Discovery of novel purine derivatives with potent and selective inhibitory activity against c-Src tyrosine kinase

Article abstract of DOI:10.1016/j.bmc.2010.05.032

We report here the discovery of novel purine derivatives with potent and selective inhibitory activity against c-Src tyrosine kinase by adopting a strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and bioass

Full text of DOI:10.1016/j.bmc.2010.05.032

Bioorganic & Medicinal Chemistry 18 (2010) 4615–4624
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel purine derivatives with potent and selective inhibitory
activity against c-Src tyrosine kinase
a
b
a,
He Huang a, , Jingui Ma b, , Jianmei Shi a, Linghua Meng b, , Hualiang Jiang , Jian Ding , Hong Liu
*
*
a Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road,
Zhangjiang Hi-Tech Park, Shanghai 201203, PR China
b Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road,
Zhangjiang Hi-Tech Park, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here the discovery of novel purine derivatives with potent and selective inhibitory activity
against c-Src tyrosine kinase by adopting a strategy integrating focused combinatorial library design, vir-
tual screening, chemical synthesis, and bioassay. Thirty two compounds were selected and synthesized.
All compounds showed potent inhibitory activity against c-Src kinase with IC50 values ranging from
Received 30 March 2010
Revised 11 May 2010
Accepted 12 May 2010
Available online 19 May 2010
3.14 lM to 0.02 lM. Compound 5i was identified as one of the most potent agent with an IC50 120 times
lower than those of the hits. The high hit rate (100%) and the potency of the new Src kinase inhibitors
demonstrated the efficiency of the strategy for the focused library design and virtual screening. The novel
active chemical entities reported here should be good leads for further development of purine-based anti-
cancer drugs targeting Src tyrosine kinase.
Keywords:
c-Src tyrosine kinase
Inhibitor
Purine derivative
Focused library
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
9H-purin-6-amine (1b, Table 1) exhibited moderate inhibitory
activity, with the IC50 values of 2.43 lM and 2.42 lM, respectively.
c-Src kinase, a non-receptor tyrosine kinase, is the best under-
stood member of a family of related kinases known as the SFKs
(Src family kinases).1 c-Src play a major role in multiple intracellular
signal transduction pathways involved in cell growth, differentia-
tion, survival, adhesion, and migration.2–5 It has been demonstrated
that Src is overexpressed or constitutively active in a variety of hu-
man tumors, including those derived from colon, breast, pancreas, li-
ver, brain, and bladder.3,6–9 Moreover, aberrant Src kinase activity is
associated with the invasive phenotype in both early and advanced
solid tumors.10 Thus, the discovery of Src kinase inhibitor holds high
potential to treat metastatic tumors.11–18
Purine analogs represent an excellent scaffold for targeting
many biosynthetic, regulatory, and signal transduction proteins
including cellular kinases, G proteins, and polymerases.19–31 Figure
1 presents some representative purine derivatives that are potent
inhibitors of the c-Src kinase.21 Based on the above findings and
the availability of abundant substituted purine compounds in our
in-house collection, we were intrigued to screening our com-
pounds on Src kinase. One of the exciting screening results is that
compounds N-(4-methoxyphenyl)-2-(piperazin-1-yl)-9H-purin-6-
amine (1a, Table 1) and N-(3-methylphenyl)-2-(piperazin-1-yl)-
Interestingly, they are different from the most reported purine-
based Src kinase inhibitors which are often the 2,6,9-trisubstituted
purines. Moreover, all the 2,6,9-trisubstituted purines and 6,9-
disubstituted purines screened by us showed poor activity against
the Src kinase (Table 1).
The focused library and virtual screening have emerged as real-
izable and cost-effective tools in drug discovery.32 A focused li-
brary is built on the basis of a lead molecule or pharmacophore
and geared toward one particular molecular target. Generally,
extracting fragments from known drugs or ligands (inhibitors or
activators) of the studied target is an effective approach for collect-
ing new building blocks.7,12,33–35 Meanwhile, druglikeness could be
introduced into the library design to reduce its size and increase its
efficiency. Herein, we described the discovery of novel purine
derivatives with potent and selective inhibitory activity against
c-Src tyrosine kinase by adopting a strategy integrated with fo-
cused combinatorial library design, virtual screening, chemical
synthesis, and bioassay.
2. Materials and methods
2.1. Focused combinatorial library design
* Corresponding authors. Fax: +86 21 50807088 (H.L.), +86 21 50806722 (L.M.).
E-mail addresses: lhmeng@mail.shcnc.ac.cn (L. Meng), hliu@mail.shcnc.ac.cn (H.
Liu).
The PIPELINE PILOT 7.0 program36 was used in the focused library
designing. Having been kept the common moiety of compounds 1a
Two authors contribute equally to this work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.032

4616
H. Huang et al. / Bioorg. Med. Chem. 18 (2010) 4615–4624
O
P
O
P
F3C
HN
HO
HO HO
NH
N
NH
N
NH
N
HO
6
O
HO
N
N
N
N
N
N
N
N
N
HO
N
H
HO
N
H
N
H
9
2
CGP79883
NVP-AAK980
AP-23236
Figure 1. Representative purine-based inhibitors of the Src kinase.
and 1b of the purine framework, the 2,6-disubstituted purine-based
Src kinase inhibitors can be divided into two parts: (A) 6-amines or -
anilines substituents; (B) 2-alkyl side chains (Fig. 2). From fragments
of Src kinase inhibitors discovered above and previously, 15 frag-
ments for part A, 8 fragments for part B, and 4 frameworks were se-
lected (Fig. 3). These fragments from different parts could react with
each other to generate a library containing 480 (15 Â 8 Â 4) mole-
cules. The druglikeness of the molecules from the combinatorial
library was then evaluated by the druglikeness filter module of PIPE-
LINE PILOT 7.0. The druglikeness descriptor set is composed of the
‘Rule of Five’ (Num_Atom > 0; N_Count + O_Count 6 11; Molecu-
lar_Weight 6 500;Num_H_Donors 6 5; Alog P 6 2.5). Other param-
eters used in the PIPELINE PILOT 7.0 were default values. In this way, 308
compounds were filtered for further evaluation.
Table 1
Inhibitory effect of the selected compounds in in-house collection against Src kinase activity by using ELISA assaya
R1
N
N
R2
N
N
R3
Compound
R1H
R2
R3
IC50
(
l
M)
% Inhibition at 10
90.5
l
Mb
1a
–H
–H
2.43 0.39
2.42 0.66
MeO
NH2
HN
N
HN
N
N
1b
86.6
NH2
MeO
MeO
NH2
NH2
HN
O
1c
1d
1e
1f
>10
>10
>10
>10
>10
>10
>10
>10
>10
22.5
16.0
35.8
20.2
10.1
16.4
13.9
6.9
N
NH2
HN
O
N
NH2
N
N
N
Br
O
NH2
O
1g
1h
1i
NH
O
NH2
–H
–H
–H
Br
O
NH2
1j
NH
1k
0
PP2c
99
a
The inhibition rate (%) was calculated using the equation: [1 À (A490/A490 control)] Â 100%. IC50 values were obtained by Logit method based on the data obtained from
three separate experiments; each compound concentration was tested in duplicate. % Inhibition rate at 10
lM.
b
Values are means of three determinations and deviation from the means is <10% of the mean value.
PP2 was used as positive control.
c

H. Huang et al. / Bioorg. Med. Chem. 18 (2010) 4615–4624
4617
A
R
NH
N
N
N
N
H
N
HN
purine framework
B
µ
1a, R = 4-OMe, IC50 = 2.43 mol/L
µ
1b, R = 3-Me, IC50 = 2.42 mol/L
Figure 2. Structures of compounds 1a–b, and three regions of them.
Figure 4. Discovery strategy and experimental flowchart in this study.
MeO
Table 2
NH MeO
Br
NH
O2N
NH
H2NO2S
NH
NH
NH
Chemical structures and Src kinase inhibitory activities of compounds 4a–i, 5a–l and
6a–ra
Cl
R
O
R
R
NH
NH
NH
N
NH
N
NH
N
N
N
N
N
N
H2NOC
NH
N
H
N
H
N
H
N
OH
N
N
N
N
HN
O2N
NH
NH
4a-i
5a-l
6a-k
AcHN
O
Compound
R
IC50 (lM)
N
NH
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
5i
3-Methoxy
4-Chloro
4-Bromo
4-Nitro
1.21 0.16
1.18 0.04
1.76 0.33
1.40 0.24
0.95 0.53
1.82 0.02
0.10 0.04
2.20 0.07
1.05 0.47
0.75 0.20
0.13 0.01
0.93 0.41
0.83 0.36
0.34 0.16
0.45 0.23
0.39 0.20
0.13 0.01
0.02 0.002
0.12 0.04
2.01 0.90
0.65 0.35
3.14 0.17
1.33 0.11
1.20 0.30
2.55 1.53
0.69 0.18
0.78 0.11
0.34 0.07
0.26 0.01
0.34 0.08
1.22 0.23
1.59 0.41
0.05 0.03
H2NO2S
NH
NH
Fragments for part A
3-Nitro
4-Aminosulfonyl
3-Aminosulfonyl
4-Carbamoyl
4-Acetamido
H
4-Methoxy
3-Methoxy
3-Methyl
4-Chloro
4-Bromo
4-Nitro
3-Nitro
4-Aminosulfonyl
3-Aminosulfonyl
4-Carbamoyl
4-Acetamido
H
4-Methoxy
3-Methoxy
3-Methyl
N
N
N
N
N
N
N
H
N
N
Cl
Frameworks
HN
N
N
N
NH
N
O
OH
O
5j
5k
5l
O
NH2
N
6a
6b
6c
6d
6e
6f
6g
6h
6i
Fragments for part B
Figure 3. Building blocks for focused library generation. The symbols ꢀ represents
the sites where fragments connect each other to form a complete structure.
4-Chloro
4-Nitro
3-Nitro
4-Aminosulfonyl
3-Aminosulfonyl
4-Carbamoyl
4-Acetamido
2.2. Virtual screening
6j
6k
A computational docking was performed by using DISCOVERY STU-
DIO 2.1 (DS 2.1) program37 in order to identify the fragments and
the possible interaction between the ligand and the Src kinase.
The X-ray crystal structure of Src kinase (PDB entry No.: 1y57)
was used as a target for the virtual screening. The binding potential
between Src kinase and molecules in the combinatorial library was
PP2b
a
The inhibition rate (%) was calculated using the equation: [1 À (A490/A490
control)] Â 100%. IC50 values were obtained by Logit method based on the data
obtained from three separate experiments; each compound concentration was
tested in duplicate. % Inhibition rate at 10
l
M.
b
PP2 was used as positive control.

4618
H. Huang et al. / Bioorg. Med. Chem. 18 (2010) 4615–4624
R
N
R
NH
N
Cl
NH
N
N
N
N
a
N
b
N
H
N
H
N
Cl
N
2
N
H
Cl
N
HN
3
1a-b, 4a-i
d
c
R
R
NH
NH
N
N
N
N
N
H
N
H
N
N
N
N
N
O
HO
6a-k
5a-l
Scheme 1. Reagents and conditions: (a) aniline (1.05 equiv.), n-pentanol, 70 °C, 5 h; (b) morpholine (5.0 equiv.), NaBF4 (1.0 equiv.), DMSO, microwave, 5 min; (c) piperazine
(5.0 equiv.), NaBF4 (1.0 equiv.), DMSO, microwave, 5 min; (d) 2-(piperazin-1-yl)ethanol (5.0 equiv.), NaBF4 (1.0 equiv.), DMSO, microwave, 5 min.
evaluated by the Ligandfit module of DS 2.1 (Forcefiled: CHARMm;
Pose Saving Maximum Poses Retained: 10). Conformational flexi-
bility of the molecules from the library was considered in the dock-
ing search. Other parameters used in the DS 2.1 were default
values. Three-hundred and eight molecules passed the druglike-
ness filter module were ranked by the scoring functions of Ligand-
fit, and analyzed for relatively lower binding energy, favorable
shape complementarity, and potential in forming hydrogen bonds
with Src kinase. In this way, 32 compounds (4a–i, 5a–l, and 6a–k)
were finally selected for synthesis and bioassay (Fig. 4).
and the plate was incubated at 37 °C for 30 min. The plate was
washed, then 100 L citrate buffer (0.1 M, pH 5.5) containing
0.03% H2O2 and 2 mg/mL o-phenylenediamine was added and sam-
ples were incubated at room temperature until color emerged. The
reaction was terminated by adding 50 lL of 2 M H2SO4, and the plate
was read using a multiwell spectrophotometer (VERSAmax™,
Molecular Devices, Sunnyvale, CA, USA) at 492 nm. The inhibitory
rate (%) was calculated with the formula: [1 À (A492/A492 control)] Â
100%. IC50 values were calculated from the inhibitory curves.
l
2.5. Molecular docking simulation
2.3. Chemical synthesis
To explore a more detail interaction mechanism between the
most active compound and Src kinase, molecular docking simula-
tion was carried out with the program AUTODOCK 4.0.1.38 The
three-dimensional (3D) structure of target protein of Src kinase is
from Protein Data Bank (PDB entry No.: 1y57). The ligand and sol-
vent molecules were removed from the crystal structure to obtain
the docking grid and the active site was defined using AutoGrid.
The grid size was set to 60 Â 60 Â 60 points with grid spacing of
0.375 Å. The grid box was centered on the center of the ligand from
the corresponding crystal structure complexes. The Lamarckian ge-
netic algorithm (LGA) is used for docking with the following set-
tings: a maximum number of 1500,000 energy evaluations, an
initial population of 50 randomly placed individuals, a maximum
number of 37,000 generations, a mutation rate of 0.02, a crossover
rate of 0.80 and an elitism value (number of top individuals that
automatically survive) of 1. The ligand was fully optimized inside
the binding site during the docking simulations. Finally, the confor-
mation with the lowest predicted binding free energy of the most
occurring binding modes in Src active pocket was selected.
On the basis of evaluation of binding potential and druglikeness,
we selected 32 compounds (4a–i, 5a–l, and 6a–k) (Table 2) for syn-
thesis and bioassay. Scheme 1 depicts the sequence of reactions
that led to the preparation of desired products using 2,6-dichloro-
purine 2 as the starting material. In general, the 2-chloro-N-aro-
matic-9H-purin-6-amine 3 were prepared by 2,6-dichloropurine
2 through a nucleophilic addition reaction with anilines in n-pent-
anol. Subsequently, replacement of the C2 chloro by reacting with
amines under microwave irradiation conditions in DMSO gave the
targeted compounds 4a–i, 5a–l, and 6a–k.
2.4. Tyrosine kinase assays
The tyrosine kinase activities of the purified proteins were
determined in 96-well ELISA plates (Corning, NY) precoated with
20
10
l
g/mL Poly(Glu,Tyr)4:1 (Sigma, St. Louis, MO). Briefly, 50
lL
lM ATP solution diluted in kinase reaction buffer (50 mM
HEPES pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2, 0.2 mM Na3VO4,
1 mM DTT) was added to each well. Then 10 L of various concen-
l
trations of test compounds or reference compound dissolved in
DMSO were added to each reaction well. The kinase reaction was
performed in triplicate and initiated by adding tyrosine kinase di-
3. Results and discussion
3.1. Focused library design and evaluation
luted in 40
for 60 min, the wells was washed three times with phosphate buf-
fered saline (PBS) containing 0.1% Tween 20 (T-PBS). Next, 100
lL of kinase reaction buffer. After incubation at 37 °C
In our preliminary study, we discovered two 2,6-disubstituted
lL
purine derivatives 1a and 1b exhibiting obvious Src kinase inhibi-
tory activities, with IC50s of 2.43
lM and 2.42 lM, respectively.
Their chemical structures contain three parts: part A is a hydro-
phobic amines head, part B is a relatively hydrophilic alkyl tail,
and framework is a linker between A and B (Fig. 2). To discover
new chemical entities of Src kinase inhibitors with more potent
anti-phosphotyrosine (PY99) antibody (1:500, Santa Cruz Biotech-
nology, Santa Cruz, CA) diluted in T-PBS containing 5 mg/mL BSA
was added and the plate was incubated at 37 °C for 30 min. After
the plate was washed three times, 100 lL horseradish peroxi-
dase-conjugated goat anti-mouse IgG (1:2000, Calbiochem, San
Diego, CA) diluted in T-PBS containing 5 mg/mL BSA was added

H. Huang et al. / Bioorg. Med. Chem. 18 (2010) 4615–4624
4619
activities, a focused library was designed using the structural frag-
ments isolated by the above and previously discovered inhibitors
(15 fragments for part A, 8 fragments for part B, and 4 frame-
works). Combining these fragments together, a 15 Â 8 Â 4 focused
library was obtained. Subsequently, the druglikeness of molecules
from the combinatorial library was evaluated. Three-hundred and
eight compounds which passed the filtration were picked out for
further evaluation. Targeting the crystal structure of Src kinase,
the focused library was then screened by using the scoring func-
tion of Ligandfit module that has been encoded in DS2.1. The dock-
ing results indicated that: (i) there is no significant difference
between diverse amine substituents, but significant difference be-
tween amines and anilines substituents in part A; (ii) morpholine,
piperazine, and 2-(piperazin-1-yl)ethanol substituents account for
more than 70% in the top 50 molecules in part B; (iii) purine scaf-
fold presents a dominant framework in the top 50 molecules.
Accordingly, as the Src kinase inhibitors, the building blocks for
parts B and the frameworks are fixed at morpholine, piperazine,
2-(piperazin-1-yl)ethanol, and purine. The fragment for part A is
a little bit flexible. Considering the drug-like and docking screening
analysis and synthetic feasibility, 32 compounds (4a–i, 5a–l, and
6a–k) were selected and synthesized for actual biological testing,
and their chemical structures and bioassay results are listed in Ta-
ble 1. These 32 compounds were synthesized through the routes
outlined in Schemes 1, and the details for synthetic procedures
and structural characterizations are described in Section 5.
2). Substitution studies revealed extremely similar results at this po-
sition. Morpholine analogs (5a–l) showed the best effect, and the
activities of both the piperazine and 2-(piperazin-1-yl)ethanol ana-
logs (4a–i and 6a–k) decrease slightly. Our attention turned next to
the modification of the substituents at the 6-position of the purine.
Overall, substituents at both the para- and meta-positions on the
amine ring are optimal for Src kinase inhibitory activity of these ana-
logs. 1a and 1b exhibited moderate inhibition, with the IC50 values of
2.43 lM and 2.42 lM, respectively. No significant change was ob-
served at the enzyme level when removing the substituentson 6-po-
sition amines (5a and 6a). The methyl or methoxy substituent can be
replaced with halogens, carbamoyl, and acetamido groups ( 4b–c,
4h–i, 5e–f, 5k–l, 6e, and 6j–k) without loss of potency, while the
substituents can be replaced with a nitro group (4d–e, 5g–h, and
6f–g) with minimal increase of potency. Introduction of a sulfon-
amide group (4f–g, 5i–j, and 6h–i) led to significant enhancement
in potency. This improved enzyme potency could result from an in-
creased donor and acceptor characters of the sulfonamide moiety
binding the hinge region.
To explore the detailed binding characteristics of the com-
pounds, the binding model of the most active compound 5i with
c-Src tyrosine kinase was constructed. Figure 5 shows the interac-
tion model of 5i to c-Src, indicating that it occupies the adenine
pocket of the ATP-binding site, and locates at the hinge region
through H-bonds with amino acid residues. The benzene sulfon-
amide head stretched deeply into the binding pocket with hydro-
phobic interaction, which was surrounded by residues Thr338,
Lys295, and Asp404. The relatively hydrophilic alkyl tail appears
to fit snugly at the entrance of the ATP-binding site. There are three
H-bonds between 5i and the hinge region of the Src kinase. 6-Posi-
tion NH group forms two hydrogen bonds with the backbone car-
bonyl and NH of Met341, respectively, at the hinge region of the
protein. The sulfonamide H atom forms an H-bond with the side-
chain nitrogen of Thr338. The binding model of the representative
compound highlights some of the critical H-bond interactions with
the active site residues which were proved to be the key for kinase
inhibitory activity. It may exert its tyrosine kinase inhibitory activ-
ity through the H-bonds to Met341 and Thr338, which are consis-
tent with the previous reports.
3.2. Effectiveness of the approach
The hit rate of our design and screening approach is 100%
regarding to binding. Of the 32 inhibitors, 18 compounds (4e, 4g,
5a–j, 5l, and 6e–i) showed potent activities in inhibiting the kinase
activity of Src (Table 2), indicating that the hit rate with IC50
s
against the enzymatic activity at submicromolar range is as high
as 56.25%. The activity of the most potent one (Compound 5i) im-
proved by more than 120 times with an IC50 of 0.02 lM. These re-
sults demonstrated that our approach not only enriched the hit
rate of active compounds but also helped to discover new enzyme
inhibitors with increased potency. Therefore, we might conclude
that our approach, focused library construction combined with
chemical synthesis and bioassay, is effective in discovering active
compounds based on the 3D structure of a target.
3.4. Biological profile of compound 5i
3.4.1. Compound 5i selectively inhibits c-Src tyrosine kinase
activity
3.3. Structure–activity relationships
To evaluate the selectivity profile, the most active inhibitor 5i
was tested in an enzymatic assay against a panel of 13 tyrosine ki-
nases. The data confirmed the potency of 5i against c-Src, and re-
vealed 5i was 100-fold and 300-fold less potent against Kit and
Fragments for part A are structurally more diverse than frag-
ments for part B. As part of a systematic SAR approach, we first inves-
tigated the importance of the alkyl side chains at 2-position (Table
Figure 5. Binding model of compound 5i in the binding cleft of Src kinase. The left part (A) and middle part (B) show the docking models of 5i into the active sites of Src
kinase. The right part (C) describes the detailed interactions between 5i and Src kinase. These images were generated using the PYMOL program (http://www.pymol.org).

4620
H. Huang et al. / Bioorg. Med. Chem. 18 (2010) 4615–4624
Table 3
were observed in the cell invasion assay (Fig. 7B). To exclude the
Effects of 5i on tyrosine kinase activities in cell-free systema
inhibitory effects of 5i on cell migration and invasion are due to its
ability to inhibit cell proliferation, MTT assays were performed in
MDA-MB-231 cells treated with the indicated concentrations of 5i
for 24 h. More than 95% of the cells were viable after 5i treatment
(data not shown). These findings revealed that the effects of 5i on
Src kinase activity resulted in decreased p-Src and p-FAK and con-
tributed to inhibition of cell migration and invasion.
Kinase
IC50
(l
M)
% Inhibition at 10 l
Mb
Src
c-Abl
Kit
0.02 0.002
6.00 1.91
1.35 0.15
>10
>10
>10
97.8
73.4
83.1
18.5
43.1
47.2
0.0
EGFR
ErbB2
KDR
VEGFR1
PDGFRb
FGFR1
FGFR2
EphA2
EphB2
c-Met
>10
>10
>10
>10
17.2
48.7
4.1
4. Conclusion
In summary, we discovered two 2,6-disubstituted purine deriv-
atives 1a and 1b exhibiting obvious Src kinase inhibitory activity in
the preliminary study. To discover novel entries of Src kinase
inhibitors with more potent activity, a focused library containing
480 compounds was designed based on the chemical structures
of the hits. Through druglikeness analysis and virtual screening,
32 compounds were selected from the focused library for chemical
synthesis and bioassay. Remarkably, all of these compounds show
Src kinase inhibitory activities, with IC50 values ranging from 3.14
>10
>10
>10
48.7
11.5
43.2
a
The inhibition rate (%) was calculated using the equation: [1 À (A490/A490
control)] Â 100%. IC50 values were determined from three separate experiments;
each compound concentration was tested in duplicate.
b
Values are means of three determinations and deviation from the means is <10%
of the mean value.
to 0.02 lM. The hit rate for inhibitors is 100%. The activity of the
most potent one (5i) improved by more than 120 times than those
of the hits. These data demonstrated that our approach could not
only enrich the hit rate of active compounds but also help to dis-
cover new inhibitors with increased the potency. In addition, fur-
ther study showed that 5i exhibited highly selectivity for Src
kinase among a panel of tyrosine kinases and inhibited phosphor-
ylation of Src and its downstream signal transduction in MDA-MB-
231 cells, which is accompanied with significant inhibition of cell
migration and invasion. These results may provide insights into
the discovery and development of novel 2,6-disubstituted purine
derivatives with potent Src kinase inhibitory activities.
5. Experimental section
5.1. Chemistry
The reagents (chemicals) were purchased from commercial
sources (Alfa, Acros, Sigma–Aldrich and Shanghai Chemical Re-
agent Company), and used without further purification. Analytical
thin layer chromatography (TLC) was HSGF 254 (0.15–0.2 mm
thickness, Yantai Huiyou Company, China). Yields were not opti-
mized. Nuclear magnetic resonance (NMR) spectra were given on
a AMX-300 (Brucker, Fällanden, Switzerland; internal standard as
tetramethylsilane). Chemical shifts were reported in parts per mil-
lion (ppm, d) downfield from tetramethylsilane. Proton coupling
patterns were described as singlet (s), doublet (d), triplet (t), quar-
tet (q), multiplet (m), and broad (br). Low- and high-resolution
(HRMS) mass spectra were given with an electric ionization and
electrospray (EI, ESI) and a LCQ-DECA spectrometer produced by
Finnigan MAT-95 (Finnigan, Santa Clara, CA, USA). Compounds
1c–k were reported previously.39 For the targeted compounds
the chemical purity was determined using an Agilent 1100 Series
HPLC. The purity of each compound was >95% (Table S1, Supple-
mentary data).
Figure 6. Effects of 5i on the phosphorylation of c-Src and FAK in MDA-MB-231
cells. Data shown are representative of three independent Western blot assays.
c-Abl, respectively. Meanwhile, 5i possessed weak inhibitory effect
on EGFR, ErbB2, KDR, VEGFR1, KDR, PDGFR, FGFR1, FGFR2, EphA2,
EphB2, and c-Met with IC50 values above 10 lM (Table 3). These re-
sults indicate that 5i is a highly selective inhibitor of c-Src kinase.
3.4.2. Compound 5i abrogates phosphorylation of c-Src and FAK
in cellular level
To explore the activity of 5i on c-Src in intact cells, MDA-MB-
231 human breast cancer cells that express high levels of c-Src
were utilized. Exposure of MDA-MB-231 cells to 5i resulted in
down-regulation of phosphorylated of c-Src and FAK, a substrate
of c-Src, in a dose-dependent manner (Fig. 6), while the total pro-
tein levels of these two proteins remain unchanged after treatment
with 5i. These results indicated that 5i inhibited c-Src activity in
tumor cells.
5.1.1. General procedure for the synthesis of 1a–b, 4a–i, 5a–l,
and 6a–k
To a stirred solution of 2,6-dichloro-purine (50 mmol) in 50 mL
n-pentanol, aniline (52.5 mmol) was added at 70 °C. Stirring was
continued until the precipitate was formed. Then the precipitate
was filtered and washed with n-pentanol to give intermediate 3.
The crude product 3 (0.3 mmol) was dissolved in DMSO (4 mL) in
a 10 mL glass vial equipped with a small magnetic stirring bar.
To this was added 5 equiv amine and 5 equiv NaBF4, and the vial
3.4.3. Compound 5i inhibits migration and invasion of MDA-
MB-231 cells
As Src kinase is involvedinregulatingcellmigration and invasion,
we further investigated the effect of 5i on these cellular processes. As
shown in Figure 7A, cell migration was dramatically inhibited in a
dose-dependent manner upon 5i treatment, with a detectable inhi-
bition at 3 lM and substantial inhibition at 10 lM. Similar results

H. Huang et al. / Bioorg. Med. Chem. 18 (2010) 4615–4624
4621
Figure 7. Compound 5i suppresses MDA-MB-231 cell migration and invasion. (A) Compound 5i inhibits cell migration. Wound-healing assay was applied to detect cell
migration. MDA-MB-231 cells were treated with various concentrations of 5i or vehicle for 24 h. Left, representative images of at least three independent experiments were
presented. Right, quantification analysis of cell migration. Columns, mean of three experiments; bars, SD. (B) Compound 5i inhibits cell invasion. Mitragel invasion chamber
was used to detect cell invasion. MDA-MB-231 cells were treated with indicated concentrations of 5i or vehicle. After 18 h, cells invading to the lower aspect of the Boyden
chamber filter were stained and photographed. Left, representative images of at least three independent experiments were presented. Right, quantification analysis of cell
invasion. Columns, mean of three experiments; bars, SD.
was tightly sealed with an aluminum/Teflon crimp top. The mix-
ture was then irradiated for 5 min at 180 °C. After completion of
the reaction, the vial was cooled to ambient temperature. Then
water was added and the precipitated product was collected by fil-
trating. The resulting residuum was washed with water and puri-
fied by flash chromatography to give 1a–b, 4a–i, 5a–l, and 6a–k.
4H), 2.80–2.74 (m, 4H). MS (EI, m/z): 329 [M]+. HRMS (EI): calcd
for C15H16ClN7 [M]+, 329.1156, found 329.1145.
5.1.1.5. N-(4-Bromophenyl)-2-(piperazin-1-yl)-9H-purin-6-
amine (4c). Prepared from 4-bromoaniline and piperazine.
Yield: 80%. Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 7.90 (s,
1H), 7.85 (d, J = 9.0 Hz, 2H), 7.48 (d, J = 9.0 Hz, 2H), 3.65–3.60 (m,
4H), 2.80–2.74 (m, 4H). MS (EI, m/z): 373 [M]+. HRMS (EI): calcd
for C15H16BrN7 [M]+, 373.0651, found 373.0647.
5.1.1.1. N-(4-Methoxyphenyl)-2-(piperazin-1-yl)-9H-purin-6-
amine (1a). Prepared from 4-methoxyaniline and piperazine.
Yield: 83%. Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 7.85 (s,
1H), 7.75 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H), 3.74 (s, 3H),
3.62–3.89 (m, 4H), 2.75–2.72 (m, 4H). MS (EI, m/z): 325 [M]+.
HRMS (EI): calcd for C16H19N7O [M]+, 325.1651, found 325.1642.
5.1.1.6. N-(4-Nitroxyphenyl)-2-(piperazin-1-yl)-9H-purin-6-
amine (4d). Prepared from 4-nitroaniline and piperazine. Yield:
76%. Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 8.20 (q,
J = 9.6 Hz, 4H), 7.99 (s, 1H), 3.71–3.67 (m, 4H), 2.87–2.82 (m, 4H).
MS (EI, m/z): 340 [M]+. HRMS (EI): calcd for C15H16N8O2 [M]+,
340.1396, found 340.1392.
5.1.1.2. N-(3-Methylphenyl)-2-(piperazin-1-yl)-9H-purin-6-
amine (1b). Prepared from 3-methylaniline and piperazine.
Yield: 79%. Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 7.88 (s,
1H), 7.79 (s, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H),
6.82 (d, J = 8.0 Hz, 1H), 3.66–3.63 (m, 4H), 2.78–2.75 (m, 4H),
2.29 (s, 3H). MS (EI, m/z): 309 [M]+. HRMS (EI): calcd for
C16H19N7 [M]+, 309.1702, found 309.1693.
5.1.1.7. N-(3-Nitroxyphenyl)-2-(piperazin-1-yl)-9H-purin-6-
amine (4e). Prepared from 3-nitroaniline and piperazine. Yield:
78%. Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 8.10 (d,
J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.87 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H),
7.58 (t, J = 8.0 Hz, 1H), 3.74–3.65 (m, 4H), 2.84–2.76 (m, 4H). MS
(EI, m/z): 340 [M]+. HRMS (EI): calcd for C15H16N8O2 [M]+,
340.1396, found 340.1390.
5.1.1.3. N-(3-Methoxyphenyl)-2-(piperazin-1-yl)-9H-purin-6-
amine (4a). Prepared from 3-methoxyaniline and piperazine.
Yield: 84%. Mp 189–191 °C; 1H NMR (300 MHz, DMSO-d6): d 7.89
(s, 1H), 7.67 (s, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 8.0 Hz,
1H), 6.57 (d, J = 8.0 Hz, 1H), 3.75 (s, 3H), 3.68–3.63 (m, 4H), 2.79–
2.73 (m, 4H). MS (EI, m/z): 325 [M]+. HRMS (EI): calcd for
C16H19N7O [M]+, 325.1651, found 325.1643.
5.1.1.8. N-(4-Aminosulfonylphenyl)-2-(piperazin-1-yl)-9H-
purin-6-amine (4f). Prepared from 4-aminobenzenesulfona-
mide and piperazine. Yield: 71%. Mp 219–221 °C; 1H NMR
(300 MHz, DMSO-d6): d 8.05 (d, J = 8.7 Hz, 2H), 7.95 (s, 1H), 7.76
(d, J = 8.7 Hz, 2H), 3.75–3.68 (m, 4H), 2.44–2.37 (m, 4H). MS (EI,
m/z): 374 [M]+. HRMS (EI): calcd for C15H18N8O2S [M]+, 374.1273,
found 374.1262.
5.1.1.4. N-(4-Chlorophenyl)-2-(piperazin-1-yl)-9H-purin-6-
amine (4b). Prepared from 4-chloroaniline and piperazine.
Yield: 87%. Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 7.90 (d,
J = 8.8 Hz, 2H), 7.89 (s, 1H), 7.36 (d, J = 8.8 Hz, 2H), 3.65–3.60 (m,
5.1.1.9. N-(3-Aminosulfonylphenyl)-2-(piperazin-1-yl)-9H-
purin-6-amine (4g). Prepared from 3-aminobenzenesulfona-

4622
H. Huang et al. / Bioorg. Med. Chem. 18 (2010) 4615–4624
mide and piperazine. Yield: 65%. Mp >250 °C; 1H NMR (300 MHz,
DMSO-d6): d 7.93 (s, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.52–7.42 (m,
2H), 7.29 (s, 1H), 3.72–3.63 (m, 4H), 2.82–2.74 (m, 4H). MS (EI,
m/z): 374 [M]+. HRMS (EI): calcd for C15H18N8O2S [M]+, 374.1273,
found 374.1263.
Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 8.21 (q, J = 9.5 Hz,
4H), 8.01 (s, 1H), 3.73–3.65 (m, 8H). MS (EI, m/z): 341 [M]+. HRMS
(EI): calcd for C15H15N7O3 [M]+, 341.1236, found 341.1232.
5.1.1.19. N-(3-Nitrophenyl)-2-morpholino-9H-purin-6-amine
(5h). Prepared from 3-nitroaniline and morpholine. Yield: 80%.
Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 8.12 (d, J = 8.0 Hz,
1H), 8.01 (s, 1H), 7.91 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.58 (t,
J = 8.0 Hz, 1H), 3.72 (br s, 8H). MS (EI, m/z): 341 [M]+. HRMS (EI):
calcd for C15H15N7O3 [M]+, 341.1236, found 341.1231.
5.1.1.10. N-(4-Carbamoylphenyl)-2-(piperazin-1-yl)-9H-purin-
6-amine (4h). Prepared from 4-carbamoylaniline and pipera-
zine. Yield: 63%. Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d
7.97 (d, J = 8.6 Hz, 2H), 7.93 (s, 1H), 7.85 (d, J = 8.6 Hz, 2H), 3.68–
3.61 (m, 4H), 2.80–2.74 (m, 4H). MS (EI, m/z): 338 [M]+. HRMS
(EI): calcd for C16H18N8O [M]+, 338.1604, found 338.1588.
5.1.1.20. N-(4-Aminosulfonylphenyl)-2-morpholino-9H-purin-
6-amine (5i). Prepared from 4-aminobenzenesulfonamide and
morpholine. Yield: 76%. Mp >250 °C; 1H NMR (300 MHz, DMSO-
d6): d 8.06 (d, J = 8.8 Hz, 2H), 7.96 (s, 1H), 7.76 (d, J = 8.8 Hz, 2H),
3.73–3.63 (m, 8H). MS (EI, m/z): 375 [M]+. HRMS (EI): calcd for
C15H17N7O3S [M]+, 375.1114, found 375.1101.
5.1.1.11. N-(4-Acetamidophenyl)-2-(piperazin-1-yl)-9H-purin-
6-amine (4i). Prepared from 4-acetamidoaniline and piperazine.
Mp >250 °C; Yield: 69%. 1H NMR (300 MHz, DMSO-d6): d 8.16 (s,
1H), 8.07 (d, J = 8.7 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H), 3.91 (br s,
4H), 3.06 (br s, 4H), 2.33 (s, 3H). MS (EI, m/z): 352 [M]+. HRMS
(EI): calcd for C17H20N8O [M]+, 352.1760, found 352.1762.
5.1.1.21. N-(3-Aminosulfonylphenyl)-2-morpholino-9H-purin-
6-amine (5j). Prepared from 3-aminobenzenesulfonamide and
morpholine. Yield: 76%. Mp >250 °C; 1H NMR (300 MHz, DMSO-
d6): d 7.96 (s, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.51–7.43 (m, 2H), 7.29
(s, 1H), 3.71–3.65 (m, 8H). MS (EI, m/z): 375 [M]+. HRMS (EI): calcd
for C15H17N7O3S [M]+, 375.1114, found 375.1110.
5.1.1.12. N-phenyl-2-morpholino-9H-purin-6-amine (5a). Pre-
pared from aniline and morpholine. Yield: 80%. Mp >250 °C; 1H
NMR (300 MHz, DMSO-d6): d 7.94 (s, 1H), 7.87 (d, J = 8.0 Hz, 2H),
7.32 (t, J = 8.0 Hz, 2H), 7.00 (t, J = 8.0 Hz, 1H), 3.71–3.62 (m, 8H).
MS (EI, m/z): 296 [M]+. HRMS (EI): calcd for C15H16N6O [M]+,
296.1386, found 296.1391.
5.1.1.22. N-(4-Carbamoylphenyl)-2-morpholino-9H-purin-6-
amine (5k). Prepared from 4-carbamoylaniline and morpholine.
Yield: 69%. Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 7.97 (d,
J = 8.4 Hz, 2H), 7.90 (s, 1H), 7.85 (d, J = 8.4 Hz, 2H), 3.73–3.64 (m,
8H). MS (EI, m/z): 339 [M]+. HRMS (EI): calcd for C16H17N7O2
[M]+, 339.1444, found 339.1437.
5.1.1.13. N-(4-Methoxyphenyl)-2-morpholino-9H-purin-6-amine
(5b). Prepared from 4-methoxyaniline and morpholine. Yield:
84%. Mp 244–246 °C; 1H NMR (300 MHz, CDCl3): d 7.60 (d,
J = 8.8 Hz, 2H), 7.50 (s, 1H), 6.90 (d, J = 8.8 Hz, 2H), 3.76 (s, 3H),
3.72–3.65 (m, 8H). MS (EI, m/z): 326 [M]+. HRMS (EI): calcd for
C16H18N6O2 [M]+, 326.1491, found 326.1481.
5.1.1.23. N-(4-Acetamidophenyl)-2-morpholino-9H-purin-6-
amine (5l). Prepared from 4-acetamidoaniline and morpholine.
Yield: 66%. Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 7.88 (s,
1H), 7.78–7.71 (m, 2H), 7.56–7.49 (m, 2H), 3.72–3.58 (m, 8H),
2.03 (s, 3H). MS (EI, m/z): 353 [M]+. HRMS (EI): calcd for
C17H19N7O2 [M]+, 353.1600, found 353.1591.
5.1.1.14. N-(3-Methoxyphenyl)-2-morpholino-9H-purin-6-amine
(5c). Prepared from 3-methoxyaniline and morpholine. Yield:
88%. Mp 242–243 °C; 1H NMR (300 MHz, DMSO-d6): d 7.91 (s, 1H),
7.65 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.57 (d,
J = 8.0 Hz, 1H), 3.74 (s, 3H), 3.69–3.65 (m, 8H). MS (EI, m/z): 326
[M]+. HRMS (EI): calcd for C16H18N6O2 [M]+, 326.1491, found
326.1496.
5.1.1.24. N-Phenyl-2-(4-(2-hydroxyethyl)piperazin-1-yl)-9H-
purin-6-amine (6a). Prepared from aniline and 2-(piperazin-1-
yl)ethanol. Yield: 74%. Mp >250 °C; 1H NMR (300 MHz, DMSO-
d6): d 7.88 (s, 1H), 7.87–7.85 (m, 2H), 7.29 (t, J = 8.4 Hz, 2H), 6.97
(t, J = 8.4 Hz, 1H), 3.70–3.62 (m, 4H), 3.53 (t, J = 6.2 Hz, 1H), 2.48–
2.45 (m, 4H), 2.41 (t, J = 6.2 Hz, 1H). MS (EI, m/z): 339 [M]+. HRMS
(EI): calcd for C17H21N7O [M]+, 339.1808, found 339.1802.
5.1.1.15. N-(3-Methylphenyl)-2-morpholino-9H-purin-6-amine
(5d). Prepared from 3-methylaniline and morpholine. Yield:
81%. Mp 238–240 °C; 1H NMR (300 MHz, DMSO-d6): d 7.90 (s,
1H), 7.77 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H),
6.82 (d, J = 8.0 Hz, 1H), 3.71–3.63 (m, 8H), 2.29 (s, 3H). MS (EI, m/
z): 310 [M]+. HRMS (EI): calcd for C16H18N6O [M]+, 310.1542, found
310.1538.
5.1.1.25. N-(4-Methoxyphenyl)-2-(4-(2-hydroxyethyl)piperazin-
1-yl)-9H-purin-6-amine (6b). Prepared from 4-methoxyaniline
and 2-(piperazin-1-yl)ethanol. Yield: 81%. Mp 240–241 °C; 1H
NMR (300 MHz, DMSO-d6): d 7.83 (s, 1H), 7.75 (d, J = 9.0 Hz, 2H),
6.88 (d, J = 9.0 Hz, 2H), 3.73 (s, 3H), 3.68–3.62 (m, 4H), 3.54 (t,
J = 6.0 Hz, 2H), 2.50–2.45 (m, 4H), 2.42 (t, J = 6.0 Hz, 2H). MS (EI,
m/z): 369 [M]+. HRMS (EI): calcd for C18H23N7O2 [M]+, 369.1913,
found 369.1908.
5.1.1.16. N-(4-Chlorophenyl)-2-morpholino-9H-purin-6-amine
(5e). Prepared from 4-chloroaniline and morpholine. Yield: 79%.
Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 7.92 (s, 1H), 7.90 (d,
J = 8.8 Hz, 2H), 7.36 (d, J = 8.8 Hz, 2H), 3.70–3.61 (m, 8H). MS (EI, m/
z): 330 [M]+. HRMS (EI): calcd for C15H15ClN6O [M]+, 330.0996,
found 330.0995.
5.1.1.26. N-(3-Methoxyphenyl)-2-(4-(2-hydroxyethyl)piperazin-
1-yl)-9H-purin-6-amine (6c). Prepared from 3-methoxyaniline
and 2-(piperazin-1-yl)ethanol. Yield: 75%. Mp 226–227 °C; 1H
NMR (300 MHz, DMSO-d6): d 7.88 (s, 1H), 7.70–7.68 (m, 1H),
7.40 (d, J = 8.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 6.54 (dd, J = 8.0 Hz,
2.3 Hz, 1H), 3.74 (s, 3H), 3.70–3.66 (m, 4H), 3.53 (t, J = 6.2 Hz,
2H), 2.49–2.44 (m, 4H), 2.41 (t, J = 6.2 Hz, 2H). MS (EI, m/z): 369
[M]+. HRMS (EI): calcd for C18H23N7O2 [M]+, 369.1913, found
369.1917.
5.1.1.17. N-(4-Bromophenyl)-2-morpholino-9H-purin-6-amine
(5f). Prepared from 4-bromoaniline and morpholine. Yield:
84%. Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 7.93 (s, 1H),
7.86 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H), 3.73–3.58 (m, 8H).
MS (EI, m/z): 374 [M]+. HRMS (EI): calcd for C15H15BrN6O [M]+,
374.0491, found 374.0479.
5.1.1.18. N-(4-Nitrophenyl)-2-morpholino-9H-purin-6-amine
(5g). Prepared from 4-nitroaniline and morpholine. Yield: 83%.

H. Huang et al. / Bioorg. Med. Chem. 18 (2010) 4615–4624
4623
5.1.1.27. N-(3-Methylphenyl)-2-(4-(2-hydroxyethyl)piperazin-1-
yl)-9H-purin-6-amine (6d). Prepared from 3-methylaniline and
2-(piperazin-1-yl)ethanol. Yield: 80%. Mp >250 °C; 1H NMR
J = 6.3 Hz, 2H), 2.50–2.45 (m, 4H), 2.42 (t, J = 6.3 Hz, 2H), 2.04 (s,
3H). MS (EI, m/z): 396 [M]+. HRMS (EI): calcd for C19H24N8O2 [M]+,
396.2022, found 396.2026.
(300 MHz, DMSO-d6):
d 7.86 (s, 1H), 7.82 (s, 1H), 7.60 (d,
5.2. Pharmacology
J = 8.0 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H),
3.71–3.64 (m, 4H), 3.53 (t, J = 6.2 Hz, 2H), 2.50–2.45 (m, 4H), 2.41
(t, J = 6.2 Hz, 2H), 2.28 (s, 3H). MS (EI, m/z): 353 [M]+. HRMS (EI):
calcd for C18H23N7O [M]+, 353.1964, found 353.1963.
5.2.1. Materials
The kinase domain of tyrosine kinase c-Src, epidermal growth
factor receptor 1 (EGFR/ErbB-1), epidermal growth factor receptor
2 (EGFR-2/ErbB-2), vascular endothelial growth factor receptor 2
(VEGFR-2/KDR), stem cell factor receptor c-kit, fibroblast growth
factor receptor-1 and -2 (FGFR-1, FGFR-2), EphA2, EphB2, and c-
Met were expressed using the Bac-to-Bac baculovirus expression
system (Invitrogen, Carlsbad, CA) and purified on Ni-NTA columns
(QIAGEN Inc., Valencia, CA) as previously described. Vascular endo-
thelial growth factor receptor 1 (VEGFR-1/Flt-1), platelet derived
growth factor receptorb (PDGFRb) and c-Abl kinase were pur-
chased from Upstate Biotechnology Inc. (Charlottesville, VA).
5.1.1.28. N-(4-Chlorophenyl)-2-(4-(2-hydroxyethyl)piperazin-1-
yl)-9H-purin-6-amine (6e). Prepared from 4-chloroaniline and
2-(piperazin-1-yl)ethanol. Yield: 79%. Mp >250 °C; 1H NMR
(300 MHz, DMSO-d6): d 7.91 (d, J = 8.6 Hz, 2H), 7.89 (s, 1H), 7.34
(d, J = 8.6 Hz, 2H), 3.69–3.62 (m, 4H), 3.53 (t, J = 6.2 Hz, 2H),
2.49–2.44 (m, 4H), 2.41 (t, J = 6.2 Hz, 2H). MS (EI, m/z): 373 [M]+.
HRMS (EI): calcd for C17H20ClN7O [M]+, 373.1418, found 373.1409.
5.1.1.29. N-(4-Nitrophenyl)-2-(4-(2-hydroxyethyl)piperazin-1-
yl)-9H-purin-6-amine (6f). Prepared from 4-nitroaniline and
2-(piperazin-1-yl)ethanol. Yield: 75%. Mp >250 °C; 1H NMR
(300 MHz, DMSO-d6): d 8.19 (q, J = 9.0 Hz, 4H), 7.98 (s, 1H),
3.72–3.66 (m, 4H), 3.54 (t, J = 6.2 Hz, 2H), 2.54–2.49 (m, 4H),
2.42 (t, J = 6.2 Hz, 2H). MS (EI, m/z): 384 [M]+. HRMS (EI): calcd
for C17H20N8O3 [M]+, 384.1658, found 384.1648.
5.2.2. Cells lines and cell culture
MDA-MB-231 human breast cancer cells were obtained from
American Type Culture Collection and cultured in Leiboviz’s L15
Medium supplemented with 10% fetal bovine serum, 100 units/mL
penicillin sodium and 100 lg/mL streptomycin sulfate. Cells were
cultured in a humidified atmosphere of 5% CO2 and 95% air at 37 °C.
5.1.1.30. N-(3-Nitrophenyl)-2-(4-(2-hydroxyethyl)piperazin-1-
yl)-9H-purin-6-amine (6g). Prepared from 3-nitroaniline and
2-(piperazin-1-yl)ethanol. Yield: 80%. Mp >250 °C; 1H NMR
(300 MHz, DMSO-d6): d 8.11 (d, J = 8.1 Hz, 1H), 7.96 (s, 1H), 7.88
(s, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.58 (d, J = 8.1 Hz, 1H), 3.79–3.72
(m, 4H), 3.35 (t, J = 5.9 Hz, 2H), 2.57–2.51 (m, 4H), 2.46 (t,
J = 5.9 Hz, 2H). MS (EI, m/z): 384 [M]+. HRMS (EI): calcd for
C17H20N8O3 [M]+, 384.1658, found 384.1648.
5.2.3. Western blot analysis
Cells (4.5 Â 105) were plated in six-well plate overnight, and then
exposedto 5i at concentrations of 1, 3, and 10 lM, or DMSOalone, for
24 h. Western blot analyses were subsequently performed with
standard procedures. Antibodies against the following were used:
p-Src (Tyr416) (#2113) (Cell Signaling Technologies, Cambridge,
MA), Src(sc-19), FAK(sc-557), andGAPDH(sc-47724)wereobtained
from (Santa Cruz Biotechnology, Santa Cruz, CA), p-FAK (Tyr861)
(44-626G) (BioSource International, Camarillo, CA).
5.1.1.31. N-(4-Aminosulfonylphenyl)-2-(4-(2-hydroxyethyl)pip-
erazin-1-yl)-9H-purin-6-amine (6h). Prepared from 4-amino-
benzenesulfonamide and 2-(piperazin-1-yl)ethanol. Yield: 62%.
Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 8.05 (d, J = 8.8 Hz,
1H), 7.95 (s, 1H), 7.76 (d, J = 8.8 Hz, 1H), 3.74–3.66 (m, 4H), 3.52
(t, J = 6.2 Hz, 2H), 2.52–2.48 (m, 4H), 2.45 (t, J = 6.2 Hz, 2H). MS
(EI, m/z): 418 [M]+. HRMS (EI): calcd for C17H22N8O3S [M]+,
418.1536, found 418.1533.
5.2.4. Wound-healing assay
Cells were plated in 24-well tissue culture plates, grown to con-
fluent. Monolayer wounds were produced using a pipette tip
scratched through the center of the well. Medium and non-adher-
ent cells were aspirated, the adherent cells were washed once, and
new medium containing various concentrations of 5i, or DMSO
alone, was added. Photographs of the initial wound were taken
for comparison. After incubation for 24 h, photographs of the same
scraped section were taken. Distances of denuded areas were mea-
sured and compared with time 0.
5.1.1.32. N-(3-Aminosulfonylphenyl)-2-(4-(2-hydroxyethyl)pip-
erazin-1-yl)-9H-purin-6-amine (6i). Prepared from 3-amino-
benzenesulfonamide and 2-(piperazin-1-yl)ethanol. Yield: 66%.
Mp >250 °C; 1H NMR (300 MHz, DMSO-d6): d 7.93 (s, 1H), 7.82
(d, J = 8.1 Hz, 1H), 7.51–7.42 (m, 2H), 7.28 (s, 1H), 3.74–3.68 (m,
4H), 3.55 (t, J = 6.2 Hz, 2H), 2.50–2.46 (m, 4H), 2.44 (t, J = 6.2 Hz,
2H). MS (EI, m/z): 418 [M]+. HRMS (EI): calcd for C17H22N8O3S
[M]+, 418.1536, found 418.1526.
5.2.5. Cell invasion assay
Transwell chamber membranes (6.5 mm diameter, 8
size; Costar, corning, NY) were coated with 100 L of 1 mg/mL
lm pore
l
Matrigel (dissolved in serum-free L15 medium; Becton Dickinson
Sciences, Franklin Lakes, NJ). 10% FBS were added to lower cham-
bers. Cells and various concentrations of compound, or DMSO
alone, were added to the upper chambers at the same time and al-
lowed to invade for 18 h at 37 °C. Cells that had not invaded were
removed from the upper chamber with a cotton swab. The remain-
ing cells were fixed, stained with 0.1% crystal violet, and photo-
graphs were taken under a microscope and measured at 595 nm
after extraction with 10% acetic acid for 10 min.
5.1.1.33. N-(4-Carbamoylphenyl)-2-(4-(2-hydroxyethyl)pipera-
zin-1-yl)-9H-purin-6-amine (6j). Prepared from 4-carbamoy-
laniline and 2-(piperazin-1-yl)ethanol. Yield: 47%. Mp >250 °C;
1H NMR (300 MHz, DMSO-d6): d 7.98 (d, J = 8.6 Hz, 2H), 7.92 (s,
1H), 7.84 (d, J = 8.6 Hz, 2H), 3.75–3.66 (m, 4H), 3.54 (t, J = 6.2 Hz,
2H), 2.54–2.51 (m, 4H), 2.44 (t, J = 6.2 Hz, 2H). MS (EI, m/z): 382
[M]+. HRMS (EI): calcd for C18H22N8O2 [M]+, 382.1866, found
382.1866.
Acknowledgments
We gratefully acknowledge financial support from the State Key
Program of Basic Research of China (Grant 2002CB512802), the Na-
tional Natural Science Foundation of China (Grants 20372069,
29725203, 20721003 and 20872153), the 863 Hi-Tech Program
of China (Grants 2006AA020602 and 2006AA020402), National
5.1.1.34. N-(4-Acetamidophenyl)-2-(4-(2-hydroxyethyl)pip-
erazin-1-yl)-9H-purin-6-amine (6k). Prepared from 4-acet-
amidoaniline and 2-(piperazin-1-yl)ethanol. Yield: 58%. Mp
>250 °C; 1H NMR (300 MHz, DMSO-d6): d 7.86 (s, 1H), 7.77 (d,
J = 8.8 Hz, 2H), 7.50 (d, J = 8.8 Hz, 2H), 3.72–3.63 (m, 4H), 3.55 (t,

4624
H. Huang et al. / Bioorg. Med. Chem. 18 (2010) 4615–4624
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2924.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.032.
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