Synthesis and structure-activity relationship studies on a novel series of naphthylidinoylureas as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT)

Article abstract of DOI:10.1016/j.bmcl.2003.12.045

The synthesis and structure-activity relationships of N-phenyl-N′-[3- (4-phenylnaphthylidinoyl)]urea derivatives 3 as a novel structural class of potent ACAT inhibitors is described. A 3-methoxy group substituted on the naphthylidinone 4-phenyl ring, toge

Full text of DOI:10.1016/j.bmcl.2003.12.045

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1309–1311
Synthesis and structure–activity relationship studies on a novel
series of naphthylidinoylureas as inhibitors of acyl-CoA:cholesterol
O-acyltransferase (ACAT)
Satoshi Ohnuma,* Masami Muraoka, Katsuhisa Ioriya and Naohito Ohashi
Research Division, Sumitomo Pharmaceuticals Co., Ltd, 1-98 Kasugade Naka 3-chome, Konohana-ku, Osaka 554-0022, Japan
Received 6 October 2003; accepted 5 December 2003
Abstract—The synthesis and structure–activity relationships of N-phenyl-N0-[3-(4-phenylnaphthylidinoyl)]urea derivatives 3 as a
novel structural class of potent ACAT inhibitors is described. A 3-methoxy group substituted on the naphthylidinone 4-phenyl ring,
together with a 1-N-nbutyl substitution, SM-32504 (3m), gave a potent ACAT inhibitor, in vitro, respectively. The most potent
compound, SM-32504 (3m), decreased the serum cholesterol level significantly in a high fat and high cholesterol-fed mouse model.
# 2004 Elsevier Ltd. All rights reserved.
Hypercholoesterolemia and cardiovascular diseases
(atherosclerosis, etc.) are major health problems in
many industrialized countries. Acyl-CoA:cholesterol
O-acyltransferase (ACAT, EC 2.3.1.26) is the primary
enzyme responsible for the intracellular esterification of
free cholesterol with fatty acyl-CoA to produce choles-
terol esters.1 In the small intestine, ACAT facilitates the
absorption of exogenous cholesterol, which is incorpo-
rated into chylomicrons.2,3 In the liver, ACAT plays an
important role in the assembly of very low density lipo-
protein (VLDL), which is secreted into the blood.4À6
Thus, ACAT inhibitors are being investigated as potent
therapeutic agents for the treatment of hypocholestero-
lemia and atherosclerosis.7
Based on the phenyl urea derivatives,10a,b we designed
N-phenyl-N0-[3-(4-phenylnaphthylidinoyl)]urea deriva-
tives 3 as a novel structural class of ACAT inhibitor.
We describe herein their synthesis and structure–activity
relationships (Fig. 1).
The general procedure for the synthesis of naphthylidi-
noylureas is shown in Scheme 1. Treatment of the dia-
nion of 2-(pivaloylamino)pyridine with substituted
benzaldehydes followed by oxidation with mangane-
se(IV)oxide afforded the aroylpyridines 4.11 Removed of
the pivaloyl group on 4 followed by cyclization with
diethylmalonate, in the presence of pyridine, gave the 2-
naphthylidinones 5.12 The crucial stepto synthesize the
target compounds 3 from 5 or 6 was a Curtius rearran-
In recent years, a number of ACAT inhibitors have
been reported and their pharmacological activities
have been evaluated in animals and humans.7À10 Most
are structurally classified into two major groups, one
being fatty acid anilide derivatives designed to mimic
acyl-CoA represented by 1 (CI-976),9 and the other
being phenyl urea derivatives.10 In the latter group,
bulky lipophilic substituents on the phenyl ring such as
2,6-diisopropyl are known to have beneficial effect on
the activity.10eÀg TMP-153 (2) and the corresponding
2-quinolone analogues have also been reported as
potent ACAT inhibitors.10a,b
* Corresponding author. Fax:+81-06-6466-5483; e-mail: ohnuma@
sumitomopharm.co.jp
Figure 1.
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.045

1310
S. Ohnuma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1309–1311
Scheme 1. (a) (1) n-BuLi, TMEDA/THF then R1C6H4CHO, (2) MnO2/CH2Cl2; (b) (1) 6N HCl, (2) CH2(CO2Et)2/pyridine; (c) NaH, R2-I/DMF;
(d) R2-I, K2CO3/DMF; (e) (1) NaOH/EtOH, (2) DPPA, Et3N/DMF then R3C6H4NH2 or R3C6H3NH2.
gement of the acylazides synthesized from the carboxylic
acids derived from esters 5 or 6 using diphenylpho-
sphorylazide (DPPA) and triethylamine13 followed by
reaction of the intermediate isocyanates (not isolated)
the treatment with substituted anilines, in DMF. The
reaction proceeded in high yield when either R3 was
alkyl (5!6!3) or hydrogen (5!7!3).
First, we examined the effect of a substituent on the
phenyl ring at the 4-position of the 2-naphthylidinone.
The result are shown in Table 1. Compounds 3a–e pos-
sessed strong inhibitory activities in the enzyme assay
and moderate inhibitory activities in the cell-based
assay. We had expected that 3a, having the same sub-
stituent as 2 would have given the highest activity,
however, it was slightly inferior to 3c and 3d. Regarding
the nature of the substituent at the 2-position of the
phenyl ring, the electron-donating methoxy group was
effective in the enzyme assay and the presence of a sub-
stituent itself was important in the cell-based assay (see
3a, 3b and 3c). Regarding the position of the methoxy
group on the phenyl ring, the 3-position was effective in
the enzyme assay and the 2-position was effective in the
cell-based assay (see 3c, 3d and 3e).
The compounds prepared were evaluated in both an
enzyme assay and a cell-based assay. In the enzyme
assay, ACAT activity was determined by measuring the
production of cholesteryl 14C-oleate while incubating
with 14C-oleoyl-CoA and rabbit liver microsomes.14 In
the cell-based assay, whole cell ACAT activity in rat
macrophages was determined by the incorporation of
an extracellular 3H-oleic acid-BSA complex into the
intracellular cholesteryl ester.15
Next, we examined the effect of substituent(s) (R3) on
the phenyl ring of the phenylureido moiety. The results
are shown in Table 2. Although it was reported that the
2,4-difluorophenyl moiety brought about potent inhibi-
tion in 2 and in some other ACAT inhibitors,10aÀd 3f
showed only weak inhibitory activity. Steric hinderance
of the substituents at the ortho-positions of the phenyl
The ACAT inhibitory activity of each compound in
these assays was expressed by an IC50 value. Desirable
compounds were required to show potent inhibitory
activities in both assay systems.
Table 1. Effect of 4-phenyl ring substitution on the biological activities
Table 2. Effect of varying phenyl substituents on the biological
activities
Compd
R1
Enzyme assay
a
Cell-based assay
IC50b (nM)
Compd
R3
Enzyme assay
IC50a (nM)
IC50 (nM)
3a
3b
3c
3d
3e
2-Cl
H
2-OMe
3-OMe
4-OMe
56
40
21
8
140
562
100
158
447
3f
2,4-F2
2-Et
>1000
>1000
>1000
76
3g
3h
3i
2-iPr
2-iPr-6-Me
2,6-iPr2
63
3d
8
a In vitro ACAT inhibition determined in rabbit liver microsomes.
b In vitro ACAT inhibition determined in rat macrophages.
a In vitro ACAT inhibition determined in rabbit liver microsomes.

S. Ohnuma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1309–1311
1311
Table 3. Effect of 1-N-substituent on the biological activities
ability, and Ms. Yoshie Nakatsuji for her technical
assistance.
References and notes
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sclerosis 2002, 161, 45. (c) Hirakawa, Y.; Shimokawa, H.
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Compd
R2
Enzyme assay
a
Cell based assay
IC50b (nM)
IC50 (nM)
3j
H
CH3
100
8
9
13
25
68
13
>1000
158
170
50
11
230
35
3d
3k
3l
3m
3n
3o
CH2CH3
(CH2)2CH3
(CH2)3CH3
CH(CH3)2
(CH2)4CH3
a In vitro ACAT inhibition determined in rabbit liver microsomes.
b In vitro ACAT inhibition determined in rat macrophages.
8. (a) Matsuda, K. Med. Res. Rev. 1994, 14, 271. (b)
McCarthy, P. A. Med. Res. Rev. 1993, 13, 139.
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Stanfield, R. L. J. Med. Chem. 1992, 35, 1609. (b) McCathy,
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2039. (e) Krause, B. R. J. Lipid Res. 1993, 34, 279. (f)
Murakami, S. Cell. Mol. Biol. 1996, 42, 865. (g) Kataoka,
K.; Yamaguchi, H. J. Med. Chem. 1996, 39, 1262.
10. (a) For examples of phenylureas, see: Meguro, K.;
Tawada, H. J. Med. Chem. 1994, 37, 2079. (b) Tawada,
H.; Meguro, K. Chem. Pharm. Bull. 1995, 43, 616. (c)
Higley, C. A.; Wexler, R. R. J. Med. Chem. 1994, 37,
3511. (d) J. Lipid Res. 1989, 30, 681. (e) Sekiya, T.;
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ring was strongly effective in enhancing the activity and
the best result was obtained with 3d, as we expected.
Finally, the effect of the 1-N-alkylsubstituents (R2) on
the 2-naphthylidione ring was investigated. The results
are shown in Table 3. Alkyl substitution was highly
effective in enhancing the inhibitory activities in both
assay systems. The effect of the length of the straight-
chain alkyl substituents was not clear in the enzyme
assay, though the effect was clearer in the cell-based
assay. The best result was obtained with n-butyl sub-
stitution, 3m. In contrast, the branched-chain alkyl
compound 3n showed diminished activity.
We also investigated the effect of the N-alkyl substituent
in the 2-methoxy analogues of 3c, however, no note-
worthy result was obtained. Furthermore, 2-methoxy
isomer 3p (R1=2-OMe, R2=nBu, R3=2,6-iPr2) showed
only about 50% of the permeability in a Caco-2 mem-
brane16 than the corresponding 3-methoxy isomer 3m.
11. Estel, L.; Linard, F.; Marsais, F.; Godard, A.; Queguiner,
G. J. Heterocyclic Chem. 1989, 26, 105.
12. Bell, S. C.; Sulkowsky, T. S.; Gochman, C.; Childness,
S. J. J. Org. Chem. 1962, 27, 562.
13. Ninomiya, K.; Shioiri, T.; Yamada, S. Tetrahedron 1974,
30, 2151.
14. Heider, J. G.; Pickens, C. E.; Kelly, L. A. J. Lipid Res.
1983, 22, 271.
15. Miyazaki, A.; Horiuchi, S. Biochim. Biophys. Acta 1992,
1126, 73.
Thus, compound 3m (SM-32504) was identified as a
potent ACAT inhibitor.17 In vivo evaluation of 3m
showed that it decreased the serum cholesterol level by
38% (at a dosage of 10 mg/kg/d for 4 d) and 40% (at a
dosage of 30 mg/kg/d for 4 d) in high fat and high cho-
lesterol-fed mice, respectively.18
16. Yamashita, S. Eur. J. Pharm. Sci. 2000, 10, 195 The
apparent permeability coefficient, Papp value for 2-
methoxy isomer 3p was 15(nm/s) compared to 29(nm/s)
for 3m.
In conclusion, we found that naphthylidinoylureas 3
were a novel series of ACAT inhibitor. In paticular,
compound 3m showed potent inhibitory activity in vitro
and excellent efficacy in vivo. Further investigation on
these inhibitors is currently in progress and the results
will be reported in the near future.
1
17. Selected spectroscopic data for 3m (SM-32504): H NMR
(300 MHz, DMSO-d6) d 0.95 (t, 3H, J=7.34 Hz), 1.01 (br,
12H), 1.35–1.47 (m, 2H), 1.64–1.75 (m, 2H), 2.86–2.95 (m,
2H), 3.76 (s, 3H), 4.51 (t, 2H, J=7.70 Hz), 6.91 (br, 2H),
7.02–7.04 (m, 2H), 7.12–7.17 (m, 1H), 7.24 (dd, 1H,
J=4.59, 7.89 Hz), 7.39 (dd, 1H, J=7.89, 8.25 Hz), 7.61
(d, 1H, J=6.60 Hz), 7.72 (d, 1H, J=3.49 Hz), 8.60 (d,
1H, J=4.59 Hz); IR (neat) 2960, 1678, 1646, 1600, 1585,
1562, 1456, 1249 cmÀ1; APCI-MS m/z 527 (MH+).
Acknowledgements
The authors thank Mr. Yukinobu Igarashi and Mr.
Yasushi Tsutsumi in measuring the cellular perme-
18. Ioriya, K.; Noguchi, T.; Muraoka, M.; Fujita, K.;
Shimizu, H.; Ohashi, N. Pharmacology 2002, 65, 18.