Novel imidazo[4,5-b]pyridine and triaza-benzo[c]fluorene derivatives: Synthesis, antiproliferative activity and DNA binding studies

Article abstract of DOI:10.1016/j.ejmech.2011.03.062

In the present paper, we have described the synthesis and biological activity of the novel derivatives of imidazo[4,5-b]pyridines and triaza-benzo[c]fluorenes (7-21, 24-26, 28-29). A preponderance of these compounds exerted strong cytostatic effects on the panel of seven human tumour cell lines in a dose-dependent manner. In particular, imidazo[4,5-b]pyridines and triaza-benzo[c]fluorenes including 2-imidazolinyl derivatives showed the most potent antitumour activity. Similarly, triaza-benzo[c]fluorenes 18 and 20 induced strong growth inhibition of tested tumour cell lines, and showed low cytotoxicity in normal human fibroblasts. DNA interaction studies of these compounds demonstrated that N-methylated 16 and 2-imidazolinyl 28 triaza-benzo[c]fluorenes bind to DNA in an intercalative mode.

Full text of DOI:10.1016/j.ejmech.2011.03.062

European Journal of Medicinal Chemistry 46 (2011) 2748e2758
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel imidazo[4,5-b]pyridine and triaza-benzo[c]fluorene derivatives: Synthesis,
antiproliferative activity and DNA binding studies
,
b
c
ꢀ ꢁ a
Marijana Hranjec a , Borka Lucic , Ivana Ratkaj , Sandra Kraljevic Pavelic , Ivo Piantanida ,
ꢁ
ꢁb
*
a,
ꢁ b
*
ꢀ
Kresimir Pavelic , Grace Karminski-Zamola
Faculty of Chemical Engineering and Technology, Department of Organic Chemistry, University of Zagreb, Marulicev trg 20, P. O. Box 177, HR-10000 Zagreb, Croatia
Department of Biotechnology, University of Rijeka, Trg brace Mazuranica 10, HR-51000 Rijeka, Croatia
“RuCer Boskovic” Institute, Division of Organic Chemistry and Biochemistry, P. O. Box 180, HR-10002 Zagreb, Croatia
a
ꢁ
b
c
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present paper, we have described the synthesis and biological activity of the novel derivatives of
imidazo[4,5-b]pyridines and triaza-benzo[c]fluorenes (7e21, 24e26, 28e29). A preponderance of these
compounds exerted strong cytostatic effects on the panel of seven human tumour cell lines in a dose-
dependent manner. In particular, imidazo[4,5-b]pyridines and triaza-benzo[c]fluorenes including
2-imidazolinyl derivatives showed the most potent antitumour activity. Similarly, triaza-benzo[c]fluo-
renes 18 and 20 induced strong growth inhibition of tested tumour cell lines, and showed low cyto-
toxicity in normal human fibroblasts. DNA interaction studies of these compounds demonstrated that
N-methylated 16 and 2-imidazolinyl 28 triaza-benzo[c]fluorenes bind to DNA in an intercalative mode.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 11 January 2011
Received in revised form
17 March 2011
Accepted 30 March 2011
Available online 6 April 2011
Keywords:
Imidazo[4,5-b]pyridines
Triaza-benzo[c]fluorenes
Antiproliferative activity
Interaction with ct-DNA
Apoptosis
Cell cycle
1. Introduction
Therefore, understanding the interactions between small organic
molecules and DNA might help lay the groundwork for rational
Imidazo[4,5-b]pyridines represent the major backbone of
numerous medical and biochemical agents possessing different
chemical and pharmacological features [1,2], which impart them
diverse biological properties like anticancer [3,4], antiviral [5e7],
antimitotic [8], anti-inflammatory [9] and tuberculostatic [10]
activity. In addition, they can act as antagonists of various biolog-
ical receptors [11], e.g. angiotensin-II1, platelet activating factor
(PAF)2 [12], metabotropic glutamate subtype V3 [13] and
AT1 receptor [14]. Importantly, imidazo[4,5-b]pyridine is a struc-
tural analogue of purine whose derivatives easily interact with
large biomolecules such as DNA, RNA or diverse proteins in vivo. It is
these particular biomolecules that represent the major targets in
current drug development strategies aimed at producing next
generation therapeutics for untreatable diseases such as cancer
[15e19]. DNA plays a central role in life processes, as it stores
complete heritage information and mediates protein biosynthesis.
development of novel and more selective anticancer agents [20,21].
We have recently reported on the synthesis and biological
activity of a series of amidino-substituted heterocyclic benzimid-
azole, benzimidazo[1,2-a]quinoline and diaza-cyclopenta[c]fluorene
derivatives [22e24]. Biological activity studies comprising cytostatic
evaluation, DNA/RNA interaction study and proteomic profiling [24]
confirmed the anticancer potential of this class of compounds and
suggested further in vivo investigation into their possible clinical
utility. The same studies also revealed imidazolinyl-substituted
benzimidazo[1,2-a]quinoline to be the most active compound
(Fig. 1) with pronounced selectivity towards colon carcinoma cells,
which inhibited human topoisomerase II and induced strong G2/M
cell cycle arrest indicative of impaired mitotic progression. We also
showed that some positively charged analogues of benzimidazo[1,2-
a]quinolines and diaza-cyclopenta[c]fluorenes intercalate into ds
DNA or RNA, which might account for their accentuated anti-
proliferative effect [22,23].
Prompted by the findings from the above mentioned studies, we
set out to explore and synthesize novel imidazo[4,5-b]pyridine and
triaza-benzo[c]fluorene derivatives with substituents on different
positions of the heterocyclic rings (Fig. 2). Cyclic imidazo[4,5-b]
* Corresponding authors. Tel.: þ385 14597245; fax: þ385 14597250.
E-mail
addresses:
mhranjec@fkit.hr
(M.
Hranjec),
gzamola@fkit.hr
(G. Karminski-Zamola).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.062

M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2748e2758
2749
SnCl2ꢁ2H2O from nitro-substituted precursors 16e17 in very good
yields. Hydrochloride salts of the above mentioned amino deriva-
tives 20e21 were prepared by protonation with HCl(g) to ensure
better solubility.
S
HN
HN
N
N
N
Cyano-substituted E-2-styryl-imidazo[4,5-b]pyridines 24e25
were prepared in very good yields by the condensation reaction
between the corresponding 2-methylimidazo[4,5-b]pyridines
22e23 and p-cyanobenzaldehyde in a sealed tube at 180 ꢀC using
previously described method [27]. Cyano-substituted triaza-benzo
[c]fluorenes 26e27 were prepared photochemically by subjecting
ethanolic solution of compounds 24e25 to irradiation with
high-pressure mercury lamp followed by UV/Vis spectroscopy. 2-
imidazolinyl-substituted triaza-benzo[c]fluorenes 28e29 were
prepared as hydrochloride salts by the Pinner reaction for amidine
synthesis [28,29].
All structures of novel E-2-styryl-imidazo[4,5-b]pyridine
derivatives 7e11 and 24e25, and derivatives of triaza-benzo[c]
fluorenes 12e21 and 26e29 were determined by the NMR anal-
ysis based on the analysis of HeH coupling constants as well as
chemical shifts. In 1H NMR spectra of E-2-styryl-imidazo[4,5-b]
pyridine derivatives 7e11 and 24e25, besides all other aromatic
protons, two doublets for trans-ethylenic protons with coupling
constants of w16.5 Hz can be observed, as well as singlet of the
NH group of imidazo[4,5-b]pyridine ring around 12 ppm. The
cyclization reaction producing “fused” triaza-benzo[c]fluorenes
caused a downfield shift of the most aromatic protons as well as
disappearance of the NH group at the imidazo[4,5-b]pyridine
ring, thus confirming the cyclic structure formation. In 1H NMR
spectra of all synthesized triaza-benzo[c]fluorenes, doublets for
two protons on fluorene ring with coupling constants of w9.5 Hz
can be observed.
N
H+Cl-
H+Cl-
N
N
2
1
Fig. 1. Structures of previously prepared 2-imidazolinyl substituted benzimidazo[1,2-
a]quinoline 1 and diaza-cyclopenta[c]fluorene 2.
pyridine derivatives related to triaza-benzo[c]fluorenes were
prepared photochemically by the dehydrocyclization reaction or by
the thermal cyclization reaction. Majority of newly synthesized
compounds were tested for their antiproliferative activity on the
panel of seven human tumour cell lines. Possible antiproliferative
mechanisms and/or targets were studied in details for compounds
18 and 28, while DNA-binding studies were performed for
compounds 16, 18 and 28.
2. Results and discussion
2.1. Chemistry
Prepared compounds (Fig. 2) were synthesized according to the
procedures shown in Schemes 1, 2 and 3 by the conventional
methods of organic synthesis for the preparation of similar
heterocyclic compounds [25,26]. Starting from the E-3-phenyl-
substituted acrylic acids 1e4 (Scheme 1), via the cyclocondensation
reaction between the corresponding 2,3-diaminopyridines 5e6
and polyphosphoric acid (PPA), E-2-styryl-imidazo[4,5-b]pyridines
7e11 were prepared. Their cyclic derivatives, namely triaza-benzo
[c]fluorenes 12e15, were prepared by the thermal reaction using
sulfolane for dehydrohalogenation cyclization at 280 ꢀC as
a mixture of two inseparable regioisomers. Unsubstituted deriva-
tive 12 was prepared by the photochemical dehydrocyclization
reaction from ethanolic solution of derivative 7. N-methylated
derivatives 16e17 were prepared from derivatives 12 and 13 using
lower yields of methyl iodide, whereas methylation of triaza-benzo
[c]derivatives 14e15 failed.
2.2. Spectroscopic properties of compounds 16, 18 and 28
Since DNA binding studies of compounds 16, 18 and 28 require
application of spectrophotometric methods; their aqueous buff-
ered solutions (proven to be stable over prolonged periods of
time) were characterized by electronic absorption (UV/Vis) and
fluorescence emission spectroscopy (Table 1). The absorbances of
buffered aqueous solutions of an each compound were propor-
tional to their concentrations up to c ¼ 2.0 ꢁ 10ꢂ5 mol dmꢂ3
,
Amino-substituted triaza-benzo[c]derivatives 18e19 were
prepared according to Scheme 2 by the reduction reaction with
changes in the UV/Vis spectra upon the temperature increase up
to 98 ꢀC were negligible, and UV/Vis spectra reproducibility after
R2
R1
X
R2
R1
H
N
Y
R1
N
N
R2
Y
N
N
N
N
X
R3
N
N
I
CH3
7 R1=H, R2=H, R3=H, X=CH, Y=H
8 R1=Cl, R2=H, R3=H, X=CH, Y=H
9 R1=Cl, R2=H, R3=NO2, X=CH, Y=H
10 R1=Cl, R2=H, R3=NO2, X=CH, Y=Br
11 R1=Cl, R2=H, R3=H, X=N, Y=H
24 R1=H, R2=CN, R3=H, X=CH, Y=H
25 R1=H, R2=CN, R3=H, X=CH, Y=Br
12 R1=H, R2=H, X=CH, Y=H
13 R1=H, R2=NO2, X=CH, Y=H
14 R1=H, R2=NO2, X=CH, Y=Br
15 R1=H, R2=H, X=N, Y=H
16 R1=H, R2=H
17 R1=H, R2=NO2
18 R1=H, R2=NH2, X=CH, Y=H
19 R1=H, R1=NH2, X=CH, Y=Br
20 R1=H, R2=NH3+Cl-, X=CH, Y=H
21 R1=H, R1=R2=NH3+Cl-, X=CH, Y=Br
26 R1=CN, R2=H, X=CH, Y=H
27 R1=CN, R2=H, X=CH, Y=Br
28 R1=2-imidazolinyl, R2=H, X=CH, Y=H
29 R1=2-imidazolinyl, R2=H, X=CH, Y=Br
Fig. 2. E-2-styryl substituted derivatives of imidazo[4,5-b]pyridines 7e11 and 24e25 and derivatives of triaza-benzo[c]fluorenes 12e21 and 26e29.

2750
M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2748e2758
R1
H2N
H2N
Y
R1
PPA
180oC
H
N
+
Y
R2
X
COOH
N
R2
X
N
5 Y=H
6 Y=Br
N
1 R1=H, R2=H, X=CH
2 R1=Cl, R2=H, X=CH
3 R1=Cl, R2=NO2, X=CH
4 R1=Cl, R2=H, X=N
7 R1=H, R2=H, X=CH, Y=H
8 R1=Cl, R2=H, X=CH, Y=H
9 R1=Cl, R2=NO2, X=CH, Y=H
10 R1=Cl, R2=NO2, X=CH, Y=Br
11 R1=Cl, R2=H, X=N, Y=H
sulfolane, 280oC
or h , O2, I2
R1
X
R1
CH3I
abs. EtOH
Y
N
N
N
N
N
N
I
12 R1=H, X=CH, Y=H
13 R1=NO2, X=CH, Y=H
14 R1=NO2, X=CH, Y=Br
15 R1=H, X=N, Y=H
CH3
16 R1=H
17 R1=NO2
Scheme 1. Synthesis of E-2-styryl substituted imidazo[4,5-b]pyridines 7e11 and derivatives of triaza-benzo[c]fluorenes 12e17.
cooling down to 25 ꢀC was excellent. Obtained results indicate
that compounds 16, 18 and 28 do not aggregate by intermolecular
stacking under applied experimental conditions (Fig. SM1).
Compounds 16, 18 and 28 exhibited strong fluorescence emission
with maximum at 386e519 nm. Fluorescence emissions of 16, 18
and 28 were proportional to their concentration in the range from
3 ꢁ 10ꢂ8 mol dmꢂ3 to 4 ꢁ 10ꢂ7 mol dmꢂ3, and their excitation
spectra were in concordance with the corresponding absorption
spectra. Absorption maxima, corresponding molar extinction
H(16) ¼ 28% (327 nm), 37% (342 nm), 38% (360 nm); H(18) ¼ 26%
(330 nm), 18% (343 nm); and H(28) ¼ 45% (347 nm), 35% (364 nm).
2.3.2. Fluorimetric titrations
Experimental conditions in UV/Vis titrations resulting in mixed
binding modes at high compound/DNA ratios (r > 0.2) were cir-
cumvented by taking advantage of strong fluorescence of aqueous
solutions of compounds 16, 18 and 28, which allowed for fluori-
metric titrations at lower compound concentrations and collection
of many data points at high ct-DNA over compound excess (r < 0.1).
Addition of ct-DNA completely quenched fluorescence of 28
(Fig. 3A), and strongly quenched fluorescence of 16 (Fig. SM3A) at
variance to fluorescence of 18, whose emission was only negligibly
changed.
coefficients (3) and fluorescence emission maxima are presented
in Table 1.
2.3. Interaction study for compounds 16, 18 and 28 with ct-DNA in
an aqueous medium
UV/Vis (18 and 28) and fluorimetric (16 and 28) titration data
were processed by using Scatchard equation [30] giving rise to log
Ks values (Table 2).
Comparison of Ks values presented in Table 2 revealed higher
affinity of compounds 16 and 28 towards ct-DNA in comparison to
free amine analogue 18. Furthermore, cumulative binding affinity
of compound 28 observed in UV/Vis titrations did not yield
significantly different Ks value in comparison to the results of
fluorimetric titration. Thus, somewhat higher value of ratio n
2.3.1. UV/Vis spectrophotometric titrations
Addition of calf-thymus DNA (ct-DNA) resulted in bathocromic
and hypochromic effects of UV/Vis spectra of studied compounds.
For compounds 16 and 28 (Fig. SM2), significant deviations from
the isosbestic points were observed pointing to the coexistence of
at least two different types of complexes. The UV/Vis spectra
obtained for compounds 16, 18 and 28 exhibited strong hypo-
chromic changes upon titration with ct-DNA as follows:
NH3*Cl-
NO2
NH2
HCl(g)
SnCl2×2H2O
Y
Y
Y
N
N
N
abs. EtOH
MeOH
HClkonc.
N
N
N
N
N
N
20 Y=H
21 Y=Br
13 Y=H
14 Y=Br
18 Y=H
19 Y=Br
Scheme 2. Synthesis of amino substituted triaza-benzo[c]derivatives 18e21.

M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2748e2758
2751
H
NC
Y
NC
N
sealed tube
180oC
+
H
N
H3C
Y
CHO
N
N
N
22 Y=H
23 Y=Br
24 Y=H
25 Y=Br
N
h , O2, I2
ethanol
NH+Cl-
NC
HN
1. HCl(g)
,
abs. EtOH
Y
Y
N
N
2. H2N(CH2)2NH2
abs. EtOH
N
N
N
N
26 Y=H
27 Y=Br
28 Y=H
29 Y=Br
Scheme 3. Synthesis of cyano substituted E-2-styryl-imidazo[4,5-b]pyridines 24e25, cyano substituted triaza-benzo[c]fluorenes 26e27 and 2-imidazolynyl substituted triaza-
benzo[c]fluorenes 28e29.
obtained for UV/Vis titrations can be attributed to agglomeration of
an excess of 28 molecules along DNA at conditions at which all
dominant binding sites are already occupied ([28]/[ct-DNA] > 0.3).
275 nm. Non-linear dependence of intensity changes at both
275 nm and 337 nm on ratio r[28]/[ct-DNA] (Fig. 4B) suggested
saturation of dominant binding sites along DNA at ratio r ¼ 0.3,
which greatly correlates withScatchard ratio n calculated from
fluorimetric titration.
2.3.3. CD spectrophotometric titrations
Non-covalent interactions at 25 ꢀC were commonly studied
through the assessment of spectroscopic properties of the
compound upon polynucleotides addition. We have, however,
chosen CD spectroscopy as a highly sensitive method for moni-
toring conformational changes in the secondary structure of
polynucleotides as to investigate the changes in polynucleotide
properties induced by small molecule binding [31]. Addition of
compound 28 resulted in increase of ct-DNA CD band at 275 nm, as
well as in an appearance of weak, negative induced CD band (ICD)
at 337 nm (Fig. 4A). Observed weak negative ICD band for
compound 28 appeared at UV maximum, which is indicative of
intercalation into ct-DNA, whereby longer axis of 28-hetero-
aromatic system is coplanar with the longer axis of adjacent DNA
base pairs [32]. Furthermore, compound 28 has another UV
maximum with a shoulder at 273 nm (see Fig. SM1 e UV/Vis
spectra of free compounds), which similarly to some hetero-
aromatic systems might be attributed to transitions along shorter
axis of a molecule [33]. Thus, upon intercalation of compound 28
into ct-DNA, such shorter axis should be perpendicular to longer
axis of adjacent DNA base pairs, and should subsequently yield
weak positive ICD effect noticed as a small increase of CD band at
A titration of ct-DNA with compound 16 yielded similar changes
to those noticed for compound 28 characterized by a weak negative
ICD band at 325 nm. At excess of compound 16 over ct-DNA
(r > 0.3), an additional weak positive ICD band at 350e400 nm
appears, which might be ascribed to non-specific agglomeration of
the compound along DNA.
A titration of ct-DNA with compound 18 revealed a small
increase of CD band at 275 nm and an absence of ICD band at
>300 nm implying small changes in the helical structure of DNA
upon compound 18 binding. An absence of ICD band might point to
the non-homogenous orientation of bound molecules of compound
18 relative to the DNA helical axis.
2.3.4. Thermal denaturation experiments
In thermal denaturation experiments, addition of cyclic deriva-
tives 16 and 28 moderately stabilized ct-DNA, with a significant non-
linear dependence of DTm values on the ratio r (Table 3). Addition of
compound 18 negligibly stabilized ct-DNA against thermal dena-
turation (
D
Tm ¼ 1e2 ꢀC for ratio r[28]/[ct-DNA] ¼ 0.2e0.3).
2.4. Discussion of interactions of 16, 18 and 28 with ct-DNA
Intercalation of 16 and 28 as a dominant binding mode was
demonstrated by a weak negative ICD band > 300 nm, significant
bathochromic and hypsochromic changes in UV/Vis titration,
thermal stabilization of DNA as well as by values of Ks > 105 Mꢂ1.At
excess of 16 or 28 over intercalative binding sites, non-specific
agglomeration along DNA double helix occurs. Surprisingly, free
amine analogue 18 induced significantly lower changes in UV/Vis
and fluorescence spectrum upon addition of ct-DNA in comparison
to 16 and 28; stabilization of ct-DNA against thermal denaturation
and appearance of the ICD band >300 nm did not occur. Moreover,
affinity was significantly lower in comparison to 28 pointing to
a much weaker, non-intercalative binding mode, which is most
likely a heterogeneous agglomeration of randomly oriented mole-
cules of compound 18 within the DNA minor groove that causes
a lack of positive ICD effect.
Table 1
Electronic absorption maxima, molar extinction coefficients and fluorescence
emission maxima of studied compounds.a
Absorption maxima
Fluorescence emission
Comp.
lmax/nm
3
ꢁ 103/dm3 molꢂ1 cmꢂ1
lmax/nm
16
360
343
327
226
342
329
262
348
239
18.67
22.72
20.66
50.86
31.60
30.67
61.91
9.87
386
406
18
519
466
28
26.16
a
Sodium cacodylate buffer I ¼ 0.05 mol dmꢂ3, pH ¼ 7.

2752
M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2748e2758
Fig. 3. A) Changes in fluorescence spectrum of 28 (c ¼ 5.8 ꢁ 10ꢂ7 mol dmꢂ3) upon titration with ct-DNA (c ¼ 2.00 ꢁ 10ꢂ2 mol dmꢂ3); B) Dependence of 28 emission at
on c(ct-DNA); All experiments were carried out at pH ¼ 7.0, (buffer sodium cacodylate I ¼ 0.05 mol dmꢂ3).
l
¼ 466 nm
2.5. Biological results and discussion of antiproliferative activity
moiety, which probably adds up to their cytotoxicty (IC50 values
for 28 and 29 were in the same range as those for cisplatin and 5-
FU) (Fig. 1). Importantly, compounds 18 and 20 showed lower
toxicity in normal human fibroblasts than other biologically active
newly synthesized compounds, cisplatin and 5-FU (Table 4).
Interestingly, both 4-pyridyl substituted compounds 11 and 15
showed the poorest antiproliferative activity among all tested
compounds.
Marked antitumour activities of compounds 18 and 28 have led
us to investigate their mechanism of action in more details, espe-
cially when having in mind that compound 18 was highly selective
for SK-BR-3 cells with low cytotoxicty in normal human fibroblasts,
and compound 28 was highly selective for two metastatic cell lines,
namely breast adenocarcinoma (SK-BR-3) and colorectal adeno-
carcinoma (SW620), which are known for their resistance to
common chemotherapeutics.
Novel imidazo[4,5-b]pyridine and triaza-benzo[c]fluorene
derivatives (1e21, 24e26 and 28e29) were evaluated for their
antiproliferative activity in vitro using the panel of seven human
cell lines derived from different tumour types including HeLa
(cervical carcinoma), SW620 (colorectal adenocarcinoma, meta-
static), MiaPaCa-2 (pancreatic carcinoma), MCF-7 (breast epithelial
adenocarcinoma, metastatic), HEp-2 (epidermoid carcinoma,
larynx) and SK-BR-3 (breast adenocarcinoma, metastatic) as well as
normal diploid human fibroblasts (WI38). Their cytostatic effect
was compared against that of two commonly used chemothera-
peutics, namely cisplatin and 5-fluorouracil (5-FU). Obtained
results are summarized and presented in Table 4, and are in line
with our previously published data showing strong antitumour
activity of several heterocyclic benzimidazole, benzimidazo[1,2-a]
quinoline and diaza-cyclopenta[c]fluorene derivatives [22,23,26].
With the exception of compounds 11 and 15 that exerted none
or modest antiproliferative activity, the majority of tested
compounds demonstrated strong cytostatic effect on all tested cell
lines in a dose-dependent manner (Fig. SM4), especially at
2.6. Cell cycle perturbations and apoptosis induction
The results of the flow cytometric analysis revealed strong
perturbations in the cell cycle of MiaPaCa-2 and SW620 cells
induced upon treatment with compound 28 (Table 5). Anti-
proliferative effect of compound 28 on MiaPaCa-2 cells could be
associated with S-phase arrest at all tested concentrations
micromolar concentrations ranging from 1 to 10 mM for E-2-styryl
substituted derivatives of imidazo[4,5-b]pyridines 7, 8, 10 and 25,
and for triaza-benzo[c]fluorenes 16, 18, 19, 20, 28 and 29 (Table 4).
Compounds 16, 18 and 20 showed selective effect on SK-BR-
3 cells. Besides the latter cell line, compound 16 specifically
inhibited the growth of MCF-7 and HeLa cells as well. Among
triaza-benzo[c]fluorenes, the strongest non-specific effect on all
accompanied by the rise in the sub-G1 population at 5 mM after
24- and 48-h treatments. Accordingly, an increase in the S-phase
arrested cell population by 22.1% and 22.3% after 24- and 48-h
exposure, respectively, concurred with the apparent decline in
G1-phase cells. Impairment of cell cycle progression in
SW620 cells treated with compound 28 for 24 h was charac-
terised by a rise in the G1 cell population by 36.2% accompanied
with a decrease in S-phase cells by 15.4%. Prolonged exposure
time (48 h) triggered completely opposite effect, namely an
increase of 3.6% in S-phase cells with a concomitant reduction in
the G1 cell population. Treatment of SW620 cells with compound
cell lines at micromolar concentrations (0.1e10 mM) was detected
for triaza-benzo[c]fluorenes 28 and 29 bearing the 2-imidazolinyl
Table 2
Binding constants (logKs), ratios n ¼ ([bound compound]/[ct-DNA]) calculated from
fluorimetric and UV/Vis titrations of 16, 18 and 28 with ct-DNA at pH ¼ 7 (buffer
sodium cacodylate, I ¼ 0.05 mol dmꢂ3).a
28 with the highest tested concentrations (10
mM) for 48 h
Compound
Fluorescence
UV/Vis
brought forth different cell cycle distribution pattern marked by
G-phase arrest and S-phase cell population up to 30% (Table 5).
Growth inhibitory effect of compound 18 on SK-BR-3 cells might
be linked with substantial rise in sub-G1 phase up to 11.2% (24 h
treatment) and the S-phase arrest upon 48-h treatment, when
reduction in the G2/M phase cell population was detected as well
(Table 6).
Morphological appearance of MiaPaCa-2, SW620 and SK-BR-
3 cells treated with compounds 28 and 18 was also examined
(Figs. 5, 6 and 7). Changes in cell morphology typical of apoptosis,
log Ks
n
log Ks
n
28
18
16
5.9
0.28
5.4c
0.44c
ꢂ0.24
b
b
4.6
ꢂ
4.9
0.20
a
Titration data were processed using the Scatchard equation, accuracy of
10e30%,consequently log Ks values vary in the same order of
obtained
n
ꢃ
magnitude.
b
Too small spectroscopicchanges or accurate calculation.
Cumulative affinity of 28 since more different complexes are simultaneously
c
present (as shown by deviation of isosbestic points on Fig. 4.).

M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2748e2758
2753
12
10
8
A
B 11
ctDNA
r=0.1
r=0.2
r=0.3
r=0.5
10
9
6
4
337 nm
275 nm
2
0
-2
-4
-6
-8
-10
-12
-0.5
-1.0
-1.5
0.0
0.1
0.2
0.3
0.4
0.5
220
240
260
280
300
320
340
360
380
400
/ nm
r [compd]/[ctDNA]
Fig. 4. A) Changes in CD spectrum of ct-DNA (c ¼ 1.0 ꢁ 10ꢂ5 mol dmꢂ3) upon titration with 28; B) Dependence of CD band of ct-DNA (lmax ¼ 274 nm) and ICD band of 28
(
lmax ¼ 337 nm) on r [28]/[ct-DNA], at pH ¼ 7, sodium cacodylate buffer, I ¼ 0.05 mol dmꢂ3
.
such as cell shrinkage and chromatin condensation, were detected
in SW620 cells treated with compound 28 (5 and 10 M) as well as
in MiaPaCa-2 cells treated with the highest tested concentration
(10 M) of the same compound. These results are in good agree-
ment with the findings of the flow cytometric analysis demon-
strating significant increase in the sub-G1 cell population indicative
of apoptotic cell death (Table 5). When treated with lower
concentrations of compound 28, the majority of MiaPaCa-2 cells
became visibly enlarged, which corresponds to S-phase arrest
revealed by the cell cycle analysis. In addition, exposure of
shown that these groups of compounds exert strong anti-
proliferative effect on all tested cell lines in a dose-dependent
manner, which makes them promising lead compounds for
further pre-clinical investigation. The strongest non-specific
antiproliferative effect was observed for triaza-benzo[c]fluo-
renes 28 and 29 bearing the 2-imidazolinyl moiety, which prob-
ably adds up to their high cytotoxicty. Contrary to these two
compounds, triaza-benzo[c]fluorenes 18 and 20 showed lower
toxicity in normal human fibroblasts when compared with
cisplatin and 5-FU used as control chemotherapeutics. Induction
of apoptosis was detected in SW620 and MiaPaCa-2 cells treated
with compound 28 at higher concentrations, while lower
concentrations gave rise to S-phase arrest. Apoptosis induction by
this compound was dose-dependent and was directly associated
with its ability to intercalate into ds DNA, as corroborated by the
spectrophotometric DNA interaction study showing N-methyl-
substituted compounds 16 and 2-imidazolinylesubstituted 28
triaza-benzo[c]derivatives to intercalate into ds DNA. Free amine
analogue 18 binds to DNA by the non-intercalative binding mode,
which might be partially responsible for observed lower cyto-
toxicity in SK-BR-3 cells and control fibroblasts. It is most likely
that compound’s 18 molecules form agglomerates within DNA
minor groove and/or interact with other biologically active
molecules involved in DNA synthesis, such as DNA replication
enzymes, leading to an arrest of DNA synthesis.
m
m
SW620 cells to the lowest tested concentration (1
mM) of this
compound resulted in cell morphology reminiscent of that found in
cells undergoing cell cycle arrest, which correlates with S-phase
arrest detected after 24 and 48 h of treatment, respectively, by flow
cytometric analysis. Based on these results, one can conclude that
compound 28 triggered apoptosis in MiaPaCa-2 and SW620 cells in
a dose-dependent manner, which could be ascribed to its ability to
intercalate into ds DNA. On the contrary, SK-BR-3 cells showed
rather low percentage of visible apoptotic cells. When treated with
compound 18, these cells became enlarged, possibly due to S-phase
arrest confirmed by flow cytometry. Weaker DNA-damaging effect
of compound 18 illustrated by an absence of apoptotic features
might stem from the lack of its intercalative properties. It is most
likely that compound’s 18 molecules form agglomerates within
DNA minor groove and/or interact with other biologically active
molecules involved in DNA synthesis (e.g. enzymes of the replica-
tion machinery) thus preventing the cells from completing the
replication of their genetic material as a prerequisite for successful
cellular division.
4. Experimental
4.1. Chemistry
4.1.1. General methods
3. Conclusions
Melting points were determined by Koffler hot-stage microscope,
and were uncorrected. IR spectra were recorded on FTIR-ATR and
PerkineElmer Spectrum 1 spectrophotometers. 1H and 13C NMR
spectra were recorded on Varian Gemini 300, Bruker Avance DPX
300 and Bruker Avance DRX 500 spectrometers using TMS as an
internal standard in DMSO-d6. Elemental analyses for carbon,
hydrogen and nitrogen were performed on PerkineElmer
2400 elemental analyzer and PerkineElmer, Series II, CHNS Analyzer
2400. Where analyses are indicated only as symbols of elements,
analytical results obtained were within 0.4% of the theoretical value.
In preparative photochemical experiments, the irradiation was per-
formed at room temperature with water-cooled immersion well
with “Origin Hanau” 400-W high-pressure mercury arc lamp using
Pyrex glass as a cut-off filter of wavelengths below 280 nm. All
compounds were routinely checked by TLC with Merck silica gel
60F-254 glass plates.
The present paper deals with the synthesis of novel derivatives
related to imidazo[4,5-b]pyridines (7e11 and 24e25) and triaza-
benzo[c]fluorenes (12e21 and 26e29). We have previously
Table 3
D
Tm valuesa (ꢀC) of ct-DNA upon addition of different ratios br of 16, 18 and 28 at
pH ¼ 7.0 (buffer sodium cacodylate, I ¼ 0.05 mol dmꢂ3).
br
16
18
28
0.1
0.2
0.3
0.5
1.60
2.60
4.25
6.10
0.70
1.40
0.20
1.30
2.70
5.10
6.40
8.40
a
Error in
D
Tm : ꢃ 0.5 ꢀC.
b
r ¼ [compound]/[polynucleotide].

2754
M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2748e2758
mM) and control compounds cisplatin and 5-fluorouracil (5-FU).
Table 4
IC50 values of compounds 7e21, 24e29 (
a
IC50
(mM)
Substance No.
Cell lines
HeLa
MCF-7
7.57
25.56
53.65
8.81
>100
75.45
>100
>100
>100
7.56
>100
72.38
61.70
55.96
67.30
12.89
7.82
HEp-2
3.89
29.29
49.36
0.91
>100
52.28
>100
31.09
>100
13.19
31.52
39.24
14.09
26.69
21.55
84.91
5.69
SK-BR-3
SW620
4.64
50.94
51.99
7.99
>100
78.28
>100
61.25
>100
14.75
26.69
34.77
38.65
25.91
40.69
MiaPaCa-2
WI38
3.87
>100
61.60
6.48
>100
47.41
75.71
>100
>100
6.27
7
8
9
2.22
31.62
45.06
3.68
>100
14.32
52.77
13.38
>100
7.52
11.43
43.36
30.91
27.16
37.73
47.06
2.88
2.06
5.79
1.80
59.92
53.14
83.52
42.74
8.11
>100
3.51
33.57
2.94
7.76
3.64
7.56
86.56
4.31
67.72
2.20
5.52
14.23
38.25
4.39
59.09
38.79
30.59
>100
80.52
>100
63.53
>100
8.56
>100
48.18
33.04
43.42
40.32
>100
8.17
10
11
12
13
14
15
16
17
18
19
20
21
24
25
26
28
29
Cisplatin
5-FU
>100
>100
85.37
>100
85.09
>100
6.76
51.47
1.768
3.71
>100
7.11
>100
39.46
0.41
1.10
0.35
10.31
>100
47.28
0.27
4.01
2.41
>100
>100
0.74
4.74
3.52
11.45
6.54
8.43
12.61
42.01
0.64
3.67
4.01
3.38
0.22
18.5
a
IC50; 50% inhibitory concentration, or compound concentration required to inhibit tumour cell proliferation by 50%.
4.1.2. General method for the synthesis of triaza-benzo[c]fluorene
derivatives (13e15)
(d,1H, J ¼ 9.6 Hz, Hfluorene), 7.82 (d,1H, J ¼ 9.6 Hz, Hfluorene), 7.66 (Abq,
1H, J1 ¼ 8.12 Hz, J2 ¼ 4.7 Hz, Harom.); b) 9.13 (d, 1H, J ¼ 8.3 Hz, Harom),
9.08 (d, 1H, J ¼ 2.5 Hz, Harom), 8.95 (d, 1H, J ¼ 9.3 Hz, Harom), 8.76 (d,
1H, J ¼ 4.6 Hz, Harom.), 8.54 (dd, 1H, J1 ¼ 9.2 Hz, J2 ¼ 2.5 Hz, Harom.),
8.28 (d, 1H, J ¼ 9.7 Hz, Hfluorene), 7.89(d, 1H, J ¼ 9.6 Hz, Harom.), 7.58
(ABq, 1H, J1 ¼ 8.3 Hz, J2 ¼ 4.7 Hz, Harom); 13C NMR (DMSO-d6,
150 MHz): 164.1, 163.2, 156.3, 149.6, 144.2, 143.9, 143.1, 142.9, 142.3,
138.7, 133.1, 128.7, 128.0, 127.0, 126.0, 125.0, 123.6, 123.2, 124.9, 124.0,
123.5, 123.4, 119.6, 118.7, 118.3, 117.6, 116.9, 115.9; Anal. (C14H8N4O2)
Calcd.: C, 63.7; H, 3.0; N, 21.0; Found: C, 63.9; H, 2.8; N, 21.2.
E-2-styryl-1H-imidazo[4,5-b]pyridine derivatives 7e11 were
dissolved in 2e3 ml of sulfolane, and reaction mixture was heated
for ½e2 h at 280 ꢀC. The cooled mixture was poured into water
(25 ml) and the resulting product was filtered off and recrys-
tallizated from ethanol to obtain triaza-benzo[c]fluorenes 13e15.
4.1.2.1. 3-Nitro-7,8,11b(11)-triazabenzo[c]fluorene 13. Compound 13
was prepared using the above described method from compound 9
(0.50 g,1.16 mmol) in sulfolane (1.5 ml) after 45 min to obtain yellow
powder (0.18 g, 57%). mp > 270 ꢀC; UV (EtOH) lmax/nm ¼ 302, 335,
4.1.2.2. Bromo-3-nitro-7,8,11b(11)-triazabenzo[c]fluorene 14. Compo-
und14 was prepared using the above described method from
compound 10 (0.25 g, 0.66 mmol) in sulfolane (1 ml) after 30 min to
351, 369; IR (diamond):
n
/cmꢂ1 ¼ 3056, 1632, 1616, 1598, 1529; 1H
NMR (DMSO-d6, 600 MHz): a) 9.93 (d, 1H, J ¼ 9.3 Hz, Harom), 9.03 (d,
1H, J ¼ 2.3 Hz, Harom), 8.40 (dd, 1H, J1¼8.1 Hz, J2¼1.3 Hz, Harom), 8.23
obtain grey powder (0.19 g, 82%). mp >270 ꢀC; UV (EtOH) lmax
/
nm ¼ 352, 305; IR (diamond):
n
/cmꢂ1 ¼ 3066, 1712, 1630, 1618, 1517;
1H NMR (DMSO-d6, 300 MHz):
d
/ppm ¼ a) 9.73 (d, 1H, J ¼ 9.3 Hz,
Harom), 9.04 (dd, 2H, J1 ¼ 9.2 Hz, J2 ¼ 2.6 Hz, Harom), 8.69 (dd, 1H,
J1 ¼ 9.3 Hz, J2 ¼ 2.6 Hz, Harom), 8.64 (d, 1H, J ¼ 2.1 Hz, Harom), 8.26 (d,
1H, J ¼ 9.2 Hz, Harom.), 7.79 (d, 1H, J ¼ 9.6 Hz, Hfluorene); b) 9.37 (d, 1H,
J ¼ 1.9 Hz, Harom), 8.95 (d, 1H, J ¼ 9.2 Hz, Harom), 8.81 (d, 1H, J ¼ 1.9 Hz,
Harom), 8.73 (d, 1H, J ¼ 2.0 Hz, Harom.), 8.48 (dd, 1H, J1 ¼ 9.2 Hz,
J2 ¼ 2.5 Hz, Harom.), 8.30 (Abq, J ¼ 9.5 Hz, Hfluorene), 7.86 (d, 1H,
Table 5
Flow cytometric analysis of the MiaPaCa-2 and SW620 cell cycle progression upon
treatment with compound 28.
Compound 28 MiaPaCa-2 Cell percentage (% ꢃ standard deviation)
Treatment
sub-G1
G1
S
G2/M
Control 24h
48.7 ꢃ 0.7
51.2 ꢃ 2.7 29.2 ꢃ 4.2 19.5 ꢃ 3.9
1
5
m
m
M 24h
M 24h
57.8 ꢃ 1.7* 41.6 ꢃ 1.8* 37.4 ꢃ 2.5* 21.3 ꢃ 2.6
53.4 ꢃ 0.3* 22.4 ꢃ 1*
59.3 ꢃ 2.1* 18.3 ꢃ 2.6
Table 6
10
m
M 24h
50.2 ꢃ 2.3
46.8 ꢃ 0.7
52.4 ꢃ 1.9 29.3 ꢃ 1.5 18.3 ꢃ 1.9
65.2 ꢃ 1.9 20.5 ꢃ 0.2 14.3 ꢃ 1.8
Flow cytometric analysis of the SK-BR-3 cell cycle progression upon treatment with
compound 18.
Control 48h
1
5
m
m
M 48h
M 48h
33.8 ꢃ 0.7* 42.4 ꢃ 1.8* 34.6 ꢃ 0.7*
23 ꢃ 2.1*
56.6 ꢃ 2.5* 42.4 ꢃ 1.5* 36.8 ꢃ 1.4* 20.8 ꢃ 1.9*
39.3 ꢃ 0.9* 26.1 ꢃ 5.1* 56.9 ꢃ 4.8* 16.9 ꢃ 2.4
Compound 18 SK-BR-3 Cell percentage (% ꢃ standard deviation)
10 mM 48h
SW620
Treatment
sub-G1
39 ꢃ 1.1
G1
S
G2/M
Control 24h
18 ꢃ 1
29.3 ꢃ 1.5 48.4 ꢃ 1.4 22.3 ꢃ 2.9
Control 24h
39.5 ꢃ 1.1
39.9 ꢃ 1.5
20.6 ꢃ 2.1
1
5
m
m
M 24h
M 24h
14.3 ꢃ 1.1* 54.3 ꢃ 2.5* 27.2 ꢃ 0.4* 18.5 ꢃ 2.5
24.4 ꢃ 0.5* 86.1 ꢃ 1.7* 11.8 ꢃ 1.5* 2.1 ꢃ 0.3*
5
m
M 24h
35.7 ꢃ 1.6* 47.1 ꢃ 0.8* 35.1 ꢃ 1.5* 17.7 ꢃ 1.9
10
50
m
m
M 24h
M 24h
45.8 ꢃ 1.7* 39.4 ꢃ 4.7
50.2 ꢃ 0.8* 33.2 ꢃ 3.7
34.5 ꢃ 2.4* 26.1 ꢃ 2.2*
48.1 ꢃ 3.1* 18.7 ꢃ 0.9
10
m
M 24
20.1 ꢃ 1.1
29.1 ꢃ 1.1
33.7 ꢃ 2.1* 47.3 ꢃ 2.8 19.2 ꢃ 0.9
Control 48h
52.7 ꢃ 0.9 31.9 ꢃ 1
16.1 ꢃ 0.9
Control 48h
M 48h
2.1 ꢃ 0.2
1.9 ꢃ 0.3
24.7 ꢃ 1.9
24.4 ꢃ 3.5
40.6 ꢃ 4.9
34.7 ꢃ 3.3
1
5
m
m
M 48h
M 48h
25.4 ꢃ 0.9* 35.1 ꢃ 2.6* 45.4 ꢃ 3.1* 19.4 ꢃ 0.7*
5
m
59.8 ꢃ 4.4* 15.8 ꢃ 3.3*
64.2 ꢃ 2.1* 14.8 ꢃ 0.2*
47.6 ꢃ 0.6* 90.9 ꢃ 3*
7.5 ꢃ 2.5* 1.5 ꢃ 0.5*
10
50
m
m
M 48h
M 48h
5.5 ꢃ 0.8* 20.9 ꢃ 1.9
10
m
M 48h
40.6 ꢃ 1.1* 39.9 ꢃ 2.4* 41.8 ꢃ 2.6* 18.2 ꢃ 1.2
3.1 ꢃ 0.1* 19.9 ꢃ 0.8* 47.6 ꢃ 1.5* 32.5 ꢃ 1.1*
(*) Statistically significant at p < 0.05.
(*) Statistically significant at p < 0.05.

M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2748e2758
2755
Fig. 5. MiaPaCa-2 cells treated with compound 28 for 24 and 48 h.
J ¼ 9.6 Hz, Harom.); 13C NMR (DMSO-d6, 75 MHz):
d
/ppm ¼ 150.2,148.1,
product was filtered off and recrystallized from ethanol or water to
obtain N-methyl-triaza-benzo[c]fluorene iodides.
143.9, 138.1, 133.6, 125.8, 125.5, 124.9, 124.1, 123.5, 119.4, 118.0, 116.6,
113.8; Anal. (C14H7N4O2Br) Calcd.: C, 49.0; H, 2.1; N, 16.3; Found: C,
49.3; H, 2.2; N, 16.2.
4.1.3.1. 8-N-Methyl-7,8,11b(11)-triazabenzo[c]fluorene iodide 16. Com-
pound 16 was prepared using the above described method from
4.1.2.3. 1,7,8,11b(11)-Tetraazabenzo[c]fluorene 15. Compound 15 was
prepared using the above described method from compound 11
(0.50 g, 2.05 mmol) in sulfolane (1.5 ml) after 45 min to obtain dark
compound 12 (0.04 g, 0.18 mmol) and methyl-iodide (12 mL,
0.18 mmol) to obtain yellow powder (0.02 g, 28%). mp >270 ꢀC; UV
(EtOH) lmax/nm ¼ 361, 344, 328, 225; IR (diamond):
n
/cmꢂ1 ¼ 2916,
yellow powder (0.15 g, 24%). mp 186e187 ꢀC; UV (EtOH) lmax
/
1661, 1619, 1552, 1433; 1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 10.67
nm ¼ 369, 351, 335, 324; IR (diamond):
n
/cmꢂ1 ¼ 3078, 1640,
(d, 1H, J ¼ 8.7 Hz, Harom.), 9.00 (dd, 1H, J1 ¼ 4.6 Hz, J2 ¼ 1.4 Hz, Harom.),
8.91 (d, 1H, J ¼ 9.7 Hz, Hfluorene), 8.80 (dd, 1H, J1 ¼ 7.5 Hz, J2 ¼ 1.4 Hz,
Harom.), 8.43 (d, 1H, J ¼ 9.7 Hz, Hfluorene), 8.41 (dd, 1H, J1 ¼ 8.2 Hz,
J2 ¼ 1.8 Hz, Harom.), 8.24 (t, 1H, J ¼ 8.1 Hz, Harom), 8.05 (dd, 1H,
J1 ¼ 8.4 Hz, J2 ¼ 4.8 Hz), 7.95 (t,1H, J ¼ 7.7 Hz, Harom.), 4.31(s, 3H, CH3);
1611 and 1555; 1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ a) 9.33 (dd,
1H, J1 ¼ 8.1 Hz, J2 ¼ 1.7 Hz, Harom.), 8.93 (dd, 1H, J1 ¼ 4.6 Hz,
J2 ¼ 1.6 Hz, Harom.), 8.73 (dd, 1H, J1 ¼ 4.7 Hz, J2 ¼ 1.6 Hz, Harom.), 8.58
(dd, 1H, J1 ¼ 7.8 Hz, J2 ¼ 1.7 Hz, Harom.), 8.12 (d, 1H, J ¼ 9.5 Hz,
H
fluorene), 7.83 (d, 1H, J ¼ 9.5 Hz, Hfluorene), 7-73-7.69 (m, 1H, Harom.),
13C NMR (DMSO-d6, 150 MHz):
133.6, 132.7, 130.4, 127.7, 126.5, 123.5, 123.5, 122.5, 118.2, 109.2; Anal.
(C15H13N3I) Calcd.: C, 49.9; H, 3.4; N, 11.7; Found: C, 50.1; H, 3.2;
N, 11.8.
d
/ppm ¼ 146.4, 143.5, 141.6, 140.9,
7.75 (dd, 1H, J1 ¼ 8.1 Hz, J2 ¼ 4.7 Hz, Harom.); b) 8.93 (dd, 1H,
J1 ¼ 4.6 Hz, J2 ¼ 1.6 Hz, Harom.), 8.68 (dd, 1H, J1 ¼ 4.4 Hz, J2 ¼ 1.3 Hz,
Harom.), 8.51 (dd, 1H, J1 ¼ 7.8 Hz, J2 ¼ 1.8 Hz, Harom.), 8.33 (dd, 1H,
J1 ¼ 8.1 Hz, J2 ¼ 1.5 Hz, Harom.), 8.05 (d, 1H, J ¼ 9.5 Hz, Hfluorene), 7.74
(d, 1H, J ¼ 9.5 Hz, Hfluorene), 7-72-7.67 (m, 1H, Harom.), 7.61 (dd, 1H,
4.1.3.2. 3-Nitro-8-N-methyl-7,8,11b(11)-triazabenzo[c]fluorene iodide
17. Compound 17 was prepared using the above described method
J1 ¼ 8.2 Hz, J2 ¼ 4.7 Hz, Harom.);13C NMR (DMSO-d6, 150 MHz):
d/
ppm ¼ a) 156.2, 150.2, 149.8, 147.5, 146.6, 138.7, 136.7, 132.3, 127.6,
121.7, 121.2, 118.6, 118.5; b) 154.2, 150.0, 149.2, 146.2, 144.3, 138.9,
132.1, 124.9, 123.0, 121.6, 119.0, 118.5, 118.1; Anal. (C13H8N4) Calcd.: C,
70.9; H, 3.7; N, 25.4; Found: C, 71.1; H, 3.8; N, 25.6.
from compound 13 (0.10 g, 0.38 mmol) and methyl-iodide (24
mL,
0.38 mmol) to obtain yellow powder (0.02 g, 15%). mp >270 ꢀC; UV
(EtOH) lmax/nm ¼ 383, 365, 297, 263, 225; IR (diamond):
n/
cmꢂ1 ¼ 3044, 2765, 1656, 1611, 1544; 1H NMR (DMSO-d6, 600 MHz):
d/ppm ¼ a) 9.21 (d, 1H, J ¼ 8.5 Hz, Harom.), 9.32 (d, 1H, J ¼ 2.6 Hz,
4.1.3. General method for the synthesis of 8-N-methyl-triaza-benzo
[c]fluorene iodide derivatives (16e17)
Equimolar amounts of triaza-benzo[c]fluorenes 12e13 and
methyl-iodide in absolute ethanol (10 ml) were stirred at room
temperature for 48 h. The solution was concentrated and obtained
Harom.), 9.21 (d, 1H, J ¼ 9.4 Hz, Harom.), 9.18 (d,1H, J ¼ 6.1 Hz, Harom.),
8.76 (d, 1H, J ¼ 9.5 Hz, Harom.), 8.70 (dd, 1H, J1 ¼ 9.3 Hz, J2 ¼ 2.7 Hz,
Harom.), 8.23 (d, 1H, J ¼ 9.5 Hz, Harom.), 8.11 (dd, 1H, J1 ¼ 8.4 Hz,
J2 ¼ 6.2 Hz, Harom.), 4.33 (s, 3H, CH3); b) 9.64 (d,1H, J ¼ 8.9 Hz, Harom.),
9.42 (d, 1H, J ¼ 2.3 Hz, Harom.), 9.37 (d, 1H, J ¼ 9.1 Hz, Harom.), 9.12 (dd,
Fig. 6. SW620 cells treated with compound 28 for 24 and 48 h.

2756
M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2748e2758
Fig. 7. SK-BR-3 cells treated with compound 18 for 24 and 48 h.
1H, J1 ¼4.9 Hz, J2 ¼ 2.4 Hz, Harom.), 9.04 (d,1H, J ¼ 4.6 Hz, Harom.), 9.00
(d,1H, J ¼ 9.2 Hz, Harom.), 8.80 (dd,1H, J1 ¼ 9.3 Hz, J2 ¼ 2.5 Hz, Harom.),
8.68 (d, 1H, J ¼ 9.5 Hz, Harom.), 4.27 (s, 3H, CH3); 13C NMR (DMSO-d6,
7.85 (d, 1H, J ¼ 9.5 Hz, Harom.), 7.54 (d, 1H, J ¼ 9.5 Hz, Harom..),
7.17e7.12 (m, 2H, Harom.), 5.57 (brs, 3H, NH2); b) 9.20 (d, 1H,
J ¼ 2.0 Hz, Harom.), 8.57 (d, 1H, J ¼ 2.1 Hz, Harom.), 8.45 (d, 1H,
J ¼ 9.2 Hz, Harom.), 7.84 (d, 1H, J ¼ 9.6 Hz, Harom.), 7.47 (d, 1H,
150 MHz):
d/ppm ¼ 151.9, 149.7, 145.2, 141.9, 138.6, 137.6, 131.8, 128.4,
126.7, 126.2, 124.3, 118.8, 118.6, 42.2; Anal. (C15H11N4O2I) Calcd.: C,
44.4; H, 2.7; N, 13.8; Found: C, 44.5; H, 2.8; N, 13.5.
J ¼ 9.6 Hz, Harom.), 7.08e7.05 (m, 2H, Harom), 5.55 (brs, 3H, NH2); 13
C
NMR (DMSO-d6, 75 MHz): a) 149.5, 147.0, 142.7, 137.9, 134.0, 126.0,
124.7, 124.5, 118.6, 117.3, 116.7, 116.5, 112.1, 111.2; b) 154.6, 148.7,
146.9, 146.7, 137.9, 134.3, 129.3, 125.6, 123.7, 118.9, 117.8, 116.9, 115.2,
111.8; Anal. (C14H9N4Br) Calcd.: C, 53.8; H, 2.9; N, 17.9; Found: C,
53.9; H, 3.1; N, 18.0.
4.1.4. General method for the synthesis of 3-amino-triaza-benzo[c]
fluorene derivatives (18e19)
Corresponding 3-nitro-triaza-benzo[c]fluorene derivatives
13e14 and solution of SnCl2x2H2O in H2O and concentrated HCl
were refluxed for 30 min. After cooling, the reaction mixture was
evaporated under vacuum and dissolved in water (50 ml). The
resulting solution was treated with 20% NaOH to pH 14. The
resulting product was filtered off and washed with water to obtain
3-amino-triaza-benzo[c]fluorenes.
4.1.5. General method for the synthesis of 3-amino-triaza-benzo[c]
fluorene hydrochlorides (20e21)
A stirred suspension of compounds 18e19 in absolute ethanol
(15 mL) was saturated with HCl(g). After 24 h of stirring, small
amount of diethylether was added, resulting product was filtered
off and washed with diethylether to obtain 3-amino-triaza-benzo
[c]fluorene hydrochlorides.
4.1.4.1. 3-Amino-7,8,11b(11)-triazabenzo[c]fluorene18. Compound 18
was prepared using the above described method from compound 13
(0.13 g, 0.49 mmol), SnCl2 ꢁ 2H2O (0.92 g, 4.00 mmol), HClconc.
(1.7 ml) and H2O (1.7 ml) to yellow powder (0.08 g, 69%). mp
206e208 ꢀC; UV (EtOH) lmax/nm ¼ 349, 335, 270, 245; IR (diamond):
4.1.5.1. 3-Amino-7,8,11b(11)-triazabenzo[c]fluorene
hydrochloride
20. Compound 20 was prepared using the above described method
from compound 18 (0.06 g, 0.26 mmol) to obtain white powder
(0.04 g, 52%). mp > 270 ꢀC; UV (EtOH) lmax/nm ¼ 347, 333, 270 and
n
/cmꢂ1 ¼ 3224, 3016, 1705, 1634, 1520; 1H NMR (DMSO-d6,
300 MHz):
d
/ppm ¼ a) 9.02 (dd, 1H, J1 ¼ 8.3 Hz, J2 ¼ 1.2 Hz, Harom.),
245; IR (diamond):
NMR (DMSO-d6, 300 MHz):
n
/cmꢂ1 ¼ 3266, 3145, 1744, 1620, 1613, 1509; 1H
8.64 (dd, 1H, J1 ¼ 4.6 Hz, J2 ¼ 1.2 Hz, Harom.), 8.49 (d, 1H, J ¼ 8.9 Hz,
Harom.), 7.85 (d, 1H, J ¼ 9.5 Hz, Harom.), 7.58 (d, 1H, J ¼ 9.5 Hz, Harom.),
7.42 (dd, 1H, J1 ¼ 8.3 Hz, J2 ¼ 4.6 Hz, Harom.), 7.16 (dd, 1H, J1 ¼ 8.9 Hz,
J2 ¼ 2.6 Hz, Harom.), 7.10 (d, 1H, J ¼ 2.5 Hz, Harom.); b) 9.49 (d, 1H,
J ¼ 8.7 Hz, Harom.), 8.57 (dd, 1H, J1 ¼ 4.7 Hz, J2 ¼ 1.4 Hz, Harom.), 8.26
(dd,1H, J1 ¼ 8.2 Hz, J2 ¼ 1.5 Hz, Harom.), 7.82 (d, 1H, J ¼ 9.4 Hz, Harom.),
7.56 (d, 1H, J ¼ 9.5 Hz, Harom.), 7.52 (dd, 1H, J1 ¼ 8.3 Hz, J2 ¼ 1.4 Hz,
Harom.), 7.14 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.6 Hz, Harom.), 7.09 (d, 1H,
J ¼ 2.6 Hz, Harom.); 13C NMR (DMSO-d6, 75 MHz): a) 148.6, 146.7,
146.3, 136.7, 133.4, 127.4, 126.5, 124.7, 123.0, 120.2, 119.7, 118.7, 118.0,
117.0; b) 156.1, 147.5, 146.6, 145.1, 142.8, 133.3, 126.1, 124.4, 122.7,
119.9, 118.7, 118.4, 117.2, 116.9; Anal. (C14H10N4) Calcd.: C, 71.8; H, 4.3;
N, 23.9; Found: C, 71.9; H,4.5; N, 24.1.
d
/ppm ¼ a) 9.80 (d, 1H, J ¼ 9.1 Hz,
Harom.), 8.88 (d, 1H, J ¼ 9.7 Hz, Harom.), 8.76 (dd, 1H, J1 ¼ 4.7 Hz,
J2 ¼ 1.4 Hz, Harom.), 8.44 (dd, 1H, J1 ¼ 9.0 Hz, J2 ¼ 1.5 Hz, Harom.), 8.41
(d, 1H, J ¼ 9.7 Hz, Harom.), 7.89 (d, 1H, J ¼ 9.5 Hz, Harom.), 7.81e7.76
(m, 2H, Harom.), 6.54 (brs, 3H, NH3þ); b) 9.52 (d, 1H, J ¼ 8.5 Hz,
Harom.), 8.84 (d, 1H, J ¼ 4.5 Hz, Harom.), 8.76 (dd, 1H, J1 ¼ 4.7 Hz,
J2 ¼ 1.3 Hz, Harom.), 8.37 (d, 1H, J ¼ 9.7 Hz, Harom.), 7.86 (d, 1H,
J ¼ 9.5 Hz, Harom.), 7.80e7.71 (m, 3H, Harom.), 6.53 (brs, 3H, NH3þ);
13C NMR (DMSO-d6, 75 MHz): a) 149.1, 145.2, 144.9, 137.5, 137.1,
131.9, 130.0, 127.7, 124.8, 124.4, 122.5, 119.1, 118.3, 115.2; b) 148.2,
143.9, 143.8, 137.0, 136.8, 129.2, 125.9, 125.1, 124.8, 124.1, 121.1, 118.8,
118.1, 114.8; Anal. (C14H11N4Cl) Calcd.: C, 62.1; H, 4.1; N, 20.7;
Found: C, 62.3; H, 4.3; N, 20.9.
4.1.4.2. 3-Amino-10-bromo-7,8,11b(11)-triazabenzo[c]fluorene
19. Com
pound 19 was prepared using the above described method from
4.1.5.2. 3-Amino-10-bromo-7,8,11b(11)-triazabenzo[c]fluorene
hydrochloride 21. Compound 21 was prepared using the above
described method from compound 19 (0.07 g, 0.22 mmol) to obtain
compound 14 (0.18 g, 0.53 mmol), SnCl2
ꢁ
2H2O (0.98 g,
yellow powder (0.03 g, 41%). mp >270 ꢀC; UV (EtOH) lmax
/
4.34 mmol), HClconc. (1.8 ml) and H2O (1.8 ml) to obtain yellow
nm ¼ 354, 340, 273, 249; IR (diamond):
n
/cmꢂ1 ¼ 3268, 3209, 3089,
powder (0.13 g, 82%). mp 252e254 ꢀC; UV (EtOH) lmax/nm ¼ 351,
1742, 1645, 1580; 1H NMR (DMSO-d6, 300 MHz):
d
/ppm ¼ 9.58 (d,
339, 274, 249; IR (diamond):
n
/cmꢂ1 ¼ 3222, 3067, 1722, 1620, 1571;
1H, J ¼ 9.1 Hz, Harom.), 8.80 (d, 1H, J ¼ 1.5 Hz, Harom.), 8.69 (d, 1H,
J ¼ 1.9 Hz, Harom.), 8.24 (d, 1H, J ¼ 9.6 Hz, Harom.), 8.01 (s, 1H, Harom),
7.86 (dd, 1H, J1 ¼ 8.9 Hz, J2 ¼ 1.9 Hz, Harom.), 7.78 (d, 1H, J ¼ 9.5 Hz,
1H NMR (DMSO-d6, 300 MHz):
d
/ppm ¼ a) 9.26 (d, 1H, J ¼ 8.9 Hz,
Harom.), 8.67 (d, 1H, J ¼ 2.0 Hz, Harom.), 8.46 (d, 1H, J ¼ 8.7 Hz, Harom.),

M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2748e2758
2757
Harom.), 7.50 (brs, 3H, NH3þ); b) 9.39 (d, 1H, J ¼ 1.6 Hz, Harom.), 8.87
(d, 1H, J ¼ 8.9 Hz, Harom.), 8.60 (d, 1H, J ¼ 1.9 Hz, Harom.), 8.21 (d, 1H,
J ¼ 9.6 Hz, Harom.), 8.00 (s, 1H, Harom.), 7.85 (dd, 1H, J1 ¼ 8.9 Hz,
J2 ¼ 1.9 Hz, Harom.), 7.78 (d, 1H, J ¼ 9.6 Hz, Harom.), 7.51 (brs, 3H,
NH3þ); 13C NMR (DMSO-d6, 75 MHz): a) 148.4, 147.4, 143.1, 134.4,
125.4, 124.5, 124.3, 123.5, 123.1, 118.1, 117.7, 116.9, 115.6, 113.8; b)
147.6, 144.6, 133.3, 125.1, 124.9, 123.8, 123.4, 118.8, 117.9, 117.6, 116.5,
115.7, 113.5, 113.2; Anal. (C14H10N4ClBr) Calcd.: C, 48.1; H, 2.9; N,
16.0; Found: C, 48.3; H, 3.0; N, 16.3.
32%). mp > 270 ꢀC; UV (EtOH) lmax/nm ¼ 366, 350, 237; IR (dia-
mond):
n
/cmꢂ1 ¼ 1603, 1620, 1713, 3013, 3295; 1H NMR (DMSO-d6,
600 MHz):
d
/ppm ¼ 11.01 (s, 2H, NH), 10.25 (s, 1H, Harom.), 8.75 (dd,
1H, J1 ¼ 4.7 Hz, J2 ¼ 1.4 Hz, Harom.), 8.44 (dd, 1H, J1 ¼ 8.1 Hz,
J2¼1.4 Hz, Harom.), 8.35 (d, 1H, J ¼ 8.2 Hz, Harom.), 8.20 (d, 1H,
J ¼ 9.5 Hz, Harom.), 8.09 (dd, 1H, J1 ¼ 8.2 Hz, J2¼1.6 Hz, Harom.), 7.92
(d, 1H, J ¼ 9.6 Hz, Harom.), 7.71 (dd, 1H, J1 ¼ 8.2 Hz, J2 ¼ 4.7 Hz,
Harom.), 4.15 (s, 4H, CH2); 13C NMR (DMSO-d6, 75 MHz):
d/
ppm ¼ 165.9, 147.4, 145.1, 141.1, 135.4, 134.1, 133.4, 130.7, 128.0,
127.3, 125.1, 124.2, 121.1, 120.2, 117.6, 45.3 (2C); Anal. (C17H14N5Cl)
Calcd.: C, 63.1; H, 4.4; N, 21.6; Found: C, 63.2; H, 4.2; N, 21.4.
4.1.6. General method for the synthesis of cyano-triaza-benzo[c]
fluorenes (26e27)
Ethanolic solution (400 ml) of compounds 24e25 was irradiated
for 11 h at room temperature with 400 W high-pressure mercury
lamp using the Pyrex filter. The air was bubbled through the solu-
tion, and the latter was concentrated. The obtained product was
filtered off to produce cyano-triaza-benzo[c]fluorenes.
4.1.7.2. 10-Bromo-2-(2-imidazolynyl)-7,8,11b-triazabenzo[c] fluorene
29. Compound 28 was prepared using the above described method
from compound 27 (0.11 g, 0.33 mmol) and ethylenediamine
(0.08 ml,1.16 mmol) to obtain light greenpowder (0.07 g, 65%). mp >
270 ꢀC; IR (diamond):
n
/cmꢂ1 ¼ 1598, 1614, 1723, 2988, 3383; UV
(EtOH) lmax/nm ¼ 355, 247; 1H NMR (DMSO-d6, 600 MHz):
d/
4.1.6.1. 2-Cyano-7,8,11b-triazabenzo[c]fluorene 26. Compound 26
was prepared using the above described method from compound
24 (0.30 g, 1.22 mmol) to obtain yellow powder (0.17 g, 57%). mp
252e254 ꢀC; UV (EtOH) lmax/nm ¼ 381, 362, 345, 234; IR (dia-
ppm ¼ 11.00 (s, 2H, NH), 10.05 (s, 1H, Harom.), 8.74 (d, 1H, J ¼ 1.0 Hz,
Harom.), 8.72 (d, 1H, J ¼ 1.0 Hz, Harom.), 8.34 (d, 1H, J ¼ 4.1 Hz, Harom.),
8.19 (d, 1H, J ¼ 4.8 Hz, Harom.), 8.08 (d, 1H, J ¼ 4.1 Hz, Harom.), 7.89 (d,
1H, J ¼ 4.8 Hz, Harom.), 4.12 (s, 4H, CH2); 13C NMR (DMSO-d6, 75 MHz):
mond):
300 MHz):
n
/cmꢂ1 ¼1614, 1640, 1966, 2224, 3067; 1H NMR (DMSO-d6,
d/ppm ¼ 165.2, 148.5, 143.5, 143.5, 137.5, 133.5, 133.0, 129.9, 126.7,
d
/ppm ¼ 10.06 (s, 1H, NH), 8.10 (dd, 1H, J1 ¼ 4.6 Hz,
125.6, 124.8, 120.2, 116.8, 115.8, 44.7; Anal. (C17H13N5ClBr) Calcd.: C,
50.8; H, 3.2; N, 17.5; Found: C, 50.9; H, 3.4; N, 17.4.
J2 ¼ 1.2 Hz, Harom.), 8.38 (dd, 1H, J1 ¼ 8.2 Hz, J2 ¼ 1.0 Hz, Harom.), 8.23
(d, 1H, J ¼ 8.1 Hz, Harom.), 8.08 (d, 1H, J ¼ 9.6 Hz, Harom.), 7.98 (d, 1H,
J ¼ 8.2 Hz, Harom.), 7.82 (d, 1H, J ¼ 9.6 Hz, Harom.), 7.64 (Abq, 1H,
J1 ¼ 8.2 Hz, J2 ¼ 4.8 Hz, Harom.); 13C NMR (DMSO-d6, 75 MHz):149.6,
145.5, 144.1,137.1,134.5, 132.3, 130.7, 129.5, 128.4, 127.9, 126.4, 121.2,
120.9, 118.9, 112.3; Anal. (C15H8N4) Calcd.: C, 73.8; H 3.3; N, 22.9;
Found: C, 73.9; H, 3.1; N, 23.1.
4.2. Spectroscopy
The electronic absorption spectra were recorded on Varian Cary
50 and Varian Cary 100 Bio spectrometer, CD spectra on Jasco J815,
in all cases using quartz cuvettes (1 cm). Fluorescence emission
spectra were recorded on Varian Eclipse fluorimeter (quartz
cuvettes, 1 cm) at 350e600 nm. The sample concentration in
fluorescence measurements had an optical absorbance below
0.05 at the excitation wavelength. Under the experimental condi-
tions used, the absorbance and fluorescence intensities of studied
compounds were proportional to their concentrations, while none
of studied compounds showed CD spectra. The measurements were
performed in an aqueous buffer solution (pH ¼ 7.0; sodium caco-
dylate buffer, I ¼ 0.05 mol dmꢂ3).
4.1.6.2. 10-Bromo-2-cyano-7,8,11b-triazabenzo[c]fluorene 27. Compo-
und 27 was prepared using the above described method from
compound 25 (0.14 g, 0.46 mmol) to obtain yellow powder (0.06 g,
42%). mp > 270 ꢀC; UV (EtOH) lmax/nm ¼ 382, 361, 345, 235; IR
(diamond):
d6, 300 MHz):
n
/cmꢂ1 ¼1536, 1570, 1625, 2226, 3076; 1H NMR (DMSO-
d
/ppm ¼ 8.41 (d, 1H, J ¼ 2.1 Hz, Harom.), 8.23 (d, 1H,
J ¼ 2.0 Hz, Harom.), 7.87 (d, 4H, J ¼ 8.4 Hz, Harom.), 7.83 (d, 1H,
J ¼ 16.6 Hz, Harom.), 7.38 (d, 1H, J ¼ 16.5 Hz, Harom.); 13C NMR (DMSO-
d6, 75 MHz):
d/ppm ¼ not soluble enough; Anal. (C15H7N4Br) Calcd.:
C, 55.8; H, 2.2; N, 17.3; Found: C, 55.9; H, 2.4; N, 17.2.
4.3. Interactions with DNA
4.1.7. General method for the synthesis of 2-imidazolinyl-triaza-
benzo[c]fluorene hydrochlorides (28e29)
The calf thymus DNA(ct-DNA)waspurchasedfromSigmaeAldrich,
dissolved in sodium cacodylate buffer, I ¼ 0.05 mol dmꢂ3, pH ¼ 7.0,
A stirred suspension of corresponding cyano-triaza-benzo[c]
fluorenes 26e27 in absolute ethanol was cooled in an ice-salt bath
and was saturated with HCl gas. The flask was then tightly stop-
pered, and the mixture was maintained at room temperature until
the nitrile band disappeared (monitored by the IR analysis at
2200 cmꢂ1). The reaction mixture was purged with N2 gas and
diluted with diethylether. The crude imidate was filtered off and
was immediately suspended in absolute ethanol. Ethylenediamine
was added and the mixture was stirred at reflux for 24 h. The crude
product was then filtered off, washed with diethylether to give
white powder, which was suspended in absolute ethanol and again
saturated with HCl (g). Reaction mixture was stirred at room
temperature for 24 h. Crude product was filtered off and washed
with small amount of diethylether to give corresponding 2-imi-
dazolynyl-triaza-benzo[c]fluorene hydrochlorides.
additionally sonicated, filtered through 0.45 mm filter, and the
concentration of the corresponding solution was determined spec-
troscopically as the concentration of phosphates [34]. The measure-
ments were performed in an aqueous buffer solution (pH ¼ 7.0;
sodium cacodylate buffer, I ¼ 0.05 mol dmꢂ3). Spectroscopic titrations
were performed by adding portions of polynucleotide solution into
the solution of the studied compound. The stability constant (Ks) and
[bound compound]/[polynucleotide phosphate] ratio (n) were calcu-
lated according to the Scatchard equation by non-linear least-square
fitting giving excellent correlation coefficients (>0.999) for obtained
values for Ks and n. Thermal denaturation curves for ct-DNA and its
complexes with studied compounds were determined as previously
described by following the absorption change at 260 nm as a function
of temperature. The absorbance of the ligand was subtracted from
every curve, and the absorbance scale was normalized. Obtained Tm
values are the midpoints of thetransition curves, determined from the
maximum of the first derivative or graphically by a tangent method.
4.1.7.1. 2-(2-Imidazolynyl)-7,8,11b-triazabenzo[c]fluorene
hydro-
chloride 28. Compound 28 was prepared using the above described
method from compound 26 (0.20 g, 0.82 mmol) and ethylenedi-
amine (0.16 ml, 2.32 mmol) to obtain light green powder (0.06 g,
Given
acid from Tm of complex. Every
average of at least two measurements, the error in
DTm values were calculated subtracting Tm of the free nucleic
D
Tm value here reported was the
D
Tm is ꢃ 0.5 ꢀC.

2758
M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2748e2758
4.4. Cell culturing and cell morphology
125-0982464-1356, 098-0982914-2918, 335-0982464-2393, and
335-0000000-3532).
The cell lines HeLa (cervical carcinoma), SW620 (colorectal
adenocarcinoma, metastatic), MiaPaCa-2 (pancreatic carcinoma),
MCF-7 (breast epithelial adenocarcinoma, metastatic), HEp-2
(epidermoid carcinoma, larynx,), SK-BR-3 (breast adenocarci-
noma, metastatic) and WI38 (normal diploid human fibroblasts)
were cultured as monolayers and maintained in Dulbecco’s modi-
fied Eagle medium (DMEM) supplemented with 10% foetal bovine
Appendix. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.03.062.
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mM. After
ꢁ
ꢁ
with10 mg/mL ofpropidiumiodide (PI)with the additionof0.2 mg/mL
of RNAse A. Stained cells were then analysed with Becton Dickinson
FACScalibur flow cytometer (10000 counts were measured). Each
test point was performed in triplicate. The percentage of the cells in
each cell cycle phase was deduced from obtained DNA histograms
using WinMDI 2.9 and Cylchred software. Statistical analysis was
performed in Microsoft Excel by using ANOVA at p < 0.05.
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Acknowledgement
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b) I. Piantanida, B.S. Palm, P. Cudic, M. Zinic, H.J. Schneider, Tetrahedron 60
(2004) 6225e6231.
Support for this study by the Croatian Ministry of Science,
Education and Sport is gratefully acknowledged (Grants number
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