Exploring the anticancer potential of pyrazolo[1,2-a]benzo[1,2,3,4] tetrazin-3-one derivatives: The effect on apoptosis induction, cell cycle and proliferation

Article abstract of DOI:10.1016/j.ejmech.2013.03.046

In order to investigate their anticancer potential, four new pyrazolo[1,2-a]benzo[1,2,3,4]tetrazinone derivatives, designed through the chemometric protocol VLAK, and three of the most active compounds of the previous series have been evaluated on some cellular events including proliferation, apoptosis induction, and cell cycle. The NCI one dose (10 μM) screening revealed that the 8,9-di-methyl derivative showed activity against Leukemia (CCRF-CEM) and Colon cancer cell line (COLO 205), reaching 81% and 45% of growth inhibition (GI), respectively. Replacement of the two methyl groups with two chlorine atoms maintained the activity toward Leukemia cell (CCRF-CEM, GI 77%) and selectively enhanced the activity against COLO 205 attaining a LD₅₀ in the μM range and against SW-620 a GI of 77%. Interestingly, an appreciable growth inhibition of 47% against therapeutically "refractory" Non-Small Cell Lung Cancer (NCI-H522) was observed. Moreover, the apoptosis induction, based on mitochondrial membrane depolarization, was found in the range EC₅₀ 3-5 μM on HeLa cell, evidencing a well defined relationship with the related in vitro cell growth inhibitory assays (MTT) performed against other selected tumor cell lines not included in the NCI tumor panel (HeLa, cervix; H292, lung; LAN-5, CNS; CaCo-2, colon; 16HBE, normal human cell lung) and against MCF-7 tumor cell line (breast). Only for the most active compounds, further cell cycle tests on HeLa displayed a cell arrest on S phase. Thus, a promising new class of anticancer candidates, acting as valuable apoptotic inductors, is proposed.

Full text of DOI:10.1016/j.ejmech.2013.03.046

European Journal of Medicinal Chemistry 64 (2013) 345e356
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Exploring the anticancer potential of pyrazolo[1,2-a]benzo[1,2,3,4]
tetrazin-3-one derivatives: The effect on apoptosis induction, cell
cycle and proliferation
,
b
b
c,1
Francesco Mingoia a , Caterina Di Sano , Francesco Di Blasi , Marco Fazzari
,
*
Annamaria Martorana c, Anna Maria Almerico c, Antonino Lauria c
a Istituto per lo Studio dei Materiali Nanostrutturati (ISMN) e CNR, Via Ugo La Malfa 153, 90146 Palermo, Italy
b Istituto di Biomedicina ed Immunologia Molecolare (IBIM) e CNR, Via U. La Malfa 153, 90146 Palermo, Italy
c Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), Sezione di Chimica Farmaceutica e Biologica e Università di
Palermo, Via Archirafi 32, I-90123 Palermo, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to investigate their anticancer potential, four new pyrazolo[1,2-a]benzo[1,2,3,4]tetrazinone
derivatives, designed through the chemometric protocol VLAK, and three of the most active compounds
of the previous series have been evaluated on some cellular events including proliferation, apoptosis
Received 2 October 2012
Received in revised form
21 March 2013
Accepted 24 March 2013
Available online 6 April 2013
induction, and cell cycle. The NCI one dose (10 mM) screening revealed that the 8,9-di-methyl derivative
showed activity against Leukemia (CCRF-CEM) and Colon cancer cell line (COLO 205), reaching 81% and
45% of growth inhibition (GI), respectively. Replacement of the two methyl groups with two chlorine
atoms maintained the activity toward Leukemia cell (CCRF-CEM, GI 77%) and selectively enhanced the
activity against COLO 205 attaining a LD50 in the mM range and against SW-620 a GI of 77%. Interestingly,
an appreciable growth inhibition of 47% against therapeutically “refractory” Non-Small Cell Lung Cancer
Keywords:
Pyrazolo[1,2-a]benzo[1,2,3,4]tetrazinone
VLAK protocol
Anticancer agents
Apoptosis inducers
Cell cycle
(NCI-H522) was observed. Moreover, the apoptosis induction, based on mitochondrial membrane de-
polarization, was found in the range EC50 3e5 mM on HeLa cell, evidencing a well defined relationship
with the related in vitro cell growth inhibitory assays (MTT) performed against other selected tumor cell
lines not included in the NCI tumor panel (HeLa, cervix; H292, lung; LAN-5, CNS; CaCo-2, colon; 16HBE,
normal human cell lung) and against MCF-7 tumor cell line (breast). Only for the most active compounds,
further cell cycle tests on HeLa displayed a cell arrest on S phase. Thus, a promising new class of anti-
cancer candidates, acting as valuable apoptotic inductors, is proposed.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
against cancer and drug resistance. Thus, deeper investigations on
molecular basis of apoptosis modulation open promising ways for a
Current advances in cancer research consider the apoptosis,
other than occurring in various physiological events, as crucially
involved in the regulation of tumor growth as well as in the
treatment response. Most of the anticancer strategies used in
more rational approach to develop new therapeutic strategies [2].
For these reasons, growing interest is currently focused on the
attempt to improve cancer therapy by mean of the modulation of
apoptosis processes.
clinical oncology such as chemotherapy,
g
-irradiation, including
Equally of interest, because strictly connected, is the exploration
of the effect on the cell cycle of active molecules. Multiple genetic
changes occur during the evolution of normal cells into cancer
ones. Cell cycle inhibitors or modulators, able to control tumor
growth, continue to be regarded as highly promising new thera-
peutic agents against human cancer [3]. As known, the loss of fi-
delity in the processes that replicate and repair the genome are
typical molecular mechanisms of cellular transformation and may
help to identify potential targets for cancer therapies [4]. Over the
last years, efforts have been made to identify the targets of cell cycle
phase that regulate several processes, but the whole view is not yet
suicide gene therapy or immunotherapy, have been related to the
activation of apoptosis signal transduction pathways in cancer cells.
The lost of such a cell event increases the cancer aggressiveness [1].
The understanding of selective induction of the apoptosis will
provide a promising basis for novel opportunities in the fight
* Corresponding author. Tel.: þ39 0916809368; fax: þ39 0916809399.
E-mail address: mingoia@pa.ismn.cnr.it (F. Mingoia).
1
Current address: Fondazione Ri.MED, Via Bandiera 11, 90133 Palermo, Italy.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.046

346
F. Mingoia et al. / European Journal of Medicinal Chemistry 64 (2013) 345e356
complete on understanding the processes involved in proliferation,
differentiation, transformation, and programmed cell death [5].
Taking into account the strictly interconnection between cell
death, cell survival, and cell progression pathways, the under-
standing of the interaction of new active compounds with key
components of these cell events may be helpful on advancement
for new therapeutic perspectives. Several components common to
apoptosis and cell cycle have been identified and constitute basic
elements to correlate different molecular linkages between cell
death, survival and cycle [6,7]. Therefore, the design of new com-
pounds able to modulate the apoptotic process, both as activators
or inhibitors, represents an important strategic therapeutic
approach in cancer disease.
In this context, our attention has been focused on a poor bio-
logically investigated nitrogen tricycle ring system, the pyrazolo
[1,2-a]benzo[1,2,3,4]tetrazinone core (PBT).
Our interest on PBT system has begun with the peculiar chem-
ical behavior observed previously in the course of reactivity studies
[8]. Under a suitable nucleophilic microenvironment, a ring open-
ing can occur generating reactive entity such as X, Y, and Z species,
as shown in Scheme 1.
Therefore, to take a deeper insight, we extended the studies
with the design and synthesis of new PBT derivatives by intro-
ducing on the phenyl moiety (ring c) some substituent (R1eR4)
(Fig. 1), exploiting the use of a valuable library of available com-
mercial nitro-anilines which are key reactants in our synthetic
approach.
The design started from an in silico analysis that took advantage
from the chemometric protocol VLAK (Virtual Lock-and-Key),
recently developed by us [15]. In the last decades, the in silico
methodologies have seen a good improvement from the point of
view of robustness and prediction capability. The molecular
modeling and the chemometric approach represented two in silico
tools that address the issue of the lead optimization from two
different points of view, ligand based drug design and structure
based drug design, respectively. Both methodologies led to good
results in our studies. For example the molecular docking was
applied in the DNA ligands design of indolopyrimidine derivatives
endowed with good antitumor activity [16]; the induced fit was
applied in the analysis of the Hsp90 and IKK binding sites [17,18].
On the other hand, the chemometric approach and the multivariate
analysis were applied on the ACAM database, in the search of the
structural features that are determinant to increase the anti-
proliferative activity [19], or in the tuning the biological activity for
Hsp90 inhibitors [20].
Moreover, by analogy with azolotetrazinones, such as mitozo-
lomide and temozolomide, effective antineoplastic agents currently
used against malignant melanoma, mycosis fungoides and brain
tumors [9e11], the PBT skeleton possesses isosteric structural fea-
tures that could induce similar behavior.
2. Results and discussions
In particular, under soft acid conditions, the first ring opening
can occur on the b ring, generating the intermediate electrophilic
diazonium species X. Depending on the chemical environment, if
loss of molecular nitrogen takes place, the aromatic carbocation
species Y could be formed and in turn could be attacked by nu-
cleophiles. Alternatively, internal rearrangement by hydrogen
transposition can afford other methylene reactive species such as
intermediate Z. All these reactive intermediates have been trapped
with charged nucleophile (Nuꢀ) or neutral nucleophile (NuH)
affording the related adducts [8].
Additionally, due to the high metabolism rate, tumor cells
evolved the ability to work in a more acid environment by
approximately 0.3e0.5 pH unit than normal cell [12,13]. Thus,
compounds possessing a peculiar chemical behavior in acid media
could display increased reactivity becoming selective toward can-
cer cell. This premise has been an additional concept, which stim-
ulated the interest toward the PBT derivatives for potential use as
cancer selective therapeutic strategy. The starting idea was to verify
“in cell” the peculiar chemical reactivity of PBT system (Scheme 1)
that in presence of different nucleophilic species (electron rich,
neutral or negatively charged molecules) such as proteins or eSH, e
NH2, eCOO-, eOH containing biomolecules, can originate adducts.
Previous structureeactivity relationships (SAR) pointed out that
the presence of a chlorine atom is well tolerated in both position 8
and 9 of the PBT ring, without remarkable difference [14]. Whereas
in the case of the methyl group, switching from the 8 to 9 position
gives rise to the most active compound, either for the number of
cell lines inhibited and for selectivity against a specific subpanel.
2.1. In silico analysis
The design of the new PBT derivatives, proposed herein, started
from the in silico tool VLAK. In recent applications [21], this protocol
was able to improve the anti-proliferative activity of annelated
pyrimidine derivatives [22,23]. Thus, with the aim to analyze the
PBT compounds already possessing good anti-proliferative activity
and to design new ones, a database of PBTs with different sub-
stituents in the C ring (Fig. 1, R1eR4), including the PBTs previously
synthesized and tested [14], was submitted to VLAK protocol.
The PBT database (an in house library of new compounds to be
processed) was built by considering the reaction pathway leading
to the PBT nucleus, which involves, as mentioned before, the use of
substituted orto-nitro aniline commercially available (see
Experimental section).
The first step of the VLAK protocol is the conversion of the
biological target in a “lock model” in which the keys (the structures)
could be “fitted” [15].
The NCI AntiCancer Agent Mechanism (ACAM) Database was
chosen as source of biological data for the construction of the VLAK
“lock models”. It consists of a repository of structures with anti-
cancer activity and a reasonably well-known mechanism of action.
Drug screening data are available for each structure as measure-
ment of their growth inhibition ability over a panel of about 60
human tumor cell lines, and all tested molecules are explicitly
designed as a training set for neural network and multivariate
analysis [24,25].
OH
OH
OH
O
Nu
a
N
H+
N
+
N
N
N
b
N+
N
N
N
N
N
H
N
Nu
+
-N2
c
Nu
PBT
X
Z
Y
Scheme 1. Ring opening under soft acid conditions.

F. Mingoia et al. / European Journal of Medicinal Chemistry 64 (2013) 345e356
347
Table 1
O
VLAK results on PBT derivatives.
a
N
b
N
N
N
Compound
MA (A%)
R1
R2
A
B
C
D
E
F
R
c
6a
6b
6c
6d
6e
6f
6g
6h
6i
52
46
55
86
79
79
74
83
78
55
81
74
62
90
77
51
52
38
51
35
46
59
43
63
75
57
73
58
55
41
74
37
56
59
41
69
86
79
84
40
53
38
51
38
47
65
37
60
76
56
65
69
79
78
76
75
72
74
72
70
40
33
39
63
63
69
56
74
62
44
69
48
R1 ¼ R4 ¼ H; R2 ¼ R3 ¼ Me
R1 ¼ R4 ¼ H; R2 ¼ R3 ¼ Cl
R1 ¼ R2 ¼ R4 ¼ H; R3 ¼ CN
R1 ¼ R2 ¼ R4 ¼ H; R3 ¼ CF3
R1 ¼ R2 ¼ R4 ¼ H; R3 ¼ Me
R1 ¼ R2 ¼ R4 ¼ H; R3 ¼ Cl
R1 ¼ R3 ¼ R4 ¼ H; R2 ¼ Cl
R4
R3
Fig. 1. Pyrazolo[1,2-a]benzo[1,2,3,4]tetrazinone derivatives (PBTs).
R1 ¼ R2 ¼ R4 ¼ H; R3 ¼ OMe
R1 ¼ R2 ¼ R4 ¼ H; R3 ¼ OH
R1 ¼ R2 ¼ R4 ¼ H; R3 ¼ F
R1 ¼ R3 ¼ R4 ¼ H; R2 ¼ Me
R1 ¼ R2 ¼ R3 ¼ R4 ¼ H
In particular, this database is constituted by 121 antitumor drugs
classified for their mechanism of action (Supplementary material),
which can be considered like the biological target in the protocol.
The NCI ACAM Database was chosen because it was demonstrated
as the molecular descriptors, by means of multivariate analysis, are
able to discriminate the mechanisms of action of the drugs [19].
The VLAK protocol assigns to PBT database compounds the
percentage of affinity (A%) with respect to six mechanisms of ac-
tion, which include alkylating agents, antimitotic agents, topo-
6j
6k
6l
MA, mechanism of action: A%, percentage of affinity; A, Alkylating Agents; B,
Antimitotic Agents; C, Topoisomerase I Inhibitors; D, Topoisomerase II Inhibitors; E,
RNA/DNA Antimetabolites; F, DNA Antimetabolites. Compounds 6eel see Ref. [14].
comparison purposes, one included in the NCI panel (MCF-7,
breast). Also for comparison reasons, three of the most active de-
rivatives previously tested at the NCI Developmental Therapeutic
Program (6ee6g) and four new PBT derivatives (6ae6d) are herein
evaluated against these cell lines. The antiproliferative activity,
isomerase
I inhibitors, topoisomerase II inhibitors, RNA/DNA
antimetabolites, and DNA antimetabolites. Table 1 lists the results
obtained for the PBTs previously reported (6ee6l) along with the
new designed ones (6ae6d) with the higher percentage of affinity
(A). The calculation procedure is detailed in the experimental sec-
tion whereas the output results for the selected PBTs, related the six
lock models representing each class of drugs (MAs), are reported in
Supplementary material.
By analyzing the results, it is possible to evidence as the PBTs of
type 6ee6l are well classified as Alkylating Agents (A) with the
exception of the derivative 6j. Among the new designed PBTs, the
top scored was the derivative 6b, classified as antimitotic agent. The
derivatives 6a, 6c were classified as topoisomerase II inhibitors and
6d as alkylating agent.
expressed as IC50
Fig. 2).
(mM), is listed in Table 2 (and graphically shown in
An evaluation of these data point out that derivative 6d con-
taining the eCF3 in position 8 was active only toward 16HBE and
LAN-5 cancer cell, in which a moderate and significant activity was
observed at 30.9 and 15.1 mM, respectively. Interestingly, beside the
strong electron withdrawing effect, the eCF3 seems to amplify the
valuable CNS tropism. Generally, according to literature data, the
incorporation of fluorine into a drug increases the lipophilicity
enhancing absorption into biological membranes [27].
Replacement of the latter group with the eCN, as in compound
(6c), a strong withdrawing group, also possessing coordinating
properties with metal ions, extended the activity to all the six
2.2. Chemistry
Thus, the new hit PBT compounds and three of the old series
were synthesized as reported in Scheme 2. Starting from
commercially available orto-nitro-anilines 1ae1g, the azo-
furanones 3ae3g were prepared in 65e85% yields, adapting the
procedures known in the literature [14] to the new reactants.
Further rearrangement at room temperature, under acid condi-
tions, afforded in 75e85% yields the corresponding nitrophenyl-
pyrazol-3-one derivatives 4ae4g. Catalytic reduction over Pd on
charcoal or chemical reduction with AcOH and Iron powder, fol-
lowed by diazotization in concentrated sulfuric acid allowed the
isolation of the PBT derivatives as colored red solid (yields 55e75%)
after treatment with bicarbonate solution.
tumor cell lines, but it remains moderate (IC50 range 27e47 mM),
probably due to the low solubility in the biological media. The
introduction of a methyl group in the same position originating
(6e), which resulted among the most active in the previous NCI
screenings [14], appears to achieve promising results only against
HeLa (IC50 ¼ 15.4
m
M) and H292 (IC50 ¼ 25.1
mM), ineffective
against LAN-5, whereas only moderately active toward the other
cell lines. The addition of a second methyl group in the position 9,
as in compound (6a) promoted a slight increase of the activity,
especially against HeLa cell line, while no substantial variation is
observed for the other ones. In parallel, in the case of the 8-chloro
derivative (6f), and with respect to our panel of tumor cell lines, it
was possible to evidence an increase of activity against the six lines
2.3. Biological assays
tested, reaching a mean value of 22 mM. By adding another chlorine
atom on position 9 of the PBT skeleton, compound (6b), the activity
In order to investigate their anticancer potential and to verify
the influence of molecular modifications on biological activity, as
selected by the VLAK protocol, derivatives 6ae6g were evaluated
for their in vitro antiproliferative and pro-apoptotic activity.
Moreover, in the attempt to correlate possible molecular relation-
ships between these cell events [6,7], three of the most active
compounds (6ae6b, 6d) were evaluated on cell cycle tests.
was enhanced as well as the selectivity toward MCF-7 and 16HBE
(IC50 13.5 and 13.9
mM, respectively), while against the other cell
lines the activity remained in the range 14.8e25.7
mM. In this latter
case the effect of doubling the substituent corresponds to an in-
crease of the activity, particularly toward MCF-7 and 16HBE,
moderately for LAN-5, while derivative 6g, the 9-chloro
substituted maintained a promising activity particularly toward
HeLa cells.
2.3.1. In vitro antiproliferative activity (MTT)
Thus, nature and position of the different substituents in the PBT
moiety affect the selectivity as well as the activity. The lipophilic
character of chlorine and fluorine substituent enhances activity
toward LAN-5 (CNS) with respect to the methyl group, conferring
affinity to HeLa and H292 tumor cells.
A preliminary bioassay was carried out by using MTT protocol
[26]. We selected several tumor cell lines, five of which not
included in the NCI panel (HeLa, cervix; H292, lung; LAN-5, CNS;
Caco-2, colon; 16HBE, lung healthy human cell), and for

348
F. Mingoia et al. / European Journal of Medicinal Chemistry 64 (2013) 345e356
O
R'
NH2
R
N2+X-
R
O
NO2
NO2
O
H
O
(i)
N
N
R'
R'
(ii)
NO2
R
2a-g
3a-g
(iii)
1a-g
a) R' = R = Me;
O
b) R' = R = Cl;
O
O
c) R' = H, R = CN;
d) R' = H, R = CF3;
e) R' = H, R = Me;
f) R' = H, R = Cl;
g) R' = Cl, R = H;
NH
N
NH
N
R
N
R
N
N
R
NO2
N
NH2
(v)
(iv)
R'
R'
R'
6a-g
5a-g
4a-g
(i) NaNO2/HCl, 0° C, (ii) HCl/ H2O rt (iii) HCl conc, H2O, (iv) H2, 60 psi, EtOH, Pd/C on
charcoal or Fe/AcOH, 60°C, 4h, (v) H2SO4/HCl, 0° C.
Scheme 2. Reaction pathway.
2.3.2. In vitro NCI one dose screening
Beside the screening tests reported above against our panel of
six cell lines, derivatives 6ae6d were submitted to Development
of interest. Based on the laboratory reactivity studies, if ring
opening of the benzotetrazine ring is the rate determining step, a
strong electron-withdrawing group should increase the biological
activity. In the case of compounds 6c and 6d, although strong
electron-withdrawing are present, the expected biological
response was not observed. Probably, these data are not conclu-
sive because of low solubility that could limit their efficacy.
Therapeutics Program (DTP) NCI protocol. A single dose (10 mM) of
the selected compounds was tested against a panel of approxi-
mately 60 tumor human cell lines grouped in nine disease sub-
panels, including leukemia, non small-cell lung, colon, central
nervous system, melanoma, ovarian, renal, prostate, and breast
tumor cell lines [28e30].
2.3.3. Apoptosis induction
Among the tested compounds, the 8,9-di-methyl derivative
showed a selective activity against the Leukemia cell line CCRF-
CEM and colon cancer cell line COLO 205, showing 81% and 45%
growth inhibition (GI), respectively. The replacement of the two
Apoptosis is endowed with a specific sequence of molecular
events that culminates in cell death. Induction of apoptosis on cells
can be promoted by different pathways. A number of methods are
used to investigate apoptotic events. The key elements of the
apoptotic pathway can involve typical membrane alterations, spe-
cific protease cascade, mitochondrial changes and DNA fragmen-
tation. Because of some of these cell changes are not specific to the
apoptotic process [31], in some cases different pathways could be
strictly connected [32].
A peculiar cell event observed in the course of apoptosis is the
mitochondrial membrane permeability alteration and apoptosis
specific protease activators are released from mitochondria. By
consequence the cytochrome C becomes redistributed to the
cytosol, followed by subsequent depolarization of the inner mito-
chondrial membrane. The release of cytochrome C in the cytosol
promotes caspase activation.
In our investigation, derivatives 6ae6g have been evaluated
on apoptotic induction against HeLa cell by flow cytometry
analysis. The results, reported in Fig. 3 show the percentage of
mitochondrial depolarization detected in the presence of PBT
compounds at different concentrations with respect to staur-
osporine treated cells (positive control) and untreated cells
(negative control).
methyl groups with two chlorine atoms led to
a valuable
enhancement of the activity and selectivity against Colon Cancer
(SW-620 and COLO 205) reaching the best result (G% ¼ ꢀ64,
meaning 64% of cell death), while toward Leukemia cell (CCRF-
CEM) growth inhibition was maintained about 80%. Interestingly,
although moderate, a significant growth inhibition (47%) against
therapeutically refractory Non-Small Cell Lung Cancer (NCI-H522)
was observed. This outcome, although could seem of modest entity,
constitutes an interesting starting point for further developments
for this refractory tumor disease (Table 3).
The most surprising results were the inactivity of the CN and
CF3 derivatives against all these cell lines. Only the di-methyl and
di-chloro substituted derivatives resulted selectively effective to-
ward some tumor cell lines, the two other derivatives were devoid
Table 2
a
Antiproliferative activity (MTT) IC50
H292 HeLa
23.4 13.2
(mM).
MCF-7
Caco-2
16-HBE
LAN-5
6a
6b
6c
6d
6e
6f
46.7
13.9
35.9
46.8
25.7
37.1
>100
50.2
24.0
38.9
13.5
46.7
30.9
36.3
17.7
27.5
95.5
25.7
28.2
15.1
93.3
31.6
48.9
As it can be observed, the eCN group on the PBT skeleton results
ineffective for the activity (line 6c) at least at concentration under
28.2
43.6
>100
25.1
23.4
25.1
14.8
27.0
>100
15.4
14.8
15.8
10
drawing effect and lipophilic character, the activity profile does not
appear dose dependent, even at low concentration (1 M) the curve
mM. Moving to the eCF3, possessing a strong electron with-
>100
42.6
20.5
29.5
m
6g
34.6
maintains constant, suggesting a saturation process (line 6d). The
replacement of the latter group in the same position of the ring
with a methyl one, led to a better defined sigmoid curve showing
a
IC50: Concentration (mM) required to inhibit tumor cell proliferation by 50%. %.
Antiproliferative activity was measured by MTT for cell viability according to the
method described by Skehan and colleagues [26]. All measurements were per-
formed in triplicate and each experiment was repeated at least three times. The IC50
value was calculated from the 50% formazan formation compared with a control
without addition of drugs. Data are expressed as the mean ꢁ SE from the dosee
response curves of at least three independent experiments.
the apoptotic induction in a range of 3e8
duction of a second methyl group on the skeleton increases once
more the activity, but reduced the range of activity to 3e4 M (line
6a). A similar trend, even more effective, is observed with the
mM (line 6e). The intro-
m

F. Mingoia et al. / European Journal of Medicinal Chemistry 64 (2013) 345e356
349
150
125
100
75
MTT LAN-5
150
125
100
75
MTT 16HBE
50
50
25
25
0
0
0,1
1
10
20
30
40
50 100 uM
0,1
1
10
20
30
40
50 100 uM
MTT Caco-2
150
150
125
100
75
MTT MCF-7
125
100
75
50
25
0
50
25
0
0,1
1
10
20
30
40
50 100 uM
0,1
1
10
20
30
40
50 100 uM
MTT H292
150
125
100
75
MTT HeLa
150
125
100
75
50
50
25
25
0
0
0,1
1
10
20
30
40
50 100 uM
0,1
1
10
20
30
40
50 100 uM
(6a)
(6b)
(6c)
(6d)
(6e)
(6f)
(6g)
Fig. 2. Curve doseeresponse for PBT derivatives 6ae6g. Data are means þ/ꢀ SEM (Standard Error of the Mean) of triplicate determination.
chlorine substituted derivatives (line 6b and 6f), in this case the
EC50 reaches 3 and 5 mM, respectively.
interference with cellular DNA replication. Indeed, derivative 6d
induced a slight effect in the sub-G0/G1 phase and G1 phase of
7.2% and ꢀ12%, respectively, but no variations in the other
phases.
2.3.4. Cell cycle analysis
To examine the influence of the more active compounds 6ae
6b, 6d, on cell cycle distribution, the HeLa cells were exposed to
four different concentrations of each compound for 72 h and,
after incubation with propidium iodide as described in experi-
mental section, the cells were evaluated using flow cytometry
[38,39]. As shown in Fig. 4, when compared with the control, 6a,
Since the order of efficacy in inducing cell death was:
6b > 6a > 6d these results are in agreement with the trend
observed in the anti-proliferative and apoptosis assays.
The S-phase checkpoint is involved with DNA repair and cell-
cycle progression. It is known to react to DNA damage by
reducing the rate of synthesis. Although it is still the least under-
stood of the cellular checkpoints, recent studies on infrared-
induced S-phase checkpoint activation have thrown valuable
insight into its role in cancer development [33].
6b (at 50
of 18% and 25.4% and a S phase-specific effect of 10% and 19.4%,
respectively. This phase specificity suggests probable
mM) showed an increased sub-G0/G1 phase (cell death)
S

350
F. Mingoia et al. / European Journal of Medicinal Chemistry 64 (2013) 345e356
Table 3
against LAN-5 (CNS) and 16HBE appeared, with IC50 of 15 and
30 M, respectively (Table 2).
Moving to the derivative 6c (8-CN) also selected and synthe-
sized for its good percentage of affinity as topoisomerase I and II
inhibitor and antimitotic agent (Table 1), it resulted poor in the
apoptotic induction assays as well as on NCI one dose screening. It
displayed only an anti-proliferative activity (MTT) in the range 27e
28 mM on HeLa and LAN-5 cells. For these reasons the cell cycle test
was not taken in consideration.
By contrast, improved results have been reached for the new
designed derivative 6a (8,9-dimethyl), classified as promising
candidates as topoisomerase I/II inhibitor and RNA-DNA antime-
tabolite (Table 1). The anti-proliferative profile of the derivative 6a,
Overview of the one dose NCI screening test results (G% for selecteda human tumor
cell lines).
m
PANEL
6a
19
6b
23
59
101
54
6c
6d
Leukemia
CCRF-CeM
MOLT-4
HOP-92
NCI H522
Colo 205
SW 620
SNB 75
100
95
103
97
104
110
96
103
91
94
82
93
97
66
94
89
96
91
84
100
97
Non-Small Cell Lung Cancer
Colon Cancer
97
ꢀ64
108
118
116
99
23
CNS Cancer
Melanoma
Ovarian Cancer
Renal Cancer
Prostate Cancer
Breast Cancer
96
SK-MEL-2
IGROV-1
UO-31
PC-3
T-47D
102
e
99
e
e
81
94
76
105
101
91
84
101
115
in the NCI tests (Table 3) performed at 10
only a 20% of growth with respect to the control (untreated cell
considered as 100% growth) reaching a IC50 of 4 M. Whereas in the
antiproliferative MTT assays the more sensitive tumor cell line were
the HeLa and the H292, displaying a IC50 13.2 and 23.4 M,
respectively. Interesting results in the range of 3e4 M were found
in the apoptosis induction. The assay has been carried out by
comparison with the positive control (0.01 M staurosporine) and
mM one dose, displayed
a
G% (Growth %). Data obtained from the NCI one dose mean graph. Interpretation
of One-Dose Data: These data have been derived from the mean graph of the percent
growth of treated cells. The number reported for the One-dose assay is growth
relative to the no-drug control, and relative to the time zero number of cells. This
allows detection of both growth inhibition (values between 0 and 100) and lethality
(negative values, less than 0). For example, a value of 100 means no growth inhi-
bition. A value of 40 would mean 60% growth inhibition. A value of 0 means no net
growth over the course of the experiment. A value of ꢀ40 would mean 40% lethality.
A value of ꢀ100 means all cells are dead (for more details see Experimental).
m
m
m
m
the negative control (the untreated cells). Toward the cell cycle on
Sub Go/G1 phase, a cell death of 18% was observed along with 10%
of S phase specific effect. It is worthy to note, that in this phase the
topoisomerase I as well as RNA-DNA antimetabolites acts according
to the classification suggested by the VLAK protocol.
The best results have been obtained with derivative 6b (8,9-
dichloro), which was classified as antimitotic agents (A% ¼ 90)
and as topoisomerase II inhibitors (A% ¼ 79). With respect to the
antiproliferative activity, either in the MTT test and in the one dose
NCI screening, some selective interesting results were obtained. In
the MTT assays (Table 2), HeLa and MCF-7 cells resulted as the more
sensitive (IC50 14.8 and 13.9 mM). Especially for MCF-7, when the
mono-chlorinated 6f was compared with the related dichloro one
6b, an increased of potency of nearly two folds was observed,
moving from 20.5 to 13.9 mM. In this case, the high A% (90) value as
antimitotic agent and as topoisomerase II inhibitor (79%), matches
well with the best biological outcomes.
Although the attempts to correlate these biological assays needs
yet to be clarified with deeper investigations, the analysis of the
data arising from the in silico and the biological experiments per-
formed, allow us to evidence some interesting results.
2.3.5. Biological data and VLAK protocol
As can be seen on Table 1, except for derivative 6j, the VLAK
protocol classified seven of the first series of compounds (6ee6l)
showing good percentage of affinity percent (A%) as alkylating
agents (range 74e83%) coupled with a good A% as RNA/DNA anti-
metabolites (range 70e79%). Among the new synthesized com-
pounds, the derivative 6d (8-CF3) even if it exhibited a valuable
score value (86%) as alkylating candidate, in the HeLa cell cycle
experiments, exhibited only a slight effect in the Sub Go/G1 and G1
phase along with an interesting apoptosis induction without a dose
dependent profile (Fig. 3). By contrast, an anti-proliferative activity
Also of interest are the results of the NCI one dose (10
mM)
testing. In Table 3 some representative data are therein reported.
Leukemia cell (CCRF-CeM) showed a growth (G%) of 23% (77% of
inhibition with respect to the untreated cell used as control). The
most sensitive was the colon tumor cell line (Colo205) exhibiting
64% of cell death. For this last tumor cell line the potency of 6b is the
highest. Although less sensitive than the previous one, other colon
tumor cell line is the SW 620 which showed only a G% ¼ 23%.
Associated to these selective valuable anti-proliferative activity,
the derivative 6b exhibits the best result either on apoptosis in-
duction (EC50 ¼ 3.5
mM) and on the cell cycle showing an effect on
Sub Go/G1 phase of 25% along with a 19% on S phase as compared
with control.
In our study, the results achieved in the case of the new
designed and synthesized compounds 6ae6d, it can be observed
that only the 8,9 dimethyl (6a) and the 8,9-dichloro derivatives (6b)
have been improved on their efficacy, while for the 8-CN (6c) and
derivatives 8-CF3 (6d) the expected activity increases were not
evidenced or only partially noticed.
Fig. 3. Percentage of mitochondrial depolarization versus dose (mM). Experiments
were performed in triplicates and replicated twice. The HeLa cells were seeded in 6
well flat-bottomed plates and incubated overnight with various drugs at serial con-
centrations. After treatment (for more details see Experimental section) cells were
detached and analyzed using a FACSCalibur flow cytometer and the signal from TMRM
was read on FL2. The data obtained were acquired, gated, compensated and analyzed
using Cell-Quest Software. Mitochondrial depolarization analysis was measured by
gating the cells treated overnight with 0.01 mM of staurosporine as positive control.
Furthermore, the average signal of the negative control (untreated cells) was
subtracted from the probe signal.
3. Conclusions
For the optimization of the PBT derivatives an in house database
(a library of PBT derivatives) was built and on which the VLAK
protocol has been applied.
Based on the main role played by the introduction of suitable
functionalities onto the PBT skeleton able to modulate the reactivity

F. Mingoia et al. / European Journal of Medicinal Chemistry 64 (2013) 345e356
351
Fig. 4. Flow cytometry profile of the more active compounds. Cells from each sample were analyzed using a FACSCalibur supported with CellQuest acquisition and data analysis
were made by comparison with the untreated cells, used as control. The effect of the DMSO were also subtracted [38,39].
as well as the selectivity toward cell lines, we selected from our in
house built database, new derivatives so as to potentiate the effects
exerted by the groups which resulted among the more influent in
modulating the activity in the previous SAR studies (6a, 6b) and
new derivatives so as to increase the electron withdrawing effect on
the “C” tricycle moiety (6c, 6d) to get some insight regarding in
chemical reactivity and biological activity.
descriptor belonging to different classes were calculated
(Supplementary material).
Thus, for each class of drugs (Alkylating Agents, Antimitotic
Agents, Topoisomerase I Inhibitors, Topoisomerase II Inhibitors,
RNA/DNA Antimetabolites, and DNA Antimetabolites), a “lock
model” was generated starting from own known drugs.
In particular, for each class of drugs with known mechanism of
action (MA) (Fig. 5a), the lock model is defined as a sequence of
molecular descriptor value ranges. Each range is defined as
Compared to the mono-substituted methyl and chlorine de-
rivatives (6e, 6f), the doubling of the substituent resulted beneficial
for the activity, in agreement with the affinity percentage shown on
Table 1. By contrast, in the case of eCN and eCF3 groups (6c, 6d), the
expected improvements of activity were not achieved despite good
prediction arisen from our virtual lock and key protocol.
The order of potency of the active compound followed the same
trend either in the evaluation of apoptosis induction and in the
viability test. In all the tests, the activity order observed was de-
rivative 6b > 6a > 6f > 6e > 6g > 6d > 6c. Curiously, this trend is
mirrored by the solubility scale (from the more to the less one).
The cell cycle data support probable interference of these
compounds with DNA replication, the main event in phase S. The
similar trend, in terms of order of activity exhibited by the com-
pounds in the different assays, suggests that the observed anti-
proliferative activity is probably exerted through apoptosis induc-
tion and cell cycle arrest in the S phase, as typical for alkylating
agents.
m
Dj(MA) ꢁ
s
Dj(MA), where
mDj(MA) is the molecular descriptor
average value and
s
Dj(MA) is the standard deviation (Fig. 5b,
Supplementary material).
When the molecular descriptor value Dj of a tested structure X
(Fig. 5c) falls within the defined range (
m
Dj ꢁ
s
Dj)
a
¼ 1 (ie D1, D3,
and Dj), otherwise
a
¼ 0 (ie D2, D4) (Fig. 5d). At the end each in
house database structure is converted in a binary sequence. In the
proposed protocol, it is supposed that the higher is the number of
fitted pins, the higher will be the potential anticancer capability of
the investigated compound. Thus, the percentage of affinity A% (eq.
(1)) for each compound belonging to in house database was defined
for each class (MA) as:
X
A% ¼
a
i; jðMAÞ=Dtot*100
(1)
where S
a
i,j(MA) is the sum of all fitted molecular descriptors for
Thus, although a lot of work still remain to make light on, a
promising new class of valuable apoptotic inductors having a PBT
scaffold is proposed herein. Further developments with properly
designed group conferring enhanced solubility properties could
help to clarify these premises.
the MA class and Dtot is the total of the molecular descriptors used
in the VLAK protocol.
In Supplementary material are reported the output results for
the selected PBTs related the lock models representing each class of
drugs (MAs).
4. Experimental
4.2. Chemistry
4.1. VLAK protocol
4.2.1. Material and methods
All melting points were taken on a Sanyo-Gallenkamp capillary
apparatus and are uncorrected; IR spectra were recorded with
bromoform on NaCl window with a Jasco FT/IR 5300 spectropho-
tometer; 1H and 13C NMR spectra were recorded in CDCl3 or DMSO-
d6 solution as below specified, using a Bruker AC-E series 200 MHz
spectrometer, unless otherwise specified. As internal reference re-
The compounds contained in the NCI AntiCancer Agents
Mechanism (NCI ACAM) [24,25,29] and those in the PBT databases,
were drawn and optimized in vacuo by ligprep of MAESTRO SUITE
[34]. The NCI ACAM Database entries containing cations or con-
sisting of a mix of two structures were excluded. Thus the starting
database was constituted from 114 compounds classified in six
mechanism of action: 30, Alkylating Agents; 13, Antimitotic Agents;
24, Topoisomerase I Inhibitors; 15, Topoisomerase II Inhibitors; 16,
RNA/DNA Antimetabolites; 16, DNA Antimetabolites (Supplemen
tary material). CODESSA PRO software was used for the molecular
descriptors calculation [35]. For all structures 286 molecular
sidual peak of the solvent was calibrated, at
d 7.26 and 2.50 ppm,
respectively for CDCl3 and DMSO-d6. Mass spectroscopy was per-
formed using a GCeMS Shimadzu QP5050 with EI (75ev). Column
chromatography was performed with Merck silica gel 230e400
Mesh ASTM. Thin layer chromatography was performed on pre-
coated (0.25 mm) silica gel GF254 plates; compounds were detected

352
F. Mingoia et al. / European Journal of Medicinal Chemistry 64 (2013) 345e356
Fig. 5. VLAK protocol applied on NCI ACAM database. MA, mechanism of action; Dj, molecular descriptors; Si, structures; T, in silico screened entry.
with 254 nm UV lamp. Microanalyses were in agreement with
theoretical values ꢁ0.4%.
and 1341 (NO2), 934 and 897 (CeCl) cmꢀ1. 1H NMR:
d
(DMSO) 1.63
0
(s, 3H, CH3), 2.45 (s, 3H, CH3), 5.82 (s, 1H, H4), 7.64 (s, 1H, H6 ), 8.59
(s, 1H, H3 ). 13C NMR:
d (DMSO) 16.65 (CH3), 18.46 (CH3), 102.63
0
4.2.2. General procedure for the preparation of 2,5-dimethyl-2-(2-
nitroarylazo)-3-oxo-2,3-dihydrofurans 3a-g
(CH), 107.76 (C), 120.26 (CH), 126.47 (CH), 134.25 (C), 136.99 (C),
142.28 (C), 144.67 (C), 191.66 (C), 194.14 (CO). M/z (EI) 191
(Mþ ꢀ139, 84%), 193 (Mþ ꢀ137, 54%), 175 (5), 177 (3), 161 (36), 163
(23), 145 (99), 147 (64), 133 (51), 135 (30), 109 (100), 111 (31) and
217 (Mþ ꢀ113, 50%), 219 (35), 202 (13), 204 (7), 172 (9), 174 (6), 161
(35), 163 (16). Anal. Calcd. For C, 43.66; H, 2.75; Cl, 21.48; N, 12.73.
Found: C, 43.70; H: 2.71; N: 12.78.
To a suspension of 2-Nitroanilines 2ae2g (10 mmol) in water
(5 mL) and hydrochloric acid (37%, 2.5 mL), a solution of sodium
nitrite (0.74 g, 10.75 mmol) in water (4 mL) at 0 ꢂC was added
dropwise. The mixture was stirred for 1 h, diluted with water
(30 mL) and made acid with concentrated hydrochloric acid (2 mL).
In the case of 2b,f the reactions were carried out at room temper-
ature. To this solution, freshly distilled 2,5-dimethyl-3-oxo-2,3-
dihydrofuran [36] (1.24 g, 11 mmol) was added and the mixture
was stirred for 1 h at room temperature. The crude residue was
collected by filtration. After drying, it was purified by column
chromatography using a dichloromethane/ethyl acetate mixture in
gradient as eluent and recrystallized from ethanol.
4.2.2.3. 2-(4-Cyano-2-nitrophenylazo)-2,5-dimethyl-3-oxo-2,3-
dihydrofuran (3c). Yield 85%. Mp 89e90 ꢂC. IR: 1715 (CO), 1539 and
1348 (NO2) cmꢀ1. 1H NMR
d (DMSO) 1.66 (s, 3H, CH3), 2.46 (s, 3H,
CH3), 5.83 (s,1H, H4), 7.46 (d,1H, J ¼ 7.3 Hz, H6’), 8.28 (dd,1H, J ¼ 7.3,
1.3 Hz, H5’), 8.79 (d, 1H, J ¼ 1.3 Hz, H3’). 13C NMR:
d (DMSO) 16.63
(CH3), 18.36 (CH3), 102.64 (CH), 108.04 (C), 114.07 (C), 116.43 (CN)
120.20 (CH), 128.92 (CH), 138.23 (CH), 145.21 (C), 145.94 (C), 186.35
(C), 188.54 (CO). M/z (EI) 148 (Mþ ꢀ138, 49%), 132 (3), 118 (5), 102
(100), 90 (14), 75 (38) and 174 (Mþ ꢀ112, 46%), 159 (20), 140 (21),
129 (8), 116 (37), 102, (17), 91 (19) and 226 (Mþ ꢀ40, 10%), 211 (46),
171 (5), 155 (4), 118 (3), 111 (2), 86 (3). Anal. Calcd. For C, 54.55; H,
3.52; N, 19.57. Found: C, 54.60; H: 3.49; N: 19.51.
4.2.2.1. 2,5-Dimethyl-2-(4,5-dimethyl-2-nitrophenylazo)-3-oxo-2,3-
dihydrofuran (3a). Yield 95%. Mp 93 ꢂC. IR: 1717 (CO), 1539 and
1321 (NO2) cmꢀ1. 1H NMR:
d
(DMSO) 1.61 (s, 3H, CH3), 2.33 (s, 3H,
CH3), 2.36 (s, 3H, CH3), 2.44 (s, 3H, CH3), 5.80 (s, 1H, H4), 7.10 (s, 1H,
H3 ), 7.94 (s, 1H, H6 ). 13C NMR:
d (DMSO) 16.64 (CH3), 18.21 (CH3),
0
0
19.06 (CH3), 19.23 (CH3), 102.67 (CH), 107.44 (C), 118.50 (CH), 124.70
(CH), 141.51 (C), 141.53 (C), 143.92 (C), 143.94 (C), 191.55 (C), 194.92
(CO). M/z (EI) 151 (Mþ ꢀ139, 100%), 135, 806), 121 (22), 105 (72) and
177 (Mþ ꢀ111, 100%), 162 (17), 148 (10), 132 (21), 119 (98), 103 (18),
91 (28). Anal. Calcd. For C, 58.13; H, 5.23; N, 14.53. Found: C, 58.17;
H: 5.19; N: 14.47.
4.2.2.4. 2,5-Dimethyl-2-(2-nitrophenylazo-4-trifluoromethyl)-3-oxo-
2,3-dihydrofuran (3d). Yield 79%. Mp 108 ꢂC. IR: 1717 (CO),1539 and
1262 (NO2) cmꢀ1. 1H NMR
d (DMSO) 1.67 (s, 3H, CH3), 2.47 (s, 3H,
CH3), 5.85 (s,1H, H4), 7.52 (d,1H, J ¼ 7.8 Hz, H6’), 7.69 (dd,1H, J ¼ 7.8,
1.2 Hz, H5’), 8.61 (d, 1H, J ¼ 1.2 Hz, H3’). 13C NMR:
d (DMSO) 16.63
(CH3), 18.32 (CH3), 102.66 (CH), 107.98 (C), 122.59 (q, J ¼ 272.8 Hz,
CF3),120.33 (CH),122.22 (q, J ¼ 3.7 Hz, CH),131.30 (q, J ¼ 33.3 Hz, C),
131.13 (q, J ¼ 3.2 Hz, CH), 145.62 (C), 145.86 (C), 191.80 (C), 194.08
4.2.2.2. 2-(4,5-Dichloro-2-nitrophenylazo)-2,5-dimethyl-3-oxo-2,3-
dihydrofuran (3b). Yield 78%. Mp 140e141 ꢂC. IR: 1715 (CO), 1532

F. Mingoia et al. / European Journal of Medicinal Chemistry 64 (2013) 345e356
353
(CO). M/z (EI) 198 (Mþ ꢀ131), 222 (Mþ ꢀ107), 269 (Mþ ꢀ60). Anal.
Compounds 4eeg were identical to literature [14].
Calcd. For C, 47.43; H, 3.06; F, 17.31; N, 12.76. Found: C, 47.38; H:
3.03; F, 17.35; N: 12.72.
4.2.4. General procedure for the preparation of: 1-(2-aminoaryl)-5-
methylpyrazol-3-ones 5aeg
Compounds 3e-g were identical to literature report [14].
Method A: A solution of nitro derivatives 4a, 4ce4d (42 mmol)
was reduced overnight over 10% Pd on charcoal in ethanol in a Parr
apparatus at 50 psi at room temperature. Removal of the catalyst
and evaporation of the solvent under reduced pressure gave a
residue which was purified by flash chromatography using a
dichloromethane/ethyl acetate mixture in gradient as eluent and
recrystallized from ethanol.
4.2.3. General method for the preparation of 5-methyl-1-(2-
nitroaryl)-pyrazol-3-ones 4aeg
To a concentrated hydrochloric acid solution (37%, 7 mL), 2-
(Arylazo)-2,5-dimethyl-3-oxo-2,3-dihydrofuran 3ae3g (10 mmol)
was added in small portions with stirring, while care is taken that
the temperature of the mixture does not exceed 30 ꢂC. After the
addition was complete the stirred mixture is kept at 20e30 ꢂC,
usually for 1 h. The cooled mixture was then poured onto ice water
and made basic with 30% aqueous sodium hydroxide. It was then
diluted with water to complete dissolution of the sodium salt of the
pyrazolone. The resultant solution was extracted with diethyl ether
(3 ꢃ 100 mL). The aqueous phase was made acid with diluted hy-
drochloric acid, with stirring and cooling to precipitate the product.
The solid was isolated by filtration, washed with water, air dried
and recrystallized from ethanol [37].
4.2.4.1. 1-(2-Aminophenyl)-4,5-dimethylpyrazol-3-one
Yield 98%. Mp 220e222 ꢂC. IR: 3439 and 3360 (NH2), 3100 (OH),
cmꢀ1 1H NMR:
(300 MHz DMSO) 1.99 (s, 3H, CH3), 2.07 (s, 3H,
CH3), 2.12 (s, 3H, CH3), 4.61 (2H, s, NH2, exchangeable), 5.47 (s, 1H,
(5a).
.
d
0
0
H4), 6.60 (s, 1H, H6 ), 6.76 (s, 1H, H3 ), 9.72 (bs, 1H, NH, exchange-
able). 13C NMR:
d
(75 MHz DMSO) 11.60 (CH3), 18.26 (CH3), 19.34
(CH3), 91.23 (CH), 117.22 (CH), 122.28 (C), 123.66 (CH), 128.10 (CH),
136.67 (C), 140.46 (C), 142.09 (C), 161.12 (CO). M/z (EI) 217 (Mþ,
100%), 202 (10), 185 (06), 173 (39), 159 (20), 147 (20), 135 (15), 120
(07), 108 (08). Anal. Calcd. For C, 66.34; H, 6.96; N, 19.34. Found: C,
66.39; H: 7.00; N: 19.31.
4.2.3.1. 1-(4,5-Dimethyl-2-nitrophenyl)-5-methyl-1,2-
dihydropyrazol-3-one (4a). Yield 96%. Mp 235 ꢂC. IR: 3195 (NH),
1524 and 1350 (NO2) cmꢀ1
.
1H NMR:
d
(DMSO) 2.12 (s, 3H, CH3),
2.34 (s, 6H, 2xCH3), 5.58 (s, 1H, H4), 7.47 (s, 1H, H3’), 7.83 (s, 1H, H6’),
9.90 (s, 1H, NH, exchangeable with D2O). 13C NMR:
(DMSO) 11.49
4.2.4.2. 1-(2-Amino-4-cyanophenyl)-5-methylpyrazol-3-one
Yield 75%. Mp 205 ꢂC. IR: 3483 (NH) 3396 and 3210 (NH2), 3140
(OH), 2226 (CN), cmꢀ1 1H NMR:
(DMSO/CDCl3) : 2.05 (s, 3H,
CH3), 5.26 (s, 2H, NH2), 5.49 (s, 1H, H4), 6.85 (dd, 1H, J ¼ 8.0, 1.2 Hz,
(5c).
d
(CH3), 18.73 (CH3), 19.22 (CH3), 93.19 (CH), 125.30 (CH), 129.38 (CH),
129.80 (C), 137.98 (C), 140.85 (C), 143.32 (C), 143.67 (C), 161.93 (CO).
M/z: (EI) 287 (Mþ 45%), 289 (Mþ þ2, 45%), 270 (17), 252 (9), 171
(17), 157 (20), 145 (17), 109 (20), 97 (100), 85 (33). Anal. Calcd. For C,
58.29; H, 5.30; N, 16.99. Found: C, 58.25; H: 5.32; N: 17.02.
.
d
d
0
0
0
H5 ), 7.03 (d, 1H, J ¼ 8.0 Hz, H6 ), 7.08 (d, 1H, J ¼ 1.2 Hz, H3 ) 9.69 (s,
1H, NH). 13C NMR:
d
(DMSO/CDCl3) 11.42 (CH3), 92.39 (CH), 110.97
(C), 118.31 (CN), 118.60 (CH), 118.79 (CH), 127.25 (CH), 127.38 (C),
140.51 (C), 144.59 (C), 161.73 (CO). M/z (EI) 214 (Mþ, 45%), 170 (20),
144 (19), 117 (23), 84 (31). Anal. Calcd. For C, 61.67; H, 4.71; N, 26.15.
Found: C, 61.71; H: 4.68; N: 26.12.
4.2.3.2. 1-(4,5-Dichloro-2-nitrophenyl)-5-methylpyrazol-3-one (4b).
Yield 89%. Mp 220 ꢂC. IR: 3104 (OH), 1530 and 1345 (NO2), 909 (Ce
Cl) cmꢀ1. 1H NMR:
d
(DMSO) 2.23 (s, 3H, CH3) 5.66 (s,1H, H4), 8.19 (s,
1H, H5’), 8.44 (s, 1H, H3’), 10.11 (s, 1H, NH). 13C NMR:
d
(DMSO) 11.38
4.2.4.3. 1-(2-Amino-4-trifluoromethylphenyl)-5-methylpyrazol-3-
one (5d). Yield 80%. Mp 216 ꢂC. IR: 3478 and 3358 (NH2), 3200
(CH3), 94.63 (CH), 126.70 (CH), 129.53 (CH), 130.83 (C), 131.55 (C),
135.78 (C), 141.65 (C), 144.54 (C), 162.59 (CO). M/z (EI) (Mþ 287,
45%), 289 (Mþþ2, 31%), 270 (16), 272 (4), 252 (7), 254 (3), 222 (6),
224 (2), 171 (18), 158 (20), 160 (15), 145 (17), 147 (10), 109 (20), 111
(13), 97 (100). Anal. Calcd. For C, 41.69; H, 2.45; Cl, 24.61; N, 14.59.
Found: C, 41.71; H: 2.43; Cl, 24.58; N: 14.56.
(OH), 1344 (CF3) cmꢀ1
.
1H NMR:
d
(250 MHz, CDCl3) 2.09 (s, 3H,
CH3), 5.59 (s, 1H, H4), 5.9e6.3 (bs, 2H, NH2), 7.01 (dd, 1H, J ¼ 7.8,
0
0
0
0.8 Hz, H6 ), 7.05 (d, 1H, J ¼ 0.8 Hz, H3 ), 7.18 (d, 1H, J ¼ 7.8 Hz, H5 ),
7.28 (bs, 1H, NH). 13C NMR:
d
(63 MHz, CDCl3) 11.55 (CH3), 92.76
(CH), 113.36 (q, J ¼ 3.7 Hz, C30), 114.72 (q, J ¼ 3.7 Hz, C50), 126.38 (q,
J ¼ 272 Hz, CF3), 126.35 (C10), 128.80 (C60), 132.08 (q, J ¼ 31.4 Hz,
C40), 142.88 (C20), 144.00 (C5), 163.41 (CO). M/z (EI) 257 (Mþ, 100%),
239 (12), 213 (28), 199 (22), 187 (21), 175 (15), 160 (16), 140 (12).
Anal. Calcd. For C, 51.37; H, 3.92; F, 22.16; N, 16.34. Found: C, 51.40;
H: 3.89; F, 22.19; N: 16.29.
4.2.3.3. 1-(4-Cyano-2-nitrophenyl)-5-methylpyrazol-3-one
Yield 75%. Mp 217 ꢂC. IR: 3445 (NH), 1694 (CO), 1541 e 1354 (NO2);
2257 (CN) cmꢀ1. 1H NMR:
(DMSO) 2.22 (s, 3H, CH3), 5.67 (s, 1H,
(4c).
d
0
00
H4), 7.83 (d, 1H, J ¼ 8.3 Hz, H6 ), 8.23 (dd, 1H, J ¼ 8.3, 1.9 Hz, H5 ),
8.42 (d, 1H, J ¼ 1.9 Hz, H3 ), 10.14 (s, 1H, NH). 13C NMR:
d (DMSO)
0
11.70 (CH3), 94.79 (CH),124.17 (CH),128.05 (CH),131.97 (CH),133.86
(CN), 134.03 (C), 141.24 (C), 145.26 (C), 162.57 (C), 166.06 (CO). Anal.
Calcd. For C, 54.10; H, 3.30; N, 22.94. Found: C, 54.1; H: 3.28; N:
22.96.
4.2.5. Method B
Iron powder (670 mg, 12 mmol) was added to a solution of 5b
(3.4 mmol) in acetic acid (30 mL). The mixture was kept at 60 ꢂC in a
steam bath for 24 h. The reaction mixture was cooled, poured onto
crushed ice and extracted with dichloromethane. The organic layer,
dried over sodium sulfate and evaporated under reduced pressure,
gave a residue which was purified by chromatography and recrys-
tallization from ethanol.
4.2.3.4. 1-(2-Nitrophenyl-4-trifluoromethyl)-5-methylpyrazol-3-one
(4d). Yield 85%. Mp 218 ꢂC. IR: 3150 (NH), 1631 (CO), 1545 and 1354
(NO2), 1325 (CF3) cmꢀ1. 1H NMR:
d
(DMSO) 2.26 (s, 3H, CH3), 5.71 (s,
0
0
1H, H4 ), 7.97 (dd, 1H, J ¼ 7.4, 1.4 Hz, H6 ), 8.17 (dd, 1H, J ¼ 7.4, 1.4 Hz,
H5 ), 8.44 (d, 1H, J ¼ 1.4 Hz, H3 ), 10.20 (1H, s, NH). 13C NMR:
4.2.5.1. 1-(2-Amino-4,5-dichlorophenyl)-5-methylpyrazol-3-one
0
0
d
(DMSO) 11.57 (CH3), 95.28 (CH), 122.79 (q, J ¼ 272 Hz, CF3), 122.64
(5b). Yield 82%. Mp 246 ꢂC. IR: 3605 (OH), 3455 and 3355 (NH2),
(q, J ¼ 3.7 Hz, CH), 128.15 (q, J ¼ 33.9 Hz, C40), 128.86 (d, C60), 129.93
(q, J ¼ 3.7 Hz, CH), 135.05 (s, C10), 141.47 (s, C5), 145.61 (s, C20),
162.82 (CO). M/z (EI) 287 (Mþ, 100%), 270 (27), 257 (12), 239 (7),
228 (8), 212 (12), 198 (16), 186 (14), 172 (11), 158 (12), 145 (48), 126
(18), 115 (17), 97 (98), 85 (31), 69 (23). Anal. Calcd. For C, 46.00; H,
2.81; F, 19.85; N, 14.63. Found: C, 45.97; H: 2.83; N: 14.66.
1624 (CO), 927 (CeCl) cmꢀ1 1H NMR:
; d (DMSO) 2.05 (s, 3H, CH3),
0
0
5.43 (s, 2H, NH2), 5.55 (s, 1H, H4), 7.02 (s, 1H, H6 ), 7.30 (s, 1H, H3 ),
9.91 (s, 1H, NH). 13C NMR:
d
(DMSO) 11.32 (CH3), 92.24 (CH), 115.85
(C), 115.99 (CH), 123.70 (C), 128.65 (CH), 130.68 (C), 141.01 (C),
144.85 (C), 161.58 (CO). M/z (EI) 257 (Mþ100), 259 (Mþþ2, 70%), 215
(41), 199 (29), 178 (24), 133 (40), 108 (23). Anal. Calcd. For C, 46.53;

354
F. Mingoia et al. / European Journal of Medicinal Chemistry 64 (2013) 345e356
H, 3.51; Cl, 27.47; N, 16.28. Found: C, 46.57; H: 3.49; Cl: 27.51; N:
16.30.
After 24 h, two plates of each cell line are fixed in situ with TCA,
to represent a measurement of the cell population for each cell line
at the time of drug addition (Tz). Experimental drugs are solubi-
lized in dimethyl sulfoxide at 400-fold the desired final maximum
test concentration and stored frozen prior to use. At the time of
drug addition, an aliquot of frozen concentrate is thawed and
diluted to twice the desired final maximum test concentration with
Compounds 5eeg were identical to literature [14].
4.2.6. General method for the preparation of 1-methyl-8,9-
substituted-3H-pyrazolo[1,2-a]benzotetrazin-3-ones 6ae6g
Concentrated sulfuric acid (0.4 mL) was added to a solution of
5ae5g, (1.9 mmol) in water (10 mL) and the mixture was diazotized
with sodium nitrite (145 mg, 2.1 mmol) in water (10 mL) at 0 ꢂC. The
reactants were stirred for further 2 h at room temperature, and
then were kept at 60 ꢂC for 3 h. The mixture was cooled to room
temperature, and made basic with solid sodium carbonate. The
solid precipitate was filtered off, and air dried.
complete medium containing 50 mg/ml gentamicin. Following drug
addition, the plates are incubated for an additional 48 h at 37 ꢂC, 5%
CO2, 95% air, and 100% relative humidity. For adherent cells, the
assay is terminated by the addition of cold TCA. Cells are fixed in
situ by the gentle addition of 50 ml of cold 50% (w/v) TCA (final
concentration, 10% TCA) and incubated for 60 min at 4 ꢂC. The su-
pernatant is discarded, and the plates are washed five times with
4.2.6.1. 1,8,9-Trimethyl-3H-pyrazolo[1,2-a]benzotetrazinꢀ-31-one (6a).
tap water and air dried. Sulforhodamine B (SRB) solution (100 ml) at
Yield 81%. Mp 142e143 ꢂC (dec.). IR: 1678 (CO) cm
.
1H NMR:
0.4% (w/v) in 1% acetic acid is added to each well, and plates are
incubated for 10 min at room temperature. After staining, unbound
dye is removed by washing five times with 1% acetic acid and the
plates are air dried. Bound stain is subsequently solubilized with
10 mM trizma base, and the absorbance is read on an automated
plate reader at a wavelength of 515 nm. For suspension cells, the
methodology is the same except that the assay is terminated by
d
(DMSO) 2.24 (s, 3H, CH3), 2.30 (s, 3H, CH3), 2.63 (s, 3H, CH3), 5.45
(s, 1H, H2) 7.26 (s, 1H, H10), 7.51 (s, 1H, H7). 13C NMR:
d
(DMSO) 14.84
(CH3),18.99 (CH3), 20.06 (CH3), 95.61 (CH),113.96 (CH),122.29 (CH),
125.21 (C), 129.30 (C), 136.27 (C), 137.35 (C), 144.53 (C), 161.51 (CO).
Anal. Calcd. For C, 63.15; H, 5.30; N, 24.55. Found: C, 62.99; H: 5.15;
N: 24.45.
fixing settled cells at the bottom of the wells by gently adding 50 ml
4.2.6.2. 8,9-Dichloro-1-methyl-3H-pyrazolo[1,2-a]benzotetrazin-3-
of 80% TCA (final concentration, 16% TCA). Using the three absor-
bance measurements [time zero, (Tz), control growth, (C), and test
growth in the presence of drug at 10 mM concentration level (Ti)],
one (6b). Yield 73%. Mp 143 ꢂC (dec.). IR: 1676 (CO), 926 (CeCl)
cmꢀ1. 1H NMR:
d
(250 MHz, CDCl3) 2.64 (s, 3H, CH3), 5.61 (s, 1H, H2),
7.34 (s, 1H, H10), 7.86 (s, 1H, H7). 13C NMR:
d
(50 MHz, CDCl3) 15.52
the percentage growth is calculated at each of the drug concen-
trations levels. Percentage growth inhibition is calculated as:
[(Ti ꢀ Tz)/(C ꢀ Tz)] ꢃ 100 for concentrations for which Ti>/ ¼ Tz
[(Ti ꢀ Tz)/Tz] ꢃ 100 for concentrations for which Ti < Tz.
Three dose response parameters are calculated for each exper-
imental agent. Growth inhibition of 50% (GI50) is calculated from
[(Ti ꢀ Tz)/(C ꢀ Tz)] ꢃ 100 ¼ 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as
measured by SRB staining) in control cells during the drug incu-
bation. The drug concentration resulting in total growth inhibition
(TGI) is calculated from Ti ¼ Tz. The LC50 (concentration of drug
resulting in a 50% reduction in the measured protein at the end of
the drug treatment as compared to that at the beginning) indicating
a net loss of cells following treatment is calculated from [(Ti ꢀ Tz)/
Tz] ꢃ 100 ¼ ꢀ50. Values are calculated for each of these three
parameters if the level of activity is reached; however, if the effect is
not reached or is exceeded, the value for that parameter is
expressed as greater or less than the maximum or minimum con-
centration tested [30].
(CH3), 96.86 (CH), 113.76 (CH), 126.86 (C), 127.76 (C), 129.75 (C),
131.03 (CH), 137.94 (C), 143.47 (C), 156.90 (CO). M/z (EI) 212
(Mþ ꢀ57, 100%), 214 (81), 185 (65), 187 (46); 144 (27), 146 (23), 111
(8), 109 (20). Anal. Calcd. For C, 44.64; H, 2.25; Cl, 26.35; N, 20.82.
Found: C, 44.71; H: 2.31; Cl: 26.42; N: 20.70.
4.2.6.3. 8-Cyano-1-methyl-3H-pyrazolo[1,2-a]benzotetrazin-3-One
(6c). Yield 67%. Mp 176e178ꢂ (dec.). IR: 1697 (CO) cmꢀ1. 1H NMR:
d
(250 MHz, DMSO) 2.63 (s, 3H, CH3), 5.75 (s, 1H, H), 7.59 (d, 1H,
J ¼ 8.7 Hz, H10), 7.98 (dd, 1H, J ¼ 8.7, 1.9 Hz, H9), 8.27 (d, 1H,
J ¼ 1.9 Hz, H7). 13C NMR:
d (50 MHz, DMSO) 14.80 (CH3), 96.26 (CH),
107.16 (C), 114.04 (C), 117.62 (CN), 132.14 (CH), 132.75 (C), 137.85
(CH), 145.47 (C), 159.97 (C), 163.82 (CO). Anal. Calcd. For C, 58.67; H,
3.13; N, 31.10. Found: C, 58.81; H: 2.99; N: 31.25.
4.2.6.4. 8-Trifluoromethyl-1-methyl-3H-pyrazolo[1,2-a]benzote-
trazin-3-One (6d). Yield 77%. Mp 237e238 ꢂC (dec.). IR: 1699 (CO),
983 (CF3) cmꢀ1. 1H NMR:
d
(250 MHz, DMSO) 2.04 (s, 3H, CH3), 5.56
(s, 1H, H2), 7.57 (s, 2H, H7 and H9, 7.83 (s, 1H, H10). 13C NMR:
(50 MHz, DMSO) 14.61 (CH3), 106.68 (CH), 118.85 (CH), 119.75
d
4.3.2. MTT assays
(CH), 124.14 (q, J ¼ 270 Hz, CF3), 128.81 (q, J ¼ 32 Hz, C), 130.98 (C),
132.19 (CH), 135.22 (C), 147.83 (C), 166.91 (CO). Anal. Calcd. For C,
49.26; H, 2.63; F, 21.25; N, 20.89. Found: C, 49.40; H: 2.61; F: 21.29;
N: 20.81.
Cytotoxicity was determined by MTT for cell viability. The MTT
assay was performed according to the method of Skehan and col-
leagues [26]. Cells were seeded in 96-well flat-bottomed microtiter
plates (30,000 cells/well). Cells were incubated overnight with
Compounds 6eeg were identical to literature report [14].
various drugs, and applied as serial dilutions (100
ml/well) at
various concentrations. Twenty microliters of MTT (5 mg/ml) were
4.3. Biology
added to each well and incubated for 3 h at 37 ꢂC. The medium was
removed and 100 ml of DMSO was added into each well. The plate
4.3.1. NCI protocol (DTP program) methodology of the in vitro
cancer screen [30]
The human tumor cell lines of the cancer screening panel are
grown in RPMI 1640 medium containing 5% fetal bovine serum and
was gently rotated on an orbital shaker for 10 min to completely
dissolve the precipitation. The absorbance was detected at 540 nm
with a Microplate Reader (Wallac Victor2,1420 Multilabel Counter).
All measurements were performed in triplicate and each experi-
ment was repeated at least three times. The IC50 value was calcu-
lated from the 50% formazan formation compared with a control
without addition of drugs. Data analysis: the viability was calcu-
lated with regard to the untreated cell control [y0], which was set to
100% viability. The resulting curves were fitted by software (Sigma
Plot 11.0) to a Hill equation with four parameters.
2 mM
L-glutamine. For a typical screening experiment, cells are
inoculated into 96 well microtiter plates in 100
mL at plating den-
sities ranging from 5000 to 40,000 cells/well depending on the
doubling time of individual cell lines. After cell inoculation, the
microtiter plates are incubated at 37 ꢂC, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of experimental drugs.

F. Mingoia et al. / European Journal of Medicinal Chemistry 64 (2013) 345e356
355
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Acknowledgments
This work was supported by CNR fund, RSTL 624/2008. Thanks
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Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.03.
046. These data include MOL files and InChiKeys of the most
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