New Imidazo[1,2-c]pyrimidin-5(6H)-Ones Derived from Cytosine: Synthesis, Structure, and Cytotoxic Activity

Article abstract of DOI:10.1002/jhet.2243

This work describes the synthesis of 8-iodoimidazo[1,2-c]pyrimidin-5(6H)-one 2 from 5-iodocytosine 1. This compound was subjected to Suzuki cross-coupling reaction with aryl and heteroarylboronic acids. After optimization, 10 products 3_a-j were

Full text of DOI:10.1002/jhet.2243

1
382
New Imidazo[1,2-c]pyrimidin-5(6H)-Ones Derived from Cytosine:
Vol 52
Synthesis, Structure, and Cytotoxic Activity
a,b
a
c
d
e,f
Josef Jansa, * Antonín Lyčka, Zdeňka Padělková, Martin Grepl, Petr Konečný,
e,f
e,f
Marián Hajdúch, and Petr Džubák *
a
Research Institute for Organic Syntheses (VUOS), Rybitví 296, CZ-533 54 Pardubice-Rybitví, Czech Republic
b
Department of Organic Chemistry, Faculty of Science, Palacký University, 17. listopadu 1192/12
CZ-771 46 Olomouc, Czech Republic
Department of General and Inorganic Chemistry, Faculty of Chemical Technology, University of Pardubice,
c
Studentská 573, CZ-532 10 Pardubice, Czech Republic
Farmak, a.s., Na Vlčinci 16/3, CZ-771 17 Olomouc, Czech Republic
d
e
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University, Olomouc,
Puškinova 6, CZ-775 20 Olomouc, Czech Republic
f
University Hospital Olomouc, Ivana Petroviče Pavlova 6, CZ-77520 Olomouc, Czech Republic
*
E-mail:josef.jansa@vuos.com; dzubakp@gmail.com
Received January 30, 2014
DOI 10.1002/jhet.2243
Published online 31 July 2014 in Wiley Online Library (wileyonlinelibrary.com).
This work describes the synthesis of 8-iodoimidazo[1,2-c]pyrimidin-5(6H)-one 2 from 5-iodocytosine 1.
This compound was subjected to Suzuki cross-coupling reaction with aryl and heteroarylboronic acids. After
optimization, 10 products 3a–j were obtained in good yields 61-90%. Cytotoxic activity of all new products
was evaluated on seven tumor cell lines including resistant variants and on normal human fibroblasts. Two
derivatives showed promising biological activity and good therapeutic index in the case of 3h.
J. Heterocyclic Chem., 52, 1382 (2015).
INTRODUCTION
Biological activity of some imidazo[1,2-c]pyrimidin-5
6H)-ones has been reported. Compounds with this scaf-
(
The reaction of 2-aminopyridine, 2-aminopyrimidine, and
-aminopyrimidine derivatives with α-halogenocarbonyl
compounds is well known to produce corresponding
imidazo-fused derivatives [1–3]. Imidazo[1,2-a]pyridine
and imidazo[1,2-a]pyrimidine scaffolds were studied for
their extensive pharmacological properties, for example,
antiviral [4,5], antitubercular [6,7], anticancer [8], cytotoxic
fold are selective GABA receptor ligands [27]. Recently,
A
4
the large family was patented as allosteric modulators of
mGluR5 receptors for treatment of neurologic and psychi-
atric disorders [28]. Nucleosides with this scaffold were
tested as antiviral agents [29] and showed activity against
hepatitis B virus [30,31] and HIV [32].
In this article, we present the access for functionalization
of imidazo[1,2-c]pyrimidin-5(6H)-one scaffold by an aryl
or heteroaryl ring. 8-Aryl derivatives 3a–j were obtained
from iododerivative 2 by Suzuki–Miyaura cross-coupling
reaction (Scheme 1). Cytotoxic activity of all products
was evaluated on tumor cell lines and normal human fibro-
blasts (Table 1).
[
9], antiparasitic [10], antibacterial [2], antifungal [11], car-
diotonic [12], and antiinflammatory [13]. Some derivatives
act as commercial drugs, from which zolpidem is widely
known nonbenzodiazepine hypnotic [14].
Imidazo[1,2-c]pyrimidines were studied for similarly
large spectrum of biological activities, such as antimicro-
bial [15], antimycobacterial [16], antitubercular [7], ino-
tropic [17], antiinflammatory, analgesic, antipyretic, and
ulcerogenic [18]. These compounds also act as Syk family
kinases inhibitors [19,20] and dopamine D4 receptor
ligands [21].
RESULTS AND DISCUSSION
Chemistry.
The starting 8-iodoimidazo[1,2-c]pyrimidin-5
Imidazo[1,2-c]pyrimidine scaffolds are also formed by re-
action of adenine and cytosine bases with α-halogenocarbonyl
compounds. Kochetkov and coworkers first reported reaction
of 9-methyladenine and 1-methylcytosine with chloroacetal-
dehyde, which produced corresponding ethenoderivatives
(6H)-one 2 was prepared in 86% yield by cyclization of 5-
iodocytosine 1 with chloroacetaldehyde in the presence of
sodium acetate. Iodine atom of 2 was substituted by an aryl or
heteroaryl ring via Suzuki–Miyaura cross-coupling [33] without
any need of protection of parent heterocycle 2 (Scheme 1).
Inspired by coupling of cytosine nucleosides with
boronic acids [34], we first selected acetonitrile–water 2 : 1
solvent system with tetrakis(triphenylphosphine)palladium
as catalyst. However, these conditions could only be used
[
22]. Because this type of reaction became an interesting
pathway for synthesis of modified nucleic acid bases with
useful biological, fluorescence, and base-pairing proper-
ties [23–26].
©
2014 HeteroCorporation

September 2015
New Imidazo[1,2-c]pyrimidin-5(6H)-nes Derived from Cytosine: Synthesis,
1383
Structure, and Cytotoxic Activity
2 2
Scheme 1. Synthesis of target imidazo[1,2-c]pyrimidin-5(6H)-ones, yields in parentheses. Reagents and conditions: (a) ClCH CHO, NaOAc, H O, 80°C, 4 h;
2 2 2 2 3
(b) ArB(OH) , Pd(dppf)Cl , EtOH/H O, Na CO , reflux, 24 h.
Table 1
Summary of cytotoxic activities (IC50, μM).
a
Compound
CCRF-CEM
CEM-DNR bulk
K562
K562-Tax
A549
HCT116
HCT116p53-/-
MRC-5
BJ
2
46.15
50.00
8.59
45.74
36.87
28.20
32.63
50.00
1.45
48.99
45.69
2.84
47.37
49.72
15.20
47.75
35.81
10.74
34.21
48.12
17.71
50.00
50.00
44.63
45.51
8.85
50.00
50.00
10.13
47.31
44.69
40.42
9.04
50.00
5.07
50.00
50.00
50.00
46.14
20.20
49.35
37.53
34.83
50.00
50.00
42.49
50.00
50.00
18.61
35.58
15.12
35.52
35.89
25.57
18.47
18.75
8.83
48.33
50.00
7.00
50.00
50.00
0.66
50.00
43.71
50.00
48.69
50.00
7.29
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
g
h
i
41.56
34.84
32.89
20.28
49.72
18.84
42.78
46.94
39.79
37.70
34.20
23.48
48.38
28.72
43.35
46.07
50.00
44.45
50.00
50.00
50.00
35.27
50.00
50.00
44.65
50.00
18.75
18.75
50.00
50.00
j
a
Cytotoxic activity was determined by MTT assay following a 3-day incubation. Values represents means of IC50 from three independent experiments
with standard deviation ranging from 10 to 25% of the average values.
Scheme 2. Numbering of atoms in compounds 2 and 3a–j for NMR purposes.
for coupling with phenylboronic and biphenyl-4-boronic
acid but not for 4-methoxyphenylboronic acid (~30%
conversion). When ethanol–water 2 : 1 was used as a solvent,
complete conversion was observed in the coupling with
Pd(dppf)Cl in ethanol–water 4 : 1 must be used to obtain com-
plete conversion to 3j. Coupled products 3a–j were obtained
in 61–90% isolated yields.
Products were identified and characterized by IR, MS, and
NMR spectroscopy (for atom numbering see Scheme 2).
5-Iodocytosine 1 showed three broad NH protons that mean
that it exist as imino tautomer in hexadeuterated dimethyl sulf-
2
4
-methoxyphenylboronic acid but lower (~80%) for cou-
pling with 4-cyanophenylboronic acid. Then, the Pd(dppf)Cl2
was used as catalyst with the aim to obtain complete
conversion in case of all used boronic acids. With 3 mol%
of this catalyst in ethanol–water 2 : 1, products 3a–i were
coupled in nearly quantitative conversions, detected by
thin-layer chromatography. Only in the coupling of 2 with
strongly deactivated 4-pyridylboronic acid, 5 mol% of
oxide (DMSO-d ) (Scheme 3). The same tautomerism was
6
observed in 5-bromo and 5-iodo-N-1-sulfonylated cytosine
1
13
derivatives [35]. Complete assignment of H and C chemical
shifts in compounds 2 and 3a–j was carried out on the basis of
1D and 2D (correlation spectroscopy, heteronuclear multiple
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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384
J. Jansa, A. Lyčka, Z. Padělková, M. Grepl, P. Konečný, M. Hajdúch, and P. Džubák
Vol 52
Scheme 3. Preferred imino tautomerism of 5-iodocytosine 1.
to CEM-DNR bulk, daunorubicin-resistant cancer cell line
expressing P-glycoprotein (adenosine triphosphate-depen-
dent transporter) and proteins from MRP family and
K562-Tax, paclitaxel-resistant cancer cell line, expressing
P-glycoprotein only [42]. Similarly, 3h is showing prefer-
ential activity to CCRF-CEM, A549, and HCT116p53À/
À cell line with deleted p53 protein and favorable thera-
peutic index to nontumor cell lines 24.32. Because P-gly-
coprotein positive cells are highly adenosine triphosphate
dependent and the functional p53 protein is contributing
to the cells survival in glucose and aminoacid deprived
cells as well [43,44], the observed activity is suggesting disrup-
tion of energy metabolic pathways of the sensitive tumor cells.
Cell cycle study of the most active compound 3h has
been performed on CCRF-CEM lymphoblasts at 1× and
quantum coherence, and heteronuclear multiple bond corre-
1
5
lation) NMR experiments (Table 2). N-NMR spectra of
selected derivatives were also measured.
Structure of product 3a was confirmed by X-ray crystal-
lography. Compound 3a (Fig. 1) crystallizes in the ortho-
rhombic space group Pbca, with eight molecules within the
unit cell. To the best of our knowledge, only one analogous
structure of imidazo(1,2-c)pyrimidin-5(6H)-one derivate in po-
sition 8 by a carbon atom the 3,4-etheno-5-methoxymethyl-2′-
deoxycytidine [36] has been reported so far. Other two
partially saturated compounds as for example N-methyl-6-(2-
chloroethyl)-5,7-dioxo-1,2,3,5,6,7-hexahydroimidazo[1,2-c]
pyrimidine-8-carboxamide [37] or methyl 2-(1-benzyl-8-formyl-
5
× IC50 concentration within 24 h treatment. Finally, we
did not observe any effect on cell cycle proliferation,
DNA and RNA synthesis even in highest concentration
(Table 3).
CONCLUSION
In conclusion, 11 new imidazo[1,2-c]pyrimidine-5(6H)-
7
3
-methoxy-5-oxo-1,2,3,5-tetrahydroimidazo[1,2-c]pyrimidin-
-yl)acetate [38] and two compounds with fused aromatic
rings [39,40] that resemble the structural motif of 3a were also
found within the crystallographic database. Typical interatomic
separations and angles for each functional group are present
within the structure of 3a [41]. A high degree of π-electron
conjugation is interrupted between atoms C2-C3 and C6-N3
where the bond lengths are bit longer than is usually found
for analogous distances in aromatic compounds. The torsion
of the phenyl ring from the plane defined by the heterocyclic
part of the molecule is 45.68(3)°.
Because of the presence of interatomic N2-H2⋯N3
interactions in the crystal packing of 3a (Fig. 2), the
chains of zig-zag oriented molecules (interplanar angle
between two neighboring imidazopyrimidine rings is 41.0
ones have been synthesized. General conditions for the
Suzuki–Miyaura cross-coupling reaction were found, and
all compounds were properly characterized. Cytotoxic ac-
tivity is dependent on the substitution of the aryl ring, in-
troduced by Suzuki–Miyaura cross-coupling reaction.
Two derivatives showed promising activity with the lowest
IC = 1.45 μM for 3h on CCRF-CEM and IC = 2.84 μM
for 3b on resistant CEM-DNR bulk cell line. Moderate ac-
tivity of some other derivatives was observed. Compound
3h showed favorable therapeutic index to nontumor cell
lines 24.32.
50
50
EXPERIMENTAL
(2)° N2-H2⋯N3 (Fig. 3) are observed within the crystal lattice.
Biological activity. The cytotoxic activity was analyzed on
human cancer cell lines, resistant variants (CCRF-CEM, CEM-
DNR, K562, K562-Tax, A549, HCT116, HCT116p53À/À,
and A549) and on normal human cells (BJ and MRC-5 –
normal cycling fibroblasts) to analyze the therapeutic index
that is based on the ratio between the IC50 for normal human
cells and cancer cell lines. The SAR ought to be evaluated for
individual cell lines independently, because it may vary
because of different tissue origin and potential molecular
targets in particular cell lines. However, in our case, the trend
of activity was very similar for all tested cell lines indicating
similar or identical target(s), and thus, we will discuss SAR
generally (Table 1).
General. The NMR spectra were measured on Bruker Avance
1
(Rheinstetten, Germany) II 400 at 400.13 MHz ( H), 100.62 MHz
1
3
15
19
(
C), 40.54 MHz ( N), and 376.50 MHz ( F) or Bruker Avance
1
13
5
00 at 500.13 MHz ( H) and 125.67 MHz ( C) in DMSO-d
6
1
13
(Rheinstetten, Germany) at ambient temperature. The H and
C
chemical shifts were referenced to the residual signal of the solvent
1
13
15
19
(
δ =2.50 for H and 39.52 for C). The N and F chemical
shifts were referred to external nitromethane in a coaxial capillary
and internal CFCl , respectively, both having δ =0.0.
3
The X-ray data for colorless crystal of 3a (Fig. 1) were
obtained at 150 K using Oxford Cryostream low-temperature de-
vice on a Nonius KappaCCD diffractometer with MoK radiation
α
(λ = 0.71073 Å), a graphite monochromator, and the ϕ and χ scan
mode. Data reductions were performed with DENZO-SMN [45].
The absorption was corrected by integration methods [46]. Struc-
tures were solved by direct methods (Sir92) [47] and refined by
Generally, the cytotoxic activity of the compounds is
rather low; only two derivatives 3b and 3h are showing
promising cytotoxic activity (the lowest IC = 1.45/
2
full matrix least square based on F (SHELXL97) [48]. Hydrogen
50
atoms were mostly localized on a difference Fourier map; how-
ever, to ensure uniformity of treatment of crystal, all hydrogen
2
.84 μM). Interestingly, 3b is showing preferential activity
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2015
New Imidazo[1,2-c]pyrimidin-5(6H)-nes Derived from Cytosine: Synthesis,
1385
Structure, and Cytotoxic Activity
Table 2
1
13
15
H, C, and some N chemical shifts (ppm) of compounds 2 and 3a–j.
2
3a 3b
3c
3d
Position
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
b
1
—
7.90
7.37
—
À122.6
114.1
132.0
À190.8
146.1
À243.3
134.2
59.8
145.2
—
—
—
—
—
7.87
7.45
—
À129.4
112.6
132.0
À189.4
145.7
À249.1
126.5
110.5
144.6
133.1
127.7
128.3
127.5
—
—
7.89
7.49
—
À129.2
112.6
129.0
À189.3
145.7
À248.9
126.5
110.0
144.5
132.2
128.1
126.6
139.1
139.6
—
7.85
7.45
—
—
—
7.85
7.45
—
—
2
3
4
112.5
132.0
—
145.7
—
125.4
110.3
144.7
125.4
128.8
113.7
158.7
54.9
112.6
132.0
—
145.6
—
126.9
109.1
144.2
131.9
129.2
128.2
131.9
—
b
5
6
—
—
—
—
—
b,c
11.81
7.58
—
—
—
—
—
—
—
11.90
7.52
—
—
—
7.97
7.42
7.33
—
11.94
7.63
—
—
—
11.81
7.44
—
—
—
7.92
6.99
—
11.95
7.57
—
7
8
8
8
8
8
8
a
—
-1′
-2′
-3′
-4′
8.11
7.75
8.02
7.44
—
—
d
X
—
3.78
—
(
Continues)
Figure 1. The molecular structure (ORTEP 50% probability level) of 3a.
Selected interatomic distances (Å), angles and interplanar angle (°): O1-
C1 1.213(2), C1-N1 1.397(2), N1-C5 1.386(2), C5-C6 1.351(2), C6-N3
Figure 2. Interatomic N2-H2∙∙∙N3 interactions of 3a, view along axis a.
Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
1
1
.390(2), N3-C2 1.320(2), C2-C3 1.439(2), C3-C4 1.352(2), C4-N2
.381(2), N2-C1 1.361(2), C3-C7 1.481(3); C6-N3-C2 105.29(14), N3-
[
C2-N1 110.60(15), C2-N1-C5 107.37(14), C2-N1-C1 126.04(15), N1-
C1-N2 112.78(15), C1-N2-C4 124.24(14); C2-C3-C4 versus C8-C7-C12
4
5.68(3). [Color figure can be viewed in the online issue, which is available
2
2
2
2
2 2
R
int = ∑∣F
o
À Fo,mean∣/∑F
o
, GOF = [∑(w(F
o
À F
c
) )/(Ndiffrs
at wileyonlinelibrary.com.]
½
Nparams)] for all data, R(F) = ∑∣∣F ∣ À ∣F ∣∣/∑∣F ∣for
o
c
o
2
2
2 2
2 2 ½
observed data, wR(F ) = [∑(w(F
o
À F
c
o
) )/(∑w(F ) )] for
all data.
Crystallographic data for structural analysis have been deposited
with the Cambridge Crystallographic Data Centre, CCDC no.
were recalculated into idealized positions (riding model) and
assigned temperature factors Hiso(H) = 1.2 Ueq(pivot atom)
with C―H 0.93 Å for hydrogen atoms in aromatic rings
and hydrogen atom for N―H bond was localized and assign
from Fourier map, standard calculated distance for N―H
bond is 0.86 Å.
9
31506 for 3a. Copies of this information may be obtained free of
charge from The Director, CCDC, 12 Union Road, Cambridge
CB2 1EY, UK (fax: +44-1223-336033; E-mail: deposit@ccdc.
cam.ac.uk or www: http://www.ccdc.cam.ac.uk).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1
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J. Jansa, A. Lyčka, Z. Padělková, M. Grepl, P. Konečný, M. Hajdúch, and P. Džubák
Vol 52
Table 2
Table 2
1
13
15
H, C, and some N c (h Ce mo ni tc i an lu se hd i)f ts (ppm) of compounds 2 and 3a–j.
a
3e
3f
3g 3h
3i
1
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
J(C,H)
—
—
—
7.89
7.49
—
—
—
7.88
7.48
—
—
—
7.88
7.46
—
—
—
7.87
7.49
—
—
7
7
.85
.45
112.7
132.0
—
112.7
132.0
—
112.7
132.0
—
112.7
132.0
—
112.8
132.0
—
196.8
190.5
—
—
—
1.96
145.6
—
128.1
108.9
144.1
—
145.6
—
—
145.6
—
—
145.5
—
—
145.4
—
—
—
182.5
—
—
1
12.09
7.78
—
—
—
8.30
7.95
—
12.02
7.71
—
—
—
8.17
7.99
—
12.10
7.79
—
—
—
8.28
7.87
—
11.91
7.68
—
—
—
7.53
7.12
7.84
—
7.69
128.5
109.1
144.0
139.2
127.8
129.5
134.8
192.6
127.9
109.4
144.2
137.5
129.3
127.4
129.4
167.2
128.7
109.5
143.9
138.0
127.9
132.2
108.5
119.0
125.0
105.7
143.4
134.5
124.7
127.1
125.4
—
—
—
137.3
—
8
7
.22
.72
128.0
125.0
127.6
124.4
168.0
167.9
182.5
—
—
—
11.01
12.90
—
a
19
15
(
δ
F) = À60.6.
b
(
δ
N).
c1 15
1
J ( N, H) = 90.1 Hz.
1
δ ( H/ C) = À/139.7, 7.76/126.7, 7.53/129.1, 7.42/127.6
d
13
The chromatographic preseparation parameters: column Luna C18,
μm, 50 × 2mm i.d. (Phenomenex, Torrance, CA), mobile phase
3
acetonitrile/water 50/50 with 0.1% of formic acid, flow rate 200 μL/
min, the column temperature 30°C. Sample preparation was obtained
using following procedure: The 1 mg of sample was dissolved in
10 mL of solvent acetonitrile/water 5/5 (1 min sonication), and then,
70 μL of this solution and 930 μL of the same solvent were added
into vial and mixed before injection of 5 μL. High-resolution mass
spectrometer Exactive based on orbitrap mass analyzer was equipped
with heated electrospray ionization. The spectrometer was tuned to
obtain maximum response for m/z 70–600. The source parameters
were set to the following values: heated electrospray ionization
temperature 250°C, spray voltage +3.0 kV (positive mode), transfer
capillary temperature 300°C, and sheath gas/aux gas (nitrogen) flow
rates 35/10. The HRMS spectra of target peaks allowed an evaluation
of their elemental composition because of high intensities of their
protonated molecules. The identification of the respective structures
was performed with less than 0.6 ppm difference between
experimental and theoretically calculated value.
Figure 3. Crystal packing of 3a, view along axis b. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
Single crystals of 3a (obtained by slow evaporation of acetone
solution) were of sufficient quality for X-ray diffraction measure-
ment. The crystallographic data and structure refinement parame-
ters for compound 3a are C12
9 3
H N O, M = 211.22, orthorhombic,
Pbca, a = 7.2310(3), b = 13.4800(9), c = 19.9031(12) Å, α = β =
3
À3
γ = 90°, Z = 8, V = 1940.03(19) Å , D = 1.446 g.cm , μ = 0.097
c
À1
mm
,
Tmin/Tmax = 0.985/0.991; À8 ≤ h ≤ 9, À17 ≤ k ≤ 14,
À25 ≤ l ≤ 25; 16,294 reflections measured (θmax = 27.50°),
IR spectra were recorded on Nicolet 6700 Fourier transform
À1
1
6,153 independent (Rint = 0.0383), 1644 with I> 2σ(I), 146
infrared spectroscopy over the range of 400–4000 cm . Elemen-
parameters, S= 1.117, R1(obs. data) = 0.0484, wR2(all data) = 0.0940;
tal analyses were performed on a Thermo Flash 2000 CHNS
experimental organic analyzer. Melting points were determined
on Stuart SMP3 apparatus (Barloworld Scientific, Staffordshire,
UK). Reaction progress was monitored by thin-layer chromatog-
raphy (dichloromethane/methanol 5:1 as mobile phase), which
À3
maximum, minimum residual electron density = 0.275, À0.211 eǺ
.
High-resolution mass spectrometry (HRMS) analyses were
measured with Thermo Exactive instrument (Thermo Scientific,
Waltham, MA, USA). The injection was performed by autosampler
of high-performance liquid chromatography apparatus Accela 1250.
was performed on SiO 60 F254 plates with ultraviolet detection
2
Table 3
Effects of compound 3h on cell cycle, apoptosis, and DNA/RNA synthesis in CCRF-CEM lymphoblasts (% of positive cells).
Ser10
pH3
Compound
<G1
G0/G1
S
G2/M
BrDU
BrU
Control
8.7
7.2
7.2
43.0
41.8
44.9
39.4
41.0
38.3
17.6
17.2
16.8
1.8
2.0
2.0
43.8
44.4
39.7
40.7
41.6
41.0
3
3
h 1× IC50
h 5× IC50
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2015
New Imidazo[1,2-c]pyrimidin-5(6H)-nes Derived from Cytosine: Synthesis,
1387
Structure, and Cytotoxic Activity
at 254 nm. Column chromatography was performed on silica gel
off and washed with 50 mL of water. Crude product was dried
on air overnight and recrystallized from dimethylformamide
6
0. All starting materials were commercially available.
(
25 mL for 3b, 50 mL for 3g). Product 3b was washed with
diethylether (2 × 10 mL), product 3g with acetone (3 × 10 mL).
-Phenylimidazo[1,2-c]pyrimidin-5(6H)-one (3a). White
crystals, yield 1.27 g (88%). mp 241–243°C, IR (KBr): 3429,
Chemistry. 5-Iodocytosine (1) [49].
Cytosine (100 g,
0
.9 mol) was dissolved in the solution of 80 g of potassium
8
hydroxide in 1500 mL of water. Then, the pulverized iodine
229 g, 0.902 mol) was added, and the mixture was stirred for
0 min at r.t. Thereafter, the reaction mixture was heated to
(
7
3
1
7
156, 3113, 3066, 2933, 2820, 2737, 2633, 1731, 1620, 1548,
511, 1494, 1445, 1411, 1288, 1274, 1247, 1142, 1114, 886,
reflux in the course of 70 min and refluxed for 2 h. After cooling
to r.t., 30 mL of 20% solution of sodium thiosulfate was added,
reaction mixture was stirred overnight, neutralized with 3.5 mL
of acetic acid, and filtered and washed with 200 mL of water.
Filter cake was suspended in 1250 mL of water and boiled for
À1
71, 760, 743, 735, 704, 668, and 648, 574 cm . HRMS m/z
+
[
M+H] Calcd. for C H N O: 212.08184, found 212.08173.
12 10
3
Anal. Calcd. for C H N O: C, 68.24; H, 4.29; N, 19.89; found:
12
9 3
C, 68.24; H, 4.25; N, 19.79.
-(Biphenyl-4-yl)imidazo[1,2-c]pyrimidin-5(6H)-one (3b). White
crystals, yield 1.78 g (81%). mp 280–287°C, IR (KBr): 3438,
241, 3146, 3127, 3080, 2940, 2898, 1732, 1618, 1544, 1485,
1407, 1286, 1278, 1262, 1243, 1144, 1109, 918, 836, 827, 758,
8
10 min. Then cooled overnight and filtered and washed with
200 mL of water and 200 mL of methanol. Product was dried at 60°
3
C/30 mbar. Yield 174.5 g (82%), off-white crystals, mp 238–243°C
dec. (lit. 225–245°C dec. ), H-NMR (400 MHz, DMSO-d ): δ
49
1
6
À1
+
7
40, 719, 693, abd 649, 576 cm . HRMS m/z [M+H] Calcd.
6.54 (br s, 1H, NH), 7.80 (br s+s, 2H, NH+CH), 10.80 (br s, 1H,
13
for C H N O: 288.11314, found 288.11309. Anal. Calcd. for
1
8 14 3
NH); C-NMR (100.6 MHz, DMSO-d
-Iodoimidazo[1,2-c]pyrimidin-5(6H)-one (2).
mixture of 50 g of 5-iodocytosine (0.211 mol) and 43.3 g
6
): δ 55.3, 149.5, 155.9, 164.7.
C
4
18
H
13
N
3
O: C, 75.25; H, 4.56; N, 14.63; found: C, 75.58; H,
.61; N, 14.74.
-(4-Methoxyphenyl)imidazo[1,2-c]pyrimidin-5(6H)-one
3c). White crystals, yield 1.61 g (90%). mp 248–250°C, IR
KBr): 3435, 3160, 3068, 2999, 2932, 2845, 1723, 1709, 1619,
8
To the
8
(
(
0.528 mol) of sodium acetate in 500 mL of water, 47.8 mL
0.422 mol) of chloroacetaldehyde (57% solution in water) was
(
(
added. The mixture was stirred for 4 h at 80°C. Then, the
mixture was cooled to r.t., product was filtered, washed with
1
1
610, 1546, 1515, 1462, 1407, 1298, 1276, 1256, 1183, 1141,
113, 1024, 882, 845, 829, 800, 742, 652, 621, 572, 563, and
4
00 mL of water and 100 mL of methanol. Product was dried,
À1
+
yield 47.4 g (86%), brown powder. Analytical sample was
recrystallized from acetone and dried at 50–60°C/30 mbar to
obtain off-white crystals, mp 230–235°C dec., IR (KBr): 3434,
519 cm
.
HRMS m/z [M+H] Calcd. for
C
H
13
12
N
O
3
2
:
242.09240, found 242.09249. Anal. Calcd. for C13
64.72; H, 4.60; N, 17.42; found: C, 64.79; H, 4.59; N, 17.32.
8-(4-Chlorophenyl)imidazo[1,2-c]pyrimidin-5(6H)-one
H
11
N
3
O
2
: C,
3
1
5
2
2
218, 3144, 3128, 3099, 2947, 1716, 1604, 1537, 1377, 1331,
269, 1246, 1165, 1138, 1116, 774, 707, 642, 622, 574, and
46 cm
(3d).
White crystals, yield 1.41 g (75%). mp 262–264°C, IR
(KBr): 3437, 3146, 3130, 3067, 2743, 1747, 1617, 1549, 1510,
1486, 1412, 1402, 1287, 1266, 1246, 1144, 1112, 1099, 922,
À1
+
.
HRMS m/z [M+H] Calcd. for
C
6
H
5
IN O:
3
61.94718, found 261.94704. Anal. Calcd. for C
6
H
4
IN
3
O: C,
À1
7.61; H, 1.54; N, 16.10. Found: C, 27.82; H, 1.51; N, 15.92.
General procedure for Suzuki–Miyaura cross-coupling. The
820, 811, 777, 747, 593, 490, and 463 cm . HRMS m/z [M
+
+H] Calcd. for C H ClN O: 246.04287, found 246.04287.
12
9
3
mixture of 2 g (7.66mmol) of 2, 11.50 mmol of appropriate boronic
Anal. Calcd. for C H ClN O: C, 58.67; H, 3.28; N, 17.10; Cl,
12 8 3
acid, 3.25g (30.66 mmol) of sodium carbonate, and 0.168g
14.43; found: C, 58.79; H, 3.23; N, 16.98; Cl, 14.55.
(
0.23 mmol) of Pd(dppf)Cl
2
(0.280g (0.38 mmol) in case of 3j)
8-[4-(Trifluoromethyl)phenyl]imidazo[1,2-c]pyrimidin-5(6H)-
one (3e). White crystals, yield 1.52 g (71%). mp 235–236°C,
IR (KBr): 3443, 3074, 2935, 2845, 2745, 2638, 1739, 1724,
1619, 1550, 1517, 1414, 1329, 1278, 1246, 1161, 1141, 1132,
1122, 1115, 1070, 1018, 893, 851, 744, 684, 652, 634, 619,
was placed to the flask, and argon atmosphere was introduced by
three vacuum/argon cycles. First, 40 mL of ethanol and then
2
in case of 3j), and an additional three vacuum/argon cycles
were carried out. Reaction mixture was heated to reflux for
2
0mL of water were added (48 mL of ethanol and 12 mL of water
À1
+
603, 586, and 413 cm . HRMS m/z [M+H] Calcd. for
C H F N O: 280.06922, found 280.06938. Anal. Calcd. for
4 h, and then, the product was isolated from reaction mixture
1
3 9 3 3
(
for isolation procedure for 3b and 3g, refer to the succeeding
C H F N O: C, 55.92; H, 2.89; N, 15.05; found: C, 56.02; H,
13 8 3 3
texts). Thereafter, the mixture was cooled to r.t., 50 mL of
water was added, and then, 6 mL of 35% hydrochloric acid was
added slowly. Ethanol was evaporated at 50°C under reduced
pressure. A little of active charcoal was added, and the mixture
was stirred at 80°C for 10 min. The mixture was filtered over
cellite and washed twice with 10 mL of 4% hydrochloric acid at
2.92; N, 14.95.
4-(5-Oxo-5,6-dihydroimidazo[1,2-c]pyrimidin-8-yl)benzaldehyde
(3f). Off-white solid, yield 1.52 g (83%). mp 267–270°C, IR
(KBr): 3438, 3147, 3131, 3068, 2859, 1748, 1695, 1615, 1607,
1568, 1550, 1503, 1487, 1411, 1395, 1293, 1279, 1248, 1215,
1167, 1143, 1126, 1112, 922, 824, 747, 685, 650, 583, 556,
À1
+
8
8
0°C. After cooling to r.t., pH of the filtrate was adjusted to
–9 by solid sodium carbonate. Precipitated product was
13 10 3 2
493, and 476 cm . HRMS m/z [M+H] Calcd for C H N O :
240.07675, found 240.07669. Anal. Calcd. for C H N O :
1
3 9 3 2
filtered, washed with 100 mL of water, and dried. Analytical
samples of all products were recrystallized from acetone
C, 65.27; H, 3.79; N, 17.56; found: C, 64.98; H, 3.91; N,
17.42.
(
3
dimethylformamide in case of 3f and 3g) and dried at 50–60°C/
0 mbar. Pure product 3j was obtained after column
4-(5-Oxo-5,6-dihydroimidazo[1,2-c]pyrimidin-8-yl)benzoic acid
(3g). Off-white solid, yield 1.45 g (74%). mp >350°C dec., IR
(KBr): 3435, 3147, 3131, 3064, 2850, 1744, 1701, 1678, 1611,
1548, 1502, 1486, 1437, 1412, 1327, 1291, 1279, 1245, 1184,
1137, 1112, 922, 845, 774, 758, 746, 701, 587, 556, 483, and
chromatography with dichloromethane/methanol 10 : 1 as a mobile
phase (for NMR chemical shifts of compounds 3a-j, refer to Table 2).
Isolation of products 3b and 3g.
mixture for 24 h (refer to the previous texts) and cooling to r.t.,
0 mL of water was added, and ethanol was evaporated at 50°C
under reduced pressure. The resulting suspension was filtered
After refluxing reaction
À1
+
442 cm . HRMS m/z [M+H] Calcd. for C H N O : 256.07167,
13 10 3 3
5
13 9 3 3
found 256.07152. Anal. Calcd. for C H N O : C, 61.18; H, 3.55;
N, 16.46; found: C, 60.82; H, 3.67; N, 16.35.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1
388
J. Jansa, A. Lyčka, Z. Padělková, M. Grepl, P. Konečný, M. Hajdúch, and P. Džubák
Vol 52
4
-(5-Oxo-5,6-dihydroimidazo[1,2-c]pyrimidin-8-yl)benzonitrile
1–4 h. After this incubation period, the formazan produced was
dissolved by the addition of 100 μL/well of 10% aq SDS (pH
5.5), followed by a further incubation at 37°C overnight. The
optical density (OD) was measured at 540nm with a Labsystem
iEMS Reader MF (Labsystem, Helsinki, Finland). Tumor cell
(
(
1
6
3h). White crystals, yield 1.38g (84%). mp 310–325°C dec., IR
KBr): 3432, 3219, 3175, 3086, 2942, 2227, 1723, 1624,
606, 1549, 1509, 1413, 1287, 1274, 1243, 1143, 844, 727,
11, 571, 544, and 500 cm . HRMS m/z [M+H] Calcd. for
À1
+
C H N O: 237.07709, found 237.07699. Anal. Calcd. for
survival (IC ) was calculated using the following equation:
1
3
9
4
50
C H N O: C, 66.10; H, 3.41; N, 23.72; found: C, 66.42; H,
IC= (ODdrug-exposed well/mean ODcontrol wells)× 100%. The
IC50 value, the drug concentration lethal to 50% of the tumor
cells, was calculated from appropriate dose–response curves.
1
3 8 4
3
.48; N, 23.35.
-(Thiophen-2-yl)imidazo[1,2-c]pyrimidin-5(6H)-one (3i). White
crystals, yield 1.01 g (61%). mp 249–255°C, IR (KBr): 3420,
8
Cell cycle and apoptosis analysis.
Subconfluent CCRF-
3
1
7
157, 3069, 2922, 2847, 2743, 1726, 1709, 1611, 1553, 1519,
499, 1405, 1287, 1250, 1241, 1141, 1114, 878, 847, 830, 731,
18, 658, 638, 591, and 571 cm . HRMS m/z [M+H] Calcd.
CEM cells (American Tissue Culture Collection), seeded at the
density of 5.10 cells/mL in 6-well panels, were cultivated with
5
À1
+
the 1× or 5× IC50 of tested compound in a humidified CO2
incubator at 37°C in RPMI 1640 cell culture medium containing
10% fetal calf serum, 10 mM glutamine, 100 U/mL penicillin,
and 100 μg/mL streptomycin. Control containing vehicle was
harvested at the same time point (24 h). Cells were washed with
cold phosphate buffered saline (PBS) and fixed in 70% ethanol
overnight at 20°C. The next day, the cells were washed in
hypotonic citrate buffer, treated with RNase (50 μg/mL), stained
with propidium iodide, and analyzed by flow cytometry using a
488 nm single beam laser (Becton Dickinson, San Jose, CA).
Cell cycle was analyzed in the program MODFITLT (Verity),
and apoptosis was measured in logarithmic model as a
percentage of the particles with propidium content lower than
for C H N OS: 218.03826, found 218.03820. Anal. Calcd. for
C
1
0
8 3
10
H
7
N
3
OS: C, 55.29; H, 3.25; N, 19.34; S, 14.76; found: C,
5
5.09; H, 3.10; N, 19.09; S, 14.56.
-(Pyridin-4-yl)imidazo[1,2-c]pyrimidin-5(6H)-one (3j). White
crystals, yield 1.01 g (62%). mp 260–263°C dec., IR (KBr):
8
3
1
1
436, 3137, 3082, 2936, 2736, 1732, 1723, 1620, 1604,
556, 1501, 1418, 1326, 1284, 1258, 1135, 1117, 1072,
008, 890, 828, 758, 679, 666, 649, 618, 579, 568, 549,
À1
+
and 497 cm . HRMS m/z [M+H] Calcd. for C H N O:
2
1
1
9
4
13.07709, found 213.07698. Anal. Calcd. for C11
H
8
N
4
O:
C, 62.26; H, 3.80; N, 26.40; found: C, 61.98; H, 3.92; N,
6.56.
Biological activity. MTT assay. As for the cytotoxic MTT
2
cells in G0/G1 phase (<G1) of the cell cycle. Half of the
assay [50], all cells were purchased from the American Tissue
Culture Collection, unless otherwise indicated: The CCRF-CEM
line are highly chemosensitive T-lymphoblastic leukemia cells,
K562 cells were derived from patient with acute myeloid
leukemia with bcr-abl translocation, A549 line is lung
adenocarcinoma, HCT116 is colorectal tumor cell line, and its p53
gene knockdown counterpart (HCT116p53-/-, Horizon Discovery,
Cambridge, UK) is a model of human cancers with p53 mutation
frequently associated with poor prognosis. The daunorubicin-
resistant subline of CCRF-CEM cells (CEM-DNR bulk) and
paclitaxel-resistant subline K562-Tax were selected in our
laboratory by the cultivation of maternal cell lines in increasing
concentrations of daunorubicin or paclitaxel, respectively, and
were characterized well in the article by Nosková et al. [42]. The
Ser10
sample was used for pH3
antibody (Sigma) labeling and
subsequent flow cytometry analysis of mitotic cells.
BrDU incorporation analysis.
Cells were cultured and
treated as for cell cycle analysis. Before harvesting, they were
pulse labeled with 10 μM 5-bromo-2-deoxyuridine (BrDU) for
3
0 min. The cells were trypsinized, fixed with ice-cold 70%
ethanol, incubated on ice for 30 min, washed with PBS, and
resuspended in 2M HCl for 30 min at room temperature to
denature their DNA. Following neutralization with 0.1M
2 4 7
Na B O , the cells were washed with PBS containing 0.5%
Tween-20 and 1% bovine serum albumin. They were then
stained with primary anti-BrdU antibody (Exbio) for 30 min at
room temperature in the dark. Cells were than washed with PBS
and stained with secondary antimouse-FITC antibody (Sigma).
The cells were then washed with PBS and incubated with
propidium iodide (0.1 mg/mL) and RNase A (0.5 mg/mL) for
CEM-DNR bulk cells overexpress
P-glycoprotein, MRP, and
LRP protein, while K562-Tax cells overexpress only P-
glycoprotein, both proteins belong to family of ABC transporters
and are involved in primary and/or acquired multidrug resistance
phenomenon. MRC-5 and BJ are nontumor human fibroblasts.
1
h at room temperature in the dark and finally analyzed by flow
cytometry using a 488 nm single beam laser (FACSCalibur,
Becton Dickinson).
2
The cells were maintained in Nunc/Corning 80cm plastic tissue
BrU incorporation analysis. Cells were cultured and treated
as for cell cycle analysis. Before harvesting, they were pulse
labeled with 1 mM 5-bromouridine (BrU) for 30 min. The cells
were fixed in 1% buffered paraformaldehyde with 0.05% of
NP-40 in room temperature for 15 min and then in the
refrigerator overnight. They were then washed in 1% glycin in
PBS, washed in PBS, and stained with primary anti-BrDU
antibody cross-reacting to BrU (Exbio) for 30 min at room
temperature in the dark. Cells were than washed with PBS and
stained with secondary antimouse-FITC antibody (Sigma).
Following the staining, the cells were washed with PBS and
fixed with 1% PBS buffered paraformaldehyde with 0.05% of
NP-40. The cells were then washed with PBS, incubated with
propidium iodide (0.1 mg/mL) and RNase A (0.5 mg/mL) for
culture flasks and cultured in cell culture medium (DMEM/RPMI
1
1
640 with 5 g/L glucose, 2 mM glutamine, 100 U/mL penicillin,
00 μg/mL streptomycin, 10% fetal calf serum, and NaHCO3).
Cell suspensions were prepared and diluted according to the
particular cell type and the expected target cell density (25,000–
3
0,000 cells/well based on cell growth characteristics). Cells were
added by pipette (80 μL) into 96-well microtiter plates. Inoculates
were allowed a preincubation period of 24h at 37°C and 5% CO2
for stabilization. Fourfold dilutions, in 20μL aliquots, of the
intended test concentration were added to the microtiter plate
wells at time zero. All test compound concentrations were
examined in duplicate. Incubation of the cells with the test
compounds lasted for 72h at 37°C, in a 5% CO atmosphere at
2
1
00% humidity. At the end of the incubation period, the cells
1
h at room temperature in the dark, and finally analyzed by
flow cytometry using 488 nm single beam laser
FACSCalibur, Becton Dickinson).
were assayed using MTT. Aliquots (10 μL) of the MTT stock
solution were pipetted into each well and incubated for further
a
(
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2015
New Imidazo[1,2-c]pyrimidin-5(6H)-nes Derived from Cytosine: Synthesis,
1389
Structure, and Cytotoxic Activity
Acknowledgments. This work was supported by Ministry of
Industry and Trade of the Czech Republic (project FR-TI1/202) and
by grants IGA UP LF_2014_010, CZ.1.05/2.1.00/01.0030 and
study stay by CZ.1.07/2.4.00/17.0015. We thank Jan Vaněček for
recording of infrared spectra and Dagmar Zvolánková for
technical help.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet