Synthesis and antibacterial activity of pyridazino[4,3-b]indole-4-carboxylic acids carrying different substituents at N-2

Article abstract of DOI:10.1016/S0014-827X(01)01173-9

The synthesis and the in vitro evaluation of antibacterial activity of new pyridazino[4,3-b]indole-4-carboxylic acids 2-4, 6 against some selected representative of Gram-positive and Gram-negative bacteria are reported. The role of the lipophilicity in th

Full text of DOI:10.1016/S0014-827X(01)01173-9

Il Farmaco 57 (2002) 63–69
www.elsevier.com/locate/farmac
Synthesis and antibacterial activity of
pyridazino[4,3-b]indole-4-carboxylic acids carrying different
substituents at N-2
Fausta Palluotto, Francesco Campagna *, Angelo Carotti, Marcello Ferappi,
Antonio Rosato, Cesare Vitali
Dipartimento Farmacochimico, Uni6ersita` di Bari, 6ia Orabona 4, 70126 Bari, Italy
Received 5 June 2001; received in revised form 9 August 2001; accepted 14 August 2001
Dedicated to Professor Giovanni Casini on the occasion of his retirement
Abstract
The synthesis and the in vitro evaluation of antibacterial activity of new pyridazino[4,3-b]indole-4-carboxylic acids 2–4, 6
against some selected representative of Gram-positive and Gram-negative bacteria are reported. The role of the lipophilicity in the
modulation of the antibacterial activity of the tested compounds is discussed. All the synthesized compounds appear quite weak
against Gram-positive bacteria, whereas have no significant activity against Gram-negative bacteria. Only derivative 2g possesses
an interesting activity against Gram-positive bacteria. © 2002 Elsevier Science S.A. All rights reserved.
Keywords: Pyridazino[4,3-b]indole; Norfloxacin; Antibacterial activity; Isatin derivatives
for a more potent and extended antibacterial activity,
we decided to enlarge our investigation by synthesizing
new lipophilic 2-aryl-3,5-dihydro-3-oxo-2H-pyridazino-
[4,3-b]indole-4-carboxylic acids 2e, h, as well as a new
series of simplified and less lipophilic N-2 alkylated
analogs 3a, i, l, 4a, i or N-2 unsubstituted 6a, i, l and
their derivatives 8 and 10 (Schemes 1–3).
1. Introduction
As a part of our ongoing research on the central
benzodiazepine receptor ligands, we recently reported
the synthesis, biological and pharmacological activities
of a large series of 2-aryl-2,5-dihydro-pyridazino[4,3-
b]indol-3(3H)-ones (PI) [1,2]. The synthetic pathway of
PIs, led us to prepare, as intermediates, a number of
2-aryl-3,5-dihydro-3-oxo-2H-pyridazino[4,3-b]indole-4-
carboxylic acids that were screened against some se-
lected representative of Gram-positive and Gram-nega-
tive bacteria [3]. Some of the tested compounds showed
an interesting antibacterial activity against Gram-posi-
tive bacteria whereas no activity was observed against
Gram-negative microorganisms. A preliminary struc-
ture–antibacterial activity relationship study suggested
the lipophilicity as an important property modulating
the biological activity.
2. Chemistry
The synthetic pathway leading to compounds 2e, h,
3a, i, l, 4a, i, 6a, i, l is described in Scheme 1. The key
intermediates diethyl-2-(3-oxo-1,3-dihydro-2H-indol-2-
ylidene)malonates 1a, h, i, l were prepared by refluxing
the corresponding isatins with phosphorus pentachlo-
ride in anhydrous benzene to give 2-chloro-3H-indol-3-
one derivatives which were then condensed with
diethylmalonate sodium salt according to a method
previously described by us [1,2].
With the aim to confirm that preliminary observation
and to better defining the main structural requirements
The intermediates 1a, h were condensed with p-
OCF3- and p-Br-phenylhydrazine hydrochloride respec-
tively to afford compounds 2e, h. Treatment of 1a, i, l
* Corresponding author.
E-mail address: campagna@farmchim.uniba.it (F. Campagna).
0014-827X/02/$ - see front matter © 2002 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01173-9

64
F. Palluotto et al. / Il Farmaco 57 (2002) 63–69
Scheme 1. Reagents: (i) R3-C6H4-NHNH2·HCl–EtOH, H2O, reflux; (ii) CH3-NHNH2–HCl, EtOH, H2O, reflux; (iii) (CH3)3C-NHNH2·HCl–
EtOH, H2O, reflux; (iv) NH2NH2·HCl–EtOH, H2O, reflux; (v) 1. NaOH–EtOH, reflux; 2. HCl, r.t.
with methylhydrazine in the presence of HCl directly
gave N-2 methyl derivatives 3a, i, l. When t-butylhy-
drazine hydrochloride was reacted with 1a, i, the N-2
t-butyl congeners 4a, i were obtained. The N-2 unsub-
stituted derivatives 6a, i, l were prepared through the
hydrolysis of the corresponding ethyl esters 5a, i, l,
obtained in turn by reacting 1a, i, l with hydrazine
hydrochloride. Spectral data of compounds 5 and 6
indicated that they exist only in the tautomeric 3-hy-
droxypyridazine form. The N-5 and O-3 ethyl deriva-
tives 8 and 10 were synthesized as depicted in Schemes
2 and 3, in order to make a proper comparison with the
unsubstituded congeners 3a and 6a, respectively. The
analytical and spectroscopic data of all the synthesized
compounds are reported in Table 1.
series of 2-aryl-3,5-dihydro-3-oxo-2H-pyridazino[4,3-
b]indole-4-carboxylic acids 2a–g the lipophilicity of the
substituent in para position is directly related to the
antibacterial activity. This relationship is more evident
for the Bacillus subtilis ATCC 6633, Enterococcus fae-
calis ATCC 29212 and Staphylococcus aureus ATCC
29213 Gram-positive organisms. Interestingly, a linear
relationship between log 1/MIC (MIC expressed in mM)
and the hydrophobic substituent constant y was found
(n=7, r2=0.73, s=0.31). However, a further strong
increase of the lipophilicity did not afford a higher
activity (compare 2h with 2d). The effect of lipophilicity
was further explored by eliminating the N-2 aryl sub-
stituent (derivatives 6) or by replacing it with alkyl
substituents of markedly different lipophilicity (deriva-
tives 3 and 4). Unexpectedly, the less lipophilic N-2
unsubstituted compound 6a elicited an activity very
close to that of the corresponding more lipophilic N-2
3. Biological results and conclusions
The MIC values reported in Table 2 showed that
compounds 2, 3, 4 and 6 are weakly active against
tested Gram-positive bacteria and substantially inactive
against Gram-negative bacteria. Compound 2g elicited
the highest activity against Gram-positive bacteria with
MIC values ranging from 0.78 to 1.6 mg/ml. MIC data
in Table 2 confirm that, with few exceptions, in the
Scheme 2. Reagents: (i) 1. NaH–an DMF, r.t.; 2. CH3CH2I–an
DMF, r.t.; (ii) 1. NaOH–EtOH, reflux; 2. HCl, r.t.

F. Palluotto et al. / Il Farmaco 57 (2002) 63–69
65
Scheme 3. Reagents: (i) 1. NaH–an DMF, r.t.; 2. CH3CH2I–an DMF, r.t.; (ii) 1. NaOH–EtOH, reflux; 2. HCl, r.t.
Table 1
Physical and spectroscopic data of compounds 2–10
Comp
m.p. (°C)
IR wmax, (cm−1
)
1H NMR, l (ppm), J (Hz) a
(crystallization
solvent)
2e
2h
323–325 dec (acetic
acid)
260–261 (methanol)
3300, 1710, 1640, 7.25–7.40 (m, 1H, Arom); 7.55–7.70 (m, 4H, Arom); 7.75–7.90 (m, 2H, Arom); 8.01 (d,
1610 1H, Arom, J=7.9); 12.20 (s, 1H, NH); 14.30 (s, 1H, COOH)
3290, 1710, 1650, 1.10–1.55 (m, 6H, 3CH2); 1.65–1.85 (m, 4H, 2CH2); 2.50–2.70 (m, 1H, CH); 7.45–7.55
1620
(m, 2H, Arom); 7.60–7.70 (m, 2H, Arom); 7.75–7.80 (m, 2H, Arom); 7.82 (s, 1H,
Arom); 12.09 (s, 1H, NH); 14.33 (s, 1H, COOH)
3a
3i
305–310 dec (dioxane) 3310, 1710, 1650, 3.94 (s, 3H, CH3); 7.25–7.40 (m, 1H, Arom); 7.55–7.65 (m, 2H, Arom); 7.99 (d, 1H,
1615
Arom, J=7.6); 12.06 (s, 1H, NH); 14.60 (s, 1H, COOH)
326–328 dec (dioxane) 3315, 1700, 1650
3.94 (s, 3H, CH3); 7.46 (dt, 1H, Arom, J=8.8, J=2.6); 7.58 (dd, 1H, Arom, J=8.8,
J=4.4); 7.84 (dd, 1 H, Arom, J=8.2, J=2.6); 12.10 (s, 1H, NH)
3l
302–303 (dioxane)
3255, 1695, 1650, 3.95 (s, 3H, CH3); 7.70 (d, 1H, J=6.0); 8.11 (d, 1H, J=8.5); 12.10 (s, 1H, NH); 14.45
1625
(s, 1H, COOH)
4a
4i
304–305 (acetonitrile) 3305, 1710, 1650
314–316 (acetonitrile) 3280, 1710, 1630
1.74 (s, 9H, 3CH3); 7.31 (dd, 1H, Arom, J=7.0, J=1.9); 7.55–7.65 (m, 2H, Arom);
8.00 (d, 1H, Arom, J=7.8); 12.00 (s, 1H, NH); 15.00 (s, 1H, COOH)
1.79 (s, 9H, 3CH3); 7.26 (dt, 1H, J=8.7, J=2.3); 7.36 (dd, 1H, J=8.7, J=4.1); 7.71
(dd, 1H, J=7.8, J=2.3); 9.92 (s, 1H, NH); 14.78 (s, 1H, COOH)
1.32 (t, 3H, CH3, J=7.0); 4.35 (q, 2H, CH2, J=7.0); 7.20–7.30 (m, 1H, Arom);
7.45–7.55 (m, 2H, Arom); 7.89 (d, 1H, Arom, J=7.8); 11.46 (s, 1H, NH), 13.19 (s,
1H, OH)
1.31 (t, 3H, CH3, J=7.2); 4.35 (q, 2H, CH2, J =7.2); 7.35 (dt, 1H, Arom, J=8.8,
J=2.5); 7.51 (dd, 1H, Arom, J=8.8, J=4.4); 7.73 (dd, 1H, Arom, J=8.2, J=2.5);
11.48 (s, 1H, NH); 13.30 (s, 1H, OH)
5a
306–307 (ethanol)
3220, 1650, 1630
3240, 1690, 1650
5i
324–326 (acetic acid)
5l
6a
6i
343–347 dec (acetic
acid)
336–345 dec (acetic
acid)
3240, 1695, 1650
3260, 1710, 1640
3285, 1690, 1640
1.31 (t, 3H, CH3, J=7.0); 4.36 (q, 2H, CH2, J=7.0); 7.61 (d, 1H, Arom, J=6.0);
7.97 (d, 1H, Arom, J=8.5); 11.54 (s, 1H, NH); 13.40 (s, 1H, OH)
7.25–7.35 (m, 1H, Arom), 7.55–7.65 (m, 2H, Arom); 8.02 (d, 1H, Arom, J=7.7); 12.08
(s, 1H, NH); 14.18 (s, 1H, OH), 14.80 (s, 1H, COOH)
7.45 (dt, 1H, Arom, J=8.8, J=2.5); 7.59 (dd, 1H, Arom, J=8.8, J=4.4); 7.87 (dd,
1H, Arom, J=8.2, J=2.5); 12.08 (s, 1H, NH); 14.25 (s, 1H, OH); 14.74 (s, 1H,
COOH)
\350 (acetic acid)
6l
7
340–350 dec (acetic
acid)
130–133
3230, 1710, 1655, 7.69 (d, 1H, Arom, J=6.0); 8.14 (d, 1H, Arom, J=8.6); 12.13 (s, 1H, NH); 14.40 (s,
1625
1H, OH), 14.80 (s, 1H, COOH)
1730, 1640, 1610
1.38 (t, 3H, CH3, J=7.2); 1.48 (t, 3H, CH3, J=7.2); 3.98 (s, 3H, CH3); 4.06 (q, 2H,
CH2, J=7.2); 4.54 (q, 2H, CH2, J=7.2); 7.20–7.35 (m, 2H, Arom); 7.55 (dt, 1H,
Arom, J=7.6, J=1.2); 8.02 (d, 1H, Arom, J=7.6)
(acetonitrile–water)
8
9
205–210 (dioxane)
3430, 1720, 1595
1.25 (t, 3H, CH3, J=7.2); 3.85 (s, 3H, CH3); 4.30 (q, 2H, CH2, J=7.2); 7.25–7.35 (m,
1H, Arom); 7.55–7.65 (m, 2H, Arom); 7.98 (d, 1H, Arom, J=7.5)
168–170
3500, 1720, 1630, 1.32 (t, 3H, CH3, J=7.1); 1.33 (t, 3H, CH3, J=7.1); 4.25 (q, 2H, CH2, J=7.1); 4.36
(acetonitrile–water)
1610
(q, 2H, CH2, J=7.1); 7.20–7.25 (m, 1H, Arom); 7.45–7.55 (m, 2H, Arom); 7.91 (d,
1H, Arom, J=7.7); 11.45 (s, 1H, NH)
10
295–305 dec (acetic
3300, 1700, 1640, 1.39 (t, 3H, CH3, J=7.1); 4.38 (q, 2H, CH2, J=7.1); 7.30–7.35 (m, 1H, Arom);
acid)
1605
7.55–7.65 (m, 2H, Arom); 8.03 (d, 1H, Arom, J=7.9); 12.07 (s, 1H, NH); 14.75 (s,
1H, COOH)
a 1H NMR spectra were recorded in DMSO-d6 with the exception of compound 4i and 7 (CDCl3).
alkyl derivatives 3a and 4a and even greater of that
of the N-2 phenyl analog 2a. As for the N-2 alky-
lated compounds, the N-2 CH3 derivatives 3 showed
an activity close to that of the corresponding more
lipophilic N-2 tert-butyl derivatives 4. The introduc-
tion of halogens in positions 7 and 8 afforded more
active compounds only in the series of N-2 unsubsti-
tuted derivatives (compare compound 6l with 6a, i).
Finally the alkylation of the indolic NH in the N-2
methyl, or phenolic OH in the N-2 unsubstituted se-
ries (derivatives 8 and 10 respectively), led to a drop
of activity.

Table 2
Antibacterial activity of pyridazino[4,3-b]indole-4-carboxylic acids
Comp
MIC (mg/ml)
B. su.
X
R1
R2
R4
R5
E. fa.
S. au. 1
S. au. 2
S. au. 3
E. co. 1
E. co. 2
P. ae.
Kl. ox.
N.D.
2a a
C6H5
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
\50
\50
6.2
12.5
25
5
6.2
12.5
3.1
25
\50
5
\50
6.2
\50
0.78
25
12.5
3.1
25
25
25
1.6
6.2
3.1
0.78
25
12.5
3.1
6.2
N.D.
N.D.
N.D.
N.D.
6.2
N.D.
1.6
25
\50
25
20
\50
\50
\50
\50
12.5
\50
\50
\50
12.5
\50
\50
\50
\50
\50
12.5
\50
\50
\50
0.8
\50
\50
\50
25
2b a
C6H4-p-F
C6H4-p-Cl
C6H4-p-Br
C6H4-p-OCF3
C6H4-p-I
C6H4-p-CH(CH3)2
C6H4-p-Br
CH3
CH3
CH3
C(CH3)3
C(CH3)3
N.D.
N.D.
N.D.
/
2c a
2d a
6.2
1.6
1.6
0.78
3.1
12.5
3.1
3.1
3.1
3.1
\50
12.5
\50
\50
\50
\50
\50
\50
\50
\50
12.5
12.5
12.5
\50
\50
0.2
2e
\50
\50
\50
\50
\50
\50
\50
\50
\50
\50
\50
25
\50
N.D.
\50
2f a
2g b
0.78
5
2h
3a
3i
3l
4a
4i
6a
6i
c C6H11
H
F
F
H
F
H
F
F
H
H
3.1
12.5
3.1
6.2
3.1
\50
\50
\50
\50
\50
\50
\50
\50
\50
\50
\50
(
\50
3.1
3
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.2
9
6.2
H
H
H
12.5
6.2
1.6
12.5
12.5
1.6
\50
\50
1.6
12.5
12.5
6.2
\50
\50
0.2
12.5
12.5
6.2
\50
\50
0.2
\50
12.5
6.2
\50
\50
0.2
6l
8
10
CH3
C2H5
\50
C2H5 \50
0.1
\50
\50
1.6
Norfloxacin
0.8
B. su., B. subtilis ATCC 6633; E. fa., E. faecalis ATCC 29212; S. au.1, S. aureus ATCC 6538; S. au. 2, S. aureus ATCC 29213; S. au. 3, S. aureus ATCC 25923; E. co. 1, Escherichia coli ATCC
8739; E. co. 2, Escherichia coli ATCC 25922; P. ae., Pseudomonas aeruginosa ATCC 27853; Kl. ox., Klebsiella oxytoca ATCC 4913. N.D., not detected.
a The MIC values [3] were reported here in order to make a proper comparative evaluation of the antibacterial activity with that of 2e, g, h.
b The synthesis was reported in Ref. [2].

F. Palluotto et al. / Il Farmaco 57 (2002) 63–69
67
From the above discussion it appears that the
lipophilicity plays an interesting role in the modulation
of the antibacterial activity of the tested compounds.
Lipophilic substituents in para position of the N-2
phenyl ring definitely enhance the activity, whereas a
marginal or no role is played by the lipophilicity of the
alkyl groups at N-2.
Most likely, other physicochemical properties, be-
sides lipophilicity, may influence the observed antibac-
terial activity, and this deserves further investigations.
J=8.5 Hz); 7.48 (d, 2H, Arom, J=8.5 Hz); 7.59 (s, 1
H, CH); 8.23 (s, 1H, NH); 8.45 (s, 1H, OH).
The
N-(4-cyclohexylphenyl)-2-hydroxymino-acet-
amide (2.45 g, 10 mmol) was added portion-wise with
stirring at room temperature (r.t.) to methylsulphonic
acid (8.85 ml). The resulting mixture was refluxed for 2
h and, after cooling, poured on ice (160 ml). The
orange precipitate was filtered, washed with cold water,
dried and recrystallized from chloroform–hexane to
give 5-cyclohexyl-1H-indole-2,3-dione. m.p. 175–
177 °C; IR, wmax (cm−1): 3120, 1765, 1735, 1710, 1625;
1H NMR (DMSO-d6) l (ppm): 1.10–1.90 (m, 10H, 5
CH2); 2.40–2.50 (m, 1H, CH, partially masked from
DMSO signals); 6.81 (d, 1H, Arom, J=8.1 Hz); 7.32
(d, 1H, Arom, J=1.5 Hz); 7.44 (dd, 1H, Arom, J=8.1
Hz, J=1.5 Hz); 10.92 (s, 1H, NH).
4. Experimental
4.1. Chemistry
Melting points were taken on a Gallenkamp MFB
595 010 M apparatus and are uncorrected. Elemental
analyses were performed on a Carlo Erba 1106 analyzer
for C, H, N; experimental results agreed to within
90.40% 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
NMR spectra were recorded on a Bruker AM 300 WB
300 MHz or on a Varian 300 Mercury spectrometers.
Chemical shifts are expressed in l (ppm) and the cou-
pling constants J in Hz. The following abbreviations
were used: s, singlet; d, doublet; t, triplet; q, quadruplet;
m, multiplet; dd, double doublet; dt, double triplet.
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, to be very
difficult to detect. Norfloxacin reference antibacterial
substance, reagents and solvents were purchased from
Sigma–Aldrich Chemie. 5-Cyclohexyl and 5-fluoro-6-
chloro isatins, not available commercially, were pre-
pared by the method of Marvel and Hiers starting from
the appropriate anilines with some modifications (see
preparations) [4–6].
4.1.2. 6-Chloro-5-fluoro-1H-indole-2,3-dione
A mixture of 3-chloro-4-fluoroaniline (7.27 g, 50
mmol) in HCl 1.8 N (35 ml) was added to a water
solution (120 ml) of chloral hydrate (9 g, 54 mmol) and
sodium sulfate decahydrate (130.0 g, 404 mmol). A
water solution (50 ml) of hydroxylamine hydrochloride
(11.0 g, 158 mmol) was then added and the resulting
mixture refluxed for 45 min. The solid obtained after
cooling was filtered, washed with water and recrystal-
lized from water to yield N-(3-chloro-4-fluorophenyl)-2-
hydroxymino-acetamide (2.16 g, 20% yield). m.p.
176–180 °C; IR, wmax (cm−1): 3400, 3350, 1660; 1H
NMR (DMSO-d6) l (ppm): 7.38 (t, 1H, Arom, J=8.8
Hz), 7.58–7.64 (m, 1H, Arom); 7.61 (s, 1H, CH, over-
lapped to aromatic signal); 7.99 (dd, 1H, Arom, J=
6.8, J=2.4 Hz); 10.38 (s, 1H, NH); 12.3 (s, 1H, OH).
The
N-(3-chloro-4-fluorophenyl)-2-hydroxymino-
acetamide (2.14 g, 10 mmol) was added portion-wise
and with stirring to sulfuric acid (6.8 ml), the tempera-
ture being kept at 60–70 °C, then raised to 80 °C for
10 min. After cooling the reaction mixture was poured
on ice (120 ml) and the resulting precipitate was
filtered, washed with cold water, dried and chro-
matographed by the flash technique using ethyl ace-
tate:chloroform mixture (4:6) to obtain 6-chloro-5-
fluoro-1H-indole-2,3-dione (Rf=0.82, 0.499 g, 25%
yield) and 4-chloro-5-fluoro-1H-indole-2,3-dione (Rf=
0.47, 0.898 g, 45% yield).
4.1.1. 5-Cyclohexyl-1H-indole-2,3-dione
A solution of cyclohexylaniline (7.0 g, 40 mmol) in
HCl 1.5 N (28 ml) and dioxane (10 ml) at 90–100 °C
was added to a water (96 ml) solution of chloral
hydrate (14.6 g, 88 mmol) and sodium sulfate decahy-
drate (104.0 g, 324 mmol) heated at 90–100 °C. A
solution of hydroxylamine hydrochloride (17.5 g, 252
mmol) in water (40 ml) was then added and the result-
ing mixture refluxed for 45 min. N-(4-cyclo-
hexylphenyl)-2-hydroxymino-acetamide was obtained
as a slurry which was decanted, washed with water and
recrystallized from chloroform–hexane (2.6 g, 26%
yield). m.p. 157–158 °C; IR, wmax (cm−1): 3360, 3330,
6-Chloro-5-fluoro-1H-indole-2,3-dione was recrystal-
lized from ethanol. m.p. 277–278 °C; IR, wmax (cm−1):
1
3170, 1750, 1710, 1620; H NMR (DMSO-d6) l (ppm):
7.06 (d, 1H, Arom, J=5.8 Hz), 7.65 (d, 1H, Arom,
J=7.6 Hz), 11.14 (s, 1H, NH).
4-Chloro-5-fluoro-1H-indole-2,3-dione was recrystal-
lized from ethanol. m.p. 243–246 °C; IR, wmax (cm−1):
1
3160, 1740, 1710, 1620 cm−1; H NMR (DMSO-d6) l
(ppm): 6.84 (dd, 1H, Arom, J=8.7 Hz, J=3.6 Hz),
7.59 (dd, 1H, Arom, J=9.8 Hz, J=8.7 Hz), 11.14 (s,
1H, NH).
1
1660; H NMR (CDCl3) l (ppm): 1.10–1.90 (m, 10H,
5CH2); 2.40–2.60 (m, 1H, CH); 7.18 (d, 2H, Arom,

68
F. Palluotto et al. / Il Farmaco 57 (2002) 63–69
4.1.3. Diethyl-2-(5-cyclohexyl-3-oxo-1,3-dihydro-2H-
indol-2-ylidene)malonate (1h) and diethyl-2-(6-chloro-
5-fluoro-3-oxo-1,3-dihydro-2H-indol-2-ylidene)-
malonate (1l)
1H, Arom); 7.65–7.70 (m, 2H, Arom); 7.98 (d, 1H,
Arom, J=7.3 Hz); 9.86 (s, 1H, NH).
The hydrolysis with ethanolic NaOH furnished the
acid 2e in almost quantitative yield.
A mixture of appropriately substituted 1H-indole-
2,3-dione (6 mmol), phosphorus (V) chloride (1.32 g,
6.3 mmol) and anhydrous benzene (2.7 ml) was refluxed
for 30 min (1h) or 2 h (1l). The solution was allowed to
cool to r.t. and the intermediate imidoylchloride was
obtained as a solid residue after the evaporation of the
solvent in vacuo (1h) or as a precipitate, which was
collected by filtration under dry nitrogen (1l). After
washing with petroleum ether, the solid was immedi-
ately dissolved in anhydrous dioxane (11 ml) and an
equimolecular solution of diethylmalonate sodium salt
in anhydrous dioxane (13 ml) was then added dropwise.
The reaction mixture was stirred at r.t. overnight. The
precipitate of sodium chloride was filtered off and the
dioxane solution evaporated in vacuo. The residue was
chromatographed on silica gel to furnish 1h (eluent
ethyl acetate–petroleum ether 3:7, Rf=0.55, 0.162 g,
7.3% yield) or recrystallized from cyclohexane to afford
1l (1.52 g, 74% yield).
4.1.5. 8-Cyclohexyl-2-(4-bromophenyl)-3-oxo-3,5-
dihydro-2H-pyridazino[4,3-b]indole-4-carboxylic acid
(2h)
4-Bromophenylhydrazine hydrochloride (0.670 g, 3
mmol) dissolved in ethanol:water (1:1, 15 ml) was
added to a solution of 1h (0.865 g, 2.5 mmol) in ethanol
(20 ml) and the resulting mixture refluxed for 8 h. The
precipitate was collected and recrystallized from
methanol to afford the carboxylic acid 2h (0.116 g, 10%
yield). The recrystallization mother liquor was evapo-
rated in vacuo and the residue chromatographed on
silica gel to give the corresponding ethyl ester (eluent
ethyl acetate–petroleum ether, 4:6, Rf=0.42, 0.865 g,
70% yield). The solid was then recrystallized from
acetonitrile. m.p. 250–252 °C; IR, wmax (cm−1): 3375,
1
1675; H NMR (CDCl3) l (ppm): 1.45 (t, 3H, CH3,
J=7.1 Hz); 1.20–1.65 (m, 6H, 3CH2, overlapped to the
CH3 signals); 1.70–2.00 (m, 4H, 2CH2); 2.50–2.70 (m,
1H, CH); 4.48 (q, 2H, CH2, J=7.1 Hz); 7.18 (d, 1H,
Arom, J=8.4 Hz); 7.36 (dd, 1H, Arom, J=8.4 Hz,
J=1.3 Hz); 7.49–7.54 (m, 2H, Arom), 7.56–7.64 (m,
2H, Arom); 7.84 (d, 1H, Arom, J=1.3 Hz); 9.78 (s,
1H, NH).
4.1.4. 3-Oxo-2-[4-(trifluoromethoxy)phenyl]-3,5-
dihydro-2H-pyridazino[4,3-b]indole-4-carboxylic acid
(2e)
4-Trifluomethoxyphenylhydrazine
hydrochloride
(0.685 g, 3 mmol) dissolved in ethanol:water (1:1, 15
ml) was added to a solution of 1a (0.722 g, 2.5 mmol)
in ethanol (5 ml) and the resulting mixture refluxed for
14 h. The precipitate was collected and recrystallized
from acetic acid to afford the carboxylic acid 2e (0.390
g, 39% yield). The reaction mother liquor was concen-
trated in vacuo to afford a precipitate which was col-
lected and chromatographed on silica gel, using ethyl
acetate:petroleum ether (4:6) as the eluent, to give
The hydrolysis with ethanolic NaOH furnished the
acid 2h in almost quantitative yield.
4.1.6. 2-Methyl-3-oxo-3,5-dihydro-2H-
pyridazino[4,3-b]indole-4-carboxylic acids (3a, i, l)
HCl 6 N (1.65 ml) and methylhydrazine (0.525 ml, 10
mmol) were added to a stirred solution of 1 (0.5 mmol)
in ethanol (10 ml) cooled at 0 °C. The mixture was
then refluxed for 22 h. The precipitate was collected,
washed with ethanol–water (1:1) and recrystallized
from dioxane to afford the carboxylic acid 3 (3a: 0.057
g, 47% yield; 3i: 0.026 g, 20% yield; 3l: 0.030 g, 20%
yield).
1H-indole-2,3-dione
3-{N-[4-(trifluoromethoxy)-
phenyl]hydrazone} (Rf=0.65, 0.158 g, 20% yield) and
ethyl 3-oxo-2-[4-(trifluoromethoxy)phenyl]-3,5-dihydro-
2H-pyridazino[4,3-b]indole-4-carboxylate
(Rf=0.27,
0.227 g, 22% yield).
1H-Indole-2,3-dione 3-{N-[4-(trifluoromethoxy)-phe-
nyl]hydrazone} was recrystallized from lygroin. m.p.
219–221 °C; IR, wmax (cm−1): 3160, 1675, 1670, 1615;
1H NMR (DMSO-d6) l (ppm): 6.91 (d, 1H, Arom,
J=7.6 Hz); 7.04 (t, 1H, Arom, J=7.2 Hz); 7.25 (dt,
1H, Arom, J=7.5 Hz, J=1.1 Hz); 7.34 (d, 2H, Arom,
J=8.5 Hz); 7.50–7.60 (m, 3H, Arom); 11.04 (s, 1H,
NH), 12.70 (s, 1H, NH).
4.1.7. 2-(tert-Butyl)-3-oxo-3,5-dihydro-2H-
pyridazino[4,3-b]indole-4-carboxylic acids (4a, i)
To a stirred solution of 1a (0.246 g, 0.85 mmol) or 1i
(0.261 g, 0.85 mmol) in ethanol–water (5:1, 18 ml),
tert-butylhydrazine hydrochloride (0.530 g, 4.25 mmol,
for 1a or 0.106 g, 0.85 mmol for 1i) was added. The
mixture was refluxed for 40 h. The precipitate was
collected, washed with ethanol–water and recrystallized
from acetonitrile to afford the carboxylic acid 4 (4a:
0.082 g, 34% yield; 4i: 0.051 g, 18% yield).
Ethyl 3-oxo-2-[4-(trifluoromethoxy)phenyl]-3,5-dihy-
dro-2H-pyridazino[4,3-b]indole-4-carboxylate was re-
crystallized from lygroin. m.p. 225–227 °C; IR, wmax
1
(cm−1): 3370, 1670, 1645, 1610; H NMR (CDCl3) l
4.1.8. Ethyl 3-hydroxy-5H-pyridazino[4,3-b]indole-
4-carboxylates (5a, i, l)
To a stirred solution of 1a (0.434 g, 1.5 mmol) or 1i
(ppm): 1.43 (t, 3H, CH3, J=7.1 Hz); 4.46 (q, 2H, CH2,
J=7.1 Hz); 7.25–7.35 (m, 4H, Arom); 7.45–7.55 (m,

F. Palluotto et al. / Il Farmaco 57 (2002) 63–69
69
(0.461 g, 1.5 mmol) or 1l (0.512 g, 1.5 mmol) in
anhydrous ethanol (30 ml), hydrazine hydrochloride
(0.123 g, 1.8 mmol for 1a and 1i or 0.307 g, 4.5 mmol
for 1l) was added. The mixture was refluxed for 22 h
and, after cooling to r.t., the precipitate was collected,
washed with ethanol–water and recrystallized from eth-
anol (5a, 0.274 g, 71% yield) or from acetic acid (5i,
0.231 g, 56% yield; 5l, 0.237 g, 51% yield).
from the corresponding ethyl ester 9 by the method
described for the synthesis of acids 6.
4.2. Microbiology
Tests of antibacterial activity were carried out by the
microdilution method according to NCCLS approved
standard [7,8] against the following American Type
Culture Collection (ATCC) microorganisms: B. subtilis
ATCC 6633, E. faecalis ATCC 29212, S. aureus ATCC
6538, S. aureus ATCC 29213, S. aureus ATCC 25923,
Escherichia coli ATCC 8739, Escherichia coli ATCC
25922, Klebsiella oxytoca ATCC 49131 and Pseu-
domonas aeruginosa ATCC 27853. The minimum in-
hibitory concentration (MIC), that is the lowest
concentration of compound that completely inhibited
bacteria growth, was determined by the method of
two-fold serial dilution technique in liquid media [7,8]
using disposable microplates. Mueller Hinton Broth
(MHB) was used as medium to prepare microdilution
samples (Difco laboratories, Accumedia, and Oxoid,
Basingstoke, UK). The tested substances were dissolved
in dimethyl sulfoxide and diluted with MHB to a final
5% dimethyl sulfoxide solution. The antibacterial activ-
ity was measured after 24 h of incubation at 37 °C.
Quinolone norfloxacin was used as reference sub-
stance in order to make a proper comparative evalua-
tion of the antibacterial activity [8,9].
4.1.9. 3-Hydroxy-5H-pyridazino[4,3-b]indole-
4-carboxylic acids (6a, i, l)
A solution of NaOH (0.200 g, 5 mmol) in ethanol (20
ml) was added with stirring to a suspension of 5 (0.5
mmol) in ethanol (5 ml) and the resulting mixture
refluxed for 1 h. The residue obtained after the evapo-
ration of the solvent in vacuo was suspended in water
(30 ml). The suspension was acidified with conc. HCl to
pH 1 and then stirred at r.t. for 1 h. The formed yellow
solid was filtered, washed with water, and dried. Re-
crystallization from acetic acid gave pure carboxylic
acids 6a, i, l in 75–83% yield.
4.1.10. Ethyl 5-ethyl-2-methyl-3-oxo-3,5-dihydro-2H-
pyridazino[4,3-b]indole-4-carboxylate (7)
Sodium hydride 97% (0.025 g, 1 mmol) was added
with stirring to a suspension of 4a (0.050 g, 0.2 mmol)
in dry DMF (2 ml) under nitrogen. After 20 min
iodoethane (0.080 ml, 1 mmol) was added and stirring
was continued for 8 h. The mixture was then poured on
ice (30 ml). The mixture was extracted twice with 30 ml
of chloroform and the organic phases washed with
water, dried on anhydrous sodium sulfate and evapo-
rated in vacuo. The obtained residue was recrystallized
from acetonitrile–water to give compound 7 (0.022 g,
36% yield).
References
[1] F. Campagna, A. Carotti, G. Casini, F. Palluotto, G. Genchi,
G.B. De Sarro, 2-Aryl-2,5-dihydropyridazino[4,3-b]indol-3(3H)-
ones: Novel rigid planar benzodiazepine receptor ligands, J.
Bioorg. Med. Chem. 1 (1993) 437–446.
[2] F. Palluotto, A. Carotti, G. Casini, F. Campagna, G. Genchi, M.
Rizzo, G.B. De Sarro, Structure–activity relationships of 2-aryl-
2,5-dihydropyridazino[4,3-b]indol-3(3H)-ones at the benzodi-
azepine receptor, J. Bioorg. Med. Chem. 4 (1996) 2091–2104.
[3] F. Palluotto, A. Carotti, G. Casini, M. Ferappi, A. Rosato, C.
Vitali, F. Campagna, Synthesis and antibacterial activity of 2-
aryl-2,5-dihydro-3(3H)-oxo-pyridazino[4,3-b]indole-4-carboxylic
acids, Il Farmaco 54 (1999) 191–194.
4.1.11. 5-Ethyl-2-methyl-3-oxo-3,5-dihydro-2H-
pyridazino[4,3-b]indole-4-carboxylic acid (8)
The carboxylic acid 8 was prepared (66% yield) by
hydrolysis of the corresponding ethyl ester 7 by the
method reported for the synthesis of acids 6.
[4] T. Sandmeyer, Sandmeyer isonitrosoacetanilide isatin synthesis,
Helv. Chim. Acta 2 (1919) 234.
4.1.12. Ethyl 3-ethoxy-5H-pyridazino[4,3-b]indole-4-
carboxylate (9)
[5] C.S. Marvel, G.S. Hiers, Isatin, Organic Synthesis, coll, vol. I,
2nd ed., 1941, pp. 327–330.
[6] R. Krishnan, S.A. Lang, M.M. Siegel, Synthesis of amino-
quinolone derivatives, J. Heterocycl. Chem. 23 (1986) 1801–1804.
[7] Antibacterial National Committee for Clinical Laboratory Stan-
dards. Methods for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically. Approved standard. M7-A4, Vil-
lanova, PA (1997).
[8] Antibacterial National Committee for Clinical Laboratory Stan-
dards. Performance standards for antimicrobial susceptibility test-
ing. Eighth informational supplement. M100-S8, Villanova, PA
(1998).
[9] Braj. B. Lohray, S. Baskaran, Bonthu Srinivasa Rao, B.
Mallesham, K.S.N. Bharath, B. Yadi Reddy, S. Venkateswarlu,
Ashok K. Sadhuklan, M. Sitaram Kumar, Hemant M. Sarnaik,
Novel quinolone derivatives as potent antibacterials, Bioorg.
Med. Chem. Lett. 8 (1998) 525–528.
Sodium hydride 97% (0.010 g, 0.4 mmol) was added
to a stirred suspension of 5a (0.064 g, 0.25 mmol) in dry
DMF (3 ml) under nitrogen. After 20 min iodoethane
(0.028 ml, 0.35 mmol) was added and stirring was
continued for 90 min. The mixture was then poured on
ice (25 ml) and the precipitate was filtered, washed with
water, dried and recrystallized from acetonitrile–water
to give compound 9 (0.052 g, 76% yield).
4.1.13. 3-Ethoxy-5H-pyridazino[4,3-b]indole-4-
carboxylic acid (10)
The carboxylic acid 10 was prepared in 77% yield