Synthesis and structure-affinity relationships at the central benzodiazepine receptor of pyridazino[4,3-b]indoles and Indeno[1,2-c]pyridazines

Article abstract of DOI:10.1016/S0968-0896(99)00093-0

A series of 2-aryl-3-chloro-2H-pyridazino[4,3-b]indoles, 2-aryl-3-methoxy-2H-pyridazino[4,3-b]indoles, and 2-aryl-2,5-dihydroindeno[1,2-c]pyridazino-3(3H)-ones has been prepared and tested for their ability to inhibit the [³H]flunitrazepam binding to the central benzodiazepine receptor. SAR are presented and discussed in comparison with existing pharmacophore models. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00093-0

Bioorganic & Medicinal Chemistry 7 (1999) 1533±1538
Synthesis and Structure±Anity Relationships at the Central
Benzodiazepine Receptor of Pyridazino[4,3-b]indoles and
Indeno[1,2-c]pyridazines
F. Campagna, a,* F. Palluotto, a A. Carotti, a G. Casini a and G. Genchi b
a
Á
Dipartimento Farmacochimico, Universita di Bari, via E. Orabona 4, I-70125 Bari, Italy
Dipartimento Farmacobiologico, Universita di Bari, via E. Orabona 4, I-70125 Bari, Italy
b
Á
Received 2 June 1998; accepted 22 December 1998
AbstractÐA series of 2-aryl-3-chloro-2H-pyridazino[4,3-b]indoles, 2-aryl-3-methoxy-2H-pyridazino[4,3-b]indoles, and 2-aryl-2,5-
dihydroindeno[1,2-c]pyridazino-3(3H)-ones has been prepared and tested for their ability to inhibit the [3H]¯unitrazepam binding
to the central benzodiazepine receptor. SAR are presented and discussed in comparison with existing pharmacophore models.
# 1999 Elsevier Science Ltd. All rights reserved.
Introduction
models,11±18 which may constitute a useful guide for the
design of new potent ligands. An interesting contribu-
tion in this ®eld came also from our recent 2-D and 3-D
QSAR studies19,20 of a new class of BZR ligands,
namely the 2-aryl-2,5-dihydropyridazino[4,3-b]-indol-3-
(3H)-ones (PIs), which are structurally related to the
well known 2-aryl-2,5-dihydropyrazolo[4,3-c]-quinoline-
3-(3H)ones (PQs).21 As a continuation of our ongoing
research on this topic we synthesized the 3-chloro (2a±d)
and 3-methoxy (3a±d) pyridazino[4,3-b]-indoles and the
indeno[1,2-c]pyridazines (8, 9a±d) (Schemes 1 and 2) to
better de®ne the in¯uence of both the indolic NH func-
tion, as a hydrogen bond donor group, and the carbonyl
group, as hydrogen bond acceptor group, on the ben-
zodiazepine receptor anity of the new BZR ligands.
Benzodiazepine receptor (BZR) ligands produce their
pharmacological e€ects by modulating the action of
GABA at GABAA-receptor/Cl ionophore supramole-
cular complex.1,2 Such a complex can be formed by dif-
ferent combinations of distinct subunits. A total of at
least 13 subunits (a1 to a6, b1 to b3, g1 to g3 and d)3±5
have been identi®ed by molecular cloning, and are
thought to assemble into a pentameric structure to form
a Cl channel. Most functional subtypes of GABAA
receptors contain a,b and g subunits, with di€erent
subtypes showing diverse sensitivity to di€erent benzo-
diazepine receptor ligands.6±8 BZR ligands bind to
allosteric modulatory sites (o sites) enhancing (agonists)
or reducing (inverse agonists) the GABA-induced Cl
ion ¯ux. A third class of ligands, called antagonists,
exhibit, per se, no relevant biological e€ects but anta-
gonize the action of agonists and inverse agonists.9
Despite the recent signi®cant advances in the structural
and functional studies of BZR,4 and in the molecular
pharmacology and physiopathology of anxiety dis-
orders,3 much remains to be achieved for the design and
development of truly innovative drugs, lacking the
numerous side e€ects of benzodiazepines.10 From a
medicinal chemistry view point, the recent years have seen
the proposal and re®nement of several pharmacophore
Results and Discussion
Chemistry
Scheme 1 outlines the synthetic route to 2-aryl-3-chloro-
2H-pyridazino[4,3-b]indoles 2a±d and 2-aryl-3-methoxy-
2H-pyridazino[4,3-b]indoles 3a±d. In short, the 2-aryl-
3,5-dihydro-pyridazino[4,3-b]indolones 1a±d were reac-
ted with phosphorus oxychloride at 90±100ꢀC to give
the corresponding 3-chloro derivatives 2a±d. The dis-
placement of chloride ion with CH3ONa in dry metha-
nol gave 3-methoxy derivatives 3a±d, which, by heating
at 200ꢀC, underwent the thermal rearrangement to the
N-5 methyl isomers 4a±d, previously reported through
the alkylation of parent compounds 1a±d.20
Key words: Benzodiazepine receptor; binding assay; structure±activity
relationships; pyridazino[4,3-b]indoles; indeno[1,2-c]pyridazines.
* Corresponding author. Fax: +39-080-544-2724; e-mail: campag-
na@farmchim.uniba.it
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00093-0

1534
F. Campagna et al. / Bioorg. Med. Chem. 7 (1999) 1533±1538
Table 1. aUV±Vis data (ethanol) of compounds 1, 2, 3 and 4
Compd
lmax (nm) (log e)
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
218 (4.43)
220 (4.52)
220 (4.51)
218 (4.52)
222 (4.32)
220 (4.42)
222 (4.41)
224 (4.40)
216 (4.45)
216 (4.47)
216 (4.70)
218 (4.35)
220 (4.51)
222 (4.51)
222 (4.44)
222 (4.50)
272 (4.60)
272 (4.67)
272 (4.66)
268 (4.62)
278 (4.56)
278 (4.56)
278 (4.54)
278 (4.54)
286 (4.69)
286 (4.63)
286 (4.86)
280 (4.52)
276 (4.68)
276 (4.64)
274 (4.58)
270 (4.59)
332 (3.77)
332 (3.92)
332 (3.92)
338 (3.86)
426 (3.30)
422 (3.35)
426 (3.31)
422 (3.31)
430 (3.36)
426 (3.33)
428 (3.58)
420 (3.28)
334 (3.85)
334 (3.91)
334 (3.84)
340 (3.85)
Scheme 1. (i) POCl3, 100ꢀC; (ii) CH3ONa, anhydrous MeOH, re¯ux;
(iii) NaH, anhydrous THF, MeI, rt; (iv) Á, 200ꢀC. a: R=H; b: R=Cl;
c: R=Br; d: R=OCH3.
a
The UV±Vis data of compounds 2 and 3 are reported together with
those of previously described congeners 1 and 4,20 for comparison.
Binding studies
All the new ligands 2, 3, 8 and 9 were evaluated for their
ability to displace [3H]¯unitrazepam from BZR (Table
3), by a previously described method.19 Data from the
present work revealed that compounds with electron
rich substituents in the para position of the N-2 phenyl
ring were, in all the series, more active than the corre-
sponding unsubstituted congeners. Binding anities of
3-chloro derivatives 2a±d were higher than the corre-
sponding 3-methoxy analogues 3a±d. This may be
ascribed, at least in part, to the formation of a hydrogen
bond with a proton-accepting group, called H1 in the
BZR pharmacophore model proposed by Cook.18
To support this hypothesis, a molecular modelling study
was undertaken. The results thereof showed that the
lower BZR anity of 3a±d with respect to 2a±d analo-
gues might indeed result from the di€erent location and
orientation of the lone pairs of oxygen and chlorine
atoms, possibly involved in a hydrogen bonding at the
H1 site. The subtle di€erences between the 3-D struc-
tures of 1a, 2a, and 3a may be better perceived by a
simple visual inspection of the molecular superposition
realized via the ®tting of three supposed receptor
anchoring points that are the N-1 atom and the cen-
troids C1 and C2 of the benzene of the benzofused
moiety and of the 2-phenyl ring, respectively (Fig. 1). In
particular, the overlay of the three ligands shows that
the oxygen lone pairs of the methoxy group (3a, violet),
unlike those of the oxygen of the carbonyl (1a, red) and
those of chlorine (2a, green) do not point toward the
H1 region. Moreover the dramatic drop of activity
observed for compounds 3a±d can be, at least in part,
attributed to the steric hindrance caused by the OCH3
group at the S1 unaccessible region, in accord with the
same pharmacophore model. Thus, as in the case of 4a±
d previously reported by us,20 both the steric hindrance
and the removal of a possible hydrogen bond with a
speci®c binding site on the receptor may be responsible
for their poor anity. The 5-deaza-analogues 8a±d and
9a±d, which possess topological features similar to 1a±d,
Scheme 2. (i) Anhydrous MeOH, rt; (ii) DCC, anhydrous CH3CN, rt;
(iii) Br2, CH3COOH, 100ꢀC. a: R=H; b: R=Cl; c: R=Br; d:
R=OCH3.
Compounds 2a±d and 3a±d show UV±Vis spectra indi-
cative of an electronic structure more conjugated than
parent compounds 1a±d and N-5 methyl homologues
4a±d (Table 1). The 2-aryl-indeno[1,2-c]pyridazinone
derivatives 8a±d and 9a±d were synthesized as indicated
in Scheme 2. In brief, 1-keto-2-indanylacetic acid 522
was reacted at room temperature with the appropriate
phenylhydrazine 6 to give the intermediate arylhydra-
zones 7a±d, which were then easily cyclized to 2-aryl-
2,4,4a,5-tetrahydroindeno[1,2-c]pyridazino-3(3H)-ones
8a±d by treatment with dicyclohexylcarbodiimide in
anhydrous acetonitrile. Compounds 8a,b, and d, had
spectroscopic and physicochemical properties consistent
with those reported by Nakao and co-workers, who
prepared the same compounds by a di€erent proce-
dure.23 Finally 2-aryl-2,5-dihydroindeno[1,2-c]pyrid-
azino-3(3H)-ones 9a±d were obtained by oxidation of
8a±d with bromine in acetic acid at 100ꢀC. Physical and
spectroscopic properties of compounds 2, 3, 7, 8, and 9
are listed in Table 2. Compounds 3 had melting points
consistent with their complete thermal rearrangement to
the corresponding N-5 methyl congeners 4.

F. Campagna et al. / Bioorg. Med. Chem. 7 (1999) 1533±1538
Table 2. Physical and spectroscopic data of compounds 2, 3, 7,a 8, and 9
1535
Compd
mp (ꢀC)
(crystallization
solvent)
IR
max (cm )
1H NMR d [ppm, J (Hz)]b
1
n
2a
2b
271±273 dec (ligroin)
280±285 dec (ligroin)
1610, 1550
7.25±7.35 (m, 1H, Arom), 7.45±7.60 (m, 5H, Arom), 7.63 (s, 1H, Arom), 7.65±7.75
(m, 1H, Arom), 7.83 (d, 1H, Arom, J=8.2), 8.19 (d, 1H, Arom, J=7.7)
7.25±7.35 (m, 1H, Arom), 7.40±7.50 (m, 2H, Arom), 7.50±7.60 (m, 2H, Arom), 7.61
(s, 1H, Arom), 7.65±7.75 (m, 1H, Arom), 7.82 (d, 1H, Arom, J=8.1), 8.16 (d, 1H,
Arom, J=7.9)
1610, 1550, 1545
2c
2d
290±300 dec (ligroin)
305±310 dec (ligroin)
1610, 1560
1600, 1560
7.30±7.45 (m, 3H, Arom), 7.62 (s, 1H, Arom), 7.65±7.75 (m, 3H, Arom), 7.82 (d, 1H,
Arom, J=7.9), 8.17 (d, 1H, Arom, J=7.7)
3.90 (s, 3H, CH3), 7.00±7.10 (m, 2H, Arom), 7.25±7.35 (m, 1H, Arom), 7.35±7.45 (m,
2H, Arom), 7.61 (s, 1H, Arom), 7.65±7.70 (m, 1H, Arom), 7.82 (d, 1H, Arom,
J=8.1), 8.18 (d, 1H, Arom, J=7.4)
3a
3b
3c
3d
7b
217±219c (methanol)
273±274c (methanol)
272±274c (methanol)
220±222c (methanol)
129±132 (methanol)
1570
4.04 (s, 3H, CH3), 7.10±7.20 (m, 1H, Arom), 7.22 (s, 1H, Arom), 7.50±7.70 (m, 7H,
Arom), 8.03 (d, 1H, Arom, J=8.0)
4.05 (s, 3H, CH3), 7.15±7.25 (m, 2H, Arom), 7.55±7.65 (m, 2H, Arom), 7.65±7,75 (m,
4H, Arom), 8.03 (d, 1H, Arom, J=7.7)
4.03 (s, 3H, CH3), 7.10±7.25 (m, 2H, Arom), 7.50±7.65 (m, 4H, Arom), 7.75±7.85 (m,
2H, Arom), 8.02 (d, 1H, Arom, J=7.5)
3.85 (s, 3H, CH3), 4.03 (s, 3H, CH3), 7.05±7.25 (m, 4H, Arom), 7.50±7.60 (m, 4H,
Arom), 8.02 (d, 1H, Arom, J=7.4)
1565
1570
1565
3310, 1715, 1605
2.00±2.15 (m, 1H, CH2), 2.70±2.95 (m, 2H, CH2), 3.30±3.40 (m, 1H, CH2), 3.60±3.75
(m, 1H, CH), 7.15±7.35 (m, 7H, Arom), 7.55±7.65 (m, 1H, Arom), 9.50 (s, 1H, NH),
12.20 (br s, 1H, OH)
7c
7d
143±144 (methanol)
115±117 (methanol)
3310, 1710, 1600
3300, 1715, 1610
2.00±2.15 (m, 1H, CH2), 2.70±2.90 (m, 2H, CH2), 3.30±3.40 (m, 1H, CH2), 3.60±3.75
(m, 1H, CH), 7.10±7.20 (m, 2H, Arom), 7.20±7.40 (m, 5H, Arom), 7.55±7.65 (m, 1H,
Arom), 9.51 (s, 1H, NH), 12.15 (br s, 1H, OH)
1.95±2.15 (m, 1H, CH2), 2.70±2.95 (m, 2H, CH2), 3.30±3.40 (m, 1H, CH2), 3.60±3.75
(m, 1H, CH), 3.68 (s, 3H, CH3, overlapped to the previous multiplet), 6.80±6.90 (m,
2H, Arom), 7.10±7.20 (m, 2H, Arom), 7.20±7.35 (m, 3H, Arom), 7.50±7.60 (m, 1H,
Arom), 9.16 (s, 1H, NH), 12.24 (br s, 1H, OH)
8a
8b
8c
8d
143±144 (methanol)
132±133 (ethanol)
152±154 (ethanol)
136±138 (ethanol)
1670, 1660
1670
2.50 (t, 1H, H-4A, J=16.1), 2.80 (dd, 1H, H-5A, J=16.3 and 5.5), 3.08 (dd, 1H, H-
4B, J=16.1 and 6.9), 3.20±3.40 (m, 1H, H-4a), 3.47 (dd, 1H, H-5B, J=16.3 and 8.5),
7.20±7.50 (m, 6H, Arom), 7.55±7.62 (m, 2H, Arom), 7.78 (d, 1H, Arom, J=7.4)
2.52 (t, 1H, H-4A, J=16.1), 2.80 (dd, 1H, H-5A, J=16.4 and 5.6), 3.07 (dd, 1H, H-
4B, J=16.1 and 6.8), 3.20±3.40 (m, 1H, H-4a), 3.47 (dd, 1H, H-5B, J=16.4 and 8.5),
7.30±7.50 (m, 5H, Arom), 7.50±7.60 (m, 2H, Arom), 7.77 (d, 1H, Arom, J=7.5)
2.51 (t, 1H, H-4A, J=16.0), 2.79 (dd, 1H, H-5A, J=16.4 and 5.5), 3.07 (dd, 1H, H-
4B, J=16.0 and 6.8), 3.20±3.35 (m, 1H, H-4a), 3.47 (dd, 1H, H-5B, J=16.4 and 8.5),
7.30±7.60 (m, 7H, Arom), 7.77 (d, 1H, Arom, J=7.4)
2.51 (t, 1H, H-4A, J=16.0), 2.79 (dd, 1H, H-5A, J=16.3 and 5.6), 3.06 (dd, 1H, H-
4B, J=16.0 and 6.9), 3.20±3.35 (m, 1H, H-4a), 3.46 (dd, 1H, H-5B, J=16.3 and 8.5),
3.81 (s, 3H, CH3), 6.90±7.00 (m, 2H, Arom), 7.25±7.50 (m, 5H, Arom), 7.77 (d, 1H,
Arom, J=7.6)
1675
1675, 1600
9a
9b
9c
9d
165±167 (methanol)
220±222 (methanol)
215±217 (methanol)
180±182 dec (methanol)
1655, 1605
1670, 1620
1670, 1620
1650, 1600
3.96 (s, 2H, CH2), 7.05±7.10 (m, 1H, H-4), 7.35±7.55 (m, 6H, Arom), 7.60±7.70 (m,
2H, Arom), 7.85±7.95 (m, 1H, Arom)
4.05 (s, 2H, CH2), 7.10±7.15 (m, 1H, H-4), 7.40±7.70 (m, 7H, Arom), 7.79 (d, 1H,
Arom, J=7.4)
4.06 (s, 2H, CH2), 7.15±7.20 (m, 1H, H-4), 7.40±7.55 (m, 2H, Arom), 7.55±7.65 (m,
3H, Arom), 7.65±7.75 (m, 2H, Arom), 7.73 (d, 1H, Arom, J=7.3)
3.81 (s, 3H, O-CH3), 4.04 (s, 2H, CH2), 7.00±7.10 (m, 2H, Arom), 7.10±7.15 (m, 1H,
H-4), 7.40±7.55 (m, 4H, Arom), 7.55±7.65 (m, 1H, Arom), 7.75±7.85 (m, 1H, Arom)
a
Physical and spectroscopic data of compound 7a have been reported in our previous paper.27
b
1H NMR spectra were recorded in CDCl3 (2, 8) or in DMSO-d6 (3, 7, 9), the abbreviations used are as follows: s, singlet; d, doublet; dd, double
doublet; t, triplet; m, multiplet(s); br, broad signal.
c
Melting point refers to the corresponding thermally rearranged compound 4.
but lack the NH indolic function, displayed an anity
for BZR six- to 10-fold lower than the corresponding
ligands 1a±d but comparable to that of 2a±d. The latter,
unlike compounds 8 and 9, have topological features
similar to the enol tautomers of 1a±d and similarly to
compounds 8 and 9 do not have a hydrogen bond donor
group. Thus the observed drop of the activity can be
substantially attributed to the removal of a conceivable
HB donation from the NH group to a proton-accepting
group in the receptor. Nonetheless, 2d, which lacks a
hydrogen bond donor group at 5-position conserves a
moderate anity (IC50=90.7 nM) for the BZR. Finally,
the higher BZR anity of 8a±d with respect to 9a±d
may indicate that the topographic planarity or quasi-
planarity of these molecules is not a crucial element for
their BZR anity, unlike that observed from Nakao et
al. for a series of higher homologues.23
A preliminary analysis of the structure±intrinsic ecacy
relationships was undertaken on the basis of the GABA
ratio values reported in Table 3. The GABA ratio can
be taken as a good estimate of agonist, antagonist and
inverse agonist activity for a BZR ligand.24,25 As repor-
ted in a previous study,19 the experimental method used

1536
F. Campagna et al. / Bioorg. Med. Chem. 7 (1999) 1533±1538
of the tetrahydroindenopyridazines 8b±d (1.20±1.47)
suggested a full agonist activity. Also for the para chloro
derivative 2b the GABA ratio indicated a similar ago-
nist pro®le as observed for the corresponding parent
compound 1b and the pyrazoloquinoline analogue (PQ)
CGS 9896.26 The GABA-ratio values of compounds 2c
and 9b±d suggested a probable antagonist activity,
unlike the corresponding congeners from the PQ and
the PI series, for which di€erent intrinsic activities have
been previously reported.19±21 Unexpectedly 2d displayed
an inverse agonist activity (GABA ratio 0.88) unlike the
corresponding congeners of the PQ and PI series for
which an antagonistic activity has been found.19,26
Conclusions
The PIs20 have been recently shown to bind with high
anities to BZR. The relatively low anity of the newly
synthesized compounds 2, 3, 8, and 9, underlines the
importance of the indolic NH function, as a hydrogen
bond donor group, for a high binding potency at the
central BZR of PI ligands 1. Analogues 3, which lack
the HB with H1 site and might be subjected to some
steric hindrance at the S1 region (see Cook model,
Ref18), are essentially inactive. Furthermore it was con-
®rmed that compounds with electron rich substituents
in the para position of the N-2 phenyl ring were, in
all the series, more active than the corresponding
unsubstituted congeners. In vivo studies have been
planned on compounds 2 and 3, which upon metabolic
transformation, could furnish the corresponding more
active compounds 1, as we have previously observed for
the N-5 methylated congener 4b, whose anticonvulsant
activity was comparable to that of diazepam.20
Figure 1. Fitted elements are C1 and C2 (aromatic centroids) and N1.
The oxygen and chlorine lone pairs, potentially involved in a HB at
the H1 site, are represented in red and in green for compounds 1a and
2a, respectively. The oxygen lone pairs of the methoxy group of com-
pound 3a (violet) are not correctly aligned to make HB at the H1 site.
Table 3. Inhibition of [3H]¯unitrazepam binding and GABA-ratioa
Compd
R
IC50 nM (‹SD)
GABA-ratiob
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
8a
8b
8c
8d
9a
9b
9c
9d
H
Cl
Br
OCH3
H
Cl
238‹18
31.1‹1
39.4‹2
23.9‹1
2490‹184
253‹36
282‹58
90.7‹8.7
[37] c
915‹48
1180‹53
3520‹800
1560‹513
2485‹449
[37] c
994‹44
2670‹440
171‹5
180‹5
180‹6
2180‹170
407‹27
293‹31
398‹16
0.94
1.12
1.13
1.02
N.D.d
1.17
1.01
Br
OCH3
H
Cl
0.88
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
1.47
1.20
1.43
N.D.
0.98
1.04
Experimental
Chemistry
Br
OCH3
H
Cl
Br
Melting points were taken on a Gallenkamp MFB 595
010 M apparatus and are uncorrected. Elemental ana-
lyses were performed on a Carlo Erba 1106 analyzer for
C, H, N; experimental results agreed to within ‹0.40%
of the theoretical values. IR spectra were recorded using
potassium bromide disks on a Perkin±Elmer 283 spec-
trophotometer, only the most signi®cant and diagnostic
absorption bands being reported. 1H NMR spectra
were recorded on a Bruker AM 300 WB 300 MHz
spectrometer. Chemical shifts are expressed in d (ppm)
and the coupling constants J in Hz. Exchange with
deuterium oxide was used to identify OH and NH
protons. UV±Vis spectra were recorded on a Hewlett±
Packard 8452A diode array spectrophotometer. Chro-
matographic separations were carried out on silica gel
columns (70±230 mesh, Merck). Phenylhydrazine free
bases, not available commercially, were prepared from
the corresponding phenylhydrazine hydrochlorides.
OCH3
H
Cl
Br
OCH3
H
Cl
Br
OCH3
1.02
a
The binding anities and GABA-ratios of the new ligands 2, 3, 8,
and 9 are reported together with those of already described cogeners 1
and 4 for comparison.
b
GABA-ratio: IC50 (compound)/IC50 (compound +20 mM-GABA).
c
Percentage of inhibition of speci®c [3H]¯unitrazepam binding at 20
mM compound concentration.
N.D.: not determined.
d
by us to measure the BZR binding led to IC50 values
generally four to ten times higher than those reported in
the literature for the same reference compounds. More-
over, in the case of agonists, our method led also to
lower GABA-ratio values. Thus the GABA ratio values
2-Aryl-3-chloro-2H-pyridazino[4,3-b]indoles (2a±d). A
mixture of 2-aryl-2,5-dihydropyridazino[4,3-b]indol-
3(3H)-one 1 (1 mmol) and phosphorus oxychloride

F. Campagna et al. / Bioorg. Med. Chem. 7 (1999) 1533±1538
1537
(5.9 mL) was heated with stirring at 90±100ꢀC for 3 h.
After cooling, petroleum ether (40 mL) was added
dropwise to the stirred reaction mixture. The oily resi-
due was separated from the ethereal solution by decan-
tation and then basi®ed with diluted NH4OH. The
resulting suspension was stirred for 10 min and then the
orange precipitate ®ltered, washed with water and dried.
Recrystallization from ligroin furnished 2a (0.234 g,
84% yield), 2b (0.223 g, 71% yield), 2c (0.262 g, 73%
yield), and 2d (0.251 g, 81% yield).
respectively. In the case of 9d the dried solid was pur-
i®ed by chromatography on silica gel (ethyl acetate/
petroleum ether, 8/2 as eluent) and then recrystallized
from methanol to give 9d (0.087 g, 30% yield).
Binding studies. The binding anities data, reported
in Table 3 were obtained by the method previously
described.19
Molecular modelling studies. Molecular models of the
heterocyclic compounds 1a, 2a, and 3a were built on a
Silicon Graphics Indigo 2 workstation from the frag-
ments library of SYBYL 6.4 molecular modelling soft-
ware (Tripos, St. Louis, MO, USA) and fully optimized
by the AM1 Hamiltonian.28 The minimum energy con-
formers selected upon a conformational search on the
torsion angle of the N-2 phenyl ring, were aligned with
the RIGIDFIT module of SYBYL by superimposing
the three supposed anchoring points that are the N-1
atom and the centroids C1 and C2 of the benzofused
moiety and of the 2-phenyl ring, respectively. The
superimposed molecules are shown in Figure 1.
2-Aryl-3-methoxy-2H-pyridazino[4,3-b]indoles (3a±d). A
solution of sodium methoxide (0.015 g, 0.28 mmol) in
dry methanol (0.8 mL) was added to a stirred suspen-
sion of 2 (0.21 mmol) in dry methanol (3 mL). The
reaction mixture was re¯uxed under nitrogen for 2.5 h.
After cooling, the solid was ®ltered and then recrys-
tallized from acetonitrile/water to give 3a (0.040 g, 68%
yield), 3b (0.056 g, 86% yield), 3c (0.037 g, 50% yield),
3d (0.049 g, 70% yield).
Thermal isomerization of 3a±d to 4a±d. Compound 3
was heated in a sealed glass tube at 200ꢀC for 10 min
and then cooled at room temperature. The solid was
recrystallized from methanol to give 4 in quantitative
yield.
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1538
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