Synthesis, crystal structure and anticancer activity of novel derivatives of ethyl 1-(4-oxo-8-aryl-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)form ate

Article abstract of DOI:10.1016/j.ejmech.2006.01.016

Synthesis and anticancer activity of ethyl 1-(4-oxo-8-aryl-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)form ates (7-12) are presented. The title compounds were obtained by two independent synthesis methods from 1-aryl-2-hydrazono-imidazolidines (1

Full text of DOI:10.1016/j.ejmech.2006.01.016

European Journal of Medicinal Chemistry 41 (2006) 539–547
http://france.elsevier.com/direct/ejmech
Short communication
Synthesis, crystal structure and anticancer activity
of novel derivatives of ethyl 1-(4-oxo-8-aryl-
4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)formate
Krzysztof Sztanke a,*, Jolanta Rzymowska b, Maciej Niemczyk b, Izabela Dybała c,
Anna E. Kozioł c
a Department of Synthesis and Technology of Drugs, Professor Feliks Skubiszewski Medical University, 6 Staszica Street, 20-081 Lublin, Poland
b Department of Biology and Genetics, Professor Feliks Skubiszewski Medical University, 6 Staszica Street, 20-081 Lublin, Poland
c Faculty of Chemistry, Maria Curie-Skłodowska University, 20-031 Lublin, Poland
Received 1 September 2005; received in revised form 3 January 2006; accepted 11 January 2006
Available online 20 March 2006
Abstract
Synthesis and anticancer activity of ethyl 1-(4-oxo-8-aryl-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)formates (7–12) are presented.
The title compounds were obtained by two independent synthesis methods from 1-aryl-2-hydrazono-imidazolidines (1-aryl-2-hydrazino-imidazo-
lines) (1–6) by cyclocondensation reaction with diethyl 2-(hydroxyimino)malonate (A) and diethyl 2-oxomalonate (B). Molecular structure of
synthesized compounds was confirmed by IR, 1H NMR, EI-MS spectra, elemental analysis and X-ray crystallography for 12. Compounds 10 and
11 exhibited anticancer activity towards following cancer cells: LS180 (ECACC 87021202, human Caucasian colon adenocarcinoma cells), SiHa
(ECACC 85060701, uterus cancer cells), T47D (ECACC 85102201, human breast carcinoma cells). Compound 10 was found to be the most
active against SiHa cancer line; its GI was 41 and 52%, respectively in both examined concentrations (10 and 50 μg ml–1), whereas compound 11
had the highest potential to reduce the growth of LS180 and SiHa cancer lines, especially in a higher dose (50 μg ml–1). Moreover, the distinctly
marked lower cytotoxicity of tested compounds against normal cell lines (HSF, human skin fibroblast cells and Vero African Green Monkey
Kidney cells, GMK clone) and almost two-times higher against cancer cell lines was confirmed. Also antibacterial activity of starting 1-(2-
1
chlorophenyl)-2-hydrazonoimidazolidine hydroiodide (4) is presented. Molecular structure of 4 was confirmed by IR, H NMR, 13C NMR, EI-
MS spectra, elemental analysis and 1H–1H COSY, HMBC and HMQC correlations. The marked antibacterial activity for this compound in
relation to Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 with equal minimal inhibitory concentration values of
15.62 and 15.62 μg ml–1 was found.
© 2006 Elsevier SAS. All rights reserved.
Keywords: Imidazo[2,1-c][1,2,4]triazines; Crystal structure; Anticancer activity; 1-aryl-2-hydrazono-imidazolidine; Antibacterial activity
1. Introduction
densed 1,2,4-triazines found application as pharmaceuticals,
herbicides, pesticides and dyes [2–6]. Pyrrolo[2,1-f][1,2,4]tria-
zines showed an interesting broad spectrum antiproliferative
activity and a pronounced in vitro growth inhibitory activity
against leukaemic cell lines, comparable to that of 9-deazaade-
nosine, whilst pyrrolo[2,1-c][1,2,4]triazines demonstrated inhi-
bitory effects on the growth of a wide range of cancer cells
generally at 10–5 M level and in some cases even at micromo-
lar concentrations [7,8]. Some of pyrazolo[5,1-c][1,2,4]tria-
zines exhibited antitumor and antifungal activity [9,10]. It is
Compounds containing the 1,2,4-triazine moiety are found
in natural sources and many of them showed important biolo-
gical activities. For instance, azaribine-antiviral drug is structu-
rally based on the 1,2,4-triazine heterocyclic system [1]. Con-
* Corresponding author.
E-mail address: krzysztof.sztanke@am.lublin.pl (K. Sztanke).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.01.016

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K. Sztanke et al. / European Journal of Medicinal Chemistry 41 (2006) 539–547
noteworthy that many potential anticancer and antiviral drugs
have been modeled on them [11–14]. Recently, there is a wide-
spread interest in the synthesis and design of novel imidazo
[2,1-c][1,2,4]triazine derivatives because of their potential bio-
logical activities associated with their skeleton. For instance
certain derivatives of imidazo[2,1-c][1,2,4]triazin-4(1H)-one
were synthesized as new bicyclic nucleosides related to 6-azai-
socytosine [15].
drazone derivatives with diethyl 2-oxomalonate (B) and libera-
tion of a water molecule (Scheme 1).
These intermediates could potentially cyclize to derivatives
of the imidazo[2,1-c][1,2,4]triazine system of type 7–12 (with
concomitant release of an ethanol molecule) or of the imidazo
[2,1-c][1,2,4]triazole one (with liberation of ethyl formate). Fi-
nally, it was found, that the two mentioned methods led to the
formation of the imidazo[2,1-c][1,2,4]triazine derivatives, as a
result of liberation of an ethanol molecule and the ring closure
and no trace of the imidazo[2,1-c][1,2,4]triazole derivatives
were detected. Two general the synthetic routes to imidazotria-
zine derivatives 7–12 are shown in Scheme 1. Based on the
spectral data (IR, NMR, MS) as well as X-ray crystallographic
studies, the concurrent course of cyclization reaction to the imi-
dazo[2,1-c][1,2,4]triazole derivatives was excluded. In conclu-
sion, the same compounds were obtained by two different syn-
thetic approaches that imply the condensation reaction of
appropriate heterocyclic hydrazone derivatives (1–6), as well
as with diethyl 2-(hydroxyimino)malonate and diethyl 2-oxo-
malonate. Therefore the structure of these compounds was con-
firmed by two independent syntheses. Mixed melting points
have not shown any depression. The IR, 1H NMR, MS spectral
data of these compounds were also identical. Both synthetic
pathways in the synthesis of ethyl 1-(4-oxo-8-aryl-4,6,7,8-tet-
Previous studies concerning the synthesis of imidazo[2,1-c]
[1,2,4]triazin-4(4H)-ones [16–19] carried out in the Depart-
ment of Synthesis and Technology of Drugs, Medical Univer-
sity of Lublin, have disclosed some compounds with various
aryl substituents at position 8, and with benzyl, arylmethyl,
methoxycarbonylmethyl and hydroxyl substituents at position
3. These compounds have revealed a significant antinocicep-
tive activity on the central nervous system in behavioral animal
tests (the “writhing syndrome” and “hot plate”), and a low
acute toxicity. Also, a derivative of imidazo[2,1-c][1,2,4]tria-
zin-4(4H)-one with a 4-chlorophenyl substituent at the 8 posi-
tion and a hydroxycarbonylmethyl substituent at the 3 position
had significant activity against all Gram-negative bacterial
strains tested [20]. Prompted by the biological interest and
our previous reports, and continuing our searching for bioac-
tive molecules [16–20] it seemed worthwhile to synthesize
some novel biheterocyclic derivatives containing a pharmaco-
phoric 1,2,4-triazine moiety. In this paper we would like to
present the preparation and biological activity assessment for
ethyl 1-(4-oxo-8-aryl-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]
triazin-3-yl)formate derivatives.
rahydroimidazo[2,1-c][1,2,4]triazin-3-yl)formate
derivatives
may be useful in view of biological interest in this class of
compounds.
1
The scrutiny of H NMR, IR, MS spectra confirms that un-
der the reaction conditions, formation of the bicyclic imidazo
[2,1-c][1,2,4]triazine ring system of type 7–12 from acyclic in-
termediates is accompanied with the liberation of an ethanol
molecule. Additionally, the structure of 12 was confirmed by
X-ray crystallography. Perspective view of the molecule 12
with atom numbering is shown in Fig. 1.
2. Chemistry
Biologically active 1-aryl-2-hydrazono-imidazolidines (1-
aryl-2-hydrazino-imidazolines) (1–6) as starting materials
were prepared by patent pending and according to Sztanke et
al. [19]. Treatment of hydrazones 1–6 with diethyl 2-(hydro-
xyimino)malonate (A) or diethyl 2-oxomalonate (B) in
refluxing n-butanol afforded the corresponding ethyl 1-(4-
oxo-8-aryl-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)
formates (7–12) after a ring closure. Reactions of 1-aryl-2-hy-
drazono-imidazolidine derivatives with diethyl 2-(hydroxyi-
mino)malonate and diethyl 2-oxomalonate have not been re-
ported in the literature as yet and these reactions represent
novel methods for preparing the polynitrogenated bicyclic imi-
dazo[2,1-c][1,2,4]triazine system. The above mentioned reac-
tions can be carried out starting both from free base of 1-
aryl-2-hydrazono-imidazolidine (method i) or from its hydroio-
dide salt in presence of triethylamine (method ii) with compar-
able yields. The reaction conditions were established experi-
mentally. The course of reactions could include the formation
of intermediate chain derivatives (2-[(1-aryl-imidazolidin-2-yli-
den)-hydrazono]malonic acid diethyl esters) as result of con-
densation of 1-aryl-2-hydrazono-imidazolidines (1–6) with
diethyl 2-(hydroxyimino)malonate (A) and concomitant release
of a hydroxylamine molecule or in consequence of condensa-
tion reaction of above mentioned 1-arylimidazolidin-2-one hy-
NMR spectral characteristic of the imidazo[2,1-c][1,2,4]tria-
zines (7–12) is included in the experimental part.
In the IR spectra of compounds 7–12 the presence of ab-
sorption bands in the range of 1684–1689 cm–1 and
1552–1561 cm–1, attainable to the triazine-C=O group and
the C=N bond at the ring junction, respectively confirmed the
formation of cyclic products, whereas the presence of absorp-
tion bands at about 1739 cm–1 was characteristic for the ester-
C=O group derived from the ethoxycarbonyl formation. From
the IR spectral data there is a clear relationship between the
frequency of the ring carbonyl group -C=O and the frequency
of the C=N bond at the ring junction and the difference in their
frequencies is of the order of 127–132 cm–1. These data are in
complete agreement with the literature data [21].
The chemical and physical data for compounds 7–12 are
given in experimental protocols.
Biologically active intermediate 1-(2-chlorophenyl)-2-hy-
drazonoimidazolidine hydroiodide (4) was obtained by a pre-
viously reported method [22] but its biological activity, physi-
cochemical and spectral data have not been reported in the
literature as yet. Its experimental details are reported in the ex-
perimental part. Although in the case of compound investi-

K. Sztanke et al. / European Journal of Medicinal Chemistry 41 (2006) 539–547
541
Scheme 1. Synthetic route to obtained compounds.
gated the hydrazone-hydrazine tautomerism is possible but in
3. Pharmacology
solution (DMSO-d6) only the presence of the hydrazone tauto-
1
mer was observed. In the H–1H COSY spectrum of free base
3.1. Antimicrobial studies
of 4 the correlation between endocyclic-NH proton signal de-
rived from the imidazolidine ring at position 3 and the H-4
methylenic protons signals were observed as expected for the
hydrazone tautomer. Also in the HMBC NMR spectrum of this
compound there is a lack of correlation between the exocyclic-
NH proton (–NHNH2) from possible hydrazinic structure and
the C-2 carbon. The HMBC and HMQC NMR spectroscopic
data for compound 4 are presented in the Table 1. NMR spec-
tral characteristic of compound 4 is given in the experimental
protocols. The chemical shift values of the C-5 and C-4 carbon
atoms exhibit unequal character as well (51.3 ppm for C-5 and
41.1 ppm for C-4) as was seen in the 13C NMR spectrum.
Determination of the in vitro antimicrobial activity of the
compound 4 was performed using the microdilution method,
according to the National Committee for Clinical Laboratory
Standards (NCCLS) [23,24] and the disc-diffusion method by
Kirby-Bauer [25,26].
The in vitro activities of the obtained compound against
pathogenic bacteria (reference strains and clinical isolates),
yeast-like fungi and moulds were compared. The microdilution
method for estimation of MIC values (the lowest concentration
of compound required to inhibit the growth of the tested micro-
organism) was applied to evaluate the antibacterial activity. In

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K. Sztanke et al. / European Journal of Medicinal Chemistry 41 (2006) 539–547
man tumor cell lines derived from various cancer types (colon,
uterus, breast): LS 180 (ECACC 87021202, human Caucasian
colon adenocarcinoma cells), SiHa (ECACC 85060701, uterus
cancer cells), T47D (ECACC 85102201, human breast carcino-
ma cells). Besides two normal cell lines were included in the
cytotoxicity study: HSF (human skin fibroblast cells—primary
cell line) and Vero (ECACC 88020401, African Green Mon-
key Kidney cells, GMK clone).
3.3. Behavioral assays
The effect of the investigated compound 10 in behavioral
animal studies was carried out on male Albino Swiss mice.
4. Results and discussion
The antimicrobial activities of the starting 1-(2-chlorophe-
nyl)-2-hydrazonoimidazolidine hydroiodide (4) against bacter-
ial, moulds and yeast-like fungi strains and the MIC values
against two reference bacterial strains (S. aureus ATCC
25923 and E. coli ATCC 25922) were tested by using the
disc-diffusion and the microdilution assay. The results from
experiments were compared with those of ampicillin and chlor-
amphenicol as references for antibacterial agents. The MIC va-
lues of the compound tested and both standard drugs are listed
in Table 3.
Fig. 1. An ORTEP [36] drawing of the molecule 12. Bond distances within the
heterocyclic ring system are: N1–N2 1.366(5); N2–C3 1.312(6); C3–C4 1.455
(7); C4–N5 1.357(6); N5–C6 1.476(6); C6–C7 1.524(7); C7–N8 1.462(6);
C8a–N8 1.345(6); N1–C8a 1.285(6); C8a–N5 1.379(6); C4–O1 1.225(6) Å.
Table 1
The HMBC and HMQC NMR spectroscopic data for free base of compound 4
The compound tested (4) in the present study was found to
have highly significant antibacterial activities against the mi-
croorganisms listed in Table 2 but no antifungal activities.
The examined compound was also inactive against moulds
Table 2
Antimicrobial activities of evaluated compound 4 against the tested bacterial
and fungal isolates using the disc diffusion method
Microorganisms
Compound
Standard
4
II
+
+
+
–
–
+
+
–
–
–
–
HMBC correlations
H-4, H-5
H-4, H-5
H-4, H-5
H-6′, H-4′ (w), H-5′ (w)
H-3′
H-4′, H-5′
H-3′, H-6′
H-6′, H-4′ (w)
H-4′, H-5′
HMQC correlations
–
H-4, H-5 (w)
H-5, H-4 (w)
–
–
–
–
I
C-2
C-4
C-5
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
E. coli ATCC 25922
Pseudomonas aeruginosa
Proteus vulgaris
Klebsiella pneumoniae
Enterobacter aerogenes
S. aureus ATCC 25923
Staphylococcus epidermidis
Streptococcus pyogenes
Streptococcus agalactiae
Candida albicans
+
+
+
–
–
+
+
–
–
–
–
++
–
–
–
–
++
+
++
++
–
H-5′
H-6′
w, weak correlation.
Aspergillus sp.
–
this method two reference strains of bacteria—Staphylococcus
aureus ATCC 25923 (Gram-positive bacteria) and Escherichia
coli ATCC 25922 (Gram-negative bacteria) were included in
this study. The antibacterial potency of the compound 4 under
conditions was compared with the activities of topical antibac-
terial drugs—ampicillin and chloramphenicol.
I, concentration of 100 μg ml–1; II, concentration of 200 μg ml–1. Zones of
growth inhibition in: –, + , ++. – : 0–10 mm (R, resistant); + :11–16 mm (I,
intermediate susceptible); ++ : 17–25 mm (S, susceptible). Standard: ampicil-
lin.
Table 3
Antibacterial activity expressed as MIC (μg ml–1) of tested compound 4
Microorganisms/code
Compound
4
Standards
C
3.2. Anticancer activity studies
A
E. coli ATCC 25922
12.5
3.91
15.62
15.62
S. aureus ATCC 25923
12.5
3.91
The newly synthesized imidazotriazines of type 10 and 11
were evaluated for their anticancer activity towards three hu-
Standards: A, ampicillin; C, chloramphenicol.

K. Sztanke et al. / European Journal of Medicinal Chemistry 41 (2006) 539–547
543
and yeast-like fungi. The 1-(2-chlorophenyl)-2-hydrazonoimi-
dazolidine hydroiodide (4) showed good activity in relation to
reference bacterial strains (S. aureus ATCC 25923 and E. coli
ATCC 25922). This compound was effective against E. coli
ATCC 25922 and S. aureus ATCC 25923 at concentrations
of 15.62 μg ml–1 and 15.62 μg ml–1, respectively. The tested
compound was found to exhibit a comparable level of activity
to ampicillin. Its antibacterial potency was fourfold lower than
that of chloramphenicol (Table 3). Compound 4 was also
found to exhibit activity against Pseudomonas aeruginosa,
Proteus vulgaris and Staphylococcus epidermidis in concentra-
tions of 100 and 200 μg ml–1 in the disc-diffusion assay
(Table 2).
In pharmacological animal tests compound 10 showed no
effect on the central nervous system of mice in behavioral tests
applied. It has been found that the investigated compound had
no antinociceptive properties. This compound given in a dose
of 0.1 LD50 did not produce a decrease in a number of animals
exhibiting pain reactivity in the “writhing syndrome” test. Its
acute toxicity was relatively low (over 1000 mg kg–1 i.p.).
Moreover compound 10 given in a dose of 0.1 LD50 did not
impair the motor coordination of mice in the rota-rod test.
In conclusion, the tested imidazo[2,1-c][1,2,4]triazine deri-
vatives (10, 11) demonstrate antiproliferative properties those
warrant further investigation as potential anticancer agents.
Further studies are in progress to define the important mechan-
isms of action of above mentioned compounds.
As a result, compound 4 was found to exhibit potent in vitro
antibacterial activity with MIC value of 15.62 μg ml–1 against
S. aureus ATCC 25923 and may be considered promising for
the development of new antibacterial agents.
5. Experimental protocols
5.1. Chemical and X-ray crystal structure analysis
Compounds 10 and 11 were evaluated for their anticancer
activity. Results for each test compounds are reported as the
growth inhibition percentage of the tested cells in comparison
to untreated ones. Compounds which reduced growth are
passed on for evaluation towards three human cancer and two
normal cell lines. Compounds 10, 11 were found to be active.
According to the data listed in the Table 4 compounds 10 and
11 have potential to reduce the growth of the uterus cancer cell
line (SiHa), colon adenocarcinoma cell line (LS180) and breast
carcinoma cell line (T47D). Compound 10 was the most active
against SiHa cancer line, because its GI was 41 and 52%, re-
spectively for both examined concentrations (10 and 50 μg
ml–1). Compound 11 was found to be the most potent against
LS180 and SiHa cancer lines, especially in a higher concentra-
tion (50 μg ml–1). Its GI values were 50 and 46% for these
cancer lines, respectively. Based on performed examination,
the distinctly marked lower cytotoxicity of tested compounds
against normal cell lines and almost two-times higher against
cancer cell lines was ascertained. Taking into consideration the
GI comparative study results concerning the influence of tested
compounds on cancer and normal cell lines it can be expected
the selective action of examined compounds. Also the antican-
cer activity of tested compounds seemed to be dose-dependent.
Chemicals (hydrazine hydrate, diethyl 2-(hydroxyimino)
malonate, diethyl 2-oxomalonate) were purchased from Merck
and Sigma-Aldrich as ‘synthesis grade’ and used without
further purification. Melting points (m. p.) were determined
on a Boetius apparatus and are given uncorrected. The IR spec-
tra were measured as potassium bromide pellets using a Perkin-
Elmer 1725X spectrometer. 1H NMR spectra for compounds 7,
8, 10 were recorded on a Bruker 200 MHz spectrometer in
1
DMSO-d6. H NMR spectra of the other ones (4, 9, 11, 12)
were recorded on a Bruker 300 MHz spectrometer in DMSO-
d6 with TMS as an external standard at 295 K. 13C NMR spec-
trum for compound 4 was recorded on a Bruker AC 200F in-
1
strument. Besides H–1H COSY, HMBC and HMQC correla-
tions were made for this compound. Mass spectroscopic
analyses for compounds 7–9 were performed on an AMD-
402 and for compounds 11–12 on Trace DSQ mass spectro-
meters for molecular ion peaks.
Diffraction data for 12 were measured at 295 K on a KM4
diffractometer using variable scan speed in the ω-2θ scan mode
and graphite monochromated CuKα radiation (λ = 1.54178 Å).
A single crystal of dimensions 0.42 × 0.21 × 0.20 mm was se-
lected from the sample crystallized from DMF/methanol (4:1)
mixture. Thin-layer chromatography was carried out on com-
mercial Merck SiO2 60 F254 plates with toluene/ethyl acetate/
methanol (1:3:0.5) eluent system and visualized in UV light
λ = 254 nm and 355 nm. Elemental analyses were performed
on a Perkin–Elmer analyzer and were in range of 0.5% for
each element analyzed (C, H, N, Cl, I). The starting 1-(2-chlor-
ophenyl)-2-hydrazonoimidazolidine hydroiodide (4) was ob-
tained by earlier described method [19,22] but its physico-
chemical and spectral data have not been described in the
literature as yet and are the following:
Table 4
Inhibition of in vitro normal and tumor cells growth by imidazotriazines 10, 11
Cytotoxicity (GI in %)
Cell line
10
11
II
I
II
I
Normal cell lines
HSF
GMK
16
20
17
25
14
20
20
25
Cancer cell lines
LS180
SiHa
30
41
26
35
52
41
30
30
13
50
46
28
T47D
1-(2-Chlorophenyl)-2-hydrazonoimidazolidine hydroiodide
(4). Recrystallization from propan-2-ol, yield 65%, m.p. 223–
225 °C. Analysis for C9H12ClIN4: IR (KBr) (ν, cm–1): 1511
(N+H), 1570 (C=N), 3457–3313 (NH + NH2); 1H NMR (δ,
ppm, DMSO-d6 , TMS): 3.77 (dd, J = 9.3 Hz, J’ = 8.1 Hz, 2H,
HSF, human skin fibroblast cells—primary cell line; Vero (GMK, ECACC
88020401, African Green Monkey Kidney cells); LS180 (ECACC
87021202), human Caucasian colon adenocarcinoma cells; SiHa (ECACC
85060701), uterus cancer cells; T47D (ECACC 85102201), human breast car-
cinoma cells; I, concentration of 10 μg ml–1; II, concentration of 50 μg ml–1
.

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K. Sztanke et al. / European Journal of Medicinal Chemistry 41 (2006) 539–547
CH2), 4.02 (dd, J = 9.3 Hz, J’ = 8.1 Hz, 2H, CH2), 4.94 (s, 2H,
NH2), 7.47–7.70 (m, 4H, CHarom.), 8.76 (s, 1H, NH), 9.39 (s,
1H, N+H); 13C NMR (δ, ppm, DMSO-d6 , TMS) 41.1 (imida-
zolidine-C-4), 51.3 (imidazolidine-C-5), 128.9 (C-5′), 130.5
(C-6′), 130.6 (C-2′), 131.1 (C-3′), 132.2 (C-4′), 133.0 (C-1′),
159.1 (imidazolidine-C-2); m/z: 338[M+].
5.1.4.1. Ethyl 1-[4-oxo-8-(4-methylphenyl)-4,6,7,8-tetrahydroi-
midazo[2,1-c][1,2,4]triazin-3-yl]formate (7). Recrystallization
from DMF; yield 61% (from reaction with A)/63% (from reac-
tion with B) (method i), 55% (from reaction with A)/52%
(from reaction with B) (method ii), m.p. 205–208 °C. Analysis
for C15H16N4O3 : IR (KBr) (ν, cm–1): 1736 (ester –C=O), 1687
1
(triazine –C=O), 1560 (C=N); H NMR (δ, ppm, DMSO-d6 ,
TMS): 1.29 (t, J = 7.1 Hz, 3H, –OCH2CH3), 2.32 (s, 3H, CH3),
4.18 (t, J = 3.1 Hz, 4H, 2CH2), 4.30 (q, J = 7.1 Hz, 2H, –O
CH2CH3), 7.27 (d, J = 8.6 Hz, 2H, ar: H-2′ and H-6′), 7.70
(d, J = 8.6 Hz, 2H, ar: H-3′ and H-5′); EIMS [70 eV, m/z
(%)]: 300 (M+, 75.37), 256 (84.29), 228 (43.70), 200
(100.00), 172 (22.43), 145 (67.56), 91 (42.89).
5.1.1. Synthesis of ethyl 1-(4-oxo-8-aryl-4,6,7,8-
tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)formates
(method i) (general procedure)
Free base of 1-aryl-2-hydrazonoimidazolidine [22] (0.05
mol) was dissolved in 80 ml of n-butanol. Diethyl 2-(hydroxyi-
mino)malonate (9.45 g, 0.05 mol) was added and the mixture
was heated under reflux for 7 h. During that time precipitation
of the solid started. The mixture was cooled overnight and the
precipitate yielded was collected and purified by recrystalliza-
tion from DMF or DMF/methanol in the proportion indicated.
5.1.4.2. Ethyl 1-[4-oxo-8-(4-methoxyphenyl)-4,6,7,8-tetrahy-
droimidazo[2,1-c][1,2,4]triazin-3-yl]formate (8). Recrystalli-
zation from DMF/methanol (2:1) mixture; yield 67% (from re-
action with A)/59% (from reaction with B) (method i), 60%
(from reaction with A)/57% (from reaction with B) (method
ii), m.p. 149–151 °C. Analysis for C15H16N4O4 : IR (KBr) (ν,
cm–1): 1740 (ester –C=O), 1689 (triazine –C=O), 1561 (C=N);
1H NMR (δ, ppm, DMSO-d6 , TMS): 1.29 (t, J = 7.1 Hz, 3H,
–OCH2CH3), 3.78 (s, 3H, OCH3), 4.17 (t, J = 3.1 Hz, 4H,
2CH2), 4.29 (q, J = 7.1 Hz, 2H, –OCH2CH3), 7.04 (d,
J = 9.1 Hz, 2H, ar: H-2′ and H-6′), 7.72 (d, J = 9.1 Hz, 2H,
ar: H-3′ and H-5′); EIMS [70 eV, m/z (%)]: 316 (M+,
100.00), 272 (21.85), 216 (92.95), 161 (42.98), 107 (4.71).
5.1.2. Synthesis of ethyl 1-(4-oxo-8-aryl-4,6,7,8-
tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)formates
(method ii) (general procedure)
Diethyl 2-(hydroxyimino)malonate (9.45 g, 0.05 mol) was
added to the suspension of appropriate 1-aryl-2-hydrazonoimi-
dazolidine hydroiodide [22] (0.05 mol) in 70 ml of n-butanol.
The mixture was stirred and triethylamine (5 ml) was added.
The reaction was carried out under reflux for 7 h. During that
time precipitation of solid started. The crude product obtained
after cooling was collected, washed off with cold methanol and
finally purified by recrystallization from DMF or DMF/metha-
nol in the proportion indicated.
5.1.4.3. Ethyl 1-[4-oxo-8-(2-chlorophenyl)-4,6,7,8-tetrahydroi-
midazo[2,1-c][1,2,4]triazin-3-yl]formate (9). Recrystallization
from DMF/methanol (3:1) mixture; yield 57% (from reaction
with A)/54% (from reaction with B) (method i), 53% (from
reaction with A)/57% (from reaction with B) (method ii), m.
p. 190–192 °C. Analysis for C14H13ClN4O3 : IR (KBr) (ν,
cm–1): 1742 (ester –C=O), 1686 (triazine –C=O), 1555 (C=N);
1H NMR (δ, ppm, DMSO-d6 , TMS): 1.29 (t, J = 7.1 Hz, 3H,
–OCH2CH3), 4.14–4.25 (m, 4H, 2CH2), 4.31 (q, J = 7.1 Hz,
2H, –OCH2CH3), 7.26–8.12 (m, 4H, ar-H); EIMS [70 eV, m/
z (%)]: 320 (M+, 42.93), 276 (100.00), 248 (52.90), 220
(83.39), 165 (59.57), 111 (39.20).
5.1.3. Synthesis of ethyl 1-(4-oxo-8-aryl-4,6,7,8-
tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)formates
(method i) (general procedure)
Free base of 1-aryl-2-hydrazonoimidazolidine [22]
(0.05 mol) was dissolved in 80 ml of n-butanol. Diethyl 2-ox-
omalonate (8.71 g, 0.05 mol) was added and the mixture was
heated under reflux for 7 h. During that time precipitation of
the solid started. The mixture was cooled overnight and the
precipitation yielded was collected and purified by recrystalli-
zation from DMF or DMF/methanol in the proportion indi-
cated.
5.1.4.4. Ethyl 1-[4-oxo-8-(3-chlorophenyl)-4,6,7,8-tetrahydroi-
midazo[2,1-c][1,2,4]triazin-3-yl]formate (10). Recrystalliza-
tion from DMF/methanol (3:1) mixture; yield 65% (from reac-
tion with A)/60% (from reaction with B) (method i), 59%
(from reaction with A)/63% (from reaction with B) (method
ii), m.p. 189–191 °C. Analysis for C14H13ClN4O3 : IR (KBr)
(ν, cm–1): 1741 (ester –C=O), 1684 (triazine –C=O), 1552
(C=N); 1H NMR (δ, ppm, DMSO-d6 , TMS): 1.29 (t,
J = 7.1 Hz, 3H, –OCH2CH3), 4.17–4.26 (m, 4H, 2CH2), 4.32
(q, J = 7.1 Hz, 2H, –OCH2CH3), 7.25–8.12 (m, 4H, ar-H);
EIMS [70 eV, m/z (%)]: 320 (M+, 26.83), 276 (100.00), 248
(44.86), 220 (72.93), 165 (31.40), 111 (31.09).
5.1.4. Synthesis of ethyl 1-(4-oxo-8-aryl-4,6,7,8-
tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)formates
(method ii) (general procedure)
Diethyl 2-oxomalonate (8.71 g, 0.05 mol) was added to the
suspension of appropriate 1-aryl-2-hydrazonoimidazolidine hy-
droiodide [22] (0.05 mol) in 70 ml of n-butanol. The mixture
was stirred and triethylamine (5 ml) was added. The reaction
was carried out under reflux for 7 h. During that time precipi-
tation of solid started. The crude product obtained after cooling
was collected, washed off with cold methanol and finally pur-
ified by recrystallization from DMF or DMF/methanol in the
proportion indicated.
5.1.4.5. Ethyl 1-[4-oxo-8-(3,4-dichlorophenyl)-4,6,7,8-tetrahy-
droimidazo[2,1-c][1,2,4]triazin-3-yl]formate (11). Recrystalli-

K. Sztanke et al. / European Journal of Medicinal Chemistry 41 (2006) 539–547
545
zation from DMF/methanol (4:1) mixture; yield 67% (from re-
5.2. Microbiology
action with A)/62% (from reaction with B) (method i), 66%
(from reaction with A)/57% (from reaction with B) (method
ii), m.p. 248–250 °C. Analysis for C14H12Cl2N4O3 : IR (KBr)
(ν, cm–1): 1738 (ester –C=O), 1687 (triazine –C=O), 1555
(C=N); 1H NMR (δ, ppm, DMSO-d6 , TMS): 1.29 (t,
J = 7.1 Hz, 3H, -OCH2CH3), 4.13–4.27 (m, 4H, 2CH2), 4.31
(q, J = 7.1 Hz, 2H, –OCH2CH3), 7.71–8.27 (m, 3H, ar-H);
EIMS [70 eV, m/z (%)]: 354 (M+, 35.76), 310 (71.85), 309
(34.13), 282 (48.26), 254 (100.00), 253 (76.47), 245 (9.02),
228 (11.29), 226 (11.69), 220 (4.05), 219 (3.41), 218 (11.22),
212 (6.33), 201 (26.17), 199 (44.84), 192 (11.77), 186 (11.26),
174 (69.48), 173 (39.55), 172 (50.82), 159 (8.38), 145 (36.85),
133 (10.59), 124 (10.67), 111 (14.50), 109 (30.39), 81 (58.93),
75 (15.28), 70 (21.88).
5.2.1. Disc diffusion assay
Assay of antimicrobial activity in vitro. Compound (4) was
tested for its antimicrobial (antibacterial and antifungal) activ-
ities by disc-diffusion method by Kirby-Bauer, using Mueller–
Hinton medium for bacteria and the same medium with 4%
glucose for fungi. The majority of test microorganisms were
obtained from clinical specimens of the Laboratory of Medical
Microbiology Department, Medical University of Lublin. The
assayed collection included the following microorganisms: Sta-
phylococcus epidermidis, Streptococcus pyogenes, Streptococ-
cus agalactiae (Gram-positive bacteria), Pseudomonas aerugi-
nosa, Proteus vulgaris, Klebsiella pneumoniae, Enterobacter
aerogenes (Gram-negative bacteria), Candida albicans, Asper-
gillus spp. Besides, two reference strains of bacteria—
S. aureus ATCC 25923 and E. coli ATCC 25922 were in-
cluded in these studies.
5.1.4.6. Ethyl 1-[4-oxo-8-(2,6-dichlorophenyl)-4,6,7,8-tetrahy-
droimidazo[2,1-c][1,2,4]triazin-3-yl]formate (12). Recrystalli-
zation from DMF/methanol (4:1) mixture; yield 59% (from re-
action with A)/61% (from reaction with B) (method i), 54%
(from reaction with A)/58% (from reaction with B) (method
ii), m.p. 232–234 °C. Analysis for C14H12Cl2N4O3 : IR (KBr)
(ν, cm–1): 1737(ester –C=O), 1686 (triazine –C=O), 1554
(C=N); 1H NMR (δ, ppm, DMSO-d6 , TMS): 1.29 (t,
J = 7.1 Hz, 3H, –OCH2CH3), 4.03–4.43 (m, 4H, 2CH2), 4.32
(q, J = 7.08 Hz, 2H, –OCH2CH3), 7.53–7.72 (m, 3H, ar-H);
EIMS [70 eV, m/z (%)]: 354 (M+, 7.20), 319 (33.94), 310
(100.00), 309 (58.84), 282 (50.49), 254 (61.57), 253 (91.14),
245 (24.18), 220 (18.95), 219 (18.98), 218 (7.40), 212 (14.12),
201 (9.30), 199 (15.69), 192 (54.84), 186 (9.18), 174 (64.39),
173(23.32), 172 (46.49), 159 (12.00), 145 (17.80), 133 (15.60),
124 (12.76), 111 (17.36), 109 (30.63), 81 (17.46), 75 (19.58),
70 (38.33).
In the disc-diffusion method, sterile paper discs (φ 5 mm)
impregnated with dissolved in DMSO compound at concentra-
tions of 100 μg ml–1 and 200 μg ml–1 were used. Discs con-
taining DMSO were used as control. The microorganisms cul-
tures were spread over the following appropriate media:
Mueller–Hinton agar for S. aureus ATCC 25923, E. coli
ATCC 25922 and Sabouraud agar for the yeast-like fungi
(Candida albicans) and for the moulds (Aspergillus spp.) in
Petri dishes. Then, the paper discs impregnated with the solu-
tions of the compound tested (4) were placed on the surface of
the media inoculated with the microorganism. The plates were
incubated at 35 °C per 24 h for the microorganisms cultures.
After incubation, the growth inhibition zones around the discs
were observed indicating that the examined compound inhibits
the growth of microorganism [25,26]. Each assay in this ex-
periment was repeated three times.
Crystal data for 12: C14H12Cl2N4O3, FW = 355.18, mono-
clinic, space group P21/c, a = 11.502(2) Å, b = 7.296(1) Å,
c = 18.574(4) Å, α = 90°, β = 105.86(3)°, γ = 90°, V = 1499.4
(5) Å3 , Z = 4, dcalc = 1.573 g cm–3, μ(CuKα) = 4.095 mm–1.
In the θ range 4.95–80.31°, 3369 reflections were collected
and corrected for absorption. The structure was solved by di-
rect methods using SHELXS-93 [27] program. The refinement
was performed by full-matrix least-squares on F2 using 3264
unique reflections (Rint = 0.051) and SHELXL-97 [28] pro-
gram. The non-hydrogen atoms were refined with anisotropic
displacement parameters. H-atom positions were located from
the geometry and were given isotropic factors of 1.2 Ueq of the
bonded C-atoms; the C–H bond ‘riding’ model was used in the
refinement. For 209 parameters refined, final discrepancy fac-
tors are R1 = 0.0412, wR2 = 0.0999 for 746 reflections with
I > 2σ(I), and R1 = 0.3192, wR2 = 0.1612 for all data,
S = 0.901.
Ampicillin was used as a standard drug. DMSO was used as
solvent control. Results were interpreted in terms of the dia-
meter of the inhibition zone and are shown in Table 2.
5.2.2. Microdilution assays
The minimal inhibitory concentration (MIC) values for
compound tested (4), defined as the lowest concentration of
the compound preventing the visible growth, were determined
by using microdilution broth method according to NCCLS
standards [23]. The inocula of microorganisms (106 cfu ml–1)
were prepared from 24 h broth cultures and suspensions were
adjusted to 0.5 McFarland standard turbidity. The test com-
pound dissolved in DMSO was first diluted to the highest con-
centration (500 μg ml–1) to be tested. Then serial twofold dilu-
tions were made in concentration ranges from 1.95 to 500 μg
ml–1 in 10 ml sterile tubes. A prepared suspension of the stan-
dard microorganisms was added to each dilution in a 1:1 ratio.
Growth (or its lack) of microorganisms was determined vi-
sually after incubation for 24 h at 37 °C. The lowest concen-
tration at which there was no visible growth (turbidity) was
taken as the MIC.
Crystallographic data have been deposited with the Cam-
bridge Crystallographic Data Centre as CCDC No. 275239.
Copies of the data can be obtained on application to The Di-
rector, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK,
(Fax: +44-1223-336-033; e-mail: deposit@ccdc.cam.ac.uk or
http://www.ccdc.cam.ac.uk).

546
K. Sztanke et al. / European Journal of Medicinal Chemistry 41 (2006) 539–547
The minimal inhibitory concentration (MIC) values were
5.4.2. The influence of compound 10 on the motor impairment
in mice
studied for reference bacterial strains: S. aureus ATCC 25923
and E. coli ATCC 25922 towards compound 4 determined in
the disc-diffusion assay. Ampicillin and chloramphenicol were
used as a standard drugs for comparison in the antibacterial
study. Control experiments using DMSO were done for anti-
bacterial activity studies. The presented results were obtained
from three independent measurements. The investigations were
carried out in the Department of Medical Microbiology, Med-
ical University, Lublin.
Motor coordination was evaluated in the rota-rod test [34].
5.4.3. Pain reactivity in the “writhing syndrome” test in mice
for compound 10
Pain reactivity was measured in mice by the “writhing syn-
drome” test of Witkin et al. [35]. The test was performed in
mice by the intraperitoneally injection of 0.6% solution of
acetic acid in a volume of 10 ml kg–1 30 min after the admin-
istration of the investigated compound 10 in a dose of 0.1
LD50. The number of writhing episodes was counted for
30 min after the injection of acetic acid.
5.3. Inhibition of tumor cell growth assay
Compounds 10, 11 were selected for in vitro screening to-
wards three human cancer cell lines: human Caucasian colon
adenocarcinoma (LS180), human uterus cancer (SiHa) and hu-
man breast carcinoma (T47D). Besides two normal cell lines:
human skin fibroblasts (HSF) and Vero (Green Monkey Kid-
ney cells) were included in the cytotoxicity study.
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5.4. Behavioral experiments
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dealer, Górzkowska, Warsaw, Poland. The animals were kept
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of 10 ml kg–1. Controls animals received the equivalent vo-
lume of solvent. Each experimental group consisted of ten ani-
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5.4.1. Acute toxicity in mice for compound 10
Acute toxicity was assessed according to Litchfield and
Wilcoxon methods [33] and presented as LD50 calculated from
the mortality of mice after 24 h.

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