Chromophore-modified bis-benzo[g]indole carboxamides: Synthesis and antiproliferative activity of bis-benzo[g]indazole-3-carboxamides and related dimers

Article abstract of DOI:10.1016/S0014-827X(03)00131-9

Tricyclic pyrazole dimers that comprise two kinds of CONH-(CH ₂)_n-N(CH₃)-(CH₂)_n-NHCO bridges to which are linked potential DNA-intercalating groups such as 1H-benzo[g]indazole, 2H-benzo[g]indazole and

Full text of DOI:10.1016/S0014-827X(03)00131-9

Il Farmaco 58 (2003) 749ꢀ763
/
www.elsevier.com/locate/farmac
Chromophore-modified bis-benzo[g]indole carboxamides: synthesis
and antiproliferative activity of bis-benzo[g]indazole-3-carboxamides
and related dimers
Ge´rard A. Pinna *, Maria A. Pirisi, Jean-Mario Mussinu, Gabriele Murineddu,
Giovanni Loriga, Amedeo Pau, Giuseppe E. Grella
Dipartimento Farmaco Chimico Tossicologico, Universita` di Sassari, via F. Muroni 23/A, 07100 Sassari, Italy
Received 20 November 2002; accepted 20 January 2003
Dedicated to the memory of Professor Piero Pratesi
Abstract
Tricyclic pyrazole dimers that comprise two kinds of CONHꢀ
/
(CH2)n ꢀ
/
N(CH3)ꢀ
/
(CH2)n ꢀ/NHCO bridges to which are linked
potential DNA-intercalating groups such as 1H-benzo[g]indazole, 2H-benzo[g]indazole and 1,4-dihydroindeno[1,2-c]pyrazole were
designed, synthesized and some of them evaluated in vitro by NCI (Bethesda, USA) against nine types of cancer cells. Compounds
2a, 2fꢀ
/
i and 2oꢀ/r demonstrated significant antiproliferative activity, all with GI50 values in the low micromolar range. Preliminary
analysis of the structureꢀ/activity relationship for dimers 2 indicated that: (i) in the ground terms (2a and 2k) antitumor activities
were strongly related to the type of chromophore, (ii) in contrast, either 1H-benzo[g]indazole- or 1,4-dihydroindeno[1,2-c]pyrazole-
dimers when bore a N1-aryl group (2g, 2h, 2i, 2o, 2p, 2q and 2r) generally showed a good level of antitumor potency and (iii) for the
most representative compounds (pairs of compounds: 2g,2h; 2o,2p and 2q,2r) the length of the bridges did not significantly
contribute to the variations in cytotoxicity. Two members of this series, 2f and 2q, were selected and tested in the hollow fiber cell
assay to evaluate in a preliminary fashion their in vivo antitumor activity. Finally, viscosity measurement of 2f with poly(dA-dT)2,
confirmed that these promising compounds behaved as typical DNA-intercalating agents.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Tricyclic pyrazole; Cytotoxic activity; Viscosity
1. Introduction
being accompanied by unwinding and elongation of the
duplex; the alkylators, which react covalently with DNA
bases; and the DNA strand breakers, which generate
reactive radicals that produce cleavage of the polinu-
cleotide strands [2].
Among intercalative substances it is also known [3]
that molecular duplication transforms mono-intercala-
tors into bifunctional intercalating agents (also called
bis-intercalators) in which there are two potential
intercalating groups (chromophores) tethered together.
In general the affinity of these compounds for DNA is
greater than that of their monointercalating counter-
parts, thus improving their antiproliferative activity.
As a result of this considerations, we have initiated a
program focused on exploring bis-intercalators obtained
by linking two benzo[g]indole groups with amino bis-
alkylamide bridges and have reported that some deri-
Cancer remains a major public health issue at the
beginning of the 21st century. Because of this critical
situation, much effort has been directed to the design of
new drugs for cancer therapeutics. DNA-interactive
drugs in current clinical use represent one of the most
important drug classes in cancer therapy [1]. In general
there are three major types of the above mentioned
clinically important drugs: the intercalators, which
insert between the base pairs of the double helix and
determine a significant change of DNA conformation
* Corresponding author. Tel.: ꢁ
8720.
/
39-7-922-8721; fax: ꢁ39-7-922-
/
E-mail address: pinger@uniss.it (G.A. Pinna).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00131-9

750
G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ763
/
Regioselective cyclization of 14 and 15 with hydrazine
hydrate, phenylhydrazine hydrochloride or 2,4-dichlor-
ophenylhydrazine hydrochloride led to tricyclic pyra-
zoles 16, 18, 19, 26, 28 and 29 in good yields. In the case
of methylhydrazine no regiospecificity was observed,
and the cyclization steps resulted in the formation of
two pairs of isomers, 17/20 and 27/30, separable by silica
gel flash-chromatography. These new compounds were
fully characterized by spectroscopic techniques, namely
1H, 13C NMR and nOe difference experiments (Table 1).
In the case of condensation of 14 the analysis of the
NMR spectra allowed us to identify the compound with
the lower Rf (55%) as the isomer 17 and the other as 20
(35%). The identification of compounds 17 and 20 was
based mainly on their 13C NMR spectra. Compound 17
shows the following features for the quaternary carbons
in the heteroaromatic region: three singlets at 137.77,
137.91 and 139.56 ppm corresponding, respectively, to
the pyrazole C-3a, C-3 and C-9b carbons. The 13C
NMR spectrum of compound 20 shows the aforemen-
tioned singlets at 135.98, 128.69 and 146.68 revealing a
high-field shift for the C-3 carbon and a low-field shift
for C-9b carbon in accord with simple mesomeric
considerations and with published data [6] for similar
systems.
Chart 1.
vatives, as represented by structure 1 (Chart 1), have
good cytotoxicity against cancer cells in vitro tests [4].
In order to improve the activity and cell line
selectivity, we planned to investigate, following the
classical ꢀ
/
CHꢁ
/
/ꢀ
/
Nꢁ
/
bioisosterism, aza-analogues 2 of
the bis-benzo[g]indoles 1, in particular exploring the
effect produced by the presence of two 1H-benzo[g]in-
dazole, 2H-benzo[g]indazole and 1,4-dihydroin-
deno[1,2-c]pirazole moieties with two different amino
bis-alkylamide chains on cellular cytotoxicity. The
influence on activity of groups at tricyclic nitrogens
has also been explored (2aꢀr, Chart 2).
/
2. Chemistry
1
In addition, the tricyclic pyrazoles showed similar H
NMR features in the aliphatic region, but differ
significantly in the aromatic one in which the aromatic
The preparation of tricyclic pyrazole bis-intercalators
2aꢀr was carried out as shown in Scheme 1.
Acids 3ꢀ11 were activated with 1,1?-carbonyldiimida-
zole (CDI), and then reacted with a stoichiometric
amount of the appropriate diamine in DMF at r.t.
/
/
C9ꢀ
remarkable feature in the 1H NMR spectrum of 20
was the downfield shift of C9ꢀH, which adsorbed at ca.
7.82 ppm.
/H doublet of 17 appeared at ca. 7.57 ppm. A
/
The carboxylic acid 3ꢀ11 were not known, with the
/
exception of 10 [5], and were for the most part prepared
using a similar method (Scheme 2).
Finally, the identity of regioisomers 17 and 20 was
unequivocally proved by nOe difference spectroscopy
analysis. Irradiation of the resonance frequency of the
protons C9-H led to the enhancement of the signal of the
methyl protons CH3ꢀ
/
N1 in the case of regioisomer 17,
whereas for regioisomer 20 no enhancement of the same
signal was observed.
When cyclocondensation was carried out with 15 and
methylhydrazine a mixture of regioisomers was formed.
After work up and on the basis of simple analysis on
silica gel thin layer chromatography (TLC) of the crude
product, we empirically correlated the structure of the
slow-moving 27 with 17 and that of 30 with 20. We were
able to establish by 1H and 13C NMR spectra the
structure 27 for the main product (55%, lower Rf) and
the structure 30 for the minor one (10%). These assign-
ments were confirmed by the nOe experiments with
isomers 27 and 30. Thus, C8ꢀ
/
H proton of the aromatic
moiety of 27 located in the close proximity of N1ꢀ
/CH3
exhibited nOe enhancement whereas no interaction was
seen between these two groups for 30.
The treatment of compounds 16ꢀ
/
20 with 2,3-di-
chloro-5,6-dicyano-p-benzoquinone (DDQ) gave the
Chart 2.

G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ
/763
751
Scheme 1. Reagents and conditions: (a) CDI, DMF, 3 h, r.t. then CH3N(CH2CH2NH2)2 or CH3N(CH2CH2CH2NH2)2, DMF, 48 h, r.t.
esters 21ꢀ
solution to afford acids 3ꢀ
The a,g-diketoesters 14 [5] and 15, isolated as hydro-
xymethylene tautomers, were prepared in excellent yield
/
25. The esters 21ꢀ
/
29 were hydrolized in basic
effect on cancer cell growth with molar concentration
ranging from 1.51 to 11.7 mM in almost all cell lines.
/
11.
The GI50 values obtained for N-aryl derivatives 2gꢀi
/
clearly showed that this substitution favourably affected
the anticellular activity being 1.32ꢀ1.59 times more
potent than the N-unsubstituted counterpart 2a. Com-
pounds 2gꢀi exhibited potent antiproliferative activity
(ꢀ90%) from the requisite benzocyclanones 12 and 13
/
/
and ethyl oxalate in the presence of sodium ethoxide.
/
against leukaemia cell lines with GI50 values up to 0.014
mM. Again, these compounds showed moderate-to-good
activities against certain solid tumor cell lines. In
addition the effect of the variation of benzo[g]indazole
conjugation could be recognized by comparing the
cytotoxicities of 2a and 2f. The 2H-benzo[g]indazole
3. Results and discussion
Compounds 2a,fꢀ
National Cancer Institute (NCI) for evaluation in an
in vitro preclinical antitumor screening program [7ꢀ9]
/
i,kꢀ
/
r were selected by the US
/
analogue 2f did have respectable cytotoxicity (GI50ꢂ
/
against ca. 60 human tumor cell lines derived from
leukemia, non-small cell lung cancer, colon cancer,
central nervous system (CNS) cancer, melanoma, ovar-
ian cancer, renal cancer, prostate cancer and breast
cancer. The results are summarized in Table 2 as
cytostatic/cytotoxic parameters.
5.01 mM), but it was less active than the ground term
2a. Compound 2f showed potent selective cytotoxicity
against all melanoma cell lines; however, 2f was not
cytotoxic against almost all other tumor cells.
Further structureꢀactivity relationship studies fo-
/
cused on the effects of variation of aromatic tricyclic
moiety of 2a were then examined. N-unsubstituted 1,4-
dihydroindeno[1,2-c]pyrazole analogue 2k was found to
The cytostatic parameters include GI50 and TGI,
which are the concentration of drug required for 50%
growth inhibition and total growth inhibition, respec-
tively. The cytotoxic parameter is the LC50, which is the
concentration required for 50% cell kill.
Table 3 lists GI50 values which, in those cell lines are
lower than the mean values over all cell lines tested.
Initial substitution of the benzo[g]indole chromo-
phore of 1 with 1H-benzo[g]indazole (1 versus 2a)
provided an active compound even if 1.38-fold less
potent than 1, with GI50 and TGI in the low micromolar
range. Moreover, 2a showed a wide-spectrum inhibitory
be moderately cytostatic (GI50ꢂ
/
18.20 mM) and only
weakly cytocidal (LC50ꢂ69.18 mM).
/
The effect of the length of the bridge connecting the
two chromophores could be recognized in this series by
comparing the cytotoxicities of 2k (GI50ꢂ
with 2l (GI50ꢂ12.02 mM) and 2m (GI50ꢂ
with 2n (GI50ꢂ11.48 mM). This indicated a slight
/
18.20 mM)
/
/
15.49 mM)
/
increase in biological activity as the length of the bridge
was increased. Moreover, a direct comparison between

752
G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ763
/
Scheme 2. Reagents and conditions: (a) EtONa, EtOOCꢀ
/
COOEt, 2ꢀ
/
8 h, r.t. (b) EtOH, RNHNH2, 2ꢀ5 h, reflux. (c) CH2Cl2, DDQ, 5 min, r.t. (d)
/
MeOH, KOH, overnight, reflux.
2k and 2m or 2l and 2n allowed a determination of the
effect of replacing the NH of 2k and 2l with an N ꢀCH3
group, since the two pairs of structures were otherwise
identical. The similar GI50 values for methyl derivatives
2l and 2n clearly showed that substitution at this
position was tolerated. Turning to the N-aryl analogues,
cancer cells (TGIꢂ
/
4.68 mM) and melanoma cancer cells
/
(TGIꢂ4.01 mM) (panel selectivity).
/
Based on in vitro anti-cancer screening results, NCI
selected compounds 2f and 2q for preliminary in vivo
hollow-fiber assay [10]. Each compound was tested
against a standard panel of 12 human tumor cell lines,
including NCI-H23, NCI-H522, MDA-MB-231, MDA-
MB-435, SW-620, COLO 205, LOX IMVI, UACC-62,
OVCAR-3, OVCAR-5, U 251 and SF-295. According to
the NCI’s protocol, compounds with a combined
intraperitoneal (IP) and subcutaneous (SC) score of
20, a SC score 8 or a net cell kill of one or more cell lines
in either implant site, are referred for xenograft testing.
compounds 2oꢀr, containing phenyl or 2,4-dichlorophe-
/
nyl groups, showed good antiproliferative activities.
Compound 2q demonstrated improved cytotoxicity
especially against leukaemia, colon cancer and mela-
noma cell lines, with GI50 values ranging from 1.17 to
2.31 mM. Moreover, TGI values supported that com-
pound 2q had high inhibitory activity against both colon

G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ
/763
753
Table 1
Determination of the regiochemistry of isomers 17/20 and 27/30
a
Comp.
n
Rf
Yield (%)
dC-k
dC-i
dC-j
dH-q
17
27
20
30
2
1
2
1
0.27
0.23
0.76
0.69
55
55
35
10
137.77
148.84
135.98
147.50
137.91
150.10
146.68
157.68
139.56
136.73
128.69
129.86
7.57
7.53
7.82
7.75
13C and 1H NMR spectra were recorded in CDCl3.
a
Rf values were determined on TLC using Polygram† SIL N-HR-/HV254 precoated plastic sheet (0.2 mm) with 4:1 petroleum ether/ethyl acetate
as eluent.
The results are: 2f, IPꢂ
/
2, SCꢂ
/
0, cell killꢂ
/
0; 2q, IPꢂ
/
binding to DNA, did not affect the viscosity signifi-
cantly [11].
The data of Fig. 1 show that the binding of 2f with
10, SCꢂ6, cell killꢂ0. Only 1,4-dihydroindeno[1,2-
/
/
c]pyrazole derivative 2q produced a moderate reduction
in the viable cell mass below the level present at the start
of implantation.
In order to determine at the molecular level whether
compounds 2 have the ability to intercalate into DNA
strands, the relative viscosity (h/h0) of polynucleotide
Poly(dA-dT)×/poly(dA-dT) caused a curve profile similar
to that of EtBr, thus demonstrating, for this class of
compounds, an intercalative mode of DNA interaction.
In summary, evaluation of a number of tricyclic
pyrazole dimers for cytotoxicity revealed that the great-
est activity resided in compounds incorporating N-aryl
substituents. In particular, 2q is a prototypic molecule
serving as a template for subsequent molecular mod-
ification with a view to increased activity and selective
toxicity for colon and melanoma neoplasms.
Poly(dA-dT)×/poly(dA-dT) in the presence of the ground
term 2f (used as 3HCl×
/3H2O derived), Ethidium bro-
mide (EtBr), and Distamycin A (Dist) were measured. It
is known that EtBr increased the viscosity of DNA, by
intercalation in DNA base pairs, while Dist, a groove
Table 2
Inhibition of in vitro tumor cell growtha by 2a, fꢀ
/i, kꢀ/r and 1
Comp.
Log GI50
GI50 (mM) b
Log TGI
TGI (mM) b
Log LC50
LC50 (mM) c
2 a
2f d
2g d
2h d
2i d
2k
ꢃ
/
5.59
5.30
5.71
5.79
5.79
4.74
4.92
4.81
4.94
5.73
5.85
5.54
5.81
5.73
2.57
5.01
ꢃ
/
5.19
4.86
5.29
5.41
5.49
4.44
4.59
4.47
4.52
5.40
5.52
4.89
5.50
5.35
6.45
13.80
5.13
ꢃ
/
4.46
4.44
4.83
4.21
5.11
4.16
4.26
4.17
4.15
5.08
5.18
4.32
5.21
4.97
34.6
36.3
ꢃ
/
ꢃ
/
ꢃ
/
ꢃ
/
1.95
1.62
ꢃ
/
ꢃ
/
14.8
61.7
ꢃ
/
ꢃ
/
3.89
3.24
ꢃ
/
ꢃ
/
1.62
ꢃ
/
ꢃ
/
7.76
ꢃ
/
18.20
12.02
15.49
11.48
1.86
ꢃ
/
36.31
25.70
33.88
30.20
3.98
ꢃ
/
69.18
54.95
67.61
70.79
8.32
2l
ꢃ
/
ꢃ
/
ꢃ
/
2m
2n e
2o
ꢃ
/
ꢃ
/
ꢃ
/
ꢃ
/
ꢃ
/
ꢃ
/
ꢃ
/
ꢃ
/
ꢃ
/
2p
2q
ꢃ
/
1.41
2.88
ꢃ
/
3.02
12.88
3.16
ꢃ
/
6.61
47.86
6.17
ꢃ
/
ꢃ
/
ꢃ
/
2r
ꢃ
/
1.55
1.86
ꢃ
/
ꢃ
/
1
ꢃ/
ꢃ/
4.47
ꢃ/
10.71
a
Expressed as meangraph midpoint (MG-MID).
GI50 and TGI are the concentration in mmoles per liter required for 50 and 100% growth inhibition, respectively (cytostatic effect).
LC50 is the concentration in mmoles per liter required for 50% lethality (cytocidal effect). The three parameters are reported both in logarithmic
b
c
and arithmetical scales.
d
As hydrochloride salt.
As fumarate salt.
e

754
G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ763
/
Table 3
Inhibition of in vitro tumor cell growth by 2a, fꢀ
/
i, kꢀ
/
r and 1
Panel/cell line
Antiproliferative activity (GI50 in mM) a
2a
2f
2g
2h
2i
2k
2l
2m
2n
2o
2p
2q
2r
1
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
2.29
ꢀ
ꢀ/
2.59
3.33
1.78
/
ꢀ
ꢀ/
/
0.014
0.035
ꢀ
/
18.1 11.1
2.18
1.47
1.24
0.87
0.31
0.36
1.08
1.74
1.17
2.00
2.12
1.62
0.60
1.41
0.54
1.32
1.20
1.48
ꢀ/
0.32
1.10 18.0
1.61
ꢀ/
1.67 12.9
12.7
2.04
6.01
5.36
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.82
1.73
MOLT-4
RPMI-8226
SR
0.38
0.20
ꢀ/
10.4
12.4
2.12
ꢀ/
0.28
ꢀ/
2.99
0.19
1.84
Non-small cell lung cancer
A549/ATCC
EKVX
2.20
1.65
2.38
ꢀ
ꢀ/
ꢀ/
4.26
/
ꢀ
ꢀ/
1.43
1.73
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
14.8
17.3
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
15.4
12.9
/
ꢀ
ꢀ/
/
ꢀ
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
1.81
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
1.46
1.81
1.75
1.84
1.68
HOP-62
HOP-92
9.09
ꢀ/
1.54
ꢀ/
ꢀ/
NCI-H226
NCI-H23
1.92
1.90
2.11
2.56
1.98
ꢀ
ꢀ/
ꢀ/
3.70
4.41
/
ꢀ
ꢀ/
ꢀ/
1.41
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
/
ꢀ
17.2
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
15.4
/
ꢀ
ꢀ/
ꢀ/
/
ꢀ
/
ꢀ
ꢀ/
ꢀ/
1.30
/
ꢀ
ꢀ/
1.77
1.51
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
1.54
1.75
NCI-H322M
NCI-H460
NCI-H522
ꢀ/
ꢀ/
ꢀ/
1.65
1.73
1.71
ꢀ
/
18.1
1.46 17.5
ꢀ/
1.71
ꢀ/
ꢀ/
ꢀ/
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
1.51
1.68
2.27
1.63
1.60
4.23
4.14
1.64
1.54
1.53
1.53
1.76
1.44
1.31
ꢀ
/
17.3
1.53 15.4
16.2
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
11.9
12.5
15.1
ꢀ
9.80
/
1.74
1.71
1.63
1.27
1.41
1.90
1.74
2.13
2.15
2.02
ꢀ
1.52
/
1.44
1.41
1.61
1.64
1.76
1.63
1.71
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
14.0
ꢀ/
2.24
1.78
1.88
ꢀ/
ꢀ/
ꢀ/
1.57 17.6
17.3
1.76
1.83
1.25
KM12
SW-620
ꢀ/
4.76
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.50
1.18
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
2.17
1.84
1.79
1.83
1.84
2.05
ꢀ
1.75
2.81
/
ꢀ
1.61
1.66
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
1.61
ꢀ
16.6
/
ꢀ
ꢀ/
/
14.7
ꢀ
ꢀ/
ꢀ/
7.22
/
1.76
1.85
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
2.75
1.75
/
ꢀ
1.17
1.19
/
ꢀ
ꢀ/
0.57
1.48
1.25
1.76
/
ꢀ/
ꢀ/
ꢀ/
14.3
11.0
ꢀ/
ꢀ/
1.48
1.81
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.54 16.8
12.5
ꢀ/
ꢀ/
ꢀ/
ꢀ/
14.4
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.32
ꢀ/
ꢀ/
ꢀ/
11.1
4.98
1.57
ꢀ/
ꢀ/
ꢀ/
Melanoma
LOX IMVI
MALME-3M
M14
1.81
1.73
1.93
1.87
2.93
2.15
2.83
1.85
1.67
1.90
1.72
1.78
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.55
/
ꢀ
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
/
ꢀ
10.3
/
ꢀ
ꢀ/
12.6
14.7
/
2.55
2.27
1.86
1.83
1.85
1.37
1.85
1.12
1.76
2.31
1.74
1.69
1.84
1.84
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
1.48
ꢀ/
ꢀ/
0.67
1.40
ꢀ/
1.78
1.86
1.70
1.64
1.71
1.77
1.60
ꢀ/
10.6
1.46
1.60
1.67
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
ꢀ/
2.00
1.75
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
16.9
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.70
1.83
1.63
1.75
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.53
12.1
14.0
15.2
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.82
1.73
ꢀ/
1.98
ꢀ/
ꢀ/
Ovarian cancer
IGROV1
1.68
1.91
2.04
1.87
2.12
2.01
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
1.70
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
16.3
14.9
15.9
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
8.56
11.4
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
1.67
1.78
1.74
1.79
1.78
1.39
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
1.47
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.74
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
0.75
0.053
ꢀ/
ꢀ/
ꢀ/
1.80
ꢀ/
ꢀ/
ꢀ/
1.79
ꢀ/
16.5
ꢀ/
ꢀ/
ꢀ/
ꢀ/
Renal cancer
786-0
1.75
2.11
1.91
ꢀ
3.06
/
1.67
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.46
/
1.54 16.0 10.9
1.43
14.5
15.3
14.5
9.13
1.70
1.86
1.72
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
2.27
2.53
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
1.54
1.53
1.62
A498
ꢀ/
ꢀ/
ꢀ/
1.62
1.88
1.40
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ACHN
CAKI-1
RXF 393
SN12C
TK-10
ꢀ/
3.65
4.89
ꢀ
/
17.1
1.58 16.7
ꢀ
ꢀ/
/
ꢀ/
1.66
2.67
ꢀ/
2.02
1.83
ꢀ/
14.8
14.9
ꢀ/
ꢀ/
1.14
ꢀ
ꢀ/
/
15.4
16.7
ꢀ
ꢀ/
/
1.43
1.86
1.72
1.54
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
10.4
UO-31
ꢀ/
4.62
ꢀ/
ꢀ/
ꢀ/
0.27
1.38

G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ
/763
755
Table 3 (Continued)
Panel/cell line
Antiproliferative activity (GI50 in mM) a
2a
2f
2g
2h
2i
2k
2l
2m
2n
2o
2p
2q
2r
1
Prostate cancer
PC-3
ꢀ
2.16
/
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
1.60
ꢀ
ꢀ/
/
17.8
15.5
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
1.59
1.32
DU-145
ꢀ/
Breast cancer
MCF7
2.07
3.13
ꢀ
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
1.56
1.59
ꢀ
ꢀ/
ꢀ/
ꢀ/
17.5
16.5
16.8
/
ꢀ
10.0
10.9
/
11.1
ꢀ
ꢀ/
ꢀ/
/
1.67
ꢀ
ꢀ/
ꢀ/
/
2.66
1.55
1.43
1.79
1.35
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.57
1.74
ꢀ/
ꢀ/
1.53
0.86
2.33
ꢀ/
ꢀ/
ꢀ/
1.25
1.29
ꢀ/
1.44
ꢀ/
ꢀ/
ꢀ/
1.02
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
MDA-MB-435
MDA-N
BT-549
1.94
1.86
1.95
2.21
ꢀ/
ꢀ/
ꢀ/
15.3
14.5
10.6
1.22
1.36
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.85
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
T-47D
2.20
4.47
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
1.17
a
Data obtained from NCI in vitro disease oriented tumor cell screen.
4. Experimental
calculated values. All reactions involving air or moist-
ure-sensitive compounds were performed under argon
atmosphere.
The general procedure for conversion to an HCl salt
was the addition of excess ethereal HCl solution to a
solution of the compound in chloroform or diethyl
ether. The solvent was evaporated and the resulting salt
was triturated with anhydrous ether and dried on
vacuum.
The general procedure for conversion to a fumarate
salt was the addition of a stoichiometric amount of a
solution of fumaric acid in dry methanol to a solution of
the compound in dry methanol. The solvent was
evaporated and the resulting salt was triturated with
anhydrous ether and dried on vacuum.
Unless otherwise specified, all materials, solvents,
reagents and precursors 12,13 were obtained from
commercial suppliers.
Melting points were obtained on an Electrothermal
IA 9100 digital melting point apparatus or on a Kofler
¨
melting point apparatus and are uncorrected. IR spectra
were recorded as thin films (for oils) or nujol mulls (for
solids) on NaCl plates with a Perkin Elmer 781 IR
spectrophotometer and are expressed in n (cmꢃ1). UVꢀ
Vis spectra were recorded as ethanolic solution with a
Perkin Elmer Lambda 5 spectrophotometer and the
absorption wavelengths are expressed as lmax in nm
followed by log o. All NMR spectra were taken on a
Varian XL-200 NMR spectrometer with 1H and 13C
being observed at 200 and 50 MHz respectively.
/
1
Chemical shifts for H and spectra were reported in d
or ppm downfield from TMS [(CH3)4Si]. Multiplicities
are recorded as s (singlet), br s (broad singlet), d
(doublet), t (triplet), q (quartet), qu (quintuplet), m
(multiplet). Elemental analyses were performed by
Laboratorio di Microanalisi, Dipartimento di Chimica,
Flash chromatography (FC) was performed using
Merck silica gel 60 (230ꢀ400 mesh ASTM). TLC was
/
Universita` di Sassari, Italy and are within 90.4% of the
/
Fig. 1. Viscometric titration of Poly(dA-dT)×poly(dA-dT) with 2f, EtBr and Dist.
/

756
G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ763
/
performed with Polygram† SIL N-HR/HV254 precoated
plastic sheets (0.2 mm).
(N1ꢀ
(C), 126.72 (C), 127.90 (CH), 128.95 (CH), 137.55 (C),
O). Anal. Calc. for
C15H16N2O2: C 70.29, H 6.29, N 10.93. Found: C 69.97,
H 6.57, N 10.76%.
/
CH3), 60.61 (CH2), 122.02 (C), 122.08 (CH), 126.45
137.74 (C), 139.48 (C), 162.72 (Cꢁ
/
4.1. General procedure for preparation of ethyl 2-oxo-2-
(1-oxo-1,2,3,4-tetrahydro-naphthalen-2-yl) acetate (14)
and ethyl 2-oxo-2-(1-oxo-2,3-dihydro-1H-inden-2-yl)
acetate (15)
20: Yield: 35%; oil; b.p. 94 8C (0.1 mmHg); Rf
(petroleum ether/ethyl acetate 8:2) 0.76; IR (film): 1715
(Cꢁ
(3.76); H NMR (CDCl3): 1.41 (t, 3H, Jꢂ
3.10 (m, 4H), 4.21 (s, 3H), 4.38 (q, 2H, Jꢂ
7.35 (m, 2H), 7.80ꢀ
7.85 (m, 2H). 13C NMR (CDCl3):
14.13 (CH3), 20.34 (CH2), 28.82 (CH2), 39.70 (N2-CH3),
60.61 (CH2), 121.88 (CH), 122.06 (C), 126.69 (CH),
127.48 (CH), 128.09 (CH), 128.69 (C), 135.98 (C),
/
O); UV (ethanol): lmax 226 (4.23), 243 (4.14), 282
1
/
7 Hz), 2.88ꢀ
/
Sodium metal (12 mmol) was added in small portion
to dry ethanol (5 ml) and stirred until all the sodium had
reacted. Ethyl oxalate (6 mmol) was added, follow by
dropwise addition of a solution of appropriate ketone
[12, 13] starting material (6 mmol) in dry ethanol (30
ml). The solution was stirred at room temperature for
/
7 Hz), 7.20ꢀ
/
/
136.22 (C), 146.68 (C), 160.33 (Cꢁ
/
O). Anal. Calc. for
2ꢀ8 h. The mixture was slowly poured over 2 N
/
C15H16N2O2: C 70.29, H 6.29, N 10.93. Found: C 70.12,
H 5.94, N 11.21%.
hydrochloride acid and the resulting precipitate was
collected by filtration and washed with a small volume
of ice-cooled ethanol and water. The air-dried residue
afforded the analytically pure product [14, 15].
4.2.3. Ethyl 1-phenyl-4,5-dihydro-1H-benzo[g]indazole-
3-carboxylate (18)
14: 98% yield; m.p. 43ꢀ
leum ether) ([12]: 44ꢀ45 8C).
15: 96% yield; m.p. 68ꢀ70 8C (triturated with petro-
leum ether) ([5]: 69ꢀ70 8C).
/
45 8C (triturated with petro-
Yield: 78%; yellowish solid; m.p. 157ꢀ
nol) ([13]: 163 8C from ethanol).
/
158 8C (etha-
/
/
/
4.2.4. Ethyl 1-(2,4-dichlorophenyl)-4,5-dihydro-1H-
benzo[g]indazole-3-carboxylate (19)
4.2. General procedure for preparation of dihydro-1H-
benzo[g]indazole-carboxylate 16ꢀ20 and dihydro-
indeno[1,2-c]pyrazole-carboxylates 26ꢀ30
Yield: 58%; yellowish solid; m.p. 131ꢀ
nol); Rf (petroleum ether/ethyl acetate 8:2) 0.59; IR
(nujol): 1710 (CꢁO); UV (ethanol): lmax 217 (4.44), 265
(4.05), 278 (4.02), 290 (3.91), 299 (3.98); 1H NMR
(CDCl3): 1.43 (t, 3H, Jꢂ7 Hz), 2.95ꢀ3.15 (m, 4H), 4.45
(q, 2H, Jꢂ7 Hz), 6.57 (d, 1H, Jꢂ7.2 Hz), 7.02 (t, 1H,
Jꢂ7.2 Hz), 7.19 (t, 1H, Jꢂ7.2 Hz), 7.28 (t, 1H, Jꢂ7.2
Hz), 7.40ꢀ7.60 (m, 3H). Anal. Calc. for C20H16Cl2N2O2:
/
132 8C (etha-
/
/
/
A stirred mixture of the appropriate diketoester (4
mmol) [14,15] and the requisite hydrazine hydrochloride
(4.6 mmol) in EtOH (28 ml) was heated under reflux for
/
/
/
/
/
/
/
2ꢀ5 h. The reaction was allowed to cool to room
/
/
temperature and the insoluble material was collected
by filtration and washed with a small volume of ice-
cooled ethanol. Purification by FC afforded the analy-
tically pure product.
C 62.03, H 4.16, Cl 18.31, N 7.23. Found: C 61.74, H
3.85, Cl 17.98, N 6.87%.
4.2.5. Ethyl 1,4-dihydro-indeno[1,2-c]pyrazole-3-
carboxylate (26)
4.2.1. Ethyl 4,5-dihydro-1H-benzo[g]indazole-3-
carboxylate (16)
Yield: 31%; yellowish solid; m.p. 168 8C (from
ethanol) ([13]: 174 8C from ethanol).
Yield: 78%; yellowish solid; m.p. 144ꢀ145 8C (tritu-
/
rated with ethyl acetate) ([13]: 152 8C from ethanol).
4.2.6. Ethyl 1-methyl-1,4-dihydro-indeno[1,2-
c]pyrazole-3-carboxylate (27) and ethyl 2-methyl-1,4-
dihydro-indeno[1,2-c]pyrazole-3-carboxylate (30)
2,4-Diketoester 15 was transformed to a mixture of
compounds 27 and 30 separated by FC eluting with
petroleum ether/ethyl acetate 8:2.
4.2.2. Ethyl 1-methyl-4,5-dihydro-1H-benzo[g]indazole-
3-carboxylate (17) and ethyl 2-methyl-4,5-dihydro-2H-
benzo[g]indazole-3-carboxylate (20)
2,4-Diketoester 14 was transformed to a mixture of
compounds 17 and 20 separated by FC eluting with
petroleum ether/ethyl acetate 8:2.
27: Yield: 55%; white solid; m.p. 178ꢀ
nol). Rf (petroleum ether/ethyl acetate 8:2) 0.23. IR
(nujol): 1730 (CꢁO). UV (ethanol): lmax 220 (4.13), 245
(4.12), 260 (4.05), 268 (3.93), 280 (3.91), 353 (3.33). H
NMR (CDCl3): 1.43 (t, 3H, Jꢂ7.2 Hz), 3.72 (s, 2H),
4.18 (s, 3H), 4.43 (q, 2H, Jꢂ7.2 Hz), 7.25ꢀ7.40 (m, 2H),
7.52ꢀ
7.56 (m, 2H). 13C NMR (CDCl3): 14.36 (CH3),
29.58 (CH2), 38.39 (N1ꢀCH3), 60.75 (CH2), 118.34
(CH), 126.35 (CH), 126.50 (CH), 126.75 (CH), 129.39
/
179 8C (etha-
17: Yield: 55%; white solid; m.p. 107ꢀ
(petroleum ether/ethyl acetate 8:2) 0.27; IR (nujol): 1710
O); UV (ethanol): lmax 252 (3.88), 260 (3.87), 270
/
108 8C; Rf
/
1
(Cꢁ
(3.74), 282 (3.69); H NMR (CDCl3): 1.43 (t, 3H, Jꢂ
Hz), 2.88ꢀ3.05 (m, 4H), 4.24 (s, 3H), 4.43 (q, 2H, Jꢂ
Hz), 7.25ꢀ7.40 (m, 2H), 7.55ꢀ
(CDCl3): 14.36 (CH3), 19.80 (CH2), 30.22 (CH2), 40.21
/
/
1
/
7
7
/
/
/
/
/
/
/
7.62 (m, 2H). 13C NMR
/

G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ
/763
757
(C), 131.38 (C), 136.73 (C), 148.84 (C), 150.10 (C),
160.03 (CꢁO). Anal. Calc. for C14H14N2O2: C 69.41, H
5.82, N 11.56. Found: C 69.47, H 5.62, N 11.47%.
30: Yield: 10%; white solid; m.p. 98ꢀ100 8C. Rf
(petroleum ether/ethyl acetate 8:2) 0.69. IR (nujol):
1710 (CꢁO). UV (ethanol): lmax 243 (4.18), 275 (3.65),
(4.36), 236 (4.28), 250 (3.98), 290 (3.62), 321 (3.59), 355
1
/
(3.62). H NMR (CDCl3): 1.51 (t, 3H, Jꢂ
/
7.0 Hz), 4.57
(q, 2H, Jꢂ7.0 Hz), 7.20ꢀ8.25 (m, 11H). Anal. Calc. for
/
/
/
C20H16N2O2: C 75.93, H 5.10, N 8.86. Found: C 75.69,
H 4.86, N 9.13%.
/
1
295 (3.72). H NMR (CDCl3): 1.43 (t, 3H, Jꢂ
3.77 (s, 2H), 4.27 (s, 3H), 4.39 (q, 2H, Jꢂ7.2 Hz), 7.25ꢀ
7.40 (m, 2H), 7.49ꢀ
7.76 (m, 2H). 13C NMR (CDCl3):
14.30 (CH3), 30.31 (CH2), 39.75 (N2ꢀCH3), 60.86 (CH2),
119.82 (CH), 125.68 (CH), 126.69 (CH), 127.01 (CH),
129.86 (C), 134.40 (C), 147.50 (2ꢄC), 157.68 (C),
O). Anal. Calc. for C14H14N2O2: C 69.41,
/7.2 Hz),
4.3.4. Ethyl 1-(2,4-dichlorophenyl)-1H-
benzo[g]indazole-3-carboxylate (24)
/
/
/
Yield: 54%; pink solid; m.p. 136ꢀ
0.54. IR (nujol): 1710 (CꢁO). UV (ethanol): lmax 219
(2.92), 241 (2.78), 276 (2.37), 306 (1.98), 320 (1.98). H
NMR (CDCl3): 1.52 (t, 3H, Jꢂ7.0 Hz), 4.58 (q, 2H,
Jꢂ7.0 Hz), 7.25ꢀ8.35 (m, 9H). Anal. Calc. for
/
137 8C. Rf (CH2Cl2)
/
/
1
/
/
160.03 (Cꢁ
/
/
/
H 5.82, N 11.56. Found: C 69.54, H 5.52, N 11.68%.
C20H14Cl2N2O2: C 62.36, H 3.66, Cl 18.41, N 7.27.
Found: C 62.62, H 3.86, Cl 18.56, N 7.58%.
4.2.7. Ethyl 1-phenyl-1,4-dihydro-indeno[1,2-c]pyrazole-
3-carboxylate (28)
Yield: 70%; yellowish solid; m.p. 111ꢀ
/
112 8C (etha-
4.3.5. Ethyl 2-methyl-2H-benzo[g]indazole-3-
carboxylate (25)
nol) ([14]: 110ꢀ112 8C from CH2Cl2/diisopropyl ether).
/
Yield: 95%; yellowish solid; m.p. 71ꢀ
roleum ether/ethyl acetate 8:2) 0.62. IR (nujol): 1710
(CꢁO). UV (ethanol): lmax 216 (3.95), 222 (3.96), 242
(3.56), 255 (3.58), 281 (3.49), 293 (3.53), 306 (3.33), 323
/
72 8C. Rf (pet-
4.2.8. Ethyl 1-(2,4-dichlorophenyl)-1,4-dihydro-
indeno[1,2-c]pyrazole-3-carboxylate (29)
Yield: 80%; yellowish solid; m.p. 165 8C (ethanol).
([15]: 165 8C triturated with ethyl acetate).
/
1
(3.23). H NMR (CDCl3): 1.52 (t, 3H, Jꢂ
(q, 2H, Jꢂ7.2 Hz), 4.53 (s, 3H), 7.50ꢀ7.68 (m, 3H),
7.80ꢀ7.95 (m, 2H), 8.55ꢀ8.65 (m, 1H). Anal. Calc. for
/
7.2 Hz), 4.50
/
/
4.3. General procedure for preparation of
/
/
benzo[g]indazole-carboxylates 21ꢀ25
/
C15H14N2O2: C 70.85, H 5.55, N 11.02. Found: C 70.99,
H 5.86, N 11.38%.
A stirred mixture of the appropriate ester (1.0 mmol)
[16ꢀ20] and of DDQ (4.2 mmol) in CH2Cl2 (8 ml) was
/
heated under reflux for 12 h. The reaction was allowed
to cool to room temperature, taken up with CH2Cl2,
washed with a 3% NH4OH aqueous solution, dried
(Na2SO4) and concentrated in vacuum. The residue was
purified by FC affording the analytically pure product.
4.4. General procedure for preparation of
benzo[g]indazole-carboxylic acids 3ꢀ
indeno[1,2-c]pyrazole-carboxylic acids 8ꢀ
/
7 and dihydro-
11
/
To appropriate ester (5 mmol) [21ꢀ29] in methanol
/
(25 ml) was added a solution of potassium hydroxide (10
mmol) in methanol (18 ml) and some drops of water.
The resulting mixture was heated under reflux over-
night. The mixture was allowed to cool to room
temperature and then poured into water and acidified
with 1 N hydrochloric acid. The precipitate was filtered,
washed with water and air-dried to yield the analytically
pure acid.
4.3.1. Ethyl 1H-benzo[g]indazole-3-carboxylate (21)
202 8C ([16]:
211 8C).
Yield: 82%; yellowish solid: M.p. 201ꢀ
/
4.3.2. Ethyl 1-methyl-1H-benzo[g]indazole-3-
carboxylate (22)
Yield: 91%; whiteꢀ
(petroleum ether/ethyl acetate 8:2) 0.24. IR (nujol): 1700
(CꢁO). UV (ethanol): lmax 223 (4.01), 254 (3.53), 267
(3.46), 278 (3.49), 303 (3.09), 319 (3.14). 1H NMR
(CDCl3): 1.51 (t, 3H, Jꢂ7.2 Hz), 4.55 (q, 2H, Jꢂ7.2
Hz), 4.63 (s, 3H), 7.60ꢀ7.75 (m, 2H), 7.99ꢀ8.05 (m, 2H),
8.20 (m, 1H), 8.40ꢀ8.50 (m, 1H). Anal. Calc. for
/
cream solid; m.p. 113ꢀ114 8C. Rf
/
/
4.4.1. 1H-Benzo[g]indazole-3-carboxylic acid (3)
/
/
Yield: 88%; white solid; m.p. 300ꢀ
MeOH 7:3) 0.30. IR (nujol): 1690 (Cꢁ
(NH), 3300ꢀ3500 (OH). UV (ethanol): lmax 210 (4.22),
/
301 8C. Rf (CHCl3/
/
/
/
O), 3100ꢀ3200
/
/
/
C15H14N2O2: C 70.85, H 5.55, N 11.02. Found: C
70.79, H 5.86, N 11.38%.
236 (4.45), 268 (4.17), 279 (4.03), 291 (4.01), 302 (3.66),
316 (3.71), 331 (3.73). 1H NMR (CDCl3): 4.87 (br s, 2H,
OH and NH exch. with D2O), 7.54ꢀ
(dd, 1H, Jꢂ2.2 and 6.4 Hz), 8.14 (d, 1H, Jꢂ
8.53 (dd, 1H, Jꢂ2.0 and 7.6 Hz). Anal. Calc. for
/
7.62 (m, 3H), 7.94
4.3.3. Ethyl 1-phenyl-1H-benzo[g]indazole-3-
carboxylate (23)
/
/9.2 Hz),
/
Yield: 86%; pink solid; m.p. 106ꢀ
/
107 8C. Rf (CH2Cl2)
C12H8N2O2: C 69.02, H 3.80, N 13.20. Found: C 68.84,
H 3.52, N 13.14%.
0.41. IR (nujol): 1710 (CꢁO). UV (ethanol): lmax 219
/

758
G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ763
/
4.4.2. 1-Methyl-1H-benzo[g]indazole-3-carboxylic acid
(4)
Anal. Calc. for C11H8N2O2: C 66.00, H 4.03, N 12.99.
Found: C 66.35, H 4.23, N 12.84%.
Yield: 79%; white solid; m.p. 261ꢀ
/
263 8C. Rf (CHCl3/
O), 3300ꢀ3500
(OH). UV (ethanol): lmax 223 (4.35), 267 (3.79), 278
MeOH 7:3) 0.67. IR (nujol): 1680 (Cꢁ
/
/
4.4.7. 1-Methyl-1,4-dihydro-indeno[1,2-c]pyrazole-3-
carboxylic acid (9)
(3.80), 304 (3.46), 319 (3.50). 1H NMR (CDCl3): 4.62 (br
Yield: 84%; yellowish solid; m.p. 198ꢀ
/
200 8C. Rf
s, 3H and OH exch. with D2O), 7.60ꢀ
(d, 1H, Jꢂ7.8 Hz), 8.19 (d, 1H, Jꢂ
1H, Jꢂ7.8 Hz). Anal. Calc. for C13H10N2O2: C 69.02,
/
7.75 (m, 3H), 8.01
(CHCl3/MeOH 7:3) 0.58. IR (nujol): 1680 (Cꢁ
/O), 3330
/
/
8.8 Hz), 8.49 (d,
(OH). UV (ethanol): lmax 217 (4.08), 246 (4.07), 248
(4.04), 271 (3.90), 279 (3.87), 333 (3.55). 1H NMR
(CDCl3, DMSO): 3.74 (br s, 2H and OH exch. with
/
H 4.46, N 12.38. Found: C 69.32, H 4.21, N 12.56%.
D2O), 4.20 (s, 3H), 7.25ꢀ
2H). Anal. Calc. for C12H10N2O2: C 67.28, H 4.71, N
13.08. Found: C 67.45, H 4.51, N 13.34%.
/
7.45 (m, 2H), 7.52ꢀ7.58 (m,
/
4.4.3. 2-Methyl-2H-benzo[g]indazole-3-carboxylic acid
(7)
Yield: 94%; white solid; m.p. 238ꢀ
/
239 8C. Rf (CHCl3/
O), 3300ꢀ3500
MeOH 7:3) 0.61. IR (nujol): 1680 (Cꢁ
/
/
(OH). UV (ethanol): lmax 215 (4.29), 221 (4.31), 246
(4.04), 288 (3.92), 306 (3.69), 321 (3.61). 1H NMR
(CDCl3, DMSO): 4.53 (br s, 3H and OH exch. with
4.4.8. 1-Phenyl-1,4-dihydro-indeno[1,2-c]pyrazole-3-
carboxylic acid (10)
Yield: 78%; yellowish solid; m.p. 249ꢀ
/
250 8C. ([5]:
D2O), 7.50ꢀ
/
7.65 (m, 3H), 7.84 (d, 1H, Jꢂ/7.6 Hz), 8.00
250 8C).
(d, 1H, Jꢂ9.2 Hz), 8.57 (d, 1H, Jꢂ
/
/
7.6 Hz). Anal. Calc.
for C13H10N2O2: C 69.02, H 4.46, N 12.38. Found: C
69.35, H 4.75, N 12.53%.
4.4.9. 1-(2,4-Dichlorophenyl)-1,4-dihydro-indeno[1,2-
c]pyrazole-3-carboxylic acid (11)
Yield: 89%; yellowish solid; m.p. 271ꢀ
/
272 8C. ([15]:
4.4.4. 1-Phenyl-1H-benzo[g]indazole-3-carboxylic acid
(5)
271ꢀ272 8C).
/
Yield: 98%; white solid; m.p. 277ꢀ
/
278 8C. Rf (CHCl3/
O), 3200ꢀ3500
(OH). UV (ethanol): lmax 224 (3.66), 239 (3.65), 276
MeOH 7:3) 0.76. IR (nujol): 1690 (Cꢁ
/
/
4.5. General procedure for preparation of 1H-
Benzo[g]indazole-carboxamides 2aꢀj and 1,4-
dihydroindeno[1,2-c]pyrazole-carboxamides 2kꢀ
/
(3.21), 304 (2.86), 319 (2.91). 1H NMR (CDCl3,
/
r
DMSO): 4.11 (br s, 1H, OH exch. with D2O), 7.34ꢀ
7.60 (m, 3H), 7.61ꢀ7.65 (m, 5H), 8.01 (d, 2H, jꢂ7.8
Hz), 8.27 (d, 1H, Jꢂ8.8 Hz). Anal. Calc. for
/
/
/
A mixture of the appropriate acid (1 mmol) [3ꢀ11]
/
/
and CDI (1.1 mmol) in DMF (2.5 ml) was stirred at r.t.
for 3 h. The reaction mixture was added of the requisite
amine [(N,N-bis(2-aminoethyl)methylamine or N,N-
bis(3-aminopropyl)methylamine)] (0.5 mmol) in DMF
(2 ml). The mixture was stirred at r.t. for 48 h, the
solvent was then removed under reduced pressure and
the residue purified as reported below to give the title
compounds.
C18H12N2O2: C 74.99, H 4.20, N 9.72. Found: C
74.82, H 4.36, N 9.84%.
4.4.5. 1-(2,4-Dichlorophenyl)-1H-benzo[g]indazole-3-
carboxylic acid (6)
Yield: 85%; yellowish solid; m.p. 268ꢀ
/
269 8C. Rf
O),
(CHCl3/MeOH 7:3) 0.76. IR (nujol): 1680 (Cꢁ
/
3300ꢀ3500 (OH). UV (ethanol): lmax 220 (4.49), 236
/
(4.41), 254 (4.35), 319 (3.54), 336 (3.59). 1H NMR
(CDCl3, DMSO): 3.46 (br s, 1H, OH exch. with D2O),
4.5.1. N3-{2-[{2-[(1H-Benzo[g]indazole-3-yl-
carbonyl)amino]ethyl}(methyl)amino]ethyl}-1H-
benzo[g]indazole-3-carboxamide (2a)
7.28ꢀ
/
7.45 (m, 3H), 7.52ꢀ
/
7.62 (m, 2H), 7.72 (d, 2H, Jꢂ
/
8.6 Hz), 8.00 (d, 1H, Jꢂ
/
8.4), 8.32 (d, 1H, Jꢂ8.8 Hz).
/
Anal. Calc. for C18H10Cl2N2O2: C 60.53, H 2.82, Cl
19.85, N 7.84. Found: C 60.85, H 2.65, Cl 19.53, N
7.56%.
Yield: 72% as an oily residue which solidified on
standing after purification by FC eluting with CHCl3/
CH3OH 9/1; yellowish solid; m.p. 149ꢀ
(CHCl3/MeOH 9:1) 0.28. IR (nujol): 1630 (Cꢁ
3120ꢀ3200 (NH), 3380ꢀ3400 (NH). UV (ethanol):
/
150 8C. Rf
/
O),
4.4.6. 1,4-Dihydro-1H-indeno[1,2-c]pyrazole-3-
/
/
carboxylic acid (8)
Yield: 98%; white-cream solid; m.p. 317ꢀ
lmax 235 (4.02), 260 (3.65), 268 (3.63), 280 (3.48), 291
(3.46), 316 (3.10), 330 (3.16). 1H NMR (CDCl3,
/
319 8C. Rf
(CHCl3/MeOH 7:3) 0.24. IR (nujol): 1720 (Cꢁ
/
O), 3380
DMSO): 2.42 (s, 3H), 2.75 (t, 4H), 3.55ꢀ
/
3.72 (m, 4H),
8.45 (m,
(NH). UV (ethanol): lmax 216 (4.04), 252 (3.83), 269
(3.70), 276 (3.67). 1H NMR (CDCl3, DMSO): 3.74 (br s,
5.50ꢀ5.90 (br s, 2H, NH exch. with D2O), 7.45ꢀ
/
/
12H and 2NH exch. with D2O). Anal. Calc. for
C29H27N7O2: C 68.69, H 5.38, N 19.39. Found: C
68.72, H 5.52, N 19.63%.
2H and OH and NH exch. with D2O), 7.25ꢀ
/
7.42 (m,
2H), 7.55 (d, 1H, Jꢂ6.8 Hz), 7.67 (d, 1H, Jꢂ
/
/6.8 Hz).

G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ
/763
759
1
4.5.2. N3-{3-[{3-[(1H-Benzo[g] indazole-3-yl-
carbonyl)amino]propyl}(methyl)amino]propyl}-1H-
benzo[g]indazole-3-carboxamide (2b)
285 (4.18), 308 (3.93), 319 (3.81). H NMR (CDCl3):
2.46 (s, 3H), 2.77 (q, 4H, Jꢂ5.6 Hz), 3.62 (q, 4H, Jꢂ
5.6 Hz), 4.13 (s, 6H), 6.70ꢀ6.85 (br s, 2H, NH exch. with
D2O), 7.10ꢀ7.18 (m, 4H), 7.40ꢀ7.60 (m, 6H), 8.25ꢀ8.35
(m, 2H). Anal. Calc. for C31H31N7O2×2C4H4O4×H2O: C
/
/
/
Yield: 27% after purification by trituration with
/
/
/
acetone; white solid; m.p. 230ꢀ
/
232 8C. Rf (CHCl3/
/
/
MeOH 9:1) 0.17. IR (nujol): 1640 (Cꢁ
/
O), 3100ꢀ3160
/
59.46, H 5.76, N 12.45. Found: C 60.07, H 5.74, N
(NH), 3360 (NH). UV (ethanol): lmax 236 (4.34), 248
(4.01), 268 (3.97), 280 (3.80), 291 (3.79), 316 (3.44), 331
12.41%.
1
(3.48). H NMR (CDCl3, DMSO): 1.82ꢀ
/
2.02 (qu., 4H),
3.40 (br s, 4H, NH exch.
3.70 (m, 4H), 7.45ꢀ8.65 (m, 10H and
4.5.6. N3-{3-[{3-[(2-Methyl-2H-benzo[g]indazole-3-
yl-carbonyl)amino]propyl}(methyl) amino]propyl}2-
methyl-2H-benzo[g]indazole-3-carboxamide (2f)
Yield: 30% as an oily residue which solidified on
standing after purification by FC eluting with CHCl3/
CH3OH 9/1; yellowish solid; m.p. 117ꢀ
rated with ethyl ether). Rf (CHCl3/MeOH 9:1) 0.30. IR
(nujol): 1660 (CꢁO), 3300 (NH). UV (ethanol): lmax 239
(4.67), 248 (4.50), 299 (4.32). 319 (4.06), 334 (3.96). H
NMR (CDCl3): 1.70ꢀ1.89 (m, 4H), 2.33 (s, 3H), 2.45ꢀ
2.65 (m, 2H), 3.45ꢀ3.60 (m, 4H), 4.30ꢀ4.15 (br s, 2H,
NH exch. with D2O), 4.30 (s, 6H), 7.25ꢀ8.50 (m, 14H),
2.29 (s, 3H), 2.57 (t, 4H), 3.10ꢀ
/
with D2O), 3.50ꢀ
/
/
2NH exch. with D2O). Anal. Calc. for C31H31N7O2: C
69.77, H 5.86, N 18.37. Found: C 69.84, H 5.63, N
18.56%.
/
118 8C (tritu-
4.5.3. N3-{2-[{2-[(1-Methyl-1H-benzo[g]indazole-3-
yl-carbonyl)amino]ethyl}(methyl)amino]ethyl}-1-
methyl-1H-benzo[g]indazole-3-carboxamide (2c)
Yield: 70% as an oily residue which solidified on
standing after purification by FC eluting with CHCl3/
/
1
/
/
/
/
/
CH3OH 9/1; white solid; m.p. 120ꢀ
with ethyl ether). Rf (CHCl3/MeOH 9:1) 0.50. IR
O), 3100ꢀ3400 (NH). UV (ethanol):
/
122 8C (triturated
10.51 (br s, 2H, NH exch. with D2O). Anal. Calc. for
C33H35N7O2: C 70.57, H 6.28, N 17.46. Found: C 70.46,
H 6.59, N 17.63%.
(nujol): 1650 (Cꢁ
/
/
lmax 221 (4.59), 254 (4.12), 269 (4.00), 280 (4.02), 304
1
(3.65), 320 (3.67). H NMR (CDCl3): 2.48 (s, 3H), 2.75
(t, 4H), 3.65 (q, 4H), 3.98 (s, 6H), 7.11 (br s, 2H, NH
4.5.7. N3-{2-[{2-[(1-Phenyl-1H-benzo[g]indazole-3-yl-
carbonyl)amino]ethyl}(methyl) amino]ethyl}1-phenyl-
1H-benzo[g]indazole-3-carboxamide (2g)
exch. with D2O), 7.25ꢀ8.35 (m, 1H). Anal. Calc. for
/
C31H31N7O2: C 69.77, H 5.86, N 18.37. Found: C 69.83,
H 5.67, N 18.53%.
Yield: 35% as an oily residue after purification by FC
eluting with CHCl3/CH3OH 9/1; yellowish oil; m.p.
118ꢀ
/
120 8C (as hydrochloride). Rf (CHCl3/MeOH 9:1)
4.5.4. N3-{3-[{3-[(1-Methyl-1H-benzo[g] indazole-3-
yl-carbonyl)amino]propyl}(methyl)amino]propyl}1-
methyl-1H-benzo[g]indazole-3-carboxamide[(E)-3-
carboxy-2-propenoate] (2d)
Yield: 53% as an oily residue which solidified on
standing after purification by FC eluting with CHCl3/
0.31. IR (nujol): 1630 (Cꢁ
/
O), 3300ꢀ3400 (NH). UV
/
(ethanol): lmax 220 (4.57), 244 (4.47), 254 (4.41), 279
(4.16), 306 (3.77), 314 (3.57), 320 (3.77). 1H NMR
(CDCl3): 2.40 (s, 3H), 2.73 (t, 4H, Jꢂ
(m, 4H, Jꢂ6.4 Hz), 7.20ꢀ7.50 (m, 16H and 2NH exch.
with D2O), 7.65 (d, 2H, Jꢂ8.8 Hz), 7.88 (d, 2H, Jꢂ8.2
Hz), 8.39 (d, 2H, Jꢂ8.8 Hz). Anal. Calc. for
H2O: C 65.77, H 5.25, Cl 9.47, N
/
6.4 Hz), 3.51ꢀ/3.69
/
/
/
/
CH3OH 8/2; white solid; m.p. 124ꢀ
Rf (CHCl3/MeOH 8:2) 0.47. IR (nujol): 1650 (Cꢁ
3300ꢀ3500 (NH). UV (ethanol): lmax 213 (4.08), 237
(4.33), 260 (3.93), 268 (3.85), 283 (2.87), 295 (3.23), 319
/
125 8C (as fumarate).
/
/O),
C41H35N7O2×
/
2HCl×
/
/
13.10. Found: C 65.87, H 5.09, Cl 9.32, N 12.81%.
1
(3.35), 333 (3.57). H NMR (CDCl3): 1.93 (qu 4H, Jꢂ
6.4 Hz), 2.33 (s, 3H), 2.59 (t, 2H, Jꢂ6.4 Hz), 3.64 (q,
4H, Jꢂ6.4 Hz), 4.39 (s, 6H), 6.68 (br s, 2H, NH exch.
with D2O), 7.05ꢀ8.35 (m, 14H). Anal. Calc. for
2C4H4O4: C 61.72, H 5.94, N 12.29.
/
4.5.8. N3-{3-[{3-[(1-Phenyl-1H-benzo[g]indazole-3-yl-
carbonyl)amino]propyl}(methyl) amino]propyl}1-
phenyl-1H-benzo[g]indazole-3-carboxamide (2h)
Yield: 20% as an oily residue after purification by FC
eluting with CHCl3/CH3OH 9/1; yellowish oil; m.p.
/
/
/
C33H35N7O2×
/
Found: C 61.69, H 6.06, N 12.64%.
124ꢀ
/
126 8C (as hydrochloride). Rf (CHCl3/MeOH 9:1)
0.41. IR (nujol): 1660 (Cꢁ
/
O), 3300ꢀ3400 (NH). UV
/
4.5.5. N3-{2-[{2-[(2-Methyl-2H-benzo[g]indazole-3-
yl-carbonyl)amino]ethyl}(methyl)amino]ethyl}2-
methyl-2H-benzo[g]indazole-3-carboxamide[(E)-3-
carboxy-2-propenoate] (2e)
Yield: 42% as an oily residue which solidified on
standing after purification by FC eluting with CHCl3/
(ethanol): lmax 212 (4.48), 223 (4.45), 242 (4.40), 253
(4.37), 273 (4.10), 307 (3.61), 320 (3.63). 1H NMR
(CDCl3): 1.79 (qu, 4H, Jꢂ
4H, Jꢂ6.4 Hz), 3.46ꢀ3.58 (m, 4H, Jꢂ
7.31 (m, 2H), 7.45ꢀ7.64 (m, 16H and 2NH exch. with
D2O), 7.83ꢀ7.95 (m, 2H), 8.42 (d, 2H,, Jꢂ9.0 Hz).
Anal. Calc. for C43H39N7O2×3HCl×3H2O: C 60.81, H
/6.4 Hz), 2.21 (s, 3H), 2.50 (t,
/
/
/
6.4 Hz), 7.21ꢀ
/
/
/
/
CH3OH 9/1; white solid; m.p. 133ꢀ
Rf (CHCl3/MeOH 9:1) 0.51. IR (nujol): 1650 (Cꢁ
3260 (NH). UV (ethanol): lmax 223 (4.55), 242 (4.36),
/
134 8C (as fumarate).
/
/
/O),
5.70, Cl 12.52, N 11.55. Found: C 60.54, H 5.47, Cl
12.35, N 11.25%.

760
G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ763
/
4.5.9. N3-{2-[2-{[{1-(2,4-Dichlorophenyl)-1H-
benzo[g]indazole-3-yl-
CH3OH 9/1; white solid. m.p. 166ꢀ
with ethyl ether). Rf (CHCl3/MeOH 9:1) 0.22. IR
O), 3100ꢀ3200 (NH), 3400 (NH). UV
(ethanol): lmax 228 (3.68), 242 (3.66), 254 (3.60), 271
1
(3.40), 278 (3.35). H NMR (CDCl3, DMSO): 1.88 (qu,
/
167 8C (triturated
carbonyl}amino]ethyl}(methyl)amino]ethyl}1-(2,4-
dichlorophenyl)-1H-benzo[g]indazole-3-carboxamide
(2i)
(nujol): 1640 (Cꢁ
/
/
Yield: 28% as an oily residue after purification by FC
eluting with CHCl3/CH3OH 9/1; yellowish oil; m.p.
4H, Jꢂ
Hz), 2.64ꢀ
3.69 (m, 4H, Jꢂ
and 2NH exch. with D2O), 7.51 (d, 2H, Jꢂ
(d, 2H, J ꢂ6.2 Hz). Anal. Calc. for C29H31N7O2: C
/
5.4 Hz), 2.30 (s, 3H), 2.50ꢀ
2.78 (br s, 2H, NH exch. with D2O), 3.51ꢀ
5.4 Hz), 3.82 (s, 4H), 7.20ꢀ7.40 (m, 4H
6.8 Hz), 7.70
/
2.63 (m, 4H, Jꢂ5.4
/
/
/
149ꢀ
/
152 8C (as hydrochloride). Rf (CHCl3/MeOH 9:1)
/
/
0.33. IR (nujol): 1660 (Cꢁ
/
O), 3300ꢀ3420 (NH). UV
/
/
(ethanol): lmax 202 (4.82), 233 (4.62), 254 (4.53), 276
(4.26), 305 (3.87), 320 (3.89). 1H NMR (CDCl3): 2.40 (s,
/
68.35, H 6.13, N 19.24. Found: C 68.47, H 6.35, N
19.52%.
3H), 2.70ꢀ
Jꢂ6 Hz), 7.17ꢀ
D2O), 7.87ꢀ7.97 (m, 1H), 8.35ꢀ
Calc. for C41H31Cl4N7O2×2HCl×H2O: C 55.55, H 3.98,
/
2.77 (m, 4H, Jꢂ
7.68 (m, 16H and 2NH exch. with
8.42 (m, 1H). Anal.
/
6 Hz), 3.51ꢀ
/
3.71 (m, 4H,
/
/
/
/
4.5.13. N3-{2-[{2-[(1-Methyl-1,4-dihydroindeno[1,2-
c]pyrazole-3-yl-
/
/
Cl 24.00, N 11.06. Found: C 55.39, H 3.64, Cl 23.98, N
11.04%.
carbonyl)amino]ethyl}(methyl)amino]ethyl}-1-methyl-
1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide (2m)
Yield: 17% as an oily residue which solidified on
standing after purification by FC eluting with CHCl3/
4.5.10. N3-{3-[3-{[{1-(2,4-Dichlorophenyl)-1H-
benzo[g]indazole-3-yl-
CH3OH 8/2; white-cream solid; m.p. 153ꢀ
rated with ethyl ether). Rf (CHCl3/MeOH 9:1) 0.47. IR
(nujol): 1670 (CꢁO), 3400 (NH). UV (ethanol): lmax 239
(4.36), 263 (4.39), 274 (4.37), 284 (4.18), 295 (4.08). H
NMR (CDCl3): 2.41 (s, 3H), 2.69 (t, 4H, Jꢂ6 Hz), 3.57
(q, 4H, Jꢂ6 Hz), 3.69 (s, 4H), 3.93 (s, 6H); 7.15ꢀ7.55
/
155 8C (tritu-
carbonyl}amino]propyl}(methyl)amino]propyl}1-(2,4-
dichlorophenyl)-1H-benzo[g]indazole-3-carboxamide
(2j)
Yield: 52% as an oily residue after purification by FC
eluting with CHCl3/CH3OH 9/1; yellowish oil. M.p.
/
1
/
/
/
156ꢀ
0.33. IR (nujol): 1660 (Cꢁ
(ethanol): lmax 234 (4.88), 254 (4.80), 274 (4.53), 290
/
157 8C (as hydrochloride). Rf (CHCl3/MeOH 9:1)
(m, 8H and 2NH exch. with D2O). Anal. Calc. for
C29H31N7O2: C 68.35, H 6.13, N 19.24. Found: C 68.52,
H 6.41, N 19.51%.
/
O), 3280ꢀ3420 (NH). UV
/
1
(4.02), 305 (4.18), 320 (4.23). H NMR (CDCl3): 1.70ꢀ
1.95 (m, 4H, Jꢂ5.2 Hz), 2.21 (s, 3H), 2.39ꢀ2.60 (t, 4H,
Jꢂ5.2 Hz), 3.40ꢀ3.60 (m, 4H, Jꢂ5.2 Hz), 7.19ꢀ7.70
(m, 16H and 2NH exch. with D2O), 7.87ꢀ8.07 (m, 1H),
8.32ꢀ8.43 (m, 1H). Anal. Calc. for C45H35Cl4N7O2×
2HCl×2H2O: C 55.38, H 4.43, Cl 22.81, N 10.51. Found
/
/
/
4.5.14. N3-{3-[{3-[(1-Methy-1,4-dihydroindeno[1,2-
c]pyrazole-3-yl-carbonyl)
/
/
/
/
/
amino]propyl}methylamino]propyl}1-methyl-1,4-
dihydroindeno[1,2-c]pyrazole-3-carboxamide[(E)-3-
carboxy-2-propenoate] (2n)
Yield: 62% as an oily residue after purification by FC
eluting with CHCl3/CH3OH 9/1; yellowish oil: m.p.
/
/
/
C 55.35, H 4.40, Cl 22.78, N 10.48%.
4.5.11. N3-{2-[{2-[(1,4-Dihydroindeno[1,2-c]pyrazole-
3-yl-carbonyl)amino]ethyl}(methyl)amino]ethyl}-1,4-
dihydroindeno[1,2-c]pyrazole-3-carboxamide (2k)
Yield: 33% as an oily residue which solidified on
standing after purification by FC eluting with CHCl3/
107ꢀ
IR (nujol): 1670 (Cꢁ
246 (4.71), 258 (4.69), 278 (4.59). H NMR (CDCl3):
1.85 (qu, 4H, Jꢂ6.4 Hz), 2.28 (s, 3H), 2.52 (t, 4H, Jꢂ
6.4 Hz), 3.55 (q, 4H, Jꢂ6.4 Hz), 3.68 (s, 4H), 4.08 (s,
6H), 7.25ꢀ7.69 (m, 8H and 2NH exch. with D2O). Anal.
H2O: C 62.39, H 6.43, N
/
108 8C (as fumarate). Rf (CHCl3/MeOH 8:2) 0.41.
/
O), 3400 (NH). UV (ethanol): lmax
1
/
/
/
CH3OH 9/1; yellowish solid; m.p. 207ꢀ
rated with ethyl ether). Rf (CHCl3/MeOH 9:1) 0.26. IR
(nujol): 1630 (CꢁO), 3120 (NH), 3400 (NH). UV
(ethanol): lmax 230 (4.00), 254 (3.91), 270 (3.74), 278
/
208 8C (tritu-
/
Calc. for C31H35N7O2×
/
C4H4O4×
/
/
14.55. Found: C 62.36, H 6.41, N 14.51%.
1
(3.69). H NMR (CDCl3): 2.37 (s, 3H), 2.65ꢀ
/2.72 (m,
4.5.15. N3-{2-[{2-[(1-Phenyl-1,4-dihydroindeno[1,2-
c]pyrazole-3-yl-
4H), 3.49ꢀ3.60 (m, 4H), 3.71 (s, 4H), 7.18ꢀ7.65 (m, 10H
/
/
and 2NH exch. with D2O).). Anal. Calc. for
C27H27N7O2: C 67.34, H 5.60, N 20.36. Found: C
67.68, H 5.62, N 20.56%.
carbonyl)amino]ethyl}(methyl)amino]ethyl}1-phenyl-
1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide (2o)
Yield: 20% as an oily residue which solidified on
standing after purification by FC eluting with CHCl3/
4.5.12. N3-{3-[{3-[(1,4-Dihydroindeno[1,2-c]pyrazole-
3-yl-carbonyl)amino]propyl}(methyl)amino]propyl}-
1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide (2l)
Yield: 25% as an oily residue which solidified on
standing after purification by FC eluting with CHCl3/
CH3OH 9/1; white solid; m.p. 135ꢀ
with ethyl ether). Rf (CHCl3/MeOH 9:1) 0.75. IR
(nujol): 1670 (CꢁO), 3380 (NH). UV (ethanol): lmax
256 (3.35), 275 (3.33), 298 (3.67). H NMR (CDCl3):
2.40 (s, 3H), 2.73 (t, 4H, Jꢂ6.2 Hz), 3.60 (q, 4H, Jꢂ6.2
/
139 8C (triturated
/
1
/
/

G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ
/763
761
Hz), 3.61 (s, 4H), 7.15ꢀ
with D2O). Anal. Calc. for C39H35N7O2: C 73.91, H
5.57, N 15.47. Found: C 73.85, H 5.42, N 15.23%.
/
7.60 (m, 18H and 2NH exch.
Cl 17.74, N 12.26. Found: C 61.87, H 4.12, Cl 17.53, N
12.45%.
4.6. Biology
4.5.16. N3-{3-[{3-[(1-Phenyl-1,4-dihydroindeno[1,2-
c]pyrazole-3-yl-
4.6.1. In vitro cytotoxicity assay
The cellular response to drugs was evaluated utilizing
the solforhodamine B assay as described [7,17]. Briefly,
the human tumor cell lines making up the NCI cancer
screening panel were routinely grown in RPMI 1640
carbonyl)amino]propyl}(methyl)amino]propyl}1-
phenyl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide
(2p)
Yield: 56% as an oily residue which solidified on
standing after purification by FC eluting with CHCl3/
medium containing 5% fetal bovine serum and 2 mM L-
glutamine. Cells were inoculated into 96-well microtiter
plates in 100 ml of complete medium at densities ranging
from 5000 to 40 000 cells/well. The microtiter plates
containing cells were incubated for 24 h prior to the
addition of experimental drugs. Following the addition
of drugs, the plates were incubated for an additional 48
h, and cells were fixed with TCA, washed, and stained
with sulforhodamine B (Sigma Chemical Co., St. Louis,
MO) at 0.4% (w/v) in 1% acetic acid. After washing with
1% acetic acid, the stain was solubilized with 10 mM
unbuffered Tris base and the absorbance was measured
CH3OH 8/2; yellowish solid; m.p. 85ꢀ
with ethyl ether). Rf (CHCl3/MeOH 8:2) 0.79. IR
(nujol): 1660 (CꢁO), 3400 (NH). UV (ethanol): lmax
235 (3.79), 248 (3.81), 281 (3.63). H NMR (CDCl3):
1.82 (qu, 4H, Jꢂ6.4 Hz), 2.28 (s, 3H), 2.52 (t, 4H, Jꢂ
6.4 Hz), 3.55 (q, 4H, Jꢂ6.4 Hz), 3.68 (s, 4H), 7.20ꢀ7.30
(m, 4H), 7.38ꢀ7.48 (m, 2H and 2NH exch. with D2O),
7.50ꢀ7.55 (m, 6H), 7.65ꢀ7.75 (m, 6H). Anal. Calc. for
/
88 8C (triturated
/
1
/
/
/
/
/
/
/
C41H39N7O2: C 74.41, H 5.94, N 14.82. Found: C 74.52,
H 5.64, N 14.56%.
on a Bio-Tek microplate reader. Doseꢀresponse para-
meters were calculated as reported [18].
/
4.5.17. N3-{2-[{2-[{1-(2,4-Dichlorophenyl)-1,4-
dihydroindeno[1,2-c]pyrazole-3-yl-
carbonyl}amino]ethyl}(methyl)amino]ethyl}1-(2,4-
dichlorophenyl)-1,4-dihydroindeno[1,2-c] pyrazole-3-
carboxamide (2q)
Yield: 37% as an oily residue which solified on
standing after purification by FC eluting with CHCl3/
4.6.2. Hollow fiber assay
The cell lines are cultived in RPMI-1640 containing
10% FBS and 2 mM glutamine. On the day preceding
hollow fiber preparation, the cells are given a supple-
mentation of fresh medium to maintain log phase
growth. For fiber preparation, the cells are harvested
by standard trypsinization technique and resuspended at
the desired cell density (varies by cell line between 2 and
CH3OH 8/2; yellowish solid; m.p. 153ꢀ
rated with ethyl ether). Rf (CHCl3/MeOH 8:2) 0.70 IR
(nujol): 1660 (CꢁO), 3420 (NH). UV (ethanol): lmax 230
/
154 8C (tritu-
/
1
10ꢄ
106 cells mlꢃ1). The cell suspension is flushed into
/
(3.92), 280 (3.72). H NMR (CDCl3): 2.38 (s, 3H), 2.70
(t, 4H, Jꢂ5.8 Hz), 3.56 (q, 4H, Jꢂ5.8 Hz), 3.84 (s, 4H),
6.96 (d, 2H, Jꢂ7.0 Hz), 7.18ꢀ7.36 (m, 8H), 7.40ꢀ7.55
/
/
1 mm ID polyvinylidene hollow fibers with a molecular
weight exclusion of 500 000 Da. The hollow fibers are
heat-sealed at 2 cm intervals and the samples generated
from these seals are placed into tissue culture medium
/
/
/
(m, 4H and 2NH exch. with D2O). Anal. Calc. for
C39H31Cl4N7O2: C 60.71, H 4.05, Cl 18.38, N 12.71.
Found: C 60.45, H 4.23, Cl 18.38, N 12.63%.
and incubated at 37 8C in 5% CO2 for 24ꢀ48 h prior to
/
implantation. A total different tumor lines are prepared
for each experiment so that each mouse receives 3 IP
implants (1 of each tumor line) and 3 SC implants (1 of
each tumor line). One the day of implantation, samples
of each tumor cell line are quantitated for viable cell
mass by a stable endpoint MTT assay so that time 0 cell
mass is known. Thus, the cytostatic and cytocidal
capacities of the test compound can be assessed. Mice
are treated with experimental agents starting on day 3 or
4 following fiber implantation and continuing once daily
for a total of four doses. Each agent is assessed by IP
injection at two dose levels with three mice/dose/experi-
ment. Vehicle controls consist of six mice receiving the
compound diluent only. The fibers are collected from
the mice on the day following the fourth compound
treatment and subjected to the stable endpoint MTT
assay. The optical density of each sample is determined
4.5.18. N3-{3-[{3-[{1-(2,4-Dichlorophenyl)-1,4-
dihydroindeno[1,2-c]pyrazole-3-yl-
carbonyl}amino]propyl}(methyl)amino]propyl}1-(2,4-
dichlorophenyl)-1,4-dihydroindeno[1,2-c] pyrazole-3-
carboxamide (2r)
Yield: 48% as an oily residue which solified on
standing after purification by FC eluting with CHCl3/
CH3OH 9/1; yellowish solid; m.p. 117ꢀ
rated with ethyl ether). Rf (CHCl3/MeOH 9:1) 0.36 IR
(nujol): 1660 (CꢁO), 3300ꢀ3420 (NH). UV (ethanol):
lmax 232 (3.86), 263 (3.76), 272 (3.72), 281 (3.87). H
NMR (CDCl3): 1.82 (qu, 4H, Jꢂ6 Hz), 2.31 (s, 3H),
2.54 (t, 4H, Jꢂ6 Hz), 3.55 (q, 4H, Jꢂ6 Hz), 3.68 (s,
4H), 6.96 (d, 2H, Jꢂ7.0 Hz), 7.18ꢀ7.35 (m, 4H), 7.40ꢀ
7.43 (m, 2H), 7.50ꢀ7.65 (m, 6H and 2NH exch. with
D2O). Anal. Calc. for C41H35Cl4N7O2: C 61.59, H 4.41,
/
118 8C (tritu-
/
/
1
/
/
/
/
/
/
/

762
G.A. Pinna et al. / Il Farmaco 58 (2003) 749ꢀ763
/
spectrophotometrically at 540 nm and the mean of each
treatment group is calculated. The percent net cell
growth in each treatment group is calculated and
compared to the percent net cell growth in the vehicle
treated controls. Each compound is assessed in a total of
in a viscometer and the relative viscosity (h/h0) of 95 mM
Poly(dA-dT)×poly(dA-dT) with respect to sodium caco-
dylate buffer was measured as a function of the
concentration of compound. The r value is definite as
molar ratio of drug to DNA base pair.
/
four experiments (3 cell lines/experimentꢄ
/4 experi-
mentꢂ12 cell lines).
/
Compounds are selected for further testing (e.g. time/
dose exposure studies, preliminary pharmacology stu-
dies, SC xenograft efficacy studies) on the basis of
several hollow fiber assay criteria. These include: (1) a
reduction in net cell growth of 50% or greater in 10 of
Acknowledgements
The authors acknowledge Professor G. Cignarella for
his insightful comment on this work.
the 48 possible test combinations (12 cell linesꢄ
/2
sitesꢄ2 compound doses); (2) a reduction in net cell
/
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