Design and synthesis of new anticancer pyrimidines with multiple-kinase inhibitory effect

Article abstract of DOI:10.1016/j.bmc.2010.04.037

A new series of N-substituted-2-aminopyrimidines based on the '4-(pyridin-3-yl)pyrimidin-2-amine' scaffold of Imatinib has been designed and synthesized. A selected group from the target compounds was tested over a panel of 60 cancer cell lines at a single dose concentration of 10 μM, and the two most active compounds, 25b and 30, were further tested in a five-dose testing mode to determine their IC₅₀ values over the 60 cell lines. Compound 30 has showed good potencies and high efficacies, and was accordingly tested at a single dose concentration of 10 μM over a panel of 54 kinases. At this concentration, the compound has showed multiple inhibitions over a number of oncogenic kinases, including ABL1, AKT1, LCK, C-SRC, PIM1, FLT3, FYN, and KDR. A molecular modeling study was made by docking of the most active compound 30 and its inactive analog 29 into the kinase domain of ABL1 to investigate their possible binding interactions.

Full text of DOI:10.1016/j.bmc.2010.04.037

Bioorganic & Medicinal Chemistry 18 (2010) 3860–3874
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of new anticancer pyrimidines with multiple-kinase
inhibitory effect
*
Ibrahim Mustafa El-Deeb, So Ha Lee
Life/Health Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
Department of Biomolecular Science, University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of N-substituted-2-aminopyrimidines based on the ‘4-(pyridin-3-yl)pyrimidin-2-amine’
scaffold of Imatinib has been designed and synthesized. A selected group from the target compounds
was tested over a panel of 60 cancer cell lines at a single dose concentration of 10 lM, and the two most
active compounds, 25b and 30, were further tested in a five-dose testing mode to determine their IC50
values over the 60 cell lines. Compound 30 has showed good potencies and high efficacies, and was
Received 22 March 2010
Revised 14 April 2010
Accepted 15 April 2010
Available online 20 April 2010
accordingly tested at a single dose concentration of 10 lM over a panel of 54 kinases. At this concentra-
Keywords:
Anticancer
Pyrimidine
Multiple-kinase
Molecular modeling
tion, the compound has showed multiple inhibitions over a number of oncogenic kinases, including ABL1,
AKT1, LCK, C-SRC, PIM1, FLT3, FYN, and KDR. A molecular modeling study was made by docking of the
most active compound 30 and its inactive analog 29 into the kinase domain of ABL1 to investigate their
possible binding interactions.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
a selective anticancer drug for treatment of chronic myeloid leuke-
mia (CML) with minimal side effects, relative to old anticancer
Cancer is thought to reflect a multi-step process, resulting from
an accumulation of inherited and/or acquired defects in genes in-
volved in the positive or negative regulation of cell proliferation
and survival. For the development of a clinically recognizable hu-
man cancer, the activation or inactivation of as many as four or five
different genes may be required.1 The human genome encodes
approximately 500 predicted protein kinases, many of them are
participating in signal transduction pathways that regulate cell
growth and survival, suggesting potential roles in cancer initiation
and progression.2 Indeed, several protein kinases have been di-
rectly implicated in human oncogenesis by virtue of being over-ex-
pressed or mutationally activated in cancer cells.3 Hence, the
protein kinases have been widely considered as drug targets for
cancer therapy, and the targeting of such kinases either with small
organic molecules4–6 or with monoclonal antibodies7,8 has been re-
cently considered as a promising tool for selective treatment of
such kinase dependent cancers.
drugs, numerous kinase inhibitors targeting oncogenic kinases
have been developed. A large number of these kinase inhibitors be-
long to the same chemical group of Imatinib, PAPs. These com-
pounds include inhibitors for receptor and non-receptor kinases
such as Bcr-Abl,10,11 CDK,12 c-KIT,13 EGFR,11,14 Lyn,11 and PLK112 ki-
nases. Accordingly, we have designed and synthesized a new series
of N-substituted-2-aminopyrimidines based on the ‘4-(pyridin-3-
yl)pyrimidin-2-amine’ scaffold of Imatinib (Fig. 1). The modifica-
tions at this scaffold were made basically at both of the amino
group of the 2-aminopyrimidine moiety and the 6-position of the
terminal pyridine ring.
A selected group (12 compounds) from the target compounds
was tested over a panel of 60 cancer cell lines at a single dose con-
centration of 10
l
M, and the two most active compounds, 25b and
R1
H
H
N
N
N
O
N
R3
N
N
R2
Phenylaminopyrimidines (PAPs) represent a large chemical
group of small molecular kinase inhibitors, and a representative
example and a lead compound of such group is the Bcr-Abl tyro-
sine kinase inhibitor Imatinib (Gleevec, STI-571).9 Imatinib, a PAP
derivative, was the first kinase inhibitor to be approved for the
treatment of cancer. After the successful approval of Imatinib as
N
N
N
N
N
R4
Imatinib (Gleevec, STI-571)
New PAP derivatives
* Corresponding author. Tel.: +82 2 958 6834; fax: +82 2 958 5189.
E-mail address: LSH6211@kist.re.kr (S.H. Lee).
Figure 1. Structure of Imatinib and the general formula of the new PAP derivatives.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.037

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
3861
30, were further tested in a five-dose testing mode to determine
their IC50 values over the 60 cell lines. Compound 30 has showed
the highest potencies and efficacies, and was accordingly tested
at a single dose concentration of 10 lM over a panel of 54 kinases,
in order to determine its kinase inhibitory profile. At this concen-
tration, the compound has showed multiple inhibitions over a
number of oncogenic kinases.
H
N
N
NH2
N
N
NH2
N
N
N
N
i
ii
N
Cl
6
7
2. Results and discussion
2.1. Chemistry
iii
iv
H
N
N
Ar
H
N
In this study, five different groups of the target compounds were
prepared by following five different synthetic schemes. In Scheme 1,
3-(dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one (1) was pre-
pared in 82% yield by heating 3-acetylpyridine with excess
N,N-dimethylformamide dimethylacetal for 4 h.15 By refluxing com-
pound 1 with guanidine hydrochloride in absolute ethanol and in
the presence of sodium ethoxide, 4-(pyridin-3-yl)pyrimidin-
2-amine (2) was obtained in a good yield.16 The benzylation of the
amino group of the pyrimidin-2-amine (2), was carried out by the
dropwise addition of 2,4-dimethylbenzyl bromide to a heated solu-
tion of 2 and K2CO3 in DMF for the preparation of compound 3 in 39%
yield.17 A group of N-mono- and N,N-dibenzoyl derivatives (4a–c)
was prepared by dropwise addition of the appropriate benzoyl chlo-
ride to the amine 2 in refluxing pyridine. For the preparation of the
N-aryl derivatives (5a–f), a modified Buchwald–Hartwig amination
protocol previously developed by our group was applied,17 where
the amine 2 was refluxed with the appropriate arylbromide in
toluene and under N2 atmosphere, in the presence of dichlorobis-
(triphenylphosphine)Pd(II) as a palladium catalyst, Xantphos as a
phosphine ligand, and NaOt-Bu as a base. By this method, the target
N-aryl derivatives were obtained in moderate yields and in a rela-
tively short time of only 8 h.
N
N
N
CF3
N
O
N
9a: Ar = 2,4-dimethylphenyl
9b: Ar = 3,5-bis(trifluoromethyl)phenyl
8
Scheme 2. Reagents and conditions: (i) 3-pyridineboronic acid, dichlorobis-
(triphenylphosphine)Pd(II), Na2CO3, acetonitrile/water (1:1), N2, 78 °C, 7 h, 74%;
(ii) 2,4-dimethylbenzylbromidee, K2CO3, DMF, 130 °C, 5 h, 43%; (iii) 3-trifluoro-
methylbenzoylchloride, pyridine, reflux, 6 h; (iv) arylbromide, dichlorobis(triphen-
ylphosphine) Pd(II), Xantphos, NaOt-Bu, toluene, N2, reflux, 8 h, 35% (9a), 68% (9b).
din-3-yl)pyrimidin-2-amine (6) instead of the amine 2 used in
Scheme 1. For the preparation of this amine, 2-amino-4-chloro-6-
methylpyridine underwent a Suzuki coupling reaction with 3-pyri-
dineboronic acid in a mixed solvent of acetonitrile and water in a
(1:1, v/v) ratio, and in the presence of dichlorobis(triphenylphos-
phine)Pd(II) and Na2CO3.18
The target of Schemes 3–5 was to obtain new pyrimidine deriv-
atives with extended structure at their pyridine tail, in order to
investigate the effect of the structural modifications at that part
of the compounds on activity. This structural extension was
achieved by substitution with aryl or heteroaryl moieties at the
6-position of the terminal pyridine ring. In order to achieve this
In Scheme 2, 4-methylpyrimidin-2-amine analogs to the benzyl,
benzoyl and N-aryl derivatives prepared in Scheme 1 were synthe-
sized, applying the same preparation protocols followed in
Scheme 1. The only difference was the use of 4-methyl-6-(pyri-
H
N
NH2
N
N
N
N
N
O
O
N
N
ii
i
iii
N
N
3
2
1
R2
H
N
N
N
N
Ar
R1
N
N
N
v
iv
N
4a: R1 = H, R2 = 3-trifluoromethylbenzoyl
4b: R1 = R2 = 3-trifluoromethylbenzoyl
5a: Ar = 5-acetamido-2-methylphenyl
5b: Ar = 3,5-bis(trifluoromethyl)phenyl
5c: Ar = 4-(N,N-dimethylamino)phenyl 4c: R1 = R2 = benzoyl
5d: Ar = 2,4-dimethylphenyl
5e: Ar = 4-methoxyphenyl
5f: Ar = 2-naphthyl
Scheme 1. Reagents and conditions: (i) N,N-dimethylformamide dimethylacetal, fusion, 4 h, 82%; (ii) guanidine-HCl, NaOEt, abs EtOH, reflux, 6 h, 73%; (iii) 2,4-
dimethylbenzyl bromide, K2CO3, DMF, 130 °C, 5 h, 39%; (iv) (substituted) benzoylchloride, pyridine, reflux, 3 h, 19% (4a), 37% (4b), 52% (4c); (v) arylbromide,
dichlorobis(triphenylphosphine)Pd(II), Xantphos, NaOt-Bu, toluene, N2, reflux, 8 h, 42% (5a), 82% (5b), 39% (5c), 31% (5d), 52% (5e), 56% (5f).

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O
O
N
N
O
O
O
N
N
O
Br
Br
ii
i
iii
NH
N
X
NH
N
N
X
ii
i
iii
Br
N
N
10
16
17
Br
10
11a: X = CH
11b: X = N
12a: X = CH
12b: X = N
H
H
N
N
N
CF3
N
NH2
H
H
N
N
O
N
N
N
N
CF3
N
NH2
CF3
iv
N
N
X
O
N
N
X
N
CF3
v
iv
HN
HN
19
18
14a: X = CH, 14b: X = N
13a: X = CH, 13b: X = N
H
N
N
CF3
N
N
H
N
N
Ar
CF3
v
N
N
X
vi
HN
15a: X = CH, Ar = 4-methoxyphenyl
20
15b: X = CH, Ar = 3,5-(trifluoromethyl)phenyl
15c: X = CH, Ar = 4-(N,N-diphenylamino)phenyl
15d: X = N, Ar = 4-(N,N-diphenylamino)phenyl
Scheme 4. Reagents and conditions: (i) aniline. dichlorobis(triphenylphos-
phine)Pd(II), dppp, aniline, N2, reflux, 12 h, 38%; (ii) N,N-dimethylformamide
dimethylacetal, fusion, 12 h, 94%; (iii) guanidine-HCl, NaOEt, abs EtOH, reflux, 8 h,
46%; (iv) 3,5-bis(trifluoromethyl)phenyl isocyanate, fusion, 2 h, 74%; (v) 3,5-
bis(trifluoromethyl)phenyl bromide, dichlorobis(triphenylphosphine)Pd(II), Xant-
phos, NaOt-Bu, toluene, N2, reflux, 12 h, 62%.
Scheme 3. Reagents and conditions: (i) n-BuLi (1.6 M/hexanes), N,N-dimethyl
acetamide, diethyl ether, N2, À78 °C, 1 h, 51%; (ii) arylboronic acid, dichlorobis(tri-
phenylphosphine)Pd(II), Na2CO3, acetonitrile/water (1:1), N2, 78 °C, 4 h, 67% (11a),
74% (11b); (iii) N,N-dimethylformamide dimethylacetal, fusion, 3 h, 99% (12a), 88%
(12b); (iv) guanidine-HCl, NaOEt, abs EtOH, reflux, 5 h, 84% (13a), 90% (13b); (v)
3,5-bis(trifluoromethyl)phenyl isocyanate, fusion, 2 h, 68% (14a), 75% (14b); (vi)
arylbromide, dichlorobis(triphenylphosphine)Pd(II), Xantphos, NaOt-Bu, toluene,
N2, reflux, 8 h, 82% (15a), 79% (15b), 31% (15c), 27% (15d).
amine 18 in an analogous method to that used for the preparation
of the amines 2, 13a and 13b. Similarly to what was made in
Scheme 3, the urea derivative 19 and the N-aryl derivative 20 were
prepared following the same procedures used for the preparation
of the urea and aryl derivatives 14a–b and 15a–d, respectively.
In Scheme 5, a number of acetyl- and acetamidophenyl analogs
to the compounds prepared in Scheme 3 were synthesized. The
reason for following a modified pathway here is the presence of
an acetyl moiety at the newly attached phenyl ring which would
interfere with the reaction of the acetyl group of 5-acetyl-2-bro-
mopyridine (10) with N,N-dimethylformamide dimethylacetal if
attached to the compound from the beginning. Therefore, 5-acet-
yl-2-bromopyridine (10) was first reacted with N,N-dimethylform-
amide dimethylacetal, followed by reaction with guanidine
hydrochloride in ethanol to yield the intermediate amine 22 in a
58% yield. This amine was directly converted to the urea derivative
23 similarly to the urea derivatives 14a and 14b. It was also used
for the preparation of the new amines 24a–d, by Suzuki coupling
with the appropriate arylboronic acids in a mixed solvent of aceto-
nitrile and water in a (1:1, v/v) ratio, and in the presence of dichlor-
obis(triphenylphosphine)Pd(II) and Na2CO3. By following similar
procedures to those used for the preparation of the urea deriva-
tives 14a–b and the N-aryl derivatives 15a–d, compounds 25a–d
and 26a–b were prepared, respectively.
target, 2,5-dibromopyridine was converted into 5-acetyl-2-bromo-
pyridine (10) in 51% yield, as showed in Scheme 1, according to
literature procedure,19 by lithiation of 2,5-dibromopyridine in
diethyl ether at À78 °C under nitrogen atmosphere, followed by
acetylation at position 5 by N,N-dimethylacetamide. The resulted
5-acetyl-2-bromopyridine was then converted to the amines 13a
and 13b following the procedures previously reported by our
group.17 The new pyrimidin-2-amines 13a and 13b were then used
for the preparation of the corresponding 3,5-bis(triflorometh-
yl)phenyl urea derivatives 14a and 14b in good yields of 68% and
75%, respectively, by fusion with 3,5-bis(trifloromethyl)phenyl iso-
cyanate for 2 h. The synthesized amines 14a and 14b were also
used for the preparation of a number of N-aryl derivatives 15a–d
using the same Buchwald–Hartwig amination protocol previously
employed for the synthesis of compounds 5a–f.17
In Scheme 4, the extension of structure at the pyridyl tail was
carried out through a nitrogen bridge, by reacting the 5-acetyl-2-
bromo-pyridine scaffold (10) with excess aniline under reflux for
12 h, in the presence of dichlorobis(triphenylphosphine)Pd(II) as
a
palladium catalyst, 1,3-bis(diphenylphosphino)propane as a
phosphine ligand, and sodium tert-butoxide as a base. The resulted
acetyl intermediate 16 was then converted to the corresponding
In order to improve the solubility of compounds 15b, 19, 25c,
and 25d in DMSO for the purpose of anticancer screening, the com-

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
3863
Table 1
H
H
N
Compounds selected for single dose cancer cell line screening and their mean
inhibitory percentages
N
N
NH2
iii
N
N
CF3
O
N
N
O
N
N
O
Compd
Structure
Inhibition %a
CF3
H
ii
i
N
N
CF3
N
N
N
5b
À0.32
Br
Br
10
Br
Br
CF3
21
22
23
N
N
iv
NH2
H
H
N
N
N
CF3
N
CF3
N
N
N
N
N
O
8
0.62
CF3
N
24a: R1 = 2-acetyl
24b: R1 = 4-acetyl
24c: R1 = 2-acetamido
24d: R1 = 3-acetamido
vi
H
N
26a: R1 = 2-acetyl
26b: R1 = 4-acetyl
N
R1
R1
N
N
9a
3.91
v
H
H
N
N
N
CF3
H
N
N
CF3
N
N
O
N
N
R2
CF3
27
0.49
25a: R1 = 2-acetyl, R2 = CF3
.
3HCl
25b: R1 = 2-acetyl, R2 = morpholino
25c: R1 = 2-acetamido, R2 = CF3
25d: R1 = 3-acetamido, R2 = CF3
R1
Scheme 5. Reagents and conditions: (i) N,N-dimethylformamide dimethylacetal,
fusion, 20 h, 88%; (ii) guanidine-HCl, NaOEt, abs EtOH, reflux, 12 h, 58%; (iii) 3,5-
bis(trifluoromethyl)phenyl isocyanate, fusion, 2 h, 79%; (iv) arylboronic acid,
dichlorobis(triphenylphosphine)Pd(II), Na2CO3, acetonitrile/water (1:1), N2, 78 °C,
12 h, 64% (24a), 61% (24b), 46% (24c), 59% (24d); (v) 3,5-bis(trifluoromethyl)phenyl
isocyanate, fusion, 2 h, 65% (25a), 66% (25b), 42% (25c), 45% (25d); (vi) 3,5-
bis(trifluoromethyl)phenyl bromide, dichlorobis(triphenylphosphine)Pd(II), Xant-
phos, NaOt-Bu, toluene, N2, reflux, 12 h, 79% (26a), 69% (26b).
H
N
N
N
N
N
15c
0.97
pounds were converted into their corresponding HCl-salts 27, 28,
29, and 30, respectively, by passing HCl gas into free base solutions
of these compounds in toluene.
H
N
H
N
N
CF3
2.2. Biological screening
N
N
O
.
2.2.1. In vitro anticancer screening
CF3
28
À6.98
The structures of the final products were submitted to National
Cancer Institute (NCI),20 Bethesda, Maryland, USA, and the 12 com-
pounds showed in Table 1 were selected on the basis of degree of
structural variation and computer modeling techniques for evalu-
ation of their antineoplastic activity. The selected compounds were
subjected to in vitro anticancer assay against tumor cells in a full
panel of 60 cell lines taken from nine different tissues (blood, lung,
colon, CNS, skin, ovary, kidney, prostate, and breast). The com-
4HCl
HN
N
H
N
CF3
N
N
pounds were tested at a single dose concentration of 10 lM, and
CF3
the percentages of growth inhibition over the sixty tested cell lines
were determined. The mean inhibition percentages of all of the
tested compounds over the full panel of cell lines are illustrated
in Table 1 and the figures showing the inhibitions exerted by each
compound over the full cell lines panel are showed in the Supple-
mentary data. As showed in Table 1, a remarkable mean inhibition
was observed with compounds 25b (38.41%) and 30 (50.92%),
while the mean inhibition was weak in the rest of the compounds.
20
0.20
HN
(continued on next page)

3864
I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
emphasizes the positive effect of substitution with the morpholino
Table 1 (continued)
moiety at the phenylurea part on activity. Similarly, a large differ-
ence in activity was observed between compounds 29 (9.74%) and
30 (50.92%) which are highly similar in structure. The only differ-
ence between these two compounds lies in the position of the acet-
amido-substituent at the distal phenyl ring, which is ortho in
compound 29 and meta in compound 30. This slight change in
the relative position of this group was associated however with a
very big difference in activity, which suggests a possible critical
binding role for this group in compound 30 in the binding site of
the molecular target of the compound. The little shift in the posi-
tion of this group is thought however to distort this critical and
important binding, which is reflected by this great drop in activity
from compound 30 to compound 29. From these observations and
by referring again to the structures of the tested compounds we
can conclude that the phenyl ring introduced to the terminal pyr-
idyl ring has a critical effect on the anticancer activity of the
compound, and that this effect is highly dependent on the type
and the position of the substituent attached. Accordingly, by com-
paring the activities of compounds 19, 25a, 29, and 30, which all bear
thesame 3,5-bis(trifluoromethyl)phenylurea moietyattachedto the
amino group, we can conclude that the presence of a 3-acetamid-
ophenyl moiety attached to the terminal pyridine ring in compound
30 is the best for activity. The substitution of one of the trifluoro-
methyl groups with a morpholine moiety was found also to have a
positive impact on activity as observed in compound 25b.
Compd
Structure
Inhibition %a
H
H
N
N
N
N
N
N
CF3
CF3
CF3
CF3
N
N
O
CF3
25a
25b
29
11.31
O
H
N
H
N
N
N
O
N
O
38.41
O
H
N
H
N
N
N
O
O
CF3
The inhibitions of the two most active compounds (25b and 30)
at 10
lM concentration over the full cell lines panel are presented
9.74
.
3HCl
together in Figure 2. As showed in Figure 2, compounds 25b and 30
have showed moderate to strong inhibitions over almost all of the
tested cell lines. The inhibitions approached 100% at many cell
lines, and exceeded the 100% inhibition limit to exert a lethal,
rather than inhibitory, effect at a number of these cell lines. The
overall inhibition of compound 30 was stronger. However, com-
pound 25b has showed stronger mean inhibitions at melanoma
and renal cancers. After the initial single dose screening of the 12
selected compounds, the compounds showing the highest activity
at the single dose (compounds 25b and 30) were further tested in a
five-dose testing mode, in order to determine their IC50 values over
the 60 tumor cell lines. For each of these two compounds, the IC50
(the concentration producing 50% inhibition), TGI (the concentra-
tion producing 100% inhibition) and LC50 (the concentration
causing 50% lethality or 50% tumor regression) were recorded.
The five-dose testing results of these 2 compounds are showed in
Table 2. Compound 30 has showed a great superiority over
compound 25b in the five-dose testing mode. The potency of
compound 30 was much higher, with IC50 values in the range of
H
N
H
N
H
N
N
N
O
.
CF3
30
50.92
3HCl
O
N
H
H
N
N
CF3
N
CF3
1–2 lM at the majority of cell lines, and below 1 lM in the breast
cancer cells T-47D and MDA-MB-468. The compound has showed
26b
À2.21
N
also high efficacies, being able to induce total growth inhibition
(TGI) at almost all of the tested cell lines, and 50% lethality (LC50
)
in 65% of the tested cell lines at concentrations below 100 lM.
O
2.2.2. In vitro kinase screening
In order to investigate the mechanism of action and the kinase
inhibitory profile of this new class of compounds, compound 30
with the highest potencies at the cancer cell lines was tested at a
a
Inhibition % represents the mean inhibition percentages over the 60 cell lines.
The inhibition percentages are calculated by subtracting the growth percentages
from 100.
single dose concentration of 10 lM over a panel of 54 kinases at
Reaction Biology Corporation using hot spot technique.21 As
showed in Figure 3, compound 30 has exerted multiple inhibitions
over a number of oncogenic serine/threonine and tyrosine kinases
at the test concentration. The inhibition was above 70% in ABL1 ki-
nase, a fusion tyrosine kinase protein responsible for 90% of
chronic myeloid leukemia (CML) cases,22 AKT1 serine/threonine ki-
nase, which is over-expressed in a number of cancers including
breast, prostate, lung, pancreatic, liver, ovarian and colorectal can-
As observed from the data, a great difference in activity was ob-
served between the tested compounds in spite of close structural
similarity. For example, a big difference in the mean inhibitory
percentages of compounds 25a (11.31%) and 25b (38.41%) was ob-
served although the only difference in between them lies in the
replacement of one of the two trifluoromethyl groups of compound
25a with a morpholino group in compound 25b. This, however,

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
3865
200
175
150
125
100
75
25b
30
50
25
0
Melanoma
Ovarian
Renal
Leukemia
Lung
Colon
CNS
Prostate
Breast
Figure 2. Multiple inhibitions of compounds 25b and 30 at 10 M concentration over the 60 cell lines panel.
l
cer,23 and LCK kinase, which is aberrantly expressed in leukemia
and colorectal cancer.24,25 In a number of other kinases, the inhibi-
tion percentages were between 60% and 70%. These kinases include
Aurora-A kinase, a mitotic serine/threonine kinase whose dysregu-
lation results in aneuploidy and is associated with high incidence
of cancers such as breast and ovarian cancers,26 C-src (CSK) tyro-
sine kinase, which is over-expressed in a number of cancers, such
as breast, colon, pancreas, lung, ovary and CNS cancers,27 and
PIM1 serine/threonine kinase, whose over-expression is reported
in prostate cancer and hematopoietic malignancies.8 In addition
to these inhibitions, compound 30 has also inhibited three other
kinases with a milder degree (with inhibition percentages ranging
between 50% and 60%). These kinases are FLT3 kinase, a type III
receptor tyrosine kinase whose mutations were reported in about
30% of cases of acute myeloid leukemia (AML),28 FYN tyrosine ki-
nase, whose over-expression aids in cellular transformation and
xenograft metastasis29 and is linked also to Alzheimer’s disease
pathology,30 and KDR (VEGFR2) kinase, which plays a critical role
in tumor angiogenesis in solid cancers.31 The compound was able
also to inhibit a number of other oncogenic kinases, as showed in
Figure 3, but the inhibitions at these kinases were below 50%.
The multiple inhibitions of compound 30 over this group of onco-
genic kinases have compromised together to yield finally the
strong and broad spectrum anticancer effect of the compound.
In order to get a better insight at the critical effect of the posi-
tion of the acetamido-substituent in compound 30 on activity and
justify for the big difference in activity between compound 30 and
compound 29, bearing the same group in the ortho-position, a
molecular modeling study was applied using MOE.2008.10 soft-
ware. In this study, both of the two compounds were docked into
the binding pocket of ABL1 kinase using the crystal structure of
the enzyme co-crystallized with Imatininb (PDB code 3K5V). The
two compounds (29 and 30) were docked over the ligand atoms
(Imatinib) in the binding site. The most stable docking poses of
the two compounds aligned with Imatinib in the binding pocket
are showed in Figure 4. Both of the two compounds occupy nearly
the same space in the binding pocket, and are overlaid well over
the skeleton of imatininb, with the ‘4-(pyridin-3-yl)pyrimidin-2-
amine’ scaffold in the same direction of that of Imatininb. It was
found that compound 30 was fit into the kinase domain through
two hydrogen bonding (HB) interactions, one of them was formed
by the HB-acceptor acetamido oxygen with Met-337 in the hinge
region, while the other was formed by the HB-donor distal urea
NH to Glu-305 inside the binding pocket. The capability of com-
pound 30 to occupy a similar pose to that of Imatininb suggests
the possibility of compound binding to the enzyme in the inactive
conformation (DGF-out conformation) similarly to Imatininb.32
Although compound 29 has showed almost a perfect overlay
over compound 30, it failed to establish the highly important
hydrogen bonding with Met-337 in the hinge region as a result
of the far positioning of its acetamido group as showed in Figures
4 and 5. This failure in initial fitting to the hinge region was asso-
ciated also with a failure in hydrogen bonding to Glu-305 inside
the binding pocket, which makes the overall binding of the com-
pound in the binding site to be so weak, and consequently diminish
the ability of the compound to competitively inhibit the enzyme.
These results emphasize the importance of the right positioning
of the acetamido group in compound 30, and give an explanation
for the lack of activity in compound 29, since similar binding
modes would be anticipated for both of the two compounds in
the other kinases inhibited by compound 30.
3. Conclusions
A new pyrimidine anticancer lead compound (compound 30)
has been developed in this study. The compound has showed good

3866
I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
Table 2
is the best for activity. Currently, further structural modifications
IC50, TGI and LC50 values in lM of compounds 25b and 30 over 60 cancer cell lines
are subjected to the urea part of compound 30 (keeping the distal
3-acetamidophenyl ring unchanged) in order to study the effect of
structural variations at that part of the compound on activity.
Cell line
25b
30
a
c
a
c
IC50
TGIb
LC50
IC50
TGIb
LC50
Leuk
HL-60(TB)
K-562
MOLT-4
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
>100 >100 >100 3.78
>100 >100 >100 1.87
>100 >100 >100 1.98
>100 >100 >100 1.48
>100 >100 >100 1.44
96.1
NA
>100 >100
>100
>100
4. Experimental
4.1. General
Non-small
cell
Lung
12.6
5.71
26.8
NA
6.94
14.6
7.07
5.85
41.9
>100
>100
NA
43.8
68.8
59.7
35.3
4.29
2.95
>100 >100 4.88
NA >100 NA
1H NMR (300 MHz) and 13C NMR (75 MHz) were recorded on a
Bruker Avance 300 spectrometer with TMS as an internal standard.
The IR spectra were recorded on Perkin Elmer Spectrum GX spec-
trometer. Melting points were taken on a Thomas-Hoover capillary
melting apparatus and are uncorrected. Chemical analyses were
carried out by EA 1108 CHNS-O of Fisons Instruments. Column
chromatography was performed on Merck Silica Gel 60 (230–400
mesh). TLC was carried out using glass sheets precoated with silica
gel 60 F254 prepared by E. Merck. All the commercially available
reagents were obtained from Aldrich and Tokyo Kasei Chemicals
and generally used without further purification.
cancer
>100 >100 >100 2.24
>100 >100 >100 2.17
>100 >100 >100 2.06
63.2
>100 >100 1.71
Colon Cancer
CNS cancer
Melanoma
>100 >100 >100 11.0
>100 >100 >100 1.60
>100 >100 >100 3.62
>100 >100 >100 3.00
>100 >100 >100 2.59
>100 >100 >100 2.30
>100 >100
4.71
15.0
8.74
8.01
5.59
23.9
6.19
18.9
17.8
30.5
5.32
NA
17.1
45.1
57.6
27.9
>100
98.7
>100
44.3
47.4
85.8
18.9
>100
NA
40.2
9.04
NA
38.7
5.35
34.9
>100 >100 4.92
>100 >100 1.27
>100 >100 5.48
>100 >100 4.57
4.2. Synthesis
27.5
>100 10.9
>100 >100 2.00
4.2.1. 3-(Dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one (1)15
>100 >100 >100 1.79
A
mixture of 3-acetylpyridine (10 g, 0.083 mol) and N,N-
NA
NA
>100 >100 2.01
>100 >100 1.83
4.48
3.76
6.80
7.74
>100
dimethylformamide dimethylacetal (13.8 g, 0.116 mol) was heated
under reflux in an oil bath for 4 h. The excess unreacted N,N-
dimethylformamide dimethylacetal was removed by distillation,
followed by removal of the residual part under vacuum. The solid
residue was triturated with water (100 mL) and then extracted
with methylene chloride (200 mL Â 3). The organic layer was sep-
arated, dried over anhydrous MgSO4, and then evaporated under
vacuum to yield the crude product. Crystallization from the mixed
solvent ethanol/chloroform (1:1, v/v) yielded pure 1 as reddish or-
ange crystals. Yield (12.0 g, 82%); mp 70–71 °C (62–70 °C);15 1H
NMR (CDCl3): d 2.90 (s, 3H), 3.12 (s, 3H), 5.62 (d, J = 12.2 Hz, 1H),
7.30 (dd, J = 3.6, 5.4 Hz, 1H), 7.79 (d, J = 12.2 Hz, 1H), 8.14 (d,
J = 7.8 Hz, 1H), 8.60 (d, J = 4.8 Hz, 1H), 9.03 (s, 1H); 13C NMR
(CDCl3): d 37.35, 45.18, 91.79, 123.25, 135.03, 135.62, 148.85,
151.38, 154.68, 186.30.
MDA-MB-
435
>100 >100 >100 2.31
SK-MEL-2
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-
RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
15.9
NA
75.5
>100 5.65
19.8
2.64
4.32
12.7
9.46
4.23
11.1
24.5
5.93
47.4
52.4
5.13
13.2
35.7
>100
9.69
39.0
50.8
26.4
>100
>100 >100 1.35
>100 >100 >100 1.69
>100 >100 >100 2.86
Ovarian
cancer
6.18
>100 >100 >100 1.85
NA >100 >100 1.57
>100 >100 >100 11.8
>100 >100 2.68
NA
NA
>100 >100 2.07
>100 >100 3.66
9.42
13.0
>100 >100 13.1
>100 >100 3.66
32.2
15.9
22.2
15.5
8.34
NA
23.8
39.8
23.2
19.6
19.7
78.8
39.9
59.2
47.1
>100
NA
57.7
94.1
61.7
47.0
62.0
Renal cancer
>100 >100 >100 6.13
6.89 >100 >100 3.83
>100 >100 >100 2.37
1.27 3.50 NA NA
>100 >100 >100 9.29
5.46 >100 >100 16.8
>100 >100 >100 5.51
RXF 393
SN12C
TK-10
4.2.2. 4-(Pyridin-3-yl)pyrimidin-2-amine (2)16
To a solution of NaOEt in absolute ethanol, made by dissolving
sodium metal (0.69 g, 30.0 mmol) in absolute ethanol (100 mL),
guanidine hydrochloride (2.86 g, 30.0 mmol) was added in one
portion and stirred at room temperature for 1 h. 3-(Dimethyl-
amino)-1-(pyridin-3-yl)prop-2-en-1-one (1) (5.28 g, 30 mmol)
was dissolved in absolute ethanol (20 mL) and added to the reac-
tion mixture. The mixture was heated under reflux for 6 h, and
was then left to cool at room temperature, followed by cooling in
an ice bath. The formed product was separated by filtration,
washed with cold ethanol (20 mL), then with water (50 mL), and
left to dry. Crystallization from ethanol yielded pure 2 as pale yel-
low needle crystals. Yield (3.8 g, 73.3%); mp 192 °C (186–188 °C);16
1H NMR (DMSO-d6): d 6.78 (s, 2H), 7.20 (d, J = 6.0 Hz, 1H), 7.52 (dd,
J = 3.0, 4.8 Hz, 1H), 8.34–8.40 (m, 2H), 8.67 (d, J = 3.6 Hz, 1H), 9.23
(s, 1H).
UO-31
Prost.
Breast cancer
DU-145
MDA-MB-
231
HS 578T
BT-549
T-47D
NA
NA
>100 >100 6.57
>100 >100 3.72
3.10
2.87
>100 >100 7.52
>100 >100 7.07
>100 >100
20.4
46.3
>100
5.85
>100 >100 >100 0.619 8.43
>100 >100 >100 0.837 2.35
MDA-MB-
468
a
b
c
IC50 is the concentration producing 50% inhibition.
TGI is the concentration producing 100% inhibition.
LC50 is the concentration causing 50% lethality (50% tumor regression), NA
means that the data is not available.
anticancer potencies and efficacies over a wide range of cancer cell
lines. The compound was found also to exert multiple inhibitions
over a number of oncogenic kinases, and it is anticipated that these
contributing inhibitions are responsible for the anticancer activity
of this compound. A structure–activity relationship study was also
concluded from the available anticancer screening data; where it
has showed the importance of the type and the position of the sub-
stituent inserted at the distal phenyl ring in determining the activ-
ity of these compounds. The good and unique activity of compound
30 within the whole series has proved that the 3-acetamido group
4.2.3. N-(2,4-Dimethylbenzyl)-4-(pyridin-3-yl)pyrimidin-2-
amine (3)17
A solution of 2,4-dimethylbenzylbromide (0.17 g, 0.87 mmol) in
DMF (5 mL) was added dropwise over a period of 4 h to a mixture
of 4-(pyridin-3-yl)pyrimidin-2-amine (2) (0.15 g, 0.87 mmol) and
K2CO3 (0.12 g, 0.87 mmol) in DMF (5 mL) while heating (130 °C)
and stirring. After complete addition of 2,4-dimethylbenzylbro-
mide, heating and stirring were maintained for 1 h. The reaction

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
3867
100
90
80
70
60
50
40
30
20
10
0
Kinase
Figure 3. Inhibition percentages of compound 30 at a single dose concentration of 10 lM over 54 kinases.
(1.31 mmol) in one portion. Reflux was maintained for 2 h, and
then the reaction mixture was cooled and poured over crushed
ice to induce precipitation of the crude product. The resulted pre-
cipitate was then filtered, dried, and purified by the suitable
method.
4.2.4.1. 3,5-Bis(trifluoromethyl)-N-(4-(pyridin-3-yl)pyrimidin-2-
yl)benzamide (4a). It was separated by column chromatography
(silica gel, ethyl acetate). Yield (38 mg, 19%); mp 238–239 °C; IR
m
/cmÀ1: 3436, 2920, 1661, 1591, 1410, 1357, 1318, 1166, 1114;
1H NMR (CDCl3):
d 7.36 (dd, J = 2.1, 4.9 Hz, 1H), 7.45 (d,
J = 5.1 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.8 (d, J = 7.5 Hz, 1H), 8.16
(d, J = 7.5 Hz, 1H), 8.23 (s, 1H), 8.29 (d, J = 7.5 Hz, 1H), 8.59 (d,
J = 3.9 Hz, 1H), 8.71 (d, J = 5.1 Hz, 1H), 9.23 (s, 1H), 9.96 (s, 1H);
13C NMR (CDCl3):
128.81, 129.39, 130.49, 131.78, 134.82, 135.45, 148.40, 151.82,
158.17, 159.40, 163.10, 164.74.
d 112.54, 121.79, 123.80, 124.85, 125.40,
Figure 4. Docking poses for compounds 29 (red) and 30 (yellow) overlaid to
Imatinib (cyan) in the kinase domain of ABL1 kinase. The docking structures show
the acetamido group of 29 shifted away from Met-337 and incapable of HB
formation.
4.2.4.2. N,N-Bis(3,5-bis(trifluoromethyl)benzoyl))-4-(pyridin-3-
yl)pyrimidin-2-amine (4b). It was separated by column chroma-
tography (silica gel, ethyl acetate). Yield (111 mg, 37%); mp 134–
mixture was left to cool to room temperature, and then poured
over ice water (50 mL). The resulted precipitate was filtered, dried,
and then purified by column chromatography (silica gel, ethyl ace-
tate/hexane, 1:1, v/v) to give 3 as brownish needles. Yield (0.10 g,
135 °C; IR
m
/cmÀ1: 3436, 1717, 1691, 1616, 1579, 1416, 1380,
1335, 1278, 1234, 1168, 1130, 1097, 1072, 818, 697; 1H NMR
(CDCl3): d 7.36 (dd, J = 3.0, 4.8 Hz, 1H), 7.53–7.60 (m, 3H), 7.77
(d, J = 7.5 Hz, 2H), 7.99–8.05 (m, 3H), 8.12 (s, 2H), 8.70–8.74 (m,
2H), 9.01 (s, 1H); 13C NMR (CDCl3): d 114.42, 121.53, 123.80,
125.14, 126.10, 126.15, 129.41, 129.50, 130.82, 131.31, 131.75,
131.98, 132.20, 134.57, 135.01, 148.34, 152.46, 159.86, 160.30,
163.77, 171.05.
39%); mp 82.5–83.5 °C; IR m
/cmÀ1: 3231, 1597, 1566, 1345, 794;
1H NMR (CDCl3): d 2.15 (s, 3H), 2.32 (s, 3H), 4.66 (d, J = 4.4 Hz,
2H), 5.86 (s, 1H), 6.98–7.11 (m, 3H), 7.26 (d, J = 7.2 Hz, 1H), 7.40
(dd, J = 2.7, 5.1 Hz, 1H), 8.30–8.33 (m, 2H), 8.69 (d, J = 3.3 Hz, 1H),
9.23 (s, 1H); 13C NMR (CDCl3): d 19.06, 21.03, 43.53, 106.42,
123.53, 126.70, 128.46, 131.30, 133.05, 133.67, 134.42, 136.31,
137.14, 148.55, 151.24, 158.99, 162.48; Anal. Calcd for C18H18N4:
C, 74.46; H, 6.25; N, 19.30. Found: C, 74.06; H, 6.25; N, 19.20.
4.2.4.3. N,N-Bis(benzoyl))-4-(pyridin-3-yl)pyrimidin-2-amine
(4c). The product was purified by crystallization from a mixed
solvent of ethanol and water (1:2, v/v). Yield (115 mg, 52%); mp
4.2.4. General procedure for the synthesis of compounds 4a–c
To a solution of the amine 2 (100 mg, 0.58 mmol) in pyridine
(5 mL) under reflux was added the appropriate benzoyl chloride
199–200 °C; IR
1255, 704; 1H NMR (CDCl3): d 7.42 (d, J = 7.8 Hz, 4H), 7.47–7.60
m
/cmÀ1: 3436, 1710, 1687, 1577, 1373, 1287,

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I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
4.2.5. General procedure for the synthesis of compounds 5a–f
A mixture of the amine 2 (150 mg, 0.87 mmol), the appropriate
arylbromide (0.96 mmol), dichlorobis(triphenylphosphine)Pd(II)
(0.061 g, 0.087 mmol), Xantphos (0.05 g, 0.087 mmol) and sodium
tert-butoxide (0.25 g, 2.61 mmol) was refluxed in toluene (10 mL)
under nitrogen atmosphere for 8 h.
4.2.5.1. N-(3-(4-(Pyridin-3-yl)pyrimidin-2-ylamino)-4-methyl-
phenyl)acetamide (5a)33
. Toluene was removed under vacuum,
and the residue was triturated with water (100 mL) and extracted
with methylene chloride (100 mL Â 2). The undissolved solid was
then filtered, and washed with cold water to yield the pure prod-
uct. Yield (117 mg, 42%); mp 220–221 °C; IR m
/cmÀ1: 3416, 3210,
3008, 1660, 1590, 1539, 1455, 1419, 800; 1H NMR (CD3OD): d
2.13 (s, 3H), 2.29 (s, 3H), 7.2 (d, J = 8.2 Hz, 1H), 7.25(d, J = 8.4 Hz,
1H), 7.35 (d, J = 5.2 Hz, 1H), 7.56 (dd, J = 3.0, 4.9 Hz, 1H), 8.04 (s,
1H), 8.45 (d, J = 5.2 Hz, 1H), 8.56 (d, J = 8.0 Hz, 1H), 8.65 (d,
J = 4.0 Hz, 1H), 9.27 (s, 1H).
4.2.5.2. N-(3,5-Bis(trifluoromethyl)phenyl)-4-(pyridin-3-yl)pyr-
imidin-2-amine (5b). Toluene was filtered while hot to remove
insoluble metal impurities. After cooling of toluene filtrate, the
pure product 5b was crystallized out as off-white crystals. The
crystals were filtered, washed with toluene (10 mL), then with
water (50 mL), and dried. Yield (274 mg, 82%); mp 233–234 °C;
IR m
/cmÀ1: 3239, 3082, 2992, 1559, 1383, 1277, 1194, 1124, 995,
880, 799, 680; 1H NMR (DMSO-d6): d 7.57–7.70 (m, 3H), 8.49 (d,
J = 6.3 Hz, 1H), 8.59 (s, 2H), 8.72–8.75 (m, 2H), 9.36 (s, 1H), 10.50
(s, 1H); 13C NMR (DMSO-d6): 109.79, 118.08, 123.90, 130.34,
130.77, 131.77, 134.31, 142.37, 148.13, 151.80, 159.48, 159.75,
161.72.
4.2.5.3. N1,N1-Dimethyl-N4-(4-(pyridin-3-yl)pyrimidin-2-yl)ben-
zene-1,4-diamine (5c)34
. After cooling of toluene, the pure prod-
uct 5c was crystallized out as yellow crystals. The crystals were
filtered, washed with toluene (10 mL), then with water (50 mL),
and dried. Yield (98 mg, 39%); mp 165–167 °C (171–174 °C);34 1H
NMR (CDCl3): d 6.81 (d, J = 8.9 Hz, 2H), 7.06 (s, 1H), 7.10 (d,
J = 5.1 Hz, 1H), 7.43 (dd, J = 3.0, 4.9 Hz, 1H), 7.51 (d, J = 8.9 Hz,
2H), 8.35 (d, J = 8.0 Hz, 1H), 8.46 (d, J = 5.1 Hz, 1H), 8.72 (d,
J = 4.7 Hz, 1H), 9.28 (d, J = 1.4 Hz, 1H).
4.2.5.4. N-(2,4-Dimethylphenyl)-4-(pyridin-3-yl)pyrimidin-2-
amine (5d)34
. Toluene was removed under vacuum, and the res-
idue was triturated with water (50 mL) and extracted with ethyl
acetate (100 mL Â 2). The organic layer was separated, dried over
anhydrous MgSO4, and then evaporated under vacuum. The crude
product was purified by column chromatography (silica gel, ethyl
acetate/hexane, 1:1, v/v) to give 5d as a yellow powder. Yield
(74 mg, 31%); mp 122–123 °C (113–115 °C);34 IR
m
/cmÀ1: 3210,
2922, 1595, 1571, 1451, 1290, 797; 1H NMR (CDCl3): d 2.32 (s,
3H), 2.34 (s, 3H), 6.94 (s, 1H), 7.08–7.14 (m, 3H), 7.42 (dd, J = 3.0,
4.8 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 8.34 (d, J = 8.1 Hz, 1H), 8.47
(d, J = 5.1 Hz, 1H), 8.71 (d, J = 3.3 Hz, 1H), 9.25 (s, 1H); 13C NMR
(CDCl3): d 18.12, 20.89, 107.77, 122.91, 123.62, 127.14, 129.84,
131.30, 132.79, 133.94, 134.50, 148.56, 151.40, 159.16, 162.56.
4.2.5.5. N-(4-Methoxyphenyl)-4-(pyridin-3-yl)pyrimidin-2-amine
Figure 5. 2D-presentation for the binding interactions in Imatinib (upper panel),
compound 30 (middle panel) and compound 29 (lower panel).
(5e)34
. Toluene was removed under vacuum, and the residue was
triturated with water (50 mL). The formed precipitate was filtered,
washed with cold water (100 mL), dried, and then crystallized from
ethanol to give 5e as yellow crystals. Yield (126 mg, 52%); mp 182–
(m, 4H), 7.87 (d, J = 7.2 Hz, 4H), 8.06 (d, J = 7.8 Hz, 1H), 8.69 (d,
J = 4.2 Hz, 1H), 8.77 (d, J = 5.1, 1H), 8.97 (s, 1H); 13C NMR (CDCl3):
113.89, 123.70, 128.73, 129.27, 131.26, 132.83, 134.50, 134.66,
148.44, 152.10, 159.64, 161.02, 163.37, 172.61.
183 °C (121–122 °C);34 IR /cmÀ1: 3435, 1575, 1509, 1423, 1242,
m
801; 1H NMR (DMSO-d6): d 3.74 (s, 3H), 6.92 (d, J = 9.0 Hz, 2H),
7.42 (d, J = 5.2 Hz, 1H), 7.59 (dd, J = 4.0, 4.5 Hz, 1H), 7.68 (d,

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
3869
J = 8.7 Hz, 2H), 8.49 (d, J = 7.8 Hz, 1H), 8.55 (d, J = 4.7 Hz, 1H), 8.73
(d, J = 4.0 Hz, 1H), 9.34 (s, 1H), 9.58 (s, 1H).
m
/cmÀ1: 3437, 1689, 1630, 1607, 1540, 1439, 1336, 1261, 1172,
1113; 1H NMR (CDCl3): d 2.65 (s, 3H), 7.43 (s, 1H), 7.46 (dd,
J = 3.0, 4.8 Hz, 1H), 7.69 (dd, J = 7.7 Hz, 1H), 7.87 (d, J = 8.1 Hz,
1H), 8.16 (d, J = 8.1 Hz, 1H), 8.22 (s, 1H), 8.45 (d, J = 7.9 Hz, 1H),
8.66 (s, 1H), 8.75 (d, J = 3.7 Hz, 1H), 9.25 (s, 1H); 13C NMR (CDCl3):
d 27.08, 115.03, 126.46, 127.68, 131.66, 133.82, 134.81, 137.86,
151.02, 154.02, 160.79, 165.03, 167.52, 172.69.
4.2.5.6. N-(Naphthalen-2-yl)-4-(pyridin-3-yl)pyrimidin-2-amine
(5f)17
. Toluene was removed under vacuum, and the residue was
triturated with water (50 mL). The formed precipitate was filtered,
washed with cold water (100 mL), dried, and then crystallized from
ethanol to give 5f as yellow plates. Yield (144 mg, 56%); mp 199–
200 °C; IR
m
/cmÀ1: 3247, 1571, 1548, 1447, 1434, 798; 1H NMR
4.2.9. General procedure for synthesis of compounds 9a, 9b
(DMSO-d6): d 7.36 (t, J = 7.8 Hz, 1H), 7.46 (t, J = 7.3 Hz, 1H), 7.56 (d,
J = 5.1 Hz, 1H), 7.63 (dd, J = 2.9, 4.8 Hz, 1H), 7.78–7.88 (m, 4 H),
8.54–8.57 (m, 2H),8.67 (d, J = 5.1 Hz, 1H), 8.76 (d, J = 4.5 Hz, 1H),
9.40 (s, 1H), 10.03 (s, 1H); 13C NMR (DMSO-d6): d 108.98, 114.43,
121.19, 124.49, 126.77, 127.46, 127.89, 128.50, 129.46, 134.18,
134.93, 138.55, 148.68, 152.03, 160.69; Anal. Calcd for C19H14N4:
C, 76.49; H, 4.73; N, 18.78. Found: C, 76.60; H, 4.70; N, 18.38.
A mixture of 4-methyl-6-(pyridin-3-yl)pyrimidin-2-amine
(0.162 g, 0.87 mmol), the appropriate arylbromide (0.96 mmol),
dichlorobis(triphenylphosphine)Pd(II) (0.061 g, 0.087 mmol), Xant-
phos (0.05 g, 0.087 mmol) and sodium tert-butoxide (0.25 g, 2.61
mmol) was refluxed in toluene (10 mL) under nitrogen atmosphere
for 8 h.
4.2.9.1. 4-Methyl-N-(2,4-dimethylphenyl)-6-(pyridin-3-yl)pyrim-
4.2.6. 4-Methyl-6-(pyridin-3-yl)pyrimidin-2-amine (6)18
idin-2-amine (9a)17
. Toluene was removed under vacuum, and
A
mixture of 2-amino-4-chloro-6-methylpyridine (2.70 g,
the residue was triturated with water (50 mL), and extracted with
ethyl acetate (100 mL Â 2). The organic layer was separated, dried
over MgSO4, and then evaporated till dryness. The crude product
was further purified by silica column using the mixed solvent of
ethyl acetate and hexane (1:1, v/v) as mobile phase to yield 9a as
18.76 mmol), 3-pyridineboronic acid (2.54 g, 20.66 mmol), dichlor-
obis(triphenylphosphine)Pd(II) (0.36 g, 0.512 mmol) and Na2CO3
(1.40 g, 13.2 mmol) was placed in a mixed solvent of acetonitrile
and water (1:1, v/v, 150 mL). N2 gas was bubbled into this mixture
for 10 min, and then the mixture was heated at 78 °C while stirring
under N2 atmosphere for 7 h. The reaction mixture was left to cool
at room temperature, poured into ice water (100 mL), and then ex-
tracted with ethyl acetate (100 mL Â 3). The organic layer was sep-
arated, washed with water (100 mL Â 3), dried over anhydrous
MgSO4, and then evaporated under vacuum to yield the crude
product which was then crystallized from ethanol to yield pure
yellow crystals of 6. Yield (2.58 g, 74%); mp 191–192 °C (192–
194 °C);18 1H NMR (CD3OD): d 2.41 (s, 3H), 7.13 (s, 1H), 7.56 (dd,
J = 2.9, 4.9 Hz, 1H), 8.49 (d, J = 8.0 Hz, 1H), 8.64 (d, J = 5.0 Hz, 1H),
9.21 (s, 1H).
an off-white powder. Yield (88 mg, 34.8%); mp 95–96 °C; IR m/cm
À1: 3215, 2921, 1597, 1581, 1552, 1448, 1343, 804; 1H NMR
(CDCl3): d 2.34 (s, 6H), 2.49 (s, 3H), 6.90 (s, 1H), 7.04–7.10 (m,
3H), 7.41 (dd, J = 3.0, 5.0 Hz, 1H) 7.97 (d, J = 8.1 Hz, 1H), 8.32 (d,
J = 8.1 Hz, 1H), 8.69 (d, J = 3.9 Hz, 1H), 9.24 (s, 1H); 13C NMR
(CDCl3): d 18.16, 20.83, 24.42, 107.49, 121.97, 123.55, 127.02,
128.68, 131.16, 133.04, 133.08, 134.49, 134.90, 148.58, 151.19,
160.79, 162.18, 169.15; Anal. Calcd for C18H18N4: C, 74.46; H,
6.25; N, 19.30. Found: C, 74.46; H, 6.50; N, 19.00.
4.2.9.2. N-(3,5-Bis(trifluoromethyl)phenyl)-4-methyl-6-(pyridin-
3-yl)pyrimidin-2-amine (9b). Toluene was filtered while hot to
remove insoluble metal impurities. After cooling of toluene filtrate,
the pure product 9b was crystallized out as pale yellow crystals. The
crystals were filtered, washed with toluene (10 mL), then with water
4.2.7. N-(2,4-Dimethylbenzyl)-4-methyl-6-(pyridin-3-yl)pyrimidin-
2-amine (7)17
The procedure used for the synthesis of compound 3 was
adapted for the synthesis of this compound. After pouring the reac-
tion mixture over ice water, the aqueous solution was extracted
with ethyl acetate (100 mL Â 2). The organic layer was separated,
dried over anhydrous MgSO4, and then evaporated under vacuum
to yield the crude product, which was then purified by column
chromatography (silica gel, ethyl acetate/hexane, 1:1, v/v) to give
7 as brown needle crystals. Yield (0.113 g, 43%); mp 125–126 °C;
(50 mL), and dried. Yield (235 mg, 68%); mp 254–255 °C; IR m :
/cmÀ1
3265, 3085, 2964, 1543, 1385, 1277, 1267, 1186, 1124, 874, 809, 701,
680; 1H NMR (DMSO-d6): d 2.08 (s, 3H), 7.56–7.60 (m, 3H), 8.47 (d,
J = 8.0 Hz, 1H), 8.61 (s, 2H), 8.73 (d, J = 4.6 Hz, 1H), 9.33 (d,
J = 1.3 Hz, 1H), 10.42 (s, 1H); 13C NMR (DMSO-d6): d 24.26, 109.66,
113.77, 118.39, 122.14, 124.27, 125.76, 130.79, 131.21, 132.43,
134.60, 143.03, 148.53, 152.03, 159.78, 161.80, 169.87.
IR m
/cmÀ1: 3254, 2920, 1604, 1557, 1341, 808; 1H NMR (CDCl3):
2.32 (s, 3H), 2.37 (s, 3H), 2.43 (s, 3H), 4.68 (d, J = 7.5 Hz, 2H), 5.41
(s, 1H), 6.90 (s, 1H), 6.98–7.02 (m, 2H), 7.25 (d, J = 7.8 Hz, 1H),
7.33 (dd, J = 3.5, 4.8 Hz, 1H), 8.31 (d, J = 7.8 Hz, 1H), 8.67 (d,
J = 4.8 Hz, 1H), d 9.22 (s, 1H); 13C NMR (CDCl3): d 19.08, 21.01,
24.38, 43.44, 106.23, 123.48, 126.66, 128.34, 131.24, 133.34,
133.94, 134.45, 136.21, 137.03, 148.55, 151.01, 162.13, 162.48,
168.95; Anal. Calcd for C19H20N4: C, 74.97; H, 6.62; N, 18.41.
Found: C, 74.99; H, 6.65; N, 18.90.
4.2.10. 5-Acetyl-2-bromopyridine (10)19
Under N2 atmosphere, n-BuLi (8.1 mL, 13 mmol, 1.6 M in hex-
ane) was added to a mixture of 2,5-dibromopyridine (3.08 g,
13 mmol) and anhydrous Et2O (50 mL) at À78 °C. The reaction
mixture was stirred for 30 min and then N,N-dimethylacetamide
(1.4 mL, 15 mmol) was added and stirred at À78 °C to ambient
temperature within 1 h. The whole mixture was poured into satu-
rated NH4Cl solution and then extracted with Et2O (150 mL Â 3).
The combined organic layer was washed with brine, dried, and
concentrated. The crude product was purified by column chroma-
tography (silica gel, ethyl acetate/hexane, 1:7.5, v/v) to give 8 as a
yellowish white solid. Yield (1.3 g, 50.5%), mp 127–129 °C (124–
128 °C);19 1H NMR (CDCl3): d 2.62 (s, 3H), 7.61 (d, J = 8.2 Hz, 1H),
8.07 (d, J = 7.6 Hz, 1H), 8.89 (s, 1H); 13C NMR (CDCl3): d 26.79,
128.44, 131.41, 137.70, 146.96, 150.42, 195.65.
4.2.8. 3-(Trifluoromethyl)-N-(4-methyl-6-(pyridin-3-yl)pyrimi-
din-2-yl)benzamide (8)
To a stirred and heated solution of 4-methyl-6-(pyridin-3-
yl)pyrimidin-2-amine (0.1 g, 0.54 mmol) in pyridine (5 mL), 3-(tri-
fluoromethyl)benzoyl chloride (0.134 g, 0.65 mmol) was added
dropwise over a period of 1 h while heating and stirring were
maintained. Reflux was maintained for 30 min, and then the reac-
tion mixture was cooled and poured over crushed ice to induce
precipitation of the crude product. The resulted precipitate was
then filtered, dried, and purified by silica column using ethyl ace-
tate as the mobile phase. Yield (56 mg, 29%), mp 148–149 °C; IR
4.2.11. General procedure for the synthesis of compounds 11a,
11b
A mixture of 5-acetyl-2-bromopyridine (10) (2.0 g, 10 mmol),
appropriate arylboronic acid (11 mmol), dichlorobis(triphenyl-

3870
I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
phosphine)Pd(II) (192 mg, 0.273 mmol) and Na2CO3 (0.75 g,
7 mmol) was placed in mixed solvent of acetonitrile and water
(1:1, 80 mL). N2 gas was bubbled into this mixture for 10 min,
and then the mixture was heated at 78 °C while stirring under N2
atmosphere for 4 h. The reaction mixture was left to cool at room
temperature, poured into ice water (100 mL), and then extracted
with methylene chloride (150 mL Â 3). The organic layer was sep-
arated, dried over anhydrous MgSO4, and then evaporated under
vacuum to yield the crude product which was then crystallized
from ethanol to yield the pure compounds 11a and 11b.
guanidine hydrochloride (114 mg, 1.185 mmol) was added in one
portion and stirred at room temperature for 1 h. The appropriate
3-(dimethylamino)-1-(6-(substituted)pyridin-3-yl)prop-2-en-1-
one (12a) or (12b) (1.185 mmol) was dissolved in absolute ethanol
(10 mL) and added to the reaction mixture. The mixture was
heated under reflux for 5 h, and was then left to cool at room tem-
perature, followed by cooling in an ice bath. The formed product
was separated by filtration, washed with cold ethanol (20 mL),
then with water (50 mL), and left to dry.
4.2.13.1. 4-(6-Phenylpyridin-3-yl)pyrimidin-2-amine (13a)17
was obtained as silver crystals. Yield (246 mg, 83.5%); mp 211–
212 °C; IR
/cmÀ1: 3317, 3156, 1648, 1590, 1571, 1542, 1478,
. It
4.2.11.1. 1-(6-Phenylpyridin-3-yl)ethanone (11a)17
tained as silvery needle crystals. Yield (1.32 g, 67%); mp 119–
120 °C (118 °C [22]); IR
/cmÀ1: 1677, 1588, 1262, 740; 1H NMR
. It was ob-
m
m
743; 1H NMR (DMSO-d6): d 6.80 (s, 2H), 7.26 (d, J = 3.0 Hz, 1H),
7.48–7.55 (m, 3H), 8.11 (d, J = 8.7 Hz, 1H), 8.17 (d, J = 7.1 Hz, 2H),
8.36 (d, J = 2.7 Hz, 1H), 8.50 (dd, J = 2.1, 6.3 Hz, 1H), 9.32 (s, 1H);
13C NMR (DMSO-d6): d 106.38, 120.48, 127.21, 129.34, 130.05,
131.54, 135.69, 138.40, 148.49, 157.84, 159.83, 161.73, 164.28;
Anal. Calcd for C15H12N4: C, 72.56; H, 4.87; N, 22.57. Found: C,
72.99; H, 4.90; N, 22.80.
(CDCl3): d 2.68 (s, 3H), 7.49–7.55 (m, 3H), 7.86 (d, J = 8.4 Hz, 1H),
8.08 (d, J = 7.2 Hz, 2H), 8.31 (dd, J = 2.2, 6.2 Hz, 1H), 9.25 (s, 1H);
13C NMR (CDCl3): d 26.76, 120.18, 127.39, 128.96, 130.11, 136.43,
138.13, 150.12, 160.60.
4.2.11.2. 1-(6-(Pyridin-3-yl)pyridin-3-yl)ethanone (11b)17
was obtained as buff plates. Yield (1.46 g, 73.6%); mp 103–
104 °C; IR
/cmÀ1: 1680, 1586, 1269, 818; 1H NMR (CD3OD): d
. It
m
4.2.13.2. 4-(6-(Pyridin-3-yl)pyridin-3-yl)pyrimidin-2-amine
2.68 (s, 3H), 7.63 (dd, J = 3.0, 4.8 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H),
8.44 (dd, J = 2.1, 6.0 Hz, 1H), 8.58 (d, J = 8.1 Hz, 1H), 8.66 (d,
J = 4.5 Hz, 1H), 9.23 (s, 1H), 9.28 (s, 1H); 13C NMR (CD3OD): d
25.49, 120.51, 124.25, 131.48, 134.36, 135.80, 137.00, 147.29,
149.29, 149.78, 157.27, 197.02; Anal. Calcd for C12H10N2O: C,
72.71; H, 5.08; N, 14.13. Found: C, 72.96; H, 5.25; N, 14.30.
(13b)17
mp >300 °C; IR
.
It was obtained as yellow plates. Yield (267 mg, 90.3%);
m
/cmÀ1: 3438, 3324, 1621, 1586, 1566, 1545,
1477, 803; 1H NMR (DMSO-d6): d 6.82 (s, 2H), 7.29 (d, J = 5.4 Hz,
1H), 7.55 (dd, J = 3.2 , 4.8 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H), 8.37 (d,
J = 5.1 Hz, 1H), 8.50–8.55 (m, 2H), 8.67 (d, J = 3.6 Hz, 1H), 9.34 (d,
J = 1.5 Hz, 1H), 9.36 (d, J = 1.5 Hz, 1H); 13C NMR (DMSO-d6): d
106.52, 121.01, 124.41, 132.14, 133.87, 134.67, 135.91, 148.35,
148.70, 150.74, 155.63, 159.92, 161.55, 164.23; Anal. Calcd for
C14H11N5: C, 67.46; H, 4.45; N, 28.10. Found: C, 67.15; H, 4.55; N,
28.30.
4.2.12. General procedure for the synthesis of compounds 12a, 12b
The appropriate 1-(6-(substituted)pyridin-3-yl)ethanone (11a)
or (11b) (2.5 mmol) and N,N-dimethylformamide dimethylacetal
(0.6 g, 5 mmol) were refluxed together in an oil bath for 3 h. The
excess unreacted N,N-dimethylformamide dimethylacetal was re-
moved under vacuum. The solid residue was triturated with water
(30 mL) and then extracted with methylene chloride (100 mL Â 2).
The organic layer was separated, dried over anhydrous MgSO4, and
then evaporated under vacuum to yield the crude product which
was used for the next step without further purification.
4.2.14. General procedure for the synthesis of compounds 14a,
14b
A mixture of the amine 13a or 13b (0.34 mmol) and 3,5-bis(tri-
fluoromethyl)phenyl isocyanate (173 mg, 0.68 mmol) was heated
without solvent on an oil bath at 120 °C for 2 h. The crude product
was then crystallized from DMSO to yield the pure urea derivatives
14a and 14b.
4.2.12.1. 3-(Dimethylamino)-1-(6-phenylpyridin-3-yl)prop-2-en-
1-one (12a)17
99%); mp 151–152 °C; IR
.
It was obtained as yellow powder. Yield (0.62 g,
4.2.14.1. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-(6-phenylpyri-
m
/cmÀ1: 1644, 1589, 1567, 1537, 1285,
din-3-yl)pyrimidin-2-yl)urea (14a). Yield (116 mg, 68%); mp
1242, 769, 745; 1H NMR (CDCl3): d 2.97 (s, 3H), 3.19 (s, 3H), 5.74
(d, J = 12.3 Hz, 1H), 7.42–7.53 (m, 3H), 7.81 (d, J = 8.3 Hz, 1H),
7.87 (d, J = 12.2 Hz, 1H), 8.05 (d, J = 7.4 Hz, 2H), 8.28 (dd, J = 1.6,
6.6 Hz, 1H), 9.18 (s, 1H); 13C NMR (CDCl3): d 37.46, 45.30, 91.71,
120.46, 127.32, 128.95, 129.84, 134.14, 136.96, 137.79, 148.10,
154.74, 158.37, 185.40; Anal. Calcd for C16H16N2O: C, 76.16; H,
6.39; N, 11.10. Found: C, 76.86; H, 6.25; N, 11.50.
>300 °C; IR m
/cmÀ1: 3094, 2971, 2911, 1722, 1585, 1411, 1388,
1277, 1177, 1127, 743; 1H NMR (DMSO-d6): d 7.51–7.58 (m, 3H),
7.76 (s, 1H), 7.90 (d, J = 5.3 Hz, 1H), 8.15–8.22 (m, 3H), 8.35 (s,
2H), 8.66 (dd, J = 2.1, 6.3 Hz, 1H), 8.85 (d, J = 4.5 Hz, 1H), 9.47 (d,
J = 1.8 Hz, 1H), 10.64 (s, 1H), 12.04 (s, 1H).
4.2.14.2. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-(6-(pyridin-3-
yl)pyridin-3-yl)pyrimidin-2-yl)urea (14b). Yield (128 mg, 75%);
4.2.12.2. 3-(Dimethylamino)-1-(6-(pyridin-3-yl)pyridin-3-yl)prop-
mp 200–201 °C; IR m
/cmÀ1: 3436, 1714, 1625, 1587, 1447, 1385,
2-en-1-one (12b)17
.
It was obtained as brown powder. Yield
1284, 1252, 1166, 1134; 1H NMR (DMSO-d6): d 7.57–7.59 (m,
1H), 7.77 (s, 1H), 7.92 (d, J = 5.1 Hz, 1H), 8.29–8.36 (m, 3H), 8.55
(d, J = 6.3 Hz, 1H), 8.67–8.69 (m, 2H), 8.85 (d, J = 4.8 Hz, 1H), 9.38
(s, 1H), 9.51 (s, 1H), 10.66 (s, 1H), 12.04 (s, 1H).
(0.56 g, 88.4%); mp 163–164 °C; IR
m
/cmÀ1: 1639, 1590, 1565,
1538, 1274, 1260, 780; 1H NMR (CDCl3): d 2.95 (s, 3H), 3.17 (s,
3H), 5.70 (d, J = 12.2 Hz, 1H), 7.40 (dd, J = 2.8, 5.0 Hz, 1H), 7.79 (d,
J = 8.2 Hz, 1H), 7.85 (d, J = 12.2 Hz, 1H), 8.27 (d, J = 8.2 Hz, 1H),
8.33 (d, J = 7.8 Hz, 1H), 8.64 (d, J = 4.5 Hz, 1H), 9.17 (s, 1H), 9.22
(s, 1H); 13C NMR (CDCl3): d 37.42, 45.29, 91.74, 120.02, 123.63,
134.30, 134.52, 136.20, 148.43, 149.29, 150.25, 154.71, 156.26,
185.70; Anal. Calcd for C15H15N3O: C, 71.13; H, 5.97; N, 16.59.
Found: C, 71.16; H, 5.99; N, 16.70.
4.2.15. General procedure for the synthesis of compounds 15a–d
A mixture of the appropriate amine 13a or 13b (0.29 mmol), the
appropriate arylbromide (0.32 mmol), dichlorobis(triphenylphos-
phine)Pd(II) (20 mg, 0.029 mmol), Xantphos (17 mg, 0.029 mmol)
and sodium tert-butoxide (42 mg, 0.44 mmol) was refluxed in tol-
uene (7 mL) under nitrogen atmosphere for 8 h. The reaction mix-
ture was left to cool at room temperature, and then cooled in an ice
bath. The formed solid was filtered, washed with cold toluene
(10 mL), then with water (50 mL).
4.2.13. General procedure for the synthesis of compounds 13a, 13b
To a solution of NaOEt in absolute ethanol, made by dissolving
sodium metal (27 mg, 1.185 mmol) in absolute ethanol (20 mL),

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
3871
4.2.15.1. N-(4-Methoxyphenyl)-4-(6-phenylpyridin-3-yl)pyrimi-
din-2-amine (15a)17
It was obtained as yellow crystalline pow-
der. Yield (84 mg, 82%); mp >300 °C; IR
/cmÀ1: 3462, 1579,
with water (50 mL) and then extracted with methylene chloride
(100 mL Â 3). The organic layer was separated, dried over anhy-
drous MgSO4, and then evaporated under vacuum to yield the
.
m
1511, 1456, 1430, 1262, 1240, 1097, 1039, 804; 1H NMR (DMSO-
d6): d 3.74 (s, 3H), 6.92 (d, J = 9.0 Hz, 2H), 7.48–7.56 (m, 4H), 7.71
(d, J = 8.7 Hz, 2H), (m, 3H), 8.55–8.58 (m, 2H), 9.41 (s, 1H), 9.57
(s, 1H); 13C NMR (DMSO-d6): d 55.65, 107.96, 114.24, 120.66,
121.38, 127.25, 129.39, 131.32, 133.90, 135.90, 138.31, 148.59,
154.83, 158.12, 159.79, 160.78; Anal. Calcd for C22H18N4O: C,
74.56; H, 5.12; N, 15.81. Found: C, 74.30; H, 5.60; N, 15.90.
product (17). Yield (0.43 g, 93.6%); mp 187–189 °C; IR m :
/cmÀ1
3283, 3038, 1635, 1583, 1547, 1496, 1422, 1387, 1318, 1253,
1115, 1061, 896, 751; 1H NMR (CDCl3): d 2.94 (s, 6H), 5.65 (d,
J = 9.0 Hz, 1H), 7.05–7.10 (m, 1H), 7.27–7.45 (m, 4H), 7.56 (s, 1H),
7.79 (d, J = 12.3 Hz, 1H), 8.07 (dd, J = 2.1, 8.7 Hz, 1H), 8.78 (s, 1H);
13C NMR (CDCl3): 38.67, 44.91, 91.38, 107.81, 120.83, 123.21,
126.69, 127.01, 129.22, 129.37, 137.20, 148.94, 153.82, 185.7.
4.2.15.2.
din-3-yl)pyrimidin-2-amine (15b). It was obtained as white
powder. Yield (105 mg, 79%); mp 113–115 °C; IR
/cmÀ1: 3276,
3098, 1578, 1558, 1383, 1275, 1184, 1129, 882, 818, 785, 739,
680; 1H NMR (DMSO-d6): d 7.49–7.57 (m, 3H), 7.63 (s, 1H), 7.72
(d, J = 5.2 Hz, 1H), 8.16–8.22 (m, 3H), 8.57–8.61 (m, 3H), 8.73 (d,
J = 5.1 Hz, 1H), 9.43 (d, J = 1.7 Hz, 1H), 10.50 (s, 1H).
N-(3,5-Bis(trifluoromethyl)phenyl)-4-(6-phenylpyri-
4.2.18. 4-(6-(Phenylamino)pyridin-3-yl)pyrimidin-2-amine (18)
To an ethanolic solution of sodium ethoxide prepared by dis-
solving sodium metal (40 mg, 1.74 mmol) in absolute ethanol
(10 mL) was added guanidine hydrochloride (170 mg, 1.74 mmol).
The mixture was stirred at room temperature for 1 h, then 3-
(dimethylamino)-1-(6-(phenylamino)pyridin-3-yl)prop-2-en-1-
one (17; 0.4 g, 1.5 mmol) in 10 mL of absolute ethanol was added.
The temperature was raised to reflux, and the mixture was heated
for 8 h. The reaction mixture was left to cool to room temperature
then cooled in ice water. The crystallized product (18) was col-
lected by filtration, washed first with cold ethanol, then with
m
4.2.15.3. N1,N1-Diphenyl-N4-(4-(6-phenylpyridin-3-yl)pyrimi-
din-2-yl)benzene-1,4-diamine (15c)17
powder. Yield (44 mg, 31%); mp 210–211 °C; IR
.
It was obtained as yellow
m
/cmÀ1: 3460,
1575, 1527, 1507, 1493, 1422, 1277, 744, 695; 1H NMR (CDCl3):
d 6.98–7.72 (m, 18H), 7.89 (d, J = 8.1 Hz, 1H), 8.10 (d, J = 6.9 Hz,
2H), 8.45 (dd, J = 2.5, 6.1 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 9.40 (s,
1H); 13C NMR (CDCl3): d 107.92, 108.12, 119.44, 120.31, 120.46,
122.30, 122.74, 123.60, 125.62, 127.13, 128.90, 129.00, 129.10,
129.60, 130.93, 134.90, 135.26, 138.58, 142.75, 147.98, 148.53,
158.92, 159.15, 160.33, 162.48; Anal. Calcd for C33H25N5: C,
80.63; H, 5.13; N, 14.25. Found: C, 80.46; H, 5.55; N, 14.20.
water. Yield (0.21 g, 46%); mp >300 °C; IR m
/cmÀ1: 3342, 3175,
1653, 1594, 1574, 1540, 1497, 1471, 1443, 1393, 1344, 1292,
806; 1H NMR (DMSO-d6): d 6.56 (s, 2H); 6.89–6.97 (m, 2H), 7.06
(d, J = 5.2 Hz, 1H), 7.29 (t, J = 7.7 Hz, 2H), 7.70 (d, J = 8.1 Hz, 2H),
8.18–8.24 (m, 2H), 8.86 (d, J = 1.9 Hz, 1H), 9.38 (s, 1H); 13C NMR
(DMSO-d6): 105.01, 110.60, 119.20, 121.70, 123.42, 129.15,
135.72, 141.45, 147.34, 157.59, 159.06, 162.36, 164.11.
4.2.19. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-(6-(phenylamino)-
pyridin-3-yl)pyrimidin-2-yl)urea (19)
4.2.15.4. N1,N1-Diphenyl-N4-(4-(6-(pyridin-3-yl)pyridin-3-yl)-
pyrimidin-2-yl)benzene-1,4-diamine (15d)17
.
It was obtained
A mixture of 4-(6-(phenylamino)pyridin-3-yl)pyrimidin-2-
as yellow powder. Yield (39 mg, 27.3%); mp 230–231 °C; IR /cm
m
amine (18; 100 mg, 0.38 mmol) and 3,5-bis(trifluoromethyl)phe-
nyl isocyanate (193 mg, 0.76 mmol) was heated without solvent
on an oil bath at 120 °C for 1 h. The mixture was then crystallized
from DMSO to yield the pure product (19). Yield (145 mg, 74%); mp
À1: 3429, 1582, 1530, 1507, 1493, 1422, 1281, 751, 695; 1H NMR
(DMSO-d6): d 6.96 (d, J = 6.9 Hz, 6H), 7.05 (d, J = 8.8 Hz, 2H),
7.24–7.30 (m, 4H), 7.56 (d, J = 5.1 Hz, 2H), 7.81 (d, J = 8.7 Hz, 2H),
8.25 (d, J = 8.1 Hz, 1H), 8.52 (d, J = 8.1 Hz, 1H), 8.60–8.68 (m, 3H),
9.35 (s, 1H), 9.45 (s, 1H), 9.82 (s, 1H); 13C NMR (CDCl3): 100.37,
108.54, 120.84, 121.17, 122.59, 123.10, 124.43, 126.18, 129.86,
131.87, 133.82, 134.72, 136.15, 136.96, 141.37, 148.00, 148.40,
148.88, 159.92, 161.57; Anal. Calcd for C32H24N6: C, 78.03; H,
4.91; N, 17.06. Found: C, 78.06; H, 4.99; N, 17.20.
278–279 °C; IR m
/cmÀ1: 3134, 3011, 2974, 1708, 1587, 1388, 1282,
1181, 1135, 739; 1H NMR (DMSO-d6): d 7.05 (d, J = 8.8 Hz, 2H), 7.35
(t, J = 7.8 Hz, 2H), 7.70–7.77 (m, 4H), 8.33 (s, 2H), 8.38 (d, J = 8.6 Hz,
1H), 8.68 (d, J = 5.4 Hz, 1H), 9.01 (s, 1H), 9.97 (s, 1H), 10.59 (s, 1H),
12.09 (s, 1H).
4.2.20. N-(3,5-Bis(trifluoromethyl)phenyl)-4-(6-(phenylamino)-
pyridin-3-yl)pyrimidin-2-amine (20)
4.2.16. 1-(6-(Phenylamino)pyridin-3-yl)ethanone (16)
A mixture of 5-acetyl-2-bromopyridine (10; 1.0 g, 5.0 mmol),
dichlorobis(triphenylphosphine)Pd(II) (140 mg, 0.2 mmol), 1,3-
bis(diphenylphosphino)propane (210 mg, 0.5 mmol) and sodium
tert-butoxide (1.2 g, 12.5 mmol) in aniline (5 mL) was refluxed un-
der N2 atmosphere for 12 h. After reaction completion, excess ani-
line was removed under vacuum, and the residue was purified
with column chromatography (silica gel, ethyl acetate/hexane
A mixture of 4-(6-(phenylamino)pyridin-3-yl)pyrimidin-2-amine-
(18; 100 mg, 0.38 mmol), 3,5-bis(trifluoromethyl)phenylbromide
(123 mg, 0.42 mmol), dichlorobis(triphenylphosphine)Pd(II) (26
mg, 0.038 mmol), Xantphos (22 mg, 0.038 mmol) and sodium tert-
butoxide (55 mg, 0.57 mmol) was refluxed in toluene (7 mL) under
nitrogen atmosphere for 12 h. The reaction mixture was left to cool
at room temperature, and then cooled in an ice bath. The formed so-
lid was filtered, washed with cold toluene (10 mL), then with water
(50 mL) to yield pure 20 as yellow crystals. Yield (112 mg, 62%); mp
1:3, v/v). Yield (0.4 g, 38%); mp 128–129 °C; IR
m
/cmÀ1: 3240,
1675, 1609, 1590, 1572, 1391, 1278, 1142, 754; 1H NMR (CDCl3):
d 2.53 (s, 3H), 6.85 (d, J = 8.9 Hz, 1H), 7.14–7.20 (m, 1H), 7.39–
7.40 (m, 4H), 7.97 (br s, 1H), 8.05 (dd, J = 2.1, 8.9 Hz, 1H), 8.78–
8.79 (m, 1H); 13C NMR (CDCl3): 26.11, 107.16, 121.93, 124.55,
124.77, 129.52, 137.62, 138.89, 151.04, 158.92, 195.33.
273–274 °C; IR m
/cmÀ1: 3288, 3221, 3133, 2994, 1591, 1552, 1524,
1372, 1281, 1172, 1123, 877, 813, 699, 681; 1H NMR (DMSO-d6): d
6.95–7.00 (m, 2H), 7.32 (t, J = 7.6 Hz, 2H), 7.51 (d, J = 5.1 Hz, 1H),
7.60 (s, 1H), 7.71 (d, J = 8.0 Hz, 2H), 8.30 (d, J = 8.9 Hz, 1H), 8.60 (s,
3H), 9.01 (s, 1H), 9.52 (s, 1H), 10.35 (s, 1H).
4.2.17. (E)-3-(Dimethylamino)-1-(6-(phenylamino)pyridin-3-
yl)prop-2-en-1-one (17)
4.2.21. (E)-1-(6-Bromopyridin-3-yl)-3-(dimethylamino)prop-2-
en-1-one (21)
A mixture of 5-acetyl-2-bromopyridine (10; 2.5 g, 12.5 mmol)
and N,N-dimethylformamide dimethylacetal (3.0 g, 25 mmol) was
heated under reflux in an oil bath for 20 h. The excess unreacted
N,N-dimethylformamide dimethylacetal was removed under vac-
A mixture of 1-(6-(phenylamino)pyridin-3-yl)ethanone (16;
0.37 g, 1.72 mmol) and N,N-dimethylformamide dimethylacetal
(0.41 g, 3.44 mmol) was heated under reflux in an oil bath for
12 h. The excess unreacted N,N-dimethylformamide dimethylace-
tal was removed under vacuum. The solid residue was triturated

3872
I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
uum. The solid residue was triturated with water (100 mL) and
then extracted with methylene chloride (200 mL Â 3). The organic
layer was separated, dried over anhydrous MgSO4, and then evap-
orated under vacuum to yield the crude product (21) which was
purified by crystallization from the mixed solvent of ethyl acetate
815, 604; 1H NMR (DMSO-d6): d 2.64 (s, 3H), 6.83 (s, 2H), 7.29
(d, J = 5.2 Hz, 1H), 8.09 (d, J = 8.4 Hz, 2H), 8.22 (d, J = 8.4 Hz, 1H),
8.32 (d, J = 8.4 Hz, 2H), 8.38 (d, J = 5.1 Hz, 1H), 8.53 (dd, J = 2.2,
6.2 Hz, 1H), 9.36 (d, J = 1.9 Hz, 1H).
and hexane. Yield (2.8 g, 88%); mp 122–123 °C; IR
m
/cmÀ1: 1645,
4.2.24.3. N-(2-(5-(2-Aminopyrimidin-4-yl)pyridin-2-yl)phenyl)-
1578, 1571, 1534, 1437, 1418, 1087, 900, 790; 1H NMR (CDCl3): d
2.86 (s, 3H); 3.10 (s, 3H), 5.52 (d, J = 12.1 Hz, 1H), 7.44 (d,
J = 8.2 Hz, 1H), 7.77 (d, J = 12.1 Hz, 1H), 7.94–7.96 (m, 1H), 8.73
(s, 1H); 13C NMR (CDCl3): 37.46, 45.32, 91.44, 127.80, 134.88,
137.62, 144.22, 149.32, 154.94, 184.89.
acetamide (24c). Yield (225 mg, 46%); mp 198–199 °C; IR m :
/cmÀ1
3893, 3456, 3324, 3176, 1674, 1587, 1534, 1457, 1322, 1241,
815, 776, 757; 1H NMR (DMSO-d6): d 6.85 (s, 2H), 7.22–7.30 (m,
2H), 7.44 (t, J = 7.7 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.99 (d,
J = 8.5 Hz, 1H), 8.21 (d, J = 8.3 Hz, 1H), 8.39 (d, J = 5.1 Hz, 1H),
8.56 (d, J = 8.1 Hz, 1H), 9.39 (s, 1H), 11.60 (s, 1H); 13C NMR
(DMSO-d6): 24.95, 106.40, 122.91, 123.49, 124.39, 130.08, 130.39,
131.13, 136.07, 137.56, 147.13, 158.92, 159.98, 161.39, 164.29,
168.67.
4.2.22. 4-(6-Bromopyridin-3-yl)pyrimidin-2-amine (22)
To an ethanolic solution of sodium ethoxide prepared by dis-
solving sodium metal (0.27 g, 11.37 mmol) in absolute ethanol
(30 mL) was added guanidine hydrochloride (1.12 g, 11.73 mmol).
The mixture was stirred at room temperature for 1 h, then
1-(6-bromopyridin-3-yl)-3-(dimethylamino)prop-2-en-1-one (21;
2.7 g, 10.6 mmol) in 30 mL of absolute ethanol was added. The
temperature was raised to reflux, and the mixture was heated for
12 h. The reaction mixture was left to cool to room temperature
then cooled in ice water. The crystallized product (22) was col-
lected by filtration, washed first with cold ethanol, then with
4.2.24.4. N-(3-(5-(2-Aminopyrimidin-4-yl)pyridin-2-yl)phenyl)-
acetamide (24d). Yield (288 mg, 59%); mp 246–247 °C; IR m :
/cmÀ1
3310, 3149, 1670, 1652, 1613, 1592, 1572, 1475, 1371, 821,
801; 1H NMR (DMSO-d6): d 2.21 (s, 3H), 5.19 (s, 2H), 7.12 (d,
J = 3.9 Hz, 1H), 7.19 (t, J = 5.6 Hz, 1H), 7.45 (t, J = 5.5 Hz, 1H), 7.71
(d, J = 5.3 Hz, 1H), 7.85 (d, J = 6.3 Hz, 1H), 8.43 - 8.46 (m, 2H),
8.56 (d, J = 6.0 Hz, 1H), 9.28 (s, 1H); 13C NMR (DMSO-d6): 24.52,
106.40, 117.75, 120.43, 121.84, 129.72, 131.64, 135.75, 138.86,
140.39, 148.44, 157.67, 159.84, 161.71, 164.28, 168.96.
water. Yield (1.54 g, 58%); mp 192–193 °C; IR
m
/cmÀ1: 3482,
3301, 3170, 1634, 1582, 1568, 1479, 1463, 1436, 1217, 1087,
815; 1H NMR (DMSO-d6): d 6.82 (s, 2H); 7.21 (d, J = 5.1 Hz, 1H),
7.79 (d, J = 8.4 Hz, 1H), 8.32–8.37 (m, 3H), 9.02 (d, J = 2.3 Hz, 1H);
13C NMR (DMSO-d6): 128.65, 132.76, 137.83, 143.61, 149.23,
160.08, 160.83, 164.19, 164.24.
4.2.25. General procedure for the synthesis of compounds 25a–d
A mixture of the appropriate amine 24a–d (0.34 mmol) and 3,5-
bis(trifluoromethyl)phenyl isocyanate (173 mg, 0.68 mmol) was
heated without solvent on an oil bath at 120 °C for 2 h. The crude
product was then purified by the suitable method to yield pure
25a–d.
4.2.23. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-(6-bromopyridin-
3-yl)pyrimidin-2-yl)urea (23)
A mixture of the amine 22 (100 mg, 0.4 mmol) and 3,5-bis(tri-
fluoromethyl)phenyl isocyanate (203 mg, 0.8 mmol) was heated
without solvent on an oil bath at 120 °C for 2 h. The mixture was
then crystallized from DMSO to yield the pure product (23). Yield
4.2.25.1. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-(6-(2-acetylphe-
nyl)pyridin-3-yl)pyrimidin-2-yl)urea (25a). The residue was dis-
solved in methylene chloride and purified by column chromato-
graphy (silica gel, ethyl acetate/hexane, 1:1, v/v). Yield (120 mg,
(160 mg, 79%); mp 139–141 °C; IR
m
/cmÀ1: 3281, 3128, 3024,
1722, 1580, 1472, 1382, 1280, 1171, 1146, 1031, 879, 683; 1H
NMR (DMSO-d6): d 7.18 (d, J = 8.4 Hz, 1H), 7.74–7.76 (m, 2H),
8.34 (s, 2H), 8.51 (d, J = 9.6 Hz, 1H), 8.72 (d, J = 5.4 Hz, 1H), 8.80
(s, 1H), 10.54 (s, 1H), 11.98 (s, 1H).
65%); mp 226–227 °C; IR m
/cmÀ1: 3092, 2963, 2925, 2853, 1724,
1695, 1584, 1410, 1387, 1286, 1185, 1126, 877, 833, 742; 1H
NMR (DMSO-d6): d 2.31 (s, 3H), 7.58–7.64 (m, 3H), 7.77 (s, 1H),
7.81 (d, J = 7.2 Hz, 1H), 7.90 (d, J = 5.4 Hz, 1H), 7.98 (t, J = 8.4 Hz,
1H), 8.33–8.36 (m, 2H), 8.67 (d, J = 8.4 Hz, 1H), 8.84 (d, J = 5.1 Hz,
1H), 9.38 (s, 1H), 10.62 (s, 1H), 11.97 (s, 1H).
4.2.24. General procedure for the synthesis of compounds 24a–d
A mixture of the amine 22 (0.4 g, 1.6 mmol), the appropriate
aryl boronic acid (1.76 mmol), dichlorobis(triphenylphosphine)-
Pd(II) (30 mg, 0.044 mmol) and Na2CO3 (120 mg, 1.12 mmol) was
placed in mixed solvent of acetonitrile and water (1:1, v/v,
20 mL). N2 gas was bubbled into this mixture for 10 min, and then
the mixture was heated at 78 °C while stirring under N2 atmo-
sphere for 12 h. The reaction mixture was left to cool at room tem-
perature, and then poured into ice water (100 mL). The resulted
solid was collected by filtration, washed with water, and crystal-
lized from ethanol to yield the pure products 24a–d.
4.2.25.2. 1-(4-(6-(2-Acetylphenyl)pyridin-3-yl)pyrimidin-2-yl)-
3-(3-(trifluoromethyl)-5-morpholinophenyl)urea
(25b). The
residue was stirred in ethyl acetate (10 mL) for 20 min, and the
undissolved solid was filtered and washed with ethyl acetate
(10 mL) to yield pure 25b. Yield (126 mg, 66%); mp 238–239 °C;
IR
m
/cmÀ1: 3092, 2968, 2917, 2856, 1713, 1687, 1619, 1586,
1568, 1447, 1416, 1239, 1118, 838, 741; 1H NMR (DMSO-d6): d
2.30 (s, 3H), 3.17 (s, 4H), 3.67 (s, 4H), 6.91 (s, 1H), 7.24 (s, 1H),
7.60–7.86 (m, 6H), 7.97 (d, J = 8.2 Hz, 1H), 8.62 (d, J = 7.8 Hz, 1H),
8.81 (d, J = 5.0 Hz, 1H), 9.36 (s, 1H), 10.41 (s, 1H), 11.68 (s, 1H);
13C NMR (DMSO-d6): 30.82, 48.21, 66.31, 106.29, 108.72, 111.60,
123.03, 127.95, 129.35, 129.70, 129.88, 130.45, 130.65, 130.84,
136.26, 137.88, 140.70, 142.08, 148.21, 151.99, 152, 42, 158.44,
159.63, 159.80, 162.26, 203.27.
4.2.24.1. 1-(2-(5-(2-Aminopyrimidin-4-yl)pyridin-2-yl)phenyl)-
ethanone (24a). Yield (296 mg, 64%); mp 155–156 °C; IR m/cm
À1: 3372, 3314, 3178, 1698, 1646, 1567, 1472, 1438, 1288, 1268,
1249, 1237, 1216, 817, 601; 1H NMR (CDCl3): d 2.31 (s, 3H); 5.16
(s, 2H), 7.03 (s, 1H), 7.39–7.72 (m, 5H), 8.41 (d, J = 2.3 Hz, 2H),
9.24 (s, 1H); 13C NMR (CDCl3): 30.64, 107.59, 122.30, 127.67,
129.10, 129.25, 130.37, 131.29, 135.28, 138.03, 141.76, 147.92,
159.27, 162.55, 163.33, 204.13.
4.2.25.3. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-(6-(2-acetami-
dophenyl)pyridin-3-yl)pyrimidin-2-yl)urea (25c). The residue
was crystallized from DMSO to yield pure 25c. Yield (80 mg,
42%); mp 284–285 °C; IR m
/cmÀ1: 3440, 3231, 3088, 2971, 1726,
4.2.24.2. 1-(4-(5-(2-Aminopyrimidin-4-yl)pyridin-2-yl)phenyl)-
1682, 1585, 1413, 2388, 1287, 1181, 1124, 879, 757, 553; 1H
NMR (DMSO-d6): d 2.05 (s, 3H), 7.28 (t, J = 7.3 Hz, 1H), 7.48 (t,
J = 7.5 Hz, 1H), 7.73 (s, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.91 (d,
ethanone (24b). Yield (283 mg, 61%); mp 268–269 °C; IR
m/cm
À1: 3374, 3297, 3158, 1682, 1636, 1589, 1574, 1471, 1365, 1266,

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
3873
J = 5.3 Hz, 1H), 8.05–8.13 (m, 2H), 8.34 (s, 2H), 8.74 (d, J = 7.0 Hz,
1H), 8.85 (d, J = 5.2 Hz, 1H), 9.51 (s, 1H), 10.56 (s, 1H), 11.32 (s,
1H), 11.89 (s, 1H).
the microtiter plates are incubated at 37 °C, 5% CO2, 95% air, and
100% relative humidity for 24 h prior to addition of experimental
drugs. After 24 h, two plates of each cell line are fixed in situ with
TCA, to represent a measurement of the cell population for each
cell line at the time of drug addition (Tz). Experimental drugs are
solubilized in dimethyl sulfoxide at 400-fold the desired final max-
imum test concentration and stored frozen prior to use. At the time
of drug addition, an aliquot of frozen concentrate is thawed and di-
luted to twice the desired final maximum test concentration with
4.2.25.4. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-(6-(3-acetami-
dophenyl)pyridin-3-yl)pyrimidin-2-yl)urea (25d). The residue
was crystallized from DMSO to yield pure 25d. Yield (86 mg,
45%); mp >300 °C; IR
m
/cmÀ1: 3094, 2971, 2923, 1719, 1534,
1473, 1412, 1387, 1280, 1175, 1134, 880, 795, 683, 554; 1H NMR
(DMSO-d6): d 2.09 (s, 3H), 7.46 (t, J = 8.0 Hz, 1H), 7.73–7.76 (m,
2H), 7.82 (d, J = 7.3 Hz, 1H), 7.90 (d, J = 5.3 Hz, 1H), 8.10 (d,
J = 8.4 Hz, 1H), 8.35 (s, 2H), 8.45 (s, 1H), 8.66 (d, J = 6.8 Hz, 1H),
8.84 (d, J = 5.3 Hz, 1H), 9.46 (s, 1H), 10.19 (s, 1H), 10.63 (s, 1H),
12.02 (s, 1H).
complete medium containing 50
four, 10-fold or ½log serial dilutions are made to provide a total
of five drug concentrations plus control. Aliquots of 100 l of these
different drug dilutions are added to the appropriate microtiter
wells already containing 100 l of medium, resulting in the re-
lg/mL gentamicin. Additional
l
l
quired final drug concentrations. Following drug addition, the
plates are incubated for an additional 48 h at 37 °C, 5% CO2, 95%
air, and 100% relative humidity. For adherent cells, the assay is ter-
minated by the addition of cold TCA. Cells are fixed in situ by the
4.2.26. General procedure for the synthesis of compounds 26a
and 26b
A mixture of the appropriate amine 24a and 24b (0.34 mmol),
3,5-bis(trifluoromethyl)phenylbromide
(111 mg,
0.38 mmol),
gentle addition of 50 ll of cold 50% (w/v) TCA (final concentration,
dichlorobis(triphenylphosphine)Pd(II) (24 mg, 0.034 mmol), Xant-
phos (20 mg, 0.034 mmol) and sodium tert-butoxide (50 mg,
0.52 mmol) was refluxed in toluene (7 mL) under nitrogen atmo-
sphere for 12 h. The reaction was filtered while hot to remove
the insoluble metal impurities. The toluene filtrate was left to cool
at room temperature, and then cooled in an ice bath. The formed
solid was filtered, washed with cold toluene (10 mL), then with
water (50 mL).
10% TCA) and incubated for 60 min at 4 °C. The supernatant is dis-
carded, and the plates are washed five times with tap water and air
dried. Sulforhodamine B (SRB) solution (100 ll) at 0.4% (w/v) in 1%
acetic acid is added to each well, and plates are incubated for 10 min
at room temperature. After staining, unbound dye is removed by
washing five times with 1% acetic acid and the plates are air dried.
Bound stain is subsequently solubilized with 10 mM trizma base,
and the absorbance is read on an automated plate reader at a wave-
length of 515 nm. For suspension cells, the methodology is the same
except that the assay is terminated by fixing settled cells at the bot-
4.2.26.1. 1-(2-(5-(2-(3,5-Bis(trifluoromethyl)phenylamino)pyr-
imidin-4-yl)pyridin-2-yl)phenyl)ethanone (26a). Yield (135 mg,
tom of the wells by gently adding 50 ll of 80% TCA (final concentra-
79%); mp 197–199 °C; IR
m
/cmÀ1: 3464, 3269, 2075, 3002, 1694,
tion, 16% TCA). Using the seven absorbance measurements [time
zero, (Tz), control growth, (C), and test growth in the presence of
drug at the five concentration levels (Ti)], the percentage growth is
calculated at each of the drug concentrations levels. Percentage
growth inhibition is calculated as:
1571, 1554, 1439, 1384, 1276, 1189, 1131, 786, 680, 594; 1H NMR
(DMSO-d6): d 2.31 (s, 3H), 7.48–7.62 (m, 4H), 7.73 (d, J = 5.2 Hz,
1H), 7.84 (d, J = 7.3 Hz, 1H), 7.99 (d, J = 8.3 Hz, 1H), 8.61–8.63 (m,
3H), 8.74 (d, J = 5.2 Hz, 1H), 9.35 (s, 1H), 10.52 (s, 1H).
4.2.26.2. 1-(4-(5-(2-(3,5-Bis(trifluoromethyl)phenylamino)pyrim-
ꢀ [(TiÀTz)/(CÀTz)] Â 100 for concentrations for which Ti P Tz.
ꢀ [(TiÀTz)/Tz] Â 100 for concentrations for which Ti<Tz.
idin-4-yl)pyridin-2-yl)phenyl)ethanone (26b). Yield (118 mg,
69%); mp >300 °C; IR
m
/cmÀ1: 3473, 3356, 1684, 1586, 1555,
1477, 1460, 1437, 1385, 1171, 1124, 815; 1H NMR (DMSO-d6): d
2.63 (s, 3H), 7.61 (s, 1H), 7.73 (d, J = 5.2 Hz, 1H), 8.07 (d, J = 8.3 Hz,
2H), 8.25 (d, J = 8.4 Hz, 1H), 8.33 (d, J = 8.3 Hz, 2H), 8.59–8.62 (m,
3H), 8.73 (d, J = 5.1 Hz, 1H), 9.46 (s, 1H), 10.51 (s, 1H).
Three dose response parameters are calculated for each experi-
mental agent. Growth inhibition of 50% (IC50) is calculated from
[(TiÀTz)/(CÀTz)] Â 100 = 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as mea-
sured by SRB staining) in control cells during the drug incubation.
The drug concentration resulting in total growth inhibition (TGI) is
calculated from Ti = Tz. The LC50 (concentration of drug resulting in
a 50% reduction in the measured protein at the end of the drug
treatment as compared to that at the beginning) indicating a net
loss of cells following treatment is calculated from [(TiÀTz)/
Tz] Â 100 = À50. Values are calculated for each of these three
parameters if the level of activity is reached; however, if the effect
is not reached or is exceeded, the value for that parameter is ex-
pressed as greater or less than the maximum or minimum concen-
tration tested.
4.3. HCl-salt formation
The free bases 15b, 19, 25c, and 25d were changed to the corre-
sponding HCl-salts 27, 28, 29, and 30, respectively, according to the
following procedure: The appropriate starting free base 15b, 19,
25c, and 25d (0.15 mmol) was dissolved in dry toluene (10 mL)
by heating and stirring under reflux. After complete dissolution
of the compound, refluxing was stopped, and the solution was sat-
urated with hydrochloride gas while stirring is maintained for
30 min. The resulted precipitate (HCl-salt) was filtered, washed
throughly with toluene (50 mL) and dried.
4.4. Cell line screening
4.5. Enzyme screening
Cell line screening was applied at the National Cancer Institute
(NCI), Bethesda, Maryland, USA.20 applying the following proce-
dure. The human tumor cell lines of the cancer screening panel
are grown in RPMI 1640 medium containing 5% fetal bovine serum
Kinase assays were performed at Reaction Biology Corporation
using the ‘HotSpot’ assay platform.21 Kinase Assay Protocol. Reac-
tion Buffer: base Reaction buffer; 20 mM Hepes (pH 7.5), 10 mM
MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4,
2 mM DTT, 1% DMSO. Reaction procedure: To a freshly prepared buf-
fer solution was added any required cofactor for the enzymatic reac-
and 2 mM
L-glutamine. For a typical screening experiment, cells
are inoculated into 96 well microtiter plates in 100
ll at plating
densities ranging from 5,000 to 40,000 cells/well depending on
tion, followed by the addition of the selected kinase at
a
the doubling time of individual cell lines. After cell inoculation,
concentration of 20 M. The contents were mixed gently, and then
l

3874
I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3860–3874
the compound under test (compound 30) dissolved in DMSO was
added to the reaction mixture in the 10 M concentration. 339-
ATP (specific activity 500 Ci/ l) was added to the mixture in order
to initiate the reaction, and the mixture was incubated at room tem-
perature for 2 h. Staurosporine was used as a control compound in a
five-dose IC50 mode with 10-fold serial dilutions starting at 20 lM,
and the reaction was carried out at 10 lM ATP concentration.
References and notes
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4.6. Molecular modeling
Docking studies were performed using ‘Molecular Operating
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Acknowledgments
This research was supported by Korea Institute of Science and
Technology. We would like to express our gratitude and thanks to
the NationalCancer Institute (NCI), Bethesda, Maryland, USA for per-
forming the anticancer testing of the new compounds. Our appreci-
ations also for Dr. Sean W. Deacon and Dr. Haiching Ma from
Reaction Biology Corporation for carrying out the kinase screening.
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Supplementary data
35. http://www.rcsb.org/pdb.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.037.