Antiproliferative and proapoptotic activities of a new class of pyrazole derivatives in HL-60 cells

Article abstract of DOI:10.1002/cbdv.200800354

A series of N-substituted pyrazole derivatives have been synthesized and tested for their anticancer effect on the HL-60 leukaemia cell line. Four were active both in cell-growth inhibition and in inducing apoptosis. The inhibition of cell growth mainly r

Full text of DOI:10.1002/cbdv.200800354

1674
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Antiproliferative and Proapoptotic Activities of a New Class of Pyrazole
Derivatives in HL-60 Cells
by Maria Anzaldia), Chiara Maccio` a), Mauro Mazzeia), Maria Bertolottob), Luciano Ottonellob),
Franco Dallegrib), and Alessandro Balbi*a)
a
`
) Dipartimento di Scienze Farmaceutiche, Universita di Genova, Viale Benedetto XV, 3,
I-16132 Genova (phone: þ39-010-35383040; fax: þ39-010-3538358; e-mail: balbi@unige.it)
b
`
) Dipartimento di Medicina Interna – Clinica di Medicina Interna 1, Universita di Genova,
Viale Benedetto XV, 6, I-16132 Genova
A series of N-substituted pyrazole derivatives have been synthesized and tested for their anticancer
effect on the HL-60 leukaemia cell line. Four were active both in cell-growth inhibition and in inducing
apoptosis. The inhibition of cell growth mainly reflects a compound-induced reduction in the number of
cells in phases from S to M, whereas the induction of apoptosis involves inhibition of expression of Bcl-2
and enhanced expression of Bax with consequent reduced activation of the proapoptotic caspase 3.
Finally, preliminary experiments carried out with tumor cells from myelogenous leukaemic patients
showed that the compounds 4c, 4l, 4m, and 4n are indeed capable of inducing apoptosis.
Introduction. – The pyrazole framework plays an essential role in numerous
pharmaceutically active compounds which indeed occupy a remarkable position in vast
areas of medicinal chemistry. From an important past, exemplified in the analgesic and
anti-inflammatory pyrazolones and pyrazolinediones, to the present with some of the
most recent important drugs, such as COX-2 inhibitors, which are not lacking severe
complications, pyrazole derivatives are synthesized and studied as antibacterial
compounds [1] [2], p38 MAP kinase inhibitors [3], antiangiogenic agent [4], and
Hsp90 and CDK2/Cyclin A inhibitors [5–8].
While the antitumor activity of pyrazole derivatives is widely discussed in literature
[4–7][9–13], only few works are focused both to antiproliferative and proapoptotic
activity [14][15].
We have already reported [16][17] that compounds which can be linked to the so-
called ꢀshort heteroretinoidsꢁ, formed by a cyclohexenyl group linked to a heterocyclic
moiety by a short ethenylic chain, possesses antimicrobial [16], anti-inflammatory, and
histoprotective properties [17]. The heterocyclic moiety (pyrazole, isoxazole, pyrimi-
dine) and the substituents present in them deeply affect the biological activities. In
particular, pyrimidine and isoxazole derivatives showed good antimicrobial activities,
while pyrazole derivatives were found to be potent inhibitors of neutrophil chemotactic
responsiveness [17].
Taking into account the above considerations and the significant differentiating
activity of the ꢀisoxazole heteroretinoidꢁ 1 [18], we started our study by evaluating our
ꢂ 2009 Verlag Helvetica Chimica Acta AG, Zꢃrich

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1675
corresponding isoxazole and pyrazole derivatives 2 and 4, respectively. Since
compound 2 showed only antimicrobial activity [16], we focused our attention on
pyrazoles 3 (see the Scheme, below) expanding this class of compounds which exhibited
promising antiproliferative properties in our preliminary experiments.
This idea proved to be a winning strategy: in fact, most of 4, to different extents,
showed antiproliferative and/or apoptotic activities. These findings, further supported
by the fact that the present compounds 4c, 4m, and 4n are indeed capable of inducing
apoptosis in tumor cells from myelogenous leukaemic patients, could pave the way to a
new class of drugs for cancer therapy. Moreover, in an attempt to understand the
mechanism of action, we found that some of them inhibit Bcl-2 and induce BAX
expression.
Results and Discussion. – Syntheses. The synthesis of pyrazole derivatives 4a–4r are
outlined in the Scheme. The key intermediate 3, which was synthesized according to our
well-tested procedure, was reacted with the hydrazine derivatives modifying the
cyclization reaction used in the past [16][17]. When bifunctional reagents were present
as hydrochlorides, they were reacted as such (Method b), while all the other hydrazine
derivatives were reacted with equimolar quantities of HCl (Method a). We were then
able to obtain the desired pyrazole derivatives in high yields and in a very short period
of time. Moreover, in nearly half of the cases, we isolated the 1,3-pyrazole derivative 5
with the predominant 1,5-pyrazoles 4. It is worth noting that compounds 4p–4q and all
the compounds 5 can only be prepared by this new procedure.
Structure Elucidation. The structural isomerism of the compounds was easily
1
determined by detailed examination of the H- and 13C-NMR spectra of the pyrazole
moiety of compounds 4r and 5r, which were chosen as an example, because the signals
the R moiety do not interfere with those needed to elucidate the structures. The
pyrazole CH signals have been assigned in both compounds on the basis of their
HECTOR (300 MHz) spectra (Table 1) and the literature data [19][20]. In particular,
the spin coupling constant (2J (H,H) of pyrazole ring) of compounds 4 is lower than
that of compounds 5. Moreover, the chemical shift of the quaternary C(5)-atom of
compounds 4 (ca. 141 ppm) is lower than that of the quaternary C(3)-atom of
compounds 5 (ca. 150 ppm), and the chemical shift of the HꢀC(5) of compounds 5 (ca.
130 ppm) is lower than that of the HꢀC(3) of compounds 4 (ca. 139 ppm). This
behavior is consistent with literature data [19][20] and is common to all the isomers we
synthesized.
Biological Results. Experiments were carried out to test the effects of different
compounds on the proliferation and apoptosis of HL60 cells. The results obtained are
collected in Table 2, showing the concentrations required for 50% inhibition of cell

1676
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Scheme. Synthesis of Pyrazole Derivatives
Table 1. Selected Chemical Shifts [ppm] of sp2 CH Groups and C-Atoms for Compounds 4r and 5r
4r
5r
d(C)
d(H)
d(C)
d(H)
C(1(ethene))
C(2(ethene))
C(3)
C(4)
C(5)
117.2
137.3
139.4
103.3
141.9
121.9
6.10
6.00
7.46
6.35
–
123.2
133.6
151.7
103.2
131.5
121.2
6.36
6.06
–
6.38
7.38
5.43
C(3’)
5.43

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1677
Table 2. Antiproliferative Effects (IC50) and Apoptosis-Inducing Effects (AC50) of Compounds 4a–4q,
5l–5n, and ATRA (¼ꢀAll Trans Retinoic Acidꢁ) on HL60 Cells
IC50 [mm]a)
AC50 [mm]a)
4a
4b
4c
4d
4e
NAb)
>40
NA
NA
27ꢁ2
53ꢁ11
NA
NA
NA
>40
4f
4g
4h
4i
28ꢁ5
15ꢁ1.9
17ꢁ4
NA
>100
NA
NA
NA
4l
5l
32ꢁ1
76ꢁ19
NA
NA
4m
5m
4n
5n
4o
4p
4q
ATRA
12ꢁ1
NA
82ꢁ17
NA
26ꢁ8
48ꢁ5
>40
NA
>40
57ꢁ25
43ꢁ14
–c)
>40
–c)
14ꢁ3
>100
c
a) Data represent the meanꢁSD of three independent experiments. b) Not active. ) Insoluble under the
experimental conditions.
proliferation (ꢁ SD) IC50 [mm] and the concentrations required to induce apoptosis in
50% of cells (ꢁ SD) AC50 [mm].
Among the compounds able to inhibit cell proliferation, four, i.e., 4c, 4l, 4m, 4n,
were found to induce cell apoptosis as well. As shown in Fig. 1, all these compounds
induced apoptosis in a dose-dependent manner.
In an attempt to investigate the mechanism of the proapoptotic effects of these
compounds, we focused our attention on Bcl-2 and Bax, known to be involved as
inhibitor and promoter of apoptosis, respectively. As shown in Fig. 2, after 24 h of
incubation with HL60 cells, compounds 4c and 4n reduced the expression of Bcl-2
(Fig. 2,b) and enhanced the expression of Bax (Fig. 2,a). Consistent with these
findings and suggesting a crucial role of expression Bcl-2 and Bax in mediating the
proapoptotic effects of the compounds, the ability of 4c and 4n to induce apoptosis was
significantly reduced by DEVD-CHO inhibitor of caspase 3 known to be the final
effector of apoptosis (Fig. 3).
Finally, to gain further insight into the mechanism of action of these compounds, the
flow cytometric analysis of the cell cycle was evaluated, measuring propidium iodide
uptake. When compared to untreated cells, cell samples treated with 4c showed a
marked increase in the percentage of apoptotic cells, and a sharp decrease in cell-
cycling capacity (Fig. 4). In particular, after 24 h of treatment the amount of cells in
phases from S to M was reduced to half.

1678
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 1. Effect of different doses of pyrazole derivatives 4c, 4l, 4m, and 4n on cell apoptosis. Data represent
the meanꢁSD of four independent experiments (n¼4).
Conclusions. – The general aim of the present work was to develop new compounds
with potent anticancer activity. In this context, among 17 pyrazole derivatives tested,
four, i.e., 4c, 4l, 4m, 4n, appear to be active both in cell-growth inhibition and in
induction of apoptosis using HL-60 cells, while the 1,3-isomers, i.e., 5l, 5m, 5n, were
devoid of any activity. Concentrations of the compounds required for 50% cell-growth
inhibition (IC50) and those required to induce 50% apoptosis (AC50) are equimolar.
Nevertheless, the possibility that these two effects merely reflect a single molecular
mechanism is far from being established. In this regard, the involvement of distinct
molecular targets responsible for the antiproliferative and apoptotic effects has indeed
suggested to explain the biological activity of synthetic retinoids only partially related
to the present compounds [17][21][22]. In the present setting, preliminary experiments
indicate that the inhibition of the cell growth mainly reflects a compound-induced
reduction in the number of cells in phases from S to M, whereas the induction of
apoptosis involves inhibition of expression of Bcl-2 and enhanced expression of Bax
with consequently reduced activation of the proapoptotic caspase 3. It is noteworthy
that compounds 4e, 4f, 4h, and 4g display antiproliferative activity but are unable to
induce apoptosis. On the contrary 4o and 4p show only proapoptotic activity. These
results indicate that the phenyl moiety alone or substituted with Cl or Br in ortho- or
para-position lead to compounds without apoptotic activity. It is worth noting that the

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1679

1680
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 3. Effect of pretreatment with DEVD-CHO for 1 h on cell apoptosis of HL60 cells exposed to
different doses of compounds 4c, 4l, 4m, and 4n. The values are presented as meanꢁSD (n¼3).
Fig. 4. Compound 4c promotes apoptosis and inhibits cell-cycle progression. DNA Content and
fragmentation were evaluated by measuring propidium iodide uptake by flow cytometry on cells
incubated for 24 h with either medium (control), or with medium added with 50 mm 4c. Percentages of
cells in apoptosis and of cells in the different phases of cell cycle are indicated. Single representative
experiments out of three.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1681
replacement of phenyl with pyridine causes an impressive change in activity. In fact, 4n
presents a remarkable activity both as an apoptosis inducer and as an antiproliferative
agent. Compounds 4a, 4d, and 4i are completely ineffective in the present setting. It is
relevant that 4a and 4d have previously been shown to exert bacterial killing [16] and to
interfere with the activation of the respiratory burst in human neutrophils [17],
consistent with an anti-inflammatory potential of these molecules.
In conclusion, the biological activity of pyrazole derivatives appears to be strongly
dependent both on the isomerism and the substituents present in the heterocyclic
moiety. In particular, the substituents present in position 1 of the pyrazole ring play a
fundamental role in determining and increasing the activity. The present results also
indicate the possibility of developing this class of molecules which could arrest cell
cycle at G1/S phase and induce apoptosis of HL-60 cells. Consistently, preliminary
experiments carried out on tumor cells from myelogenous leukaemic patients show that
the compounds 4c, 4l, 4m, and 4n are indeed capable of efficiently inducing apoptosis
(Table 3).
Table 3. Induction of Apoptosis in Blast Cells from Myelogenous Leukaemia Patients. Results are
expressed as (% apoptosis in the presence of the drugs) – (% apoptosis in the absence of the drugs).
Patient 1
Patient 2
4c
4l
4m
4n
50
52
60
84
52
48
58
75
We would like to thank Prof. Francesco Lucchesini, University of Genoa, Italy, for the 1H- and
13C-NMR analyses. Financial support of this research by the Ateneo of Genoa is gratefully acknowledged.
Experimental Part
General. The purity of all compounds was checked by TLC (SiO2 60-F-254 pre-coated plates;
visualization by UV light or by vanillin in H2SO4. M.p.: Fisher-Johns apparatus; uncorrected. IR Spectra:
1
film or KBr disks, on a Perkin-Elmer 398 spectrometer. H- and 13C-NMR spectra: Varian Gemini 200
(1H: 200 MHz, 13C: 50 MHz) or Bruker DPX 300 (300 MHz) instrument, in CDCl3 or (D6)DMSO;
chemical shifts (d) in ppm from the peak for internal TMS; coupling constants (J) in Hz. Elemental
analyses: Carlo Erba 1106 Elemental Analyser in the Microanalysis Laboratory of our Department.
Compound 3 was prepared as described in [16].
General Procedure (GP) for the Preparation of Compounds 4 and 5. The hydrazines (Method a) or
hydrazine hydrochlorides (Method b) (2 mmol) were added in one portion to a stirred soln. of compound
3 (0.5 g, 2 mmol) in acidified EtOH (10 ml containing 0.17 ml of HCl 37%) (Method a) or EtOH (10 ml)
(Method b). The resulting soln. was stirred for 1 h at different temps. After cooling, compounds 4i, 4p,
and 4q were collected by filtration from the mixture and crystallized. All the other compounds were
obtained by SiO2 chromatography of the residue after removal of the solvent.
5-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazole (4a). From NH2NH2 ·H2O at 508
(Method a); chromatographic eluent: toluene. Thick oil. 89% Yield. For IR, and 1H- and 13C-NMR
data, see [17].
2-{5-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}ethanol (4b) and 2-{3-[2-(2,6,6-
Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}ethanol (5b). From 2-hydrazinoethanol at 258
(Method a). After evaporation to dryness, the residue was chromatographed on SiO2 eluting first with

1682
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
toluene/AcOEt 9 :1 and then with AcOEt. The first eluate gave 5b as thick yellow oil in 10% yield. The
second eluted fraction gave 4b in 70% yield as a thick yellow oil.
Data of 4b. For IR, and 1H- and 13C-NMR data, see [17].
Data of 5b. IR (film): 3303, 2956, 1630, 1508, 1375, 1363. 1H-NMR (200 MHz, CDCl3): 0.86 (s, 3 H);
0.92 (s, 3 H); 1.16–1.22 (m, 3 H); 1.41–1.51 (m, 3 H); 1.60 (s, 3 H); 1.99–2.03 (m, 2 H); 2.27 (d, J ¼ 9.4,
1 H); 3.85 (s, OH); 3.96–4.03 (m, 2 H); 4.21–4.27 (m, 2 H); 5.42–5.46 (m, 1 H); 6.16 (dd, J ¼ 9.4, 16.0,
1 H); 6.31 (d, J ¼ 2.6, 1 H); 6.35 (d, J ¼ 16.0, 1 H); 7.36 (d, J ¼ 2.6, 1 H). 13C-NMR (50 MHz, CDCl3):
22.4; 22.4; 26.2; 27.1; 30.7; 31.6; 54.0; 55.1; 62.0; 103.0; 122.3; 123.5; 131.4; 133.7; 134.4; 151.9. Anal. calc.
for C16H24N2O: C 73.81, H 9.29, N 10.76; found: C 74.18, H 9.12, N 10.63.
N,N-Dimethyl-5-[2-(2,6,6-trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazole-1-carbothioamide (4c).
From thiosemicarbazide at 258 (Method a). After evaporation to dryness, the residue was chromato-
graphed on SiO2 eluting with toluene, red oil. Yield 55%. For IR, and 1H- and 13C-NMR data, see [17].
4-Amino-5-{5-[2-(2,6,6-trimethylcyclohex-1-en-1-yl)ethenyl]pyrazol-1-yl}-4H-[1,2,4]triazole-3-thiol
(4d). From Purpaldꢄ at reflux (Method a). After evaporation to dryness, the residue was chromato-
graphed on SiO2 eluting first with toluene and then with toluene/AcOEt 9 :1. The resulting yellow solid
was filtered, washed with H2O, dried, and crystallized from cyclohexane. Yield 80%. M.p. 138–1398. For
IR, and 1H- and 13C-NMR data, see [17].
1-Phenyl-5-[2-(2,6,6-trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazole (4e) and 1-Phenyl-3-[2-
(2,6,6-trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazole (5e). From PhNHNH2 ·HCl at 258 (Method
b). After evaporation to dryness, the residue was chromatographed on SiO2 eluting with toluene. The
first eluate gave 5e in 10% yield as thick oil. The second eluted fraction gave 4e in 80% yield as a thick oil.
Data for 4e. For IR, and 1H- and 13C-NMR data, see [17].
Data for 5e. IR (film): 2958, 2915, 1600, 1518, 1457, 1384. 1H-NMR (200 MHz, CDCl3): 0.88 (s, 3 H);
0.94 (s, 3 H); 1.17–1.23 (m, 1 H); 1.43–1.53 (m, 1 H); 1.63 (s, 3 H); 2.01–2.05 (m, 2 H); 2.29 (d, J ¼ 9.1,
1 H); 5.41–5.48 (m, 1 H); 6.14 (dd, J ¼ 9.1, 15.8, 1 H); 6.50 (d, J ¼ 15.8, 1 H); 6.53 (d, J ¼ 2.6, 1 H); 7.22–
7.28 (m, 1 H); 7.39–7.45 (m, 2 H); 7.64–7.68 (m, 2 H); 7.83 (d, J ¼ 2.6, 1 H). 13C-NMR (50 MHz, CDCl3):
22.3; 22.3; 26.2; 27.0; 30.7; 31.7; 53.9; 103.6; 118.0; 120.5; 122.4; 125.3; 126.8; 128.6; 133.1; 133.6; 139.3;
151.7. Anal. calc. for C20H24N2: C 82.15, H 8.27, N 9.58; found: C 81.89, H 8.33, N 9.45.
1-(2-Chlorophenyl)-5-[2-(2,6,6-trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazole (4f) and 1-(2-
Chlorophenyl)-3-[2-(2,6,6-trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazole (5f). From 2-chlorophenyl-
hydrazine hydrochloride at reflux (Method b). After evaporation to dryness, the residue was
chromatographed on SiO2 eluting with toluene. The first eluate gave 5f in 10% yield as a red thick oil.
The second eluted fraction gave 4f in 87% yield as a red thick oil.
Data of 4f. For IR, 1H- and 13C-NMR data, see [17].
Data of 5f. IR (film): 2910, 2854, 1890, 1593, 1495, 1382, 1364. 1H-NMR (200 MHz, CDCl3): 0.89 (s,
3 H); 0.92 (s, 3 H); 1.22–1.26 (m, 1 H); 1.40–1.46 (m, 1 H); 1.63 (s, 3 H); 2.01–2.05 (m, 2 H); 2.30 (d, J ¼
8.6, 1 H); 5.42–5.47 (m, 1 H); 6.20 (dd, J¼ 8.6, 15.8, 1 H); 6.40 (d, J ¼ 15.8, 1 H); 6.54 (d, J ¼ 2.4, 1 H);
7.24–7.34 (m, 3 H); 7.36–7.40 (m, 1 H); 7.82 (d, J ¼ 2.4, 1 H). 13C-NMR (50 MHz, CDCl3): 22.2; 22.3;
26.2; 27.3; 30.6; 31.7; 54.0; 104.1; 122.0; 122.5; 127.9; 129.5; 130.5; 130.8; 131.3; 131.4; 133.7; 136.7; 138.0;
154.0. Anal. calc. for C20H23ClN2: C 73.49, H 7.09, Cl 10.85, N 8.57; found: C 73.44, H 7.12, Cl 10.90, N 8.68.
1-(4-Chlorophenyl)-5-[2-(2,6,6-trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazole (4g) and 1-(4-
Chlorophenyl)-3-[2-(2,6,6-trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazole (5g). From 4-chlorophenyl-
hydrazine hydrochloride at 258 (Method b). According to the GP the first eluate gave 5g as yellow thick
oil, yield 10%. The second eluate gave 4g as red oil. Yield 78%.
Data of 4g. For IR, 1H- and 13C-NMR data, see [17].
Data of 5g. IR (film): 2920, 2857, 1522, 1498, 1383, 1363. 1H-NMR (200 MHz, CDCl3): 0.88 (s, 3 H);
0.94 (s, 3 H); 1.23–1.27 (m, 1 H); 1.41–1.45 (m, 1 H); 1.63 (s, 3 H); 2.00–2.03 (m, 2 H); 2.40 (d, J ¼ 8.6,
1 H); 5.45–5.51 (m, 1 H); 6.12 (dd, J ¼ 8.6, 16.1, 1 H); 6.48 (d, J ¼ 16.1, 1 H); 6.52 (d, J ¼ 2.6, 1 H); 7.37
(d, J ¼ 8.8, 2 H); 7.60 (d, J ¼ 8.8, 2 H); 7.78 (d, J ¼ 2.6, 1 H). 13C-NMR (50 MHz, CDCl3): 22.2; 22.3; 26.1;
27.1; 30.6; 31.7; 53.9; 104.1; 118.4; 119.5; 120.6; 122.1; 126.7; 131.7; 133.1; 134.2; 138.6; 151.8. Anal. calc.
for C20H23ClN2: C 73.49, H 7.09, Cl 10.85, N 8.57; found: C 73.45, H 7.17, Cl 10.86, N 8.63.
1-(4-Bromophenyl)-5-[2-(2,6,6-trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazole (4h) and 1-(4-Bro-
mophenyl)-3-[2-(2,6,6-trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazole (5h). From 4-bromophenylhy-

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1683
drazine hydrochloride at 258 (Method b). According to the GP, the first eluate gave 5h as yellow solid,
8% yield. M.p. 68–708 from cyclohexane. The second eluate gave 4h as thick red oil. Yield 88%.
Data of 4h. For IR, and 1H- and 13C-NMR data, see [17].
Data of 5h. IR (KBr): 2918, 2856, 1592, 1523, 1496, 1382. 1H-NMR (200 MHz, CDCl3): 0.88 (s, 3 H),
0.94 (s, 3 H); 1.17–1.23 (m, 1 H); 1.42–1.46 (m, 1 H); 1.63 (s, 3 H); 2.02–2.05 (m, 2 H); 2.26 (d, J ¼ 8.5,
1 H); 5.43–5.49 (m, 1 H); 6.14 (dd, J ¼ 8.5, 15.8, 1 H); 6.48 (d, J ¼ 15.8, 1 H); 6.54 (d, J ¼ 2.6, 1 H); 7.54
(s, 4 H); 7.80 (d, J ¼ 2.6, 1 H). 13C-NMR (50 MHz, CDCl3): 22.3; 22.4; 26.2; 27.1; 30.7; 31.8; 53.9; 104.1;
118.5; 119.4; 120.6; 122.1; 126.8; 131.6; 132.9; 134.3; 138.2; 152.0. Anal. calc. for C20H23BrN2: C 64.69, H,
6.24, Br 21.52, N 7.54; found: C 64.55, H 6.06, Br 21.43, N 7.45.
4-{5-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}benzoic Acid (4i). From 4-hy-
drazinobenzoic acid (Method a) at 258. The resulting white solid was crystallized from AcOEt. Yield
1
93%. M.p. 209–2108. IR (KBr): 2917, 2508, 1704,1606, 1512, 1420, 1384, 1260. H-NMR: 0.85 (s, 3 H);
0.88 (s, 3 H); 1.18–1.22 (m, 1 H); 1.38–1.42 (m, 1 H); 1.58 (s, 3 H); 1.99–2.03 (m, 2 H); 2.25 (d, J ¼ 7.4,
1 H); 5.40–5.44 (m, 1 H); 6.08–6.22 (m, 2 H); 6.72 (d, J ¼ 1.6, 1 H); 7.57 (d, 2 H); 7.67 (d, J ¼ 1.6, 1 H);
8.1 (d, 2 H). 13C-NMR: 23.3; 23.5; 27.4; 28.2; 31.6; 32.9; 54.7; 105.9; 115.1; 119.1; 122.1; 125.2; 130.4;
130.7; 131.2; 133.7; 137.02; 141.6; 143.4; 167.3. Anal. calc. for C21H24N2O2: C 74.97, H 7.19, N 8.33; found: C
74.74, H 7.09, N 8.40.
Ethyl 4-{5-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}benzoate (4l) and Ethyl 4-
{3-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}benzoate (5l). From ethyl 4-hydrazi-
nobenzoate at 258 (Method a). After evaporation to dryness, the residue was chromatographed on SiO2
eluting with toluene. The first eluate gave 5l as yellow oil. Yield 7%. The second eluted fraction gave 4l as
yellow oil. Yield 92%.
Data of 4l. IR (film): 2958, 2921, 1715, 1607, 1515, 1463, 1366, 1247. 1H-NMR: 0.91 (s, 3 H); 0.95 (s,
3 H); 1.26–1.28 (m, 1 H); 1.40–1.48 (m, 4 H); 1.63 (s, 3 H); 1.99–2.03 (m, 2 H); 2.22 (d, J ¼ 8.4, 1 H);
4.40 (q, J ¼ 7.2, 2 H); 5.40–5.44 (m, 1 H); 6.10 (dd, J ¼ 8.4, 15.4, 1 H); 6.25 (d, J ¼ 15.4, 1 H); 6.50 (d, J ¼
2.2, 1 H); 7.62 (d, J ¼ 8.4, 2 H); 7.64 (d, J ¼ 2.2, 1 H); 8.18 (d, J ¼ 8.4, 2 H). 13C-NMR: 14.4; 23.1; 23.1;
26.9; 27.9; 31.5; 32.8; 54.9; 61.4; 105.0; 118.8; 122.0; 124.5; 129.3; 130.6; 133.1; 137.0; 140.1; 140.9; 143.3;
165.1. Anal. calc. for C23H28N2O2: C 75.79, H 7.74, N 7.69; found: C 75.82, H 7.70, N 7.64.
Data of 5l. IR (film): 2959, 2926, 1715, 1608, 1524, 1367, 1276. 1H-NMR (200 MHz, CDCl3): 0.88 (s,
3 H); 0.94 (s, 3 H); 1.22–1.26 (m, 1 H); 1.42 (t, J ¼ 7.2, 3 H); 1.49–1.53 (m, 1 H); 1.63 (s, 1 H); 2.01–2.05
(m, 2 H); 2.30 (d, J ¼ 7.8, 1 H); 4.38 (q, J ¼ 7.2, 2 H); 5.44–5.48 (m, 1 H); 6.16 (dd, J ¼ 7.8, 16.0, 1 H);
6.50 (d, J ¼ 16.0, 1 H); 6.57 (d, J ¼ 2.6, 1 H); 7.74 (d, J ¼ 9.0, 2 H); 7.90 (d, J ¼ 2.6, 1 H); 8.10 (d, J ¼ 9.0,
2 H). 13C-NMR (50 MHz, CDCl3): 13.6; 22.3; 22.4; 26.2; 27.0; 30.6; 31.8; 53.9; 60.3; 104.7; 116.9; 120.7;
122.1; 126.9; 130.3; 132.9; 134.6; 142.4; 152.5; 165.2. Anal. calc. for C23H28N2O2: C 75.79, H 7.74, N 7.69;
found: C 75.80, H 7.71, N 7.63.
4-({5-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}methyl)phenol (4m) and 4-
({[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}methyl)phenol (5m). From 3-hydrox-
ybenzylhydrazine dihydrochloride at 258 (Method b). The residue was chromatographed on SiO2 eluting
with toluene. The first eluate gave 5m as a yellow oil. Yield 10%. The second eluate gave 4m as already
pure yellow crystals in 87% Yield.
1
Data of 4m. M.p. 128–1308 from AcOEt. IR (KBr): 3149, 2959, 2865, 1602, 1458, 1282. H-NMR
(200 MHz, CDCl3): 0.78 (s, 3 H); 0.87 (s, 3 H); 1.15–1.19 (m, 1 H); 1.33–1.37 (m, 1 H); 1.48 (s, 3 H);
1.98–2.02 (m, 2 H); 2.17 (d, J ¼ 8.97, 1 H); 5.24 (s, 2 H); 5.41–5.45 (m, 1 H); 5.98 (dd, J ¼ 9.0, 15.9, 1 H);
6.15 (d, J ¼ 15.9, 1 H); 6.31 (d, J ¼ 2.0, 1 H); 6.44 (s, 1 H); 6.61 (dd, J ¼ 2.2, 7.8, 1 H); 6.67 (dd, J ¼ 2.2,
7.8, 1 H); 7.13 (t, J ¼ 7.8, 1 H); 7.35 (d, J ¼ 2.0, 1 H). 13C-NMR (50 MHz, CDCl3): 22.0; 22.3; 26.0; 27.1;
30.5; 31.7; 52.2; 53.9; 102.4; 112.8; 114.5; 116.7; 116.9; 121.1; 128.9; 132.3; 136.4; 137.3; 137.5; 140.8; 156.8.
Anal. calc. for C21H26N2O: C 78.22, H 8.13, N 8.69; found: C 78.20, H 8.10, N 8.47.
Data of 5m. IR (film): 3260, 2958, 2923, 1602, 1459, 1280. 1H-NMR: 0.84 (s, 3 H); 0.91 (s, 3 H); 1.17–
1.23 (m, 1 H); 1.39–1.45 (m, 1 H); 1.58 (s, 3 H); 1.98–2.03 (m, 2 H); 2.20 (d, J ¼ 8.9, 1 H); 5.17 (s, 2 H);
5.23–5.27 (m, 1 H); 6.03 (dd, J ¼ 8.9, 16.1, 1 H); 6.31 (d, J ¼ 2.4, 1 H); 6.35 (d, J ¼ 16.1, 1 H); 6.42 (s,
1 H); 6.70 (dd, J ¼ 1.8, 7.1, 2 H); 7.12 (t, J ¼ 7.1, 1 H); 7.28 (d, J¼2.4, 1 H). 13C-NMR: 23.0; 23.0; 26.9;
27.8; 31.5; 32.5; 54.6; 55.4; 102.9; 114.2; 115.6; 118.7; 121.3; 122.6; 129.8; 130.9; 133.8; 133.9; 137.7; 151.0;
157.2. Anal. calc. for C21H26N2O: C 78.22, H 8.13, N 8.69; found: C 78.23, H 8.09, N 8.64.

1684
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
2-{5-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}pyridine (4n) and 2-{3-[2-
(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}pyridine (5n). From 2-hydrazinopyridine
dihydrochloride at 508 (Method b). The residue was chromatographed on SiO2 eluting with toluene. The
first eluate gave 5n as a yellow oil. Yield 11%. The second eluate gave 4n as yellow oil. Yield 72%.
Data of 4n. IR (film): 2900, 2915, 2864, 1590, 1470, 1436, 1378. 1H-NMR (200 MHz, CDCl3): 0.91 (s,
3 H); 0.96 (s, 3 H); 1.18–1.22 (m, 1 H); 1.42–1.47 (m, 1 H); 1.68 (s, 3 H); 1.99–2.03 (m, 2 H); 2.18 (d, J ¼
9.1, 1 H); 5.25–5.27 (m, 1 H); 6.07 (dd, J ¼ 9.1, 15.8, 1 H); 6.51 (d, J ¼ 1.8, 1 H); 7.12 (d, J ¼ 15.8, 1 H);
7.20–7.28 (m, 1 H); 7.62 (d, J ¼ 1.8, 1 H); 7.82–7.86 (m, 2 H); 8.44–8.46 (m, 1 H). 13C-NMR (50 MHz,
CDCl3): 23.2; 23.2; 27.1; 27.9; 31.6; 32.7; 54.7; 105.4; 117.1; 121.0; 121.5; 121.5; 134.7; 135.5; 138.5; 140.9;
142.6; 147.9; 151.9. Anal. calc. for C19H23N3: C 77.78, H 7.90, N 14.32; found: C 77.74, H 7.50, N 14.17.
Data of 5n. IR (film): 2900, 2914, 2862, 1592, 1470, 1436, 1383. 1H-NMR (200 MHz, CDCl3): 0.88 (s,
3 H); 0.94 (s, 3 H); 1.18–1.22 (m, 1 H); 1.49–1.55 (m, 1 H); 1.62 (s, 3 H); 2.00–2.05 (m, 2 H); 2.33 (d, J ¼
9.3, 1 H); 5.44–5.48 (m, 1 H); 6.17 (dd, J ¼ 9.3, 16.3, 1 H); 6.50 (d, J ¼ 16.3, 1 H); 6.53 (d, J ¼ 2.8, 1 H);
7.08–7.16 (m, 1 H); 7.72–7.82 (m, 1 H); 7.94 (d, J ¼ 6.9, 1 H); 8.30–8.40 (m, 1 H); 8.46 (d, J ¼ 2.8, 1 H).
13C-NMR (50 MHz, CDCl3): 22.3; 22.4; 26.2; 27.0; 30.8; 31.8; 53.98; 104.0; 111.4; 120.2; 120.6; 122.3;
127.1; 132.9; 134.6; 137.9; 147.1; 150.6; 152.8. Anal. calc. for C19H23N3: C 77.78, H 7.90, N 14.32; found: C
77.72, H 7.57, N 14.20.
4-(5-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl)benzenesulfonic Acid (4o) and
4-(3-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl)-1H-pyrazol-1-yl)benzenesulfonic Acid (5o). From 4-
hydrazinobenzenesulfonic acid hemihydrate at reflux (Method a). The residue was chromatographed on
SiO2 eluting first with toluene/AcOEt 1:1 and then with EtOH. The first eluate gave 5o as an already
pure yellow solid. Yield 16%. The second eluate gave 4o as a yellow oil. Yield 49%.
Data of 4o. IR (film): 3445, 2959, 2917, 1599, 1502, 1387, 1182. 1H-NMR (200 MHz, CDCl3): 0.80 (s,
3 H); 0.84 (s, 3 H); 1.17–1.21 (m, 1 H); 1.37–1.42 (m, 1 H); 1.51 (s, 3 H); 1.94–1.98 (m, 2 H); 2.19 (d, J ¼
8.9, 1 H); 5.38–5.42 (m, 1 H); 6.05–6.13 (m, 2 H); 6.49 (d, J ¼ 1.8, 1 H); 7.45 (d, J ¼ 8.4, 2 H); 7.68 (d,
J ¼ 1.8, 1 H); 7.89 (d, J ¼ 8.4, 2 H); 8.70 (s, 1 H, D2O exchangeable). 13C-NMR (50 MHz, CDCl3): 22.2;
22.2; 26.1; 27.0; 30.7; 31.8; 54.1; 103.6; 117.7; 121.1; 124.1; 124.1; 126.2; 126.2; 132.2; 136.3; 139.7; 140.2;
140.8; 142.7. Anal. calc. for C20H24N2O3S: C 64.49, H 6.49, N 7.52, S 8.61; found: C 64.69, H 6.40, N 7.64, S
8.50.
Data of 5o. M.p. 234–2378 from AcOEt. IR (KBr): 3447, 2960, 2912, 1601, 1521, 1384, 1195. 1H-NMR
(200 MHz, CDCl3): 0.81 (s, 3 H); 0.84 (s, 3 H); 1.12–1.16 (m, 1 H); 1.40–1.45 (m, 1 H); 1.56 (s, 3 H);
1.94–1.98 (m, 2 H); 2.26 (d, J ¼ 8.7, 1 H); 5.39–5.43 (m, 1 H); 6.12 (dd, J ¼ 7.4, 15.5, 1 H); 6.39 (d, J ¼
15.5, 1 H); 6.72 (d, J ¼ 2.6, 1 H); 7.66–7.74 (m, 4 H); 8.32 (d, J ¼ 2.6, 1 H); 8.72 (s, 1 H, D2O
exchangeable). 13C-NMR (50 MHz, CDCl3): 22.6; 22.6; 26.6; 27.4; 30.8; 31.9; 53.8; 105.0; 118.3; 121.2;
124.2; 124.2; 126.4; 126.4; 126.8; 132.9; 135.7; 139.4; 140.1; 151.5. Anal. calc. for C20H24N2O3S: C 64.49, H
6.49, N 7.52, S 8.61; found: C 64.60, H 6.43, N 7.66, S 8.51.
4-{5-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}benzonitrile (4p). From 4-cy-
anophenylhydrazine hydrochloride at 508 (Method b). After evaporation to dryness, the residue was
washed and crystallized from cyclohexane. Yield 49%. White solid. M.p. 97–1008. IR (KBr): 3421, 2956,
2911, 2229, 1607, 1509, 1391. 1H-NMR (200 MHz, CDCl3): 0.88 (s, 3 H); 0.92 (s, 3 H); 1.22–1.27 (m, 1 H);
1.39–1.43 (m, 1 H); 1.60 (s, 3 H); 1.99–2.04 (m, 2 H); 2.25 (d, J ¼ 8.8, 1 H); 5.45–5.49 (m, 1 H); 6.02–
6.16 (m, 2 H); 6.48 (d, J ¼ 1.8, 1 H); 7.59–7.67 (m, 3 H); 7.76 (d, J ¼ 8.8, 2 H). 13C-NMR (50 MHz,
CDCl3): 22.3; 22.3; 26.1; 27.1; 30.6; 31.9; 54.1; 55.5; 104.9; 110.2; 117.5; 117.5; 121.4; 124.2; 124.2; 131.9;
132.4; 132.4; 137.1; 140.5; 140.9; 141.0. Anal. calc. for C21H23N3: C 79.46, H 7.30, N 13.24; found: C 79.27, H
7.57, N 13.29.
4-{5-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}benzenesulfonamide (4q).
From (4-sulfamoylphenyl)hydrazine hydrochloride at reflux (Method b). The resulting white solid was
filtered and washed with EtOH. Yield 23%. M.p. 179–1808. IR (KBr): 3305, 2952, 2922, 1596, 1500, 1339,
1165. 1H-NMR (200 MHz, DMSO): 0.77 (s, 3 H); 0.80 (s, 3 H); 1.10–1.14 (m, 1 H); 1.22–1.26 (m, 1 H);
1.49 (s, 3 H); 1.87–1.92 (m, 2 H); 2.21 (d, J ¼ 8.8, 1 H); 5.34–5.38 (m, 1 H); 6.00–6.22 (m, 2 H); 6.65 (d,
J ¼ 2.0, 1 H); 7.44 (s, 2 H, D2O exchangeable); 7.54–7.60 (m, 3 H); 7.89 (d, J ¼ 8.4, 2 H). 13C-NMR
(50 MHz, DMSO): 22.6; 22.6; 26.5; 27.4; 30.9; 32.1; 53.9; 55.5; 104.9; 118.0; 121.2; 124.6; 124.6; 126.7;

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1685
126.7; 132.7; 136.3; 140.6; 140.7; 141.4; 142.8. Anal. calc. for C20H25N3O2S: C 64.66, H 6.78, N 11.31, O
8.61, S 8.63; found: C 64.41, H 6.86, N 11.32, O 8.61, S 8.88.
Ethyl 2-{5-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl]-1H-pyrazol-1-yl}ethanoate (4r) and Ethyl
2-{3-[2-(2,6,6-Trimethylcyclohex-2-en-1-yl)ethenyl)-1H-pyrazol-1-yl}ethanoate (5r). From ethyl hydrazi-
noacetate hydrochloride at 508 (Method b). The residue was chromatographed on SiO2 eluting with
toluene/AcOEt 9 :1. The first eluate gave 5r as an already pure white solid. Yield 18%. The second eluate
gave 4r as a yellow oil. Yield 72%.
Data of 4r. IR (film): 2959, 2915,1758, 1464, 1375, 1206. 1H-NMR (200 MHz, CDCl3): 0.84 (s, 3 H);
0.91 (s, 3 H); 1.20–1.24 (m, 1 H); 1.25 (t, J ¼ 7.3, 3 H); 1.41–1.46 (m, 1 H); 1.60 (s, 3 H); 2.00–2.06 (m,
2 H); 2.25 (d, J ¼ 8.6, 1 H); 4.20 (q, J ¼ 7.3, 2 H); 4.91 (s, 2 H); 5.42–5.46 (m, 1 H); 6.00 (dd, J ¼ 8.6, 15.8,
1 H); 6.11 (d, J ¼ 15.8, 1 H); 6.32 (d, J ¼ 1.9, 1 H); 7.44 (d, J ¼ 1.9, 1 H). 13C-NMR (50 MHz, CDCl3):
14.1; 22.9; 23.0; 26.9; 27.7; 31.4; 32.5; 50.9; 54.9; 61.8; 103.3; 117.2; 121.9; 132.9; 137.3; 139.4; 141.9; 167.7.
Anal. calc. for C18H26N2O2: C 71.49, H 8.67, N 9.26; found: C 71.45, H 8.57, N 9.47.
Data of 5r. M.p. 45–468 from toluene. IR (KBr): 2954, 2914, 1741, 1464, 1373, 1235. 1H-NMR
(200 MHz, CDCl3): 0.85 (s, 3 H); 0.91 (s, 3 H); 1.15–1.19 (m, 1 H); 1.25 (t, J ¼ 7.5, 3 H); 1.41–1.46 (m,
1 H); 1.60 (s, 3 H); 1.98–2.03 (m, 2 H); 2.23 (d, J ¼ 8.8, 1 H); 4.22 (q, J ¼ 7.5, 2 H); 4.84 (s, 2 H); 5.40–
5.44 (m, 1 H); 6.02 (dd, J ¼ 8.8, 16.0, 1 H); 6.37 (d, J ¼ 16.0, 1 H); 6.37 (d, J ¼ 2.4, 1 H); 7.36 (d, J ¼ 2.4,
1 H). 13C-NMR (50 MHz, CDCl3): 14.1; 23.1; 23.1; 26.9; 27.8; 31.5; 32.5; 52.9; 54.7; 61.8; 103.2; 121.2;
123.2; 131.5; 133.6; 133.9; 151.7; 167.9. Anal. calc. for C18H26N2O2: C 71.49, H 8.67, N 9.26; found: C 71.34,
H 8.71, N 9.34.
Cell Culture. HL-60 Cells (promyelocitic cell line) were grown in RPMI 1640 (Gibco, Grand Island,
NY, USA) containing 10% FCS (ICN), 100 Uml penicillin (Gibco), 100 mg/ml streptomycin (Gibco),
and 2 mm l-glutamine (Gibco) in 5% CO2 at 378. To evaluate their viability, cells were stained with
trypan blue and counted with a phase-contrast microscopy. Cells which showed trypan blue uptake were
considered nonviable.
Morphological Evaluation of Apoptosis and Necrosis. HL-60 Cells were exposed to different
concentrations of each compound. After a 48-h period of culture, the effects of each compound on
apoptosis were determined morphologically by fluorescent microscopy after labelling with acridine
orange (Sigma) and ethidium iodide (EI). Cells (3ꢂ105) were centrifuged and resuspended in the dye
mixture; 10 ml of the cell suspension was placed on a microscope slide and examined with a fluorescence
microscope. Living cells were determined by the uptake of acridine orange (green fluorescence) and
exclusion of ethidium bromide (EB; red fluorescence) stain. Apoptotic cells were identified by
perinuclear condensation of chromatin stained with acridine orange and by the formation of apoptotic
bodies (percentage of apoptotic cells). Necrotic cells were identified by uniform labelling of the cells
with EB.
Analysis of Cell Proliferation (MTT Assay). Cell proliferation was measured using mitochondrial
respiration. It was assessed by a colorimetric test that detects the conversion of 3-(4,5-dimethyl-2H-
thiazol-2-yl)-2,5-diphenyltetrazolium hydrobromide (MTT) into formazan product by enzymes of the
respiratory chain. Briefly, 50,000 cells/well were seeded in 96-well plates in 100 ml of medium alone and
exposed to appropriate doses of each compound for 48 h. At the end of the incubation time, MTT soln.
was added to the cell cultures at the final concentration of 5 mg/ml, and cells were incubated for an
additional 4 h. Thereafter, cells were lysed in DMSO. The conversion of MTT to formazan,
corresponding to mitochondrial respiratory activity, was monitored by automated microplate reader at
590 nm. The number of cells proliferating is calculated using different concentrations of cells cultured in
96-well plate under the same experimental conditions (calibration curve).
Caspase Studies. Caspase-3 inhibitor (DEVD-CHO, Ac-Asp-Glu-Val-Asp-aldehyde from Bachem,
20 mm) was added to cells 1 h before cell exposure to more active compounds among those tested.
Experiments were carried out using the same experimental conditions of apoptosis assays.
Cell-Cycle Analysis. The technique of Nicoletti et al. [23] was used, with minor changes, to evaluate
cell cycle and apoptosis. Briefly, after washing, 106 cells were permeabilized by adding 200 ml of PBS plus
0.1% NP40 to cell pellet. Samples were gently shaken, and, after 30 s, 200 ml of PBS plus 50 mg/ml
propidium iodide (PI) and 100 mg/ml RNase (both from Sigma-Aldrich, St. Louis, MO) were added.
Samples were incubated in the dark at r.t. for 10 min and then analyzed on a flow cytometer

1686
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
(FACScalibur, BD Biosciences, San Jose, CA). Ten thousand events were analyzed per sample. The
amount of cells in the different phases of the cell cycle and the amount of cells in apoptosis was evaluated
by measuring right and left shift from normal diploid DNA content, resp.
Immunocytochemistry. Bax and Bcl-2 protein expression was investigated by immunocytochemistry
as described by Dibbert [24] with slight modification. Briefly, HL60 cells, collected after incubation with
or without the more active compounds among those tested, were cytocentrifuged (Cytospinꢄ). After
rehydration in PBS, spots were submerged in peroxidase-quenching soln. for 10 min to neutralize
endogenous peroxidase activity. Then, the slides were incubated with anti-human Bax polyclonal
antibody (1 mg/ml, Santa-Cruz, CA) or with anti-human Bcl-2 mAb (1 mg/ml, Santa-Cruz, CA) diluted
1:200 in PBS. The secondary biotinylated IgG antibody (Kit Histostain SP, Zymed Laboratories, San
Francisco, CA) was used. After washing and subsequent incubation with biotin-strepatavidin-peroxidase
(Kit Histostain SP), slides were incubated at r.t. for 5 min with the peroxidase-substrate soln. (Kit
Histostain SP), rinsed with PBS, and counter-stained with ematoxylin. Then, cytospins were mounted in
Eukitt (Merk), examined by light microscopy (Leica, Cambridge, UK), and evaluated by image analysis
(Leica). Isotype-matched mAbs (R&D Systems) of irrelevant specificity were tested as negative control.
Image Analysis. Image analysis was performed by the Leica Q500 MC image analysis system (Leica).
For each sample, 100 cells were randomly analyzed, and the optical density of the signal was quantified by
computer analysis. The video image was digitalized for image analysis at 256 grey levels. Imported data
were analyzed quantitatively by Q500 MC Software-Qwin (Leica). The operator randomly selected
single cells using the cursor. Then, the positive area was analyzed automatically. The same optical
threshold and filter combination were used throughout all of these experiments.
REFERENCES
[1] A. Tanitame, Y. Oyamada, K. Ofuji, H. Terauchi, M. Kawasaki, M. Wachi, J. Yamagishi, Bioorg.
Med. Chem. Lett. 2005, 15, 4299.
[2] A. Tanitame, Y. Oyamada, K. Ofuji, M. Fujimoto, K. Suzuki, T. Ueda, H. Terauchi, M. Kawasaki, K.
Nagai, M. Wachi, J. Yamagishi, Bioorg. Med. Chem. 2004, 12, 5515.
[3] J. Regan, S. Breitfelder, P. Cirillo, T. Gilmore, A. G. Graham, E. Hickey, B. Klaus, J. Madwed, M.
Moriak, N. Moss, C. Pargellis, S. Pav, A. Proto, A. Swinamer, L. Tang, C. Torcellini, J. Med. Chem.
2002, 45, 2994.
[4] A. H. Abadi, A. A. H. Eissa, G. S. Hassan, Chem. Pharm. Bull. 2003, 51, 838.
[5] X. Barril, M. C. Beswick, A. Collier, M. J. Drysdale, B. W. Dymock, A. Fink, K. Grant, R. Howes,
A. M. Jordan, A. Massey, A. Surgenor, J. Wayne, P. Workman, L. Wright, Bioorg. Med. Chem. Lett.
2006, 16, 2543.
[6] B. W. Dymock, X. Barril, P. A. Brough, J. E. Cansfield, A. Massey, E. McDonald, R. E. Hubbard, A.
Surgenor, S. D. Roughley, P. Webb, P. Workman, L. Wright, M. J. Drysdale, J. Med. Chem. 2005, 48,
4212.
[7] K.-M. J. Cheung, T. P. Matthews, K. James, M. G. Rowlands, K. J. Boxall, S. Y. Sharp, A. Maloney,
S. M. Roe, C. Prodromou, L. H. Pearl, G. W. Aherne, E. McDonald, P. Workman, Bioorg. Med.
Chem. Lett. 2005, 15, 3338.
[8] P. Pevarello, M. G. Brasca, R. Amici, P. Orsini, G. Traquandi, L. Corti, C. Piutti, P. Sansonna, M.
Villa, B. S. Pierce, M. Pulici, P. Giordano, K. Martina, E. L. Fritzen, R. A. Nugent, E. Casale, A.
Cameron, M. Ciomei, F. Roletto, A. Isacchi, G. Fogliatto, E. Pesenti, W. Pastori, A. Marsiglio, K. L.
Leach, P. M. Clare, F. Fiorentini, M. Varasi, A. Vulpetti, M. A. Warpehoski, J. Med. Chem. 2004, 47,
3367.
[9] W. E. Kirkpatrick, T. Okabe, I. W. Hillyard, R. K. Robins, A. T. Dren, T. Novinson, J. Med. Chem.
1977, 20, 387.
[10] P. Furet, P. Imbach, T. M. Ramsey, A. Schlapbach, D. Scholz, G. Caravatti, PCT Int. Appl. WO
20045,282 (Chem. Abstr. 2004, 140, 111424v).
[11] D. Raffa, G. Daidone, B. Maggio, S. Cascioferro, F. Plescia, D. Schillaci, Farmaco 2004, 59, 215.
[12] S. A. F. Rostom, Bioorg. Med. Chem. 2006, 14, 6475.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1687
[13] G. M. Allan, C. Bubert, N. Vicker, A. Smith, H. J. Tutill, A. Purohit, M. J. Reed, B. V. L. Potter, Mol.
Cell. Endocrinol. 2006, 248, 204.
[14] S. Ludwig, O. Planz, H.-H. Sedlacek, S. Pleschka, PCT Int. Appl. WO 2004085682 (Chem. Abstr.
2004, 141, 307497).
[15] E. M. Perchellet, M. M. Ward, A.-L. Skaltsounis, I. K. Kostakis, N. Pouli, P. Marakos, J.-P. H.
Perchellet, Anticancer Res. 2006, 26, 2791.
[16] M. Anzaldi, E. Sottofattori, R. Rizzetto, B. Granello di Casaleto, A. Balbi, Eur. J. Med. Chem. 1999,
34, 837.
[17] A. Balbi, M. Anzaldi, M. Mazzei, M. Miele, M. Bertolotto, L Ottonello, F. Dallegri, Bioorg. Med.
Chem. 2006, 14, 5152.
[18] S. Manfredini, D. Simoni, G. Caminiti, S.Vertuani, F. P. Invidiata, B. Moscato, S. Hates, E. De Clercq,
J. Balzarini, Med. Chem. Res. 1998, 8, 291.
[19] M. Begtrup, G. Boyer, P. Cabildo, C. Cativiela, R. Claramunt, J. Elguero, J. I. Garcꢅa, C. Toiron, P.
Vedsø, Magn. Reson. Chem. 1993, 31, 107.
[20] A. R. Katritzky, C. W. Rees, J. M. Lagowski, in ꢀComprehensive Heterocyclic Chemistryꢁ, Pergamon
Press, 1984, Vol. 5, pp. 182–195.
[21] R. G. Keedwell, Y. Zhao, L. A. Hammond, S. Qin, K.-Y. Tsang, A. Reitmair, Y. Molina, Y. Okawa,
L. I. Atangan, D.-L. Shurland, K. Wen, D. M. A. Wallace, R. Bird, R. A. S. Chandraratna, G. Brown,
Cancer Res. 2004, 64, 3302.
[22] D. Simoni, M. Roberti, F. P. Invidiata, R. Rondanin, R. Baruchello, C. Malagutti, A. Mazzali, M.
Rossi, S. Grimaudo, F. Capone, L. Dusonchet, M. Meli, M. V. Raimondi, M. Landino, N.
DꢁAlessandro, M. Tolomeo, D. Arindam, S. Lu, D. M. Benbrook, J. Med. Chem. 2001, 44, 2308.
[23] I. Nicoletti, G. Migliorati, M. C. Pagliacci, F. Grignani, C. Riccardi, J. Immunol. Methods 1991, 139,
271.
[24] B. Dibbert, I. Daigle, D. Braun, C. Schranz, M. Weber, K. Blaser, U. Zangemeister-Wittke, A.
Akbar, H. Simon, Blood 1998, 92, 778.
Received December 31, 2008