3-Heptylamino-5-phenylpyridazine derivatives as analogues of Acyl-CoA: Cholesterol acyltransferase inhibitors containing the N-heptyl-N′- arylureidic moiety

Article abstract of DOI:10.1002/ardp.200600106

A series of novel Acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors 8a-f was synthesized; the substances were characterized by the presence of a 2,5-dimethylpyrazin-3-yl moiety at one end and a 3-heptylamino-5- phenylpyridazine system at the other o

Full text of DOI:10.1002/ardp.200600106

Arch. Pharm. Chem. Life Sci. 2006, 339, 645–651
A. Gelain et al.
645
Full Paper
3-Heptylamino-5-Phenylpyridazine Derivatives as Analogues of
Acyl-CoA: Cholesterol Acyltransferase Inhibitors Containing
the N-Heptyl-N 9-Arylureidic Moiety
Arianna Gelain1, Daniela Barlocco1, Byong-Mog Kwon2, Tae-Sook Jeong2, Kyung-Ran Im2,
Laura Legnani3, and Lucio Toma3
1 Istituto di Chimica Farmaceutica e Tossicologica, Universitꢀ di Milano, Milano, Italy
2 Korea Research Institute of Bioscience & Biotechnology, Taejeon, South Korea
3 Dipartimento di Chimica Organica, Universitꢀ di Pavia, Pavia, Italy
A series of novel Acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors 8a–f was synthesized;
the substances were characterized by the presence of a 2,5-dimethylpyrazin-3-yl moiety at one
end and a 3-heptylamino-5-phenylpyridazine system at the other one, linked through linear
alkyl spacers of different length. The new derivatives were designed based on the hypothesis
that the 3-amino-5-phenylpyridazine moiety could mimic the aryl substituted urea, which was
present in a number of ACAT inhibitors previously described. The choice of the 2,5-dimethylpy-
razin-3-yl substituent was supported by a preliminary investigation, which indicated that this
moiety is the most powerful in conferring ACAT inhibitory properties to the new series. The
pharmacological results proved the idea to be sound. Finally, compounds 9a–c, lacking the phe-
nylpyridazine moiety were prepared and tested to further strengthen our hypothesis.
Keywords: ACAT inhibitors / Hypercholesterolemia / Pyridazine derivatives / Ureido analogues /
University of Pavia (FAR 2005
Received: June 30, 2006; accepted: September 15, 2006
DOI 10.1002/ardp.200600106
Introduction
Acyl-CoA: cholesterol acyltransferase (E.C.2.3.1.26, ACAT)
is an integral membrane protein, localized in the endo-
plasmic reticulum that catalyzes the intracellular esteri-
Figure 1. Structures of the ureido I and pyridazino II derivatives.
fication of cholesterol by using CoA-activated fatty acids
[1]. ACAT-mediated esterification plays a role in intestinal
cholesterol absorption, hepatic production of very low
density lipoproteins (VLDL) and unregulated deposition
of cholesteryl esters in atherosclerotic lesions [2]. There-
fore, ACAT inhibition represents an attractive target for
the treatment of hypercholesterolemia, atherosclerosis,
coronary diseases [3, 4] and, possibly, also for Alzheimer's
disease [5].
In previous papers [6–10], we reported our results on
the design, the synthetic approach, the enzyme inhibi-
tion assay and the conformational study of novel ACAT
inhibitors. In particular, a number of 5,6-diphenylpyrida-
zines carrying an n-alkylamino substituent at position 3
were investigated. They resulted, in several cases,
endowed with good inhibitory activity. Further studies
showed that the alkyl chain of the substituent at position
3 could end with a group such as a substituted urea. This
gave rise to compounds of the general formula I, which
were still equipped with interesting inhibitory proper-
ties (Fig. 1).
Correspondence: Daniela Barlocco, Istituto di Chimica Farmaceutica e
Tossicologica, V. le Abruzzi 42, I-20131 Milano, Italy.
E-mail: daniela.barlocco@unimi.it
Fax: +39 02 503-17565
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646
A. Gelain et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 645–651
Table 1. Chemical data and ACAT inhibition of compounds 1–5
and GERI-BP001 M.
Table 2. Chemical data of compounds 6 and 7.
Compound
R
%
Formula
% Inh.a)
Yield
(100 lg/mL)
Compound
n
R
R1
% Yield
Formula
1
2
H
55
16
C16H22N4
72
83
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
3
4
5
6
7
8
3
4
5
6
7
8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
90
88
89
87
29
23
73
71
70
69
28
34
C9H16N4
C10H18N4
C11H20N4
C12H22N4
C13H24N4
C14H26N4
C16H30N4
C17H32N4
C18H34N4
C19H36N4
C20H38N4
C21H40N4
C20H23ClN6
(CH2)6CH3
(CH2)6CH3
(CH2)6CH3
(CH2)6CH3
(CH2)6CH3
(CH2)6CH3
3
4
5
3
4
C21H26N6O
C32H32N6
C22H28N6
83
88
chain, were synthesized to evaluate the role of the linker
flexibility vs. rigidity (Table 4).
31
89
82
Finally, to confirm the validity of our preliminary
investigation on the efficiency of different heterocycles
at N1, we prepared and tested the analogue of the most
potent compound of the series 8d, still retaining the 5,6-
diphenylpyridazine system (compound 15), which char-
acterized the ureido derivatives I [11] (Table 3 and
Scheme 2).
GERI-BP001 M
a)
Inhibition determined in rat liver microsomes. All values are
means from three experiments, which differ by less than
10%.
Based on the hypothesis that the aryl substituted urea
could be mimicked by a 3-amino-5-phenyl-pyridazine
moiety in the binding ability and in the orientation of
the groups, we planned the synthesis of a series of novel
pyridazinyldiaminoalkyl derivatives of the general for-
mula II (Fig. 1).
Results and discussion
All compounds were tested for their inhibitory proper-
ties towards ACAT extracted from rat liver microsomes.
Their activity, expressed as inhibition percentage at
100 lg/mL, is reported in Tables 1, 3, and 4. GERI-BP001
M was used as reference compound [12].
In order to define which heterocycle should be intro-
duced in this new series, we performed a preliminary
investigation aimed at exploring the efficiency of various
systems at N1. Since the data we obtained indicated the
2,5-dimethylpyrazin-3-yl derivative 5 as the best com-
pound both in terms of inhibitory activity and chemical
yield (Table 1 and Scheme 1), we synthesized compounds
8a–f, all bearing this group on the left side and the 3-hep-
tylamino-5-phenylpyridazine system on the right side,
the two moieties being linked through a linear alkyl
spacer of different length. To better verify the real role of
the phenylpyridazine ring, a few derivatives 9a–c lack-
ing this moiety were also prepared (Tables 2, 3 and
Scheme 1). In addition, compounds 12a, b having either a
piperazinyl or a homopiperazinyl linker between the
two heterocyclic moieties, instead of the linear alkyl
As shown in Table 3, the length of the chain proved to
have some influence on the ACAT inhibitory activity,
though it could not easily be rationalized through an
expression of activity as a function of chain length. In
particular, the medium-length tertiary amine 8d was the
most active (95% inhibition) when compared to its higher
and lower homologues (8a–c, 8e–f) which showed per-
centage inhibition in the range 58–87%. By contrast, the
presence of a ring (12a, b) instead of the flexible spacer
dramatically reduced the activity. In fact, inhibition
values of 57 and 62% were found for 12a and 12b, respec-
tively (Table 4). However, this could also depend on the
distance between the two nitrogen atoms, even shorter
than in 8a, the compound with the shortest alkyl linker,
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Arch. Pharm. Chem. Life Sci. 2006, 339, 645–651
3-Heptylamino-5-phenylpyridazine Derivatives
647
Scheme 1. Synthesis of N1-substituted-N6-(5-phenylpyridazin-3-yl)-1,6-diaminohexane derivatives 1–5 and N1-(2,5-dimethylpyrazin-
3-yl)-diaminoalkyl-Nx-substituted derivatives 6–9.
Scheme 2. Synthesis of N1-(5,6-diphenylpyridazin-3-yl)-N6-(5-phenylpyridazin-3-yl)-N6-hepthyl-1,6-diaminohexane 15.
which was in turn one of the weakest representatives of tion percentages lower than 60% were found, thus con-
the series. It should be noted that, when the pyridazine firming the effective role of the heterocycle as a potential
moiety was replaced by a second n-heptyl residue (9 ser- substitute for the ureido group in this series of ACAT
ies), the activity always dropped significantly and inhibi- inhibitors. Finally, the 5,6-diphenylpyridazinyl deriv-
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648
A. Gelain et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 645–651
Table 3. Chemical data and ACAT inhibition of compounds 8, 9, 15, and GERI-BP001 M.
Compound
8a
n
R
R1
% Yield
Formula
% Inh.a)
(100 lg/mL)
3
(CH2)6CH3
17
C26H36N6
70
8b
4
(CH2)6CH3
15
C27H38N6
87
8c
5
6
(CH2)6CH3
(CH2)6CH3
17
17
C28H40N6
79
95
8d
C29H42N6
8e
8f
7
(CH2)6CH3
(CH2)6CH3
21
31
C30H44N6
58
76
8
C31H46N6
9a
9b
9c
3
4
5
(CH2)6CH3
(CH2)6CH3
(CH2)6CH3
(CH2)6CH3
(CH2)6CH3
(CH2)6CH3
40
43
39
C23H44N4
C24H46N4
C25H48N4
52
55
58
15
6
(CH2)6CH3
18b)
C39H46N6
79
82
GERI-BP001 M
a)
In vitro% ACAT inhibition determined in rat liver microsomes. All values are means from three experiments, which differ by less
than 10%.
From 14.
b)
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3-Heptylamino-5-phenylpyridazine Derivatives
649
Table 4. Chemical data and ACAT inhibition of compounds 12
and GERI-BP001 M.
vent as internal standard. Merck F-254 commercial plates
(Merck, Germany) were used for analytical TLC to follow the
course of reaction and check product purity. Silica gel 60 (Merck,
230–400 mesh) was used for flash chromatography. The struc-
tures of all compounds were consistent with their analytical and
spectroscopic data.
General synthesis of N1-substituted-N6-(5-phenyl-
pyridazin-3-yl)-1,6-diaminohexane derivatives 1–5
3-Chloro-5-phenylpyridazine (6.8 mmol, 1.30 g) was added,
under stirring to 1,6-diaminohexane 10d (68 mmol, 7.90 g) and
the mixture was heated for 1 h at 1508C. After cooling, the resi-
due was purified by flash chromatography (eluent: CH2Cl2/
CH3OH-NH3, ratio 9:1) to give 1 (1.00 g, 55%). The appropriate
heterocyclic chloride (2.1 mmol) and TEA (2.1 mmol) were added
to 1 (2.1 mmol, 0.57 g) and the mixture stirred at 1308C for 2 h.
Compounds 2–5 were purified by flash chromatography (elu-
ent: CH2Cl2/CH3OH-NH3, ratio 98:2) (for details see Tables 1, 5
and Scheme 1).
Compound
n
R
%
Yield
Formula % Inh.a)
(100 lg/mL)
11a
11b
1
2
H
H
91
88
C10H16N4
C11H18N4
12a
12b
1
2
46
46
C20H22N6 57
C21H24N6 62
General synthesis of N1-(2,5-dimethylpyrazin-3-yl)-
diaminoalkyl-Nx-substituted derivatives 6–9
3-Chloro-2,5-dimethylpyrazine (5.0 mmol, 0.71 g) was added
under stirring to the required amine (10a–f, 0.05 mol) and the
mixture was heated at 1508C for 2 h. After cooling, the residue
was purified by flash chromatography (eluent: CH2Cl2/CH3OH-
NH3, ratio 9:1) to give the mono substituted amines 6a–f which
were heated at 1508C for 1.5 h, under stirring, with equimolar 1-
bromoheptane and TEA. After purification by flash chromato-
graphy (eluent: CH2Cl2/CH3OH-NH3, ratio 98 : 2), the intermedi-
ates 7a–f were treated with equimolar amounts of either 3-
chloro-5-phenylpyridazine or 1-bromoheptane in the presence
of excess TEA at 1508C for 4 h. Compounds 8a–f and 9a–c were
finally purified by flash chromatography (eluent: CH2Cl2/CH3OH-
NH3, ratio 98:2) (for details see Tables 2, 3, 5 and Scheme 1).
Compounds 12a and 12b, having a cyclic spacer instead of the
linear alkyl chain, were prepared in a similar manner starting
from piperazine or homopiperazine, respectively (for details see
Tables 4, 5).
GERI-BP001 M
82
a)
In vitro% ACAT inhibition determined in rat liver micro-
somes. All values are means from three experiments, which
differ by less than 10%.
ative (15, 79% inhibition) was much weaker than its 2,5-
dimethylpyrazinyl analogue (8d, 95% inhibition).
In conclusion, our initial hypothesis that the pyrida-
zino moiety could possibly replace the ureido moiety pre-
sent in a series of ACAT inhibitors we previously
described [11], was supported fairly well by the data
shown in Table 3. The new 2,5-dimethylpyrazin-3-yl
derivatives 8 have ACAT inhibitory activity fully compar-
able to that previously reported for ureido counterparts.
In addition, the choice based on a preliminary screening
of the 2,5-dimethylpyrazinyl moiety instead of the 5,6-
diphenylpyridazinyl ring, which characterized the
ureido derivatives, proved to be successful, as clearly evi-
denced by the comparison between 15 and 8d.
Synthesis of N1-(5,6-diphenylpyridazin-3-yl)-N6-(5-
phenylpyridazin-3-yl)-N6-hepthyl-1,6-diaminohexane 15
a) To 1,6-diaminohexane 10d (18.7 mmol, 2.18 g), 3-chloro-5,6-
diphenylpyridazine [11] (1.87 mmol, 0.50 g) was added under
stirring and the mixture was heated for 1 h at 1508C. After cool-
ing, the residue was purified by flash chromatography (eluent:
CH2Cl2/CH3OH-NH3, ratio 9:1) to give 13 (78%). C22H26N4.
b) Compound 13 (1.46 mmol, 0.5 g) was heated at 1508C for
1.5 h, under stirring, with 1-bromoheptane (1.46 mmol, 0.26 g)
and TEA (1.46 mmol, 0.15 g). After purification by flash chroma-
tography (eluent: CH2Cl2/CH3OH-NH3, ratio 98:2), 14 was
obtained (16%). C29H40N4.
This work was partially supported by Italy-Korea Joint
Research Program and KRIBB Initiative Research Program. The
Authors also acknowledge the financial support from the Uni-
versity of Milan (FIRST 2005) and Pavia (FAR 2005).
c) Compound 14 (0.23 mmol, 0.10 g) was treated with 3-chloro-5-
phenylpyridazine (0.23 mmol, 0.044 g) in the presence of excess
TEA (0.46 mmol, 0.047 g) at 1508C for 4 h. After purification by
flash chromatography (eluent: CH2Cl2/CH3OH-NH3, ratio 98:2) 15
was obtained (18%). C39H46N6 (see Table 5 and Scheme 2 for
details).
Experimental
Chemistry
1H-NMR spectra were recorded on a Bruker AC200 spectrometer
(Bruker); chemical shifts are reported as d (ppm), using the sol-
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650
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Arch. Pharm. Chem. Life Sci. 2006, 339, 645–651
Table 5. 1H-NMR data of compounds 1–15 (CDCl3; chemical shifts in ppm).
Compound 1H-NMR
1
2
3
4
5
1.30–1.55 (m, 6H + 2H, ex), 1.65–1.75 (m, 2H), 2.65–2.75 (t, 2H), 3.40–3.50 (m, 2H), 4.80–4.90 (brs, 1H, ex), 6.75 (s, 1H), 7.40–
7.55 (m, 3H), 7.55–7.65 (d, 2H), 8.80 (s, 1H)
1.30–1.55 (m, 4H), 1.60–1.80 (m, 4H), 3.30–3.50 (m, 4H), 4.85 (brs, 2H, ex), 6.60 (d, 1H), 6.70 (s, 1H), 7.05 (d, 1H), 7.25–7.55 (m,
3H), 7.60–7.70 (m, 2H), 8.80 (s, 1H)
1.45–1.55 (m, 2H), 1.55–1.75 (m, 6H), 3.35–3.50 (m, 4H), 3.95–4.00 (s, 3H), 4.30 (brs, 1H, ex), 4.80 (brs, 1H, ex), 6.65 (d, 1H), 6.70
(s, 1H), 7.05 (d, 1H), 7.45–7.55 (m, 3H), 7.60–7.70 (m, 2H), 8.80 (s, 1H)
1.50–1.60 (m, 4H), 1.65–1.80 (m, 4H), 3.40–3.50 (m, 4H), 5.00 (brs, 2H, ex), 6.60 (s, 1H), 6.75 (s, 1H), 7.10–7.35 (m, 11H), 7.40–
7.50 (m,2H), 7.50–7.60 (m,2H), 8.75 (s,1H)
1.45–1.55 (m, 4H), 1.60–1.80 (m, 4H), 3.30 (s, 3H), 3.35 (s, 3H), 3.40–3.50 (m, 4H), 4.20 (brs, 1H, ex), 4.85 (brs, 1H, ex), 6.75 (s, 1H),
7.45–7.50 (m, 3H), 7.55 (s, 1H), 7.60–7.65 (m, 2H), 8.80 (s, 1H)
6a
6b
6c
6d
1.75–1.85 (m, 2H + 2H, ex), 2.30 (s, 3H), 2.35 (s, 3H), 2.60 (t, 2H), 3.40 (t, 2H), 5.95 (brs, 1H, ex), 7.50 (s, 1H)
1.50–1.70 (m, 4H + 2H, ex), 2.30 (s, 3H), 2.35 (s, 3H), 2.55 (t, 2H), 3.40 (t, 2H), 4.50 (brs, 1H, ex), 7.50 (s, 1H)
1.35–1.40 (m, 2H + 2H, ex), 1.50–1.55 (m, 4H), 2.30 (s, 3H), 2,35 (s, 3H), 2.60 (t, 2H), 3.40 (t, 2H), 4.20 (brs, 1H, ex), 7.55 (s, 1H)
1.30–1.45 (m, 6H + 2H, ex), 1.50–1.65 (m, 2H), 2.20 (s, 3H), 2.25 (s, 3H), 2.50–2.70 (m, 2H), 3.30–3.45 (m, 2H), 4.25 (brs, 1H, ex),
7.45 (s, 1H)
6e
6f
1.30–1.50 (m, 6H), 1.50–1.70 (m, 4H + 2H, ex), 2.30 (s, 3H), 2.35 (s, 3H), 2.60–2.70 (t, 2H), 3.35–3.50 (m, 2H), 4.20 (brs, 1H, ex),
7.50 (s, 1H)
1.20–1.50 (m, 10H + 2H, ex), 1.55–1.70 (m, 2H), 2.30 (s, 3H), 2.35 (s, 3H), 2.60–2.70 (t, 2H), 3.35–3.45 (m, 2H), 4.20 (brs, 1H, ex),
7.50 (s, 1H)
7a
7b
7c
7d
7e
7f
0.85 (t, 3H), 1.20–1.40 (m, 8H), 1.50–1.60 (m, 2H + 1H, ex), 1.75–1.90 (m, 2H), 2.30 (s, 3H), 2.35 (s, 3H), 2.60 (t, 2H), 2.80 (t, 2H),
3.50–3.60 (m,2H), 6.05 (brs, 1H,ex), 7.75 (s, 1H)
0.85 (t, 3H), 1.20–1.35 (m, 8H), 1.40–1.50 (m, 2H), 1.50–1.75 (m, 4H + 1H, ex), 2.30 (s, 3H), 2.35 (s, 3H), 2.60 (t, 2H), 2.65–2.75 (t,
2H), 3.40–3.50 (m, 2H), 4.55 (brs, 1H, ex), 7.55 (s, 1H)
0.85 (t, 3H), 1.20–1.40 (m, 8H), 1.40–1.60 (m, 6H + 1H, ex), 1.60–1.70 (m, 2H), 2.30 (s, 3H), 2.35 (s, 3H), 2.55–2.65 (m, 4H), 3.40–
3.50 (m, 2H), 4.20 (br s, 1H, ex), 7.55 (s, 1H)
0.85 (t, 3H), 1.20–1.30 (m, 8H), 1.35–1.40 (m, 4H), 1.50–1.60 (m, 4H), 1.60–1.70 (m, 2H + 1H, ex), 2.25 (s, 3H), 2.30 (s, 3H), 2.50–
2.70 (m,4H), 3.35–3.50 (m, 2H), 4.20 (brs, 1H, ex), 7.55 (s, 1H)
0.80–0.90 (t, 3H), 1.20–1.40 (m, 12H + 1H, ex), 1.40–1.50 (m, 4H), 1.55–1.70 (m, 4H), 2.30 (s, 3H), 2.35 (s, 3H), 2.55–2.69 (m, 4H),
3.40–3.50 (m, 2H), 4.20 (brs, 1H, ex), 7.55 (s, 1H)
0.85 (t, 3H), 1.20–1.40 (m, 16H), 1.40–1.55 (m, 4H), 1.60–1.65 (m, 2H), 1.90 (brs, 1H, ex), 2.30 (s, 3H), 2.35 (s, 3H), 2.55–2.65 (m,
4H), 3.40–3.50 (m, 2H), 4.20 (brs, 1H, ex), 7.45 (s, 1H)
8a
8b
8c
8d
8e
8f
0.85 (t, 3H), 1.20–1.40 (m, 8H), 1.60–1.70 (m, 2H), 1.90–2.00 (m, 2H), 2.30 (s, 3H), 2.50 (s, 3H), 3.40–3.50 (m, 2H), 3.50–3.55 (m,
2H), 3.85 (t. 2H), 5.90 (brs, 1H, ex), 6.80 (s,1H), 7.45–7.55 (m, 4H), 7.55–7.60 (m,2H), 8.85 (s, 1H)
0.85 (t, 3H), 1.20–1.40 (m, 8H), 1.60–1.70 (m, 4H) 1.70–1.80 (m, 2H), 2.30 (s, 3H), 2.35 (s, 3H), 3.45–3.60 (m, 4H), 3.80 (t, 2H), 4.50
(brs, 1H, ex), 6.80 (s, 1H), 7.45–7.60 (m, 6H), 8.75 (s, 1H)
0.85 (t, 3H), 1.20–1.40 (m, 8H),1.40–1.50 (m, 6 H), 1.60–1.80 (m, 2 H), 2.30 (s, 3H), 2.35 (s, 3H), 3.40–3.55 (m, 4H), 3.65 (t, 2H), 4.20
(brs, 1H, ex), 6.80 (s, 1H), 7.45–7.55 (m, 4H), 7.55–7.60 (m, 2H), 8.75 (s, 1H)
0.85 (t, 3H), 1.20–1.40 (m, 8H), 1.40–1.50 (m, 4H), 1.60–1.80 (m, 6H), 2.25 (s, 3H), 2.30 (s, 3H), 3.40–3.50 (m, 2H), 3.50–3.60 (m,
2H), 3.60–3.70 (m, 2H), 4.20 (brs, 1H, ex), 6.75 (s, 1H), 7.45–7.50 (m, 3H), 7.55 (s, 1H), 7.60–7.65 (m, 2H), 8.70 (s, 1H)
0.85 (t, 3H), 1.20–1.50 (m, 14H) 1.50–1.70 (m, 6H), 2.30 (s, 3H), 2.35 (s, 3H), 3.40–3.50 (m, 2H), 3.50–3.70 (m, 4H), 4.20 (brs, 1H,
ex), 6.80 (s, 1H), 7.45–7.50 (m, 3H), 7.55 (s, 1H), 6.60–7.65 (m, 2H), 8.75 (s, 1H)
0.85 (t, 3H), 1.20–1.40 (m, 16H), 1.40–1.55 (m, 4H), 1.55–1.65 (m, 2H), 2.30 (s, 3H), 2.35 (s, 3H), 3.40–3.45 (m, 2H), 3.50–3.60 (m,
4H), 4.20 (brs, 1H, ex), 6.75 (s, 1H), 7.40–7.50 (m, 3H), 7.55 (s, 1H), 7.60–7.65 (m, 2H), 8.70 (s, 1H)
0.85 (t, 6H), 1.15–1.35 (m, 16H), 1.40–1.50 (m, 4H), 1.70–1.85 (m, 2H), 2.30 (s, 3H), 2.35 (s, 3H), 2.40–2.50 (m, 4H), 2.55–2.65 (m,
2H), 3.45–3.55(m, 2H), 6.35 (brs, 1H, ex), 7.30 (s,1H)
9a
9b
9c
0.85 (t, 6H), 1.20–1.35 (m,16H), 1.35–1.55 (m, 4H), 1.55–1.70 (m, 4H), 2.30 (s, 3H), 2.35 (s.3H), 2.35–2.50 (m, 6H), 3.40–3.50 (m,
2H), 4.35 (brs, 1H, ex), 7.55 (s, 1H)
0.85 (t, 6H), 1.20–1.35 (m, 16H), 1.35–1.55 (m, 8H), 1.60–1.70 (m, 2H), 2.30 (s, 3H), 2.35 (s, 3H), 2.35–.2.45 (m, 6H), 3.40–3.50 (m,
2H), 4.20 (brs, 1H, ex), 7.55 (s, 1H)
11a
11b
12a
1.85 (s, 1H, ex), 2.35 (s, 3H), 2.45 (s, 3H), 2.95–3.00 (m, 4H), 3.10–3.15 (m, 4H), 7.85 (s, 1H)
1.80–1.90 (m, 2H), 2.20 (brs, 1H, ex), 2.30 (s, 3H), 2.45 (s, 3H), 2.80–2.85 (m, 2H), 3.00–3.10 (m, 2H), 3.50–3.65 (m, 4H), 7.75 (s, 1H)
2.40 (s, 3H), 2.55 (s, 3H), 3.30–3.40 (m, 4H), 3.85–3.90 (m, 4H), 7.10 (s, 1H), 7.45–7.55 (m, 3H), 7.60–7.70 (m, 2H), 7.95 (s, 1H), 8.85
(s, 1H)
12b
2.05–2.15 (m, 2H), 2.35 (s, 3H), 2.45 (s, 3H), 3.45–3.50 (m, 2H), 3.70–3.80 (m, 2H), 3.80–3.90 (m, 2H), 4.10–4.15 (m, 2H), 6.90 (s,
1H), 7.45–7.55 (m, 3H), 7.60–7.65 (m, 2H), 7.80 (s, 1H), 8.80 (s, 1H)
13
14
1.30–1.50 (m, 6H), 1.60–1.70 (m, 2H), 1.75–1.90 (brs, 3H, ex), 2.65 (t, 2H), 3.35–3.45 (m, 2H), 6.60 (s, 1H), 7.10–7.35 (m, 10H)
0.85 (t, 3H), 1.20–1.35 (m, 10H), 1.40–1.55 (m, 4H + 1H, ex), 1.60–1.75 (m, 4H), 2.50–2.65 (m, 4H), 3.40–3.50 (m, 2H), 4.85 (brs,
1H, ex), 6.60 (s, 1H), 7.10–7.35 (m, 10H)
15
0.85 (t, 3H), 1.20–1.40 (m, 10H), 1.40–1.60 (m, 4H), 1.60–1.80 (m, 4H + 1H, ex), 3.40–3.55 (m, 4H), 3.60–3.70 (m, 2H), 6.70 (s, 1H),
6.80 (s, 1H), 7.10–7.35 (m, 11H), 7.45–7.55 (m, 2H), 7.55–7.60 (m, 2H), 8.70 (s, 1H)
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2006, 339, 645–651
3-Heptylamino-5-phenylpyridazine Derivatives
651
Enzyme assays
% Inhibition ¼
In vitro assay against rat ACAT
ꢀ
ꢁ
Sample ðcpmÞ ꢀ Background ðcpmÞ
Control ðcpmÞ ꢀ Background ðcpmꢁ
Microsomes prepared from rat liver were used as a source of the
enzyme. The activity of the ACAT inhibitors against rat ACAT
was measured following a previously described method [12].
GERI-BP001 M was used as reference compound. Accordingly,
the reaction mixture, containing 4 lL of microsomes (10 mg/mL
protein), 20 lL of 0.5 M potassium-phosphate buffer (pH 7.4,
10 mM dithiothreitol), 15 lL of bovine serum albumin (fatty
acid free, 40 mg/mL), 2 lL of cholesterol in acetone (20 mg/mL,
added last), 41 lL of water, and 10 mL of test sample in a total
volume of 92 lL, was preincubated for 30 min at 378C. The reac-
tion was initiated by the addition of 8 lL of [1-14C]oleoyl-CoA
solution (0.05 lCi, final conc. 10 lM). After 15 min of incubation
at 378C, the reaction was stopped by the addition of 1.0 mL of iso-
propanol-heptane (4:1; v/v) solution. A mixture of 0.6 mL of hep-
tane and 0.4 mL of 0.1 M potassium-phosphate buffer (pH 7.4,
2 mM dithiothreitol) was then added to the terminated reaction
mixture. The above solution was mixed and allowed to phase
separation under gravity for 2 min. Cholesterol oleate was recov-
ered in the upper heptane phase (total volume 0.9–1.0 mL). The
radioactivity in 100 mL of the upper phase was measured in a
7 mL liquid scintillation vial with 4 mL of scintillation cocktail
(Lipoluma, Lumac Co., Basel, Switzerland) using a liquid scintil-
lation counter (Packard Delta-2000; Hewlett Packard). Back-
ground values were obtained by preparing heat inactivated
microsomes. Since some of the radioactivity recovered in the
upper phase could be due to enzymatic incorporation of radioac-
tive fatty acid into other products than cholesteryl oleate, the
partitioned heptane phases were purified by preparative TLC
(eluent n-hexane/diethyl ether/acetic acid; 90:10:1) and their
radioactivity measured as described above. Percent inhibition of
ACAT activity was calculated by subtracting the background
values from both control and test sample values according to
the following equation:
1006 1 ꢀ
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i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com