Structure-activity relationships of n-(3,5-dimethoxy-4-n-octyloxycinnamoyl)-N′-(3,4-dimethylphenyl) piperazine and analogues as inhibitors of acyl-CoA: Cholesterol O- acyltransferase

Article abstract of DOI:10.1248/cpb.49.830

A novel series of acyl-CoA: cholesterol O-acyltransferase (ACAT) inhibitors were synthesized from a lead compound, 1-(4-hydroxy-3-methoxyphenyl)-7-phenylhept-1-en-3-one (1, Yakuchinone B) through a modification of three regions (A, B, C) in the molecule. In this study, the compounds prepared were tested for in vitro inhibitory activity on microsomal ACAT from the liver of rats and for in vivo hypocholesterolemic activity in rats given a high cholesterol diet. N-(3,5-Dimethoxy-4- n-octyloxycinnamoyl)-N′-(3,4-dimethylphenyl)piperazine (45), which belongs to the amide compounds, has finally been discovered. Compound 45 inhibited rat hepatic ACAT in a more striking manner than CI-976, an amide compound ACAT inhibitor, and it exhibited a high level of hypocholesterolemic activity in vivo. Since 45 strongly inhibited both microsomal ACAT prepared from HepG2 (a cell line derived from human hepatocarcinoma) and Caco2 (a cell line derived from human colon adenocarcinoma), there is speculation that 45 might have the ability to inhibit ACAT in both the human intestine and liver independent of the difference in the distribution of ACAT isozymes. On the other hand, 45 did not induce adrenotoxicity in subacute toxicity studies in rats. These results suggest that it has promise for development as a new therapeutic agent for hypercholesterolemia and atherosclerosis.

Full text of DOI:10.1248/cpb.49.830

830
Chem. Pharm. Bull. 49(7) 830—839 (2001)
Vol. 49, No. 7
Structure–Activity Relationships of N-(3,5-Dimethoxy-4-n-
octyloxycinnamoyl)-N؅-(3,4-dimethylphenyl)piperazine
and Analogues as Inhibitors of Acyl-CoA: Cholesterol O-Acyltransferase
Kenji OHISHI,* Ritsuo AIYAMA, Hiroshi HATANO, Yasuto YOSHIDA, Yasue WADA, Wakae YOKOI,
Haruji SAWADA, Tsunekazu WATANABE, and Teruo YOKOKURA
Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186–8650, Japan.
Received January 10, 2001; accepted March 23, 2001
A novel series of acyl-CoA: cholesterol O-acyltransferase (ACAT) inhibitors were synthesized from a lead
compound, 1-(4-hydroxy-3-methoxyphenyl)-7-phenylhept-1-en-3-one (1, Yakuchinone B) through a modification
of three regions (A, B, C) in the molecule. In this study, the compounds prepared were tested for in vitro in-
hibitory activity on microsomal ACAT from the liver of rats and for in vivo hypocholesterolemic activity in rats
given a high cholesterol diet. N-(3,5-Dimethoxy-4-n-octyloxycinnamoyl)-N؅-(3,4-dimethylphenyl)piperazine (45),
which belongs to the amide compounds, has finally been discovered. Compound 45 inhibited rat hepatic ACAT in
a more striking manner than CI-976, an amide compound ACAT inhibitor, and it exhibited a high level of hypo-
cholesterolemic activity in vivo. Since 45 strongly inhibited both microsomal ACAT prepared from HepG2 (a cell
line derived from human hepatocarcinoma) and Caco2 (a cell line derived from human colon adenocarcinoma),
there is speculation that 45 might have the ability to inhibit ACAT in both the human intestine and liver inde-
pendent of the difference in the distribution of ACAT isozymes. On the other hand, 45 did not induce adrenotoxi-
city in subacute toxicity studies in rats. These results suggest that it has promise for development as a new thera-
peutic agent for hypercholesterolemia and atherosclerosis.
Key words acyl-CoA: cholesterol O-acyltransferase inhibitor; Yakuchinone B; amide compound; hypocholesterolemic activity;
structure–activity relationship study
Since the pioneering results of the Framingham study dis- of plasma cholesterol levels in rats given a high cholesterol
closed in 1971,1) hypercholesterolemia has been recognized diet.
as a major risk factor for the development of coronary heart
The present paper will describe the structure–activity rela-
disease (CHD).2,3) Agents controlling total plasma choles- tionships and biological activities of these novel ACAT in-
terol levels are expected to serve as an effective therapeutic hibitors.
method for atherosclerosis,4) since lowering plasma choles-
Chemistry Diarylheptanoids (1—7) were synthesized
terol levels has been proven to reduce mortality from my- by condensation of 1-phenyl-5-hexanone with corresponding
ocardial infarction.5) Acyl-CoA: cholesterol O-acyltrans- benzaldehydes according to the previously described
ferase (ACAT, EC 2.3.1.26)6,7) is an intracellular enzyme re- method.17)
sponsible for catalyzing the esterification of free cholesterol
As shown in Chart 1, phenylalkylamides (11, 13—15) or
with fatty acyl-CoA to produce cholesteryl esters. This en- anilides (12, 17, 18) were prepared by alkylation of 8, fol-
zyme plays important roles in the absorption of dietary cho- lowed by amidation with appropriate phenylalkylamine or
lesterol from the small intestine, the secretion of very low- aniline by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
density lipoprotein (VLDL) from the liver, and the accumu- hydrochloride (EDC) in CHCl3.
lation of cholesteryl esters in atherosclerotic lesions. Inhibi-
Phenylpiperamides (16, 19—46) can be synthesized from
tion of ACAT should reduce the absorption of cholesterol, 8 via intermediates 9a, b or 10a, b (Chart 1). In the case of
lower plasma cholesterol levels,8—12) and should arrest the the modification of region C in the molecule (Fig. 1),
progression and promote the regression of atherosclerotic phenylpiperamides (16, 30—41) were prepared by alkylation
plaque.13,14) Therefore, ACAT inhibitors are a prime objective of 8 with corresponding halide, followed by amidation with
in the development of new therapeutic agents for hypercho- appropriate phenylpiperazine. With modification of region A
lesterolemia and atherosclerosis.15)
in the molecule (Fig. 1), phenylpiperamides (19—29, 42—
On this basis, metabolites were screened from about three 46) were prepared by amidation of 8 with corresponding
thousand fungi and numerous plant components that produce phenylpiperazine, followed by alkylation with appropriate
an ACAT inhibitor. Of these, it was found that 1-(4-hydroxy- halide.
3-methoxyphenyl)-7-phenylhept-1-en-3-one (1, Yakuchinone
B), which is a component of the seeds of Alpinia oxyphylla measured by incubating [1-14C]oleoyl-CoA with microsome
MIQUEL (Zingiberaceae),16) has ACAT inhibitory activity.
fractions prepared from the liver of Sprague–Dawley rats
Biology The ability to inhibit hepatic ACAT in vitro was
In order to obtain ACAT inhibitors that exhibit a high level given a normal diet. ACAT inhibitory activity was expressed
of hypocholesterolemic activity in vivo, the structure–activity by IC50.
relationships of three structural moieties of Yakuchinone B
In vivo hypocholesterolemic activity was assessed in these
(1) as the lead compound (Fig. 1) were examined. The com- rats by admixing a test compound into their diet at a concen-
pounds prepared were tested for the ability to inhibit the mi- tration of 0.005 or 0.1% for four days. For the next three days
crosomal ACAT from rat liver and to suppress the elevation the rats were fed a high cholesterol diet containing a test
To whom correspondence should be addressed. e-mail: kenji-ooishi@yakult.co.jp
© 2001 Pharmaceutical Society of Japan

July 2001
831
Chart 1. Preparation of Target Molecules 11—46
compound at the same concentration (nϭ8). For this investi-
gation the dosages of each compound calculated from the
body weight and the food consumption during the study pe-
riod were about 5 and 70—80 mg/kg/d for each concentra-
tion. Cholesterol lowering effect expressed as an inhibitory
rate (%) was calculated as follows: cholesterol lowering ef-
fect (%)ϭ[(BϪA)/(BϪC)]ϫ100 (A, B, and C represent
plasma total cholesterol levels in the drug-treated, control,
and normal groups, respectively).
Fig. 1. Three Structural Regions Examined in Structure–Activity Rela-
tionship Studies
residues at R1 and R3 and a benzyl residue at R2 in the A-
phenyl ring exhibited excellent ACAT inhibitory activity. On
the other hand, compounds 1—7 were not able to suppress a
rise in the plasma total cholesterol levels when the derivative
was mixed in the diet at a concentration of 0.1% and admin-
istered at a dose of 70—80 mg/kg/d to rats given a high cho-
lesterol diet.
The reason for the weak hypocholesterolemic activity in
vivo for these compounds is not clear, but may be related to
poor efficacy for ACAT inhibition and/or to low bioavailabil-
ity. Next, we synthesized the derivatives of compound 7 and
examined the ACAT inhibitory activity in vitro and hypocho-
lesterolemic activity in vivo.
Results and Discussion
To obtain ACAT inhibitors that exhibit a high level of
hypocholesterolemic activity in vivo, the structure–activity
relationships for three structural regions (Fig. 1) of Yakuchi-
none B (1) were examined as a lead compound.
The Structure–Activity Relationships for Region A
The biological data for Yakuchinone B (1) and its derivatives
(2—7), substituted on the A-phenyl ring, has been shown in
Table 1. Compound 1 inhibited rat hepatic ACAT with an
IC50 value of 20.6 mM. When an acetyl residue was intro-
duced at R2 of 1 (2), ACAT inhibitory activity increased by
2.1 times. Compound 3, which had a benzyl residue at R2,
showed higher activity than 2. However, compound 4, which
had a benzoyl residue at R2, exhibited only a weak ACAT in-
hibitory activity. The ACAT inhibitory activity of 5, which
did not have a methoxy residue like that at R1 of 3, was re-
markably lower than that of 3. Compound 6, which had an
ethoxy residue instead of a methoxy residue of 3 at R1, ex-
hibited almost the same activity as 3. Compound 7, which
had another methoxy residue like that at R3 of 3, provided
greater inhibition of ACAT than 3.
The Structure–Activity Relationships of Regions B and
C
The amide compounds CI-9768,18,19) and FR12916920)
(Fig. 2) are excellent ACAT inhibitors. Therefore, introduc-
tion of the amino units into region B of 7 was investigated in
order to further increase ACAT inhibitory activity. As shown
in Table 2, introduction of a n-propylamino-linker in region
B (11) increased ACAT inhibitory activity by 2 times, as we
expected. Additionally, derivative 12 in which an amino-
linker or 16 in which a tertiary amine of a piperazino-linker
was introduced in region B had higher inhibitory activity of
These data indicate that compound 7 with methoxy

832
Vol. 49, No. 7
Table 1. Effect of Region A Phenyl Substituent on Biological Activities
ACAT inhibition
IC50 (nM)a)
Cholesterol lowering
(% reduction)b)
No.
R1
R2
R3
1
2
3
CH3O–
CH3O–
CH3O–
H
H
H
H
20600
9800
2700
5
2
6
CH3CO–
4
5
6
CH3O–
H
H
H
H
38000
21600
3500
Ϫ2
5
CH3CH2O–
3
7
CH3O–
CH3O–
1700
2
a) In vitro ACAT inhibition was measured using hepatic microsomes isolated from rats fed a normal diet. Each determination was performed in triplicate. b) Sprague–Daw-
ley rats were fed a diet containing each compound at a concentration of 0.1% (70—80 mg/kg/d).
Fig. 2. ACAT Inhibitors
rat hepatic ACAT than 11. ACAT inhibitory activity de- and 4 in the C-phenyl ring, significantly inhibited the eleva-
creased in the following order: amino (12)Ͼpiperazino tion of plasma total cholesterol levels in rats given a high
(16)Ͼn-propylamino (11)Լn-butylamino (15)ϾϾethylamino cholesterol diet when administered as a dietary admixture at
(14)ϭn-butyl (7)ϾϾmethylamino (13).
a concentration of 0.1%. On the other hand, 17, which had
Further, ACAT inhibitory activity markedly increased with methyl residues at positions 2 and 4 in the C-phenyl ring like
the introduction of methyl residues to the C-phenyl ring of 19 but which had an amino-linker in region B, did not exhibit
12 and 16 (17—20). In particular, 19, which had a piper- hypocholesterolemic activity. Moreover, 20, which was a
azino-linker in region B and methyl residues at positions 2 structural isomer of 19 with a similar ACAT inhibitory pro-

July 2001
833
Table 2. Effect of X-Linker and Region C Phenyl Substituent on Biologi-
cal Activities
ACAT increased in a striking manner as we expected. How-
ever, these compounds did not exhibit hypocholesterolemic
activity. Conversion of benzyl residue of 19 into an n-heptyl
(26), n-octyl (27), n-nonyl (28), or n-decyl (29) residue but
not an n-hexyl (25) residue significantly increased ACAT in-
hibitory activity.
In addition, the hypocholesterolemic activity of n-alkyl de-
rivatives increased according to the length of the alkyl chain,
and n-octyl (27), n-nonyl (28), and n-decyl (29) derivatives
produced significant hypocholesterolemic activity when ad-
ministered as a dietary admixture at a concentration of
0.005%. Although 23 and 24 inhibited ACAT in a more strik-
ing fashion than 29, these derivatives did not exhibit hypoc-
holesterolemic activity. Based on these results, it is assumed
that the elevation in this activity with the introduction of a
long n-alkyl residue is related not only to an increase in their
specific activity with respect to ACAT but also to other fac-
tors such as their increased bioavailability.
On the other hand, 30, which had an n-decyl residue in the
A-phenyl ring but no residue in the C-phenyl ring, did not
lower plasma cholesterol level. This result suggests that ap-
propriate substituents in the C-phenyl ring are needed to ex-
hibit hypocholesterolemic activity.
The structure–activity relationship studies of the deriva-
tives with substituents in the C-phenyl ring, which had an n-
decyl residue at R2 in the A-phenyl ring (29—42), indicate
the following. Results for monomethyl-substituted com-
pounds (31—33) demonstrated that a 4-methyl residue was
very important in exhibiting a high level of hypocholes-
terolemic activity in vivo, while a 2-methyl or 3-methyl
ACAT
Cholesterol
lowering
No.
X
R4
inhibition
IC50 (nM)a) (% reduction)b)
7
11
12
13
14
15
–(CH2)4–
–NH(CH2)3–
–NH–
H
H
H
H
H
H
1700
840
2
9
260
7
–NHCH2–
–NH(CH2)2–
–NH(CH2)4–
3000
1700
900
Ϫ4
2
Ϫ11
16
17
18
H
550
52
3
Ϫ4
3
2,4-(CH3)2
3,4-(CH3)2
47
19
20
2,4-(CH3)2
3,4-(CH3)2
62
60
63**
7
a, b) See corresponding footnotes of Table 1. Significantly different from drug- residue was not.
untreated control using unpaired, two-tailed Student’s t-test, ** pϽ0.01.
With respect to a 4-substituent in the C-phenyl ring,
methyl (33), tert-butyl (34), methoxy (35), and fluorine (36)
analogs had a higher level of ACAT inhibitory activity than a
nitro analog (37). Therefore, the electrical nature of the sub-
stituents did not clearly correlate with their ACAT inhibitory
activity. On the other hand, methyl (33), tert-butyl (34), or
methoxy (35) analogs, which were electron-donating groups,
had a higher level of hypocholesterolemic activity in vivo
than those with fluorine (36) or nitro (37), which were elec-
tron-withdrawing groups. These results indicated that al-
though the electrical properties of C-phenyl substituents did
not affect specific activity with respect to ACAT, the effec-
tiveness of lowering plasma cholesterol level in vivo was
largely influenced by the electrical properties of those sub-
stituents. The supposition is that the introduction of electron-
donating groups in the C-phenyl ring at position 4 influenced
their pharmacokinetics, such as absorption, distribution, and
clearance, as with the introduction of a long-chain alkyl
residue at R2 in the A-phenyl ring, so in vivo hypocholes-
terolemic activity increased. Factors determining the potency
of ACAT inhibitors of in vivo hypocholesterolemic activity
are being analyzed through the comparison of the pharmaco-
kinetics of the ACAT inhibitors that were synthesized. These
results will be reported later.
file in vitro, did not exhibit hypocholesterolemic activity.
The Structure–Activity Relationships of 19 and Its De-
rivatives Derivatives of 19 were synthesized with substitu-
tion in the A- and C-phenyl rings as their ability to inhibit
microsomal ACAT from the liver of rats and hypocholes-
terolemic activity in vivo was investigated. In addition, the
concentration of the derivatives added to the diet was set at
0.005% to select derivatives that were more potent than 19.
For this investigation, the dosage of each derivative was
about 5 mg/kg/d. Biological data for 19 and its derivatives
21—46 is shown in Table 3.
Although 19 inhibited the elevation in plasma total choles-
terol levels when administered as a dietary admixture at a
concentration of 0.1% (Table 2), it did not exhibit hypocho-
lesterolemic activity when administered at a concentration of
0.005% (Table 3).
In respect to derivatives of 19 with substitution in the A-
phenyl ring, conversion of benzyl residue of 19 into a 3-
phenylpropyl (21) or phenoxyethyl (22) residue decreased
ACAT inhibitory activities in vitro, and these derivatives did
not produce significant hypocholesterolemic activity. Next,
benzyl residue of 19 was converted into a geranyl, farnecyl
or n-alkyl residue, since it seemed that the ACAT inhibitory
activity might increase by the introduction of a long-chain
aliphatic moiety with a structure similar to an acyl residue of
an acyl-CoA which was one substrate for ACAT.
With respect to dimethyl-substituted compounds (29, 38—
42), a 3,4-dimethyl-substituted derivative (42) had more of
an effect in lowering cholesterol level than a 4-methyl substi-
tuted derivative (33) or a 2,4-dimethyl substituted derivative
(29).
When benzyl residue of 19 was converted into a geranyl
(23) or farnecyl (24) residue, inhibitory activity on hepatic
With respect to A-phenyl substituted derivatives that had

834
Vol. 49, No. 7
Table 3. Effect of Regions A and C Phenyl Substituent of 19 on Biological Activities
ACAT inhibition
IC50 (nM)a)
Cholesterol lowering
No.
R2
R4
(% reduction)b)
19
21
22
23
2,4-(CH3)2
2,4-(CH3)2
2,4-(CH3)2
2,4-(CH3)2
62
100
790
6.4
2
17
20
2
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
2,4-(CH3)2
2,4-(CH3)2
2,4-(CH3)2
2,4-(CH3)2
2,4-(CH3)2
2,4-(CH3)2
H
4.5
79
2
n-C6H13–
n-C7H15–
n-C8H17–
n-C9H19–
n-C10H21–
n-C10H21–
n-C10H21–
n-C10H21–
n-C10H21–
n-C10H21–
n-C10H21–
n-C10H21–
n-C10H21–
n-C10H21–
n-C10H21–
n-C10H21–
n-C10H21–
n-C10H21–
n-C6H13–
n-C7H15–
n-C8H17–
n-C9H19–
12
15
25
15
38**
49**
65***
2
19
22
52
2-CH3
18
27
3-CH3
38
2
4-CH3
23
55***
28*
50**
10
4-C(CH3)3
4-OCH3
35
37
4-F
47
4-NO2
180
24
Ϫ2
2,3-(CH3)2
2,5-(CH3)2
2,6-(CH3)2
3,5-(CH3)2
3,4-(CH3)2
3,4-(CH3)2
3,4-(CH3)2
3,4-(CH3)2
3,4-(CH3)2
23
316
650
93
31*
Ϫ3
20
26
70**
20
54
38
46**
79***
59***
11
20
a) See corresponding footnotes of Table 1. b) Sprague-Dawley rats were fed a diet containing each compound at a concentration of 0.005% (about 5 mg/kg/d). Significantly
different from drug-untreated control using unpaired, two-tailed Student’s t-test, * pϽ0.05, ** pϽ0.01, *** pϽ0.001.
two methyl residues in the C-phenyl ring at positions 3 and 4 sized.
(42—46), n-heptyl (44), n-octyl (45), n-nonyl (46), and n-
Biological Activities of 45 As shown in Table 4, 45
decyl (42) derivatives produced significant hypocholes- more strongly inhibited rat hepatic ACAT than did other
terolemic activity. For these derivatives, unlike those with a ACAT inhibitors (an amide compound CI-976 and a urea
2,4-dimethyl residue in the C-phenyl ring (25—29), the drug compound YM75021)) and had a level of hypocholes-
efficacy did not increase according to the length of the n- terolemic activity in vivo that was as high as that with CI-976
alkyl chain at R2 in the A-phenyl ring, and the n-octyl deriva- and YM750.
tive (45) had the highest level of hypocholesterolemic activ-
ity.
A number of potent ACAT inhibitors that exhibit excellent
hypocholesterolemic activity in various species have been
From the structure–activity relationship studies on three synthesized for the purpose of pharmaceutical develop-
structural regions using Yakuchinone B (1) as a lead com- ment,22—24) and some of these have demonstrated highly sat-
pound, 45, N-(3,5-dimethoxy-4-n-octyloxycinnamoyl)-NЈ- isfactory results in model animals. Clinical development of
(3,4-dimethylphenyl)piperazine (Fig. 2) was finally synthe- ACAT inhibitors, however, has not been successful,25,26) be-

July 2001
835
Table 4. Biological Activities of 45
cause of poor clinical results and/or the induction of toxicity
in the adrenal glands of several animal species.27—38) Re-
cently, ACAT has been classified into two isozymes, ACAT-1
and ACAT-2, and research suggests that they differ in terms
of their distribution in the body.39—43) Two reasons are be-
lieved to be involved in why the numerous ACAT inhibitors
that have been developed thus far have not been clinically
successful.
ACAT inhibitory activity
Hypocholesterolemic
IC50 (nM)a)
No.
activity
ED50 (mg/kg/d)b)
Rat Liver HepG2
Caco2
45
CI-976
YM750
11
98
55
88
112
32
63
94
18
2.4
5.1
4.2
One is the relationship between adrenotoxicity and the in-
hibition of ACAT-1, which was found ubiquitously through-
out the body including macrophages and adrenal glands.
Tanaka et al. reported that since a compound with a high
level of macrophage ACAT (ACAT-1) inhibitory activity in-
duced adrenal toxicity, there might be a positive correlation
between specific activity with respect to ACAT-1 and adreno-
toxicity.37) On the other hand, Dominick et al.29) and
Sliskovic et al.33) reported that the adrenotoxicity observed
with ACAT inhibitors was likely to be independent of ACAT
inhibition. Thus, it is still unclear as to whether or not
adrenotoxicity is related to specific activity for ACAT-1 inhi-
bition. As shown in Table 4, 45 strongly inhibited microso-
mal ACAT from HepG2, in which ACAT-1 was predomi-
nantly expressed.42) On the other hand, in subacute toxicity
studies of 45 using rats, which abundantly expressed ACAT-1
in adrenal glands,43) no adrenotoxicity was observed when it
was orally administered via a stomach tube once a day to the
animals at a dose of 500 mg/kg/d for four weeks (data not
shown). Reindel et al. reported that in 2 weeks of oral toxic-
ity studies on PD 132301-2, an urea compound ACAT in-
hibitor, principal toxicity occurred in adrenals and that the
susceptibility for adrenal toxicity of this compound differed
among species: rank order of the toxicity (minimal toxic
dose, mg/kg) was dogs (6)Ͼguinea pigs (30)Ͼrabbits
(30)Ͼmonkeys (50)Ͼrats (1000)Ͼhamsters (no lesions at
1000 mg/kg).38) From this report, it suggests that subacute
toxicity of compound 45 prefers to assess in other species,
such as dogs and guinea pigs, because susceptibility for
adrenal toxicity of this compound also seems different be-
tween species like that of PD 132301-2.
a) In vitro ACAT inhibition was measured using microsomes isolated from rat liv-
ers, HepG2, or Caco2. Each determination was performed in triplicate. b) Hypocho-
lesterolemic activity expressed as an effective dose to reduce plasma total cholesterol
levels by 50% of the control value (nϭ8).
both ACAT-1 and ACAT-2 are expressed to the same extent
as in human intestinal tissue.42) Because the IC50 values for
the inhibition of microsomal ACAT derived from the two cell
lines were the same, it is assumed that 45 might have suffi-
cient ability to inhibit ACAT in both the human intestines
and liver regardless of the pattern of expression of ACAT
isozymes.
Although there remains much to explore through clinical
studies of 45 as to whether the inhibition of intestinal ACAT
or of hepatic ACAT plays a greater role in the treatment of
hypercholesterolemia in humans, the results mentioned sug-
gest that 45 might be an effective therapeutic agent for this
condition.
Conclusion
A novel series of amide compounds were synthesized to
find a new ACAT inhibitor, which exhibited a high level of
hypocholesterolemic activity in vivo, using Yakuchinone B
(1) as a starting material. Among the compounds synthe-
sized, N-(3,5-dimethoxy-4-n-octyloxycinnamoyl)-NЈ-(3,4-di-
methylphenyl)piperazine (45) strongly inhibited rat hepatic
ACAT to a level higher than that with CI-976 and YM750,
and it exhibited a high level of in vivo hypocholesterolemic
activity equivalent to that of CI-976 and YM750. Moreover,
since 45 strongly inhibited the microsomal ACAT prepared
from human cell lines (Caco2, HepG2), there is speculation
that it might have the ability to inhibit ACAT in both the
human intestines and the liver. Thus, 45 has fulfilled expecta-
tions for its use as a new therapeutic drug for hypercholes-
terolemia and atherosclerosis.
Another reason why numerous ACAT inhibitors did not
provide clinical success seems to relate to the large differ-
ences in the distribution of ACAT-1 and ACAT-2 in the liver
and intestines with respect to humans and rodents. In the
liver and intestines of mice, ACAT-2 is predominantly ex-
pressed41) and produces ACAT activity. Additionally, in the
liver of rats, it seems that ACAT-2 is predominantly ex-
Experimental
General Procedures All melting points (mp) were determined on an
Ishii micromelting point apparatus. IR spectra were recorded on a Shimadzu
pressed, whereas the expression of ACAT-1 is detected in the IR435 spectrophotometer as KBr disks. 1H-NMR spectra were measured
intestines.43)
with a JEOL JNM-A400 (400 MHz) instrument. Chemical shifts were re-
ported in d units from tetramethylsilane as the internal standard. Coupling
constants (J) were reported in hertz. Mass spectra were obtained with a Hi-
On the other hand, research has shown that ACAT-1 is pre-
dominantly expressed in the human liver and that both
tachi M-80B mass spectrometer using electron impact (EI) for ionization,
ACAT-1 and ACAT-2 play a major role in human in-
testines.42) Based on these reports, one can speculate that
with selective inhibitors for isozymes, the results in model
animals such as mice and rats might be quite different from
those of clinical studies in humans. As shown in Table 4, 45
strongly inhibited microsomal ACAT prepared from HepG2
(human hepatocarcinoma), in which ACAT-1 isozyme is ex-
pressed predominantly as in human liver tissue.42) On the
other hand, 45 also strongly inhibited microsomal ACAT pre-
pared from differentiating Caco2 (human colon adenocarci-
noma) with properties like intestinal epithelial cells, in which
and data were exhibited as m/z. Elemental analyses were carried out on a
CHN Coder MT-3 (Yanagimoto MFG. Co., Ltd., Kyoto, Japan). The
reagents and solvents used were obtained from commercial suppliers with-
out further purification. Column chromatography was performed on silica
gel (Merck; particle size 0.063—0.200 mm). Reaction progress was checked
by TLC analysis on silica gel coated glass plates (Kieselgel 60 F254, thick-
ness of 0.25 mm). Visualization was with UV light (254 nm) or iodine.
Yakuchinone B (1),17) CI-97618) and YM75021) were synthesized accord-
ing to the previously described method in our laboratories. 4-tert-Butyl-
phenylpiperazine, 2,6-dimethylphenylpiperazine, 3,4-dimethylphenylpiper-
azine, and 3,5-dimethylphenylpiperazine were prepared from bis(2-chloro-
ethyl)-N-(ethoxycarbonyl)amine and corresponding anilines according to the
procedure described by Mishani et al.44)

836
Vol. 49, No. 7
1-(4-Acetyloxy-3-methoxyphenyl)-7-phenylhept-1-en-3-one (2) Ten and sodium hydride (1.81 g, 45.2 mmol) was then very carefully added to
milliliters of 10% KOH aq. solution was added to the 1-phenyl-5-hexanone this solution. 1-Bromodecane (10.00 g, 45.2 mmol) was added to this mix-
(2.64 g, 15.0 mmol) in EtOH (100 ml) and stirred at room temperature for an ture portionwise and the mixture was stirred at room temperature for 96 h.
hour. Then 4-acetyloxy-3-methoxybenzaldehyde (2.91 g, 15.0 mmol) was The reaction mixture was poured into water (5 l) and was adjusted at pH 3
added to the reaction mixture and stirred overnight at room temperature. The with 1 N aq. HCl. Ethyl 4-n-decyloxy-3,5-dimethoxycinnamate was extracted
resulting precipitate was collected by filtration and the precipitate was re- with CHCl3 (2 l) and the extracts were dried with sodium sulfate anhydrous.
crystallized from CHCl3–EtOH to give 2 as white crystals (3.15 g, yield The solvent was removed in vacuo to give ethyl 4-n-decyloxy-3,5-di-
59.6%). mp 74—76 °C. 1H-NMR (CDCl3) d: 1.65—1.78 (4H, m), 2.32 (3H, methoxycinnamate as a clear oil which was used for the next reaction with-
s), 2.62—2.72 (4H, m), 3.87 (3H, s), 6.66 (1H, d, Jϭ16.4 Hz), 7.03—7.35 out purification. To the solution of ethyl 4-n-decyloxy-3,5-dimethoxycinna-
(8H, m), 7.48 (1H, d, Jϭ16.4 Hz). IR (KBr) cmϪ1: 1763, 1653, 1227. MS
m/z: 352 (Mϩ), 309, 118. Anal. Calcd for C22H24O4: C, 74.98; H, 6.86.
Found: C, 75.02; H, 6.85.
mate in EtOH (250 ml) was added 1 N aq. NaOH (65 ml, 65 mmol) followed
by stirring for 4 d at room temperature. Then, 1 N aq. HCl was added to the
reaction mixture and concentrated. This solution was then poured into water
The following compounds 3—7 were prepared in a manner similar to that (2 l). The resulting precipitate was collected by filtration, washed with water,
described for 2 from 1-phenyl-5-hexanone and the appropriate benzalde- and dried to give 9b as pale yellow crystals (4.44 g, yield 35.0%). mp 79—
1
hyde.
81 °C. H-NMR (CDCl3) d: 0.88 (3H, t, Jϭ7.0 Hz), 1.27—1.44 (14H, m),
1-(4-Benzyloxy-3-methoxyphenyl)-7-phenylhept-1-en-3-one (3): Yield 1.75 (2H, m), 3.88 (6H, s), 4.01 (2H, t, Jϭ6.8 Hz), 6.36 (1H, d, Jϭ15.9 Hz),
1
78.0%. mp 84—87 °C. H-NMR (CDCl3) d: 1.60—1.76 (4H, m), 2.62 (4H,
m), 3.86 (3H, s), 5.13 (2H, s), 6.58 (1H, d, Jϭ16.1 Hz), 6.83 (1H, d,
6.78 (2H, s), 7.71 (1H, d, Jϭ15.9 Hz). IR (KBr) cmϪ1: 2919, 1683, 1281.
N-(3,5-Dimethoxy-4-hydroxycinnamoyl)-N؅-(2,4-dimethylphenyl)-
Jϭ8.3 Hz), 7.01 (1H, dd, Jϭ8.3, 2.0 Hz), 7.05 (1H, d, Jϭ2.0 Hz), 7.10— piperazine (10a) EDC (8.55 g, 44.6 mmol) and 1-(2,4-dimethylphenyl)-
7.42 (10H, m), 7.45 (1H, d, Jϭ16.1 Hz). IR (KBr) cmϪ1: 1639, 1267. MS piperazine (8.49 g, 44.6 mmol) were added to a solution of 8 (10.00 g,
m/z: 400 (Mϩ), 309, 118, 92. Anal. Calcd for C27H28O3: C, 80.97; H, 7.05. 44.6 mmol) in CHCl3 (600 ml) and stirred overnight at room temperature.
Found: C, 81.35; H, 7.07.
The reaction mixture was directly purified by silica gel column chromatogra-
1-(4-Benzoyloxy-3-methoxyphenyl)-7-phenylhept-1-en-3-one (4): Yield phy (CHCl3) and recrystallized from EtOH to give 10a as pale yellow crys-
1
1
68.3%. mp 112—114 °C. H-NMR (CDCl3) d: 1.70—1.73 (4H, m), 2.63— tals (5.06 g, yield 28.6%). mp 213—215 °C. H-NMR (CDCl3) d: 2.20 (3H,
2.72 (4H, m), 3.85 (3H, s), 6.69 (1H, d, Jϭ16.1 Hz), 7.15—7.21 (6H, m), s), 2.24 (3H, s), 2.79 (4H, s), 3.32 (4H, s), 3.80 (6H, s), 6.90—7.01 (5H, m),
7.25—7.30 (2H, m), 7.48—7.55 (3H, m), 7.52—7.65 (1H, m), 8.21 (2H, d, 7.13 (1H, d, Jϭ15.4 Hz), 7.44 (1H, d, Jϭ15.4 Hz). IR (KBr) cmϪ1: 3110,
Jϭ8.3 Hz). IR (KBr) cmϪ1: 1734, 1655, 1261. MS m/z: 414 (Mϩ), 309, 118,
92. Anal. Calcd for C27H26O4: C, 78.24; H, 6.32. Found: C, 77.89; H, 6.31.
1636, 1233. MS m/z: 397 (Mϩ), 208, 161, 133, 91.
Compound 10b was prepared in a manner similar to that described for
1-(4-Benzyloxyphenyl)-7-phenylhept-1-en-3-one (5): Yield 72.5%. mp 10a from 8 and 1-(3,4-dimethylphenyl)piperazine.
1
99—100 °C. H-NMR (CDCl3) d: 1.60—1.76 (4H, m), 2.65 (4H, m), 5.09
N-(3,5-Dimethoxy-4-hydroxycinnamoyl)-NЈ-(3,4-dimethlphenyl)piper-
(2H, s), 5.14 (2H, m), 6.61 (1H, d, Jϭ16.1 Hz), 6.98 (1H, d, Jϭ8.8 Hz), azine (10b): Yield 46.0%. mp 215—217 °C. 1H-NMR (CDCl3) d: 2.20 (3H,
7.05—7.52 (10H, m), 7.45 (1H, d, Jϭ16.1 Hz), 7.83 (1H, d, Jϭ8.8 Hz). IR s), 2.24 (3H, s), 3.17 (4H, s), 3.88 (4H, br s), 3.94 (6H, s), 5.74 (1H, s),
(KBr) cmϪ1: 2928, 1686, 1248. MS m/z: 370 (Mϩ), 309, 118, 92. Anal. 6.70—6.77 (5H, m), 7.05 (1H, d, Jϭ8.0 Hz), 7.63 (1H, d, Jϭ15.4 Hz). IR
Calcd for C26H26O2: C, 84.29; H, 7.07. Found: C, 84.59; H, 6.96.
(KBr) cmϪ1: 3200, 1638, 1192. MS m/z: 397 (Mϩ), 208, 161, 134.
1-(4-Benzyloxy-3-ethoxyphenyl)-7-phenylhept-1-en-3-one (6): Yield
N-(3-Phenyl)propyl-(2E)-3-(4-benzyloxy-3,5-dimethoxyphenyl)-2-
61.8%. mp 110—112 °C. 1H-NMR (CDCl3) d: 1.48 (3H, t, Jϭ7.1 Hz), propenamide (11) EDC (12.20 g, 63.6 mmol) and 1-amino-3-phenyl-
1.60—1.76 (4H, m), 2.62—2.68 (4H, m), 4.14 (2H, q, Jϭ7.1 Hz), 5.19 (2H,
s), 6.58 (1H, d, Jϭ16.1 Hz), 6.88 (1H, d, Jϭ8.3 Hz), 7.04 (1H, dd, Jϭ8.3, mmol) in CHCl3 (320 ml). The mixture was stirred overnight at room tem-
2.2 Hz), 7.09 (1H, d, Jϭ2.2 Hz), 7.15—7.20 (3H, m), 7.25—7.48 (8H, m). perature. The reaction mixture was washed with 1 N aq. HCl and saturated
IR (KBr) cmϪ1: 2939, 1655, 1269. MS m/z: 414 (Mϩ), 353, 118, 92. Anal. NaCl solution, and then dried with sodium sulfate anhydrous. The mixture
propane (12.90 g, 95.4 mmol) were added to a solution of 9a (10.00 g, 31.8
Calcd for C28H30O3: C, 81.13; H, 7.29. Found: C, 80.88; H, 7.35.
was purified by silica gel column chromatography (CHCl3) and recrystal-
1-(4-Benzyloxy-3,5-dimethoxyphenyl)-7-phenylhept-1-en-3-one (7): Yield
lized from EtOH to give 11 as white crystals (0.50 g, yield 3.6%). mp 115—
76.7%. mp 93—95 °C. 1H-NMR (CDCl3) d: 1.64—1.79 (4H, m), 2.63— 116 °C. 1H-NMR (CDCl3) d: 1.89 (2H, m), 2.68 (2H, t, Jϭ7.6 Hz), 3.41
2.71 (4H, m), 3.85 (6H, s), 5.05 (2H, s), 6.62 (1H, d, Jϭ16.1 Hz), 6.75 (2H, (2H, q, Jϭ6.6 Hz), 3.81 (6H, s), 5.03 (2H, s), 5.72 (1H, br t, Jϭ5.4 Hz), 6.26
s), 7.15—7.20 (3H, m), 7.25—7.37 (5H, m), 7.42—7.49 (3H, m). IR (KBr) (1H, d, Jϭ15.4 Hz), 6.69 (2H, s), 7.15—7.52 (11H, m). IR (KBr) cmϪ1
:
cmϪ1: 2937, 1693, 1265. MS m/z: 430 (Mϩ), 369, 118, 92. Anal. Calcd for 3276, 1648, 1238. MS m/z: 431 (Mϩ), 340, 207, 91. Anal. Calcd for
C28H30O4: C, 78.11; H, 7.02. Found: C, 77.90; H, 7.11.
C27H29NO4: C, 75.15; H, 6.77; N, 3.25. Found: C, 75.43; H, 6.80; N, 3.25.
4-Benzyloxy-3,5-dimethoxycinnamic Acid (9a) 3,5-Dimethoxy-4-hy-
The following compounds 12—15, 17, 18 were prepared in a manner sim-
droxycinnamic acid 8 (124.8 g, 556.6 mmol) was dissolved in EtOH (1.5 l) ilar to that described for 11 from 9a and the appropriate aniline or pheny-
and H2SO4 (50 ml) was added dropwise to this mixture under an ice-cold lalkylamine.
condition and then refluxed at 100 °C for 5.5 h. After cooling to room tem-
N-Phenyl-(2E)-3-(4-benzyloxy-3,5-dimethoxyphenyl)-2-propenamide
perature, the reaction mixture was concentrated and poured into water (4.5 l) (12): Yield 71.5%. mp 109—110 °C. 1H-NMR (CDCl3) d: 3.64 (6H, s), 5.00
and the resulting precipitate was collected by filtration and dried in vacuo to (2H, s), 6.59 (2H, s), 6.66 (1H, d, Jϭ15.4 Hz), 7.06 (1H, t, Jϭ7.1 Hz),
give ethyl 3,5-dimethoxy-4-hydroxycinnamate (131.4 g, yield 93.6%). Ethyl 7.22—7.32 (5H, m), 7.40—7.72 (5H, m), 8.75 (1H, s). IR (KBr) cmϪ1
:
3,5-dimethoxy-4-hydroxycinnamate (30.00 g, 118.9 mmol) was dissolved in 3247, 1654, 1232. MS m/z: 389 (Mϩ), 280, 132, 91. Anal. Calcd for
N,N-dimethylformamide (DMF) (500 ml) and then sodium hydride (10.88 g C24H23NO4: C, 74.02; H, 5.95; N, 3.60. Found: C, 74.20; H, 5.94; N, 3.44.
of 60% in oil, 272.2 mmol) was added very carefully to this solution. Benzyl
N-Benzyl-(2E)-3-(4-benzyloxy-3,5-dimethoxyphenyl)-2-propenamide
chloride (18.05 g, 142.2 mmol) was added to this mixture portionwise and (13): Yield 15.1%. mp 147—148 °C. 1H-NMR (CDCl3) d: 3.80 (6H, s), 4.54
the mixture was stirred at room temperature for 96 h. The reaction mixture (2H, d, Jϭ5.6 Hz), 5.02 (2H, s), 6.08 (1H, br t, Jϭ5.8 Hz), 6.34 (1H, d,
was poured into water (2 l) and the resulting precipitate was collected by fil- Jϭ15.6 Hz), 6.69 (2H, s), 7.24—7.48 (10H, m), 7.56 (1H, d, Jϭ15.6 Hz). IR
tration. The precipitate was purified by silica gel column chromatography (KBr) cmϪ1: 3252, 1652, 1276. MS m/z: 403 (Mϩ), 312, 226, 92. Anal.
(CHCl3) and dried in vacuo to give ethyl 4-benzyloxy-3,5-dimethoxycinna- Calcd for C25H25NO4: C, 74.42; H, 6.25; N, 3.47. Found: C, 74.36; H, 6.46;
mate as white crystals (40.12 g, yield 99.3%). 1 N aq. NaOH (65 ml, N, 3.24.
65 mmol) was added to a solution of ethyl 4-benzyloxy-3,5-dimethoxycinna-
mate (1.97 g, 5.8 mmol) in ethanol (100 ml). The mixture was stirred for 7 d namide (14): Yield 46.5%. mp 116—119 °C. 1H-NMR (CDCl3) d: 2.88 (2H,
at room temperature. Then, the reaction mixture was acidified with 1 N aq. t, Jϭ6.8 Hz), 3.66 (2H, q, Jϭ6.6 Hz), 3.82 (6H, s), 5.02 (2H, s), 5.69 (1H,
N-(2-Phenyl)ethyl-(2E)-3-(4-benzyloxy-3,5-dimethoxyphenyl)-2-prope-
HCl and was concentrated. This solution was then poured into water (2 l). br t, Jϭ5.6 Hz), 6.23 (1H, d, Jϭ15.4 Hz), 6.69 (2H, s), 7.20—7.55 (11H, m).
The resulting precipitate was collected by filtration, washed with water, and IR (KBr) cmϪ1: 3291, 1648, 1275. MS m/z: 417 (Mϩ), 326, 226, 91. Anal.
dried to give 9a as white crystals (1.63 g, yield 89.5%). mp 110—112 °C. Calcd for C26H27NO4: C, 74.80; H, 6.52; N, 3.35. Found: C, 75.04; H, 6.68;
1H-NMR (CDCl3) d: 3.85 (6H, s), 5.06 (2H, s), 6.36 (1H, d, Jϭ15.9 Hz), N, 3.26.
6.76 (2H, s), 7.25—7.48 (5H, m), 7.70 (1H, d, Jϭ15.9 Hz). IR (KBr) cmϪ1
3049, 1626, 1281. MS m/z: 314 (Mϩ), 223, 164.
4-n-Decyloxy-3,5-dimethoxycinnamic Acid (9b) Ethyl 3,5-dimethoxy- 1.72 (4H, m), 2.63 (2H, t, Jϭ7.4 Hz), 3.38 (2H, q, Jϭ6.5 Hz), 3.80 (6H, s),
4-hydroxycinnamate (8.77 g, 34.8 mmol) was dissolved in DMF (500 ml) 5.02 (2H, s), 5.78 (1H, br t, Jϭ5.2 Hz), 6.29 (1H, d, Jϭ15.4 Hz), 6.69 (2H,
:
N-(4-Phenyl)butyl-(2E)-3-(4-benzyloxy-3,5-dimethoxyphenyl)-2-prope-
namide (15): Yield 4.2%. mp 133—135 °C. 1H-NMR (CDCl3) d: 1.52—

July 2001
837
s), 7.13—7.54 (11H, m). IR (KBr) cmϪ1: 3279, 1648, 1239. MS m/z: 445
(Mϩ), 354, 226, 91. Anal. Calcd for C28H31NO4: C, 75.48; H, 7.01; N, 3.14.
Found: C, 75.60; H, 7.23; N, 3.08.
N-(2,4-Dimethylphenyl)-(2E)-3-(4-benzyloxy-3,5-dimethoxyphenyl)-2-
propenamide (17): Yield 30.5%. mp 215—216 °C. 1H-NMR (CDCl3) d:
2.26 (3H, s), 2.30 (3H, s), 3.84 (6H, s), 5.05 (2H, s), 6.48 (1H, d,
Jϭ15.4 Hz), 6.74 (2H, s), 7.00—7.50 (8H, m), 7.65 (1H, d, Jϭ15.4 Hz),
7.75 (1H, br s). IR (KBr) cmϪ1: 3201, 1664, 1239. MS m/z: 417 (Mϩ), 326,
120, 92. Anal. Calcd for C26H27NO4: C, 74.80; H, 6.52; N, 3.35. Found: C,
75.05; H, 6.45; N, 3.56.
N-(3,4-Dimethylphenyl)-(2E)-3-(4-benzyloxy-3,5-dimethoxyphenyl)-2-
propenamide (18): Yield 55.3%. mp 148—149 °C. 1H-NMR (CDCl3) d:
2.21 (3H, s), 2.22 (3H, s), 3.80 (6H, s), 5.04 (2H, s), 6.48 (1H, d,
Jϭ15.6 Hz), 6.70 (2H, s), 7.07 (1H, d, Jϭ8.0 Hz), 7.26—7.48 (8H, m), 7.63
(1H, d, Jϭ15.6 Hz). IR (KBr) cmϪ1: 3310, 1658, 1271. MS m/z: 417 (Mϩ),
326, 120, 91. Anal. Calcd for C26H27NO4: C, 74.80; H, 6.52; N, 3.35. Found:
C, 74.67; H, 6.54; N, 3.57.
N-(4-Benzyloxy-3,5-dimethoxycinnamoyl)-N؅-phenylpiperazine (16)
EDC (6.40 g, 33.4 mmol) and 1-phenylpiperazine (8.80 g, 54.2 mmol) were
added to a solution of 9a (10.00 g, 31.9 mmol) in CHCl3 (500 ml) and stirred
overnight at room temperature. The reaction mixture was directly purified by
silica gel column chromatography (CHCl3) and recrystallized from EtOH to
give 16 as pale yellow crystals (9.64 g, yield 66.0%). mp 98—99 °C. 1H-
NMR (CDCl3) d: 3.16 (4H, br s), 3.81 (4H, br s), 3.82 (6H, s), 5.03 (2H, s),
6.74 (2H, s), 6.81 (1H, d, Jϭ15.4 Hz), 6.85—6.92 (3H, m), 7.23—7.48 (7H,
m), 7.62 (1H, d, Jϭ15.4 Hz). IR (KBr) cmϪ1: 1646, 1336, 1272. MS m/z:
458 (Mϩ), 367, 208, 132. Anal. Calcd for C28H30N2O4: C, 73.34; H, 6.59; N,
6.11. Found: C, 73.74; H, 6.83; N, 5.87.
(3H, s), 2.90 (4H, br s), 3.81 (4H, br s), 3.88 (6H, s), 4.57 (2H, d, Jϭ7.0 Hz),
5.05—5.13 (2H, m), 5.57 (1H, t, Jϭ7.3 Hz), 6.75 (2H, s), 6.82 (1H, d,
Jϭ15.4 Hz), 6.88 (1H, d, Jϭ8.0 Hz), 6.97 (1H, dd, Jϭ8.0, 2.1 Hz), 7.02 (1H,
d, Jϭ2.1 Hz), 7.62 (1H, d, Jϭ15.4 Hz). IR (KBr) cmϪ1: 2956, 1636, 1241.
MS m/z: 600 (Mϩ), 397, 208, 161. Anal. Calcd for C38H52N2O4: C, 75.96; H,
8.72; N, 4.66. Found: C, 76.02; H, 8.66; N, 4.63.
N-(3,5-Dimethoxy-4-n-hexyloxycinnamoyl)-NЈ-(2,4-dimethylphenyl)-
1
piperazine (25): Yield 71.0%. mp 113—114 °C. H-NMR (CDCl3) d: 0.90
(3H, t, Jϭ6.7 Hz), 1.30—1.50 (6H, m), 1.70—1.78 (2H, m), 2.26 (3H, s),
2.29 (3H, s), 2.88 (4H, br s), 3.81 (4H, br s), 3.86 (6H, s), 3.99 (2H, t,
Jϭ6.7 Hz), 6.76 (2H, s), 6.85 (1H, d, Jϭ15.4 Hz), 6.87 (1H, d, Jϭ8.0 Hz),
6.96 (1H, dd, Jϭ8.0, 2.1 Hz), 7.01 (1H, d, Jϭ2.1 Hz), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2924, 1636, 1243. MS m/z: 480 (Mϩ), 255,
190, 161. Anal. Calcd for C29H40N2O4: C, 72.47; H, 8.39; N, 5.83. Found: C,
72.23; H, 8.56; N, 5.68.
N-(3,5-Dimethoxy-4-n-heptyloxycinnamoyl)-NЈ-(2,4-dimethylphenyl)-
piperazine (26): Yield 51.9%. mp 88—89 °C. 1H-NMR (CDCl3) d 0.89 (3H,
t, Jϭ6.8 Hz), 1.27—1.48 (8H, m), 1.72—1.80 (2H, m), 2.28 (3H, s), 2.31
(3H, s), 2.91 (4H, br s), 3.81 (4H, br s), 3.86 (6H, s), 3.99 (2H, t, Jϭ6.7 Hz),
6.76 (2H, s), 6.82 (1H, d, Jϭ15.4 Hz), 6.89 (1H, d, Jϭ8.0 Hz), 6.98 (1H, dd,
Jϭ8.0, 2.1 Hz), 7.02 (1H, d, Jϭ2.1 Hz), 7.62 (1H, d, Jϭ15.4 Hz). IR (KBr)
cmϪ1: 3002, 1636, 1242. MS m/z: 494 (Mϩ), 305, 190, 161. Anal. Calcd for
C30H42N2O4: C, 72.84; H, 8.56; N, 5.66. Found: C, 72.82; H, 8.75; N, 5.44.
N-(3,5-Dimethoxy-4-n-octyloxycinnamoyl)-NЈ-(2,4-dimethylphenyl)-
1
piperazine (27): Yield 68.2%. mp 107—109 °C. H-NMR (CDCl3) d: 0.88
(3H, t, Jϭ6.8 Hz), 1.26—1.48 (10H, m), 1.72—1.80 (2H, m), 2.27 (3H, s),
2.30 (3H, s), 2.89 (4H, br s), 3.81 (4H, br s), 3.87 (6H, s), 3.99 (2H, t,
Jϭ6.8 Hz), 6.76 (2H, s), 6.83 (1H, d, Jϭ15.4 Hz), 6.88 (1H, d, Jϭ8.0 Hz),
6.97 (1H, dd, Jϭ8.0, 2.1 Hz), 7.01 (1H, d, Jϭ2.1 Hz), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 3000, 1636, 1241. MS m/z: 508 (Mϩ), 319,
208, 161. Anal. Calcd for C31H44N2O4: C, 73.19; H, 8.72; N, 5.51. Found: C,
73.26; H, 8.66; N, 5.72.
N-(4-Benzyloxy-3,5-dimethoxycinnamoyl)-N؅-(2,4-dimethylphenyl)-
piperazine (19) Compound 10a (5.00 g, 12.6 mmol) was dissolved in
DMF (400 ml) and then sodium hydride (0.66 g, 16.4 mmol) was very care-
fully added to this solution. Benzyl chloride (2.07 g, 16.4 mmol) was added
to this mixture portionwise and the mixture was stirred at room temperature
for 96 h. The reaction mixture was poured into water (4 l) and neutralized
with 1 N aq. HCl. The resulting precipitate was collected by filtration. The
precipitate was purified by silica gel column chromatography (CHCl3) and
recrystallized from EtOH to give 19 as pale yellow crystals (3.24 g, yield
N-(3,5-Dimethoxy-4-n-nonyloxycinnamoyl)-NЈ-(2,4-dimethylphenyl)-
1
piperazine (28): Yield 70.4%. mp 100—101 °C. H-NMR (CDCl3) d: 0.88
(3H, t, Jϭ6.8 Hz), 1.23—1.47 (12H, m), 1.72—1.78 (2H, m), 2.28 (3H, s),
2.31 (3H, s), 2.91 (4H, br s), 3.82 (4H, br s), 3.88 (6H, s), 3.99 (2H, t,
Jϭ6.8 Hz), 6.75 (2H, s), 6.80 (1H, d, Jϭ15.4 Hz), 6.90 (1H, d, Jϭ8.0 Hz),
6.98 (1H, dd, Jϭ8.0, 2.1 Hz), 7.03 (1H, d, Jϭ2.1 Hz), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2916, 1636, 1242. MS m/z: 522 (Mϩ), 333,
190, 161. Anal. Calcd for C32H46N2O4: C, 73.53; H, 8.87; N, 5.36. Found: C,
73.45; H, 8.85; N, 5.51.
1
52.9%). mp 102—104 °C. H-NMR (CDCl3) d: 2.28 (3H, s), 2.31 (3H, s),
2.91 (4H, br s), 3.82 (4H, br s), 3.86 (6H, s), 5.04 (2H, s), 6.74 (2H, s), 6.80
(1H, d, Jϭ15.4 Hz), 6.90 (1H, d, Jϭ8.0 Hz), 6.98 (1H, dd, Jϭ8.0, 2.1 Hz),
7.03 (1H, d, Jϭ2.1 Hz), 7.27—7.36 (3H, m), 7.47—7.49 (2H, m), 7.61 (1H,
d, Jϭ15.4 Hz). IR (KBr) cmϪ1: 1636, 1338, 1273. MS m/z: 486 (Mϩ), 395,
190, 161. Anal. Calcd for C30H34N2O4: C, 74.05; H, 7.04; N, 5.76. Found: C,
73.71; H, 7.25; N, 5.54.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(2,4-dimethylphenyl)-
piperazine (29): Yield 53.1%. mp 97—99 °C. 1H-NMR (CDCl3) d: 0.88
(3H, t, Jϭ6.8 Hz), 1.22—1.48 (14H, m), 1.71—1.77 (2H, m), 2.28 (3H, s),
2.31 (3H, s), 2.91 (4H, br s), 3.82 (4H, br s), 3.88 (6H, s), 3.99 (2H, t,
Jϭ6.7 Hz), 6.75 (2H, s), 6.80 (1H, d, Jϭ15.4 Hz), 6.90 (1H, d, Jϭ8.0 Hz),
6.98 (1H, dd, Jϭ8.0, 2.1 Hz), 7.03 (1H, d, Jϭ2.1 Hz), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2915, 1636, 1241. MS m/z: 536 (Mϩ), 347,
208, 161. Anal. Calcd for C33H48N2O4: C, 73.84; H, 9.01; N, 5.22. Found: C,
73.67; H, 9.12; N, 5.44.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-N؅-phenylpiperazine (30)
EDC (1.58 g, 8.2 mmol) and 1-phenylpiperazine (1.34 g, 8.2 mmol) were
added to a solution of 9b (3.00 g, 8.2 mmol) in CHCl3 (200 ml) and stirred
for 3 d at room temperature. The reaction mixture was directly purified by
silica gel column chromatography (CHCl3) and recrystallized from EtOH to
give 30 as pale yellow crystals (1.29 g, yield 30.9%). mp 77—78 °C. 1H-
NMR (CDCl3) d: 0.88 (3H, t, Jϭ6.8 Hz), 1.22—1.49 (14H, m), 1.73—1.79
(2H, m), 1.86 (6H, s), 3.19 (4H, br s), 3.83 (4H, br s), 4.00 (2H, t,
Jϭ6.8 Hz), 6.76 (2H, s), 6.82 (1H, d, Jϭ15.4 Hz), 6.86—6.94 (3H, m),
7.24—7.30 (2H, m), 7.63 (1H, d, Jϭ15.4 Hz). IR (KBr) cmϪ1: 2949, 1642,
1236. MS m/z: 508 (Mϩ), 347, 208, 132. Anal. Calcd for C31H44N2O4: C,
73.19; H, 8.72; N, 5.51. Found: C, 73.20; H, 8.65; N, 5.63.
The following compounds 21—29 were prepared in a manner similar to
that described for 19 from 10a and the appropriate halide.
N-[3,5-Dimethoxy-4-(3-phenyl)propyloxycinnamoyl]-NЈ-(2,4-di-
methylphenyl)piperazine (21): Yield 96.8%. mp 103—104 °C. 1H-NMR
(CDCl3) d: 2.06 (2H, m), 2.28 (3H, s), 2.31 (3H, s), 2.84 (2H, t, Jϭ7.7 Hz),
2.91 (4H, br s), 3.82 (4H, br s), 3.87 (6H, s), 4.04 (2H, t, Jϭ6.3 Hz), 6.75
(2H, s), 6.81 (1H, d, Jϭ15.4 Hz), 6.87—7.04 (3H, m), 7.15—7.31 (5H, m),
7.62 (1H, d, Jϭ15.4 Hz); IR (KBr) cmϪ1: 2908, 1636, 1241. MS m/z: 514
(Mϩ), 325, 190, 161. Anal. Calcd for C32H38N2O4: C, 74.68; H, 7.44; N,
5.44. Found: C, 74.50; H, 7.29; N, 5.63.
N-[3,5-Dimethoxy-4-(2-phenoxy)ethyloxycinnamoyl]-NЈ-(2,4-di-
methylphenyl)piperazine (22): Yield 20.9%. mp 123—124 °C. 1H-NMR
(CDCl3) d: 2.27 (3H, s), 2.30 (3H, s), 2.91 (4H, br s), 3.82 (4H, br s), 3.83
(6H, s), 4.27 (2H, t, Jϭ5.1 Hz), 4.39 (2H, t, Jϭ5.0 Hz), 6.74 (2H, s), 6.80
(1H, d, Jϭ15.4 Hz), 6.87—7.05 (6H, m), 7.23—7.30 (2H, m), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2934, 1643, 1241. MS m/z: 516 (Mϩ), 327,
208, 161. Anal. Calcd for C31H36N2O5: C, 72.07; H, 7.02; N, 5.42. Found: C,
71.88; H, 6.78; N, 5.63.
N-(3,5-Dimethoxy-4-geranyloxycinnamoyl)-NЈ-(2,4-dimethylphenyl)-
piperazine (23): Yield 84.1%. mp 69—71 °C. 1H-NMR (CDCl3) d: 1.59
(3H, s), 1.66 (3H, s), 1.67 (3H, s), 1.93—2.10 (4H, m), 2.28 (3H, s), 2.31
(3H, s), 2.91(4H, br s), 3.81 (4H, br s), 3.89 (6H, s), 4.57 (2H, d, Jϭ7.0 Hz),
5.08 (1H, t, Jϭ6.0 Hz), 5.56 (1H, t, Jϭ6.5 Hz), 6.75 (2H, s), 6.81 (1H, d,
Jϭ15.4 Hz), 6.89 (1H, d, Jϭ8.0 Hz), 6.98 (1H, dd, Jϭ8.0, 2.1 Hz), 7.03 (1H,
d, Jϭ2.1 Hz), 7.62 (1H, d, Jϭ15.4 Hz). IR (KBr) cmϪ1: 2930, 1636, 1241.
MS m/z: 532 (Mϩ), 397, 208, 161. Anal. Calcd for C33H44N2O4: C, 74.40; H,
8.33; N, 5.26. Found: C, 74.42; H, 8.13; N, 5.22.
The following compounds 31—41 were prepared in a manner similar to
that described for 30 from 9b and the appropriate phenylpiperazine.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(2-methylphenyl)piper-
1
azine (31): Yield 33.3%. mp 85—87 °C. H-NMR (CDCl3) d: 0.88 (3H, t,
Jϭ6.8 Hz), 1.22—1.48 (14H, m), 1.72—1.79 (2H, m), 2.34 (3H, s), 2.94
(4H, br s), 3.82 (4H, br s), 3.88 (6H, s), 3.99 (2H, t, Jϭ6.8 Hz), 6.76 (2H, s),
6.82 (1H, d, Jϭ15.4 Hz), 6.97—7.22 (4H, m), 7.63 (1H, d, Jϭ15.4 Hz). IR
(KBr) cmϪ1: 2915, 1647, 1238. MS m/z: 522 (Mϩ), 347, 208, 147. Anal.
Calcd for C32H46N2O4: C, 73.53; H, 8.87; N, 5.36. Found: C, 73.48; H, 8.96;
N, 5.40.
N-(3,5-Dimethoxy-4-farnecyloxycinnamoyl)-NЈ-(2,4-dimethylphenyl)-
piperazine (24): Yield 68.8%. mp 58—59 °C. 1H-NMR (CDCl3) d: 1.59
(6H, s), 1.66 (3H, s), 1.68 (3H, s), 1.94—2.12 (8H, m), 2.27 (3H, s), 2.30
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(3-methylphenyl)piper-
1
azine (32): Yield 17.4%. mp 76—77 °C. H-NMR (CDCl3) d: 0.88 (3H, t,

838
Vol. 49, No. 7
C33H48N2O4: C, 73.84; H, 9.01; N, 5.22. Found: C, 74.00; H, 8.93; N, 5.12.
N-(4-Benzyloxy-3,5-dimethoxycinnamoyl)-N؅-(3,4-dimethylphenyl)-
piperazine (20) Compound 10b (7.00 g, 17.7 mmol) was dissolved in
DMF (700 ml) and then sodium hydride (0.92 g, 23.0 mmol) was very care-
fully added to this solution. Benzyl chloride (2.90 g, 23.0 mmol) was added
to this mixture portionwise followed by stirring at room temperature for
96 h. The reaction mixture was poured into water (5 l) and the resulting pre-
cipitate was collected by filtration. The precipitate was purified by silica gel
column chromatography (CHCl3) and recrystallized from EtOH to give 20
as pale yellow crystals (7.50 g, yield 87.1%). mp 99—100 °C. 1H-NMR
(CDCl3) d: 2.19 (3H, s), 2.23 (3H, s), 3.14 (4H, m), 3.82 (4H, br s), 3.84
(6H, s), 5.04 (2H, s), 6.68 (1H, dd, Jϭ8.0, 2.4 Hz), 6.74 (2H, s), 6.75 (1H, d,
Jϭ2.4 Hz), 6.80 (1H, d, Jϭ15.4 Hz), 7.04 (1H, d, Jϭ8.0 Hz), 7.22—7.50
(5H, m), 7.61 (1H, d, Jϭ15.4 Hz). IR (KBr) cmϪ1: 1644, 1334, 1272. MS
m/z: 486 (Mϩ), 395, 190, 161. Anal. Calcd for C30H34N2O4: C, 74.05; H,
7.04; N, 5.76. Found: C, 73.71; H, 7.27; N, 5.56.
Jϭ6.7 Hz), 1.22—1.48 (14H, m), 1.71—1.79 (2H, m), 2.32 (3H, s), 3.19
(4H, br s), 3.85 (4H, br s), 3.87 (6H, s), 4.00 (2H, t, Jϭ6.8 Hz), 6.70—6.77
(5H, m), 6.81 (1H, d, Jϭ15.4 Hz), 7.16 (1H, t, Jϭ7.7 Hz), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2989, 1642, 1240. MS m/z: 522 (Mϩ), 347,
208, 147. Anal. Calcd for C32H46N2O4: C, 73.53; H, 8.87; N, 5.36. Found: C,
73.29; H, 8.85; N, 5.53.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(4-methylphenyl)piper-
1
azine (33):Yield 6.7%. mp 108—110 °C. H-NMR (CDCl3) d: 0.88 (3H, t,
Jϭ6.8 Hz), 1.22—1.48 (14H, m), 1.72—1.78 (2H, m), 2.27 (3H, s), 3.15
(4H, br s), 3.84 (4H, br s), 3.90 (6H, s), 3.99 (2H, t, Jϭ6.8 Hz), 6.75 (2H, s),
6.80 (1H, d, Jϭ15.4 Hz), 6.82 (2H, d, Jϭ8.2 Hz), 7.09 (2H, d, Jϭ8.2 Hz),
7.62 (1H, d, Jϭ15.4 Hz). IR (KBr) cmϪ1: 2917, 1642, 1238. MS m/z: 522
(Mϩ), 347, 208, 147. Anal. Calcd for C32H46N2O4: C, 73.53; H, 8.87; N,
5.36. Found: C, 73.56; H, 8.96; N, 5.24.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(4-tert-butylphenyl)piper-
1
azine (34): Yield 2.0%. mp 122—123 °C. H-NMR (CDCl3) d: 0.88 (3H, t,
The following compounds 42—46 were prepared in a manner similar to
that described for 20 from 10b and the appropriate halide.
Jϭ6.8 Hz), 1.23—1.48 (23H, m), 1.72—1.78 (2H, m), 3.19 (4H, m), 3.84
(4H, br s), 3.88 (6H, s), 3.99 (2H, t, Jϭ6.8 Hz), 6.75 (2H, s), 6.80 (1H, d,
Jϭ15.4 Hz), 6.86—6.91 (2H, m), 7.28—7.34 (2H, m,), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2950, 1642, 1229. MS m/z: 564 (Mϩ), 347,
208, 189. Anal. Calcd for C35H52N2O4: C, 74.43; H, 9.28; N, 4.96. Found: C,
74.29; H, 9.41; N, 4.93.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(3,4-dimethylphenyl)-
1
piperazine (42): Yield 78.8 %. mp 100—101 °C. H-NMR (CDCl3) d: 0.88
(3H, t, Jϭ6.7 Hz), 1.22—1.48 (14H, m), 1.70—1.78 (2H, m), 2.19 (3H, s),
2.24 (3H, s), 3.15 (4H, m), 3.85 (4H, br s), 3.87 (6H, s), 3.99 (2H, t,
Jϭ6.8 Hz), 6.69 (1H, dd, Jϭ8.0, 2.4 Hz), 6.75 (2H, s), 6.77 (1H, d,
Jϭ2.4 Hz), 6.81 (1H, d, Jϭ15.4 Hz), 7.04 (1H, d, Jϭ8.0 Hz), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2917, 1642, 1240. MS m/z: 536 (Mϩ), 347,
208, 161. Anal. Calcd for C33H48N2O4: C, 73.84; H, 9.01; N, 5.22. Found: C,
73.75; H, 8.98; N, 5.45.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(4-methoxyphenyl)piper-
azine (35): Yield 17.8%. mp 113—114 °C. 1H-NMR (CDCl3) d: 0.88 (3H, t,
Jϭ6.8 Hz), 1.22—1.48 (14H, m), 1.75 (2H, m), 3.08 (4H, br s), 3.76 (3H, s),
3.84 (4H, br s), 3.87 (6H, s), 3.99 (2H, t, Jϭ6.8 Hz), 6.76 (2H, s), 6.80—
6.93 (5H, m), 7.62 (1H, d, Jϭ15.4 Hz). IR (KBr) cmϪ1: 2949, 1643, 1223.
MS m/z: 538 (Mϩ), 347, 208, 163. Anal. Calcd for C32H46N2O5: C, 71.34; H,
8.61; N, 5.20. Found: C, 71.29; H, 8.57; N, 5.11.
N-(3,5-Dimethoxy-4-n-hexyloxycinnamoyl)-NЈ-(3,4-dimethylphenyl)-
piperazine (43): Yield 48.5%. mp 92—93 °C. 1H-NMR (CDCl3) d: 0.90
(3H, t, Jϭ6.8 Hz), 1.30—1.50 (6H, m), 1.72—1.80 (2H, m), 2.19 (3H, s),
2.24 (3H, s), 3.16 (4H, m), 3.86 (4H, br s), 3.88 (6H, s), 4.00 (2H, t,
Jϭ6.8 Hz), 6.70 (1H, dd, Jϭ8.0, 2.4 Hz), 6.75 (2H, s), 6.77 (1H, d,
Jϭ2.4 Hz), 6.80 (1H, d, Jϭ15.4 Hz), 7.04 (1H, d, Jϭ8.0 Hz), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2918, 1642, 1246. MS m/z: 480 (Mϩ), 291,
207, 160. Anal. Calcd for C29H40N2O4: C, 72.47; H, 8.39; N, 5.83. Found: C,
72.45; H, 8.46; N, 5.80.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(4-fluorophenyl)piper-
1
azine (36): Yield 40.2%. mp 65—66 °C. H-NMR (CDCl3) d: 0.88 (3H, t,
Jϭ6.8 Hz), 1.21—1.49 (14H, m), 1.72—1.79 (2H, m), 3.12 (4H, m), 3.84
(4H, br s), 3.87 (6H, s), 4.00 (2H, t, Jϭ6.8 Hz), 6.76 (2H, s), 6.81 (1H, d,
Jϭ15.4 Hz), 6.85—7.01 (4H, m), 7.62 (1H, d, Jϭ15.4 Hz). IR (KBr) cmϪ1
:
2914, 1641, 1236. MS m/z: 526 (Mϩ), 347, 208, 151. Anal. Calcd for
C31H43FN2O4: C, 70.69; H, 8.23; N, 3.61. Found: C, 70.70; H, 8.36; N, 3.63.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(4-nitrophenyl)piperazine
(37): Yield 44.8%. mp 117—119 °C. 1H-NMR (CDCl3) d: 0.88 (3H, t,
Jϭ6.8 Hz), 1.22—1.48 (14H, m), 1.72—1.80 (2H, m), 3.52 (4H, br s), 3.88
(6H, s), 3.91 (4H, br s), 4.00 (2H, t, Jϭ6.7 Hz), 6.76—6.85 (5H, m), 7.65
(1H, d, Jϭ15.2 Hz), 8.08 (2H, d, Jϭ9.2 Hz). IR (KBr) cmϪ1: 2916, 1643,
1599, 1246. MS m/z: 553 (Mϩ), 347, 208, 178. Anal. Calcd for C31H43N3O6:
C, 67.25; H, 7.83; N, 7.59. Found: C, 67.29; H, 7.95; N, 7.55.
N-(3,5-Dimethoxy-4-n-heptyloxycinnamoyl)-NЈ-(3,4-dimethylphenyl)-
1
piperazine (44): Yield 46.0%. mp 102—104 °C. H-NMR (CDCl3) d: 0.89
(3H, t, Jϭ6.5 Hz), 1.26—1.48 (8H, m), 1.72—1.80 (2H, m), 2.19 (3H, s),
2.24 (3H, s), 3.16 (4H, m), 3.85 (4H, br s), 3.88 (6H, s), 3.99 (2H, t,
Jϭ6.7 Hz), 6.70 (1H, dd, Jϭ8.0, 2.4 Hz), 6.75 (2H, s), 6.77 (1H, d,
Jϭ2.4 Hz), 6.80 (1H, d, Jϭ15.4 Hz), 7.04 (1H, d, Jϭ8.0 Hz), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2916, 1641, 1245. MS m/z: 494 (Mϩ), 305,
208, 161. Anal. Calcd for C30H42N2O4: C, 72.84; H, 8.56; N, 5.66. Found: C,
72.76; H, 8.63; N, 5.63.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(2,3-dimethylphenyl)-
piperazine (38): Yield 30.4%. mp 98—100 °C. 1H-NMR (CDCl3) d: 0.88
(3H, t, Jϭ6.7 Hz), 1.22—1.48 (14H, m), 1.72—1.78 (2H, m), 2.26 (3H, s),
2.28 (3H, s), 2.91 (4H, br s), 3.82 (4H, br s), 3.87 (6H, s), 3.99 (2H, t,
Jϭ6.84 Hz), 6.75 (2H, s), 6.82 (1H, d, Jϭ15.4 Hz), 6.87 (1H, d, Jϭ7.8 Hz),
6.93 (1H, d, Jϭ7.8 Hz), 7.08 (1H, t, Jϭ7.7 Hz), 7.62 (1H, d, Jϭ15.4 Hz). IR
(KBr) cmϪ1: 2914, 1644, 1205. MS m/z: 536 (Mϩ), 347, 208, 161. Anal.
Calcd for C33H48N2O4: C, 73.84; H, 9.01; N, 5.22. Found: C, 73.59; H, 9.30;
N, 5.24.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(2,5-dimethylphenyl)-
piperazine (39): Yield 29.9%. mp 66—67 °C. 1H-NMR (CDCl3) d: 0.88
(3H, t, Jϭ6.8 Hz), 1.22 (14H, m), 1.72—1.78 (2H, m), 2.29 (6H, s), 2.92
(4H, br s), 3.83 (4H, br s), 3.87 (6H, s), 3.99 (2H, t, Jϭ6.8 Hz), 6.76 (2H, s),
6.78—6.86 (3H, m), 7.07 (1H, d, Jϭ15.4 Hz), 7.62 (1H, d, Jϭ15.4 Hz). IR
(KBr) cmϪ1: 2988, 1646, 1237. MS m/z: 536 (Mϩ), 347, 208, 161. Anal.
Calcd for C33H48N2O4: C, 73.84; H, 9.01; N, 5.22. Found: C, 73.65; H, 8.96;
N, 5.49.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(2,6-dimethylphenyl)-
piperazine (40): Yield 27.6%. mp 65—67 °C. 1H-NMR (CDCl3) d: 0.88
(3H, t, Jϭ6.8 Hz), 1.23—1.48 (14H, m), 1.72—1.78 (2H, m), 2.32 (6H, s),
3.13 (4H, br s), 3.85 (4H, br s), 3.87 (6H, s), 3.99 (2H, t, Jϭ6.8 Hz), 6.76
(2H, s), 6.83 (1H, d, Jϭ15.4 Hz), 6.94—7.02 (3H, m), 7.63 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2913, 1636, 1243. MS m/z: 536 (Mϩ), 347,
208, 161. Anal. Calcd for C33H48N2O4: C, 73.84; H, 9.01; N, 5.22. Found: C,
73.84; H, 9.23; N, 5.03.
N-(4-n-Decyloxy-3,5-dimethoxycinnamoyl)-NЈ-(3,5-dimethylphenyl)-
piperazine (41): Yield 36.8%. mp 79—80 °C. 1H-NMR (CDCl3) d: 0.88
(3H, t, Jϭ7.0 Hz), 1.22—1.48 (14H, m), 1.72—1.78 (2H, m), 2.29 (6H, s),
3.20 (4H, m), 3.84 (4H, br s), 3.88 (6H, s), 4.00 (2H, t, Jϭ6.8 Hz), 6.57 (3H,
s), 6.75 (2H, s), 6.80 (1H, d, Jϭ15.4 Hz), 7.62 (1H, d, Jϭ15.4 Hz). IR (KBr)
cmϪ1: 2949, 1642, 1230. MS m/z: 536 (Mϩ), 347, 208, 161. Anal. Calcd for
N-(3,5-Dimethoxy-4-n-octyloxycinnamoyl)-NЈ-(3,4-dimethylphenyl)-
1
piperazine (45): Yield 49.5%. mp 104—106 °C. H-NMR (CDCl3) d: 0.88
(3H, t, Jϭ6.7 Hz), 1.28—1.50 (10H, m), 1.72—1.80 (2H, m), 2.19 (3H, s),
2.24 (3H, s), 3.16 (4H, t, Jϭ5.0 Hz), 3.86 (4H, br s), 3.88 (6H, s), 3.99 (2H,
t, Jϭ6.7 Hz), 6.70 (1H, dd, Jϭ8.0, 2.4 Hz), 6.75 (2H, s), 6.77 (1H, d,
Jϭ2.4 Hz), 6.79 (1H, d, Jϭ15.4 Hz), 7.05 (1H, d, Jϭ8.0 Hz), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2916, 1641, 1245. MS m/z: 508 (Mϩ), 319,
208, 161. Anal. Calcd for C31H44N2O4: C, 73.19; H, 8.72; N, 5.51. Found: C,
73.18; H, 8.81; N, 5.46.
N-(3,5-Dimethoxy-4-n-nonyloxycinnamoyl)-NЈ-(3,4-dimethylphenyl)-
piperazine (46): Yield 41.4%. mp 98—100 °C. 1H-NMR (CDCl3) d: 0.88
(3H, t, Jϭ6.8 Hz), 1.23—1.48 (12H, m), 1.70—1.78 (2H, m), 2.20 (3H, s),
2.24 (3H, s), 3.17 (4H, m), 3.86 (4H, br s), 3.88 (6H, s), 3.99 (2H, t,
Jϭ6.8 Hz), 6.70 (1H, dd, Jϭ8.0, 2.4 Hz), 6.75 (2H, s), 6.77 (1H, d,
Jϭ2.4 Hz), 6.79 (1H, d, Jϭ15.4 Hz), 7.05 (1H, d, Jϭ8.0 Hz), 7.62 (1H, d,
Jϭ15.4 Hz). IR (KBr) cmϪ1: 2917, 1641, 1245. MS m/z: 522 (Mϩ), 333,
208, 161. Anal. Calcd for C32H46N2O4: C, 73.53; H, 8.87; N, 5.36. Found: C,
73.50; H, 8.90; N, 5.32.
Assay of ACAT Activity Microsome fractions were prepared from liv-
ers of male Sprague–Dawley rats given a normal diet (F2: Funabashi Farmer
Co., Ltd., Chiba, Japan). Microsome fractions were also prepared from cell
lines including Caco2 (Riken Cell Bank No. RCB0988; human colon adeno-
carcinoma) that were cultured for 14 d after confluence and from HepG2
(Riken Cell Bank No. RCB0459; human hepatocarcinoma). Microsomes
were prepared according to the method described by Field and Salome.45)
ACAT activity was measured using [1-14C]oleoyl-CoA as a substrate accord-
ing to the method described by Helgerud et al.46)
Plasma Cholesterol Levels Male Sprague–Dawley rats (5-weeks-old)
were given a diet, F2 (Funabashi Farmer Co., Ltd., Chiba, Japan), containing

July 2001
839
0.5% cholic acid, 10% sucrose, 10% coconut oil, 0.005 % 6-propyl-2- 24) Sliskovic D. R., Trivedi K. K., Curr. Med. Chem., 1, 204—225 (1994).
thiouracil and 0.005 or 0.1% test compounds for four days. For the three fol- 25) Roark W. H., Roth B. D., Exp. Opin. Invest. Drugs, 3, 1143—1152
lowing days, the rats were given the F2 diet, which contained 1.5% choles-
(1994).
terol, 0.5% cholic acid, 10% sucrose, 10% coconut oil, 0.005% 6-propyl-2- 26) Lee H. T., Picard J. A., Exp. Opin. Ther. Patents, 5, 397—416 (1995).
thiouracil and 0.005 or 0.1% test compounds. Normal group rats were fed 27) Trivedi B. K., Purchase T. S., Holmes A., Augelli-Szafran C. E., Es-
the F2 diet containing 0.5% cholic acid, 10% sucrose, 10% coconut oil and
senburg A. D., Hamelehle K. L., Stanfield R. L., Bousley R. F., Krause
0.005% 6-propyl-2-thiouracil. The rats were bled via the abdominal aorta
B. R., J. Med. Chem., 37, 1652—1659 (1994).
while under pentobarbital anesthesia. The total plasma cholesterol concen- 28) Dominick M. A., Bobrowski W. A., MacDonald J. R., Gough A. W.,
tration was determined enzymatically using a commercially available kit
(Determiner TC555, Kyowa Medex Co., Tokyo, Japan).
Toxicol. Pathol., 21, 54—62 (1993).
29) Dominick M. A., McGuire E. J., Reindel J. F., Bobrowski W. F., Bocan
T. M. A., Gough A. W., Fundam. Appl. Toxicol., 20, 217—224 (1993).
30) Reindel J. F., Dominick M. A., Bocan T. M. A., Gough A. W.,
McGuire E. J., Toxicol Pathol., 22, 510—518 (1994).
31) Smith C., Ashton M. J., Bush R. C., Facchini V., Harris N. V., Hart T.
W., Jordan R., McKenzie R., Riddell D., Bioorg. Med. Chem. Lett., 6,
47—50 (1996).
References
1) Kannel W. B., Castelli W. P., Gordon T., McNamara P. M., Ann. Intern.
Med., 74, 1—12 (1971).
2) Martin M. J., Hulley S. B., Browner W. S., Kuller L. H., Wentworth D.,
Lancet, 2, 933—936 (1986).
3) Badimon J. J., Fuster V., Chesebro J. H., Badimon L., Circulation, 87
(Suppl. II), 3—16 (1993).
4) McCarthy P. A., Med. Res. Rev., 13, 139—159 (1993).
5) Brown G., Albers J. J., Fischer L. D., Schaefer S. M., Lin J. T., Kaplan
C., Zhao X. Q., Bisson B. D., Fitzpatrick V. F., Dodge H. T., N. Engl. J.
Med., 323, 1289—1298 (1990).
32) White A. D., Creswell M. W., Chucholowski A. W., Blankey C. J., Wil-
son M. W., Bousley R. F., Essenburg A. D., Hamelehle K. L., Krause
B. R., Stanfield R. L., Dominick M. A., Neub M., J. Med. Chem., 39,
4382—4395 (1996).
33) Sliskovic D. R., Picard J. A., O’Brien P. M., Liao P., Roark W. H., Roth
B. D., Anderson M. A., Mueller S. B., Bocan T. M. A., Bousley R. F.,
Hamelehle K. L., Homan R., Reindel J. F., Stanfield R. L., Turluck D.,
Krause B. R., J. Med. Chem., 41, 682—690 (1998).
6) Spector A. A., Mathur S. N., Kaduce T. L., Prog. Lipid Res., 18, 31—
53 (1979).
7) Suckling K. E., Stange E. F., J. Lipid Res., 26, 647—671 (1985).
8) Krause B. R., Anderson M., Bisgaier C. L., Bocan T., Bousley R., De-
Hart P., Essenburg A., Hamelehle K., Homan R., Kieft K., McNally
W., Stanfield R., Newton R. S., J. Lipid Res., 34, 279—294 (1993).
9) Largis E. E., Wang C. H., DeVries V. G., Schaffer S. A., J. Lipid Res.,
30, 681—690 (1989).
10) Carr T. P., Rudel L. L., Arteriosclerosis, 10, 823a (1990).
11) Carr T. P., Parks J. S., Rudel L. L., Arterioscler. Thromb., 12, 1274—
1283 (1992).
34) Warner G. J., Stoudt G., Bamberger M., Johnson W. J., Rothblat G. H.,
J. Biol. Chem., 270, 5772—5778 (1995).
35) Matsuo M., Hashimoto M., Suzuki J., Iwanami K., Tomoi M., Shimo-
mura K., Toxicol. Appl. Pharmacol., 140, 387—392.
36) Tanaka A., Terasawa T., Hagihara H., Sakuma Y., Ishibe N., Sawada
M., Takasugi H., Tanaka H., J. Med. Chem., 41, 2390—2410 (1998).
37) Tanaka A., Terasawa T., Hagihara H., Ishibe N., Sawada M., Sakuma,
Y., Hashimoto M., Takasugi H., Tanaka H., J. Med. Chem., 41, 4408—
4420 (1998).
12) Burrier R. E., Deren S., McGregor D. G., Hoos L. M., Smith A. A., 38) Reindel J. F., Dominick M. A., Krause B. R., Toxicol. Pathol., 20,
Davis H. R., Jr., Biochem. Pharmacol., 47, 1545—1551 (1994).
642—643 (1992).
13) Brown M. S., Goldstein J. L., Annu. Rev. Biochem., 52, 223—261
39) Joyce C., Skinner K., Anderson R. A., Rudel L. L., Curr. Opin.
Lipidol., 10, 89—95 (1999).
(1983).
14) Gillies P. J., Robinson C. S., Rathgeb K. A., Atherosclerosis, 83, 177—
40) Miyazaki A., Sakashita N., Lee O., Takahashi K., Horiuchi S., Haka-
mata H., Morganelli P. M., Chang C. C., Chang T. Y., Arterioscler.
Thromb. Vasc. Biol., 18, 1568—1574 (1998).
185 (1990).
15) Sliskovic D. R., White A. D., Trends Pharmacol. Sci., 12, 194—199
(1991).
41) Cases S., Novak S., Zheng Y. M., Myers H. M., Lear S. R., Sande E.,
Welch C. B., Lusis A. J., Spencer T. A., Krause B. R., Erickson S. K.,
Farese R. V., Jr., J. Biol. Chem., 273, 26755—26764 (1998).
42) Chang C. C., Sakashita N., Ornvold K., Lee O., Chang E. T., Dong R.,
Lin S., Lee C. Y., Strom S., Kashyap R., Fung J., Farese J., Patoiseau J.
F., Delhon A., Chang T. Y., J. Biol. Chem., 275, 28083—28092 (2000).
16) Itokawa H., Aiyama R., Ikuta A., Phytochemistry, 21, 241—243
(1982).
17) Itokawa H., Aiyama R., Ikuta A., Chem. Pharm. Bull., 31, 2491—
2496 (1983).
18) Roth B. D., Blankley C. J., Hoefle M. L., Holmes A., Roark W. H.,
Trivedi B. K., Essenburg A. D., Kieft K. A., Krause B. R., Stanfield R. 43) Matsuda H., Hakamata H., Kawasaki T., Sakashita N., Miyazaki A.,
L., J. Med. Chem., 35, 1609—1617 (1992).
Takahashi K., Shichiri M., Horiuchi S., Biochim. Biophys. Acta, 1391,
19) Field F. J., Albright E., Mathur S., Lipids, 26, 1—8 (1991).
193—203 (1998).
20) Sakuma Y., Hagihara H., Ohne K., Nagayoshi A., Mutoh S., Ito Y., 44) Mishani E., Dence C. S., McCarthy T. J., Welch M. J., Tetrahedron
Notsu Y., Okuhara M., Jpn. J. Pharmacol., 70, 35—41 (1996) Lett., 37, 319—322 (1996).
21) Ito T., Matsuda K., Iwaoka K., Japan patent provisional publication, 45) Field F. J., Salome R. G., Biochim. Biophys. Acta, 712, 557—570
No. 170727/93, (1993).
(1982).
22) Picard J. A., Curr. Opin. Ther. Pat., 3, 151—160 (1993).
23) Matsuda K., Med. Res. Rev., 14, 271—305 (1994).
46) Helgerud P., Saarem K., Norum K. R., J. Lipid Res., 22, 271—277
(1981).