Synthesis and biological evaluation of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines as new ligands of central and peripheral benzodiazepine receptors

Article abstract of DOI:10.1016/S0014-827X(02)00017-4

A large number of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines were synthesised and tested to evaluate their binding affinities at both central (CBR) and peripheral (PBR) benzodiazepine receptors. Relatively good PBR binding affinities were found for ligands belonging to the 3-arylmethyloxy-pyridazinoindole series, whereas only 2-aryl-indenopyridazines 7a, 8a and 10a display a weak binding affinity for CBR. To find out the main structural determinants affecting PBR affinity, a molecular modelling study based on the comparative analysis of the three-dimensional properties of four properly selected derivatives 24a, 3b, 18a and 10d, with those of highly active and selective PBR ligands, taken as reference, was performed.

Full text of DOI:10.1016/S0014-827X(02)00017-4

Il Farmaco 58 (2003) 129Á140
/
www.elsevier.com/locate/farmac
Synthesis and biological evaluation of pyridazino[4,3-b]indoles and
indeno[1,2-c]pyridazines as new ligands of central and peripheral
benzodiazepine receptors
Francesco Campagna a,*, Fausta Palluotto a, Maria Paola Mascia b,
Elisabetta Maciocco b, Carla Marra b, Angelo Carotti a, Antonio Carrieri a
a Dipartimento Farmacochimico, Universita` di Bari, via E. Orabona 4, 70126 Bari, Italy
b Dipartimento di Biologia Sperimentale, Universita` di Cagliari e Istituto CNR di Neuroscienze, Sezione di Neuropsicofarmacologia, Cagliari, Italy
Received 29 June 2002; accepted 5 September 2002
Abstract
A large number of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines were synthesised and tested to evaluate their binding
affinities at both central (CBR) and peripheral (PBR) benzodiazepine receptors. Relatively good PBR binding affinities were found
for ligands belonging to the 3-arylmethyloxy-pyridazinoindole series, whereas only 2-aryl-indenopyridazines 7a, 8a and 10a display
a weak binding affinity for CBR. To find out the main structural determinants affecting PBR affinity, a molecular modelling study
based on the comparative analysis of the three-dimensional properties of four properly selected derivatives 24a, 3b, 18a and 10d,
with those of highly active and selective PBR ligands, taken as reference, was performed.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Pyridazino[4,3-b]indoles; Indeno[1,2-c]pyridazines; Peripheral benzodiazepine receptor; Binding assay; StructureÁactivity relationships
/
1. Introduction
Benzodiazepines, b-carbolines, imidazopyridines, pyr-
azoloquinolines, and imidazoquinoxalines represent the
major families of compounds with high affinity for
CBR. In addition to their anxiolitic properties, most of
the drugs in clinical use, such as diazepam, possess
anticonvulsant, sedative, and muscle relaxant effects [2].
In the central nervous system (CNS), diazepam also
binds to PBR [7]. This receptorial protein is distinct
from the CBR protein even if both modulate the activity
of g-aminobutyric acid in the brain. PBR, initially
discovered in a wide range of peripheral tissues (kidney,
liver and lung), has been found to occur in particularly
high density in steroid-producing tissues, such as
adrenal, testis, placenta and brain tissues and it is
Benzodiazepines bind two main classes of receptors,
namely the central (CBR) and the peripheral (PBR)
benzodiazepine receptor. CBR is present exclusively in
the central nervous system where benzodiazepines
produce their pharmacological effects by modulating
the action of GABA at GABAA-receptor/Clꢀ iono-
phore supramolecular complex [1,2], a pentameric
protein, formed by different combinations of distinct
subunits called a, b, d, g and r [3Á
/5].
CBR ligands bind at allosteric modulatory sites (v
sites) enhancing (agonists) or reducing (inverse agonists)
the GABA-induced Clꢀ ion flux. The antagonists,
exhibit, per se, no relevant biological effects but
antagonise the action of agonists and inverse agonists
[6].
localised mainly in the mitochondria [8Á10].
/
PBRs are involved in several functions such as cell
proliferation, immune response modulation, regulation
of mitochondrial oxidative phosphorylation and regula-
tion of steroidogenesis [11Á14].
/
Many compounds bind to PBR with varying affi-
nities. Among them, isoquinoline carboxamides
(PK11195), benzodiazepines (Ro 5-4864), imidazopyr-
* Corresponding author.
E-mail address: campagna@farmchim.uniba.it (F. Campagna).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 0 0 1 7 - 4

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F. Campagna et al. / Il Farmaco 58 (2003) 129Á140
/
idines (alpidem and zolpidem), 3-indole-acetamides
(FGIN-1-27), and pyrrolobenzoxazepines (NF 182) dis-
play outstanding binding affinities. From a medicinal
chemistry point of view, the synthesis of new, PBR
ligands should prove useful to determine some key
structural features for high affinity and selectivity and
to better characterise the pharmacological and thera-
peutic roles of PBR.
In the search for novel anxiolitic agents devoid of the
undesirable side effects of classical benzodiazepines, we
extensively explored a new class of CBR ligands, namely
the 2-aryl-2,5-dihydropyridazino[4,3-b]indol-3(3H)ones
(I, Plate 1), which showed a wide range of pharmaco-
logical activity spanning from agonist to inverse agonist
Fig. 1. Proposed pharmacophore model for CBR [18]. The main
binding sites for pyrazoloquinoline CGS-8216 are indicated as HB1
and HB2 (HB donor sites), A2 (HB acceptor site), L1 and L2
(hydrophobic sites). L3 is another lipophilic region reached by the 5-
phenyl ring of classical benzodiazepines. S1 represents a sterically
inaccessible region. Binding to HB2 and A2 is not necessary for inverse
agonist activity.
[15Á17].
/
According to the pharmacophore proposed by Cook
[18], these compounds can efficiently interact with the
lipophilic regions L1 and L2 as well as with HB1 and
HB2 hydrogen-bond donors sites and finally with the
A2 hydrogen-bond acceptor site (Fig. 1).
During our investigation of new classes of CBR
ligands, we observed that, contrary to many derivatives
of I, the esters II [15] (Plate 1), intermediates for the
synthesis of I, showed no [3H]flunitrazepam displace-
ment from its CBR binding site up to a 20 mM
concentration.
We hypothesised that the steric hindrance of the
ethoxycarbonyl group at the S1 region of the CBR (Fig.
1), might probably be responsible for the lack of affinity
of II and that, on the basis of the actual knowledge on
the structural requirements for affinity at PBR, this
moiety should not instead be detrimental for binding to
this receptor. The esters II, in fact, may possess the
structural features required for the interaction with
PBR, that are three lipophilic regions (L1, L3 and L4),
and a hydrogen-bond donor group (HB2), generally a
carbonyl oxygen, as reported in several 3D QSAR and
pharmacophore models [19,24,25] (Fig. 2).
In order to validate our hypothesis, we tested the
ability of esters 3a and b (Table 2) to displace the high
affinity radioligand [3H]PK11195 from its PBR binding
site in rat cortex homogenate. The experimental binding
data showed that 3b bound with a relatively good
Fig. 2. Schematic representation of PBR pharmacophore model as
proposed by different groups [19,24,25]. The molecular structure of
PK1195 is sketched to help interpretation.
but, unexpectedly, no significant binding was found for
3a.
In order to explain this difference and to gain further
information on the molecular determinants for both
high affinity and selectivity at PBR, within our classes of
compounds, we synthesised a new series of pyrida-
zino[4,3-b]indoles and indeno[1,2-c]pyridazines having
the general formula III (Plate 2) and evaluated their
binding affinities at both CBR and PBR.
The structural modifications were mainly made on the
2, 3, 4 and 5 positions, bearing in mind that two
aromatic rings and an electron rich group are the most
likely ligand anchoring points in the interaction model
with PBR.
affinity and a high selectivity to PBR (IC50ꢁ610 nM),
/
Plate 1.
Plate 2.

F. Campagna et al. / Il Farmaco 58 (2003) 129Á
/
140
131
2. Chemistry
equimolar mixture of 21 and sodium hydride in dry
dimethylformamide (DMF) gave the 3-arylmethyloxy
derivative 22a, b, f, and 23a, b, which were further
ethylated at N-5 by means of sodium hydride and
iodoethane to give 24a, b, f. Finally, the diethylamide
26a was prepared from the acid 25a by standard
methods. Physical and spectroscopic data of the newly
synthesised compounds are reported in Table 1.
2-Aryl-2,5-dihydropyridazino[4,3-b]indol-3(3H)ones
(1Á4) were prepared according to methods previously
/
reported by us [15,16]. Carboxamide derivatives 5b and
6b were synthesised by standard procedures starting
from the corresponding parent carboxylic acid [15] as
described in Section 5. Compound 8a was obtained by
oxidation of the indenopyridazine 7a described in ref.
[17]. Scheme 1 outlines the synthetic route to 2-aryl-
indeno[1,2-c]pyridazine-4-carboxylic acid ethyl esters
10a, b, d and 11a.
3. Binding and SAR studies
All the compounds were evaluated for their in vitro
binding affinity at the CBR and PBR by means of a
In short, compounds 10a, b, and d were synthesised
refluxing the 2-hydroxy-2-(1-oxo-indan-2-yl)malonic
acid diethyl ester (9), prepared according to the method
reported in ref. [20], with the appropriate phenyl
hydrazine hydrochloride in ethanol. The oxidation of
10a by means of chromic anhydride in refluxing acetic
acid gave the corresponding 3,5-dioxo derivative 11a.
The new 3-aryl-pyridazino[4,3-b]indole-4-carboxylic
acid ethyl esters 15a, e and 18a were synthesised as
indicated in Scheme 2. In brief, 3-oxo-2-(3-oxo-1,3-
dihydro-indol-2-ylidene)-3-aroyl-propionic acid ethyl
esters 14a, e were prepared by refluxing isatin 12 with
phosphorus pentachloride in anhydrous benzene to give
2-chloro-3H-indol-3-one (13), which was then con-
densed with appropriate ethylaroylacetate sodium salt
according to the method previously described by us
[15,16]. The cyclization with hydrazine hydrate in
anhydrous ethanol easily furnished the pyridazine
derivatives 15a, e. Treatment of 15a with sodium
hydride and ethyl iodide gave the N-5 ethyl derivative
18a. Carboxamide 17e was prepared starting from the
carboxylic acid 16e by standard methods as described in
Section 5.
binding
[3H]PK11195, respectively, as radioligands and cerebral
cortices from male SpragueÁDawley rats as receptor
assay
using
[3H]Flunitrazepam
and
/
source [22,23]. The binding data are expressed as IC50 or
as percentage of inhibition of specific radioligand
binding at 1 mM compound concentration (reported in
square brackets in the tables). The measured binding
affinities for CBR and PBR are reported in Tables 2Á4
/
for condensed 2-arylpyridazine, condensed 3-arylpyri-
dazine and 3-arylmethyloxy pyridazinoindole deriva-
tives, respectively.
2-Aryl-pyridazino[4,3-b]indoles 1a, bÁ2a, c and 2-
/
aryl-indeno[1,2-c]pyridazines 7a and 8a, both devoid of
carbonyl group at C-4, display an appreciable affinity
for CBR. A lower CBR potency was found for
indenopyridazines, as already observed by us [17], and
no significant affinity for PBR was detected. On the
contrary, ligands 3a, b, 4a, 10a, b, d, 11a, and 5b, 6b,
containing an ethoxycarbonyl or a carboxamido group,
respectively, bind sometimes weakly, but selectively at
PBR. The highest potency was measured for 3b (IC50ꢁ
/
610 nM). These results confirm that in the series of
condensed pyridazine derivatives bearing an aryl sub-
stituent in position 2, a bulky substituent at C-4 is not
only well-tolerated but also necessary for binding to
PBR. In addition to that, taking into account the
relatively higher affinity of halogenated compounds 3b
3-Arylmethyloxy derivatives 22a, b, f, 23a, b, 24a, b, f,
25a and 26a were prepared starting from 3-hydroxy-5H-
pyridazino[4,3-b]indole-4-carboxylic acid ethyl ester (21)
[21], as outlined in Scheme 3. In short, treatment of the
appropriate benzyl or halophenylmethylhalide with an
and 10d (IC50ꢁ610 and 730 nM, respectively), it seems
/
reasonable to hypothesise that the halogen substituent
at the N-2 phenyl ring could interact with the L1
lipophilic region, determining an appreciable improve-
ment of the PBR affinity. The p-chloroderivative 10b is
the only exception to this trend. The replacement of the
ethoxycarbonyl group of 3b with carboxamide func-
tions, even similar to those present in PK11195, results
in a marked loss of PBR affinity in 5b and 6b (7 and
16%, respectively at 1 mM). These data suggest that our
ligands may bind to a different less lipophilic, or less
accessible, receptor region. Like the 2-aryl-pyrida-
zino[4,3-b]indoles and 2-aryl-indeno[1,2-c]pyridazines,
3-aryl analogues 15a, e, 17e and 19, although showing
selective binding at PBR, do not possess a good PBR
Scheme 1.

132
F. Campagna et al. / Il Farmaco 58 (2003) 129Á
/
140
Scheme 2.
affinity. A modest, but significant increase of the affinity
of 15a (31% [3H] PK11195 displacement at 1 mM), is
PBR affinity was substantially conserved in the meta-
halophenylmethyloxy derivatives 22f and 24f.
observed by ethylation of its N-5 leading to 18a (IC50ꢁ
/
658 nM). An analogous substantial enhancement of the
PBR affinity resulted from the N-5 alkylation of the 3-
aryloxy derivatives 22a and 22f leading to 24a and 24f,
respectively. The former is endowed with the highest
binding potency observed for all the tested compounds
4. Molecular modelling studies
To find out the main structural determinants affecting
the measured PBR binding affinities, a molecular
modelling study based on the analysis of three-dimen-
sional properties of four properly selected derivatives
was performed.
The convergent pharmacophore models recently de-
veloped and referred by different groups [19,24,25] for
PBR ligands were taken into account to interpret, in a
(IC50ꢁ240 nM). This result might indicate that the N-5
/
alkyl group probably reaches a lipophilic pocket close to
the receptor region accommodating the C-4 carbonyl
group. The replacement of the ethoxycarbonyl group
with a carboxamide function in the case of 24a, as well
as of 2-aryl-pyridazinoindole analogue 3b, yields a
marked drop of PBR affinity.
qualitative manner, the structureÁaffinity relationships
/
The introduction of a chloro substituent on the
phenyl ring of the benzyloxy substituent determined,
in the case of para-halophenylmethyloxy derivatives
22b, 24b and diversely from the 2-aryl-pyridazinoindole
and 2-aryl indenopyridazine analogues 3b and 10d, a
decrease of the activity compared with the correspond-
ing unsubstituted parent compounds 22a and 24a. The
(SAFIR). These models are all characterised by four
relevant features: three hydrophobic moieties (L1, L3
and L4) and a hydrogen-bond acceptor group (HB2) as
schematically indicated in Fig. 2.
In the present study, three highly active and selective
PBR ligands namely isoquinoline carboxamide
PK11195, pyrrolo[2,1-d][1,5]benzoxazepin-7yl diethyl-

F. Campagna et al. / Il Farmaco 58 (2003) 129Á
/
140
133
Scheme 3.
carbamate (27) and (2E) 1-oxo-1,2-dihydro-3H-pyr-
rolo[3,4-b]quinolin-3-ylidene acetamide (28) from ref.
[19] (Plate 3) were chosen as molecular templates to
compare their structural features with those shown by
our selected molecules, that are 24a, 3b, 18a and 10d.
Relevant distances among the proposed pharmaco-
pounds 3b, 18a and 10d, ligand 24a present a L1Á
/
L3
distance very close to those observed in the templates
˚
(5.98 versus 5.13Á
/
6.38 A) and moreover, even the L1Á
/
HB2 distance is compatible with those measured in the
˚
templates (6.11 versus 4.88Á6.59 A). This may justify, at
/
least in part, the higher affinity of 24a. The difference of
about two orders of magnitude of its IC50 compared
with that of the templates could be ascribed to an
phore features, namely L1Á
/
L3, L1Á
/
HB2 and L3ÁHB2,
/
were measured for low energy conformers of our
molecules and compared with those seen on the selected
templates. The centroid characterising the important,
but not essential hydrophobic region L4, was not taken
into account due to its less precise spatial location. Since
our ligands bind less tightly to PBR with respect to the
templates, some significant differences in the intramole-
cular distances of the pharmacophoric points would
have been expected.
unfavourable very low value of the HB2Á
/
L3 distance
6.44 A) and/or to a large ‘excluded
˚
(3.68 versus 5.86Á
/
volume’ from its tricyclic ring system that may impact a
probably forbidden zone of the receptor, as can be seen
in the molecular superposition onto PK11195 reported
in Fig. 2.
It is worth noting that the low active ligand 18a does
not fit the geometrical features required for an efficient
The analysis of the distances reported in Table 5
binding to PBR, especially the HB2ÁL3 distance.
/
suggests that the L1Á
geometrical feature for an efficient binding process at
least within our set of ligands. Indeed, unlikely com-
/
L3 distance might be a crucial
The low activity of derivative 10d could also arise
from the presence of additional chemical groups (i.e. the
carbonyl of the dihydropyridazinone moiety and the

134
F. Campagna et al. / Il Farmaco 58 (2003) 129Á140
/
Table 1
Physical and spectroscopic data of compounds 5, 6, 8, 10, 11, 14Á
/
18, 22Á/26
a
Comp. m.p. (8C) (crystallisation IR nmax (cmꢀ1
solvent)
)
1H NMR, d(ppm), J (Hz)
5b
283Á
/
285 (ethanol)
3075, 1655, 1630,
1.17 (t, 3H, CH3, Jꢁ
2H, CH2, Jꢁ7.1); 7.10Á
2H, Arom); 7.84 (d, 1H, Arom, Jꢁ
0.75 (t, 3H, CH3, Jꢁ7.3); 1.00 (t, 3H, CH3, Jꢁ
2H, CH2, Jꢁ7.3); 3.53 (t, 2H, CH2, Jꢁ7.3); 7.18 (t, 2H, Arom, Jꢁ
3H, Arom); 7.55Á7.65 (m, 2H, Arom); 7.91 (d, 1H, Arom, Jꢁ7.8); 9.00 (s, 1H, NH)
7.35 (s, 1H, CH); 7.40Á7.50 (m, 1H, Arom); 7.50Á7.65 (m, 4H, Arom); 7.67 (dt, 1H,
Arom, Jꢁ7.2, 0.7); 7.85 (dt, 1H, Arom, Jꢁ7.4. 0.7); 7.80Á7.95 (m, 2H, Arom)
1.35 (t, 3H, CH3, Jꢁ7.3); 4.15 (s, 2H, CH2), 4.35 (q, 2H, CH2, Jꢁ
Arom), 7.79 (d, 1H, Jꢁ7.5)
1.41 (t, 3H, CH3, Jꢁ7.3); 4.17 (s, 2H, CH2); 4.45 (q, 2H, CH2, Jꢁ
Arom); 7.60Á7.70 (m, 2H, Arom), 7.85Á7.95 (m, 1H, Arom)
1.42 (t, 3H, CH3, Jꢁ7.3); 4.35 (q, 2H, CH2, Jꢁ7.3); 7.40Á7.55 (m, 3H, Arom); 7.55Á
(m, 4H, Arom); dd (1H, Arom, Jꢁ6.6, 1.7)
1.33 (t, 3H, CH3, Jꢁ7.1); 4.41 (q, 2H, CH2, Jꢁ
(m, 3H, Arom)
1.15 (t, 3H, CH3, Jꢁ
(m, 5H, Arom); 7.90Á
1.06 (t, 3H, CH3, Jꢁ7.1); 4.18 (q, 2H, CH2, Jꢁ
1H, Arom, Jꢁ8.2); 7.44 (d, 1H, Arom, Jꢁ7.4); 7.59 (dt, 1H, Arom, Jꢁ
8.20 (m, 2H, Arom); 8.25Á8.35 (m, 2H, Arom)
3200br, 1700, 1600 0.99 (t, 3H, CH3, Jꢁ7.1); 4.20 (q, 2H, CH2, Jꢁ
1H, Arom, Jꢁ7.8); 7.55Á7.70 (m, 3H, Arom); 8.57 (d, 1H, Arom, Jꢁ
NH)
3260 br, 1700, 1595 1.04 (t, 3H, CH3, Jꢁ
1H, Arom, Jꢁ8.2); 7.70 (dt, 1H, Arom, Jꢁ
(m,2H, Arom); 8.59 (d, 1H, Arom, Jꢁ7.8); 9.81 (s, 1H, NH)
3070 br, 1630, 1585, 7.46 (dt, 1H, Arom, Jꢁ7.3, 1.0); 7.72 (dt, 1H, Arom, Jꢁ7.2, 1.1); 7.80 (d, 1H, Arom, Jꢁ
7.95 (m, 2H, Arom); 8.30Á8.40 (m, 2H, Arom); 8.45 (d, 1H, Arom, Jꢁ7.7);
12.14 (s, 1H, NH); 14.10 (s, 1H, COOH)
0.64 (t, 3H, CH3, Jꢁ7.0); 1.12 (t, 3H, CH3, Jꢁ
(m, 1H, CH), 3.10Á3.30 (m, 1H, CH), 3.85Á3.95 (m, 1H, CH), 7.43 (t, 2H, Arom, Jꢁ
7.61 (t, 1H, Arom, Jꢁ7.7); 8.15 (d, 2H, Arom, Jꢁ8.6); 8.35 (d, 2H, Arom, Jꢁ
(d, 1H, Arom, Jꢁ7.9); 9.38 (s, 1H, NH)
1.04 (t, 3H, CH3, Jꢁ7.1); 1.42 (t, 3H, CH3, Jꢁ
2H, CH2, Jꢁ7.1); 7.45Á7.55 (m, 5H, Arom); 7.65Á
Arom, Jꢁ7.7)
1.31 (t, 3H, CH3, Jꢁ
Arom); 7.50Á7.60 (m, 2H, Arom); 7.91 (d, 1H, Arom, Jꢁ
1.31 (t, 3H, CH3, Jꢁ7.0); 4.35 (q, 2H, CH2, Jꢁ7.0); 5.40 (s, 2H, CH2); 7.20Á
Arom); 7.30Á7.45 (m, 4H, Arom); 7.52 (d, 2H, Arom, Jꢁ4.0); 7.91 (d, 1H, Arom, Jꢁ
7.8); 11.50 (s, 1H, NH)
1.32 (t, 3H, CH3, Jꢁ7.1); 4.35 (q, 2H, CH2, Jꢁ
Arom); 7.30Á7.40 (m, 3H, Arom); 7.50Á7.55 (m, 2H, Arom); 7.92 (d, 1H, Arom, Jꢁ
11.50 (s, 1H, NH)
0.91 (t, 3H, CH3, Jꢁ
7.09 (dd, 2H, Arom, Jꢁ
1.0); 8.00 (d, 1H, Arom, Jꢁ
0.94 (t, 3H, CH3, Jꢁ7.2); 4.02 (q, 2H. CH2, Jꢁ
7.11 (d, 2H, Arom, Jꢁ8.5); 7.25Á7.45 (m, 8H, Arom); 7.55 (dt, 1H, Arom, Jꢁ
8.00 (d, 1H, Arom, Jꢁ7.7)
1.22 (t, 3H, CH3, Jꢁ7.2); 1.32 (t, 3H, CH3, Jꢁ
2H, CH2, Jꢁ7.2); 5.41 (s, 2H, CH2); 7.25Á7.40 (m, 6H, Arom); 7.50Á
7.95 (d, 1H, Arom, Jꢁ7.4)
1.22 (t, 3H, CH3, Jꢁ7.1); 1.32 (t, 3H, CH3, Jꢁ
2H, CH2, Jꢁ7.1); 5.40 (s, 2H, CH2); 7.30Á7.50 (m, 5H, Arom); 7.50Á
7.96 (d, 1H, Arom, Jꢁ7.7)
1.33 (t, 3H, CH3, Jꢁ7.2); 1.42 (t, 3H, CH3, Jꢁ
2H, CH2, Jꢁ7.2); 5.44 (s, 2H, CH2); 7.15Á7.30 (m, 4H, Arom); 7.35Á
7.44 (s, 1H, Arom); 7.51 (t, 1H, Arom, Jꢁ7.9); 8.00 (d, 1H, Arom, Jꢁ
/
7.1); 1.32 (t, 3H, CH3, Jꢁ
7.20 (m, 2H, Arom); 7.35Á
7.8); 9.60 (s, 1H, NH)
7.3); 1.60Á
/
7.1), 3.44 (q, 2H, CH2, Jꢁ
/
7.1); 3.64 (q,
1620
/
/
/7.50 (m, 3H, Arom); 7.55Á
/7.65 (m,
/
6b
242Á
/
246 (ligroin)
1650, 1620, 1610
/
/
/1.80 (m, 4H, 2CH2); 3.36 (t,
/
/
/
7.4); 7.40Á7.50 (m,
/
/
/
8a
230Á
149Á
148Á
162Á
155Á
196Á
202Á
/
235 dec. (ethanol)
150 (ethanol)
150 (ethanol)
163 (ethanol)
157 (ethanol)
198 (ethanol)
204 (ethanol)
1720, 1655, 1625
1710, 1670, 1625
1715, 1680
/
/
/
/
/
10a
10b
10d
11a
14a
14e
/
/
/7.3); 7.40Á
/
7.65 (m, 8H,
7.55 (m, 5H,
/
/
/
/7.3); 7.40Á
/
/
/
/
1700, 1665
/
/
/
/
7.65
7.95
7.60
/
/
1740, 1665, 1630
/
/
7.1); 7.45Á
/
7.70 (m, 6H; Arom); 7.85Á
/
/
3380, 1705, 1675,
1655, 1600
/
7.1); 4.21 (q, 2H, CH2, Jꢁ
8.00 (m, 2H, Arom); 9.26 (s, 1H, NH)
7.1); 6.97 (t, 1H, Arom, Jꢁ
7.7, 1.0); 8.15Á
/
7.1); 6.90Á
/
7.00 (m, 2H, Arom); 7.40Á
/
/
/
3380, 1720, 1680,
1610, 1600
/
/
/
7.4); 7.30 (d,
/
/
/
/
/
15a
15e
16e
17e
210Á
/
212 (CH3CN)
/
/
7.1); 7.45Á
/
7.50 (m, 4H, Arom); 7.53 (d,
/
/
/
7.7); 9.71 (s, 1H,
241Á
/
243 (chloroform/
/
7.1), 4.25 (q, 2H, CH2, Jꢁ
/7.1); 7.48 (t, 1H, Arom, Jꢁ
/
7.2); 7.58 (d,
hexane)
/
/
7.7, 1.2); 7.75Á
/
7.85 (m,2H, Arom); 8.30Á
/
8.40
/
ꢀ/350
/
/
/
1570
8.1); 7.85Á
/
/
/
314Á
/
316 (ethyl acetate)
3230, 1611, 1600
/
/
7.0); 2.60Á
/
2.75 (m, 1H, CH), 2.85Á
/
3.00
/
/
/7.7);
/
/
/8.1); 8.52
/
18a
169Á
/
170 (ethanol)
1720, 1575
/
/
7.1); 4.20 (q, 2H, CH2, Jꢁ
/
7.1), 4.38 (q,
/
/
/7.75 (m, 3H, Arom); 8.63 (d, 1H,
/
22a
22b
194Á
/
196 (acetonitrile/
3360, 1670, 1640,
1605
/
7.1); 4.35 (q, 2H, CH2, Jꢁ
/
7.1); 5.42 (s, 2H, CH2); 7.20Á
7.7); 11.50 (s, 1H, NH)
7.30 (m, 1H,
/
7.35 (m, 6H,
water)
/
/
184Á
/
186 (acetonitrile)
3520, 3340, 1660,
1630
/
/
/
/
/
/
22f
23a
23b
24a
24b
24f
142Á
/
145 (acetonitrile/
3360, 1700, 1590
1720, 1640, 1620
1720, 1640, 1600
1715, 1640, 1605
1700, 1635, 1605
/
/7.1); 5.41 (s, 2H, CH2); 7.20Á
/
7.30 (m, 2H,
7.6);
water)
/
/
/
166Á
/
168 (acetonitrile/
/
7.1); 4.00 (q, 2H. CH2, Jꢁ
7.3, 1.5); 7.20Á7.40 (m, 10H, Arom); 7.54 (dt, 1H, Arom, Jꢁ
7.4)
/7.1); 5.30 (s, 2H, CH2); 5.42 (s, 2H, CH2);
water)
/
/
/
7.7,
/
203Á
/
205 (acetonitrile)
/
/7.2); 5.29 (s, 2H, CH2); 5.41 (s, 2H, CH2);
/
/
/7.7, 1.2);
/
174Á
/
175 (acetonitrile/
/
/
7.2); 4.03 (q, 2H, CH2, Jꢁ
/
7.2); 4.38 (q,
water)
/
/
/
7.65 (m, 2H, Arom);
/
181Á
/
183 (acetonitrile)
162 (acetonitrile/
/
/
7.1); 4.01 (q, 2H, CH2, Jꢁ
/
7.1); 4.37 (q,
/
/
/
7.65 (m, 2H, Arom);
/
160Á
/
3425, 1720, 1630,
1610
/
/
7.2); 4.02 (q, 2H, CH2, Jꢁ
7.40 (m, 1H, Arom);
7.5)
/
7.2); 4.47 (q,
water)
/
/
/
/
/

F. Campagna et al. / Il Farmaco 58 (2003) 129Á
/
140
135
Table 1 (Continued)
a
Comp. m.p. (8C) (crystallisation IR nmax (cmꢀ1
solvent)
)
1H NMR, d(ppm), J (Hz)
25a
26a
228Á
/
234 (dioxane)
3400, 1720
1615, 1630
1.25 (t, 3H, CH3, Jꢁ
Arom); 7.50Á7.65 (m, 2H, Arom); 7.97 (d, 1H, Arom, Jꢁ
1.13 (t, 3H, CH3, Jꢁ7.0); 1.29 (t, 3H, CH3, Jꢁ7.0); 1.33 (t, 3H, CH3, Jꢁ
2H, CH2, Jꢁ7.1, 2.3); 3.40Á3.55 (m, 1H, CH); 3.70Á3.85 (m, 1H, CH); 3.85Á
CH); 4.05Á4.20 (m, 1H, CH); 5.45 (d, 1H, HA, CH2ÁPh, AB system, JAB 19.6); 5.51(d,
1H, HA, CH2-Ph, AB system, JAB 19.6); 7.10Á7.40 (m, 5H, Arom); 7.40Á7.55 (m, 3H,
Arom); 8.00 (d, 1H, Arom, Jꢁ7.7)
/
7.0); 4.24 (q, 2H, CH2, Jꢁ
/
7.0); 5.45 (s, 2H, CH2); 7.25Á
/
7.40 (m, 6H,
/
/
7.6); 14.01 (br, 1H, COOH)
7.0); 3.31 (dq,
4.05 (m, 1H,
Low melting point
/
/
/
/
/
/
/
/
/
ꢁ/
ꢁ/
/
/
/
a
1H NMR spectra were recorded in CDCl3 (5b, 6b, 10b, 10d, 14a, 15a, 15e, 17e, 18a, 26a) or in DMSO-d6 (8a, 10a, 11a, 14e, 16e, 22a, 22b, 22f,
23a, 23b, 24a, 24b, 24f, 25a).
benzene of the indole ring) which might generate some
unfavourable steric and/or electrostatic interactions with
the receptor (see Fig. 3).
5. Experimental
5.1. Chemistry
In summary, the present modelling study allowed us
to understand the main reasons underlying the relatively
low PBR affinity displayed by our ligands and suggested
proper structural modifications for the synthesis of more
active compounds. We are presently working on this
and drastic molecular simplifications have been planned
to confer to the new molecules the essential, and
possibly minimum, structural requirements for a high
PBR binding affinity.
Melting points were taken on a Gallenkamp MFB 595
010 M apparatus and are uncorrected. Elemental
analyses were performed on a Carlo Erba 1106 analyser
for C, H, N; experimental results agreed to within 9
/
0.40% of the theoretical values. IR spectra were
recorded using potassium bromide disks on a Perkin-
Elmer 283 spectrophotometer, only the most significant
and diagnostic absorption bands being reported. 1H
Table 2
Binding data of condensed 2-arylpyridazine derivatives
a
b
Comp.
X
Y
R
CBR
PBR
1a
1b
2a
2c
3a
3b
4a
5b
6b
NH
NH
H
H
238
31
[0%]
[10%]
[3%]
[0%]
[5%]
610
H
4-Cl
H
NÃ
NÃ
/
CH3
H
1560
994
/CH3
H
4-OCH3
H
NH
NH
COOC2H5
COOC2H5
COOC2H5
CON(C2H5)2
CON(nC3H7)2
H
[0%]
[0%]
N.D.
[0%]
[0%]
2180
4-Cl
H
NÃ/CH3
[16%]
[7%]
[16%]
[11%]
[0%]
[43%]
[30%]
730
NH
NH
CH2
CO
4-Cl
4-Cl
H
c
7a
8a
H
H
[30%] 20 mM
[45%] 20 mM
[0%]
10a
10b
10d
11a
CH2
CH2
CH2
CO
COOC2H5
COOC2H5
COOC2H5
COOC2H5
H
4-Cl
4-Br
H
[0%]
[0%]
[18%]
a
Inhibition of [3H] flunitrazepam binding expressed as IC50 (nM) or as percentage of inhibition at 1 mM concentration.
Inhibition of [3H] PK11195 binding expressed as IC50 (nM) or as percentage of inhibition at 1 mM concentration.
Reported in ref. [17].
b
c

136
F. Campagna et al. / Il Farmaco 58 (2003) 129Á140
/
Table 3
Binding data of condensed 3-arylpyridazine derivatives
5.1.1. 2-Chlorophenyl-3,5-dihydro-3-oxo-2H-
pyridazino[4,3-b]indole-4-carboxylic acid dialkylamides
(5b, 6b)
Catalytic amount of anhydrous DMF was added to a
mixture of 2-chlorophenyl-3,5-dihydro-3-oxo-2H-pyri-
dazino[4,3-b]indole-4-carboxylic acid, obtained from the
ester 3b as reported in ref. [15], (0.051 g, 0.15 mmol) and
thionyl chloride (20.5 mmol, 1.5 ml). The resulting
mixture was refluxed for 7 h. The unreacted thionyl
chloride was evaporated and to the residue suspended in
anhydrous THF (2 ml) the appropriate amine (0.55
mmol) was added. The mixture was stirred at room
temperature (r.t.) for 2 h. In the case of 5b, the residue,
obtained after evaporation of the solvent in vacuo, was
chromatographed on silica gel using ethyl acetate/
a
b
Comp.
X
Y
R
H
CBR
[0%]
PBR
15a
15e
17e
18a
NH
NH
NH
COOC2H5
COOC2H5
CON(C2H5)2
COOC2H5
COOC2H5
[31%]
[9%]
[13%]
658
NO2 [0%]
NO2 [0%]
NÃ
CO
/C2H5
H
H
[3%]
[0%]
c
19
2300
petroleum ether 9:1 (5b, Rfꢁ0.6, 0.03 g, 52% yield).
/
a
Inhibition of [3H] flunitrazepam binding expressed as IC50 (nM)
or as percentage of inhibition at 1 mM concentration.
Inhibition of [3H] PK11195 binding expressed as IC50 (nM) or as
In the case of 6b, the final suspension was filtered, the
resulting solution evaporated in vacuo and the residue
crystallised from ligroin 6b (0.060 g, 57% yield).
b
percentage of inhibition at 1 mM concentration.
Reported in ref. [26].
c
NMR spectra were recorded on a Bruker AM 300 WB
300 MHz or on a Varian 300 Mercury spectrometers.
Chemical shifts are expressed in d (ppm) and the
coupling constants J in Hz. The following abbreviations
were used: s, singlet; d, doublet; t, triplet; m, multiplet;
dd, double doublet. Exchange with deuterium oxide was
used to identify OH and NH protons, which in some
cases gave broad signals spread widely on the base line
and these were very difficult to detect. Reagents and
solvents were purchased from Sigma-Aldrich Chemie.
5.1.2. 2-Phenyl-2H-indeno[1,2-c]pyridazine-3,5-dione
(8a)
CrO3 (0.140 g, 1 mmol) was added to a solution of 2-
phenyl-2,5-dihydro-indeno[1,2-c]pyridazine-3-one (7a)
[17] (0.260 g, 1 mmol) in acetic acid (4.6 ml) and the
resulting mixture refluxed for 2 h. After cooling, the
reaction mixture was poured on ice (40 ml) and the
precipitate filtered, dried and chromatographed, using
chloroform/ethyl acetate 9:1 as an eluant, to obtain 8a
(Rfꢁ
/
0.67, 49% yield).
Table 4
Binding data of 3-arylmethyloxypyridazinoindole derivatives
a
b
Comp.
X
Y
R
CBR
PBR
22a
22b
22f
23a
23b
24a
24b
24f
26
NH
NH
NH
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
CON(C2H5)2
H
[0%]
[0%]
[0%]
[0%]
[0%]
[0%]
[0%]
[0%]
[0%]
[27%]
[26%]
1470
1570
[18%]
240
4-Cl
3-Cl
H
NÃ
NÃ
NÃ
NÃ
NÃ
NÃ
/
CH2C6H5
/
CH2ÃC6H4Ã
/
/4Cl
4-Cl
H
/
C2H5
C2H5
C2H5
C2H5
/
4-Cl
3-Cl
H
[26%]
770
[26%]
/
/
a
Inhibition of [3H] flunitrazepam binding expressed as IC50 (nM) or as percentage of inhibition at 1 mM concentration.
Inhibition of [3H] PK11195 binding expressed as IC50 (nM) or as percentage of inhibition at 1 mM concentration.
b

F. Campagna et al. / Il Farmaco 58 (2003) 129Á
/
140
137
Plate 3.
5.1.4. 3,5-Dioxo-2-phenyl-3,5-dihydro-2H-indeno[1,2-
c]pyridazine-4-carboxylic acid ethyl ester (11a)
Table 5
Intramolecular distances (A) between pharmacophoric elements
˚
CrO3 (0.07 g, 0.50 mmol) was added to a solution of
3-oxo-2-phenyl-3,5-dihydro-2H-indeno[1,2-c]pyrida-
zine-4-carboxylic acid ethyl ester (10a) (0.166 g, 0.50
mmol) in acetic acid (2.3 ml) and the mixture refluxed
for 2 h. After cooling, the reaction mixture was poured
on ice (25 ml) and the resulting precipitate filtered, dried
and chromatographed using chloroform/ethyl acetate
Comp.
pIC50
L1Á
/
L3
L1Á
/HB2
HB2ÁL3
/
a
PK11195 (R)
a
8.70
9.17
8.72
6.62
6.21
6.18
6.14
5.13
6.38
6.20
5.98
7.36
4.29
7.38
6.59
5.81
4.88
6.11
6.96
4.54
6.74
5.86
6.17
6.44
3.68
6.63
3.66
6.52
27
28
a
24a
3b
18a
10d
5:5 as an eluant to obtain 11a (Rfꢁ
/
0.82, 0.104 g, 60%
yield).
a
Ref. [19].
5.1.5. 3-Oxo-2-(3-oxo-1,3-dihydro-indol-2-yl)-3-aryl-
propionic acid ethyl esters (14a, e)
5.1.3. 3-Oxo-2-aryl-3,5-dihydro-2H-indeno[1,2-
c]pyridazine-4-carboxylic acid ethyl esters (10a, b, d)
The appropriate arylhydrazine hydrochloride (1.2
mmol) was added to a solution of 2-hydroxy-2-(1-oxo-
indan-2-yl)-malonic acid diethyl ester (9) [20] (0.307 g, 1
mmol) in ethanol (6 ml) and the resulting mixture
A mixture of 1H-indole-2,3-dione (12) (10.00 g, 68
mmol), phosphorus (V) chloride (15.00 g, 81.6 mmol)
and anhydrous benzene (30 ml) was refluxed for 4 h.
After cooling, the precipitate (imidoylchloride) was
collected by filtration under dry nitrogen. After washing
with petroleum ether, the solid was immediately dis-
solved in anhydrous dioxane (100 ml) and the equimo-
lecular solution of ethylaroylacetate sodium salt in
anhydrous dioxane (120 ml), was added dropwise. The
reaction mixture was stirred at r.t. overnight. The
precipitate sodium chloride was filtered off and the
solution evaporated in vacuo. The residue was triturated
refluxed 8Á19 h (16 h for 10a, 19 h for 10b, 8 h for
/
10d). The resulting precipitate was collected and chro-
matographed on silica gel using ethyl acetate/petroleum
ether 3:7 as an eluant to give the esters 10a (Rfꢁ
0.086 g, 26% yield), 10b (Rfꢁ0.33, 0.260 g, 71% yield)
and 10d (Rfꢁ0.47, 0.296 g, 72% yield).
/0.50,
/
/

138
F. Campagna et al. / Il Farmaco 58 (2003) 129Á140
/
dissolved in water (40 ml), acidified with conc. HCl to
pH 1 and stirred at r.t. for 30 min. The yellow
precipitate was filtered, washed with water and dried
to give 16e (0.286 g, 90% yield).
5.1.8. 3-(4-Nitro-phenyl)-5H-pyridazino[4,3-b]indole-4-
carboxylic acid diethylamide (17e)
A mixture of 3-(4-nitro-phenyl)-5H-pyridazino[4,3-
b]indole-4-carboxylic acid (16e) (0.159 g, 0.5 mmol) and
thionyl chloride (4 ml) was refluxed for 6 h. The
unreacted thionyl chloride was evaporated in vacuo
and to the residue suspended in anhydrous THF (4 ml),
diethylamine (0.192 ml) was added. The reaction
mixture was stirred at r.t. for 24 h. The solid, obtained
after evaporation of the solvent, was suspended in
chloroform and filtered. The filtrate was evaporated in
vacuo and the residue chromatographed on silica gel
using ethyl acetate as an eluant to obtain 17e (Rfꢁ
/0.70,
0.035 g, 18% yield).
5.1.9. 3-Phenyl-5-ethyl-5H-pyridazino[4,3-b]indole-4-
carboxylic acid ethyl ester (18a)
Sodium hydride 95% (0.020 g, 0.80 mmol) was added
to a stirred suspension of 3-phenyl-5H-pyridazino[4,3-
b]indole-4-carboxylic acid ethyl ester (15a) (0.162 g, 0.50
mmol) in dry DMF (3 ml) under nitrogen. After 1 h,
iodoethane (0.064 ml, 0.80 mmol) was added and
stirring was continued for 4 h. The mixture was then
poured on ice (40 ml) and the precipitate was filtered,
dried and chromatographed on silica gel using chloro-
Fig. 3. Molecular superposition of PK11195 (black), 24a (white) and
10d (grey) generated as follows: the centroid of the benzene ring of
isoquinoline, the centroid of the ortho-substituted phenyl ring and the
carbonyl oxygen of PK11195 were fitted onto the centroid of the
pyridazine ring, the centroid of the benzyloxy and the carbonyl oxygen
of the ester 24a and the centroid of the benzene ring of indole, the
centroid of the 2-phenyl and the carbonyl oxygen of the ester for 10d,
respectively.
form/ethyl acetate 8:2 as an eluant to give 18a (Rfꢁ
0.47, 0.040 g, 23% yield).
/
with diethyl ether to give a solid which was recrystallised
from ethanol to afford 14a (6.55 g, 30% yield), and 14e
(6.23 g, 25% yield).
5.1.10. 3-Arylmethyloxy-5H-pyridazino[4,3-b]indole-4-
carboxylic acid ethyl esters (22a, b, f and 23a, b)
5.1.6. 3-Aryl-5H-pyridazino[4,3-b]indole-4-carboxylic
acid ethyl esters (15a, e)
Sodium hydride 95% (0.038 g, 1.5 mmol) was added
to a stirred suspension of 3-hydroxy-5H-pyridazino[4,3-
b]indole-4-carboxylic acid ethyl ester (21) [21] (0.384 g,
1.5 mmol) in dry DMF (13 ml) under nitrogen. After 30
min, benzyl bromide or 4-chlorobenzylchloride or 3-
chlorobenzyl bromide (1.5 mmol) was added and
stirring was continued for 3 h. The mixture was then
poured on ice (150 ml) and the precipitate filtered,
washed with water and dried. Chromatographic purifi-
cation of the crude solid on silica gel using ethyl acetate/
petroleum ether 5:5 as an eluant furnished 22a and 23a
Hydrazine hydrate (0.265 ml, 5.4 mmol) was added to
a stirred solution of 3-oxo-2-(3-oxo-1,3-dihydro-indol-2-
yl)-3-aryl-propionic acid ethyl ester (14a or 14e) (5
mmol) in anhydrous ethanol (250 or 500 ml, respec-
tively) and stirring was continued for 3 h at r.t. The
solvent was evaporated in vacuo and the residue crystal-
lised from methanol/water and then from acetonitrile to
afford 15a (0.400 g, 25% yield) or triturated with ethanol
and then crystallised from chloroform/hexane to afford
15e (0.905 g, 50% yield).
(23a: Rfꢁ
g, 43% yield) or 22b and 23b (23b: Rfꢁ
10% yield; 22b: Rfꢁ0.40, 0.383 g, 67% yield). The
/
0.76, 0.052 g, 8% yield; 22a: Rfꢁ/0.47, 0.224
/0.68, 0.076 g,
5.1.7. 3-(4-Nitro-phenyl)-5H-pyridazino[4,3-b]indole-4-
carboxylic acid (16e)
/
crystallisation of the crude precipitate from acetonitrile/
water furnished 22f (0.241 g, 40% yield).
A solution of NaOH (0.500 g, 14 mmol) in ethanol (47
ml) was added to a stirred suspension of 3-p-nitrophe-
nyl-5H-pyridazino[4,3-b]indole-4-carboxylic acid ethyl
ester (15e) (0.346 g, 1 mmol) in ethanol (10 ml) and then
refluxed for 3 h. The reaction mixture was cooled to r.t.
and the solvent evaporated in vacuo. The residue was
5.1.11. 3-Arylmethyloxy-5-ethyl-5H-pyridazino[4,3-
b]indole-4-carboxylic acid ethyl ester (24a, b, f)
Sodium hydride 95% (0.009 g, 0.35 mmol) was added
to a stirred suspension of 3-arylmethyloxy-5H-pyrida-

F. Campagna et al. / Il Farmaco 58 (2003) 129Á
/
140
139
zino[4,3-b]indole-4-carboxylic acid ethyl ester (22a, b, f)
(0.21 mmol) in dry DMF (1.5 ml) under nitrogen. After
40 min, iodoethane (0.028 ml, 0.35 mmol) was added
and stirring was continued for 2 h (in the case of 22a, f)
or overnight (in the case of 22b). The mixture was then
poured on ice (20 ml). In the case of 22a, b, the
precipitate was filtered, washed with water, dried and
chromatographed on silica gel using ethyl acetate/
Conformational analysis of reference ligands and
compounds 3b, 10d, 18a, 24a was performed using the
BEST SEARCH module implemented in Catalyst
[27,28]. All the calculations were run on a SGI-O2
R10000 workstation.
petroleum ether 5:5 as an eluant to furnish 24a (Rfꢁ
/
References
0.63, 0.049 g, 61% yield) or 24b (Rfꢁ0.58, 0.058 g, 67%
/
yield). In the case of 22f, the mixture was extracted twice
with 20 ml of chloroform and the organic phases washed
with water, dried on anhydrous sodium sulphate and
evaporated in vacuo. The obtained residue was chro-
matographed on silica gel using ethyl acetate/petroleum
[1] I.B. Kerr, J. Ong, GABA agonists and antagonists, Med. Res.
Rev. 12 (1992) 593Á
[2] R. Squires, GABA and Benzodiazepine Receptors, vol. 1Á
Press, Boca Raton, FL, USA, 1988.
/
636.
/2, CRC
[3] E.A. Bernard, in: G. Biggio, E. Sanna, E. Costa (Eds.), GABAA
Receptor and Anxiety. From Neurobiology to Treatment, Raven
ether 5:5 as an eluant to give 24f (Rfꢁ
26% yield).
/
0.65, 0.022 g,
Press, New York, 1995, pp. 1Á
[4] G.B. Smith, W.R. Olsen, Functional domains of GABAA
receptors, Trends Pharmacol. Sci. 16 (1995) 162Á168.
[5] T.M. De Lorey, R.W. Olsen, g-Aminobutyric acidA receptor
structure and function, J. Biol. Chem. 262 (1992) 6747Á6750.
[6] J. Tulinsky, L.B. Gammill, The chemistry and pharmacology at
GABA-A and GABA-B ligands, Curr. Med. Chem. 3 (1994) 226Á
/
16.
/
5.1.12. 3-Benzyloxy-5-ethyl-5H-pyridazino[4,3-
b]indole-4-carboxylic acid (25a)
/
A solution of NaOH (0.100 g, 2.5 mmol) in ethanol
(10 ml) was added with stirring to a suspension of 3-
benzyloxy-5-ethyl-5H-pyridazino[4,3-b]indole-4-car-
boxylic acid ethyl ester (24a) (0.094 g, 0.25 mmol) in
ethanol (2 ml) and the resulting mixture refluxed for 2 h.
After cooling, the precipitate was filtered, washed with
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