimera package.15
Demethylation of 25a,b with BBr3 at low temperature gave
the corresponding phenolic compounds 10a,b. Alkylation of
10a,b using three different methods C−E gave the desired
compounds 27−37. Method C: Alkylation was carried out
C
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
using chloro compound R2N-(CH2)n-CH2Cl under basic
condition in acetone/DMF mixture. Method D: Alkylation
was carried out using mesityl ester R2N-(CH2)n-CH2OMs
under basic condition in DMF. Method E: Alkylation was
carried out using chlorobromo compound Br-(CH2)n-Cl under
basic condition in acetone/DMF mixture, followed by reaction
with the amine R2-NH in DMF (Scheme 3).
compared to lead 4 (Table 2). Moreover, removal of the phenyl
ring and replacement with a hydrogen atom (20), methyl group
Table 2. SAR Investigation of the Urea Functional Group of
Lead 4
For compound 26, 2-hydroxy-1-phenyl-ethanone (11) was
converted to 12 in a manner similar to that reported earlier,12
which was later reacted with 4-(2-amino-ethyl)-phenylamine
and phenyl isocyanate, in a manner similar to 25 (Scheme 4).
RESULTS AND DISCUSSION
Aurora kinase A
HCT-116 EC50
LigandFit
b
■
a
a
compd
413
R
IC50 (nM)
(nM)
score
Before we set out to optimize the lead 4, we solved the X-ray
co-crystal structure of 4−Aurora A complex structure (PDB ID:
3M11) to determine the important structural features essential
for activity and to identify possible modification sites.13 The X-
ray structure revealed that 4 binds to the Aurora A ATP
binding pocket with the furano-pyrimidine core forming two
essential hydrogen bonds with the hinge region Ala213 (Figure
2). In addition to these hydrogen bonds, the urea carbonyl
group forms a hydrogen bond interaction with Lys162 of
Aurora A. Disruption of this hydrogen bond interaction, by
replacement of the urea function of 4 with either an amide
(13)13 or sulphonamide (14)13 linkage, led to loss of potency
at enzyme as well as cellular level (Table 1). Moreover, moving
Ph
43
160
929
203
141
367
123
400
822
155.858
148.505
127.233
135.981
147.692
140.318
153.602
19
CH2Ph
H
20
>10000
1786
756
21
CH3
22
cC6H11
p-OMe-Ph
p-F-Ph
2317
2417
2201
770
a
Values are expressed as the mean of at least two independent
b
determinations and are within 15%. LigandFit score calculated by
docking of compounds to Aurora kinase A X-ray crystal structure
(PDB ID: 3M11) using DS/LigandFit program (Discovery studio
2.1).
Table 1. SAR Investigation of the Side Chain of Lead 4
(21), or cyclohexyl group (22) all led to decreased activity, with
20 being completely inactive in the cellular assay. These results
indicate the importance of aromatic hydrophobic interactions
in the back pocket for cellular inhibition.
Introduction of either an electron donating (23)17 or
withdrawing group (24)17 in the terminal phenyl ring was
found to be detrimental to activity, suggesting a strict steric
requirement to exist in the back pocket region for Aurora
kinase inhibition. To better understand the SAR trend for
Aurora kinase inhibition of compounds 19−24, they were
docked to the Aurora kinase A X-ray crystal structure (PDB ID:
3M11) using the DS/LigandFit program (Discovery studio
2.1). The LigandFit scores are shown in Table 2 and further
emphasize the importance of aromatic hydrophobic interaction
(4 vs 20−22) and limited steric bulk accommodation (4 vs 23)
in the back pocket region of the enzyme.
Having found that the modifications in the urea side chain of
4 could not improve the cellular activity, we turned our
attention toward the two phenyl groups appended to the furan
ring of the furano-pyrimidine core. The X-ray co-crystal study
showed both to form hydrophobic interactions with the
surrounding amino acid residues. More importantly, the 6-
position phenyl group in the furano-pyrimdine ring is directed
toward the solvent exposed part of the enzyme (Figure 2). On
the basis of this information, we envisioned introduction of
solubilizing functional groups bearing amino functionality into
this phenyl ring. It has been shown previously that the presence
of basic amino functionality can improve aqueous solubility,
decrease lipophilic character, and enhance activity by improving
interactions between the ligand and the enzyme.18−20
Aurora kinase A
HCT-116 EC50
a
a
compd
n
R
IC50 (nM)
(nM)
413
1313
1413
15
2
2
2
2
0
1
3
p-NH(CO)NHPh
p-NH(CO)Ph
43
400
304
>10000
>10000
>10000
2747
p-NHSO2Ph
5831
b
m-NH(CO)NHPh
p-NH(CO)NHPh
p-NH(CO)NHPh
p-NH(CO)NHPh
>10000
16
201
852
17
>10000
>10000
b
18
>10000
a
Values are expressed as the mean of at least two independent
b
determinations and are within 15%. Inhibition <50% at 10 μM.
the urea functional group from the para-position (4) to the
meta- (15), closer from the furano-pyrimidine core (16, 17), or
further away (18) through chain length reduction or
elongation, all led to loss of activity. The decreased activity of
13−18 emphasizes the importance of the location of the urea
group for potent Aurora kinase activity. Only when positioned
as in 4 can the key hydrogen-bond interaction with Lys162
observed in the X-ray co-crystal study of 4 be made.
The X-ray co-crystal study of 4 showed that the terminal
phenyl ring of the urea functional group enters the region
commonly referred to as the “back pocket” in kinase literature,
where it forms hydrophobic interactions with the surrounding
amino acid residues.16 To establish the importance of this
hydrophobic interaction in the back pocket, the terminal phenyl
group was moved away from the urea function by introduction
of a carbon linker between the urea and the Ph group to give
19, which was less active in both enzyme and cell-based assays
Before attempting the introduction of solubilizing function-
ality, we were interested to know the importance of phenyl
rings (attached to the furano-pyrimidine ring) for maintaining
the activity. In particular, we were concerned about the increase
in molecular weight that would result by the introduction of
solubilizing functionality to the lead 4 (Ml. Wt, 526). With the
D
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 3. Effect of Solubilizing Group Introduction on Physicochemical Properties, Enzyme Inhibition, and Cellular
Antiproliferative Activity
a
b
Values are expressed as the mean of at least two independent determinations and are within 15%. Values are expressed as the mean of at least
three independent determinations and are within 15%. NHAC: non-hydrogen atom count (calculated from ChemDraw Ultra 7.0). −RT ln IC50
=
0.592 ln(IC50 in mol). LE calculation for 4: NHAC = 40. −RT ln IC50 = 0.592 × ln(4.30 × 10−8) = 10.0415. LE = −RT ln IC50/NHAC = 10.0415/
40 = 0.25.
E
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
aim of decreasing the molecular weight, we investigated the role
played by the two phenyl rings attached to the furano-
pyrimidine core of 4 in maintaining activity. Accordingly,
compound 25 (without the 5-position phenyl ring) and
compound 26 (without the 6-position phenyl ring) were
synthesized through a simplification strategy (Table 3).
Surprisingly, 25 showed a 2-fold improvement in antiprolifer-
ative activity, though a 3-fold loss of Aurora A inhibition, and
26 showed 8-fold loss of Aurora A inhibition, suggesting that a
phenyl ring at the 6- but not 5- position of the furano-
pyrimidine core is essential for interaction with the enzyme.
Moreover, 25 had a similar level of Aurora kinase B enzyme
inhibition as that of the lead 4. The improved cellular activity is
probably due to improved cellular permeability, resulting from
decreased Log D and molecular weight for 25, compared to 4.
It should be noted that the ligand efficiency (LE) for 25 is
superior to that of 4, even though the Aurora kinase activity is
lowered. Similarly the lipophilicity efficiency (LipE) is also
better for 25 than 4.
LE is used to compare binding efficacy of two inhibitors/
ligands relative to their size and is used extensively in fragment-
based lead optimization programs and also used as a criteria in
selecting suitable candidates for moving them up the drug
discovery pipeline.21−23 Use of LE for comparison instead of
IC50 would remove the bias due to the increased size of a
ligand, as bulkier ligands have more interaction points with the
enzyme/receptor. Hence, use of the LE constitutes an
opportunity to compare the binding efficacy between two
molecules equating their size. Similarly, LipE is used as
comparative binding efficacy measure where the lipophilicity
of two molecules are equated. Although 25 showed marginally
improved in vitro activity and physicochemical properties, when
tested in vivo at 50 mg/kg iv dose in the HCT-116 tumor
xenograft mouse model, significant tumor growth suppression
was not observed (data not shown).
Figure 3. Docking study of compounds 25 and 25a−c into the X-ray
co-crystal structure of Aurora kinase A in complex with inhibitor 4
(PDB ID: 3M11). Binding energy values were calculated to identify
the compound with the most exothermic binding energy in order to
guide further chemical modification in this series. Figure was created
using UCSF Chimera package.15
Using compound 25 as our template, it was decided to
introduce basic amino functional groups through two or three
atom carbon linkers into the 6-position phenyl group on the
furano-pyrimidine core; the carbon linkers would be tethered to
the phenyl ring through an ether bridge. To identify a potential
position for installation of the solubilizing group, a docking
study was first carried out. For this, the X-ray co-crystal
structure of 4−Aurora A protein (PDB ID: 3M11) was used to
dock compounds 25 (no substitution) and 25a−c (bearing a
methoxy group attached to para-, meta-, and ortho-position of
the phenyl ring). The docking information (Figure 3) reveals
that the para-OMe compound, 25a, has the most favorable
binding energy compared to compound 25 and the other two
analogues 25b and 25c. This was further substantiated by the
better Aurora kinase A inhibition of 25a compared to 25 (Table
S1, Supporting Information). This result suggests that the
amino group-bearing side chain in the para-position would be
better suited to enter the solvent exposed region of the enzyme.
Hence, further structure−activity explorations were achieved by
introducing the solubilizing side chain to the para-position of
the 6-position phenyl ring in the furano-pyrimidine core.
First, various secondary amines of different basicities (pKa)
and steric sizes were explored in order to affect the overall
lipophilicity of the molecule (estimated as LogD7.4 using ACD’s
structure designer suit) Initially, N,N-dimethyl amino sub-
stitution (27)14 was introduced through a two-carbon linker at
the para-position of the phenyl ring, which resulted in a 2-fold
improvement in Aurora kinase A and B activity (Table 3). Most
importantly, these improvements in enzyme inhibition were
maintained at the cellular level by 7-fold improvement in
antiproliferative activity in HCT-116 cells for 27 compared to
the lead 4. A concomitant improvement in LE for 27 (0.26),
compared to 4 (0.25), was also noted. Interestingly,
introduction of the basic amino functionality reduced the
LogD, resulting in huge jump in the LipE for 27 (4.78),
compared to the lead 4 (1.75). Also, 27 showed about 250-fold
improvement in aqueous solubility compared to the lead 4
(30.28 ng/mL vs 0.12 ng/mL).
Having successfully improved both the activity and
physicochemical properties, steric effects were examined. The
amine side chain length was increased by introducing N,N-
diethyl amine (28) and N,N-dibutyl amine (29) groups;
introduction of the latter resulted in loss of activity at the
enzyme and cellular levels compared to 28. The N,N-diethyl
(28) analogue showed only a 2-fold loss of activity in the
antiproliferative assay compared to 27, still over 3-fold better
than the initial lead 4. We also investigated the effect of
introducing cyclic secondary amines such as pyrollidine (30),
piperidine (31), N-methylpiperazine (32), and morpholine
(33) as the amino functionality. Compounds 31−33 exhibited
over 2−3-fold improved antiproliferative activity compared to
the initial lead 4, although their Aurora A enzyme inhibition
levels were similar to 4, due to their lower lipophilicity.
Second, introduction of a hydroxy group to the terminal
amino side chain was contemplated, with the aim of altering
physicochemical properties and activity level. Introduction of a
F
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
hydroxy group to the side chain of 28 and 31 resulted in 34 and
35 (IBPR002).14 Comparison of these four compounds shows
that the introduction of a hydroxy group resulted in improved
activity against Aurora B for compounds 34/35, compared to
28/31. However, the change in enzyme inhibition profile was
not reflected in the antiproliferative activity for 34/35, which is
in a range similar to that for compounds 28/31.
Third, the effect of increasing the length of the linker carbon
chain bearing the solubilizing amino functionality was
investigated. When the linker carbon length was increased
from two carbons (in compound 27) to three carbons (to give
36), the activity level decreased at the enzyme and cellular level
compared to 27. Moreover, when the solubilizing side chain of
27 was moved from the para-position to the meta-position
(37), the activity decreased for both Aurora A and B enzymes,
which is reflected in around 4-fold decreased antiproliferative
activity for 37 compared to 27 (Table 3). This indicates that
the solubilizing side chain in the para-position is better suited
to enter the solvent exposed region of the enzyme, which is
consistent with our docking studies with 25a−c (Figure 3).
Overall, smaller amino groups are well tolerated, when placed
at para-position of the phenyl ring through a two-carbon side
chain linker. All the compounds bearing the solubilizing
functional group showed improved antiproliferative activity
compared to the initial lead 4. In particular, compounds with
higher Aurora A enzyme inhibition and lower lipophilicity (Log
D) demonstrated better antiproliferative activity. More
interestingly, within this series of compounds, good correlation
was observed between Aurora kinase A enzyme inhibition and
HCT-116 antiproliferative activity (Table 3). In addition, any
discrepancies between the activity levels could be further
justified based on Aurora B enzyme inhibition levels. For
example, 27 and 28, despite possessing similar level of Aurora A
inhibition, displayed a 2-fold difference in cellular activity,
which could be due to lower Aurora B activity for 28 compared
to 27.
Compound 27, the best in terms of LE and LipE, was
selected for further in vitro testing to evaluate its potential as
anticancer agent. Treatment of HCT-116 with 27 has resulted
in reduced histone H3 phosporylation and Aurora A (Thr288)
autophosporylation (Figure 4A), indicating the inhibition of
Aurora kinase B and A activity, respectively. This inhibition
effect of 27 is similar to that of 1 but was much more
pronounced (1 exhibited only negligible inhibition at 50 nM
concentration; data not shown).
Functional loss of Aurora A reportedly delays mitotic
progression24 while inactivating Aurora B overrides the mitotic
checkpoint, thereby causing premature chromosome decon-
densation without separation of the duplicated chromatids.25
We tested the properties of compound 27 in mitotic
progression using HeLa cells released from double thymidine
block. Thymidine treatment results in blockade of DNA
synthesis and cell cycle arrest in the S phase. Once thymidine is
removed, the cells progress through cell cycle in a synchronized
manner and can be used to study the effect of drugs on the cell
cycle progression by calculating the mitotic index (number of
cells in mitosis/total number of cells). Compared to DMSO-
treated control, the addition of >500 nM of 27 delayed mitotic
progression for approximately 4 h (Figure 4B), suggesting
Aurora kinase A inhibition.26,27 These cells that were delayed in
mitotic progression were finally flattened with decondensed
chromosomes and reformed nuclear envelope without cell
division/cytokinesis (Figure 4C). Consistently, accumulated
Figure 4. Functional study of 27 on mitotic progression. (A) Western
blot analysis for phosphorylation inhibition of Aurora substrates in
HCT-116 cells treated with 27 for 2 h. Compound 27 inhibited
phospho Aurora A (Thr288) and phospho histone H3 (Ser10)
formation in HCT-116 cells in a dose dependent manner, suggesting
inhibition of kinase activity for both Aurora A and B. (B) Mitotic index
from HeLa cells treated with mock (DMSO) or with various
concentrations of 27 following release from the double thymidine
block. Mitotic index was scored every 2 h between 10 and 18 h after
the second thymidine release by quantifying cells that harbor
condensed chromosomes using the MetaMorph software (Molecular
Devices). Errors in SEM were obtained from triplicate experiments.
(C) HeLa cells released from double thymidine block were treated
with the control DMSO (Mock) or 1.0 μM of 27. After 18 h of
treatment, cells were fixed and subjected for immunofluorescence
staining. Nuclear membrane was marked with lamin B1 (green)
staining. DNA was stained with Hoechst33342 (red). Bars: 10 μm.
(D) Flow cytometry analysis for DNA content in HCT-116 cells
treated with 27 at various concentrations for 48 h.
G
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
multinucleated cells with 4N or 8N DNA content were
observed in a flow cytometry analysis of HCT-116 cells treated
with compound 27 for 48 h, an indicator of mitotic checkpoint
override by the inhibition of Aurora kinase B (Figure 4D).28,29
This effect could be observed at much lower concentrations of
27 than 1, emphasizing the superior potency of 27 compared
with 1 and consistent with the improved antiproliferative
activity observed for 27 compared to 1. These experiments
suggest potent in vitro efficacy of 27 to both Aurora kinase A
and B inhibition.
Further to determine the selectivity, 27 was tested against a
panel of 31 therapeutically important kinases (Table S2,
Supporting Information). At a dose of 1 μM, 27 was found to
inhibit seven kinases with >50% inhibition, including Auroras
A, B, and C. Except for CHK2, the other three kinases (Flt3,
NTRK1, RAF1) showed inhibition levels lower than Aurora
kinases, suggesting 27 to be a potent and selective Aurora
kinase inhibitor.
Once 27 had been confirmed as a suitable candidate for
further development, it was tested in an in vivo mouse acute
tolerability assay. The hydrochloride salt of 27 (free form has
poor solubility in the dosing formulation) of 27 was injected
intravenously into three mice at a dose of 50 mg/kg QD for
every day for five days. The mice were observed for another 5
days; no notable changes in activity were observed, and none of
the mice died. Building on this preliminary investigation, HCT-
116 tumor bearing nude mice were administered 27 at a dose of
50 and 10 mg/kg iv QD, and 20 mg/kg iv BID for 5 days in
two cycles, with a two-day break between the cycles. Similarly,
1 was administered at 50 mg/kg iv using the same schedule and
treatment cycle. Significant tumor growth suppression was
observed for 27 when administered at 50 mg/kg QD as well as
at 20 mg/kg BID, similar to that of 1 at 50 mg/kg QD (Figure
5).
the aim of improving in vitro activity and identifying in vivo
active compounds. SAR exploration of the urea side chain
revealed that modification in this part is detrimental to activity.
On the basis of the X-ray co-crystal structure of 4 in complex
with Aurora A, as well as a docking study, various solubilizing
functional groups were introduced to the 6-position phenyl
group attached to the furano-pyrimidine core to optimize both
the activity (Aurora kinase A/B inhibition and HCT-116
antiproliferation) and properties (mol wt and lipophilicity).
Several secondary amino groups with varying size and basicity
(pKa) were introduced through a linker chain to the phenyl
group attached to the furano-pyrimidine core. All the
compounds bearing the solubilizing functional group showed
improved antiproliferative activity compared to the initial lead
4. Overall, smaller amino groups are well tolerated when placed
at para-position of the phenyl ring through a two-carbon side
chain linker. In particular, compound 27 bearing an N,N-
dimethyl amino group showed a 2-fold improvement in Aurora
kinase A and B activity compared to the lead 4. Most
importantly, these improvements in enzyme inhibition were
reflected at the cellular level by 7-fold improvement in
antiproliferative activity in HCT-116 cells for 27 compared to
the lead 4. Furthermore, compound 27 possessed the best
ligand efficiency (LE = 0.26) and lipophilicity efficiency (LipE
= 4.78) among the significantly active compounds, and was
therefore selected for further in vitro and in vivo profiling.
Compound 27 inhibited the kinase activity of both Aurora A
and B, interfered with the cell cycle progression, and affected
HURP (hepatoma up-regulated protein) phosphorylation for
the assembly of mitotic spindles.14 Moreover, preliminary in
vivo evaluation of 27 in a HCT-116 xenograft mouse model
suggested it holds great promise as an anticancer agent. Further
preclinical investigations are underway to determine the full
potential of this compound as an anticancer therapy.
EXPERIMENTAL SECTION
CONCLUSION
■
■
■
Our studies toward targeted anticancer agents have included
the disclosure of the novel furano-pyrimdine 4 as an Aurora
kinase inhibitor. Modification of the lead 4 was initiated with
GENERAL METHODS
All commercial chemicals and solvents are of reagent grade and
were used without further purification unless otherwise stated.
All reactions were carried out under dry nitrogen atmosphere
and were monitored for completion by TLC using Merck 60
F254 silica gel glass backed plates (5 cm × 10 cm); zones were
detected visually under UV irradiation (254 nm) or by spraying
with phosphomolybdic acid reagent (Aldrich) followed by
heating at 80 °C. Flash column chromatography was carried out
using silica gel (Merck Kieselgel 60, no. 9385, 230−400 mesh
ASTM). 1H and 13C NMR spectra were obtained with a Varian
Mercury-300 or Varian Mercury-400 spectrometers, and the
chemical shifts were recorded in parts per million (ppm, δ) and
reported relative to TMS or the solvent peak. High-resolution
mass spectra (HRMS) were measured with a Finnigan (MAT-
95XL) electron impact (EI) or by using Finnigan/Thermo
Quest MAT 95XL FAB mass spectrometer. Low-resolution
mass spectra (LRMS) data were measured on an Agilent MSD-
1100 ESI-MS/MS system. Purity of the final compounds were
determined with an Hitachi 2000 series HPLC system using C-
18 column (Agilent ZORBAX Eclipse XDB-C18 5 μm, 4.6 mm
× 150 mm) operating at 25 °C. Elution was carried out using
acetonitrile as mobile phase A and water containing 0.1%
formic acid +10 mmol NH4OAc as mobile phase B. Elution
condition: at 0 min, phase A 10% + phase B 90%; at 45 min,
Figure 5. In vivo antitumor effect of 27 in human colon cancer (HCT-
116) xenograft nude mice model. The growth of HCT-116 tumor
xenograft is inhibited by 27 (50 mg/kg, iv, QD; 20 mg/kg, iv, BID)
and 1 (50 mg/kg, iv, QD) with *p < 0.05. Drug treatments on days 1−
5 and 8−12. For all groups except for treatment with 27 at 10 mg/kg,
n = 8 mice; n = 7 mice for the treatment group 27 at 10 mg/kg.
H
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
phase A 90% + phase B 10%; at 50 min, phase A 10% + phase B
90%; at 60 min, phase A 10% + phase B 90%. The flow-rate of
the mobile phase was 0.5 mL/min, and the injection volume of
the sample was 5 μL. Peaks were detected at 210 nm. Purity of
all the tested compounds were found to be >95% unless
otherwise stated.
7.01−6.94 (m, 2H), 6.72 (d, J = 7.6 Hz, 1H), 4.68 (bt, J = 5.6
Hz, 1H), 3.67 (q, J = 6.4 Hz, 2H), 2.72 (t, J = 6.4 Hz, 2H). 13
C
NMR (100 MHz, CDCl3) δ 164.5 (C), 157.4 (C), 153.7 (C),
146.7 (C), 139.5 (C), 138.8 (C), 138.4 (C), 131.8 (C), 129.6
(CH), 129.4 (CH), 129.2 (CH), 129.1 (C), 129.0 (CH), 128.9
(CH), 128.4 (CH), 126.2 (CH), 123.4 (CH), 120.2 (CH),
119.9 (CH), 118.0 (CH), 114.9 (C), 103.1 (C), 41.7 (CH2),
34.9 (CH2). HRMS (EI) calcd for C33H27N5O2 [M]+,
525.2165; found, 525.2155.
1-{4-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]-
phenyl}-3-phenylurea 16. Method A, using 7c and phenyl-
isocyanate to give 16. Purification: column chromatography
over silica gel column using MeOH:CH2Cl2 (1:20). Yield, 73%.
1H NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H), 8.62 (s, 1H),
8.48 (s, 1H), 7.68−7.63 (m, 6H), 7.51−7.48 (m, 2H), 7.43−
7.36 (m, 8H), 7.28−7.24 (m, 3H), 6.71 (s, 1H). LRMS (ESI)
m/z: 498 [M + H]+.
1-(4-{[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]-
methyl}phenyl)-3-phenylurea 17. Method A, using 7d and
phenylisocyanate to give 17. Purification: column chromatog-
raphy over silica gel column using MeOH:CH2Cl2 (1:20).
Yield, 82%. 1H NMR (300 MHz, CDCl3) δ 8.44 (s, 1H), 7.86−
7.83 (m, 3H), 7.59−7.45 (m, 13H), 7.29−7.24 (m, 4H), 7.14
(d, J = 7.2 Hz, 2H), 4.94 (t, J = 5.7 Hz, 1H), 4.64 (d, J = 5.7 Hz,
1H). LRMS (ESI) m/z: 512 [M + H]+.
1-(4-{3-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]-
propyl}phenyl)-3-phenylurea 18. Method A, using 7e and
phenylisocyanate to give 18. Purification: column chromatog-
raphy over silica gel column using MeOH:CH2Cl2 (1:20).
Yield, 78%. 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 7.59−
7.50 (m, 8H), 7.35−7.23 (m, 6H), 7.14−7.11 (m, 1H), 7.01 (d,
J = 8.4 Hz, 2H), 6.56 (d, J = 8.4 Hz, 2H), 4.67 (bt, J = 5.6 Hz,
1H), 3.43 (q, J = 6.8 Hz, 2H), 2.47 (t, J = 6.8 Hz, 2H), 1.73 (q,
J = 6.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 164.5 (C),
157.6 (C), 153.9 (C), 153.9 (CH), 146.6 (C), 138.4 (C), 136.3
(C), 136.1 (C), 132.3 (C), 129.7 (CH), 129.6 (CH), 129.3
(C), 129.0 (CH), 128.9 (CH), 128.8 (CH), 128.5 (CH), 128.4
(CH), 126.2 (CH), 123.3 (CH), 120.2 (CH), 114.8 (C), 103.2
(C), 49.9 (CH2), 32.0 (CH2), 30.7 (CH2). HRMS (EI) calcd
for C34H29N5O2 [M]+, 539.2321; found, 539.2327. HPLC
purity: 92%.
1-Benzyl-3-(4-{2-[(5,6-diphenylfuro[2,3-d]pyrimidin-4-yl)-
amino]ethyl}phenyl)urea 19. Method A, using 7a and
benzylisocyanate to give 19. Purification: column chromatog-
raphy over silica gel column using MeOH:CH2Cl2 (1:30).
Yield, 86%. 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 7.48−
7.37 (m, 6H), 7.32−7.23 (m, 9H), 7.17 (d, J = 8.4 Hz, 2H),
6.93 (d, J = 8.4 Hz, 2H), 6.37 (brs, 1H), 5.05 (bt, J = 5.6 Hz,
1H), 4.64 (bt, J = 5.2 Hz, 1H), 4.46 (d, J = 5.6 Hz, 2H), 3.67
(q, J = 6.4 Hz, 2H), 2.73 (t, J = 6.4 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ 164.6 (C), 157.4 (C), 156.2 (C), 153.9 (CH),
146.6 (C), 138.9 (C), 137.2 (C), 133.3 (C), 132.0 (C), 129.6
(CH), 129.5 (CH), 129.3 (C), 129.1 (CH), 128.8 (CH), 128.5
(CH), 128.4 (CH), 128.4 (CH), 127.2 (CH), 126.3 (CH),
120.5 (CH), 114.8 (C), 103.1 (C), 44.0 (CH2), 41.9 (CH2),
34.3 (CH2). HRMS (EI) calcd for C34H29N5O2 [M]+,
539.2321; found, 539.2320.
N-[2-(4-Aminophenyl)ethyl]-5,6-diphenylfuro[2,3-d]-
pyrimidin-4-amine 7a. 4-Chloro-5,6-diphenylfuro[2,3-d]-
pyrimidine (5) (0.200 g, 65.2 mmol) and 4-(2-aminoethyl)-
aniline (6a) (0.107 g, 70.8 mmol) in ethanol (5 mL) were
heated at reflux for 8 h. The reaction mixture was concentrated
under vacuum, and the residue partitioned between water and
ethyl acetate. The organic layer separated, dried over Na2SO4,
concentrated, and the crude compound was purified by silica
gel column chromatography using a mixture of MeOH:CH2Cl2
(1:40), to give 7a (0.195 g, 74%). 1H NMR (300 MHz,
CDCl3): δ 8.43 (s, 1H), 7.50−7.23 (m, 10H), 6.79 (d, J = 8.4
Hz, 2H), 6.59 (d, J = 8.4 Hz, 2H), 4.68 (t, J = 5.2 Hz, 1H), 3.65
(q, J = 6.4 Hz, 2H), 3.62 (bs, 2H), 2.67 (t, J = 6.4 Hz, 2H).
HRMS (EI) calcd for C26H22N4O [M]+, 406.1794; found,
406.1789.
Compounds 7b−e were prepared from 5 and appropriate
amines (6b−e), in a manner similar to 7a.
N-[2-(3-Aminophenyl)ethyl]-5,6-diphenylfuro[2,3-d]-
pyrimidin-4-amine 7b. Yield, 86%. 1H NMR (300 MHz,
CDCl3) δ 8.43 (s, 1H), 7.50−7.21 (m, 12H), 7.00 (t, J = 8.1
Hz, 1H), 6.55 (ddd, J = 8.1, 1.2, 0.9 Hz, 1H), 6.38−6.31 (m,
2H), 4.72 (t, J = 5.7 Hz, 1H), 3.68 (q, J = 6.3 Hz, 2H), 2.70 (t, J
= 6.3 Hz, 2H). LRMS (ESI) m/z: 407 [M + H]+.
N-(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)-benzene-1,4-di-
amine 7c. Yield, 80%. 1H NMR (300 MHz, DMSO-d6) δ 8.39
(s, 1H), 7.67−7.61 (m, 6H), 7.50−7.46 (m, 2H), 7.40−7.34
(m, 3H), 6.99 (d, J = 6.9 Hz, 2H), 6.49 (d, J = 6.9 Hz, 2H),
6.44 (s, 2H). LRMS (ESI) m/z: 379 [M + H]+.
N-(4-Aminobenzyl)-5,6-diphenylfuro[2,3-d]pyrimidin-4-
amine 7d. Yield, 85%. 1H NMR (300 MHz, CDCl3) δ 8.44 (s,
1H), 7.54−7.44 (m, 8H), 7.27−7.25 (m, 2H), 6.93 (d, J = 6.9
Hz, 2H), 6.10 (d, J = 6.9 Hz, 2H), 4.87 (t, J = 5.7 Hz, 1H), 4.51
(d, J = 5.7 Hz, 2H), 3.65 (brs, 2H). LRMS (ESI) m/z: 393 [M
+ H]+.
N-[3-(4-Aminophenyl)propyl]-5,6-diphenylfuro[2,3-d]-
pyrimidin-4-amine 7e. Yield, 50%. 1H NMR (400 MHz,
CDCl3) δ 8.41 (s, 1H), 7.58−7.49 (m, 8H), 7.29−7.25 (m,
2H), 6.83 (d, J = 8.0 Hz, 2H), 6.60 (d, J = 8.0 Hz, 2H), 4.66 (t,
J = 5.2 Hz, 1H), 3.57 (bs, 2H), 3.41 (q, J = 6.8 Hz, 2H), 2.38 (t,
J = 6.8 Hz, 2H), 1.69 (quin, J = 6.8 Hz, 2H). HRMS (EI) calcd.
for C27H24N4O [M]+, 420.1950; found, 420.1949.
Urea Side Chain Installation in Compounds 15−24.
Method A. The urea side chain was installed by reaction
between 7b−e and appropriate phenylisocyanates to give the
final products in a manner similar to that for 4 reported by us.13
Method B. A mixture of 7a−e (1.0 mmol) and carbon-
yldiimdazole (CDI, 2.0 mmol) in THF (10 mL) was stirred at
ambient temperature for 4 h, followed by addition of the
required amine (2.0 mmol) and heating to reflux for another 16
h. After cooling the reaction, the mixture was evaporated and
purified by silica gel column chromatography.
1-(3-{2-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]-
ethyl}phenyl)-3-phenylurea 15. Method A, using 7b and
phenylisocyanate to give 15. Purification: column chromatog-
raphy over silica gel column using MeOH:CH2Cl2 (1:20).
Yield, 82%. 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 7.45−
7.27 (m, 11H), 7.24−7.16 (m, 4H), 7.09 (t, J = 7.2 Hz, 1H),
1-(4-{2-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]-
ethyl}phenyl)urea 20. Method B, using 7a and aqueous
ammonia to give 20. Purification: column chromatography over
silica gel column using MeOH:CH2Cl2:NH4OH (5:100:1).
Yield, 78%. 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 7.47−
7.38 (m, 5H), 7.32−7.28 (m, 2H), 7.25−7.18 (m, 5H), 6.97 (d,
I
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
J = 8.4 Hz, 2H), 4.79 (brs, 2H), 4.65 (bt, J = 5.6 Hz, 1H), 3.69
(q, J = 6.4 Hz, 2H), 2.75 (t, J = 6.4 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ 164.7 (C), 157.4 (C), 157.2 (C), 154.0 (C),
146.6 (C), 136.8 (C), 134.2 (C), 132.0 (C), 129.6 (CH), 129.5
(CH), 129.3 (C), 129.2 (CH), 128.8 (CH), 128.4 (CH), 126.3
(CH), 121.2 (CH), 114.8 (C), 103.1 (C), 41.9 (CH2), 34.4
(CH2). HRMS (FAB) calcd for C27H24N5O2 [M + H]+,
450.1930; found, 450.1933. HPLC purity: 86%.
1H), 7.08 (s, 1H), 7.10 (ddd, J = 9.0, 2.1, 1.2 Hz, 1H), 3.90 (s,
3H). LCMS (ESI) m/z: 261 [M + H]+.
1-Phenyl-3-(4-{2-[(6-phenylfuro[2,3-d]pyrimidin-4-yl)-
amino]ethyl}phenyl)urea 25. The mixture of 9a (2.0 g, 8.69
mmol), 2-(4-aminophenyl)-ethyl amine (1.3 mL, 10.43 mmol),
and triethyl amine (3.56 mL, 26.07 mmol) was heated to reflux
in EtOH (40 mL) for 5 h. The precipitate formed after cooling
to room temperature was filtered, washed with EtOH, and then
dried under vacuum. The solid product obtained was dissolved
in CH2Cl2 (45 mL) and phenyl isocyanate (1.38 mL, 12.76
mmol) was added slowly. After stirring at room temperature for
8 h, the precipitate formed was filtered and washed with
CH2Cl2 to obtain 25 as a white solid. Yield, 88% (two steps).
1H NMR (300 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.59 (s, 1H),
8.28 (s, 1H), 8.10 (br, 1H), 7.78 (d, J = 7.2 Hz, 2H), 7.52−7.37
(m, 8H), 7.28−7.17 (m, 4H), 6.94 (t, J = 7.2 Hz, 1H), 3.70 (q,
J = 7.2 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H). 13C NMR (75 MHz,
DMSO-d6) δ 165.7 (C), 157.0 (C), 153.9 (CH), 152.6 (C),
150.2 (C), 139.8 (C), 137.9 (C), 132.8 (C), 129.2 (CH), 129.0
(CH), 128.8 (CH × 2), 124.1 (CH), 121.7 (CH), 118.3 (CH),
118.1 (CH), 102.6 (C), 98.2 (CH), 42.1 (CH2), 34.4 (CH2).
HRMS (FAB) m/z: calcd for C27H24N5O2 ([M + H]+),
450.1930; found, 450.1936.
1-(4-{2-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]-
ethyl}phenyl)-3-methylurea 21. Method B, using 7a and
methylamine to give 21. Purification: column chromatography
over silica gel column using MeOH:CH2Cl2:NH4OH
1
(5:100:1). Yield, 72%. H NMR (400 MHz, CDCl3) δ 8.43
(s, 1H), 7.49−7.39 (m, 6H), 7.33−7.30 (m, 2H), 7.27−7.23
(m, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 8.0 Hz, 2H),
6.31 (brs, 1H), 4.67 (bq, J = 4.8 Hz, 1H), 4.65 (bt, J = 5.6 Hz,
1H), 3.69 (q, J = 6.4 Hz, 2H), 2.86 (d, J = 4.8 Hz, 3H), 2.75 (t,
J = 6.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 164.7 (C),
157.4 (C), 156.9 (C), 154.0 (CH), 146.6 (C), 137.3 (C), 133.5
(C), 132.0 (C), 129.6 (CH), 129.5 (CH), 129.4 (C), 129.2
(CH), 128.8 (CH), 128.5 (CH), 128.4 (CH), 126.3 (CH),
120.8 (CH), 114.9 (C), 103.1 (C), 41.9 (CH2), 34.4 (CH2),
26.9 (CH3). HRMS (FAB) m/z: calcd for C28H26N5O2 ([M +
H]+), 464.2087; found, 464.2096.
1-[4-(2-{[6-(4-Methoxyphenyl)furo[2,3-d]pyrimidin-4-yl]-
amino}ethyl)phenyl]-3-phenylurea 25a. Synthesized from 9b
1-Cyclohexyl-3-(4-{2-[(5,6-diphenylfuro[2,3-d]pyrimidin-4-
yl)amino]ethyl}phenyl)urea 22. Method B, using 7a and
cyclohexylamine to give 22. Purification: column chromatog-
raphy over silica gel column using MeOH:CH2Cl2 (1:20).
1
in a manner similar to 25. Yield, 85%. H NMR (300 MHz,
DMSO-d6) δ 8.62 (s, 1H), 8.59 (s, 1H), 8.25 (s, 1H), 8.01 (bs,
1H), 7.73 (d, J = 9.0 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.37 (d,
J = 8.1 Hz, 2H), 7.27 (t, J = 8.4 Hz, 2H), 7.20−7.16 (m, 3H),
7.07 (d, J = 9.0 Hz, 2H), 6.95 (t, J = 7.2 Hz, 1H), 3.81 (s, 3H),
3.69 (q, J = 7.2 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H). LCMS (FAB)
m/z: 480 [M + H]+.
1
Yield, 75%. H NMR (300 MHz, CDCl3): δ 8.56 (s, 1H),
7.44−7.38 (m, 4H), 7.30−7.17 (m, 10H), 4.66 (t, J = 5.4 Hz,
NH), 3.68−3.60 (m, 2H), 2.67 (t, J = 6.6 Hz, 2H), 2.17−1.93
(m, 1H), 1.70−1.56 (m, 4H), 1.41−1.26 (m, 4H), 1.17−1.06
(m, 2H). LCMS (ESI) m/z: 532 [M + H]+.
1-[4-(2-{[6-(3-Methoxyphenyl)furo[2,3-d]pyrimidin-4-yl]-
1-(4-{2-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]-
ethyl}phenyl)-3-(4-methoxyphenyl)urea 23.17 Method B,
using 7a and p-amino-anisole to give 23. Purification: column
chromatography over silica gel column using MeOH:CH2Cl2
amino}ethyl)phenyl]-3-phenylurea 25b. Synthesized from 9c
in a manner similar to 25. Yield, 79%. H NMR (300 MHz,
1
CDCl3+CD3OD): δ 8.30 (s, 1H), 7.44−7.30 (m, 9H), 7.19 (d,
J = 6.3 Hz, 2H), 7.09−7.01 (m, 2H), 7.00 (s, 1H), 6.93−6.89
(m, 1H), 3.88 (s, 3H), 3.82 (t, J = 6.9 Hz, 2H), 3.37 (brs, 2H),
2.96 (t, J = 6.9 Hz, 2H). LCMS (ESI) m/z: 480 [M + H]+.
1-[4-(2-{[6-(4-Hydroxyphenyl)furo[2,3-d]pyrimidin-4-yl]-
amino}ethyl)phenyl]-3-phenylurea 10a. Treated 25a (1.0
mmol) with BBr3 (9 mL, 1.0 M in CH2Cl2) in CH2Cl2 (10
mL) at 0 °C for 1 h, then H2O (10 mL) was added to quench
1
(1:20). Yield, 70%. H NMR (300 MHz, CDCl3): δ 8.42 (s,
1H) 7.46−7.43 (m, 4H), 7.41−7.22 (m, 10H), 6.90 (q, J = 6.8
Hz, 4H), 4.49 (t, J = 5.4 Hz, 1H), 3.80 (s, 3H), 3.68 (q, J = 6.0
Hz, 2H), 2.73 (t, J = 6.8 Hz, 2H). LCMS (ESI) m/z: 556 [M +
H]+.
1-(4-{2-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]-
ethyl}phenyl)-3-(4-fluorophenyl)urea 24.17 Method B, using
7a and p-fluoro-aniline to give 24. Purification: column
chromatography over silica gel column using MeOH:CH2Cl2
the reaction and neutralized by the addition of NaHCO3(aq)
.
The precipitate formed was filtered and purified by silica gel
column chromatography using MeOH:CH2Cl2 (1:20). Yield,
1
1
(1:20). Yield, 78%. H NMR (300 MHz, CDCl3) δ 8.43 (s,
96%. H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 8.66 (s,
1H), 7.47−7.39 (m, 4H), 7.34−7.29 (m, 4H), 7.25−7.22 (m,
5H), 7.03−6.93 (m, 4H), 6.88 (s, 1H), 4.67 (t, J = 5.4 Hz, 1H),
3.69 (q, J = 6.0 Hz, 2H), 2.74 (t, J = 6.0 Hz, 2H). LCMS (ESI)
m/z: 544 [M + H]+.
1H), 8.63 (s, 1H), 8.23 (s, 1H), 8.01 (bs, 1H), 7.61 (d, J = 8.4
Hz, 2H), 7.43 (d, J = 7.2 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H),
7.27 (t, J = 7.6 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 7.11 (s, 1H),
6.95 (t, J = 7.6 Hz, 1H), 6.88 (d, J = 8.4 Hz, 2H), 3.68 (q, J =
7.6 Hz, 2H), 2.86 (t, J = 7.6 Hz, 2H). LCMS (ESI) m/z: 466
[M + H]+.
4-Chloro-6-(4-methoxyphenyl)furo[2,3-d]pyrimidine 9b.
Synthesized in a manner similar to 9a12 starting from 2-
1
bromo-1-(4-methoxy-phenyl)-ethanone (8b) in 28% yield. H
1-[4-(2-{[6-(3-Hydroxyphenyl)furo[2,3-d]pyrimidin-4-yl]-
amino}ethyl)phenyl]-3-phenylurea 10b. Synthesis carried out
from 25b in a manner similar to 10a. Purification: column
chromatography over silica gel column using MeOH:CH2Cl2
NMR (300 MHz, CDCl3) δ 8.71 (s, 1H) 7.85 (d, J = 9.0 Hz,
2H), 7.02 (d, J = 9.0 Hz, 2H), 6.93 (s, 1H), 3.89 (s, 3H).
LCMS (ESI) m/z: 261 [M + H]+.
1
4-Chloro-6-(3-methoxyphenyl)furo[2,3-d]pyrimidine 9c.
(1:20). Yield, 89%. H NMR (400 MHz, CDCl3 + CD3OD) δ
Synthesized in a manner similar to 9a12 starting from 2-
8.23 (s, 1H), 7.43−7.30 (m, 9H), 7.18 (d, J = 8.4 Hz, 2H),
7.06−6.99 (m, 3H), 6.93−6.89 (m, 1H), 3.82 (q, J = 7.6 Hz,
2H), 2.96 (t, J = 7.6 Hz, 2H). LCMS (ESI) m/z: 466 [M +
H]+.
1
bromo-1-(3-methoxy-phenyl)-ethanone (8c) in 24% yield. H
NMR (300 MHz, CDCl3) δ 8.75 (s, 1H) 7.50 (ddd, J = 9.0, 1.5,
1.2 Hz, 1H), 7.44 (dd, J = 2.1, 1.5 Hz, 1H), 7.39 (t, J = 9.0 Hz,
J
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1-Phenyl-3-(4-{2-[(5-phenylfuro[2,3-d]pyrimidin-4-yl)-
amino]ethyl}phenyl)urea 26. Compound 26 was prepared
from 1212 using 4-(2-amino-ethyl)-phenylamine and PhNCO
6.0 Hz, 2H), 2.56 (q, J = 7.2 Hz, 4H), 0.98 (t, J = 7.2 Hz, 6H).
LCMS (ESI) m/z: 565 [M + H]+.
1-(4-{2-[(6-{4-[2-(Dibutylamino)ethoxy]phenyl}furo[2,3-d]-
pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 29. Meth-
od D using 10a and methanesulfonic acid 2-dibutylamino-ethyl
ester to give 29. Yield, 30%. 1H NMR (400 MHz, DMSO-d6) δ
8.63 (s, 1H), 8.60 (s, 1H), 8.25 (s, 1H), 8.01 (brs, 1H), 7.71 (d,
J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz,
2H), 7.27 (t, J = 8.0 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.05 (d,
J = 9.2 Hz, 2H), 6.95 (t, J = 7.2 Hz, 2H), 4.07 (2H), 3.69 (q, J =
6.8 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.79 (brs, 2H), 2.47 (brs,
4H), 1.39 (t, J = 6.4 Hz, 4H), 1.28 (m, J = 7.2 Hz, 4H), 0.87 (t,
J = 7.2 Hz, 6H). LCMS (ESI) m/z: 621 [M + H]+.
1-Phenyl-3-(4-{2-[(6-{4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-
furo[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)urea 30. Meth-
od D using 10a and methanesulfonic acid 2-pyrrolidin-1-yl-
ethyl ester to give 30. Yield, 32%. 1H NMR (400 MHz, DMSO-
d6) δ 8.62 (s, 1H), 8.59 (s, 1H), 8.25 (s, 1H), 8.01 (brs, 1H),
7.71 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.38 (d, J =
8.4 Hz, 2H), 7.27 (t, J = 8.0 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H),
7.08 (d, J = 8.8 Hz, 2H), 6.95 (t, J = 7.2 Hz, 2H), 4.13 (t, J =
6.0 Hz, 2H), 3.69 (q, J = 6.0 Hz, 2H), 2.89−2.82 (m, 4H), 2.54
(brs, 4H), 1.79 (m, 4H). LCMS (ESI) m/z: 563 [M + H]+.
HPLC purity: 94% .
1-Phenyl-3-(4-{2-[(6-{4-[2-(piperidin-1-yl)ethoxy]phenyl}-
furo[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)urea 31. Meth-
od D using 10a and methanesulfonic acid 2-piperidin-1-yl-ethyl
ester to give 31. Yield, 27%. 1H NMR (300 MHz, DMSO-d6) δ
8.62 (s, 1H), 8.59 (s, 1H), 8.25 (s, 1H), 8.00 (brs, 1H), 7.71 (d,
J = 9.0 Hz, 2H), 7.45−7.37 (m, 4H), 7.27 (t, J = 7.8 Hz, 2H),
7.18 (d, J = 8.1 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H), 6.95 (t, J =
7.2 Hz, 2H), 4.13 (t, J = 5.7 Hz, 2H), 3.67 (q, J = 6.3 Hz, 2H),
2.87 (t, J = 7.2 Hz, 2H), 2.54 (brs, 2H), 2.27 (brs, 4H), 1.50
(m, 4H), 1.39 (m, 2 H). LCMS (ESI) m/z: 577 [M + H]+.
1-(4-{2-[(6-{4-[2-(4-Methylpiperazin-1-yl)ethoxy]phenyl}-
furo[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea
32. Method E using 10a, bromochloroethane, and 4-
methylpiperazine to give 32. Yield, 40%. 1H NMR (300
MHz, DMSO-d6) δ 8.72 (s, 1H), 8.70 (s, 1H), 8.25 (s, 1H),
8.00 (brs, 1H), 7.71 (d, J = 9.0 Hz, 2H), 7.44 (d, J = 7.8 Hz,
2H), 7.38 (d, J = 8.7 Hz, 2H), 7.26 (t, J = 7.6 Hz, 2H), 7.21−
7.16 (m, 3H), 7.07 (d, J = 8.7 Hz, 2H), 6.95 (t, J = 6.6 Hz, 1H),
4.13 (t, J = 5.7 Hz, 2H), 3.68 (q, J = 6.6 Hz, 2H), 2.87 (t, J =
6.6 Hz, 2H), 2.70 (t, J = 6.0 Hz, 2H), 2.35−2.25 (m, 8H), 2.14
(s, 3H). LCMS (ESI) m/z: 592 [M + H]+.
1-(4-{2-[(6-{4-[(2-Morpholin-4-yl)ethoxy]phenyl}furo[2,3-
d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 33.
Method C using 10a and 4-(2-chloro-ethyl)-morpholine to
give 33. Yield, 29%. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s,
1H), 8.59 (s, 1H), 8.25 (s, 1H), 8.01 (brs, 1H), 7.71 (d, J = 8.4
Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H),
7.27 (t, J = 8.0 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.08 (d, J =
8.8 Hz, 2H), 6.95 (t, J = 7.2 Hz, 2H), 4.15 (t, J = 6.0 Hz, 2H),
3.68 (q, J = 6.0 Hz, 2H), 3.58 (t, J = 4.8 Hz, 4H), 2.87 (t, J =
7.2 Hz, 2H), 2.71 (t, J = 6.0 Hz, 2H), 2.48 (brs, 4H). LCMS
(ESI) m/z: 579 [M + H]+.
1
in a manner similar to 25. Yield, 92% (two steps). H NMR
(400 MHz, CDCl3) δ 8.44 (s, 1 H), 7.50 (brs, 1H), 7.49 (brs,
1H), 7.36−7.29 (m, 6H), 7.25−7.21 (m, 4H), 7.19 (d, J = 8.4
Hz, 2H), 7.04−7.00 (m, 1H), 6.93 (d, J = 8.4 Hz, 2H), 5.00 (t,
J = 6.4 Hz, 1H), 3.72 (q, J = 6.4 Hz, 2H), 2.77 (t, J = 6.4 Hz,
2H). HRMS (FAB) calcd for C27H24N5O2 [M + H]+, 450.1930;
found, 450.1937. HPLC purity: 86%.
Solubilizing Group Installation in Compounds 27−37.
Method C. A mixture of 10a,b (1.0 mmol), potassium
carbonate (3.0 mmol), potassium iodide (1.0 mmol), and
R2N(CH2)2Cl (1.5 mmol) in 10 mL of DMF:acetone (1:1) was
heated to reflux for 6 h. Then the mixture was concentrated,
water was added, and then extracted with ethyl acetate. The
organics were separated, concentrated under vacuum, and the
solid obtained was purified by silica gel column chromatog-
raphy using MeOH:CH2Cl2:NH4OH (10:200:1) to get the
desired product.
Method D. A mixture of 10a (0.50 mmol) and potassium
carbonate (1.60 mmol) in 5 mL of DMF was heated to 75 °C,
then R2N(CH2)2OMs (0.75 mmol) in 4 mL of DMF was
slowly added. After stirring overnight, the resulting mixture was
filtered, then water was added and extracted with ethyl acetate.
The organics were separated and concentrated under vacuum,
and the solid obtained was purified by silica gel column
chromatography using MeOH:CH2Cl2:NH4OH (10:200:1) to
get the desired product.
Method E. A mixture of 10a (1.0 mmol), potassium
carbonate (3.0 mmol), potassium iodide (1.5 mmol), and
Br(CH2)nCl (1.5 mmol) in 10 mL of DMF:acetone (1:1) was
heated to reflux for 6 h. Then added R2NH (2.0 mmol) and
refluxed for another 8 h. After cooling, the mixture was
concentrated, then H2O (20 mL) was added and then extracted
with ethyl acetate. The organics were separated, concentrated
under vacuum, and the solid obtained was purified by silica gel
column chromatography using MeOH:CH2Cl2:NH4OH
(10:200:1) to get the desired product.
1-(4-{2-[(6-{4-[2-(Dimethylamino)ethoxy]phenyl}furo[2,3-
d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 27.14
Method C using 10a and 2-(chloroethyl)dimethyl amine to
1
give 27. Yield, 52%. H NMR (300 MHz, CD3OD) δ 8.20 s,
1H), 8.01 (brs, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.4
Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 7.26 (t, J = 7.8 Hz, 2H), 7.19
(d, J = 8.4 Hz, 2H), 7.04 (d, J = 9.0 Hz, 2H), 6.99 (s, 1H),
6.98−6.96 (m, 1H), 4.15 (t, J = 5.4 Hz, 2H), 3.78 (t, J = 7.2 Hz,
2H), 2.91 (t, J = 7.2 Hz, 2H), 2.78 (t, J = 5.4 Hz, 2H), 2.35 (s,
+
6H). LCMS (ESI) m/z: 537 [M + H] .
The HCl salt of 27 was prepared by dissolving 27 in
CH2Cl2/MeOH mixture and then passing HCl gas (prepared
by reacting concentrated H2SO4 with NaCl) into the mixture
for 2 h with stirring at room temperature. Solvent was
evaporated under reduced pressure to afford HCl salt of 27.
1-(4-{2-[(6-{4-[2-(Diethylamino)ethoxy]phenyl}furo[2,3-d]-
pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 28. Meth-
od D using 10a and methanesulfonic acid 2-diethylamino-ethyl
ester to give 28. Yield, 41%. 1H NMR (400 MHz, DMSO-d6) δ
8.63 (s, 1H), 8.60 (s, 1H), 8.25 (s, 1H), 8.01 (brs, 1H), 7.71 (d,
J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz,
2H), 7.27 (t, J = 7.6 Hz, 2H), 7.18 (d, J = 8 Hz, 2H), 7.06 (d, J
= 8.4 Hz, 2H), 6.95 (t, J = 7.2 Hz, 2H), 4.07 (t, J = 6.0 Hz, 2H),
3.69 (q, J = 6.8 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.79 (t, J =
1-[4-(2-{[6-(4-{2-[Ethyl(2-hydroxyethyl)amino]ethoxy}-
phenyl)furo[2,3-d]pyrimidin-4-yl]amino}ethyl)phenyl]-3-phe-
nylurea 34. Method E using 10a, bromochloroethane, and 2-
hydroxyethyl-ethylamine to give 34. Yield, 38%. 1H NMR (400
MHz, CD3OD) δ 8.22 (s, 1 H), 7.75 (s, 1 H), 7.74 (s, 1 H),
7.41 (d, J = 8.4 Hz, 2 H), 7.35 (d, J = 8.4 Hz, 2 H), 7.28 (m, 2
H), 7.21 (d, J = 8.4 Hz, 2 H), 7.02 (m, 4H), 4.15 (t, J = 5.6 Hz,
K
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2 H), 3.78 (t, J = 7.2 Hz, 2 H), 3.67 (t, J = 6.4 Hz, 2 H), 2.97
(m, 4 H), 2.75 (m, 4H), 1.11 (t, J = 7.2 Hz, 2 H). LCMS (ESI)
m/z: 581 [M + H]+.
0.04% Triton X-100, the proteins were aliquoted and stored at
−80 °C.
Aurora Kinase A Enzyme Inhibition Assay.30 Test
compounds, enzyme, substrate-tetra(LRRWSLG), DTT, and
ATP were dissolved in Aur buffer (50 mM Tris-HCl pH 7.4, 10
mM NaCl, 10 mM MgCl2, 100 μg/mL BSA) individually
before the assay. Compounds were series diluted from 10 mM
stock (for single dose, compounds were diluted from 10 mM
stock to 100 and 20 μM; for IC50, 5× serial dilution was made
from 100 to 0.16 μM) in Aur buffer. Diluted compounds (25
μL) were preincubated with purified 105 ng (10 μL) of GST-
tAurora A (123−401aa) fusion protein at 25 °C for 15 min into
96-well U-bottomed plates (268152, NUNC). Then 5 μM ATP
(5 μL), 1 mM DTT (5 μL), and 0.1 mM tetra(LRRWSLG)
peptide substrate (5 μL) were added into the reactions of test
compounds and GST-tAurora A. The reactions were incubated
at 37 °C for 90 min. Then 50 μL of Kinase-Glo Plus reagent
(V3771, Promega) was added into the reactions, followed by
the incubation at 25 °C for 20 min. Finally, 70 μL of reactions
were transferred to 96-well black plates (237108, NUNC) to
quantify the ATP remaining in the solution. The luminescence
was recorded by vector2 V (1420 multilable HTS counter,
Perkin-Elmer).
1-[4-(2-{(6-{4-[2-(4-Hydroxypiperidin-1-yl)ethoxy]phenyl}-
furo[2,3-d]pyrimidin-4-yl)amino}ethyl)phenyl]-3-phenylurea
35.14 Method E using 10a, bromochloroethane, and 2-
hydroxypiperidine to give 35. Yield, 40%. 1H NMR (300
MHz, CD3OD) δ 8.29 (s, 1H), 7.70 (d, J = 9.3 Hz, 2H), 7.44−
7.26 (m, 7H), 7.13 (d, J = 8.4 Hz, 2H), 7.05−7.00 (m, 1H),
6.94 (d, J = 8.7 Hz, 2H), 6.77 (s, 1H), 4.14 (t, J = 5.7 Hz, 2H),
3.81−3.78 (m, 4H), 3.41−3.39 (m, 1H), 3.23−3.17 (m, 2H),
2.95−2.92 (m, 2H), 2.85 (t, J = 5.7 Hz, 2H), 1.99−1.90 (m,
2H), 1.44−1.37 (m, 2H). LCMS (ESI) m/z: 593 [M + H]+.
1-(4-{2-[(6-{4-[3-(Dimethylamino)prooxy]phenyl}furo[2,3-
d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 36.
Method E using 10a, bromochloropropane, and dimethylamine
to give 36. Yield, 35%. 1H NMR (300 MHz, CD3OD) δ 8.22 (s,
1H), 8.03 (s, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.4 Hz,
2H), 7.35 (d, J = 8.7 Hz, 2H), 7.27 (t, J = 7.8 Hz, 2H), 7.21 (d,
J = 8.4 Hz, 2H), 7.05 (d, J = 9.0 Hz, 2H), 7.00 (s, 1H), 6.98−
6.96 (m, 1H), 4.15 (t, J = 5.4 Hz, 2H), 3.78 (t, J = 7.2 Hz, 2H),
2.91 (t, J = 7.2 Hz, 2H), 2.78 (t, J = 5.4 Hz, 2H), 2.35 (s, 6H),
1.83 (m, 2H). LCMS (ESI) m/z: 551 [M + H]+. HPLC purity:
93%.
Aurora Kinase B Enzyme Inhibition Assay.30 Test
compounds, recombinant human Aurora kinase B (PV3970,
purchased from Invitrogen, Carlsbad, CA, USA), peptide
substrate tetra(LRRASLG), DTT, and ATP were dissolved in
Aur buffer (50 mM Tris-HCl pH 7.4, 10 mM NaCl, 10 mM
MgCl2, 100 μg/mL BSA) individually before the assay.
Compounds were series diluted from 10 mM stock (for single
dose, compounds were diluted from 10 mM stock to 10 μM
and 1 μM; for IC50, 4× serial dilution was made from 10 μM to
0.16 nM) in Aur buffer. Diluted compounds were preincubated
with 40 ng Aurora B proteins at 25 °C for 15 min into 96 well
U-bottomed plate (268152, NUNC), 5 μM ATP (5 μL), 1 mM
DTT (5 μL), and 0.1 mM tetra(LRRASLG) peptide substrate
(5 μL) were added into the reactions of test compound and
Aurora B mixture. The reactions were incubated at 37 °C for 90
min. Then 50 μL of Kinase-Glo Plus reagent (V3771, Promega,
Madison, WI, USA) was added into the reactions, followed by
the incubation at 25 °C for 20 min. Then 70 μL of reactions
were transferred to 96-well black plates (237108, NUNC) to
quantify the ATP remaining in the solution. The luminescence
was recorded by vector2 V (1420 multilable HTS counter,
Perkin-Elmer).
HCT-116 Antiproliferative Assay, Flow Cytometry
Analysis, and Western Blot Analysis. HCT-116 antiproli-
ferative assay of newly synthesized compounds and flow
cytometry analysis of HCT-116 cells treated with compound 27
was carried out as reported in our previous publication.13
Western blot analysis of HCT-116 cells treated with compound
27 was performed as reported in our previous publication.10
Mitotic Progression Assay. To determine mitotic
progression, HeLa cells were first synchronized in S phase
with double thymidine block. Accordingly, cells were treated
with 2.5 mM thymidine for 16 h, washed with 1× phosphate
buffered saline (PBS), trypsinized, and reseeded in culture dish.
After 12 h, cells were treated again with 2.5 mM thymidine for
another 12 h. Then cells were released from the second
thymidine block by washing twice with culture medium. To
determine the mitotic index, cells were stained with
Hoechst33342, and the cell-cycle content was scored from
cells harvested every 2 h between 10 and 18 h after the second
1-(4-{2-[(6-{3-[2-(Dimethylamino)ethoxy]phenyl}furo[2,3-
d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 37.
Method C using 10b and 2-(chloroethyl)dimethylamine to
1
give 37. Yield, 38%. H NMR (300 MHz, CD3OD) δ 8.31 (s,
1H), 8.01 (d, J = 5.7 Hz, 2H), 7.39−7.18 (m, 9H), 7.00−6.93
(m, 2H), 6.95 (s, 1H), 6.86−6.85 (m, 1H), 4.04 (t, J = 5.7 Hz,
2H), 3.66 (t, J = 6.3 Hz, 2H), 2.80 (t, J = 6.3 Hz, 2H), 2.72 (t, J
= 5.7 Hz, 2H), 2.34 (s, 6H). LCMS (ESI) m/z: 537 [M + H] +.
HPLC purity: 90%.
Aqueous Solubility Determination of 4 and 27.
Solubility tests were carried out by suspending 10 mg of test
compound in double distilled water (1 mL), followed by
stirring the mixture at rt for 24 h. The mixture was filtered to
remove undissolved test compound. The amount of test
compound dissolved in the filtrate was determined by HPLC.
Calibration curves were prepared by dissolving known
concentration of the test compounds in DMSO and
determining the AUC of the peaks obtained from HPLC.
HPLC conditions were same as that used for the purity
determination of the compounds.
Aurora Kinase A Protein Purification. The GST-tAurora
A (123−401aa) fusion protein was produced by baculovirus
expression system. The Aurora A catalytic domain with an N-
terminal GST tag was constructed in pBacPAK8 plasmid and
expressed in sf9 cells. Recombinant baculovirus infected sf9
cells were harvested by centrifugation, and the pellets were
resuspended in PBS buffer (PBS, pH 7.3, 0.2 mM PMSF, 0.5
mM Na3VO4, 0.5 mM EDTA, 2 mM DTT, Complete Protease
Inhibitor Cocktail table (1125700, Roche). Cells were lysed by
sonication, and lysates were cleared by centrifugation at 15000
rpm for 30 min. The supernatants were loaded into 1 mL of
GST Sepharose 4 Fast Flow (17-5132-01, GE healthcare)
column previously washed with PBS buffer. The column were
washed with 30 volumes of PBS buffer and then eluted by
elution buffer (50 mM Tris (pH 8.0), 10 mM glutathione). To
concentrate GST-tAurora A, buffer was replaced with Tris
buffer (100 mM Tris (pH 7.5), 300 mM NaCl, 1 mM EDTA, 4
mM DTT) using Amicon ultra-15 (MWCO:30K, Millipore) to
2.4 mg/mL. After the addition of equal volume of glycerol and
L
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
thymidine release by the cell cycle analysis using MetaMorph
software (Molecular Devices).
AUTHOR INFORMATION
Corresponding Author
*Phone: +886-37-246-166 ext 35708. Fax: +886-37-586-456. E-
mail: hphsieh@nhri.org.tw.
■
Immunofluorescence Staining and Confocal Micros-
copy. The immunofluorescence staining and confocal
microscopy were performed as described previously.14 In
brief, cells were fixed in 4% paraformaldehyde in PBS,
permeabilized with 0.1% TritonX-100, and immunostained
with a goat antilamin B1 antibody (Santa Cruz). After three
washes with PBS, cells were probed with Alexa-488-conjugated
secondary antibody (Invitrogen). Cell nuclei were counter-
stained with Hoechst33342 (Invitrogen). Cells were mounted
onto glass slides with ProLong Gold antifade reagent
(Invitrogen) and were visualized using a Leica TCS SP5
confocal microscope. Images were processed by the Imaris 7.2.1
(Bitplane) software.
Docking of Ligands to Aurora Kinase A Co-crystal
Structure. X-ray co-crystal structure of Aurora A protein in
complex with inhibitor 4 (PDB ID: 3M11) was used for
docking utilizing Discovery Studio 2.1.31 The docking of the
ligands was done by DS/LigandFit program with CHARMm
forcefield.32 To bring the docked ligand−protein complex to
equilibrium, the DS/Simulation program was used to run
molecular dynamics simulation. The Minimization convergent
was used in two step methods: Steepest Descent with RMS
gradient convergent to 0.1, and the final step was Conjugate
Gradient with RMS Gradient convergent to 0.0001. Default set
of parameters were used for the simulation, except for the
Production parameter, which was set as 100000 steps. All the
minimization process was in water environment by the
Distance-Dependent Dielectrics methods. After molecular
dynamics simulation, the binding energy was calculated by
DS/Calculate Binding Energies protocol.
In Vivo Pharmacological Evaluation of 27 in HCT-116
Subcutaneous Xenograft Nude Mice Model. Adapted
from the previous report,33 adult male athymic nu/nu nude
mice (BioLASCO, Ilan, Taiwan) were sc implanted with human
colorectal HCT 116 (1 × 106) cancer cells per mouse. Human
colorectal HCT 116 cancer cells were propagated in McCoy’s
5A Medium containing 10% fetal bovine serum (Gibco,
Gaitherburg, MD). Tumor sizes were measured using an
electronic caliper and calculated in length × (width)2/2. Tumor
size and animal body weight were monitored twice a week. The
animals were treated with the HCl salt of compound 27 (10
and 50 mg kg−1, QD; 20 mg kg−1, BID) and compound 1 (50
mg kg−1, QD) when the tumor size reached 100−200 mm3.
The compounds were dissolved in a mixture of DMSO/
Cremophor EL/saline (10/20/70% in v/v/v) and administered
iv into the tumor-bearing nude mice once or twice daily for 5
days per week for 2 weeks. Animal body weight and tumor size
were measured twice a week. For all groups except for
treatment with 27 at 10 mg/kg, n = 8 mice; n = 7 mice for the
treatment group 27 at 10 mg/kg. Data were subjected to
ANOVA followed by Student−Newman−Keuls test using SPSS
software (SPSS, Chicago, IL, USA). A p value <0.05 between
groups was considered statistically significant.
Author Contributions
∥These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from National Health Research Institutes and
National Science Council (NSC-101-2325-B-400-003 and
NSC-101-2113-M-400-002-MY4 for H.P.H.; NSC-99-2113-M-
400-002-MY3, for H.Y.S.), Taiwan and Science & Engineering
Research Board (SR/FT/LS-64/2011 for M.S.C.), India, are
gratefully acknowledged.
DEDICATION
■
Dedicated to Professor Biing-Jiun Uang on the occasion of his
60th birthday.
ABBREVIATIONS USED
■
BID, twice a day; Flt3, Fms-like tyrosine kinase 3; HCT-116,
human colon cancer-116 cell line; HeLa, human cervical cancer
cells; HURP, hepatoma up-regulated protein; LipE, lipophilic
efficiency; Log D, logarithm of distribution coefficient; NHAC,
non-hydrogen atom count; NTRK1, neurotrophic tyrosine
kinase receptor type 1; PDB ID, Protein Data Bank
identification number; pKa, negative logarithm of acid
dissociation constant; QD, once a day; RAF1, v-raf-1 murine
leukemia viral oncogene homologue 1; SEM, standard error of
the mean
REFERENCES
■
(1) Noble, M. E.; Endicott, J. A.; Johnson, L. N. Protein kinase
inhibitors: insights into drug design from structure. Science 2004, 303,
1800−1805.
(2) Druker, B. J.; Lydon, N. B. Lessons learned from the
development of an abl tyrosine kinase inhibitor for chronic
myelogenous leukemia. J. Clin. Invest. 2000, 105, 3−7.
(3) Chahrour, O.; Cairns, D.; Omran, Z. Small molecule kinase
inhibitors as anti-cancer therapeutics. Mini-Rev. Med. Chem. 2012, 12,
399−411.
(4) Fu, J.; Bian, M.; Jiang, Q.; Zhang, C. Roles of Aurora kinases in
mitosis and tumorigenesis. Mol. Cancer Res. 2007, 5, 1−10.
(5) Gautschi, O.; Heighway, J.; Mack, P. C.; Purnell, P. R.; Lara, P.
N., Jr.; Gandara, D. R. Aurora kinases as anticancer drug targets. Clin.
Cancer Res. 2008, 14, 1639−1648.
(6) Carmena, M.; Earnshaw, W. C. The cellular geography of aurora
kinases. Nature Rev. Mol. Cell Biol. 2003, 4, 842−854.
(7) 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.; Miller, K. M. VX-
680, a potent and selective small-molecule inhibitor of the Aurora
kinases, suppresses tumor growth in vivo. Nature Med. 2004, 10, 262−
267.
(8) Oslob, J. D.; Romanowski, M. J.; Allen, D. A.; Baskaran, S.; Bui,
M.; Elling, R. A.; Flanagan, W. M.; Fung, A. D.; Hanan, E. J.; Harris, S.;
Heumann, S. A.; Hoch, U.; Jacobs, J. W.; Lam, J.; Lawrence, C. E.;
McDowell, R. S.; Nannini, M. A.; Shen, W.; Silverman, J. A.; Sopko, M.
M.; Tangonan, B. T.; Teague, J.; Yoburn, J. C.; Yu, C. H.; Zhong, M.;
Zimmerman, K. M.; O’Brien, T.; Lew, W. Discovery of a potent and
selective aurora kinase inhibitor. Bioorg. Med. Chem. Lett. 2008, 18,
4880−4884.
ASSOCIATED CONTENT
■
S
* Supporting Information
Aurora kinase A inhibition of 25a, kinase selectivity profile, and
mice body weight data for in vivo antitumor evaluation of 27.
This material is available free of charge via the Internet at
http://pubs.acs.org.
M
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(9) Coumar, M. S.; Wu, J. S.; Leou, J. S.; Tan, U. K.; Chang, C. Y.;
Chang, T. Y.; Lin, W. H.; Hsu, J. T.; Chao, Y. S.; Wu, S. Y.; Hsieh, H.
P. Aurora kinase A inhibitors: identification, SAR exploration and
molecular modeling of 6,7-dihydro-4H-pyrazolo-[1,5-a]pyrrolo[3,4-
d]pyrimidine-5,8-dione scaffold. Bioorg. Med. Chem. Lett. 2008, 18,
1623−1627.
(10) Coumar, M. S.; Leou, J. S.; Shukla, P.; Wu, J. S.; Dixit, A. K.; Lin,
W. H.; Chang, C. Y.; Lien, T. W.; Tan, U. K.; Chen, C. H.; Hsu, J. T.;
Chao, Y. S.; Wu, S. Y.; Hsieh, H. P. Structure-based drug design of
novel Aurora kinase A inhibitors: structural basis for potency and
specificity. J. Med. Chem. 2009, 52, 1050−1062.
(11) Lin, W. H.; Song, J. S.; Chang, T. Y.; Chang, C. Y.; Fu, Y. N.;
Yeh, C. L.; Wu, S. H.; Huang, Y. W.; Fang, M. Y.; Lien, T. W.; Hsieh,
H. P.; Chao, Y. S.; Huang, S. F.; Tsai, S. F.; Wang, L. M.; Hsu, J. T.;
Chen, Y. R. A cell-based high-throughput screen for epidermal growth
factor receptor pathway inhibitors. Anal. Biochem. 2008, 377, 89−94.
(12) Coumar, M. S.; Tsai, M. T.; Chu, C. Y.; Uang, B. J.; Lin, W. H.;
Chang, C. Y.; Chang, T. Y.; Leou, J. S.; Teng, C. H.; Wu, J. S.; Fang,
M. Y.; Chen, C. H.; Hsu, J. T.; Wu, S. Y.; Chao, Y. S.; Hsieh, H. P.
Identification, SAR studies, and X-ray co-crystallographic analysis of a
novel furanopyrimidine aurora kinase A inhibitor. ChemMedChem
2010, 5, 255−267.
(13) Coumar, M. S.; Chu, C. Y.; Lin, C. W.; Shiao, H. Y.; Ho, Y. L.;
Reddy, R.; Lin, W. H.; Chen, C. H.; Peng, Y. H.; Leou, J. S.; Lien, T.
W.; Huang, C. T.; Fang, M. Y.; Wu, S. H.; Wu, J. S.; Chittimalla, S. K.;
Song, J. S.; Hsu, J. T.; Wu, S. Y.; Liao, C. C.; Chao, Y. S.; Hsieh, H. P.
Fast-forwarding hit to lead: aurora and epidermal growth factor
receptor kinase inhibitor lead identification. J. Med. Chem. 2010, 53,
4980−4988.
(14) Wu, J. M.; Chen, C. T.; Coumar, M. S.; Lin, W. H.; Chen, Z. J.;
Hsu, J. T.; Peng, Y. H.; Shiao, H. Y.; Lin, W. H.; Chu, C. Y.; Wu, J. S.;
Lin, C. T.; Chen, C. P.; Hsueh, C. C.; Chang, K. Y.; Kao, L. P.; Huang,
C. Y.; Chao, Y. S.; Wu, S. Y.; Hsieh, H. P.; Chi, Y. H. Aurora kinase
inhibitors reveal mechanisms of HURP in nucleation of centrosomal
and kinetochore microtubules. Proc. Natl. Acad. Sci. U. S. A. 2013, 110,
E1779−E1787.
(15) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimeraa
visualization system for exploratory research and analysis. J. Comput.
Chem. 2004, 25, 1605−1612.
(16) Liao, J. J. Molecular recognition of protein kinase binding
pockets for design of potent and selective kinase inhibitors. J. Med.
Chem. 2007, 50, 409−424.
(17) Ke, Y. Y.; Shiao, H. Y.; Hsu, Y. C.; Chu, C. Y.; Wang, W. C.;
Lee, Y. C.; Lin, W. H.; Chen, C. H.; Hsu, J. T.; Chang, C. W.; Lin, C.
W.; Yeh, T. K.; Chao, Y. S.; Coumar, M. S.; Hsieh, H. P. 3D-QSAR-
assisted drug design: identification of a potent quinazoline-based
Aurora kinase inhibitor. ChemMedChem 2013, 8, 136−148.
(18) Poulsen, A.; Blanchard, S.; Soh, C. K.; Lee, C.; Williams, M.;
Wang, H.; Dymock, B. Structure-based design of PDK1 inhibitors.
Bioorg. Med. Chem. Lett. 2012, 22, 305−307.
(19) Cui, J. J.; Tran-Dube, M.; Shen, H.; Nambu, M.; Kung, P. P.;
Pairish, M.; Jia, L.; Meng, J.; Funk, L.; Botrous, I.; McTigue, M.;
Grodsky, N.; Ryan, K.; Padrique, E.; Alton, G.; Timofeevski, S.;
Yamazaki, S.; Li, Q.; Zou, H.; Christensen, J.; Mroczkowski, B.;
Bender, S.; Kania, R. S.; Edwards, M. P. Structure based drug design of
crizotinib (PF-02341066), a potent and selective dual inhibitor of
mesenchymal-epithelial transition factor (c-MET) kinase and ana-
plastic lymphoma kinase (ALK). J. Med. Chem. 2011, 54, 6342−6363.
(20) Mortlock, A. A.; Foote, K. M.; Heron, N. M.; Jung, F. H.;
Pasquet, G.; Lohmann, J. J.; Warin, N.; Renaud, F.; De Savi, C.;
Roberts, N. J.; Johnson, T.; Dousson, C. B.; Hill, G. B.; Perkins, D.;
Hatter, G.; Wilkinson, R. W.; Wedge, S. R.; Heaton, S. P.; Odedra, R.;
Keen, N. J.; Crafter, C.; Brown, E.; Thompson, K.; Brightwell, S.;
Khatri, L.; Brady, M. C.; Kearney, S.; McKillop, D.; Rhead, S.; Parry,
T.; Green, S. Discovery, synthesis, and in vivo activity of a new class of
pyrazoloquinazolines as selective inhibitors of aurora B kinase. J. Med.
Chem. 2007, 50, 2213−2224.
(21) Bembenek, S. D.; Tounge, B. A.; Reynolds, C. H. Ligand
efficiency and fragment-based drug discovery. Drug Discovery Today
2009, 14, 278−283.
(22) Perola, E. An analysis of the binding efficiencies of drugs and
their leads in successful drug discovery programs. J. Med. Chem. 2010,
53, 2986−2997.
(23) Hann, M. M.; Keseru, G. M. Finding the sweet spot: the role of
nature and nurture in medicinal chemistry. Nature Rev. Drug Discovery
2012, 11, 355−365.
(24) Glover, D. M.; Leibowitz, M. H.; McLean, D. A.; Parry, H.
Mutations in aurora prevent centrosome separation leading to the
formation of monopolar spindles. Cell 1995, 81, 95−105.
(25) Gadea, B. B.; Ruderman, J. V. Aurora kinase inhibitor
ZM447439 blocks chromosome-induced spindle assembly, the
completion of chromosome condensation, and the establishment of
the spindle integrity checkpoint in Xenopus egg extracts. Mol. Biol. Cell
2005, 16, 1305−1318.
(26) Marumoto, T.; Honda, S.; Hara, T.; Nitta, M.; Hirota, T.;
Kohmura, E.; Saya, H. Aurora-A kinase maintains the fidelity of early
and late mitotic events in HeLa cells. J. Biol. Chem. 2003, 278, 51786−
51795.
(27) Manfredi, M. G.; Ecsedy, J. A.; Meetze, K. A.; Balani, S. K.;
Burenkova, O.; Chen, W.; Galvin, K. M.; Hoar, K. M.; Huck, J. J.;
LeRoy, P. J.; Ray, E. T.; Sells, T. B.; Stringer, B.; Stroud, S. G.; Vos, T.
J.; Weatherhead, G. S.; Wysong, D. R.; Zhang, M.; Bolen, J. B.;
Claiborne, C. F. Antitumor activity of MLN8054, an orally active
small-molecule inhibitor of Aurora A kinase. Proc. Natl. Acad. Sci. U. S.
A. 2007, 104, 4106−4111.
(28) Kallio, M. J.; McCleland, M. L.; Stukenberg, P. T.; Gorbsky, G.
J. Inhibition of aurora B kinase blocks chromosome segregation,
overrides the spindle checkpoint, and perturbs microtubule dynamics
in mitosis. Curr. Biol. 2002, 12, 900−905.
(29) Hauf, S.; Cole, R. W.; LaTerra, S.; Zimmer, C.; Schnapp, G.;
Walter, R.; Heckel, A.; van Meel, J.; Rieder, C. L.; Peters, J. M. The
small molecule Hesperadin reveals a role for Aurora B in correcting
kinetochore−microtubule attachment and in maintaining the spindle
assembly checkpoint. J. Cell Biol. 2003, 161, 281−294.
(30) Koresawa, M.; Okabe, T. High-throughput screening with
quantitation of ATP consumption: a universal non-radioisotope,
homogeneous assay for protein kinase. Assay Drug Dev. Technol. 2004,
2, 153−160.
(31) Discovery Studio, version 2.1; Accelyrs Inc.: San Diego, CA,
2008.
(32) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.;
Swaminathan, S.; Karplus, M. CHARMM: A program for macro-
molecular energy minimization and dynamics calculations. J. Comput.
Chem. 1983, 4, 187−217.
(33) Hu, C. B.; Chen, C. P.; Yeh, T. K.; Song, J. S.; Chang, C. Y.;
Chuu, J. J.; Tung, F. F.; Ho, P. Y.; Chen, T. W.; Lin, C. H.; Wang, M.
H.; Chang, K. Y.; Huang, C. L.; Lin, H. L.; Li, W. T.; Hwang, D. R.;
Chern, J. H.; Hwang, L. L.; Chang, J. Y.; Chao, Y. S.; Chen, C. T.
BPR0C261 is a novel orally active antitumor agent with antimitotic
and anti-angiogenic activities. Cancer Sci. 2011, 102, 182−191.
N
dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX