5, 6-Diphenylpyridazine derivatives as acyl-CoA:cholesterol acyltransferase inhibitors

Article abstract of DOI:10.1021/jm010807h

Alkyl-5, 6-diphenylpyridazine derivatives combining several main features of ACAT inhibitors, such as a long alkyl side chain linked to a heterocycle and the o-diphenyl system, were synthesized and tested. Moreover, modeling studies on representative terms were performed. Some compounds displayed ACAT inhibition in the micromolar range, both on the enzyme isolated from rat liver microsomes and in cell-free homogenate of murine macrophages.

Full text of DOI:10.1021/jm010807h

4292
J . Med. Chem. 2001, 44, 4292-4295
5,6-Dip h en ylp yr id a zin e Der iva tives a s Acyl-CoA:Ch olester ol Acyltr a n sfer a se
In h ibitor s
Maria Paola Giovannoni,*,§ Vittorio Dal Piaz,§ Byoung-Mog Kwon,# Mi-Kyung Kim,# Young-Kook Kim,#
Lucio Toma,† Daniela Barlocco,‡ Franco Bernini, and Monica Canavesi
Dipartimento di Scienze Farmaceutiche, Universita` di Firenze, via G. Capponi 9, 50121 Firenze, Italy, Korea Research
Institute of Bioscience & Biotechnology, 52 Uen-Dong Yusung-Ku, Taejeon 305-600, South Korea, Dipartimento di Chimica
Organica, Universita` di Pavia, Via Taramelli 10, 27100 Pavia, Italy, Istituto di Chimica Farmaceutica e Tossicologica,
Universita` di Milano, Viale Abruzzi 42, 20131 Milano, Italy, and Istituto di Farmacologia e Farmacognosia, Universita` di
Parma, Viale delle Scienze, 43100 Parma, Italy
Received J anuary 5, 2001
Alkyl-5,6-diphenylpyridazine derivatives combining several main features of ACAT inhibitors,
such as a long alkyl side chain linked to a heterocycle and the o-diphenyl system, were
synthesized and tested. Moreover, modeling studies on representative terms were performed.
Some compounds displayed ACAT inhibition in the micromolar range, both on the enzyme
isolated from rat liver microsomes and in cell-free homogenate of murine macrophages.
Ch a r t 1
In tr od u ction
Coronary heart disease is the major cause of death
in western industrialized countries,1 and hypercholes-
terolemia was found to be the leading risk factor for its
development.2 Acyl-CoA:cholesterol O-acyltransferase
(ACAT) is an intracellular enzyme responsible for the
catalysis of the esterification of free cholesterol in
various tissues.3 Moreover it plays an important role
in the absorption4 of cholesterol, in secretion of very low-
density lipoproteins (VLDL),5 and in the accumulation
of cholesteryl ester in foam cells, which were recovered
in atheromatous plaques.6 Therefore the inhibition of
ACAT enzyme could represent a good therapeutic ap-
proach.7 Among different chemical classes of ACAT
inhibitors,8,9 interesting activity was shown by anilidic
derivatives of fatty acids, in which the long alkylic
chains mimic the natural substrate of ACAT, and by
ureas derivatives.10,11 Moreover, quite interesting re-
sults were obtained by Rhone-Poulenc Rorer with the
synthesis of a class of 2-substituted-4,5-diphenyl-1H-
imidazole in which a long side alkyl chain, which can
be also embodied in a cycle, is linked to the heteroaro-
matic ring through a sulfur atom with different oxida-
tion state.12 Among these derivatives, compound RP73163
is a representative member.13 In addition, several
compounds with the same pharmacological activity such
as GERI-BP001,14 were identified from microbial origin
(Chart 1). The pyridazine ring has often proved to be a
suitable moiety for biologically active compounds,15,16
but to our knowledge, it has never been exploited as a
possible substrate for ACAT inhibitors. On these bases
we report here the synthesis and biological properties
of new compounds (2, 3, 5, 7-10) which combine some
of the characteristics of the reported ACAT inhibitors,
namely a long alkylic side chain linked to a heterocyclic
ring by a heteroatom of different nature and the
o-diphenyl system.
Ch em istr y
2-Substituted-5,6-diphenylpyridazin-3(2H)-ones (2)
were obtained from 117 by alkylation with the appropri-
ate bromo derivative in dimethylformamide and in the
presence of anhydrous potassium carbonate. Alkaline
hydrolysis of the ester 2k -m and subsequent acidifica-
tion led to the corresponding acids 3a -c. The pyridazine
derivatives 5a -j were obtained by condensation of 417
and the required alkylamines or sodium alcoholates.
The amidic derivative 5e was obtained from 4 by
replacement of the chlorine in 3-position by an amino
group using aqueous ammonia, followed by acylation
with hexanoyl chloride. The regiochemistry of the last
1
step was assigned on the basis of the H NMR data of
the compound, and eventually confirmed by reduction
of 5e to 5a by LiAlH4 in anhydrous diethyl ether. (See
Scheme 1.)
The thioderivatives 7-10 were prepared from thione
(6).18 Alkylation with the appropriate bromo derivative
gave compounds 7. Treatment of 7a -d by 30% H2O2 in
CH3COOH at room temperature afforded the sulfoxides
8, which were further oxidized to 9 by m-chloroperben-
zoic acid in dichloromethane at room temperature.
Compounds 10 were easily obtained from 7e-h by
alkaline hydrolysis (Scheme 2).
* To whom correspondence should be addressed. Tel: +39-55-
2757290. Fax: +39-55-240776. E-mail: mariapaola.giovannoni@unifi.it.
§ Dipartimento di Scienze Farmaceutiche.
#
Korea Research Institute.
† Dipartimento di Chimica Organica.
‡ Istituto di Chimica Farmaceutica e Tossicologica.
Istituto di Farmacologia e Farmacognosia.
10.1021/jm010807h CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/26/2001

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4293
Sch em e 1a
Ta ble 2. Chemical Data and ACAT Inhibition of Pyridazine
Derivatives
%
mp
(°C)
IC50
compd
X
n
R
yielda
formulab
(µM)c
5a
5b
5c
5d
5e
5f
5g
5h
5i
NH
5 CH3
6 CH3
7 CH3
8 CH3
64
68
50
61
81
95
84
70
70
62
79
91
77
74
120-122d C22H25N3
104-107g C23H27N3
113-115d C24H29N3
111-113d C25H31N3
217-219h C22H23N3O
24
55
40
18
>300
73
72
58
104
180
62
76
130
116
NH
NH
NH
NHCO 4 CH3
O
O
O
O
O
S
S
4 CH3
5 CH3
6 CH3
7 CH3
8 CH3
5 CH3
6 CH3
7 CH3
8 CH3
5 CH3
6 CH3
7 CH3
8 CH3
5 CH3
6 CH3
oil
C21H22N2O
C22H24N2O
C23H26N2O
C24H28N2O
C25H30N2O
C22H24N2S
C23H26N2S
C24H28N2S
C25H30N2S
C22H24N2OS 75
C23H26N2OS 77
C24H28N2OS 92
C25H30N2OS 87
C22H24N2O2S 60
C23H26N2O2S 49
C24H28N2O2S 36
C25H30N2O2S 76
oil
oil
oil
oil
Sch em e 2a
5j
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
10a
10b
10c
10d
66-67d
oil
S
S
oil
oil
SO
SO
SO
SO
SO2
SO2
SO2
SO2
S
77e 95-97d
81e oil
79e oil
75c oil
91f oil
88f oil
7 RdCH3 80f oil
8 CH3
5 COOH
6 COOH
7 COOH
8 COOH
70f oil
62
58
48
57
120-122d C22H22N2O2S >300
126-128d C23H24N2O2S >300
97-100g C24H26N2O2S >300
S
S
S
oil
C
25H28N2O2S >300
Ta ble 1. Chemical Data and ACAT Inhibition of Pyridazinone
Derivatives
RP73163
GERI-BP001M
0.1
42
a
b
From 5. C, H, N analysis within (0.4%. c In vitro ACAT
inhibition determined in rat liver microsomes. IC50 values are
means from three experiments. Ethanol. e From 6. f From 7.
d
g
h
Ethanol/water 1:1. Cyclohexane.
%
mp
(°C)
IC50
modated. Longer chains up to 9 carbon atoms as well
as shortening by one carbon atom were well tolerated
(compounds 2f-h , 2d ). On the contrary, the presence
of shorter alkyl chains as well as cyclic residues and
terminal carboxylic acid groups always led to very weak
or completely inactive compounds. As far as the py-
ridazine series is concerned (see Table 2), compounds
bearing an oxygen or a sulfur atom at position 3, as well
as its oxidized derivative SO, showed a quite similar
trend. In fact, the best activity was displayed when n
) 5, 6 while it tended to decrease for longer alkyl
substituents. Also in this case, the presence of a
terminal carboxy group (compounds 10a -d ) led to
inactive compounds. Further oxidation to SO2 deriva-
tives gave better inhibitors (9b, 9c; IC50 ) 49 and 36
µM, respectively). However, the most potent compounds
were found in the amino derivative series, and the two
most active (5a , 5d ; IC50 ) 24 and 18 µM) have the
shortest (n ) 5) and the longest (n ) 8) chains,
respectively. Substitution of the amino by an amido
group (compound 5e) resulted in an inactive derivative.
To better define the pharmacological properties of this
series, three significant compounds (5c,d ; 5h ), were
further evaluated on macrophages.19 Compound 58-0359
was used as a control.
compd
R
n
yielda
formulab
(µM)c
2a
2b
2c
2d
2e
2f
2g
2h
2i
CH3
2
0
3
4
5
6
7
8
0
0
55 oil
56 138-140 C19H18N2O
C19H18N2O
230 ( 15
152 ( 8.5
176 ( 11
66 ( 4.6
57 ( 2.2
86 ( 5.1
80 ( 5.2
85 ( 3.7
119 ( 9.6
CH(CH3)2
CH3
58 oil
59 oil
86 oil
72 oil
87 54-56
77 oil
C20H20N2O
C21H22N2O
C22H24N2O
C23H26N2O
C24H28N2O
C25H30N2O
CH3
CH3
CH3
CH3
CH3
cyclopentyl
cyclohexyl
55 155-157 C21H20N2O
35 161-163 C22H22N2O >300
58 oil 23H26N2O 56 ( 2.5
70 135-137 C23H24N2O3 >300
75 112-115 C24H26N2O3 >300
0.1
2j
2n
3a
3b
C
COOH
COOH
6
7
RP73163
GERI-BP001M
42 ( 1.7
From 1. C, H, N analysis within (0.4%. c In vitro ACAT
inhibition determined in rat liver microsomes. IC50 values are
mean from three experiments.
a
b
Resu lts a n d Discu ssion
All compounds were tested for their inhibitory prop-
erties toward ACAT extracted from rat liver mi-
crosomes.14 In the 2-substituted pyridazinone series
(Table 1), activity was displayed by compounds bearing
a long linear alkyl chain, the optimum being found for
n ) 5 (compound 2e; IC50 ) 57 µM). Introduction of a
methyl group at position 4 of this compound (compound
2n ) left the activity completely unchanged, suggesting
that a small lipophilic group can easily be accom-
The three compounds inhibit cholesterol esterification
in macrophages enriched with cholesterol, an in vitro
model of foam cells,20 without affecting cholesterol efflux
and cell viability21 (Table 3). These results, together

4294 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Brief Articles
Ta ble 3. Other Biological Properties of Compounds 5c,d and
5h
Ta ble 4. Molecular Electrostatic Potential Values (kcal/mol) on
the Molecular Surface, Calculated for the AM1 Optimized
Conformations
cholesterol efflux
[3H]-cholesterol
in the medium
(dpm × 103/mg
protein) (µM)
% inhibition
of cell viability
murine
macrophages
(µM)
macrophage
ACAT inhibitory
activitya
2e
2n
5a
5g
7a
8a
9a
V(N1) -50.1 -51.2 -66.7 -64.0 -62.5 -59.3 -54.7
compd
control
(IC50; µM)
12.9 ( 0.1
from its influence on the electrostatic properties of the
heterocyclic moiety. For the global minima of all the
modeled compounds, the molecular electrostatic poten-
tial was calculated on the molecular surface and the
value of the electrostatic potential minima generated
by the heteroatoms of the heterocyclic moiety was
determined. Once again, no safe correlations appear
possible, as can be seen, for example, in Table 4, where
the value of the potential in proximity of the N1 nitrogen
atom of pyridazine or pyridazinone is reported. How-
ever, 5a was the one that presents the more negative
electrostatic potential minimum near the N1 nitrogen
atom of pyridazine.
58-035b 12.8 ( 1.4 (2.15)
0.6 ( 0.01
3.2 ( 0.32
5c
5d
5h
12.9 ( 1.6 (5)
11.5 ( 0.8 (25)
11.5 ( 3.5 (1)
9.9 ( 1.6 (5)
3.7 ( 1.7 (10)
4.8 ( 3.7 (25)
5.9 ( 1.9 (1)
2.7 ( 2.7 (5)
<1 (10)
14.1 ( 1.8 (5)
13.8 ( 4.8 (25)
11.0 ( 0.34
5.1 ( 1.16
<1 (25)
12.6 ( 1.3 (5)
11.8 ( 0.6 (25)
<1 (1)
<1 (5)
<1 (10)
<1 (25)
a
b
IC50 values are means from three experiments. See ref 9 for
its structure.
In conclusion, though not very potent, the compounds
presented show appreciable preference toward ACAT in
macrophages than in rat liver. Since accumulation of
cholesteryl esters in macrophages results in foam cell
formation and in a final atherosclerotic lesion, this class
could represent a novel model to be investigated.
Finally, our results seem to suggest that an easier
rotation of the alkyl chain and a more negative electro-
static potential near the pyridazine ring are elements
that favor a good activity.
with the observed ability of the tested compounds to
reduce cholesterol esterification when added to cell-free
homogenates, suggest that they influence cholesterol
metabolism by specifically inhibiting ACAT activity. The
differences in the compounds’ activity on ACAT assay
in macrophages and in rat liver microsomes, could be
interpreted on the basis of the different properties of
ACAT isoenzymes.22,23 Moreover, it was reported that
disruption of the gene for ACAT (Acact) in mice resulted
in a marked reduction in cholesteryl ester levels in the
adrenal glands and peritoneal macrophages; in contrast,
the livers of these mice still contained significant
amount of cholesteryl esters and exhibited no reduction
in cholesterol esterification activity and in sterol ab-
sorption in the intestine.24
We performed modeling studies on several represen-
tative terms. The effect of the chain length has been
discussed above and appears quite intriguing; in fact,
while most of the results suggest a linear C5 or C6 alkyl
chain as the optimal length, in some cases this is not
true. An explanation of this behavior could reside in the
nature of the X atom (or group) that could exert its
effects by forcing the alkyl chain to assume different
orientations or influencing the movements of the same
chain with respect to the heterocyclic ring. Thus, a
different chain length could be necessary in each series
to accommodate it into the same binding site. The
modeling study of compounds 2e/2n /5a /5g/7a /8a /9a /
was performed at the semiempirical level with the AM1
program;25 the conformational space of each compound
was fully explored, and the conformers of minimum
energy were located. The 5- and 6-phenyl groups can
rotate, but they present similar energy minima and
similar barriers to rotation in all the cases; on the
contrary, when we determined the energy profiles for
rotation around the single bond N2-C1′ (for 2e and 2n )
or C3-X (for the other compounds), we found a large
heterogeneity in the energy profiles. The energy barriers
to rotation ranged from about 3 kcal/mol in 5a to about
6 kcal/mol in 7a . It is not easy to correlate these profiles
with activity; however, the most active compound (5a )
is the one that presents the better easiness of rotation.
A different effect of the atom X on activity could derive
Exp er im en ta l Section
Ch em istr y. All melting points were determined on a Bu¨chi
apparatus and are uncorrected. 1H NMR spectra were recorded
with Varian Gemini 200 instruments. Chemical shifts are
reported in ppm, using the solvent as internal standard.
Extracts were dried over Na2SO4, and the solvents were
removed under reduced pressure. Merck F-254 commercial
plates were used for analytical TLC to follow the course of
reaction. Silica gel 60 (Merck 70-230 mesh) was used for
column chromatography.
Gen er a l P r oced u r e for Com p ou n d s 2a -m , 7a -h , 3a -
c, a n d 10a -d . A mixture of 5,6-diphenylpyridazin-3(2H)-one
(1)17 or its thioanalogue (6)18 (1.6 mmol), anhydrous K2CO3 (1.8
g, 13 mmol), and the appropriate (cyclo)alkyl bromide (15
mmol) or alkyl bromoester in anhydrous DMF (6 mL) was
heated at 40-80 °C under stirring for 2-48 h. After dilution
with cold water (150 mL), the suspension was extracted with
CH2Cl2 (3 × 50 mL). Removal of the solvent afforded a crude
oil which crystallized by treatment with EtOH for compounds
2b, 2i, and 7a ; all other compounds were purified by column
chromatography. Eluents: cyclohexane/ethyl acetate 2:1. Es-
ters 2k -m and 7e-h were recovered as crude products and
hydrolyzed in an alkaline medium to give compounds 3a -c
and 10a -d .
2-Hexyl-4-m eth yl-5,6-d ip h en ylp yr id a zin -3(2H)-on e 2n .
The 5,6-diphenyl-4,5-dihydropyridazin-3(2H)-one17 (0.4 g, 1.6
mmol) was heated with 40% aqueous CH2O in 5% ethanolic
KOH (20 mL) at 100 °C for 30 min. After cooling, acidification
with 6 N HCl afforded the crude 5,6-diphenyl-4-methylpy-
ridazin-3(2H)-one,26 which was recovered by suction and
reacted with n-hexylbromide as above-reported for 2a -j to give
2n , finally purified by column chromatography (cyclohexane/
ethyl acetate 2:1).
Gen er a l P r oced u r e for Am in es 5a -d . 3-Chloropy-
ridazine (4; 1.5 mmol) and the appropriate alkylamine (150
mmol) were heated at 160 °C in a sealed tube for 3 h. After
dilution with water (10 mL), the mixture was extracted with
CH2Cl2 (3 × 20 mL) and concentrated in vacuo. The oil residue

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J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4295
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crystallized by treatment with ice-ethanol (5a ,b) or ethanol/
water 1:1 (5c,d ).
N-(5,6-Dip h en ylp yr id a zin -3-yl)h exa n oic Am id e 5e.
3-Chloropyridazine (4; 0.1 mmol) and NH3 30% (6 mL) were
heated in a reactor at 150 °C for 3 h. The 3-amino derivative
was recovered by suction from the still hot solution and washed
with a small amount of water. The dried compound was stirred
at room temperature with hexanoyl chloride (10 mmol) for 22
h. The mixture was cautiously diluted with water and ex-
tracted with CH2Cl2 (3 × 30 mL). After evaporation of the
solvent under vacuum, the oil residue crystallized by treatment
with ethanol.
Gen er a l P r oced u r e for Eth er s 5f-j. Sodium (0.2 mmol)
was dissolved in the appropriate alcohol (0.5-0.8 mL) at room
temperature. 3-Chloropyridazine (4, 0.1 mmol) was added
under stirring for 1 h maintaining the temperature at 120-
140 °C. After dilution with water, the mixture was extracted
with CH2Cl2 (3 × 20 mL). Distillation under vacuum (10-2
mmHg at 140 °C) afforded compounds 5f-j, which were
purified by column chromatography. Eluents: cyclohexane/
ethyl acetate 3:1.
Gen er a l P r oced u r e for Su lfoxid es 8a -d . To a suspen-
sion of 7a -d (0.5 mmol) in 50% (w/v) AcOH (10 mL) was added
35% (w/v) H2O2 (0.5 mL). The mixture was stirred for 11-30
h at room temperature. After standard workup of the reaction
mixture, compounds 8a -d were recovered and purified by
column chromatography. Eluents: cyclohexane/ethyl acetate
1:2.
Gen er a l P r oced u r e for Su lfon es 9a -d . A solution of
8a -d (0.5 mmol) and MCPBA (1.5 mmol) in CH2Cl2 (6 mL)
was stirred at room temperature for 1-3 h. The solution was
extracted with 5% (w/v) NaHCO3 (2 × 15 mL), and the organic
layer was washed with water (15 mL) and dried. Removal of
the solvent afforded the crude compounds 9a -d , which were
purified by column chromatography. Eluents: cyclohexane/
ethyl acetate 3:1.
Biologica l Assa ys. Compounds were tested according to
previously reported procedures: ACAT assay,14,19 cholesterol
esterification in cell culture,19 cellular cholesterol efflux,19 and
evaluation of cell viability.21
Su p p or tin g In for m a tion Ava ila ble: Additional spectral
data for synthesized compounds and biological methods. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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