Synthesis and aldose reductase inhibitory activity of a new series of benzo[h]cinnolinone derivatives

Article abstract of DOI:10.1016/S0014-827X(00)00035-5

Following our previous studies on pyridazinone carboxylic acids as potent and selective aldose reductase (ALR2) inhibitors, a new series of benzo[h]cinnolinone carboxylic acids, variously substituted at the positions 4, 7-10 and differently modified both

Full text of DOI:10.1016/S0014-827X(00)00035-5

www.elsevier.nl/locate/farmac
Il Farmaco 55 (2000) 544–552
Synthesis and aldose reductase inhibitory activity of a new series
of benzo[h]cinnolinone derivatives
Luca Costantino a, Giulio Rastelli a, Giorgio Cignarella b, Daniela Barlocco b,*
a Dipartimento di Scienze Farmaceutiche, Via G. Campi, 183-41100 Modena, Italy
b Istituto Chim. Farmaceutico e Tossicologico, Uni6ersita` di Milano, Viale Abruzzi, 42-20131 Milan, Italy
Received 30 November 1999; accepted 28 February 2000
Dedicated to Professor Pietro Pratesi
Abstract
Following our previous studies on pyridazinone carboxylic acids as potent and selective aldose reductase (ALR2) inhibitors, a
new series of benzo[h]cinnolinone carboxylic acids, variously substituted at the positions 4, 7–10 and differently modified both at
the central ring and at the acidic side chain, were synthesized and tested as inhibitors of ALR2. Comparison with previously
synthesized compounds allows us to define more precisely structure–activity relationships for this class of compounds. In fact, in
addition to the importance of the acidic side chain, their properties are highly influenced by the substituents present on the
benzo[h]cinnolinone nucleous, with potency ranging from that of Sorbinil to very weakly active compounds. © 2000 Elsevier
Science S.A. All rights reserved.
Keywords: Aldose reductase; Benzo[h]cinnolinone carboxylic acids; Diabetic complications; Sorbinil
1. Introduction
In a previous paper [2] we reported that pyridazinone
carboxylic acids of general formula (A) are selective
inhibitors of ALR2, with a potency comparable to
Sorbinil.
According to these studies, the substituents present on
the benzene ring, limited to 8,9-(OCH3)2 and 7,9-(CH3)2
were found to influence the activity in different ways
depending on the acidic side chain. In fact, while elonga-
tion of the latter to the propionic homolog always led to
less active derivatives, the corresponding butanoic
analogs showed different properties.
In this study we extend the SAR for this class of
compounds by the synthesis of numerous derivatives,
differently substituted at the phenyl ring, carrying either
acetic or longer acidic chains. Moreover, the 5,6-unsatu-
rated substrate as well as compounds having an addi-
tional substituent at position 4 were evaluated as ARIs.
Aldose reductase (alditol:NADP+ oxidoreductase, EC
1.1.1.21, ALR2) is an enzyme that catalyzes the conver-
sion of glucose to sorbitol, which is in turn converted to
fructose by sorbitol dehydrogenase. The increased glu-
cose flux through this metabolic pathway has been linked
to the development of late-onset diabetic complications
such as neuropathy, nephropathy, retinopathy and
cataract. Inhibitors of ALR2 thus seem to have the
potential to prevent or treat diabetic complications,
without the need of a strict control of glycemia [1].
2. Chemistry
The target compounds 1–26 (see Table 1) were pre-
pared from the corresponding benzocinnolinones 29–42
by treatment with the required ethyl bromoesters; the
* Corresponding author. Tel.: +39-02-2950 2223; fax: +39-02-
2951 4197.
E-mail address: daniela.barlocco@unimi.it (D. Barlocco).
0014-827X/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(00)00035-5

L. Costantino et al. / Il Farmaco 55 (2000) 544–552
545
compounds thus obtained were easily converted into
the corresponding carboxylic acids by alkaline hydroly-
sis followed by acidification (Scheme 1). The cyano
derivative 27 (see Table 1) was obtained from 43 [2] by
condensing with chloroacetonitrile and subsequently
converted to the corresponding tetrazole 28 (see Table
1) according to literature methods (Scheme 1). The
8-methansulfonamido derivative 16 was obtained from
the 8-amino-5,6-dihydrobenzo[h]cinnolin-3(2H)one [3]
which was transformed into the corresponding ethyl
ester, then condensed with methanesulfonyl chloride
according to normal procedures and finally hydrolized.
Compound 20 was prepared from 41, in turn ob-
tained by transesterification, of the corresponding ethyl
ester [2], by treatment with benzyl bromoacetate fol-
lowed by catalytic reduction.
aminosubstituted (12) being almost inactive. A further
increase by a C atom of the side chain in compounds 1,
3, 5 to give 2, 4, 6, respectively, did not significantly
alter their profile.
Finally, insertion of an insaturation into the acidic
chain (22–24) gave potent compounds, 24 being the
most interesting term of this new series. On the con-
trary, branched chains proved to be detrimental (25,
26).
4. Experimental
4.1. Chemistry
Melting points were determined on a Bu¨chi 510
capillary melting point apparatus and are uncorrected.
Elemental analyses for the tested compounds were
The still unknown intermediate methoxy- and benzyl-
oxy-5,6-dihydrobenzo[h]cinnolin-3(2H)-ones
(29–36)
1
were prepared according to a general procedure previ-
ously reported [2] starting from the appropriate tetra-
lones 44–51 (Scheme 2). However, while the 7-, 8- and
9-methoxysubstituted-4,4a-dihydroderivatives (58–60)
were easily dehydrogenated using m-nitrobenzenesul-
fonate in alkaline medium, the low yield obtained in the
case of 7-benzyloxy-5,6-dihydrobenzocinnolinone (61)
suggested to use DDQ in xylene for the remaining 8-
and 9-benzyloxyderivatives (62, 63). Moreover, it
should be noted that in the case of the 8-substituted
tetralones 47 and 51, the reaction with glyoxylic acid in
alkaline medium yielded the corresponding a-hydroxy-
2-tetraloneacetic acids, which allowed the synthesis of
10-substituted compounds 32, 36 by direct reaction
with hydrazine hydrate in acetic acid (see Scheme 2).
within +/− 0.4% of the theoretical values. H NMR
spectra were recorded on a Bruker AC200 spectrome-
ter; chemical shifts are reported as l (ppm) relative to
tetramethylsilane as internal standard. DMSO-d6 was
used as solvent unless otherwise noted. TLC on silica
gel plates was used to check product purity. Silica gel
60 (Merck, 70–230 mesh) was used for column chro-
matography. Tolrestat was synthesized according to a
procedure reported in the literature [4]. Sorbinil was
kindly provided by Pfizer. 8-Methoxytetralone (47) was
synthesized from 8-hydroxytetralone [5,6] as described
by Bilger et al. [7]. 5-, 6- and 7-Phenylmethoxyte-
tralones (48–50) as well as 8-phenylmethoxytetralone
(51) were synthesized according to previously reported
methods [8,9]. Benzo[h]cinnolin-3(2H)-one and 4-
aminobenzo[h]cinnolin-3(2H)-one were synthesized
from 5,6-dihydrobenzo[h]cinnolin-3(2H)-one [10]. 8-
Amino (37), 8- and 9-Acetylamino-5,6-dihydroben-
zo[h]cinnolin-3(2H)-one (38,39) were synthesized as
described in Ref. [3].
3. Results and discussion
The ALR2 inhibition data reported in Table 2 clearly
show that the activity of this class is influenced both by
the nature and by the position of the substituents on
the benzo[h]cinnolinone nucleous. Their potency ranges
from values comparable to that of Sorbinil to very
weak derivatives.
The presence of substituents at position 7 and 8 of
the model I led to compounds (8, 9, 13) provided with
similar activity (for I [2], IC50=12.6 mM). Insertion of
a double bond at position 5–6 of I (compound 19) still
retained good properties. On the contrary, substituents
at position 9 and 10 (10, 11, 17) as well as at the 4
position of the pyridazinone ring (20, 21) lowered the
activity. Replacement of the carboxylic function with a
tetrazole (28) caused a marked decrease in potency
while the cyano derivative (27) was inactive.
4.1.1. Benzo[h]cinnolin-3(2H)-one-2-aliphatic acids
(1–26) and compound 27
4.1.1.1. General method. (a) A mixture of the required
pyridazinone (29–42) (0.01 mol), the appropriate ethyl
bromoester (0.02 mol) and potassium carbonate (0.02
mol) in acetone (40 ml) was refluxed overnight (in the
case of 20 benzyl ethylbromoacetate was used). After
cooling, the inorganic salts were filtered off, the solvent
evaporated to give the esters, which were purified by
flash chromatography. Compound 27 was prepared in
an analogous way, using chloroacetonitrile.
(b) A mixture of the appropriate ester (0.01 mol) and
sodium hydroxide (0.04 mol) in 95% ethanol (40 ml)
was stirred at room temperature (r.t.) for 2 h. After
evaporation of the solvent, the residue was acidified
with hydrochloric acid in diethyl ether, diluted with
Among the butanoic homologs, the most potent was
the 9-methoxyderivative (5; IC50=4.79 mM). All the
other compounds were less active than I, the 8-

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L. Costantino et al. / Il Farmaco 55 (2000) 544–552
Table 1
Physico-chemical properties of compounds 1–28
Compd
R
H
R1
R2
% Yield a Formula
77 a
1H NMR
1
7-OCH3
ꢀ(CH2)3ꢀCOOH
C17H18N2O4
2.0 (2H, m), 2.3 (2H, m), 2.8
(4H, s), 3.8 (3H, s), 4.2 (2H,
t), 6.9 (1H, s), 7.0 (1H, d),
7.3 (1H, t), 7.6 (1H, d), 12.2
(1H, s).
2
H
7-OCH3
ꢀ(CH2)4ꢀCOOH
70
C18H20N2O4
1.8 (4H, m), 2.3 (2H, t), 2.8
(4H, s), 3.8 (3H, s), 4.1 (2H,
t), 6.9 (1H, s), 7.0 (1H, d),
7.3 (1H, t), 7.6 (1H, d), 12.2
(1H, s).
3
4
5
6
H
H
H
H
8-OCH3
8-OCH3
9-OCH3
9-OCH3
ꢀ(CH2)3ꢀCOOH
ꢀ(CH2)4ꢀCOOH
ꢀ(CH2)3ꢀCOOH
ꢀ(CH2)4ꢀCOOH
77
69
66
70
C17H18N2O4
C18H20N2O4
C17H18N2O4
C18H20N2O4
2.0 (2H, m), 2.3 (2H, t), 2.8
(4H, s), 3.8 (3H, s), 4.2
(2H, t), 6.8 (1H, s), 7.0 (2H,
d), 7.9 (1H, d), 12.2 (1H, s).
1.6 (2H,m), 1.8 (2H, m), 2.3
(2H, t), 2.8 (4H, s), 3.8
(3H, s), 4.1 (2H, t), 6.8 (1H,
s), 6.9 (2H, d), 7.9 (1H, d).
(CDCl3): 2.0 (2H, m), 2.3
(2H, t), 2.8 (4H, s), 3.8
(3H, s), 4.3 (2H, t), 6.8 (2H,
m), 7.1 (1H, d), 7.5 (1H, s).
(CDCl3): 1.6 (2H, m), 1.8
(2H, m), 2.3 (2H, t), 2.8 (4H,
s), 3.8 (3H, s), 4.1 (2H, t),
6.80 (2H, m), 7.10 (1H, d),
7.50 (1H, s).
7
H
10-OCH3
ꢀ(CH2)3ꢀCOOH
68
C17H18N2O4
(CDCl3): 2.2 (2H, m), 2.4
(2H, m), 2.8 (4H, m), 3.9
(3H, s), 4.2 (2H, m), 6.7 (1H,
s), 6.8 (2H, m), 7.2 (1H, m).
2.9 (4H, m), 4.8 (2H, s), 5.2
(2H, s), 6.9 (1H, s), 7.4
(8H, m), 13.1 (1H, s).
2.9 (4H, s), 4.8 (2H, s), 5.2
(2H, s), 6.9 (1H, s), 7.0
(2H, m), 7.4 (5H, m), 7.8
(1H, m), 13.0 (1H, s).
8
9
H
H
7-OCH2ꢀC6H5
8-OCH2ꢀC6H5
ꢀCH2ꢀCOOH
ꢀCH2ꢀCOOH
75
70
C21H18N2O4
C21H18N2O4
10
11
12
13
H
H
H
H
9-OCH2ꢀC6H5
ꢀCH2ꢀCOOH
69
70
72
77
C21H18N2O4
C21H18N2O4
C16H17N3O3
C16H15N3O4
2.8 (4H, s), 4.8 (2H, s), 5.2
(2H, s), 6.9 (1H, s), 7.0
(1H, dd), 7.4 (7H, m), 13.0
(1H, s).
2.8 (4H, m), 4.7 (2H, s), 5.2
(3H, s), 6.9 (1H, s), 7.0
(1H, d), 7.2 (1H, d), 7.3 (4H,
m), 7.6 (2H, m), 13.1 (1H, s).
2.3 (2H, m), 2.7 (4H, m), 4.1
(2H, m), 5.5 (2H, s), 6.4
(1H, s), 6.6 (1H, d), 6.8
(1H, s), 7.6 (1H, d).
10-OCH2ꢀC6H5 ꢀCH2ꢀCOOH
8-NH2
ꢀ(CH2)3ꢀCOOH
ꢀCH2ꢀCOOH
8-NHCOCH3
2.1 (3H, s), 2.8 (4H, s), 4.8
(2H, s), 6.9 (1H, s), 7.5
(1H, d), 7.7 (1H, s), 7.9 (1H,
d), 10.1 (1H, s), 13.0 (1H, s).

L. Costantino et al. / Il Farmaco 55 (2000) 544–552
547
Table 1 (Continued)
Compd
R
H
R1
R2
% Yield a Formula
1H NMR
14
8-NHCOCH3
ꢀ(CH2)2ꢀCOOH
40
72
40
77
72
C17H17N3O4
2.1 (3H, s), 2.8 (4H, m), 3.3 (4H,
s), 4.3 (2H, t), 6.9 (1H, s), 7.5
(1H, d), 7.7 (1H, s), 7.9 (1H,
d), 10.1 (1H, s).
2.1 (4H, m), 2.3 (2H, t), 2.8 (3H,
s), 4.1 (2H, t), 6.4 (1H, s), 6.9
(1H, s), 7.5 (1H, d), 7.7 (1H,
s), 7.9 (1H, d), 10.2 (1H, s).
15
16
17
18
H
H
H
H
8-NHCOCH3
8-NHSO2CH3
9-NHCOCH3
9-NHCOCH3
ꢀ(CH2)3ꢀCOOH
ꢀ(CH2)3ꢀCOOH
ꢀCH2ꢀCOOH
C18H19N3O4
C17H19N3O5S (CD3OD): 2.1 (2H, m), 2.5 (2H,
t), 2.9 (4H, s), 3.1 (3H, s), 4.3
(2H, t), 6.8 (1H, s), 7.2 (2H,
dd), 8.1 (1H, d).
C16H15N3O4
(CD3OD): 2.2 (3H, s), 3.0 (4H,
s), 5.0 (2H, s), 6.9 (1H, s), 7.3
(1H, d), 7.6 (1H, dd), 8.2 (1H,
s).
(CD3OD):1.3 (2H, m), 2.1 (3H,
m), 2.4 (2H, t), 2.9 (4H, s), 4.3
(2H, t), 6.8 (1H, s), 7.2 (1H,
d), 7.6 (1H, dd), 8.2 (1H, s).
4.8 (2H, s); 7.2 (1H, s); 7.4 (AB
system, 2H), 7.6 (2H, m), 7.8
(1H, m); 8.5 (1H, m).
ꢀ(CH2)3ꢀCOOH
C18H19N3O4
19
20
H
H
H
ꢀCH2ꢀCOOH 5,6-double bond 74
C14H10N2O3
C19H20N2O5
ꢀCOOꢀ(CH2)3ꢀCH3
ꢀCH2ꢀCOOH
71
0.9 (3H, t), 1.3 (2H, m), 1.5 (2H,
m), 2.7 (4H, m), 4.4 (2H, t),
4.8 (2H, s), 7.4 (3H, m), 8.0
(1H, m).
21
ꢀNH2
H
ꢀCH2ꢀCOOH 5,6-double bond 74
C14H11N3O3
4.8 (2H, s); 7.3 (2H, s, exch.
with D2O); 7.5 (2H, AB sys-
tem); 7.6 (2H, m); 7.7 (1H, m);
8.6 (1H, m).
22
23
H
H
7-OCH3
9-OCH3
ꢀCH2ꢀCHꢁCHꢀCOOH
ꢀCH2ꢀCHꢁCHꢀCOOH
77
77
C17H16N2O4
C17H16N2O4
2.8 (4H, s), 3.8 (3H, s), 4.9 (2H,
d), 5.7 (1H, d), 7.0 (2H, m),
7.3 (1H, q), 7.7 (2H, m).
2.8 (4H, s), 3.8 (3H, s), 5.0 (2H,
d), 5.8 (1H, d), 6.8 (1H, s), 6.9
(1H, dd), 7.1 (2H, m), 7.6 (1H,
s).
24
H
7-OCH3
ꢀCHꢁCHꢀCH2ꢀCOOH
71
C17H16N2O4
(CDCl3): 1.3 (3H, t), 2.8 (4H,
m), 3.3 (2H, d), 3.9 (3H, s), 4.2
(2H, q), 6.5 (1H, m), 6.80 (1H,
s), 7.0 (1H, d), 7.3 (1H, t), 7.7
(2H, m).
25
26
H
H
7-OCH3
9-OCH3
ꢀC(CH3)2ꢀCOOH
77
75
C17H18N2O4
C17H18N2O4
1.8 (6H, d), 2.8 (4H, s), 3.8 (3H,
s), 6.9 (1H, s), 7.1 (1H, d), 7.9
(1H, d), 8.3 (1H, d).
0.9 (3H, t), 2.2 (2H, q), 2.8 (4H,
s), 3.8 (3H, s), 5.3 (1H, m), 6.9
(2H, m), 7.2 (1H, d), 7.4 (1H,
s).
ꢀCH(CH2CH3)ꢀCOOH
27
28
H
H
H
H
ꢀCH2ꢀCN
75
76
C14H11N3O
C14H12N6O
(CDCl3): 2.9 (4H, s), 5.1 (2H, s),
6.8 (1H, s), 7.2 (1H, s), 7.4
(2H, m), 8.1 (1H, m).
2.9 (4H, s), 3.9 (1H, br. s, exch.
with D2O), 5.5 (2H, s), 6.9
(1H, s), 7.4 (3H, m), 7.9 (1H,
m).
ꢀCH2ꢀtetrazole
a Yields are based on the 2-unsubstituted pyridazinones.
water and extracted with dichloromethane (3×10 ml).
the residue was purified by trituration with diethyl ether
After drying (Na2SO4) and evaporation of the solvent,
or crystallized from acetone/petroleum ether (see Table

548
L. Costantino et al. / Il Farmaco 55 (2000) 544–552
1 for chemical data). In the case of 20, catalytic dehy-
drogenation was performed instead of hydrolysis.
4.1.5. 6-Methoxy-1,2,3,4-tetrahydro-1-oxo-2-naphtyli-
deneacetic acid (53)
Yield 50%, m.p. 170°C; H NMR: 2.9 (2H, t), 3.3
1
(2H, dt), 3.9 (3H, s), 6.6 (1H, t), 7.0 (2H, m), 7.9 (1H,
d), 13.0 (1H, s).
4.1.2. 2-Tetrazolylmethyl-5,6-dihydrobenzo[h]cinnolin-
3(2H)-one (28)
Sodium azide (0.326 g, 5.0 mmol) was added to a
solution of trimethyltin chloride (1g, 5.0 mmol) in water
(3 ml) and stirred for 3 h, then added to the cyano
derivative 27 (0.1 g, 0.4 mmol) dissolved in toluene (3
ml) and the whole refluxed in a nitrogen athmosphere.
After 24 h, the solvent was removed under reduced
pressure and the residue was purified by flash chro-
matography (CH2Cl2:CH3OH 8:2)
4.1.6. 7-Methoxy-1,2,3,4-tetrahydro-1-oxo-2-naphtyli-
deneacetic acid (54)
Yield 68%, m.p. 160°C; H NMR: 2.9 (2H, t), 3.3
(2H, dt), 3.8 (3H, s), 6.7 (1H, t), 7.2 (1H, dd), 7.3 (1H,
d), 7.4 (1H, d), 13.0 (1H, s).
1
4.1.7. 5-Phenylmethoxy-1,2,3,4-tetrahydro-1-oxo-2-
naphtylideneacetic acid (55)
1
Yield 29%, m.p. 163–5°C, H NMR: 2.9 (2H, t), 3.3
4.1.3. 5-, 6-, 7-Methoxy- or -phenylmethoxy-
1,2,3,4-tetrahydro-1-oxo-2-naphtylideneacetic acids
(52–57)
(2H, dt), 5.2 (2H, s), 6.6 (1H, t), 7.5 (8H, m), 13.0 (1H,
broad s).
A solution of sodium hydroxide (23.2 mmol) in
water (12 ml) and ethanol (6 ml) was added to a
mixture of substituted tetralone (4.7 mmol) and gly-
oxylic acid (19.0 mmol) in water (8 ml) at r.t. The
mixture was stirred at r.t. for 1 h, then heated at reflux
for 2 h. After cooling, the solvent was removed under
reduced pressure, the residue acidified with HCl 10 N,
and the solid collected and purified by column chro-
4.1.8. 6-Phenylmethoxy-1,2,3,4-tetrahydro-1-oxo-2-
naphtylideneacetic acid (56)
Yield 16%, m.p. 145–8°C, H NMR: 2.9 (2H, t), 3.3
(2H, broad t), 5.2 (2H, s), 6.7 (1H, t), 7.0 (2H, m), 7.5
(5H, m), 7.9 (1H, m), 13.0 (1H, broad s).
1
4.1.9. 7-Phenylmethoxy-1,2,3,4-tetrahydro-1-oxo-2-
naphtylideneacetic acid (57)
Yield 30%, m.p. 138–40°C. 1H NMR: 2.9 (2H, t), 3.3
(2H, broad t), 5.2 (2H, s), 6.7 (1H, t), 7.4 (8H, m), 12.9
(1H broad s).
matography
12.4:0.4).
(CH2Cl2:CH3OH:Acetic
acid
87.2:
4.1.4. 5-Methoxy-1,2,3,4-tetrahydro-1-oxo-2-naphtyli-
deneacetic acid (52)
4.1.10. 5-, 6-, 7-Methoxy- or -phenylmethoxy-4,4a,5,6-
tetrahydrobenzo[h]cinnolin-3(2H)-ones (58–63)
1
Yield 59%, m.p. 140°C; H NMR: 2.9 (2H, t), 3.3
(2H, dt,), 3.9 (3H, s), 6.6 (1H, t), 7.3 (1H, dd,), 7.4 (1H,
t), 7.6 (1H, dd,), 13.0 (1H, s).
A mixture of the appropriate naphtylideneacetic acid
(1.36 mmol) and zinc dust (2.75 mmol) in acetic acid (6
Scheme 1.

L. Costantino et al. / Il Farmaco 55 (2000) 544–552
549
Scheme 2. (a) Glyoxylic acid/OH−/D; (b) Zn/CH3COOH, D; (c) NH2ꢀNH2·H2O/EtOH/D; (d) m-nitrobenzensulfonate/OH− or DDQ/Xylene; (e)
NH2ꢀNH2·H2O/CH3COOH/D.
ml) and water (3 ml) was heated at 70°C for 1 h.
The insoluble was filtered off, water (10 ml) was
added to the solution and the mixture was then
extracted with dichloromethane (4×15 ml). The
organic layer was dried (Na2SO4) and the solvent
evaporated under reduced pressure. The residue was
dissolved in ethanol (8 ml), added of hydrazine
hydrate (1.94 mmol) and refluxed for 3 h. After

550
L. Costantino et al. / Il Farmaco 55 (2000) 544–552
Table 2
ALR2 inhibition data of compounds 1–28
Compd
R
R1
R2
ALR2 inh. activity a
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
7-OCH3
7-OCH3
8-OCH3
8-OCH3
9-OCH3
9-OCH3
10-OCH3
7-OCH2-C6H5
8-OCH2-C6H5
9-OCH2-C6H5
10-OCH2-C6H5
8-NH2
8-NHCOCH3
8-NHCOCH3
8-NHCOCH3
8-NHSO2CH3
9-NHCOCH3
9-NHCOCH3
H
ꢀ(CH2)3ꢀCOOH
ꢀ(CH2)4ꢀCOOH
ꢀ(CH2)3ꢀCOOH
ꢀ(CH2)4ꢀCOOH
ꢀ(CH2)3ꢀCOOH
ꢀ(CH2)4ꢀCOOH
ꢀ(CH2)3ꢀCOOH
ꢀCH2ꢀCOOH
ꢀCH2ꢀCOOH
ꢀCH2ꢀCOOH
ꢀCH2ꢀCOOH
ꢀ(CH2)3ꢀCOOH
ꢀCH2ꢀCOOH
ꢀ(CH2)2ꢀCOOH
ꢀ(CH2)3ꢀCOOH
ꢀ(CH2)3ꢀCOOH
ꢀCH2ꢀCOOH
ꢀ(CH2)3ꢀCOOH
ꢀCH2ꢀCOOH 5,6–double bond
ꢀCH2ꢀCOOH
ꢀCH2ꢀCOOH 5,6-double bond
ꢀCH2ꢀCHꢁCHꢀCOOH
ꢀCH2ꢀCHꢁCHꢀCOOH
ꢀCHꢁCHꢀCH2ꢀCOOH
ꢀC(CH3)2ꢀCOOH
ꢀCH(CH2CH3)ꢀCOOH
ꢀCH2ꢀCN
19.62 (15.58–24.70)
23.62 (19.51–28.59)
25.81 (20.50–32.49)
17.39 (12.89–23.46)
4.79 (3.63–6.31)
7.94 (6.32–9.97)
73.84 (56.01–97.34)
6.37 (5.04–8.06)
7.85 (6.09–10.119
38.75 (32.23–46.59)
30.47 (22.59–41.11)
31% inh. (140mM)
14.07 (11.68–16.95)
32% inh. (100 mM)
56.02 (44.50–70.52)
39.56 (33.13–47.23)
46.50 (35.27–61.30)
58.86 (49.07–70.60)
6.53 (5.30–8.05)
34.35 (25.64–46.02)
35.36 (28.74–43.50)
4.25 (3.65–4.95)
8.99 (7.98–10.13)
3.90 (3.13–4.86)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
H
H
H
ꢀCOOꢀ(CH2)3ꢀCH3
ꢀNH2
H
H
H
H
H
H
H
H
H
7-OCH3
9-OCH3
7-OCH3
7-OCH3
9-OCH3
H
H
112. 0 (89.6–140)
33% inh. (71 mM)
24% inh. (79 mM)
45.57 (32.19–64.52)
I
H
H
ꢀCH2-COOH
12.6 (7.14-22.1) b
1.19 (1.04–1.36)
0.096 (0.079–0.117)
Sorbinil
Tolrestat
a IC50 (95% C.L.) (mM) or percent inhibition (at a given mM concentration).
b From Ref. [2].
cooling, the precipitate was collected and washed with
water.
4.1.13. 9-Methoxy-4,4a,5,6-tetrahydrobenzo[h]cinnolin-
3(2H)-one (60)
Yield 73%, m.p. 155°C; H NMR: 1.5 (1H, m), 2.2
(3H, m), 2.8 (3H, m), 3.8 (3H, s), 6.9 (1H, dd), 7.2 (1H,
d), 7.5 (1H, d), 10.9 (1H, s).
1
4.1.11. 7-Methoxy-4,4a,5,6-tetrahydrobenzo[h]cinnolin-
3(2H)-one (58)
1
Yield 87%, m.p. 178°C (dec.); H NMR: 1.5 (1H, m),
2.3 (4H, m), 2.5 (1H, m), 3.1 (1H, dt), 3.8 (3H, s), 6.9
(1H, dd), 7.2 (1H, t), 7.6 (1H, dd), 10.9 (1H, s).
4.1.14. 7-Phenylmethoxy-4,4a,5,6-tetrahydrobenzo[h]-
cinnolin-3(2H)-one (61)
4.1.12. 8-Methoxy-4,4a,5,6-tetrahydrobenzo[h]cinnolin-
3(2H)-one (59)
Yield 72%, m.p. 217°C; 1H NMR: 1.5 (1H, m),
2.3 (5H, m), 2.8 (1H, m), 5.1 (2H, s), 7.1 (1H, d),
7.2 (1H, t), 7.4 (5H, m), 7.7 (1H, d), 10.9 (1H, broad
s).
1
Yield 77%, m.p. 180°C (dec.); H NMR: 1.5 (1H, m),
2.3 (3H, m), 2.8 (3H, m), 3.8 (3H, s), 6.8 (2H, m), 7.9
(1H, d), 10.8 (1H, s).

L. Costantino et al. / Il Farmaco 55 (2000) 544–552
551
4.1.15. 8-Phenylmethoxy-4,4a,5,6-tetrahydrobenzo[h]-
cinnolin-3(2H)-one (62)
Yield 82%, m.p. 220°C; H NMR: 1.5 (1H, m), 2.6
(6H, m), 5.1 (2H, s), 6.9 (2H, m), 7.4 (5H, m), 7.9 (1H,
d), 10.8 (1H, broad s).
4.1.24. 9-Phenylmethoxy-5,6-dihydrobenzo[h]cinnolin-
3(2H)-one (35)
Yield 41%, m.p. 215–8°C, H NMR: 2.8 (4H, s), 5.2
(2H, s), 6.8 (1H, s), 7.0 (1H, dd), 7.4 (7H, m), 13.8 (1H,
broad s).
1
1
4.1.16. 9-Phenylmethoxy-4,4a,5,6-tetrahydrobenzo[h]-
cinnolin-3(2H)-one (63)
4.1.25. 10-Substituted-5,6-dihydrobenzo[h]cinnolin-
3(2H)-one (32, 36)
Yield 70%, m.p. 142–5°C; 1H NMR: 1.5 (1H, m), 2.5
(6H, m), 5.1 (2H, s), 7.0 (1H, dd), 7.2 (1H, d), 7.1 (5H,
m), 7.6 (1H, d), 10.9 (1H, broad s).
A solution of sodium hydroxide (23.2 mmol) in water
(12 ml) and ethanol (6 ml) was added to a mixture of
substituted tetralone 47 or 51 (4.7 mmol) and glyoxylic
acid (19.0 mmol) in water (8 ml) at r.t. The mixture was
stirred at r.t. for 1 h, then heated at reflux for 2 h. After
cooling, the solvent was removed under reduced pres-
sure, the residue acidified with 10 N HCl, and the solid
collected to give the corresponding a-hydroxy-2-te-
tralone acetic acid, which was dissolved in EtOH (10
ml). Hydrazine hydrate (5.47 mmol) was added and the
solution refluxed for 3 h. After cooling the solvent was
concentrated under reduced pressure. The precipitate
thus formed was constituted by 32 or 36.
4.1.17. 7-, 8-, and 9-Methoxy-and 7-phenylmethoxy-
5,6-dihydrobenzo[h]cinnolin-3(2H)-ones (29–31, 33)
The corresponding 4,4a-dihydroderivatives were
transformed into 29–31 and 33 by a standard proce-
dure, using sodium m-nitrobenzenesulfonate in alkaline
medium [3].
4.1.18. 7-Methoxy-5,6-dihydrobenzo[h]cinnolin-
3(2H)-one (29)
1
Yield 97%, m.p. 270°C, H NMR: 2.8 (4H, s), 3.8
(3H, s), 6.8 (1H, s), 7.0 (1H, dd), 7.3 (1H, t), 7.6 (1H,
dd), 12.9 (1H, s).
4.1.26. 10-Methoxy-5,6-dihydrobenzo[h]cinnolin-
3(2H)-one (32)
Yield 78%, m.p. 186–7°C; 1H NMR (CDCl3): 2.8
(4H, m), 3.8 (3H, s), 6.8 (1H, s), 7.1 (3H, m), 13.0 (1H,
s).
4.1.19. 8-Methoxy-5,6-dihydrobenzo[h]cinnolin-
3(2H)-one (30)
1
Yield 95%, m.p. 235°C, H NMR: 2.8 (4H, s), 3.8
(3H, s), 6.7 (1H, s), 6.9 (2H, m), 7.8 (1H, d), 12.9 (1H,
s).
4.1.27. 10-Phenylmethoxy-5,6-dihydrobenzo[h]cinnolin-
3(2H)-one (36)
Yield 27.5%, m.p. 180–2°C, H NMR (CDCl3): 2.8
1
4.1.20. 9-Methoxy-5,6-dihydrobenzo[h]cinnolin-
3(2H)-one (31)
Yield 90%, m.p. 245°C; H NMR: 2.8 (4H, s), 3.8
(4H, m), 5.2 (2H, s), 6.8 (1H, s), 7.2 (8H, m), 11.6 (1H,
s).
1
(3H, s), 6.8 (1H, s), 6.9 (1H, dd), 7.2 (1H, d), 7.4 (1H,
d), 13.0 (1H, s).
4.2. Enzyme section
Calf lenses for the purification of ALR2 were ob-
tained locally from freshly-slaughtered animals. The
capsule was incised and the frozen lenses were sus-
pended in sodium potassium phosphate buffer, pH 7
(standard buffer) containing 5 mM DTT (1 g tissue/3.5
ml) and stirred in an ice-cold bath for 1 h. The suspen-
sion was then centrifuged at 22 000×g at 4°C for 40
min and the supernatant was subjected to ion exchange
chromatography on DE52 [2]. Enzyme activity for all
tested enzymes was measured by monitoring the change
in absorbance at 340 nm, which accompanies the oxida-
tion of NADPH catalyzed by ALR2. The assay was
performed at 37°C as previously described, using 4.7
4.1.21. 7-Phenylmethoxy-5,6-dihydrobenzo[h]cinnolin-
3(2H)-one (33)
Yield 6.7%, m.p. 192°C, H NMR: 2.8 (4H, m), 5.2
1
(2H, s), 6.8 (1H, s), 7.4 (8H, m), 12.9 (1H, broad s).
4.1.22. 8- and 9-Phenylmethoxy-5,6-dihydrobenzo[h]-
cinnolin-3(2H)-one (34,35)
A mixture of 62 or 63 (0.10 g, 0.32 mmol) and
2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) (0.070
g, 0.31 mmol) in xylene (5 ml) was refluxed for 12 h.
After cooling, the precipitate was collected and purified
by column chromatography (CH2Cl2:CH3OH 9.5:0.5)
mM
D,L-glyceraldehyde as substrate in 0.25 M sodium
4.1.23. 8-Phenylmethoxy-5,6-dihydrobenzo[h]cinnolin-
3(2H)-one (34)
Yield 51%, m.p. 200–3°C, H NMR: 2.8 (4H, s), 5.2
(2H, s), 6.8 (1H, s), 7.0 (2H, m), 7.4 (5H, m), 7.8 (1H,
m), 13.8 (1H, broad s).
phosphate buffer pH 6.8, containing 0.38 M ammo-
nium sulphate and 0.11 mM NADPH. The sensitivity
of ALR2 to inhibition by different ARIs was tested in
the above assay conditions by including the inhibitors
dissolved in DMSO at the desired concentration in the
1

552
L. Costantino et al. / Il Farmaco 55 (2000) 544–552
pyridazinones: higher homologues of the antihypertensive and
antithrombotic 5H-indeno[1,2-c]pyridazinones, J. Med. Chem.
32 (1989) 2277–2282.
reaction mixture. DMSO in the assay mixture was kept
at constant concentration of 1%. A reference blank
containing all the above reagents except the substrate
was used to correct for the non enzymatic oxidation of
NADPH. IC50 values (the concentration of the in-
hibitor required to produce 50% inhibition of the en-
zyme catalyzed reaction) were determined from least
squares analyses of the linear portion of the log dose-
inhibition curves. Each curve was generated using at
least three concentrations of inhibitor causing an inhibi-
tion between 20 and 80% with two replicates at each
concentration [2]. The 95% confidence limits (95% CL)
were calculated from T-values for n−2, where n is the
total number of determinations [11].
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