Discovery, synthesis, and investigation of the antitumor activity of novel piperazinylpyrimidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.02.057

Protein kinases play several pertinent roles in cell proliferation, and targeting these proteins has been shown to be a successful strategy toward controlling different malignancies. Despite the great discovery stories during the last two decades, there i

Full text of DOI:10.1016/j.ejmech.2011.02.057

European Journal of Medicinal Chemistry 46 (2011) 2043e2057
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery, synthesis, and investigation of the antitumor activity of novel
piperazinylpyrimidine derivatives
*
Hassan M. Shallal, Wade A. Russu
Department of Pharmaceutics & Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, 3601 Pacific Ave., Stockton, CA 95211, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein kinases play several pertinent roles in cell proliferation, and targeting these proteins has been
shown to be a successful strategy toward controlling different malignancies. Despite the great discovery
stories during the last two decades, there is still a demand for anticancer small molecules with the
potential of being selective on both the protein kinase and/or the cellular level. A series of novel
piperazinylpyrimidine compounds were synthesized and tested for their potential to selectively inhibit
the growth of certain tumor cell lines included within the NCI-60 cell line panel. MDA-MB-468, a triple-
negative/basal-like breast carcinoma, cell line was among the most sensitive cell lines towards
compounds 4 and 15. The three most interesting compounds identified in cellular screens (4, 15, and 16)
were subjected to kinase profiling and found to have an interesting selective tendency to target certain
kinase subfamily members; PDGFR, CK1, RAF and others. Compound 4 showed a selective tendency to
bind to and/or inhibit the function of certain KIT and PDGFRA mutants compared to their wild-type
isoforms. Piperazinylpyrimidine based derivatives represent a new class of selective kinase inhibitors.
Significantly 4 is more potent at inhibiting oncogenic mutant forms of PDGFR family kinases, which is
relevant in terms of its potential use in treating tumors that have become resistant to treatment or driven
by such mutations. The clinical demand for agents useful in the control of triple-negative/basal-like
breast cancer justifies our interest in compound 15 which is a potent growth inhibitor of MDA-MB-468
cell line.
Received 22 November 2010
Received in revised form
19 February 2011
Accepted 22 February 2011
Available online 3 March 2011
Keywords:
Piperazinylpyrimidine
NCI-60
Kinases
MDA-MB-468
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
molecularly targeted anticancer agents; a statement that is easily
verified by noting that approximately 14 kinase targeting agents
The human kinome, represented by 518 kinases, is probably one
of the most investigated protein families on biological, chemical,
and clinical levels. This is mainly due to the fact that protein kinases
are involved in the majority of signal transduction pathways
regulating the cell machinery of survival, proliferation and main-
tenance. Accordingly, several pathological abnormalities are corr-
elated with aberrations in the operational integrity and accuracy of
certain kinases inside the cell. This makes kinases very attractive
targets for treating, diagnosing and establishing personalized
therapies of various disorders such as different malignancies,
neurodegenerative disorders, rheumatoid arthritis, autoimmune
diseases, and others [1e4]. Kinase inhibitors, either small mole-
cules or monoclonal antibodies, represent a very strategic class of
have earned FDA approval during the last two decades as either
anticancer or antiangiogenic agents. In comparison, not as many
agents targeting other caner-relevant families such as Bcl-2
proteins, histone deacetylases, and phosphatases, have gained
regulatory approval. Furthermore, the discovery, preclinical, and
clinical development of novel kinase inhibitors currently absorb the
lion’s share of pharmaceutical industry developmental budgets and
research efforts especially those who are seeking novel and effec-
tive cancer controlling agents [5,6].
As commonly believed by the kinase focusing research
community, the design of selective kinase inhibitors has proven to
be quite a challenging task due to the conservation of ATP-binding
site, targeted by most inhibitors, among different kinases. Several
design strategies, both computer assisted, bioinformatics aided,
and structural based, have been implemented to tailor more
selective kinase inhibitors against a subset of kinases or certain
kinase subfamilies [2,7e13]. One successful example is lapatinib
which is known to be a selective inhibitor against many wild-type
* Corresponding author. Tel.: þ1 209 946 2339; fax: þ1 209 946 2160.
E-mail address: wrussu@pacific.edu (W.A. Russu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.057

2044
H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
and mutant EGFR subfamily members and is currently utilized
clinically in combination with capecitabine for metastatic breast
cancer. In contrast, sunitinib, another successfully marketed kinase
inhibitor, has been shown in several studies to be a highly
promiscuous agent capable of interacting with more than 15% of
kinases with a very high affinity (Kd < 100 nM) [14,15]. The selec-
tivity of lapatinib compared to the promiscuity of sunitinib is
usually rationalized by the observation that lapatinib is a type-II
inhibitor which binds to the ATP-binding site as well as penetrating
the adjacent allosteric binding site of its target kinases, whereas
sunitinib is a type-I inhibitor that binds mainly to the ATP-binding
site of several kinases [16,17]. However, it is unjustified, according
to several published reports, to claim that every type-I kinase
inhibitor is promiscuous and that every type-II inhibitor is selective
[15,18]. Additionally, a given small molecule kinase inhibitor
usually tends to recognize a given conformational ensemble of its
target kinase(s) that may happen to differentially belong to either
the active and/or the inactive state. That is to say that a type-I
inhibitor, for example, may still bind to the inactive form of its
kinase but with less affinity than with the active state. Considering
another dimension of variability, some inhibitors interact potently
with both the active and the inactive forms of their targeted
kinases. For example, MK-2461 is able to bind with considerable
potency to both the phosphorylated and the unphosphorylated
forms of c-MET kinase with a measured binding constant (Kd) of 4.4
and 27.2 nM respectively [19]. Contrary to the behavior of MK-2461,
sunitinib exhibits a strong differential selectivity towards the
unactivated wild-type KIT versus the active form [16]. Generally
speaking, the clinical fact that both lapatinib and sunitinib have
successfully helped save or at least improve the life quality of
certain cancer patient populations illustrates that, when it comes to
kinase inhibitors, it is arguable that selectivity is always a virtue and
non-selectivity is a constant drawback. In fact, some kinase
modulating agents may achieve better clinical outcomes via tar-
geting several kinases whereas others can cause troublesome side
effects even while being selective [20]. Moreover, small molecules
generally and kinase inhibitors specifically are usually promiscuous
hitters of several protein families and that could explain why not all
potent kinase inhibitors survive through the drug development
process [21]. The phenomenon of kinase inhibitors being mostly
non-selective has inspired the founding of several high-throughput
kinase profiling screens in order to help determining intended as
well as off-target kinases affected by a given kinase inhibitor
[15,22,23]. These screens have made it possible to investigate the
kinase binding potential or the functional inhibitory activity of
a given small molecule against a large panel of kinases and thereby
have facilitated uncovering some of the structural features relevant
to promiscuity. Considering the current information we have about
both kinases and their inhibitors, it is unreasonable to claim that
a given small molecule is either selective or promiscuous kinase
inhibitor without experimental support via performing a larger
scale kinase profiling. However, it is now appreciated that the
kinome space along with its multidimensional conformational
space depends on structural features, at the primary, secondary and
tertiary levels, that make a given kinase more promiscuous than
another even with a difference of only a few amino acids [24].
Attention has been brought to the necessity of a deeper under-
standing of the promiscuity from the perspective of the protein
kinases which has revealed very interesting correlations between
the ATP-binding sites of certain subfamilies that are not closely
related based on sequence similarity [25]. Resistance through
several mechanisms, most frequently single point mutation, has
been developed by cancer cells towards several kinase inhibitors;
those that target proliferation and/or angiogenesis including both
selective and non-selective kinase inhibitors [26e28].
The major aim of this report is to identify new lead molecules,
from a series of novel piperazinylpyrimidine derivatives, which
inhibit the growth of cancer cell lines. This series design is ratio-
nally based on targeting kinases. The kinase binding and inhibition
activity of the most potent molecules is presented.
2. Results & discussion
2.1. Design
Our major aim was to investigate the potential of novel piper-
azinylpyrimidine derivatives to achieve antiproliferative activity
and/or kinase binding. Piperazinylpyrimidine moiety was chosen
because of its synthetic accessibility, availability of relevant starting
materials, and its ability to provide four H-bond accepting nitro-
gens symmetrically oriented in space. In the current investigation,
a systematic attachment of a quinazoline ring, recognized as
a kinase privileged fragment, to the piperazinylpyrimidine moiety,
through linkers that position the two aforementioned fragments in
different relative orientations, has been adopted by our group as
a strategy in our quest to discover novel anticancer agents and/or
prototype kinase inhibitors. Towards achieving this goal, a novel
series of 16 compounds, that are easily synthesized, were designed
to target the human kinome via linking the two major fragments,
mentioned above, either directly, group-I, or through an orientation
linker, group-II, which offers more rigidity (n ¼ 0) or more flexi-
bility (n ¼ 1) (Fig. 1). It is important to mention that only aromatic
linkers based on benzene ring were chosen in this study to avoid
excessive conformational flexibility inherent to aliphatic linkers
and to facilitate deriving spatial-activity relationships. Additionally,
considering that synthetic accessibility is one of the major factors
that should be considered during the early design phase, starting
materials allowing the incorporation of aromatic linkers are much
more readily available and cheaper than their aliphatic analogs. The
designed derivatives were tailored to localize to the hinge region of
kinases via their kinase privileged fragment and extend either
through the ATP-binding groove or towards the adjacent allosteric
site so as to be either type-I or type-II inhibitors respectively or
perhaps even adopt a novel binding mode. There are two major
rationales upon which this design strategy is based i) certain
kinases within the massive kinome space, 518 kinases adopting
a wide range of conformational ensembles of both active and
inactive states, may be able to accommodate the piperazinylpyr-
imidine scaffold attached directly or indirectly to the quinazoline
moiety; the relevant question to be addressed is whether this is
true or not and if it is true, which kinases are sensitive to these
designed compounds, and ii) exploiting the steric space of a given
class of small molecules with very similar chemical features that
are positioned differently within the binding site may generate
more than one lead kinase inhibitor with different respective
Fig. 1. Design strategy of final compounds 3e6 and 13e24.

H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
2045
kinase selectivity profiles and/or anticancer cellular activity. We
decided to narrow down the compounds to be kinome profiled by
using anticancer cellular assays as primary screening followed by
kinase profiling of only those compounds with encouraging cellular
activity profiles and that incorporated different linkers. Such an
order of experiments would avoid missing possible cytotoxic or
cytostatic lead compounds which may fail to interact with their
intended targets and happen to interact with another protein
family that wasn’t anticipated. This initial set of designed
compounds was intended to be followed by a latter optimization
cycle if they failed to show any promising activity on both the
cellular and protein levels or to be further investigated if they
exhibited interesting profiles on either the cellular or the protein
level.
Scheme 2.
were initially accepted for screening against the NCI-60 at a single
concentration of 10 M. Eight out of the above compounds (3e4,13,
15e16, and 19e21) showed a significant antiproliferative activity at
a 10 M dose and were accordingly evaluated at five concentrations
2.2. Chemistry
m
The final compounds were derived from two major piper-
azinylpyrimidine key intermediates (Schemes 1 and 2). The avail-
ability of starting materials made the execution of two different
schemes for synthesizing two otherwise structurally similar inter-
mediates convenient. Intermediate 1 was reported in the literature
and the deprotection step was performed using HCl/MeOH method
[29]. Intermediate 2 was synthesized using palladium catalyzed
Suzuki cross coupling followed by deprotection as illustrated in
Scheme 2. Group-1 compounds (3e6) were prepared directly by
allowing the respective 4-chloroquinazoline derivative to react
with one of the piperazinylpyrimidine intermediates (Scheme 3).
Group-I compounds represent the direct attachment between the
ATP-binding site localizing quinazoline fragment and the selectivity
inducing piperazinylpyrimidine scaffold. Intermediates 7e12 were
synthesized by allowing m-aminobenzoic acid, p-aminobenzoic
acid, or p-aminomethylbenzoic acid to react with the correspond-
ing quinazoline derivative using similar reaction conditions to
those implemented in preparing group-I compounds (Scheme 4).
Scheme 4 demonstrates the connection of the quinazoline frag-
ment with the different linkers implemented in the design of
group-II compounds (13e24). Group-II derivatives were prepared
via coupling the carboxylic acid group of the intermediates (7e12)
with the secondary aliphatic amine functionality of either inter-
mediate 1 or intermediate 2 using EDC. HCl and triethylamine
mixture as outlined in Scheme 5. Group-II is structurally different
from group-I via the incorporation of a linker between the two
major fragments. Such a linker strategy offers three different
options; m-aminophenylcarbonyl and p-aminophenylcarbonyl
provide similar rigidity level while orienting the fragments differ-
ently whereas p-aminomethylphenylcarbonyl provides more flex-
ibility that allows the compound to cover more conformational
space in orienting the major fragments.
m
in order to determine their dose-response behavior and calculate
their GI50 (dose inhibiting 50% of the growth compared to the
control), TGI (dose inhibiting the growth completely), and LC50
(dose killing 50% of the starting cell population) values. Table 1
shows the GI50, TGI, and LC50 values for compounds 4, 15, and 16.
Three compounds (3e4 and 15) showed an interesting pattern of
selective cytotoxic activity and were submitted for retesting in
order to confirm the reproducibility of their cellular actions. The
three compounds gave reproducible results and compound 15 has
been chosen by the DTP-BEC (Developmental Therapeutic
Program-Biological Evaluation Committee) to advance to testing in
vivo [30e32]. Compounds 3 and 4 gave very similar profile against
the NCI-60 according to their structural similarity. However, 3
showed an overall partially weaker cytotoxic activity than 4 and
that’s why the former was neither considered in any further
investigation nor will it be mentioned throughout the rest of the
current report.
Compound 16 (NSC: D-750776) showed the highest overall
potency and non-selectivity against the cell lines and it did not
display a large difference between its cytostatic markers (mean
GI50 ¼ 1.05
m
M and mean TGI ¼ 3.24
mM) and cytotoxic indicator
M). These data could be projected into potential
(LC50 ¼ 22.91
m
toxicity against normal cells resulting in a narrow therapeutic index
which may validate the decision made by the NCI-BEC not to
advance 16 into animal testing. On the other hand, 4 (NSC:
D-750901) and 15 (NSC: D-750905) displayed a very interesting
pattern of selective cytostatic potency against certain cell lines
scattered among tissues of different origin. For example, 15 was
very potent against RPMI-8226 (leukemia, GI50 ¼ 68 nM), NCI-H23
(lung, GI50 ¼ 90 nM), SK-MEL-5 (skin, GI50 ¼ 61 nM), and MDA-MB-
468 (breast, GI50 ¼ 30 nM) cancer cell lines. These two derivatives,
4 and 15, also showed a remarkable difference between their
cytostatic markers (mean GI50 & mean TGI) and cytotoxic indicator
(mean LC50). These data may classify 4 and 15 as cytostatic
compounds whereas 16 may be described as a cytotoxic agent.
According to the data presented in Table 1, and the testing reports
2.3. In vitro cellular screening
Target compounds, 3e6 and 13e24, were screened in-house
against A549 (NSCLC) and MCF7 (breast cancer) cell lines to test the
target compounds’ cytotoxic potential and to reduce the number of
compounds to be further investigated (data not shown). Structures
of the active compounds were submitted for screening against the
NCI-60 cell lines panel. Compounds (3e4, 13, 15e16, and 19e23)
Scheme 1.
Scheme 3.

2046
H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
Therefore, the overall selectivity of compounds 4 and 15 against the
NCI-60 cell line panel accompanied with their potent cytostatic
action against MDA-MB-468 cell line represents an interesting
phenomenon to be investigated. MDA-MB-468 cell line is known to
overexpress EGFR receptor [34]. Therefore, gefitinib, known as an
EGFR inhibitor, was included in the study as a positive control.
According to the histogram shown in Fig. 3, the antiproliferative
activity of a given test compound was directly proportional to the
treatment time and the concentration. The ranking of activity
according to the obtained IC50 values is as follows: 4 and 15 are both
more antiproliferative than 16 than gefitinib against this specific
cell line. A more in-depth biologically oriented investigation of the
molecular mechanism of growth inhibitory action of 15 against
MDA-MB-468 is currently ongoing and its results will be reported
in due time.
Scheme 4.
From the above described in vitro cellular screening of several
piperazinylpyrimidine derivatives, it can be concluded that some
compounds belonging to this class exhibit different yet interesting
behaviors against different cancer cell lines. This foundation
prompted us to further investigate the promising derivatives and
halt any further optimization effort for the meantime. 4, 15, and 16
were chosen for other experiments because they showed the most
selective, 4 and 15, or global, 16, antitumor activity and because
they represent the three major structural scenarios of this small
class. At this point during the investigation, the question was
whether these three compounds, shown to be optimized for
promising cellular activity, are able to bind to kinases.
obtained from the NCI-60 screening, the relationships between the
structure variations of the piperazinylpyrimidine derivatives and
their cytostatic/cytotoxic activities can be summarized as shown in
Fig. 2. A benzyl group in the 5-position of pyrimidine ring leads to
more antiproliferative activity compared to ethyl substitution.
Derivatives based on 2-phenylquinazoline show overall weaker
antiproliferative potential compared to the unsubstituted quina-
zoline based derivatives. Concerning the linkers in group-II
compounds, the para orientation shows the least cytotoxic activity
whereas the meta positioning leads to the most potent derivatives
with 16 being a clear example of this summation. Incorporation of
a flexibility inducing element has refined the series into the very
selective cytostatic performance of 15. The strong correlation
between the cellular profile of 4 (group-I) and 15 (group-II) in both
terms of cellular selectivity and cytostatic rather than cytotoxic
action raises the hypothesis that they both can affect similar set of
targets in the cell and that 15, through its flexible methylene group,
is able to attain a conformational ensemble very similar to those
attained by the more rigid derivative, 4, devoid of a linker.
The concentration and time dependencies of the anti-
proliferative activity of compounds 4, 15, and 16 against MDA-MB-
468 cell line were further investigated by our group using MTT
assay. Several reasons have promoted us to give attention to this
specific cell line. Firstly, this cell line represents the triple-negative/
basal-like breast carcinoma which is more prevalent in younger
women and women of African-American decent, is usually
accompanied by frequent relapses and poor survival. As reported in
the literature, there is a current therapeutic demand for efficient yet
safe agents to be used in this specific clinical setting. Secondly,
MDA-MB-468 cell line is significantly sensitive to DNA-damaging
agents which are known to be highly toxic and non-selective [33].
2.4. Kinase profiling of 4, 15, and 16
The presented series of novel compounds were structurally
designed with the intention to interfere with certain members of
the human kinome. Two major questions needed to be tackled.
Firstly, can these compounds act as kinase inhibitors? And if they
can, which kinases can they affect? Compounds 4, 15, and 16 were
selected because they represent the three major structural deter-
minants of our design and also because they were either selective
cytostatic or potent cytotoxic on the cellular level. Compound
4 represents the direct attachment of the quinazoline ring with the
piperazinylpyrimidine scaffold (group-I). Compound 15 exemplifies
the use of a more flexible linker whereas compound 16 has a more
rigid incorporated linker and both belong to group-II (Fig. 1). There
are many commercial services established to facilitate screening
against large panels of kinases. However, each method has its own
benefits as well as limitations. Accordingly, two different kinase
experiments were chosen for the current study. One is classified as
a binding or non-biochemical assay while the other is functional
and biochemically oriented [23].
In order to test the potential of 4, 15, and 16 to bind with the
human kinome to which they were designed to target, a kinase
binding assay was used to examine the ability of 10
16 to interfere with the binding of a given kinase with an immo-
bilized, ATP directed agent [15,35e40]. A single dose of 10 M was
mM of 4, 15, or
m
chosen to allow for detecting kinases able to bind to the test
compounds. In this binding assay, a set of 243 kinases were
included. This set of kinases covers many possibilities i) wild-type
versus mutant kinases ii) phosphorylated versus non-phosphory-
lated set of either wild-type or mutant ABL1 kinases (Table 2). For
a complete list of the kinases tested, refer to the supporting infor-
mation (Table S1). Details of the experiment are provided in the
experimental section. It is however relevant to mention that a given
interaction is represented by a numerical value called binding
percentage of control (B-POC). The lower the B-POC value, the
stronger the test compound binds with the test kinase. Only results
with B-POC ꢀ 50 are shown in Table 3. The kinases are ranked from
Scheme 5.

H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
2047
Table 1
The GI50 (dose inhibiting 50% of the growth compared to control), TGI (dose inhibiting the growth completely), and LC50 (Dose killing 50% of the starting number of cells) values
result from screening of compounds 4, 15, and 16 against the NCI-60 cell line panel after 48 h treatment. All values are given in
mM. Cells highlighted in cyan have GI50values:
0 < GI50 ꢀ 0.1
m
M, while cells highlighted in yellow have values: 0.1 < GI50 ꢀ 1
mM.
Tissue
Cell line
Compound 4
Compound 15
Compound 16
GI50
TGI
LC50
GI50
TGI
60.90
38.60
>100
27.60
14.30
>100
61.40
>100
80.09
>100
33.30
>100
30.70
55.40
38.20
48.90
25.00
LC50
GI50
TGI
ND
LC50
ND
6.76
>100
>100
3.89
8.13
>100
7.41
>100
7.94
6.92
>100
>100
5.13
Leukemia
CCRF-CEM
HL-6O(TB)
K-562
10.30
11.90
0.47
8.88
0.32
7.82
0.39
16.40
5.43
11.10
0.44
24.70
10.20
13.80
30.40
15.90
0.27
19.40
15.50
12.60
12.80
19.50
0.31
14.40
12.30
16.50
11.20
0.86
12.70
13.40
2.64
64.40
41.80
>100
>100
>100
>100
>100
10.00
11.20
0.37
4.02
0.07
12.20
0.20
18.20
9.91
5.96
0.09
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
ND
0.35
0.37
0.30
0.21
1.17
1.45
1.62
ND
1.54
2.23
1.34
0.60
3.09
4.90
3.47
M0LT-4
40.60
21.80
35.70
33.10
51.60
24.20
48.40
30.80
RPMI-8226
A549/ATCC
EKVX
97.30
Non-small cell lung cancer
>100
>100
>100
HOP-62
HOP-92
62.00
ND
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
>100
>100
>100
0.42
1.07
2.04
1.51
0.62
1.41
1.78
1.07
1.35
3.24
0.79
1.91
2.57
1.00
1.95
2.82
2.34
2.04
1.15
1.26
1.00
0.95
1.26
1.48
0.31
1.29
0.48
0.52
1.62
0.43
2.34
0.40
0.28
1.82
2.34
0.63
1.58
0.59
2.88
2.29
2.45
0.43
0.66
2.24
0.85
2.45
2.45
0.44
0.24
0.74
1.05
2.09
2.69
4.79
4.27
2.09
2.75
3.98
3.55
3.55
9.33
2.40
4.27
>100
23.70
40.30
48.90
5.06
4.59
55.00
>100
>100
>100
36.40
>100
79.20
50.10
59.90
>100
>100
56.10
>100
>100
54.10
44.30
89.20
51.60
91.40
70.30
Colon cancer
>100
14.10
13.50
0.25
15.80
14.00
11.00
12.50
27.50
0.22
36.40
11.00
9.32
9.49
0.53
6.25
11.40
1.78
24.20
22.70
0.06
5.50
8.91
60.90
13.20
63.20
35.10
25.10
27.70
57.20
26.00
28.50
>100
9.33
>100
6.61
>100
5.01
>100
>100
>100
>100
>100
>100
HT29
KM12
83.20
24.30
45.10
>100
23.10
>100
>100
58.20
>100
28.70
42.20
32.70
>100
>100
>100
53.80
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX lMVl
MALME-3M
M14
>100
>100
>100
>100
>100
>100
>100
97.30
>100
93.70
CNS cancer
Melanoma
>100
2.24
4.90
8.91
5.89
6.61
2.45
2.57
2.14
2.14
2.69
2.82
1.20
2.63
1.86
1.82
8.13
1.95
5.75
3.31
8.51
4.47
6.61
5.37
4.57
1.91
11.48
7.08
6.17
1.82
2.88
6.46
2.29
2.95
7.41
1.74
7.59
3.47
3.24
>100
78.20
24.60
19.60
33.60
26.30
27.50
35.80
99.30
13.90
16.80
25.90
63.30
23.20
5.13
5.25
4.68
4.57
5.89
5.25
3.47
5.37
5.37
5.01
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROVI
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786e0
>100
>100
>100
18.20
22.00
0.15
>100
37.70
41.40
63.10
2.90
20.80
0.50
1.97
8.86
26.80
80.90
49.80
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10.60
19.80
11.20
0.09
19.40
5.90
Ovarian cancer
Renal cancer
>100
48.20
>100
>100
71.30
66.40
>100
>100
50.10
16.70
15.40
0.16
29.50
9.16
>100
58.80
>100
>100
21.50
>100
54.80
>100
82.50
>100
7.59
>100
>100
>100
>100
>100
>100
>100
4.47
>100
>100
39.81
4.37
>100
>100
5.62
>100
69.80
25.60
17.60
0.63
0.68
25.50
12.80
11.70
12.00
3.79
17.20
10.90
13.70
13.30
15.60
11.80
3.24
15.60
20.00
1.26
0.71
0.06
>100
63.80
10.70
37.50
3.61
5.44
14.30
7.24
10.90
4.90
10.50
16.00
1.94
17.00
49.60
0.34
36.70
24.20
42.40
39.30
57.90
24.40
60.80
38.40
A498
ACHN
CAKI-1
RXF 393
SN12C
>100
>100
>100
94.80
41.60
59.00
54.80
TK-10
UO-31
>100
>100
>100
79.80
>100
92.80
>100
43.00
>100
60.50
>100
>100
72.80
84.90
>100
10.70
>100
Prostate cancer
Breast cancer
PC-3
DU-145
MCF7
>100
30.70
58.20
38.10
54.80
18.40
45.50
2.73
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
>100
>100
5.37
5.62
>100
22.91
>100
>100
81.28
1.25
0.03
4.46
2.22
51.28
Mean
5.24
37.15
the most to the least sensitive kinase towards binding with a given
test compound. Table 3 illustrates that within the set of tested
kinases, certain members of CDK, CK1, PDGFR, DDR, ABL, p38, RAF,
or RIPK subfamilies were more or less recognized by the three test
compounds. Some members of CK1 subfamily were commonly
recognized by the three test compounds. 10 mM of compound 15
showed almost equal affinity to bind to several members of CK1
subfamily whereas CSNK1D was almost equivalently sensitive to
the three derivatives; B-POC ¼ 26 against 4, B-POC ¼ 23 against 15,
and B-POC ¼ 22 against 16. Another CKI member, CSNK1E, was
found to be sensitive towards 15 and 16 more than towards 4.
CSNKIE has been found to be significantly expressed in several
cancer types compared to their normal tissues [41]. The above
results lead to the hypothesis that concerning CK1 subfamily
members, the combination of piperazinylpyrimidine scaffold with
quinazoline ring may be relevant for binding irrespective to the

2048
H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
Table 2
The phosphorylation status of kinases used in the single-dose kinase binding assay.
Phosphorylated
Non-phosphorylated
Wild-type
Mutants
1
8
198
36
Accordingly,
a biochemical functional screening of the test
compounds was decided to be pursued within the current inves-
tigation. In terms of lead kinase subfamilies, it is interesting that
4 was able to target PDGFR wild-type and mutants without
affecting ABL1 or ABL2 kinases (only phosphorylated ABL1(F317I)
mutant was targeted by 4). It is very common for inhibitors binding
ABL subfamily to also target PDGFR subfamily and vice versa with
Imatinib being
a prime example of such behavior [43,44].
Compound 4 represents a prototype structure that can discriminate
between PDFGR and ABL subfamilies. The test compounds, as
prototype kinase binders, have excellent overall selectivity towards
certain kinase subfamilies. Although 4 lacks the anilino NH group
which is known to be one of the fundamental structural features of
many kinase inhibitors, it shows clear potential to bind to certain
kinases which supports the hypothesis that it is hard to have
a common set of rules when designing kinase inhibitors [12]. Based
on the results of this primary single-dose screen, we investigated
the concentration-dependent potential of compound 4 to bind to
FLT3, KIT, KIT(D816H), or KIT(D816V). This was achieved via
determining the Kd (dissociation constant) of the four interactions
(Table 3). The dose-response curves of compound 4 against FLT3,
KIT, KIT(D816H), or KIT(D816V) are included in the supporting
information (Fig. S2). The ability of 4 to differentially discriminate
between certain KIT and FLT3 wild-type and mutant isoforms will
be discussed latter.
Fig.
2. The
overall
structure-antiproliferative
activity
relationships
of
piperazinylpyrimidines.
presence of linkers or a specific linker. The above observation
suggests for CK1 family kinases, especially CSNK1D, there exists
a common binding site which is able to accommodate piper-
azinylpyrimidine derivatives of different molecular sizes and
linkers. In retrospect, the three test compounds have some struc-
tural similarities with purine derivatives found to be dual CDK/CK1
inhibitors [42]. Compound 4 exhibited obvious tendency to bind
strongly with certain wild-type and/or mutant members of the CDK
and PDGFR subfamilies. This could be attributed to a steric factor
based on that 4 is smaller in size than both 15 and 16 and that the
targeted binding site of these subfamilies, CDK and PDGFR, is
unable to accommodate these larger derivatives (15 and 16). It is
interesting to observe that 15 bound with higher affinity to BRAF
and its common V600E mutant whereas 4 and 16 were recognized
by RIPKI, another TKL (tyrosine kinase like) kinase. Among the
tested unphosphorylated and phosphorylated derivatives of ABL1,
only two close mutants were bound by the test compounds. ABL1
(F317I) was moderately recognized by 4 (B-POC ¼ 30) and 16
(B-POC ¼ 20) whereas ABL1(F317L) was more strongly recognized
by 15 (B-POC ¼ 6.8). Since phosphorylated kinases are presumed to
exist preferentially in the active state, the last observation raises the
possibility of the ability of the test compounds to interact with
active kinases and inhibit their phosphotransferase function.
In order to evaluate whether the molecules could inhibit the
phosphotransferase kinase function, a radiometric based assay was
used to measure the ability of 1 mM of compounds 4, 15, or 16 to
Table 3
The results of the kinase binding assay. The B-POC values of 10 mM of compounds
4, 15, and 16 against the most sensitive kinases tested within this binding experi-
ment are given in the table (B-POC ꢀ 50). Both the B-POC and Kd values are the
average of duplicate measurements. ND indicates not determined.
Kinase
Cmpd-4
Cmpd-15
Cmpd-16
B-POC
Kd
(
m
M)
B-POC
B-POC
ABL1(F317I)-phosphorylated
ABL1(F317L)-phosphorylated
BRAF
BRAF(V600E)
CDK8
CDK11
CSF1R
CSNK1A1
CSNK1D
30
>50
>50
>50
3.4
0
31
>50
26
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.53
ND
ND
ND
2.5
1.3
0.11
ND
ND
ND
ND
ND
ND
>50
6.8
34
13
>50
47
>50
36
23
29
18
20
>50
>50
>50
>50
>50
>50
>50
22
23
43
>50
28
>50
>50
>50
>50
>50
>50
>50
>50
>50
25
CSNK1E
45
CSNK1G2
CSNK1G3
DDR1
>50
>50
47
8.4
46
49
4
9.1
30
0.5
9.8
4.3
>50
21
24
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
FLT3
FLT3(D835H)
FLT3(ITD)
FLT3(K663Q)
KIT
KIT(D816H)
KIT(D816V)
KIT(L576P)
KIT(V559D)
p38-gamma
PDGFRA
Fig. 3. The results of the MTT assay in which 4, 15, 16, and gefitinib were screened
against MDA-MB-468 cell line. The Histogram shows the average IC50 values in mM. The
IC50 values were calculated using non-linear regression analysis as described in the
experimental section. The error bars represent the 95% confidence interval calculated
for 9n (three independent experiments, each is a triplicate).
>50
>50
19
PDGFRB
RIPK1
4.1
3.5

H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
2049
Table 5
interfere with the function of a set of 55 protein kinases; 39 wild-
type and 16 mutants (Table S2) [45,46]. This set of tested kinases
was chosen based on the results of the binding assay described
The potential of compound 4 to either bind to or inhibit the function of certain KIT or
PDGFRA isoforms. The B-POC of 10 M and F-POC of 1 M of compound 4 measured
in the binding and function inhibition experiments respectively. JM denotes for
juxtamembrane domain, and GK denotes gatekeeper residue. ND indicates not
determined.
m
m
above. Again the ability of 1 mM of a given test compound to inhibit
the phosphotransferase function of a given kinase is expressed by
functional percentage of control (F-POC) value which is also
inversely proportional to the potency of the test compound, at that
specific concentration, for a given kinase. The most promising
interactions, at a single dose, were further investigated to deter-
mine the IC50 values (Table 4). The IC50 of compound 4 was deter-
mined against KIT, KIT(D816H), KIT(D816V), KIT(V560G), FLT3,
PDGFRA(V561D), and PDGFRA(D842V) (Table 4). Doses-response
curves were used to determine the IC50 values (Fig. S3). The three
test compounds, once again, weakly inhibited the same member of
CK1 subfamily, CSNK1D. Compound 4 demonstrated the ability to
inhibit the function of some PDGFR subfamily members; both wild-
type and mutants, without affecting ABL subfamily members. On
the other hand, 15 and 16 showed weak inhibitory action against
ABL kinase and some members of PDGFR subfamily.
Both kinase profiling experiments, binding and functional,
revealed a very interesting pattern by which compound 4 shows
more potential to either bind to or inhibit the function of some of the
KIT and PDGFRA mutants compared to their wild-type isoforms.
Table 5 can be used to illustrate this phenomenon and aid in its
analysis. The location of each mutation within the kinase different
domains is included [16,47]. It has been reported that different gain-
of-function mutations of the KIT kinase lead to constitutive catalytic
activity independent of activation by extracellular stem cell factor.
The occurrence of these mutations was detected in cases where
marketed KIT inhibitors (imatinib, dasatinib and sunitinib) face
resistance by the target cancer cells and become inefficacious in the
treatment of chronic myeloid leukemia (CML) and/or gastrointes-
tinal stromal tumors (GIST) [16,48,49]. Compound 4 showed higher
activity against the KIT A-loop mutants; D816H and D816V
compared to its activity against wild-type KIT, a foundation that is
supported by both the Kd values in the binding screen and the IC50
values in the functional assay. As common with many other kinase
inhibitors, mutations of the gatekeeper residue of KIT could render
the protein less liable to binding by 4; an observation reinforced by
the significant increase in the B-POC value of the juxtamembrane
mutant KIT(V559D) upon mutation of the gatekeeper in the double
mutant KIT(V559D, T670I). This shows that the gatekeeper residue
and probably the adjacent hinge region are critical in binding of 4 to
Kinase
Location
B-POC
Kd
(
m
M)
F-POC
IC50 (mM)
KIT(D816V)
KIT(D816H)
KIT(A829P)
KIT(V559D)
KIT(L576P)
KIT(V559D,T670I)
KIT(V560G)
KIT
A-loop
A-loop
A-loop
JM
JM
JM and GK
JM
Wild-type
JM
A-loop
Wild-type
0.5
30
84
0.11
1.3
ND
ND
ND
ND
ND
2.5
ND
ND
ND
83
30
2.82
0.316
ND
ND
ND
ND
ND
36
94
20
25
116
4.3
9.8
66
ND
9.1
ND
ND
21
ND
ND
ND
0.699
>10.0
0.295
0.316
ND
PDGFRA(V561D)
PDGFRA(D842V)
PDGFRA
the same location, could originate from the difference in the
preferred conformational ensembles and highlights the potential
effect of a single amino acid mutation upon the probability distri-
bution of the protein’s conformations to be selected for binding by
a given small molecule like 4. The ability of 4 to efficiently bind to
certain KIT mutants, that are resistant to other kinase inhibitors,
more than it binds to the wild-type isoform makes it valuable as
a structural prototype and adds another virtue to its global selective
kinase profile. Accordingly, 4 can be selective towards cancer
phenotypes harboring and depending on such mutant KIT forms
versus normal cells depending mainly on wild-type KIT to function.
Compound 4 also exhibited a similar fashion of selectivity towards
certain PDGFRA mutants (V561D in the juxtamembrane or D842V in
the A-loop) as illustrated by the F-POC and IC50 determination. Such
PDGFRA mutants exhibit differential resistance towards certain
kinase inhibitors like imatinib and nilotinib and become common in
certain forms of resistant cancer phenotypes [47,50e52].
Compound 15 demonstrated a similar trend of selectivity
towards the mutant BRAF(V600E) versus the wild-type BRAF
(Table 3). BRAF(V600E) mutant is commonly found in several kinds
of cancer especially melanoma and GIST. This mutation has
attracted the attention of research groups to develop specific
inhibitors against it [50,53]. ABL1 mutants have been generally
shown to affect the sensitivity towards ABL kinase inhibitors. Point
mutations in codon 317 of ABL1 have been found to specifically
impart resistance towards dasatinib [54e56]. Compound 4 and 16
have moderate affinity for the phosphorylated form of ABL1(F317I)
while 15 has stronger binding affinity towards the phosphorylated
form of ABL1(F317L). This observation again highlights the value of
these test compounds’ potential to be discretely selective towards
certain kinase mutants.
KIT. It is clear that 4 was able to bind the KIT(D816V) (Kd ¼ 0.11
more than KIT(D816H) (Kd ¼ 1.3 M) whereas it inhibited the
M) more than KIT(D816V)
M). Such opposite effects, on twodifferent mutations at
mM)
m
function of KIT(D816H) (IC50 ¼ 0.316
m
(IC50 ¼ 2.82
m
Table 4
As revealed by Table 3 and Table 4, although compounds 4 and
15 have certain common kinase targets, they don’t share an overall
similar selectivity profile in that 4 is more active towards the PDGFR
subfamily. This is contrary to the striking correlation of their
cellular profiles against the NCI-60 panel. It could be the case that
4 and 15, very similarly, affect other kinases that are not included in
the kinase profiling experiments reported here. Another possible
scenario is that 4 and 15 could equally inhibit other protein(s) that
belong to a different protein family other than protein kinases. It
also may be possible that 4 and 15 target the same signaling
transduction pathway at different points. The biological investiga-
tion of other possible targets is beyond the scope of the current
report and we leave it for future studies that can investigate the
global genomic and/or proteomic differences induced by 4 and 15
on the cellular level.
The results of the kinase function inhibition assay. The F-POC of 1 mM of compounds
4, 15, and 16 against selected kinases tested within this experiment. Both the F-POC
and IC50 values are the average of duplicate measurement. ND indicates not
determined.
Kinase
Cmpd-4
Cmpd-15
Cmpd-16
F-POC
IC50
(
m
M)
F-POC
F-POC
ABL
CSNK1D
FLT3
KIT
KIT(D816H)
KIT(D816V)
KIT(V560G)
MAPK1
MET
PDGFRA(D842V)
PDGFRA(V561D)
92
90
74
94
30
83
36
100
75
25
20
89
ND
ND
6.692
>10.0
0.316
2.823
0.699
ND
ND
0.316
0.295
ND
80
87
80
87
74
95
100
100
100
86
73
100
96
100
83
100
100
100
99
100
100
100
100
82
A question emerged at this level of investigation; how does
4 bind to its kinase targets generally and to wild-type and mutant
PKB
b

2050
H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
KIT versions specifically? To tackle this question, we compared the
observed binding affinity of 4 against the wild-type KIT and its
D816V mutant, obtained from the binding data, to the respective
binding affinity of 38 kinase inhibitors against the same two
kinases observed under very similar experimental conditions
(Table 6) [15]. DDGꢁ, as a measure of the differential binding
potential against the mutant KIT(D816V) versus the wild-type KIT,
was calculated according to the literature [57,58]. A negative DDGꢁ
indicates preferential binding to the mutant whereas a positive
DDGꢁ indicates increased resistance by the mutant against the
specific inhibitor. Analysis of Table 6 revealed a very interesting
feature; the seven reference kinase inhibitors, that have negative
DDGꢁ, are known to be type-I kinase inhibitors which bind to the
same binding site occupied by ATP [22,59e64]. The above obser-
vation agrees with several published computational as well as
experimental reports demonstrating that an aspartic acid to valine
mutation in the position 816 of KIT destabilizes the inactive (DFG-
out) conformations of KIT. The same mutation would consequently
decrease the binding to type-II kinase inhibitors which mainly bind
preferentially to the inactive (DFG-out) conformational ensemble
of KIT [16,65,66]. The above notation coupled with the experi-
mental tendency of 4 to bind better to the V816KIT compared to the
D816KIT supports the hypothesis that 4 may be a type-I kinase
inhibitor that targets the ATP-binding site in contrast to type-II
inhibitors which exploit the adjacent allosteric binding site.
2.5. Molecular docking
In order to visualize and generate a binding hypothesis of 4, 15,
and 16 to their candidate kinases, a rigid docking experiment was
performed where the mode of binding of those compounds to
CSNK1D was simulated using Autodock 4 software [67]. The results
of the static simulation show that 4, 15, or 16 can bind to CSNK1D as
classical type-I inhibitors which usually occupies the ATP-binding
site surrounded by the hinge region, P-loop,
b6 strand and b9
strand [68]. None of the docked ligands exploited the allosteric
binding site commonly occupied by type-II inhibitors [18]. CSNK1D
was selected for the described docking session because it is equally
capable of binding to 10
mM of any of the tested compounds
according to the kinase binding experiment (Table 3).It is important
to mention that the grid box was designed to be large enough to
allow the software to choose between different modes of binding;
type-I or type-II. Not only the interaction pattern predicted by this
docking experiment agrees with our primary rationale of using the
quinazoline ring as a hinge region localizing fragment, but also it
supports the earlier mentioned hypothesis that 4 binds with KIT
kinase as a typical type-I inhibitor. A clear steric complementarity
between the available space in the ATP-binding site of CSNK1D and
the size of the tested derivatives can be easily confirmed by the
results of the above rigid docking (Fig. 4). Concerning the electro-
static complementarity, as commonly observed in the binding of
type-I quinazoline based kinase inhibitors, a hydrogen bonding
interaction was predicted, by the docking experiment, between the
quinazoline nitrogen of a given ligand (4, 15 or 16) as a hydrogen
bond acceptor and the backbone NH of LEU88 residue in the hinge
region as a hydrogen bond donor. Another H-bond was suggested
between the carbonyl oxygen of 15 (acceptor) and the NH backbone
of SER20 (donor) lies in the P-loop. Fig. 4 also shows the likelihood
Table 6
Comparison of the behavior of 4 towards KIT and its mutant KIT(D816V) with a set of
38 structurally diverse kinase inhibitors. The Kd values of the reference kinase
inhibitors were determined experimentally against the two kinases similarly to that
of 4 and were obtained from literature [15]. DDGꢁ (Kcal.molꢂ1) ¼ R.T.ln [Kd(mutant)/
Kd(wild-type)] where R ¼ 0.001985 kcal molꢂ1 Kꢂ1 and T ¼ 298 K.
Kinase inhibitor
Kd (nM)
KIT
DDGꢁ
KIT(D816V)
Staurosporine
PKC-412
4
LY-333531
Erlotinib
Gefitinib
Flavopiridol
CI-1033
ZD-6474
19
220
2500
0.64
7.7
110
920
1600
4300
4600
3900
290
290
2500
2.6
310
29
6200
81
820
ꢂ2.01
ꢂ1.98
ꢂ1.847
<ꢂ1.4
<ꢂ1.08
<ꢂ0.49
<ꢂ0.45
ꢂ0.41
0.06
0.11
0.19
0.85
1.36
1.4
2.03
2.19
2.41
>2.41
2.48
2.78
3.07
3.09
3.31
3.68
4.1
>10,000
>10,000
>10,000
>10,000
7800
of a
p-cation interaction between the pyrimidine ring of either
15 or 16 and the protonated LYS133 side chain located in the
b6
strand. It can be easily speculated that under more realistic
dynamic conditions of interaction, the last mentioned interaction
260
240
1800
0.62
31
2.7
200
2
14
170
5.4
3.7
2.8
7.5
could be a mix between
p-cation interaction and H-bonding
VX-680
between one of the symmetric pyrimidine ring nitrogens in the
bigger ligands (15 or 16) and the side chain quaternary amino group
of LYS133. Accordingly, piperazinylpyrimidine can be hypothesized
to interact with their respective kinase candidates as type-I inhib-
itors, a hypothesis that still needs experimental investigation using
either x-ray crystallography or NMR spectroscopy.
JNJ-7706621
Dasatinib
Sorafenib
MLN-518
CHIR-265
ABT-869
Imatinib
BIRB-796
AST-487
AMG-706
GW-786034
CHIR-258
AZD-1152HQPA
SU-14813
Sunitinib
PTK-787
BMS-387032
CP-690550
CP-724714
EKB-569
GW-2580
Lapatinib
MLN-8054
PI-103
Roscovitine
SB-202190
SB-203580
SB-431542
VX-745
>10,000
360
2.6. Summary
410
500
1400
4600
340
380
In summary a primary lead discovery investigation identified
4, 15, and 16 to be highly optimized for selective cytostatic, as in the
cases of 4 and 15, or global cytotoxic activity, as in the case of 16,
against the NCI-60 panel. Consequently, 15 grabbed the attention of
the NCI and is currently being investigated in animal models of
cancer. Also noted is the very interesting phenomenon of 4 and 15
exhibiting reproducible selective cytostatic activity against MDA-
MB-468 breast cancer cell line which represents a model for
a clinically distinctive group of triple negative/basal-like breast
cancer patients. This interesting selective antiproliferative activity
against MDA-MB-468 cell line is concentration and time dependent
and its mechanism at the molecular pathways level is currently
being investigated. Kinase profiling experiments were chosen to
detect the ability of 4, 15, and 16 to either bind to or inhibit the
function of a large panel of kinases. Both kinase binding and
function inhibition experiments showed an overall selective
17
0.68
0.37
5.1
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>4.48
e
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
e
e
e
e
e
e
e
e
e
e
e
e

H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
2051
Fig. 4. The results from the docking simulation of 4 (a),15 (b), and 16 (c), with CSNK1D (PDB: 1CKI). The protein is represented as a solid ribbon. The SER20, LEU88, LYS133, 4,15, and 16
are shown as ball and stick representation. The hinge region is colored green, the 6 strand is colored magenta, the 9 strand is colored in cyan, the P-loop is colored in red, and the A-
b
b
a
helix is colored inyellow. The pictures were prepared using DS ViewerPro 6.0 software. The predicted binding mode of both 15 and 16 was the same among the top ranked energycluster
whereas that of 4 belongs tothe second ranked energycluster. (For interpretation of the references to colour in this figure legend, the reader is referred tothe webversion of this article.).
behavior of the three compounds toward binding and inhibiting the
function of certain kinase subfamilies. Compound 4 showed an
additional interesting feature of binding to or inhibiting the func-
tion of certain KIT and PDGFR mutants compared to their wild-type
isoforms. The last observation strengthen the hypothesis that
4 may act as a type-I kinase inhibitor based on a comparison with
38 kinase inhibitors and molecular docking experiments. Piper-
azinylpyrimidine based derivatives have been shown through this
report to have the potential of being lead selective kinase inhibitors
as well as optimized selective antiproliferative agents.
commercial suppliers and used without further purification.
Reactions requiring anhydrous conditions were performed in dry
solvents under Argon atmosphere. Reactions were monitored
by thin layer chromatography (TLC) on precoated silica gel F254
plates (EMD) using a UV detector for visualization. Flash column
chromatography was performed with EMD 230e400 mesh silica
gel 60 Å. Yields are of purified products. Melting points were
determined using Thomas Hoover melting point apparatus and
are uncorrected. 1H NMR and 13C-NMR spectra were recorded on
a Jeol JNMꢂECA600 spectrometer. The obtained FID of each
experiment was processed using Delta NMR 1D processor soft-
ware. The chemical shifts are expressed in parts per million (ppm,
3. Experimental section
d) down field from TMS. Spin multiplicities are designated
as s (singlet), d (doublet), dd (doublet of doublets), ddd (doublet
of doublet of doublets), t (triplet), q (quartet), m (multiplet),
and br (broad). Mass spectra (MS) were determined by MALDI
3.1. Chemistry
3.1.1. General methods
All temperatures are expressed in ꢁC (degrees centigrade). All
reagents, solvents, and starting materials were obtained from
instrument. 1
mL test compound solution in acetonitrile is
mixed with an equal amount of the matrix solution (saturated

2052
H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
2,5-dihydroxybenzoic acid in 50% ethanol) on the MALDI plate.
MALDI spectra were recorded in positive reflectron mode on an
AXIMA curved-field reflectron (CFR) MALDI-TOF mass spectrom-
eter (Kratos/Shimadzu, Columbia, MD, USA). Analytical HPLC was
was obtained as a pale yellow solid (2.2 gm, 80% yield); mp:
108e110 ꢁC 1H NMR (DMSO-d6):
1.89 (s, 1H), 2.93 (t, J ¼ 4.8 Hz,
4H), 3.76 (t, J ¼ 4.8 Hz, 4H), 3.79 (s, 2H), 7.17e7.29 (m, 5H), 8.18 (s,
2H). 13C NMR (DMSO-d6):
35.59, 44.96, 45.92, 121.79, 126.34,
d
d
performed using
a
C18 Phenomenex quantitative
5
m
m
128.48, 128.6, 140.1, 157.8, 160.5. MS: calcd 254.15 for C15H18N4
4.6 mm ꢃ 150 mm column with a 30 min gradient of solvent from
10% to 90% CH3CN in H2O. Retention time (Rt) was recorded
in minutes and purity was indicated as percentage of the
target compound calculated from % area under the peak (AEP).
All final purified compounds showed purity levels greater
than 95%.
[M]þ; found, 255.15 [M þ 1]þ.
3.1.4. 4-(4-(5-Ethylpyrimidin-2-yl)piperazin-1-yl)quinazoline (3)
A mixture of 1 (72 mg, 0.377 mmol), 4-chloroquinzoline (61 mg,
0.377 mmol), and triethylamine (38 mg, 0.377 mmol) in iso-
propanol (5 ml) was refluxed at 80 ꢁC for 1.5 h. The solid separated
out of the reaction was filtered, washed with hot solvent and dried
to afford compound 3 which was obtained as a yellow solid (61 mg,
50% yield); mp: >250 ꢁC. The purity of 3 was confirmed using HPLC
3.1.2. 5-Ethyl-2-(piperazin-1-yl)pyrimidine (1)
a) A mixture of tert-butyl piperazine-1-carboxylate (6.5 gm,
35.2 mmol), 2-chloro-4-ethylpyrimidine (5 gm, 35.2 mmol), trie-
thylamine (3.55 gm, 35.2 mmol), and absolute ethanol (60 ml)
were refluxed for 12 h and then cooled. Ice water was added and
the resulting precipitate was filtered, washed with cold water, and
finally dried to afford the desired crude intermediate, tert-butyl
(97.27%, Rt ¼ 9.987). 1H NMR (DMSO-d6):
1.13 (t, J ¼ 7.56 Hz, 3H),
d
2.45 (q, J ¼ 7.56 Hz, 2H), 3.95 (t, J ¼ 5.28 Hz, 4H), 4.16 (t, J ¼ 5.28 Hz,
4H), 7.68 (ddd, J ¼ 1.38 Hz, J ¼ 6.84 Hz, J ¼ 8.4 Hz, 1H), 7.96 (m, 2H),
8.22 (d, J ¼ 8.4 Hz, 1H), 8.31 (s, 2H), 8.82 (s, 1H). 13C NMR (DMSO-
d6):
d 15.63, 21.91, 42.85, 48.78, 113.28, 121.66, 125.04, 126.69,
4-(5-ethylpyrimidin-2-yl)piperazine-1-carboxylate,
as
white
126.88,134.78,143.5,149.89,157.19,159.95,162.51. MS: calcd 320.17
flakes (9.24 gm, 90% yield); mp: 67e68 ꢁC 1H NMR (DMSO-d6):
for C18H20N6 [M]þ; found, 320.39 [M]þ.
d
1.19 (t, J ¼ 7.56 Hz, 3H), 1.46 (s, 9H), 2.47 (q, J ¼ 7.56 Hz, 2H), 3.49
(t, J ¼ 4.8 Hz, 4H), 3.76 (t, J ¼ 4.8 Hz, 4H), 8.19 (s, 2H). 13C NMR
3.1.5. 4-(4-(5-Benzylpyrimidin-2-yl)piperazin-1-yl)quinazoline (4)
Compound 4 was prepared from 2 (90 mg, 0. 354 mmol) and
4-chloroquinzoline (58 mg, 0. 354 mmol) following the same
procedure for the preparation of 3. The product was a yellow solid
(86 mg, 63%); mp: >250 ꢁC. The purity of 4 was confirmed using
(DMSO-d6):
d 15.59, 22.66, 28.4, 43.76, 43.77, 79.8, 124.95, 154.84,
157.08, 160.05. MS: calcd 292.19 for C15H24N4O2 [M]þ; found,
293.19 [M
1]þ. b) HCl/MeOH mixture was prepared by
þ
adding10 ml acetyl chloride dropwise to 100 ml methanol at 0 ꢁC.
The resulting mixture was warmed to room temperature. The
intermediate from step a (9 gm, 30.8 mmol) was then added to the
HCl/MeOH and stirred for 12 h at room temperature. Methanol
was removed using a rotary evaporator. The residue was dissolved
in water; the solution was neutralized using solid NaHCO3. The
mixture was extracted with CHCl3 (3 ꢃ 200 mL). The combined
organic layers was washed successively with saturated NaHCO3,
and saturated brine and then dried with anhydrous Na2SO4.
Concentration of the organic layer afforded the desired crude
product 1 as a white solid (4.8 gm, 81% yield); mp: 115e116 ꢁC 1H
HPLC (97.59%, Rt ¼ 15.025).1H NMR (DMSO-d6):
d 3.8 (s, 2H), 3.96
(t, J ¼ 5.16 Hz, 4H), 4.26 (t, J ¼ 5.16 Hz, 4H), 7.17e7.3 (m, 5H), 7.71
(ddd, J ¼ 2.22 Hz, J ¼ 6.36 Hz, J ¼ 8.4 Hz, 1H), 8.01 (m, 2H), 8.25 (d,
J ¼ 8.4 Hz, 1H), 8.34 (s, 2H), 8.86 (s, 1H). 13C NMR (DMSO-d6):
d
34.51, 42.6, 48.69, 112.49, 119.73, 123.03, 126.11, 126.98, 127.32,
128.32, 128.54, 135.36, 140.86, 140.96, 148.74, 157.84, 159.77,
162.12. MS: calcd 382.19 for C23H22N6 [M]þ; found, 383.35
[M þ 1]þ.
3.1.6. 4-(4-(5-Ethylpyrimidin-2-yl)piperazin-1-yl)-2-phenylqui-
nazoline (5)
NMR (DMSO-d6):
d
1.20 (t, J ¼ 7.56 Hz, 3H), 2.48 (q, J ¼ 7.56 Hz,
2H), 3.09 (t, J ¼ 5.16 Hz, 4H), 3.94 (t, J ¼ 5.16 Hz, 4H), 8.19 (s, 2H).
Compound 5 was prepared from 1 (72 mg, 0.377 mmol) and
2-phenyl-4-chloroquinzoline (90 mg, 0.377 mmol) following the
same procedure for the preparation of 3. The product was a white
solid (100 mg, 67%); mp: 137e138 ꢁC. The purity of 5 was
confirmed using HPLC (96.43%, Rt ¼ 15.09). 1H NMR (DMSO-d6):
13C NMR (DMSO-d6):
d 15.53, 22.67, 42.76, 44.30, 125.52, 157.15,
160.09.
3.1.3. 5-Benzyl-2-(piperazin-1-yl)pyrimidine (2)
a) A mixture of K2CO3 (6 gm, 43.5 mmol, 3 equiv) and Pd(PPh3)4
(1 gm, 0.86 mmol) were stirred at room temperature in 20 mL THF/
water (1:1) for 30 min. Tert-butyl 4-(5-bromopyrimidin-2-yl)
piperazine-1-carboxylate (5 gm, 14.5 mmol, 1 equiv) was then
added with B-benzyl-9-BBN (2 equiv, 49 ml of a 0.5 M solution in
THF). The resulting mixture was heated with stirring under argon
atmosphere in an oil bath adjusted at 100e110 ꢁC until the reaction
had reached completion, as monitored by TLC. The reaction mixture
was extracted with CHCl3 (3 ꢃ 200 mL). The combined organic
layers were then dried with anhydrous Na2SO4. Chloroform was
evaporated under reduced pressure and the residue was purified
using flash column chromatography (initially with 4:1 hexane/
ethylacetate and finally with 1:1 hexane/ethylacetate). Tert-butyl
4-(5-benzylpyrimidin-2-yl)piperazine-1-carboxylate was obtained
as a yellow solid (3.8 gm, 74% yield); mp: 121e123 ꢁC 1H NMR
d
1.14 (t, J ¼ 7.56 Hz, 3H), 2.44 (q, J ¼ 7.56 Hz, 2H), 3.93e3.98 (m,
8H), 7.50e7.54 (m, 4H), 7.83 (ddd, J ¼ 1.38 Hz, J ¼ 6.84 Hz,
J ¼ 8.28 Hz, 1H), 7.9 (dd, J ¼ 0.96 Hz, J ¼ 8.28 Hz, 1H), 8.1 (dd,
J ¼ 0.96 Hz, J ¼ 8.28 Hz, 1H), 8.3 (s, 2H), 8.51 (m, 2H). 13C NMR
(DMSO-d6):
d 15.6, 21.93, 43.33, 48.85, 114.75, 119.3, 123.55,
125.36, 125.4, 127.96, 128.37, 130.31, 132.9, 138.03, 152.11, 157.13,
158.08, 160.36, 164.12. MS: calcd 396.21 for C24H24N6 [M]þ; found,
397.4 [M þ 1]þ.
3.1.7. 4-(4-(5-Benzylpyrimidin-2-yl)piperazin-1-yl)-2-phenylqu-
inazoline (6)
Compound 6 was prepared from 2 (90 mg, 0.354 mmol) and
2-phenyl-4-chloroquinzoline(84mg,0.354mmol)followingthesame
procedure for the preparation of 3. The product was a white solid
(160 mg, 98%); mp: 135e137 ꢁC.The purity of 6 was confirmed using
(DMSO-d6):
d
1.47 (s, 9H), 3.47 (t, J ¼ 4.68 Hz, 4H), 3.75 (t,
HPLC (98.57%, Rt ¼ 19.707). 1H NMR (DMSO-d6):
d 3.79 (s, 2H),
J ¼ 4.68 Hz, 4H), 3.78 (s, 2H), 7.14e7.29 (m, 5H), 8.16 (s, 2H). 13C
3.91e3.96 (m, 8H), 7.17e7.30 (m, 5H), 7.49e7.54 (m, 4H), 7.82 (ddd,
J ¼ 0.78 Hz, J ¼ 6.6 Hz, J ¼ 7.92 Hz, 1H), 7.9 (d, J ¼ 8.28 Hz, 1H), 8.08 (d,
NMR (DMSO-d6):
d 28.4, 35.61, 43.70, 79.92, 122.29, 126.41, 128.51,
128.66, 140.02, 154.84, 157.87, 160.69. MS: calcd 354.21 for
C20H26N4O2 [M]þ; found, 355.21 [M þ 1]þ. b) 3.7 gm of the obtained
product from step a was then deprotected using HCl/MeOH as
described above in the preparation of 1. As a result, compound 2
J¼ 8.28Hz,1H), 8.32 (s, 2H), 8.51 (m, 2H).13C NMR (DMSO-d6):
d34.56,
43.24, 48.81,114.74,122.82,125.35,125.4,126.11,127.95,128.35,128.56,
130.32,132.89,138.02,140.9,152.11,157.81,158.06,160.26,164.09. MS:
calcd 458.22 for C29H26N6 [M]þ; found, 458.28 [M]þ.

H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
2053
3.1.8. General preparation of quinazoline-4-ylaminobenzoic acid
and quinazoline-4-ylaminomethylbenzoic acid intermediates
(7e12)
3.1.15. (4-(5-Ethylpyrimidin-2-yl)piperazin-1-yl)(3-(quinazolin-4-
ylamino)phenyl)methanone (13)
Intermediate 1 (72 mg, 0.377 mmol) was reacted with inter-
mediate 7 (99 mg, 0.377 mmol) in the presence of equivalent
amounts of both EDC.HCl (72 mg, 0.377 mmol) and TEA (38 mg,
0.377 mmol) using 5 ml of CH2Cl2 as a solvent. The reaction mixture
was stirred under room temperature overnight. The solvent was
evaporated under pressure and the title compound was purified
using flash column chromatography (2.5% MeOH in CH2Cl2). The
product was a white solid (107 mg, 64%); mp: 146e147 ꢁC. The
purity of 13 was confirmed using HPLC (97.1%, Rt ¼ 11.075). 1H NMR
Either 4-chloroquinzoline or 2-phenyl-4-chloroquinzoline was
reacted with an equivalent amount of the corresponding amino-
benzoic acid or aminomethylbenzoic acid derivative in the pres-
ence of an equivalent amount of triethylamine using anhydrous
isopropanol as a solvent. The reaction was performed under 80 ꢁC
until the starting materials were consumed. The solid furnished out
of the reaction, after solvent evaporation, was washed with hot
water to remove triethylamine HCl and used in the next step
without any further purification.
(DMSO-d6):
d
1.12 (t, J ¼ 7.44 Hz, 3H), 2.43 (q, J ¼ 7.44 Hz, 2H),
3.35e3.79 (br, m, 8H), 7.2 (dd, J ¼ 1.2 Hz, J ¼ 7.74 Hz, 1H), 7.48 (t,
J ¼ 7.92 Hz, 1H), 7.65 (ddd, J ¼ 1.38 Hz, J ¼ 7.38 Hz, J ¼ 8.28 Hz, 1H),
7.8 (dd, J ¼ 1.2 Hz, J ¼ 7.38 Hz,1H), 7.87 (ddd, J ¼ 1.38 Hz, J ¼ 7.08 Hz,
J ¼ 8.28 Hz, 1H), 7.98 (ddd, J ¼ 0.72 Hz, J ¼ 1.8 Hz, J ¼ 8.28 Hz, 1H),
8.02 (t, J ¼ 1.68 Hz,1H), 8.27 (s, 2H), 8.57 (dd, J ¼ 0.72 Hz, J ¼ 7.74 Hz,
3.1.9. 3-(quinazolin-4-ylamino)benzoic acid (7)
This intermediate is commercially available and is also reported
[69e71]. 1H NMR (DMSO-d6):
d
7.61 (t, J ¼ 7.92 Hz, 1H), 7.84e7.88
(m, 2H), 8.00e8.1 (m, 3H), 8.38 (t, J ¼ 1.74 Hz, 1H), 8.91 (s, 1H), 8.96
1H), 8.63 (s, 1H), 9.93 (s, 1H). 13C NMR (DMSO-d6):
d 15.58, 21.9,
(d, J ¼ 8.28, 1H). 13C NMR (DMSO-d6):
d 113.69, 121.06, 124.49,
41.49, 43.34, 43.87, 46.95, 115.16, 120.82, 122.17, 122.97, 123.18,
125.01, 126.38, 127.85, 128.67, 133.13, 135.88, 139.23, 149.7, 154.36,
157.13, 157.67, 160.24, 169.02. MS: calcd 439.21 for C25H25N7O [M]þ;
found, 439.12 [M]þ.
124.86, 126.55, 128.02, 128.34, 128.74, 131.25, 135.47, 137.31, 140.61,
151.42, 159.4, 166.62. MS: calcd 265.09 for C15H11N3O2 [M]þ; found,
265.6 [M]þ.
3.1.10. 4-(quinazolin-4-ylamino)benzoic acid (8)
3.1.16. (4-(5-Ethylpyrimidin-2-yl)piperazin-1-yl)(4-(quinazolin-4-
ylamino)phenyl)methanone (14)
This intermediate is commercially available and is also reported
[70e72].1H NMR (DMSO-d6):
d
7.75e7.77 (m,1H), 7.91e8.05 (m, 6H),
Compound 14 was prepared from intermediate 1 (72 mg,
0.377 mmol) and intermediate 8 (99 mg, 0.377 mmol) following the
same procedure utilized in the preparation of 13. The product
obtained was a white solid (100 mg, 60%); mp: 248e249 ꢁC. The
purity of 14 was confirmed using HPLC (95%, Rt ¼ 10.753). 1H NMR
8.83e8.84 (m, 2H). 13C NMR (DMSO-d6):
d
115.08, 123.12, 124.43,
124.66, 127.32, 128.13, 130.44, 135.32, 142.76, 144.92, 153.12, 159.25,
167.34. MS: calcd 265.09 for C15H11N3O2 [M]þ; found, 265.7 [M]þ.
(DMSO-d6):
d
1.12 (t, J ¼ 7.56 Hz, 3H), 2.43 (q, J ¼ 7.56 Hz, 2H),
3.1.11. 4-((quinazolin-4-ylamino)methyl)benzoic acid (9)
3.33e3.84 (br, m, 8H), 7.5 (d, J ¼ 8.64 Hz, 2H), 7.66 (ddd, J ¼ 1.38, Hz,
J ¼ 7.2 Hz, J ¼ 8.28 Hz, 1H), 7.82 (d, J ¼ 8.28 Hz, 1H), 7.88 (ddd,
J ¼ 1.38 Hz, J ¼ 7.2 Hz, J ¼ 8.28 Hz, 1H), 8.02 (d, J ¼ 8.64 Hz, 2H), 8.27
(s, 2H), 8.59 (d, J ¼ 8.46 Hz, 1H), 8.66 (s, 1H), 9.93 (s, 1H). 13C NMR
This intermediate is commercially available and is also reported
[73]. 1H NMR (DMSO-d6):
d
4.86 (d, J ¼ 5.88 Hz, 2H), 7.46 (d,
J ¼ 8.64 Hz, 2H), 7.54 (ddd, J ¼ 1.2 Hz, J ¼ 7.08 Hz, J ¼ 8.28 Hz,1H), 7.7
(dd, J ¼ 0.84 Hz, J ¼ 7.56 Hz, 1H), 7.78 (ddd, J ¼ 1.2 Hz, J ¼ 6.9 Hz,
J ¼ 8.22 Hz,1H), 7.89 (d, J ¼ Hz, 2H), 8.31 (dd, J ¼ 0.84 Hz, J ¼ 7.56 Hz,
1H), 8.44 (s, 1H), 8.93 (t, J ¼ 5.88 Hz, 1H). 13C NMR (DMSO-d6):
(DMSO-d6):
d 15.53, 21.87, 41.55, 43.6, 46.95, 115.19, 121.34, 122.99,
124.92, 126.37, 127.62, 127.78, 127.84, 130.37, 133.12, 140.57, 149.72,
154.2, 157.1, 157.55, 160.2, 169.08. MS: calcd 439.21 for C25H25N7O
d
46.91, 115.41, 123.19, 126.35, 127.64, 128.07, 129.83, 129.95, 133.23,
þ
[M]þ; found, 439.16 [M]
.
145.23,149.66,155.54,159.91,167.73. MS: calcd 279.1 for C16H13N3O2
[M]þ; found, 279.5 [M]þ.
3.1.17. (4-(5-Ethylpyrimidin-2-yl)piperazin-1-yl)(4-((quinazolin-4-
ylamino)methyl)phenyl)methanone (15)
3.1.12. 3-(2-Phenylquinazolin-4-ylamino)benzoic acid (10)
Compound 15 was prepared from intermediate 1 (77 mg,
0.405 mmol) and intermediate 9 (113 mg, 0.405 mmol) following
the general procedure used in the preparation of 13. The product
obtained was a white solid (40 mg, 21%); mp: 170e171 ꢁC. The
purity of 15 was confirmed using HPLC (95.48%, Rt ¼ 11.288). 1H
This intermediate is commercially available and is also reported
[74]. 1H NMR (DMSO-d6):
d 7.54e7.80 (m, 6H), 7.94e7.96 (m, 2H),
8.19e8.21 (m, 1H), 8.52e8.54 (m, 2H), 8.67e8.69 (m, 1H), 8.93 (s,
1H). MS: calcd 341.12 for C21H15N3O2 [M]þ; found, 341.6 [M]þ.
NMR (DMSO-d6):
d
1.11 (t, J ¼ 7.56 Hz, 3H), 2.42 (q, J ¼ 7.56 Hz, 2H),
3.34e3.76 (br, m, 8H), 4.84 (d, J ¼ 5.82 Hz, 2H), 7.4 (d, J ¼ 8.4 Hz,
2H), 7.44 (d, J ¼ 8.4 Hz, 2H), 7.54 (ddd, J ¼ 1.2 Hz, J ¼ 6.72 Hz,
J ¼ 8.28 Hz, 1H), 7.7 (dd, J ¼ 0.84 Hz, J ¼ 7.56 Hz, 1H), 7.78 (ddd,
J ¼ 1.2 Hz, J ¼ 6.72 Hz, J ¼ 8.28 Hz, 1H), 8.26 (s, 2H), 8.31 (dd,
J ¼ 0.84 Hz, J ¼ 7.56 Hz, 1H), 8.46 (s, 1H), 8.9 (t, J ¼ 5.82 Hz, 1H). 13C
3.1.13. 4-(2-Phenylquinazolin-4-ylamino)benzoic acid (11)
This intermediate is commercially available and is also reported
[74,75]. 1H NMR (DMSO-d6):
d
7.55e7.58 (m, 3H), 7.7e7.73 (m, 1H),
7.96e8.08 (m, 6H), 8.39e8.4 (m, 2H), 8.73e8.74 (s, 1H). 13C NMR
(DMSO-d6): 114.10, 122.91, 124.38, 127.07, 127.71, 129.11, 129.32,
d
NMR (DMSO-d6): d 15.58, 21.89, 43.25, 43.75, 46.92, 114.88, 122.63,
130.56, 132.29, 135.26, 142.99, 158.77, 158.99, 167.41. MS: calcd
341.12 for C21H15N3O2 [M]þ; found, 341.7 [M]þ.
124.97, 125.77, 127.09, 127.23, 127.57, 132.67, 134.27, 141.11, 149.17,
155.05, 157.12, 159.37, 160.18, 169.13. MS: calcd 453.23 for
þ
C26H27N7O [M]þ; found, 453 [M]
.
3.1.14. 4e((2-Phenylquinazolin-4-ylamino)methyl)benzoic acid (12)
The product was a white solid (37%); mp: 272 ꢁC 1H NMR
3.1.18. (4-(5-Benzylpyrimidin-2-yl)piperazin-1-yl)(3-(quinazolin-
4-ylamino)phenyl)methanone (16)
(DMSO-d6):
2H), 7.89 (m, 3H), 8.31 (m,1H), 8.40 (m, 2H), 8.85 (t, J ¼ 5.82 Hz,1H).
13C NMR (DMSO-d6):
43.66, 113.66, 122.49, 125.18, 127.01, 127.15,
d
4.97 (d, J ¼ 5.82 Hz, 2H), 7.44e7.56 (m, 4H), 7.79 (m,
Compound 16 was prepared from intermediate 2 (90 mg,
0.354 mmol) and intermediate 7 (93 mg, 0.354 mmol) following the
same procedure utilized in the preparation of 13. The product
obtained was a white solid (125 mg, 70%); mp: 120e122 ꢁC. The
purity of 16 was confirmed using HPLC (97.17%, Rt ¼ 15.488). 1H
d
127.68, 127.91, 129.01, 129.13, 129.73, 130, 132.53, 138.46, 144.41,
149.91, 159.08, 159.51, 167.05. MS: calcd 355.13 for C22H17N3O2
[M]þ; found, 355.6 [M]þ.

2054
H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
NMR (DMSO-d6):
d
3.34e3.78 (br, m, 10H), 7.17e7.29 (m, 6H), 7.48
of 20 was confirmed using HPLC (96.02%, Rt ¼ 14.823). 1H NMR
(t, J ¼ 7.92 Hz, 1H), 7.65 (ddd, J ¼ 0.96 Hz, J ¼ 6.96 Hz, J ¼ 8.88 Hz,
1H), 7.8 (d, J ¼ 7.56 Hz, 1H), 7.87 (m. 1H), 7.98 (m, 1H), 8.02 (s, 1H),
8.31 (s, 2H), 8.56 (d, J ¼ 7.92 Hz, 1H), 8.62 (s, 1H), 9.92 (s, 1H). 13C
(DMSO-d6): d 1.12 (t, 3H), 2.43 (q, 2H), 3.32e3.78 (m, 8H), 7.51e7.65
(m, 6H), 7.89 (m, 2H), 8.12 (m, 2H), 8.27 (s, 2H), 8.47 (m, 2H), 8.6 (d,
J ¼ 8.28 Hz, 1H), 10.0 (s, 1H). 13C NMR (DMSO-d6):
d 15.54, 21.88,
NMR (DMSO-d6):
d
34.53, 41.5, 43.25, 43.78, 46.9, 115.15, 120.82,
43.59, 114.04, 121.22, 123.07, 124.92, 126.07, 127.91, 128.17, 128.47,
130.28, 130.33, 133.36, 138.16, 140.74, 150.53, 157.1, 157.77, 158.94,
160.22, 169.11. MS: calcd 515.24 for C31H29N7O [M]þ; found, 515.89
122.17, 122.96, 123.18, 126.12, 126.38, 127.86, 128.34, 128.56, 128.67,
133.13, 139.23, 140.87, 149.7, 154.36, 157.67, 157.81, 160.17, 169.02.
MS: calcd 501.23 for C30H27N7O [M]þ; found, 501.95 [M] þ
.
[M] þ
.
3.1.19. (4-(5-Benzylpyrimidin-2-yl)piperazin-1-yl)(4-(quinazolin-
4-ylamino)phenyl)methanone (17)
3.1.23. (4-(5-Ethylpyrimidin-2-yl)piperazin-1-yl)(4e((2-phenyl-
quinazolin-4 ylamino)methyl)phenyl)methanone (21)
Compound 17 was prepared from intermediate 2 (90 mg,
0.354 mmol) and intermediate 8 (93 mg, 0.354 mmol) following the
procedure used in the preparation of compound 13. The product
obtained was a white solid (135 mg, 76%); mp: 245e247 ꢁC. The
purity of 17 was confirmed using HPLC (96.73%, Rt ¼ 14.952). 1H
Compound 21 was prepared from intermediate 1 (120 mg,
0.625 mmol) and intermediate 12 (221 mg, 0.625 mmol) following
the same procedure utilized in the preparation of 13. The product
obtained was a white solid (105 mg, 20%); mp: 207e208 ꢁC. The
purity of 21 was confirmed using HPLC (97.08%, Rt ¼ 15.735). 1H
NMR (DMSO-d6):
d
3.05e3.8 (br, m, 10H), 7.18e7.31 (m, 5H), 7.5 (d,
NMR (DMSO-d6): d 1.1 (t, 3H), 2.41 (q, 2H), 3.34e3.76 (m, 8H), 4.97
J ¼ 8.64 Hz, 2H), 7.67 (ddd, J ¼ 1.38, Hz, J ¼ 7.2 Hz, J ¼ 8.1 Hz, 1H),
7.82 (d, J ¼ 8.28 Hz, 1H), 7.89 (ddd, J ¼ 1.2 Hz, J ¼ 6.96 Hz,
J ¼ 8.28 Hz, 1H), 8.04 (d, J ¼ 8.64 Hz, 2H), 8.32 (s, 2H), 8.65 (d,
J ¼ 8.1 Hz, 1H), 8.67 (s, 1H), 10.01 (s, 1H). 13C NMR (DMSO-d6):
(d, J ¼ 5.82 Hz, 2H), 7.4e7.55 (m, 8H), 7.79 (m, 2H), 8.25 (s, 2H), 8.33
(m, 1H), 8.43 (m, 2H), 8.98 (t, J ¼ 5.82 Hz, 1H). 13C NMR (DMSO-d6):
d
15.56, 21.88, 41.4, 43.26, 43.56, 46.85, 113.79, 122.68, 124.94,
125.45, 127.24, 127.31, 127.84, 128.2, 130.3, 132.83, 134.26, 138.52,
141.51, 149.98, 157.1, 159.15, 159.59, 160.16, 169.11. MS: calcd 529.26
d
34.50, 43.52, 45.38, 115.20, 121.73, 122.87, 123.11, 126.08, 126.35,
þ
for C32H31N7O [M]þ; found, 529.88 [M]
.
127.75, 127.8, 128.3, 128.51, 130.34, 133.12, 140.59, 140.83, 149.69,
154.27, 157.57, 157.78, 160.13, 169.09. MS: calcd 501.23 for
C30H27N7O [M]þ; found, 501.74 [M] þ
.
3.1.24. (4-(5-Benzylpyrimidin-2-yl)piperazin-1-yl)(3-(2-phenylq-
uinazolin-4-ylamino)phenyl)methanone (22)
3.1.20. (4-(5-Benzylpyrimidin-2-yl)piperazin-1-yl)(4-((quinazolin-
4-ylamino)methyl)phenyl)methanone (18)
Compound 22 was prepared from intermediate 2 (120 mg,
0.472 mmol) and intermediate 10 (160 mg, 0.472 mmol) following
the same procedure utilized in the preparation of 13. The product
obtained was a white solid (135 mg, 23%); mp: 214e215 ꢁC. The
purity of 22 was confirmed using HPLC (95.38%, Rt ¼ 18.948). 1H
Compound 18 was prepared from intermediate 2 (102 mg,
0.405 mmol) and intermediate 9 (113 mg, 0.405 mmol) following
the general procedure used in the preparation of 13. The product
obtained was a white solid (40 mg, 19%); mp: 99e101 ꢁC. The purity
of 18 was confirmed using HPLC (96.14%, Rt ¼ 15.95). 1H NMR
NMR (DMSO-d6):
d 3.37e3.83 (br, m, 10H), 7.2e7.32 (m, 6H),
7.44e7.67 (m, 5H), 7.91 (m, 2H), 8.1 (ddd, J ¼ 0.9 Hz, J ¼ 2.1 Hz,
(DMSO-d6):
d
3.37e3.83 (br, m, 10H), 4.89 (d, J ¼ 5.82 Hz, 2H),
J ¼ 8.28 Hz, 1H), 8.2 (m, 1H), 8.32 (s, 2H), 8.47 (m, 2H), 8.62 (d,
7.22e7.34 (m, 5H), 7.43 (d, J ¼ 8.28 Hz, 2H), 7.49 (d, J ¼ 8.28 Hz, 2H),
7.6 (ddd, J ¼ 1.2 Hz, J ¼ 6.72 Hz, J ¼ 8.28 Hz,1H), 7.76 (dd, J ¼ 0.84 Hz,
J ¼ 7.56 Hz, 1H), 7.84 (ddd, J ¼ 1.2 Hz, J ¼ 6.72 Hz, J ¼ 8.28 Hz, 1H),
8.34 (s, 2H), 8.36 (d, J ¼ 7.92 Hz, 1H), 8.51 (s, 1H), 8.94 (t, J ¼ 5.82 Hz,
J ¼ 8.28 Hz, 1H), 10.0 (s, 1H). 13C NMR (DMSO-d6):
d 34.53, 41.46,
43.84, 46.95, 113.99, 120.65, 121.84, 122.89, 122.98, 123.02, 126.07,
126.11, 127.88, 128.17, 128.33, 128.54, 128.66, 128.78, 130.3, 131.43,
132.01, 133.34, 136.11, 138.18, 139.54, 140.85, 150.49, 157.77, 157.85,
158.92, 160.15, 169.14. MS: calcd 577.26 for C36H31N7O [M]þ; found,
1H). 13C NMR (DMSO-d6):
d 34.49, 43.23, 114.86, 122.61, 122.89,
125.74, 126.09, 127.07, 127.2, 127.54, 128.31, 128.53, 132.62, 134.23,
140.83, 141.09, 149.17, 155.02, 157.77, 159.36,þ160.1, 169.1. MS: calcd
577.55 [M] þ
.
515.24 for C31H29N7O [M]þ; found, 515 [M]
.
3.1.25. (4-(5-Benzylpyrimidin-2-yl)piperazin-1-yl)(4-(2-phenylq-
uinazolin-4-ylamino)phenyl)methanone (23)
3.1.21. (4-(5-Ethylpyrimidin-2-yl)piperazin-1-yl)(2-(3-phenylquin-
azolin-4-ylamino)phenyl)methanone (19)
Compound 23 was prepared from intermediate 2 (120 mg,
0.472 mmol) and intermediate 11 (160 mg, 0.472 mmol) following
the same procedure utilized in the preparation of 13. The product
obtained was a white solid (70 mg,12%); mp: >250 ꢁC. The purity of
23 was confirmed using HPLC (95%, Rt ¼ 18.98). 1H NMR (DMSO-
Compound 19 was prepared from intermediate 1 (120 mg,
0.625 mmol) and intermediate 10 (213 mg, 0.625 mmol) following
the same procedure utilized in the preparation of 13. The product
obtained was a white solid (210 mg, 40%); mp: 204e205 ꢁC. The
purity of 19 was confirmed using HPLC (97.67%, Rt ¼ 15.123). 1H
d6): d 3.38e3.82 (br, m, 10H), 7.21e7.33 (m, 6H), 7.53e7.69 (m, 5H),
7.93 (m, 2H), 8.15 (m. 2H), 8.34 (s, 2H), 8.51 (m, 2H), 8.64 (d,
NMR (DMSO-d6):
d
1.09 (t, 3H), 2.4 (q, 2H), 3.16e3.71 (m, 8H), 7.18
J ¼ 8.28 Hz, 1H), 10.06 (s, 1H). 13C NMR (DMSO-d6):
d 34.56, 43.53,
(d, J ¼ 7.56 Hz, IH), 7.41e7.59 (m, 5H), 7.83 (m, 2H), 8.02 (m, 1H),
114.08, 121.28, 122.95, 123.11, 126.16, 127.98, 128.21, 128.38, 128.54,
128.6, 128.75, 130.29, 130.42, 131.47, 133.44, 138.19, 140.8, 140.89,
150.57, 157.83, 159, 160.18, 169.19. MS: calcd 577.26 for C36H31N7O
8.13 (m, 1H), 8.21 (s, 2H), 8.4 (m, 2H), 8.54 (d, J ¼ 8.22 Hz, 1H), 9.87
(s, 1H). 13C NMR (DMSO-d6):
d 15.36, 21.85, 42.66, 44.25, 114.01,
þ
[M]þ; found, 577.69 [M]
.
120.67, 121.79, 122.86, 125.02, 125.97, 127.9, 128.16, 128.27, 128.6,
130.12, 130.2, 133.23, 136.23, 138.3, 139.59, 150.56, 157.03, 157.92,
159.02, 160.3, 169.2. MS: calcd 515.24 for C31H29N7O [M]þ; found,
3.1.26. (4-(5-Benzylpyrimidin-2-yl)piperazin-1-yl)(4-((2-phenyl-
quinazolin-4-ylamino)methyl)phenyl)methanone (24)
þ
515.84 [M]
.
Compound 24 was prepared from intermediate 2 (120 mg,
0.472 mmol) and intermediate 12 (167 mg, 0.472 mmol) following
the same procedure utilized in the preparation of 13. The product
obtained was a white solid (59 mg, 10%); mp: 216e217 ꢁC. The
purity of 24 was confirmed using HPLC (97.27%, Rt ¼ 19.543). 1H
3.1.22. (4-(5-Ethylpyrimidin-2-yl)piperazin-1-yl)(2-(4-phenylquin-
azolin-4-ylamino)phenyl)methanone (20)
Compound 20 was prepared from intermediate 1 (120 mg,
0.625 mmol) and intermediate 11 (213 mg, 0.625 mmol) following
the same procedure utilized in the preparation of 13. The product
obtained was a white solid (180 mg, 34%); mp: >250 ꢁC. The purity
NMR (DMSO-d6):
d
3.37e3.79 (br, m, 10H), 4.99 (d, J ¼ 5.82 Hz, 2H),
7.19e7.31 (m, 5H), 7.42e7.66 (m, 8H), 7.82 (m, 2H), 8.3 (s, 2H), 8.35

H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
2055
(d, J ¼ 8.22 Hz, 1H), 8.44e8.47 (m, 2H), 9.02 (t, J ¼ 5.82 Hz, 1H). 13
C
treatment and measurement were performed for cells prior to
treatmentand were assigned TZ (T-zero). The % growth wascalculated
using the following equation: growth ¼ ðTi ꢂ TzÞ ꢃ 100=ðC ꢂ TzÞ;
where Ti is the reading for the treated well after two days of the
treatment with a certain concentration of a specific compound. C is
the average reading measured in the untreated wells after two days of
treatment with the vehicle. TZ is the average reading measured prior
to treatment. GI50 is the dose that causes 50% inhibition of the growth
after the treatment time compared to the TZ and calculated from
ꢂ50 ¼ ðTi ꢂ TzÞ ꢃ 100=ðC ꢂ TzÞ: TGI is the dose that causes a total
inhibition of the growth and can be computed using the equation:
Ti ¼ TZ. LC50 is the dose that kills 50% of the cells compared to the cell
density at the treatment time and calculated from ꢂ50 ¼ ðTi ꢂ TzÞ ꢃ
100=Tz: This experiment was performed by the National Cancer
Institute (NCI).
NMR (DMSO-d6):
d 34.51, 43.24, 43.56, 113.79, 122.68, 122.88,
125.45, 126.1, 127.23, 127.3, 127.84, 128.2, 128.31, 128.53, 130.04,
132.83, 134.24, 138.52, 140.83, 141.51, 149.98, 157.77, 159.15, 159.58,
160.09, 169.12. MS: calcd 591.27 for C37H33N7O [M]þ; found, 591.69
þ
[M]
.
3.2. Antiproliferative cellular screening
3.2.1. MTT viability assay
MDA-MB-468 human breast cells were obtained from ATCC and
cultured in RPMI 1640 medium (ATCC) supplemented with 10% FBS
(Invitrogen), and 2% antibiotic-antimycotic mixture of Penicillin-G,
Streptomycin sulfate, and Amphotericin B (Invitrogen) in a 5% CO2
e 95% humidity incubator at 37 ꢁC. 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazoliumbromide (MTT) assay was chosen to
determine the IC50 (the concentration required to decrease cell
viability by 50%) of the test compounds (4, 15, and 16) against the
MDA-MB-468 cell line using gefitinib as a positive control [76].
5000 cells, in 100 uL of the culture medium, were plated per well in
96-well plates. Cells were allowed to adhere for 1 day. Cells were
then treated with fresh culture medium containing one of several
3.3. Kinase targeting investigation
3.3.1. Kinase binding screening
Biotinylated kinase ligand was immobilized onto streptavidin
coated magnetic beads and washed with blocking buffer. The beads
were then incubated with DNA-tagged kinase in the presence of
test compound for 1 h at room temperature. Binding reactions were
carried out in 20% Seablock, 0.17 ꢃ PBS, 0.005% Tween 20, 6 mM
DTT, in a final volume of 0.04 mL. Beads were then washed with
wash buffer (PBS, 0.05% Tween 20), and then incubated with
elution buffer (PBS, 0.05% Tween 20, 0.5 M non-biotinylated kinase
ligand) and eluted. The kinase concentration in the eluate was then
measured by quantitative PCR. The detected signal is proportional
to the number of eluted DNA-tagged kinase molecules and hence to
the binding potency of a given test compound. The results of this
single dose screening are reported in the form of binding
percentage of control (B-POC) which is defined as follows:
B ꢂ POC ¼ ½ðtest compound signal ꢂ positive control signal Þ ꢃ
100ꢄ=ðnegative control signal ꢂ positive control signalÞ: The neg-
ative control signal is obtained by the vehicle and corresponds to
B-POC ¼ 100 %. The positive signal control is detected by a binding
positive compound and its B-POC is 0%. The B-POC results are the
averageofduplicate measurements. The picturesshowinginteraction
with the kinome were generated using the TREEspot data visualiza-
tion (Fig. S1). In order to determine the Kd, the above procedure is
followed at 11-point three fold serial dilution of the test compound,
concentrations of the test compounds; 100, 10, 1, 0.1, 0.01, 0.001 mM
(final DMSO concentration was 1% irrelevant to the test comp-
ounds’ concentration). The plates were processed after 48, 72, or
120 h via discarding the media and adding 100 uL 0.5 mg/ml MTT
reagent in fresh culture media. The plates were then incubated for
4 h under 10% CO2 at 37 ꢁC. The media was then removed and
100 uL of DMSO was added to each well to dissolve the formed
formazone crystals. Plates were kept at room temperature over
night away from light. The optical density of the formed violet color
was quantified using an automatic spectrophotometer (Tristar LB
941) at a wavelength of 562 nm. The experiment was carried out
with triplicate for each concentration of a given compound. Three
independent experiments were conducted on three different dates.
The concentration required for 50% reduction in the optical density,
and hence the viability compared to control vehicle treated wells,
was estimated for each compound against the studied cell line by
non-linear regression analysis of the Log10 concentration in moles
versus % viability curves. Curve fitting was performed using the
variable slope-four parameter module implemented in the
GraphPad Prism software (version 5.02).
from 30 mM to 0.508 nM, with the result of a standard dose-response
curve. The Kd is then calculated according to the following equation:
3.2.2. Growth inhibition against NCI-60 panel
The SRB (sulforhodamine B) assay was used to measure the ability
of a test compound to inhibit the growth of a given cell line [31]. The
cell lines used in the NCI-60 are maintained using RPMI 1640 medium
slope
response
þdosehill slope; hill slope
¼
background þ signal ꢂ background=1 þ Kdhill
¼ ꢂ1.
supplemented with5%fetalbovine serum and 2 mM
L-glutamine. Cell
During the Kd calculation, the final DMSO % was fixed at 2.5%
regardless of the test compound concentration [35].
lines are seeded into a series of 96-well microtiter plates, with varied
inoculation densities, depending on the growth rate and doubling
time of each cell line. The plates are then incubated at 37 ꢁC, 5 % CO2,
95 % air and 100 % relative humidity for 24 h before treating with the
test compounds. On the treatment day, experimental agents are
addedtothecellswithaseriesoftargetfinalconcentrations:100,10,1,
A non-linear least square fit with the LevenbergeMarquardt
algorithmwasimplementedtofitthe curves (Fig. S2). Kinasebinding
assays were performed by Ambit Bioscience (San Diego, CA, USA).
3.3.2. Functional kinase inhibition assay
0.1, and 0.01
m
M (final DMSO concentration was 0.25% irrelevant of
This experiment was conducted by mixing the test kinase,10
m
m
M
M
the test compound’s concentration). After 48 h treatments while
being incubated under the above conditions, the cells are fixed with
trichloroacetic acid (concentration differs according to the nature of
the cell line; adherent or suspension) for one hour at 4 ꢁC. After
removal of the supernatant, the plates are washed with water several
g-
33P-ATP, positively charged substrate, magnesium acetate, 1
test compound, and reaction buffer at room temperature. The
reaction is then stopped by the addition of phosphoric acid solu-
tion. 10 mL of the reaction is filtered through a P30 filament, washed
properly, dried, and finally subjected to scintillation counting [45].
DMSO was used as a vehicle for test compounds and its concen-
tration was 2% regardless of the serial dilution of tested agents. The
inhibition of a given kinase is represented by the function
percentage of control (F-POC) which is calculated as follows:
times and dried completely in the air. Then 100 mL of 0.4% sulfo-
rhodamine B (SRB) solution in 1% acetic acid is added for 10 min. The
unbound dye is removed and the plates are washed with 1% acetic
acid. The bound dye is then solubilized with 10 mM trizma base, and
the absorbance is measured using a plate reader at 515 nm. The same
F ꢂ POC
¼ ½meanðcounts ꢂ blanksÞꢄtest ꢃ 100=½meanðcountsꢂ

2056
H.M. Shallal, W.A. Russu / European Journal of Medicinal Chemistry 46 (2011) 2043e2057
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3.4. Molecular docking
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̀
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