Crystal structure, antitumour and antimetastatic activities of disubstituted fused 1,2,4-triazinones

Article abstract of DOI:10.1016/j.bmcl.2009.07.036

Molecular structure of 3,8-disubstituted 7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-ones (8-14) was confirmed by X-ray crystallography of 14. All the compounds were evaluated for their antitumour and antimetastatic activities in vitro. Furthermore, their cytotoxicities towards human normal cell line-HSF cells were established, allowing us to point out some structure-activity relationships. Among them, imidazotriazinone 12, revealing remarkable dose-dependent viability decreases in human myeloma RPMI 8226 cells, was found to be completely non-toxic towards normal HSF cells. In addition, heterobicycles 8-12 were proved to exhibit significant antimetastatic potentials in the motility assay.

Full text of DOI:10.1016/j.bmcl.2009.07.036

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5095–5100
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Crystal structure, antitumour and antimetastatic activities of disubstituted
fused 1,2,4-triazinones
a
a
b
Krzysztof Sztanke a, , Kazimierz Pasternak , Małgorzata Sztanke , Martyna Kandefer-Szerszen ,
*
´
Anna E. Kozioł c, Izabela Dybała c
a Chair and Department of Medical Chemistry, Medical University, 4 Staszica Street, 20-081 Lublin, Poland
b Department of Virology and Immunology, Maria Curie-Skłodowska University, 19 Akademicka Street, 20-033 Lublin, Poland
c Faculty of Chemistry, Maria Curie-Skłodowska University, 20-033 Lublin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Molecular structure of 3,8-disubstituted 7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-ones (8–14) was
confirmed by X-ray crystallography of 14. All the compounds were evaluated for their antitumour and
antimetastatic activities in vitro. Furthermore, their cytotoxicities towards human normal cell line—
HSF cells were established, allowing us to point out some structure–activity relationships. Among them,
imidazotriazinone 12, revealing remarkable dose-dependent viability decreases in human myeloma RPMI
8226 cells, was found to be completely non-toxic towards normal HSF cells. In addition, heterobicycles 8–
12 were proved to exhibit significant antimetastatic potentials in the motility assay.
Received 29 May 2009
Revised 2 July 2009
Accepted 2 July 2009
Available online 10 July 2009
Keywords:
Fused 1,2,4-triazine
Imidazo[2,1-c][1,2,4]triazine nucleus
Crystal structure
Ó 2009 Elsevier Ltd. All rights reserved.
Antitumour activity
Antimetastatic potential
The 1,2,4-triazine nucleus is a prominent structural core system
present in numerous biologically active compounds. Azanucleo-
sides, structurally based on the 1,2,4-triazine scaffold, such as
6-azacytosine and 6-azauracil, have been found to reveal antitu-
mour,1,2 antiviral,3,4 and antifungal5 activities. 6-Azaisocytosine
(e.g., 3-amino-1,2,4-triazin-5(2H)-one), an isosteric isomer of
6-azacytosine and 6-azauracil, is of biological interest due to its
resistance to deaminase, while the well-known antiviral, anti-
fungal and antipsoriatic drug, azaribine, is structurally associated
with the 1,2,4-triazine moiety.6
Moreover, various condensed 1,2,4-triazines have been re-
ported to be extremely potent and the majority of them found bio-
logical applications.7–12 Pyrrolo[2,1-f][1,2,4]triazines as congeners
of substituted nucleic acid purines have revealed an interesting
broad spectrum of antiproliferative activity, as well as pronounced
in vitro growth inhibitory activities against human leukaemic cell
lines, comparable to that of 9-deazaadenosine.13 Pyrrolo[2,1-
c][1,2,4]triazines have also been found to possess inhibitory effects
on the growth of a wide range of cancer cells generally at 10À5 M
level, and in some cases, even at micromolar concentrations.14,15
Furthermore, some of pyrrazolo[5,1-c][1,2,4]triazines have been
reported to reveal in vitro remarkable antitumoural and antifungal
activities.16–22
Certain synthetic derivatives of the imidazo[2,1-c][1,2,4]triazin-
4(1H)-one have been suggested as novel bicyclic nucleosides re-
lated to 6-azaisocytosine.23 On the other hand, previously reported
by us polyazaheterocycles, containing the dihydroimidazo[2,1-
c][1,2,4]triazin-4(6H)-one core system, have acquired considerable
interest, because of their distinctly lower cytotoxicity towards nor-
mal cells and several-times higher against cancer cell lines;24,25
a
significant effect in tumour cells and simultaneously a lack of
toxicity towards normal cells.26,27 And so, our findings suggest that
imidazotriazinones are able to selectively inhibit the growth of tu-
mour cells, and therefore they might be promising drug candidates
having a beneficial side-effect profile.
Prompted by these facts and in continuation of our attempt to
obtain medicinally important heterocycles,24–31 herein we report
the molecular structure, antitumour and antimetastatic activities
in vitro of 3,8-disubstituted 7,8-dihydroimidazo[2,1-c][1,2,4]tria-
zin-4(6H)-ones (8–14).
The bicyclic polyazaheterocycles (8–13), bearing the bridge-
head nitrogen atom, have been generated in a straightforward
manner via an alternative synthetic approach,32 which obviate
the use of 1-arylimidazolidin-2-one hydrazone and
a
-oxoacid,
such as phenylglyoxylic acid (B), instead of its
a-oxoester—ethyl
phenylglyoxylate34,35 previously used. In this new approach
improved yields for the desired compounds 8–13 were obtained.
The target imidazotriazinone 14 has been obtained via the condensa-
tion/cyclization reaction of 1-(2,5-dichlorophenyl)imidazolidin-2-one
* Corresponding author. Tel.: +48 81 5357362; fax: +48 81 5357361.
E-mail address: krzysztof.sztanke@am.lublin.pl (K. Sztanke).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.036

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K. Sztanke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5095–5100
hydrazone with phenylglyoxylic acid, according to general proce-
dure for the synthesis of heterobicycles 8–14.32,33 This novel hete-
robicycle (14) has not been previously described in the literature.
The structural determination of this compound was achieved from
its elemental analysis and spectral data (1H NMR, IR).36 The as-
signed structure of 14 was further validated by single-crystal
X-ray analysis.
The
target
3,8-disubstituted
7,8-dihydroimidazo[2,1-
c][1,2,4]triazin-4(6H)-ones (8–14) were obtained in good yields
(Table 1) in one-pot synthetic approach by treating of hydrazones
1–7 with phenylglyoxylic acid in inert organic solvents under re-
flux for 5–7 h (Scheme 2). These polyazaheterocycles could be pre-
pared starting both from hydroiodides of 1-arylimidazolidin-2-one
hydrazone in the triethylamine presence (method i),32 as well as
from their free bases (method ii).33 1H NMR and IR spectroscopic
data of the target imidazotriazinones (8–14), obtained by the two
above-mentioned methods, were also identical. Highest yields for
imidazotriazinones of the 8–14 type were achieved, when appro-
priate 1-arylimidazolidin-2-one hydrazone hydroiodides, without
conversion to their free bases, were used in the condensation with
phenylglyoxylic acid (Table 1, method i).
Forming the dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-one
core of the molecules 8–14 is proposed to proceed by a stepwise
mechanism. In the first step the condensation of an appropriate
hydrazone of the 1–7 type with phenylglyoxalic acid (B) took place
to afford an intermediate— the open-chain compound as the result
of loss of a water molecule. Because of the presence of the hydro-
gen atom on the cyclic N-3 nitrogen of the imidazolidine ring, the
subsequent cyclocondensation of this intermediate was attempted
to give the bicyclic polyazaheterocycle of the 8–14 type, with con-
comitant loss of water and subsequent ring closure. The concurrent
course of the cyclization reaction leading to the dihydroimi-
dazo[2,1-c][1,2,4]triazole core system (with concomitant loss of
formic acid molecule) was excluded, because spectral data
1H NMR and IR spectra of all the imidazotriazinones 8–14 were
considerably different in comparison to those of their starting
materials (entries 1–7). Above all, there were no signals of protons
derived from the endocyclic imidazolidine-NH and exocyclic @N–
NH2 formations in the 1H NMR spectra of heterobicycles 8–14.
Simultaneously, the number of aromatic protons had increased
by five due to the additional unsubstituted phenyl ring present
at the 3-position of the polyazaheterobicyclic structure of 8–14.
In the IR spectra the presence of absorption bands of 1687 and
1554 cmÀ1, ascribable to the triazine-C@O group and the C@N
bond at the ring junction confirmed the formation of the cyclic
products. In the fully conjugated core system of the 8–14 type,
where the double bond is contained within the triazinone ring,
the difference in C@O and C@N frequencies was found to be in
the order of 131–133 cmÀ1. These data are in a full agreement with
those provided by Le Count and Greer for other fully conjugated
fused 1,2,4-triazin-5-ones.37
Biologically active 1-arylimidazolidin-2-one hydrazones24,25
(entries 1–7), useful as key building blocks for the synthesis of tar-
get core heterobicycles 8–14, were prepared in gram quantities
using reliable patent pending methodology proposed by Sztanke.30
The four-step synthetic pathway,26 leading to these compounds is
depicted in Scheme 1. In the first step, commercially available
substituted anilines were converted into N-arylethylenediamines
by the Lehmann procedure38 or by the classical Knoevenagel and
Mercklin method using the modification of Takeda.39,40 Their
further condensation with carbon disulfide, in an inert organic
solvent—xylene, led to the formation of 1-arylimidazolidine-2-thi-
ones, followed by the cyclization of intermediate dithiocarbaminic
acid derivatives, with concomitant loss of hydrogen sulfide mole-
cule and the subsequent ring closure. Because of the existence of
thiol–thione tautomerism, the alkylation of respective thiols with
one equivalent of methyl iodide in methanolic medium was possi-
ble, to afford 1-aryl-2-methylthioimidazoline hydroiodides in 75–
85% yields,28 those in turn were refluxed with hydrazine hydrate
in methanol to give 1-aryl-2-hydrazinoimidazoline hydroiodides
in good yields (62–75%).30
Table 1
Physicochemical properties of 3,8-disubstituted 7,8-dihydroimidazo[2,1-c][1,2,4]tria-
zin-4(6H)-ones (8–14)
Compound
R
Yield (%)
Mp (°C)
Molecular weight
log kw
(i)
(ii)
8
9
4-CH3O
3-CH3
2-Cl
3-Cl
4-Cl
75
67
71
75
73
70
67
63
62
65
69
60
66
62
249–251
217–218
230–231
213–214
274–276
278–280
230–232
320.35
304.35
324.76
324.76
324.76
359.21
359.21
3.01
3.48
3.22
3.82
3.80
4.59
nd
10
11
12
13
14
3,4-Cl2
2,5-Cl2
Log kW values (HPLC)54 were determined on an octadecyl silica column (LC-18) with
mobile phase of the type MeOH–H2O according to the procedure55 therein reported.
nd—not determined.
R
R
S
R
R
a
N
N
NH
SH
d
NH2
NH
N
NH2
e
b
R
c
R
R
NH
CN
N
N
SCH3
HI
f
NHNH2
HI
N
N
Scheme 1. Synthetic pathway for the preparation of starting 1-aryl-2-hydrazinoimidazolines. Reagents and conditions: (a) aziridine, AlCl3, dry toluene, (b) HCHO, Na2S2O5,
NaCN, water, reflux; (c) H2, NiRa, MeOH/NH3, 100 °C; (d) CS2, xylene, rt, 20 min, reflux, 7 h; (e) CH3I/MeOH, rt, 48 h, reflux, 6 h; (f) hydrazine hydrate/MeOH, reflux, 24 h.

K. Sztanke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5095–5100
5097
R
N
N
+
NH2
C
NH
1-7
O
HOOC
B
i or ii -H2O
R
R
N
N
N
N
N
- HCOOH
N
NH
HOOC
N
-H2O
R
N
N
N
N
O
8-14
Scheme 2. One-pot approach for the synthesis of imidazotriazinones 8–14. 1, 8: R = 4-CH3O; 2, 9: R = 3-CH3; 3, 10: R = 2-Cl; 4, 11: R = 3-Cl; 5, 12: R = 4-Cl; 6, 13: R = 3,4-Cl2;
7, 14: R = 2,5-Cl2. Reagents and conditions: (i) (1–7) HI + B, triethylamine, n-butanol, reflux, 7 h; (ii) (1–7) + B, DMF, reflux, 5 h.
distinctly support the 7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4-
(6H)-one nucleus formation and argue against an alternative cycli-
zation process.
while Cl1Á Á ÁCl244 interactions connect adjacent dimers into a rib-
bon (Fig. 2). Associations of ribbons through the C–HÁ Á ÁO@C hydro-
Finally, the molecular structure of 3,8-disubstituted 7,8-dihy-
droimidazo[2,1-c][1,2,4]triazin-4(6H)-ones was confirmed by
X-ray crystallography of 14.41 Its structure was solved by direct
methods using the SHELXS-97 program42 and refined by full-matrix
least-squares on F2 using SHELXL-97 program.43 A perspective view
of the molecule 14 with atom numbering is shown in Figure 1.
Bond lengths in the dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-
one system indicate localized double bonds between C8a–N1 and
N2–C3 atoms. The presence of an aryl substituent at the C3 atom
does not allow for electron-delocalization in the C3–C4–O4 frag-
ment, which is indicated by the bond length distribution. The
endocyclic torsion-angle values ( 4°) indicate planarity of the
six-membered triazine ring, while the dihydroimidazole ring is
considerable non-planar, adopting an envelope conformation. The
phenyl substituents at the C3 and N8 atoms are not coplanar with
the heterocyclic nucleus (het); observed interplanar angles are 29°
and 64° for het/C3-phenyl and het/N8-phenyl, respectively. Due to
the molecular structure of 14 the molecular packing in the solid
state is stabilized by weak C–HÁ Á ÁO, C–HÁ Á ÁCl and ClÁ Á ÁCl contacts.
Intermolecular C4p–HÁ Á ÁCl2 bonds link molecules to form a dimer
Figure 1. The molecular structure of heterobicycle 14. Bond distances within 7,8-
dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-one system are: N1–N2 1.381(2); N2–C3
1.306(3); C3–C4 1.472(3); C4–N5 1.372(3); N5–C6 1.459(3); C6–C7 1.520(4); C7–
N8 1.473(3); N8–C8a 1.358(3); C8a–N1 1.307(3); C8a–N5 1.357(3); C4–O4 1.219(3)
Å.

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K. Sztanke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5095–5100
of their promising biological properties an alternative synthetic ap-
proach leading to the formation of 3,8-disubstituted 7,8-dihydro-
imidazo[2,1-c][1,2,4]triazin-4(6H)-ones (8–14) might be
considered as a useful method for the preparation of these biolog-
ically active compounds because of the affordability of the starting
materials, good yields obtained and straightforward product
isolation.
An attempt was made to determine the action of target hetero-
bicycles (8–14) on the viability of human peripheral blood myelo-
ma cells (RPMI 8226 cells),45 being a recognized model for multiple
myelomas,46,47 in order to select compounds having promising
anticancer activity and a clean toxicity profile. In the experiment
conducted RPMI 8226 cells and a normal cell line—HSF cells48 were
incubated in the presence of imidazotriazinones 8–14 and with
culture medium alone. Antiproliferative activities in vitro of the
tested compounds49 were determined by a colourimetric assay
MTT based, proposed by Takenouchi and Munekata.50
From the results of this biological assay (Table 2) certain struc-
ture–activity relationships can be derived. It is especially worth
noting, that the presence of electron-withdrawing chlorine substi-
tuent at position-para of the phenyl ring was found to be essential
for the imidazotriazinone to be active against human myeloma
RPMI 8226 cells and to have selective action. Thus, the most
interesting heterobicycle 12, having a log kw value of 3.80, with a
para-chlorophenyl moiety at position 8 of heterobicyclic system,
revealed remarkable dose-dependent viability decreases in myelo-
ma RPMI 8226 cells. Simultaneously, this compound was found to
be completely non-toxic for the normal cell line used—HSF cells,
independently from the concentration applied. These experimental
data suggest a beneficial toxicity profile of the imidazotriazinone
12.
Figure 2. Packing diagram showing C–HÁ Á ÁCl and ClÁ Á ÁCl44 intermolecular contacts
within the ribbon.
gen bonds and Cl1Á Á ÁCl1 contacts completes three-dimensional
crystal network.
Different arrangement of molecules is observed in the crystal of
2,6-dichlorophenyl-7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-
one derivative,24 where O@C groups are involved in numerous
C–HÁ Á ÁO hydrogen bonds and orthogonal multipolar interactions
to the heterocyclic system. In this case, antiparallel orientation of
the C–Cl bonds of neighbouring molecules was observed.
Spectral (1H NMR, IR) and analytical data of all the synthesized
heterobicycles, given in References and notes and those provided
in Supplementary data, were in full agreement with proposed
structures (Table 1).
Replacement of the electron-withdrawing para-Cl substituent,
that of the phenyl ring, in 12 for the electron-withdrawing
meta-Cl group, leading to its positional analogue 11, with a log kw
In view of current interest in the design of heterobicyclic deriv-
atives containing the 1,2,4-triazine moiety, particularly on account
Table 2
Percentage of viable human skin fibroblasts—HSF cells and human peripheral blood myeloma RPMI 8226 cells following 24 h treatment with the tested concentrations of
heterobicycles 8–14
Compound
R
Concentration in
l
M
Cell viability in normal HSF cells (in %)
Cell viability in cancer RPMI 8226 cells (in %)
8
4-CH3O
1
50
100
88 5.9
64 5.7
73 6.6
106 6.2
57 4.5
43 1.8
9
10
11
12
13
14
3-CH3
2-Cl
1
50
100
100 4.1
94 3.8
88 1.4
107 5.6
79 4.7
71 5.7
1
50
100
108 3.1
104 2.0
104 3.9
103 5.6
103 6.2
101 7.7
3-Cl
1
50
100
108 3.8
85 5.0
88 5.0
105 9.3
70 4.1
60 2.4
4-Cl
1
50
100
104 3.3
96 6.7
102 6.5
101 5.5
56 1.2
39 2.3
3,4-Cl2
2,5-Cl2
1
50
100
93 1.7
48 1.5
47 1.4
108 5.8
69 5.3
58 4.0
1
50
100
87 1.6
31 2.9
17 3.2
109 6.6
43 4.1
41 2.6
Cell viability after exposure for 24 h was evaluated by means of MTT (tetrazolium salt reduction) assay.
Cell viability in control—100%.
Data represent the mean SD of at least three independent measurements.
Human skin fibroblast cells—HSF cells—primary cell line.
Human peripheral blood myeloma cells—RPMI 8226 cells—cancer cell line.
The examined compounds 8–14 were dissolved in DMSO prior to dilution into the biological assay.

K. Sztanke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5095–5100
5099
value of 3.82, led to the viability increase in RPMI 8226 myeloma
120
100
80
60
40
20
0
cells, that is the reduction of antiproliferative action. Incorporation
of an additional chlorine atom into the phenyl ring, leading to di-
chloro-substituted imidazotriazinone derivatives (13 and 14),
was found to be responsible for the complete loss of their selectiv-
ity. An especially large cytotoxicity increase in normal cells was
observed for the 2,5-dichloro derivative (14), which at a concentra-
tion of 100 lM caused over twofold greater viability decrease in
normal HSF cells compared to cancer ones. This compound
revealed remarkable dose-dependent viability decreases in normal
cells. Replacement of the one chloro group, that of the phenyl ring,
in the most promising 2,5-dichloro-substituted derivative (14), by
a hydrogen atom leading to its 2-chloro analogue (10), having a
log kw value of 3.22, appeared to be unfavourable, because this
structural change resulted in the complete loss of activity of 10.
As well, the replacement of electron-withdrawing para-chloro
group at the phenyl ring in heterobicycle 12, for either electron-
withdrawing ortho-chloro one, leading to compound 10, resulted
in the complete disappearance of biological potency. Replacement
of para-Cl substituent at the phenyl ring, with strong electron-
donating para-OCH3 group, leading to heterobicycle 8 (with a
log kw value of 3.01), had no influence on the viability in myeloma
RPMI 8226 cells. Simultaneously, this structural change led to cyto-
toxicity increase towards normal cells. Furthermore, it has been
noted, that replacement of electron-withdrawing 3-Cl substituent,
that at the phenyl ring, in an imidazotriazinone 11 for a weakly
electron-donating 3-CH3 one, leading to the analogue 9, with a
log kw value of 3.48, caused only slight viability changes in normal
and cancer cell lines.
C
8
9
10
C – control – cell motility in culture medium alone – 100 %
HeLa B cells were treated with 25 µM of heterobicycles 8-14 for 24 h
11
12
13
14
Figure 3. Antimetastatic activities of the examined imidazotriazinones 8–14.
Because the ability of cancer cells to migrate is associated with
their invasive metastatic potential,53 these experimentaldatasuggest
that five imidazotriazinones 8–12 exert remarkable antimetastatic
activities. According to the literature search, this is the first report re-
lated to antimetastatic activities of these imidazotriazinones.
In conclusion, our findings revealed that the incorporation of
para-chlorophenyl moiety into position 8 of heterobicyclic scaffold,
in the series of imidazotriazinone derivatives is essential and
favourable for antitumour activity and selectivity. Taking into con-
sideration the influence of the best compound (12) on human
peripheral blood myeloma cells and normal ones, selective action
can be expected. The anticancer effect of heterobicycle 12 is attrib-
uted to decreased cell division and inhibition of cell migration.
Thus, imidazotriazinone 12 may be considered as a basis for the
design of novel antitumour agents without affect normal cells.
Since this compound is showing promising results in vitro, studies
to establish its in vivo efficacy and safety are being planned. In
addition, our findings suggest that imidazotriazinones 8–12 are
able to inhibit or limit the metastatic process, and therefore these
ones might be powerful candidates as preventive agents against
cancer metastasis.
It may be concluded that the presence of the chlorine atom at
para-position on the phenyl ring was the best choice of substitu-
tion in the series of imidazotriazinone compounds (8–14) giving
rise to the most promising compound 12.
In the next series of experiments, the effect of heterobicycles 8–
14 on tumour cells was studied using the cell motility assay pro-
posed by Yang et al.51 In this assay, human cervix carcinoma cells
(HeLa B cells),52 were exposured to the culture medium alone and
to 25 lM of an appropriate imidazotriazinone of the 8–14 type for
24 h. The results are presented as percentage of cell motility in
comparison to control. Cell motility in the absence of the tested
imidazotriazinone derivative was considered as 100%. This assay
showed that five heterobicycles 8–12, those bearing monosubsti-
tuted phenyl ring (Table 3 and Fig. 3) could significantly inhibit
the migration ability of cervix carcinoma cells in vitro, inasmuch
as a smaller percentage of these cells migrated to the wound area
was found in HeLa B cells exposured to compounds 8–12 (Table 3
and Fig. 3). Introduction of an additional chlorine atom to the
phenyl ring appeared to be unfavourable for the antimetastatic
activity, because this structural change led to a dramatic decrease
(see 3,4-dichloro derivative 13) or even to the completely lack of
biological activity (2,5-dichloro analogue 14).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.036.
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Compound
R
Cell motility (% of control)
8
9
4-CH3O
3-CH3
2-Cl
3-Cl
4-Cl
59 3.1
68 2.1
59 3.1
52 6.2
50 9.3
90 3.1
103 15
10
11
12
13
14
3,4-Cl2
2,5-Cl2
14. Diana, P.; Barraja, P.; Lauria, A.; Montalbano, A.; Almerico, A. M.; Dattolo, G.;
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Cell motility after 24 h in culture medium alone (control)–100%.
Data represent the mean SD of at least three independent measurements.
HeLa B (ECACC 85060701)—human Negroid cervix carcinoma cells.

5100
K. Sztanke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5095–5100
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to The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44 1223
336 033; e-mail: deposit@ccdc.cam.ac.uk or www: http://
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www.ccdc.cam.ac.uk). Single crystals of compound 14 were obtained from
DMF/MeOH (4:1 v/v) mixture. All the crystals were grown by slow evaporation
of solvents, at a temperature of about 293 K.
42. Sheldrick, G. M. SHELXS-97: Program for Crystal Structure Solution, University of
Göttingen: Germany, 1997.
22. Belskaia, N. P.; Deryabina, T. G.; Koksharov, A. V.; Kodess, M. I.; Dehaen, W.;
Lebedev, A. T.; Bakulev, V. A. Tetrahedron Lett. 2007, 48, 9128.
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from Diffraction Data; University of Göttingen: Germany, 1997.
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1994, 116, 4910.
24. Sztanke, K.; Rzymowska, J.; Niemczyk, M.; Dybała, I.; Kozioł, A. E. Eur. J. Med.
Chem. 2006, 41, 539.
45. Human peripheral blood myeloma cells—RPMI 8226 cells (ATCC CCL-155)—
were obtained from the American Type Culture Collection. This cell line
25. Sztanke, K.; Rzymowska, J.; Niemczyk, M.; Dybała, I.; Kozioł, A. E. Eur. J. Med.
Chem. 2006, 41, 1373.
26. Sztanke, K.; Pasternak, K.; Rzymowska, J.; Sztanke, M.; Kandefer-Szerszen´ , M.
derived from the peripheral blood of
myeloma. RPMI 8226 cells were maintained in RPMI 1640 medium
supplemented with 10% FBS and 2 mM -glutamine. Cells were kept at 37 °C
a 61-year-old male with multiple
L
Bioorg. Med. Chem. 2007, 15, 2837.
27. Sztanke, K.; Pasternak, K.; Rzymowska, J.; Sztanke, M.; Kandefer-Szerszen´ , M.
Eur. J. Med. Chem. 2008, 43, 1085.
in a humidified atmosphere consisting of 95% air and 5% CO2. Cells were
maintained at a density of 3 Â 105 cells per mL.
46. Matsuoka, Y.; Moore, G. E.; Yagi, Y.; Pressman, D. Proc. Soc. Exp. Biol. Med. 1967,
125, 1246.
28. Sztanke, K.; Pasternak, K.; Sidor-Wójtowicz, A.; Truchlin´ ska, J.; Józ´wiak, K.
Bioorg. Med. Chem. 2006, 14, 3635.
29. Sztanke, K.; Pasternak, K.; Rajtar, B.; Sztanke, M.; Majek, M.; Polz-Dacewicz, M.
Bioorg. Med. Chem. 2007, 15, 5480.
30. Sztanke, K. PL Patent, Appl. No. 373877, 2005.
31. Sztanke, K.; Fidecka, S.; Ke˛dzierska, E.; Karczmarzyk, Z.; Pihlaja, K.; Matosiuk,
47. Farmer, L. J.; Zhi, L.; Jeong, S.; Lamph, W. W.; Osburn, D. L.; Croston, G.; Flatten,
K. S.; Heyman, R. A.; Nadzan, A. M. Bioorg. Med. Chem. Lett. 2003, 13, 261.
48. HSF cells—human skin fibroblasts, human normal cell line. HSF cells were
obtained by the standard trypsinization of a skin forearm fragment (1 mm2)
derived from healthy person. HSF cells were routinely grown in Dulbecco’s
modified Eagle’s medium (DMEM), supplemented with 10% foetal bovine
D. Eur. J. Med. Chem. 2005, 40, 127.
32. Synthesis of 8–14 (method i): Phenylglyoxylic acid (7.51 g, 0.05 mol) was
added to the suspension of an appropriate 1-arylimidazolidin-2-one hydrazone
hydroiodide (0.05 mol) in 60 cm3 of n-butanol. The mixture was stirred
vigorously, and triethylamine (5 mL) was added. The reaction was carried out
under reflux for 7 h. The mixture was kept overnight in a refrigerator and
during that time, precipitation of solid started. The crude product was
collected, washed off with cold methanol, and finally purified by
recrystallization from DMF. Physicochemical data of the synthesized
compounds are collected in Table 1.
serum (FBS), 100 l
g mLÀ1 of streptomycin, 100 U mLÀ1 of penicillin in plastic
tissue culture flasks. HSF cells were plated into 96-well plastic plates (Nunc,
Denmark) at a density of 1 Â 105 cells per well in appropriate media with 10%
FBS to obtain the confluent growth of cells. These ones were grown at 37 °C in a
humidified atmosphere consisting of 5% CO2.
49. The effect of the examined concentrations (1, 50 and 100 lM) of heterocycles
(8–14) on the cell viability was estimated by a colorimetric assay MTT based
(the succinate dehydrogenase inhibition, SDI test) proposed by Takenouchi and
Munekata.50 The cell proliferation was assessed after exposure for 24 h in cell
33. Synthesis of 8–14 (method ii): An appropriate 1-arylimidazolidin-2-one
hydrazone (0.05 mol) was dissolved in 50 mL of DMF. Then phenylglyoxylic
acid (7.51 g 0.05 mol) was added and the mixture was heated under reflux for
5 h. The mixture was kept overnight in a refrigerator, the precipitate yielded
was collected by filtration, and finally purified by recrystallization from DMF.
Physicochemical data of the synthesized compounds are collected in Table 1.
34. Sztanke, K. Acta Pol. Pharm. Drug Res. 2004, 61, 373.
populations via incubation with
a
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), that was reduced into purple, water-
insoluble formazan product by the mitochondrial dehydrogenases of viable
cells. The production of formazan is measured spectrophotometrically
following its solubilization. MTT reduction cell viability assay was performed
using cells cultured in 96-well plates. The absorbance of each well was read
using E-max microplate reader at 570 nm wavelength. The obtained results
were presented as percentage of cell viability in comparison to control. The
presented results were obtained from three independent measurements and
provided with the standard deviation. The investigations were carried out in
the Department of Virology and Immunology, Maria Curie-Skłodowska
University, Lublin, Poland.
´
35. Sztanke, K.; Tkaczynski, T. Proc. 1st World Meeting APGI/APV on
Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology, Budapest,
9–11 May, 1995, 107.
36. 3-Phenyl-8-(2,5-dichlorophenyl)-7,8-dihydroimidazo[2,1-c][1,2,4]triazin-
4(6H)-one (14). Anal. Calcd. for C17H12Cl2N4O: C, 56.84; H, 3.37; Cl, 19.74; N,
15.60. Found: C, 56.77; H, 4.42; Cl, 19.66; N, 15.65. 1H NMR (d, ppm, DMSO-d6,
300 MHz, TMS): 4.10 (dd, J = 8.9 Hz, J’ = 3.3 Hz, 2H, CH2), 4.32 (dd, J = 8.8 Hz,
50. Takenouchi, T.; Munekata, E. Peptides 1998, 19, 365.
51. Yang, S. F.; Chu, S. C.; Liu, S. J.; Chen, Y. C.; Chang, Y. Z.; Hsieh, Y. S. J.
Ethnopharmacol. 2007, 110, 483.
52. Human Negroid cervix carcinoma cells—HeLa B cells (ECACC 85060701)—were
obtained from the European Collection of Cell Cultures. HeLa B cells were
J’ = 3.2 Hz, 2H, CH2), 7.40–8.08 (m, 8H, CHarom.); IR (KBr) (
(triazine-C@O), 1553 (C@N).
m
, cmÀ1): 1686
37. Le Count, D. J.; Taylor, P. J. Tetrahedron 1975, 31, 433.
38. Lehmann, D.; Faust, G. DD Patent 1,556,141, 1982; Chem. Abstr. 1983, 98,
125599g.
39. Knoevenagel, E.; Mercklin, E. Chem. Ber. 1904, 37, 4087.
40. Takeda, A. J. Org. Chem. 1957, 22, 1096 (and references cited therein).
41. Crystal data for 3-phenyl-8-(2,5-dichlorophenyl)-7,8-dihydroimidazo[2,1-
c][1,2,4]triazin-4(6H)-one (14). C17H12Cl2N4O, FW = 359.21, monoclinic, C2/c,
a = 24.459(6), b = 9.133(2), c = 14.388(3) Å, b = 100.37(3)°, the intensities were
maintained in RPMI 1640 medium supplemented with 2 mM
L-glutamine and
10% FBS. Cells were kept at 37 °C in a humidified atmosphere consisting of 95%
air and 5% CO2. Cells were maintained at a density of 3 Â 105 cells per mL.
53. Rzeski, W.; Matysiak, J.; Kandefer-Szerszen´ , M. Bioorg. Med. Chem. 2007, 15,
3201.
54. Tuzimski, T.; Sztanke, K. J. Planar Chromatogr.-Mod. TLC 2005, 18, 274.
55. HPLC experimental.54 HPLC analysis of the target imidazotriazinones (8–13)
was performed using a Shimadzu LC-10 AT chromatograph (Kyoto, Japan),
measured at room temperature, using CuK
a radiation and a single crystal of
dimensions 0.30 Â 0.28 Â 0.27 mm. 6814 Reflections were measured, of which
equipped with 5
AV UV–vis detector operated at 254 nm and 366 nm, and
injector with 20 loop (Cotati, CA, USA). All chromatographic
l
m Supelcosil LC-18 column (150 Â 4.6 mm i.d.), an SP-10
3469 were independent (Rint = 0.0693). Final discrepancy factors are
a
Rheodyne
R1 = 0.0435, wR2 = 0.1025 for I > 2r(I) and R1 = 0.1044, wR2 = 0.1225 for all
a
lL
data, S = 1.001. Full crystallographic details of 14 have been deposited with the
Cambridge Crystallographic Data Centre and allocated the deposition number
CCDC 728254. Copies of the data can be obtained, free of charge, on application
experiments were carried out at ambient temperature (22 1 °C) with
eluent flow rate of 1.0 mL minÀ1
Shimadzu Class-VP program.
. Chromatograms were registered using