Synthesis and antiproliferative activity of 3-(2-chloroethyl)-5-methyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4-(3H)-one

Article abstract of DOI:10.1016/j.ejmech.2015.04.004

Based on the encouraging results found for 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4-(3H)-one 7 previously tested by us, as well as the consideration that heterocycle fused tetrazepinones bearing the 2-chloroethyl substituent show a better cytotoxic profile than temozolomide and mitozolomide against human cancer cell lines which express the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), in this paper we report the multistep synthesis and the biological study of 3-(2-chloroethyl)-5-methyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4-(3H)-one 10. Like compound 7, it was active on P-glycoprotein expressing cells (MDR) HL60 and on K562 cell line that are resistant to apoptosis induced by different stimuli, showing GI50 values of 14 and 18 μM respectively. As an antiproliferative agent against the above cells compound 10 was about 2.2 times more active than compound 7. Compound 10 was also tested against WiDR cells which are overexpressing the DNA repair protein MGMT, showing a GI50 value of 2.3 μM. Finally, concerning the effect on cell cycle we observed an evident difference between compounds 7 and 10. In fact, compound 7 induces a block of cell cycle in G0-G1, therefore acting as phase-specific drug, in contrast, compound 10 is a not phase-specific agent. Both the compounds are able to increase the apoptotic sub G0-G1 peak of cell cycle.

Full text of DOI:10.1016/j.ejmech.2015.04.004

European Journal of Medicinal Chemistry 96 (2015) 98e104
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiproliferative activity of 3-(2-chloroethyl)-5-methyl-
6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]
tetrazepin-4-(3H)-one
,
,
Benedetta Maggio a, Maria Valeria Raimondi a *, Demetrio Raffa a *, Fabiana Plescia a,
Stella Cascioferro a, Gabriella Cancemi a, Manlio Tolomeo b, Stefania Grimaudo c,
Giuseppe Daidone a
a
ꢀ
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Universita degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
b Centro Interdipartimentale di Ricerca in Oncologia Clinica e Dipartimento Biomedico di Medicina Interna e Specialistica, Sezione di Malattie Infettive,
ꢀ
Universita degli Studi di Palermo, Via del Vespro 129, 90127 Palermo, Italy
c
ꢀ
Sezione di Gastroenterologia, Dipartimento di Biomedicina Interna e Specialistica, Universita degli Studi di Palermo, Via del Vespro 129, 90127 Palermo,
Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the encouraging results found for 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-
dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4-(3H)-one 7 previously tested by us, as well as the consider-
ation that heterocycle fused tetrazepinones bearing the 2-chloroethyl substituent show a better cytotoxic
profile than temozolomide and mitozolomide against human cancer cell lines which express the DNA
repair protein O6-methylguanine-DNA methyltransferase (MGMT), in this paper we report the multistep
synthesis and the biological study of 3-(2-chloroethyl)-5-methyl-6-phenyl-8-(trifluoromethyl)-5,6-
dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4-(3H)-one 10. Like compound 7, it was active on P-glycopro-
tein expressing cells (MDR) HL60 and on K562 cell line that are resistant to apoptosis induced by
Received 6 August 2014
Received in revised form
31 March 2015
Accepted 3 April 2015
Available online 6 April 2015
Keywords:
1,2,3,5-Tetrazepinones
Pyrazolo[3,4-f][1,2,3,5]-tetrazepinones
Drug resistance
different stimuli, showing GI50 values of 14 and 18 mM respectively. As an antiproliferative agent against
the above cells compound 10 was about 2.2 times more active than compound 7. Compound 10 was also
tested against WiDR cells which are overexpressing the DNA repair protein MGMT, showing a GI50 value
Apoptosis
Antiproliferative activity
of 2.3 mM. Finally, concerning the effect on cell cycle we observed an evident difference between com-
pounds 7 and 10. In fact, compound 7 induces a block of cell cycle in G0-G1, therefore acting as phase-
specific drug, in contrast, compound 10 is a not phase-specific agent. Both the compounds are able to
increase the apoptotic sub G0-G1 peak of cell cycle.
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
However, clinical efficacy of temozolomide 1 is limited by drug
resistance which is the principal reason for the failure of anti-
Temozolomide,
or
8-carbamoyl-3-methylimidazo[5,1-d]
cancer therapies. In particular, chemoresistance to alkylating
agents may result from the cells' capacity to repair cytotoxic le-
sions. O6-methylguanine-DNA methyltransferase (MGMT), a DNA
repair protein, is known to remove alkyl groups from O6-guanine,
thus regenerating intact DNA [9,10]. So, cell lines SF-188 (Merþ)
and WiDR (Merþ), expressing elevated levels of MGMT, show sig-
nificant resistance to the action of alkylating agents like temozo-
lomide 1 than the MGMT-deficient tumor cells SF-126 (Mer-) and
BE (Mer-) [11].
[1,2,3,5]tetrazin-4-(3H)-one 1, active principle of Temodar® and
Temodal®, is a chemotherapeutic drug used in patients with
recurrent and refractory high-grade glioblastoma, gliomas, lym-
phomas and melanoma [1e6]. Temozolomide 1 is a prodrug
affording under physiological conditions
a methyl-diazonium
cation which alkylate the O6-position of guanine in DNA to form
O6-methylguanine-DNA [7,8].
The effort to overcome drug resistance to temozolomide 1, led to
the discovery of some fused 1,2,3,5-tetrazepin-4-(3H)-ones 2, 3 and
4 which, like temozolomide 1, contain the atomic sequence N]
* Corresponding authors.
E-mail addresses: mariavaleria.raimondi@unipa.it (M.V. Raimondi), demetrio.
raffa@unipa.it (D. Raffa).
http://dx.doi.org/10.1016/j.ejmech.2015.04.004
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

B. Maggio et al. / European Journal of Medicinal Chemistry 96 (2015) 98e104
99
NeN(R)COeN (Fig. 1) but their cytotoxic effects is independent of
the cell phenotypes, showing similar activity on SF-188 (Merþ) and
WiDR (Merþ) as well as SF-126 (Mer-) and BE (Mer-) cell lines [11].
Tetrazepinones act with a mechanism of action, not yet fully
understood, that is different than the one of temozolomide 1. In
fact, they are weak alkylating agents but are able to induce signif-
icant levels of DNA single strand breaks in cancer cells. Tetrazepi-
nones do not appreciably hydrolize to produce an alkylating
monomethyltriazene as temozolomide does, indeed they produce a
major benzimidazole derivative which has a weak effect on the
cells. As a consequence, the cytotoxic activity is due to tetrazepi-
none molecule itself or probably to a short-lived species afforded
during its degradation [11e15].
Due to the significant activity of 1,2,3,5-tetrazepin-4-(3H)-ones
on Mer þ cell lines, expressing elevated levels of MGMT, and the
low number of example in the literature of tetrazepinones fused
with an heterocyclic ring [11,14], we reported in earliest papers
[16,17] the synthesis of the two pyrazolo[3,4-f][1,2,3,5]tetrazepi-
nones 6 and 7 (Fig. 2), as well as the antiproliferative activity and
the antiapoptotic effects against K562, K562R (imatinib mesilate
resistant), HL60 and multidrug resistant (MDR) HL60 cell lines of 7.
Compound 7 showed antiproliferative and apoptotic effects
against K562, K562R (imatinib mesilate resistant), HL60 and
multidrug resistant (MDR) HL60 cell lines. In particular, the pro-
apoptotic activity against HL60 and K562 resistant cell lines was
markedly higher than etoposide and busulfan respectively. Finally,
flow cytometry studies carried out on K562 cells allowed to
establish that 7 induces G0-G1 phase arrest followed by apoptosis.
Considering the encouraging results found for compounds 7
[17], it was thought interesting to investigate a different substitu-
tion at the 3-position of the tetrazepinone ring of 7. At this point we
selected the 2-chloroethyl group as the 3-substituent of a new
tetrazepinone derivative. The choice was based on the fact that
heterocycle fused tetrazepinones such as 8 and 9 (Fig. 3) bearing
the 2-chloroethyl substituent, show a better cytotoxic profile than
temozolomide and mitozolomide [11] when they are tested against
human cancer cell lines which express the DNA repair protein
MGMT. In this paper we report the multi-step synthesis and the
biological study of 3-(2-chloroethyl)-5-methyl-6-phenyl-8-(tri-
Fig. 2. Structure formulas of 3,5,8-trimethyl-6-phenyl-5,6-dihydropyrazolo[3,4-f]
[1,2,3,5]tetrazepin-4-(3H)-one 6 and 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-
dihydropyrazolo [3,4-f][1,2,3,5]tetrazepin-4-(3H)-one 7.
tetrazepin-4-(3H)-one 10 was carried out as outlined in Scheme 1.
The starting pyrazole-5-amine derivative 11 was N-formylated
by formic acid to give the formamide derivative 12 which in turn
was reduced to secondary amine 13. This compound was trans-
formed to the diamine derivative 14 by nitrosation of the pyrazole
nucleus of 13 and subsequent reduction of the nitrous group [17].
The 4-amino group of 14 was first protected with the tert-butox-
ycarbonyl group, by treatment with di-tert-butyl dicarbonate
(Boc)2O, affording 15. Compound 15 was then reacted with chlor-
oethyl isocyanate at room temperature for 20 days to give com-
pound 16. Attempts to obtain 16 by heating the reaction mixture
were unsuccessful. At this point the compound was deprotected by
treatment with trifluoroacetic acid in dichloromethane to give 1-
[4-amino-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]-3-(2-
chloroethyl)-1-methylurea 17. Finally, diazotation of derivative 17
with sodium nitrite in HCl 6 N and subsequent adjustment to pH 8,
afforded
the
3-(2-chloroethyl)-5-methyl-6-phenyl-8-(tri-
fluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-
4(3H)-one 10. This last transformed rapidly to pyrazolotriazole
derivative 18 when it was heated in chloroform according to the
decomposition mechanism previously reported [17].
All the above new compounds were identified on the basis of
satisfactory elemental and spectroscopic data. The 1H NMR of 12
indicated that the compound exists as a Z/E mixture at room
temperature. When the spectrum was registered at 100 ꢀC three
distinct singlets (broad) were showed for NH, CHO and pyrazole H-
4. Compounds 10, 15, 16 and 17 showed signals attributable to the
fluoromethyl)-5,6-dihydropyrazole
(3H)-one 10 (Fig. 4).
[3,4-f][1,2,3,5]tetrazepin-4-
methyl group and to aromatic protons in the range 2.73e2.90
7.42e8.18 respectively. Moreover, compounds 15, 16, showed
signals, exchangeable with D2O, for two NH in the ranges 5.50e8.18
and 6.69e8.72 respectively. Compound 17 showed also signals,
exchangeable with D2O, for NH2 and NH at 4.44 and 6.77
respectively. Finally, two triplet at 3.81 and 4.32 were showed
for compound 10 attributable to the chloroethyl moiety.
d and
d
d
2. Chemistry
d
d
d
d
The synthesis of the novel 3-(2-chloroethyl)-5-methyl-6-
phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]
Fig. 1. Some examples of active 1,2,3,5-tetrazepin-4-(3H)-ones from the literature bearing, like Temozolomide 1, the sequence N]NeN(R)COeN.

100
B. Maggio et al. / European Journal of Medicinal Chemistry 96 (2015) 98e104
Fig. 3. Designed modification of compound 7 to improve the cytotoxic activity.
3. Pharmacological results and discussion
(concentration able to inhibit 50% of cell growth) and AC50 (con-
centration able to induce apoptosis in 50% of cells) respectively
(Table 1).
3.1. Cytotoxicity
As shown in Table 1, the antiproliferative and apoptotic activities
of compound 10 were similar in sensitive HL60 and MDR HL60-R
cell lines, respectively. This indicated that the presence of P-
glycoprotein in cells does not modify the activity of compound 10.
Compared to compound 7, that was also active in P-glycoprotein
expressing cells, compound 10 was 2.3 and 2.1 times more active as
antiproliferative agent and 1.9 and 1.7 times more active as
apoptotic agent in HL60 and HL60-R cells respectively (Tables 1 and
2).
Compound 10 was tested against sensitive HL60 cells (acute
promyelocytic leukemia), P-glycoprotein expressing (MDR) HL60-R
cells, K562 cells (a Bcr-Abl expressing leukemia) and HepG2 cells
(human hepatocellular carcinoma). Cells were exposed to different
concentrations of compound 10 and the number of living cells and
the percentage of apoptotic cells were determined after 48 h as
reported in section “Experimental protocols”. Antiproliferative and
apoptosis-inducing
activities
were
expressed
as
GI50
Scheme 1. Synthetic route to obtain the pyrazoletetrazepinone 10.

B. Maggio et al. / European Journal of Medicinal Chemistry 96 (2015) 98e104
101
Table 1
GI50( M) and AC50 (mM) values of compound 7 and 10 in HL60, HL60-R, K562 and HepG2 cell lines and GI50(mM) values of Daunorubicin and Etoposide in HL60 and HL60-R.
m
Compounds
HL60
HL60-R
IC50
K562
IC50
HepG2
IC50
IC50
AC50
AC50
AC50
AC50
7
10
21 3.3
36 5.5
19 3.1
n.t.
30 4.8
14 2.2
38 5.3
>400
38 2.8
22 2.8
n.t.
40 6.2
18 2.8
n.t.
57 8.2
37 5.2
n.t.
n.t.
26 4.0
n.t.
n.t.
43 6.7
n.t.
9
1.6
Daunorubicin
Etoposide
0.1 0.024
0.4 0.03
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
Table 2
Finally, compound 10 was tested on human colon carcinoma
WiDr cell line which express high levels of O6-methylguanine-DNA
methyltransferase (MGMT) and which is resistant toward alkylat-
ing agents such as temozolomide [11]. Our tests confirmed that
temozolomide is ineffective as cytotoxic agents in WiDr cell line,
Percentage of cells in G0-G1, S, G2-M after 24 h and 48 h exposure of K562 cells to
compound 10. These percentages were calculated on the cycling cells and do not
include the subG0-G1 fraction. The percentage of subG0-G1 cells was calculated on
the total cell number.
Time of drug exposure G0-G1(%)
S (%)
G2-M (%)
SubG0-G1
3.21 0.4
showing an IC50 of 1720( 123) mM. Interestingly, compound 10 was
quite active in this cell line showing an IC50 of 2.3( 0.3) mM.
0 (control)
24 h
44,65 6.8 43.19
43.96 7.2 43.59 6.3 12.45 3.1
7
12.16 2.2
15
2
48 h
40.97 5.3 44.24 3.9 14.79 1.8 38.35 5.2
3.2. Cell cycle
When compared to other chemotherapeutic drugs commonly
used in acute leukemias, such as daunorubicin or etoposide, com-
pound 10 was substantially less active in sensitive HL60 cells but
more active than daunorubicin and markedly more active than
etoposide in HL60-R cells (Table 1). Of interest, compound 10 was
also active in K562 cells, a cell line expressing the oncogene Bcr-Abl
which confers resistance toward drug-induced apoptosis, and in
human hepatocellular carcinoma HepG2 (Table 1).
The effect of compound 10 on cell cycle distribution was
analyzed in K562 cells. Cells were cultured in the presence of 30
mM
compound 10, and flow cytometric analysis of cell cycle was carried
out after 24 h and 48 h of treatment as described in section
“Experimental protocols”. As shown in Fig. 4 and Table 2, com-
pound 10 did not cause important modifications in the percentage
of cell cycle distribution. A proportional decrease in G0eG1, S, and
G2eM peaks and a proportional increase of the apoptotic sub-
Fig. 4. Effects of compound 10 on DNA content/cell following treatment of K562 cells for 24 h (b) and 48 h (c). The cells were cultured without compound (control, panel a) or with
30 M compound 10 as reported in section “Experimental protocols”. SubG0-G1 (A), G0-G1, S and G2-M cells are indicated in panel a.
m

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B. Maggio et al. / European Journal of Medicinal Chemistry 96 (2015) 98e104
G0eG1 peak was correlated to the time of exposure (24 or 48 h).
This indicate that compound 10 do not acts on a specific phase of
cell cycle but is able to kill cells in G0-G1, S and G2-M (decrease in
G0eG1, S, and G2eM peaks) causing a proportional increase in sub
G0-G1 peak. Therefore, compound 10 could be effective in cancers
with different kinetics. This is particularly interesting, because it
could be useful alone or in combination with other anticancer
agents to decrease the percentage of minimal residual disease
caused by kinetic factors.
As regards the slight difference in the antiproliferative activity
and cell cycle effects between 7 and 10, it possibly could be
explained considering the minor alkylating mechanism of tetra-
zepinones (see Fig. 5). The chloroethyldiazonium cation 20b
derived from 10 could afford alkylation as well as cross-linking in
the DNA double strand, causing more damage than the mono
alkylating methyldiazonium cation 20a.
evaporator under reduced pressure. All melting points were
determined on a Büchi 530 capillary melting point apparatus and
are uncorrected. IR spectra were recorded with a Perkin Elmer
Spectrum RXI FT-IR System spectrophotometer as solid in KBr disc
or nujol mull supported on NaCl disks. 1H and 13C NMR spectra (250
and 62.90 MHz respectively) were obtained using a Bruker AC-E
250 spectrometer (tetramethylsilane as an internal standard):
chemical shifts are expressed in
d values (ppm). Mass spectra at
70 eV were obtained using an Autospec Ultima Ortogonal T.O.F.T.
(Micromass) spectrometer or a GCeMS Varian Star 3400cx Saturn
III spectrometer. Merck silica gel (Kiesegel 60/230-400 mesh) was
used for flash chromatography columns. Microanalyses data (C, H,
N) were obtained by a Elemental Vario EL III apparatus and are
within 0.4% of the theoretical values. Yields refer to purified
products. Names of compounds were generated by ACD/chem-
sketch (free ware) 2012 software, file version 14.01.
5.1.2. Preparation of N-[1-phenyl-3-(trifluoromethyl)-1H-pyrazol-
5-yl]formamide 12
4. Conclusion
A solution of 1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-amine
13 [17] (1 g, 4.40 mmol) in 95% formic acid (5 mL) was refluxed for
1 h. The reaction mixture was cooled to room temperature and
basified with 10% aqueous sodium hydroxide until pH ¼ 8. The solid
was filtered off and crystallized from diethyl ether/cyclohexane.
Yield: 80%; m.p.: 84e86 ꢀC; MS (m/z) 255 (Mþ); 1H NMR (CDCl3)
The data reported here show that the 3-(2-chloroethyl)-5-
methyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f]
[1,2,3,5]tetrazepin-4(3H)-one 10 is an antiproliferative agent and
inducer of apoptosis in vitro both on leukemia cells (HL60, HL60-R,
K562) and on solid tumor (HepG2). It seems that the substitution of
the methyl group at the 3 position of the pyrazolo[3,4-f][1,2,3,5]
d
6.49 and 7.00 (1H, 2 s, pyrazole H); 7.42e7.71 (6H, a set of signals,
C6H5 and NH, exchange for 1H with D2O); 8.25 and 8.41(1H, 2 s,
formyl proton). Singlets at 6.49/7.00 and 8.25/8.41 collapsed to
respectively when the
tetrazepin-4(3H)-one system in compound
7 with the 2-
chloroethyl moiety in compound 10 had a positive effect on activ-
ity. Compound 10 is found to be active on cells that express the
MDR phenotype but with an antiproliferative as well as pro-
apoptotic activity 2.1 and 1.7 times higher than compound 7
respectively. Of interest, compound 10 is active on WiDr, a cell line
overexpressing MGMT and markedly resistant to temozolomide.
Finally, the effects of compound 10 on cell cycle is different from
compound 7. In fact, while 7 acts as phase-specific drug blocking
cells in the G0-G1 phase, compound 10 do not modify the per-
centage of cells in each phase of cell cycle but induce a proportional
decrease of G1-S-G2M peaks with a concomitant increase of the
subG0-G1 apoptotic peak. The slight difference in the anti-
proliferative activity and cell cycle effects between 7 and 10 might
be explained considering the potential minor alkylating mecha-
nism of tetrazepinones.
d
two very broad singlets at 6.82 and 8.30
d
spectra was registered at 100 ꢀC. Anal Calcd for C11H8F3N3O: C,
51.77; H, 3.16; N, 16.47. Found C, 51.99; H, 3.36; N, 16.21.
5.1.3. Preparation of N-methyl-1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-5-amine 13
To a solution of N-[1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-
yl]formamide 12 (0.2 g, 0.78 mmol) in anhydrous diethyl ether
(2.3 mL) a suspension of aluminum lithium hydride (90 mg,
2.4 mmol, in 4.5 mL of anhydrous diethyl ether) was added. After
24 h of reflux under stirring, the reaction mixture was cooled to
room temperature and quenched with methanol and water. Then
the solvent was evaporated, the aqueous residue was extracted
with diethyl ether and the organic layer was evaporated to give an
oily product that was identical in all respect with an authentic
specimen of N-methyl-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-
5-amine 13 (IR, 1H NMR, TLC) [16]. Yield: 95%.
5. Experimental protocols
5.1. Chemistry
5.1.1. General
5.1.4. The N5-methyl-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-
4,5-diamine 14
It was obtained starting from 13 and following a literature
method [17].
Reaction progress was monitored by TLC on silica gel plates
(Merck 60, F254, 0.2 mm). Organic solutions were dried over
Na2SO4. Evaporation refers to removal of solvent on a rotary
Fig. 5. Potential minor mechanism of action of tetrazepinones 7 and 10: formation of DNA-alkylating diazonium oins 20a,b.

B. Maggio et al. / European Journal of Medicinal Chemistry 96 (2015) 98e104
103
5.1.5. Preparation of tert-butyl [5-(methylamino)-1-phenyl-3-
(trifluoromethyl)-1H-pyrazol-4-yl]carbamate 15
sodium nitrite solution was added dropwise, keeping the temper-
ature at 0 ꢀC. Stirring was continued at the above temperature for
1 h and then the mixture was extracted with cold dichloromethane
(0 ꢀC) (2 x 5 mL). The aqueous layer was neutralized with a cold 20%
aqueous potassium hydroxide solution (0 ꢀC), keeping the tem-
perature at 0 ꢀC, and then the pH was adjusted to 8 with a cold
saturated aqueous sodium hydrogen carbonate solution (0 ꢀC). The
resulting solution was extracted again with cold dichloromethane
(5 ꢂ 10 mL). The combined extracts were dried over anhydrous
sodium sulfate, filtrated and evaporated under reduced pressure at
room temperature to give 10 as a yellow solid. The product was
dissolved at room temperature in diethyl ether, and the solution
was scratched until crystals were formed. The suspension was
cooled at ꢁ20 ꢀC for 30 min and then filtered off.
To a magnetically stirred mixture of 0.46 g (1.8 mmol) of N5-
methyl-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4,5-diamine 14
and 1.8 mL of water, 0.454 mL (0.432 g, 1.98 mmol) of di-tert-butyl-
dicarbonate were added at room temperature. Stirring was
continued overnight and then the mixture was diluted with cold
water (15 mL) and stirred until solidification. The solid separated
was filtered off and then recrystallized to afford tert-butyl (5-
(methylamino)-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)
carbamate 15.
15: yield 85%; mp 98e100 ꢀC (ethyl acetate/petroleum ether,
b.p. 40e60 ꢀC); MS (m/z) 300 (Mþꢁ CH2]C(CH3)2); IR (KBr) (cmꢁ1
)
3428, 3395, 3319 (2 ꢂ NH), 1720, 1702 (CO); 1H NMR (DMSO-d6)
d
1.43 (9H, s, t-butyl); 2.73 (3H, d, J ¼ 5.1 Hz, NHeCH3); 5.50 (1H, q,
10: Yield 37.7%; mp 72e74 ꢀC dec (diethyl ether); MS (m/z) 267
(Mþꢁ ClCH2CH2NCO); IR (KBr) (cmꢁ1) 1670 (CO); 1H NMR (CDCl3)
J ¼ 5.1 Hz, exchangeable slowly with D2O, NHeCH3); 7.45e7.58 (5H,
a set of signals, C6H5); 8.18 (1H, s, exchangeable with D2O, NH). Anal
Calcd for C16H19F3N4O2: C, 53.93; H, 5.37; N, 15.72. Found C, 53.63;
H, 5.57; N, 15.45.
d
2.84 (3H, s, NeCH3); 3.81 (2H, t, J ¼ 6.60 Hz, CH2CH2Cl); 4.32 (2H,
t, J ¼ 6.60 Hz, CH2CH2Cl); 7.48e7.58 (5H, a set of signals, C6H5). Anal
Calcd for C14H12ClF3N6O: C, 45.11; H, 3.25; N, 22.55. Found C, 45.50;
H, 3.35; N, 22.90.
5.1.6. Preparation of tert-butyl [5-{[(2-chloroethyl)
carbamoyl](methyl)amino}-1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-4-yl]carbamate 16
5.2. Pharmacology
To a solution of 0.950 g (2.7 mmol) of tert-butyl (5-(methyl-
amino)-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)carbamate
15 in 25 mL of anhydrous benzene, 2.3 mL (2.81 g, 26.7 mmol) of 2-
chloroethyl isocyanate were added, and the mixture was stirred for
20 days at room temperature. Then the mixture was filtered and the
filtrate was evaporated at 30 ꢀC by an oil vacuum pump to remove
the excess of chloroethyl isocyanate and solvent. A white crystalline
residue was obtained which was recrystallized to give pure 16.
16: yield 83%; mp 80e81 ꢀC (benzene); MS (m/z) 461; IR (KBr)
(cmꢁ1) 3440, 3323 (2 ꢂ NH), 1742, 1656 (2 ꢂ CO); 1H NMR (DMSO-
5.2.1. Cytotoxicity assays [18].
To evaluate the number of live and dead neoplastic cells, the
cells were stained with trypan blue and counted on a hemocy-
tometer. To determine the growth inhibitory activity of the drug
tested, 2 ꢂ 105 cells were plated into 25 mm wells (Costar, Cam-
bridge, UK) in 1 mL of complete medium and treated with different
concentrations of the compound to test (freshly prepared DMSO
solution, the concentration of DMSO in the medium never excee-
ded 0.3% v/v). After 48 h of incubation, the number of viable cells
was determined and expressed as the percentage of control
proliferation.
d6)
d 1.43 (9H, s, t-butyl); 2.87 (3H, s, NeCH3); 3.10e3.50 (4H, m,
2 ꢂ CH2), 6.90 (1H, br s, exchangeable with D2O, NH); 7.50e7.55
(5H, a set of signals, C6H5); 8.72 (1H, br s, exchangeable with D2O,
NH). Anal Calcd for C19H23ClF3N5O3: C, 49.41; H, 5.02; N, 15.16.
Found C, 49.30; H, 5.32; N, 14.90.
5.2.2. Morphological evaluation of apoptosis and necrosis [19].
Drug-induced apoptosis and necrosis was determined
morphologically after labeling with acridine orange and ethidium
bromide. Cells (2 ꢂ 105) were centrifuged (300 ꢂ g) and the pellet
5.1.7. Preparation of 1-[4-amino-1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-5-yl]-3-(2-chloroethyl)-1-methylurea 17
was resuspended in 25 mL of the dye mixture. Ten microliters of the
mixture were examined in oil immersion with a 100 ꢂ objective
using a fluorescence microscope. Live cells were determined by the
uptake of acridine orange (green fluorescence) and exclusion of the
ethidium bromide (red fluorescence) stain. Live and dead apoptotic
cells were identified by the perinuclear condensation of chromatin,
1 g of tert-butyl [5-{[(2-chloroethyl)carbamoyl](methyl)amino}-
1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]carbamate 16 was
deprotected using 35 mL of a mixture of 10% trifluoroacetic acid/
dichloromethane for 1 h under stirring at room temperature. The
reaction mixture was evaporated, and the residue was solubilized
with dichloromethane and washed with a saturated aqueous so-
dium hydrogen carbonate solution. The organic layer was dried
over anhydrous sodium sulfate and then evaporated to afford a
residue which was crystallized to give compound 17.
stained by acridine orange (100
mg/mL) or ethidium bromide
(100 g/mL), respectively, and by the formation of apoptotic bodies.
m
The percentage of apoptotic cells was determined after counting at
least 300 cells.
17: yield 64%; mp 139e140 ꢀC (diethyl ether); MS (m/z) 361; IR
5.2.3. Flow cytometric analysis of cell cycle distribution and
apoptosis [20].
(KBr) (cmꢁ1) 3447e3265 (multiple bands, NH2, NH), 1660 (CO); 1H
NMR (DMSO-d6)
d
2.90 (3H, s, NeCH3); 3.10e3.40 (4H, m, 2 ꢂ CH2);
The effects of compound 10 on cell cycle distribution were
studied on K562 cells (myeloblastic leukemia) by flow cytometric
analysis after staining with propidium iodide. Cells were exposed
24 h to compound 10. After treatment cells were washed once in
ice-cold phosphate buffered saline medium and resuspended at
106/mL in a hypotonic fluorochromone solution of propridium io-
4.44 (2H, s, exchangeable with D2O, NH2); 6.77 (1H, br s,
exchangeable slowly with D2O, NH); 7.42e7.50 (5H, a set of signals,
C6H5). Anal Calcd for C14H15ClF3N5O: C, 46.48; H, 4.18; N, 19.36.
Found C, 46.65; H, 4.57; N, 19.60.
5.1.8. Preparation of 3-(2-chloroethyl)-5-methyl-6-phenyl-8-
(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-
4(3H)-one 10
dide (50 mg/mL) and nonidet P-40 (Sigma) [0.03% (v/v)] in 0.1%
sodium citrate. After 30min of incubation, the fluorescence of each
sample was analyzed as single-parameter frequency histograms by
using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
The distribution of cells in the cell cycle and the apoptotic subG0-
G1 peak was analyzed with the ModFit LT3 program (Verity Soft-
ware House, Inc.).
To a magnetically stirred cold solution (T ¼ ꢁ5 ꢀC) of 0.25 g
(0.69 mmol) of 1-(4-amino-1-phenyl-3-(trifluoromethyl)-1H-pyr-
azol-5-yl)-3-(2-chloroethyl)-1-methylurea 17 in 10 mL of 6 N
aqueous hydrochloric acid solution, 0.25 mL of a 20% aqueous

104
B. Maggio et al. / European Journal of Medicinal Chemistry 96 (2015) 98e104
for the treatment of glioma, Neuro-Oncol. 11 (2009) 69e79.
Acknowledgment
[9] I. Passagne, A. Evrard, P. Depeille, P. Cuq, D. Cupissol, L. Vian, O6-methyl-
guanine-DNA methyltransferase (MGMT) overexpression in melanoma cells
induces resistance to nitrosoureas and temozolomide but sensitizes to mito-
mycin C, Toxicol. Appl. Pharm. 211 (2006) 97e105.
Financial support from “Fondo di Finanziamento della Ricerca di
Ateneo (ex 60%)” 2007 is gratefully acknowledged.
[10] C.H. Fan, W.L. Liu, H. Cao, C. Wen, L. Chen, G. Jiang, O6-methylguanine-DNA
methyltransferase as a promising target for the treatment of temozolomide-
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Appendix A. Supplementary data
[11] B.J. Jean-Claude, A. Mustafa, A. Watson, Z. Damian, D.E. Vasilescu, T.H. Chan,
B. Leyland-Jones, Tetrazepinones are equally cytotoxic to Merþ and mer-
human tumor cell lines, J. Pharm. Exp. Ther. 288 (1999) 484e489.
[12] B.J. Jean-Claude, A. Mustafa, Z. Damian, J. De Marte, D.E. Vasilescu, R. Yen,
T.H. Chan, B. Leyland-Jones, Cytokinetics of a novel 1,2,3-triazene-containing
heterocycle, 8-nitro-3-methyl-benzo-1,2,3,5-tetrazepin-4(3H)-one (NIME), in
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(1999) 753e762.
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2015.04.
004. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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