6-Substituted 2-(N-trifluoroacetylamino)imidazopyridines induce cell cycle arrest and apoptosis in SK-LU-1 human cancer cell line

Article abstract of DOI:10.1016/j.ejmech.2009.11.049

A series of 6-substituted 2-(N-trifluoroacetylamino)imidazopyridines have been synthesized and their bioactivities were evaluated. Compounds 6a, 6c, and 11a were the most active compounds with modest cytotoxic activity against six human cancer cell lines U251 (glioma), PC-3 (prostate), K-562 (leukemia), HCT-15 (colon), MCF7 (breast) and SK-LU-1 (lung). The cell cycle analysis showed that compounds 6a, 6c, and 11a induce a G2/M phase cell cycle arrest on SK-LU-1 cell line where inhibition of CDK-1 and CDK-2 may be implicated.

Full text of DOI:10.1016/j.ejmech.2009.11.049

European Journal of Medicinal Chemistry 45 (2010) 1211–1219
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
6-Substituted 2-(N-trifluoroacetylamino)imidazopyridines induce cell cycle arrest
and apoptosis in SK-LU-1 human cancer cell line
a
,
b
a
a
*
´
´
´
´
Miguel Angel Martınez-Urbina , Alejandro Zentella , Miguel Angel Vilchis-Reyes , 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 6-substituted 2-(N-trifluoroacetylamino)imidazopyridines have been synthesized and their
bioactivities were evaluated. Compounds 6a, 6c, and 11a were the most active compounds with modest
cytotoxic activityagainstsix humancancercelllines U251 (glioma), PC-3(prostate), K-562(leukemia), HCT-15
(colon), MCF7 (breast) and SK-LU-1 (lung). The cell cycle analysis showed that compounds 6a, 6c, and 11a
induce a G2/M phase cell cycle arrest on SK-LU-1 cell line where inhibition of CDK-1 and CDK-2 may be
implicated.
Received 9 March 2009
Received in revised form
19 November 2009
Accepted 23 November 2009
Available online 11 December 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Imidazo[1,2-a]pyridine derivatives
Cell cycle
CDKs
Apoptosis
1. Introduction
anxioselective activities [7]. They work as a-amyloid formation
inhibitors [8] and constitute a novel class of orally active nonpeptide
bradykinin B2 receptor antagonists [9]. Recently [10] it was described
that 2-aminoimidazo[1,2-a]-pyridine scaffold represents a novel
structuralclassofproteinserine/threoninekinaseinhibitorsandthese
kinds of compounds affect potently the cyclin-dependent kinases by
competing with ATP for binding to a catalytic subunit of the protein.
In addition, Jaramillo et al. [11] disclose a detailed SAR study of 2-
aminoimidazo[1,2-a]pyridines around the role of substituent at 3-
position, and with respect the optimal spacer between phenyl and
imidazopyridine rings, in this study the presence of an sp2 carbon
was shown to be necessary in order to keep activity. Carbonyl, vinyl,
or Z-configurated cyanovinyl substituents, were also efficient;
nevertheless activity was lost partially when either E-configurated
olefins or bulkier substituents such as tetrazole were introduced.
Because most of the actual SAR reports over this subject have
been made on substituents 2 and 3 of imidazopyridine ring as well
as the spacer between the aromatic bicyclic moiety, it appears to
be interesting to determine the effect of removing the substituent
at 3-position, and the effect of other groups such as heterocyclic
compounds at 6-position of the imidazo[1,2-a]pyridine ring. Herein
we described the synthesis of a series of 6-substituted 2-(N-tri-
fluoroacetylamino)imidazopyridines compounds 6a–e and 11a–c,
which displayed cytotoxicity against six human cancer cell lines
U251 (glioma), PC-3 (prostate), K-562 (leukemia), HCT-15 (colon),
MCF7 (breast) and SK-LU-1 (lung). The effects on the cell cycle,
Advances in molecular and cellular biology through the last
decades have revealed some of the many process implicated in
cancer approach. However, at the present time the prognosis against
of most human malignancies has not been improved significantly.
The continuous search for novel agents which target pathological
processes of human carcinogenesis has lead to the synthesis of small
molecules which may modulate cell cycle and apoptotic pathways
[1]. Special interest hasbeen focused on molecules that can arrest cell
cycle mechanism by the inhibition effect for cyclin-dependent
kinases (CDK) family, because perturbation of the cell cycle has been
related to human neoplastic diseases [2]. From various subtypes of
CDKs, the most important explored targets in cancer therapy have
been CDK-1, -2, and -4 [3].
Nitrogen-bridgehead fused heterocycles containing an imidazole
ring have been shown as a common structural moiety in pharmaco-
logically important molecules, displaying a wide range of activities
and spreadingon diversenumberof targets. Probably the mostwidely
used heterocyclic system from this group is imidazo[1,2-a]pyridine
[4]. Imidazo[1,2-a]pyridines shown anticytomegalo-zoster and anti-
varicella-zoster virus [5], antibacterial [6], hypnoselective, and
* Corresponding authors. Tel.: þ52 55 56 22 44 21; fax: þ52 55 56 16 22 17.
E-mail addresses: urbinarepowered@yahoo.com (M.A. Martı´nez-Urbina),
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.2009.11.049

1212
M.A. Mart´ınez-Urbina et al. / European Journal of Medicinal Chemistry 45 (2010) 1211–1219
apoptosis induction, and inhibitory activity on CDK-1 and CDK-2 of
selected compounds were also studied.
with trifluoroacetic anhydride in refluxing methylene chloride.
Metalation of compound 5 with i-propyl magnesium chloride, and
then reaction with 4 equivalents of the corresponding fluorinated
aldehyde with further oxidation of the crude reaction with
manganese dioxide afforded diarylketones 6a–e in yields over
30%. Synthesis of compounds 8a–c is summarized in Scheme 2.
Compound 7a was obtained by classical Friedel-Crafts acylation of
N-methylindole with 6-chloronicotinyl chloride. Reaction of N-
methylimidazole with n-Butyllithium and quenching with 6-
chloro-N-methoxy-N-methylnicotinamide (Weinreb amide) leads
to the desired compound 7b. Diarylketone 7c was obtained by
acylation of imidazole with 6-chloronicotinyl chloride under
conditions reported by Bastiaansen and Godefroi [13]. Finally,
treatment of 7a–c with ammonia yields corresponding amines
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 6a–e (Scheme 1) was accom-
plished according to the previous reported proceedings [12]. The
protection of 2-amino-5-iodo-pyridine 2 with p-tolylsulfonyl
chloride in pyridine and subsequent treatment with iodoaceta-
mide in the presence of diisopropylethylamine in DMF provided
compound 4. Conversion of
4
to the desired 2-(N-tri-
fluoroacetylamino)imidazopyridine 5 was achieved by treatment
Tos
NH
i
ii
I
NH2
I
NH2
N
3
N
1
N
2
iii
Tos
O
N
CF3
N
O
iv
NH
N
N
I
I
NH2
5
4
v, vi
6a R= 3-Fluoro-4-methylphenyl
6b R= 3-Fluoro-4-(trifluoromethyl)phenyl
6c R= 2-Fluoro-5-methylphenyl
6d R= 2-Fluoro-5-(trifluoromethyl)phenyl
6e R= 2-Fluoro-4-(trifluoromethyl)phenyl
11a R= 1-methyl-1H-indole-3-yl
O
CF3
N
NH
R
N
11b R= 1-methyl-1H-imidazole-2-yl
11c R= 1H-imidazole-2-yl
O
iv
Tos
N
O
R
Tos
NH
iii
O
9a-c
R
N
N
NH2
O
10a-c
ii
O
R
NH2
8a-c
N
Scheme 1. Reagents and conditions i) I2/HIO4/H2SO4/CH3COOH, 80 ꢀC; ii) TsCl, py, 85 ꢀC; iii) ICH2CONH2, DIPEA, DMF, r.t.; iv) TFAA, CH2Cl2 reflux; v) i-PrMgCl, THF, ꢁ40 ꢀC,
aldehyde; vi) MnO2, CH2Cl2.

M.A. Mart´ınez-Urbina et al. / European Journal of Medicinal Chemistry 45 (2010) 1211–1219
1213
effect was observed when substitution with trifluoromethyl instead
methyl attached to phenyl ring occurs (compounds 6a and 6c vs. 6b
and 6d). When heterocyclic substituents at 6-position were intro-
duced (compounds 11a–c) comparable or improved cytotoxic
activity was detected (Tables 1 and 2).
In order to examine the effect on cell cycle, apoptosis induction,
and enzyme inhibition we selected the most active compounds (in
the cytotoxicity assay) representing each substitution pattern
(compounds 6a, 6c, and 11a) and we used the most sensitive cell
lines, MCF7 and SK-LU-1 (Table 2). Furthermore, the origin of these
cell lines is representative of the most frequent cancer types
worldwide [14].
Cl
O
i
+
N
Cl
N
N
Cl
N
N
O
7a
Cl
N
N
ii, iii
N
N
O
7b
Cl
N
O
N
iv
N
+
Cl
N
H
N
H
Cl
N
O
2.2.2. Cell cycle analysis
Asynchronously growing MCF7 and SK-LU-1 cells were exposed
to the chosen compounds at concentrations of 2ꢂIC50 for 24 h,
stained with propidium iodide and analyzed by flow cytometry in
order to determine the total population distribution in the different
phases (G0/G1, S, and G2/M). Control cells treated only with DMSO
using the highest amount into the experiments proceeded through
a normal cell cycle (Fig. 1a, and Table 3). However, under the same
7c
O
R
O
R
v
Cl
NH2
N
N
7a-c
8a-c
Scheme 2. Reagents and conditions i) AlCl3, CH2Cl2; ii) n-BuLi, THF, ꢁ70 ꢀC; iii) 6-chloro-
N-methoxy-N-methylnicotinamide, ꢁ70 ꢀC to r.t.; iv) (C2H5)3N/Pyridine r.t. to reflux;
v) NH3 anh./EtOH 150 ꢀC.
conditions, treatment of MCF7 cells with compound 6a (95
not show any difference compared with DMSO control (Fig. 1b, and
Table 3). When MCF7 cells were treated with compound 6c (22 M)
mM) did
m
it was observed a statistically significant increase of cell population
in phase G0/G1 from 57.2% for control to 74.1% after treatment.
Nevertheless for S and G2/M phase a decreasing of cell population
was displayed (Fig. 1c, and Table 3). Treatment of MCF7 cells with
8a–c. Synthesis of compounds 11a–c (Scheme 1) was performed
just as it was described for synthesis of 5, using as starting
material the amines 8a–c instead of 2-amino-5-iodo-pyridine 3.
compound 11a (89 mM) led to the enrichment of G0/G1 cell pop-
2.2. Biological activities
ulation going from 57.2% for control to 70.6% after treatment, while
a decreasing of cells in S phase was observed, remaining cells in G2/
M phase unchanged (Fig. 1d, and Table 3). The use of Olomoucine as
control did not have any effect on cell cycle of MCF7 cells (Table 3)
at the selected concentration for these experiments (112 mM).
Control (DMSO), SK-LU-1 cell cycle histograms and percentage of
population distribution are shown in Fig. 1e and Table 3. When SK-
2.2.1. Cytotoxicity
All compounds were evaluated for cytotoxic activity in vitro
against six human cell lines (glia carcinoma U251, prostate PC-3,
leukemia K-562, colon HCT-15, human breast MCF7 and lung
carcinoma SK-LU-1). To compare cytotoxic effects between a known
CDK inhibitor and the compounds herein described we used Olo-
mucine as a positive control and DMSO as solvent control. A starting
LU-1 cells were treated with compound 6a (106 mM) a statistically
significant accumulation of cell population in G2/M phase was
observed with 16.7% for control to 60.6% for treated cells. With
respect to cells in S phase, they were unchanged and population in
G0/G1 decreased significantly from 61.7% for control to 22.6% for
screening with a fixed concentration of 50 mM showed citotoxicity
for everyone of these six human cell lines (Table 1) with growth
inhibition activity greatest than the observed for intermediary 5 and
Olomucine. Compounds 6a, 6b, 6c, 6e, 11a, and 11c displayed to be
the most active. In addition, the IC50 values for these compounds
were determined and the results are summarized in Table 2.
As shown in Table 2, MCF7 and SK-LU-1 displayed to be the most
sensitive cell lines. Comparing data of compounds 6a–e (Tables 1 and
2) it is possible to observe that substitution with fluorine at 2-posi-
tion (6c, 6d and 6e) of the phenyl ring shown better cytotoxic activity
than those having a substitution at 3-position (6a and 6b). Negligible
treated cells (Fig. 1f, and Table 3). Compound 6c (70 mM) causes
a most evident arrest at G2/M phase with a 75.7% of overall cell
population in this phase only a 10.4% of cells in G0/G1 phase and
cells in S phase were observed without change (Fig.1g, and Table 3).
Treatment with compound 11a (50 mM) induced weaker cell cycle
alteration with arrest in G2/M phase (47.5%) decreasing the pop-
ulation in G0/G1 phase (32.9%) and displaying S phase without
change (Fig.1h, and Table 3). Olomucine treatment (64 mM) showed
a decrease of cells in G0/G1 phase and a weak arrest of cells in G2/M
phase while cells in S phase remained unchanged. Differences
observed with the stage of arrest between MCF7 and SK-LU-1 cells
could be due to the genetic differences of each both cell line.
Table 1
Cytotoxicity of target compounds against six human cancer cell lines. Primary
screening at 50
m
M.a
Compound
Cell line (growth inhibition %)
U251
PC-3
K-562
HCT-15
MCF7
SK-LU-1
2.2.3. Apoptosis
5
6a
6b
6c
6d
6e
11a
11b
20.8
37.9
38.4
61.5
59.3
80.1
92.7
11.9
80.2
32.0
50.9
30.7
32.6
59.2
61.5
92.6
63.7
6.1
33.1
52.2
50.1
92.6
88.0
90.2
95.5
45.3
88.3
19.8
56.3
65.5
59.2
91.2
91.7
100
78.0
NA
36.7
57.3
56.5
66.3
65.1
97.2
80.4
30.8
60.7
37.6
25.6
81.5
48.3
71.7
70.5
100
69.2
29.2
97.5
36.9
In this work we also examined the effect of compounds 6a, 6c
and, 11a on cell death (apoptosis) for SK-LU-1 cells. The appearance
of phosphatidylserine (PS) residues (normally hidden within the
plasma membrane) on the surface of the cell is used as a parameter
to detect and measure apoptosis. The presence of PS on the cell
surface creates one of the specific signals for recognition and
removal of apoptotic cells by macrophages. These PS changes can
be detected with the anticoagulant (annexin V), which has shown
a high affinity for binding to PS. As the apoptotic process prog-
resses, cell membrane integrity is lost. Using DNA specific viability
11c
Olomucine
58.1
22.7
47.8
28.9
a
Each experiment was independently performed two times at a time of 48 h. NA,
not active.

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M.A. Mart´ınez-Urbina et al. / European Journal of Medicinal Chemistry 45 (2010) 1211–1219
Table 2
Cytotocicity IC50 values for compounds 6a, 6c, 6e, 11a and Olomucine against six cancer cell lines.a
Compound
Cell line IC50
U251
(mM)
PC-3
K-562
HCT-15
MCF7
SK-LU-1
6a
6b
6c
6e
80.6 ꢃ 2.1
84.2 ꢃ 4.5
51.9 ꢃ 2.3
52.3 ꢃ 4.6
27.8 ꢃ 1.5
37.7 ꢃ 5.5
81.5 ꢃ 2.1
ND
83.6 ꢃ 0.1
83.2 ꢃ 3.1
10.9 ꢃ 2.0
56.6 ꢃ 3.3
54.6 ꢃ 5.4
51.5 ꢃ 3.3
89.3 ꢃ 1.9
56.2 ꢃ 2.7
57.1 ꢃ 3.2
10.5 ꢃ 0.8
25.0 ꢃ 1.5
65.1 ꢃ 4.3
56.8 ꢃ 8.5
62.5 ꢃ 3.0
47.5 ꢃ 0.9
50.8 ꢃ 1.3
10.7 ꢃ 1.6
24.9 ꢃ 2.1
44.3 ꢃ 0.1
42.4 ꢃ 4.1
55.9 ꢃ 1.1
53.0 ꢃ 7.7
57.1 ꢃ 3.1
11.2 ꢃ 1.0
35.1 ꢃ 4.3
24.8 ꢃ 0.4
25.1 ꢃ 1.1
68.1 ꢃ 0.41
69.1 ꢃ 1.5
36.3 ꢃ 3.0
28.0 ꢃ 1.9
ND
11a
11c
Olomucine
52.3 ꢃ 2.5
81.0 ꢃ 7.4
a
Results express the mean IC50 ꢃ SD obtained from three independent experiments performed at 48 h. ND, not determinate at tested concentrations.
dyes, such as Propidium Iodide (PI) it is possible to distinguish
between early apoptotic, late apoptotic, and dead cells. In order to
prove these findings, selected cells were treated with compounds
quantitative determination at IC50 of Olomoucine (7
m
M) [15]. We
found a strong inhibition effect for cyclin B/CDK-1 activity when
compound 6c and compound 11a were presented, 83% and 61%
respectively (Table 4). Both compounds were most active than
Olomoucine. A weaker inhibition of cyclin B/CDK-1 was observed
with compound 6a (Table 4). Evaluation of cyclin E/CDK-2 inhibi-
tion activities enabled us to detect inhibition percents of 44, 49, 45
and 51% for compounds 6a, 6c, 11a, and Olomoucine respectively
(Table 4). These results allow us to assume that compounds 6a, 6c,
and 11a may affect the cell cycle via modifying cyclin B/CDK-1, and
cyclin E/CDK-2 activities; even though other targets can be involved
so further experiments are necessary to make sure the exact
mechanism.
6a, 6c and 11a for 48 h at 106 mM, 70 mM and 50 mM respectively
(2ꢂIC50). FACS analyses (Fig. 2) showed populations of annexin V-
stained cells (early apoptotic) and annexin V-PI stained cells (late
apoptotic) with the drug concentrations used. Control cells treated
only with DMSO alone showed a 5% of apoptosis. However when
cells were treated with compound 6a an average of 39.5% of cells
underwent apoptosis, apoptosis was evident inclusive at 24 h of
exposition as shown by the appearance of an evident sub-G1 peak
in cell cycle analysis histograms (Fig. 1f), while compound 6c and
compound 11a causes a 20% and 24.1% of apoptotic death respec-
tively (Fig. 3). A weaker effect was observed at 24 h of treatment
with compounds 6c and 11a as shown in cell cycle analysis histo-
grams (Fig. 1g and h).
3. Conclusion
The antiproliferative activity of 2-(N-trifluoroacetylamino)-
imidazopyridines 6-substituted against a variety of cancer cell lines
was evaluated. Most of synthesized compounds displayed modest
cytotoxic activity in the micromolar range, especially against MCF7
and SK-LU-1. Compounds 6a, 6c, and 11a showed significant arrest in
G2/M phase, followed by apoptotic cell death in SK-LU-1 cells.
Compounds 6a, 6c, and 11a could affect the cell cycle via inhibition of
cyclin B/CDK-1, and cyclin A/CDK-2 activities but since synthesized
compounds are each basically equitoxic against MCF7 and SK-LU-1
2.2.4. Inhibiting CDK-1 and CDK-2 activities
Since imidazo[1,2-a]pyridines have shown both cytotoxic
activity and inhibition against CDK-2 and CDK-1 in vitro [10,11] we
examined the activities for cell cycle relative complex cyclin B/CDK-
1, and cyclin E/CDK-2 in presence of compounds 6a, 6c, and 11a. In
order to compare the inhibition grade of the activity of cyclin B/
CDK-1, and cyclin E/CDK-2 complexes by tested compounds with
that of Olomoucine (a CDK inhibitor), we performed a one-point
Fig. 1. Cell cycle distribution by flow cytometry in MCF7 cells treated with a) DMSO control, b) 6a (95
mM), c) 6c (22 mM), d) 11a (89 mM); and SK-LU-1 cells treated with e) DMSO
control, f) 6a (106 M), g) 6c (70 M), h) 11a (50
m
m
mM). Compounds were tested at 2ꢂIC50. Histograms are the most representative of three independent experiments.

M.A. Mart´ınez-Urbina et al. / European Journal of Medicinal Chemistry 45 (2010) 1211–1219
1215
Table 3
Cell cycle distribution percentage by flow cytometry in MCF7 and SK-LU-1 cells treated for 24 h at 2ꢂIC50
a
.
Compound
MCF7
SK-LU-1
%G0/G1
%G0/G1
%S
%G2/M
%S
%G2/M
Control
6a
6c
11a
Olomucine
57.2 ꢃ 3.4
63.5 ꢃ 4.9
74.0 ꢃ 2.4*
70.6 ꢃ 3.2
60.6 ꢃ 3.1
17.0 ꢃ 1.2
14.5 ꢃ 3.3
7.1 ꢃ 2.1*
6.3 ꢃ 1.2*
15.0 ꢃ 2.4
22.1 ꢃ 0.1
18.1 ꢃ 3.5
13.4 ꢃ 2.4
20.2 ꢃ 0.3
23.2 ꢃ 0.4
61.7 ꢃ 2.9
18.2 ꢃ 1.5
16.2 ꢃ 5.3
21.2 ꢃ 1.8
14.7 ꢃ 6.5
19.7 ꢃ 8.5
16.7 ꢃ 0.9
60.6 ꢃ 1.1*
75.7 ꢃ 11.5*
47.5 ꢃ 7.7*
31.8 ꢃ 3.6*
22.6 ꢃ 5.7*
10.4 ꢃ 11.9*
32.9 ꢃ 6.1*
44.2 ꢃ 2.9*
a
Results express the means ꢃ SD obtained from three independent experiments. *P < 0.05 as compared with solvent (DMSO) on 24 h treatment. Tested concentrations for
M), 6c (22 M), 11a (89 M), and for SK-LU-1 cells 6a (106 M), 6c (70 M), 11a (50 M).
MCF7 cells were 6a (95
m
m
m
m
m
m
cells (Table 2) but have minimal cell cycle effects in MCF7 cells and
significant cell cycle effects in SK-LU-1 cells we could consider that
other targets may be involved and further experiments are necessary
to assess the exact mechanism.
with 10% aqueous NaOH, dried (Na2SO4), and concentrated in
vacuum. The residue was purified by flash chromatography on silica
gel. Recrystallization from ethanol gave pale yellow prisms of 2
(4.75 g, 86% yield).
1H NMR (300 MHz, CDCl3):
d
¼ 4.51 (s, 2H), 6.35 (d, 1H, J ¼ 8 Hz),
7.62 (d, 1H, J ¼ 8 Hz), 8.21 (s, 1H). C5H5IN2, MW calcd. 220.96.
4. Experimental protocols
4.1.2. 5-Chloro-N-tosylpyridin-2(1H)-imine (3)
All reactions involving air-sensitive reagents were performed
under inert atmosphere (N2 or argon) using syringe–septum cap
techniques. All glassware was oven-dried prior to use. All melting
points were determined with a MEL-TEMPÒ capillary melting point
apparatus and were uncorrected. Infrared spectra were determined
in a spectrophotometer Perkin–Elmer 282-B and Nicolet FT-IR
magna 55ꢂ. The mass spectra were recorded on a JEOL JMS-SX 10217
instrument (EI). The 1H NMR spectra were recorded on a Varian
Gemini 200 MHz, Varian Unity 300 MHz. High resolution spectra
were recorded using a Varian Inova 500 MHz (1H NMR and 125 MHz
2-Amino-5-iodo-pyridine 2 (2.2 g, 10 mmol) was dissolved in
anhydrous pyridine (10 mL). p-Toluensulphonyl chloride (2.1 g,
11 mmol) was added and the solution was heated at 90 ꢀC under
Nitrogen overnight. Pyridine was removed in vacuo to yield a brown
solid. Water (200 mL) was added and the mixture was stirred for
1 h. The solid was collected and dried in vacuum to give 3.52 g (94%
yield) of 3 as a beige solid; m.p. 177–178 ꢀC.
1H NMR (200 MHz, CDCl3):
d
¼ 2.34 (s, 3H), 6.92 (d, 1H,
J ¼ 8.7 Hz), 7.56 (AA0BB0 system, 4H, J ¼ 8.1 Hz), 7.98 (dd, 1H, J ¼ 8.6
and 2.1 Hz), 8.34 (d,1H, J ¼ 2.2 Hz),11.23 (bs,1H, NH). C12H11IN2O2S,
MW calcd. 374.2. Mass (FABþ) m/z ¼ 374.9 (M þ H)þ.
13C NMR) spectrometers. Chemical shifts are expressed as
d values
relative to TMS as internal standard, J values are given in Hz and
spectra were recorded in CDCl3, DMSO-d6 or a mixture of both. Flash
chromatography was performed on silica gel 60 (230–400 mm) by
Merck.
All the aldehydes and reagents herein used were acquired from
Sigma–Aldrich (St. Louis, MO) and used without further purification
unless otherwise stated. THF was distilled from Na–benzophenone
under N2.
4.1.3. (E)-2-(5-iodo-2-(tosylimino)pyridin-1(2H)-yl)acetamide (4)
To a suspension of 3 (3.7 g, 10 mmol) in anhydrous DMF (20 mL)
was added i-Pr2Net (1.42 g, 11 mmol) under argon. To the solution
was added 2-iodoacetamide (2.1 g, 11 mmol) and the mixture was
stirred at r.t. overnight. The solution was poured onto water
(50 mL), filtered, washed with water (200 mL) and dried in vacuum
to give 4 as a white solid (3.8 g, 88% yield) m.p. 245 ꢀC.
1H NMR (200 MHz, DMSO-d6):
d
¼ 2.32 (s, 3H), 4.75 (s, 2H), 7.15
4.1. Synthesis
(d, 1H, J ¼ 9.4 Hz), 7.38 (bs, 1H, NH), 7.43 (AA0BB0 system, 4H,
J ¼ 8.3 Hz), 7.77 (bs, 1H, NH), 7.90 (dd, 1H, J ¼ 9.5 and 2.2 Hz), 8.35
(d, 1H, J ¼ 2.2 Hz). C14H14IN3O3S, MW calcd. 431.25.
4.1.1. 2-Amino-5-iodo-pyridine (2)
A mixture of 2-aminopyridine 1 (2.4 g, 25 mmol), periodic acid
dihydrate (0.86 g, 3.75 mmol), and iodine (2.7 g, 10.7 mmol) was
heated in a mixed solution of acetic acid (60 mL), water (3 mL), and
sulphuric acid (0.5 mL) at 80 ꢀC for 4 h. The reaction mixture was
then poured into 10% aqueous Na2S2O3 solution to quench any
unreacted iodine and extracted with ether. The extract was washed
4.1.4. 6-Iodo-2-(trifluoroacetamido)imidazo[1,2-a]pyridine (5)
To a suspension of 4 (2.16 g, 5 mmol) in anhydrous dichloro-
methane (40 mL) was added trifluoroacetic anhydride until disso-
lution and the solution was refluxed for 6 h. Solvents were removed
Fig. 2. Apoptosis induced in SK-LU-1 cell line. Apoptosis was assessed by phosphatidylserine exposure on the cell membrane after 48 h treatment with a) DMSO control, b)
compound 6a (106
experiments.
m
M), c) compound 6c (70
m
M), d) compound 11a (50
m
M). Concentrations are 2ꢂIC50. Histograms are the most representative of three independent

1216
M.A. Mart´ınez-Urbina et al. / European Journal of Medicinal Chemistry 45 (2010) 1211–1219
103.6 (CH), 115.2 (CH), 115.3 (q, JCF ¼ 288.98 Hz), 115.4 (d, CH,
SK-LU-1
50
40
30
20
10
0
JCF ¼ 24 Hz), 122.3 (C), 124.3 (CH), 129.5 (d, C, JCF ¼ 17.50 Hz), 129.9
(d, CH, JCF ¼ 1.7 Hz), 131.2 (d, CH, JCF ¼ 4.7 Hz), 131.5 (CH), 136.2 (d,
CH, JCF ¼ 6.4 Hz),140.7 (C),141.6 (C),154.1 (q, JCF ¼ 38.7 Hz),160.2 (d,
CF, JCF ¼ 246.6 Hz), 190.8 (C). C17H11F4N3O2, MW calcd. 365.28. Mass
(m/z, %): 365 (Mþ, 100), 296 (35), 256 (20), 137 (22).
4.1.7. 2,2,2-Trifluoro-N-(6-(3-fluoro-4-(trifluoromethyl)benzoyl)-
imidazo[1,2-a]pyridin-2-yl)acetamide (6b)
Compound 6b was prepared starting from 2-fluoro-5-tri-
fluoromethylbenzaldehyde as electrophile and isolated as a yellow
solid (126 mg, 30% yield); m.p. 120 ꢀC.
1H NMR (500 MHz, CDCl3 þ DMSO-d6):
d
¼ 7.35 (dd, 1H,
JHH ¼ 7.5 and JHF ¼ 7.5 Hz), 7.44 (dd, 1H, J ¼ 2 and 8 Hz), 7.47 (dd, 1H,
J ¼ 2 and 7.5 Hz), 7.48 (d, 1H, J ¼ 7 Hz), 7.57 (dd, 1H, J ¼ 1.5 and
9.0 Hz), 8.36 (bs, 1H), 8.89 (bs, 1H), 12.21 (bs, 1H). 13C NMR
Compound
Fig. 3. Apoptosis percentage induced in SK-LU-1 cells. Apoptosis was assessed by
phosphatidylserine exposure on the cell membrane after 48 h treatment at 2ꢂIC50. The
(125.7 MHz, CDCl3 þ DMSO-d6): ¼ 102.4 (CH), 115.0 (CH), 115.4 (q,
d
percentages were 5.1% for DMSO control, 39.5% for compound 6a (106
m
M), 20.0% for
JCF ¼ 287.2 Hz), 115.6 (d, CH, JCF ¼ 24 Hz), 122.1 (C), 122.3 (q,
JCF ¼ 288.9 Hz) 124.3 (CH), 128.9 (d, C, JCF ¼ 17.50 Hz), 129.6 (d, CH,
JCF ¼ 1.7 Hz), 131.2 (d, CH, JCF ¼ 4.7 Hz), 131.5 (CH), 136.2 (d, CH,
JCF ¼ 6.4 Hz), 140.7 (C), 141.6 (C), 154.3 (q, JCF ¼ 38.7 Hz), 160.6 (d, CF,
JCF ¼ 246.6 Hz), 189.8 (C). C17H8F7N3O2, MW calcd. 419.25. Mass (m/
z, %): 419 (Mþ, 100), 400 (15), 350 (95), 191 (63). IR (KBr, cmꢁ1):
3012, 2874, 1750, 1472, 1309.
compound 6c (70 mM), and 24.1% for compound 11a (50 mM). Percentages are the
means of three independent experiments.
in vacuum and the solid was suspended in EtOAc (100 mL) and
stirred for 30 min. The solid was collected and again stirred in water
for 30 min. The solid was collected and dried in vacuum to give 1.2 g
(65% yield) of 5 m.p. 245–246 ꢀC.
1H NMR (200 MHz, CDCl3 þ DMSO-d6):
d
¼ 7.28 (d, 1H,
4.1.8. 2,2,2-Trifluoro-N-(6-(2-fluoro-5-methylbenzoyl)imidazo[1,2-
a]pyridin-2-yl)acetamide (6c)
Compound 6c was prepared starting from 2-fluoro-5-methyl-
benzaldehyde as electrophile and isolated as a yellow solid
(128 mg, 35% yield); m.p. 208–210 ꢀC.
J ¼ 9.5 Hz), 7.39 (dd, 1H, J ¼ 1.5 and 9.5 Hz), 8.17 (s, 1H), 8.69 (dd, 1H,
J ¼ 0.5 and 1.5 Hz), 12.10 (bs, 1H, NH).
13C NMR (125.7 MHz, CDCl3 þ DMSO-d6):
¼ 74.6 (C), 102.1 (C),
d
115.3 (q, JCF ¼ 287.2 Hz, COCF3), 116.7 (CH), 130.7 (CH), 132.3 (CH),
139.32 (CH), 139.7 (C), 154.0 (q, JCF ¼ 38.6, COCF3). C9H5F3IN3O, MW
calcd. 355.06. Mass (FABþ): m/z ¼ 356 (M þ H)þ.
1H NMR (500 MHz, DMSO-d6):
d
¼ 2.36 (s, 3H, CH3), 7.29 (dd,1H,
J ¼ 1.5 and 8.5 Hz), 7.43 (dd, 1H, J ¼ 2.0 and 4.5 Hz), 7.49 (qddd, 1H,
J ¼ 0.5, 2.0, 7 and 8.5 Hz), 7.63 (t, 1H, J ¼ 1 and 2 Hz), 7.69 (dd, 1H,
J ¼ 2 and 7.5 Hz), 8.41 (s, 1H), 9.16 (dd, 1H, J ¼ 1 and 1.5 Hz), 12.58
4.1.5. General procedure for the synthesis of compounds 6a–e
To a stirred solution of 5 (354.9 mg, 1 mmol) in THF (10 mL) at
ꢁ40 ꢀC under argon was added 2 M i-PrMgCl in THF (1 mL, 2 mmol)
over a period of 2 min. Stirring was continued for 1 h at ꢁ40 ꢀC
before triethylamine (0.140 mL, 2 mmol) was added. After 15 min
corresponding aldehyde (4 mmol) was added. The reaction mixture
was stirred overnight at r.t. before quenching with sat. NH4Cl
(10 mL). Water (5 mL) was added to dissolve the formed precipitate
and the mixture was extracted with CH2Cl2 (3 ꢂ 10 mL). The
combined organic phases were dried (Na2SO4) and column chro-
matography on silica gel afforded 6a–e.
(bs,1H, NH). 13C NMR (125.7 MHz, DMSO-d6):
d
¼ 20.0 (CH3), 104.31
(CH), 115.56 (CH), 116.10 (d, CH, J ¼ 22.12 Hz), 123.10 (C), 123.8 (CH),
125.6 (C), 130.4 (CH), 133.48 (CH), 133.9 (d, CH, J ¼ 7.29 Hz), 134.12
(C), 140.7 (C), 142.0 (C), 154.12 (q, J ¼ 77.3 Hz), 157.45 (d,
JCF ¼ 246.75 Hz), 189.82 (C). 19F NMR (282.2 MHz, DMSO-d6)
d
¼ ꢁ117.28 (td, F, J ¼ 2.0, 7 and 8.5 Hz), ꢁ72.0 (CF3). C17H11F4N3O2,
MW calcd. 365.28. Mass (m/z, %): 365 (Mþ, 100), 296 (35), 256 (20),
137 (22). IR (KBr, cmꢁ1): 3258, 3093, 1718, 1615, 1564, 1212, 1147.
4.1.9. 2,2,2-Trifluoro-N-(6-(2-fluoro-5-(trifluoromethyl)benzoyl)-
imidazo[1,2-a]pyridin-2-yl)acetamide (6d)
Compound 6d was prepared starting from 2-fluoro-5-tri-
fluoromethylbenzaldehyde as electrophile and isolated as a yellow
solid (126 mg, 30% yield); m.p. 145–147 ꢀC.
4.1.6. 2,2,2-Trifluoro-N-(6-(3-fluoro-4-methylbenzoyl)imidazo[1,2-
a]pyridin-2-yl)acetamide (6a)
Compound 6a was prepared starting from 3-fluoro-4-methyl-
benzaldehyde as electrophile and isolated as a yellow solid
(109 mg, 30% yield); m.p. 210 ꢀC.
1H NMR (500 MHz, DMSO-d6):
d
¼ 7.25 (dd, 1H, J ¼ 1.5 and
8.5 Hz), 7.39 (dd, 1H, J ¼ 2.0 and 4.5 Hz), 7.46 (qddd, 1H, J ¼ 0.5, 2.0,
7 and 8.5 Hz), 7.53 (t, 1H, J ¼ 1 and 2 Hz), 7.69 (dd, 1H, J ¼ 2 and
7.5 Hz), 8.41 (s, 1H), 9.12 (dd, 1H, J ¼ 1 and 1.5 Hz), 12.48 (bs, 1H,
1H NMR (500 MHz, CDCl3 þ DMSO-d6):
d
¼ 2.31 (d, 3H,
J ¼ 1.5 Hz), 7.33 (dd,1H, JHH ¼ 7.5 and JHF ¼ 7.5 Hz), 7.41 (dd,1H, J ¼ 2
and 8 Hz), 7.46 (dd, 1H, J ¼ 2 and 7.5 Hz), 7.47 (d, 1H, J ¼ 7 Hz), 7.60
(dd,1H, J ¼ 1.5 and 9.0 Hz), 8.26 (bs,1H), 8.92 (bs,1H),12.09 (bs,1H).
NH). 13C NMR (125.7 MHz, DMSO-d6):
d
¼ 104.19 (CH), 115.26 (CH),
115.80 (d, CH, J ¼ 22.12 Hz), 123.10 (C), 123.8 (CH), 124.10 (q,
J ¼ 246.75 Hz) 126.6 (C), 131.4 (CH), 133.48 (CH), 133.9 (d, CH,
J ¼ 7.29 Hz), 134.12 (C), 141.2 (C), 142.2 (C), 153.9 (q, J ¼ 246.75 Hz),
157.45 (d, J ¼ 77.3 Hz), 188.61 (C). C17H8F7N3O2, MW calcd. 419.25.
Mass (m/z, %): 419 (Mþ, 100), 400 (15), 350 (95). IR (KBr, cmꢁ1):
3014, 2836, 1725, 1670, 1492, 1217.
13C NMR (125.7 MHz, CDCl3 þ DMSO-d6):
¼ 14.2 (d, CH, J ¼ 2.8 Hz),
d
Table 4
Enzyme inhibition data for compounds 6a, 6c, and 11a.a
CDK/cyclin
Compound (% inhibition)
6a
6c
11a
Olomucine
4.1.10. 2,2,2-Trifluoro-N-(6-(2-fluoro-4-(trifluoromethyl)benzoyl)-
imidazo[1,2-a]pyridin-2-yl)acetamide (6e)
Compound 6e was prepared starting from 2-fluoro-4-tri-
fluoromethylbenzaldehyde as electrophile and isolated as a yellow
solid (134 mg, 32% yield); m.p. 100–102 ꢀC.
CDK-1/B
CDK-2/E
29
44
83
49
61
45
50
51
a
Values are the means for two experiments performed at 7
m
M of each tested
compound.

M.A. Mart´ınez-Urbina et al. / European Journal of Medicinal Chemistry 45 (2010) 1211–1219
1217
1H NMR (500 MHz, CDCl3):
d
¼ 7.53 (d, 1H, J ¼ 9 Hz), 7.55 (d, 1H,
4.1.14. (6-Aminopyridin-3-yl)(1-methyl-1H-indol-3-yl)methanone
(8a)
J ¼ 9 Hz), 7.63 (d, 1H, J ¼ 8.5 Hz), 7.73 (t, 1H, J ¼ 7 Hz), 7.77 (dt, 1H,
J ¼ 1 and 9 Hz), 8.26 (s, 1H), 11.20 (bs, 1H). 13C NMR (125.7 MHz,
Anhydrous NH3 (15 mL) was added to a suspension of 7a
(541.5 mg, 2 mmol) in ethanol (15 mL) in a Parr reactor, the mixture
was stirred at 150 ꢀC for 8 h. Excess ammonia was permitted to
evaporated and the product was purified by silica gel column
chromatography (422 mg, 84% yield) m.p. 222–224 ꢀC.
CDCl3):
d
¼ 103.73 (CH), 114.19 (CH), 115.57 (q, CF3, J ¼ 289.9 Hz),
116 (CH), 121.85 (CH), 122.5 (q, CF3, J ¼ 289.9 Hz), 124 (C), 125 (CH),
129.18 (C), 131.30 (CH), 131.38 (CH), 135.6 (C), 140.8 (C), 142.70 (C),
154.7 (CO), 159.30 (d, JCF ¼ 254.4 MHz), 188.3 (CO). C17H8F7N3O2,
MW calcd. 419.25. Mass (m/z, %): 419 (Mþ, 100), 400 (15), 350 (95),
191 (63). IR (KBr, cmꢁ1): 3033, 2866, 2832, 1726, 1630, 1572, 1332,
1217, 1137.
1H NMR (200 MHz, DMSO-d6):
d
¼ 3.87 (s, 3H, CH3), 6.52 (d, 1H,
J ¼ 8.8 Hz), 6.65 (bs, 2H, NH2), 7.22 (t, 1H, J ¼ 5.8 Hz), 7.29 (t, 1H,
J ¼ 5.2 Hz), 7.54 (dd, 1H, J ¼ 1.8 and 5.4 Hz), 7.84 (dd, 1H, J ¼ 2.4 and
6.2 Hz), 8.07 (s, 1H), 8.19 (dd, 1 H, J ¼ 1.6 and 4.6 Hz), 8.45 (d, 1H,
J ¼ 2.2). C15H13N3O, MW calcd. 251.28. Mass (m/z, %): 251 (Mþ, 100),
158 (70). IR (KBr, cmꢁ1): 3213, 3044, 1586, 1398, 1371.
4.1.11. (6-Chloropyridin-3-yl)(1-methyl-1H-indol-3-yl)
methanone (7a)
A suspension of aluminium(III)chloride (10.67 g, 80 mmol) in
anhydrous dichloromethane (15 mL) was cooled to 0 ꢀC and 6-
chloronicotinyl chloride (7.74 g, 44 mmol) was slowly added over
5 min. The resulting mixture was then allowed to warm to r.t. over
30 min. The resulting suspension was then re-cooled to 0 ꢀC and
a solution of N-methylindole (5 g, 40 mmol) in dichloromethane
(25 mL) was added dropwise over 15 min and then heated to reflux
for 4 h. The reaction mixture was poured onto ethyl acetate
(300 mL) and NaOH 2 N (100 mL) was added. Resulting precipitate
was filtered and the organic phase was dried over NaSO4. Solvents
were removed in vacuo and the solid was purified by silica gel
column chromatography (9.7 g, 90% yield), m.p. 89 ꢀC.
4.1.15. (6-Aminopyridin-3-yl)(1-methyl-1H-imidazol-2-yl)-
methanone (8b)
Compound 8b was prepared as described for 8a, starting from
(6-chloropyridin-3-yl)(1-methyl-1H-imidazol-2-yl)methanone 7b,
and purified by silica gel column chromatography (323 mg, 80%
yield), m.p. 210–211 ꢀC.
1H NMR (200 MHz, DMSO-d6):
J ¼ 0.8, 8.2 Hz), 6.94 (s, 2H, NH2), 7.14 (d, 1H, J ¼ 1.0 Hz), 7.49 (d, 1H,
J ¼ 0.8 Hz), 8.21 (dd, 1H, J ¼ 2.4 and 6.4 Hz), 9.02 (dd, 1H, J ¼ 0.4 and
1.8 Hz). C10H10N4O, MW calcd. 202.1.
d
¼ 3.93 (s, 3H, CH3), 6.46 (dd,1H,
1H NMR (200 MHz, CDCl3):
d
¼ 3.88 (s, 3H, CH3), 7.37 (t, 1H,
4.1.16. (6-Aminopyridin-3-yl)(1H-imidazol-2-yl)methanone (8c)
Compound 8c was prepared as described for 8a, starting from
(6-chloropyridin-3-yl)(1H-imidazol-2-yl)methanone 7c, and puri-
fied by silica gel column chromatography (320 mg, 85% yield), m.p.
225 ꢀC.
J ¼ 4.8 Hz), 7.40 (t, 1H, J ¼ 5.2 Hz), 7.45 (d, 1H, J ¼ 0.8 Hz), 7.49 (d,1H,
J ¼ 0.8 Hz), 7.53 (s, 1H), 8.10 (dd, 1H, J ¼ 2.4 and 5.8 Hz), 8.39 (dt, 1H,
J ¼ 1.6 Hz), 8.81 (d, 1H, J ¼ 2.4 Hz). C15H11ClN2O, MW calcd. 270.71.
Mass (m/z, %): 270 (Mþ, 78), 158 (100). IR (KBr, cmꢁ1): 3048, 1631,
1524, 1364, 1100.
1H NMR (200 MHz, CDCl3 þ DMSO-d6):
d
¼ 5.56 (bs, 2H), 6.56
(dd, 1H, J ¼ 0.6 and 8.4 Hz), 7.28 (bs, 2H), 8.66 (dd, 1H, J ¼ 2.2 and
6.4 Hz), 9.34 (d, 1H, J ¼ 1.8 Hz), 12.49 (bs, 1H). C9H8N4O, MW calcd.
188.19. Mass (m/z, %): 188 (Mþ, 55), 160 (70), 121 (35). IR (KBr,
cmꢁ1): 3447, 3303, 2919, 1604, 1411, 1314, 1107.
4.1.12. (6-Chloropyridin-3-yl)(1-methyl-1H-imidazol-2-
yl)methanone (7b)
1-Methyl-1H-pyrrole (2.07 g, 25.21 mmol) was dissolved in
anhydrous dichloromethane (50 mL) and cooled to ꢁ60 ꢀC under
argon atmosphere. To the stirred solution was added dropwise n-
Butyllithium 1.6 M (18.9 mL, 30.25 mmol) over a 15 min period. After
1 h at ꢁ60 ꢀC a solution of Weinreb amide 6-chloro-N-methoxy-N-
methylnicotinamide (6.06 g, 30.25 mmol) in dichloromethane
(20 mL) was added dropwise maintaining the temperature below
ꢁ50 ꢀC. The mixture was stirred for 4 h at r.t. before quenching with
a saturated ammonium chloride solution (20 mL). Organic layer was
dried over Na2SO4 and purified by silica gel column chromatography
to yield 7b (4.6 g, 82% yield) m.p. 104–105 ꢀC.
4.1.17. 4-Methyl-N-(5-(1-methyl-1H-indole-3-carbonyl)pyridin-2-yl)
benzenesulfonamide (9a)
(6-Aminopyridin-3-yl)(1-methyl-1H-indol-3-yl)methanone 8a
(628.2 mg, 2.5 mmol) was dissolved in anhydrous pyridine (10 mL).
p-Toluensulphonyl chloride (567.4 mg, 3 mmol) was added and the
solution was heated at 90 ꢀC under Nitrogen overnight. Pyridine
was removed in vacuum to yield a brown solid. Water (20 mL) was
added and the mixture was stirred for 1 h. The solid was collected
and dried in vacuum to give 9a as a white solid (882 mg, 87% yield),
m.p. 205 ꢀC.
1H NMR (200 MHz, DMSO-d6):
d
¼ 4.11 (s, 3H, CH3), 7.17 (s, 1H),
7.26 (d, 1H, J ¼ 1.8 Hz), 7.44 (dd, 1H, J ¼ 0.6 and 8.2 Hz), 8.64 (dd, 1H,
J ¼ 2.6, 5.8 Hz), 9.29 (dd, 1H, J ¼ 0.6 and 1.8 Hz). C10H8ClN3O, MW
calcd. 221.64. Mass (m/z, %): 220 (Mþ ꢁ 1, 100), 192 (85), 186 (55). IR
(KBr, cmꢁ1): 3106, 1642, 1406, 1262, 1180, 900, 776.
1H NMR (200 MHz, CDCl3 þ DMSO-d6):
d
¼ 2.40 (s, 3H, CH3),
3.87 (s, 3H, CH3), 7.26–7.41 (m, 6H), 7.62 (s, 1H), 7.89 (dd, 2H, J ¼ 1.6
and 4.8 Hz), 8.03 (dd, 1H, J ¼ 2.4 and 6.4 Hz), 8.37 (m, 1H), 8.59 (bs,
1H). C22H19N3O3S, MW calcd. 405.47.
4.1.13. (6-Chloropyridin-3-yl)(1H-imidazol-2-yl)methanone (7c)
6-Chloronicotinyl chloride (544.6 mg, 8 mmol) is added drop-
wise to a solution of imidazole (2.82 g, 16 mmol) and triethylamine
(1.62 g, 16 mmol) in pyridine (5 mL) at 0 ꢀC under Nitrogen. The
mixture was stirred for 4 h at r.t. and then treated with 2 N NaOH
(80 mL). Subsequent refluxing for 1 h, and addition of water
(10 mL), and cooling leads to precipitation of the product which
was purified by column chromatography. White solid (997 mg, 60%
yield), m.p. 192–193 ꢀC.
4.1.18. 4-Methyl-N-(5-(1-methyl-1H-imidazole-2-carbonyl)-
pyridin-2-yl)benzenesulfonamide (9b)
Compound 9b was prepared as described for 9a, starting from
8b and was obtained as a brown solid (731 mg, 82% yield) m.p.
180 ꢀC.
1H NMR (200 MHz, CDCl3 þ DMSO-d6):
d
¼ 2.39 (s, 3H, CH3),
4.04 (s, 3H, NCH3), 7.12 (s, 1H), 7.18 (s, 1H), 7.26 (d, 2H, J ¼ 8 Hz), 7.35
(bs, 1H, NH), 7.86 (d, 2H, J ¼ 8.2 Hz), 8.47 (dd, 1H, J ¼ 2.2 and 7 Hz),
9.30 (d, 1H, J ¼ 2.2). C17H16N4O3S, MW calcd. 356.4.
1H NMR (200 MHz, CDCl3 þ DMSO-d6):
d
¼ 7.46 (d, 1H, J ¼ 0.6),
7.48 (s, 1H), 7.51 (d, 1H, J ¼ 0.6), 8.88 (dd, 1H, J ¼ 2.4 and 6.0 Hz),
9.45 (dd, 1H, J ¼ 0.6 and 1.8). C9H6ClN3O, MW calcd. 207.62. Mass
(m/z, %): 207 (Mþ, 20), 179 (100), 144 (35). IR (KBr, cmꢁ1): 3275,
3114, 1639, 1409, 1135, 776.
4.1.19. N-(5-(1H-imidazole-2-carbonyl)pyridin-2-yl)-4-methyl-
benzenesulfonamide (9c)
Compound 9c was prepared as described for 9a, starting from 8c
and was obtained as a white solid (642 mg, 75% yield), m.p. 176 ꢀC.

1218
M.A. Mart´ınez-Urbina et al. / European Journal of Medicinal Chemistry 45 (2010) 1211–1219
1H NMR (200 MHz, DMSO-d6):
d
¼ 2.34 (s, 3H, CH3), 7.27 (dd,1H,
dissolution and the solution was refluxed for 5 h. Solvents were
J ¼ 0.8 and 7.2 Hz), 7.28 (d, 1H, J ¼ 7.2), 7.35 (d, 2H, J ¼ 8), 7.52 (dd,
1H, J ¼ 0.8, 1.8 Hz), 7.80 (d, 2H, J ¼ 8.2 Hz), 8.49 (dd, 1H, J ¼ 2.2 and
7.2 Hz), 9.40 (d, 1H, J ¼ 1.6 Hz), 13.50 (bs, 1H). C16H14N4O3S, MW
calcd. 342.37. Mass (m/z, %): 342 (Mþ þ 1, 5), 277 (100).
removed in vacuum and the solid was purified by silica gel column
chromatography (202 mg, 60% yield) m.p. 218–220 ꢀC.
1H NMR (500 MHz, DMSO-d6):
d
¼ 4.0 (s, 3H, CH3), 7.25 (s, 1H),
7.57 (d, 1H, J ¼ 9.5 HZ), 7.63 (s, 1H), 7.98 (dd, 1H, J ¼ 1.5 and 9.5 Hz),
8.44 (s, 1H), 9.83 (t,), 12.54 (bs, 1H, NH). 13C NMR (125.7 MHz,
4.1.20. (Z)-2-(5-(1-methyl-1H-indole-3-carbonyl)-2-(tosylimino)-
pyridin-1(2H)-yl)acetamide (10a)
DMSO-d6):
d
¼ 36.5 (CH3), 104.60 (CH), 114.98 (CH), 116.1 (q,
JCF ¼ 287.25), 123.0 (C), 141.1 (C), 142.25 (C), 142.52 (C), 154.54 (d,
JCF ¼ 38.38), 179.84 (CO). C14H10F3N5O2, MW calcd. 337.26. Mass (m/
z, %): 337 (Mþ, 100), 268 (20), 240 (15). IR (KBr, cmꢁ1): 3432, 3296,
2925, 2852, 1724, 1637, 1567, 1406, 1153.
To a suspension of 9a (446 mg, 1.1 mmol) in anhydride DMF
(5 mL) was added i-Pr2Net (2.1 mL, 1.21 mmol) under argon. To the
solution was added 2-iodoacetamide (219 mg, 1.2 mmol) and the
mixture was stirred at r.t. overnight. The solution was poured onto
water (20 mL), filtered, washed with water (50 mL) and dried in
vacuum to give 10a as a yellow solid (382 mg, 75% yield), m.p.
275 ꢀC.
4.1.25. N-(6-(1H-imidazole-2-carbonyl)imidazo[1,2-a]pyridin-2-yl)
-2,2,2-trifluoroacetamide (11c)
To
a suspension of 10c (400 mg, 1 mmol) in anhydrous
1H NMR (200 MHz, CDCl3 þ DMSO-d6):
d
¼ 2.39 (s, 3H, CH3),
dichloromethane (10 mL) was added trifluoroacetic anhydride until
dissolution and the solution was refluxed for 5 h. Solvents were
removed in vacuum and the solid was purified by silica gel column
chromatography (181 mg, 56% yield), m.p 302–304 ꢀC.
3.88 (s, 3H, NCH3), 4.97 (s, 2H, NH2), 6.47 (bs, 1H, R2NH), 7.23–7.42
(m, 5H), 7.60 (d,1H, J ¼ 9.4), 7.74 (bs,1H), 7.81 (d,1H, J ¼ 8.2), 7.84 (s,
1H), 8.0 (dd, 1H, J ¼ 2.2 and 7.2 Hz), 8.27 (d, 1H, J ¼ 2.4), 8.31 (d, 1H,
J ¼ 1.8). C24H22N4O4S, MW calcd. 462.52.
1H NMR (200 MHz, DMSO-d6):
d
¼ 7.47 (s, 2H), 7.62 (d, 1H,
J ¼ 9.6), 8.13 (dd, 1H, J ¼ 1.8 and 7.8 Hz), 8.48 (s, 1H), 10.09 (bs, 1H),
12.60 (bs, 1H). C13H8F3N5O2, MW calcd. 323.2. Mass (m/z, %): 323
(Mþ, 100), 254 (75), 226 (40).
4.1.21. (Z)-2-(5-(1-methyl-1H-imidazole-2-carbonyl)-2-
(tosylimino)pyridin-1(2H)-yl)acetamide (10b)
Compound 10b was prepared as described for 10a, starting from
9b and was obtained as a white solid (355 mg, 78% yield), m.p. 218–
220 ꢀC.
4.2. Biology
1H NMR (200 MHz, DMSO-d6):
d
¼ 2.34 (s, 3H, CH3), 3.96 (s, 3H,
4.2.1. Cell culture and cytotoxicity assays
NCH3), 4.93 (bs, 2H), 7.21 (d, 1H, J ¼ 0.8 Hz), 7.29 (d, 1H, J ¼ 0.4 Hz),
7.37 (s, 1H), 7.42 (bs, 1H, NH), 7.61 (d, 1H, J ¼ 0.4), 7.7 (d, 2H,
J ¼ 8.4 Hz), 7.82 (bs,1H), 8.46 (dd,1H, J ¼ 2.2 and 9.6 Hz), 9.18 (d,1H,
J ¼ 2.0 Hz). C19H19N5O4S, MW calcd. 413.45.
All tested substances were dissolved in DMSO and diluted to
tested concentrations. Cell lines were cultured in RPMI-1640
medium supplemented with 10% fetal bovine serum (FBS) and 2 mM
L
-glutamine at 37 ꢀC under a 5% CO2 atmosphere. For each cell line,
70% confluent cell culture flask was trypsinized and cells were
seeded in 96 well plates at a density of 5000 cells/well; 24 h after
seeding, the cells were treated with the different compounds for 48 h
and viability was accessed by sulphorhodamine B (SRB) method.
When incubation with tested compounds was finished,
adherent cell cultures were fixed out 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
4.1.22. (Z)-2-(5-(1H-imidazole-2-carbonyl)-2-(tosylimino)pyridin-
1(2H)-yl)acetamide (10c)
Compound 10c was prepared as described for 10a, starting from
9c and was obtained as a white solid (294 mg, 67% yield) m.p. 214–
215 ꢀC.
1H NMR (200 MHz, DMSO-d6):
d
¼ 2.34 (s, 3H, CH3), 4.94 (s, 2H),
7.29 (d, 2H, J ¼ 8.4 Hz), 7.43 (d, 1H, J ¼ 4.6 Hz), 7.44 (bs, 2H), 7.45 (d,
1H, J ¼ 5 Hz), 7.70 (d, 2H, J ¼ 8.2), 7.84 (bs, 1H), 8.60 (dd, 1H, J ¼ 2.2
and 7.4 Hz), 9.40 (d,1H, J ¼ 2.0 Hz),13.50 (bs,1H). C18H17N5O4S, MW
calcd. 399.42.
and left dry to the air. The fixed cells were stained with 100 mL of
0.4% SRB solution. Protein-bonded dye was solubilized with 10 mM
unbuffered Tris base and the optical density was achieved on
a microplate reader (EIx 808; Bio-Tek Instruments, Inc., Winooski,
VT, USA) using a test wavelength of 515 nm. Preliminary screening
4.1.23. 2,2,2-Trifluoro-N-(6-(1-methyl-1H-indole-3-carbonyl)-
imidazo[1,2-a]pyridin-2-yl)acetamide (11a)
was made at 50 mM for tested compounds using 5-Fluorouracil as
To a suspension of 10a (463.5 mg, 1 mmol) in anhydrous
dichloromethane (10 mL) was added trifluoroacetic anhydride until
dissolution and the solution was refluxed for 6 h. Solvents were
removed in vacuum and the solid was purified by silica gel column
chromatography (278 mg, 72% yield), m.p. 235–237 ꢀC.
a positive control. A dose response curve was plotted for each most
active compound and the IC50 was estimated from non-linear
regression using JMP software (version 3.2.1; SAS Institute Inc.,
Cary, NC, USA).
1H NMR (500 MHz, DMSO-d6):
d
¼ 3.90 (s, 3H, CH3), 7.29 (td, 1H,
4.2.2. Cell cycle analysis
J ¼ 1.0, 1.5 and 7.0 Hz), 7.34 (td, 1H, J ¼ 1.5, 7 and 7 Hz), 7.59 (dd, 1H,
J ¼ 1.5 and 7 Hz), 7.61 (d, 1H, J ¼ 9.5 Hz), 7.67 (dd, 1H, J ¼ 1.5 and
9.5 Hz), 8.26 (td, 1H, J ¼ 0.5, 1.5 and 7.0 Hz), 8.29 (s, 1H), 8.36 (s, 1H),
9.22 (dd,1H, J ¼ 1.0,1.5 Hz),12.55 (bs,1H, NH). 13C NMR (125.7 MHz,
Log phase MCF7 and SK-LU-1 cells were seeded out using RPMI-
1640 þ 10% FBS at 1 ꢂ106 cells per 10 cm petri dish, and the cells
were allowed to attach for 24 h at 37 ꢀC. The compounds were
added to the cells and incubated for an additional 24 h. After
incubation, the cells were harvested with PBS-EDTA and then
centrifuged at 3000 r.p.m. for 5 min. The PBS-EDTA was removed,
and the cell pellet was washed with 1ꢂ PBS, followed by centri-
fugation at 3000 r.p.m. for 5 min. The supernatant was discarded,
and the pellet was resuspended in 5 mL of ice-cold 70% ethanol. The
cells were then held at ꢁ20 ꢀC for 24–48 h. The ethanol-fixed cells
were centrifuged at 3000 r.p.m. for 5 min, the supernatant was
removed, and the cell pellet was washed with 1ꢂ PBS. Following
another centrifugation at 3000 r.p.m. for 5 min, the PBS was
removed, and each sample pellet was mixed with 1 mL of staining
DMSO-d6):
d
¼ 33.23 (CH3), 103.85 (CH), 110.67 (CH), 113.51 (C),
115.36 (CH), 115.65 (q, JCF ¼ 288.25), 121.50 (CH), 122.32 (CH),
123.26 (CH), 125.16 (CH), 125.38 (C), 126.64 (C), 129.61 (CH), 137.36
(C), 139.55 (CH), 140.13 (C), 141.58 (C), 154.00 (q, JCF ¼ 37.83), 185.54
(CO). C19H13F3N4O2, MW calcd. 386.33.
4.1.24. 2,2,2-Trifluoro-N-(6-(1-methyl-1H-imidazole-2-carbonyl)-
imidazo[1,2-a]pyridin-2-yl)acetamide (11b)
To
a suspension of 10b (415 mg, 1 mmol) in anhydrous
dichloromethane (10 mL) was added trifluoroacetic anhydride until

M.A. Mart´ınez-Urbina et al. / European Journal of Medicinal Chemistry 45 (2010) 1211–1219
1219
solution (0.1% Triton X-100, 0.2 mg/mL DNase-free RNase A, 20
m
g/
10% poliacrilamide gels and revealed in a Typhoon 9400 (Amer-
sham Biosciences, Inc.). Band quantifications were made using
a Kodak MI software.
mL propidium iodide in PBS). The cells DNA content of cells was
measured on a FACScan Flow Cytometer (Becton Dickinson, USA)
and analysis was made with Flowjo software V 7.2.5 (Tree Star Inc.,
Ashland, OR, USA).
References
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propidium iodide (PI) in binding buffer (Sigma–Aldrich, St. Louis,
MO, USA), and analyzed using a FACScan Flow Cytometer (Becton
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4.2.4. Kinase assays
All reactions were run in 20
for 30 min at 37 ꢀC and 7
M of each tested compound. The cyclin
B/CDK-1 assay conditions were 50 mM Tris–HCl (pH ¼ 7.4), 10 mM
MgCl2, 50 mM NaF, 500 M Na3VO4, 8 mM -glicerophosphate,
1 mM DTT, 400 Ci
mL containing 2.5% DMSO
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IACR Press, Lyon, 2004.
m
m
b
m
M. ATP (Sigma–Aldrich, St. Louis, MO, USA), 4 m
ATPg32, 250 ng recombinant Histone 1.2 (Calbiochem, San Diego,
CA, USA), and 220 ng cyclin B/CDK-1 (Biofin, Kassel, Germany)
enzyme. The cyclin E/CDK-2 assay conditions were 50 mM Tris–
HCl (pH ¼ 7.4), 10 mM MgCl2, 50 mM NaF, 500
-glicerophosphate, 1 mM DTT, 400 M. ATP (Sigma–Aldrich, St.
Louis, MO, USA), 4
Ci ATPg32, 250 ng recombinant Histone 1.2
mM Na3VO4, 8 mM
b
m
m
(Calbiochem, San Diego, CA, USA), and 135 ng cyclin E/CDK-2
(Biofin, Kassel, Germany) enzyme. All reactions were terminated
ꢀ
with 5
m
L
b
-Merchaptoethanol and the samples were resolved by
[15] J. Vesely, J. Havlicek, M. Strnad, et al., Eur. J. Biochem. 224 (1994) 771–786.