A novel class of antihyperlipidemic agents with low density lipoprotein receptor up-regulation via the adaptor protein autosomal recessive hypercholesterolemia

Article abstract of DOI:10.1021/jm901909p

We have previously reported compound 2 as a inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) and up-regulator of the low density lipoprotein receptor (LDL-R) expression. In this study we focused on compound 2, a unique LDL-R up-regulator,

Full text of DOI:10.1021/jm901909p

3284 J. Med. Chem. 2010, 53, 3284–3295
DOI: 10.1021/jm901909p
A Novel Class of Antihyperlipidemic Agents with Low Density Lipoprotein Receptor
Up-Regulation via the Adaptor Protein Autosomal Recessive Hypercholesterolemia
Shigehiro Asano,*,† Hitoshi Ban,*,‡ Norie Tsuboya,‡ Shinsaku Uno,‡ Kouichi Kino,† Katsuhisa Ioriya,†
Masafumi Kitano,† and Yoshihide Ueno‡
†Drug Research Division, Dainippon Sumitomo Pharma Co., Ltd., 3-1-98 Kasugade Naka, Konohana-ku, Osaka 554-0022, Japan, and
‡Drug Research Division, Dainippon Sumitomo Pharma Co., Ltd., 33-94 Enoki, Suita, Osaka 564-0053, Japan
Received December 23, 2009
We have previously reported compound 2 as a inhibitor of acyl-coenzyme A:cholesterol O-acyltrans-
ferase (ACAT) and up-regulator of the low density lipoprotein receptor (LDL-R) expression. In this
study we focused on compound 2, a unique LDL-R up-regulator, and describe the discovery of a novel
class of up-regulators of LDL-R. Replacement the methylene urea linker in compound 2 with an
acylsulfonamide linker kept a potent LDL-R up-regulatory activity, and subsequent optimization work
gave compound 39 as a highly potent LDL-R up-regulator (39; EC25 = 0.047 μM). Compound 39
showed no ACAT inhibitory activity even at 1 μM. The sodium salts of compound 39 reduced plasma
total and LDL cholesterol levels in a dose-dependent manner in an experimental animal model of
hyperlipidemia. Moreover, we revealed in this study using RNA interference that autosomal recessive
hypercholesterolemia (ARH), an adaptor protein of LDL-R, is essential for compound 39 up-regulation
of LDL-R expression.
Introduction
Coronary heart disease (CHDa) is one of the leading causes
of death in industrialized nations, and its burden on health-
care resources is continuously increasing worldwide.1 Epide-
miological evidence indicates that hypercholesterolemia is an
important risk factor for CHD;2 consequently, significant
efforts have been undertaken to mitigate this condition.
Low density lipoprotein (LDL) is a vehicle lipoprotein that
transports cholesterol from the liver to peripheral tissues, and
can be retained there by arterial proteoglycans triggering the
formation of plaques. Increased LDL levels in the blood are
known to be associated with a variety of cardiovascular
conditions, including atherosclerosis, stroke, and peripheral
vascular diseases. Accordingly, evidence has shown that low-
ering total cholesterol (TC) and LDL cholesterol (LDL-C)
levels can reduce the incidence of CHD.3 It is known that the
Figure 1
LDL receptor (LDL-R) plays a central role in LDL incor-
in 1982,5 its human cDNA cloned shortly thereafter,6 and its
poration. The existence of this receptor, which is a ubiquitous
gene isolated in 1985.7
cell surface glycoprotein of 839 amino acids, was demon-
We have previously reported a novel antihyperlipidemic agent
strated in 1974 by Goldstein and Brown.4 In subsequent
1 (SMP-797)8 and described 1,4-diarylpiperidine-4-methylurea
research, LDL-R was purified from bovine adrenal glands
derivative 29 as its back-up compounds (Figure 1). Compounds
1 and 2 possess dual activity consisting of inhibition of choles-
terol O-acyltransferase (ACAT) and up-regulation of LDL-R.
*To whom correspondence should beaddressed. For S.A. - Telephone:
þ81-6-6466-5185. Fax: þ81-6-6466-5287. E-mail: shigehiro-asano@
Although inhibition of ACAT is believed to reduce the absorp-
tion of dietary cholesterol in the small intestine,10,11 thereby
ds-pharma.co.jp. For H. B. - Telephone: þ81-6-6337-5815. Fax: þ81-6-
lowering serum lipid level,12 and to prevent the progression of
6337-6010. E-mail: hitoshi-ban@ds-pharma.co.jp.
a Abbreviations: ACAT, acyl-coenzyme A:cholesterol O-acyltransferase;
atherosclerotic lesions,13 numerous clinical trial studies of potent
CHD, coronary heart disease; TC, total cholesterol; HMG-CoA, 3-hydro-
ACAT inhibitors have shown poor efficacy,14 with no ACAT
inhibitor in clinical use so far.15 Safety concerns have also been
reported with some ACAT inhibitors.16
xy-3-methylgultryl coenzyme A; BINAP, 2,20-bis(diphenylphosphino)-1,10-
binaphthyl: TBS, tert-butyl dimethylsilyl; SAR, structure-activity relation-
ships; DiI-LDL, 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine
perchlorate-labeled LDL; Cmax, maximum drug concentration; AUC, area
under the curve; SEM, standard error of the mean; siRNA, short-interfering
RNA; HRMS, high-resolution mass spectrometer.
On the other hand, numerous clinical trial studies of
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase
r
pubs.acs.org/jmc
Published on Web 03/31/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3285
Scheme 1. Synthesis of Compounds 9;17a
a Reagents and Conditions: (a) 6 N KOH solution, DMSO; (b) concentrated hydrochloric acid solution; (c) (1) (COCl)2, N,N-dimethylformamide,
CH2Cl2; (2) 19, NaH, THF or 2,6-diisopropylniline or 26-31, NEt3, CH2Cl2.
Scheme 2. Synthesis of Sulfamide Compound 19 and Sulfamate
Compounds 26-31a
inhibitors, known as statins, have shown strong cholesterol-
lowering effects; therefore, hypercholesterolemia is presently
treated by statins. Statins block the rate-limiting step of choles-
terol biosynthesis, up-regulate the LDL-R, and clear LDL
particles from the blood.17 In previous reports, we have shown
that up-regulation of LDL-R by 1 or its back-up compounds is
independent of the ACAT inhibitory activity of these compounds
and that, unlike statins, 1 and its back-up compounds have no
effect on cholesterol biosynthesis.
On the basis of this background information, we believe
that modification of compound 2 can lead to the discovery of
new compounds that possess LDL-R up-regulatory activity
without ACAT inhibition. Such compounds are expected to
be novel antihyperlipidemic agents without the side effects
associated with ACAT inhibition. In this paper, we report the
identification and preliminary optimization of a series of
piperidine-based compounds as potent LDL-R up-regulators
without ACAT inhibition.
Chemistry
a Reagents and Conditions: (a) NH2SO2Cl, N-methyl-2-pyrrolidi-
none; (b) (1) ClSO2NCO, heptane; (2) H2O.
The compounds synthesized in this study and their syn-
thetic methods are shown in Schemes 1-4. The nitriles 3 and
49 were hydrolyzed with aqueous 6 N KOH solution at 130 °C
in DMSO to give the carboxamides 5 and 6, respectively. The
carboxamides, thus obtained, were converted to the corres-
ponding carboxylic acids (7 and 8) by acidic hydrolysis with
concentrated hydrochloric acid solution at 90 °C. Direct
hydrolysis of the nitriles to the carboxylic acids in concen-
trated hydrochloric acid solution did not proceed quickly,
leading to generation of byproduct. Conversion of the car-
boxylic acids 7 and 8 to the corresponding acylchloride using
oxalyl chloride, followed by condensation with the appropri-
ate nucleophile, such as 2,6-diisopropylaniline, 19, or 26-31,
afforded the desired amide 9, acylsulfamide 10, or acylsulfo-
namides 11-17 (Scheme 1).
Preparation of the sulfamide compound 19 and the sulfa-
mate compounds 26-31 used in the above condensation
reaction are illustrated in Scheme 2. The 2,6-diisopropyl-
phenylsulfamide 19 was prepared by condensation of 2,6-
diisopropylaniline (18) with sulfamoyl chloride,18 which was
easily synthesized from chlorosulfonyl isocyanate and for-
mic acid. Phenyl sulfamates 26-31 with mono or dialkyl
substituents on the phenyl ring were readily synthesized from
the commercially available phenols by a method described in
the literature.19
Compounds 32-44 containing various substituents on the
nitrogen in the piperidine were prepared according to the
method described in Scheme 3. Preparation of compound 32
as a key intermediate was carried out by deprotection of
compound 17 over 20% palladium hydroxide on carbon under
medium hydrogen atmosphere (0.4 MPa). Compound 32 was
converted to the N-methylpiperidine 33 by reductive amina-
tion using formaldehyde and sodium cyanoborohydride. Acy-
lation of 32 with acid chlorides in pyridine gave the N-acylated
compounds 34 and 35. Coupling reaction of 32 with aryl
bromides, such as bromobenzene, 2-bromo-3-methoxypyri-
dine, or 2-bromobenzothiazole under microwave irradiation in
the presence of tris(dibenzylidene acetone)dipalladium(0) and
(S)-(-)-BINAP gave the corresponding N-arylpiperidine
compounds 36-38. Nucleophilic addition of 32 to appropriate
2-chloropyrimidines gave 39-42 in good yield. Selective bro-
mination of 39 with N-bromosuccinimide in THF produced 43

3286 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Scheme 3. Synthesis of Compounds 32-44a
Asano et al.
a Reagents and Conditions: (a) 20% Pd(OH)2/C, H2, AcOH, N,N-dimethylformamide; (b) NaBH3CN, AcOH, 37% HCHO aq, methanol;
(c) R1COCl, 4-(N,N-dimethylamino)pyridine, pyridine; (d) R1Br, sodium tert-butoxide, Pd2(dba)3, (S)-(-)-BINAP, toluene; (e) R1Cl, K2CO3,
N,N-dimethylformamide; (f) N-bromosuccinimide, THF; (g) (1) sodium tert-butoxide, diethylether; (2) CH3I, N,N-dimethylformamide.
Scheme 4. Synthesis of Compounds 46-49a
a Reagents and Conditions: (a) BBr3, CH2Cl2; (b) C2H5I or C3H7I, K2CO3, N,N-dimethylformamide; (c) (1) Br(CH2)2OTBS or Br(CH2)3OTBS,
K2CO3, N,N-dimethylformamide, KI; (2) tetrabutylammonium fluoride, THF.
in excellent yield. Treatment of compound 39 with sodium tert-
butoxide in methanol, followed by N-alkylation of the formed
sodium salt gave compound 44. The structure of 44 was deter-
minded to be an N-alkyl compound by HMBC experiment and
MS/MS analysis (see Supporting Information).
removed by tetrabutylammonium fluoride (TBAF) in THF
at the final step. The structure of 46-49 was determined to be
O-alkyl compounds by HMBC experiment (see Supporting
Information).
As shown in Scheme 4, demethylation of compound 39
using boron tribromide in CH2Cl2 afforded the phenol deri-
vative 45. Alkylation of compound 45 with commercially
available alkyl halides provided various alkoxy derivatives
(46-49). In the case of compound 48 and 49, the tert-butyl
dimethylsilyl (TBS) moiety as the protective group was
Results and Discussion
As ACAT inhibitors generally contain 2,6-diisopropylphe-
nylurea as a common structure, we hypothesized that mod-
ification of this part can be effective in reducing ACAT
inhibitory activity. Based on this hypothesis, we changed the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3287
Table 1. Effects of Substitution in the Phenyl Group (C-part) and the Linker Part (L) on Biological Activity
compound
L
R2
LDL-R up-regulation EC25 (μM)a
ACAT inhibition IC50 (μM)b
2
Ntc
>10
>10
>10
3.88
4.07
0.69
>10
0.37
0.49
0.018
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
0.41
9
CONH
2,6-diiPr
2,6-diiPr
H
10
11
12
13
14
15
16
1
CONHSO2NH
CONHSO3
CONHSO3
CONHSO3
CONHSO3
CONHSO3
CONHSO3
2-Me
2-nPr
2-cPentyl
2,6-di-Me
2,6-diiPr
0.33
0.031
a Effects of compounds on DiI-LDL uptake in human HepG2 cells. The up-regulatory activity of 1 at 10 μM was assumed as EC100, EC25 value means
concentration at which LDL-R expression is up-regulated by 25% (n = 1). b Inhibitory activity for ACAT derived from rat macrophages (n = 1). c Not tested.
C-part and the linker part (L, Figure 1) of 2 to obtain potent
LDL-R up-regulators without ACAT inhibition. The biological
activityofcompounds1,2,and9-16aresummarizedinTable 1.
Replacement of the methylene urea linker in 2to an amide (9) or
an acylsulfamide (10) gave complete loss of LDL-R up-regula-
tory activity. On the other hand, this activity remained in the
acylsulfonamide 16 with an EC25 value comparable to that of 1.
Interestingly, LDL-R up-regulatory activity was enhanced by
introduction of sterically hindered substituents at the ortho-
position on the phenyl moiety in the C-part (compounds
11-16). As expected, ACAT inhibitory activity of compound
16 was about 20-fold weaker than that of compound 2 (2;
IC50 = 0.018 μM versus 16; IC50 = 0.33 μM). On the basis of
these findings, we could confirm that the mechanism of LDL-R
up-regulation is independent of ACAT inhibition. As we have
previously disclosed the structure-activity relationship (SAR)
of A-part (Figure 1) and shown that introduction of 2-methox-
yphenyl group in this part provides favorable results,9 our work
in this study was mainly focused on using an alkoxyphenyl or an
alkoxypyridyl as substituent. An aromatic group in the A-part is
essential for ACAT inhibition. In order to improve the LDL-R
up-regulation and to reduce ACAT inhibition of 16, we exam-
ined the SAR of the A-part in 16 using various substituents.
The results are summarized in Table 2. LDL-R up-regulatory
activity was diminished in compounds 32-35, but conserved in
compound 36, suggesting that the aryl substituent on the
nitrogen in part-A is important. The 2-(3-methoxy)pyridyl 37
showed a loss of LDL-R up-regulatory activity, and this result
was similar to the SAR information previously provided for
urea derivatives. Surprisingly, the 2-pyrimidyl 39 demonstrated
the most potent LDL-R up-regulatory activity with an EC25
value about 10-fold greater than that of 16 (16;EC25 = 0.37 μM
versus 39;EC25 = 0.047 μM). In addition, compound 39 did not
inhibit ACAT enzyme even at 1 μM, selectivity (LDL-R up-
regulation/ACAT inhibition) of 39 compared to compound 16.
The 2-benzothiazolyl 38 showed an LDL-R up-regulatory
activity comparable to that of 16. These findings suggest that
two hetero atoms in the aromatic ring might be essential for
LDL-R up-regulation. Next, we introduced several substituents
onto the pyrimidine ring. As shown in Table 2, compounds
40-43 gave diminished LDL-R up-regulatory activity, a finding
that could not be explained by electronic effect or substituent
size. Additionally, we examined the acylsulfonamide part of the
linker (L, Figure 1) and found that compound 44 with N-Me had
no LDL-R up-regulatory activity. These findings indicate that
the hydrogen atom in the linker is necessary to keep the activity.
In addition to the SAR of the A-part, we investigated in this
study the effect of an alkoxy substituent on the phenyl moiety in
the B-part. The results are summarized in Table 3. Compound
45 with a phenolic hydroxyl group showed no activity, and
replacement of the methoxy group with an ethoxy or propoxy
group (46 or 47), or a hydroxyalkoxy group (48 or 49) was also
unfavorable, giving decreased activity due to elongation of the
alkyl chain. These findings suggested that a methoxy group
could be the most suitable substituent at this position, a result
which does not agree with our previous work.9
Before examining cholesterol-lowering effect of the selected
compounds 16 and 39, we confirmed the oral bioavalability of
these compounds. As compound 16 oral bioavailability in
male hamsters was poor (maximum drug concentration
(Cmax) = 0.013 μg/mL at 10 mg/kg), hence we prepared the
hydrochlorideand sodium salt of this compound. Asshownin
Figure 2, both salts showed improved oral bioavailability
compared to the free base, in particular the sodium salt was
better than the hydrochloride as indicated by Cmax and area
under the curve (AUC)0-360 at 10 mg/kg (hydrochloride;
C
max = 0.30 μg/mL, AUC0-360 = 0.69 μg h/mL versus sodium
3
salt; Cmax = 0.42 μg/mL, AUC0-360 = 1.16 μg h/mL). We
3
therefore used the sodium salts of 16 and 39 for in vivo evaluation.
Comparing to compound 16, compound 39 has approximately
8-fold potent activity in terms of in vitro LDL-R up-regulation,
and 39 showed 3-10-fold potent lipid lowering effect. Addition-
ally, pharmacokinetics properties of compounds 16 and 39 were
similar, thus these results will be helpful for a better under-
standing of the relationship of a unique LDL-R up-regulatory
activity (in vitro) and cholesterol-lowering effect in plasma
(in vivo) (sodium salts of 39; Cmax = 0.29 μg/mL, AUC0-360
=
1.09 μg h/mL).
3
For further in vivo characterization, the cholesterol-lowering
effect of the sodium salts of 16 and 39 was examined in male
hamster fed a cholesterol-rich diet. The test-compounds were
orally given at 1, 3, 10, or 30 mg/kg once a day for 7 consecutive

3288 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Asano et al.
Table 2. Effects of Substitution in the Nitrogen of the Piperidine (A-part) on Biological Activity
LDL-R up-regulation
EC25, (μM)a
ACAT inhibition
IC50, (μM)b
solubility at pH = 7.4
(mg/mL)c
compound
R1
R3
2
Nte
>10
>10
>10
>10
1.52
0.37
>10
0.26
0.047
0.50
>10
>10
0.61
>10
0.49
0.018
>1.0
>1.0
>1.0
>1.0
>1.0
0.33
0.022
Nte
32
33
34
35
36
16
37
38
39
40
41
42
43
44
1
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Nte
Ac
Nte
Nte
0.18
benzoyl
phenyl
2-OMe-C6H4
0.010
Nte
2-(3-OMe)pyridyl
2-benzothiazolyl
2-pyrimidyl
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
0.031
0.003
0.23d
0.20
Nte
Nte
2-(4-Me)pyrimidyl
2-(4-CF3)pyrimidyl
2-(5-nPr)pyrimidyl
2-(5-Br)pyrimidyl
2-pyrimidyl
<0.0001
<0.0003
0.010
a Effects of compounds on DiI-LDL uptake in human HepG2 cells (n = 1). b Inhibitory activity for ACAT in rat macrophages (n = 1). c Solubility in
phosphate buffer solution at room temperature was determined by HPLC analysis. d Sodium salt. e Not tested.
Table 3. Effects of Substitution in the 4-Phenyl Moiety (B-part) on
that of 1, which possesses both ACAT inhibitory activity and
Biological Activity
LDL-R up-regulatory activity. These findings were encoura-
ging as they supported our hypothesis; i.e. compounds that can
up-regulate LDL-R without inhibiting ACAT may be effective
in lowering TC and LDL-C in vivo.
Finally, we confirmed that compound 39 even at a concen-
tration of 1 μM had no effect on cholesterol synthesis. This
concentration is much higher than that needed for LDL-R up-
regulation in HepG2 cells, where up-regulation of LDL-R
expression by acylsulfonamide compounds is not due to
transcriptional modulation of LDL-R gene. Hence, this me-
chanism of action is completely different from that of HMG-
CoA reductase inhibitors, which up-regulate LDL-R by indirect
action, resulting from cholesterol starvation. Recently, it has
been reported that LDL-R expression and activity are regu-
lated not only by the transcriptional modulation of LDL-R
gene but also by other mechanisms, such as stability of LDL-R
mRNA,20 LDL-R adaptor protein,21 and degradation of
LDL-R protein.22,23 Therefore, we speculate that acylsulfon-
amide compounds may regulate LDL-R expression by a mec-
hanism other than transcriptional regulation of LDL-R gene.
Identification of the molecular defect responsible for the
recessive form of hypercholesterolemia that clinically resem-
bles familial hypercholesterolemia has provided new insights
into LDL-R physiology. Autosomal recessive hypercholester-
olemia (ARH) is a rare Mendelian dyslipidemia characterized
by markedly elevated plasma LDL levels. ARH patients show
increased cell-surface LDL binding, and impaired LDL de-
gradation, consistent with a defect in LDL-R internalization.
Recently, the disorder was shown to be caused by mutation in
a phosphotyrosine binding domain protein, ARH, which is
required for internalization of LDL in the liver.24 M. Arca
et al. reported that transient loss of the adaptor protein, ARH,
by short-interfering RNA (siRNA) results in failure of LDL
endocytosis and almost complete absence of LDL/LDL-R
LDL-R up-regulation solubility at pH = 7.4
(mg/mL)b
compound
R4
EC25, (μM)a
45
39
46
47
48
49
1
H
Me
>10
0.047
3.1
0.20
0.23c
Et
nPr
0.069
>10
1.5
<0.0003
>0.20
>0.20
0.010
(CH2)2OH
(CH2)3OH
>10
0.49
a Effects of compounds on DiI-LDL uptake in human HepG2 cells
(n = 1). b Solubility in phosphate buffer solution at room temperature
was determined by HPLC analysis. c Sodium salts.
days based on cholesterol plasma levels reached in a prelimin-
ary in vivo pharmacokinetics study. Both compounds reduced
plasma TC and LDL-C levels in a dose-dependent manner
(Figure 3). Particularly, the lipids-lowering effect of compound
39 was approximately 10-fold more potent than that of com-
pound 16. In vitro experiments also supported this result.
Statistically significant TC and LDL-C lowering effects were
seen at a dose over 3 mg/kg/day for 39, and pharmacokinetics
data indicated that 39 plasma concentration could keep over
EC25 for several hours following administration of this com-
pound at by 10 mg/kg. These findings suggest that the EC25
value is sufficient potency for lipids-lowering efficacy. Interest-
ingly, cholesterol-lowering effect of 39 was almost the same as

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3289
Figure 2. (A) Serum concentration of free base, hydrochloride, and sodium salt of compound 16 after oral administration (10 or 30 mg/kg) to
male hamsters. (B) Serum concentration of sodium salt of compound 39 after oral administration (10 mg/kg) to male hamsters. Each point with
vertical bar represents the mean ( standard error of the mean (SEM) (n = 3).
Figure 3. (A) Effects of compound 16 (blue column), 39 (red column), and 1 (white column) on plasma TC levels in hamster fed a rich diet for
1 week. (B) Effects of compounds 16 (blue column), 39 (red column), and 1 (white column) on plasma LDL-C levels in hamster fed a rich diet for
1 week. All compounds were orally administered to hamster by gavage once a day for 1 week. Each column with vertical bar represents the
mean ( SEM (compound 16 and 39; n = 5, 1; n = 12). Significantly different from control using unpaired two-tailed t-test: * p < 0.05, ** p <
0.01, *** p < 0.001.
complex in the internal compartment of hepatocytes.25 Addi-
tionally, immunofluorescence analysis revealed that most LDL
resides on the cell surface in hepatocytes lacking ARH. These
findings suggest that ARH is required to promote internaliza-
tion of LDL/LDL-R complex into hepatocytes. In order to
clarify the mechanism of LDL-R up-regulation by compound
39, we evaluated the activity of 39 on ARH protein expression
in HepG2 cells. The expression of ARH was suppressed by
addition of ARH-specific siRNA. Under these conditions,
compound 39 did not up-regulate LDL-R even at a concentra-
tion of 1 μM, while under the same condition, Atrovastatin, an
HMG-CoA reductase inhibitor, up-regulated LDL-R. ARH
could therefore be the target molecule of compound 39 for
LDL-R up-regulation, indicating that the mechanism by which
compound 39 acts on LDL-R expression is different from that
of HMG-CoA reductase inhibitors.
endocytosis of LDL/LDL-R complex or recycling of LDL-R
by quick degradation of the LDL/LDL-R complex taken into
cells. As far as we know, no report has investigated such a
unique mechanism of antihyperlipidemic drugs. To reduce
plasma LDL, combination therapy with compound 39 and an
HMG-CoA reductase inhibitor may be useful, because the
mechanism of action for LDL-R up-regulation is different
between these two compounds.
Conclusion
Based on our previous work we tried in this study to find
compounds that possess LDL-R up-regulating activity with-
out ACAT inhibition. We started this approach by modifica-
tion of 1,4-diarylpiperidine-4-methylurea 2. Replacement of
the methyleneurea linker (2) with the acylsulfonamide (16)
was effective in keeping the up-regulatory activity for LDL-R
expression and reducing ACAT inhibitory activity. Particu-
larly, conversion of the 2-methoxyphenyl group (16), which is
essential for ACAT inhibition, into a 2-pyrimidyl group (39)
enhanced LDL-R up-regulatory activity and abolished
ACAT inhibitory activity. Additionally, the sodium salt of
Compound 39 not only up-regulated LDL-R expression in
HepG2 cells, but also increased degradation of [I125]-labeled
LDL cholesterol in a concentration-dependent manner (119%
@ 0.01 μM, 136% @ 0.1 μM, 140% @ 1 μM). Accordingly,
we speculate that compound 39 up-regulatory activity for
LDL-R, which is via ARH protein, is due to acceleration of

3290 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Asano et al.
the selected compounds 16 and 39 showed good oral pharma-
cokinetics properties in hamsters, and reduced plasma TC and
LDL-C levels in a dose-dependent manner in an experimental
animal model of hyperlipidemia. These results indicate that
LDL-R up-regulation, but not ACAT inhibition, is important
for plasma lipids reduction. Finally, we clarified the mecha-
nism of action of 39 toward LDL-R up-regulation using ARH-
specific RNA interference, and revealed that ARH, an adaptor
protein of LDL-R, is a potential target for LDL-R up-regula-
tion. The results of this study indicate that compound 39 with
its unique mechanism of action might replace 1 or 2 as a novel
antihyperlipidemic agent.
with 1 N sodium hydroxide solution (2ꢀ), and then the aqueous
layer was neutralized with hydrochloric acid solution. The mixture
was extracted with ethyl acetate (2ꢀ), washed by brine, and dried
over anhydrous magnesium sulfate. After filtration, the solvent was
removed in vacuo, and the residue was purified by silica-gel column
chromatography to give 7 (1.31 g, 77%) as a white solid. Mp
1
156-158 °C; H NMR (DMSO-d6, 300 MHz) δ 1.93 (2H, m),
2.49 (2H, m), 2.69 (2H, m), 3.32 (2H, m), 3.75 (3H, s), 3.76 (3H, s),
6.81-6.94 (6H, m), 7.00 (1H, d, J = 7.9 Hz), 7.28 (1H, dd, J = 8.1,
8.1 Hz), 12.57 (1H, s); IR (ATR) 1707, 1599, 1589, 1497 cm-1
;
HRMS (ESI) m/z Calcd for C20H24NO4 342.1700; Found 342.1694
(Δ = -1.75 ppm).
1-(Diphenylmethyl)-4-(3-methoxyphenyl)piperidine-4-carboxylic
acid (8). Compound 8 was prepared from 6 in a manner similar to
that described for compound 7 with a yield of 67% as a white solid.
Mp 215-217 °C; 1H NMR (DMSO-d6, 300 MHz) δ 1.86 (2H, m),
2.05 (2H, m), 2.35 (2H, m), 2.64 (2H, m), 3.73 (3H, s), 4.27 (1H, s),
6.83 (1H, dd, J = 8.2, 2.5 Hz), 6.88 (1H, d, J = 2.5 Hz), 6.97 (1H,
d, J= 8.2 Hz), 7.17 (2H, m), 7.26 (5H, m), 7.40 (4H, m), 12.44 (1H,
s); IR (ATR) 1701, 1581, 1489 cm-1; HRMS (ESI) m/z Calcd for
C26H28NO3 402.2064; Found 402.2056 (Δ = -1.86 ppm).
N-(2,6-Diisopropylphenyl)-1-(2-methoxyphenyl)-4-(3-methoxy-
phenyl)piperidine-4-carboxamide (9). To a solution of compound
7 (171 mg, 0.50 mmol) in CH2Cl2 (3.0 mL) was added N,N-
dimethylformamide (5.0 μL) and oxaryl chloride (89.9 μL, 1.00
mmol) at 0 °C, and then the mixture was stirred at room
temperature for 3 h. The solvent was removed in vacuo, and then
the residue was azeotropied with toluene (2x). To a solution of
the acid chloride thus obtained in CH2Cl2 (3.0 mL) was added
2,6-diisopropylaniline (104 μL, 0.55 mmol) and triethylamine
(279 μL, 2.00 mmol) at room temperature, and then the mixture
was stirred for 6 h. The reaction was quenched by adding water,
and then the mixture was extracted with chloroform. The organic
layer was washed with brine and dried over anhydrous magne-
sium sulfate. After filtration, the solvent was removed in vacuo,
and the residue was purified by silica gel column chromatography.
The solvent was removed in vacuo, and the residue obtained was
triturated with diethylether/methanol to give 9 (181 mg, 72%) as a
white solid. Mp 144-146 °C; 1H NMR (DMSO-d6, 300 MHz) δ
0.91 (12H, m), 2.08 (2H, m), 2.68 (2H, m), 2.87 (2H, m), 2.91 (2H,
m), 3.26 (2H, m), 3.78 (3H, s), 3.79 (3H, s), 6.89 (3H, m), 6.94 (2H,
m), 7.07 (2H, m), 7.13-7.21 (3H, m), 7.31 (1H, dd, J = 8.1,
8.1 Hz), 8.87 (1H, s); IR (ATR) 3365, 1643, 1599, 1497 cm-1; MS
(ESI) m/z 501 (M þ 1); Anal. Calcd for C32H40N2O3: C, 76.77; H,
8.05; N, 5.60. Found: C, 76.81; H, 8.10; N, 5.58; LC: 97.6% pure,
tR = 9.01 min (Method A).
Experimental Section
Melting points were determined on an electrothermal appara-
tus without correction. IR spectra were recorded on a JEOL JIR-
SPX60 spectrometer as ATR. NMR spectra were recorded on a
JEOL JNM-LA300 spectrometer or on a Varian Mercury-vx
spectrometer. Chemical shifts (σ) are given in parts per million,
and tetramethylsilane was used as internal standard for spectra
obtained in DMSO-d6 and CDCl3. All J values are given in Hz.
Mass spectra were recorded on a Bruker Daltonics esquire 3000plus
and high-resolution mass spectra (HRMS) were recorded on a
Thermo Fisher Scientific LTQ orbitrap Discovery MS equipment.
Elemental analysis was performed on a CE Instrument EA1110
and a Yokokawa analytical system IC7000. Reagents and solvents
were used as obtained from commercial suppliers without further
purification. Column chromatography was carried out using a
Yamazen W-prep system and performed using prepacked silica-gel
or amino silica-gel columns. Reaction progress was determined by
TLC analysis on a silica-gel or an amino silica-gel coated glass
plate. Visualization was done with UV light (254 nm) or iodine. All
reactions were carried out under a nitrogen atmosphere unless
otherwise mentioned. HPLC was performed using a Shimadzu
Corporation system. The sample was dissolved in methanol/0.1%-
TFA solution, applied on a Myghtysil RP-18 GP column (4.6 mm
ꢀ 150 mm, 5 μM), and eluted at 1 mL/min with a 30 min gradient
(from 60% B to 90% B), where solvent A is water (0.1% TFA
solution) and solvent B is methanol (Method A) or acetonitrile
(Method B). The purity of test compounds was determined by
HPLC and was g95%.
1-(2-Methoxyphenyl)-4-(3-methoxyphenyl)piperidine-4-car-
boxamide (5). To a solution of 3 (3.22 g, 0.01 mol) in DMSO
(15 mL) was added 6 N KOH solution (15 mL), and the mixture
was stirred at 120 °C for 6 h. The reaction was quenched by
adding water (60 mL) at room temperature, and the mixture was
neutralized with hydrochloric acid solution. The resulting solid
was filtered, and the solid was washed with diethylether to give 5
(3.03 g, 89%) as a white solid. Mp 139-141 °C; 1H NMR
(DMSO-d6, 300 MHz) δ 1.91 (2H, m), 2.53 (2H, m), 2.74 (2H,
m), 3.17 (2H, m), 3.74 (3H, s), 3.76 (3H, s), 6.79-6.90 (5H, m),
6.95 (3H, m), 7.17 (1H, s), 7.25 (1H, dd, J = 8.0, 8.0 Hz); IR
(ATR) 3426, 3197, 1664 cm-1; HRMS (ESI) m/z Calcd for
C20H25N2O3 341.1860; Found 341.1854 (Δ = -1.57 ppm).
1-(Diphenylmethyl)-4-(3-methoxyphenyl)piperidine-4-carbox-
amide (6). Compound 6 was prepared from 4 in a manner similar
to that described for compound 5 with a yield of 100% as a white
N-{[(2,6-Diisopropylphenyl)amino]sulfonyl}-1-(2-methoxyphenyl)-
4-(3-methoxyphenyl)piperidine-4-carboxamide (10). To a solution of
compound 7 (171 mg, 0.50 mmol) in CH2Cl2 (3.0 mL) was added
N,N-dimethylformamide (5.0 μL) and oxaryl chloride (89.9 μL,
1.00 mmol) at 0 °C, and then the mixture was stirred at room
temperature for 3 h. The solvent was removed in vacuo, and then
the residue was azeotropied with toluene (2x). To a solution of
N-(2,6-diisopropylphenyl)sulfamide 19 (385 mg, 1.50 mmol) in
THF (3.0 mL) was added 55% sodium hydride with mineral oil
(65.6 mg, 1.50 mmol) at 0 °C, and then the mixture was stirred.
After 15 min, to the mixture was added THF (4.0 mL) solution of
the acidchloride obtainedaboveat 0°C, andthenthe mixturewas
stirred for 16 h at room temperature. The reaction was quenched
by adding water, and then the mixture was extracted with ethyl
acetate. The organic layer was washed with brine and dried over
anhydrous magnesium sulfate. After filtration, the solvent was
removed in vacuo, and the residue was purified by silica gel
column chromatography. The solvent was removed in vacuo, and
the residue obtained was triturated with diethylether/hexane to
give10(181 mg, 62%) asa white solid. Mp164-166 °C;1H NMR
(DMSO-d6, 300 MHz) δ 0.98 (12H, d, J = 6.8 Hz), 2.09 (2H, m),
2.62(2H, m), 2.86 (2H, m), 3.22 (4H, m), 3.77 (3H, s), 3.78 (3H, s),
6.84-6.92 (5H, m), 7.06 (4H, m), 7.20 (1H, m), 7.32 (1H, dd, J =
7.8, 7.8 Hz), 9.28 (1H, s), 11.18 (1H, s); IR (ATR) 3230, 1684,
1
solid. Mp 78-80 °C; H NMR (DMSO-d6, 300 MHz) δ 1.77
(2H, m), 2.03 (2H, m), 2.35 (2H, m), 2.52 (2H, m), 3.68 (3H, s),
4.17 (1H, s), 6.76 (1H, m), 6.83-6.90 (3H, m), 7.00 (1H, s), 7.12
(2H, m), 7.17-7.25 (5H, m), 7.34 (4H, m); IR (ATR) 3475, 3213,
1680 cm-1; HRMS (ESI) m/z Calcd for C26H29N2O2 401.2224;
Found 401.2213 (Δ = -2.54 ppm).
1-(2-Methoxyphenyl)-4-(3-methoxyphenyl)piperidine-4-carboxylic
acid (7). To compound 5 (1.70 g, 5.0 mmol) was added concentrated
hydrochloric acid solution (17 mL), and the mixture was stirred at
100 °C for 2 days. The mixture was concentrated, and then a dilution
was made of the residue with toluene. The mixture was extracted

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3291
1597, 1495, 1338, 1242 cm-1; MS (ESI) m/z 580 (M þ 1); Anal.
Calcd for C32H41N3O5S: C, 66.29; H, 7.13; N, 7.25. Found:
C, 66.46; H, 7.17; N, 7.26; LC: 98.1% pure, tR = 10.73 min
(Method A).
diethylether solution (220 μL, 0.22 mmol) at room temperature,
and the mixture was stirred for 1 h. The solvent was removed in
vacuo, and the residue was triturated with diethylether. The
resulting solid was filtered, and washed by diethylether to give
hydrochloride (115 mg, 93%) as a white solid. Mp 165-167 °C;
1H NMR (DMSO-d6, 60 °C, 300 MHz) δ 1.05 (12H, d, J =
6.8 Hz), 2.48 (2H, m), 2.75 (2H, m), 3.40 (2H, m), 3.55 (2H, m),
3.65 (2H, m), 3.78 (3H, s), 3.86 (3H, s), 6.92 (1H, m), 7.04 (3H, m),
7.12-7.19 (4H, m), 7.20 (2H, m) 7.66 (1H, m); IR (ATR) 2972,
1608, 1492, 1454, 1382, 1292 cm-1; HRMS (ESI) m/z Calcd for
C32H39N2O6S 579.2534; Found 579.2534 (Δ = 0.02 ppm); Anal.
Phenyl {[1-(2-Methoxyphenyl)-4-(3-methoxyphenyl)piperidin-
4-yl]carbonyl}sulfamate (11). Compound 11 was prepared from
7 in a manner similar to that described for compound 9 with a
yield of 37% as a white solid (methanol). Mp 185-187 °C; 1H
NMR (DMSO-d6, 60 °C, 300 MHz) δ 2.12 (2H, m), 2.65 (2H,
m), 3.18 (2H, m), 3.36 (2H, m), 3.75 (3H, s), 3.85 (3H, s),
6.88 (1H, m), 6.96-7.31 (12H, m); IR (ATR) 1570, 1490,
1327, 1255 cm-1; MS (ESI) m/z 497 (M þ 1); Anal. Calcd for
C26H28N2O6S: C, 62.89; H, 5.68; N, 5.64. Found: C, 62.81; H,
5.68; N, 5.61; LC: 99.5% pure, tR = 3.50 min (condition A).
2-Methylphenyl {[1-(2-Methoxyphenyl)-4-(3-methoxyphenyl)-
piperidin-4-yl]carbonyl}sulfamate (12). Compound 12 was pre-
pared from 7 in a manner similar to that described for compound 9
with a yield of 24% as a white solid (methanol). Mp 191-193 °C;
1H NMR (DMSO-d6, 60 °C, 300 MHz) δ 2.10 (2H, m), 2.23 (3H,
s), 2.64 (2H, m), 3.10 (2H, m), 3.34 (2H, m), 3.76 (3H, s), 3.84 (3H,
s), 6.89 (1H, m), 6.97-7.31 (11H, m); IR (ATR) 1574, 1491, 1321,
1255 cm-1; MS (ESI) m/z 511 (M þ 1); Anal. Calcd for
C27H30N2O6S: C, 63.51; H, 5.92; N, 5.49. Found: C, 63.68; H,
6.00; N, 5.46; LC: 99.8% pure, tR = 4.77 min (Method A).
2-Propylphenyl {[1-(2-Methoxyphenyl)-4-(3-methoxyphenyl)-
piperidin-4-yl]carbonyl}sulfamate (13). Compound 13 was pre-
pared from 7 in a manner similar to that described for com-
pound 9 with a yield of 44% as a white solid (methanol). Mp
187-188 °C; 1H NMR (DMSO-d6, 60 °C, 300 MHz) δ 0.85 (3H,
t, J = 7.3 Hz), 1.52 (2H, m), 2.10 (2H, m), 2.59 (2H, m), 2.64
(2H, m), 3.10 (2H, m), 3.38 (2H, m), 3.76 (3H, s), 3.84 (3H, s),
6.89 (1H, m), 6.97-7.20 (10H, m), 7.29 (1H, dd, J = 8.1, 8.1 Hz);
IR (ATR) 1570, 1485, 1302, 1255 cm-1; MS (ESI) m/z 539
(M þ 1); Anal. Calcd for C29H34N2O6S: C, 64.66; H, 6.36; N,
Calcd for C32H41ClN2O6S 1.25H2O: C, 60.08; H, 6.85; N, 4.38.
3
Found: C, 59.93; H, 6.75; N, 4.49; LC: 96.6% pure, tR = 14.72
min (Method A).
Sodium Salt of Compound 16. To a suspension of 16 (116 mg,
0.20 mmol) in methanol (3 mL) was added sodium tert-butoxide
(18.8 mg, 0.196 mmol) at room temperature, and the mixture
was stirred for 30 min. The solvent was removed in vacuo, and
the residue was triturated with diethylether. The resulting solid
was filtered, and washed by diethylether to give sodium salt
1
(115 mg, 96%) as a white solid. Mp 256-258 °C; H NMR
(DMSO-d6, 300 MHz) δ 1.01 (12H, d, J = 6.8 Hz), 1.78 (2H, m),
2.52 (2H, m), 2.81 (2H, m), 3.17 (2H, m), 3.61 (2H, m), 3.72 (3H,
s), 3.76 (3H, s), 6.74 (1H, m), 6.82-6.89 (4H, m), 6.99 (5H, m),
7.18 (1H, dd, J = 8.1, 8.1 Hz); IR (ATR) 1597, 1498, 1439,
1290 cm-1; HRMS (ESI) m/z Calcd for C32H39N2O6S 579.2534;
Found 579.2536 (Δ = 0.25 ppm); Anal. Calcd for C32H39N2-
NaO6S 2H2O: C, 60.17; H, 6.79; N, 4.39. Found: C, 59.75; H,
3
6.23; N, 4.44; LC: 96.7% pure, tR = 14.94 min (Method A).
2,6-Diisopropylphenyl {[1-(Diphenylmethyl)-4-(3-methoxyphenyl)-
piperidin-4-yl]carbonyl}sulfamate (17). Compound 17 was prepared
from 8 in a manner similar to that described for compound 9 with a
yield of 65% as a white solid (hexane/ethanol). Mp 212-214 °C; 1H
NMR (DMSO-d6, 300 MHz) δ 0.99 (12H, d, J = 6.8 Hz), 1.95
(1.4H,m),2.41(0.6H,m),2.63(1.4H,m),2.85(0.6H,m),3.03(1.4H,
m), 3.22 (2H, m), 3.33 (0.6H, m), 3.50 (0.6H, m), 3.62 (1.4H, m),
3.70 (3H, s), 5.65 (0.7H, m), 5.81 (0.3H, m), 6.79 (0.7H, m), 6.85
(0.3H, m), 6.92 (2H, m), 7.02 (3H, m), 7.18-7.50 (7H, m), 7.63-7.71
(4H, m), 9.71 (0.7H, s), 9.95 (0.3H, s); IR (ATR) 1556, 1423,
1321, 1255 cm-1; MS (ESI) m/z 641 (M þ 1); Anal. Calcd for
C38H44N2O5S: C, 71.22; H, 6.92; N, 4.37. Found: C, 70.88; H, 6.98;
N, 4.48.
5.20. Found: C, 64.42; H, 6.42; N, 5.15; LC: 99.9% pure, tR
=
9.68 min (Method A).
2-Cyclopentylphenyl {[1-(2-Methoxyphenyl)-4-(3-methoxyphenyl)-
piperidin-4-yl]carbonyl}sulfamate (14). Compound 14 was prepared
from 7 in a manner similar to that described for compound 9 with
a yield of 36% as a white solid (methanol). Mp 192-194 °C; 1H
NMR (DMSO-d6, 60 °C, 300 MHz) δ 1.48 (2H, m), 1.60 (2H, m),
1.73 (2H, m), 1.95 (2H, m), 2.10 (2H, m), 2.49 (1H, m), 2.64 (2H,
m), 3.10 (2H, m), 3.40 (2H, m), 3.76 (3H, s), 3.84 (3H, s), 6.89 (1H,
m), 6.98-7.32 (11H, m); IR (ATR) 1568, 1331, 1255 cm-1; MS
(ESI) m/z 565 (M þ 1); Anal. Calcd for C31H36N2O6S: C, 65.94;
H, 6.43; N, 4.96. Found: C, 65.84; H, 6.44; N, 4.99; LC: 99.5%
pure, tR = 12.57 min (Method A).
2,6-Dimethylphenyl {[1-(2-Methoxyphenyl)-4-(3-methoxyphenyl)-
piperidin-4-yl]carbonyl}sulfamate (15). Compound 15 was prepared
from 7 in a manner similar to that described for compound 9 with a
yield of 32% as a white solid (methanol). Mp 188-189 °C; 1HNMR
(DMSO-d6, 60 °C, 300 MHz) δ 2.13 (2H, m), 2.18 (6H, s), 2.64 (2H,
m), 3.36 (4H, m), 3.77 (3H, s), 3.85 (3H, s), 6.87 (1H, m), 6.99-7.09
(9H, m), 7.28 (1H, dd, J = 8.1, 8.1 Hz); IR (ATR) 1564, 1481, 1321,
1255 cm-1; MS (ESI) m/z 525 (M þ 1); Anal. Calcd for C28H32-
N2O6S: C, 64.10; H, 6.15; N, 5.34. Found: C, 63.93; H, 6.16; N, 5.35;
LC: 99.3% pure, tR = 6.74 min (Method A).
N-(2,6-Diisopropylphenyl)Sulfamide (19). To a solution of
sulfamoyl chloride18 (1.15 g, 0.01 mol) in N-methyl-2-pyrrolidi-
none (10 mL) was added 2,6-diisopropylaniline (942 μL, 5.0 mmol)
at 0 °C, and the mixture was stirred at room temperature for 6 h.
The mixture was poured into water, and then was extracted with
ethyl acetate. The organic layer was washed with brine and dried
over anhydrous magnesium sulfate. After filtration, the solvent
was removed in vacuo, and residue was purified by silica gel
column chromatography to give 19 (981 mg, 77%) as a white
solid. Mp 130-132 °C; 1H NMR (DMSO-d6, 300 MHz) δ 1.11
(12H, d, J = 6.8 Hz), 3.59 (2H, m), 6.72 (2H, s), 7.13 (2H, d, J =
7.3 Hz), 7.20 (1H, m), 8.36 (1H, s); IR (ATR) 3365, 3334, 3273,
1373, 1155 cm-1; HRMS (ESI) m/z Calcd for C12H21N2O2S
257.1318; Found 257.1315 (Δ = -1.37 ppm).
Phenyl Sulfamate (26). To a solution of phenol (0.44 mL,
5.0 mmol) in heptane (20 mL) was added chlorosulfonylisocyanate
(0.48 mL, 5.5 mmol) at room temperature, and the mixture was
stirred at reflux for 10 h. The mixture was added water (0.7 mL) at
room temperature, and stirred at reflux for 2 h. The reaction was
quenched by adding water, and then the mixture was extracted
with ethyl acetate. The organic layer was washed twice with water,
and dried over anhydrous magnesium sulfate. After filtration, the
solvent was removed in vacuo, and residue was triturated with
hexane to give 26 (536 mg, 62%) as a white solid. Mp 77-79 °C; 1H
NMR (DMSO-d6, 300 MHz) δ 7.26-7.34 (3H, m), 7.46 (2H, m),
7.98 (2H, s); IR (ATR) 3417, 3304, 1360, 1142 cm-1; HRMS
(APCI) m/z Calcd for C6H6NO3S 172.0074; Found 172.0068 (Δ =
-3.18 ppm).
2,6-Diisopropylphenyl {[1-(2-Methoxyphenyl)-4-(3-methoxy-
phenyl)piperidin-4-yl]carbonyl}sulfamate (16). Compound 16
was prepared from 7 in a manner similar to that described for
compound 9 with a yield of 45% as a white solid (methanol). Mp
1
181-183 °C; H NMR (DMSO-d6, 60 °C, 300 MHz) δ 1.05
(12H, d, J = 6.8 Hz), 2.17 (2H, m), 2.69 (2H, m), 3.48 (4H, m),
3.77 (3H, s), 3.86 (3H, s), 6.88 (1H, m), 6.97-7.19 (9H, m), 7.30
(1H, m); IR (ATR) 1566, 1464, 1317, 1255 cm-1; MS (ESI) m/z
581 (M þ 1); Anal. Calcd for C32H40N2O6S 0.25H2O: C, 65.67;
3
H, 6.98; N, 4.79. Found: C, 65.62; H, 6.98; N, 4.89; LC: 97.4%
pure, tR = 14.63 min (Method A).
Hydrochloride of Compound 16. To a suspensionof 16 (116 mg,
0.20 mmol) in methanol (2 mL) was added 1 N hydrochloride

3292 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Asano et al.
2-Methylphenyl Sulfamate (27). Compound 27 was prepared
from 2-methylphenol in a manner similar to that described for
compound 26 with a yield of 82% as a white solid (hexane). Mp
36-38 °C; 1H NMR (DMSO-d6, 300 MHz) δ 2.28 (3H, s),
7.16-7.31 (4H, m), 8.01 (2H, s); IR (ATR) 3379, 3275, 1342,
1153 cm-1; HRMS (APCI) m/z Calcd for C7H8NO3S 186.0230;
Found 186.0224 (Δ = -3.59 ppm).
2-Propylphenyl Sulfamate (28). Compound 28 was prepared
from 2-propylphenol in a manner similar to that described for
compound 26 with a yield of 61% as a white solid (hexane). Mp
56-58 °C; 1H NMR (DMSO-d6, 300 MHz) δ 0.91 (3H, t, J =
7.3 Hz), 1.58 (2H, tq, J = 7.7, 7.3 Hz), 2.64 (2H, t, J = 7.7 Hz),
7.19-7.34 (4H, m), 8.04 (2H, s); IR (ATR) 3367, 3280, 1373,
1153 cm-1; HRMS (APCI) m/z Calcd for C9H12NO3S 214.0543;
Found 214.0535 (Δ = -4.00 ppm).
J = 6.8 Hz), 1.80 (2H, m), 2.70 (2H, m), 2.75 (3H, s), 2.92 (2H, m),
3.44 (2H, m), 3.65 (2H, m), 3.76 (3H, s), 6.81 (1H, m), 6.95 (2H, m),
7.04 (3H, m), 7.22 (1H, dd, J =8.0, 8.0 Hz), 9.08 (1H, s);IR(ATR)
1549, 1302, 1161 cm-1; MS (ESI) m/z 489 (M þ 1); Anal. Calcd for
C26H36N2O5S 0.5H2O: C, 62.75; H, 7.49; N, 5.63. Found: C, 62.45;
3
H, 7.30; N, 5.68; LC: 96.6% pure, tR = 12.67 min (Method A).
2,6-Diisopropylphenyl {[1-Acetyl-4-(3-methoxyphenyl)piperidin-
4-yl]carbonyl}sulfamate (34). To a solution of compound 32
(95 mg, 0.20 mmol) in pyridine (1.0 mL) was added acetyl chloride
(28.5 μL, 0.40 mmol) and 4-(N,N-dimethylamino)pyridine (2.4 mg,
0.020 mmol) at room temperature, and the mixture was stirred for
6 h. The solvent was removed in vacuo, the residue was distilled with
ethyl acetate, and the mixture was washed with saturated ammo-
nium chloride solution, brine and dried over anhydrous magnesium
sulfate. After filtration, the solvent was removed in vacuo, and the
residue was purified by silica gel column chromatography. The
solvent was removed in vacuo, and the residue was triturated with
hexane/diethylether to give 34 (62.3 mg, 60%) as a white solid. Mp
168-170 °C; 1H NMR (DMSO-d6, 300 MHz) δ 1.04 (12H, d, J =
6.8 Hz), 1.75 (1H, m), 1.90 (1H, m), 2.00 (3H, s), 2.49 (2H, m), 2.86
(1H, m), 3.23 (1H, m), 3.31 (2H, m), 3.72 (1H, m), 3.75 (3H, s), 4.13
(1H, m), 6.87-6.95 (3H, m), 7.18 (3H, m), 7.31 (1H, m); IR (ATR)
1718, 1605, 1369 cm-1; MS (ESI) m/z 517 (M þ 1); Anal. Calcd
2-Cyclopentylphenyl Sulfamate (29). Compound 29 was pre-
pared from 2-cyclopentylphenol in a manner similar to that
described for compound 26 with a yield of 38% as a white solid
1
(hexane). Mp 103-105 °C; H NMR (DMSO-d6, 300 MHz) δ
1.51 (2H, m), 1.64 (2H, m), 1.78 (2H, m), 1.99 (2H, m), 3.40 (1H,
m), 7.21-7.32 (3H, m), 7.40 (1H, m), 8.04 (2H, s); IR (ATR)
3365, 3261, 1352, 1155 cm-1; HRMS (APCI) m/z Calcd for
C11H14NO3S 240.0700; Found 240.0689 (Δ = -4.35 ppm).
2,6-Dimethylphenyl Sulfamate (30). Compound 30 was pre-
pared from 2,6-dimethylphenol in a manner similar to that
described for compound 26 with a yield of 61% as a white solid
for C27H36N2O6S 1.25H2O: C, 60.15; H, 7.20; N, 5.20. Found:
3
C, 59.99; H, 6.85; N, 5.31; LC: 96.7% pure, tR = 16.72 min
(Method A).
1
(hexane). Mp 102-104 °C; H NMR (DMSO-d6, 300 MHz) δ
2,6-Diisopropylphenyl {[1-Benzoyl-4-(3-methoxyphenyl)piperidin-
4-yl]carbonyl}sulfamate (35). Compound 35 was prepared with
benzoyl chloride in a manner similar to that described for
compound 34 with a yield of 65% as a white solid. Mp 150-
152 °C; 1H NMR (DMSO-d6, 60 °C, 300 MHz) δ 1.05 (12H, d,
J = 7.0 Hz), 1.78 (2H, m), 2.48 (2H, m), 3.21 (2H, m), 3.52 (2H,
m), 3.74 (3H, s), 3.90 (2H, br), 6.82 (1H, m), 6.98 (2H, m), 7.12
(3H, m), 7.24 (1H, dd, J = 8.3, 8.3 Hz), 7.35-7.44 (5H, m); IR
(ATR) 1718, 1632, 1448, 1250 cm-1; MS (ESI) m/z 579 (M þ 1);
2.31 (6H, s), 7.09 (3H, m), 8.04 (2H, s); IR (ATR) 3352, 3267,
1338, 1144 cm-1; HRMS (APCI) m/z Calcd for C8H10NO3S
200.0387; Found 200.0378 (Δ = -4.55 ppm).
2,6-Diisopropylphenyl Sulfamate (31). Compound 31 was
prepared from 2,6-diisopropylphenol in a manner similar to that
described for compound 26 with a yield of 78% as a white solid
1
(hexane). Mp 101-103 °C; H NMR (DMSO-d6, 300 MHz) δ
1.13 (12H, d, J = 7.0 Hz), 3.49 (2H, m) 7.17-7.21 (3H, m), 8.07
(2H, s); IR (ATR) 3380, 3278, 1371, 1190 cm-1; HRMS (APCI)
m/z Calcd for C12H18NO3S 256.1013; Found 256.1002 (Δ =
-4.06 ppm).
Anal. Calcd for C32H38N2O6S 0.75H2O: C, 64.90; H, 6.72; N,
3
4.73. Found: C, 64.96; H, 6.39; N, 4.76; LC: 97.1% pure, tR
19.92 min (condition A).
=
2,6-Diisopropylphenyl {[4-(3-Methoxyphenyl)Piperidin-4-yl]-
carbonyl}sulfamate (32). To a solution of compound 17 (3.84 g,
6.0 mmol) in N,N-dimethylformamide (60 mL) was added 20%
palladium hydroxide on carbon (50% wet, 600 mg), and the
mixture was stirred under hydrogen atmosphere at room tem-
perature for 1 h. The reaction mixture was filtered through Celite,
and the filtrate was concentrated. To a solution of the residue in
1,4-dioxane (50 mL) was added activated carbon (100 mg), and
then the mixture was stirred for 30 min. The mixture was filtered
through Celite, and the filtrate was concentrated. The residue was
purified by recrystallization with diethylether to give 32 (2.73 g,
2,6-Diisopropylphenyl {[4-(3-Methoxyphenyl)-1-phenylpiperidin-
4-yl]carbonyl}sulfamate (36). To a solution of compound 32
(95 mg, 0.20 mmol) in 1,4-dioxane (1.0 mL) was added bromo-
benzene (42.1 μL, 0.40 mmol), (S)-(-)-BINAP (7.5 mg, 0.012
mmol), tris(dibenzylideneacetone)dipalladium(0) (5.5 mg, 0.006
mmol), and sodium tert-butoxide (57.7 mg, 0.60 mmol) at room
temperature, and the mixture was stirred for 2 h at 120 °C under
microwave irradiation. The reaction was quenched by adding
saturated ammonium chloride solution, and then the mixture was
extracted with ethyl acetate. The organic layer was washed with
brine and dried over anhydrous magnesium sulfate. After filtra-
tion, the solvent was removed in vacuo, and the residue was
purified by silica gel column chromatography. The solvent was
removed in vacuo, and the residue was triturated with methanol
to give 36 (95 mg, 86%) as a pale-yellow solid. Mp 165-167 °C;
1H NMR (DMSO-d6, 60 °C, 300 MHz) δ 1.05 (12H, d, J =
6.8 Hz), 2.07 (2H, m), 2.67 (2H, m), 3.01 (2H, m), 3.38 (2H, m),
3.58 (2H, m), 3.77 (3H, s), 6.89 (2H, m), 7.00 (4H, m), 7.13-7.27
(5H, m), 7.31 (1H, dd, J = 8.1, 8.1 Hz); IR (ATR) 1560, 1489,
1263 cm-1; MS (ESI) m/z 551 (M þ 1); Anal. Calcd for C31H38-
1
96%) as a white solid. Mp 219-221 °C; H NMR (DMSO-d6,
300 MHz) δ 1.01 (12H, d, J = 6.8 Hz), 1.80(2H, m), 2.53 (2H, m),
2.93 (2H, m), 3.22 (2H, m), 3.56 (2H, m), 3.72 (3H, s), 6.81 (1H,
m), 6.93 (2H, m), 7.04 (3H, m), 7.22 (1H, dd, J = 7.7, 7.7 Hz), 8.31
(2H, s); IR (ATR) 3076, 1608, 1599, 1552, 1330, 1257 cm-1
;
HRMS (ESI) m/z Calcd for C25H35N3O5S 475.2261; found
475.2246 (Δ = -3.13 ppm); Anal. Calcd for C25H34N2O5S
3
0.5H2O: C, 62.09; H, 7.29; N, 5.79. Found: C, 61.86; H, 7.21; N,
5.82; LC: 97.2% pure, tR = 12.48 min (Method A).
N2O5S 1.5H2O: C, 64.45; H, 7.15; N, 4.85. Found: C, 64.19; H,
2,6-Diisopropylphenyl {[4-(3-Methoxyphenyl)-1-methylpiperidin-
4-yl]carbonyl}sulfamate (33). To a suspension of compound 32
(95 mg, 0.20 mmol) in methanol (2.0 mL) was added 35% for-
maldehyde solution (47.6 μL, 0.60 mmol), acetic acid (45.8 μL,
0.80 mmol), and sodium cyanoborohydride (50.3 mg, 0.80 mmol)
at room temperature, and the mixture was stirred for 1 day. The
reaction was quenched by adding brine, and then the mixture
was extracted with chloroform (2ꢀ). The organic layer was dried
over anhydrous magnesium sulfate. After filtration, the solvent
was removed in vacuo, and the residue was purified by recrystalli-
zation with methanol to give 33 (29.9 mg, 31%) as a white solid.
Mp 222-224 °C; 1H NMR (DMSO-d6, 300 MHz) δ 1.04 (12H, d,
3
7.11; N, 5.22; LC: 97.2% pure, tR = 15.43 min (Method A).
2,6-Diisopropylphenyl {[4-(3-Methoxyphenyl)-1-(3-methoxy-
pyridin-2-yl)piperidin-4-yl]carbonyl}sulfamate (37). Compound
37 was prepared with 2-bromo-3-methoxypyridine in a manner
similar to that described for compound 36 with a yield of 72%
as a white solid. Mp 195-197 °C; 1H NMR (DMSO-d6, 60 °C,
300 MHz) δ 1.06 (12H, d, J = 6.8 Hz), 2.02 (2H, m), 2.59 (2H, m),
3.18(2H, m), 3.39 (2H, m), 3.76 (3H, s), 3.80 (2H, m), 3.83 (3H, s),
6.90 (2H, m), 7.02 (2H, m), 7.15 (3H, m), 7.27 (2H, m), 7.74 (1H,
d, J = 5.1 Hz); IR (ATR) 1618, 1540, 1460, 1325, 1246 cm-1; MS
(ESI) m/z 582 (M þ 1); Anal. Calcd for C31H39N3O6S 2H2O: C,
3

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3293
60.27; H, 7.02; N, 6.80. Found: C, 59.89; H, 6.75; N, 6.75; LC:
97.8% pure, tR = 13.99 min (Method A).
m/z 621 (M þ 1); Anal. Calcd for C30H35F3N4NaO5S: C, 58.05;
H, 5.68; N, 9.03. Found: C, 57.76; H, 5.63; N, 8.96; LC: 99.6%
pure, tR = 19.25 min (Method B).
2,6-Diisopropylphenyl {[1-(1,3-Benzothiazol-2-yl)-4-(3-methoxy-
phenyl)piperidin-4-yl]carbonyl}sulfamate (38). Compound 38 was
prepared with 2-bromobenzothiazole in a manner similar to that
described for compound 36 with a yield of 65% as a pale-yellow
solid. Mp 181-183 °C; 1H NMR (DMSO-d6, 60 °C, 300 MHz) δ
1.07 (12H, d, J = 7.0 Hz), 2.08 (2H, m), 2.62 (2H, m), 3.39 (2H, m),
3.42 (2H, m), 3.77 (3H, s), 3.91 (2H, m), 6.90 (1H, m), 7.02 (2H, m),
7.07 (1H, m), 7.18 (3H, m), 7.30 (2H, m), 7.46 (1H, d, J = 8.1 Hz),
2,6-Diisopropylphenyl {[4-(3-Methoxyphenyl)-1-(5-propylpyri-
midin-2-yl)piperidin-4-yl]carbonyl}sulfamate (42). Compound 42
was prepared with 2-chloro-5-propylpyrimidine in a manner
similar to that described for compound 39 with a yield of 41%
1
as a white solid. Mp 89-91 °C; H NMR (DMSO-d6, 60 °C,
300 MHz) δ 0.87 (3H, t, J = 7.3 Hz), 1.05 (12H, d, J = 6.8 Hz),
1.52 (2H, m), 1.83 (2H, m), 2.37 (2H, t, J = 7.4 Hz), 2.50 (2H, m),
3.26 (2H, m), 3.48 (2H, m), 3.74 (3H, s), 4.33 (2H, m), 6.83 (1H,
m), 6.98 (2H, m), 7.12(3H, m), 7.25 (1H, dd, J = 8.2, 8.2 Hz), 8.21
(2H, s);IR(ATR) 1585, 1464, 1363, 1248 cm-1; MS(ESI) m/z 595
7.75 (1H, d, J = 7.9 Hz); IR (ATR) 1618, 1576, 1454, 1259 cm-1
;
MS (ESI) m/z 608 (M þ 1); Anal. Calcd for C32H37N3O5S2 2H2O:
3
C, 59.70; H, 6.42; N, 6.53. Found: C, 59.56; H, 6.09; N, 6.61; LC:
99.3% pure, tR = 11.18 min (Method B).
(M þ 1); Anal. Calcd for C32H42N4O5S 1H2O: C, 62.72; H, 7.24;
3
2,6-Diisopropylphenyl {[4-(3-Methoxyphenyl)-1-pyrimidin-2-
ylpiperidin-4-yl]carbonyl}sulfamate (39). To a solution of com-
pound 32 (950 mg, 2.0mmol) inN,N-dimethylformamide (10mL)
was added 2-chloropyrimidine (275 mg, 2.40 mmol) and potas-
sium carbonate (553 mg, 4.0 mmol) at room temperature, and the
mixture was stirred at 60 °C for 3 h. The reaction was quenched by
adding water, and then the mixture was extracted with ethyl
acetate. The organic layer was washed with brine and dried over
anhydrous magnesium sulfate. After filtration, the solvent was
removed in vacuo, and the residue was purified by silica gel
column chromatography. The solvent was removed in vacuo,
and the residue was washed with diethylether/hexane to give 39
(1.05 g, 95%) as a white solid. Mp 201-203 °C; 1H NMR
(DMSO-d6, 300 MHz) δ 1.01 (12H, d, J = 6.8 Hz), 1.60 (2H,
m), 2.50 (2H, m), 3.20 (2H, m), 3.58 (2H, m), 3.71 (3H, s), 4.42
(2H, m), 6.55 (1H, dd, J =4.8, 4.8 Hz), 6.76(1H, m), 6.96 (2H, m),
7.05 (3H, m), 7.18 (1H, dd, J = 8.2, 8.2 Hz), 8.32 (2H, d, J =
4.8 Hz); IR (ATR) 1585, 1286 cm-1; MS (ESI) m/z 553 (M þ 1);
N, 9.14. Found: C, 62.74; H, 7.06; N, 9.09; LC: 97.7% pure, tR =
13.56 min (Method B).
2,6-Diisopropylphenyl {[1-(5-Bromopyrimidin-2-yl)-4-(3-meth-
oxyphenyl)piperidin-4-yl]carbonyl}sulfamate (43). To a solution
of compound 39 (100 mg, 0.181 mmol) in THF (2.0 mL) was
added N-bromosuccinimide (32.2 mg, 0.181 mmol) at 0 °C, and
the mixture was stirred for 1 h. The reaction was quenched by
adding saturated sodium hydrogen carbonate solution, and then
the mixture was extracted with ethyl acetate. The organic layer
was washed with brine and dried over anhydrous magnesium
sulfate. After filtration, the solvent was removedinvacuo, andthe
residue was purified by silica gel column chromatography. The
solvent was removed in vacuo, and the residue was triturated with
diethylether to give 43 (95.6 mg, 84%) as a white solid. Mp
106-108 °C; 1H NMR(DMSO-d6, 300 MHz) δ 1.01(12H, d, J =
6.8 Hz), 1.58 (2H, m), 2.50 (2H, m), 3.20 (2H, m), 3.62 (2H, m),
3.70 (3H, s), 4.35 (2H, m), 6.75 (1H, d, 8.1 Hz), 6.96 (2H, m), 7.03
(3H, s), 7.17 (1H, dd, J = 8.1, 8.1 Hz), 8.41 (2H, s); IR (ATR)
1578, 1362, 1250 cm-1; MS (ESI) m/z 631 (M þ 1); Anal. Calcd
Anal. Calcd for C29H36N4O5S 1.1H2O: C, 60.84; H, 6.73; N, 9.79.
3
for C29H35BrN4O5S 2.5H2O: C, 51.48; H, 5.96; N, 8.28. Found:
Found: C, 60.55; H, 6.33; N, 9.71; LC: 99.4% pure, tR = 18.99
min (Method A).
3
C, 51.33; H, 5.59; N, 8.19; LC: 99.8% pure, tR = 19.66 min
(Method B).
Sodium Salt of Compound 39. Sodium salts of compound 39
was prepared in a manner similar to that described for sodium
salt of compound 16 with a yield of 95% as a white solid.
Mp >280 °C; 1H NMR (DMSO-d6, 300 MHz) δ 1.01 (12H, d,
J = 7.0 Hz), 1.55 (2H, m), 2.50 (2H, m), 3.19 (2H, m), 3.62 (2H,
m), 3.70 (3H, s), 4.41 (2H, m), 6.54 (1H, dd, J = 4.8, 4.8 Hz), 6.74
(1H, dd, J = 8.1, 2.2 Hz), 6.96 (2H, m), 7.02 (3H, s), 7.16 (1H,
dd, J = 8.1, 8.1 Hz), 8.31 (1H, d, J = 4.8 Hz); IR (ATR) 1585,
1282 cm-1; HRMS (ESI) m/z Calcd for C29H35N4O5S 551.2334;
Found 551.2335 (Δ = 0.27 ppm); Anal. Calcd for C29H35N4-
2,6-Diisopropylphenyl {[4-(3-Methoxyphenyl)-1-pyrimidin-2-
ylpiperidin-4-yl]carbonyl}methylsulfamate (44). To a solution
of sodium salt of compound 39 (100 mg, 0.174 mmol) in N,N-
dimethylformamide (2.0 mL) was added methyl iodide (217 μL,
3.48 mmol) at room temperature, and the mixture was stirred at
60 °C for 20 h. The reaction was quenched by adding water, and
then the mixture was extracted with ethyl acetate. The organic
layer was washed with brine and dried over anhydrous magne-
sium sulfate. After filtration, the solvent was removed in vacuo,
and the residue was purified by silica gel column chromato-
graphy to give 44 (43.6 mg, 44%) as a white amorphous. 1H NMR
(DMSO-d6, 300 MHz) δ 1.11 (12H, d, J = 6.8 Hz), 1.97 (2H, m),
2.46 (2H, m), 2.92 (3H, s), 3.16 (4H, m), 3.77 (3H, s), 4.58 (2H, m),
6.63 (1H, dd, J = 4.6, 4.6 Hz), 6.87-6.97 (3H, m), 7.25 (3H, m),
7.41 (1H, dd, J = 8.0, 8.0 Hz), 8.36 (2H, d, J = 4.6 Hz); IR (ATR)
1705, 1583, 1365, 1253 cm-1; MS (ESI) m/z 567 (M þ 1); Anal.
NaO5S 0.75H2O: C, 59.22; H, 6.25; N, 9.53. Found: C, 58.90;
3
H, 6.24; N, 9.29; LC: 99.1% pure, tR = 19.03 min (Method A).
2,6-Diisopropylphenyl {[4-(3-Methoxyphenyl)-1-(4-methylpyri-
midin-2-yl)piperidin-4-yl]carbonyl}sulfamate (40). Compound 40
was prepared with 2-chloro-4-methylpyrimidine in a manner
similar to that described for compound 39 with a yield of 72%
as a white solid. Mp 164-166 °C; 1H NMR (DMSO-d6, 60 °C,
300 MHz) δ 1.06 (12H, d, J = 6.8 Hz), 1.87 (2H, m), 2.28 (3H, s),
2.54 (2H, m), 3.28 (2H, m), 3.41 (2H, m), 3.76 (3H, s), 4.37 (2H,
m), 6.49 (1H, d, J = 5.0 Hz), 6.88(1H, m), 6.98 (2H, m), 7.17(3H,
m), 7.28 (1H, dd, J = 8.1, 8.1 Hz), 8.20 (1H, d, J = 5.0 Hz); IR
(ATR) 3292, 1728, 1578, 1417, 1257 cm-1; MS (ESI) m/z 567 (M
Calcd for C30H38N4O5S 0.25H2O: C, 63.08; H, 6.79; N, 9.81.
3
Found: C, 62.83; H, 6.71; N, 9.76; LC: 97.6% pure, tR = 13.82
min (Method B).
2,6-Diisopropylphenyl {[4-(3-Hydroxyphenyl)-1-pyrimidin-2-
ylpiperidin-4-yl]carbonyl}sulfamate (45). To a solution of com-
pound 39 (1.0 g, 1.81 mmol) in CH2Cl2 (15 mL) was added 1 M
boron tribromide dichloromethane solution (5.43 mL, 5.42
mmol) at 0 °C, and the mixture was stirred at room temperature
for 20 h. The reaction mixture was poured into ice water, and the
mixture was extracted with chloroform (2x). The organic layer
was dried over anhydrous magnesium sulfate. After filtration,
the solvent was removed in vacuo, and the residue was purified
by silica gel column chromatography. The mixture obtained was
washed by methanol to give 45 (850 mg, 87%) as a white solid.
Mp 193-195 °C; 1H NMR (DMSO-d6, 300 MHz) δ; 1.05 (12H,
d, J = 6.8 Hz), 1.85 (2H, m), 2.54 (2H, m), 3.28 (4H, m), 4.38
(2H, m), 6.63 (1H, dd, J = 4.8, 4.8 Hz), 6.70 (1H, m), 6.80 (2H,
m), 7.19 (4H, m), 8.36 (2H, d, J = 4.8 Hz), 9.46 (1H, s); IR
þ 1); Anal. Calcd for C30H38N4O5S 0.25H2O: C, 63.08; H, 6.79;
3
N, 9.81. Found: C, 62.86; H, 6.82; N, 9.65; LC: 99.3% pure, tR =
17.61 min (Method A).
2,6-Diisopropylphenyl ({4-(3-Methoxyphenyl)-1-[4-(trifluoro-
methyl)pyrimidin-2-yl]piperidin-4-yl}carbonyl)sulfamate (41).
Compound 41 was prepared with 2-chloro-4-trifuluoromethyl-
pyrimidine in a manner similar to that described for compound
1
39 with a yield of 90% as a white solid. Mp 174-176 °C; H
NMR (DMSO-d6, 60 °C, 300 MHz) δ 1.05 (12H, d, J = 6.8 Hz),
1.89 (2H, m), 2.58 (2H, m), 3.36 (2H, m), 3.46 (2H, m), 3.75 (3H,
s), 4.37 (2H, m), 6.86 (1H, m), 6.96 (1H, d, J = 5.0 Hz), 6.99 (2H,
m), 7.14 (3H, m), 7.27 (1H, dd, J = 8.0, 8.0 Hz), 8.66 (1H, d, J =
5.0 Hz); IR (ATR) 3311, 1722, 1589, 1448, 1327 cm-1; MS (ESI)

3294 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Asano et al.
(ATR) 3491, 3300, 1714, 1585, 1414, 1369, 1215 cm-1; HRMS
(ESI) m/z Calcd for C28H35N4O5S 539.2323; Found 539.2304
6.61; N, 9.54. Found: C, 61.21; H, 6.56; N, 9.47; LC: 99.0% pure,
tR = 15.81 min (Method A).
2,6-Diisopropylphenyl ({4-[3-(3-Hydroxypropoxy)Phenyl]-1-
(Δ = -3.52 ppm). Anal. Calcd for C28H34N4O5S 0.25H2O: C,
3
61.92; H, 6.40; N, 10.31. Found: C, 61.93; H, 6.26; N, 10.35; LC:
98.4% pure, tR = 15.40 min (Method A).
pyrimidin-2-ylpiperidin-4-yl}carbonyl)sulfamate (49). Compound
49 was prepared with 3-bromopropyl tert-butyldimethylsilyl ether
in a manner similar to that described for compound 48 with a yield
of 89% as a white solid. Mp 112-114 °C; 1H NMR (DMSO-d6,
300 MHz) δ 1.01 (12H, d, J = 7.0 Hz), 1.59 (2H, m), 1.83 (2H, m),
3.21 (2H, m), 3.30 (2H, m), 3.54 (2H, t, J = 6.2 Hz), 3.60 (2H, m),
3.98 (2H, t, J = 6.4 Hz), 4.40 (2H, m), 6.54 (1H, dd, J = 4.7,
4.7 Hz), 6.73 (1H, d, J = 7.5 Hz), 6.95 (2H, m), 7.04 (3H, m),
7.16 (1H, m), 8.32 (2H, d, J = 4.7 Hz); IR (ATR) 1583, 1458,
1363, 1242 cm-1; MS (ESI) m/z 597 (M þ 1); Anal. Calcd for
2,6-Diisopropylphenyl {[4-(3-Ethoxyphenyl)-1-pyrimidin-2-
ylpiperidin-4-yl]carbonyl}sulfamate (46). To a solution of com-
pound 45 (108 mg, 0.20 mmol) in N,N-dimethylformamide
(1.0 mL) was added ethyl iodide (32.0 μL, 0.40 mmol) and
cesium carbonate (196 mg, 0.60 mmol) at room temperature,
and the mixture was stirred at 60 °C for 4 h. The reaction was
quenched by adding water, and then the mixture was extracted
with ethyl acetate. The organic layer was washed with brine
and dried over anhydrous magnesium sulfate. After filtration,
the solvent was removed in vacuo, and the residue was purified
by preparative TLC. The crude was washed with diethylether
to give 46 (88.7 mg, 78%) as a white solid. Mp 173-175 °C;
1H NMR (DMSO-d6, 60 °C, 300 MHz) δ 1.04 (12H, d, J =
6.8 Hz), 1.31 (3H, t, J = 6.9 Hz), 1.69 (2H, m), 2.52 (2H, m),
3.27 (2H, m), 3.58 (2H, m), 4.00 (2H, q, J = 6.9 Hz), 4.38 (2H,
m), 6.54 (1H, dd, J = 4.8, 4.8 Hz), 6.76 (1H, m), 6.97 (2H, m),
7.07 (3H, m), 7.19 (1H, dd, J = 8.2, 8.2 Hz), 8.32 (2H, d, J =
4.8 Hz); IR (ATR) 1587, 1367, 1281 cm-1; MS (ESI) m/z 567
C31H40N4O6S 0.5H2O: C, 61.47; H, 6.82; N, 9.25. Found:
3
C, 61.38; H, 6.84; N, 9.02; LC: 98.6% pure, tR = 16.69 min
(Method A).
Effects of Compounds on LDL-R Activity in Human Hepatoma
Cell Line. Preparation of 1,10-dioctadecyl-3,3,30,30-tetramethy-
lindocarbocyanine perchlorate (DiI)-labeled LDL (DiI-LDL)
was carried out as follows. DiI (invitrogen, U.S.A) was mixed
with numan LDL (CHEMICON, U.S.), and the mixture was
kept for 8 h at 37 °C. Next, a specific density solution (d = 1.182)
was added and mixed, and the whole was centrifuged (32 krpm,
14 h; using Beckman Ti55.2 rotator). After centrifugation, the
DiI-labeled LDL fraction was collected and dialyzed against
saline. The effect of each compound on LDL-R activity was
determined as the amount of DiI-LDL up-taken into HepG2
cells used as indicator. Namely hepatoma cell line HepG2 used
for the experiment. HepG2 cells were plated on a 96 well plate
and incubated with Dulbecco’s modified Eagle/F-12 medium
(DMEM/F-12) containing 10% fetal bovine serum and anti-
biotics for 2-3 days at 37 °C in a CO2 incubator. After washing
the cells, DMEM/F-12 media containing the test compound,
10% delipoprotein serum, and antibiotics was added thereto,
and the cells were incubated for 19 h at 37 °C. DiI-LDL was next
added, and the cells were incubated for an additional 5 h at 37 °C
in a CO2 incubator. For measurement of the amount of non-
specific DiI-LDL uptake, 30-50 times the nonlabeled LDL was
added to each well. After washing the cells with Dulbecco’s
phosphate buffer (SIGMA, U.S.), Dulbecco’s phosphate buffer
was added to each well, and the fluorescence was measured with
a fluorescence plate reader to determine the amount of DiI-LDL
up-taken into the cells. After measuring the fluorescence, the
Dulbecco’s phosphate buffer was removed. Cells were then
dissolved in 1 N sodium hydroxide solution. Using part of the
solution, protein amount was measured. The effect of each
compound on LDL-R was estimated on the basis of 100% of
the control group, using the value given by subtracting the
nonspecifically uptaken amount from the calculated fluores-
cence value/protein amount.
(M þ 1); Anal. Calcd for C30H38N4O5S 0.75H2O: C, 62.10; H,
3
6.86; N, 9.66. Found: C, 61.70; H, 6.44; N, 9.59; LC: 96.6%
pure, tR = 11.49 min (Method B).
2,6-Diisopropylphenyl {[4-(3-Propoxyphenyl)-1-pyrimidin-2-
ylpiperidin-4-yl]carbonyl}sulfamate (47). Compound 47 was pre-
pared with 1-propyl iodide in a manner similar to that described
for compound 46 with a yield of 60% as a white solid. Mp
188-190 °C; 1H NMR (DMSO-d6, 60 °C, 300 MHz) δ 0.96 (3H,
t, J = 7.4 Hz), 1.03 (12H, d, J = 6.8 Hz), 1.60 (2H, m), 1.72 (2H,
qt, J = 7.4, 6.5 Hz), 2.50 (2H, m), 3.25 (2H, m), 3.67 (2H, m),
3.88 (2H, t, J = 6.5 Hz), 4.38 (2H, m), 6.52 (1H, dd, J = 4.7,
4.7 Hz), 6.73 (1H, m), 6.97 (2H, m), 7.03 (3H, m), 7.14 (1H, dd,
J = 7.9, 7.9 Hz), 8.31 (2H, d, J = 4.7 Hz); IR (ATR) 1585, 1464,
1363, 1248 cm-1; MS (ESI) m/z 581 (M þ 1); Anal. Calcd for
C31H40N4O5S 2H2O: C, 60.37; H, 7.19; N, 9.08. Found: C,
3
60.07; H, 6.88; N, 9.06; LC: 99.0% pure, tR = 13.97 min
(Method B).
2,6-Diisopropylphenyl ({4-[3-(2-hydroxyethoxy)phenyl]-1-pyri-
midin-2-ylpiperidin-4-yl}carbonyl)sulfamate (48). To a solution of
compound 45 (108 mg, 0.20 mmol) in N,N-dimethylformamide
(1.0 mL) was added 2-bromoethyl tert-butyldimethylsilyl ether
(86.1 μL, 0.40 mmol) and cesium carbonate (196 mg, 0.60 mmol)
at room temperature, and the mixture was stirred at 60 °C for 4 h.
The reaction was quenched by adding water, andthen the mixture
was extracted with ethyl acetate. The organic layer was washed
with brine and dried over anhydrous magnesium sulfate. After
filtration, the solvent was removed in vacuo, and the residue was
purified by preparative TLC to give tert-butyldimethylsilyl ether
derivative (125 mg) as a white solid. To a solution of the tert-
butyldimethylsilyl ether derivative (125 mg, 0.179 mmol) in THF
(2.5 mL) was added 1 M tetrabutylammonium fluoride (540 μL,
0.538 mmol) at room temperature, the mixture was stirred for 2 h.
The reaction was quenched by adding saturated ammonium
chloride solution, and then the mixture was extracted with ethyl
acetate. The organic layer was washed with 0.2 N hydrochloric
acid solution (2x), brine and dried over anhydrous magnesium
sulfate. After filtration, the solventwas removedinvacuo, and the
residue was purified by silica gel column chromatography. The
solvent was removed in vacuo, and the residue was triturated with
diethylether/hexane to give 48 (67.0 mg, 57%) as a white solid.
Mp 143-145 °C; 1H NMR (DMSO-d6, 300 MHz) δ 1.03 (12H, d,
J = 6.8 Hz), 1.87 (2H, m), 2.56 (2H, m), 3.25 (2H, m), 3.34 (2H,
m), 3.69 (2H, t, J = 5.1 Hz), 3.99 (2H, t, J = 5.1 Hz), 4.38 (2H,
m), 6.61 (1H, dd, J = 4.7, 4.7 Hz), 6.88 (1H, m), 6.96 (2H, m), 7.19
(3H, m), 7.29 (1H, dd, J = 8.3, 8.3 Hz), 8.35 (2H, d, J = 4.7 Hz);
IR (ATR) 3510, 1703, 1589, 1495, 1255 cm-1; MS (ESI) m/z 583
Acknowledgment. We are grateful to Ms. K. Bando for
performing the elemental analysis, Ms. I. Taoka for recording
MS spectra, and Mr. T. Ueda for 2D-NMR experiments.
Supporting Information Available: NMR spectroscopic data
for compounds 44 and 46, and MS/MS analytical data for
compound 44. This material is available free of charge via the
Internet at http://pubs.acs.org.
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(M þ 1); Anal. Calcd for C30H38N4O6S 0.25H2O: C, 61.36; H,
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