Synthesis of new pyrazolo[5,1-c][1,2,4] benzotriazines, pyrazolo[5,1-c]pyrido[4,3-e][1,2,4] triazines and their open analogues as cytotoxic agents in normoxic and hypoxic conditions

Article abstract of DOI:10.1016/j.bmc.2008.09.055

The synthesis and antitumor activity in normoxic and hypoxic conditions of a series of pyrazolo[5,1-c][1,2,4]benzotriazine and its related analogues are reported. All compounds were tested on human colorectal adenocarcinoma cell line HCT-8 and for compounds 15 and 20, which show to have selective cytotoxicity in hypoxic and in normoxic conditions respectively, ROS production, cell cycle, and DNA fragmentation were measured. This preliminary study encouraged us to consider 15 and 20 as interesting leads for further optimization.

Full text of DOI:10.1016/j.bmc.2008.09.055

Bioorganic & Medicinal Chemistry 16 (2008) 9409–9419
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of new pyrazolo[5,1-c][1,2,4] benzotriazines,
pyrazolo[5,1-c]pyrido[4,3-e][1,2,4] triazines and their open analogues
as cytotoxic agents in normoxic and hypoxic conditions
b
a
a
b
Giovanna Ciciani a, , Marcella Coronnello , Gabriella Guerrini , Silvia Selleri , Miriam Cantore ,
*
Paola Failli b, Enrico Mini b, Annarella Costanzo a
a Dipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente attivi (HeteroBioLab) Università degli Studi di Firenze,
Via U. Schiff, 6, 50019 Polo Scientifico, Sesto Fiorentino, Firenze, Italy
b Dipartimento di Farmacologia Preclinica e Clinica Aiazzi-Mancini, Università degli Studi di Firenze, Viale Pieraccini, 6, 50139 Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2008
Revised 16 September 2008
Accepted 23 September 2008
Available online 26 September 2008
The synthesis and antitumor activity in normoxic and hypoxic conditions of a series of pyrazolo[5,
1-c][1,2,4]benzotriazine and its related analogues are reported. All compounds were tested on human
colorectal adenocarcinoma cell line HCT-8 and for compounds 15 and 20, which show to have selective
cytotoxicity in hypoxic and in normoxic conditions respectively, ROS production, cell cycle, and DNA frag-
mentation were measured. This preliminary study encouraged us to consider 15 and 20 as interesting
leads for further optimization.
Keywords:
Tricyclic heteroaromatic system
Pyrazole derivatives
Ó 2008 Elsevier Ltd. All rights reserved.
Pyrazolo[51-c][1,2,4] benzotriazine
Pyrazolo[51-c]pyrido[4,3-e][1,2,4]triazine
Antitumor activity
1. Introduction
Since several bioreductive agents contain nitro and/or N-oxide
group, in their aromatic or heteroaromatic planar system, it could
In the last 50 years, the extensive in vitro and in vivo screening
of natural products and chemical compounds has led to the devel-
opment of the anticancer drugs currently utilized in the clinic.
Despite many therapeutic successes, cancer is the second-
most-frequent cause of death1,2 and may become the most com-
mon in the near future. Thus, the search for new drugs continues
to be a great challenge for medical science.
In this work, the attention has been focused on solid tumours,
whose common condition is the presence of hypoxic cells,3–5
which not only accelerate malignant progression and increase
metastasis, but also negatively influence responsiveness to radio-
therapy and chemotherapy.6,7
be very attractive to explore if the pyrazolo[5,1-c][1,2,4]benzotri-
azine system possesses a similar activity.
Our research group has been involved in designing derivatives
of the pyrazolo[5,1-c][1,2,4] benzotriazine system which have been
extensively studied over a period of time in our laboratory for the
synthesis of benzodiazepine receptor ligands. These studies have
led us to individuate high affinity ligands endowed with inverse
agonist pharmacological efficacy,25 anxioselective agents,26 and
selective anticonvulsant activity.27 On the other hand this planar
system can be considered a ‘useful chemical space’ for various bio-
logical activity too. In fact, in previous papers27,28 we pointed out
that the 3-nitropyrazolo[5,1-c][1,2,4]benzotriazine system, 7,8-
disubstituted possesses cytotoxic activity in the micromolar range
in normoxic conditions, on leukemia cell lines more then against
solid tumours.
The aim of the present study was to synthesize and develop a
set of new compounds containing the pyrazolo[5,1-c][1,2,4]benzo-
triazine and pyrazolo[5,1-c]pyrido[4,3-e][1,2,4]triazine system (A)
and their open analogues (B and C, Chart 2) in order to evaluate
their potential anticancer properties in normoxic and hypoxic con-
ditions on human colorectal adenocarcinoma cell line HCT-8. In
this study, two previously synthesized compounds, 8-iodopyrazol-
The approach proposed by Sartorelli’s group8 in the early 1970s
that ‘tumour hypoxia can be turned to advantage by exploiting it to
activate bioreductive prodrugs within tumours’, has led to the
development of several hypoxia selective agents. The main catego-
ries of these bioreductive drugs belong to nitroaromatics (I),5,6,9,10
quinones (MMC),11 aromatic (II–V),12–17 or aliphatic-N-oxide
(VI)18–20 derivatives and metal complexes (VII)21–24 (Chart 1).
* Corresponding author. Tel.: +39 055 4573767; fax +39 055 4573671.
E-mail address: giovanna.ciciani@unifi.it (G. Ciciani).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.055

9410
G. Ciciani et al. / Bioorg. Med. Chem. 16 (2008) 9409–9419
NO2
O
CONH2
NH
N
O
O
O2N
I
H2N
N
O
MMC
O
Cl
Cl
SN23862
H2N
O-
O-
N+
N+
NH2
NH
N
H
Cl
N
N
N+
O-
N+
O-
II
Q85 HCl
III
TIRAPAZAMINE (TPZ)
O-
O
N+
O
N
CH3
IV
N
N+
O-
SN29751
H3C
N
N+
HO
NH
O
O
N
N
O-
N
N
V
N+
O-
RB90740
N+
HO
HN
O-
VI
CN
O-
H
N
N+
O
N+
N+
VO
O
N+
VII
Vanadyl complexes
O-
N
H
CN
Chart 1.
o[5,1-c][1,2,4]benzotriazine 5-oxide, a4329 and 8-hydroxypyrazol-
o[5,1-c][1,2,4]benzotriazine 5-oxide a4530, were tested for the
structure activity relationships (SARs) discussion.
In this preliminary stage, reactive oxygen species (ROS) produc-
tion, cell cycle and DNA damage in normoxic and hypoxic condi-
tions for the most interesting compounds, were evaluated.
Mitomicyn C (MMC) was our reference compound in all performed
tests.
2
1
N
3
9
R8
N
R3
8
A
X= N
X= CH
X
N
4
5N+
O-
7
6
N
N
N
OH
2. Chemistry
HN
R4
X
The synthetic pathways to obtain desired final compounds (1–3,
5–20) are illustrated in Schemes 1–3 and chemical-physical data
are shown in Tables 1 and 2.
N+
O-
R4
N
N
NH2
NO2
Compounds 1 and 3 were achieved from 3-nitro-4-hydrazino-
pyridine31 and 2-cyano-3-ethoxypropeneate or 2-ethoxy-1,1-
ethenedicarbonitrile in ethanol. The previously synthesized com-
pound 4,30 utilized as starting material in this study, was obtained
from 2,4-dinitrophenylhydrazine. Compound 1, 5-aminopyrazole-
B
C
X= N
X= C-NO2
Chart 2.

G. Ciciani et al. / Bioorg. Med. Chem. 16 (2008) 9409–9419
9411
NH2
NH
R= CN
EtO
R
R= COOEt
CN
X
NO2
X= N
X= C-NO2
1
2
3
X=N
X=N
X=N
R4=CN
R4=CONH2
R4=COOEt
N
i
N
R4
4ª
X= C-NO2 R4=COOEt
NH2
X
NO2
N
N
3-4
ii
2
ii
N
N
CONH2
N
COOH
N
COOEt
N
N
N+
O-
N
N
N
iii
N+
O-
OH
N
N
iv
N+
O-
HN
X
N+
O-
N
COOEt
5
6
7
8
9
X= N
X= C-NO2
Scheme 1. Reagents: (i) H2SO4, 60 °C, ammonia; (ii) NaOH 10%; (iii) NaNO2/H2SO4; (iv) EtOH/ H2SO4; (a) see Ref. 30.
N
N
N
Cl
R3
N
R8
R3
i
N
N+
O-
N
N+
X
R3= H,a NO2,b Ph,c
10
11
12
13
14
15
16
R3= H
X=O R8= N-methylpiperazine
X=-- R8= N-methylpiperazine
X=O R8= Morpholine
X=O R8= O(CH2)2NMe2
X=O R8= O(CH2)4CH3
R3= H
R3= H
R3= H
R3= H
R3= NO 2 X=O R8= N-methylpiperazine
R3= Ph X=O R8= N-methylpiperazine
Scheme 2. Reagents: (i) N-methylpiperazine or morpholine in ethoxyethanol (for compounds 10 and 12); N-methylpiperazine as reagent/solvent and N2 (for compound 11);
dimethyletanolamine and sodium hydroxide 10% (for compound 13), pentanol, sodium hydroxide 40% and tetra-butylamonium bromide in dichloromethane (for compound
14); N-methylpiperazine, potassium carbonate in toluene (for compounds 15 and 16), (a) see Ref. 30; (b) see Ref. 27; (c) see Ref. 35.
4-carbonitrile derivative, was easily converted into 4-carboxyam-
ide, 2, for simple treatment with concentrated sulfuric acid at
60 °C and then precipitated with ammonia solution.
Het/Ar-N (pyrazole) bond has produced. The recovered products
were 5-(4-hydroxypyridin-3-yl-ONN-azoxy)-1H-pyrazole and 5-
(3-nitro-6-hydroxyphenyl-ONN-azoxy)-1H-pyrazole 4-substituted,
8–9. This type of reactivity was previously observed on analogues30
and the fact that 3 behaves in the same manner could be due to
electronic feature of the pyridine ring that resembles, in terms of
The next treatment with sodium hydroxide solution of the
5-aminopyrazoles 2-4 gave two different types of derivatives.
Compound 2, the 5-aminopyrazole-4-carboxyamide derivative, under-
went the ring closure to pyrazolobenzotriazine 5-oxide system
following the traditional intramolecular cyclization between the
nitro and amino group under basic conditions.32,33,30 The obtained
product 5 was in turn transformed into 3-carboxyderivative 6 and
then into 3-ethoxycarbonylderivative 7, following a previously de-
scribed method. Instead the 4-ethoxycarbonyl-5-aminopyrazoles 3
and 4,30 in addition to the expected condensation between the ni-
tro and the amino group, underwent the nucleophilic attack of the
OHꢀ species at the heteroaromatic-/aromatic-C1 and a cleavage of
r
parameters, a 2,4-dinitrophenyl ring, see Scheme 1.
Compounds 10–16 were obtained by exploiting the reactivity of
the chlorine atom at position 8 to nucleophilic aromatic substitu-
tion, as previously reported.30,34 The starting material, 8-chloropy-
razolo[5,1-c][1,2,4]benzotriazine 5-oxide variously substituted at
position 330,27,35 was reacted with nucleophilic reagents (N-meth-
ylpiperazine, morpholine, dimethylethanolamine, and pentanol) in
different conditions able to permit the nucleophilic substitution
(see Section 6, for details). In particular, when the N-methylpiper-

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G. Ciciani et al. / Bioorg. Med. Chem. 16 (2008) 9409–9419
N
N
R9
N
R8
H
N
R8
R3
i
N
N
N
X
N
X
a43a
11
R8= I
X=O
17
18
19
R8= Phenyl
R8= p-OMe-Phenyl
R8= Thienyl
X=O R3= H R9= H
X=O R3= H R9= H
X=O R3= H R9= H
R8= N-methylpiperazine X=--
20
R8= N-methylpiperazine X=--
R3= NO2 R9= NO2
Scheme 3. Reagents: (i) for compounds 17–19, Suzuki coupling conditions: ArB(OH)2, sodium carbonate, tetrakis (Pd(PPh3)4), tetrahydrofurane; (a) see Ref. 29. (i) For
compound 20: HNO3 65%/H2SO4.
Table 1
Chemical data of 5-aminopyrazoles 1–3 and –ONN-azoxy derivatives, 8-9
N
N
OH
HN
N
R4
X
N+
O-
R4
N
NH2
X
NO2
Compounds
R4
X
MF (MW)
Yield (%)
Mp °C (recryst. solvent)
1
2
3
8
9
CN
N
N
N
N
C9H6O2N6 (230.18)
C9H8O3N6 (248.20)
C11H11O4N5 (277.24)
C11H11O4N5 (277.24)
C12H11O6N5 (321.25)
35
80
40
45
43
198–199 (ethanol)
CONH2
COOEt
COOEt
COOEt
220–221 (methoxyethanol)
158–159 (ethanol 80%)
260–262 (boiled in acetone)
251–252 (water)
C-NO2
Table 2
Chemical data of pyrazolo[5,1-c]pyrido[4,3-e][1,2,4]triazines 5-oxides and pyrazolo[5,1-c] [1,2,4]benzotriazines 5-oxides
N
R9
N
R8
R3
X
N
N+
O-
No.
R3
X
R8
R9
MF (MW)
Yield (%)
Mp °C (recryst. solvent)
5
6
7
CONH2
COOH
COOEt
H
H
H
H
H
NO2
Ph
H
N
N
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
C9H6O2N6 (230.17)
C9H5O3N5 (231.17)
C11H9O3N5 (259.22)
C14H16ON6 (284.32)
C14H16N6 (268.32)
60
70
50
25
20
85
25
35
65
20
75
90
87
50
>300 (water)
>300 (water)
197–198 (ethanol)
238–239 (ethanol)
176–177 (ethoxyethanol)
226–227 (ethanol)
68–69 (cyclohexane)
93–94 (ethanol)
264–265 (methoxyethanol)
210–211 (ethanol)
175–176° (ethanol)
195–196° (ethanol)
209–210° (ethanol)
245–246° (methoxyethanol)
10
11°
12
13
14
15
16
17
18
19
20°
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N-Methylpiperazine
N-Methylpiperazine
Morpholine
O(CH2)2NMe2
O(CH2)4CH3
N-Methylpiperazine
N-Methylpiperazine
Ph
C13H13O2N5 (271.28)
C13H15O2N5 (273.29)
C14H16O2N4 (272.30)
C14H15O3N7 (329.31)
C20H20ON6 (360.41)
C15H10ON4 (262.27)
C16H12O2N4 (292.29)
C13H8ON4S (268.30)
C14H14O4N8 (358.31)
H
H
NO2
p-OMe-Ph
2-Tienyl
N-Methylpiperazine
a
N5-deoxide compounds.
azine was used as solvent/reagent in nitrogen atmosphere, the
oxide30 underwent both the 8-nucleophilic substitution and the
starting material 8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-
N-5 deoxidation and derivative 11 was recovered; it is plausible

G. Ciciani et al. / Bioorg. Med. Chem. 16 (2008) 9409–9419
9413
that the N-methylpiperazine behaves as a reducing agent, see
Scheme 2.
Notably, all compounds caused antiproliferative effects with IC50
values in the range 5–65 M. In details, compound 20, 16, and 7
showed IC50 values of 5.98 0.04 M, 10 1.20 M, and 23.8
1.0 M, respectively; it is also interesting to note that compounds
20 and 7 had IC90 values of 18.5 0.08 M and 49.1 0.33. From
these data, compound 20, 8-(4-methylpiperazin-1-yl)-3,9-dinitro-
pyrazolo[5,1-c][1,2,4] benzotriazine, emerges as the most potent
and active cytotoxic agent under normoxic conditions in the range
of concentrations used in both tests.
l
Scheme 3 depicts the synthetic approach either to obtaining
derivatives 17–19 or compound 20. The Suzuki cross-coupling
reaction has been applied on starting product a43, 8-iodopyrazol-
o[5,1-c][1,2,4]benzotriazine 5-oxide;29 its reaction with phenylbo-
ronic-, p-OMe-phenylboronic-, and 2-thienylboronic acid
respectively gave, with good yield, the desired compounds 17–
19, useful, as a43, for the next biological screening.
l
l
l
l
Finally, 3,9-dinitroderivative 20, was obtained by nitration with
concentrated nitric acid/sulfuric acid from 11 (Scheme 3).
Figure 1 indicates that the most hypoxic-selective agent was
compound 15, 8-(4-methylpiperazin-1-yl)-3-nitropyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide. MMC has similar cytotoxic activity
in both conditions on HCT-8 cells as reported in the literature.37
3. Results
3.2. ROS
3.1. In vitro normoxic and hypoxic cytotoxicity
Previously synthesized compounds a4329 and a4530 and newly
compounds 2–3, 6–10, 12–20, were evaluated for cytotoxicity on
oxygenated HCT-8 colon carcinoma cells, using the sulforhodamine
B (SRB) colorimetric assay as previously described.36 All com-
Compounds 15 and 20 were the most interesting and thus were
evaluated on reactive oxygen species (ROS) production in normox-
ic and hypoxic conditions at 10 lM in HCT-8 cells. The involve-
ment of ROS, in fact, in the bioreductive drugs action and in the
DNA damage is well known.38–41
pounds were tested in normoxic conditions at 10 and 100 lM fol-
Under normoxia and hypoxia conditions (Fig. 2), treatment with
15 and 20 caused similar and significantly greater ROS production
than the untreated control (P < 0.05). MMC produced a slight
enhancement of ROS production in a similar manner in both con-
dition. It can be excluded that the ‘differential cytotoxic-activity’
shown by 15 and 20 in normoxic and hypoxic conditions depended
on ROS production.
lowing 72 h of incubation and the percentages of cell inhibition are
reported in Table 3.
Before studying the cytotoxic effects of our compounds in hy-
poxia, the effect of hypoxia on HCT-8 cell proliferation (data not
shown), was investigated after 72 h incubation using SRB test.36
Cell proliferation was slightly less than normal for the first 24 h
of hypoxic treatment, which was chosen for drug exposure with
drug washout (DW) and reoxygenation at 24–48 h.
3.3. Cell-cycle
Figure 1 reports the cytotoxic effects under normoxia and hy-
poxia of our compounds at 10 lM following 24 h drug exposure
To examine the effect of compounds 15, 20, and MMC on HCT-8
cell cycle phases, flowcytometric analysis was performed, as
described in Section 6. Tables 5 and 6 show the percentages of
G0/G1, S-, G2/M cell phases, and DNA fragmentation determined
by sub-G1 peak quantitation under normoxic and hypoxic condi-
tions, respectively.
and 48 h drug washout (48 h DW). In Figure 1 and Table 3, all com-
pounds exhibit a time-related cytotoxicity, with the exception of
compound 20 that does not follow this trend (% inhib. 70.25 at
24 h vs % inhib. 73.85 at 72 h).
From the evaluation of normoxic data in Figure 1 and Table 3
compounds 3, 7, 9, 15, 16, and 20 were selected on the basis of
No significant modifications of the cell cycle phases were ob-
served under the experimental normoxic conditions, except for a
slight increase in the S-phase cell fraction with MMC (52.6% vs
35.7% of control T24DW) as reported in Table 5. The hypodiploid
peak, representing the cells with DNA fragmentation, was detect-
able for 15, 20 (39.0% and 42.0%, respectively) and was even more
evident for MMC (71.2%) after 24 h exposure. At a later time point
(T24DW) decreased cell death (25%) was observed for compound 15
and this trend was maintained at T48DW (27.0%), while for com-
pound 20 the hypodiploid peak increased from 42% at 24 h to
59% of the total cell population at 48 h-DW with an intermediate
degree of DNA fragmentation at T24DW (33%).
After 24 h hypoxic exposure (Table 6) and at later time points
(T24-, T48DW) no compound seemed to cause any significant cell-
cycle perturbation. Only compound 15 and MMC caused a signifi-
cant increase in DNA fragmentation at T24DW (58.3% and 71.4%,
respectively) and T48DW (60% and 99%, respectively) as compared
to untreated control (15.6% and 16%). Conversely, with compound
20 the dead cell fraction was decreased to 21% at T48DW. It is inter-
esting to note that despite the initial death of control (T24, 37%), it
recuperated vitality after 24 h of incubation in reoxygenated
conditions. According to reported data42–45 MMC induces cell cycle
perturbations and apoptosis in cell lines.
their higher cytotoxic activity (% inhibition P50 at 10
P60 at 100 M concentration), and were subsequently tested on
a range of concentrations from 10 to 100 M to obtain dose-
lM and
l
l
response curves. The IC50 and IC90 values are listed in Table 4.
Table 3
Percentage of growth inhibition on HCT-8 cell line determined after 72 h continuous
exposure
Compound
% Cell inhibition
10
l
M
100 lM
2
3
6
7
8
9
10
12
13
14
15
16
17
18
19
20
a43a
a45b
9.72 0.28
24.38 1.73
13.22 0.86
12.73 3.8
3.87 0.83
11.72 1.0
3.19 1.67
3.27 1.3
ꢀ2.36 1.25
ꢀ6.17 3.0
25.06 3.18
53.02 6.7
13.22 2.55
11.02 0.35
10.86 0.1
73.85 1.43
ꢀ1.62 0.6
ꢀ4.21 0.17
22.35 3.75
69.72 0.31
11.36 0.61
97.44 0.17
31.54 1.7
60.4 3.46
32.41 2.5
25.79 1.09
42.31 2.0
38.04 1.31
93.22 0.48
69.6 2.98
35.49 1.75
21.59 1.06
48.47 2.43
98.68 0.018
55.54 2.3
19.68 0.63
4. Discussion
In previous papers27,28 the critical role of the nitro group at po-
sition 3 on the pyrazolobenzotriazine system, independently on
substituents at position 7 and 8, for normoxic cytotoxic activity
a
See Ref. 29.
See Ref. 30.
b

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G. Ciciani et al. / Bioorg. Med. Chem. 16 (2008) 9409–9419
115
95
75
55
35
15
-5
MMC
MMC
20
15
16
20
19
7
6
9
2
9
15
6
19
7
8
2
a45
a43
12
3
3
10
8
16
13
14
17
17
14
13
18
10
12
a45
18
a43
-25
Normoxic conditions
Hypoxic conditions
Figure 1. Percentage of growth inhibition in normoxic and hypoxic conditions on HCT-8 cell line, following 24 h incubation of synthesized compounds at 10 lM. The
cytotoxic effect was checked by sulforhodamine B (SRB) test. See Section 6. MMC was the reference compound.
Table 4
was demonstrated. In the present study the synthesis and
preliminary pharmacological evaluation of three chemical series:
pyrazolo[5,1-c][1,2,4]benzotriazine, pyrazolo[5,1-c]pyrido[4,3-e]
[1,2,4]triazine and their open analogues (A, B, and C, Chart 2) is
reported.
With the aim of evaluating the influence of the hydro/lipophilic
balance13,46,47 on pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide sys-
tem some substituents present in the main categories of cytotoxic
agents,3,6,12,15,17were introduced.
IC50 and IC90 values of new compounds determined after 72 h continuous exposure
Compound
IC50
(
l
M)
IC90
(lM)
3
7
9
58.2 2.45
23.8 1.0
65.2 2.50
62.5 2.30
10 1.20
>100
49.1 0.33
>100
>100
>100
15
16
20
MMC
5.98 0.04
0.083 0.002
18.5 0.08
5.7 0.75
The mono-substitution at position 8 with groups endowed with
lipophilic features such as aromatic- or heteroaromatic rings (17,
18, and 19), pentyloxy chain (14), iodine atom (a43)29, or the intro-
duction in the same position of an dimethylaminoethanolic chain
(13), methylpiperazinyl- and morpholinyl group (10 and 12,
respectively), hydroxyl group (a45)30 with hydrophilic features,
gave inactive compounds.
While the introduction of a nitro group in position 3 of 8-(4-
methylpiperazin-1-yl)derivatives permits identification of a hyp-
oxic agent (15), the introduction of a phenyl ring, (16), yielded a
time-dependent normoxic cytotoxic agent (28.55% at 24 h and
53.02% at 72 h, see Table 1 and Fig. 1) that will be the object of a
further study. The synthesis of a dinitro derivative 3,9-dinitro-8-
(4-methylpiperazin-1-yl)pyrazolo[5,1,-c][1,2,4] benzotriazine 20,
gave a selective cytotoxic normoxic agent.
*
250
200
150
100
50
*
*
normoxia
hypoxia
*
*
*
Derivatives containing pyrazolo[5,1-c]pyrido[4,3-e][1,2,4]tri-
azine 5-oxide system, were synthesized since these compounds
could be considered 7-aza-analogues of the pyrazolo[5,1-
c][1,2,4]benzotriazine system, but they were devoid of cytotoxicity
in normoxic and hypoxic conditions. The same results occurred for
the open analogues (B and C, Chart 2) 2, 3, 8, 9 synthesized with
0
20
DMSO
15
MMC
Figure 2. Effects of compounds 15, 20, and MMC on reactive oxygen species under
normoxic and hypoxic conditions as described in Section 6. Bars are means SEM of
three separate experiments. *P < 0.05 versus untreated control (one-way ANOVA
test with Bonferroni post test).
Table 5
Percentage of HCT-8 cells in each phase of the cell cycle and percentage of DNA fragmentation at concentration of 10
l
M in normoxic conditions as described in Section 6
% DNA fragmentation
Compound
% Cells
GoG1
T24DW
S
G2M
T24
T48DW
T24
T24DW
T48DW
T24
T24DW
T48DW
T24
T24DW
T48DW
15
20
40.3
40.5
50.6
40.3
48
39.8
35.1
38.6
nd
36.9
37.3
38.6
36.9
39
38
35.7
52.6
35.3
35.1
28.6
nd
22.8
22.2
10.8
22.8
13
24.9
35.1
32.8
nd
39
42
6.8
71.2
25
33
15
43.6
27
59
16
44
48.6
46.7
28.2
13.4
17.6
19.2
Control
Mitomycin

G. Ciciani et al. / Bioorg. Med. Chem. 16 (2008) 9409–9419
9415
Table 6
Percentage of HCT-8 cells in each phase of the cell cycle and percentage of DNA fragmentation at concentration of 10
l
M in hypoxic conditions as described in Section 6
% DNA fragmentation
Compound
% Cells
GoG1
T24DW
S
G2M
T24
T48DW
T24
T24DW
T48DW
T24
T24DW
T48DW
T24
T24DW
T48DW
15
20
34.6
43.8
42.8
36.3
36.8
51.2
46
47
40.7
40.5
38.4
49.1
43
40.4
45.9
37
24.7
15.6
18.8
14.6
20.2
10.6
20.4
14.1
12.6
23
15.5
nd
46.3
42.8
37
58.3
29.3
15.6
71.44
60
21
16
99
31.1
47.5
nd
38.1
33.6
42.7
Control
Mitomycin
43.2
nd
42.8
Table 7
ClogP value of tested compounds
N
R9
N
N
R8
N
N
OH
N+
R3
R4
HN
X
N
X
X
NH2
N+
O-
R4
N
NO2
O-
A
B
C
Series
Compound
R4 or R3
X
R8
R9
ClogPa
B
B
A
A
C
C
A
A
A
A
A
A
A
A
A
A
A
A
2
3
6
7
8
9
10
12
13
14
15
16
17
18
19
20b
a43c
a45d
CONH2
COOEt
COOH
COOEt
COOEt
COOEt
H
H
H
H
NO2
Ph
H
H
H
NO2
H
N
N
N
N
N
ꢀ1.30 1.43
0.81 1.43
ꢀ0.9 1.52
ꢀ0.06 1.52
0.49 1.31
3.30 1.10
0.50 1.36
0.06 1.35
0.69 1.40
2.96 1.32
0.23 1.53
2.26 1.53
2.68 1.32
2.49 1.33
2.48 1.34
1.63 1.62
1.95 0.91
0.18 1.31
H
H
H
H
C–NO2
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N-Methylpiperazine
Morfoline
H
H
H
H
H
H
H
H
O(CH2)2NMe2
O(CH2)4CH3
N-Methylpiperazine
N-Methylpiperazine
Ph
p-OMe-Ph
2-Tienyl
N-Methylpiperazine
I
OH
H
NO2
H
H
H
a
The ClogP values were determined using the ACD/logP software.
N5-Deoxide compound.
See Ref. 29.
b
c
d
See Ref. 30.
the aim of clarifying if the planarity of the system was necessary to
cytotoxicity.
ably the N-oxide function is necessary for preferential hypoxic
cytotoxicity of 15; while 20, a N-deoxide dinitroderivative, shows
preferential cytotoxicity in normoxic conditions. While overall
our experimental results reveal a great killing potential for both
compounds 15 and 20, as determined by certain DNA fragmenta-
tion and ROS production, the differential cytotoxicity in normoxic
and hypoxic conditions was not clarified. This suggests that other
differences in the metabolism or physiology of hypoxic and norm-
oxic cells are responsible for the differential sensitivities of hypoxic
and normoxic cells to compound 15 and 20.
In this preliminary in vitro study, Clog P values of tested com-
pounds were calculated (see Table 7) using the ACD/logP software48
because ClogP could be an important parameter predicting a
molecule’s ability for cellular uptake. From the analysis of ClogP
data it could be speculated that, for all studied compounds, this
parameter is not indicative of final activity. In fact, compound 10,
8-(4-methylpiperazin-1-yl)pyrazolo[5,1,-c][1,2,4]benzotriazine 5-
oxide (ClogP 0.50 1.36) is inactive and its 3-nitro derivative, 15,
(ClogP 0.23 1.53) is hypoxic-selective, despite a similar lipophilic-
ity parameter. On the other hand, compounds a43,30 inactive and 20,
the normoxic-selective, also have similar ClogP value (1.95 0.91 vs
1.63 1.62). Therefore, the presence of suitable substituents, such as
nitro-, N-methylpiperazinyl-, and N-oxide, opportunely combined
to exert cytotoxicity, seems to be necessary.
Additional experiments with other methodologies are required
to understand the mechanism involved in the cell death induced
by our compounds in normoxic and hypoxic conditions.
6. Experimental
6.1. Chemistry
5. Conclusion
Melting points were determined with a Gallenkamp apparatus
and were uncorrected. Silica gel plates (Merk F254) and silica gel
60 (Merk 70–230 mesh) were used for analytical and column
From these preliminary results, two potential selective hypoxic
and normoxic lead compounds, 15 and 20 can be identified. Prob-

9416
G. Ciciani et al. / Bioorg. Med. Chem. 16 (2008) 9409–9419
chromatography, respectively. The structures of all compounds
were supported by their IR spectra (KBr pellets in nujol mulls,
Perkin-Elmer 1420 spectrophotometer) and 1H NMR data
(measured with a Bruker 400 MHz). Chemical shifts were ex-
pressed in d ppm, using DMSO-d6 or CDCl3 as solvent. The coupling
constant values (JH6-H7, H7-H6; JH7-H9, H9-H7) were in agreement with
the assigned structure. The chemical and physical data of new
compounds are shown in Tables 1–3; Microanalyses were
performed with a Perkin-Elmer 260 analyzer for C, H, N, and the
results were within 0.4% of the theoretical value.
6.3.2. Ethyl 5-(4-hydroxypyridin-3-yl-ONN-azoxy)-1H-pyrazole
4-carboxylate (8)
From 3 at 30 °C for 2 h. Yellow crystals. TLC eluent: dichloro-
methane/methanol 10:0.5 v/v; IR
m
cmꢀ1 3340, 1687; 1H NMR
(DMSO-d6) d 13.60 (br s, 1H, NH, exch.); 11.58 (br s, 1H, OH, exch);
9.20 (s, 1H, H-20); 8.82 (d, 1H, H-60); 8.58 (d, 1H, H-50); 8.40 (s, 1H,
H-3); 4.24 (q, 2H, CH2); 1.38 (t, 3H, CH3). Anal. C, H, N.
6.3.3. Ethyl 5-(3-nitro-6-hydroxyphenyl-ONN-azoxy)-1H-
pyrazole 4-carboxylate (9)
From 430 at 30 °C for 1 h. Orange crystals. TLC eluent: dichloro-
6.2. General procedure for the synthesis of 1 and 3
methane/methanol 10:0.5 v/v; IR m
cmꢀ1 3440, 3100, 1710; 1H
NMR (CDCl3) d 12.60 (br s, 1H, OH, exch.); 9.20 (d, 1H, H-20);
8.40 (dd, 1H, H-40); 8.14 (s, 1H, H-3); 7.35 (d, 1H, H-50); 4.46 (q,
2H, CH2); 1.43 (t, 3H, CH3). Anal. C, H, N.
A suspension of 3-nitro-4-hydrazinopyridine31 (1.5 mmol) and
2-ethoxy-1,1-ethenedicarbonitrile (1.5 mmol) or 2-cyano-3-eth-
oxypropeneate (1.5 mmol) in ethanol (50 ml) were refluxed until
the starting material disappeared (4 h). The work up of final
solution gave the 1-(3-nitropyridin-4-yl)-5-aminopyrazole-4-car-
bonitrile or the ethyl-1-(3-nitropyridin-4-yl)-5-aminopyrazole-4-
carboxilate 1a and 1c, respectively.
6.3.4. 3-Carboxypyrazolo[5,1-c]pyrido[4,3-e][1,2,4]triazine 5-
oxide (6)
A suspension of 5 (2.0 mmol) in 10 ml of concentrated sulfuric
acid was rapidly cooled to 0 °C and treated with cooled sodium ni-
trite solution (3 g in 6 ml of water), with stirring. The final syrup
solution was poured into ice/water and the precipitate was fil-
tered; it was pure enough for the next step. Yellow crystals. TLC
6.2.1. 1-(3-Nitropyridin-4-yl)-5-aminopyrazole-4-carbonitrile
(1)
From 3-nitro-4-hydrazinopyridine31 and 2-ethoxy-1,1-ethen-
edicarbonitrile. Yellow crystals. TLC eluent: toluene/ethyl acetate
eluent: toluene/ethyl acetate/acetic acid 8:2.1 v/v/v; IR m
cmꢀ1
2700–2600, 1700, 1550; 1H NMR (DMSO-d6) d 9.52 (s, 1H, H-6);
8:2 v/v; IR
m
cmꢀ1 3300, 3200, 2215; 1H NMR (DMSO-d6) d 9.27
9.00 (d, 1H, H-8); 8.38 (s, 1H, H-2); 8.20 (d, 1H, H-9). Anal. C, H, N.
(s, 1H, H-20); 8.95 (d, 1H, H-60); 7.90 (s, 1H, H-3); 7.86 (d, 1H, H-
50); 7.24 (br s, 2H, NH2, exch.). Anal. C, H, N.
6.3.5. 3-Ethoxycarbonylpyrazolo[5,1-c]pyrido[4,3-
e][1,2,4]triazine 5-oxide (7)
6.2.2. Ethyl 1-(3-nitropyridin-4-yl)-5-aminopyrazol-4-
carboxylate (3)
A suspension of 0.43 mmol of acid 6 in 20 ml of anhydrous eth-
anol, containing 1 ml of concentrated sulfuric acid, was refluxed for
2 h until the starting material disappeared in TLC. After cooling the
solution was evaporated and the residue was purified by recrystal-
lization. Yellow crystals. TLC eluent: toluene/ethyl acetate/acetic
From 3-nitro-4-hydrazinopyridine31 and 2-cyano-3-ethoxypro-
peneate. Yellow crystals. TLC eluent: dichloromethane/methanol
10:0.5 v/v; IR
m
cmꢀ1 3300, 3200, 1687; 1H NMR (CDCl3) d 9.20
(s, 1H, H-20); 8.88 (d, 1H, H-60); 7.82 (s, 1H, H-3); 7.64 (d, 1H, H-
50); 5.40 (br s, 2H, NH2, exch.); 4.24 (q, 2H, CH2); 1.38 (t, 3H,
CH3). Anal. C, H, N.
acid 8:2:1 v/v/v; IR m
cmꢀ1 1650, 1550; 1H NMR (CDCl3) d 9.80
(s, 1H, H-6); 9.10 (d, 1H, H-8); 8.60 (s, 1H, H-2); 8.25 (d, 1H, H-
9); 4.50 (q, 2H, CH2); 1.45 (t, 3H, CH3). Anal. C, H, N.
6.2.3. 1-(3-Nitropyridin-4-yl)-5-aminopyrazole-4-carboxiamide
(2)
6.4. General procedure for the synthesis of 10–13
A solution of compound 1 (1.0 mmol) in 5 ml of concentrated
sulfuric acid was heated at 60 °C with stirring for 2 h. After cooling
the solution was treated with ice/water and neutralized with con-
centrated ammonia. The solid was filtered and recrystallized by
suitable solvent. Yellow crystals. TLC eluent: toluene/ethyl acetate/
A solution of 8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide30 (0.5 mmol) in 15 ml of ethoxyethanol was reacted with
an excess of N-methylpiperazine, morpholine or N,N-dimethyleth-
anolamine and maintained between 60 and 100 °C for 2–12 h. The
reaction was monitored by TLC and when the starting material dis-
appeared, the final solution was worked-up.
acetic acid 8:2:1 v/v/v; IR
m
cmꢀ1 3350, 3200, 1659; 1H NMR
(DMSO-d6) d 9.20 (s, 1H, H-20); 8.94 (d, 1H, H-60); 7.95 (s, 1H, H-
3); 7.84 (d, 1H, H-50); 7.48 (br s, 1H, NH, exch.); 6.98 (br s, 1H,
NH, exch.); 6.75 (br s, 2H, NH2, exch.). Anal. C, H, N.
The crude final product was obtained and purified by suitable
solvent by recrystallization.
6.4.1. 8-(4-Methylpiperazin-1-yl)pyrazolo[5,1-
6.3. General procedure for the synthesis of 5, 8, and 9
c][1,2,4]benzotriazine 5-oxide (10)
From starting material30 and N-methylpiperazine, 2.5 ml, at
80 °C for 2 h. Yellow crystals. TLC eluent: chloroform/methanol
9:2 v/v; 1H NMR (CDCl3) d 8.38 (d, 1H, H-6); 8.08 (d, 1H, H-2);
7.50 (d, 1H, H-9); 7.08 (dd, 1H, H-7); 6.68 (d, 1H, H-3); 3.70 (m,
4H, –N(CH2)2–); 2.75 (m, 4H, Me-N(CH2)2–); 2.50 (s, 3H, N-CH3).
Anal. C, H, N.
A suspension of 1.0 mmol of suitable 5-aminopyrazole 2, 3, and
430 in 10% solution of sodium hydroxide (10 ml) was stirred. In
case of compound 5 the precipitate was filtered, instead the final
solution was made slightly acid in case of compound 8 and acidi-
fied with conc hydrochloric acid in case of compound 9. The raw
products were recrystallized by suitable solvent.
6.4.2. 8-(4-Methylpiperazin-1-yl)pyrazolo[5,1-
6.3.1. 3-Carbamoylpyrazolo[5,1-c]pyrido[4,3-e][1,2,4]triazine 5-
oxide (5)
c][1,2,4]benzotriazine (11)
From starting material30 and N-methylpiperazine, 8 ml, at
100 °C for 2 hours, under nitrogen flow. Yellow crystals. TLC elu-
ent: chloroform/methanol 9:2 v/v; 1H NMR (CDCl3) d 8.38 (d, 1H,
H-6); 8.18 (d, 1H, H-2); 7.56 (d, 1H, H-9); 7.24 (m, 2H, H-7 and
H-3); 3.70 (m, 4H, –N(CH2)2–); 2.75 (m, 4H, Me-N(CH2)2–); 2.50
(s, 3H, N-CH3). Anal. C, H, N.
From 2 at room temperature for 2 h. Orange crystals. TLC elu-
ent: toluene/ethyl acetate/acetic acid 8:2.1 v/v/v; IR
m
cmꢀ1 3440,
3100, 1550; 1H NMR (DMSO-d6) d 9.58 (s, 1H, H-6); 9.15 (d, 1H,
H-8); 8.65 (s, 1H, H-2); 8.28 (d, 1H, H-9); 7.65 (br s, 1H, NH, exch.);
7.25 (br s, 1H, NH). Anal. C, H, N.

G. Ciciani et al. / Bioorg. Med. Chem. 16 (2008) 9409–9419
9417
6.4.3. 8-Morpholinpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide
(12)
From starting material30 and morpholine, 3 ml, at 60 °C for 12 h.
Yellow crystals. TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1
v/v/v; 1H NMR (CDCl3) d 8.41 (d, 1H, H-6); 8.08 (d, 1H, H-2); 7.52
(d, 1H, H-9); 7.09 (dd, 1H, H-7); 6.68 (d, 1H, H-3); 3.92 (m, 4H,
O(CH2)2–); 3.58 (m, 4H, N(CH2)2–). Anal. C, H, N.
6.7. General procedure for the synthesis of 15 and 16
solution of 8-chloro-3-nitropyrazolo[5,1-c][1,2,4]benzotri-
A
azine 5-oxide28 and 8-chloro-3-phenylpyrazolo[5,1-c][1,2,4]ben-
zotriazine 5-oxide35 (0.5 mmol) in toluene (10 ml), potassium
carbonate (60 mg) and N-methylpiperazine (0.1 ml) was kept at re-
flux temperature for 20 h. When the reaction was concluded the
suspension was treated with water and the organic layer was sep-
arated, dried and evaporated. The final product was purified by
suitable solvent.
6.4.4. 8-(N,N-Dimethylaminoethyloxy)pyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (13)
From starting material30 and N,N-dimethylaminoethanol, 3 ml,
10% sodium hydroxide solution 1.0 ml at 50 °C for 12 h. Yellow
crystals. TLC eluent: chloroform/methanol 10:1 v/v; 1H NMR
(CDCl3) d 8.47 (d, 1H, H-6); 8.09 (d, 1H, H-2); 7.74 (d, 1H, H-9);
7.20 (dd, 1H, H-7); 6.72 (d, 1H, H-3); 4.35 (t, 2H, OCH2); 2.90 (t,
2H, NCH2); 2.45 (s, 3H, CH3). Anal. C, H, N.
6.7.1. 8-(4-Methylpiperazin-1-yl)-3-nitropyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (15)
From 8-chloro-3-nitropyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide28. Red crystals. TLC eluent: toluene/ethyl acetate/methanol
8:3:2 v/v/v; 1H NMR (CDCl3) d 8.64 (s, 1H, H-2); 8.37 (d, 1H,
H-6); 7.44 (d, 1H, H-9); 7.22 (dd, 1H, H-7); 3.75 (m, 4H,
–N(CH2)2–); 2.68 (m, 4H, Me-N(CH2)2–); 2.45 (s, 3H, N-CH3). Anal.
C, H, N.
6.5. 8-n-Pentyloxypyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide
(14)
A mixture of starting compound30 (100 mg, 0.288 mmol), 10 ml
of dichloromethane, 5 ml of 40% sodium hydroxide solution,
0.1 mole of tetra-butylammonium bromide and n-propanol in large
excess (5 ml) was vigorously stirred at 30–50 °C for 12 h. The or-
ganic layer was then separated and the aqueous layer extracted
twice with 10 ml of dichloromethane. The combined organic ex-
tracts were evaporated and the residue was recovered with isopro-
pyl ether and recrystallized by the suitable solvent. Yellow crystals.
TLC eluent: toluene/ethyl acetate 8:2 v/v; 1H NMR (CDCl3) d 8.47
(d, 1H, H-6); 8.10 (s, 1H, H-2); 7.71 (d, 1H, H-9); 7.15 (dd, 1H, H-
7); 6.73 (d, 1H, H-3); 4.24 (t, 2H, CH2O); 1.92 (m, 2H, CH2); 1.49
(m, 4H CH2CH2); 0.98 (t, 3H, CH3). Anal. C, H, N.
6.7.2. 8-(4-Methylpiperazin-1-yl)-3-phenylpyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (16)
From 8-chloro-3-phenylpyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide.35 Red-orange crystals. TLC eluent: toluene/ethyl acetate/
methanol 8:3:2 v/v/v; 1H-NMR (CDCl3) d 8.37 (m, 3H, H-6, H-2,
and H-9); 8.01 (d, 2H, Ph); 7.48 (m, 2H, Ph); 7.32 (t, 1H, Ph); 7.07
(dd, 1H, H-7); 3.68 (m, 4H, –N(CH2)2–); 2.68 (m, 4H, Me-
N(CH2)2–); 2.45 (s, 3H, N-CH3). Anal. C, H, N.
6.8. 8-(4-Methylpiperazin-1-yl)-3,9-dinitropyrazolo[5,1-
c][1,2,4]benzotriazine (20)
To a suspension of 7 (0.3 mmol) in cooled conc. sulfuric acid
(6 ml), was added drop wise fuming nitric acid (0.5 ml): the red
solution was stirred at 0 °C for 2 h. Addition of ice/water and 10%
sodium hydroxide solution yields a precipitate which was filtered
and purified by recrystallization. Red crystals. TLC eluent: chloro-
form/methanol 9:2 v/v; 1H NMR (CDCl3) d 8.80 (s, 1H, H-2); 8.72
(d, 1H, H-6); 7.60 (d, 1H, H-7); 3.60 (m, 4H, –N(CH2)2–); 2.68 (m,
4H, Me-N(CH2)2–); 2.43 (s, 3H, N-CH3). Anal. C, H, N.
6.6. General procedure for the synthesis of 17–19
Tetrakis-(triphenylphpsphine)palladium(0) (30 mg, 0.026 mmol)
and 8-iodopyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (0.32 mmol)29
were combined in anhydrous tetrahydrofurane (4.0 ml). The
suitable boronic acid (0.62 mmol) in absolute ethanol (2.5 ml)
and aqueous sodium carbonate (2 M, 4 ml) were added and the
reaction was heated to reflux for 12 h and monitored by TLC. The
suspension was treated with water, the crude product was filtered,
and recrystallized by the suitable solvent.
6.9. Pharmacological methods
Puck’s EDTA solution without phenol red: 140 mM NaCl, 5 mM
KCl, 5.5 mM glucose, 4 mM NaHCO3, 0.8 mM EDTA, and 9 mM
Hepes buffer (pH 7.3).49
6.6.1. 8-Phenylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (17)
Starting material29 and phenylboronic acid. Green crystals. TLC
eluent: isopropyl ether/ciclohexane 8:3 v/v; 1H NMR (CDCl3) d 8.62
(m, 2H, H-6 and H-9); 8.12 (s, 1H, H-2); 7.88 (dd, 1H, H-7); 7.80 (m,
2H, Ph); 7.55 (m, 3H, Ph); 6.80 (d, 1H, H-3). Anal. C, H, N.
Chemicals: 20,70-diclorofluoresceinadiacetato (DCFDA) was from
the Eastman Kodak Company (Rochester, NY). Stock solutions of
DCFDA (50 lM) were prepared in PBS (phosphate saline buffer)
and stored at 4 °C.
Compounds were solubilized in dimethyl sulfoxide (DMSO) at
100-times the desired maximum test concentration (maximum fi-
nal DMSO concentration of 0.1%; this concentration was not toxic)
and stored frozen. Compounds were then diluted with complete
media to obtain the 10ꢁ desired final maximum test concentra-
tion. Additional serial dilutions were made to provide a total of five
drug concentrations plus control.
6.6.2. 8-(4-methoxyphenyl)pyrazolo[5,1-c][1,2,4]benzotriazine
5-oxide (18)
Starting material29 and 4-methoxyphenylboronic acid. Yellow
crystals. TLC eluent: isopropyl ether/ciclohexane 8:3 v/v; 1H NMR
(CDCl3) d 8.58 (d, 1H, H-6); 8.55 (d, 1H, H-9); 8.13 (s, 1H, H-2);
7.84 (dd, 1H, H-7); 7.75 (d, 2H, Ph); 7.08 (d, 2H, Ph); 6.78 (d, 1H,
H-3); 3.92 (s, 3H, OCH3). Anal. C, H, N.
6.9.1. Cell cultures
6.6.3. 8-(Thien-2-yl)pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide
(19)
The cell line used in this study was a human colorectal carci-
noma, HCT-8 obtained from the American Type Culture Collection
(Rockville, MD). The HCT-8 cell line was maintained in RPMI 1640
(Euroclone Ltd, UK) supplemented with 10% fetal calf serum (FCS)
(Euroclone Ltd, UK) and antibiotics (penicillin, 100 U/ml; strepto-
Starting material29 and 2-thienylboronic acid. Dark yellow crys-
tals. TLC eluent: isopropyl ether/ciclohexane 8:3 v/v; 1H NMR
(CDCl3) d 8.56 (m, 2 H, H-6 and H-9); 8.14 (s, 1H, H-2); 7.86 (dd,
1H, H-7); 7.68 (m, 1H, H-50 8-tienyl); 7.54 (m, 1H, H-30 8-tienyl);
7.14 (m, 1H, H-40 8-tienyl); 6.78 (d, 1H, H-3). Anal. C, H, N.
mycin, 100
lg/ml). Cells were incubated at 37 °C, 5% CO2/95% air
and subcultured twice weekly. We optimized the growth of tumor

9418
G. Ciciani et al. / Bioorg. Med. Chem. 16 (2008) 9409–9419
cells under hypoxic conditions by increasing the glucose concen-
tration to 4.5 g/L to the culture medium.
At experimental times (T24, T24DW, and T48hDW) floating cells
were collected, adherent cells were harvested with Puck’s EDTA
and pooled with the floating ones and fixed in 70% ice-cold ethanol
and stored at 4 °C. Cells were then rehydrated in PBS and stained in
6.9.2. Cytotoxicity assay
Before studying the cytotoxic effects of our compounds in hy-
poxia we investigated the effect of hypoxia on HCT-8 cells prolifer-
propidium iodide (PI, 50 lg/ml) solution containing RNase A (5 U/
ml) for 30 min.51 PI-stained cells were analyzed for DNA content
using a flow cytometer (Coulter XL, Coulter Cytometry, Hialeah,
FL, USA). The red fluorescence emitted by PI was collected by a
620 nm long-pass filter. The percentage of cells in cycle phases
and the percentage of those with fragmentated DNA, hypodiploid
peak(sub-G1) was determined using WinMDI2.8 Windows Multi-
ple Document Interface Flow Cytometry Application (Cylchred
Windows 95 Version 1.02).
ation (data not shown) using sulforhodamine
B (SRB) test
according to the procedure described by Skehan et al.36 Cell prolif-
eration was slightly less than normal for the first 24 h of hypoxic
treatment, then we have chosen 24 h drug exposure with drug
washout and reoxygenation at 24–48 h.
To identify a normoxia/hypoxia-selective cytotoxin, cells were
seeded at 5000 cells/well and cultured at 37 °C in normoxia in a
humidified incubator containing 5% CO2 for 24 h to assure com-
plete adherence of the cells to the plates before addition of exper-
imental drugs in complete medium. To evaluate the cytotoxic
effects in normoxia at established times (72 or 24 h exposure),
the compounds were diluted to desired concentrations in complete
medium and in replicates of eight wells/condition were added. To
evaluate the cytotoxic effects in hypoxia, cells and drug solutions
were allowed to equilibrate for 2 h into a Ruskinn Concept 400
annormoxic incubator flushed with the 0.1% O2 mixture (95% N2
and 5% CO2). Then drug solutions were added to wells for 24 h
and after drug washout cells were reoxygenated for 48 h. In every
assay 0.1% DMSO (negative control) and MMC (positive control)
were tested. At the end of incubation the cells are fixed in situ
6.9.5. Statistical analysis
The results were represented as mean SEM and statistical
analysis was performed using one-way Anova test and Bonferroni’s
multiple comparison test (GraphPad Prism software, Inc., CA, USA).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.09.055.
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