Synthesis, structure elucidation and identification of antitumoural properties of novel fused 1,2,4-triazine aryl derivatives

Article abstract of DOI:10.1016/j.ejmech.2007.07.009

Synthesis, structure elucidation and anticancer activities of novel fused 1,2,4-triazine aryl derivatives containing the ethoxycarbonyl (6-10) and carbohydrazide formations (11-15) are presented. Molecular structures of the synthesized compounds were conf

Full text of DOI:10.1016/j.ejmech.2007.07.009

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European Journal of Medicinal Chemistry 43 (2008) 1085e1094
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, structure elucidation and identification of antitumoural
properties of novel fused 1,2,4-triazine aryl derivatives
a
b
Krzysztof Sztanke a, , Kazimierz Pasternak , Jolanta Rzymowska ,
*
a
c
´
Ma1gorzata Sztanke , Martyna Kandefer-Szerszen
a Chair and Department of Medical Chemistry, Professor Feliks Skubiszewski Medical University, 4 Staszica Street, 20-081 Lublin, Poland
b Chair and Department of Biology and Genetics, Professor Feliks Skubiszewski Medical University, 6 Staszica Street, 20-081 Lublin, Poland
c Department of Virology and Immunology, Maria Curie-Skłodowska University, 19 Akademicka Street, 20-033 Lublin, Poland
Received 3 April 2007; received in revised form 4 July 2007; accepted 9 July 2007
Available online 29 July 2007
Abstract
Synthesis, structure elucidation and anticancer activities of novel fused 1,2,4-triazine aryl derivatives containing the ethoxycarbonyl (6e10)
and carbohydrazide formations (11e15) are presented. Molecular structures of the synthesized compounds were confirmed by IR, 1H NMR, 13
C
NMR, EI-MS spectra and elemental analyses. Antitumour activities in vitro for heterobicyclic hydrazides of the type 11e14 were evaluated by
BrdU method for human LS180, SiHa and T47D carcinoma cells. Amongst them, hydrazide 14 has exhibited remarkable inhibitory effect
against SiHa and LS180 tumour cells, and simultaneously was found to be non-toxic towards the human normal cell line e HSF cells. Further-
more, the pulse field gel electrophoresis experiment was performed for characterizing DNA-cleaving activity of heterobicycle 14. The DNA
fragments of 2500, 2000 and 500 kilobase pairs (kbp) were commonly detected in the cancer cell lines (SiHa, LS180 and T47D) treated
with compound 14. DNA fragmentation pattern, since three types of fragments induced by the tested hydrazide of the type 14 were detected,
suggesting a way of interaction with DNA. It is worth pointing out, that DNA strand breaks were also produced in human breast cancer (T47D)
cells, a cell line where the induction of DNA fragmentation is very difficult. Moreover, the statistically significant apoptotic activity in T47D
human breast cancer cells for the tested heterobicycle 14 was proved using the annexin V e binding assay. The antiproliferative properties
in vitro for compounds 6e14 were evaluated by MTT method for human leukaemic Jurkat cells. Significant viability decreases in Jurkat cells
treated with different concentrations of compounds 10 and 11 were observed, suggesting that these derivatives have antiproliferative activities.
Their acute toxicities were established. For these compounds the influence on the central nervous system of mice in behavioural tests was
1
examined. Molecular structure for free base of the intermediate 4 was confirmed by He1H COSY, HMBC and HMQC correlations.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Fused 1,2,4-triazines; Imidazo[2,1-c][1,2,4]triazine nucleus; Ethoxycarbonyl group; Carbohydrazide formation; A novel DNA strand breaking agent;
Anticancer activity; Apoptotic activity
1. Introduction
impressive array of biological activities, among which antitu-
mour [1,2], antiviral [3,4], and antifungal [5] properties have
been cited in the scientific literature.
In addition, 6-azaisocytosine (e.g., 3-amino-1,2,4-triazin-5
(2H )-one), an isosteric isomer of 6-azacytosine and 6-azauracil,
is of great biological interest due to its resistance to deaminase,
while azaribine e the well-known antiviral drug is structurally
associated with the 1,2,4-triazine moiety [6].
The 1,2,4-triazine ring is a prominent structural core system
found in numerous biologically active compounds. For instance
certain azanucleosides (6-azacytosine, 6-azauracil), structurally
based on the 1,2,4-triazine nucleus, have displayed an
Various condensed 1,2,4-triazines found application as phar-
maceuticals, herbicides, pesticides and dyes [7e12]. Pyrrolo
* Corresponding author. Tel.: þ48 81 5324002; fax: þ48 81 5323998.
E-mail address: krzysztof.sztanke@am.lublin.pl (K. Sztanke).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.07.009

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K. Sztanke et al. / European Journal of Medicinal Chemistry 43 (2008) 1085e1094
[2,1-f][1,2,4]triazines, congeners of substituted nucleic acid pu-
rines, have been synthesized and screened in vitro, revealing an
interesting broad spectrum antiproliferative activity and a pro-
nounced in vitro growth inhibitory activity against leukaemic
cell lines, comparable to that of 9-deazaadenosine. Pyrrolo
[2,1-c][1,2,4]triazines demonstrated inhibitory effects on the
growth of a wide range of cancer cells generally at 10ꢀ5 M level,
and in some cases, even at micromolar concentrations [13,14].
Some of pyrazolo[5,1-c][1,2,4]triazines have acquired consid-
erable importance because of their remarkable antitumour and
antifungal activities. It is noteworthy that many potential anti-
cancer and antiviral drugs have been modelled on them [15e20].
Recently, there is a widespread interest in the synthesis and
design of novel imidazo[2,1-c][1,2,4]triazine derivatives be-
cause of their potential biological activities associated with
their skeleton. For instance certain synthetic derivatives of
the imidazo[2,1-c][1,2,4]triazin-4(1H )-one have been de-
signed and obtained as novel bicyclic nucleosides related to
6-azaisocytosine [21], while the others displayed the distinctly
marked lower cytotoxicity towards normal cells (HSF, GMK)
and several-times higher against cancer cell lines [22,23]. An-
other previously obtained derivatives containing the imi-
dazo[2,1-c][1,2,4]triazine nucleus revealed a strong affection
to tumour cells and simultaneously were found to be non-toxic
towards the normal cell line investigated e GMK cells [24].
In continuation of our attempt to achieve medicinally impor-
tant organic molecules [22e28], and having in mind the prom-
ising anticancer activities showed by previously obtained 1,2,
4-triazine fused heterocycles [13e20,22e24], it seemed worth-
while to synthesize some novel heterobicyclic derivatives bear-
ing the 1,2,4-triazine moiety as an expected pharmacophore.
The presented herein novel fused 1,2,4-triazine aryl derivatives
containing the ester (6e10) and hydrazide formations (11e15)
hold the aza skeleton of the known pyrrolo[2,1-c][1,2,4]triazine
scaffold, the derivatives of which showed good antiproliferative
activities with IC50 values in the range of 5.5e88 mM [13]. In an
attempt to achieve new heterobicycles with possible anticancer
properties, we designed and synthesized the above-mentioned
fused 1,2,4-triazines to evaluate their anticancer activities. Fur-
thermore, some behavioural pharmacological tests (acute toxic-
ity and ‘‘writhing syndrome’’) were performed to check the
influence of the compounds on the central nervous system of
the laboratory animals.
R
R
N
N
N
NH
NH2
NH2
NH
1-5
N
i
R
N
N
N
N
COOC2H5
O
6-10
ii or iii or iv
R
N
N
N
N
CONHNH2
O
11-15
1, 6, 11: R=H; 2, 7, 12: R=4-CH3; 3, 8, 13: R=4-CH3O; 4, 9, 14: R=3-Cl;
5, 10, 15: R=3,4-Cl2
Scheme 1. Syntheticroutetoobtainedheterobicycles.Reagentsandconditions: i e
(1e5) þ diethyl 2-oxomalonate, n-butanol, reflux, 7 h; ii e (6e10) þ hydrazine
hydrate (100%), n-butanol, reflux, 5 h; iii e (6e10) þ hydrazine hydrate
(100%), etanol, reflux, 6 h; iv e (6e10) þ hydrazine hydrate (100%), the alloy
bath, 90 ꢁC, 7 h.
products (11e15) in the highest yield, three different synthetic
methods were applied. Thus, the condensation of above-men-
tioned esters (6e10) with hydrazine hydrate was carried out:
(a) in n-butanol at the temperature of boiling for 5 h (method ii),
(b) in ethanolic medium under reflux for 6 h (method iii), and
(c) without any solvent by heating in the alloy bath in the tem-
perature of ca. 90 ꢁC for 7 h (method iv). Substrates were mixed
together in molar ratio 1:1 (method ii), 1:3 (method iii) and 1:10
(method iv) (Scheme 1). It was found that all these methods led
directly to the formation of the desired hydrazides 11e15
(with concomitant loss of ethanol molecule). Highest yields
(on an average 74%) for hydrazides 11e15 were achieved
when the reaction is performed in ethanolic medium in excess
of hydrazine hydrate (method iii). Mixed melting points of the
final products (11e15) obtained by three above-mentioned
2. Chemical part
The synthetic pathway used in the preparation of the com-
pounds is outlined in Scheme 1. Biologically active starting
materials 1-aryl-2-hydrazonoimidazolidines (1-aryl-2-hydrazi-
noimidazolines) [22,23] (1e5) were prepared by patent pend-
ing and according to Sztanke [29]. Treatment of hydrazones
1e5 with diethyl 2-oxomalonate in refluxing n-butanol af-
forded the corresponding ethyl 1-(4-oxo-8-aryl-4,6,7,8-tetrahy-
droimidazo[2,1-c][1,2,4]triazin-3-yl)formates (6e10) (method i),
which in turn were refluxed with hydrazine hydrate to obtain
8-aryl-4-oxo-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazine-
3-carbohydrazides (11e15). In an attempt to prepare the final
1
methods have not shown any depression. The IR, H NMR,
13C NMR and MS spectral data of these compounds were
also identical.

K. Sztanke et al. / European Journal of Medicinal Chemistry 43 (2008) 1085e1094
1087
In view of continuous and widespread interest in the design
of novel heterobicyclic derivatives containing the 1,2,4-triazine
moiety, particularly on account of their pharmacological prop-
erties the synthetic approach leading to the formation of novel
fused 1,2,4-triazine aryl derivatives containing the ester (6e10)
and hydrazide formations (11e15), described by us, might be
considered as a useful method for the preparation of these bio-
logically active compounds because of the affordability of the
starting materials, good yields obtained and straightforward
product isolation.
NMR spectral characteristic of the synthesized heterobicyclic
hydrazides (11e15) revealed in their 1H NMR spectra signals of
the H-7 and H-6 as a broad singlet at ca 4.19 ppm (11e14) or as
two double-doublet signals at 3.60 and 4.03 ppm with the cou-
pling constants of J ¼ 8.5 Hz, J0 ¼ 7.5 Hz and J ¼ 8.6 Hz,
J0 ¼ 7.5 Hz, respectively (15). Furthermore, the 1H NMR spectra
of hydrazides 11e15 displayed no signals belonging to the e
OCH2CH3 group; instead new signals derived from hydrazide
structure appeared between 4.40e4.63 ppm (eNHNH2) and
9.40e9.70 ppm (eNHNH2) integrating for two protons and
one proton, respectively (controlled by changing with D2O).
Their further 1H NMR spectroscopic details are included in the
Section 5.
The 13C NMR chemical shift values of the C-7 and C-6 car-
bon atoms confirmed unequal character of both methylene
groups as well (ca. 45.8 ppm for C-7 and 40.5 ppm for C-6).
Moreover, in the 13C NMR spectra of hydrazides of the type
11e15, the quaternary carbon signals derived from the exocy-
clic hydrazide group (eCONHNH2) were recorded in the range
of 152.4e152.5 ppm. Additionally the endocyclic triazine-
CaO (triazine-C-4), triazine-C-3 and C-8a signals were seen
at about 143.8, 150.8 and 161.0 ppm, respectively.
In the IR spectra of carbohydrazides 11e15, the presence
of absorption bands in the range of 1685e1690 and 1553e
1562 cmꢀ1, attainable to the endocyclic triazine-CaO group
at position 4 and the CaN bond at the ring junction, respec-
tively, confirmed the formation of final products. Also, the
IR spectra of compounds 11e15 revealed an additional peak
at about 1740 cmꢀ1 due to the exocyclic-carbonyl function de-
rived from the hydrazide structure. From the IR spectral data
there is a clear relationship between the frequency of the
ring carbonyl group eCaO and the frequency of the CaN
bond at the ring junction and the difference in their frequen-
cies is of the order of 124e132 cmꢀ1. These data are in com-
plete agreement with the literature data [30].
are presented in this paper for the first time. Further physicochem-
ical, experimental and spectroscopic details are reported in the
Section 5. Although in the case of the compound investigated
the hydrazoneehydrazine tautomerism is possible but in solution
(DMSO-d6) only the presence of the hydrazone tautomer was ob-
served. In the 1He1H COSY spectrum of free base of 4 the corre-
lation between endocyclic-NH proton signal derived from the
imidazolidine ring atposition 3 and theH-4 methylenic protonsig-
nals 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 (eNHNH2) from
possible hydrazinic structure and the C-2 carbon. The HMBC
and HMQC NMR spectroscopic data for compound 4 are pre-
sented in Table 1. NMR spectral characteristic of compound 4 is
given in the Section 5. The chemical shift values of the C-5 and
C-4 carbon atoms exhibit unequal character as well (51.3 ppm
for the C-5 and 40.9 ppm for the C-4) as was seen in the 13C
NMR spectrum.
3. Results and discussion
The series of heterobicyclic hydrazides of the type 11e14
were evaluated for their antitumour activities. Results for each
test compound are reported as the growth inhibition percentage
of the treated cells when compared to the untreated control ones.
Compounds 11e14 were tested against three human tumour cell
lines derived from various cancer types (colon, uterus, breast):
LS 180 (ECACC 87021202, human Caucasian colon adenocar-
cinoma cells), SiHa (ECACC 85060701, uterus cancer cells),
T47D (ECACC 85102201, human breast carcinoma cells).
Moreover, two normal cell lines e HSF (human skin fibroblast)
cells and Vero (ECACC 88020401, African Green Monkey Kid-
ney cell line, GMK clone) were included in the cytotoxicity
study.
According to the data summarized in Table 2, the final heter-
obicyclic hydrazide of the type 14, with electron-withdrawing
Table 1
The HMBC and HMQC NMR spectroscopic data for free base of intermediate 4
4'
3'
5'
Cl
6'
1'
2'
N
N
5
4
NH2
2
EI-MS of heterobicyclic hydrazides (11e15) provided mo-
lecular ion peaks at m/z 272, 286, 302, 306, 340, respectively,
with determined intensities confirming their molecular weights.
The most typical fragmentation pattern involved the cleavage of
the 31 molecular unit fragment of the side chain that most prob-
ably was N2H3. Further details are presented in Section 5.
All the chemical and physical data for compounds 11e15
are given in Section 5.
The intermediates e tautomeric 1-aryl-2-hydrazonoimidazoli-
dine hydroiodides (1e5) were obtained by a previously reported
method [24,29,31]. However, the HMBC, HMQC correlations
and 13C NMR spectroscopic data of free base of intermediate 4
NH
HMBC correlations
HMQC correlations
C-2
C-4
C-5
C-10
C-20
C-30
C-40
C-50
C-60
H-4, H-5
H-4, H-5
e
H-4, H-5 (w)
H-4, H-5
H-5, H-4 (w)
e
H-20
H-20, H-50, H-60
H-40, H-60
H-20, H-40, H-60
H-50, H-60
H-60, H-20
H-20, H-40
e
H-40
H-50
H-60
w e Weak correlation.

1088
K. Sztanke et al. / European Journal of Medicinal Chemistry 43 (2008) 1085e1094
substituent, such as a chloro group at position 3 of the phenyl
ring, was found to be the most active of the series. It has shown
45 and 63% growth inhibition, respectively, against uterus can-
cer (SiHa) cells in both examined concentrations: 32.6 mM
(10 mg mLꢀ1) and 163.0 mM (50 mg mLꢀ1). Similarly, this het-
erobicycle has exhibited 42 and 54% growth inhibition against
human colon adenocarcinoma (LS180) cells in both tested con-
centrations, respectively. However, the remaining human breast
cancer (T47D) cell line has shown the least susceptibility level
to this compound (the following growth inhibition values were
analysed: 26 and 40% for both its tested concentrations).
It is worth pointing out that heterobicycle 14 was found to
be non-toxic towards the human cell line investigated e HSF
cells. Furthermore, the distinctly marked lower cytotoxicity of
compound 14 towards one monkey normal cell line e GMK
cells and almost two-times higher against SiHa and LS180
cancer cells was ascertained.
Furthermore, DNA damage in the examined cancer cells after
incubation with the most potent hydrazide 14 at a concentration
of 32.6 mM was analyzed using field gel electrophoresis through
a 1% agarose gel. This method is applied for the analysis of
DNA-fragmenting activity of various antitumour agents [32e34].
Compound 14 was found to be effective in inducing DNA dam-
age. The additional DNA fragments of 2500, 2000 and 500 kilo-
base pairs (kbp) appeared in the cancer cell lines (SiHa, LS180,
T47D) treated with compound 14 in comparison to DNA of con-
trol cells, suggesting strongly that the examined heterobicycle
induced DNA cleavage (see Fig. 1). DNA fragmentation pattern,
since three types offragments induced by the tested hydrazide of
the type 14 were detected, suggesting a way of interaction with
DNA. Endonucleases that mediate this DNA cleavage may be
classified by substrate specificity. Upregulation of endonucle-
ases, their intranuclear and intracellular redistribution and pri-
mary changes of chromatin structure are three major pathways
of DNA fragmentation.
It is worth pointing out that DNA strand breaks were also pro-
duced in human breast cancer cells, a cell line where the induc-
tion of DNA fragmentation is very difficult [35,36]. In
conclusionthetested hydrazide of type 14revealed the damaging
influence on DNA molecules of cancer cells. The examined het-
erobicycle was found to possess the efficiency for DNA strand
breakage of cancer cell lines such as the cytotoxic antibiotic e
bleomycin [37], isolated from Streptomyces verticillus and effec-
tive in combination therapies against certain types of skin cancer,
testicular carcinoma and lymphomas. DNA strand breakage
caused by compound 14 can have dramatic effects on higher-or-
der chromatin structure because of its supercoiling and tight
packaging within the nucleus.
Taking into consideration the growth inhibition comparative
study results concerning the influence of the tested heterobi-
cycle (14) on cancer and normal cell lines, the selective action
of the examined compound can be expected. Also the anticancer
activity of the investigated heterobicycle seemed to be dose-
dependent.
From the point of a structureeactivity relationship in the class
of novel tested 8-aryl-4-oxo-4,6,7,8-tetrahydroimidazo[2,1-c]
[1,2,4]triazine-3-carbohydrazides (11e14), the results obtained
clearly indicated that a chloro substituent at position 3 in the phe-
nyl ring played a vital role for exerting cytotoxic effect on human
tumour cell lines. Generally, an antitumour property increases as
a result of the replacement of hydrogen, 4-methyl and 4-methoxy
substituents in 11e13 with a 3-chloro substituent in 14. Thus, the
highest growth inhibitory activity was observed for compound
14, having a 3-chlorophenyl substituent at position 8 of the
heterobicycle. Compounds 11e13 containing phenyl, 4-methyl-
phenyl and 4-methoxyphenyl groups in position 8 of the hetero-
bicycle demonstrated only slightly antiproliferative effects against
the tested cancer cell lines. Simultaneously, heterobicycles 12
and 13 were strictly speaking non-toxic for normal cell lines
investigated e HSF and GMK cells, whereas hydrazide 11 was
found to be non-toxic for GMK cells.
In the next series of experiments, the apoptotic activity of the
heterobicyclic hydrazide 14 in human breast cancer (T47D) cells
was proved using the apoptosis assay based on the annexin
V/propidium iodide staining. When T47D human breast cancer
cells were grown in the presence of compound 14 (32.6 mM),
a substantial percentage of apoptosis became evident (8.5%,
compared to2% inthecontrol)(Table3andPhoto1). Onthecon-
trary, when the normal cells e human skin fibroblast (HSF) cells
were grown in the presence of derivative 14 (32.6 mM), the
Table 2
Inhibition of in vitro normal and tumour cells growth by heterobicycles 9 and 11e14
Cell line
Cytotoxicity (growth inhibition in %)
9 [22]
11
12
13
14
I
II
I
II
I
II
I
II
I
II
Normal cell lines
HSF
GMK
16
20
17
25
16
2
20
0
0
2
0
0
0
0
0
20
0
29
0
0
Cancer cell lines
LS180
SiHa
30
41
26
35
52
41
0
0
4
15
0
0
11
5
14
9
1
19
1
1
18
0
42
45
26
54
63
40
T47D
9
8
HSF e human skin fibroblast cells e primary cell line; Vero (GMK, ECACC 88020401, African Green Monkey Kidney cells); LS180 (ECACC 87021202) e human
Caucasian colon adenocarcinoma cells; SiHa (ECACC 85060701) e uterus cancer cells; T47D (ECACC 85102201)e human breast carcinoma cells; I e concentration
of 10 mg mLꢀ1, which corresponds to following concentration values: 31.2 mM (9), 36.7 mM (11), 34.9 mM (12), 33.1 mM (13), 32.6 mM (14); II e concentration of
50 mg mLꢀ1, which corresponds to following concentrationvalues: 155.9 mM (9), 183.6 mM (11), 174.6 mM (12), 165.4 mM (13), 163.0 mM (14); compound 15 was not
tested due to its incomplete solubility.

K. Sztanke et al. / European Journal of Medicinal Chemistry 43 (2008) 1085e1094
1089
2500 kbp
2000 kbp
500 kbp
C
14
GMK
C
14
C
14
LS180
C
14
T47D
SiHa
C - control
Fig. 1. Induction of DNA damage in the tested cell lines after incubation with
heterobicycle 14 in concentration of 32.6 mM. Cells, prepared as agarose
plugs, were lysed and subjected to pulsed field gel electrophoresis through
a 1% agarose gel with propidium iodide.
quantity of apoptotic cells was found to be 1%, while in the con-
trol 0.6% (see Table 3 and Photo 2). Based on the performed ex-
amination, statistically significant apoptotic activity of the tested
hydrazide of the type 14 in human breast cancer cells was
ascertained.
Photo 1. The apoptotic effects of compound 14 (32.6 mM) in T47D human
breast cancer cells after 72 h of exposure. The assay is based on annexin
V/propidium iodide staining. Apoptotic cells: cells with nuclei stained yellow
(early stage of apoptosis) or red (late stage of apoptosis). The remaining
unstained cells represent the living cells. Magnification 400ꢃ. (For interpreta-
tion of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
Although breast cancer is most often treated with conven-
tional cytotoxic agents, it was proved difficult to induce apo-
ptosis in breast cancer cells [36]. Therefore, search for new
agents that are effective in activating apoptosis, such as 14,
seemed to be of great interest. Finally, our present data suggest
that a novel heterobicyclic hydrazide of the type 14 could be
useful in stimulating apoptosis in human breast cancer cells.
Owing to above-mentioned biological activity, compound 14
may be promising for the development of novel agents that in-
duce the DNA strand breakage, and therefore may be used as
the basis for the design of novel non-toxic anticancer drugs.
However, the results obtained from the tested ester and hy-
drazide derivative of 8-(3-chlorophenyl)-4-oxo-4,6,7,8-tetrahy-
droimidazo[2,1-c][1,2,4]triazine (9 and 14), showed that an
improved antitumour activity against LS180 and SiHa cancer
cells is due to the conversion of the ethoxycarbonyl formation
present in the position 3 of heterobicycle 9 into the carbohydra-
zide group located in the same position (in compound 14). The
carbohydrazide formation present in 14 renders this compound
highly soluble and hydrophilic. Probably, the decrease in lipo-
philicity, better water solubility profile and increasing percent-
age of nitrogen element could be a qualitative explanation of
profitable changes in its growth inhibitory properties in relation
to 9 as observed in Table 4.
Inthenextseriesofexperiments, wehavealsoinvestigatedthe
action of the designed heterobicycles (6e14) on the viability of
human leukaemic Jurkat cells in order to select compounds hav-
ingpromisingantiproliferativeactivities. Jurkatcellsareahuman
leukaemic cell line, which express CD4 clusters and has the abil-
itytoproduceseveral cytokinesinresponse tostimuli. Itisawell-
established model for the study of apoptotic death pathways of
cancer cells [38,39]. Therefore, in the conducted experiment
Jurkat cells were incubated in the presence of heterobicycles
6e14. Compounds 6e9 and 12e14 in three different concentra-
tions of the compound solutions (1, 50, 100 mM) nearly did not
change the viability of human leukaemic Jurkat cells. Simulta-
neously, so slight viability changes seemed to be mainly concen-
tration-independent (Table 5). However, the results summarized
in Table 5 pointed out that two of the examined derivatives: 10
and 11 exhibit evident antiproliferative potencies. The highest
viability decreases (to 28 and 23%, respectively, after exposure
Table 3
The effect of 32.6 mM heterocycle 14 on the level of apoptotic cells in the
tested normal and cancer cells
Cell lines
The amount of apoptotic cells in %
Normal cell line
HSF in control
0.6 ꢂ 0.05
1.0 ꢂ 0.1
HSF þ compound 14
Cancer cell line
T47D in control
2.0 ꢂ 0.1
T47D þ compound 14
HSF e human skin fibroblast cells e primary cell line; T47D (ECACC
8.5* ꢂ 0.5
85102201) human breast carcinoma cells; *statistical significance
e
( p < 0.001); apoptosis was determined using an annexin V-based assay; all ex-
periments were conducted in triplicate and gave similar results; the data are
presented as a mean ꢂ SD of three independent experiments.
Photo 2. The effect of the examined heterobicyclic hydrazide of the type 14
(32.6 mM) on human skin fibroblast cells stained with annexin and propidium
iodide. Apoptosis was measured at 72 h of incubation. Magnification 400ꢃ.

1090
K. Sztanke et al. / European Journal of Medicinal Chemistry 43 (2008) 1085e1094
Table 4
Calculated water solubility at 25 ꢁC, log P values and percentages of nitrogen
element for compounds 9 and 14
Table 6
Tumor cell growth inhibition expressed as IC50 (mM) in the presence of the
most active compounds (10, 11 and 14)
Compound
Solubilitya (mg Lꢀ1
)
log Pb
N (%)
Cell line
IC50 (in mM)
9
13.62
265.70
2.39
0.73
17.47
27.40
10
11
14
14
a
Adherent
LS180
SiHa
Water solubility values were calculated using EPI Suite for Windows v.
3.11. USEPA, Washington, DC, 2000.
>100
>100
>100
>100
>100
>100
>100
75.4
>100
b
The log P values were calculated using Pallas 3.1.1.2. software (distributed
by CompuDrug) 2003.
T47D
Leukaemic
Jurkat
32.2
85.5
>100
IC50 e the molar concentration that inhibits tumour cell growth to 50%; Vero
(GMK,ECACC88020401, AfricanGreenMonkeyKidneycells);LS180(ECACC
87021202) e human Caucasian colon adenocarcinoma cells; SiHa (ECACC
85060701) e uterus cancer cells; T47D (ECACC 85102201) e human breast car-
cinoma cells; Jurkat (ECACC 88042803) e human leukaemic T cell lymphoblast.
for24 h)inJurkat cellstreatedwith 50and100 mM heterobicycle
10 were observed. The toxic effect of the investigated compound
seemed to be dose-dependent.
In our previous studies compound 10 has shown 50 and
46% growth inhibition against LS180 and SiHa cancer cell
lines, respectively, in concentration of 50 mg mLꢀ1. Moreover,
its distinctly marked lower cytotoxicity against the examined
normal cell lines (HSF, GMK) and almost two-times higher
against cancer cells was ascertained. Full details of the method
of testing have been described elsewhere [22].
examination, it can be found from Table 5, that derivatives 10
and 11 were proved to reveal strong effects against the examined
Jurkat cells. Simultaneously, these heterobicycles demonstrated
slight toxic effects towards human skin fibroblast (HSF) cells.
Taking into consideration the influence of both investigated
compounds (10 and 11) on leukaemic Jurkat cells and a normal
cell line e human skin fibroblast (HSF) cells, the selective ac-
tion of the examined derivatives can be expected.
Also, compound 11 applied in a concentration of 100 mM
evoked a significant viability decrease (to 33%) in leukaemic
Jurkat cells after exposure for 24 h. Based on performed
IC50 values for the most active compounds (10, 11 and 14)
calculated from the doseesurvival curves obtained after 72 h
agent treatment (14) from 5-bromo-20-deoxy-uridine (BrdU)
test and from the doseesurvival curves obtained after 24 h
drug treatment (10 and 11) from MTT test, are shown in Table
6. Heterobicycle 10 was found to be effective against leukaemic
Jurkat cells with the best IC50 value of 32.2 mM. Also, derivative
11 with an IC50 value of 85.5 mM, was found to be moderately
active against the above-mentioned Jurkat cells, but it was al-
most 2.7-fold less potent than 10. Heterobicyclic hydrazide of
the type 14 exhibited moderate activity against SiHa cancer cells
with an IC50 value of 75.4 mM, but it was completely non-toxic
towards human normal cell line e HSF cells.
Due to effectiveness of the tested heterobicycles (10 and 11)
against leukaemic Jurkat cells, some behavioural tests were per-
formed to exclude or confirm their influence on the central ner-
vous system. It has been proved that compound 10 showed no
effect on the central nervous system of mice in behavioural tests
applied. This one given in a dose of 0.1 LD50 did not produce the
decrease in number of animals exhibiting pain reactivity in the
‘‘writhing syndrome’’ test. However, the heterobicyclic hydra-
zide of the type 11 exhibited only weak analgesic activity in
the ‘‘writhing test’’. The acute toxicities of both compounds
were relatively low (over 1000 mg kgꢀ1 i.p.). Furthermore,
two investigated heterobicycles revealed no neurotoxic proper-
ties, because, given in a dose of 0.1 LD50, did not impair the
motor coordination of mice in the rota-rod test.
Table 5
Percentage of viable normal human skin fibroblast (HSF) cells and human leu-
kaemic Jurkat cells following 24 h treatment with the tested concentrations of
heterobicycles 6e14
Compound Concentration Cell viability in normal Cell viability in cancer
in mM
HSF cells (in %)
Jurkat cells (in %)
6
1
50
100
1
91.6 ꢂ 4.7
98.1 ꢂ 3.8
77.1 ꢂ 5.4
91 ꢂ 2.9
82 ꢂ 6.8
103 ꢂ 8.0
105 ꢂ 7.6
83 ꢂ 5.2
7
50
100
1
87 ꢂ 2.8
89 ꢂ 4.5
82 ꢂ 3.0
93 ꢂ 6.2
8
103.5 ꢂ 5.6
100 ꢂ 5.8
97 ꢂ 4.0
118 ꢂ 4.3
110 ꢂ 2.9
110 ꢂ 4.7
114 ꢂ 3.9
111 ꢂ 3.0
107 ꢂ 3.2
91 ꢂ 6.4
50
100
1
9
104 ꢂ 5.0
103 ꢂ 3.6
98 ꢂ 3.9
50
100
1
10
11
12
13
14
102 ꢂ 4.5
84 ꢂ 1.8
50
100
1
28 3.8
85 ꢂ 1.9
23 2.4
89 ꢂ 7.0
82 ꢂ 1.4
50
100
1
83 ꢂ 3.2
82 ꢂ 4.2
82 ꢂ 1.4
33 4.0
93 ꢂ 6.7
109 ꢂ 7.3
111 ꢂ 10.9
95 ꢂ 7.4
50
100
1
87 ꢂ 9.3
79 ꢂ 10.6
97 ꢂ 11.3
97 ꢂ 12.7
100 ꢂ 10.4
100 ꢂ 10.7
116 ꢂ 10.8
115 ꢂ 11.1
98 ꢂ 3.0
50
100
1
102 ꢂ 4.4
100 ꢂ 5.3
108 ꢂ 9.6
113 ꢂ 5.9
105 ꢂ 6.8
4. Conclusion
50
100
In this report, easy and useful methods to obtain biologically
active fused 1,2,4-triazine aryl derivatives containing the ester
or hydrazide formations have been presented. It can be found
Cell viability after exposure for 24 h was evaluated by means of MTT (tetra-
zolium salt reduction) assay; cell viability in control e 100%; compound 15
was not tested due to its incomplete solubility.

K. Sztanke et al. / European Journal of Medicinal Chemistry 43 (2008) 1085e1094
1091
from Tables 2e6 that there is one compound (14) that possesses
efficiency for DNA strand breakage of the examined cancer cells
and has a strong affinity to SiHa cancer cells and reveals statis-
tically significant apoptotic activity in T47D carcinoma cells
and two heterobicycles (10 and 11) that exhibit strong effects to
human leukaemic Jurkat cells. So both 8-phenyl and
8-(3-chlorophenyl)-4-oxo-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]-
triazine-3-carbohydrazides (11 and 14) and one ethyl 1-(4-
oxo-8-(3,4-dichlorophenyl)-4,6,7,8-tetrahydroimidazo[2,1-c]
[1,2,4]triazin-3-yl)formate (10) may have potential antitumou-
ral activities. Further optimisation of these identified chemical
leads can possibly lead to more active molecules. Since
above-mentioned heterobicycles (10, 11 and 14) are showing
promising results, studies to establish their in vivo efficacy and
safety are being planned for their further development. Further
studies are in progress to define the important mechanisms of
action of the above-mentioned compounds.
d6, TMS) 40.9 (imidazolidine-C-4), 51.3 (imidazolidine-C-5),
122.9 (C-40), 124.4 (C-60), 126.8 (C-20), 131.2 (C-50), 132.2
(C-30), 141.8 (C-10), 158.5 (imidazolidine-C-2); m/z: 338[Mþ].
5.1.1. General procedure for synthesis of ethyl
1-(4-oxo-8-aryl-4,6,7,8-tetrahydroimidazo[2,1-c]
[1,2,4]triazin-3-yl)formates (6e10) (method i)
Freebaseof1-aryl-2-hydrazonoimidazolidine[23](0.05 mol)
was dissolved in 80 mL of n-butanol. Diethyl 2-oxomalonate
(8.71 g, 0.05 mol) was added and the mixture was refluxed for
7 h. During that time precipitation of the solid started. The mix-
ture was cooled overnight, the precipitate yielded was collected
and purified by recrystallization from DMF or DMF/methanol
mixture in the proportion indicated [22]. Physicochemical, ana-
lytical and spectral (IR, 1H NMR, 13C NMR, MS) characteristics
of the obtained compounds, given in Ref. [22], are in full agree-
ment with proposed structures.
5. Experimental protocols
5.1.2. General procedure for synthesis of 8-aryl-4-
oxo-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazine-3-
carbohydrazides (11e15) (method ii)
5.1. Instrumentations and general materials
A solution of respective ethyl 1-(4-oxo-8-aryl-4,6,7,8-tetra-
hydroimidazo[2,1-c][1,2,4]triazin-3-yl)formate (6e10) [22]
(0.03 mol) and 0.03 mol of hydrazine hydrate (100%) in 50
mL of n-butanol were refluxed for 5 h. The excess of solvent
was distilled under reduced pressure. The mixture was cooled
overnight, the precipitate yielded was collected and finally puri-
fied by recrystallization from DMF.
Chemicals (hydrazine hydrate, 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 uncor-
rected. The IR spectra were measured as potassium bromide pel-
lets using a Perkin-Elmer 1725X spectrometer. 1H NMR spectra
for compounds 11e14wererecordedona Bruker 200 MHz spec-
trometerin DMSO-d6 with TMS as an external standard at 295 K.
1H NMR spectrum for compound 15 was recorded on a Bruker
300 MHz spectrometer in DMSO-d6 with TMS as an external
standard at 295 K. 13C NMR spectra for compounds 4, 11e15
were recorded on a Bruker AC 200F instrument. Besides
1He1H COSY, HMBC and HMQC correlations were made for
free base ofintermediate 4. Mass spectroscopic analyses for com-
pounds 4 and 11e15 were performed at 70 eVon an AMD-402
and a Trace DSQ (Thermo Finnigan) mass spectrometers, respec-
tively, for molecular ion peaks. The molecular ion for compounds
14 and 15 was calculated according to 35Cl isotope.
5.1.3. General procedure for synthesis of 8-aryl-4-
oxo-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazine-3-
carbohydrazides (11e15) (method iii)
A solution of the corresponding ethyl 1-(4-oxo-8-aryl-4,6,
7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)formate (6e10)
[22] (0.05 mol) and 0.15 mol of hydrazine hydrate (100%) in
80 mL of ethanol was refluxed for 6 h. The excess of solvent
was removed by evaporation. Upon cooling the precipitate
was collected, washed with 2 ꢃ 10 mL cold ethanol and finally
purified by recrystallization from DMF.
Thin-layer chromatography was carried out on commercial
Merck SiO2 60 F254 plates having fluorescence indicator. The
spots were visualized in UV light l ¼ 254 and 355 nm. Elemen-
tal analyses were performed on a Perkin-Elmer analyzer and
were in range of ꢂ0.4% for each element analyzed (C, H, N,
I, Cl). The starting 1-(3-chlorophenyl)-2-hydrazonoimidazoli-
dine hydroiodide (4) was obtained by earlier described
method [29]. Its physicochemical and spectral data are the
following:
1-(3-Chlorophenyl)-2-hydrazonoimidazolidine hydroiodide
(4) [23]: recrystallized from propan-2-ol, yield 62%, m.p.
55e56 ꢁC. Spectroscopic data for C9H12ClIN4: IR (KBr) (n,
cmꢀ1): 1509 (NþH), 1571 (CaN), 3460e3315 (NH þ NH2);
1H NMR (d, ppm, DMSO-d6, TMS): 3.95 (dd, J ¼ 9.3 Hz,
J0 ¼ 8.1 Hz, 2H, CH2), 4.22 (dd, J ¼ 9.3 Hz, J0 ¼ 8.1 Hz, 2H,
CH2), 5.22 (s, 2H, NH2), 7.25e7.53 (m, 4H, CHarom.), 8.65
(s, 1H, NH), 9.26 (s, 1H, NþH); 13C NMR (d, ppm, DMSO-
5.1.4. General procedure for synthesis of 8-aryl-4-
oxo-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazine-3-
carbohydrazides (11e15) (method iv)
A solution of the corresponding ethyl 1-(4-oxo-8-aryl-4,6,
7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)formate (6e10)
[22] (0.05 mol) and 0.5 mol of hydrazine hydrate (100%) were
heated on the alloy bath in the temperature of ca. 90 ꢁC for
7 h. The excess of hydrazine hydrate was distilled under reduced
pressure. The reaction mixture was cooled, formed precipitate
was collected, washed with 2 ꢃ 20 mL cold ethanol and finally
purified by recrystallization from DMF.
5.1.4.1. 8-Phenyl-4-oxo-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]
triazine-3-carbohydrazide (11). Recrystallization from DMF;
yield 71% (method ii)/79% (method iii)/66% (method iv), m.p.
293e294 ꢁC. Spectroscopic data for C12H12N6O2: IR (KBr) (n,
cmꢀ1): 1744 (hydrazide-CaO), 1685 (triazine-CaO), 1561

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K. Sztanke et al. / European Journal of Medicinal Chemistry 43 (2008) 1085e1094
1
(CaN); H NMR (d, ppm, DMSO-d6, TMS): 4.21 (br s, 4H,
2CH2), 4.59 (s, 2H, NH2), 7.17e7.86 (m, 5H, ar-H), 9.68
(s, 1H, NH); 13C NMR (d, ppm, DMSO-d6, TMS): 40.5 (imidazo-
lidine-C-6), 45.4 (imidazolidine-C-7), ar C: [119.3 (CH), 124.2
(2CH), 129.0 (2CH), 138.3 (C)], 143.7 (triazine-C-4), 151.0 (tri-
azine-C-3), 152.4 (hydrazide-CaO), 160.9 (C-8a); EI-MS
[70 eV, m/z (%)]: 273 (8.5), 272 (Mþ, 55.0), 244 (11.9), 243
(14.6), 242 (100.0), 241 (85.4), 215 (5.5), 214 (17.9), 213 (4.9),
211 (9.1), 187 (4.2), 186 (27.5), 185 (31.2), 170 (4.2), 160
(4.5), 159 (16.3), 158 (56.1), 145 (11.3), 144 (22.7), 143 (5.3),
133 (3.4), 132 (30.1), 131 (41.0), 129 (3.2), 128 (5.0), 119
(4.7), 118 (13.8), 117 (15.9), 116 (4.0), 107 (4.6), 106 (19.9),
105 (16.6), 104 (40.9), 92 (3.3), 91 (14.9), 90 (4.8), 81 (6.4), 78
(7.9), 77 (78.3), 70 (27.0), 69 (4.5), 65 (15.6), 64 (4.1), 63 (3.8),
54 (5.0), 51 (17.1), 50 (3.5), 42 (25.3), 41 (3.6), 39 (4.9).
(13.5), 135 (28.6), 134 (22.4), 133 (18.3), 132 (8.2), 121 (10.8),
120 (48.1), 118 (5.5), 117 (6.1), 108 (9.5), 107 (7.1), 105 (7.2),
104 (6.5), 92 (18.8), 91 (6.3), 78 (8.4), 77 (20.6), 70 (9.8), 65
(9.8), 64 (8.9), 63 (6.3), 54 (5.4), 42 (13.4).
5.1.4.4. 8-(3-Chlorophenyl)-4-oxo-4,6,7,8-tetrahydroimidazo
[2,1-c][1,2,4]triazine-3-carbohydrazide (14). Recrystallization
from DMF; yield 68% (method ii)/73% (method iii)/64% (method
iv), m.p. 239e241 ꢁC. Spectroscopic data for C12H11ClN6O2: IR
(KBr) (n, cmꢀ1): 1739 (hydrazide-CaO), 1685 (triazine-CaO),
1
1553 (CaN); H NMR (d, ppm, DMSO-d6, TMS): 4.20 (br s,
4H, 2CH2), 4.59 (s, 2H, NH2), 7.24e7.73 (m, 4H, ar-H), 9.70 (s,
1H, NH); 13C NMR (d, ppm, DMSO-d6, TMS): 40.5 (imidazoli-
dine-C-6), 45.3 (imidazolidine-C-7), ar C: [117.3 (CH), 118.8
(CH), 123.7 (CH), 130.6 (CH), 133.4 (C), 139.8 (C)], 144.3 (tri-
azine-C-4), 150.8 (triazine-C-3), 152.4 (hydrazide-CaO), 160.8
(C-8a); EI-MS [70 eV, m/z (%)]: 308 (18.8), 307 (9.9), 306 (Mþ,
59.7), 279 (6.2), 278 (43.2), 277 (39.0) 276 (100.0), 275 (86.2),
250 (8.3), 249 (8.6), 248 (24.0), 222 (10.8), 221 (12.8), 220
(32.2), 219 (29.2), 212 (8.8), 211 (33.4), 194 (14.2), 193 (9.2),
192 (35.1), 184 (9.2), 179 (7.1), 178 (14.5), 168 (6.4), 167
(10.1), 166 (19.5), 165 (27.3), 158 (9.7), 157 (8.8), 152 (7.4),
142 (7.8), 141 (6.2), 140 (29.9), 139 (15.0), 138 (36.4), 125
(10.9), 117 (10.9), 113 (20.2), 112 (8.2), 111 (63.8), 99 (12.0),
90 (7.8), 81 (12.4), 77 (8.1), 76 (7.3), 75 (29.2), 70 (39.0), 63
(6.6), 54 (7.7), 42 (33.8).
5.1.4.2. 8-(4-Methylphenyl)-4-oxo-4,6,7,8-tetrahydroimidazo
[2,1-c][1,2,4]triazine-3-carbohydrazide (12). Recrystallization
from DMF; yield 67% (method ii)/74% (method iii)/68% (method
iv), m.p. 270e272 ꢁC. Spectroscopic data for C13H14N6O2: IR
(KBr) (n, cmꢀ1): 1738 (hydrazide-CaO), 1690 (triazine-CaO),
1562 (CaN); 1H NMR (d, ppm, DMSO-d6, TMS): 2.31 (s, 1H,
CH3), 4.19 (br s, 4H, 2CH2), 4.63 (s, 2H, NH2), 7.26
(d, J ¼ 8.5 Hz, 2H, ar: H-20 and H-60), 7.71 (d, J ¼ 8.4 Hz, 2H,
ar: H-30 and H-50), 9.67 (s, 1H, NH); 13C NMR (d, ppm,
DMSO-d6, TMS): 20.4 (-CH3), 40.5 (imidazolidine-C-6), 45.5
(imidazolidine-C-7), ar C: [119.4 (2CH), 129.4 (2CH), 133.4
(C), 135.8 (C)], 143.4 (triazine-C-4), 151.0 (triazine-C-3), 152.4
(hydrazide-CaO), 161.0 (C-8a); EI-MS [70 eV, m/z (%)]: 287
(11.5), 286 (Mþ, 72.8), 271 (6.1), 258 (12.5), 257 (15.8), 256
(98.4), 255 (100.0), 229 (6.7), 228 (14.6), 227 (5.2), 211 (8.4),
201 (7.9), 200 (43.7), 199 (26.9), 184 (4.6), 174 (5.6), 173
(24.7), 172 (76.7), 159 (8.5), 158 (18.8), 157 (7.1), 147 (4.5),
146 (42.0), 145 (46.3), 144 (6.2), 143 (5.2), 132 (12.6), 131
(19.9), 130 (5.5), 121 (4.4), 120 (20.5), 119 (19.2), 118 (39.7),
117 (13.8), 116 (6.7), 105 (8.9), 104 (6.7), 92 (6.9), 91 (75.4),
90 (9.5), 89 (11.8), 81 (4.4), 79 (7.0), 78 (6.9), 77 (16.1), 70
(16.9), 65 (25.3), 51 (4.6), 42 (17.4), 41 (4.8).
5.1.4.5. 8-(3,4-Dichlorophenyl)-4-oxo-4,6,7,8-tetrahydroimidazo
[2,1-c][1,2,4]triazine-3-carbohydrazide (15). Recrystallization
from DMF; yield 70% (method ii)/66% (method iii)/61% (method
iv), m.p. 157e158 ꢁC. Spectroscopic data for C12H10Cl2N6O2: IR
(KBr) (n, cmꢀ1): 1739 (hydrazide-CaO), 1683 (triazine-CaO),
1554 (CaN); 1H NMR (d, ppm, DMSO-d6, TMS): 3.60
(J ¼ 8.5 Hz, J0 ¼ 7.5 Hz, dd, 2H, CH2), 4.03 (J ¼ 8.6 Hz,
J0 ¼ 7.5 Hz, dd, 2H, CH2), 4.40 (s, 2H, NH2), 7.46e8.26 (m,
3H, ar-H), 9.40 (s, 1H, NH); 13C NMR (d, ppm, DMSO-d6,
TMS): 40.3 (imidazolidine-C-6), 47.0 (imidazolidine-C-7), ar C:
[118.2 (CH), 120.2 (C), 123.2 (CH), 129.9 (CH), 130.8 (C),
140.2 (C)], 144.8 (triazine-C-4), 150.2 (triazine-C-3), 152.5 (hy-
drazide-CaO), 161.2 (C-8a); EI-MS [70 eV, m/z (%)]: 342
(11.3), 340 (Mþ, 18.4), 312 (25.7), 311 (23.2), 310 (35.4), 309
(31.8), 256 (12.1), 255 (11.2), 254 (16.5), 253 (10.6), 245
(11.7), 230 (13.7), 228 (23.6), 226 (11.0), 189 (11.0), 187
(18.7), 186 (18.5), 178 (11.6), 177 (13.7), 176 (53.1), 175
(26.6), 174 (96.3), 173 (15.4), 172 (20.8), 165 (12.0), 163
(56.6), 162 (11.5), 161 (100.0), 159 (12.5), 151 (11.1), 147
(20.0), 145 (32.0), 143 (13.2), 139 (15.0), 133 (11.8), 126
(14.8), 125 (14.2), 111 (17.8), 109 (25.9), 99 (18.7), 90 (18.6),
75 (14.5), 74 (10.9), 70 (22.7), 69 (13.4), 63 (18.9), 62 (10.6),
43 (15.8), 42 (32.8), 41 (11.1).
5.1.4.3. 8-(4-Methoxyphenyl)-4-oxo-4,6,7,8-tetrahydroimidazo
[2,1-c][1,2,4]triazine-3-carbohydrazide (13). Recrystallization
from DMF; yield 65% (method ii)/70% (method iii)/63% (method
iv), m.p. 301e302 ꢁC. Spectroscopic data for C13H14N6O3: IR
(KBr) (n, cmꢀ1): 1742 (hydrazide-CaO), 1688 (triazine-CaO),
1556 (CaN); 1H NMR (d, ppm, DMSO-d6, TMS): 3.78 (s, 3H,
eOCH3), 4.17 (br s, 4H, 2CH2), 4.58 (s, 2H, NH2), 7.02
(d, J ¼ 8.5 Hz, 2H, ar: H-20 and H-60), 7.73 (d, J ¼ 8.5 Hz, 2H,
ar: H-30 and H-50), 9.67 (s, 1H, NH); 13C NMR (d, ppm,
DMSO-d6, TMS): 40.5 (imidazolidine-C-6), 46.0 (imidazoli-
dine-C-7), 55.3 (-CH3O), ar C: [114.1 (2CH), 121.5 (2CH),
131.2 (C), 156.2 (C)], 143.0 (triazine-C-4), 151.1 (triazine-C-3),
152.4 (hydrazide-CaO), 161.0 (C-8a); EI-MS [70 eV, m/z (%)]:
303 (11.7), 302 (Mþ, 74.6), 287 (10.8), 274 (12.1), 273 (15.6),
272 (98.1), 271 (100.0), 245 (5.8), 244 (10.2), 243 (5.7), 228
(6.0), 217 (12.8), 216 (41.8), 215 (15.3), 189 (25.5), 188 (44.2),
175 (5.4), 174 (18.6), 173 (7.3), 162 (37.0), 161 (34.8), 160
(6.3), 158 (9.4), 149 (6.8), 148 (10.4), 147 (20.1), 146 (7.2), 136
5.2. Inhibition of tumour cell growth assay
The newly synthesized heterobicyclic hydrazides of the type
11e14, with exception of 15, due to its incomplete solubility,
were evaluated for their antitumoural properties in vitro towards
three human cancer cell lines: human Caucasian colon

K. Sztanke et al. / European Journal of Medicinal Chemistry 43 (2008) 1085e1094
1093
adenocarcinoma (LS180), human uterus cancer (SiHa) and
human breast carcinoma (T47D). Moreover, two normal cell
lines e HSF (human skin fibroblast) cells and Vero (Green
Monkey Kidney cells) were included in the cytotoxicity study.
In thecurrentprotocoleach cell linewas inoculated at104 cells
per mL density and preincubated on a microtiter plate. Test agents
were then added at double examined concentrations (10 and
50 mg mLꢀ1) and culture incubated for 72 h. End-point determi-
nations were made with 5-bromo-20-deoxy-uridine (BrdU) label-
ing and detection kit III [40e43] on Elisa reader (BIO-TEC
Instruments USA).
The growth percentage was evaluated spectrophotometrically
versus untreated controls with used cell viability of growth assay.
Results for each spectrophotometrical measure were noticed as
per cent of growth inhibition. All the investigated compounds
were dissolved in DMSO prior to dilution into the biological as-
say. All experiments were done in triplicates. The investigations
were carried out in the Department of Biology and Genetics,
Medical University, Lublin, Poland.
procedure [38] and are the following. Cells were maintained in
RPMI 1640 medium, supplemented with 10% FBS, 2 mM L-glu-
tamine and 10 mg mLꢀ1 gentamycin. Cells were grown at 37 ꢁC
inahumidifiedatmosphereconsistingof5%CO2. Cellswerepas-
saged three times weekly and maintained at a density of
2 ꢃ 105 cells per mL. Cells used in the experiment were in loga-
rithmic growth phase. The medium used for experiment had the
same constituents as that used for cell passage, unless otherwise
indicated.
MTTreductioncell viability assay was performed using Jurkat
cells cultured in 96-well plates. Compounds 6e14 were dissolved
in DMSO priortodilutioninto the biological assay. Heterobicycle
15 was not tested due to its incomplete solubility. The effect of the
examined concentrations of heterocycles 6e14 on the cell viabil-
ity was estimated by a MTT based colorimetric assay (the succi-
natedehydrogenaseinhibition, SDI test) describedbyTakenouchi
and Munekata [50]. The metabolic activity was measured in cell
populations via incubation with a 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) that was reduced into
a coloured, water-insoluble formazan salt by viable cells. Addi-
tions were made to the culture medium for 24 h. Cells were
washed twice with HEPES-buffered incubation medium
(140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.1 mM MgCl2,
1.2 mM CaCl2, 5.5 mM glucose and 20 mM HEPES, pH 7.4),
and incubated for 45 min at 37 ꢁC in HBM containing MTT
(0.5 mg mLꢀ1). After this period, the HBM was carefully re-
moved and the blue formazan product was solubilized in
300 mL of 100% DMSO. The absorbance of each well was read
in an ELISA microplate reader at 570 nm. The obtained results
were presented as percentage of cell viability in comparison to
control. The presented results were obtained from three indepen-
dentmeasurements. TheinvestigationswerecarriedoutintheDe-
partment of Virology and Immunology, Maria Curie-Sk1odowska
University, Lublin, Poland.
5.3. Detection of induction of DNA damage in the tested
cell lines after incubation with heterobicycle 14
Following treatment of heterobicyclic hydrazide 14(32.6 mM),
cells were washed twice with phosphate buffered saline (PBS). In
the next step the DNAwas isolated by using the lysing buffer and
proteolytic enzyme e proteinase K [44]. After purification, DNA
samples were spread over 1% agarose gel with addition of propi-
dium iodide. Electrophoresis was performed at 120 V for 1.5 h at
the room temperature. All experiments were repeated three times.
The investigations were carried out in the Department of Biology
and Genetics, Medical University, Lublin, Poland.
5.4. Apoptosis assay
To estimate the apoptotic effect of compound 14 on the nor-
mal cell line e HSF cells and human breast carcinoma (T47D)
cells, the annexin VeFITC apoptosis detection kit (Sigma) was
used. The procedure consists of the binding of annexin V, having
a strong Ca2þ-dependent affinity for phosphatidylserine (PS) in
the membrane of cells, which is the beginning the apoptotic pro-
cess and the binding of propidium iodide to the cellular DNA in
cells where the cell membrane is totally compromised [45e49].
For apoptosis quantitation, the amount of apoptotic cells could
be observed under a confocal microscope Nicon Eclipse (Zeiss,
Germany) by using a filter l ¼ 460e490 nm. A total number of
1000 cells in three independent measurements were subjected to
analysis. The investigations were carried out in the Department
of Biology and Genetics, Medical University, Lublin, Poland.
5.6. Behavioural experiments
Behavioural experiments were performed on male Albino
Swiss mice (body weights of 20e25 g) purchased from licensed
´
dealer, Gorzkowska, Warsaw, Poland. Eight to ten animals were
kept in a cage, at room temperature of 20 ꢂ 1 ꢁC, on a 12/12 h
lightedark cycle (light on from 7 a.m. to 7 p.m.). Standard food
(Bacutil, Motycz, Poland) and water were available ad libitum.
Experimental and control groups consisting of 10 animals each
were selected by means of a randomized schedule. The experi-
ments were performed between 8 a.m. and 3 p.m. The investigated
compounds (10 and 11) were administered intraperitoneally (i.p.)
as suspension in 3% Tween 80 in a constant volume of 10 mL
kgꢀ1. Control animals received the equivalent volume of solvent.
Each experimental group consisted of ten animals.
5.5. Action of novel heterobicycles (6e14) on the
viability of human leukaemic Jurkat cells
The investigations were carried out in the Department of
Toxicology, Medical University, Lublin, Poland.
Human peripheral blood leukaemia, T cell line (Jurkat) was
obtained from the Institute of Immunology and Experimental
Therapy, Polish Academy of Sciences, Wroc1aw. The experi-
ments were conducted according to the previously described
5.6.1. Acute toxicity in mice for compounds 10 and 11
Acute toxicity was assessed according to Litchfield and
Wilcoxon methods [51] and presented as LD50 calculated
from the mortality of mice after 24 h.

1094
K. Sztanke et al. / European Journal of Medicinal Chemistry 43 (2008) 1085e1094
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