1-Methyl-3H-pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-ones. Design, synthesis, and biological activity of new antitumor agents

Article abstract of DOI:10.1021/jm049075u

1-Methylpvrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-ones 4, synthesized in good to excellent yields, were designed as novel alkylating agents because of their peculiar chemical behavior. All derivatives showed antiproliferative activity against more than 50 t

Full text of DOI:10.1021/jm049075u

J. Med. Chem. 2005, 48, 2859-2866
2859
1-Methyl-3H-pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-ones. Design, Synthesis, and
Biological Activity of New Antitumor Agents
Anna Maria Almerico,*,† Francesco Mingoia,‡ Patrizia Diana,† Paola Barraja,† Antonino Lauria,†
Alessandra Montalbano,† Girolamo Cirrincione,† and Gaetano Dattolo†
Dipartimento Farmacochimico, Tossicologico e Biologico, Universita` di Palermo, Via Archirafi 32, 90123 Palermo, Italy, and
Istituto per lo Studio dei Materiali Nanostrutturati, CNR Sezione di Palermo, Via Ugo La Malfa 153, 90146 Palermo, Italy
Received November 17, 2004
1-Methylpyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-ones 4, synthesized in good to excellent yields,
were designed as novel alkylating agents because of their peculiar chemical behavior. All
derivatives showed antiproliferative activity against more than 50 types of tumor cell lines
with GI50 reaching sub-micromolar values. SAR studies revealed that the presence of a chlorine
atom is well-tolerated in both positions 8 and 9, whereas in the case of the methyl group,
switching from the 8 to the 9 position gives rise to the most active compound of the series, 4g,
either for the number of cell lines inhibited and for selectivity against leukaemia and renal
cancer subpanels. COMPARE and 3D-MIND computations indicate, for compounds 4, an activity
profile analogous to rifamycins and cytidine analogues.
Introduction
Azolotetrazinones have been the focus of medicinal
chemists in the past decades because of the outstanding
antineoplastic activity exhibited by several derivatives
incorporating the imidazole moiety. In particular, mi-
tozolomide (1) and temozolomide (2) (recently marketed
as Temodal, effective against malignant melanoma,
mycosis fungoides, and brain tumors1) have attracted
Figure 1.
remarkable attention.
Compounds 1 and 2 are prodrugs that undergo, in a
antitumor agents and report the synthesis and the
nucleophilic microenvironment provided by a sequence
evaluation of the antiproliferative activity of a panel of
of guanine residues together with associated water
derivatives belonging to this series.
molecules, in the major groove of DNA, ring opening
Although apparently derivatives 4 do not share
following the nucleophilic attack at C-4 by an activated
similarity with temozolomide and other related azo-
molecule of water to afford a monoalkyltriazene species,
lotetrazinones, since they cannot generate a monoalkyl-
a bioactive alkylating agent. Such a reactive entity
triazene species, our interest in this class of heterocycles
likely undergoes an SN2 alkylation of the nucleophilic
arises from the very peculiar behavior of the ring system
residues in the immediate vicinity such as N-7 and/or
(vide infra) that makes the compounds capable of
O-6 sites of guanine, eliminating nitrogen and 5-ami-
generating cation intermediates that in turn might be
noimidazole-4-carboxamide.2
reactive toward biologically important nucleophiles.
Recently our research group has reported the inter-
esting antitumor activity of a new class of pyrrolo[2,1-
d][1,2,3,5]tetrazinones 3 that possess the deaza skeleton
of temozolomide. Most compounds of the series were
found to have GI50 values in the low micromolar or sub-
micromolar range and reaching, in the case of compound
3 (R ) CN, R1 ) Me, R2 ) Ph, R3 ) 4-Cl-C6H4),
nanomolar concentrations.3 Therefore, it seems that a
tetrazine ring can be an effective pharmacophore in
several classes of antitumor agents.
In this context we decided to investigate if the
presence of an isomeric 1,2,3,4-tetrazine moiety can also
give rise to classes of biologically active compounds.
Therefore, in this paper we propose 1-methylpyrazolo-
[1,2-a]benzo[1,2,3,4]tetrazin-3-ones of type 4 as new
Chemistry
The benzo[1,2,3,4]tetrazine system has not been
extensively studied and to date only few reports (26)
are present in the literature dealing with its synthesis
and chemical behavior. The number of known benzotet-
razine condensed with other heterocycles is, if possible,
even lower, and only three references can be found in
Scifinder Scholar on imidazo- and pyrazolobenzotetra-
zinones.4-6
The synthetic approach to the pyrazolo[1,2-a]benzo-
[1,2,3,4]tetrazinones was first undertaken by one of us
years ago6 and involved the use of the furan derivative
6 as starting material (Scheme 1). The reaction sequence
that successfully led to the parent compound 4a is now
extended to the preparation of several derivatives of the
class by using the commercially available 2-nitroanilines
5a-h. The diazonium salts obtained from 5, upon
treatment with sodium nitrite in aqueous hydrochloric
* Corresponding author. Tel: +39-0916161606. Fax:+39-0916169999.
E-mail almerico@unipa.it.
† Universita` di Palermo.
‡ CNR Sezione di Palermo.
10.1021/jm049075u CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/15/2005

2860 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Almerico et al.
several other nucleophiles (Nu ) â-naphthol, N3-) in
acid conditions to originate deep-red azo compound 14
and azide 15, respectively (Scheme 2). Similar behavior
is of course evidenced now for all the other derivatives
of the series without any significant difference attribut-
able to the nature of R and R′ substituents. This means
that compounds of type 4 behave as masked diazonium
salts, able to generate a species of type 10 that reacts
as the classical aromatic ones in coupling reactions in
the presence of acid, with or without loss of nitrogen.
Under thermal conditions, loss of nitrogen gives rise to
the cation 11, which intramolecularly rearranges to the
methylene intermediate 12. The same intermediate can
be formed under milder conditions (i.e. room tempera-
ture), although more slowly, as testified by NMR experi-
ments. In fact, the 1H NMR spectra of compounds 4a-
h, measured in DMSO-d6 in the presence of 10% H2O,
showed over a period of nearly 15 days a total conversion
of pyrazolo-tetrazinones into hydroxymethylenepyra-
zolinones 13a-h (Nu ) OH), as demonstrated by the
appearance of a signal at ca. 4.40 ppm, due to the
presence of the methylene.
Scheme 1
Therefore, the chemical mechanism that successfully
led to alkylation under laboratory conditions can con-
stitute the basis for creating an agent that could react
with biologically important nucleophiles (Nu ) DNA,
enzymes) in an analogous fashion and, by the interme-
diacy of the exocyclic methylene species 12, irreversibly
alkylate them. With these presumptions, 1-methylpyra-
zolo[1,2-a]benzotetrazin-3-ones are designed as novel
alkylating agents. Of course, the nature and position of
the different substituents in the benzotetrazine moiety
can affect each step of the ring-opening process outlined
in Scheme 2 and therefore the alkylating ability: our
choice of groups in the 8 or 9 position allows wide
exploitation of the chemistry.
Biology
acid, were coupled with the furan 6 to give the 2-arylazo
derivatives 7a-h. Rearrangement of these upon treat-
ment with concentrated hydrochloric acid afforded the
2-arylpyrazolones 8 through a well-established path-
way7 that can be envisaged as a ring opening-ring
closure sequence. Electron-withdrawing substituents on
the phenyl ring markedly favor the conversion 7f8,
whereas steric effects seem to be unimportant, since the
presence of the nitro group in the 2 position did not
preclude the rearrangement.
The reduction of the nitro group was carried out by
standard procedures, either with hydrogen and Pd at
room temperature or with iron in acetic acid at 60 °C
in the case of substrates bearing a chlorine. In all the
cases the 2-(2-aminoaryl)pyrazolones 9 were isolated in
yields from good to nearly quantitative. The amines
9a-h were treated with sodium nitrite in concentrated
sulfuric acid at 0 °C, and after keeping the reaction
mixture at 60 °C for 3 h it was possible to isolate, upon
neutralization, the 1-methylpyrazolo[1,2-a]benzo[1,2,3,4]-
tetrazin-3-ones 4a-h in yields from good to excellent.
The chemical behavior of this class of pyrazolo-
tetrazinones toward selected nucleophiles was investi-
gated under different reaction conditions. The parent
compound6 was already demonstrated to react with
alcohols under reflux to give the alkoxymethylpyrazoli-
none 13 (R ) R′)H, Nu ) MeO, EtO, CD3O) and with
The pyrazolo[1,2-a]benzo[1,2,3,4]tetrazinone deriva-
tives 4a-h were selected by the National Cancer
Institute (Bethesda, MD) for testing in the developmen-
tal therapeutics program (DTP) against a panel of
approximately 60 tumor cell lines that have grouped in
disease subpanels, including leukaemia, non-small-cell
lung, colon, central nervous system, melanoma, ovarian,
renal, prostate, and breast tumors cell lines. The in vitro
test system and the information, encoded by the activity
pattern over all cell lines, were obtained (see experi-
mental) according to the previously reported methodol-
ogy.8 The antitumor activity of a test compound is given
by three parameters for each cell line; pGI50 value (GI50
is the molar concentration of the compound that inhibits
50% net cell growth), pTGI value (TGI is the molar
concentration of the compound leading to total inhibition
of net cell growth), and pLC50 value (LC50 is the molar
concentration of the compound that induces 50% net cell
death). Moreover, a mean graph midpoint (MG•MID)
is calculated for each of the mentioned parameters,
giving an average activity parameter over all cell lines.
For the calculation of the MG•MID, insensitive cell
lines are included with the highest concentration tested.
The discovery of compounds with new selectivity pat-
terns is one of the targets of the screening program.
Selectivity of a compound with respect to a certain cell

Antitumor Pyrazolobenzotetrazinones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2861
Scheme 2
Table 1. Overview of the Results of the in Vitro Antitumor Screening for Compounds 4a
pTGIc
pLC50
b
d
pGI50
no.
no. giving
no. giving
no. giving
compd studiede positive resultse
range
MG•MIDf positive resultse
range
MG•MID positive resultse
range
MG•MID
4a
4b
4c
4e
4f
4g
4h
55
53
55
55
53
54
54
53
53
54
55
51
54
53
5.68-4.53
5.85-4.50
5.75-4.50
6.28-4.59
5.75-4.67
6.76-4.46
6.05-4.50
4.75
5.11
4.90
5.05
4.96
5.41
4.95
50
52
50
54
52
51
52
5.23-4.08
5.53-4.07
5.45-4.00
5.72-4.10
5.47-4.34
6.14-4.29
5.53-4.02
4.41
4.76
4.59
4.71
4.67
5.05
4.61
40
49
48
50
50
46
45
4.26-4.06
5.25-4.11
5.19-4.05
5.27-4.11
5.21-4.01
5.36-4.01
5.25-4.05
4.14
4.44
4.30
4.41
4.34
4.69
4.29
a Data obtained from the NCI’s in vitro disease-oriented human tumor cells screen. b pGI50 is the -log of the molar concentration that
inhibits 50% net cell growth. c pTGI is the -log of the molar concentration giving total growth inhibition. d pLC50 is the -log of the molar
concentration leading to 50% net cell death. e Refers to the number of cell lines. f MG•MID ) mean graph midpoint ) arithmetical mean
value for all tested cancer cell lines. If the indicated effect was not attainable within the used concentration interval, the highest tested
concentration was used for the calculation.
line of the screen is characterized by a high deviation
of the particular cell line parameter compared to the
MG•MID value. The data from these in vitro screenings
can be presented in different formats; herein we report
in Table 1 the results in terms of number of cell lines
investigated and activity range, together with the
MG•MID values.
With respect to the tumor subpanel, the pyrazolo[1,2-
a]benzo[1,2,3,4]tetrazinones were particularly effica-
cious against all kind of leukemia. In fact, the calculated
pGI50 MG•MID values of the leukemia subpanel are
always higher than the overall cell line MG•MID values
(∆MG•MID 0.57-0.93). In the non-small cell lung
subpanel, the most sensitive cell line was NCI H522,
which was inhibited by all the pyrazolo[1,2-a]benzo-
[1,2,3,4]tetrazinone derivatives (range 5.77-5.23),
whereas in the colon cancer one, the activity against
COLO-205 and SW-620 was remarkable (range 5.85-
4.73 and 5.78-5.06, respectively). The SF 539 (of the
CNS subpanel) and OVCAR 3 (of the ovarian subpanel)
were inhibited by derivatives 4b,c and 4f-h, generally
in the low micromolar range (pGI50 > 4.90 and up to
6.05).
Excellent response was obtained in the renal subpanel
in which most of the derivatives had GI50 in the low
micromolar range, the most sensitive renal cancer lines
being 786-0, CAKI-1, TK-10, and UO-31.
Pyrazolo[1,2-a]benzo[1,2,3,4]tetrazinones were also
efficacious against breast cancer lines with particular
respect to MCF7 and T-47D (with pGI50 reaching 5.85).
Results and Discussion
An evaluation of the data reported in the table
revealed that, with the sole exception of compound 4d,
which was inactive in all the screening tests, all the new
pyrazolo[1,2-a]benzo[1,2,3,4]tetrazinones showed anti-
proliferative activity against every type of tumor cell
lines investigated. Considering the MG•MID values,
the most active compound of the series was demon-
strated to be derivative 4g, at either GI50 and TGI level,
followed by 4b and 4e. Compound 4f inhibited the 51
cell lines at micromolar concentrations at the GI50 level
(range 5.75-4.67) and only in a few cases was less active
(p ≈ 4.50) at the TGI level. The other two derivatives,
4c and 4h, although less potent, inhibited a large
number of the investigated cell lines, the parent com-
pound being the least active. Only at the LC50 level was
the number of cell lines giving positive response lower
(40-50).
The most interesting derivative of the pyrazolo[1,2-
a]benzo[1,2,3,4]tetrazinone series 4g, besides the activ-
ity against renal cancer, showed the best selectivity

2862 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Almerico et al.
against all the leukemia and colon cancer cell lines,
being also remarkably active against melanoma and
breast cancer subpanel at GI50 level, whereas when TGI
is considered, the leukemia, colon, and melanoma
anticancer activity was retained. Analogous consider-
ations can be drawn when LC50 is considered.
As far as structure-activity relationships are con-
cerned, it seems that the presence of a chlorine atom is
well-tolerated in both positions 8 and 9 of the polycyclic
ring, without any remarkable difference. Whereas in the
case of the methyl group, switching from the 8 to 9
position gives rise to the most active compound of the
series, either for the number of cell lines inhibited and
for selectivity against a certain subpanel.
compounds showed a Pearson Correlation Coefficient
(PCC) of 0.674, 0.656, and 0.734, at GI50 level, and a
lower value, 0.617, 0.553, and 0.640, at TGI level. In
all the cases the first rank was with compound S133100
(rifamycin SV), which however does not have a reported
mechanism of action. We also utilized the new facility
3D-MIND12 (drug discovery and data mining informa-
tion for new directions) that allows a simultaneous
examination of the information found in the DTP
antitumor drug screen by using “self-organizing maps”
(SOMs), which cluster these data in the high-dimen-
sional GI50 space and provide a means of its visual
translation into a two-dimensional anticancer map.
SOM anticancer maps organize the data from tested
agents into regions that share the same pattern of
growth inhibition and which substantially reflect their
molecular targets and modes of action. The projected
cluster list gives the NSC number and the location
corresponding to the projected compound. The results
of this analysis on derivative 4g indicate its location in
the Q3 region map where the cytidine analogues are
found and in particular derivative NSC626155. These
data suggest that this class of compounds is worthy of
great attention. It is our intention to undertake studies
directed to elucidate the biochemical mechanism of
action of this series of pyrazolo[1,2-a]benzo[1,2,3,4]-
tetrazinones.
The pyrazolo[1,2-a]benzo[1,2,3,4]tetrazinone deriva-
tives 4b,c,e-h were evaluated as anticancer agents in
an in vivo animal model in which polyvinylidene fluoride
hollow fibers containing various human cancer cell
cultures were implanted intraperitoneally (ip) and
subcutaneously (sc) into mice and compounds were
administrated by the ip route.9 The effects of the
compounds on reduction of viable cancer cell mass
compared to those of controls were determined. All
compounds were tested in the hollow fiber assay against
a 6-cell line panel consisting of the hematopoietic cell
lines (K-562, MOLT-4, HL-60, SR, RPMI 8226, CCRF-
CEM) as described previously.10 The compounds were
solubilized in 10% DMSO in saline/Tween-80R and
administrated ip once daily for a total of four doses at
each of two dose levels (150 and 100 mg/kg). The day
after the last compound dose, the fibers were collected
and assessed for viable cell mass. Only compounds 4c
(RdOMe), despite the poor MG•MID value reported in
in vitro tests, achieved a total score 18 (out of a
maximum of 24) and gave rise to a net cell kill.
The data above-described suggest that the pyrazolo-
[1,2-a]benzo[1,2,3,4]tetrazinones 4 do behave as anti-
tumor agent. With the aim of investigating the probable
mechanism of action, preliminary DNA binding assays
were carried out (data not shown), but the results were
controversial, since some of the derivatives seem to
interact with DNA, although it was impossible to
evidence any covalent binding. Moreover, to model the
biological action we carried out reaction with a purine
and a pyrimidine base. However, under laboratory
conditions (i.e. phosphate buffer, pH ≈8) no reaction
with guanine or adenine was evidenced and again
compounds of type 13 (Nu ) OH) were obtained.
Certainly these data are not conclusive, since solubility
problems can limit the reactivity of derivatives 4 toward
the nucleophile present in lower concentration. On the
other hand, still other biological nucleophiles could be
the final target of the generated cation, or pyrazoloben-
zotetrazinones may need bioactivation in vivo, a feature
common to many other alkylating agents (i.e. dacarba-
zine or mitomycin). If ring opening of the benzotetrazine
ring is the rate-determining step, an electron-withdraw-
ing group should enhance the biological activity, but still
the bioactivation can occur with a different mechanism.
Conclusions
The series of pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-
ones described in this paper were primarily designed
to target DNA (as alkylating agents) because of their
chemical reactivity. In the in vitro assays they showed
inhibition of a large number of tumor cell lines at
micromolar concentrations. However, their mechanism
of action needs to be further elucidated, and other
possible targets to be alkylated could not be excluded.
Experimental Section
Methodology of the in Vitro Cancer Screen. The human
tumor cell lines of the cancer screening panel are grown in
RPMI 1640 medium containing 5% fetal bovine serum and 2
mM L-glutamine. For a typical screening experiment, cells are
inoculated into 96-well microtiter plates in 100 µL at plating
densities 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 °C, 5%
CO2, 95% air, and 100% relative humidity for 24 h prior to
addition of experimental drugs. After 24 h, two plates of each
cell line are fixed in situ with TCA, to represent a measure-
ment of the cell population for each cell line at the time of
drug addition (Tz). Experimental drugs are solubilized in
DMSO at 400-fold the desired final maximum test concentra-
tion 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 complete
medium containing 50 µg/mL gentamicin. Additional 4-fold,
1
10-fold, or /2 log serial dilutions are made to provide a total
of five drug concentrations plus control. Aliquots of 100 µL of
these different drug dilutions are added to the appropriate
microtiter wells already containing 100 µL of medium, result-
ing in the required final drug concentrations.
Following drug addition, the plates are incubated for an
additional 48 h at 37 °C, 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 µL of cold 50% (w/v) TCA (final concentration,
10% TCA) and incubated for 60 min at 4 °C. The supernatant
To predict the probable mechanism of action, a
computerized analysis COMPARE11 was performed for
the derivatives most active in vitro and in vivo, com-
pounds 4g,b,c, respectively. When tested as seeds
against the NCI “Standard Agents” Database, the

Antitumor Pyrazolobenzotetrazinones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2863
is discarded, and the plates are washed five times with tap
water and air-dried. Sulforhodamine B (SRB) solution (100 µL)
at 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 fixing settled cells at the bottom of
the wells by gently adding 50 µL of 80% TCA (final concentra-
tion, 16% TCA). Using the seven absorbance measurements
[time zero (Tz), control growth (C), and test growth in the
presence of drug at the five concentration levels (Ti)], the
percentage growth is calculated at each of the drug concentra-
tions levels. Percentage growth inhibition is calculated as
2,5-Dimethyl-2-(4-methyl-2-nitrophenylazo)-3-oxo-2,3-
dihydrofuran (7b) was recrystallized from ethanol (yield
80%): mp 71 °C; IR 1714 (CO), 1531 and 1340 (NO2) cm-1; 1H
NMR δ 1.62 (3H, s, CH3), 2.46 (6H, bs, 2 × CH3), 5.81 (1H, s,
H-4), 7.28 (1H, d, J ) 8.8 Hz, H-6′), 7.59 (1H, dd, J ) 8.8, 1.0
Hz, H-5′), 7.95 (1H, d, J ) 1.0 Hz, H-3′); 13C NMR ppm 16.65
(q), 18.2 (q), 20.56 (q), 102.7 (d), 107.5 (s), 118.1 (d), 124.2 (d),
134.2 (d), 140.9 (s), 143.4 (s), 146.7 (s), 191.7 (s), 194.9 (s).
2,5-Dimethyl-2-(4-methoxy-2-nitrophenylazo)-3-oxo-
2,3-dihydrofuran (7c) was isolated as an oil that solidified
on standing (yield 98%): mp 40 °C; IR 1712 (CO), 1535 and
1
1338 (NO2) cm-1; H NMR (CDCl3) δ 1.73 (3H, s, CH3), 2.43
(3H, s, CH3), 3.90 (3H, s, OCH3), 5.54 (1H, s, H-4), 7.07 (1H,
dd, J ) 8.8, 1.5 Hz, H-5′), 7.33-7.40 (2H, m, H-3′ and H-6′);
13C NMR (CDCl3) ppm 16.8 (q), 18.3 (q), 56.0 (q), 102.7 (d),
107.5 (s), 108.3 (d), 118.4 (d), 119.4 (d), 137.3 (s), 148.6 (s),
161.5 (s), 190.7 (s), 196.1 (s).
[(Ti - Tz)/(C - Tz)] × 100
2,5-Dimethyl-2-(4-hydroxy-2-nitrophenylazo)-3-oxo-
2,3-dihydrofuran (7d) was recrystallized from ethanol (yield
93%): mp 139 °C; IR 3292 (broad OH), 1688 (CO), 1585 and
for concentrations for which Ti g Tz
1346 (NO2) cm-1 1H NMR δ 1.57 (3H, s, CH3), 2.43 (3H, s,
;
[(Ti - Tz)/Tz] × 100
for concentrations for which Ti < Tz
CH3), 5.76 (1H, s, H-4), 7.11 (1H, dd, J ) 8.8, 2.9 Hz, H-5′),
7.37 (1H, d, J ) 2.9 Hz, H-3′), 7.42 (1H, d, J ) 8.8 Hz, H-6′),
11.28 (1H, s, OH); 13C NMR ppm 16.7 (q), 18.1 (q), 102.6 (d),
107.0 (s), 109.9 (d), 119.5 (d), 119.8 (d), 134.6 (s), 149.8 (s),
161.4 (s), 191.5 (s), 195.6 (s).
Three dose-response parameters are calculated for each
experimental agent. Growth inhibition of 50% (GI50) is calcu-
lated from [(Ti - Tz)/(C - Tz)] × 100 ) 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 incubation. The drug concentration resulting in total
growth inhibition (TGI) is calculated from Ti ) 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] ×
100 ) -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 concentration tested.
2,5-Dimethyl-2-(4-chloro-2-nitrophenylazo)-3-oxo-2,3-
dihydrofuran (7e) was recrystallized from ethanol (yield
99%): mp 95 °C; IR 1715 (CO), 1537 and 1342 (NO2) cm-1; 1H
NMR δ 1.62 (3H, s, CH3), 2.46 (3H, s, CH3), 5.82 (1H, s, H-4),
7.41 (1H, d, J ) 8.8 Hz, H-6′), 7.87 (1H, d, J ) 8.8 Hz, H-5′),
8.34 (1H, s, H-3′); 13C NMR ppm 16.8 (q), 18.4 (q), 102.7 (d),
107.8 (s), 120.2 (d), 124.4 (d), 134.0 (d), 136.3 (s), 141.5 (s),
147.1 (s), 191.9 (s), 194.5 (s).
2,5-Dimethyl-2-(4-fluoro-2-nitrophenylazo)-3-oxo-2,3-
dihydrofuran (7f) was recrystallized from ethanol (yield
96%): mp 108 °C; IR 1715 (CO), 1537 and 1341 (NO2) cm-1
;
1H NMR δ 1.63 (3H, s, CH3), 2.46 (3H, s, CH3), 5.81 (1H, s,
H-4), 7.51 (1H, dd, JHH ) 8.8 Hz, JFH ) 4.9 Hz, H-6′), 7.69
(1H, ddd, JHH ) 8.8, 2.9 Hz, JFH ) 8.3 Hz, H-5′), 8.19 (1H, dd,
JHH ) 2.9 Hz, JFH ) 8.3 Hz, H-3′); 13C NMR ppm 16.7 (q), 18.3
(q), 102.7 (d), 107.6 (s), 112.2 (d, JCF ) 9.6 Hz), 120.8 (d, JCF
) 28.3 Hz), 121.0 (d, JCF ) 23.1 Hz), 139.5 (s, JCF ) 3.9 Hz),
147.7 (s, JCF ) 10.5), 165.2 (s, JCF ) -254.1 Hz), 191.8 (s),
194.8 (s).
Adriamycin was tested with each screening run as a quality
control measure. This is done to establish the ability of the
screen to generate information relevant to mechanism of
growth inhibition/cell killing. It is not a reference compound
that has unique importance for the compounds.
Chemistry. All melting points were taken on a Buchi-
Tottoli capillary apparatus and are uncorrected; IR spectra
were determined in bromoform with a Jasco FT/IR 5300
2,5-Dimethyl-2-(5-methyl-2-nitrophenylazo)-3-oxo-2,3-
dihydrofuran (7g) was recrystallized from ethanol (yield
82%): mp 89 °C; IR 1713 (CO), 1522 and 1342 (NO2) cm-1; 1H
NMR δ 1.64 (3H, s, CH3), 2.43 (3H, s, CH3), 2.46 (3H, s, CH3),
5.82 (1H, s, H-4), 7.08 (1H, s, H-6′), 7.57 (1H, d, J ) 8.8 Hz,
H-4′), 8.05 (1H, d, J ) 8.8 Hz, H-3′); 13C NMR ppm 16.7 (q),
18.3 (q), 20.8 (q), 102.7 (d), 107.6 (s), 118.5 (d), 124.6 (d), 132.1
(d), 143.6 (s), 143.9 (s), 145.6 (s), 191.7 (s), 194.7 (s).
2,5-Dimethyl-2-(5-chloro-2-nitrophenylazo)-3-oxo-2,3-
dihydrofuran (7h) was purified by column chromatography
using light petroleum ether (bp 40-60 °C):ethyl acetate 8:2
as eluant (yield 70%): mp 87 °C; IR 1716 (CO), 1537 and 1342
(NO2) cm-1; 1H NMR (CDCl3) δ 1.79 (3H, s, CH3), 2.46 (3H, s,
CH3), 5.58 (1H, s, H-4), 7.18 (1H, d, J ) 2.0 Hz, H-6′), 7.54
(1H, dd, J ) 8.8, 2.0 Hz, H-4′), 7.98 (1H, d, J ) 8.8 Hz, H-3′);
13C NMR (CDCl3) ppm 17.1 (q), 18.7 (q), 103.1 (d), 108.4 (s),
118.9 (d), 125.8 (d), 130.4 (d), 134.0 (2s), 145.9 (s), 191.1 (s),
196.2 (s).
General Method for the Preparation of 5-Methyl-1-(2-
nitroaryl)pyrazol-3-ones 8a-h. 2,5-Dimethyl-2-(arylazo)-3-
oxo-2,3-dihydrofuran 7a-h (10 mmol) was added in small
portions, with stirring, to concentrated hydrochloric acid (37%,
7 mL), taking care that the temperature of the mixture does
not exceed 30 °C. After the addition was complete, the stirred
mixture is kept at 20-30 °C, usually for 1 h. The cooled
mixture was then poured onto ice water and basified with 30%
aqueous sodium hydroxide. It was then diluted with water to
complete dissolution of the sodium salt of the pyrazolone. The
resultant solution is extracted with diethyl ether (3 × 100 mL).
1
spectrophotometer; H and 13C NMR spectra were measured
respectively in DMSO-d6 solution, unless otherwise specified
(TMS as internal reference), at 200 and 50.3 MHz, using a
Bruker AC-E series 200 MHz spectrometer. Column chroma-
tography was performed with Merck silica gel 230-400 mesh
ASTM. Microanalyses were in agreement with theoretical
values (0.4%.
General Method for the Preparation of 2,5-Dimethyl-
2-(2-nitroarylazo)-3-oxo-2,3-dihydrofurans 7a-h. To a
suspension of the 2-nitroanilines 5a-h (20 mmol) in water (10
mL) was added hydrochloric acid (37%, 5 mL), and the mixture
was diazotized with a solution of sodium nitrite (1.48 g, 21.5
mmol) in water (8 mL) at 0 °C (only in the case of 5e was the
diazotization carried out at room temperature). The mixture
was stirred for 1 h, diluted with water (50 mL), and added
with concentrated hydrochloric acid (3 mL). Then, freshly
distilled 2,5-dimethyl-3-oxo-2,3-dihydrofuran13 (6) (2.47 g, 22
mmol) was added and the mixture was stirred for 1 h at room
temperature. The solid precipitated was filtered off, washed
with water, and air-dried. It was then purified by recrystal-
lization or by column chromatography.
2,5-Dimethyl-2-(2-nitrophenylazo)-3-oxo-2,3-dihydro-
furan (7a) was recrystallized from ethanol (yield 90%): mp
74 °C; IR 1680 (CO), 1535 and 1340 (NO2) cm-1 1H NMR
;
(CDCl3) δ 1.76 (3H, s, CH3), 2.40 (3H, s, CH3), 5.50 (1H, s, H-4),
7.10-8.00 (4H, m, C6H4); 13C NMR (CDCl3) ppm 17.1 (q), 18.7
(q), 103.0 (d), 108.1 (s), 118.8 (d), 124.3 (d), 130.8 (d), 133.5 (d)
140.4 (s), 143.1 (s), 146.0 (s), 190.9 (s).

2864 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Almerico et al.
The aqueous phase was acidified with diluted hydrochloric
acid, with stirring and cooling, to precipitate the product. The
solid was filtered off, washed with water, and air-dried.
5-Methyl-1-(2-nitrophenyl)pyrazol-3-one (8a) was re-
crystallized from ethanol (yield 94%): mp 219 °C; IR 3200
(OH), 1535 and 1345 (NO2) cm-1; 1H NMR δ 2.24 (3H, s, CH3),
5.66 (1H, s, H-4), 7.60-8.10 (4H, m, C6H4), 9.90 (1H, s, NH);
13C NMR ppm 11.4 (q), 93.6 (d), 124.8 (d), 128.4 (d), 128.6 (d),
131.8 (s), 133.2 (d), 140.8 (s), 145.9 (s), 162.0 (s).
1-(4-Methyl-2-nitrophenyl)-5-methylpyrazol-3-one (8b)
was recrystallized from ethanol (yield 88%): mp 235 °C; IR
3200 (OH), 1537 and 1345 (NO2) cm-1; 1H NMR δ 2.15 (3H, s,
CH3), 2.45 (3H, s, CH3), 5.61 (1H, s, H-4), 7.55-7.64 (2H, m,
H-6′ and H-5′), 7.86 (1H, s, H-3′), 9.97 (1H, s, NH); 13C NMR
ppm 11.6 (q), 20.4 (q), 93.6 (d), 125.0 (d), 128.5 (d), 129.6 (s),
133.8 (d), 139.4 (s), 141.0 (s), 145.9 (s), 162.1 (s).
1-(4-Methoxy-2-nitrophenyl)-5-methylpyrazol-3-one (8c)
was recrystallized from ethanol (yield 75%): mp 194 °C; IR
3200 (OH), 1535 and 1345 (NO2) cm-1; 1H NMR δ 2.11 (3H, s,
CH3), 3.90 (3H, s, OCH3), 5.58 (1H, s, H-4), 7.33-7.63 (3H, m,
C6H3), 9.90 (1H, s, NH); 13C NMR ppm 11.5 (q), 56.2 (q), 92.9
(d), 110.0 (d), 118.7 (d), 124.8 (s), 130.2 (d), 141.2 (s), 147.1
(s), 158.8 (s), 161.9 (s).
4.90 (2H, s, NH2), 5.52 (1H, s, H-4), 6.40-7.32 (4H, m, C6H4),
9.70 (1H, s, NH); 13C NMR ppm 11.5 (q), 91.1 (d), 115.2 (2d),
124.2 (s), 127.3 (d), 128.5 (d), 140.3 (s), 144.4 (s), 161.2 (s).
1-(2-Amino-4-methylphenyl)-5-methylpyrazol-3-one (9b)
was recrystallized from ethanol (yield 72%): mp 235 °C; IR
3476 and 3381 (NH2), 3150 (OH) cm-1; 1H NMR δ 2.00 (3H, s,
CH3), 2.20 (3H, s, CH3), 4.85 (2H, s, NH2), 5.49 (1H, s, H-4),
6.41 (1H, d, J ) 7.4 Hz, H-5′), 6.61 (1H, s, H-3′), 6.87 (1H, d,
J ) 7.4 Hz, H-6′), 9.77 (1H, s, NH); 13C NMR ppm 11.6 (q),
21.0 (q), 91.3 (d), 116.2 (d), 116.8 (d), 122.2 (s), 127.3 (d), 138.1
(s), 140.6 (s), 144.3 (s), 161.2 (s).
1-(2-Amino-4-methoxyphenyl)-5-methylpyrazol-3-
one (9c) was recrystallized from ethanol (yield 88%): mp 223
°C; IR 3480 and 3382 (NH2), 3138 (OH) cm-1; 1H NMR δ 1.99
(3H, s, CH3), 3.70 (3H, s, OCH3), 4.90 (2H, s, NH2), 5.49 (1H,
s, H-4), 6.20-6.37 (2H, m, H-5′ and H-3′), 6.90 (1H, d, J ) 7.1
Hz, H-6′), 9.77 (1H, s, NH); 13C NMR ppm 11.5 (q), 54.9 (q),
91.1 (d), 100.2 (d), 101.8 (d), 118.1 (s), 128.6 (d), 140.8 (s), 145.9
(s), 159.7 (s), 161.7 (s).
1-(2-Amino-4-hydroxyphenyl)-5-methylpyrazol-3-one
(9d) was recrystallized from ethanol (yield 66%): mp 190 °C;
IR 3476 and 3385 (NH2), 3140 (OH), 2971 (OH) cm-1; 1H NMR
δ 2.01 (3H, s, CH3), 4.74 (2H, s, NH2), 5.47 (1H, s, H-4), 6.03
(1H, dd, J ) 8.8, 2.0 Hz, H-5′), 6.23 (1H, d, J ) 2.0 Hz, H-3′),
6.78 (1H, d, J ) 8.8 Hz, H-6′), 9.28 (1H, s, NH), 9.70 (1H, broad
OH); 13C NMR ppm 11.5 (q), 91.0 (d), 101.8 (d), 103.6 (d), 116.9
(s), 128.5 (d), 140.7 (s), 145.9 (s), 157.9 (s), 161.1 (s).
1-(4-Hydroxy-2-nitrophenyl)-5-methylpyrazol-3-one (8d)
was recrystallized from ethanol (yield 92%): mp 201 °C; IR
1
3200 (broad OH), 1535 and 1345 (NO2) cm-1; H NMR δ 2.09
(3H, s, CH3), 5.55 (1H, s, H-4), 7.16 (1H, dd, J ) 8.8, 2.9 Hz,
H-5′), 7.35 (1H, d, J ) 2.9 Hz, H-3′), 7.48 (1H, d, J ) 8.8 Hz,
H-6′), 10.29 (2H, broad, NH and OH); 13C NMR ppm 11.5 (q),
92.7 (d), 11.2 (d), 119.9 (d), 123.5 (s), 130.5 (d), 141.3 (s), 147.1
(s), 157.7 (s), 161.7 (s).
1-(2-Amino-4-fluorophenyl)-5-methylpyrazol-3-one (9f)
was purified by chromatography using dichloromethane:
methanol 95:05 as eluant and recrystallized from ethanol (yield
90%): mp 216 °C; IR 3478 and 3383 (NH2), 3200 (OH) cm-1
;
1H NMR δ 2.01 (3H, s, CH3), 5.25 (2H, s, NH2), 5.53 (1H, s,
H-4), 6.38 (1H, ddd, JHH ) 8.8, 2.9 Hz, JFH ) 8.4 Hz, H-5′),
6.60 (1H, dd, JHH ) 2.9 Hz, JFH )8.4 Hz, H-3′), 7.03 (1H, dd,
JHH ) 8.8 Hz, JFH ) 5.9 Hz, H-6′), 9.87 (1H, s, NH); 13C NMR
ppm 11.4 (q), 91.7 (d), 101.5 (d, JCF ) 25.0 Hz), 102.2 (d, JCF
) 23.2 Hz), 120.7 (s, JCF ) 2.2 Hz), 129.3 (d, JCF ) 11.3 Hz),
1-(4-Chloro-2-nitrophenyl)-5-methylpyrazol-3-one (8e)
was recrystallized from ethanol (yield 76%): mp 220 °C; IR
3150 (OH), 1535 and 1344 (NO2) cm-1; 1H NMR δ 2.11 (3H, s,
CH3), 5.66 (1H, s, H-4), 7.77 (1H, d, J ) 7.3 Hz, H-6′), 7.90
(1H, d, J ) 7.3 Hz, H-5′), 8.21 (1H, s, H-3′), 10.10 (1H, s, NH);
13C NMR ppm 11.6 (q), 94.2 (d), 125.0 (d), 129.8 (d), 130.9 (s),
132.5 (d), 133.2 (s), 141.4 (s), 146.4 (s), 162.5 (s).
140.9 (s), 146.8 (s, JCF ) 12.5 Hz), 160.0 (s), 164.8 (s, JCF
-241.4 Hz).
)
1-(4-Fluoro-2-nitrophenyl)-5-methylpyrazol-3-one (8f)
was recrystallized from ethanol (yield 95%): mp 218 °C; IR
3150 (OH), 1537 and 1364 (NO2) cm-1; 1H NMR δ 2.17 (3H, s,
CH3), 5.64 (1H, s, H-4), 7.71 (1H, ddd, JHH ) 8.8, 2.9 Hz, JFH
) 8.8 Hz, H-5′), 7.81 (1H, dd, JHH ) 8.8 Hz, JFH ) 4.9 Hz,
H-6′), 8.07 (1H, dd, JHH ) 2.9 Hz, JFH ) 8.8 Hz, H-3′), 10.06
1-(2-Amino-5-methylphenyl)-5-methylpyrazol-3-one (9g)
was recrystallized from ethanol (yield 88%): mp 205 °C; IR
3449 and 3364 (NH2), 3140 (OH) cm-1; 1H NMR δ 2.02 (3H, s,
CH3), 2.17 (3H, s, CH3), 4.74 (2H, s, NH2), 5.52 (1H, s, H-4),
6.73 (1H, d, J ) 7.8 Hz, H-3′), 6.83 (1H, d, J ) 2.0 Hz, H-6′),
6.92 (1H, dd, J ) 7.8, 2.0 Hz, H-4′), 9.61 (1H, s, NH); 13C NMR
ppm 11.6 (q), 19.9 (q), 91.5 (d), 116.1 (d), 124.4 (s), 124.8 (s),
127.7 (d), 129.4 (d), 140.5 (s), 142.0 (s), 161.3 (s).
Method B. Iron powder (670 mg, 12 mmol) was added to a
solution of nitro derivative 8e,h (3.4 mmol) in acetic acid (30
mL). The mixture was kept at 60 °C 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 that was purified by by recrystallization or by column
chromatography.
1-(2-Amino-4-chlorophenyl)-5-methylpyrazol-3-one (9e)
was recrystallized from ethanol (yield 90%): mp 216 °C; IR
3478 and 3383 (NH2), 2924 (OH) cm-1; 1H NMR δ 2.02 (3H, s,
CH3), 5.29 (2H, s, NH2), 5.53 (1H, s, H-4), 6.60 (1H, dd, J )
8.8, 2.9 Hz, H-5′), 6.85 (1H, d, J ) 2.9 Hz, H-3′), 7.00 (1H, d,
J ) 8.8 Hz, H-6′), 9.88 (1H, s, NH); 13C NMR ppm 11.5 (q),
91.9 (d), 114.8 (d), 115.2 (d), 123.0 (s), 129.0 (d), 132.9 (s), 140.8
(s), 146.1 (s), 161.5 (s).
1-(2-Amino-5-chlorophenyl)-5-methylpyrazol-3-one (9h)
was purified by chromatography using light petroleum ether
(bp 40-60 °C):ethyl acetate 8:2 as eluant (yield 90%): mp 210
°C; IR 3418 and 3343 (NH2), 2950 (OH) cm-1; 1H NMR δ 2.04
(3H, s, CH3), 5.16 (2H, s, NH2), 5.54 (1H, s, H-4), 6.82 (1H, d,
J ) 8.8 Hz, H-3′), 7.10 (1H, dd, J ) 8.8, 2.0 Hz, H-4′), 7.16
(1H, d, J ) 1.95 Hz, H-6′), 9.93 (1H, s, NH); 13C NMR ppm
11.5 (q), 92.0 (d), 117.0 (d), 118.5 (s), 124.8 (s), 126.9 (d), 128.6
(d), 140.9 (s), 143.8 (s), 161.6 (s).
(1H, s, NH); 13C NMR ppm 11.5 (q), 93.8 (d), 118.9 (d, JCF
)
28.3 Hz), 120.4 (d, JCF ) 22.2 Hz), 128.7 (s, JCF ) 3.9 Hz),
130.7 (d, JCF ) 8.7 Hz), 141.4 (s), 146.6 (s, JCF ) 9.6 Hz), 162.3
(s), 162.7 (s, JCF ) -249.8 Hz).
1-(5-Methyl-2-nitrophenyl)-5-methylpyrazol-3-one (8g)
was recrystallized from ethanol (yield 84%): mp 217 °C; IR
3200 (OH), 1535 and 1345 (NO2) cm-1; 1H NMR δ 2.16 (3H, s,
CH3), 2.45 (3H, s, CH3), 5.61 (1H, s, H-4), 7.44 (1H, dd, J )
8.8, 1.0 Hz, H-4′), 7.52 (1H, d, 1.0 Hz, H-6′), 7.92 (1H, d, J )
8.8 Hz, H-3′), 9.98 (1H, s, NH); 13C NMR ppm 11.5 (q), 20.8
(q), 93.6 (d), 124.9 (d), 129.0 (d), 129.2 (d), 132.1 (s), 140.9 (s),
143.8 (s), 144.6 (s), 162.1 (s).
1-(5-Chloro-2-nitrophenyl)-5-methylpyrazol-3-one (8h)
was recrystallized from ethanol (yield 65%): mp 228 °C; IR
3150 (OH), 1541 and 1346 (NO2) cm-1; 1H NMR δ 2.23 (3H, s,
CH3), 5.72 (1H, s, H-4), 7.73 (1H, d, J ) 8.8 Hz, H-3′), 7.95
(1H, d, J ) 2.0 Hz, H-6′), 8.07 (1H, dd, J ) 8.8, 2.0 Hz, H-4′),
10.12 (1H, s, NH); 13C NMR ppm 11.5 (q), 94.5 (d), 126.7 (d),
128.2 (d), 128.7 (d), 133.4 (s), 137.5 (s), 141.5 (s), 144.7 (s),
162.6 (s).
Preparation of 1-(2-aminoaryl)-5-methylpyrazol-3-
ones 9a-h. Method A. A solution of nitro derivatives 8a-
d,f,g (42 mmol) in ethanol was reduced overnight over 10%
Pd on charcoal 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 that was
purified by recrystallization or by column chromatography.
1-(2-Aminophenyl)-5-methylpyrazol-3-one (9a) was re-
crystallized from benzene (yield 70%): mp 203 °C; IR 3480
and 3380 (NH2), 3100 (OH) cm-1; 1H NMR δ 2.04 (3H, s, CH3),
General Method for the Preparation of 1-Methyl-8-R-
9-R′-3H-pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-ones 4a-

Antitumor Pyrazolobenzotetrazinones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2865
h. Concentrated sulfuric acid (0.4 mL) was added to a solution
of the amine 9a-h (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 °C. The reactants were stirred for 2 h
further at room temperature, and then were kept at 60 °C 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.
1-Methyl-3H-pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-
one (4a) (yield 91%): mp 190 °C (dec); IR 1660 (CO) cm-1; 1H
NMR δ 2.50 (3H, s, CH3), 6.08 (1H, s, H-2), 7.70-8.20 (2H, m,
H-9 and H-10), 8.30-8.70 (2H, m, H-7 and H-8); 13C NMR ppm
13.3 (q), 101.2 (d), 109.7 (s), 128.6 (d), 131.8 (d), 136.5 (d), 140.6
(s), 145.1 (d), 150.0 (s), 165.7 (s).
References
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I. R.; Lewis, P.; Stevens, M. F. G. Temozolomide: A new oral
cytotoxic chemotherapeutic agent with promising activity against
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Newlands, E. S.; Blackledge, G. R. P.; Slack, J. A.; Rustin, G. J.
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trial of temozolomide (CCRG 81045: M&B 39831: NSC 362856).
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(2) (a) Stevens, M. F. G.; Hickman, J. A.; Langdon, S. P.; Chubb,
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Slack, J. A.; Newton, C. G.; Lunt, E.; Fizames, C.; Lavelle, F.
Antitumor activity and pharmacokinetics in mice of 8-carbamoyl-
3-methyl-imidazo[5,1-d]-1,2,5-tetrazin-4(3H)-one: (CCRG 81045;
M&B 39831) A novel drug with potential as an alternative to
dacarbazine. Cancer Res. 1987, 47, 5846-5852; (b) Clark, A. S.;
Stevens, M. F. G.; Sansom, C. E.; Schwalbe, C. H. Antitumor
imidazotetrazines. Part XXI. Mitozolomide and temozolomide:
Probes for the major groove of DNA. Anticancer Drug Des. 1990,
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Stevens, M. F. G.; Clark, A. S. Antitumor imidazotetrazines. 25.
Crystal structure of 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-
tetrazin-4(3H)-one (temozolomide) and structural comparison
with the related drugs mitozolomide and DTIC. J. Med. Chem.
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1,8-Dimethyl-3H-pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-
3-one (4b) (yield 91%): mp 147 °C (dec); IR 1678 (CO) cm-1
;
1H NMR δ 2.35 (3H, s, CH3), 2.62 (3H, s, CH3), 5.57 (1H, s,
H-2), 7.23-7.49 (2H, m, H-9 and H-10), 7.64 (1H, s, H7); 13C
NMR ppm 14.7 (q), 20.0 (q), 93.5 (d), 113.5 (d), 122.7 (s), 125.3
(s), 128.2 (s), 129.1 (d), 134.9 (d), 144.9 (s), 156.7 (s).
1-Methyl-8-methoxy-3H-pyrazolo[1,2-a]benzo[1,2,3,4]-
tetrazin-3-one (4c) (yield 90%): mp 155 °C (dec); IR 1660
1
(CO) cm-1; H NMR (CDCl3) δ 2.63 (3H, s, CH3), 3.87 (3H, s,
OCH3), 5.51 (1H, s, H-2), 7.03 (1H, dd, J ) 8.8, 2.9 Hz, H-9),
7.25 (1H, d, J ) 8.8 Hz, H-10), 7.37 (1H, d, J ) 2.9 Hz, H-7);
13C NMR (CDCl3) ppm 15.3 (q), 55.9 (q), 85.0 (d), 98.8 (s), 113.4
(d), 113.7 (d), 116.8 (s), 120.5 (d), 125.5 (s), 142.4 (s), 157.4 (s).
8-Hydroxy-1-methyl-3H-pyrazolo[1,2-a]benzo[1,2,3,4]-
tetrazin-3-one (4d) (yield 60%): mp >290 °C; IR 3160 (broad
1
OH), 1597 (CO) cm-1; H NMR δ 2.16 (3H, s, CH3), 5.50 (1H,
s, H-2), 6.84-7.63 (3H, m, H7, H-9 and H-10), 9.79 (1H, broad
OH); 13C NMR ppm 18.6 (q), 92.5 (d), 115.4 (d), 125.5 (d), 128.5
(d), 131.6 (s), 139.3 (s), 140.5 (s), 156.1 (s), 160.9 (s).
8-Chloro-1-methyl-3H-pyrazolo[1,2-a]benzo[1,2,3,4]-
tetrazin-3-one (4e) (yield 80%): mp 158 °C (dec); IR 1680
(CO) cm-1; 1H NMR δ 2.67 (3H, s, CH3), 5.70 (1H, s, H-2), 7.69
(1H, d, J ) 8.8 Hz, H-10), 7.58 (1H, d, J ) 8.8 Hz, H-9), 7.96
(1H, s, H-7); 13C NMR ppm 15.5 (q), 96.2 (d), 113.3 (d), 129.8
(d), 129.9 (s), 133.7 (d), 135.5 (s), 143.5 (s), 157.1 (s), 197.3 (s).
8-Fluoro-1-methyl-3H-pyrazolo[1,2-a]benzo[1,2,3,4]-
tetrazin-3-one (4f) (yield 77%): mp 143 °C (dec); IR 1672 (CO)
cm-1; 1H NMR δ 2.62 (3H, s, CH3), 5.60 (1H, s, H-2), 7.48 (1H,
ddd, JHH ) 8.8, 2.9 Hz, JFH ) 9.8 Hz, H-9), 7.57 (1H, dd, JHH
) 8.8 Hz, JFH ) 4.9 Hz, H-10), 7.75 (1H, dd, JHH ) 2.9 Hz, JFH
) 8.8 Hz, H-7); 13C NMR ppm 14.7 (q), 93.5 (d), 115.5 (d, JCF
) 18.3 Hz), 115.7 (d, JCF ) 15.3 Hz), 121.1 (d, JCF ) 23.3 Hz),
124.6 (s, JCF ) 3.1 Hz), 129.3 (s, JCF ) 9.0 Hz), 145.6 (s), 156.5
(s), 161.3 (s, JCF ) -238.8 Hz).
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M.; Dattolo, G.; Cirrincione, G. Pyrrolo[2,1-d][1,2,3,5]tetrazine-
4(3H)-ones, a new class of azolotetrazines with potent antitumor
activity. Bioorg. Med. Chem. 2003, 11, 2371-2380.
(4) Severina, I. S.; Axenova, L. N.; Veselovsky, A. V.; Pyatakova,
N. V.; Buneeva, O. A.; Ivanov, A. S.; Medvedev, A. E. Nonselec-
tive inhibition of monoamine oxidases a and b by activators of
soluble guanylate cyclase. Biochemistry (Translation of Bio-
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dihydrofurans. Useful intermediates in the synthesis of 1-aryl-
5-methyl-3-pyrazolones. Synthesis 1979, 283-287.
1,9-Dimethyl-3H-pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-
3-one (4g) (yield 77%): mp 151 °C (dec); IR 1680 (CO) cm-1
;
(8) (a) Grever, M. R.; Sherpartz, S. A.; Chabner, B. A. The National
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1
H NMR δ 2.40 (3H, s, CH3), 2.65 (3H, s, CH3), 5.59 (1H, s,
H-2), 7.15 (1H, d, J ) 7.8 Hz, H-8), 7.36 (1H, s, H-10), 7.67
(1H, d, J ) 7.8 Hz, H-7);13C NMR ppm 14.9 (q), 21.7 (q), 94.2
(d), 113.6 (d), 126.4 (d), 127.4 (s), 128.9 (d), 145.0 (s), 145.9
(2s), 156.7 (s).
9-Chloro-1-methyl-3H-pyrazolo[1,2-a]benzo[1,2,3,4]-
tetrazin-3-one (4h) (yield 60%): mp 137 °C (dec); IR 1663
(CO) cm-1; 1H NMR δ 2.66 (3H, s, CH3), 5.66 (1H, s, H2), 7.38
(1H, d, J ) 7.8 Hz, H-8), 7.49 (1H, s, H-10), 7.78 (1H, d, J )
7.8 Hz, H-7); 13C NMR ppm 14.5 (q), 94.9 (d), 113.4 (d), 125.7
(d), 126.1 (s), 128.6 (s), 130.4 (d), 138.4 (s), 145.9 (s), 156.5 (s).
Acknowledgment. This work was financially sup-
ported by Ministero dell’Istruzione dell’Universita` e
della Ricerca. We thank the National Cancer Institute
(Bethesda, MD) and especially Dr V.L. Narayanan and
his team for the antitumor tests reported in this paper.
Supporting Information Available: Elemental analyses
and tables of full biological data. This material is available
free of charge via the Internet at http://pubs.acs.org
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D. A.; Rubistein, L.; Plowman, J.; Boyd, M. R., J. Display and
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JM049075U