Synthesis and biological evaluation of novel ethyl 2-amino-6-ferrocenyl-1, 6-dihydropyrimidine-5-carboxylates and ethyl 2-amino-6-ferrocenylpyrimidine-5- carboxylates

Article abstract of DOI:10.1016/j.jorganchem.2012.02.016

Reactions of ethyl 2-acyl-3-ferrocenylacrylates (acyl = acetyl, benzoyl, p-nitrobenzoyl) with guanidine and 1,1-dimethylguanidine furnish ethyl 2-amino-6-ferrocenyl-4-methyl(aryl)-1,6-dihydropyrimidine-5-carboxylates. Their oxidative dehydrogenation with

Full text of DOI:10.1016/j.jorganchem.2012.02.016

Journal of Organometallic Chemistry 708-709 (2012) 37e45
Contents lists available at SciVerse ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis and biological evaluation of novel ethyl 2-amino-6-ferrocenyl-1,6-
dihydropyrimidine-5-carboxylates and ethyl 2-amino-6-ferrocenylpyrimidine-
5-carboxylates
,
a
a
b
Elena I. Klimova a , Jessica J. Sanchez García , Tatiana Klimova , Teresa Ramírez Apan ,
*
Eduardo A. Vázquez López a, Marcos Flores-Alamo a, Marcos Martínez García b
a Facultad de Química, Universidad Nacional Autónoma de México, Cd. Universitaria, Coyoacán, C.P. 04510, México D.F., Mexico
b Universidad Nacional Autónoma de México, Instituto de Química, Cd. Universitaria, Coyoacán, C.P. 04510, México D.F., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Reactions of ethyl 2-acyl-3-ferrocenylacrylates (acyl ¼ acetyl, benzoyl, p-nitrobenzoyl) with guanidine
and 1,1-dimethylguanidine furnish ethyl 2-amino-6-ferrocenyl-4-methyl(aryl)-1,6-dihydropyrimidine-5-
carboxylates. Their oxidative dehydrogenation with PhI(OAc)2 results in the corresponding ethyl 2-
amino-6-ferrocenyl-4-methyl(aryl)pyrimidine-5-carboxylates. The structures of the synthesized
compounds were established on the basis of the data from 1H and 13C NMR spectroscopy and confirmed
by X-ray diffraction analysis. All compounds were tested in vitro against six human tumor cell lines U-
251, PC-3,K-562, HCT-15, MCF-7 and SKLU-1 to assess their in vitro antitumor activity. The results suggest
Received 30 November 2011
Received in revised form
13 February 2012
Accepted 15 February 2012
Keywords:
Ferrocene
Ferrocenyl-1,6-dihydropyrimidines
Ferrocenylpyrimidines
Oxidation
biological specificity towards PC-3 and K-562 cells for compounds 4aec at doses 50 mM, which are lower
than cis-platin IC’50s in the two cell lines. Additionally, peritoneal mouse macrophages (MB) were also
evaluated for compounds 4aef and 5aef.
Ó 2012 Elsevier B.V. All rights reserved.
Antitumor activity
Macrophages
1. Introduction
benzimidazole, benzindazole, pyrazoline, pyrazole, pyrimidone,
tetrahydropyridazine derivatives, etc., were reported [10ce19] to
Pyrimidines are widespread among natural bases [1], they are
abundant constituents of medicines possessing pronounced anti-
viral [2], gastric antisecretory [3], diuretic [4], antimalarial [5], anti-
HIV-1 [6,7] activities, etc. It is also known that in recent years,
bioorganometallic chemistry has developed as a rapidly growing
and maturing area which links classical organometallic chemistry
to biology, medicine, and molecular biotechnology [8]. The prop-
erties of ferrocene and its derivatives attract steady interest of
researchers in many fields of organic and organometallic chemistry.
Wide range of studies of applied properties of ferrocene is due to its
specific physical and chemical properties: high thermal stability,
high vapor pressure, low toxicity, good solubility in organic
solvents, and diverse chemical transformations [9]. The incorpo-
ration of ferrocenyl substituents into molecules of organic
compounds frequently brings about the enhancement of the bio-
logical activity of the original compounds [10]. Many ferrocenyl-
substituted nitrogen heterocycles, such as quinuclidine, triazole,
pertain to biologically active compounds. Therefore, the interest in
the synthesis of novel ferrocenylpyrimidines is quite justified.
The starting compounds for the synthesis in the pyrimidine
series usually bear two amino or an amino and an imino groups at
the same carbon atom [10c] (urea, guanidine, amidine derivatives),
which react with
derivatives, -enones, etc. The first ferrocenyl-substituted tetra-
hydropyrimidines of the type 1 were prepared by the coupling of
thiourea [20e22] and urea [23] with ferrocenyl- -enones under
sonication or in the presence of bases (Scheme 1).
In the last years, new publicationes about the synthesis of fer-
rocenylpyrimidines from -ferrocenylenones and derivatives of
b-dicarbonyl compounds, ethyl cyanoacetate
a,b
a,b
a,b
guanidines and amidines have appeared [24e26]. However, the
approaches to the synthesis of ferrocenylpyrimidines and their
properties remain largely unexplored so far.
In the present work, we accomplished the synthesis of func-
tionalized ferrocenyldihydropyrimidines and ferrocenylpyr-
imidines by the coupling of guanidine and 1,1-dimethylguanidine
with ethyl 2-acyl-3-ferrocenylacrylates. The use of 2-acylacrylates
for the preparation of pyrimidine derivatives has not hitherto
been documented.
* Corresponding author. Tel./fax: þ52 555622 5371.
E-mail address: eiklimova@yahoo.com.mx (E.I. Klimova).
0022-328X/$ e see front matter Ó 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2012.02.016

38
E.I. Klimova et al. / Journal of Organometallic Chemistry 708-709 (2012) 37e45
20e30 min (4aec) or w40 min (4def), room temperature) to afford
the corresponding pyrimidines (5aef) in ca. 65e75% yields (see
Scheme 2).
Ethyl 2-amino-6-ferrocenylpyrimidine-5-carboxylates 5aef
isolated by column chromatography on alumina represent red
substances stable on storage in solid state and in solutions (e.g., in
dichloromethane, ethanol, and acetone).
The structures of these products were established based on the
data from IR spectroscopy, 1H and 13C NMR spectroscopy, mass
spectrometry, and elemental analysis. The principal NMR spectral
features of pyrimidines 5aef are as follows. The signals for the four
protons of the C5H4 ring of ferrocene appear as two multiplets
located in a lower field compared to the singlets of the unsubstituted
cyclopentadienyl fragments. The 13C NMR spectra contain two
singlets for four carbon atoms of the substituted cyclopentadienyl
fragments of ferrocene, one at a lower field and the other at a higher
field compared with the position of the singlet for the five carbon
atoms of the unsubstituted cyclopentadienyl fragments of ferro-
cene. The signals for the CipsoFc, CH3 groups, and ethoxycarbonyl
substituents are upfield shifted compared to the corresponding
signals of amino(ferrocenyl)dihydropyrimidines 4aef.
Scheme 1.
2. Results and discussion
2.1. Chemistry
Ethyl (E)- and (Z)-2-acetyl- (2a, 2b), (E)-2-benzoyl- (2c), and (E)-
2-(p-nitrobenzoyl)-3-ferrocenylacrylates (2d) served as the start-
ing compounds (Fig. 1).
They were prepared by coupling ferrocenecarbaldehyde with
ethyl acetoacetate, ethyl benzoylacetate, and ethyl (p-nitrobenzoyl)
acetate, respectively, in the presence of piperidinium acetate
[27,28].
We found that the reaction of ethyl 2-acyl-3-ferrocenylacrylates
2aed with guanidinium carbonate (3a) and 1,1-dimethylguanidinium
sulfate (3b) in aqueous ethanol in the presence of Na2CO3 at ca.
80e85 ꢀC affords ethyl 2-amino-6-ferrocenyl-1,6-dihydropyrimidine-
5-carboxylates (4aef) (ca. 35e42%) along with some other condensa-
tion and decomposition products of the starting acrylates (Scheme 2).
Studies of their compositions and structures are currently in progress.
Ferrocenyldihydropyrimidines were isolated by column chro-
matography on alumina as orange substances stable on storage in
solid state. The structures of compounds 4aef were established
based on the data from IR spectroscopy, 1H and 13C NMR spec-
troscopy, mass spectrometry, and elemental analysis.
The systematic comparative analysis of the 1H and 13C NMR
spectra of dihydropyrimidine and pyrimidine derivatives, 4aef and
5aef, reveals the effect of aromatization. The distinct differences in
the NMR characteristics make it possible to use them for the
identification of these types of compounds from their 1H and 13C
NMR spectra (Fig. 2).
The spatial structures of compounds 4a, 4e, and 5e were
determined by X-ray diffraction of their single crystals. Crystals of
4a were obtained by crystallization from dimethyl sulfoxide, crys-
tals of 4e and 5e were obtained by crystallization from dichloro-
methane and chloroform, respectively.
The general views of molecules 4a, 4e, and 5e are shown in
Figs. 3e5, respectively, the principal geometric parameters are lis-
ted in Table 1. The data from X-ray diffraction analysis confirmed
the dihydropyrimidine structures for compounds 4a and 4e and an
aromatic structure for compound 5e.
The central fragment of the molecule 5e is a flat six-membered
ring with two nitrogen atoms. Data from the X-ray analysis show
that the C(17)eC(18) bond in the pyrimidine ring is somewhat
longer [d ¼ 1.408(2) Å] than the standard value of 1.38 Å (Ref. [1]).
The lengths of the CeFe and CeC bonds in the ferrocenyl substit-
uent as well as the geometric parameters of the ferrocene sandwich
are close to the standard values [29] (Table 2).
The following putative reaction scheme seems to rationalize the
formation of 1,6-dihydropyrimidines 4aef (Scheme 3). The two
nucleophilic sites of guanidines 3a, b attack sequentially or
simultaneously the carbon atoms in positions 3 of ethyl 2-acyl-3-
ferrocenylacrylates 2aed and the carbon atoms of the carbonyl
groups of the acyl substituents followed by dehydration of inter-
mediates (6aef).
The mechanism for the oxidative dehydrogenation with
a PhI(OAc)2/K2CO3 system proposed by Richardson et al. [30] seems
to be applicable for compounds 4aef (Scheme 4).
Thus the 1H NMR spectra of the dihydropyrimidines contain
singlets for the methine protons of the FcCH-fragments and
broadened singlets for the NH protons. The protons for the
substituted cyclopentadienyl ring of ferrocene appear as four
multiplets located in a higher field compared to the singlets of the
unsubstituted cyclopentadienyl ring. The 1H NMR spectra of
compounds 4aef contain also the signals for the methyl, aryl,
amino and ethoxycarbonyl substituents.
The 13C NMR spectra of these compounds corroborate
completely their 1,6-dihydropyrimidine structure. They contain
d ca. 90e94 ppm; four signals for the carbon
atoms of the C5H4 ring appear upfield compared to the singlet of
the carbon atoms of the C5H5 ring. The methylene carbon atoms of
the eCO2Et substituents resonate in the region of d ca. 59e62 ppm.
We found further that ferrocenyldihydropyrimidines 4aef
underwent smooth oxidative dehydrogenation with diacetox-
yiodobenzene in dichloromethane in the presence of K2CO3 (ca.
signals for CipsoFc at
Initially, PhI(OAc)2 reacts with the dihydropyrimidines to form
N(1)-[acetoxy(phenyl)-l
3-iodo]-derivatives (7aef). Their trans-
formations to the target pyrimidines 5aef occurs presumably via
intermediates (8aef) with elimination of PhI. The oxidative dehy-
drogenation of dihydropyrimidines 4aef proceeds fast and is not
virtually accompanied by the formation of side products. However,
the reduction of the yields of pyrimidines 5aec with the free amino
group is observed even upon insignificant increase in the reaction
time (the maximum duration is 30 min), presumably, due to the
involvement of the free amino group in the oxidation (the forma-
tion of nitrenes and their subsequent transformations).
Fig. 1. Structures of the starting compounds.

E.I. Klimova et al. / Journal of Organometallic Chemistry 708-709 (2012) 37e45
39
Scheme 2.
3. Pharmacology
in vitro against six human tumor cell lines: U-251 (glioma), PC-3
(prostate), K-562 (leukemia), HCT-15 (colon), MCF-7 (breast) and
SKLU-1 (lung). A primary screening at a fixed concentration of
In order to examine the applicability of four types of compounds
(4aec, 4def, 5aec and 5def) as antitumor agents, they were tested
50 mM showed cytotoxicity against the six human tumor cell lines
Fig. 2. Characteristic fragments of 1H and 13C NMR spectra: (a) and (b) of 4b, respectively; (c) and (d) of 5e.

40
E.I. Klimova et al. / Journal of Organometallic Chemistry 708-709 (2012) 37e45
4. Conclusion
The six novel 2-amino-6-ferrocenyl-1,6-dihydropyrimidine-5-
carboxylates 4aef and six novel 2-amino-6-ferrocenylpyrimidine-
5-carboxylates 5aef were prepared and structurally characterized
using spectroscopic techniques. Most of synthesized compounds
displayed modest cytotoxic activity in the micromolar range.
Compounds containing 1,6-dihydropyrimidyl moiety 4aef
appeared to be the most active against six human tumor cell lines
than the pyrimidine-5-carboxylates 5aef. Compounds 4b and 4c
exhibited the highest activity against almost tumoral cell lines y
cisplatin, which was used as reference. These results identified that
ferrocenyl(dihydro)pyrimidines are new leads in antitumor
chemotherapy. The study suggest the beneficial potential of these
leads that need to be further explored in order to discover and
develop better and yet safety therapeutic antitumor agents.
5. Experimental
Fig. 3. X-ray crystal structure of 4a.
All the solvents were dried according to standard procedures
[31] and were freshly distilled before use. Column chromatography
and TLC were carried out on alumina (Brockmann activity III). The
1H and 13C NMR spectra were recorded on a Unity Inova Varian
spectrometer (300 and 75 MHz, compounds 4def, 5b,c) and Varian
400-MR spectrometer (400 and 100 MHz, compounds 4aec, 5a,
5def) for solutions in CDCl3 and DMSO-d6, with Me4Si as the
internal standard. The IR spectra were measured with an FTIR
spectrophotometer (Spectrum RXI PerkineElmer instruments)
using KBr pellets. UV spectra were recorded on a Specord UVeVIS
spectrophotometer. The mass spectra were obtained on a Varian
MAT CH-6 instrument (EI MS, 70 eV). Elementar Analysensysteme
LECO CHNS-900 was used for elemental analyses.
tested. Cisplatin was used at the same concentration as a positive
control. Peritoneal mouse macrophages (MØ) were also deter-
mined at 50
Compounds 4b and 4c showed a 100 percent inhibition of
cellular growth at 50 M for six human tumor cell lines, compounds
mM in DMSO (Table 3).
m
4a and 4d showed better activity than cisplatin for K-562. In
addition, the IC50 values for these compounds were determined
and the results are summarized in Table 4.
The cytotoxic in vitro activity of the ferrocene derivatives varied
from IC50 > 100
for 5e and 5f to IC50 ¼ 6.6
4c and IC50 ¼ 8.7 M (PC-3) and IC50 ¼ 7.4
values, being lower than those for cisplatin (IC50 ¼ 15.9
M for K-562), make compounds 4c and 4b as
m
M (in the case of PC-3 and K-562 cancer cell lines)
M (PC-3) and IC50 ¼ 8.5 M (K-562) for
M (K-562) for 4b. These
M for PC-3
m
m
m
m
The unit cell parameters and the X-ray diffraction intensities of
4a, 4e and 5e were recorded on a Gemini (detector Atlas CCD,
Cryojet N2) diffractometer. The structure of compounds 4a, 4e and
5e were solved by the direct method (SHELXS-97 [32]) and refined
using full-matrix least-squares on F2.
m
and IC50 ¼ 15.2
m
promising antiproliferative agents against two human tumor cell
lines: PC-3 (prostate) and K-562 (leukemia).
The following reagents were purchased from Aldrich: ferroce-
necarbaldehyde, 99%; ethyl acetoacetate, 99þ%; ethyl benzoylace-
tate, 90%; ethyl 4-nitrobenzoylacetate; guanidinium carbonate, 99%;
1,1-dimethylguanidinium sulfate, 97%; diacetoxyiodobenzene, 98%.
Ethyl 2-acyl-3-ferrocenylacrylates 2aed were prepared by
condensation of ferrocenecarbaldehyde with ethyl acetoacetate,
ethyl benzoylacetate and ethyl 4-nitrobenzoylacetate, respectively,
in benzene in the presence of piperidinium acetate. The physical
and 1H NMR spectroscopic characteristics of compounds 2a, 2b, 2c
and 2d were in accord with the literature data [27,33,34].
5.1. Synthesis
5.1.1. Reactions of ethyl 2-acyl-3-ferrocenylacrylates 2aed with
guanidinium carbonate (3a) (general procedure)
A mixture of compound 2a (2bed) (10 mmol), guanidinium
carbonate 3a (1.35 g, 7.5 mmol), ethanol (60 ml), H2O (10 ml) and
2.0 g Na2CO3 was stirred for 8 h at 80 ꢀC. The solvents were
removed in vacuo and the residue was dissolved in dichloro-
methane (50 ml). The solution was mixed with Al2O3 (activity III)
(20 g) and the solvent was evaporated in air. This sorbent was
applied onto a column with Al2O3 (the height of alumina is ca.
20 cm) and the reaction products were eluted from the column first
with petroleum ether, then with a 1:2 dichloromethane e petro-
leum ether and 1:10 methanol e dichloromethane solvent system
to give dihydropyrimidines 4aec.
Ethyl 2-amino-6-ferrocenyl-4-methyl-1,6-dihydropyrimidine-
5-carboxylate (4a), orange crystals, yield 0.74 g (40%, from 2a),
Fig. 4. . X-ray crystal structure of 4e.

E.I. Klimova et al. / Journal of Organometallic Chemistry 708-709 (2012) 37e45
41
Fig. 5. X-ray crystal structure of 5e.
0.77 g (42%, from 2b), m.p. 126e128 ꢀC. IR (KBr):
n
486, 502, 607,
(CH), 59.49 (CH2), 68.62 (C5H5), 64.66, 66.09, 67.26, 67.51 (C5H4),
92.21 (CipsoFc), 102.60, 153.14, 162.12, 165.17 (4C). Anal. Calcd. for
C18H21FeN3O2: C, 58.87; H, 5.76; Fe, 15.21; N, 11.44. Found: C, 58.72;
H, 5.83; Fe, 15.34; N, 11.29%. MS: m/z 367 [M]þ.
771, 803, 821, 953, 1004, 1020, 1052, 1077, 1104, 1152, 1198, 1219,
1306, 1338, 1371, 1480, 1571, 1667, 1721, 2252, 2767, 2900, 2980,
3324 cmꢁ1 1H NMR (400 MHz, DMSO-d6):
d 1.32 (3H, t, CH3,
J ¼ 7.2 Hz), 2.32 (3H, s, CH3), 4.20 (2H, q, CH2, J ¼ 7.2 Hz), 4.28 (5H, s,
C5H5), 4.01 (1H, m, C5H4), 4.11 (1H, m, C5H4), 4.13 (1H, m, C5H4),
4.22 (1H, m, C5H4), 5.20 (1H, s, CH), 5.33 (1H, bs, NH), 7.16 (2H, bs,
Table 2
Crystal data and structure refinement parameters for compounds 4a, 4e and 5e.
NH2). 13C NMR (100 MHz, DMSO-d6):
d 14.32, 21.79 (2CH3), 47.57
Data
4a
4e
5e
Molecular formula
C18H21FeN3O2$
(CH3)2SO
C25H27FeN3O2
C25H25FeN3O2
Table 1
Selected bond lengths and angles for compounds 4a, 4e, and 5e.
Formula weight
445.36
457.35
455.33
(g molꢁ1
)
Bond lengths [Å]
Bond angles [ꢀ]
Temperature (K)
Crystal system
Space group
a(Å)
b(Å)
c(Å)
130(2)
Monoclinic
P21/c
10.0339(5)
19.6886(7)
10.8598(5)
90
103.751(4)
90
2083.90(16)
4
1.420
1.54180
6.952
130(2)
Triclinic
P-1
130(2)
Monoclinic
P21/c
7.3017(2)
26.4303(6)
10.8992(2)
90
97.251(2)
90
2086.57(8)
4
4a
N(1)eH(1N)
C(11)eH(11)
N(1)eC(11)
N(1)eC(18)
N(2)eC(18)
C(12)eC(13)
C(12)eC(11)
N(2)eC(13)
N(3)eC(18)
4e
0.82(3)
1.0000
N(1)eC(11)eH(11)
C(18)eN(1)eH(1N)
N(1)eC(18)eN(2)
C(18)eN(2)eC(13)
N(2)eC(13)eC(12)
C(13)eC(12)eC(11)
C(12)eC(11)eN(1)
C(11)eN(1)eC(18)
N(1)eC(18)eN(3)
108.9
115.0 (19)
123.04(17)
117.07 (16)
122.92(18)
117.89(17)
107.27(15)
119.45(16)
117.98(18)
10.3102(7)
10.7919(7)
11.6861(8)
115.227
111.697(6)
92.325(5)
1062.13(15)
2
1.463(2)
1.348(2)
1.332(3)
1.370(3)
1.525(3)
1.376(3)
1.336(3)
a
b
g
(ꢀ)
(ꢀ)
(ꢀ)
V(Å3)
Z
D calc. (mg/m3)
Wavelength (Å)
Absorption coefficient
1.430
1.54184
5.911
1.449
0.71073
0.751
N(1)eH(1N)
H(19)eC(19)
N(1)eC(19)
N(1)eC(20)
N(2)eC(20)
N(2)eC(17)
N(3)eC(20)
C(17)eC(18)
C(18)eC(19)
5e
0.85(3)
1.0000
N(1)eC(19)eH(19)
C(20)eN(1)eH(1N)
C(18)eC(17)eN(2)
C(19)eC(18)eC(17)
N(1)eC(19)eC(18)
C(17)eN(2)eC(20)
N(2)eC(20)eN(3)
N(3)eC(20)eN(1)
N(2)eC(20)eN(1)
109.3
123 (2)
1.476(3)
1.342(3)
1.342(3)
1.360(3)
1.341(3)
1.388(3)
1.511(3)
122.4(2)
114.3(2)
105.49(18)
116.5(2)
118.4(2)
119.4(2)
122.2(2)
(mmꢁ1
F(000)
range (ꢀ)
)
936
4.49e68.08
13,559
3799
0.0271
R1 ¼ 0.0306
wR2 ¼ 0.0825
R1 ¼ 0.0338
wR2 ¼ 0.0834
266
1.065
0.326/ꢁ0.324
480
4.63e67.82
6432
3833
0.0328
R1 ¼ 0.0400
wR2 ¼ 0.1106
R1 ¼ 0.0440
wR2 ¼ 0.1127
288
1.076
0.788/ꢁ0.347
952
3.54e26.06
14,970
4110
0.0244
R1 ¼ 0.0286
wR2 ¼ 0.0691
R1 ¼ 0.0345
wR2 ¼ 0.0708
283
1.027
0.353/ꢁ0.311
q
Reflections collected
Reflections independent
Rint
Final R indices [I > 2
s(I)]
N(1)eC(20)
N(1)eC(17)
N(2)eC(20)
N(2)eC(19)
N(3)eC(20)
C(17)eC(18)
C(18)eC(19)
1.346(2)
1.343(2)
1.347(2)
1.336(2)
1.354(2)
1.408(2)
1.399(2)
C(18)eC(17)eN(1)
C(17)eC(18)eC(19)
N(1)eC(20)eN(2)
C(19)eN(2)eC(20)
N(2)eC(19)eC(18)
N(3)eC(20)eN(1)
N(2)eC(20)eN(3)
121.29(15)
116.59(15)
125.84(15)
116.46(14)
122.56(15)
116.66(15)
117.50(15)
R indices (all data)
Refinable parameters
Goodness-of-fit on F2
Maximum/minimum
residual electron
density (eÅꢁ3
)

42
E.I. Klimova et al. / Journal of Organometallic Chemistry 708-709 (2012) 37e45
Scheme 3.
Ethyl 2-amino-6-ferrocenyl-4-phenyl-1,6-dihydropyrimidine-
5-carboxylate (4b), orange crystals, yield 0.95 g (40%), m.p.
198e199 ꢀC. IR (KBr):
483, 511, 621, 780, 812, 821, 973, 1001, 1019,
5.1.2. Reactions of ethyl 2-acyl-3-ferrocenylacrylates 2aed with
1,1-dimethylguanidine 3b
n
Following the general procedure, the reactions of 2aed
(5 mmol) with 1,1-dimethylguanidinium sulfate 3b (2.04 g,
7.5 mmol) in a mixture of ethanol (70 ml) and water (10 ml) in the
presence of Na2CO3 (2.5 g) (6 h at 80 ꢀC) afforded compounds 4d,
4e, and 4f.
1042,1083,1101,1161, 1200,1223,1314,1337,1392,1475, 1548,1573,
1682, 1716, 2259, 2781, 2910, 3004, 3341 cmꢁ1 1H NMR (400 MHz,
DMSO-d6):
d
0.83 (3H, t, CH3, J ¼ 6.9 Hz), 3.76 (2H, q, CH2, J ¼ 6.9 Hz),
4.23 (5H, s, C5H5), 3.99 (1H, m, C5H4), 4.06 (1H, m, C5H4), 4.09 (1H,
m, C5H4), 4.18 (1H, m, C5H4), 5.12 (1H, s, CH), 6.42 (1H, bs, NH), 7.10
(2H, bs, NH2), 7.16 (2H, m, C6H5), 7.22 (3H, m, C6H5). 13C NMR
Ethyl 2-dimethylamino-6-ferrocenyl-4-methyl-1,6-dihydropy
rimidine-5-carboxylate (4d), orange crystals, yield 0.87 g (45%,
(100 MHz, DMSO-d6):
d
13.89 (CH3), 47.82 (CH), 56.46 (CH2), 69.04
2a), 0.81 g (41%, from 2b), m.p. 145e146 ꢀC. IR (KBr):
n 495, 796,
(C5H5), 66.01, 66.66, 67.93, 68.29 (C5H4), 89.18 (CipsoFc), 121.05,
126.99, 128.72 (C6H5), 99.13,134.87,147.42,157.84,160.11 (5C). Anal.
Calcd. for C23H23FeN3O2: C, 64.35; H, 5.40; Fe, 13.01; N, 9.79%.
Found: C, 64.48; H, 5.29; Fe, 13.12; N, 9.82%. MS: m/z 429 [M]þ.
826, 947, 1000, 1068, 1098, 1120, 1183, 1208, 1231, 1245, 1337, 1373,
1477, 1563, 1640, 1724, 2903, 2938, 2976, 3077, 3091, 3333 cmꢁ1 1H
NMR (300 MHz, CDCl3):
d
1.30 (3H, t, CH3, J ¼ 7.2 Hz), 2.28 (3H, s,
CH3), 3.15 (6H, s, 2CH3), 4.17 (2H, q, CH2, J ¼ 7.2 Hz), 4.13 (5H, s,
C5H5), 3.94 (1H, m, C5H4), 4.01 (1H, m, C5H4), 4.08 (1H,m, C5H4),
4.26 (1H, m, C5H4), 5,21 (1H, s, CH), 5.38 (1H, bs, NH). 13C NMR
Ethyl
rimidine-5-carboxylate (4c), orange crystals, yield 0.95 g (40%),
m.p. 195e196 ꢀC. IR (KBr):
486, 503, 609, 784, 806, 821, 969, 1002,
2-amino-6-ferrocenyl-4-(4-nitrophenyl)-1,6-dihydropy
n
(75 MHz, CDCl3): d 14.66, 24.25, 36.96 (4CH3), 49.32 (CH), 59.12
1021, 1039, 1080, 1105, 1152, 1202, 1217, 1315, 1337, 1389, 1479, 1547,
(CH2), 68.26 (C5H5), 64.95, 67.26, 67.34, 67.67 (C5H4), 94.41 (CipsoFc),
99.92, 154.32, 158.43, 167.46 (4C).Anal. Calcd. for C20H25FeN3O2: C,
60.77; H, 6.38; Fe,14.13; N,10.63. Found: C, 60.61; H, 6.49; Fe, 14.25;
N, 10.58%. MS: m/z 395 [M]þ.
1570, 1678, 1712, 2264, 2779, 2908, 3006, 3325 cmꢁ1 1H NMR
(400 MHz, DMSO-d6):
d
0.85 (3H, t, CH3, J ¼ 7.2 Hz), 3.80 (2H, q, CH2,
J ¼ 7.2 Hz), 4.23 (5H, s, C5H5), 3.99 (1H, m, C5H4), 4.07 (1H, m, C5H4),
4.09 (1H, m, C5H4), 4.19 (1H, m, C5H4), 5.14 (1H, s, CH), 6.56 (1H, bs,
NH), 7.20 (2H, bs, NH2), 7.38 (2H, d, C6H4, J ¼ 8.7 Hz), 8.13 (2H, d,
Ethyl 2-dimethylamino-6-ferrocenyl-4-phenyl-1,6-dihydropy
rimidine-5-carboxylate (4e), orange crystals, yield 0.92 g (40%),
C6H4, J ¼ 8.7 Hz). 13C NMR (100 MHz, DMSO-d6):
d
13.83 (CH3),
m.p. 159e160 ꢀC. IR (KBr):
n 491, 699, 778, 808, 816, 999, 1001, 1021,
48.01 (CH), 58.54 (CH2), 68.45 (C5H5), 65.02, 65.83, 67.45, 67.57
(C5H4), 94.28 (CipsoFc), 122.65, 129.75 (C6H4), 99.46, 146.29, 149.75,
156.03, 158.04, 165.68 (6C). Anal. Calcd. for C23H22FeN4O4: C, 58.25;
H, 4.68; Fe, 11.77; N, 11.80%. Found: C, 58.34; H, 4.57; Fe, 11.87; N,
11.69%. MS: m/z 474 [M]þ.
1037, 1055, 1089, 1152, 1217, 1282, 1325, 1344, 1522, 1571, 1620,
1687,1737, 2933, 2975, 3074, 3303 cmꢁ1 1H NMR (300 MHz, CDCl3):
d
0.9 (3H, t, CH3, J ¼ 6.9 Hz), 3.11 (6H, s, 2CH3), 3.91 (2 H, q, CH2,
J ¼ 6.9 Hz), 4.19 (5H, s, C5H5), 4.06 (2H, m, C5H4), 4.14 (1H, m, C5H4),
4.38 (1H, m, C5H4), 5.32 (1H, s, CH), 5.54 (1H, s, NH), 7.30 (3H, m,
Scheme 4.

E.I. Klimova et al. / Journal of Organometallic Chemistry 708-709 (2012) 37e45
43
Table 3
Inhibition of human tumor cells lines by compounds (4aef, 5aef) and peritoneal mouse macrophages (MØ) at 50
m
M in DMSO.a
Compounds % of growth inhibition
U-251
PC-3
K-562
HCT-15
MCF-7
SKLU-1
MØ
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
65.4 ꢂ 4.6
100
100
75.3 ꢂ 1.1
100
100
94.6 ꢂ 5.4
100
100
68.9 ꢂ 15.0
100
100
77.7 ꢂ 6.7
100
100
59.6 ꢂ 4.4
100
100
35.6 ꢂ 4.7
34.0 ꢂ 2.8
72.5 ꢂ 3.1
71.5 ꢂ 2.9
69.5 ꢂ 4.2
31.5 ꢂ 0.3
24.5 ꢂ 4.5
36.6 ꢂ 3.4
42.9 ꢂ 1.6
34.1 ꢂ 5.3
31.3 ꢂ 4.3
26.2 ꢂ 1.3
22.9 ꢂ 5.9
51.8 ꢂ 5.7
42.1 ꢂ 6.5
57.8 ꢂ 6.6
31.6 ꢂ 3.5
37.7 ꢂ 5.0
65.3 ꢂ 1.7
43.8 ꢂ 8.0
15.5 ꢂ 0.5
11.0 ꢂ 1.9
89.9 ꢂ 8.1
89.7 ꢂ 10.2
71.9 ꢂ 10.3
71.8 ꢂ 28.2
81.1 ꢂ 18.9
63.9 ꢂ 13.0
65.0 ꢂ 12.2
52.3 ꢂ 6.9
37.4 ꢂ 2.3
32.4 ꢂ 0.1
86.7 ꢂ 4.1
91.3 ꢂ 8.7
53.85 ꢂ 2.1
69.3 ꢂ 6.1
64.3 ꢂ 9.3
64.1 ꢂ 10.5
56.6 ꢂ 2.1
72.4 ꢂ 4.6
15.65 ꢂ 0.8
12.1 ꢂ 4.7
74.4 ꢂ 2.1
30.2 ꢂ 4.4
35.4 ꢂ 11.8
62.0 ꢂ 10.1
77.9 ꢂ 17.9
54.98 ꢂ 9.7
67.3 ꢂ 0.2
44.6 ꢂ 4.3
29.5 ꢂ 1.2
14.8 ꢂ 2.7
81.8 ꢂ 7.1
78.9 ꢂ 7.6
42.9 ꢂ 12.0
63.4 ꢂ 6.6
74.3 ꢂ 9.2
40.98 ꢂ 4.7
39.8 ꢂ 0.005
56.1 ꢂ 12.5
42.2 ꢂ 4.0
34.0 ꢂ 2.9
77.9 ꢂ 2.5
40.5 ꢂ 3.6
44.8 ꢂ 4.1
36.6 ꢂ 15.4
38.9 ꢂ 3.6
46.74 ꢂ 4.3
50.5 ꢂ 1.3
43.6 ꢂ 4.2
38.4 ꢂ 7.6
25.3 ꢂ 3.2
95.8 ꢂ 2.1
Cisplatin
a
Results express mean ꢂ standard error (SEM) obtained from 2 independent experiments performed at 48 h.
C6H5), 7.39(2H, m, C6H5). 13C NMR (75 MHz, CDCl3):
d
13.98, 37.30
III) (10 g) and the solvent was evaporated in air. This sorbent was
applied onto a column with Al2O3 (the height of alumina is ca.
20 cm) and the reaction products were eluted from the column
with a 1:2 ether e petroleum ether solvent system to give
compounds 5aef, respectively.
(3CH3), 50.17 (CH), 59.28 (CH2), 68.49 (C5H5), 65.32, 67.50, 67.66,
68.04 (C5H4), 93.84 (CipsoFc), 127.24, 127.96, 128.92 (C6H5), 100.03,
129.06, 142.13, 154.42, 167.64 (5C). Anal. Calcd. for C25H27FeN3O2: C,
65.66; H, 5.95; Fe, 12.21; N, 9.18. Found: C, 65.53; H, 6.07; Fe, 12.30;
N, 9.06%. MS: m/z 457 [M]þ.
Ethyl 2-amino-6-ferrocenyl-4-methylpyrimidine-5-carboxylate
Ethyl
dihydropyrimidine-5-carboxylate (4f), orange crystals, yield
0.88 g (35%), m.p. 158e159 ꢀC. IR (KBr):
486, 530, 708, 757, 812,
2-dimethylamino-6-ferrocenyl-4-(4-nitrophenyl)-1,6-
(5a), red crystals, yield 1.4 g (70%), m.p. 120e121 ꢀC. IR (KBr):
n
491, 793, 820, 923, 1002, 1061, 1086, 1120, 1181, 1200, 1233, 1232,
1331, 1368, 1472, 1565, 1642, 1719, 2931, 2934, 2971, 3065, 3018,
n
861, 965, 1002, 1021, 1033, 1083, 1105, 1152, 1226, 1287, 1335, 1363,
3321 cmꢁ1 1H NMR (400 MHz, CDCl3):
d
1.34 (3H, t, CH3, J ¼ 7.2 Hz),
1387, 1470, 1515, 1570, 1658, 1718, 2940, 3096, 3225 cmꢁ1 1H NMR
2.34 (3H, s, CH3), 4.36 (2H, q, CH2, J ¼ 7.2 Hz), 4.11 (5H, s, C5H5), 4.40
(300 MHz, CDCl3):
d
0.93 (3H, t, CH3, J ¼ 6.9 Hz), 3.16 (6 H, s, 2CH3),
(2H, m, C5H4), 4.80 (2H, m, C5H4), 5.32 (2H,bs, NH2). 13C NMR
3.92 (2H, q, CH2, J ¼ 6.9 Hz), 4.19 (5H, s, C5H5), 4.05 (1H, m, C5H4),
4.10 (1H, m, C5H4), 4.17 (1H, m, C5H4), 4.37 (1H, m, C5H4), 5.33 (1H,
s, CH), 5.48 (1H, bs, NH), 7.49 (2H, d,C6H4, J ¼ 8.7 Hz), 8.15 (2H, d,
(100 MHz, CDCl3): d 13.99, 22.18 (2CH3), 61.47 (CH2), 70.11 (C5H5),
69.29, 70.55 (C5H4), 80.38 (CipsoFc), 115.94, 161.52, 164.95, 165.28,
169.62 (5C). Anal. Calcd. for C18H19FeN3O2: C, 59.20; H, 5.24; Fe,
15.30; N,11.50. Found: C, 59.27; H, 5.16; Fe,15.43; N,11.39%. MS: m/z
365 [M]þ.
C6H4, J ¼ 8.7 Hz). 13C NMR (75 MHz, CDCl3):
d 14.09, 37.26 (3 CH3),
50.03 (CH), 59.56 (CH2), 68.51 (C5H5), 67.51, 67.84, 68.23, 68.92
(C5H4), 93.58 (CipsoFc), 122.65, 129.75 (C6H4), 100.57, 127.09, 147.29,
149.45, 154.51, 166.68 (6C). Anal. Calcd. for C25H26FeN4O4: C, 59.77;
H, 5.22; Fe, 11.12; N, 11.15. Found: C, 59.65; H, 5.27; Fe, 11.08; N,
11.20%. MS: m/z 502 [M]þ.
Ethyl 2-amino-6-ferrocenyl-4-phenylpyrimidine-5-carboxylate
(5b), red crystals, yield 1.8 g (72%), m.p. 149e150 ꢀC. IR (KBr):
n
482, 531, 706, 760, 811, 858, 962, 1001, 1018, 1032, 1080, 1105, 1147,
1223, 1281, 1335, 1362, 1374, 1449, 1499, 1664, 1715, 2924, 3066,
3231 cmꢁ1 1H NMR (300 MHz, CDCl3):
d
0.99 (3H, t, CH3, J ¼ 7.2 Hz),
5.1.3. Reactions of ethyl 2-dimethylamino-6-ferrocenyl-4-methyl(or
-aryl)-1,6-dihydropyrimidine-5-carboxylates 4aef with
diacetoxyiodobenzene
4.10 (2H, q, CH2, J ¼ 7.2 Hz), 4.14 (5H, s, C5H5), 4.44 (2H, m, C5H4),
4.91 (2H, m, C5H4), 5. 23 (2H, bs, NH2), 7.43 (3H, m, C6H5), 7.58 (2H,
m, C6H5). 13C NMR (75 MHz, CDCl3):
d 13.79 (CH3), 61.23 (CH2),
A mixture of compounds 4aef (3 mmol), diacetoxyiodobenzene
(1.3 g, 4 mmol), dichloromethane (60 ml), and 1.5 g Na2CO3 was
stirred for 1 h at 20 ꢀC. The mixture was mixed with Al2O3 (activity
69.67 (C5H5), 69.34, 70.35 (C5H4), 81.14 (CipsoFc), 128.17, 128.37,
129.15 (C6H5), 104.91, 139.64, 161.73, 165.43, 166.29, 169.81 (6C).
Anal. Calcd. for C23H21FeN3O2: C, 64.65; H, 4.56; Fe, 13.07; N, 9.83.
Found: C, 64.73; H, 4.47; Fe, 13.12; N, 9.71%. MS: m/z 427 [M]þ.
Table 4
Ethyl
carboxylate (5c), red crystals, yield 1.8 g (72%), m.p. 163e164 ꢀC.
IR (KBr): 477, 534, 711, 762, 812, 852, 960, 1002, 1021, 1038,
1079, 1100, 1148, 1229, 1291, 1339, 1363, 1395, 1472, 1523, 1568,
1661,1718, 2942, 3096, 3229 cmꢁ1 1H NMR (300 MHz, CDCl3):
1.06
2-amino-6-ferrocenyl-4-(4-nitrophenyl)pyrimidine-5-
Inhibitory concentrationa (IC50 mM) values obtained in PC-3 and K-562 cell lines for
compounds 4aef and 5aef in DMSO.
n
Compound
Cell line IC50
PC-3
(m
M)a
K-562
d
(3H, t, CH3, J ¼ 7.2 Hz), 4.13 (2H, q, CH2, J ¼ 7.2 Hz), 4.15 (5H, s, C5H5),
4.48 (2H, m, C5H4), 4.91 (2H, m, C5H4), 5.18 (2H, bs, NH2), 7.74 (2H, d,
C6H4, J ¼ 8.4 Hz), 8.30 (2H, d, C6H4, J ¼ 8.4 Hz). 13C NMR (75 MHz,
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
12.7 ꢂ 1.1
8.7 ꢂ 0.7
6.6 ꢂ 0.5
27.3 ꢂ 2.4
48.2 ꢂ 3.7
43.8 ꢂ 4.0
64.9 ꢂ 5.9
49.4 ꢂ 1.4
17.1 ꢂ 1.5
31.2 ꢂ 2.1
>100
38.3 ꢂ 1.6
7.4 ꢂ 0.1
8.5 ꢂ 0.2
21.0 ꢂ 1.2
55.5 ꢂ 5.3
21.6 ꢂ 2.0
34.0 ꢂ 0.5
24.5 ꢂ 2.3
18.0 ꢂ 0.9
31.0 ꢂ 1.5
>100
CDCl3): d 13.82 (CH3), 61.92 (CH2), 70.40 (C5H5), 69.88, 71.26 (C5H4),
79.96 (CipsoFc), 123.64, 129.35 (C6H4), 115.65, 137.60, 147.23, 154.87,
161.75, 163.15, 168.97 (7C). Anal. Calcd. for C23H20FeN4O4: C, 58.49;
H, 4.27; Fe, 11.82; N, 11.86. Found: C, 58.57; H, 4.19; Fe, 11.74; N,
11.75%. MS: m/z 472 [M]þ.
Ethyl
carboxylate (5d), red crystals, yield 1.4 g (70%), m.p. 55e56 ꢀC. IR
(KBr): 482, 503, 596, 647, 730, 797, 812, 893, 1021, 1044, 1085,
2-dimethylamino-6-ferrocenyl-4-methylpyrimidine-5-
>100
15.9 ꢂ 1.2
>100
15.2 ꢂ 1.4
Cisplatin
n
a
1104, 1167, 1200, 1222, 1286, 1329, 1368, 1413, 1439, 1525, 1571,
Results express the mean IC50 ꢂ standard error (SEM) obtained from three
independent experiments performed at 48 h.
1704, 2897, 2928, 2979, 3085 cmꢁ1 1H NMR (400 MHz, CDCl3):

44
E.I. Klimova et al. / Journal of Organometallic Chemistry 708-709 (2012) 37e45
d
1.29 (3H, t, CH3, J ¼ 7.2 Hz), 2.36 (3H, s, CH3), 3.24 (6H, s, 2CH3),
4.30 (2H, q, CH2, J ¼ 7.2 Hz), 4.08 (5H, s, C5H5), 4.35 (2H, m, C5H4),
4.80 (2H, m, C5H4). 13C NMR (100 MHz, CDCl3):
14.01, 22.67, 36.76
trichloroacetic acid. The plates were incubated at 4 ꢀC for 1 h,
washed with tap H2O, and air-dried. The trichloroacetic-acid-fixed
cells were stained by the addition of 0.4% SRB. Free SRB solution
was the removed by washing with 1% aqueous acetic-acid. The
plates were the air-dried, and the bound dye was solubilized by the
d
(4CH3), 61.13 (CH2), 70.01 (C5H5), 69.41, 69.93 (C5H4), 82.09 (Cip-
soFc), 113.16, 160.85, 164.11, 164.46, 170.26 (5C). Anal. Calcd. for
C20H23FeN3O2: C, 61.10; H, 5.89; Fe, 14.20; N, 10.68. Found: C, 60.97;
H, 5.94; Fe, 14.14; N, 10.73%. MS: m/z 393 [M]þ. Ethyl 2-
dimethylamino-6-ferrocenyl-4-phenylpyrimidine-5-carboxylate
addition of 100 ml 10 mM-unbuffered Tris base. The plates were
placed on a shaked for 5 min, prior analysis. Optical densities were
determined in an Ultra Microplated Reader (Elx 808: Bio-Tek
Instruments, Inc., Winooski, VT, USA) using test wavelength of
515 nm.
(5e), red crystals, yield 1.55 g (68%), m.p. 114e116 ꢀC. IR (KBr):
n 476,
497, 529, 641, 699, 761, 810, 821, 999, 1024, 1037, 1041, 1070, 1127,
1168,1199,1217,1293,1354,1383,1404,1441,1492,1518,1549,1582,
1710, 2864, 2927, 2958, 3074, 3002 cmꢁ1 1H NMR (400 MHz,
5.2.2. Isolation and culture of primary peritoneal macrophages
Isolation and culture of primary peritoneal macrophages were
conducted as described elsewhere [36]. Swiss female mice,
25e30 g, were treated in accord with the Animal Care and Use
Committee (NOM-062-Z00-1999). Mice were injected intraperito-
neally with 1 ml of 3% (wt volꢁ1) thioglycollate 3 days before har-
vesting. Peritoneal exudate cells were harvested, washed and
suspended in DMEM. Peritoneal exudate cells were seeded into
48 well plates (Becton Dickinson, Oxnard, CA, USA) at a density of
1 ꢃ 106 cells mlꢁ1, and then incubated for 2 h at 37 ꢀC in a 5% CO2
incubator. Non-adherent cells were washed off and cultured in
DMEM supplemented with 10% FCS.
CDCl3):
d
0.99 (3H, t, CH3, J ¼ 7.2 Hz), 3.29 (6H, s, 2 CH3), 4.08 (2H, q,
CH2, J ¼ 7.2 Hz), 4.13 (5H, s, C5H5), 4.38 (2H, m, C5H4), 4.93 (2H, m,
C5H4), 7.41 (3H, m, C6H5), 7.63(2H, m, C6H5). 13C NMR (100 MHz,
CDCl3):
d 13.60, 36.86 (3CH3), 61.20 (CH2), 70.402 (C5H5), 69.85,
70.16 (C5H4), 81.92 (CipsoFc), 128.11, 128.17, 129.13 (C6H5), 112.96,
139.48, 160.89, 164.74, 164.85, 170.15 (6C). Anal. Calcd. for
C25H25FeN3O2: C, 65.95; H, 5.53; Fe, 12.27; N, 9.22. Found: C, 70.06;
H, 5.61; Fe, 12.34; N, 9.16%. MS: m/z 455 [M]þ.
Ethyl 2-dimethylamino-6-ferrocenyl-4-(4-nitrophenyl)pyrimi-
dine-5-carboxylate (5f), red crystals, yield 1.8 g (72%), m.p.
156e157 ꢀC. IR (KBr):
n 482, 503, 596, 730, 797, 812, 893, 1021, 1044,
1085, 1103, 1167, 1200, 1222, 1286, 1368, 1410, 1439, 1525, 1570,
Cell viability was determined by the MTT colorimetric assay.
1704, 2897, 2927, 2979, 3085 cmꢁ1 1H NMR (300 MHz, CDCl3):
Briefly, 10
ml MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,3-diphenylte-
d
1.04 (3H, t, CH3, J ¼ 7.2 Hz), 3.29 (6H, s, 2CH3), 4.10 (2H, q, CH2,
trazolium bromide) was added to the medium after 48 h incubation
with the test samples. After 4 h culture, the medium was removed
and DMSO were added to dissolved the formazan solution
produced in the cells. The optical density of the formazan solution
was measured with a microplated reader at 414 nm [37].
J ¼ 7.2 Hz), 4.13 (5H, s, C5H5), 4.42 (2H, m, C5H4), 4.91 (2H, m, C5H4),
7.79 (2H, d, C6H4, J ¼ 8.4 Hz), 8.27 (2H, d, C6H4, J ¼ 8.4 Hz). 13C NMR
(75 MHz, CDCl3):
d 13.68, 36.86 (3CH3), 61.48 (CH2), 70.10 (C5H5),
69.83, 70.45 (C5H4), 81.37 (CipsoFc), 123.31, 129.30 (C6H4), 112.75,
145.67, 148.14, 160.72, 162.41, 165.82, 169.54 (7C). Anal. Calcd. for
C25H24FeN4O4: C, 60.02; H, 4.83; Fe, 11.16; N, 11.20. Found: C, 59.96;
H, 4.76; Fe, 11.24; N, 11.11%. MS: m/z 500 [M]þ.
Acknowledgements
This work was supported by the DGAPA (Mexico.grant IN
211112) and CONACyT (Mexico, grant 100970).
5.2. Biology
5.2.1. Cytotoxicity assay
Appendix A. Supplementary material
The compound were screened in vitro against human cancer cell
lines: HCT-15 (human colorectal adenocarcinoma), MCF-7 (human
mammary adenocarcinoma), K-562 (human chronic myelogenous
leukemia), U-251 (human glioblastoma), PC-3 (human prostatic
adenocarcinoma), SKLU-1 (human lung adenocarcinoma), cell lines
were supplied by National Cancer Institute (USA). The human
tumor cytotoxicity was determined using the protein-binding dye
sulforhodamine B (SRB) in microculture assay to measure cell
growth, as described in the protocols established by the NCI (Monks
et al. [35],) The cell lines were cultured in RPMI-1640 medium
CCDC 851259, 851260 and 851261 contain the supplementary
crystallographic data for this paper. These data can be obtained free
of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
References
[1] D.J. Brown, The Pyrimidines, John Wiley& Sons, New York, 1994.
[2] L.M. Beauchamp, B.L. Serling, J.E. Kelsey, K.K. Biron, P. Collins, J. Selway, J.-
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m
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