Mono- and di-substituted 5,6-diphenyl-3-alkylaminopyridazines active as ACAT inhibitors

Article abstract of DOI:10.3987/COM-01-9351

A series of mono- or di-para-substituted 5,6-diphenyl-3-alkylaminopyridazines were synthesized and their inhibitory activity against acyl-CoA:cholesterol acyltransferase (ACAT) was tested on the enzyme prepared from rat liver microsomes. The compound which combines a chlorine atom on the 6-phenyl ring and a n-hexylamino chain showed a significant enhancement of activity with respect to the unsubstituted derivative. Attempts to correlate the activity of the compounds to their structural features, also through theoretical calculations, are reported.

Full text of DOI:10.3987/COM-01-9351

HETEROCYCLES, Vol. 57, No. 1, 2002, pp. 39 - 46, Received, 1st October, 2001
MONO- AND DI-SUBSTITUTED 5,6-DIPHENYL-3-ALKYLAMINO-
PYRIDAZINES ACTIVE AS ACAT INHIBITORS
Lucio Toma,a, Maria Paola Giovannoni,b Vittorio Dal Piaz,b Byoung-Mog
*
Kwon,c Young-Kook Kim,c Arianna Gelain,d and Daniela Barloccod,
*
[a] Dipartimento di Chimica Organica, Università di Pavia, Via Taramelli 10,
27100 Pavia, Italy; [b] Dipartimento di Scienze Farmaceutiche, Università di
Firenze, Via Capponi 9, 50121 Firenze, Italy; [c] Korea Research Institute of
Bioscience & Biotechnology, 52 Uen-Dong Yusung-Ku, Taejeon 305-600, Korea;
[d] Istituto di Chimica Farmaceutica e Tossicologica, Università di Milano, Viale
Abruzzi 42, 20131 Milano, Italy
Abstract - A series of mono- or di-para-substituted 5,6-diphenyl-3-
alkylaminopyridazines were synthesized and their inhibitory activity against acyl-
CoA:cholesterol acyltransferase (ACAT) was tested on the enzyme prepared from
rat liver microsomes. The compound which combines a chlorine atom on the 6-
phenyl ring and a n-hexylamino chain showed a significant enhancement of
activity with respect to the unsubstituted derivative. Attempts to correlate the
activity of the compounds to their structural features, also through theoretical
calculations, are reported.
INTRODUCTION
Hypercholesterolemia is recognized as an important risk factor for the development of coronary heart
disease (CHD).1 Since acyl-CoA:cholesterol acyltransferase (ACAT) is responsible for the production of
cholesteryl esters, ACAT inhibitors have been identified as useful targets in the treatment of
hypercholesterolemia.2-7 As a continuation of previous studies8 on 5,6-diphenyl derivatives active as
ACAT inhibitors, we recently reported9 the synthesis and the inhibiting properties of a series of 5,6-
dinitrophenyl- and 5-aminophenyl-6-nitrophenyl-3-alkylaminopyridazines. Theoretical studies on these
compounds suggested that an enhancement of their inhibition properties could be attained by introduction
of substituents that make the pyridazine ring more electron-rich. To verify this hypothesis, we have now
synthesized a new series of derivatives, having methoxy- or chloro-substituents in the para position of
either one or both the phenyl rings. Moreover, the n-hexyl- and n-nonylamino groups were chosen as

substituents on the 3-position since the most interesting compounds in the unsubstituted series were found
to have these side-chains.8 We report here the synthesis of a number of derivatives that were tested for
their inhibition of ACAT from rat liver microsomes. In addition, attempts to correlate their activity to
their structural features through a theoretical investigation are discussed.
CHEMISTRY
The series of derivatives (1a-f) and (2a-f), para-substituted on one or both the phenyl rings with groups
which are notoriously electron-releasing, namely the methoxy and chloro substituents, were synthesized
according to the procedure reported in Scheme 1. The suitable phenylacetic acid was transformed into the
corresponding acyl chloride and condensed with the appropriately substituted benzene in the presence of
aluminum chloride at 0 °C. The so obtained keto derivative (3) easily led to the corresponding keto ester
(4) by treatment with ethyl bromoacetate in dimethyl sulfoxide and in the presence of sodium hydride.
Hydrolysis in basic medium to 5 and subsequent cyclization by hydrazine hydrate in refluxing ethanol led
to the desired dihydropyridazinones (6), which were dehydrogenated to 7 by stirring at 100 °C in an
alkaline medium with sodium m-nitrobenzenesulfonate. Finally, heating with POCl3 at 60 °C gave the
desired chloropyridazine (8), which was directly condensed with n-hexylamine to give 1 or with n-
nonylamine to give 2.
ENZYME ASSAY
All the compounds obtained were tested for their potency to inhibit ACAT extracted from rat liver
microsomes, according to a previously reported method.10 The unsubstituted derivatives (1g) and (2g)
were used as reference. IC50 values are listed in Table 1.
All the tested compounds were active at micromolar range. They all presented about the same activity
(IC50 ≈ 10-30 µM), the only positive exceptions being in the n-hexyl series the compound chlorinated on
the 6-phenyl group (1e) (IC50 = 1.0 µM) and in the n-nonyl series the doubly chlorinated compound (2f)
(IC50 = 2.5 µM). A negative exception is represented by 2e (IC50 = 80 µM) the compound corresponding
to 1e but with a longer n-alkyl chain.
THEORETICAL CALCULATIONS
Since previous studies suggested9 a correlation between the activity of this class of compounds and the
electrostatic properties near the nitrogen atoms of the pyridazine ring, we similarly decided to determine
the same properties in the series of compounds (1a-f) and (2a-f). Thus, the conformational space of all the
compounds was explored at the semiempirical AM1 level11 allowing to determine their energy minima.
Their geometrical features appeared quite similar as the presence of one or two substituents on the para

Scheme 1
R
O
CH2CO2H
CH2COCl
a
b
R1
R1
R1
3
c
R
R
R
N
O
O
NH
e
d
CO2H
CO2Et
O
R1
R1
R1
6
5
4
f
R
R
1
2
h
N
N
NH
N
g
O
Cl
i
R1
R1
7
8
a) SOCl2, CH2Cl2, reflux; b) R-C6H5, AlCl3, CH2Cl2, 0 °C, then rt; c) NaH, BrCH2CO2Et, DMSO,
.
toluene; d) 25% NaOH in EtOH/H2O 1:1; e) NH2NH2 H2O, EtOH, reflux; f) sodium m-nitro-
benzensulfonate, NaOH, reflux; g) POCl3, 60 °C; h) n-hexylamine; i) n-nonylamine.
positions of the phenyl rings does not influence the conformational behaviour of the molecules. For the
global minimum of each compound the molecular electrostatic potential (MEP) was calculated on the
molecular surface of the optimized conformations at the AM1 level. The values of the electrostatic
potential minima generated by the two nitrogen atoms of the pyridazine ring and the maximum generated
by the NH group, taken as significant points of the core region of the molecules potentially involved in
favorable interactions at the active site of the enzyme, resulted in all the cases quite similar (Table 1).
These results confirmed our initial hypothesis that a more electron-rich pyridazine ring is necessary to
confer ACAT inhibitory properties to this class. In fact, the average values of the electrostatic potentials
of this new series are quite close to those of the most potent derivatives in the previously reported amino/

Table 1. Biological data and molecular electrostatic potential data of compounds (1a-g) and (2a-g).
R
N
N
NH(CH2)nMe
R1
1, 2
a
compd
n
R
R1
IC50
V(NH)b
V(N1)b
V(N2)b
5
8
5
8
5
8
5
8
5
8
5
8
H
H
33
22
11
8.9
25
11
19
18
35.2
34.9
35.4
35.0
34.4
34.4
37.1
37.1
36.9
36.2
38.6
38.6
-65.9
-65.9
-65.3
-65.4
-65.1
-64.8
-64.3
-64.2
-62.8
-62.7
-61.2
-61.2
-65.2
-65.6
-64.3
-64.2
-65.5
-64.9
-63.0
-62.9
-62.7
-62.6
-61.2
-61.2
1a
2a
1b
2b
1c
2c
1d
2d
1e
2e
1f
OMe
OMe
H
OMe
OMe
OMe
OMe
H
H
Cl
Cl
Cl
H
OMe
OMe
Cl
Cl
H
H
Cl
Cl
1.0
80
19
Cl
2.5
2f
5
8
H
H
24
18
35.8
35.7
-66.4
-65.9
-74.8
-65.0
1g
2g
H
H
a) In vitro ACAT inhibition determined in rat liver microsomes. IC50 values are from three experiments,
which agreed within 10%.
b) MEP values (V, kcal/mol) on the molecular surface, calculated for the AM1 optimized conformations.
nitro-substituted analogs.9 In addition, the data of activity seem to suggest a very important role of the
chlorine atom in the interaction with the active site of the enzyme. This interaction might be influenced
by the length of the n-alkyl chain, which could control the way of presentation of the molecules in the
active site. In fact, though compounds (1e) and (2e) differ only in the length of the alkyl chain and hence
have practically identical electrostatic properties, they are, respectively, the most and the least active
compounds. The combination present in 1e, i. e. a chlorine atom on the para-position of the 6-phenyl ring
together with the shorter (n=5) alkyl chain, seems to be very favorable, since it produces a significant
enhancement in activity with respect to the reference compounds (1g) and (2g). On the contrary, the
longer homolog (2e) (n=8) is less active with respect to both 1e and the reference compounds, though by
different degrees.
Further work appears necessary to better define the required structural features of this class of ACAT
inhibitors, but compound (1e) could represent a good model for the search of more active compounds.

EXPERIMENTAL
Melting points were determined on a Büchi 510 capillary melting points apparatus and are uncorrected.
1H-NMR spectra were recorded on a Bruker AC200 spectrometer; chemical shifts are reported as δ (ppm)
relative to tetramethylsilane. TLC on silica gel plates was used to check product purity. Silica gel 60
(Merck; 230-400 mesh) was used for flash chromatography. The structures of all compounds were
consistent with their analytical and spectroscopic data. Compound (3c) (desoxyanisoin) was purchased
from Aldrich Co.
General method for the synthesis of compounds (3)
A solution of the required phenylacetic acid (0.012 mol) and SOCl2 (2.1 g, 0.029 mol) in CH2Cl2 (5 mL)
was refluxed overnight. After evaporating the excess thionyl chloride, the obtained acyl chloride was
dissolved in CH2Cl2 (20 mL) together with the appropriately substituted benzene (0.040 mol), added
dropwise to an ice-cooled suspension of AlCl3 (4.9 g, 0.037 mol) in CH2Cl2 (10 mL), and stirred
overnight at rt. The reaction mixture was poured onto a mixture of ice (27 g) and HCl (11 mL). The
organic phase was separated, dried over sodium sulfate, the solvent evaporated, and the crude product
purified by flash chromatography by eluting with cyclohexane/ethyl acetate 9:1 (see Table 2 for data). For
1
3a, H-NMR (CDCl3) δ: 3.8 (s, 3H), 4.2 (s, 2H), 6.8 (d, 2H, J= 8 Hz), 7.2 (d, 2H, J=8 Hz), 7.4-7.6 (m,
3H), 8.0 (d, 2H, J=7.5 Hz).
General method for the synthesis of compounds (4)
To a suspension of dry NaH (95%; 0.16 g, 0.0064 mol) in anhydrous DMSO (3.3 mL) under nitrogen
atmosphere, a solution of the appropriate ketone (3) (0.0064 mol) in anhydrous DMSO (8.2 mL) was
added dropwise. After the reaction subsides, ethyl bromoacetate (0.7 mL, 0.0064 mol) in toluene (5 mL)
was added dropwise and the mixture stirred overnight. The mixture was then acidified by 2N HCl, diluted
with water (30 mL) and exctracted by dichloromethane (3 × 20 mL). After drying over sodium sulfate,
the solvent was evaporated and the residue purified by flash chromatography, eluting with
cyclohexane/ethyl acetate 9:1, to give as the first run the unreacted 3, followed by the desired 4 (see Table
1
2 for data). For 4a, H-NMR (CDCl3) δ: 1.2 (t, 3H, J=7 Hz), 2.7 (dd, 1H, J=17.5, 5 Hz), 3.3 (dd, 1H,
J=17.5, 9.5 Hz), 3.7 (s, 3H), 4.2 (q, 2H, J=7 Hz), 5.1 (dd, 1H, J=9.5, 5 Hz), 6.8 (d, 2H, J=8 Hz), 7.2 (d,
2H, J=8 Hz), 7.3-7.6 (m, 3H), 8.0 (d, 2H, J=7.5 Hz).
General method for the synthesis of compounds (6)
a) The appropriate ester (4) (0.0023 mol) was hydrolized by 25% NaOH in ethanol/water 1:1 (15 mL,
0.094 mol), by stirring at rt for 2 h. After evaporation of ethanol, the residue was diluted with water (30
mL) and extracted with CH2Cl2 (3 × 10 mL). The aqueous layer was then acidified by 6N HCl and
extracted with CH2Cl2 (3 × 20 mL). After drying over sodium sulfate, evaporation of the solvent gave the
acid (5) which was used as such for the next step.

Table 2. Chemical properties of compounds (1-8).
compound
R
R1
Yield %
mp/°C
1a
2a
3a
4a
5a
6a
7a
8a
1b
2b
3b
4b
5b
6b
7b
8b
1c
2c
4c
5c
6c
7c
8c
1d
2d
3d
4d
5d
6d
7d
8d
1e
2e
3e
4e
5e
6e
7e
8e
1f
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
77.7
71.4
68.0
43.7
88.7
46.0
94.2
87.8
82.0
84.0
30.0
50.6
96.7
36.4
90.0
92.2
56.7
85.7
43.4
65.5
41.0
99.0
81.6
95.1
94.0
55.7
33.9
81.0
63.6
85.7
83.0
72.9
65.4
12.0
37.9
78.4
56.5
89.7
85.4
83.2
75.3
21.0
46.3
73.0
48.5
56.7
82.0
102-104
105-106
92-93
oil
115-116
202-203
210-213
95-96
99-100
94-96
74-75
oil
118-120
175-176
182-185
85-86
112-114
110-112
oil
120-122
155-156
235-237
145-148
97-99
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
94-96
130-131
oil
145-146
197-198
205-206
120-122
135-136
133-134
135-136
oil
132-133
208-209
221-222
114-115
138-140
132-134
97-98
H
H
H
Cl
Cl
Cl
Cl
Cl
2f
3f
4f
5f
6f
7f
8f
Cl
Cl
Cl
Cl
Cl
Cl
Cl
oil
118-119
172-173
236-238
94-95
Cl
Cl
Cl

b) A mixture of the required acid (5) (0.0017 mol) and hydrazine monohydrate (0.17 mL, 0.0035 mol) in
ethanol (13 mL) was refluxed overnight. After cooling, the precipitated dihydropyridazinone (6) was
1
filtered under suction and thoroughly washed with ethanol (see Table 2 for data). For 6a, H-NMR
(CDCl3)δ: 2.8 (br d, 1H, J=17 Hz), 3.0 (dd, 1H, J=17, 7.5 Hz), 3.7 (s, 3H), 4.4 (br d, 1H, J=7.5 Hz), 6.8 (d,
2H, J=8.5 Hz), 7.1 (d, 2H, J=8.5 Hz), 7.4 (m, 3H), 7.7 (m, 2H), 8.6 (s, 1H, exch. with D2O).
General method for the synthesis of compounds (7)
A mixture of the required dihydropyridazinone (6) (0.0007 mol), sodium m-nitrobenzensulfonate (0.156 g,
0.00069 mol), and 0.5N NaOH (8 mL, 0.0040 mol) was refluxed for 4 h. After cooling, the solution was
acidified to pH 1 by 6N HCl. The so formed precipitate was collected by suction and thoroughly washed
1
with water (see Table 2 for data). For 7a, H-NMR (CDCl3) δ: 3.8 (s, 3H), 6.8 (d, 2H, J=8 Hz), 6.9 (s,
1H), 7.1 (d, 2H, J=8 Hz), 7.2-7.4 (m, 5H), 11.0 (s, 1H, exch. with D2O).
General method for the synthesis of compounds (1a-f) and (2a-f)
a) A mixture of the appropriate pyridazinone (7) (0.00065 mol) and POCl3 (1.5 mL, 0.016 mol) was
stirred at 60 °C for 3 h. After cooling, the mixture was poured onto ice/water (10 mL), its pH was brought
to 6 by 5N NaOH, and then it was extracted with CH2Cl2 (3 × 15 mL). After drying over sodium sulfate,
evaporation of the solvent gave 8, which was used as such for the next step (see Table 2 for data).
b) The required chloropyridazine (8) (0.1 mmol) was heated overnight at 160 °C with n-hexyl- or n-
nonylamine (0.2 mmol). The crude product was purified by flash chromatography, eluting with
1
cyclohexane/ethyl acetate 6:4 (see Table 1 for data). For 1a, H-NMR (CDCl3) δ: 0.9 (t, 3H, J=6.5 Hz),
1.2-1.5 (m, 6H), 1.6-1.7 (m, 2H), 3.4 (q, 2H, J=6.5 Hz), 3.8 (s, 3H), 5.0 (t, 1H, J=5.5 Hz, exch. with D2O),
6.6 (s, 1H), 6.8 (d, 2H, J=8.5 Hz), 7.1 (d, 2H, J=8.5 Hz), 7.3-7.4 (m, 5H).
1a: Anal. Calcd for C23H27N3O: C, 76.42; H, 7.53; N, 11.62. Found: C, 76.73; H, 7.33; N, 11.69.
2a: Anal. Calcd for C26H33N3O: C, 77.38; H, 8.24; N, 10.41. Found: C, 77.25; H, 8.09; N, 10.23.
1b: Anal. Calcd for C23H27N3O: C, 76.42; H, 7.53; N, 11.62. Found: C, 76.38; H, 7.46; N, 11.54.
2b: Anal. Calcd for C26H33N3O: C, 77.38; H, 8.24; N, 10.41. Found: C, 77.55; H, 8.40; N, 10.11.
1c: Anal. Calcd for C24H29N3O2: C, 73.63; H, 7.47; N, 10.73. Found: C, 73.50; H, 7.30; N, 10.67.
2c: Anal. Calcd for C27H35N3O2: C, 74.79; H, 8.14; N, 9.69. Found: C, 74.48; H, 8.21; N, 9.83.
1d: Anal. Calcd for C22H24N3Cl: C, 72.22; H, 6.61; N, 11.48. Found: C, 72.01; H, 6.56; N, 11.60.
2d: Anal. Calcd for C25H30N3Cl: C, 73.60; H, 7.41; N, 10.30. Found: C, 73.64; H, 7.22; N, 10.42.
1e: Anal. Calcd for C22H24N3Cl: C, 72.22; H, 6.61; N, 11.48. Found: C, 72.15; H, 6.75; N, 11.55.
2e: Anal. Calcd for C25H30N3Cl: C, 73.60; H, 7.41; N, 10.30. Found: C, 73.40; H, 7.35; N, 10.15.
1f: Anal. Calcd for C22H23N3 Cl2: C, 66.00; H, 5.79; N, 10.50. Found: C, 66.22; H, 5.71; N, 10.68.
2f: Anal. Calcd for C25H29N3 Cl2: C, 67.87; H, 6.61; N, 9.50. Found: C, 68.02; H, 6.53; N, 9.58.

Enzyme Assay
Microsomes prepared from rat liver were used as a source of the enzyme. The activity of the ACAT
inhibitors was measured according to a previously described method.10
Theoretical calculations
Theoretical calculations were performed with the SPARTAN package12 using the AM1 semiempirical
method11 and were carried out at the RHF level. The geometry of all the compounds investigated was
fully optimized and energy minimized. Several optimizations from different starting geometries were
performed to take into account the possible conformers of each compound. However, the n-hexyl and n-
nonyl chains were always taken in the straight conformation.
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