Novel 3-Arylamino- and 3-Cycloalkylamino-5,6-diphenyl-pyridazines Active as ACAT Inhibitors

Article abstract of DOI:10.1002/ardp.200290010

A new series of pyridazine derivatives, structurally related to the previously reported ACAT inhibitors 3-(cyclo)alkylamino-5,6-diphenyl-pyridazines, were synthesized and tested for their inhibitory properties. Substitution of the 3-alkylamino chain with a phenylamino group maintains activity. In contrast, the presence of either substituents on the phenylamino group or aliphatic rings having more or less than six carbon atoms lowers it.

Full text of DOI:10.1002/ardp.200290010

Arch. Pharm. Pharm. Med. Chem. 2002, 11, 563–566
ACAT Inhibitors 563
Lucio Tomaa,
Novel 3-Arylamino- and
3-Cycloalkylamino-5,6-diphenyl-pyridazines Active
as ACAT Inhibitors
Maria Paola Giovannonib,
Claudia Vergellib,
Vittorio Dal Piazb,
Byoung-Mog Kwonc,
Young-Kook Kimc,
Arianna Gelaind,
A new series of pyridazine derivatives, structurally related to the previously reported
ACAT inhibitors 3-(cyclo)alkylamino-5,6-diphenyl-pyridazines, were synthesized
and tested for their inhibitory properties. Substitution of the 3-alkylamino chain with
a phenylamino group maintains activity. In contrast, the presence of either substitu-
ents on the phenylamino group or aliphatic rings having more or less than six carbon
atoms lowers it.
Daniela Barloccod
a
Dipartimento di Chimica
Organica,
Università di Pavia,
Via Taramelli 10,
27100 Pavia, Italy
Key Words: Hypercholesterolemia, Acyl-CoA:cholesterol acyltransferase (ACAT),
ACAT inhibitors, 5,6-Diphenylpyridazine derivatives
b
Dipartimento di Scienze
Farmaceutiche,
Università di Firenze,
Via Capponi 9,
Received: May 31, 2002 [FP704]
50121 Firenze, Italy
Korea Research Institute of
c
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
system [5]. Further investigation on the model I demon-
strated the ability of substituents on the 5- and 6-phenyl
Introduction
Acetyl-CoA:cholesterol
O-acyl
transferase
(EC
rings to modulate the inhibitory properties of this series
[6, 7]. In addition, the substitution of the n-alkylamino
group at C-3 with a cyclohexylamino group retained the
activity [8].We have now directed our studies to verify the
importance of the n-alkyl- or cyclohexylamino side chain
at position 3 of I.For this purpose, we synthesized a new
series of derivatives (1 a–p), where either a cycloalkyl
moiety of size different from cyclohexyl or a (substitut-
ed)phenyl was present.This paper reports their synthe-
sis and the results of the enzyme assay. In addition, at-
tempts to correlate the activity of the compounds to their
structural features are commented upon.
2.3.1.26; ACAT) is a microsomal enzyme that catalyzes
the formation of long chain fatty acid cholesterol esters
[1, 2].Inhibition of ACAT reduces intracellular cholesterol
esters that are substrates for steroidogenesis in adrenal
cells [3].Though the adrenal side effects of ACAT inhibi-
tors remain a key point for their development as anti-
atherosclerotic agents, a number of them are currently in
preclinical and clinical studies [4].With the aim of finding
compounds possibly devoid of such unwanted side ef-
fects, we have recently undertaken a study on a series of
pyridazine derivatives (I, Chart 1), which combine two
main characteristics of the reported ACAT inhibitors,
namely a long alkylic side chain and an ortho-diphenyl
Chemistry
The compounds were prepared from 3-chloro-5,6-
diphenylpyridazine [9] by heating at 140 °C in a sealed
tube with the appropriate amine in a molar ratio of 1:2.
Usual workup gave the desired compounds which were
finally crystallized from an appropriate solvent (see
Scheme 1).
Correspondence: Lucio Toma, Dipartimento di Chimica
Organica, Università di Pavia, Via Taramelli 10, 27100 Pavia,
Italy. Phone: +39 0382 507843, fax: +39 0382 507323, e-mail:
toma@chifis.unipv.it. Daniela Barlocco, Istituto di Chimica
Farmaceutica e Tossicologica, Università di Milano, Viale
Abruzzi 42, 20131 Milano, Italy. Phone: +39 02 503 17515, fax:
+39 02 503 17565, e-mail: daniela.barlocco@unimi.it.
© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/11/0563 $ 17.50+.50/0

564 Toma et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 563–566
Enzyme assay
All the compounds obtained were tested for their poten-
cy to inhibit ACAT extracted from rat liver microsomes,
according to a previously reported method [10]. GERI-
BP001 M was used as reference. Inhibition values are
listed in Tables 1 and 2.
Chart 1
Results and discussion
The substitution of the n-alkylamino with a cyclo-
alkylamino chain, which was reported by us to retain the
ACAT inhibition in the case of the cyclohexyl derivative
1 a [8], was further evaluated in the series of compounds
reported in Table 1. The compounds bearing a cyclo-
pentyl (1 b) and a cycloheptyl (1 c) moiety show a
Scheme 1
Table 1. Chemical and biological data of compounds 1 a–e.
a
Compd.
R
R1
Reaction
time [h]
Yield
[%]
Formula
Mp
[°C]
IC50
[µM]
1 a
1 b
1 c
1 d
1 e
H
H
H
CH3
H
cyclohexyl
cyclopentyl
cycloheptyl
cyclohexyl
C6H5
5
4
4
7
3
42
95
60
80
95
C22H23N3
C21H21N3
C23H25N3
C23H25N3
C22H17N3
182–184
180–182
198–199
160–162
232–234
3.6
46
25
31
4.5
a In vitro ACAT inhibition determined in rat liver microsomes.IC50 values are from three experiments which agreed within
10 %. In the same test GERI-BP001 M showed an IC50 value of 42 µM.
Table 2. Chemical and biological data of compounds 1 f–p.
Compd.
R
R1
Reaction
time [h]
Yield
[%]
Formula
Mp
[°C]
%
Inhibitiona
1 f
H
H
H
H
H
H
H
H
3-OCH3-C6H4
4-OCH3-C6H4
3-CF3-C6H4
4-CF3-C6H4
3-Cl-C6H4
4-Cl-C6H4
4-Br-C6H4
4-i-C3H7-C6H4
4-n-C7H15-C6H4
C6H5
3
48
2
3
3
2.5
6.5
3.5
3
10
48
72
68
14
92
57
65
33
36
80
95
82
C23H19N3O
C23H19N3O
C23H16N3F3
C23H16N3F3
C22H16N3Cl
C22H16N3Cl
C22H16N3Br
C25H23N3
200–202
227–229
>300
83
79
69
92 (36)
76
77
79
80
72
1 g
1 h
1 i
1 j
1 k
1 l
1 m
1 n
1 o
1 p
>300
233–235
276–279
278–280
153–155
154–157
139–141
178–180
H
CH3
H
C29H31N3
C23H19N3
C23H19N3
94 (39)
36
CH2C6H5
a
In vitro percent inhibition of ACAT from rat liver microsomes at a final concentration of 200 µg/mL. In parenthesis
IC50/µM.Values are from three experiments, which agreed within 10 %.In the same test GERI-BP001 M showed an in-
hibition percentage of 88 and 1 e showed an inhibition percentage of 85.

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 563–566
ACAT Inhibitors 565
decrease in activity by about one degree of magnitude
with respect to 1 a. A similar decrease also results in the
case of the tertiary amino compound 1 d.Conversely, ar-
omatization of the ring to 1 e maintains activity (IC50 = 4.5
µM as against 3.6 µM in the case of 1 a). Attempts to
modulate the activity by introduction of substituents on
the 3-phenylamino moiety were also undertaken. For
this purpose, a series of substituted-phenyl derivatives
were tested at a concentration of 200 µg/mL.The values
of their inhibition percentages are reported in Table 2. At
this concentration two compounds, 1 i and 1 o, present-
ed good values of the inhibition percentages while the
other compounds were poorer inhibitors than 1 e. How-
ever, a net decrease of activity was evident also for 1 i
and 1o when their IC50 values were determined. Thus,
the overall data inTable 2 show that the electronic nature
of the substituents on the phenyl ring of the side chain
does not play an important positive role. This was also
confirmed by the fact that theoretical studies on the com-
pounds failed to establish any correlation of the molecu-
lar electrostatic potential on several points of their mo-
lecular surface (Table 3) with the inhibitory activity. It
should be noted that the substitution of the phenyl ring by
a benzyl group led to an almost inactive compound (1 p),
thus indicating a possible conjugation of the system.
However, this would not explain why the phenyl- and the
cyclohexyl derivatives have almost the same activity. On
the contrary, the importance of the secondary amine
seems to be clearly evidenced in both classes, as shown
by the considerable loss of potency of the N-Me deriva-
tives 1 d and 1 o compared to 1 a and 1 e, respectively.
In conclusion, the data of these new series of ACAT in-
hibitors seem to suggest that the long alkyl chain, which
is supposed to mimic the natural substrate of ACAT [11],
is not an essential requirement for this class of pyri-
dazine inhibitors.In fact, they indicate a high tolerance of
the enzyme both to phenyl and cyclohexyl moieties.
However, the inhibitory activity is lowered by substitu-
ents on the phenylamino group and by cycloalkyl deriva-
tives of ring size different from six.This seems to suggest
that some limitations connected to steric hindrance are
operative in the modulation of activity.
1
Table 4. H-NMR data of compounds 1 a–p (CDCl3;
chemical shifts in ppm).
Compd.
1H-NMR
1 a
1.20–2.20 (m, 10 H); 3.70–3.80 (m, 1 H); 5.60
(br s, 1 H, exch with D2O); 6.70 (s, 1 H);
7.10–7.40 (m, 10 H)
1 b
1 c
1.40–2.20 (m, 8 H); 4.00–4.20 (m, 1 H); 4.90
(br s, 1 H, exch with D2O); 7.0 (s, 1 H);
7.20–7.80 (m, 10 H)
1.40–2.20 (m, 12 H); 3.90–4.00 (m, 1 H); 6.0
(br s, 1 H, exch with D2O); 6.75 (s, 1 H);
7.10–7.50 (m, 10 H)
1 d
1 e
1 f
1.00–2.00 (m, 10 H); 3.10 (s, 3 H); 4.50–4.75
(m, 1 H); 6.75 (s, 1 H); 7.10–7.40 (m, 10 H)
7.05 (s, 1 H); 7.20–7.60 (m, 15 H); 11.90 (br s,
1 H, exch with D2O)
3.80 (s, 3 H); 6.85–7.15 (m, 5 H); 7.20–7.50
(m, 10 H); 11.70 (br s, 1 H, exch with D2O)
3.90 (s, 3 H); 6.95–7.20 (m, 5 H); 7.25–7.50
(m, 10 H); 11.70 (br s, 1 H, exch with D2O)
7.20–7.50 (m, 15 H);11.50 (br s, 1 H, exch with
D2O)
1 g
1 ha
1 ia
1 j
Table 3. Molecular electrostatic potential on the molecu-
lar surface of compounds 1 e–n, p.Values of the electro-
static potential minima (V, kcal/mol) generated by the
two nitrogen atoms of the pyridazine ring (N1 and N2)
and of the maximum generated by the NH group.
6.65 (d, 2 H); 7.20–8.00 (m, 13 H); 11.80 (br s,
1 H, exch with D2O)
7.00 (s, 1 H); 7.10–7.55 (m, 14 H); 12.00 (br s,
1 H, exch with D2O)
1 k
1 l
6.95 (s, 1 H); 7.00–7.25 (m, 14 H); 11.40 (br s,
1 H, exch with D2O)
7.00 (s, 1 H); 7.15 (d, 2 H); 7.20–7.50 (m,
10 H); 7.60 (d, 2 H); 11.90 (br s, 1 H, exch with
D2O)
1.30 (d, 6 H); 2.90 (m, 1 H); 7.00–7.50 (m,
15 H); 11.55 (br s, 1 H, exch with D2O)
0.95 (m, 3 H); 1.20–1.40 (m, 8 H); 1.50–1.70
(m, 2 H); 2.60 (t, 2 H); 7.10–7.45 (m, 15 H);
8.65 (br s, 1 H, exch with D2O)
3.80 (s, 3 H);6.85 (s, 1 H);7.00–7.55 (m, 15 H)
4.70 (d, 2 H); 6.90 (s, 1 H); 7.00–7.50 (m,
15 H); 7.80 (br s, 1 H, exch with D2O)
Compd.
V (NH)
V (N1)
V (N2)
1 e
1 f
1 g
1 h
1 i
1 j
1 k
1 l
38.7
39.4
38.3
43.4
45.4
40.7
41.5
42.0
38.2
38.4
32.9
–65.4
–63.5
–65.4
–61.2
–60.7
–63.5
–63.5
–63.2
–65.6
–65.5
–66.4
–65.6
–62.8
–65.3
–59.0
–58.6
–62.7
–62.6
–62.2
–66.2
–66.1
–64.3
1 m
1 n
1 o
1 p
1 m
1 n
1 p
a
DMSO-d6.

566 Toma et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 563–566
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