Synthesis and cytotoxic activity of 2-methylimidazo[1,2-a]pyridine- and quinoline-substituted 2-aminopyrimidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.10.002

A series of 2-methylimidazo[1,2-a]pyridine- and quinoline-substituted 2-aminopyrimidines derivatives were synthesized using a convenient synthetic route. We evaluate the isosteric replacement of methyl groups in 4-(2-methylimidazo[1,2-a]pyridin-3-yl)-N-p-

Full text of DOI:10.1016/j.ejmech.2009.10.002

European Journal of Medicinal Chemistry 45 (2010) 379–386
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and cytotoxic activity of 2-methylimidazo[1,2-a]pyridine- and
quinoline-substituted 2-aminopyrimidine derivatives
,
b
a
a
Miguel Angel Vilchis-Reyes a , Alejandro Zentella , Miguel Angel Martınez-Urbina , Angel Guzman ,
*
´
´
´
´
b
a
b
a,
*
´
´
´
Omar Vargas , Marıa Teresa Ramırez Apan , Jose Luis Ventura Gallegos , Eduardo Dıaz
a Instituto de Qu´ımica, Universidad Nacional Auto´noma de Me´xico, Circuito Exterior, Ciudad Universitaria, Me´xico 04510, D. F., Me´xico
b Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, Circuito Exterior, Ciudad Universitaria, Me´xico 04510, D. F., Me´xico
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-methylimidazo[1,2-a]pyridine- and quinoline-substituted 2-aminopyrimidines derivatives
were synthesized using a convenient synthetic route. We evaluate the isosteric replacement of methyl
groups in 4-(2-methylimidazo[1,2-a]pyridin-3-yl)-N-p-tolylpyrimidin-2-amine (compound 1) by tri-
fluoromethyl groups and the isosteric substitution of the 2-methylimidazo[1,2-a]pyridin-3-yl scaffold by
quinolin-4-yl or quinolin-3-yl moieties. The replacement of hydrogen by fluorine does not affect notably
the cytotoxic activity and CDK inhibitor activity in this series. Quinolin-4-yl-substituted compound, 8,
presents cytotoxic activity and is most effective and selective against CDK1/CycA than against CDK2/CycB.
Compound 11, which has a quinolin-3-yl moiety is CDK inhibitor but presents null cytotoxic activity.
Quinolin-4-yl-substituted compounds constitute a new lead of cytotoxic and CDK inhibitor compounds
from which more compelling and selective inhibitors can be designed.
Received 9 March 2009
Received in revised form
28 September 2009
Accepted 1 October 2009
Available online 13 October 2009
Keywords:
Imidazo[1,2-a]pyridine
Quinoline
Bioisosteric replacement
CDK
Apoptosis
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
nature of current cancer chemotherapy and drug resistance has
initiated a search for more selective approaches.
Cancer is a leading cause of death. Approximately, one in four
persons dies of cancer in the United States [1] and cancer is the
third most common cause of dead worldwide [2]. The incidence of
cancer has not dropped over the last decades; the most compli-
cated cases are present in developing countries and it is expected
that in the following years its incidence will be bigger than the
incidence of cardiovascular diseases [2].
Regardless of the use of surgical treatment and irradiation,
chemotherapy still remains an important option for the treatment
of solid cancers. Despite extensive research efforts, the therapeutic
treatment of tumor patients is still not satisfying. Chemothera-
peutic drugs should preferentially target tumor cells without
harming normal cells or tissues. Unfortunately, in most cases,
chemotherapy remains a treatment modality with harsh side
effects due to the toxicity of the drugs. Toxic effects on rapidly
proliferating cells such as intestinal cells and bone marrow can
cause life-threatening infections and other side effects, which
seriously affect the quality of life of the patient. The indiscriminate
Imidazo[1,2-a]pyridine scaffold has been extensively studied for
their pharmacological properties, such as hypnotic [3], antiviral [4],
antiparasitic [5] and proton pump inhibitor [6]. This moiety has
revealed inhibitory properties on several kinases. Some examples
are IRK-4 [7], GSK3 [8] and MAPK/ERK kinase [9]. Recently,
2-aminopyrimidines substituted with 2-methylimidazo[1,2-
a]pyridine have showed excellent cytotoxic and inhibitory activity
against cyclin-dependent kinase 2 (CDK2) [10,11]. Meridianines,
indole-substituted 2-aminopyrimidines, are kinase inhibitors [12],
showing that the replacement of the imidazo[1,2-a]pyridine is
possible. As a part of our search for new antitumor compounds,
a new series of imidazo[1,2-a]pyridine- and quinoline-substituted
2-aminopyrimidines were synthesized as potential antitumoral
compounds. At the same time and with the same objective we
explore the bioisosteric replacement in compound 1 (Table 1) of the
hydrogen atoms in the methyl group of 2-methylimidazo[1,2-
a]pyridine and in the tolyl part of the molecule by fluorine. In
addition, since it is known that the bioisosteric substitution of the
2-methylimidazo[1,2-a]pyridine by a quinoline ring gave good
antagonist compounds when applied to bradykinin B2 receptor
[13], we prepared compounds 7–12. The synthesized compounds
(Table 1) were tested against the following tumor human cell lines:
U251 (glioma), PC-3 (prostate), K562 (Leukemia), HCT-15 (colon),
* Corresponding authors. Tel.: þ52 55 56 22 44 21; fax: þ52 55 56 16 22 17.
E-mail addresses: vilchis90@yahoo.com (M.A. Vilchis-Reyes), maudiaz@servidor.
unam.mx (E. Dı´az).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.002

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M.A. Vilchis-Reyes et al. / European Journal of Medicinal Chemistry 45 (2010) 379–386
Table 1
Table 1 (continued)
In vitro susceptibility of SK-LU-1 and MCF7 cell lines of synthesized imidazo[1,2-a]
pyridine and quinoline-substituted 2-aminopyrimidines.
Compound Ar
R
IC50 (m
M)a
SK-LU-1
MCF7
H
N
N
R
N
12
NT
>50.0
N
F3C
Ar
(m
M)a
NT ¼ Not tested.
Compound Ar
R
IC50
SK-LU-1
a
The results show the concentration producing 50% of growth inhibition. Data
MCF7
are presented as mean ꢀ SD of three independent experiments.
MCF7 (breast) and SK-LU-1 (lung). The effect on the cell cycle, cell
death and inhibition of CDK2/CycA and CDK1/CycB of selected
compounds was analyzed.
N
N
1
0.0197 ꢀ 0.009 0.059 ꢀ 0.007
0.034 ꢀ 0.016 0.066 ꢀ 0.002
N
N
N
2. Chemistry
2
3
Synthesis of compounds 1–12 was achieved starting with the
corresponding aldehyde (Scheme 1). 3- and 4-quinolinecarbox-
aldehyde are commercially available, 2-methyilimidazo[1,2-
a]pyridine-3-carboxaldehyde (15) and 2-(trifluoromethyl)imidazo
[1,2-a]pyridine-3-carboxaldehyde (16) were prepared through
reaction of 2-aminopyridine with bromoacetone or 1,1,1-trifluoro-
3-bromoacetone in ethanol-dioxane solution to afford compounds
13 and 14 (Scheme 2). Vilsmeier–Haack reaction of these with
chloromethyliminium chloride yielded compounds 15 and 16
(Scheme 2).
Addition of ethynyl magnesium bromide 0.5 M in THF to the
aromatic aldehydes 15, 16, 17 and 18 gave the propargylic alcohol
derivatives 19, 20, 21 and 22. These were oxidized with MnO2 to
afford the corresponding alkynyl ketone 23, 25 and 26. Oxidation of
20 with MnO2 produce unsatisfactory results, but the use of calcium
hypochlorite under phase-transfer catalyst condition produced 24.
Cyclocondensation of 23, 24, 25 and 26 with guanidine sulfate
afforded imidazo[1,2-a]pyridines-substituted 2-aminopyrimidines
27 and 4 and quinoline-substituted 2-aminopyrimidines 7 and 10.
Then the desired products 1, 2, 3, 5, 6, 8, 9,11, and 12 wereobtained in
excellent yields afterthe coupling between 27, 4, 7 and 10 and an aryl
bromide or iodide under modified Buchwald–Hartig conditions [14].
All synthesized compounds were characterized by 1H NMR,13C NMR
and mass spectroscopy.
F3C
N
CHO
1.540 ꢀ 0.14
4.270 ꢀ 0.36
S
N
CF3
CF3
CF3
4
5
6
H
56.510 ꢀ 1.40 53.680 ꢀ 21.62
0.175 ꢀ 0.005 0.044 ꢀ 0.02
N
N
N
N
N
0.745 ꢀ 0.18
0.290 ꢀ 0.24
40.25 ꢀ 0.25
0.600 ꢀ 0.04
1.650 ꢀ 0.06
F3C
7
8
9
H
>50
N
3. Biological results and discussion
In this study we evaluated (1) the bioisosteric replacement in
compound
1 of the methyl hydrogen atoms of 2-methyl-
imidazo[1,2-a]pyridine by fluorine in compounds 4, 5 and 6, the
substitution of methyl hydrogen atoms of the tolyl groups by
fluorine in compound 2, 6, 9 and 12. Fluorine presents the advan-
tage of having a van der Waals radius comparable to that of
hydrogen [15]. (2) The bioisosteric substitution of the complete
2-methylimidazo[1,2-a]pyridine scaffold by the isoelectronic
quinoline-4-yl (compounds 7, 8, and 9) and (3) the regioisomer
quinoline-3-yl (compounds 10, 11 and 12). In compound 3 the
1.400 ꢀ 0.15
1.720 ꢀ 0.13
N
N
F3C
entire toluene group was replaced with
a thiazol ring. All
compounds were evaluated for their cytotoxic activity in vitro
against the tumor human cell lines U251 (glioma), PC-3 (prostate),
K562 (leukemia), HCT-15 (colon), MCF7 (breast) and SK-LU-1
10
11
H
>50.0
>50.0
w50.0
N
(lung). A primary screening at a fixed concentration of 50 mM
showed cytotoxicity against the six human tumor cell lines
(Table 2) as a positive control we used 5-fluorouracil at the same
concentration. Although the type of cell lines tested differs among
them in origin and genetic background some regularity could be
observed. Compounds 1–9 showed better activity than 5-fluoro-
uracil. In contrast, less activity or no activity was found for the
NT
N

M.A. Vilchis-Reyes et al. / European Journal of Medicinal Chemistry 45 (2010) 379–386
381
N
NH2
O
OH
iii
O
ii
i
N
Ar
Ar
Ar
H
Ar
4, 7, 10, 27
15-18
19-22
23-26
Ar
Compound (Yield/%)
iv
N
N
15 (44), 19 (92), 23 (80), 27 (75)
CH3
CF3
N
N
H
N
N
R
4 (29), 16 (52), 20 (94), 24 (NI)
17 (CA), 21 (NI), 25 (NI), 7 (20)
N
Ar
1-3, 5,6,8,9,11,12
N
18 (CA), 22 (NI), 26 (NI), 10 (10)
N
Scheme 1. Reagents: (i) Ethynyl magnesium bromide 0.5 M; (ii) MnO2; for compound 20 Ca(ClO)2, CH2Cl2, water, tetrabutylammonium bromide (catalytic); (iii) Guanidine sulfate,
K2CO3, (iv) Xantphos (3%), Pd(dba)2 (3%), Cs2CO3, toluene, aryl halide (1-iodo-4-methylbenzene, 1-iodo-4-(trifluoromethyl)benzene or 5-bromothiophene-2-carboxaldehyde). CA,
commercially available; NI, not isolated.
compounds with the quinoline-3-yl substituent 10, 11 and 12. The
most sensitive tumor human cell lines are MCF7 and SK-LU-1. In
MCF7 cell line most of the synthesized compounds showed a 100
compound 2 is 50 times more active in SK-LU-1 cell line and 25
times more active than compound 9. The cytotoxic activity
exhibited by compounds 11 and 12 was not significant compared
with compounds 8 and 9. This last result indicates that quinolin-4-
yl group is better than quinolin-3-yl group to replace the 2-methyl
imidazo[1,2-a]pyridine-3-yl scaffold. Compounds 4 and 5 showed
poor activity than compounds with and N-aryl moiety, e.g. 5 and 8.
Next, in order to evaluate the effect of the selected compound
(1, 3, and 8) on the grown and division of asynchronous SK-LU-1
and MCF-7 cell lines (Table 3, Fig. 1) we used flow cytometry,
measuring the DNA content of each cell line after a 24 h treatment.
As a positive control we used the CDK2 inhibitor Olomoucine and as
a negative control DMSO alone at the highest quantity used in the
experiments. All compounds were used at a concentration of
approximately twice IC50 value in each cell line; except for
compound 8 in SK-LU-1 cell line. The results show the proportion of
cells emitting fluorescence proportional to the DNA content. In SK-
LU-1 cells compound 1, 3 and 8 significantly arrest cells in the G2/M
phase of the cell cycle (Fig. 1) with a significant decrease in the
proportion of cells in the G0/G1 phase in comparison with control
percent inhibition of cellular grown at 50 mM. We decided to
determine the IC50 value of compounds 1–12 in the most sensitive
cell lines MCF7 and SK-LU-1. Results are depicted in Table 1. The
most potent compounds are 1 and 2 in both cell lines. The change of
the tolyl group for a thiazolyl group reduces the biological activity
of compound 3. The bioisosteric replacement of the hydrogen
atoms in the methyl group of the tolyl moiety do not affect
significantly the biological activity of the compounds 2 and 9
comparing with 1 and 8 respectively. The substitution of the
hydrogen atoms of the methyl group in position 2 of the imi-
dazo[1,2-a]pyridine by fluorine, compound 5, produces a nine fold
decrement of potency in SK-LU-1 cells and a 0.74 fold decrement of
potency in MCF7 cells than compound 1. The replacement by
fluorine in both methyl groups, tolyl and 2-methylimidazo[1,2-
a]pyridin-3-yl moieties, produces a slight decrement in activity in
MCF7 cell lines, and a moderate decrement in SK-LU-1 cells, this
replacements cause an increase in the lipophilicity and an electron
withdrawing effect which alter the biological properties of
a molecule. Having a trifluoromethyl group in the molecule instead
of methyl group increments the metabolic stability of the molecule.
This could represent an advantage during in vivo studios.
Table 2
Inhibition of human tumor cells lines by compounds 1–12.a
Compound
% of growth inhibition
Compounds 7 and 8 which enclose a quinolin-4-yl group instead
of the imidazo[1,2-a]pyridin-3-yl scaffold, have modest cytotoxic
activity in both SK-LU-1 and MCF7 cell lines. Comparing, compound
1 is 71 times more active in SK-LU-1 cell line and 10 times more
active in MCF7 cell line than compound 8; on the other hand,
U251
PC-3
K562
HCT-15
MCF7
SK-LU-1
1
2
3
4
5
6
7
8
9
10
11
12
100.0
85.6
57.8
94.2
64.5
49.5
74.0
100.0
92.0
77.4
100.0
87.3
31.9
59.5
22.9
45.4
89.4
84.8
88.1
56.3
100.0
94.2
9.4
50.9
NA
NA
95.2
88.6
85.4
274.0
100.0
94.4
NA
100.0
100.0
NA
88.5
83.4
75.9
100.0
100.0
100.0
71.0
100.0
95.0
92.2
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
80.9
100.0
100.0
100.0
100.0
91.8
38.5
87.7
49.8
64.2
78.2
100.0
100.0
59.3
46.3
35.4
NA
NA
73.7
30.3
24.0
71.3
5-fluorouracil
65.1
70.5
Scheme 2. Reagents: (i) Bromoacetone or 3-bromo-1,1,1-trifluoroacetone (ii) triethyl-
amine; (iii) POCl3, DMF; (iv) water.
NA ¼ Not active.
a
50 mM; mean of three experiments with three replicates each one.

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M.A. Vilchis-Reyes et al. / European Journal of Medicinal Chemistry 45 (2010) 379–386
Table 3
Cell cycle analysis of SK-LU-1 and MCF7 cell lines after treatment with 1, 3, and 8. DMSO was used as vehicle control and Olomoucine, a CDK-2 inhibitor, as a positive control.
Compound
Cell cycle phase (%)a
SK-LU-1
MCF7
G1
G1
S
G2/M
S
G2/M
b
1
3
8
4.7 ꢀ 0.4b
42.5 ꢀ 0.4b
8.1 ꢀ 9.6b
61.7 ꢀ 2.9
44.2 ꢀ 2.9
20.9 ꢀ 10.7
13.9 ꢀ 4.2
22.0 ꢀ 19.3
18.2 ꢀ 1.5
19.7 ꢀ 8.6b
77.5 ꢀ 5.6
60.8 ꢀ 1.8
54.5 ꢀ 6.3
18.7 ꢀ 5.6b
57.0 ꢀ 3.7
60.6 ꢀ 3.1
25.3 ꢀ 4.3
15.7 ꢀ 4.6
15.1 ꢀ 6.7
17.0 ꢀ ꢀ 1.2
15.1 ꢀ 2.3
14.7 ꢀ 7.0
22.2 ꢀ 1.6
64.7 ꢀ 1.4b
23.2 ꢀ 0.4
23.2 ꢀ 0.4
48.0 ꢀ 8.5b
74.0 ꢀ 11.4b
16.1 ꢀ 0.9
DMSO
Olomoucine
31.8 ꢀ 3.7b
a
Results are expressed as means ꢀ SD of three independent experiments. Cells were incubated with the compounds for 24 h.
P < 0.05 as compared with solvent (DMSO) on 24 h treatment.
b
cells (DMSO, Fig. 1), S phase presents no difference with respect to
apoptotic cell death. The upper left corner of the quadrant repre-
sents debris, lower left are live cells, upper right are late apoptotic
or necrotic cells and lower right are apoptotic cells. After an incu-
bation period of 24 h with the mentioned compounds, apoptosis
was not significantly superior to control experiment. However, our
data shows that compounds 1, 3, 5 and 8 are effective apoptotic
inducers on SK-LU-1 cell line after a 48 h treatment (Fig. 2),
showing a significant increase in apoptotic death cells comparing
with DMSO treated cells. Not significant necrotic cells were found
in cells treated with molecules 1, 3, 5 and 8. If a molecule induces
death via an apoptotic pathway, then this molecule will cause fewer
toxic side reactions in vivo than if it induces death via necrosis.
It has been reported that substituted 2-aminopyrimidines are
kinase inhibitors, especially of CDK2 [10–12]. We next analyzed the
activity of compounds 1, 2, 3, 8, and 11 on recombinant CDK2/CycA
and CDK1/CycB (Table 4). We used as positive control Olomoucine, it
has been reported that the IC50 on CDK2/CycA of this compound is
7 mM [16]; for this reason we tested all compounds at the same
concentration. In general all compounds tested are better than Olo-
moucine in both CDK2/CycA and CDK1/CycB. Compound 1, 2 and 3
inhibit CDK1/CycB at the same extend at 7 mM. Interestingly,
compound with a quinolinyl moiety present better inhibitory
potency against CDK1/CycB, this observation could explain the G2/M
arrest induced in MCF7 cell line when compounds 1 and 3 failed to
exhibit this phenomena on this cell line (Fig. 1, Table 3). On CDK2/
control. Olomoucine has significant effect on S and G2/M phase of
cell cycle of SK-LU-1 at the concentration used in these experiments
(Table 3). MCF7 cell line shows a cell cycle pattern after treatment
with compound 1 and 3 which possess a 2-methylimidazo[1,2-
a]pyridine scaffold, similar to control cells, differences are statisti-
cally not significant. In contrast, compound 8 shows a strong arrest
in G2/M phase of the cell cycle after a 24 h treatment in comparison
with control cells (Table 3, Fig. 1) and a diminution in the G1/G0
phase of the cell cycle; S phase not presents significant differences
in comparison with control cells. Olomoucine has no effect on cell
cycle of MCF7 cells at the concentration used in these experiments
(Table 3). The cell enlargement characteristic of a G2/M arrest was
also evident as an enhancement in the FSC vs. SSC dot plots
generated by all the tested compounds in SK-LU-1 cells and by
compound 8 in MCF7.
Resistance to cell apoptosis is one of the mechanisms that is
important in cancer drug resistance. To determine whether G2/M
arrest was followed by induction of apoptosis, asynchronously
growing SK-LU-1 cell line was exposed to compounds 1, 3, 5 and 8
for 24 and 48 h. Cell death was monitored by flow cytometry, using
the binding of fluorescein isothiocyanate-labeled annexin V to
phosphatidylserine (Annexin V-FITC). Apoptotic cells show positive
fluorescence for annexin V-FITC; subsequently, an augment in the
green fluorescence in the FLH-1 channel indicates an increase in
Fig. 1. Cell cycle phase effect of compound 1, 3, and 8 on SK-LU-1 and MCF7 cell lines. DMSO was used as solvent control (A, SK-LU-1; E, MCF7). Olomoucine was used as a positive
control (not shown). Treatment with 0.04 M compound 1 (B), 8.4 M compound 3 (C), 1 M compound 8 (D), and 70 M Olomoucine were utilized on SK-LU-1 cells. 0.118
compound 1 (F), 8.4 M compound 3 (G), 1 M compound 8 (H), and 60 M Olomoucine were used on MCF7 cells. Representative experiment out of three is shown.
m
m
m
m
mM
m
m
m

M.A. Vilchis-Reyes et al. / European Journal of Medicinal Chemistry 45 (2010) 379–386
383
Fig. 2. Induction of apoptosis on SK-LU-1 after treatment with 0.04
m
M compound 1, 8.4
mM compound 3, 0.4
mM compound 5 and 1
m
M compound 8 for 48 h, DMSO was used as
a negative control (A). Detection of apoptosis through annexin V and PI staining after treatment with 1 (B), 3 (C), 5 (D), and 8 (E) on SK-LU-1 cells. Representative experiment out of
three is shown.
CycAmostactive compoundsare 2 and3, albeitcompound3 does not
exhibit the highest cytotoxic value compared with 1 and 2. It is
important to note that compound 11 was never significantly cyto-
toxic;however it shows inhibition on CDKs similarto 8 in both CDK1/
CycB and CDK2/CycA (Table 1, Table 4). These differences in cytotoxic
and CDKs inhibitory activity may be reflecting variations in cellular
permeability, in intracellular distribution, and in metabolism.
In conclusion we have synthesized a series of 2-methyl-
imidazo[1,2-a]pyridine- and quinoline-substituted 2-amino-
pyrimidines which inhibit CDK1/CycB and CDK2/CycA. Among
4.1. Chemistry
4.1.1. General method of synthesis of compounds 13 and 14
2-Aminopyridine is dissolved in a 3:1 mixture of ethanol–
dioxane and 1.1 equivalents of the appropriated bromoacetone are
added slowly. The reaction mixture is vigorously mixed during 2 h,
a solid is formed. Then three equivalents of triethylamine are added
and the mixture is refluxed overnight. The volatiles are removed
under vacuum and water is added to dissolve the formed salts.
the compounds containing
a
2-methylimidazo[1,2-a]pyridine,
4.1.1.1. 2-Methylimidazo[1,2-a]pyridine (13). The aqueous suspen-
sion is extracted with AcOEt. The organic phase is dried with
anhydrous Na2SO4 and the solvent is evaporated in a Rotavapor
evaporator. The oily residue is distillated under vacuum to give the
title compound as yellow pale oil which solidifies and is stable at
4 ꢁC. Yield 50%. B. p. 108–110 ꢁC/6 mmHg. 1H NMR (CDCl3,
hydrogen replacement by fluorine does not affect notably the
cytotoxic activity. Quinoline-substituted compound, 8 and 11, are
most potent and selective against CDK1/CycB than against CDK2/
CycA. Quinolin-4-yl-substituted compounds constitute
a new
family of cytotoxic and CDK inhibitor compounds from which more
potent and selective inhibitors can be designed. The compounds
synthesized broaden the knowledge of the activity of these kinds of
derivatives.
200 MHz)
d: 2.46 (d, 3H, J ¼ 0.8 Hz), 6.71 (td, 1H, J ¼ 1.2, 6.6 Hz), 7.10
(ddd, 1H, J ¼ 1.2, 5.6, 9.0 Hz), 7.33 (s, 1H), 7.50 (dd, J ¼ 0.8, 9.0 Hz),
8.02 (dt, 1H, J ¼ 1.2, 6.8 Hz). C8H8N2 calculated m. w.: 132.16. MS
[EIþ] m/z: 132 [M]þ (100), 131 [M ꢂ H]þ (80).
4. Experimental protocols
4.1.1.2. 2-(Trifluoromethyl)imidazo[1,2-a]pyridine (14). A solid is
formed after adding water which is separated by suction. The solid
is purified by column chromatography with EtOAc–Hexane 1:1.
Recrystallization from hexane yields 75% of a white solid. M. p. 91–
Melting points were determined on a MEL-TEMPÒ capillary
melting point apparatus and are uncorrected. 1H NMR spectra were
recorded on a Varian Gemini 200 MHz, Varian Unity 300 MHz, and
Varian Inova 500 MHz (1H NMR and 125 MHz 13C NMR). Chemical
92 ꢁC. 1H NMR (CDCl3, 300 MHz)
d
: 6.92 (td, 1H, J ¼ 1.2, 6.9 Hz),
7.31 (ddd, 1H, J ¼ 1.2, 6.9, 9.3 Hz), 7.89 (s, 1H), 8.15 (td, 1H, J ¼ 1.2,
shifts are expressed as
d
values relative to TMS as internal standard,
6.9 Hz). 13C NMR (CDCl3, 75.5 MHz),
d: 111.46, 113.96, 118.70,
J values are given in Hz; spectra were recorded in CDCl3, or
a mixture of CDCl3 and DMSO-d6 when problems of solubility were
presented (compounds 2, 3, 7, 10 and 12). Mass spectra were
recorded on a JEOL JMS-SX 10217 spectrometer by electron impact
(EI). All reagents were acquired from Sigma-Aldrich unless other-
wise stated. The solvents were distilled before being used. Tetra-
hydrofuran (THF) and toluene were dried with sodium using
benzophenone as indicator. Bromoacetone was obtained from
bromide and acetone and distilled under vacuum prior to use [17].
Silica gel (mesh 230–400) was purchased from Merck.
Table 4
Percent of inhibition of human CDK/Cyc enzymes by compounds 1, 2, 3, 8 and 11.a
CDK/Cyc
Compound
Olomoucine
1
2
3
8
11
CDK1/CycB
CDK2/CycA
48
43
59
58
60
69
52
76
75
45
73
49
a
7 mM; mean of two independent experiments.

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M.A. Vilchis-Reyes et al. / European Journal of Medicinal Chemistry 45 (2010) 379–386
122.00 (q, J ¼ 267.5 Hz), 126.25, 126.52, 135.96 (q, J ¼ 38 Hz), 145.4.
C8H5F3N2 calculated m. w.: 186.13 MS [EIþ] m/z: 186 [M]þ (100),
167 [M ꢂ F]þ (15).
d
: 7.00 (d, 1H, J ¼ 5.4 Hz), 7.02 (dd, 1H, J ¼ 1.2, 9.2 Hz), 7.44 (td, 1H,
J ¼ 1.4, 9.2 Hz), 7.78 (dd, 1H, J ¼ 1.2, 9.2 Hz), 8.40 (d, 1H, J ¼ 5.4 Hz),
9.20 (dd, 1H, J ¼ 1.2, 7.2 Hz). C12H8F3N5 calculated m. w.: 279.22 MS
[EIþ] m/z: 279 [M]þ (100), 278 [M ꢂ H]þ (55), 258 [M ꢂ H2F]þ (20).
4.1.2. General method of synthesis of compounds 15 and 16
Over excess of anhydrous DMF at 0 ꢁC are added slowly 2.5
equivalents of POCl3 and the mixture is mixed during 15 min.
Posterior, one equivalent of compound 13 or 14 is added and the
reaction mixture is stirred vigorously by 1 h. Then the reaction is
warmed at 60 ꢁC by 24 h or until no starting material is detected by
TLC. Chopped ice was added and the mixture is neutralized with
ammonium hydroxide.
4.1.3.2. 4-(Quinolin-4-yl)pyrimidin-2-amine (7). Purified by column
chromatography with EtOAc–Hexane 9:1 to obtain 10% of the title
compound. Yellow solid, m. p. 228–229 ꢁC (desc.) 1H NMR
(CDCl3 þ DMSO-d6, 500 MHz)
d: 6.30 (s, 2H); 6.78 (d, 1H,
J ¼ 5.0 Hz); 7.45 (d,1H, J ¼ 4.5 Hz); 7.50 (ddd, 1H, J ¼ 1.5, 6.5, 8.5 Hz),
7.68 (ddd, 1H, J ¼ 1.4, 6.8 Hz, 8.6 Hz); 8.04 (dd, 1H, J ¼ 0.5 Hz,
8.0 Hz); 8.16 (dd, 1H, J ¼ 1.0, 8.5 Hz); 8.35 (d, 1H, J ¼ 5.0 Hz), 8.90 (d,
1H, J ¼ 4.5 Hz). 13C NMR (CDCl3 þ DMSO-d6, 125.7 MHz)
d: 110.35,
4.1.2.1. 2-Methylimidazo[1,2-a]pyridine-3-carbaldehyde
(15). Oil
120.22, 124.64, 125.17, 126.45, 128.92, 129.14, 143.91, 148.05, 149.37,
158.45, 163.15, 164.06. C13H10N4 calculated m. w.: 222.25 MS [EIþ]
m/z: 222 [M]þ (70), 221 [M ꢂ H]þ (100).
was obtained which was solubilized in hot hexane, after cooling
a solid was formed. Recrystallized from hexane to obtain a white
solid. Yield 44% m. p. 119–120 ꢁC. 1H NMR (CDCl3, 300 MHz)
d: 2.73
(s, 3H), 7.07 (td, 1H, J ¼ 1.2, 6.8 Hz), 7.52 (ddd, 1H, J ¼ 1.2, 6.8,
4.1.3.3. 4-(Quinolin-3-yl)pyrimidin-2-amine (10). Title compound
was purified by column chromatography using EtOAc–Hexane 8:2
to yield 20% of a yellow solid, M. p. 252–253 ꢁC (desc.), 1H NMR
9.0 Hz), 7.68 (td, 1H, J ¼ 1.2, 9.0 Hz), 10.01 (s, 1H). 13C NMR (CDCl3,
75.5 MHz) d: 114.46, 114.83, 116.63, 121.19, 128.31, 130.01, 147.65,
157.08, 176.96. C9H8N2O calculated m. w.: 160.17 MS [EIþ] m/z: 160
(CDCl3 þ DMSO-d6, 200 MHz)
d: 5.64 (ws, 2H), 7.21 (d, 1H,
[M]þ (100), 159 [M ꢂ H]þ (98), 131 [M ꢂ CHO]þ (25).
J ¼ 5.2 Hz), 7.62 (td, 1H, J ¼ 1.2, 7.0 Hz), 7.79 (td, 1H, J ¼ 1.2, 6.8 Hz),
7.97 (dd, 1H, J ¼ 0.6, 8.2 Hz), 8.14 (d, 1H, J ¼ 8.4 Hz), 8.43 (d, 1H,
J ¼ 5.2 Hz, H-8), 8.81 (d, 1H, J ¼ 1.8 Hz), 9.52 (d, 1H, J ¼ 2.4 Hz).
C13H10N4 calculated m. w.: 222.25 MS [EIþ] m/z: 222 [M]þ (85), 221
[M ꢂ H]þ (100).
4.1.2.2. 2-(Trifluoromethyl)imidazo[1,2-a]pyridine-3-carbaldehyde
(16). Purified by column chromatography (EtOAc–Hexane 1:9) and
recrystallized from hexane to obtain 52% yield of white plaques. M.
p.116–118 ꢁC. 1H NMR (CDCl3, 200 MHz) 7.28 (td, 1H, J ¼ 1.2, 7.0 Hz),
7.68 (ddd, 1H, J ¼ 1.2, 6.8, 9.0 Hz), 7.90 (dt, 1H, J ¼ 1.2, 9.0 Hz), 9.65
4.1.3.4. 4-(2-Methylimidazo[1,2-a]pyridin-3-yl)pyrimidin-2-amine
(27). Purified by column chromatography using EtOAc–acetone 1:1
to yield 75% of the yellow solid. M. p. 167 ꢁC, 1H NMR (CDCl3,
(dt, 1H, J ¼ 1.2, 7 Hz), 10.22 (s, 1H). 13C (CDCl3, 75.5 MHz)
d: 114.46,
114.83, 116.63,122.00 (q, J ¼ 267.5 Hz),128.31,130.01,147.65,157.08,
176.96. C9H5F3N2O calculated m. w.: 214.14 MS [EIþ] m/z: 214 [M]þ
(100), 195 [M ꢂ F]þ (75), 185 [M ꢂ CHO]þ (20).
300 MHz)
d
: 3.10 (s, 3H), 6.87 (d, 1H, J ¼ 5.4 Hz), 6.88 (td, 1H, J ¼ 1.2,
6.9 Hz), 7.30 (ddd, 1H, J ¼ 1.2, 6.6, 9.0 Hz), 7.61 (dt, 1H, J ¼ 1.2,
9.0 Hz), 9.62 (dt, 1H, J ¼ 1.2, 9.0 Hz). 13C NMR (CDCl3, 75.5 MHz)
d:
4.1.3. General method of synthesis of compounds 4, 7, 10 and 27
The corresponding aldehyde 15, 16, 17 or 18 was reacted with 2
equivalents of ethynyl magnesium bromide 0.5 M in anhydrous THF
at ꢂ70 ꢁC under nitrogen atmosphere. The mixture was stirred by
30 min and allowed to reach room temperature. A saturated NH4Cl
solution was added and the aqueous layer was separated and
extracted with THF; this reaction is quantitative.
The propargylic alcohol derivatives obtained 19, 21 and 22 were
oxidized with 35 equivalents of MnO2 in reducing agents free
acetone mixing for 30 min. After the completion of reaction, the
MnO2 was separated by suction filtration and washed with acetone.
16.76, 108.95, 112.58, 116.64, 126.26, 127.85, 128.62, 146.22, 147.07,
158.01, 158.21, 162.62. C12H11N5 calculated m. w.: 225.25 MS [EIþ]
m/z: 225 [M]þ (85), 224 [M ꢂ H]þ (100).
4.1.4. General procedure to synthesize compounds 1, 2, 3, 5, 6, 8, 9,
11, and 12
In an well oven-dried Schlenk flask were added 1 equivalent of
4, 7, 10 or 27, 1.1 equivalents of the proper aryl halide, 3–5% Xant-
phos, 3–5% Pd(dba)2 and 3 equivalents of anhydrous Cs2CO3. The
flask was evacuated/backfilled with nitrogen at least three times
and anhydrous toluene was added into the flask, the flask was
evacuated/backfilled for others two times. The reaction mixture
was warmed at 80 ꢁC until the starting materials were completely
consumed or the reaction did not proceed anymore (4–12 h). Then,
the reaction was allowed to get room temperature and the solids
were removed by vacuum filtration and rinsed plenty with EtOAc.
All compounds were purified by column chromatography.
Concentration of solvent in
a Rotavapor evaporator yielded
compounds 23, 25 or 26. Compound 20 was oxidized employing 5
equivalents of Ca(ClO)2 in CH2Cl2–water and using 7% of tetrabu-
tylammonium hydrogen sulfate as phase-transfer catalyst, the
mixture was stirred vigorously and heated to reflux for one hour,
the reaction mixture is diluted with CH2Cl2 and extracted with
brine. Concentration of the organic layer under vacuum afforded
compound 24. The alkynyl ketones 23–26 were used in the
following reaction without further purification.
Compounds 23–26 were reacted with 2 equivalents of guani-
dine sulfate in presence of 3 equivalents of K2CO3 in n-butanol,
reactions were heated in an oil bath to reflux overnight or until the
reaction was over by TLC. All volatiles were removed under vacuum
and the solid form was extracted with acetone. The suspended solid
was removed by filtration and discarded. The solvent was removed
under vacuum, and the resulting solid was purified. Following this
procedure the following compounds were obtained.
4.1.4.1. 4-(2-Methylimidazo[1,2-a]pyridin-3-yl)-N-p-tolylpyrimidin-
2-amine (1). Eluted with EtOAc to obtain 80% of a pale green solid.
M. p. 190 ꢁC (desc.); 1H NMR (CDCl3, 200 MHz)
d: 2.36 (s, 3H), 2.74
(s, 3H), 6.78 (td, 1H, J ¼ 1.4, 7.0 Hz), 6.95 (d, 1H, J ¼ 5.4 Hz), 7.17 (d,
2H, J ¼ 8.2 Hz), 7.29 (ddd, 1H, J ¼ 1.2, 6.8, 9.0 Hz), 7.49 (d, 2H,
J ¼ 8.4 Hz), 7.61 (dt, 1H, J ¼ 1.2, 9.0 Hz), 8.44 (d, 1 H, J ¼ 5.6 Hz), 9.59
(dt, 1H, J ¼ 1.2, 7.0 Hz); 13C NMR (CDCl3, 75.5 MHz)
d: 17.07, 20.85,
109.30,112.55,116.66,120.92,126.38,128.28,129.44,132.85,136.64,
146.26, 147.34, 157.62, 157.91, 160.08; C19H17N5 calculated m. w.:
315.37 MS [EIþ] m/z: 315 [M]þ (95), 314 [M ꢂ H]þ (100).
4.1.3.1. 4-(2-(Trifluoromethyl)imidazo[1,2-a]pyridin-3-yl)pyrimidin-
2-amine (4). Purified by column chromatography with EtOAc. Yield
29% of a yellow solid. M. p. 230–231 ꢁC; 1H NMR (CDCl3, 200 MHz)
4.1.4.2. 4-(2-Methylimidazo[1,2-a]pyridin-3-yl)-N-(4-(trifluoromethyl)-
phenyl)-pyrimidin-2-amine (2). Eluted with EtOAc to obtain 95% of
the title compound as a yellow powder. M. p. 201–202 ꢁC (desc.); 1H

M.A. Vilchis-Reyes et al. / European Journal of Medicinal Chemistry 45 (2010) 379–386
385
NMR (CDCl3, 200 MHz)
d
: 2.76 (s, 3H), 6.87 (td, 1H, J ¼ 1.2, 6.8 Hz),
(q, J ¼ 3.8 Hz), 127.40, 129.77, 130.21, 142.40, 143.78, 148.95, 158.78,
159.71, 165.21. C20H13F3N4 calculated m. w.: 366.34 MS [EIþ] m/z:
366 [M]þ (80), 365 [M ꢂ H]þ (100).
7.06 (d, 1H, J ¼ 5.4 Hz), 7.35 (ddd, 1H, J ¼ 1.4, 7.0, 9 Hz), 7.38 (ws,1H),
7.59 (d, 2H, J ¼ 8.2 Hz), 7.65 (dt, 1H, J ¼ 1.2, 9.0 Hz), 7.79 (d, 2H,
J ¼ 8.4 Hz), 8.52 (d, 1H, J ¼ 5.4 Hz), 9.55 (dt, 1H, J ¼ 1.2, 7.0 Hz), 13C
NMR (CDCl3 þ DMSO-d6, 75.5 MHz)
d
: 16.43, 109.64, 112.23, 116.05,
4.1.4.8. 4-(Quinolin-3-yl)-N-p-tolylpyrimidin-2-amine (11). Eluted
with EtOAc–Hexane 1:1 and recrystallized from acetonitrile to
obtain 83% of a yellow solid. M.p. 209–210 ꢁC; 1H NMR (CDCl3,
118.37, 119.85 (q, J ¼ 265.76 Hz), 125.31 (q, J ¼ 4.53 Hz), 126.09,
127.72, 143.03, 145.70, 146.73, 156.94, 157.28, 159.27. C19H14F3N5
calculated m. w.: 369.34 MS [EIþ] m/z: 369 [M]þ (100), 368
[M ꢂ H]þ (92).
200 MHz)
d
: 2.36 (s, 3H), 7.29 (d, 2H, J ¼ 8.4 Hz), 7.28 (d, 1H,
J ¼ 5.0 Hz), 7.33 (ws, 1H), 7.60 (d, 2H, J ¼ 8.6 Hz), 7.62 (td, 1H, J ¼ 1.2,
8.0 Hz), 7.79 (ddd, 1H, J ¼ 1.4, 7.0, 8.4 Hz), 7.95 (dd, 1H, J ¼ 1.2,
8.2 Hz), 8.17 (d, 1H, J ¼ 8.2), 8.53 (d, 1H, J ¼ 5.2 Hz), 8.81 (d, 1H,
4.1.4.3. 5-(4-(2-Methylimidazo[1,2-a]pyridin-3-yl)pyrimidin-2-yl-
amino)thiophene-2-carbaldehyde (3). Eluted with EtOAc–Hexane
6:4 obtaining 65% of dark green flakes. M. p. 235 ꢁC (desc.); 1H NMR
J ¼ 1.8 Hz), 9.61 (d, 1H, J ¼ 2.2 Hz); 13C (CDCl3, 75.5 MHz)
d: 20.83,
108.15, 119.82, 127.26, 127.48, 128.74, 128.99, 129.37, 129.49, 129.74,
130.76, 132.48, 134.73, 136.68, 149.04, 159.02, 160.52, 162.50;
C20H16N4 calculated m. w.: 312.37 MS [EIþ] m/z: 312 [M]þ (85), 311
[M ꢂ H]þ (100).
(CDCl3 þ DMSO-d6, 200 MHz) : 2.68 (s, 3H), 6.83 (d, 1H, J ¼ 4.2 Hz),
d
6.92 (td, 1H, J ¼ 1.2, 6.8 Hz), 7.02 (d, 1H, J ¼ 5.4 Hz), 7.33 (ddd, 1H,
J ¼ 1.0 Hz, J ¼ 6.8 Hz, J ¼ 8.8 Hz), 7.55 (d, 1H, J ¼ 4.2 Hz), 7.57 (d, 1H,
J ¼ 8.8 Hz), 8.53 (d,1H, J ¼ 5.4 Hz), 9.65 (s,1H), 9.67 (d,1H, J ¼ 8.6 Hz),
11.05 (s, 1H); 13C (CDCl3 þ DMSO-d6, 75.5 MHz)
d: 16.18, 110.22,
4.1.4.9. 4-(Quinolin-3-yl)-N-(4-(trifluoromethyl)phenyl)pyrimidin-2-
110.93, 112.48, 115.80, 117.62, 126.62, 127.97, 131.70, 136.61, 145.51,
146.54, 152.01, 156.74, 156.86, 181.89. C17H13N5OS calculated m. w.:
335.38 MS [EIþ] m/z: 335 [M]þ (100), 209 [M ꢂ C5H4ONS]þ (18).
amine (12). Eluted with EtOAc–Hexane 1:1 to obtain 91% of a white
solid. M. p. 258 ꢁC (desc.) 1H NMR (CDCl3 þ DMSO-d6, 200 MHz)
d:
7.44 (d, 1H, J ¼ 5.4 Hz), 7.59 (d, 2H, J ¼ 8.6 Hz), 7.66 (ddd, 1H, J ¼ 1.2,
6.8, 8.0 Hz), 7.83 (ddd, 1H, J ¼ 1.4, 6.8, 8.4 Hz), 8.02 (d, 2H,
J ¼ 8.6 Hz), 8.02 (d, 1H, J ¼ 8.4 Hz), 8.16 (d, 1H, J ¼ 8.6 Hz), 8.62 (d,
1H, J ¼ 5.2 Hz), 8.87 (d, 1H, J ¼ 2.2 Hz), 9.31 (ws, 1H), 9.64 (d, 1H,
J ¼ 2.2 Hz). C20H13F3N4 calculated m. w.: 366.34 MS [EIþ] m/z: 366
[M]þ$ (5), 365 [M ꢂ H]þ (10), 263 [M ꢂ C7H5N]þ(100).
4.1.4.4. N-p-Tolyl-4-(2-(trifluoromethyl)imidazo[1,2-a]pyridin-3-yl)-
pyrimidin-2-amine (5). Eluted with EtOAc to yield 83% of a yellow
solid. M. p. 133–134 ꢁC; 1H NMR (CDCl3, 200 MHz)
d: 2.34 (s, 3H),
6.94 (td, 1H, J ¼ 7.0, 1.4 Hz) 7.11 (dq, 1H, J ¼ 5.2, 1.0 Hz), 7.15 (d, 2H,
J ¼ 8.4 Hz), 7.08 (d, 1H), 7.42 (ddd, 1H, J ¼ 1.4, 6.4, 8.2 Hz), 7.49 (d,
2H, J ¼ 8.4 Hz), 7.78 (ddd, 1H, J ¼ 1.0, 6.2, 8.2 Hz), 8.54 (d,1H,
J ¼ 5.2 Hz), 9.21 (dd, 1H, J ¼ 1.2, 7.2 Hz). C19H14F3N5 calculated m.
w.: 369.34 MS [EIþ] m/z: 329 [M]þ (75), 328 [M ꢂ H]þ (100).
4.2. Biology
4.2.1. Cell culture and assay for cytotoxic activity
The cytotoxic activity of 1–12 was assayed by the protein-
binding method of sulphorhodamine B (SRB). Cell lines were
cultured in RPMI-1640 medium, supplemented with 10% FCS, 2 mM
4.1.4.5. 4-(2-(Trifluoromethyl)imidazo[1,2-a]pyridin-3-yl)-N-(4-
(trifluoromethyl)phenyl)-pyrimidin-2-amine (6). Eluted with EtOAc–
Hexane 9:1 to obtain 92% of a white solid. M. p. 217–218 ꢁC; 1H NMR
L
-glutamine, 100 IU mLꢂ1 penicillin G, 100 g mLꢂ1 streptomycin
m
(CDCl3, 500 MHz)
d
: 6.98 (td, 1H, J ¼ 1.5, 7.0 Hz), 7.22 (dd, 1H, J ¼ 1.0,
sulfate, and 0.25 m
g mLꢂ1 amphotericin B. The cell line cultures
5.0 Hz), 7.46 (ddd, 1H, J ¼ 1.2, 6.5, 9.0 Hz), 7.48 (ws, 1H), 7.58 (d, 2H,
J ¼ 8.5), 7.77 (d, 2H, J ¼ 8.5 Hz), 7.81 (dt,1H, J ¼ 1.0, 9 Hz), 8.63 (d,1H,
J ¼ 5.5 Hz), 9.14 (dt, 1H, J ¼ 1.5, 7.5 Hz); 13C NMR (CDCl3, 125.7 MHz)
were maintained in a 5% CO2, 95% humidity atmosphere at 37 ꢁC. In
a 96-well plate were seeded 5–10 ꢃ 103 cells/well and incubated for
24 h. Then the cultured cell lines were grown in the presence of the
putative cytotoxic compound for an additional 48 h. Test
substances were dissolved in DMSO to create a stock solution,
fluorouracil was used as a positive control. DMSO was added to
control wells at the highest concentration used and no effect in cell
grown was observed.
d: 113.63,114.62,118.64,118.78,118.89,126.32,126.94,127.91,142.19,
145.56, 155.40, 159.28, 159.37. C19H11F6N5 calculated m. w.: 423.31
MS [EIþ] m/z: 423 [M]þ (85), 422 [M ꢂ H]þ (100).
4.1.4.6. 4-(Quinolin-4-yl)-N-p-tolylpyrimidin-2-amine (8). Eluted
with EtOAc–Hexane 1:1 and recristallized from acetonitrile to
obtain 75% of a pale yellow solid. M. p. 205–207 ꢁC (desc.) 1H
After the incubation with test compounds was over, adherent
cell cultures were fixed in situ by adding 50 mL of cold 50% (wt/vol)
trichloroacetic acid and incubated at 4 ꢁC for 1 h. The supernatant
was discarded and the plates were washed with water and left dry
NMR (CDCl3, 200 MHz)
d
: 2.32 (s, 3H), 7.00 (d, 1H, J ¼ 5.1 Hz); 7.13
(d, 1H, J ¼ 8.4 Hz), 7.36 (ws, 1H,), 7.53 (d, 1H, J ¼ 4.2 Hz), 7.54 (d,
2H, J ¼ 8.4 Hz), 7.58 (ddd, 1H, J ¼ 1.2, 6.8, 8.4 Hz); 7.78 (ddd, 1H,
J ¼ 1.4, 6.8, 8.4 Hz); 7.8 (d, 2H, J ¼ 8.0 Hz), 8.23 (dd, 1H, J ¼ 0.8,
8.6 Hz); 8.24 (dd, 1H, J ¼ 1.0, 8.4 Hz); 8.58 (d, 1H, J ¼ 5.0 Hz); 9.02
to the air. The fixed cells were stained with 100 mL of 0.4% SRB
solution. Protein-bonded dye was solubilized with 10 mM unbuf-
fered Tris base and the optical density was read on a microplate
reader (Elx 808; Bio-Tek Instruments, Inc., Winooski, VT, USA) with
a test wavelength of 515 nm. A preliminary screening was made at
(d, 1H, J ¼ 4.4 Hz). 13C NMR (CDCl3, 200 MHz)
d: 20.79, 112.67,
119.66, 120.89, 125.35, 127.25, 129.49, 129.66, 130.06, 132.56,
136.51, 144.17, 148.86, 149.99, 158.73, 160.28, 164.97. C20H16N4
calculated m. w.: 312.37 MS [EIþ] m/z: 312 [M]þ (95), 311
[M ꢂ H]þ (100).
50 mM and the IC50 was determined in the most sensible cell lines. A
dose-response curve was plotted for each compound, and the IC50
was estimated from non-linear regression using JMP software
(version 3.2.1.; SAS Institute Inc. Cary, NC, USA).
4.1.4.7. 4-(Quinolin-4-yl)-N-(4-(trifluoromethyl)phenyl)pyrimidin-2-
amine (9). Eluted with EtOAc–Hexane 1:1 to obtain the title
compound (96%) as a white solid. M. p. 187–188 ꢁC, 1H NMR (CDCl3,
4.2.2. Cell cycle analysis (Flow cytometry)
MCF7 and SK-LU-1 cells were subcultured to a final density of
1 ꢃ106 cells in 100 ꢃ 20 mm culture Petri dishes. After a 24 h
period of regular culture conditions, cell were cultured in the
500 MHz)
d
: 7.13 (d, 1 H, J ¼ 5.5 Hz), 7.55 (d, 1H, J ¼ 4 Hz), 7.56 (d,
2H, J ¼ 9.0 Hz), 7.59 (ddd, 1H, J ¼ 1.2, 6.5, 8.5 Hz), 7.70 (ws, 1H), 7.79
(ddd, 1H, J ¼ 1.2, 7.0, 8.5 Hz), 7.81 (d, 2H, J ¼ 8.5 Hz), 8.23 (dd, 1H,
J ¼ 1.0, 7.5 Hz), 8.24 (dd, 1H, J ¼ 1.0, 8.0 Hz), 8.66 (d, 1H, J ¼ 5.0 Hz),
presence of compounds at approximately 2 times the IC50 (0.04
m
M
compound 1, 8.4
utilized for SK-LU-1 cells and 0.118
compound 3 and 1
m
M compound 2 and 1
m
M compound 8 were
9.05 (d, 1H, J ¼ 4.5 Hz); 13C NMR (CDCl3, 125.7 MHz)
d: 113.83,
mM compound 1, 8.4 m
M
118.41, 120.92, 124.27 (q, J ¼ 33.18), 124.33 (q, J ¼ 270.6 Hz), 126.24
mM compound 10 were used with MCF7 cells)

386
M.A. Vilchis-Reyes et al. / European Journal of Medicinal Chemistry 45 (2010) 379–386
for 24 h. After these treatment periods, cells were harvested and
centrifuged for five minutes at 3000 rpm. The pellet was resus-
pended in PBS (pH ¼ 7.4) and centrifuged again for five minutes at
3000 rpm. Cells were fixed with 70% ethanol at 4 ꢁC for at least 12 h.
Ethanol was eliminated by centrifugation and the pellet was
5 mL of b-mercaptoethanol were added to the samples to stop
the reaction. Samples were boiled 3 min in a screw-cap micro-
centrifuge tube to reduce the risk of vaporizing radioisotope. The
samples were run on a 10% polyacrylamide gel. The amount of
incorporated label was measured using a densitometry of the
exposed film obtained with a Typhoon 9400 apparatus (Amersham
Biosciences, Inc.) by using Kodak MI software (Carestream Health,
Rochester, N.Y.).
washed with PBS. DNA was labeled with 5 m
g mLꢂ1 propidium
iodide (PI) solution. Cell cycle analysis was made using a FACScan
cytometer (BD Biosciences, San Jose, CA) and CELLQuest software
(BD Biosciences). The cell cycle profile was obtained by analyzing
10,000 events using the FlowJo software Version 7.2.5 (Tree Star
Inc, Ashland OR, USA).
Acknowledgements
We thank CONACyT for a doctoral grant to M. A. Vilchis-Reyes,
and we gratefully acknowledge the helpful assistance of the spec-
4.2.3. Annexin V-FITC apoptosis assay
Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich) was
used to measure apoptosis. The kit contains annexin V labeled with
a fluorophore that can identify apoptotic cells by binding phos-
phatidylserine exposed on the cytoplasmic surface of the cell
membrane of apoptotic cells. In addition, the kit includes a red
fluorescent propidium iodide (PI) nucleic acid binding dye that
stains dead cells. Briefly, 2.5 ꢃ105 SK-LU-1 cells in 2 mL medium
were plated to each well of the six-well plate. After 24 h, cells were
treated with either DMSO alone at the highest concentration used
´
troscopic staff of Instituto de Quımica (UNAM). The authors
´
acknowledge Prof. Jose Solano Becerra, M. Sc. Carlos Villarreal and
M. Sc. Noemi Baranda for the revision of the original manuscript.
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as control or with 0.04
mM compound 1, 8.4
mM compound 3,
0.4 M compound 5 and 1
m
mM compound 8 for 48 h. At the end of
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the treatment, adherent and nonadherent cells were harvested and
washed twice with PBS and then resuspended with 0.5 mL 1ꢃ
annexin-binding buffer. Cells were incubated in the dark with 5 mL
of annexin V and 10 mL of the PI solution for 10 minutes at room
temperature. After incubation, the samples were analyzed with
FACScan flow cytometry (BD Biosciences, San Jose, CA) using Cell-
Quest Software (BD Biosciences). The percentage of apoptotic cells
in the cell samples was obtained by analyzing 10,000 events using
the FlowJo software Version 7.2.5 (Tree Star Inc, Ashland OR, USA).
4.2.4. CDK inhibition measurement
CDK/Cyclin activity was assayed by a protein kinase test using
histone H1.2 as substrate. 220 ng of recombinant CDK1/CycB
(Biaffin, Kassel, Germany) or 135 ng of recombinant Cdk2/CycA
(Biaffin, Kassel, Germany) in 20
mL of kinase reaction buffer
(pH ¼ 7.4) Tris-HCl 50 mM, MgCl2 10 mM, NaF 50 mM, Na3VO4
32
500
mM, b-glicerofosfate 8 mM, DTT 1 mM, ATP 400 m
M and ATPg
4
m
Ci. (Perkin-Elmer, Wellesley, MA) is mixed with 0.5 m
L 10 mg/ml
recombinant Histone H1.2. (Calbiochem, San Diego, CA) and incu-
bated 30 minutes at 30 ꢁC. The CDK/Cyclin complexes were
inhibited at 7 mM by compound 1, 2, 3, 8, and 11. 7 mM Olomoucine
was used as a positive control, vehicle solvent (DMSO) was used as
a negative control at a 2.5% concentration or less.
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