2-Azetidinone cholesterol absorption inhibitors: Structure-activity relationships on the heterocyclic nucleus

Article abstract of DOI:10.1021/jm960405n

A series of azetidinone cholesterol absorption inhibitors related to SCH 48461 ((-)-6) has been prepared, and compounds were evaluated for their ability to inhibit hepatic cholesteryl ester formation in a cholesterol-fed hamster model. Although originally

Full text of DOI:10.1021/jm960405n

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3684
J . Med. Chem. 1996, 39, 3684-3693
2-Azetid in on e Ch olester ol Absor p tion In h ibitor s: Str u ctu r e-Activity
Rela tion sh ip s on th e Heter ocyclic Nu cleu s
J ohn W. Clader,* Duane A. Burnett, Mary Ann Caplen, Martin S. Domalski, Sundeep Dugar, Wayne Vaccaro,
Rosy Sher, Margaret E. Browne, Hongrong Zhao, Robert E. Burrier, Brian Salisbury, and Harry R. Davis, J r.
Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New J ersey 07033-0539
Received J une 7, 1996X
A series of azetidinone cholesterol absorption inhibitors related to SCH 48461 ((-)-6) has been
prepared, and compounds were evaluated for their ability to inhibit hepatic cholesteryl ester
formation in a cholesterol-fed hamster model. Although originally designed as acyl CoA:
cholesterol acyltransferase (ACAT) inhibitors, comparison of in vivo potency with in vitro activity
in a microsomal ACAT assay indicates no correlation between activity in these two models.
The molecular mechanism by which these compounds inhibit cholesterol absorption is unknown.
Despite this limitation, examination of the in vivo activity of a range of compounds has revealed
clear structure-activity relationships consistent with a well-defined molecular target. The
details of these structure-activity relationships and their implications on the nature of the
putative pharmacophore are discussed.
In tr od u ction
In a preliminary report, we disclosed that 2-azetidi-
nones such as SCH 48461 ((-)-6) are effective inhibitors
of cholesterol absorption in a cholesterol-fed hamster
model.11 Subsequently, SCH 48461 has been shown to
reduce serum cholesterol in human clinical trials.12
Although this class of compounds was initially designed
as ACAT inhibitors, early structure-activity studies
demonstrated a striking divergence of in vitro ACAT
inhibition and in vivo activity in the cholesterol-fed
hamster. A detailed examination of the hypocholester-
olemic activity of (-)-6 indicates that it acts at the
intestintal wall to inhibit cholesterol absorption through
a novel and as yet undetermined mechanism.13 Initial
follow-up studies on SCH 48461 have shown that the
azetidinone nucleus is a critical element for in vivo
activity (Chart 1). For instance, neither the thioazeti-
dinone 7 nor amino acid 8 derived from SCH 48461
possess any significant activity in the hamster, and
other lactams show, at best, severely attenuated activ-
ity.14 On the basis of these findings, we initiated an
investigation of structure-activity relationships around
the 2-azetidinone nucleus. These studies, the first of
which is the subject of this report, have revealed clear
structure-activity relationships (SAR) for cholesterol
absorption inhibition which are distinct from the modest
ACAT inhibitory activity shown by these compounds.
Thus, these compounds appear to be acting via a novel
mechanism which may be fundamentally important in
the intestinal absorption of cholesterol.
Atherosclerotic coronary artery disease (CAD) is a
major cause of death and morbidity as well as a
significant and preventable drain on healthcare re-
sources in the western world.1 There is now a clear
association between reduction in serum lipids and
decreased incidence of CAD. Although reducing dietary
fat and cholesterol is still considered the appropriate
first-line therapy, the advent of more effective pharma-
cological agents has resulted in increased use of drug
therapy to control serum cholesterol.2 Serum choles-
terol can be reduced by inhibiting endogenous choles-
terol biosynthesis, promoting hepatic cholesterol clear-
ance from the plasma, and inhibiting the absorption of
dietary and biliary cholesterol from the intestines.3
Several agents are available which inhibit biosynthesis
and/or promote clearance. However, of the variety of
agents shown to inhibit cholesterol absorption in ani-
mals, only the bile acid sequestrants have shown
sufficient efficacy and safety to warrant extensive
clinical use.4 Even here, the modest efficacy and
unpleasant side effects associated with resins have
limited their use.
Several classes of compounds have been investigated
as cholesterol absorption inhibitors (Figure 1). Acyl
CoA:cholesterol acyltransferase (ACAT) inhibitors such
as CI-976 (1),5 CL 277082 (2),6 and SCH 46442 (3),7
which inhibit absorption by blocking the formation of
intestinal cholesteryl esters, have received considerable
preclinical attention, but none has shown clinical ef-
ficacy. Recently, it has been suggested that cholesterol
absorption can be decreased by inhibiting pancreatic
cholesteryl ester hydrolase.8 Compounds such as WAY
121,898 (4)9 have been effective in rodent models, but
their effects in clinical trials remain to be determined.
Saponins such as CP 88,818 (5a ) have been effective
clinically, but the gram-quantity doses required in
compounds disclosed to date make these less attractive.
It remains to be seen if more potent analogs such as
CP 148,623 (5b) will be as effective at a more reasonable
dose.10
Ch em istr y
3-Substituted 2-azetidinones such as 6 were prepared
as described in Scheme 1. Ester enolate-imine reaction
as previously described11,15 gave primarily the 3,4-cis
isomer (method A). Subsequent treatment with potas-
sium tert-butoxide (method B) converted this to a
mixture of isomers from which the major trans isomer
was separated either chromatographically or by crystal-
lization. Subsequently, we have found that N-arylaze-
tidinones can be prepared with high trans diastereose-
lectivity via the ketene-imine reaction (method C).16
4,4-Disubstituted 2-azetidinone 9 was prepared from
the corresponding imine via a modified ketene-imine
X Abstract published in Advance ACS Abstracts, August 1, 1996.
S0022-2623(96)00405-0 CCC: $12.00 © 1996 American Chemical Society

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2-Azetidinone Cholesterol Absorption Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3685
F igu r e 1. Cholesterol absorption inhibitors.
Ch a r t 1
Sch em e 1. Preparation of C-3-Monosusbtituted
2-Azetidinones
OCH3
Method A
R1
O
R3
Ar
R1
1. LDA, THF, –78 °C
2. R3CH NAr
N
CO2Et
N
O
KO-t-Bu
THF, 0 °C
Method B
OCH3
6
Method C
R1
R1
O
R3
SCH 48461 (–)
SCH 47949 (±)
R3CH NAr
toluene,
n-Bu3N
N
COCl
Ar
OCH3
OCH3
Sch em e 2. Preparation of C-3-Disubstituted
2-Azetidinones
O
R2
N
HN
OH
R1
R3
1. LDA,
THF:DMPU
R1
R3
S
2. R2X
N
N
OCH3
OCH3
O
Ar
O
Ar
7
8
R1
R2
R3
R2
R3
cholesterol-fed hamster, % changea
1. LDA,
THF:DMPU
serum
cholesterol
cholesteryl
esters
N
N
2. R1X
no.
O
Ar
O
Ar
6
7
8
-29
0
-8
-77.5
-23
0
Compounds with an acyl, alkyl, or phenylalkyl group
on nitrogen were prepared by acylation or alkylation of
1(H)-2-azetidinone (74) (methods E and F) as shown in
Scheme 3. The azetidinone 74 was prepared by ester
enolate-imine condensation using an N-(trimethylsilyl)-
imine followed by in situ hydrolysis of the N-TMS-
azetidinone.15 Additional 2-azetidinones were prepared
by standard functional group manipulations as de-
scribed in the Experimental Section.
reaction with 5-phenylvaleric acid in the presence of
phenyl dichlorophosphate as activating agent.17 Ad-
ditional 4,4-disubstituted 2-azetidinones 10 and 11 were
prepared from the corresponding imine via methods C
and A. In all three cases, the hindered imine starting
material was prepared from 4-methoxyaniline and the
corresponding ketone in the presence of TiCl4.
3,3-Disubstituted 2-azetidinones were prepared as
shown in Scheme 2. Alkylation of 2-azetidinone eno-
lates derived from C-3-monosubstituted compounds
(method D) proceeded with high diastereofacial selectiv-
ity to give compounds which result from alkylation on
the face opposite from the C-4 aryl moiety. By exchang-
ing the acid chloride or ester in the azetidinone syn-
thesis and the subsequent alkylating agent, either
diastereomer of the product could be obtained.
Biology
Compounds were evaluated for cholesterol absorption
inhibitory activity using a 7-day cholesterol-fed hamster
model. Some compounds were also evaluated for in
vitro activity in a rat liver microsomal ACAT assay.
Details of both models have been described previously.18
The change in hepatic cholesterol ester content is the
most sensitive and reproducible end point in the cho-
lesterol-fed hamster model. This end point has also

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3686 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Clader et al.
Sch em e 3. Preparation of N-Alkyl- and
N-Acyl-2-azetidinones
cant activity in the cholesterol-fed hamster. Compound
11, which substitutes a trifluoromethyl group for the
C-4 hydrogen was also nearly devoid of activity, sug-
gesting that disubstitution at C-4 is not tolerated.
OCH3
1. LDA, THF–HMPA,
Ph(CH2)3
–70 °C to rt
With the importance of the C-4 substituent estab-
lished, we began a detailed study of the effect of the
substituent pattern on the C-4 phenyl moiety on in vivo
and in vitro activity. The results of this study are given
in Table 1. These data show that activity in the
cholesterol-fed hamster is extremely sensitive to both
the nature and pattern of substituents at C-4. For
example, the 3-methoxyphenyl (15 and 16), 3,4-dimethox-
yphenyl (18 and 19), and 3,4-(methylenedioxy)phenyl
(20 and 21) analogs are all significantly less active than
4-methoxyphenyl compound 6, while the 2,4-dimethoxy
derivative 22 is equipotent with 6. Except for benzyl
ether 33, a variety of ethers (23-32) are well tolerated,
although in compounds such as 23 and 24 there is also
an increase in the ACAT inhibitory activity which may
contribute to the overall reduction in cholesteryl ester
content. Phenolic derivatives 34 and 35 are slightly less
active than the corresponding methyl ethers. This may
be partly due to reduced absorption of these poorly
soluble compounds. Trifluoromethyl ether 37 is signifi-
cantly less active than the corresponding methyl ether.
Insertion of an extra methylene unit to give a hy-
droxymethyl (39) or methoxymethyl (40) group also
reduces activity. These data suggest that a properly
placed hydrogen-bonding moiety in this position is
critical for activity. Consistent with this idea, the
4-methyl derivative 38 shows markedly reduced activity.
However, even slightly perturbing the location of the
alkoxy moiety as in 39 and 40 or substitution with other
potential hydrogen bond acceptor or donor groups at the
para position (compounds 41-50) reduces activity com-
pared to 6.
Ph
H3CO
2.
CO2Et
N
O
H
N TMS
Method E
Method F
OCH3
OCH3
Ph
Ph
N
R
O
N
R
O
O
Ch a r t 2
OCH3
OCH3
N
O
OCH3
9
OCH3
OCH3
CF3
OCH3
N
N
O
O
Studies on (-)-6 and related optically pure com-
pounds have indicated that activity resides predomi-
nantly in a one absolute stereochemistry at C-4.19 As
a part of the investigation of substituents on the C-4
phenyl group, the importance of the relative stereo-
chemistry between C-3 and C-4 was also investigated.
The data in Table 1 indicate no consistent preference
for the relative stereochemistry between C-3 and C-4.
Since several early leads showed better dose-response
curves in the 3R,4S (trans) series, most of the remaining
structure-activity work focused on trans isomers with
a 4-methoxyphenyl group at C-4.
OCH3
OCH3
10
cholesterol-fed hamster, % changea
11
serum
cholesterol
cholesteryl
esters
no.
9
10
11
-9
-22
0
0
-22
-11
been shown to correlate well with the degree of inhibi-
tion of cholesterol absorption. Unlike most ACAT
inhibitors, potent azetidinone cholesterol absorption
inhibitors also show a substantial reduction in serum
cholesterol in this model. This difference provided one
of the first indications that the mechanism of action of
these compounds did not involve ACAT inhibitions.
In Figure 2, the ACAT inhibitory activity of the
compounds in Table 1 is compared with their activity
in the cholesterol-fed hamster model. Not only does the
in vivo activity not correlate with the in vitro activity
but the level of ACAT activity in many instances is
inconsistent with any level of in vivo activity due to
ACAT inhibition.20 As a result, ACAT activity was no
longer routinely determined, and subsequent SAR stud-
ies focused entirely on activity in the cholesterol-fed
hamster.
Resu lts
Str u ctu r e-Activity Rela tion sh ip s a t C-4. Early
work in this series11 established that the p-methoxyphe-
nyl group at C-4 of the azetidinone was critical for in
vivo activity in the cholesterol-fed hamster model. The
same study also demonstated that in vivo activity
resided primarily in a single configuration at C-4. In a
limited follow-up study (Chart 2), several C-4-disubsti-
tuted compounds were prepared, including two sym-
metrically disubstitued compounds, 9 and 10, which
combine the 4(S)- and 4(R)-p-methoxyphenyl moiety in
a single molecule. Neither compound showed signifi-
Str u ctu r e-Activity Rela tion sh ip s a t N-1. Unlike
the phenyl group at C-4, the phenyl group on nitrogen
was found to tolerate a variety of substituents or could
be unsubstituted with no loss in activity (Table 2). For
example, 3- and 2-methoxyphenyl derivatives 51 and
52 as well as the unsubstituted phenyl derivative 65
are each at least as active as 6 in the cholesterol-fed

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2-Azetidinone Cholesterol Absorption Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3687
Ta ble 1. Structure-Activity Relationships at the C-4 Phenyl Group
X
R2
R1
N
O
OCH3
cholesterol-fed hamster, % changeb
ACAT % inhib
serum
cholesterol
cholesteryl
esters
no.
R1
R2
X
method
formulaa
mp (°C)
at 50 µM
6
12
Ph(CH2)3
H
H
4-CH3O
B
A
A
A
B
A
A
B
A
B
A
C
B
A
C
A
C
A
C
A
C
B
B
c
c
c
C
B
c
c
c
C
c
E
c
C26H27NO3
C26H27NO3
C25H25NO2
C25H25NO2
C26H27NO3
C26H27NO3
C26H27NO3
C27H29NO4
C27N29NO4
C26H25NO4
C26H25NO4
C27H29NO4
C27H29NO3
C27H29NO3
C28H31NO3
C28H31NO3
C28H31NO3
C28H31NO3
C29H33NO3
C29H33NO3
C29H33NO3
C31H29NO3
C32H31NO3
C25H25NO3
C25H25NO3
C24H23NO3
96.0-97.5
90-93
33
38
22
28
21
39
-29
-45
-6
-77.5
-95
-15
0
-24
-54
0
Ph(CH2)3 4-CH3O
H
Ph(CH2)3
H
Ph(CH2)3 3-CH3O
13 Ph(CH2)3
14
15 Ph(CH2)3
16
17 Ph(CH2)3
18 Ph(CH2)3
19
20 Ph(CH2)3
21
22 Ph(CH2)3
23 Ph(CH2)3
24
25 Ph(CH2)3
26
27 Ph(CH2)3
28
29 Ph(CH2)3
30
31 Ph(CH2)3
32 Ph(CH2)3
33 Ph(CH2)3
34 Ph(CH2)3
H
H
92.0-92.5
120.5-121.5
109.5-110.0
90.5-91.0
115-117
70.5-71.5
86.0-87.0
79.5-81.0
144.0-144.5
oil
H
0
3-CH3O
-17
-19
0
H
H
H
2-CH3O
3,4-CH3O
53
-16
-36
-28
-11
-33
H
Ph(CH2)3 3,4-CH3O
H
Ph(CH2)3 3,4-(OCH2O)
H
H
0
0
0
0
3,4-(OCH2O)
-19
0
H
d
2,4-CH3O
4-CH3CH2O
-26
-52
-48
-40
-37
-38
-28
-34
-15
-26
-37
-15
-16
-31
0
-78
-98
-93
-95
-91
-94
-78
-64
-70
-80
-77
-48
-48
-75
-39e
-29
-21
-46
-56
0
85-87
84-85
68
79
H
Ph(CH2)3 4-CH3CH2O
4-nC2H7O
Ph(CH2)3 4-nC3H7
4-iC3H7O
Ph(CH2)3 4-iC3H7O
4-nC4H9O
H
92.5-93.5
96.0-97.0
96.5-97.5
96-97
H
H
59
d
H
H
79-82
H
Ph(CH2)3 4-nC4H9O
71.0-72.5
101.5-102.5
103.5-105.5
111-112
170.0-170.5
152.2-155.0
54-55
H
H
H
H
4-tC4H9O
4-PhO
4-PhCH2O
4-OH
8
20
35
H
Ph(CH2)3 4-OH
d
36 Ph(CH2)3
37 Ph(CH2)3
38 Ph(CH2)3
39 Ph(CH2)3
40 Ph(CH2)3
41 Ph(CH2)3
42 Ph(CH2)3
43 Ph(CH2)3
44 Ph(CH2)3
H
H
H
H
H
H
H
H
H
3,4-OH
4-CF3O
4-CH3
4-HOCH2
4-CH3OCH2
4-NH2
4-N(CH3)2
4-NHSO2CH3
4-NHCOCH3
C26H24F3NO3 66-67
0
C26H27NO2
C26H27NO3
C27H29NO3
C25H26N2O2
C27H30N2O2
74.5-77.0
18
-6
-20
-21
-27
0
112-113
oil
d
155-157
96.5-97.5
63
-29
-60
-46
-26
0
C26H28N2O4Sd oil
-8
d
C27H28N2O3
C25H25NO2S
C26H27NO2S
C26H27NO3S
C26H27NO4S
C25H24N2O4
C25H24FNO2
153-154
-4
45
H
Ph(CH2)3 4-SH
134-135
104.5-105.5
111-115
57-58
-19
0
46 Ph(CH2)3
47 Ph(CH2)3
48 Ph(CH2)3
49 Ph(CH2)3
50 Ph(CH2)3
H
H
H
H
H
4-CH3S
A
c
c
A
A
0
4-CH3SO
4-CH3SO2
4-NO2
-17
-26
-11
0
-32
-75
0
116-117
83.0-84.0
21
-27
4-F
12
a
b
Except where noted, elemental analysis within 0.4% of theoretical values was obtained. All compounds were tested at 50 mg/kg/day
unless otherwise stated. c Prepared by functional group changes as described in the Experimental Section. High-resolution mass spectrum
d
and/or NMR data availabile from the author. e Tested at 10 mg/kg/day.
hamster model. Hydrogen-bonding potential does not
appear to be important, as evidenced by the 4-(trifluo-
romethoxy)phenyl, phenyl, 4-fluorophenyl, and 4-me-
thylphenyl derivatives 57, 65, 66, and 72. On the other
hand, the reduced activity of the 4-phenoxyphenyl
derivative 58 suggests some steric limitations on the
N-aryl group.
While a variety of substituted phenyl groups are
tolerated on nitrogen, the aryl moiety itself is critical
for activity (Table 3). Desphenyl analog 74 is completely
inactive. Compounds with either small alkyl groups
(75-78) or even larger cycloalkyl analogs (79, 80) on
nitrogen are either weak or inactive, as are benzyl
derivatives 81 and 82. These data show that the
aromatic moiety on nitrogen is an essential part of the
pharmacophore. The only exception is the N-cyclopropyl
derivative 83. The π character of the cyclopropyl moiety
may be contributing to the modest activity of this
compound. N-Acyl substitution (84, 85) dramatically
reduces activity, suggesting that activation of the aze-
tidinone toward nucleophiles not only is not required
but is detrimental to activity.
Str u ctu r e-Activity Rela tion sh ip s a t C-3. Lastly,
the effect of substitution at C-3 was examined (Table
4). This study examined both the nature of the sub-
stituent at C-3 and the degree of substitution at this
position. Decreasing the length of the phenylalkyl chain
at C-3 (compounds 86 and 87) reduces activity. In-
creasing the chain length to four or five carbons (88-
90) causes a modest decrease in activity, while a six-
carbon chain (91, 92) results in a marked reduction in
activity. Replacing the phenyl alkyl group with a simple
alkyl group (93-96) or saturation of the phenyl group
(97, 98) both result in a reduction in activity. Thus, a
properly positioned phenylalkyl moiety at C-3 is also
an important part of the pharmacophore.