Synthesis and induction of G0-G1 phase arrest with apoptosis of 3,5-dimethyl-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.2008.01.007

The multistep synthesis of 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4(3H)-one 15 has been carried out. The compound showed antiproliferative and apoptotic effects against K562, K562-R (imatinib mesilate resistant), HL60 and multidrug resistant (MDR) HL60 cell lines. Compound 15 showed a pro-apoptotic activity against HL60 and K562 resistant cell lines markedly higher than etoposide and busulfan, respectively. Flow cytometry studies carried out on K562 cells allowed to establish that 15 induces G0-G1 phase arrest followed by apoptosis.

Full text of DOI:10.1016/j.ejmech.2008.01.007

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2386e2394
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and induction of G0eG1 phase arrest with apoptosis of
3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo
[3,4-f][1,2,3,5]tetrazepin-4(3H )-one
Benedetta Maggio a, Demetrio Raffa a, Maria Valeria Raimondi a, Stella Cascioferro a,
Fabiana Plescia a, Manlio Tolomeo b, Eleonora Barbusca b, Giuliana Cannizzo b,
Salvatrice Mancuso b, Giuseppe Daidone a,
*
a Dipartimento di Chimica e Tecnologie Farmaceutiche, Universita` degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
b Divisione di Ematologia e Servizio AIDS, Universita` degli Studi di Palermo, Policlinico, via del Vespro 129, 90127 Palermo, Italy
Received 8 August 2007; received in revised form 3 January 2008; accepted 3 January 2008
Available online 25 January 2008
Abstract
The multistep synthesis of 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4(3H )-one 15 has been
carried out. The compound showed antiproliferative and apoptotic effects against K562, K562-R (imatinib mesilate resistant), HL60 and multi-
drug resistant (MDR) HL60 cell lines. Compound 15 showed a pro-apoptotic activity against HL60 and K562 resistant cell lines markedly higher
than etoposide and busulfan, respectively. Flow cytometry studies carried out on K562 cells allowed to establish that 15 induces G0eG1 phase
arrest followed by apoptosis.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: 1,2,3,5-Tetrazepinones; Pyrazolo[3,4-f][1,2,3,5]tetrazepinones; Drug resistance; G0eG1 arrest; Apoptosis; Antiproliferative activity
1. Introduction
(human colon cancer), OVCAR-3 (epithelial ovarian cancer)
and MCF-7 (human breast cancer). All the experimental results
so far obtained suggest that tetrazepinones follow a mechanism
of action different from that of temozolomide [6], but still it is
not well established [7].
Drug resistance in cancer treatment calls for the availability of
new chemotherapeutic agents able to overcome this phenome-
non. In the literature is reported the synthesis of some benzo-
1,2,3,5-tetrazepin-4(3H )-ones of type 1 (see Fig. 1) which, like
temozolomide 2 [1,2], contain the N]NeN(CH3)COeN atomic
sequence [3e5]. Benzotetrazepinones suffer from instability
unless they bear in the benzene moiety an electron-withdrawing
group. In fact, the above group destabilizes the diazonium ion
which is afforded during the heterolytic opening of the N2eN3
bond of the tetrazepinone ring and, consequently, stabilizes the
parent tetrazepinone [4] (see Scheme 1).
Literature provides few examples of derivatives containing
the tetrazepinone ring fused to a heterocyclic nucleus [3,8]
and therefore we thought that it would be of interest to synthe-
size new derivatives of this class, containing other heterocy-
cles, in order to gain more insight into the structureeactivity
relationship of fused 1,2,3,5-tetrazepinones. Recently we
have described the synthesis of the pyrazolo[3,4-f][1,2,3,5]tet-
razepinone derivative 3, but owing to its inherent instability at
r.t. it could not be tested in vitro to evaluate its possible antipro-
liferative activity [9]. In order to circumvent the instability of
the above pyrazolotetrazepinone we designed the derivative
15 which contains the pyrazole nucleus substituted at the 3-
position with a trifluoromethyl group, able to exert a strong
inductive electron-withdrawing effect on the 4-position of the
Tetrazepinones were shown to be much more active than
temozolomide when tested against a variety of alkylating agent
resistant cell lines such as SF-188 (human brain cancer), WiDR
* Corresponding author. Tel.: þ39 091 6236116; fax: þ39 091 6236119.
E-mail address: gdaidone@unipa.it (G. Daidone).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.007

B. Maggio et al. / European Journal of Medicinal Chemistry 43 (2008) 2386e2394
2387
melting point, probably following the same decomposition
pathway of benzotetrazepinones [4]. All the above compounds
were identified on the basis of satisfactory elemental and spec-
troscopic data. In particular, the 4-nitroso structure of the green
compound 10 was based on the lack of the H-4 pyrazole signal
1
in the H NMR spectrum, which ruled out an N-nitrosamine
structure, and on the absorption band in the visible spectrum
at 600 nm, with low molar absorptivity value (96), indicative
of a nitrous group [14]. The 13C NMR spectrum of 15 accorded
fully with the other spectral data. In fact, it showed, among the
other signals, two singlets at 36.08 and 38.52 d, respectively,
for two N-methyl groups, the singlet at 155.91 d for a carbonyl
group and, finally, a quadruplet at 121.67 d (JCeF ¼ 269.53 Hz)
for a trifluoromethyl group.
Fig. 1.
above nucleus and, as a consequence, to increase the stability
of 15 than 3 (see above) [4]. Finally, due to our interest in
antileukemic compounds [10] and considering that no tetraze-
pinone derivative has been so far tested against leukemic cell
lines, we tested 15 against some sensitive and resistant ones.
3. Pharmacological results and discussion
2. Chemistry
3.1. Cytotoxicity
The synthesis of the novel tetrazepinone 15 was carried out
as outlined in Scheme 2. The starting material 4, prepared ac-
cording to literature procedures from ethyl (1-hydroxy-2,2,2-
trifluoroethyl)ether by a three-step route [11], was hydrolyzed
with potassium hydroxide in order to obtain the 5-amino-pyra-
zole-4-carboxylic acid derivative 5 (Scheme 1). Compound 5
was decarboxylated at 215 ꢀC affording the 5-aminopyrazole
derivative 6 [12] which, in turn, was acetylated. The acetyl
derivative 7 was methylated with methyl iodide in the presence
of potassium hydroxide to give 8 which was transformed into
the 5-methylaminopyrazole derivative 9 by hydrolysis with
potassium hydroxide solution under reflux for 10 h. Nitrosation
of 9 with nitrosyl hydrogen sulphate, obtained in situ by so-
dium nitrite and concentrated sulphuric acid, afforded the
nitrous derivative 10. The attempt to nitrosate 9 with sodium
nitrite in acetic acid [13] was unsuccessful as the 4-position
of the pyrazole nucleus is deactivated by the electron-
withdrawing trifluoromethyl group. Compound 10 was reduced
with tin(II) chloride in 36.5% aqueous hydrochloric acid to
give the corresponding 4-amino derivative 11. At this point
the amino group of 11 was protected with a benzyloxycarbonyl
group, by treatment with benzyl chloroformate, affording 12
which was then reacted with methyl isocyanate. The obtained
ureido derivative 13 was deprotected by treatment with hydro-
gen and 10% PdeC to give the derivative 14 which, in turn, was
diazotized at 0 ꢀC with nitrous acid. Finally, the diazotization
solution of 14 was adjusted to pH 8 to give the desired 3,5-di-
methyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-
f][1,2,3,5]tetrazepin-4(3H )-one 15. The latter transformed eas-
ily to pyrazolotriazole derivative 16 when it was heated until its
Compound 15 was tested against sensitive HL60 cells (acute
promyelocytic leukemia), P-glycoprotein expressing (MDR)
HL60-R cells, K562 cells (a Bcr-Abl expressing leukemia)
and K562-R cells that are resistant to the Bcr-Abl tyrosine-
kinase inhibitor imatinib mesilate (GleevecÒ). Cells were ex-
posed to different concentrations of compound 15 and the num-
ber of living cells and the percentage of apoptotic cells were
determined after 48 h as reported in Section 5. Antiproliferative
and apoptosis-inducing activities were expressed as IC50 (con-
centration able to inhibit 50% of cell growth) and AC50 (concen-
tration able to induce apoptosis in 50% of cells), respectively
(Table 1). As shown in Fig. 2(a) and (b) and in Table 1, the anti-
proliferative and apoptotic activities of compound 15 were sim-
ilar in sensitive HL60 and HL60-R cell lines, respectively. This
indicated that the presence of P-glycoprotein in cells does not
modify the activity of compound 15. Compared to other chemo-
therapeutic drugs commonly used in acute leukemias, such as
daunorubicin (17) or etoposide (18) (see Fig. 3), that show in
HL60 cells AC50 values of 0.1 mM and 0.4 mM, respectively
(data not shown), compound 15 was substantially less active
(AC50 36 mM). However, as shown in Fig. 4, in MDR cells the
apoptotic inducing activity of 15 was markedly higher than eto-
poside (AC50 >400 mM) and identical to that of daunorubicin
(AC50 38 mM). Similar results were obtained in K562 and
K562-R cells. K562 is a cell line resistant to apoptosis induced
by different stimuli including chemotherapeutic agents [15] but
it is sensitive toward apoptosis induced by imatinib mesilate (19)
which is the lead drug used in all Bcr-Abl expressing leukemias.
K562-R is a cell line derived from K562 by exposure to increas-
ing concentrations of imatinib mesilate. As shown in Fig. 5(a)
Scheme 1. Suggested decomposition pathway of benzotetrazepinones [4].

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B. Maggio et al. / European Journal of Medicinal Chemistry 43 (2008) 2386e2394
Scheme 2. Synthetic pathway for the formation of tetrazepinone 15. Reagents: (a) EtOH/KOH, reflux, 30 min; HCl; (b) heating (215 ꢀC), 1 h; (c) (CH3CO)2O,
24 h; (d) CH3I/KOH, reflux, 1 h; (e) KOH, H2O/EtOH, reflux, 10 h; (f) NaNO2/H2SO4; (g) SnCl2/HCl; (h) 2 M NaOH/PhCH2OCOCl; (i) CH3NCO, reflux,
190 h; (j) H2/10% PdeC, 20 h; (k) NaNO2/2 N HCl; NaHCO3; (l) heating (105 ꢀC), 5 min.

B. Maggio et al. / European Journal of Medicinal Chemistry 43 (2008) 2386e2394
2389
Table 1
IC50 (inhibition concentration 50%) and AC50 (concentration able to induce 50%
apoptosis) of compound 15 in sensitive and resistant HL60 and K562 cell lines
of different concentrations of compound 15. Flow cytometric
analysis of cell cycle was carried out after 24 h and 48 h of
culture as described in Section 5. As shown in Fig. 7, com-
pound 15 caused a dose dependent arrest of cells in G0eG1
phase after 24 h of treatment. No subG0eG1 apoptotic peak
was observed after 24 h of treatment (Fig. 7). After 48 h the
G0eG1 peak decreased and an evident apoptotic subG0eG1
peak appeared. The percentage of cells in subG0eG1 phase
at 48 h was correlated to the concentration of compound 15
used and to the percentage of cells recruited in G0eG1 after
24 h. As shown in Fig. 7, after 24 h of treatment with com-
pound 15 (50 mM), the percentage of cells in G0eG1 phase
increased from 35.8% (control) to 60.7%. After 48 h the per-
centage of cells in G0eG1 decreased to 28.7% with a concom-
itant increase of apoptotic cells (subG0eG1 peak) from 5%
(24 h) to 52.2% (48 h). In contrast, the modifications in the per-
centage of cells in S and G2eM phases observed at 24 h and
48 h of treatment were less evident. This indicates that com-
pound 15 induces a block of cells in G0eG1 and, afterward,
it kills these cells by activating apoptosis. Most chemothera-
peutic drugs used in the treatment of malignancies act in S or
G2eM phase of cell cycle but not in G0eG1. Thus, a variable
percentage of cells in G0eG1 could escape from the cytotoxic
effects of the therapy (kinetic resistance) causing a relapse of
the disease months or years after therapy [16]. Recently,
several compounds capable of arresting neoplastic cells in
G0eG1 have been described, nevertheless they don’t kill them.
Cells
IC50
(mM)
AC50
(mM)
HL60
21
30
40
38
36
38
57
62
HL60-R
K562
K562-R
IC50 and AC50 were calculated after 48 h drug exposure.
and (b), compound 15 was effective as antiproliferative and ap-
optotic agent in both K562 and K562-R cell lines. Fig. 6 shows
the apoptotic effect of compound 15 compared to busulphan (20)
(a drug commonly used in Bcr-Abl positive chronic myeloge-
nous leukemia) and to imatinib mesilate in K562-R cells. Com-
pound 15 (AC50 56 mM) was markedly more active than
busulphan (AC50 >800 mM) and slightly less active than imati-
nib mesilate (AC50 62 mM).
3.2. Cell cycle
The effect of compound 15 on cell cycle distribution was
analyzed in K562 cells. Cells were cultured in the presence
4. Conclusion
To summarize, the tetrazepinone 15 was able to act on
sensitive and resistant HL60 and K562 cell lines. This is prob-
ably due to the fact that tetrazepinones follow a new mecha-
nism of action and therefore they are able to overcome the
defences of neoplastic cells. Finally, the compound showed
the advantage, than most chemotherapeutic drugs, to block
cells in the G0eG1 phase and, in the same time, to kill them.
The above results encourage the research of other heterocy-
cle-fused tetrazepinones.
5. Experimental protocols
5.1. Chemistry
5.1.1. General
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 evaporator under reduced pressure. All melting points
were determined on a Buchi 530 capillary melting point appa-
¨
ratus and are uncorrected. IR spectra were recorded with
a Perkin Elmer Spectrum RXI FT-IR System spectrophotome-
ter 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 (tetra-
methylsilane as an internal standard): chemical shifts are
expressed in d values (ppm). Mass spectra at 70 eV were
Fig. 2. Antiproliferative (a) and apoptotic (b) effects of compound 15 on HL60
and HL60-R cells. The antiproliferative activity was evaluated by counting liv-
ing cells after 48 h exposure to different concentrations of compound 15. The
number of living cells was expressed as percentage of the control. The percent-
age of apoptotic cells was determined after 48 h exposure to different concen-
trations of compound 15 as reported in Section 5. Bars: ꢁSE.

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B. Maggio et al. / European Journal of Medicinal Chemistry 43 (2008) 2386e2394
Fig. 3. Structural formulas of commercial drugs tested for comparison.
obtained using an Autospec Ultima Orthogonal T.O.F.T. (Mi-
cromass) spectrometer or a GCeMS Varian Star 3400cx Sat-
urn III spectrometer. Merck silica gel (Kiesegel 60/230e400
mesh) was used for flash chromatography columns. Micro-
analyses data (C, H, N) were obtained by an Elemental Vario
EL III apparatus and are within ꢁ0.4% of the theoretical
values. Yields refer to purified products.
(1.56 g of KOH in 1.12 ml of water) was added, and the reac-
tion mixture was refluxed for 30 min. The above mixture was
evaporated and the solid residue was dissolved in 3.2 ml of
water adjusting its pH with aqueous 37% hydrochloric acid
until acidity. The white solid which separated was filtered
off and then crystallized to give compound 5. Yield 78%.
Compound 5. Mp 215e216 ꢀC (ethyl acetate/petroleum
ether b.p. 40e60 ꢀC). Anal Calcd for C11H8F3N3O2: C,
48.72; H, 2.97; N, 15.49. Found: C, 48.51; H, 3.10; N,
15.30; MS (m/z) 227 (Mþ ꢂ CO2); IR (KBr) (cmꢂ1) 3440,
3336 (NH2), 3039e2660 (OH) 1664 (CO); 1H NMR
(DMSO-d6) (d) 6.66 (2H, s, exchangeable with D2O, NH2);
7.51e7.59 (5H, a set of signals, C6H5); 12.78 (1H, br s,
exchangeable with D2O, OH).
5.1.2. 5-Amino-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-
carboxylic acid [5]
To 2.47 g (8.7 mmol) of methyl 5-amino-1-phenyl-3-(tri-
fluoromethyl)-1H-pyrazole-4-carboxylate (4) [10] dissolved
in 3.4 ml of ethanol, a solution of potassium hydroxide
5.1.3. 1-Phenyl-3-(trifluoromethyl)-1H-pyrazol-5-amine [6]
The compound 5-amino-1-phenyl-3-(trifluoromethyl)-1H-
pyrazole-4-carboxylic acid 5 (1 g, 3.7 mmol) was allowed to
stand at its melting point for 1 h, then the crude product was
crystallized. Yield 30%.
Compound 6. Mp 101e102 ꢀC (ethanol). Anal Calcd for
C10H8F3N3: C, 52.87; H, 3.55; N, 18.50. Found: C, 52.95;
H, 3.70; N, 18.71; MS (m/z) 227 (Mþ); IR (KBr) (cmꢂ1
)
3452, 3324 (NH2); 1H NMR (CDCl3) (d) 3.96 (2H, br s,
exchangeable with D2O, NH2); 5.81 (1H, s, pyrazole H-4);
7.40e7.54 (5H, a set of signals, C6H5). [Lit. [11]: 3.92 (2H,
br, s), 5.87 (1H, s), 7.40e7.58 (5H, m).]
5.1.4. N-[1-Phenyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]-
acetamide [7]
A mixture of 1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-
amine 6 (1 g, 4.40 mmol) and acetic anhydride (4 ml) was
Fig. 4. Apoptotic effects of compound 15 compared to daunorubicin and eto-
poside in HL60-R cells. The percentage of apoptotic cells was determined
after 48 h exposure to different concentrations of each compound as reported
in Section 5. Bars: ꢁSE.

B. Maggio et al. / European Journal of Medicinal Chemistry 43 (2008) 2386e2394
2391
Fig. 6. Apoptotic effects of compound 15 compared to imatinib mesilate and
busulphan in K562-R cells. The percentage of apoptotic cells was determined
after 48 h exposure to different concentrations of each compound as reported
in the Section 5. Bars: ꢁSE.
with diethyl ether (3 ꢃ 25 ml). Evaporation of the extracts
allowed to obtain an oily residue which was crystallized to
give 8. Yield 66%.
Compound 8. Mp 58e60 ꢀC (petroleum ether b.p. 40e
60 ꢀC). Anal Calcd for C13H12F3N3O: C, 55.12; H, 4.27; N,
14.84. Found: C, 55.30; H, 4.45; N, 14.60; MS (m/z) 283
1
(Mþ); IR (KBr) (cmꢂ1) 1690 (CO); H NMR (CDCl3) (d)
1.85 (3H, s, CH3); 3.12 (3H, s, CH3); 6.60 (1H, s, pyrazole
H-4); 7.45e7.49 (5H, a set of signals, C6H5).
Fig. 5. Antiproliferative (a) and apoptotic (b) effects of compound 15 on K562
and K562-R cells. The antiproliferative activity was evaluated by counting liv-
ing cells after 48 h exposure to different concentrations of compound 15. The
number of living cells was expressed as percentage of the control. The percent-
age of apoptotic cells was determined after 48 h exposure to different concen-
trations of compound 15 as reported in Section 5. Bars: ꢁSE.
5.1.6. N-Methyl-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-
amine [9]
To a solution of 1 g (3.53 mmol) of N-methyl-N-[1-phenyl-
3-(trifluoromethyl)-1H-pyrazol-5-yl]acetamide 8 in 6 ml of
ethanol, 3.4 ml of a 5 N aqueous potassium hydroxide solution
was added, and the mixture was refluxed for 10 h. The mixture
was filtered off, and the filtrate when evaporated left an oily
residue which was chromatographed following the flash chro-
matography procedure [17]: external diameter of the column
4 cm, ethyl acetate/petroleum ether b.p. 40e60 ꢀC (3:7 v/v)
as eluent (1.5 l). Fractions 12e17 (each 50 ml) were collected
and evaporated to give 6 as a pure brown oil. Yield 82%.
Compound 9. Anal Calcd for C11H10F3N3: C, 54.77; H,
4.18; N, 17.42. Found: C, 54.90; H, 4.36; N, 17.55; MS (m/
z) 241 (Mþ); IR (hexachloro-1,3-butadiene) (cmꢂ1) 3200e
stirred at room temperature for 24 h, and then cold water was
added until precipitation of an oily material which easily solid-
ified. The solid was filtered off and then crystallized to give 7.
Yield 88%.
Compound 7. Mp 146e148 ꢀC (ethyl acetate). Anal Calcd
for C12H10F3N3O: C, 53.54; H, 3.74; N, 15.61. Found: C,
53.60; H, 3.90; N, 15.40; MS (m/z) 269 (Mþ); IR (KBr)
1
(cmꢂ1) 3241 (NH), 1673 (CO); H NMR (CDCl3) (d) 2.01
(3H, s, CH3); 6.79 (1H, s, pyrazole H-4); 7.35e7.48 (5H, a set
of signals, C6H5); 7.82 (1H, s, exchangeable with D2O, NH).
1
3420 (multiple bands, NH); H NMR (CDCl3) (d) 2.81 (3H,
d, CH3, J ¼ 5.20 Hz); 3.89 (1H, br s, exchangeable with
D2O, NH); 5.73 (1H, s, pyrazole H-4); 7.35e7.46 (5H, a set
of signals, C6H5).
5.1.5. N-Methyl-N-[1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]-
acetamide [8]
To 1 g (3.71 mmol) of N-[1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-5-yl]acetamide 7 dissolved in 13.6 ml of acetone was
added 1 g of powdered potassium hydroxide and then the
mixture was refluxed gently for 10 min. After this time
0.35 ml (5.62 mmol) of iodomethane in 2 ml of acetone was
added and reflux was continued for 1 h. The mixture was
filtered off, the filtrate was evaporated affording a residue
which was treated with water (25 ml) and extracted three times
5.1.7. N-Methyl-4-nitroso-1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-5-amine [10]
To a magnetically stirred cold solution (ice bath 5 ꢀC) of
1 g (4.15 mmol) of N-methyl-1-phenyl-3-(trifluoromethyl)-
1H-pyrazol-5-amine 9 in 3.1 ml of acetic acid, a mixture of
0.31 g (4.49 mmol) of sodium nitrite in 0.82 ml of 98%
sulphuric acid was added in portions. Stirring was continued

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B. Maggio et al. / European Journal of Medicinal Chemistry 43 (2008) 2386e2394
5.1.8. N5-Methyl-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-
4,5-diamine [11]
To a magnetically stirred cold solution (T ¼ 5e10 ꢀC) of
3.18 g of tin(II) chloride in 6.52 ml of 37% aqueous hydro-
chloric acid, 1.5 g (5.55 mmol) of N-methyl-4-nitroso-1-phe-
nyl-3-(trifluoromethyl)-1H-pyrazol-5-amine 10 was added in
portions. Stirring was continued overnight at room tempera-
ture and then the mixture was diluted with 66 ml of cold water.
After adjusting its pH with a 40% aqueous sodium hydroxide
solution until alkalinity, the aqueous layer was extracted with
diethyl ether (3 ꢃ 50 ml) and the combined extracts were dried
over anhydrous sodium sulphate and then evaporated to give
N5-methyl-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4,5-di-
amine 11 as a pure oily residue. Yield 90%.
Compound 11. Anal Calcd for C11H11F3N4: C, 51.56; H,
4.33; N, 21.87. Found: C, 51.76; H, 4.50; N, 21.55; MS (m/
z) 256 (Mþ); IR (KBr) (cmꢂ1) 3222e3435 (multiple bands,
NH and NH2); 1H NMR (CDCl3) (d) 2.71 (3H, s, CH3);
3.11 (3H, s, exchangeable with D2O, NH and NH2); 7.36e
7.56 (5H, a set of signals, C6H5).
5.1.9. Benzyl [5-(methylamino)-1-phenyl-3-(trifluoro
methyl)-1H-pyrazol-4-yl]carbamate [12]
To a magnetically stirred cold solution (T ¼ 0e5 ꢀC) of
0.256 g (1 mmol) of N5-methyl-1-phenyl-3-(trifluoromethyl)-
1H-pyrazole-4,5-diamine 11 in 2 ml of 2 M sodium hydroxide
water/dioxane (1:1) (v/v) solution and 0.157 ml (1.1 mmol) of
benzyl chloroformate were added dropwise. Stirring was con-
tinued overnight at room temperature and then the mixture was
diluted with cold water and extracted with diethyl ether
(3 ꢃ 3 ml). After evaporation of the extracts the obtained
oily residue was crystallized to give 12. Yield 82%.
Compound 12. Anal Calcd for C19H17F3N4O2: C, 58.46; H,
4.39; N, 14.35. Found: C, 58.29; H, 4.37; N, 14.28; mp 9ꢂ5e1
96 ꢀC (diethyl ether); MS (m/z) 390 (Mþ); IR (KBr) (cm
)
1
3386e3198 (multiple bands, 2 ꢃ NH), 1740 (CO); H NMR
(CDCl3) (d) 2.69 (3H, s, CH3); 3.51 (1H, s, exchangeable
with D2O, NH); 5.19 (2H, s, CH2); 6.14 (1H, br s, exchange-
able with D2O, NH); 7.36e7.57 (10H, a set of signals,
2 ꢃ C6H5).
Fig. 7. Effects of compound 15 on DNA content/cell following treatment of
K562 cells for 24 h and 48 h. The cells were cultured without compound (con-
trol, panels a and e) or with the compound at the following concentrations:
25 mM (panels b and f), 50 mM (panels c and g), 75 mM (panels d and h).
Cell cycle distribution was analyzed by the standard propidium iodide proce-
dure as described in Section 5. SubG0eG1 (A), G0eG1, S, and G2eM cells
are indicated in panel a.
5.1.10. Benzyl (5-{methyl[(methylamino)carbonyl]-
amino}-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)
carbamate [13]
overnight at room temperature and then the mixture was
poured in crushed ice. The green solid separated was filtered
off, washed with a saturated aqueous sodium hydrogen
carbonate solution until pH of washing filtrate was neutral
and then crystallized to afford N-methyl-4-nitroso-1-phenyl-
3-(trifluoromethyl)-1H-pyrazol-5-amine 7. Yield 38%.
To a solution of 0.46 g (1.18 mmol) of benzyl [5-(methyla-
mino)-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]carba-
mate 12 in 12 ml of anhydrous chloroform, 2.4 ml
(36.9 mmol) of methyl isocyanate was added, and the mixture
was refluxed for 190 h. The solution was evaporated obtaining
an oily residue which was crystallized to give 13. Yield 41%.
Compound 13. Anal Calcd for C21H20F3N5O3: C, 56.37; H,
4.51; N, 15.65. Found: C, 56.45; H, 4.60;þN, 15.78; mp 16ꢂ4e1
Compound 10. Anal Calcd for C11H9F3N4O: C, 48.89; H,
3.36; N, 20.73. Found: C, 48.80; H, 3.50; N, 20.93; mp
138e140 ꢀC (ethyl acetate/petroleum ether, b.p. 40e60 ꢀC);
MS (m/z) 270 (Mþ); IR (KBr) (cmꢂ1) 3231 (NH); vis (meth-
166 ꢀC (diethyl ether); MS (m/z) 447 (M ); IR (KBr) (cm
)
1
1
anol) lmax nm (3) 600 (96), 360 (6859), 284 (7214); H NMR
(CDCl3) (d) 2.54 (3H, s, CH3); 7.49e7.54 (5H, a set of signals,
C6H5); 9.72 (1H, s, exchangeable with D2O, NH).
3421 and 3214 (2 ꢃ NH), 1716 and 1663 (2 ꢃ CO); H NMR
(CDCl3) (d) 2.65 (3H, br s, CH3); 2.86 (3H, s, CH3); 5.17 (3H,
broad signal exchangeable with D2O for 1H, CH2 and NH);

B. Maggio et al. / European Journal of Medicinal Chemistry 43 (2008) 2386e2394
2393
6.21 (1H, s, exchangeable with D2O, NH); 7.36e7.46 (10H,
a set of signals, 2 ꢃ C6H5).
Compound 16. Anal Calcd for C11H8F3N5: C, 49.44; H,
3.02; N, 26.21. Found: C, 49.66; H, 3.22; N, 26.45; MS (m/
z) 267 (Mþ); 1H NMR (CDCl3) (d) 4.16 (3H, s, CH3);
7.49e7.59 (5H, a set of signals, C6H5).
5.1.11. 1-[4-Amino-1-phenyl-3-(trifluoromethyl)-
1H-pyrazol-5-yl]-1,3-dimethylurea [14]
5.2. Pharmacology
To a solution of 0.5 g (1.11 mmol) of benzyl (5-{methyl
[(methylamino)carbonyl]amino}-1-phenyl-3-(trifluoromethyl)-
1H-pyrazol-4-yl)carbamate 13 in 50 ml of methanol, 50 mg of
10% PdeC as a catalyst was added. The mixture was left
under hydrogenation in a Parr apparatus at 50 psi for 20 h.
After this time the suspension was filtered, and the filtrate
was evaporated affording an oily residue which was crystal-
lized to give 14. Yield 58%.
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 hemo-
cytometer. To determine the growth inhibitory activity of the
drug tested, 2 ꢃ 105 cells were plated into 25 mm wells (Co-
star, Cambridge, 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 exceeded 0.3% v/v). After 48 h of incubation,
the number of viable cells was determined and expressed as
the percentage of control proliferation.
Compound 14. Anal Calcd for C13H14F3N5O: C, 49.84; H,
4.50; N, 22.36. Found: C, 49.70; H, 4.6þ5; N, 22.50; mp 16ꢂ4e1
165 ꢀC (benzene); MS (m/z) 313 (M ); IR (KBr) (cm
)
1
3452e3316 (multiple bands, NH2 and NH), 1656 (CO); H
NMR (CDCl3) (d) 2.82 (3H, d, NeCH3, J ¼ 4.12 Hz); 2.92
(3H, s, CH3); 3.58 (2H, br s, exchangeable with D2O, NH2);
4.91 (1H, br s, NH); 7.37e7.44 (5H, a set of signals, C6H5).
5.2.2. Morphological evaluation of apoptosis
and necrosis [19]
Drug-induced apoptosis and necrosis were determined
morphologically after labeling with acridine orange and ethid-
ium bromide. Cells (2 ꢃ 105) were centrifuged (300 ꢃ g) and
the pellet was resuspended in 25 ml of the dye mixture. Ten
microliters of the mixture were examined in oil immersion
with a 100ꢃ objective using a fluorescence microscope. Live
cells were determined by the uptake of acridine orange (green
fluorescence) and exclusion of the ethidium bromide (red fluo-
rescence) stain. Live and dead apoptotic cells were identified
by the perinuclear condensation of chromatin, stained by acri-
dine orange (100 mg/ml) or ethidium bromide (100 mg/ml),
respectively, and by the formation of apoptotic bodies. The
percentage of apoptotic cells was determined after counting
at least 300 cells.
5.1.12. 3,5-Dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydro-
pyrazolo[3,4-f][1,2,3,5]tetrazepin-4(3H )-one [15]
To a magnetically stirred cold solution (T ¼ ꢂ5 ꢀC) of 0.3 g
(0.96 mmol) of 1-[4-amino-1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-5-yl]-1,3-dimethylurea 14 in 2.8 ml of 2 N aqueous
hydrochloric acid solution, 0.35 ml of a 20% aqueous sodium
nitrite solution was added dropwise, keeping the temperature
at 0 ꢀC. Stirring was continued at the above temperature for
1 h and then the mixture was extracted with cold dichlorome-
thane (0 ꢀC) (3 ꢃ 5 ml). The pH of aqueous layer was adjusted
to 8 with a saturated aqueous sodium hydrogen carbonate solu-
tion (0 ꢀC), and the resulting solution was extracted again with
cold dichloromethane (5 ꢃ 5 ml). The combined extracts were
dried over anhydrous sodium sulphate, filtered and evaporated
under reduced pressure (rotary vacuum pump) at r.t. to give 15
as a pure yellow powder. The product was dissolved at room
temperature in diethyl ether, and the solution was scratched un-
til crystals were formed. The suspension was cooled at ꢂ20 ꢀC
for 30 min and then the crystal product was filtered off. Yield
58%.
5.2.3. Flow cytometric analysis of cell cycle distribution
and apoptosis [20]
The effects of compound 15 on cell cycle distribution were
studied on K562 cells (myeloblastic leukemia) by flow cyto-
metric analysis after staining with propidium iodide. Cells
were exposed for 24 h to compound 15. After treatment cells
were washed once in ice-cold phosphate buffered saline
medium and resuspended at 106/ml in a hypotonic fluorochro-
mone solution of propidium iodide (50 mg/ml) and nonidet P-
40 (Sigma) [0.03% (v/v)] in 0.1% sodium citrate. After 30 min
of incubation, the fluorescence of each sample was analyzed as
single-parameter frequency histograms by using an FACScan
flow cytometer (Becton Dickinson, San Jose, CA). The distri-
bution of cells in the cell cycle and the apoptotic subG0eG1
peak were analyzed with the ModFit LT3 program (Verity
Software House, Inc.).
Compound 15. Anal Calcd for C13H11F3N6O: C, 48.15; H,
3.42; N, 25.92. Found: C, 48.35; H, 3.52; N, 25.70; mp 108e
109 ꢀC dec.; MS (m/z) 324 (Mþ); IR (KBr) (cmꢂ1) 1656 (CO);
1H NMR (CDCl3) (d) 2.83 (3H, s, CH3); 3.49 (3H, s, CH3);
7.48e7.57 (5H, a set of signals, C6H5). 13C NMR (CDCl3)
(d) 35.66 (CH3), 38.11 (CH3), 121.67 (q, JCeF ¼ 269.53,
CF3), 123.57, 124.29, 126.57, 131.15, 131.31, 138.97,
139.05, 155.91 (CO).
5.1.13. 3-Methyl-4-phenyl-6-(trifluoromethyl)-3,
4-dihydropyrazolo[3,4-d][1,2,3]triazole [16]
The compound 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-
5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4(3H )-one 15
(0.05 g, 0.15 mmol) was allowed to stand at its melting point
for 5 min affording quantitatively 16 as a pure oily product.
Acknowledgement
Financial support from MIUR (ex 60% fund) is gratefully
acknowledged.

2394
B. Maggio et al. / European Journal of Medicinal Chemistry 43 (2008) 2386e2394
[11] K. Tanaka, T. Suzuki, S. Maeno, K. Mitsuhashi, J. Heterocycl. Chem. 23
(1986) 1535.
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Osamu, Takanobu Masahito, Ishizuka Hitoshi, Manabe Yukiaki, Jpn.
Kokai Tokkyo Koho, Patent No. JP 08208620 (1996).
[13] E. Gonzales, R. Sarlin, J. Elguero, J. Heterocycl. Chem. 12 (1975)
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