Synthesis and antiproliferative activity of 3-amino-N-phenyl-1H-indazole-1-carboxamides

Article abstract of DOI:10.1016/j.ejmech.2008.03.023

A series of new 3-amino-N-phenyl-1H-indazole-1-carboxamides 10 have been prepared from commercially available phenyl isocyanate precursors 8 and 3-aminoindazole 9. Some of the synthesized compounds were evaluated for their in vitro antineoplastic activity

Full text of DOI:10.1016/j.ejmech.2008.03.023

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European Journal of Medicinal Chemistry 44 (2009) 165e178
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiproliferative activity of 3-amino-N-phenyl-
1H-indazole-1-carboxamides
a
a
a
Demetrio Raffa a, , Benedetta Maggio , Stella Cascioferro , Maria Valeria Raimondi ,
Domenico Schillaci a, Giorgio Gallo a, Giuseppe Daidone a, Salvatore Plescia a,
Fiorella Meneghetti b, Gabriella Bombieri b, Antonietta Di Cristina c,
Rosaria M. Pipitone c, Stefania Grimaudo c, Manlio Tolomeo c
*
a Dipartimento di Chimica e Tecnologie Farmaceutiche, Universita` degli Studi di Palermo, Via Archirafi, 32, 90123 Palermo, Italy
b Istituto di Chimica Farmaceutica e Tossicologica ‘‘P. Pratesi’’, Universita` degli Studi di Milano, Viale Abruzzi, 42, 20131 Milano, Italy
c Divisione di Ematologia e Servizio AIDS, Policlinico Universitario ‘‘Paolo Giaccone’’, Universita` degli Studi di Palermo,
Via del Vespro, 129, 90127 Palermo, Italy
Received 7 August 2007; received in revised form 20 February 2008; accepted 20 March 2008
Available online 8 April 2008
Abstract
A series of new 3-amino-N-phenyl-1H-indazole-1-carboxamides 10 have been prepared from commercially available phenyl isocyanate pre-
cursors 8 and 3-aminoindazole 9. Some of the synthesized compounds were evaluated for their in vitro antineoplastic activity against 60 human
cell lines derived from seven clinically isolated cancer types (lung, colon, melanoma, renal, ovarian, brain, and leukemia) according to the NCI
standard protocol. The test results indicated that 3-amino-1H-indazole-1-carboxamides 10 were endowed with an interesting antiproliferative
activity. The most active compounds of this series, 10d,e, were able to inhibit cell growth of many neoplastic cell lines at concentrations lower
than 1 mM (0.0153 mM in SR leukemia) causing a block in G0eG1 phase of cell cycle. Analysis of pRb expression showed that these two com-
pounds increased the ratio between underphosphorylated pRb and total pRb. The X-ray structure of 10w, confirmed the 3-amino-N-phenyl-1H-
indazole-1-carboxamide structure of compounds 10.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: 1H-Indazole-1-carboxamides; Crystallographic study; G0eG1 arrest; pRb
1. Introduction
animal models [3], and pyrimido[1,2-b]indazoles 4 are potential
anticancer agents against A-549 cell line [4]. Finally, lonidamine
5, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid, is
a relatively new non-conventional anticancer drug that selec-
tively inhibits the energy metabolism of neoplastic cells and
increases the cell membranes permeability [5].
Recently our research group has reported the synthesis
of new indazole derivatives owing to their interesting antipro-
liferative activity. In particular, we have linked the indazole
moiety to the quinazolin-4(3H )-one and benzotriazin-4(3H )-
one nuclei with the aim to evaluate the pharmacological pro-
file of the indazol-3-yl and indazol-5-yl derivatives [6,7].
The pharmacological screening showed that 3-(indazol-3-yl)-
7-chloro-2-ethyl-quinazolin-4(3H )-one 6 was the most active
Indazole nucleus represents a very attractive scaffold to
obtain new molecules endowed with antineoplastic activity.
As examples (Fig. 1), N2-(substituted benzyl)-3-(4-methyl-
phenyl)-2H-indazoles 1 exhibit antiangiogenic activity [1] and
4-methoxy-N-(3-chloro-indazol-7-yl)benzenesulfonammide 2
possesses antiproliferative activity against L1210 murine leuke-
mia cells [2]. Moreover, N-[4-(3-amino-1H-indazol-4-yl)-
phenyl]-N0-(3-methylphenyl)-urea (ABT-869) 3 has shown
significant tumor growth inhibition in multiple preclinical
* Corresponding author. Tel.: þ39 091 6236117.
E-mail address: demraffa@unipa.it (D. Raffa).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.023

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D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
K562 cells as well as on the underphosphorylated and total ret-
inoblastoma protein (pRb) ratio.
Due to the presence of the indazole nucleus, the N-phenyl-
3-(phenylureido)-1H-indazole-1-carboxamides
by-products
11 and the 1-(1H-indazol-3-yl)-3-phenylureido derivatives 12
(see Scheme 1) were also considered as potential antiprolifer-
ative agents.
2. Chemistry
The synthesis [10] of the 3-amino-N-phenyl-1H-indazole-
1-carboxamide derivatives 10 started from the commercially
available phenyl isocyanate precursors 8 and 3-aminoindazole
9 [11]. As reported in Fig. 2, this reaction should give all the
possible substitution products, that is the monosubstituted de-
rivatives 10, 12 and 13 as well as the disubstituted ones 11 and
14. However, as outlined in Scheme 1, only the products
10e12 were obtained owing to the experimental conditions:
(A) refluxing of a suitable substituted phenyl isocyanate 8
with 3-aminoindazole 9 (isocyanate:indazole 1:1) in tetrahy-
drofuran (1e6 h); (B) refluxing of a suitable substituted
phenyl isocyanate 8 with 3-aminoindazole 9 (isocyanate:inda-
zole 2:1) in tetrahydrofuran (1e6 h); and (C) stirring at 25 ꢀC
of a suitable substituted phenyl isocyanate 8 and the 3-aminoin-
dazole 9 (isocyanate:indazole 1:1) in tetrahydofuran for 24 h.
The attempt to obtain derivatives 10 from 8a,d and 9 ac-
cording to the procedure A failed, affording a complex multi-
component mixture. Later on, the mixture was monitored for
the presence of compounds 10a,d by TLC using the authentic
specimens of 10a,d, 11a,d and 12a,d obtained from the proce-
dures B and C; TLC revealed the presence of small amounts of
10a,d and 11a,d in the reaction mixture.
By varying the ratio of reactants as outlined in procedure B,
the workup and purification of the reaction mixture afforded one
of the compounds 11 or 12 as the major product. In particular,
isocyanates 8a,dei,l,n,qes gave the compounds 11a,dei,
l,n,qes whereas 8b,c,o,p the 1-(1H-indazol-3-yl)-3-phenylur-
eido derivatives 12b,c,o,p. Moreover, attempts to obtain deriva-
tives 10 by hydrolysis of derivatives 11a,dei,l,n,q,r gave the
1-(1H-indazol-3-yl)-3-phenylureido derivatives 12a,dei,l,n,q,r.
Finally, according to the procedure C, stirring at room tem-
perature for 24 h a mixture of substituted phenyl isocyanate 8
and 3-aminoindazole 9 in tetrahydofuran allowed us to obtain
the expected derivatives 10.
Inprinciplethisreactionshouldgiveboththe3-amino-N-phe-
nyl-1H-indazole-1-carboxamide derivative 10 and the 3-amino-
N-phenyl-2H-indazole-2-carboxamide isomer 13 (Fig. 2). The
analyticalas well as spectroscopicaldata of the reactionproducts
well correlated with both the structures 10 and 13, therefore, for
a conclusive attribution of the 3-amino-N-phenyl-1H-indazole-
1-carboxamide structure 10, compound 10w was analyzed by
X-ray diffraction techniques (Fig. 3).
Fig. 1. Chemical structure of some examples of antineoplastic agents which
posses the indazole nucleus.
derivative with a GI50 recorded on K562 and K562-R cell lines
of 10.7 and 11.7 mM, respectively. Moreover, we synthesized
some new triazenoindazoles which showed in vitro cytotoxic
activity against K562, HL-60, L1210 and MCF7 cell lines.
Among the tested compounds, 3-(30,30-diethyltriazeno)-5-
chloroindazole 7 was the most active one against all the cell
lines [8]. These data clearly indicated that triazenoindazoles
act in vitro with a different mechanism with respect to that
of dacarbazine which is generally based on metabolic activa-
tion in vivo [9].
Finally, owing to our interest to the chemistry and pharma-
cology of indazole derivatives, and considering that the chem-
ical class of 3-amino-N-phenyl-1H-indazole-1-carboxamides
10 possesses some novel structural elements, we decided to in-
vestigate whether these compounds can give rise to biological
activity. Later these were, therefore, synthesized and evaluated
for their antineoplastic activity. The most active compounds
were evaluated for their effects on cell cycle distribution in
On the basis of the crystallographic results of 10w and con-
sidering the sterical hindrance of the phenylureido moiety in
the position 3, we have attributed to the product of diacylation
the 1H-indazole-1-carboxamide structure 11 instead of 2H-in-
dazol-2-carboxamide one 14.

D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
167
Scheme 1. Synthetic pathway for the formation of the compounds 10, 11 and 12.
The structures of the new compounds were characterized
3. Crystallography
by analytical and spectroscopic measurements. The 1H
NMR spectra of 10 showed both the NH and NH2 signals
of the 3-amino-N-phenyl-1H-indazole-1-carboxamide struc-
ture. In fact, the 1-carboxamide NH proton appeared as sin-
glet at 8.99e9.89 d, while the amino protons were found
The crystal structure of 10w is shown in Fig. 3 as ORTEP
[12] view. The overall conformation of the molecule is defined
by the torsion angles t1 C(12)eO(2)eC(15)eC(16) of 76(1)ꢀ,
t2 C(14)eC(9)eN(4)eC(8) of 29(1)ꢀ and t3 C(21)eC(16)e
C(15)eO(2) of 50(1)ꢀ, showing a significant tilting of the dis-
tal phenyl moiety with respect to the rest of the molecule. The
indazole ring has an inclination of 35(1)ꢀ compared to the
1
as a broad singlet at 6.31e6.70 d. H NMR spectra of 11
are consistent with an N-phenyl-3-(phenylureido)-1H-inda-
zole-1-carboxamide structure. The 1-carboxamide NH
protons appeared as singlets at 9.42e10.11 d, while the
phenylureido NH protons were found at 9.67e10.26
d and 10.10e10.70 d, respectively. Finally, the 1H NMR
spectra of 12 demonstrated the 1-(1H-indazol-3-yl)-3-phe-
nylureido structure. The 1H-indazole NH proton appeared
as a singlet at 12.47e12.69 d, while the phenylureido NH
protons were found at 9.30e10.30 d and 9.79e10.72 d,
respectively.
˚
proximal phenyl group (centroids distance 6.9(1) A) and of
80(1)ꢀ with the distal phenyl group (centroids distance
˚
10.9(1) A). The two phenyl groups are almost perpendicular
ꢀ
˚
with a dihedral angle of 81(1) (centroids distance 5.1(1) A).
The main packing determinants are hydrogen bonds N(3)e
0
ꢀ
˚
H/O(2) , with distance of 2.18(1) A and angle 163(1) (at
3/2ꢁx, 1/2ꢁy, 1/3ꢁz) with consequent formation of dimers
involving centrosymmetrically related molecules. Further

168
D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
Fig. 2. Possible substitution products that can be derived from compounds 8 and 9.
0
˚
interactions between C(11)eH/N(1) at 2.73(1) A with angle
molar concentration inducing 50% net cell death). The GI50
values for the test compounds were reported in the Table 1.
133(1)ꢀ contribute to the dimer’s stability (Fig. 3).
4. Pharmacological results
4.2. Cell cycle analysis
4.1. Tumor cell line screen
The effects of the most active compounds (10d,e,y, 12i,l)
on cell cycle distribution were analysed in K562 cells. Cells
were cultured for 24 h in the presence of each compound at
the GI50 concentration evaluated after 24 h of treatment
(10d 1.20 mM, 10e 15.9 mM, 10y 180 mM, 12i 140 mM and
12l 42.7 mM). Flow cytometric analyses of cell cycle, carried
out as described in Section 7, are reported in Fig. 4. Com-
pounds 10y, 12i,l induced a recruitment of cells in S and
G2M phase while compounds 10d,e caused a marked increase
of cells in G0eG1 phase and a decrease of cells either in S
or G2eM phase. Since most of chemotherapeutic drugs act
on S or G2M phase and are ineffective in G0eG1 phase,
we considered compounds 10d,e of interest for deep
investigations.
3-Amino-N-phenyl-1H-indazole-1-carboxamides 10a,d,e,
v,y, N-phenyl-3-(phenylureido)-1H-indazole-1-carboxamide
11d, and the 1-(1H-indazol-3-yl)-3-phenylureido derivatives
12g,i,l were selected by the National Cancer Institute (NCI)
to evaluate their in vitro antiproliferative activity against 60
human cell lines derived from seven clinically isolated cancer
types (lung, colon, melanoma, renal, ovarian, brain, and leuke-
mia) according to the NCI standard protocol [13].
The antitumor activity is given by three parameters for each
cell line; GI50 (compound’s molar concentration inhibiting
50% net cell growth), TGI (compound’s molar concentration
for total inhibition of net cell growth), and LC50 (compound’s

D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
169
Fig. 3. Left: ORTEP [12] view of the compound 10w, showing the atom-numbering scheme (ellipsoids are at 40% probability and H-atoms are shown as spheres of
ꢀ
˚
arbitrary radius). Right: intermolecular interactions along the crystallographic c axis. Selected bond lengths [A] and angles [ ]: O(1)eC(8), 1.212(4); O(2)eC(12),
1.384(4); O(2)eC(15), 1.439(5); N(1)eC(1), 1.313(5); N(1)eN(2), 1.403(4); N(2)eC(7), 1.388(5); N(2)eC(8), 1.399(5); N(3)eC(1), 1.372(5); N(4)eC(8),
1.357(5); N(4)eC(9), 1.414(5); O(1)eC(8)eN(4), 126.9(4); O(1)eC(8)eN(2), 121.2(4); N(1)eN(2)eC(7), 110.8(3); N(1)eN(2)eC(8), 120.3(3); N(1)eC(1)e
N(3), 121.0(4); N(2)eC(8)eN(4), 111.9(3); N(3)eC(1)eC(2), 126.8(4); N(2)eN(1)eC(1), 105.6(3).
4.3. Effects on retinoblastoma protein (pRb)
Therefore, in both cases the ratio between underphosphory-
lated pRb and total pRb was increased.
The retinoblastoma gene encodes a nuclear phosphoprotein
(pRb) expressed in the majority of vertebrates’ normal cells and
acts as a tumor suppressor factor which plays a key role in the
control of cell division [14]. The pRb underphosphorylated
form is mainly found in resting cells, whereas the highly phos-
phorylated forms are present in proliferating cells. Growth
factors act to promote cells through the G1 and S phase of
cell cycle and, during this time, pRb undergoes sequential
phosphorylation. The pRb underphosphorylated form binds
to critical regulatory proteins (including E2F) and represses
the transcription of genes involved in cell cycle progression
from the G1 to the S phase [15]. Fig. 5 shows the effects of
10d,e on total Rb and underphosphorylated pRb levels in
K562 cells.
5. Discussion
All the compounds 10a,d,e,v,y, 11d, 12g,i,l tested by NCI
were found to possess low cytotoxicity. On the other hand,
they turn out to be inhibitor of cell growth with GI50 values
ranging from 0.0153 to 98.8 mM with the exception of 12g
which was totally inactive (Table 1). Among these, the best
antiproliferative agent resulted compound 10d which showed
antiproliferative activity against every type of tumor cell lines
investigates. As it can be seen in Table 2 it was particularly
active against all kinds of leukemia and mostly active against
some other cell lines such as non-small cell lung (NCI-H460,
NCI-H522), colon cancer (HTC-15, HT29, KM12), CNS can-
cer (SF-295, SF-539), melanoma (LOXIMVI, M14, SK-MEL-
5, UACC-62), renal cancer (ACHN, UO-31), prostate cancer
(DU-145) and breast cancer (MCF7, MDA-MB-231/ATCC,
MDA-MB-435, T-47D). Good activity has also been shown
Flow cytometric analyses of pRb and underphosphorylated
pRb were carried out as described in Section 7. Compounds
10d,e caused a decrease of total pRb but the level of under-
phosphorylated pRb was similar to that of the control.

170
D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
Table 1
Compound’s molar concentration inhibiting 50% net cell growth (GI50, mM) of 10a,d,e,v,y, 11d, 12g,i,l against 60 human cell lines derived from seven clinically
isolated cancer types
Cancer types
Leukemia
Panel/cell line
10a
10d
10e
0.213
2.23
15.9
10y
10v
36.7
11d
12g
59.6
12i
12l
CCRF-CEM
HL-60 (TB)
K-562
nt
nt
>100
nt
>100
nt
nt
33.2
<100
0.466
1.20
47.2
>100
54.0
27.9
41.2
39.4
21.1
29.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
17.9
42.7
59.1
52.0
40.5
63.2
>100
16.3
MOLT-4
RPMI-8226
SR
32.7
16.1
12.9
0.636
1.86
0.0153
4.48
2.72
86.0
92.5
16.0
55.4
0.0489
Non-small cell
lung cancer
A549/ATCC
EKVX
HOP-62
48.5
84.0
51.0
17.3
98.5
95.6
12.9
23.5
13.3
20.8
18.6
21.2
19.4
20.6
15.7
27.4
>100
44.8
>100
30.2
>100
45.8
54.1
75.2
24.6
83.1
83.5
>100
>100
59.7
18.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
15.7
48.2
19.6
12.9
26.1
50.0
81.6
15.0
33.9
11.2
13.7
14.2
13.5
16.8
27.8
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
>100
>100
>100
>100
>100
>100
<100
>100
33.4
55.1
0.408
2.90
78.9
49.5
34.5
34.9
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
<100
13.4
13.9
19.0
19.9
15.5
13.5
11.3
18.3
>100
66.2
>100
73.6
>100
39.1
>100
85.9
61.1
43.5
44.1
48.2
67.9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
71.8
55.9
16.2
36.2
26.1
23.4
36.7
95.8
34.8
40.9
74.6
86.2
38.3
4.27
7.03
1.08
2.39
2.94
3.62
KM12
SW-620
>100
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB75
U251
36.2
54.8
31.4
4.66
0.530
0.495
25.8
21.7
17.8
42.7
28.6
30.5
>100
56.6
60.9
>100
49.6
37.6
95.9
75.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
16.2
39.0
30.5
78.3
17.5
31.1
>100
>100
>100
<100nt
22.8
>100
17.0
13.9
37.0
98.7
30.6
61.7
33.8
70.2
40.1
96.1
49.3
<100
74.7
<100
>100
Melanoma
LOX IMVI
MALME-3M
M14
0.477
19.2
0.772
12.2
14.7
18.6
nt
92.1
49.3
71.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
22.5
76.9
22.5
81.4
42.2
28.7
47.2
31.7
>100
>100
nt
>100
>100
64.9
37.2
>100
>100
>100
>100
>100
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
36.4
11.5
56.3
39.7
71.7
43.9
32.6
15.2
29.2
14.7
>100
>100
>100
>100
>100
2.22
10.0
0.353
27.9
73.6
52.3
41.6
Ovarian cancer
IGROV1
7.70
6.98
6.23
35.5
18.8
30.7
20.0
35.9
44.0
27.3
18.1
92.0
63.2
45.5
39.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.758
4.59
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
65.7
57.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
90.8
37.7
34.3
29.6
10.7
<100
44.2
<100
31.3
21.9
36.9
>100
>100
30.3
58.3
>100
95.2
>100
>100
38.7
>100
31.1
Renal cancer
786-0
A498
<100
31.4
3.76
16.7
1.69
21.0
19.0
22.8
28.3
29.6
23.1
24.2
17.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
82.4
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
50.1
45.3
20.2
>100
>100
21.1
22.2
12.9
27.2
31.0
24.3
72.5
37.9
17.4
45.7
>100
74.3
>100
30.4
<100
<100
18.6
>100
>100
>100
92.4
28.5
0.347
13.5
1.02
Prostate cancer
Breast cancer
PC-3
DU-145
<100
62.3
14.9
25.0
83.1
>100
35.5
73.1
>100
>100
>100
>100
>100
31.4
51.7
80.0
MCF7
60.0
56.5
98.8
73.0
54.9
28.8
68.7
0.555
7.87
0.847
14.5
23.7
13.8
20.2
19.1
14.0
14.8
0.269
90.7
29.5
37.6
53.6
43.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
18.2
1.75
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
>100
>100
>100
>100
>100
44.2
17.9
19.4
51.5
76.4
37.5
46.1
33.8
47.1
74.2
>100
17.2
2.82
13.5
1.55
MDA-MB-435
BT-549
T-47D
>100
28.0
>100
>100
25.9
33.0
>100
>100
0.215
nt: Not tested.

D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
171
Fig. 5. Effects of compounds 10d,e on total pRb or underphosphorylated pRb
(under-P-pRb) expressions. The cells were cultured without any compounds or
with each compound (10d ¼ 14 mM, 10e ¼ 34 mM). Black filled line: cells
stained with an isotype monoclonal antibody (control); thick line: cells stained
with an anti-pRb or an anti-underphosphorylated-pRb monoclonal antibody;
thin line: cells stained with an anti-pRb or an anti-underphosphorylated-pRb
monoclonal antibody after 24 h treatment with each compound.
The effects of 10d,e on cell cycle distribution showed how
these compounds induced a marked recruitment of cells in
G0eG1. Concerning the effects on retinoblastoma protein
(pRb), compounds 10d,e caused a decrease in total pRb with-
out any modification of the underphosphorylated pRb level.
Table 2
In vitro antiproliferative activity (mM) against the cell lines in which the com-
pound 10d resulted mostly active
Cancer types
Panel/cell line
10d
GI50
TGI
15.2
LC50
54.9
Leukemia
HL-60 (TB)
K-562
0.466
1.20
33.8
20.4
25.8
82.2
>100
>100
Fig. 4. Effects of compounds 10d (b), 10e (c), 10y (d), 12i (e), 12l (f) on DNA
content/cell following the treatment of K562 cells for 24 h. The cells were cul-
tured without any compound (a) or with each compound used at the concen-
tration corresponding to the GI50. Cell cycle distribution was analyzed by
the standard propidium iodide procedure as described in Section 7. Sub-
G0eG1 (A), G0eG1, S, and G2eM cells are indicated in panel a.
MOLT-4
RPMI-8226
SR
0.636
1.86
0.0153
84.0
34.8
Non-small cell
lung cancer
NCI-H460
NCI-H522
0.408
2.90
13.0
84.6
>100
>100
17.6
>100
>100
56.3
Colon cancer
HCT-15
HT29
KM12
1.08
2.39
2.94
from compound 10e even if it resulted less active with respect
to 10d. In fact, it was efficacious against the leukemic cell
lines with particular respect to CCRF-CEM, HL-60, MOLT-
4 and RPMI-8226 (Table 3) being a micromolar inhibitor in
all the other ones (Table 1).
Moreover, 10d,e were the only compounds endowed of
significant TGI values ranging from 1.25 to 97.9 mM against
almost all cell lines of the NCI panel and, in some cases, of
LC50 value ranging from 34.3 to 96.4 mM (Tables 2 and 3).
Significant results were also showed from the compounds
10y, 12i,l although only in a very limited numbers of cell lines;
with respect to leukemic cell line SR compounds 12l showed
a remarkable activity, the compound 10y was very active
against MCF7 while the IGROV1 and T-47D were inhibited
by 12i (Table 4).
>100
>100
CNS cancer
Melanoma
SF-295
SF-539
0.530
0.495
>100
>100
>100
5.36
16.1
>100
8.64
>100
33.4
>100
28.3
8.82
43.9
>100
>100
LOX IMVI
M14
0.477
0.772
2.22
61.5
>100
34.3
SK-MEL-5
UACC-62
0.353
>100
Renal cancer
ACHN
UO-31
1.69
0.347
>100
>100
Prostate cancer
Breast cancer
DU-145
1.02
>100
MCF7
0.555
0.847
2.82
89.1
MDA-MB-231/ATCC
MDA-MB-435
T-47D
>100
>100
>100
1.55

172
D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
Table 3
In vitro antiproliferative activity (mM) of 10e
over Na SO . All melting points were determined on a Buchi
¨
2
4
530 capillary apparatus and are uncorrected. IR spectra were
recorded with a Perkin Elmer Spectrum RXI FT-IR System
spectrophotometer in KBr disc. 1H NMR spectra were
obtained with a Bruker AC-E 250-MHz spectrometer (tetra-
methylsilane as an internal standard): chemical shifts are
expressed in d values (ppm). Merck silica gel (Kiesegel 60/
230e400 mesh) was used for flash chromatography columns.
Microanalyses (C, H, N) performed at REDOX snc Monza
(MI) Italy are within ꢂ0.4% of the theoretical values. Yields
refer to purified products and are not optimized.
Cancer type
Leukemia
Panel/cell line
10e
GI50
TGI
LC50
CCRF-CEM
HL-60 (TB)
MOLT-4
0.213
2.23
4.48
2.72
17.5
30.4
46.2
30.8
>100
>100
>100
>100
RPMI-8226
Table 4
In vitro antiproliferative activity (mM) of 10y, 12i and 12l
Compound
Cancer types
Panel/cell line
GI50
TGI
64.6
LC50
7.1.2. Starting material
All phenyl isocyanates 8aez were purchased from Aldrich
Chemical Company and were used as received.
10y
12i
Breast
MCF7
IGROV1
T-47D
SR
0.269
0.758
0.215
0.0489
>100
>100
>100
>100
Ovarian
Breast
Leukemia
>100
>100
24
12l
7.1.3. Synthesis of 3-amino-N-phenyl-1H-indazole-1-
carboxamides 10
This suggests that they could act by inducing a decrease in the
phosphorylated form of pRb. A similar activity has recently
been described for compound tyrphostin AG1024 in mela-
noma cells [16]. Tyrphostin AG1024 caused the loss
of phosphorylated forms of pRb that was not associated with
suppression of cyclin-dependent kinases 2 and 4 activity but
rather with proteasomal and nonproteasomal degradation of
phosphorylated pRb. Because mutations in the retinoblastoma
gene (RB-1) have been described in a wide variety of neo-
plasm, and constitutive phosphorylation/inactivation of pRb
has been implicated in conferring uncontrolled growth to
many neoplastic cells [17], compounds able to decrease the
level of phosphorylated pRb could be of interest in the treat-
ment of different malignancies.
7.1.3.1. General method. A mixture of 14 mmol of substituted
phenyl isocyanate 8 and 14 mmol of 3-aminoindazole 9 in
10 ml of THF was stirred at room temperature for 24 h. At
the completion of the reaction the solvent was removed under
reduced pressure, the solid residue was washed with ethanol
and then crystallized from a suitable solvent.
7.1.3.1.1. 3-Amino-N-(2,6-difluorophenyl)-1H-indazole-1-
carboxamide 10a. 2,6-Difluorophenyl isocyanate 8a was al-
lowed to react with 3-aminoindazole 9 as described in Section
7.1.3.1 to give 10a. Purified by flash chromatography on silica
gel [25] with chloroform as eluant, and crystallized from diox-
ane, 65% yield; mp 165e168 ꢀC; I.R. (KBr, cmꢁ1) 3486, 3359
(NH, NH2), 1688 (CO); 1H NMR (DMSO) d 6.40 (s, 2H, NH2,
exchangeable); 7.19e8.15 (a set of signals, 7H, aromatic pro-
tons); 9.30 (s, 1H, NH, exchangeable). Anal. (C14H10F2N4O)
C, H, N.
6. Conclusion
3-Amino-N-phenyl-1H-indazole-1-carboxamides 10 here
described were primarily designed considering the well-known
biological activity of the indazole nucleus and their structural
novelty. The effects on cell cycle distribution showed how
these compounds induced a marked recruitment of cells in
G0eG1 but not in G2eM. This is a very encouraging result
considering that currently, drugs capable of blocking and/or
killing cancer cells in the G0eG1 phase are considered of pri-
mary importance because most chemotherapeutic drugs used
in the treatment of malignancies act in the S or G2eM phase
of the cell cycle but not in G0eG1. Thus, a variable percent-
age of cells in G0eG1 could escape from the cytotoxic effects
of the therapy (minimal residual disease) causing a relapse of
the disease months or years later [18e24].
7.1.3.1.2. 3-Amino-N-(2,5-dichlorophenyl)-1H-indazole-1-
carboxamide 10b. 2,5-Dichlorophenyl isocyanate 8b was
allowed to react with 3-aminoindazole 9 as described in Sec-
tion 7.1.3.1 to give 10b. Purified by preparative TLC (silica
gel plate, layer thickness 2 mm, chloroform as eluant), and
crystallized from ethanol, 45% yield; mp 185e188 ꢀC; I.R.
1
(KBr, cmꢁ1) 3439, 3341 (NH, NH2), 1697 (CO); H NMR
(DMSO) d 6.67 (s, 2H, NH2, exchangeable); 7.21e8.40
(a set of signals, 7H, aromatic protons). 9.47 (s, 1H, NH,
exchangeable). Anal. (C14H10Cl2N4O) C, H, N.
7.1.3.1.3. 3-Amino-N-(4-benzylphenyl)-1H-indazole-1-car-
boxamide 10d. 4-Benzylphenyl isocyanate 8d was allowed
to react with 3-aminoindazole 9 as described in Section
7.1.3.1 to give 10d. The solid collected was crystallized
from dioxane, 26% yield; mp 158e160 ꢀC; I.R. (KBr, cmꢁ1
)
7. Experimental protocols
3456, 3344 (NH, NH2), 1699 (CO); 1H NMR (DMSO)
d 3.91 (s, 2H, CH2); 6.35 (s, 2H, NH2, exchangeable);
7.18e8.19 (a set of signals, 13H, aromatic protons). 9.44 (s,
1H, NH, exchangeable). Anal. (C21H18N4O) C, H, N.
7.1. Chemistry
7.1.1. General
Reaction progress was monitored by TLC on silica gel
plates (Merck 60, F254, 0.2 mm). Organic solutions were dried
7.1.3.1.4. 3-Amino-N-(4-butoxyphenyl)-1H-indazole-1-car-
boxamide 10e. 4-Butoxyphenyl isocyanate 8e was allowed
to react with 3-aminoindazole 9 as described in Section

D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
173
1
7.1.3.1 to give 10e. The solid collected was crystallized from
ethanol, 98% yield; mp 138e141 ꢀC; I.R. (KBr, cmꢁ1) 3428e
3224 (NH, NH2), 1676 (CO); 1H NMR (DMSO) d 0.93 (t, 3H,
CH3); 1.43 (m, 2H, CH2); 1.67 (m, 2H, CH2); 3.94 (t, 2H,
CH2); 6.31 (s, 2H, NH2, exchangeable); 6.89e8.18 (a set of
signals, 8H, aromatic protons). 9.35 (s, 1H, NH, exchange-
able). Anal. (C18H20N4O2) C, H, N.
7.1.3.1.5. N-(4-Acetylphenyl)-3-amino-1H-indazole-1-car-
boxamide 10f. 4-Acetylphenyl isocyanate 8f was allowed to
react with 3-aminoindazole 9 as described in Section 7.1.3.1
to give 10f. The solid collected was crystallized from dioxane,
98% yield; mp 228e230 ꢀC; I.R. (KBr, cmꢁ1) 3469e3185
(NH, NH2), 1703 (CO),1660 (CO); 1H NMR (DMSO)
d 2.55 (s, 3H, CH3); 6.42 (s, 2H, NH2, exchangeable);
7.28e8.22 (a set of signals, 8H, aromatic protons). 9.89 (s,
1H, NH, exchangeable) Anal. (C16H14N4O2) C, H, N.
3353 (NH, NH2), 1703 (CO); H NMR (DMSO) d 4.04 (s,
2H, CH2); 6.41 (s, 2H, NH2, exchangeable); 7.08e8.15 (a
set of signals, 13H, aromatic protons). 9.06 (s, 1H, NH, ex-
changeable). Anal. (C21H18N4O) C, H, N.
7.1.3.1.11. 3-Amino-N-(2-methoxy-5-nitrophenyl)-1H-inda-
zole-1-carboxamide 10u. 2-Methoxy-5-nitrophenyl isocyanate
8u was allowed to react with 3-aminoindazole 9 as described in
Section 7.1.3.1 to give 10u. The solid collected was crystallized
from dioxane, 76% yield; mp 245e249 ꢀC; I.R. (KBr, cmꢁ1
)
3463e3336 (NH, NH2), 1704 (CO); 1H NMR (DMSO) d 4.08
(s, 3H, OCH3); 6.66 (s, 2H, NH2, exchangeable); 7.28e9.15
(a set of signals, 7H, aromatic protons). 9.34 (s, 1H, NH, ex-
changeable). Anal. (C15H13N5O4) C, H, N.
7.1.3.1.12. 3-Amino-N-(3,4-dimethoxyphenyl)-1H-indazole-
1-carboxamide 10v. 3,4-Dimethoxyphenyl isocyanate 8v was
allowed to react with 3-aminoindazole 9 as described in Sec-
tion 7.1.3.1 to give 10v. Purified by flash chromatography on
silica gel [25] with chloroform as eluant, and crystallized
7.1.3.1.6.
3-Amino-N-(2-chloro-4-nitrophenyl)-1H-inda-
zole-1-carboxamide 10j. 2-Chloro-4-nitrophenyl isocyanate
8j was allowed to react with 3-aminoindazole 9 as described
in Section 7.1.3.1 to give 10j. The solid collected was crystal-
lized from ethanol, 22% yield; mp 191e193 ꢀC; I.R. (KBr,
cmꢁ1) 3426e3311 (NH, NH2), 1720 (CO); 1H NMR
(DMSO) d 6.70 (s, 2H, NH2, exchangeable); 7.27e8.59 (a
set of signals, 7H, aromatic protons). 9.70 (s, 1H, NH, ex-
changeable). Anal. (C14H10ClN5O3) C, H, N.
from ethanol, 45% yield; mp 158e159 ꢀC; I.R. (KBr, cmꢁ1
)
3445e3249 (NH, NH2), 1680 (CO); 1H NMR (DMSO)
d 3.74 (s, 3H, OCH3); 3.78 (s, 3H, OCH3); 6.32 (s, 2H,
NH2, exchangeable); 6.91e8.20 (a set of signals, 7H, aromatic
protons). 9.33 (s, 1H, NH, exchangeable). Anal. (C16H16N4O3)
C, H, N.
7.1.3.1.13. 3-Amino-N-[4-(benzyloxy)phenyl]-1H-indazole-
1-carboxamide 10w. 4-(Benzyloxy)phenyl isocyanate 8w
was allowed to react with 3-aminoindazole 9 as described in
Section 7.1.3.1 to give 10w. The solid collected was crystal-
lized from ethanol, 95% yield; mp 127e128 ꢀC; I.R. (KBr,
cmꢁ1) 3458, 3368 (NH, NH2), 1706 (CO); 1H NMR
(DMSO) d 5.09 (s, 2H, OCH2); 6.32 (s, 2H, NH2, exchange-
able); 6.99e8.19 (a set of signals, 13H, aromatic protons).
9.39 (s, 1H, NH, exchangeable). Anal. (C21H18N4O2) C, H, N.
7.1.3.1.14. 3-Amino-N-(2,4-dimethoxyphenyl)-1H-indazole-
1-carboxamide 10x. 2,4-Dimethoxyphenyl isocyanate 8x was
allowed to react with 3-aminoindazole 9 as described in Section
7.1.3.1 to give 10x. The solid collected was crystallized from
7.1.3.1.7. 3-Amino-N-1-naphthyl-1H-indazole-1-carboxa-
mide 10k. 1-Naphthyl isocyanate 8k was allowed to react
with 3-aminoindazole 9 as described in Section 7.1.3.1 to give
10k. The solid collected was crystallized from ethanol, yield
60%; mp 191e193 ꢀC; I.R. (KBr, cmꢁ1) 3405e3325 (NH,
1
NH2), 1678 (CO); H NMR (DMSO) d 6.51 (s, 2H, NH2,
exchangeable); 7.27e8.19 (a set of signals, 11H, aromatic pro-
tons). 9.70 (s, 1H, NH, exchangeable). Anal. (C14H10ClN5O3)
C, H, N.
7.1.3.1.8.
3-Amino-N-(2-methyl-5-nitrophenyl)-1H-inda-
zole-1-carboxamide 10m. 2-Methyl-5-nitrophenyl isocyanate
8m was allowed to react with 3-aminoindazole 9 as described
in Section 7.1.3.1 to give 10m. The solid collected was crys-
tallized from dioxane, 74% yield; mp 250e253 ꢀC; I.R.
ethanol, 57% yield; mp 140e142 ꢀC; I.R. (KBr, cmꢁ1
)
3412e3234 (NH, NH2), 1686 (CO); 1H NMR (DMSO) d 3.78
(s, 3H, OCH3); 3.91 (s, 3H, OCH3); 6.52 (s, 2H, NH2, ex-
changeable); 6.55e8.18 (a set of signals, 7H, aromatic pro-
tons). 8.99 (s, 1H, NH, exchangeable). Anal. (C16H16N4O3)
C, H, N.
1
(KBr, cmꢁ1) 3466e3371 (NH, NH2), 1703 (CO); H NMR
(DMSO) d 2.44 (s, 3H, CH3); 6.56 (s, 2H, NH2, exchange-
able); 7.26e8.84 (a set of signals, 7H, aromatic protons).
9.26 (s, 1H, NH, exchangeable). Anal. (C15H13N5O3) C, H, N.
7.1.3.1.9. 3-Amino-N-(2-ethoxyphenyl)-1H-indazole-1-car-
boxamide 10r. 2-Ethoxyphenyl isocyanate 8r was allowed to
react with 3-aminoindazole 9 as described in Section 7.1.3.1
to give 10r. The solid collected was crystallized from ethanol,
60% yield; mp 143e145 ꢀC; I.R. (KBr, cmꢁ1) 3423, 3348
(NH, NH2), 1702 (CO); 1H NMR (DMSO) d 1.46 (t, 3H,
CH3); 4.19 (q, 2H, CH2); 6.56 (s, 2H, NH2, exchangeable);
6.97e8.29 (a set of signals, 8H, aromatic protons). 9.37 (s,
1H, NH, exchangeable). Anal. (C16H16N4O2) C, H, N.
7.1.3.1.10. 3-Amino-N-(2-benzylphenyl)-1H-indazole-1-car-
boxamide 10t. 2-Benzylphenyl isocyanate 8t was allowed to
react with 3-aminoindazole 9 as described in Section 7.1.3.1
to give 10t. The solid collected was crystallized from ethanol,
61% yield; mp 135e136 ꢀC; I.R. (KBr, cmꢁ1) 3442, 3374,
7.1.3.1.15. 3-Amino-N-(2-methoxyphenyl)-1H-indazole-1-
carboxamide 10y. 2-Methoxyphenyl isocyanate 8y was al-
lowed to react with 3-aminoindazole 9 as described in Section
7.1.3.1 to give 10y. Purified by flash chromatography on silica
gel [25] with chloroform as eluant, and crystallized from diox-
ane, 89% yield; mp 163e165 ꢀC; I.R. (KBr, cmꢁ1) 3431e3342
(NH, NH2), 1708, 1691 (CO); 1H NMR (DMSO) d 3.94 (s, 3H,
OCH3); 6.59 (s, 2H, NH2, exchangeable); 6.97e8.29 (a set of
signals, 8H, aromatic protons). 9.26 (s, 1H, NH, exchangeable).
Anal. (C15H14N4O2) C, H, N.
7.1.3.1.16. 3-Amino-N-(2,5-dimethoxyphenyl)-1H-indazole-
1-carboxamide 10z. 2,5-Dimethoxyphenyl isocyanate 8z was
allowed to react with 3-aminoindazole 9 as described in Sec-
tion 7.1.3.1 to give 10z. The solid collected was crystallized

174
D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
from dioxane, 98% yield; mp 172e176 ꢀC; I.R. (KBr, cmꢁ1
)
exchangeable); 10.26 (s, 1H, NH, exchangeable); 10.47 (s, 1H,
NH, exchangeable). Anal. (C25H21N5O4) C, H, N.
3450, 3352 (NH, NH2), 1691 (CO); 1H NMR (DMSO)
d 3.74 (s, 3H, OCH3); 3.87 (s, 3H, OCH3); 6.56 (s, 2H,
NH2, exchangeable); 6.59e8.20 (a set of signals, 7H, aromatic
protons). 9.25 (s, 1H, NH, exchangeable). Anal.
(C16H16N4O3$1/2H2O) C, H, N.
7.1.4.1.5.
N-(4-Methoxyphenyl)-3-({[(4-methoxyphenyl)
amino]carbonyl}amino)-1H-indazole-1-carboxamide 11g. 4-
Methoxyphenyl isocyanate 8g was heated to reflux with 3-
aminoindazole 9 for 1 h as described in Section 7.1.4.1 to
give 11g. The solid collected was crystallized from dioxane,
27% yield; mp 240e242 ꢀC; I.R. (KBr, cmꢁ1) 3412e3127
(NH), 1687 (br, CO); 1H NMR (DMSO) d 3.75 (s, 3H,
OCH3); 3.77 (s, 3H, CH3); 6.93e8.31 (a set of signals,
12H, aromatic protons); 9.72 (s, 1H, NH, exchangeable);
10.10 (s, 2H, 2 ꢃ NH, exchangeable). Anal. (C23H21N5O4)
C, H, N.
7.1.4.1.6. N-(4-Trifluoromethoxyphenyl)-3-({[(4-(trifluoro-
methoxy)phenyl)amino]carbonyl}amino)-1H-indazole-1-car-
boxamide 11h. 4-(Trifluoromethoxy)phenyl isocyanate 8h
was heated to reflux with 3-aminoindazole 9 for 1 h and
30 min as described in Section 7.1.4.1 to give 11h. The solid
collected was crystallized from dioxane, 36% yield; mp
257e261 ꢀC; I.R. (KBr, cmꢁ1) 3417e3135 (NH), 1718
(CO), 1690 (CO); 1H NMR (DMSO) d 7.36e8.33 (a set of sig-
nals, 12H, aromatic protons); 9.96 (s, 1H, NH, exchangeable);
10.25 (s, 1H, NH, exchangeable); 10.38 (s, 1H, NH, exchan-
geable).Anal. (C23H15F6N5O4) C, H, N.
7.1.4. Synthesis of N-phenyl-3-(phenylureido)-1H-indazole-
1-carboxamides 11
7.1.4.1. General method. Substituted phenyl isocyanate 8
(14 mmol) was added drop wise to a solution of 3-aminoin-
dazole 9 (7 mmol) in 10 ml of THF then the mixture was
heated to reflux for a suitable time. After this time, the so-
lution was concentrated under reduced pressure, the residue
washed with ethanol and then crystallized from a suitable
solvent.
7.1.4.1.1. N-(2,6-Difluorophenyl)-3-({[(2,6-difluoropheny-
l)amino]carbonyl}amino)-1H-indazole-1-carboxamide 11a.
2,6-Difluorophenyl isocyanate 8a was heated to reflux with 3-
aminoindazole 9 for 1 h as described in Section 7.1.4.1 to
give 11a. The solid collected was crystallized from dioxane,
30% yield; mp 201e202 ꢀC; I.R. (KBr, cmꢁ1) 3384e3127
(NH), 1714 (CO), 1690 (CO); 1H NMR (DMSO) d 7.20e8.34
(a set of signals, 10H, aromatic protons); 9.42 (s, 1H, NH, ex-
changeable); 10.02 (s, 1H, NH, exchangeable); 10.70 (s, 1H,
NH, exchangeable). Anal. (C21H13F4N5O2) C, H, N.
7.1.4.1.7.
fluoro-5-(trifluoromethyl)phenyl)amino]carbonyl}amino)-1H-
indazole-1-carboxamide 11i. 2-Fluoro-5-(trifluoromethyl)
N-(2-Fluoro-5-trifluoromethylphenyl)-3-({[(2-
7.1.4.1.2. N-(4-Benzylphenyl)-3-({[(4-benzylphenyl)amino]
carbonyl}amino)-1H-indazole-1-carboxamide 11d. 4-Benzyl-
phenyl isocyanate 8d was heated to reflux with 3-aminoindazole
9 for 1 h as described in Section 7.1.4.1 to give 11d. The solid
collected was crystallized from ethanol, 38% yield; mp 160e
163 ꢀC; I.R. (KBr, cmꢁ1) 3410e3129 (NH), 1716 (CO), 1691
phenyl isocyanate 8i was heated to reflux with 3-aminoinda-
zole 9 for 1 h as described in Section 7.1.4.1 to give 11i.
The solid collected was crystallized from ethanol, 15%
yield; mp 250e252 ꢀC; I.R. (KBr, cmꢁ1
) 3408e3146
(NH), 1701 (br, CO); 1H NMR (DMSO) d 7.38e8.53 (a
set of signals, 10H, aromatic protons); 9.79 (br s, 1H,
NH, exchangeable); 9.88 (br s, 1H, NH, exchangeable);
10.52 (br s, 1H, NH, exchangeable).Anal. (C23H13F8N5O2)
C, H, N.
1
(CO); H NMR (DMSO) d 3.91 (s, 2H, CH2); 3.93 (s, 2H,
CH2); 7.20e8.30 (a set of signals, 22H, aromatic protons);
9.76 (s, 1H, NH, exchangeable); 10.10 (s, 2H, 2 ꢃ NH, ex-
changeable). Anal. (C35H29N5O2$1/2H2O) C, H, N.
7.1.4.1.8.
N-(2-Trifluoromethoxyphenyl)-3-({[(2-trifluo-
7.1.4.1.3.
N-(4-Butoxyphenyl)-3-({[(4-butoxyphenyl)ami-
romethoxyphenyl)amino]carbonyl}amino)-1H-indazole-1-car-
boxamide 11l. 2-(Trifluoromethoxy)phenyl isocyanate 8l was
heated to reflux with 3-aminoindazole 9 for 3 h as described
in Section 7.1.4.1 to give 11l. The solid collected was crystal-
lized from ethanol, 35% yield; mp 195e200 ꢀC; I.R.(KBr,
no]carbonyl}amino)-1H-indazole-1-carboxamide 11e. 4-Bu-
toxyphenyl isocyanate 8e was heated to reflux with
3-aminoindazole 9 for 1 h as described in Section 7.1.4.1 to
give 11e. The solid collected was crystallized from dioxane,
23% yield; mp 225e229 ꢀC; I.R. (KBr, cmꢁ1) 3311e3130
(NH), 1687 (br, CO); 1H NMR (DMSO) d 0.93 (t, 6H,
2 ꢃ CH3); 1.44 (q, 4H, 2 ꢃ CH2); 1.63e1.73 (m, 4H,
2 ꢃ CH2); 3.95 (q, 4H, 2 ꢃ CH2); 6.91e8.30 (a set of signals,
12H, aromatic protons). 9.70 (s, 1H, NH, exchangeable);
10.08 (s, 1H, NH, exchangeable); 10.11 (s, 1H, NH, exchange-
able). Anal. (C29H33N5O4) C, H, N.
7.1.4.1.4. N-(4-Acetylphenyl)-3-({[(4-acetylphenyl)amino]-
carbonyl}amino)-1H-indazole-1-carboxamide 11f. 4-Acetyl-
phenyl isocyanate 8f was heated to reflux with 3-aminoindazole
9 for 1 h as described in Section 7.1.4.1 to give 11f. The solid col-
lected was crystallized from dioxane, 23% yield; mp 250e
253 ꢀC; I.R. (KBr, cmꢁ1) 3270e3116 (NH), 1715 (CO), 1672
(CO); 1H NMR (DMSO) d 2.54 (s, 6H, 2 ꢃ CH3); 7.38e8.31 (a
set of signals, 12H, aromatic protons); 10.11 (s, 1H, NH,
1
cmꢁ1) 3418e3141 (NH), 1722 (CO), 1689 (CO); H NMR
(DMSO) d 7.25e8.31 (a set of signals, 12H, aromatic
protons); 9.64 (s, 1H, NH, exchangeable); 9.72 (s, 1H, NH,
exchangeable); 10.50 (s, 1H, NH, exchangeable).Anal.
(C23H15F6N5O4) C, H, N.
7.1.4.1.9. N-(3,4-Dichlorophenyl)-3-({[(3,4-dichloropheny-
l)amino]carbonyl}amino)-1H-indazole-1-carboxamide 11n. 3,4-
Dichlorophenyl isocyanate 8n was heated to reflux for
10 min with 3-aminoindazole 9 then stirred at room temper-
ature for 24 h as described in Section 7.1.4.1 to give 11n. The
solid collected was washed with ethanol and crystallized
from dioxane, 98% yield; mp 268e270 ꢀC; I.R. (KBr,
1
cmꢁ1) 3419e3122 (NH), 1727 (CO), 1696 (CO); H NMR
(DMSO) d 7.36e8.30 (a set of signals, 10H, aromatic pro-
tons); 10.01 (br s, 1H, NH, exchangeable); 10.32 (br s, 1H,

D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
175
NH, exchangeable); 10.44 (br s, 1H, NH, exchangeable).
Anal. (C21H13Cl4N5O2) C, H, N.
carbonyl}amino)-1H-indazole-1-carboxamide 11a was hydro-
lyzed as described in Section 7.1.5.1 (Method A) to give 12a.
The solid collected was crystallized from dioxane, 52% yield;
mp 216e218 ꢀC; I.R. (KBr, cmꢁ1) 3278 (br, NH), 1651 (CO);
1H NMR (DMSO) d 7.07e7.99 (a set of signals, 7H, aromatic
protons); 9.38 (s, 1H, NH, exchangeable); 9.88 (s, 1H, NH, ex-
changeable); 12.54 (s, 1H, NH, exchangeable). Anal.
(C14H10F2N4O) C, H, N.
7.1.4.1.10. N-(4-Bromophenyl)-3-({[(4-bromophenyl)ami-
no]carbonyl}amino)-1H-indazole-1-carboxamide
11q. 4-
Bromophenyl isocyanate 8q was heated to reflux with
3-aminoindazole 9 for 1 h as described in Section 7.1.4.1 to
give 11q. The solid collected was crystallized from N,N,-di-
methylformamide, 60% yield; mp 278e280 ꢀC; I.R. (KBr,
1
cmꢁ1) 3415e3120 (NH), 1692 (CO), 1681 (CO); H NMR
7.1.5.1.2. N-(2,5-Dichlorophenyl)-N0-(1H-indazol-3-yl)urea
12b. 2,5-Dichlorophenyl isocyanate 8b was allowed to react
with 3-aminoindazole 9 as described in Section 7.1.5.1 (Method
B) to give 12b. Purified by flash chromatography on silica gel
[25] with chloroform as eluant, and crystallized from ethanol,
96% yield; mp 188e189 ꢀC; I.R. (KBr, cmꢁ1) 3406e3225
(DMSO) d 7.36e8.32 (a set of signals, 12H, aromatic
protons); 9.88 (s, 1H, NH, exchangeable); 10.17 (s, 1H, NH,
exchangeable); 10.27 (s, 1H, NH, exchangeable). Anal.
(C21H15Br2N5O2) C, H, N.
7.1.4.1.11. N-(2-Ethoxyphenyl)-3-({[(2-ethoxyphenyl)ami-
1
no]carbonyl}amino)-1H-indazole-1-carboxamide
11r. 2-
(br, NH), 1694 (CO); H NMR (DMSO) d 7.07e8.51 (a set
Ethoxyphenyl isocyanate 8r was heated to reflux with
3-aminoindazole 9 for 3 h as described in Section 7.1.4.1 to
give 11r. The solid collected was crystallized from dioxane,
30% yield; mp 241e243 ꢀC; I.R. (KBr, cmꢁ1) 3394e3111
of signals, 7H, aromatic protons); 10.30 (s, 1H, NH, exchange-
able); 10.70 (s, 1H, NH, exchangeable); 12.68 (s, 1H, NH,
exchangeable). Anal. (C14H10Cl2N4O) C, H, N.
7.1.5.1.3. N-(4-Phenoxyphenyl)-N0-(1H-indazol-3-yl)urea
12c. 4-Phenoxyphenyl isocyanate 8c was allowed to react
with 3-aminoindazole 9 as described in Section 7.1.5.1 (Method
B) to give 12c. The solid collected was crystallized from diox-
ane, 21% yield; mp 212e214 ꢀC; I.R. (KBr, cmꢁ1) 3369e3092
(NH), 1692 (CO); 1H NMR (DMSO) d 6.98e8.03 (a set of sig-
nals, 13H, aromatic protons); 9.61 (s, 1H, NH, exchangeable);
9.94 (s, 1H, NH, exchangeable); 12.53 (s, 1H, NH, exchange-
able). Anal. (C20H16N4O2) C, H, N.
1
(NH), 1716 (CO), 1683 (CO); H NMR (DMSO) d 1.04 (t,
3H, CH3);1.21 (t, 3H, CH3); 3.97e4.07 (m, 4H, 2 ꢃ CH2);
6.97e8.34 (a set of signals, 12H, aromatic protons); 9.38 (s,
1H, NH, exchangeable); 9.67 (s, 1H, NH, exchangeable);
10.51 (s, 1H, NH, exchangeable). Anal. (C25H25N5O4) C, H, N.
7.1.4.1.12. N-(4-Butylphenyl)-3-({[(4-butylphenyl)amino]-
carbonyl}amino)-1H-indazole-1-carboxamide 11s. 4-Butyl-
phenyl isocyanate 8s was heated to reflux with 3-aminoindazole
9 for 3 h as described in Section 7.1.4.1 to give 11s. The solid
collected was crystallized from dioxane, 32% yield; mp 238e
241 ꢀC; I.R. (KBr, cmꢁ1) 3417e3126 (NH), 1723 (CO), 1690
7.1.5.1.4.
N-(4-Benzylphenyl)-N0-(1H-indazol-3-yl)urea
12d. N-(4-Benzylphenyl)-3-({[(4-benzylphenyl)amino]carbo-
nyl}amino)-1H-indazole-1-carboxamide 11d was hydrolyzed
as described in Section 7.1.5.1 (Method A) to give 12d. The
solid residue collected was crystallized from dioxane, 98%
yield; mp 236e238 ꢀC; I.R. (KBr, cmꢁ1) 3291e3140 (NH),
1686 (br CO); 1H NMR (DMSO) d 3.90 (s, 2H, CH2);
7.06e8.03 (a set of signals, 13H, aromatic protons); 9.57 (s,
1H, NH, exchangeable); 9.88 (s, 1H, NH, exchangeable);
12.51 (s, 1H, NH, exchangeable). Anal. (C21H18N4O) C, H, N.
1
(CO); H NMR (DMSO) d 0.89 (t, 6H, 2 ꢃ CH3); 1.27e1.35
(m, 4H, 2 ꢃ CH2); 1.52e1.62 (m, 4H, 2 ꢃ CH2); 2.52e2.59
(m, 4H, 2 ꢃ CH2); 7.15e8.32 (a set of signals, 12H, aromatic
protons); 9.78 (s, 1H, NH, exchangeable); 10.09 (s, 1H, NH,
exchangeable); 10.12 (s, 1H, NH, exchangeable). Anal.
(C29H33N5O2) C, H, N.
7.1.5. Synthesis of 1-(1H-indazol-3-yl)-3-phenylureidic
derivatives 12
7.1.5.1.5.
N-(4-Butoxyphenyl)-N0-(1H-indazol-3-yl)urea
12e. N-(4-Butoxyphenyl)-3-({[(4-butoxyphenyl)amino]carbo-
nyl}amino)-1H-indazole-1-carboxamide 11e was hydrolyzed
as described in Section 7.1.5.1 (Method A) to give 12e. The
solid collected was crystallized from ethanol, 98% yield; mp
216e217 ꢀC; I.R. (KBr, cmꢁ1) 3349e3145 (NH), 1676 (CO);
1H NMR (DMSO) d 0.93 (t, 3H, CH3); 1.38e1.48 (m, 2H,
CH2); 1.65e1.71 (m, 2H, CH2); 3.92 (t, 2H, CH2); 6.88e8.06
(a set of signals, 8H, aromatic protons); 9.59 (s, 1H, NH, ex-
changeable); 9.85 (s, 1H, NH, exchangeable); 12.50 (s, 1H,
NH, exchangeable). Anal. (C18H20N4O2) C, H, N.
7.1.5.1. General methods. Method A. Substituted N-Phenyl-
3-(phenylureido)-1H-indazole-1-carboxamide 11 (1.16 mmol)
was dissolved in 50 ml of ethanol and heated to reflux for
10 min. After this time 50 ml of 2 N NaOH was added and
the reflux resumed for 15 min. At the completion of the reac-
tion the ethanol was removed under reduced pressure, the
mixture cooled at room temperature in ice bath and the pH
adjusted to 4.0 with aqueous concentrated HCl. The solid
was filtered, washed first with water then with ethanol before
being recrystallized from a suitable solvent.
Method B. A mixture of 14 mmol of substituted phenyl iso-
cyanates 8 and 14 mmol of 3-aminoindazole 9 in 10 ml of THF
was heated to reflux for 3 h. At the completion of the reaction
the solvent was removed under reduced pressure, washed
with hot ethanol and recrystallized from a suitable solvent.
7.1.5.1.1. N-(2,6-Difluorophenyl)-N0-(1H-indazol-3-yl)urea
12a. N-(2,6-Difluorophenyl)-3-({[(2,6-difluorophenyl)amino]
7.1.5.1.6.
N-(4-Acetylphenyl)-N0-(1H-indazol-3-yl)urea
12f. N-(4-Acetylphenyl)-3-({[(4-acetylphenyl)amino]carbonyl}-
amino)-1H-indazole-1-carboxamide 11f was hydrolyzed as
described in Section 7.1.5.1 (Method A) to give 12f. The
solid collected was crystallized from dioxane, 95% yield;
mp 252e254 ꢀC; I.R. (KBr, cmꢁ1) 3305e3101 (NH), 1671
1
(CO), 1700 (CO); H NMR (DMSO) d 2.53 (s, 3H, CH3);
7.05e8.02 (a set of signals, 8H, aromatic protons); 9.68 (s,
1H, NH, exchangeable); 10.14 (s, 1H, NH, exchangeable);

176
D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
12.60 (s, 1H, NH, exchangeable). Anal. (C16H14N4O2) C, H,
N.
3-aminoindazole 9 as described in Section 7.1.5.1 (Method
B) to give 12o. The solid collected was crystallized frꢁom1
7.1.5.1.7. N-(4-Methoxyphenyl)-N0-(1H-indazol-3-yl)urea
12g. N-(4-Methoxyphenyl)-3-({[(4-methoxyphenyl)amino]
carbonyl}amino)-1H-indazole-1-carboxamide 11g was hydro-
lyzed as described in Section 7.1.5.1 (Method A) to give 12g.
The solid collected was crystallized from ethanol, 88% yield;
mp 228e230 ꢀC; I.R. (KBr, cmꢁ1) 3325e3089 (NH), 1671
DMF/H2O, 40% yield; mp 288e290 ꢀC; I.R. (KBr, cm
)
1
3397e3086 (NH), 1698 (CO); H NMR (DMSO) d 7.08e
8.26 (a set of signals, 8H, aromatic protons); 9.75 (s, 1H,
NH, exchangeable); 10.31 (s, 1H, NH, exchangeable); 12.67
(s, 1H, NH, exchangeable). Anal. (C14H11N5O3) C, H, N.
7.1.5.1.13. N-(2-Methyl-4-nitrophenyl)-N0-(1H-indazol-3-
yl)urea 12p. 2-Methyl-4-nitrophenyl isocyanate 8p was al-
lowed to react with 3-aminoindazole 9 as described in Section
7.1.5.1 (Method B) to give 12p. The solid collected was crys-
tallized from DMF, 75% yield; mp 264e265 ꢀC; I.R. (KBr,
cmꢁ1) 3479e3334 (NH), 1704 (CO); 1H NMR (DMSO)
d 2.48 (s, 3H, CH3); 7.06e8.55 (a set of signals, 7H, aromatic
protons); 10.27 (s, 1H, NH, exchangeable); 10.50 (br s, 1H,
NH, exchangeable); 12.69 (s, 1H, NH, exchangeable). Anal.
(C15H13N5O3) C, H, N.
1
(CO); H NMR (DMSO) d 3.73 (s, 3H, OCH3); 6.89e8.05
(a set of signals, 8H, aromatic protons); 9.52 (s, 1H, NH,
exchangeable); 9.79 (s, 1H, NH, exchangeable); 12.47 (s,
1H, NH, exchangeable). Anal. (C15H14N4O2) C, H, N.
7.1.5.1.8. N-[4-(Trifluoromethoxy)phenyl]-N0-(1H-indazol-
3-yl)urea
12h. N-(4-(Trifluoromethoxy)phenyl)-3-({[(4-
(trifluoromethoxy)phenyl)amino]carbonyl}amino)-1H-inda-
zole-1-carboxamide 11h was hydrolyzed as described in
Section 7.1.5.1 (Method A) to give 12h. The solid collected
was crystallized from ethanol, 98% yield; mp 241e245 ꢀC;
I.R. (KBr, cmꢁ1) 3448e3127 (NH), 1692 (CO); 1H NMR
(DMSO) d 7.31e7.67 (a set of signals, 8H, aromatic protons);
9.64 (s, 1H, NH, exchangeable); 10.00 (s, 1H, NH, exchange-
able); 12.57 (s, 1H, NH, exchangeable). Anal.
(C15H11F3N4O2) C, H, N.
7.1.5.1.14.
N-(4-Bromophenyl)-N0-(1H-indazol-3-yl)urea
12q. N -(4-Bromophenyl)-3-({[(4-bromophenyl)amino]carbo-
nyl}amino)-1H-indazole-1-carboxamide 11q was hydrolyzed
as described in Section 7.1.5.1 (Method A) to give 12q. The
solid collected was crystallized from ethanol, 90% yield; mp
235e237 ꢀC; I.R. (KBr, cmꢁ1) 3429e3115 (NH), 1689
1
7.1.5.1.9. N-[2-Fluoro-5-(trifluoromethyl)phenyl]-N0-(1H-
(CO); H NMR (DMSO) d 7.05e8.03 (a set of signals, 8H,
indazol-3-yl)urea
12i. N-(4-(Trifluoromethoxy)phenyl)-3-
aromatic protons); 9.64 (s, 1H, NH, exchangeable); 9.97 (s,
1H, NH, exchangeable); 12.56 (s, 1H, NH, exchangeable).
Anal. (C14H11BrN4O) C, H, N.
({[(4-(trifluoromethoxy)phenyl)amino]carbonyl}amino)-1
H-indazole-1-carboxamide 11i was hydrolyzed as described in
Section 7.1.5.1 (Method A) to give 12i. The solid collected was
crystallized from dioxane, 98% yield; mp 220e222 ꢀC; I.R.
(KBr, cmꢁ1) 3402e3140 (NH), 1674 (CO); 1H NMR
(DMSO) d 7.07e8.75 (a set of signals, 7H, aromatic protons);
10.12 (s, 1H, NH, exchangeable); 10.54 (br s, 1H, NH,
exchangeable); 12.69 (s, 1H, NH, exchangeable). Anal.
(C15H10F4N4O) C, H, N.
7.1.5.1.15.
N-(2-Ethoxyphenyl)-N0-(1H-indazol-3-yl)urea
12r. N -(2-Ethoxyphenyl)-3-({[(2-ethoxyphenyl)amino]carbo-
nyl}amino)-1H-indazole-1-carboxamide 11r was hydrolyzed
as described in Section 7.1.5.1 (Method A) to give 12r. The solid
collected was crystallized from ethanol, 93% yield; mp 182e
1
183 ꢀC; I.R. (KBr, cmꢁ1) 3333e3131 (NH), 1678 (CO); H
NMR (DMSO) d 1.50 (t, 3H, CH3); 4.12 (q, 2H, CH2); 6.63e
8.31 (a set of signals, 8H, aromatic protons); 10.00 (s, 1H,
NH, exchangeable); 10.61 (br s, 1H, NH, exchangeable);
12.48 (s, 1H, NH, exchangeable). Anal. (C16H16N4O2) C, H, N.
7.1.5.1.10. N-[2-(Trifluoromethoxy)phenyl]-N0-(1H-indazol-
3-yl)urea
12l. N-(2-(Trifluoromethoxy)phenyl)-3-({[(2-
(trifluoromethoxy)phenyl)phenylamino]carbonyl}amino)-1
H-indazole-1-carboxamide 11l was hydrolyzed as described in
Section 7.1.5.1 (Method A) to give 12l. The solid collected was
crystallized from ethanol, 98% yield; mp 192 ꢀC; I.R. (KBr,
7.2. X-ray structure determination
cmꢁ1) 3300 (br, NH), 1665 (CO); H NMR (DMSO) d 7.06e
Crystals of 10w were obtained from an ethanol solution at
room temperature as yellow prisms. The intensity data were
collected on Enraf Nonius CAD-4 diffractometer with
1
8.45 (a set of signals, 8H, aromatic protons); 10.18 (br s, 1H,
NH, exchangeable); 10.72 (br s, 1H, NH, exchangeable);
12.58 (br s, 1H, NH, exchangeable). Anal. (C15H11F3N4O2) C,
H, N.
˚
Mo Ka radiation (l ¼ 0.71073(A)) at room temperature. The
lattice parameters were determined by least-squares refine-
ments of 25 high angle reflections. Crystal system: monoclinic
7.1.5.1.11.
N-(3,4-Dichlorophenyl)-N0-(1H-indazol-3-
˚
yl)urea 12n. N-(3,4-Dichlorophenyl)-3-({[(3,4-dichlorophe-
nyl)amino]carbonyl}amino)-1H-indazole-1-carboxamide 11n
was hydrolyzed as described in Section 7.1.5.1 (Method A)
to give 12n. The solid collected was crystallized from ethanol,
98% yield; mp 244e250 ꢀC; I.R. (KBr, cmꢁ1) 3439e3122
(NH), 1693 (CO); 1H NMR (DMSO) d 7.08e7.98 (a set of sig-
nals, 7H, aromatic protons); 9.67 (s, 1H, NH, exchangeable);
9.99 (s, 1H, NH, exchangeable); 12.58 (s, 1H, NH, exchange-
able). Anal. (C14H10Cl2N4O) C, H, N.
(I2/c), cell dimensions (A) a ¼ 23.227(4), b ¼ 5.801(1), c ¼
27.825(5), b ¼ 111.76(2)ꢀ, Final R indices [I > 2s(I )] R1 ¼
0.088, wR2 ¼ 0.178 for 3205 independent reflections. The
structure was solved by direct methods and the refinement
was carried out with SHELX-97 [26]. All non-H-atoms were
refined anisotropically. The H-atoms positions were detected
in a difference Fourier synthesis and refined with isotropic
thermal factors.
CCDC-644638 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
7.1.5.1.12.
12o. 4-Nitrophenyl isocyanate 8o was allowed to react with
N-(4-Nitrophenyl)-N0-(1H-indazol-3-yl)urea

D. Raffa et al. / European Journal of Medicinal Chemistry 44 (2009) 165e178
177
Cambridge Crystallographic Data Centre, 12, Union Road,
Cambridge CB21EZ, UK; fax: þ44 1223 336 033; or
deposit@ccdc.cam.ac.uk).
calculated from [(Ti ꢁ Tz)/(C ꢁ Tz)] ꢃ 100 ¼ 50, which is the
drug concentration resulting in a 50% reduction in the net pro-
tein increase (as measured by SRB staining) in control cells dur-
ing the drug incubation. The drug concentration resulting in total
growth inhibition (TGI) is calculated from Ti ¼ Tz. The LC50
(concentration of drug resulting in a 50% reduction in the mea-
sured protein at the end of the drug treatment as compared to that
at the beginning) indicating a net loss of cells following treat-
ment is calculated from [(Ti ꢁ Tz)/Tz] ꢃ 100 ¼ ꢁ50. Values
are calculated for each of these three parameters if the level of
activity is reached; however, if the effect is not reached or is ex-
ceeded, the value for that parameter is expressed as greater or
less than the maximum or minimum concentration tested.
7.3. Biology
7.3.1. Methodology of the in vitro cancer screen
The human tumor cell lines of the cancer screening panel
are grown in RPMI 1640 medium containing 5% foetal bovine
serum and 2 mM L-glutamine. For a typical screening exper-
iment, cells are inoculated into 96 well microtiter plates in
100 ml at plating densities ranging from 5,000 to 40,000
cells/well depending on the doubling time of individual cell
lines. After cell inoculation, the microtiter plates are incu-
bated at 37 ꢀC, 5% CO2, 95% air and 100% relative humidity
for 24 h prior to addition of experimental drugs. After 24 h,
two plates of each cell line are fixed in situ with TCA, to rep-
resent a measurement of the cell population for each cell line
at the time of drug additions (Tz). Experimental drugs are sol-
ubilized in dimethyl sulfoxide at 400-fold the desired final
maximum test concentration and stored frozen prior to use.
At the time of drug addition, an aliquot of frozen concentrate
is thawed and diluted to twice the desired final maximum test
concentration with complete medium containing 50 mg/ml
gentamicin. Additional four, 10-fold or 1/2 log serial dilutions
are made to provide a total of five drug concentrations plus
control. Aliquots of 100 ml of these different drug dilutions
are added to the appropriate microtiter wells already contain-
ing 100 ml of medium, resulting in the required final drug con-
centrations. Following drug addition, the plates are incubated
for an additional 48 h at 37 ꢀC, 5% CO2, 95% air, and 100%
relative humidity. For adherent cells, the assay is terminated
by the addition of cold TCA. Cells are fixed in situ by the gen-
tle addition of 50 ml of cold 50% (w/v) TCA (final concentra-
tion, 10% TCA) and incubated for 60 min at 4 ꢀC. The
supernatant is discarded, and the plates are washed five times
with tap water and air dried. Sulforhodamine B (SRB) solu-
tion (100 ml) at 0.4% (w/v) in 1% acetic acid is added to
each well, and plates are incubated for 10 min at room tem-
perature. After staining, unbound dye is removed by washing
five times with 1% acetic acid and the plates are air dried.
Bound stain is subsequently solubilized with 10 mM trizma
base, and the absorbance is read on an automated plate reader
at a wavelength of 515 nm. For suspension cells, the method-
ology is the same except that the assay is terminated by fixing
settled cells at the bottom of the wells by gently adding 50 ml
of 80% TCA (final concentration, 16% TCA). Using the seven
absorbance measurements [time zero, (Tz), control growth, C,
and test growth in the presence of drug at the five concentra-
tion levels (Ti)], the percentage growth is calculated at each of
the drug concentrations levels. Percentage growth inhibition
is calculated as:
7.3.2. Flow cytometric analysis of cell cycle distribution
The effects of the most active compounds of the series on
cell cycle distribution were studied on K562 cells (myeloblastic
leukemia) by flow cytometric analysis after staining with propi-
dium iodide. Cells were exposed for 24 h to each compound.
After treatment cells were washed once in ice-cold phosphate
buffered saline medium (PBS e Sigma) and resuspended at
1 ꢃ 106 ml in a hypotonic fluorochrome solution containing
propidium iodide (Sigma, St. Louis, Mo) 50 mg/ml in 0.1% so-
dium citrate plus 0.03% (v/v) nonidet P-40 (Sigma). After
30 min of incubation, the fluorescence of each sample was an-
alyzed as single-parameter frequency histograms by using
a FACScan flow cytometer (Becton Dickinson, San Jose,
CA). The distribution of cells in the cell cycle was analyzed
with the ModFit LT3 program (Verity Software House, Inc.).
7.3.3. Flow cytometric evaluation of pRb
Cells (1.5 ꢃ 106) were washed twice with PBS (Sigma) and
resuspended in 100 ml Cytofix/Cytoperm solution (Becton
Dickinson). After 20 min cells were washed twice with BD
Perm/Wash Ô buffer solution (Becton Dickinson) and incu-
bated with 20 ml FITC conjugated anti-pRb or Pe conjugated
anti-underphosphorylated pRb monoclonal antibodies (Becton
Dickinson) at 4 ꢀC. After 30 min cells were washed twice and
analysed by flow cytometry.
Acknowledgement
FinancialsupportfromMIUR(PRIN2004)andMIUR(PRIN
2006) is gratefully acknowledged. The NCI Cell results were ob-
tained through screening services provided by the Developmen-
tal Therapeutics Program of the National Cancer Institute;
additional information can be found at http://dpt.nci.nih.gov/.
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