New ACAT Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7713
(5) Joyce, C.; Skinner, K.; Anderson, R. A.; Rudel, L. L. Acyl-
coenzyme A: cholesteryl acyltransferase 2. Curr. Opin. Lipidol.
1999, 10, 89-95.
(6) Rudel, L. L.; Lee, R. G.; Cockman, T. L. Acyl coenzyme A:
cholesterol acyltransferase types 1 and 2: structure and function
in atherosclerosis. Curr. Opin. Lipidol. 2001, 12, 121-127.
(7) Song, B. L.; Qi, W.; Yang, X. Y.; Chang, C. C.; Zhu, J. Q.; Chang,
T. Y.; Li, B. L. Organization of human ACAT-2 gene and its cell-
type-specific promoter activity. Biochem. Biophys. Res. Commun.
2001, 282, 580-588.
(8) Giovannoni, M. P.; Dal Piaz, V.; Kwon, B. M.; Kim, M. K.; Kim
Y. K.; Toma, L.; Barlocco, D.; Bernini, F.; Canavesi, M. 5,6-
Diphenylpyridazine derivatives as acyl-CoA: cholesterol
acyltransferase inhibitors. J. Med. Chem. 2001, 44, 4292-
4295.
(9) Toma, L.; Nava, D.; Celentano, G.; Giovannoni, M. P.; Dal Piaz,
V.; Kwon, B. M.; Kim, M. K.; Kim, Y. K.; Barlocco, D. 5,6-
Dinitrophenyl and 5-aminophenyl-6-nitrophenyl analogues of the
ACAT inhibitor 5,6-diphenyl-3-alkylaminopyridazines. Hetero-
cycles 2000, 53, 2709-2718.
(10) Toma, L.; Giovannoni, M. P.; Dal Piaz, V.; Kwon, B. M.; Kim, Y.
K.; Gelain, A.; Barlocco, D. Mono- and di-substituted 5,6-
diphenyl-3-alkylamino-pyridazines active as ACAT inhibitors.
Heterocycles 2002, 57, 39-46.
(11) Toma, L.; Giovannoni, M. P.; Vergelli, C.; Dal Piaz, V.; Kwon,
B. M.; Kim, Y. K.; Gelain, A.; Barlocco, D. Novel 3-arylamino-
and 3-cycloalkylamino-5,6-diphenyl-piridazines active as ACAT
inhibitors. Arch. Pharm. Pharm. Med. Chem. 2002, 11, 563-
566.
(12) Higley, C. A.; Wilde, R. G.; Maduskuie, T. P.; Johnson, A. L.;
Pennev, P.; Billheime, J. T.; Robinson, C. S.; Gilles, P. J.; Wexler,
R. R. Acyl CoA: cholesterol acyltransferase (ACAT) inhibitors:
synthesis and structure-activity relationship studies of a new
series of trisubstituted imidazoles. J. Med. Chem. 1994, 37,
3511-3522.
(13) Coates, W. J.; McKillop, A. One-pot preparation of 6-substi-
tuted 3(2H)-pyridazinones from ketones. Synthesis 1993, 3, 334-
342.
1.25-1.35 (m, 8H); 1.45-1.55 (m, 2H); 1.60-1.65 (m, 2H);
1.70-1.80 (m, 2H); 1.80-1.85 (m, 2H); 3.25-3.40 (m, 4H);
4.10-4.15 (m, 2H); 6.50 (br s, 1H); 6.80-6.95 (m, 4H); 7.20-
7.45 (m, 8H); 7.95-8.05 (m, 2H); 12.60 (s, 1H).
General Procedure for the Synthesis of the Pyrid-
azinethiols 9a-c. To a solution of the appropriate pyrid-
azinone8,13,14 (2 mmol) in toluene (120 mL) was added Lawes-
son’s reagent (0.5 g, 12.4 mmol), and the solution was stirred
at 130 °C for 3 h. After cooling, the solvent was evaporated
under vacuum and the product purified by flash chromatog-
raphy (eluent petroleum ether/EtOAc 8/2).
3-Mercapto-5,6-diphenylpyridazine 9a (Y ) 60%), 1H NMR
(CDCl3) δ: 7.10-7.40 (m, 10H); 7.80 (s, 1H); 12.40 (br s, 1H).
General Procedure for the Synthesis of the Chloro-
pyridazines 10a-c. A mixture of the appropriate pyridazi-
none8,13,14 (0.65 mmol) and POCl3 (1.5 mL, 16 mmol) 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 5 N NaOH,
and then it was extracted with CH2Cl2 (3 × 15 mL). After
drying over sodium sulfate, evaporation of the solvent gave
10a-c, which was used as such for the next step.
3-Chloro-5,6-diphenylpyridazine 10a (Y ) 70%): 1H NMR
(CDCl3) δ: 7.15 (d, 2H); 7.30-7.40 (m, 8H); 7.55 (s, 1H).
Enzyme Assays. In Vitro Assay against Rat ACAT. Mi-
crosomes prepared from rat liver were used as a source of the
enzyme. The activity of the ACAT inhibitors against rat ACAT
was measured according to a previously described method.16
GERI-BP001 M16 was used as reference compound.
In vitro assay against hACAT-1 and hACAT-2. Microsomal
fractions of Hi5 cells containing baculovirally expressed ACAT-1
or -2 were used as the sources of enzymes.17 The activity of
the hACAT-1 and hACAT-2 was measured according to the
method of Brecher and Chan18 with slight modification.17
Modeling. Computational Methods. All calculations were
carried out using the Gaussian 0319 program package. The
conformational space of compounds 14-18 was explored
through optimizations at the B3LYP level with the 6-31G*
basis set. The energy profiles for rotation around the C3-X
bond were initially determined on simplified molecules un-
substituted at the 5 and 6 positions of the heterocyclic ring.
Then, after addition of the 5- and 6-phenyl groups to the
minima located in the profiles, the structures were fully
optimized allowing to determine the minimum energy confor-
mations of compounds 14-18.
(14) Wermuth, C. G.; Bourguignon, J. J.; Schlewer, G.; Gies, J. P.;
Schoenfelder, A.; Melikian, A.; Bouchet, M. J.; Chantreux, D.;
Molimard, J. C.; Heaulme, M. Synthesis and structure-activity
relationships of a series of aminopyridazine derivatives of
γ-aminobutyric acid acting as selective GABA-A antagonists. J.
Med. Chem. 1987, 30, 239-249.
(15) Burnett, J. R.; Wilcox, L. J.; Huff, M. W. Acyl coenzyme A:
cholesterol acyltransferase inhibition and hepatic apolipoprotein
B secretion. Clin. Chim. Acta 1999, 286, 231-242.
(16) Jeong, T. S.; Kim, S. U.; Son, K. H.; Kwon, Y. K.; Choi, M. U.;
Bok, S. H. GERI-BP001 compounds, new inhibitors of acyl-
CoA: cholesterol acyltransferase from Aspergillus fumigatus F37
I. Production, isolation and physicochemical and biological
properties. J. Antibiot. 1995, 48, 751-756.
(17) Cho, K. H.; An, S.; Lee, W. S.; Paik, Y. K.; Kim, Y. K.; Jeong, T.
S. Mass-production of human ACAT-1 and ACAT-2 to screen
isoform-specific inhibitor: a different substrate specificity and
inhibitory regulation. Biochem. Biophys. Res. Commun. 2003,
309, 864-872.
Acknowledgment. This work was partially sup-
ported by Italy-Korea Joint Research Program and
KRIBB Initiative Research Program. The authors also
aknowledge the financial support from the University
of Milan (FIRST 2004) and Pavia (FAR 2004).
(18) Brecher, P.; Chan, C. T. Properties of acyl-CoA: cholesterol
O-acyltransferase in aortic microsomes from atherosclerotic
rabbits. Biochim. Biophys. Acta 1980, 617, 458-471.
(19) Gaussian 03, Revision B.02, Frisch, M. J.; Trucks, G. W.;
Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.;
Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.;
Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci,
B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji,
H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene,
M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo,
C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala,
P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J.
J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M.
C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.;
Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz,
P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham,
M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P.
M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople,
J. A., Gaussian, Inc., Pittsburgh, PA, 2003.
Supporting Information Available: Physical properties
and 1H NMR data of compounds. This material is available
References
(1) American Heart Associaton. 1999 Heart and Stroke Statistical
Update; American Heart Association: Dallas, TX, 1998.
(2) Norata, G. D.; Catapano, A. L. Lipid lowering activity of drugs
affecting cholesterol absorption. Nutr. Metab. Cardiovas. Dis.
2004, 14, 42-51.
(3) Kushwaha, R. S.; Vandeberg, J. F.; Rodriguez, R.; Vandeberg,
J. L. Cholesterol absorption and hepatic acyl-coenzyme A:cho-
lesterol acyltransferase activity play major roles in lipemic
response to dietary cholesterol and fat in laboratory opossums.
Metabolism 2004, 53, 817-822.
(4) Chang, C. C. Y.; Lee, C. G..; Chang, E. T.; Cruz, J. C.; Levesque,
M. C.; Chang, T. Y. Recombinant acyl-CoA: cholesterol acyl-
transferase-1 (ACAT-1) purified to essential homogeneity utilizes
cholesterol in mixed micelles or in vesicles in a highly cooperative
manner. J. Biol. Chem. 1998, 273, 35132-35141.
JM050703X