Synthesis of substituted 3-amino-N-phenyl-1H-indazole-1-carboxamides endowed with antiproliferative activity

Article abstract of DOI:10.1016/j.ejmech.2010.10.032

Several new N-phenyl-1H-indazole-1-carboxamides 1c-h and 4l,m were prepared by reacting phenyl isocyanate derivatives 3a,b with 3-amino-1H-indazole derivatives 2c,e,g or 1H-indazole 2l respectively. Chemical transformations of compounds 1a,b and 1g,h gave

Full text of DOI:10.1016/j.ejmech.2010.10.032

European Journal of Medicinal Chemistry 46 (2011) 168e174
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of substituted 3-amino-N-phenyl-1H-indazole-1-carboxamides
endowed with antiproliferative activity
Benedetta Maggio a, Maria Valeria Raimondi a, Demetrio Raffa a, Fabiana Plescia a, Stella Cascioferro a,
Salvatore Plescia a, Manlio Tolomeo b, Antonietta Di Cristina b, Rosaria Maria Pipitone b,
,
Stefania Grimaudo b, Giuseppe Daidone a
*
a Dipartimento di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi, 32, 90123-Palermo, Italy
b Centro Interdipartimentale di Ricerca in Oncologia Clinica e Dipartimento Biomedico di Medicina Interna e Specialistica, Università di Palermo, Palermo, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Several new N-phenyl-1H-indazole-1-carboxamides 1ceh and 4l,m were prepared by reacting phenyl
isocyanate derivatives 3a,b with 3-amino-1H-indazole derivatives 2c,e,g or 1H-indazole 2l respectively.
Chemical transformations of compounds 1a,b and 1g,h gave 3-acetamido-N-phenyl-1H-indazole-1-
carboxamide derivatives 5a,b, and 3,5-diamino-N-phenyl-1H-indazole-1-carboxamide derivatives 4i, j
respectively. Finally, 3,5-diacetamido-N-phenyl-1H-indazole-1-carboxamide derivatives 6a,b were
prepared by acetylation of 4i, j. Some of synthesized compounds were evaluated for their in vitro anti-
proliferative activity against the full NCI tumor cell lines panel derived from nine clinically isolated
cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast).
Compound 1c, the most active of the series, was able to inhibit cell growth showing GI50 values in the
Received 21 April 2010
Received in revised form
15 October 2010
Accepted 29 October 2010
Available online 10 November 2010
Keywords:
3-amino-N-phenyl-1H-indazole-1-
carboxamides
0.041e33.6 mM range, mean GI50 1.90 mM, being very effective against colon and melanoma cell lines. Cell
G0eG1 arrest
cycle analysis in K562 cells showed that 1c causes a marked increase of cells in G0eG1 phase. Moreover,
it increases the ratio between hypophosphorylated pRb and total pRb.
Ó 2010 Elsevier Masson SAS. All rights reserved.
pRb
Antiproliferative agents
1. Introduction
phenyl-1H-indazole-1-carboxamide derivatives of type 1a and 1b,
substituted in the indazole nucleus, in order to estimate as different
In the literature are reported several examples of indazole
derivatives endowed with antineoplastic activity [1e7].
substituents can influence the antiproliferative activity. Moreover,
the influence of the 3-amino group on the activity was evaluated by
comparing the activity of 1b in respect of 4m which is devoid of this
group. Finally, we verified whether the substitution of the amino
group with the acetamido one in compounds 1b and 4j may afford
some advantage for the antiproliferative activity.
Recently our research group has reported the synthesis of some
3-amino-N-phenyl-1H-indazole-1-carboxamide derivatives of type
1 (Fig. 1). Among these, compounds 1a and 1b were able to inhibit
at low micromolar concentrations the cell growth of many
neoplastic cell lines of the NCI 60 human cell lines panel, being the
benzyl derivative 1a quite more active than the butoxy analogue 1b
[8]. Compounds 1a,b caused a marked arrest of cells in G0eG1
phase and a decrease of the phosphorylated form of retinoblastoma
protein (pRb) which is involved in conferring uncontrolled growth
to many neoplastic cells [9e11].
The above compounds are not substituted in the indazole
nucleus and this gives the hope to obtain more active compounds if
a suitable substituent is bore by the above nucleus. In fact, it is well
documented in the literature that substitution may greatly influ-
ence the activity. For this reason, we synthesized new 3-amino-N-
2. Results
2.1. Chemistry
The 1H-indazole-1-carboxamide derivatives 1ceh, 4iem, 5a,b
and 6a,b were obtained as reported in the Scheme 1. In particular,
derivatives 1ceh and 4l,m were synthesized reacting 3-amino-
indazoles 2c,e,gor indazole 2l respectively with the isocyanates 3a,b
under the same experimental conditions utilized for the obtainingof
1a,b [8]. Products of type 7 derived from the reaction of the indazole
3-amino group with one molecule of isocyanate derivative
3 were not isolated from the reaction mixtures. As regards
the 3,5-diaminoderivatives 4i, j , they were obtained reducing with
hydrogen and 10% PdeC the parent 5-nitroderivatives 1g,h. Finally,
* Corresponding author. Tel.: þ39 91 23891916.
E-mail address: gdaidone@unipa.it (G. Daidone).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.032

B. Maggio et al. / European Journal of Medicinal Chemistry 46 (2011) 168e174
169
spectra of four representative compounds (4m, 1f, 4j, and 6a)
confirmed the proposed structures.
2.2. Biology
2.2.1. Tumor cell line screening
Among the synthesized indazole derivatives, that is 1ceh,
4iem, 5a,b and 6a,b , eight of them (1c,d, 4i, j,m, 5b, 6a,b) were
selected by the National Cancer Institute (NCI), Bethesda, USA, and
tested initially at a single dose (10 mM) in the full NCI cells panel
derived from nine clinically isolated cancer types (leukemia, non-
small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and
breast) [12]. Compounds 1c and 4i resulted to be quite more active
than the remaining compounds 1d, 4j,m, 5b and 6a,b. Moreover,
they satisfied the NCI pre-determined threshold inhibition criteria
and therefore were also evaluated in the 5-dose screening (0.01, 0.1,
Fig. 1. Structure of 3-amino-N-phenyl-1H-indazole-1-carboxamide derivatives.
the acetyl derivatives 5a,b and 6a,b were obtained from the reaction
of acetic anhydride with 1a,b and 4i, j respectively.
All the above compounds were identified on the basis of satis-
factory elemental and spectroscopic data. In particular, the struc-
ture assignation for compounds 1ceh and 4i, j was based on the
presence of the 3-NH2 signal as a singlet integrated for two protons
1, 10, 100 mM). The antiproliferative activity of 1c, 4i and the
previously synthesized derivative 1a, considered for comparison
purposes, is showed in Table 1. The antiproliferative activity of a test
compound 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 molar concentration inducing 50%
net cell death).
in the 1H NMR spectra at 6.03e6.07
d which, as a consequence,
ruled out a structure of type 7. All the above results accorded with
our previously reported data for the 3-amino-N-phenyl-1H-inda-
zole-1-carboxamide derivatives which are not substituted in the
indazole nucleus, e.g. 1a,b [8]. In the case of 4i, j the spectra showed
also the signal for the further 5-amino group at about 5.06
the 1H NMR spectra of all compounds showed the signal for
the 1-carboxamide NH proton in the 9.17e10.33
range. 13C NMR
d
. Finally,
2.2.2. Cell cycle analysis
In consideration of the above results for 1c, and in order to
obtain more insight on its mechanism of action, we thought it
d
Scheme 1. Synthetic pathway of compounds 1ceh, 4iem, 5a,b and 6a,b.

170
B. Maggio et al. / European Journal of Medicinal Chemistry 46 (2011) 168e174
Table 1
In vitro antiproliferative activity (
m
M) for 1a, 1c and 4i against the full NCI cell lines panel derived from nine clinically isolated human cancer types.
Cancer types
1a
1c
4i
Panel/cell line
GI50
TGI
LC50
GI50
TGI
34.4
42.1
14.7
55.3
46.5
LC50
GI50
TGI
18.8
LC50
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF539
SNB-19
SNB75
U251
Nt
Nt
Nt
54.9
>100
>100
84.0
1.23
>100
>100
>100
>100
>100
>100
>100
2.07
>100
72.2
>100
>100
>100
>100
0.466
1.20
0.636
1.86
12.9
11.2
13.7
14.2
13.5
16.8
27.8
0.408
2.90
13.4
4.27
7.03
1.08
2.39
2.94
3.62
4.66
0.530
0.495
22.8
17.0
13.9
0.477
19.2
0.772
Nt
36.4
11.5
2.22
10.0
0.353
6.98
6.23
10.7
31.3
21.9
Nt
36.9
3.76
16.7
1.69
21.1
22.2
12.9
27.2
0.347
13.5
1.02
0.555
0.847
17.2
13.5
1.55
15.2
33.8
20.4
25.8
48.6
92.0
46.5
42.6
58.2
83.5
>100
13.0
>100
37.7
35.9
>100
>100
17.6
>100
41.5
32.3
>100
5.36
>100
47.4
30.2
16.1
64.1
>100
Nt
>100
>100
8.64
50.9
>100
48.8
63.1
40.4
>100
63.9
Nt
>100
26.2
31.5
33.4
58.0
58.2
28.0
>100
>100
51.1
28.3
8.82
43.9
71.8
39.6
>100
2.62
0.0357
2.41
2.58
2.52
3.19
1.21
10.7
15.4
5.34
18.2
0.0645
15.6
0.588
0.238
0.467
0.0458
1.15
0.0726
0.0565
3.46
2.24
4.48
5.14
6.58
17.2
11.4
24.3
35.0
57.1
36.7
27.0
24.4
38.3
39.5
27.5
23.8
39.6
26.5
14.8
20.8
14.2
17.2
15.1
18.7
30.7
15.1
22.1
27.3
31.5
20.9
16.0
89.8
17.8
23.8
48.5
27.8
19.8
29.7
12.6
54.1
28.3
24.1
60.5
Non-small cell lung
>100
>100
>100
>100
>100
>100
>100
84.6
>100
>100
>100
>100
>100
56.3
>100
>100
>100
>100
>100
>100
>100
65.9
>100
97.6
16.4
33.7
66.7
68.4
>100
14.7
>100
23.8
1.53
25.1
>100
24.0
>100
76.5
47.7
24.2
11.5
43.3
32.3
27.0
18.4
54.8
14.0
14.0
96.1
59.7
11.2
10.4
16.1
16.9
13.0
4.64
14.7
13.1
65.3
57.4
91.4
92.4
58.3
>100
>100
53.9
40.9
50.0
50.5
46.0
41.3
58.5
72.5
45.4
51.8
53.6
57.1
52.0
46.7
>100
>100
>100
>100
60.6
>100
>100
>100
77.2
>100
79.1
>100
>100
>100
>100
38.8
>100
74.5
58.4
53.2
>100
>100
>100
>100
>100
Colon
4.17
6.79
2.75
4.51
3.34
4.48
13.0
3.96
8.70
13.9
17.4
6.53
3.38
18.0
CNS
13.0
0.0515
1.51
14.6
14.0
12.5
Melanoma
LOX IMVI
MALME-3M
M14
61.5
0.0503
6.69
>100
>100
Nt
>100
>100
34.3
>100
>100
>100
>100
>100
>100
>100
Nt
>100
0.0888
0.0813
33.6
18.5
0.283
4.92
0.0412
13.7
6.66
5.73
4.59
7.36
51.9
65.5
90.1
56.2
46.4
60.8
39.3
>100
66.7
63.2
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
>100
>100
>100
26.1
13.7
6.52
14.5
2.90
16.4
12.1
8.29
22.6
20.2
17.4
15.3
12.4
14.4
3.37
4.30
21.4
13.4
19.4
4.72
4.33
10.9
21.1
80.2
>100
>100
>100
20.8
34.3
>100
>100
39.1
50.5
28.2
60.7
17.4
>100
>100
>100
Ovarian
48.2
>100
>100
>100
>100
>100
18.8
>100
>100
>100
14.9
11.0
15.8
3.92
16.1
0.0468
0.217
13.5
14.6
15.6
3.12
2.90
10.4
>100
75.1
37.7
25.9
28.4
13.7
26.8
43.9
30.9
46.0
32.6
24.0
24.0
26.5
22.7
43.0
33.5
22.8
>100
92.7
59.6
92.8
Renal
94.4
54.3
56.1
39.6
>100
90.1
71.1
>100
>100
87.0
52.6
85.4
54.3
>100
76.7
66.0
>100
>100
>100
73.4
>100
96.3
>100
>100
80.0
84.0
>100
>100
>100
>100
60.6
>100
>100
>100
>100
89.1
>100
>100
>100
>100
>100
31.5
42.9
38.7
84.5
44.3
28.9
18.8
37.6
41.7
44.0
27.6
Prostate
Breast
0.395
5.33
13.3
5.88
0.0741
6.92
7.74
17.3
14.6
5.80
MDA-MB-231/ATCC
HS 578T
BT-549
>100
>100
>100
T-47D
Nt ¼ not tested, GI50 ¼ compound’s molar concentration inhibiting 50% net cell growth, TGI ¼ compound’s molar concentration for total inhibition of net cell growth,
LC50 ¼ compound’s molar concentration inducing 50% net cell death.
would be of interest to study the effect of this compound on the
cell cycle distribution. The effect of 1c on cell cycle distribution
was analyzed in K562 cells. Cells were cultured for 24 h in the
presence of 1c used at 20, 40 and 60 mM and then evaluated after
this time. Flow cytometric analysis of cell cycle was carried out as
described in the Experimental Section and is reported in Fig. 2.
2.2.3. Effects of retinoblastoma protein (pRb)
The retinoblastoma gene encodes a nuclear phosphoprotein
(pRb) expressed in the majority of vertebrates normal cells and acts
as a tumor suppressor factor, playing a key role in the control of cell
division [9]. The pRb hypophosphorylated form is mainly found in
resting cells, whereas the highly phosphorylated 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
The analysis of cell cycle performed at 60 mM showed a marked
block in G0eG1 phase.

B. Maggio et al. / European Journal of Medicinal Chemistry 46 (2011) 168e174
171
Fig. 2. Effects of compound 1c on DNA content following the treatment of K562 cells for 24 h. The cells were cultured a) without compounds, or with each compound used at the
following concentrations: b) 20
cells are indicated in panel a).
mM; c) 40 mM; d) 60 mM. Cell-cycle distribution was analyzed by the standard propidium iodide procedure. Sub-G0eG1 (A), G0eG1, S, and G2eM
undergoes sequential phosphorylation. The pRb hypophosphory-
lated form binds to critical regulatory proteins (including E2F) and
represses the transcription of genes involved in cell cycle
progression from the G0eG1 to the S phase [10]. Fig. 3 shows the
effects of 1c on total pRb and hypophosphorylated pRb levels in
K562 cells. Flow cytometric analysis of pRb and hypophosphory-
lated pRb were carried out as described in the Experimental
Section. Compound 1c induced a marked increase of hypo-
phosphorylated pRb form and only a slow increase of total pRb. This
is congruent with the G0eG1 block observed in Fig. 2.
Growth inhibition data of 1c against the most sensitive cell lines are
reported in Table 2.
The ratio obtained by dividing the compound’s GI50 full panel
mean graph midpoint (MG-MID, the average sensitivity of all cell
lines against a test compound) by its individual sub-panel mean
GI50 value (the average sensitivity of each sub-panel against the
same compound) is considered as a measure of compound selec-
tivity. Ratios between 3 and 6 refer to moderate selectivity, ratio >6
indicate high selectivity towards the corresponding cancer type,
while compounds meeting neither of these criteria are rated non-
selective [13,14]. Compound 1c resulted to be highly selective
against colon cancer with selectivity index of 9.5 and a sub-panel
3. Discussion
mean GI50 value of 0.20 mM.
As regards the 5-amino derivative 4i, it proved to be non-
selective and, though less active than 1c, showed good activity
against all the cancer cell lines, with the exception of NCI-H460, SK-
MEL-2 and RXF 393. The best results were observed against
leukemia cell, with GI50 values in the 2.07e5.14 mM range (see Table
1). The most significant values of GI50 of 4i against sensitive cell
lines are reported in Table 2.
The pharmacological results of all the tested compounds
showed that the 5-chloro derivative 1c produced the best anti-
proliferative profile. The remarkable difference of activity between
1c and the analogous derivative 1d is due only to the different
nature of substituent R4: benzyl for 1c and butoxy for 1d. As the
benzyl group is more lipophilic in character than the butoxy one, it
seem that lipophilicity plays a role in the antiproliferative activity
of 1c,d. This trend was previously observed for the compounds 1a
and 1b [8]. Compound 1c showed antiproliferative activity against
almost every type of tumor cell lines studied. It was particularly
efficacious against all types of colon cancer cell lines and some
other cell lines such as leukemia (K562), non-small cell lung cancer
(NCI-H460), CNS cancer (SF-295), melanoma (LOX IMVI, M14, MDA-
MB-435, SK-MEL-5, UACC-G2), renal cancer (ACHN, CAKI-1), breast
cancer (MCF7, T-47D). Good activity was also showed, with the
exception of UO-31 and SK-MEL-2, against the remaining cell lines,
The decreased activity of 4i than 1c is due to the substitution in
1c of the chloro atom with the amino group. As the chloro atom is
lipophilic in character (
essentially hydrophilic (
of activity between 1c and 4i be correlated to higher lipophilicity of
1c than that of 4i. This agrees with the pharmacological results
observed for 1a/1b [8] and 1c/1d.
P
¼ 0.71) whereas the amino group is
P
¼ ꢀ1.23), it is possible that the difference
The presence of the 3-amino group in the indazole derivatives is
on the whole of some advantage for the activity. In fact, comparing
the full panel mean growth percent values at 10
mM of 1b (80.93%)
being the GI50 values in the 1.21e18.8
mM range (see Table 1).
and 4m (95.19%) we can observe that 4m, devoid of the amino

172
B. Maggio et al. / European Journal of Medicinal Chemistry 46 (2011) 168e174
Table 2
In vitro antiproliferative activity (mM) against the cell lines in which the compounds
1c and 4i resulted mostly active.
Panel/cell
line
1c
4i
GI50
TGI
LC50
GI50 TGI LC50
Leukemia
CCRF-CEM
K-562
1.23
34.4 >100
14.7 >100
2.07 18.8 >100
2.24 11.4 >100
0.0357
0.0645
0.588
0.238
0.467
0.0458 >100
1.15
0.0726 >100
0.0565
0.515
Non-small cell NCI-H460
Colon cancer
14.7
23.8 >100 13.1 26.5
1.53 >100 4.17 14.8
25.1 77.5 6.79 20.8
60.6 4.64 23.8 >100
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-602
SF-295
LOX IMVI
M14
MDA-MB-435 0.0813 >100
SK-MEL-5
UACC-62
53.9
40.9
50.0
50.5
46.0
41.3
58.5
45.4
46.7
51.9
65.5
46.4
39.3
63.2
39.6
>100
2.75 14.2
24.0
79.1 4.51 17.2
>100
3.34 15.1
4.48 18.7
3.96 15.1
76.5 >100
24.2 >100
18.4
14.5 >100
>100
CNS cancer
Melanoma
0.0503
0.0888
53.2 3.38 16.0
4.59 17.8
7.36 23.8
0.2834
0.0412 >100
5.73
0.0468
0.217 >100
2.90
0.395
0.0741
21.1
80.2 6.52 19.8
>100
34.3 >100
17.4 >100
>100
44.3 >100
18.8 84.0 6.92 26.5
27.6 >100 5.80 22.8
2.90 12.6
8.29 24.1
3.37 13.7
Ovarian cancer OVCAR-4
Renal cancer
ACHN
CAKI-1
4.30 26.8 >100
4.33 24.0 87.0
Prostate cancer PC-3
Breast cancer
MCF7
T-47D
85.4
66.0
GI50
¼
compound’s molar concentration inhibiting 50% net cell growth,
TGI ¼ compound’s molar concentration for total inhibition of net cell growth,
LC50 ¼ compound’s molar concentration inducing 50% net cell death.
hypophosphorylate pRb/total pRb ratio. In fact, The pRb hypo-
phosphorylated form binds to critical regulatory proteins (including
E2F) and represses the transcription of genes involved in cell cycle
progression from the G0eG1 to the S phase [10].
4. Conclusions
The biological data obtained for 1c have demonstrated that the
presence of the chloro atom in the indazole nucleus increases the
antiproliferative activity of the parent compound 1a, accomplishing
in this way the aim of our research. The ability of 1c to block cancer
cells in the G0eG1 phase by inhibiting the phosphorylation of pRb
can be considered of interest because mutations in the retino-
blastoma gene (RB-1) have been described in a wide variety of
neoplasms and constitutive phosphorylation of pRb has been
implicated in conferring uncontrolled growth to many cancer cells
[11,15].
Fig. 3. Effects of 1c on total Rb and hypophosphorylated Rb expression evaluated by
flow cytometry after staining K562 cells with a fluorescein isothiocyanate (FITC)-
conjugated anti-Rb mAb, or
hypophosphorylated Rb. a) Expression of total Rb in cells treated with (dark-gray line)
or without (light-gray line) 60 M 1c. b) Expression of hypophosphorylated Rb in cells
treated with (dark-gray line) or without (light-gray line) 60 M 1c.
a phycoerythrin (PE)-conjugated mAb raised against
m
m
group, is less active than 1b. Moreover, acetylation of the 3-amino
group of 1b to give 5b (mean GI%¼85.29) does not take any
advantage for activity. Analogous results have been obtained with
the contemporary 3 and 5-acetylation of 4j to give 6b (mean GI
% ¼ 94.98).
5. Experimental
5.1. Chemistry
Compounds 1c and 4j were tested for their toxicity towards
normal human fibroblast cells showing GI50 values of 36 and 32
mM
5.1.1. General
respectively. The above values, when compared than the GI50 values
of 1c and 4j obtained for the most sensitive tumor cell lines (see
Table 2), indicated a selective effect of these compounds towards
the tumor cell lines. Compound 1c was particularly selective,
showing very valuable therapeutic indexes (1020e6.28 range).
To understand the mechanism/s of action of 1c, cell cycle analysis
was performed. The effect of 1c on cell cycle was studied on K562 to
avoid the appearance of a sub-G0eG1 apoptotic peak that could
mask the real effect of the compound on the G0eG1eSeG2eM
phases of cell cycle. In fact, K562 cells are resistant to early drug-
induced apoptosis. Compound 1c induced an increase of cells in
G0eG1 peak and a decrease of cells in S a G2eM phases when used at
Reaction progress was monitored by TLC on silica gel plates
(Merck 60, F254, 0.2 mm). Organic solutions were dried over
Na2SO4. Evaporation refers to the removal of solvent on a rotary
evaporator under reduced pressure. All melting points were
determined on a Büchi 530 capillary melting point apparatus and
are uncorrected. IR spectra were recorded with a Perkin Elmer
Spectrum RXI FT-IR System spectrophotometer as solid in KBr disc.
1H NMR and 13C NMR spectra were obtained in DMSO-d6 at 300.13
and 75.47 MHz respectively, using a Bruker AC series 300 MHz
spectrometer (tetramethylsilane 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.
Analyses indicated by the symbols of the elements were within
ꢁ0.3% of the theoretical values. They were obtained by an
a concentration of or higher than 40
mM. This could be due to a block
of G1eS transition caused by the ability of 1c to increase the

B. Maggio et al. / European Journal of Medicinal Chemistry 46 (2011) 168e174
173
Elemental Vario EL. III apparatus. Yields refer to purified products
and are not optimized. The name of the compounds was obtained
using the ACD/I-Lab Web service (ACD/IUPAC Name Free 8.05).
signals, 7H, aromatic protons), 9.60 (s, 1H, exchangeable NH). Anal.
(C18H19N5O4) C,H,N.
5.1.2.7. N-(4-Benzylphenyl)-1H-indazole-1-carboxamide (4l). Yield:
5.1.2. General procedure for the preparation of compounds
1ceh,l,m
80%; m.p.: 92e94 ꢂC (ethanol); IR (KBr) [cmꢀ1]: 3382 (NH), 1727
n
(CO); 1H NMR (DMSO-d6)
d [ppm]: 3.93 (s, 2H, CH2), 7.19e8.51 (a set
A solution of equimolar amounts of indazole derivative 2c,e [16],
g [17] or indazole 2l [18] and phenyl isocyanate derivative 3a,b [18]
(7 mmol) in THF (20 mL) was stirred at room temperature for 24 h.
Then, the solvent was removed under reduced pressure and the
solid residue was crystallized from a suitable solvent or purified as
described below.
of signals, 14H, aromatic protons), 10.33 (s, 1H, exchangeable NH).
Anal. (C21H17N3O) C,H,N.
5.1.2.8. N-(4-butoxyphenyl)-1H-indazole-1-carboxamide
(4m). Yield: 60%; m.p.: 54e56 ꢂC (ethanol); IR (KBr)
n
[cmꢀ1]:
d [ppm]: 0.94 (t,
3373e3359 (NH), 1722 (CO); 1H NMR (DMSO-d6)
3H, CH3), 1.43 (m, 2H, CH2), 1.69 (m, 2H, CH2), 3.95 (t, 2H, CH2),
5.1.2.1. 3-Amino-N-(4-benzylphenyl)-5-chloro-1H-indazole-1-car-
boxamide (1c). The solid collected was crystallized from ethanol
the first time and then from methanol. Yield: 55%; m.p.:
6.93e8.49 (a set of signals, 9H, aromatic protons), 10.23 (s, 1H,
slowly exchangeable, NH). 13C NMR (DMSO-d6)
d [ppm]: 13.64
(CH3), 18.70 (CH2), 30.73 (CH2), 67.18 (CH2), 114.08 (CH), 114.28 (2ꢃ
CH), 121.55 (CH), 122.43 (CH), 123.35 (2ꢃ CH), 125.49 (C), 128.90
(CH), 130.52 (C), 138.01 (CH), 138.75 (C), 149.16 (C), 155.22 (CO).
Anal. (C18H19N3O2) C,H,N.
142e143 ꢂC; IR (KBr)
NMR (DMSO-d6)
n
[cmꢀ1]: 3459e3352 (NH, NH2),1700 (CO); 1H
d
[ppm]: 3.91 (s, 2H, CH2), 6.39 (s, 2H, slowly
exchangeable, NH2,), 7.18e8.18 (a set of signals, 12H, aromatic
protons), 9.47 (s, 1H, exchangeable, NH). Anal. (C21H17ClN4O) C,H,N.
5.1.3. General procedure for the preparation of compounds 4i, j
To a solution of 1.3 mmol of compound 4g,h in 250 mL of warm
solvent (ethyl acetate for 4g and methanol for 4h), 50 mg of 10%
PdeC as catalyst was added. The mixture was left under hydroge-
nation in a Parr apparatus at 50 psi for 20 h. The suspension was
filtered and the filtrate was evaporated affording the crude
compound 4i, j which was purified by flash chromatography and
crystallization as described below.
5.1.2.2. 3-Amino-N-(4-butoxyphenyl)-5-chloro-1H-indazole-1-car-
boxamide (1d). The product was purified by crystallization from
ethanol and preparative TLC (silica gel plate, layer thickness 2 mm,
chloroform as eluent). Yield: 55%; m.p.: 116e117 ꢂC (ethanol); IR
(KBr)
d6)
n
[cmꢀ1]: 3385e3303 (NH, NH2), 1698 (CO); 1H NMR (DMSO-
d
[ppm]: 0.93 (t, 3H, CH3), 1.44 (m, 2H, CH2), 1.68 (m, 2H, CH2),
3.94 (t, 2H, CH2), 6.37 (s, 2H, exchangeable, NH2,), 6.89e8.18 (a set
of signals, 7H, aromatic protons), 9.43 (s, 1H, slowly exchangeable,
NH). Anal. (C18H19ClN4O2) C,H,N.
5.1.3.1. 3,5-Diamino-N-(4-benzylphenyl)-1H-indazole-1-carbox-
amide (4i). Flash chromatography procedure on silica gel
(0.040e0.063 mm): external diameter of the column 6 cm, ethyl
acetate/petroleum ether b.p. 40e60 ꢂC (8:2 v/v) as eluent (2.3 L).
Fractions 26e46 (each 25 mL) were collected and evaporated
affording a product which was crystallized from diethyl ether to
5.1.2.3. 3-Amino-N-(4-benzylphenyl)-6-chloro-1H-indazole-1-car-
boxamide (1e). The product was purified by crystallization from
ethanol and preparative TLC (silica gel plate, layer thickness 2 mm,
chloroform as eluent). Yield: 73%; m.p.: 140e142 ꢂC (ethanol); IR
(KBr)
d6)
n
[cmꢀ1]: 3454e3352 (NH, NH2), 1693 (CO); 1H NMR (DMSO-
give 4i. Yield: 93%; m.p.: 129e131 ꢂC; IR (KBr)
n
[cmꢀ1]: 3456e3232
d [ppm]: 3.89 (s, 2H,
d
[ppm]: 3.88 (s, 2H, CH2), 6.43 (s, 2H, exchangeable, NH2,),
(2ꢃ NH2, NH), 1684 (CO); 1H NMR (DMSO-d6)
7.16e8.19 (a set of signals, 12H, aromatic protons), 9.47 (s, 1H,
exchangeable, NH). Anal. (C21H17ClN4O) C,H,N.
CH2), 5.07 (s, 2H, exchangeable, NH2), 6.06 (s, 2H, exchangeable,
NH2), 6.84e7.85 (a set of signals, 12H, aromatic protons), 9.17 (s, 1H,
exchangeable, NH). Anal. (C21H19N5O) C,H,N.
5.1.2.4. 3-Amino-N-(4-butoxyphenyl)-6-chloro-1H-indazole-1-car-
boxamide (1f). The product was purified by crystallization from
ethanol and preparative TLC (silica gel plate, layer thickness 2 mm,
chloroform as eluent). Yield: 56%; m.p.: 124e126 ꢂC (ethanol); IR
5.1.3.2. 3,5-Diamino-N-(4-butoxyphenyl)-1H-indazole-1-carbox-
amide (4j). Flash chromatography procedure on silica gel
(0.040e0.063 mm): external diameter of the column 4 cm, ethyl
acetate as eluent (1 L). Fractions 15e20 (each 25 mL) were collected
and evaporated affording a product which was crystallized from
(KBr)
d6)
n
[cmꢀ1]: 3386e3305 (NH, NH2), 1701 (CO); 1H NMR (DMSO-
d
[ppm]: 0.93 (t, 3H, CH3), 1.43 (m, 2H, CH2), 1.67 (m, 2H, CH2),
3.91 (t, 2H, CH2), 6.43 (s, 2H, slowly exchangeable, NH2,), 6.84e8.21
ethanol. to give 4j. Yield: 86%; m.p.: 133e134 ꢂC; IR (KBr)
3428e3211 (2ꢃ NH2, NH), 1706 (CO); 1H NMR (DMSO-d6)
n
[cmꢀ1]:
d [ppm]:
(a set of signals, 7H, aromatic protons), 9.46 (s, 1H, exchangeable,
NH). 13C NMR (DMSO-d6)
d
[ppm]: 13.67 (CH3), 18.70 (CH2), 30.74
0.92 (t, 3H, CH3), 1.42 (m, 2H, CH2), 1.65 (m, 2H, CH2), 3.92 (t, 2H,
CH2), 5.06 (s, 2H, exchangeable, NH2), 6.03 (s, 2H, exchangeable,
NH2), 6.86e7.88 (a set of signals, 7H, aromatic protons), 9.12 (s, 1H,
(CH2), 67.19 (CH2), 113.47 (CH), 114.32 (2ꢃ CH), 117.60 (C), 121.78
(2ꢃ CH), 122.29 (CH), 122.49 (CH), 130.80 (C), 134.10 (C), 139.81 (C),
149.15 (C), 151.10 (C), 154.89 (CO). Anal. (C18H19ClN4O2) C,H,N.
exchangeable, NH). 13C NMR (DMSO-d6)
d [ppm]: 13.63 (CH3), 16.69
(CH2), 30.73 (CH2), 67.16 (CH2), 102.00 (CH), 114.32 (3ꢃ CH), 118.62
(CH), 119.81 (C), 121.16 (2ꢃ CH), 131.38 (C), 132.50 (C), 144.07 (C),
149.16 (C), 150.88 (C), 154.46 (CO). Anal. (C18H21N5O2) C,H,N.
5.1.2.5. 3-Amino-N-(4-benzylphenyl)-5-nitro-1H-indazole-1-carbox-
amide (1g). Yield: 66%; m.p.: 231e233 ꢂC (ethyl acetate); IR (KBr)
n
[cmꢀ1]: 3469e3342 (NH, NH2), 1716 (CO); 1H NMR (DMSO-d6)
[ppm]: 3.92 (s, 2H, CH2), 6.74 (s, 2H, exchangeable, NH2),
d
5.1.4. General procedure for the preparation of compounds 5a,b
A mixture of compound 1a,b [8] (1 mmol), and acetic anhydride
(16 mL) was stirred at room temperature overnight. The solid
collected was washed with diethyl ether, then chromatographed
and/or crystallized from a suitable solvent.
7.20e9.05 (a set of signals, 12H, aromatic protons), 9.70 (s, 1H,
exchangeable, NH). Anal. (C21H17N5O3) C,H,N.
5.1.2.6. 3-Amino-N-(4-butoxyphenyl)-5-nitro-1H-indazole-1-car-
boxamide (1h). Yield: 72%; m.p.: 181e183 ꢂC (ethyl acetate); IR
(KBr)
d6)
3.93 (t, 2H, CH2), 6.70 (s, 2H, exchangeable NH2), 6.89e9.04 (a set of
n
[cmꢀ1]: 3454e3365 (NH, NH2), 1716 (CO); 1H NMR (DMSO-
5.1.4.1. 3-(Acetylamino)-N-(4-benzylphenyl)-1H-indazole-1-carbox-
d
[ppm]: 0.93 (t, 3H, CH3), 1.43 (m, 2H, CH2), 1.68 (m, 2H, CH2),
amide (5a). Yield: 89%; m.p.: 174e175 ꢂC (ethyl acetate); IR (KBr)
n
[cmꢀ1]: 3383e3264 (NH), 1727 (carboxamide CO), 1676 (CO); 1H

174
B. Maggio et al. / European Journal of Medicinal Chemistry 46 (2011) 168e174
NMR (DMSO-d6)
d
[ppm]: 2.23 (s, 3H, CH3), 3.92 (s, 2H, CH2),
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., Top-
sham, Me, USA).
7.25e8.33 (a set of signals, 13H, aromatic protons), 9.88 (s, 1H,
exchangeable carboxamide NH), 10.77 (s, 1H, exchangeable, NH).
Anal. (C23H20N4O2) C,H,N.
5.1.4.2. 3-(Acetylamino)-N-(4-butoxyphenyl)-1H-indazole-1-carbox-
amide (5b). The product was purified by preparative TLC (silica gel
plate, layer thickness 2 mm, ethyl acetate/petroleum ether b.p.
40e60 ꢂC (1:1 v/v) as eluent) and crystallization from ethyl acetate.
5.2.2. 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 Dick-
inson). After 20 min cells were washed twice with BD Perm/WashÔ
buffer solution (Becton Dickinson) and incubated with 20 L FITC
conjugated anti-pRb or Pe conjugated anti-hypophosphorylated
pRb monoclonal antibodies (Becton Dickinson) at 4 ꢂC. After 30 min
cells were washed twice and analyzed by flow cytometry.
Yield: 82%; m.p.: 142e144 ꢂC; IR (KBr)
n
[cmꢀ1]: 3378e3250 (NH),
m
1728 (carboxamide CO), 1672 (acetamido CO); 1H NMR (DMSO-d6)
[ppm]: 0.96 (t, 3H, CH3), 1.46 (m, 2H, CH2), 1.70 (m, 2H, CH2), 2.23
d
(s, 3H, CH3), 3.96 (t, 2H, CH2), 6.96e8.32 (a set of signals, 8H,
aromatic protons), 9.80 (s, 1H, exchangeable, carboxamido NH),
10.73 (s, 1H, exchangeable, NH). Anal. (C20H22N4O3) C,H,N.
5.2.3. Toxicity evaluation on normal cells
Human normal fibroblasts (2 ꢃ 105) were seeded in 1 mL of
complete medium constituted by RPMI 1640, fetal calf serum (10%),
penicillin (1%) and streptomycin (1%) in 16 mm wells (tissue culture
cluster 24 wells, Costar) and incubated at 37 ꢂC in a CO2 atmo-
sphere. Compounds were added after 24 h. The number of living
cells was evaluated after 72 h by trypan blue exclusion method and
expressed as percent of control proliferation.
5.1.5. General procedure for the preparation of compounds 6a,b
A mixture of compound 4i, j (1 mmol) and acetic anhydride
(4 mL) was stirred at room temperature overnight, and then cold
water was added until precipitation. The solid was filtered off and
then crystallized from dioxane to give 6a,b.
5.1.5.1. 3,5-Bis(acetylamino)-N-(4-benzylphenyl)-1H-indazole-1-car-
boxamide (6a). Yield: 82%; m.p.: 236e238 ꢂC; IR (KBr)
n
[cmꢀ1]:
d [ppm]:
Acknowledgement
3373e3259 (3ꢃ NH), 1727, 1679 (CO); 1H NMR (DMSO-d6)
2.07 (s, 3H, CH3), 2.19 (s, 3H, CH3), 3.92 (s, 2H, CH2), 7.24e8.19 (a set
of signals, 12H, aromatic protons), 9.87 (s, 1H, exchangeable, car-
boxamido NH), 10.16 (s, 1H, exchangeable, NH), 10.71 (s, 1H,
Financial support from MIUR (60% fund) is gratefully acknowl-
edged. The authors wish to thank The National Cancer Institute of
the United States of America for performing the screening of
compounds.
exchangeable, NH). 13C NMR (DMSO-d6)
d [ppm]: 22.89 (CH3), 23.83
(CH3), 40.42 (CH2), 111.37 (CH), 114.12 (CH), 120.24 (C), 120.55 (2ꢃ
CH), 122.57 (CH), 125.84 (CH), 128.32 (2ꢃ CH), 128.54 (2ꢃ CH),
128.84 (2ꢃ CH), 134.57 (C), 135.63 (C), 136.01 (C), 136.76 (C), 141.32
(C), 143.40 (C), 148.69 (CO), 168.20 (CO), 169.06 (CO). Anal.
(C25H23N5O3) C,H,N.
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5.1.5.2. 3,5-Bis(acetylamino)-N-(4-butoxyphenyl)-1H-indazole-1-
carboxamide (6b). Yield: 74%; m.p.: 200e203 ꢂC; IR (KBr)
3277e3177 (3ꢃ NH), 1712, 1654 (CO); 1H NMR (DMSO-d6)
n
[cmꢀ1]:
[ppm]:
d
0.94 (t, 3H, CH3), 1.45 (m, 2H, CH2), 1.69 (m, 2H, CH2), 2.08 (s, 3H,
CH3), 2.20 (s, 3H, CH3), 3.95 (s, 3H, CH3), 6.93e8.19 (a set of signal,
7H, aromatic protons), 9.83 (s, 1H, exchangeable, carboxamido NH),
10.12 (s, 1H, exchangeable, NH), 10.67 (s, 1H, exchangeable, NH).
Anal. (C22H25N5O4) C,H,N.
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5.2.1. Flow cytometric analysis of cell cycle distribution
The effects of compound 1c on cell cycle distribution were
studied on K562 cells (myeloblastic leukemia) by flow cytometric
analysis after staining with propidium iodide. Cells were exposed
24 h. After treatment cells were washed once in ice-cold phosphate
buffered saline medium (PBSeSigma) and resuspended at 106/mL
in a hypotonic fluorochrome solution of propidium iodide (Sigma,
St Louis, Mo) (50 mg/mL) and nonidet P-40 (Sigma) [0.03% (v/v)]
in 0.1% sodium citrate. After 30 min of incubation, the fluorescence
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[18] The compounds are commercially available.
of each sample was analyzed as single-parameter frequency
[19] The methodology can be found at http://dtp.nci.nih.gov/.