New low-density lipoprotein receptor upregulators acting via a novel mechanism

Article abstract of DOI:10.1021/jm960153q

The synthesis and biological activity of a new series of benzamides and related compounds that upregulate the expression of the low-density lipoprotein (LDL) receptor in human hepatocytes (HepG2 cells) by a novel mechanism are described. The lead compound, N-[5-[(3- cyclohexylpropionyl)amino]-2-methylphenyl]-4-hydroxybenzamide (1, RPR102359), increased the expression of the LDL receptors in HepG2 cells by 80% when tested at a concentration of 3 μM. Mevinolin (lovastatin) was found to increase the LDL receptor expression by 70% at the same concentration. In contrast to mevinolin, 1 was found to have no effect on cholesterol biosynthesis in liver homogenates or in HepG2 cells at doses where substantial upregulation of the LDL receptor was observed and thus stimulated LDL receptor expression by a novel mechanism.

Full text of DOI:10.1021/jm960153q

J . Med. Chem. 1996, 39, 3343-3356
3343
New Low -Den sity Lip op r otein Recep tor Up r egu la tor s Actin g via a Novel
Mech a n ism
Michael J . Ashton, Thomas J . Brown, Garry Fenton, Frank Halley,* Mark F. Harper, Peter M. Lockey,
Barry Porter, Alan G. Roach, Keith A. J . Stuttle, Nigel Vicker, and Roger J . A. Walsh
Rhoˆne-Poulenc Rorer, Dagenham Research Centre, Rainham Road South, Dagenham, Essex RM10 7XS, U.K.
Received February 26, 1996X
The synthesis and biological activity of a new series of benzamides and related compounds
that upregulate the expression of the low-density lipoprotein (LDL) receptor in human
hepatocytes (HepG2 cells) by a novel mechanism are described. The lead compound, N-[5-[(3-
cyclohexylpropionyl)amino]-2-methylphenyl]-4-hydroxybenzamide (1, RPR102359), increased
the expression of the LDL receptors in HepG2 cells by 80% when tested at a concentration of
3 µM. Mevinolin (lovastatin) was found to increase the LDL receptor expression by 70% at
the same concentration. In contrast to mevinolin, 1 was found to have no effect on cholesterol
biosynthesis in liver homogenates or in HepG2 cells at doses where substantial upregulation
of the LDL receptor was observed and thus stimulated LDL receptor expression by a novel
mechanism.
In tr od u ction
Elevated levels of plasma cholesterol are associated
with increased risk of premature atherosclerosis, par-
ticularly when associated with certain lipoprotein frac-
tions including low-density lipoprotein (LDL).1 The
liver is responsible for clearance of approximately 80%
of LDL from the circulation via specific LDL receptors
on the plasma membrane of hepatocytes.2 On binding
LDL, the receptor is internalized in endocytic vesicles,
the LDL particle is degraded, and the receptor is
recycled. The capacity of the liver to control plasma
LDL levels is critically dependent on the number of LDL
receptors expressed on the cell membrane.
Familial hypercholesterolemia (FH) is a condition
resulting from mutant alleles of the LDL receptor gene.
F igu r e 1. Hypothesis of transcriptional regulation and site
of drug intervention.
Patients show abnormally high levels of plasma LDL
as a consequence of either severely reduced numbers
or a complete absence of hepatic LDL receptors. In the
homozygote, accelerated atherosclerosis can lead to
coronary heart disease even in childhood. Transplanta-
tion of normal liver into an FH patient resulted in a
normalization of plasma cholesterol levels.3 Hence the
liver is the major target organ for hypolipidemic drugs
which act by upregulation of LDL receptor expression.
LDL receptor expression is controlled primarily by
feedback repression of the LDL receptor gene by cho-
lesterol or its metabolites.4 Hepatic cholesterol comes
from two main sources: LDL uptake via the LDL
receptor and synthesis from acetate, the rate-limiting
step of which is catalyzed by 3-hydroxy-3-methylglu-
taryl-coenzyme A (HMG CoA) reductase. Cholesterol
is secreted from the liver as a component of lipoproteins
or in the bile as either neutral or acidic sterols. Excess
cholesterol is esterified and stored in the cytoplasm as
lipid droplets.
or an oxysterol regulates the function of a sterol
response element-binding protein (SREBP) to down-
regulate expression of the LDL receptor and HMG CoA
reductase genes. Consequently inhibition of cholesterol
biosynthesis by HMG CoA reductase inhibitors includ-
ing mevinolin, or inhibition of cholesterol oxidation to
regulatory oxysterols by ketoconazole,5 results in en-
hanced expression of LDL receptors. Additionally evi-
dence now exists for regulation of LDL receptor expres-
sion independent of intracellular cholesterol-regulated
pathways. Enhanced expression of LDL receptors in the
presence of suppressive concentrations of 25-hydroxy-
cholesterol has been observed with insulin,6 growth
factors,7 and cyclic AMP.8 Pharmacological regulation
of hepatic LDL receptor expression by a sterol indepen-
dent mechanism would represent a novel and attractive
therapeutic target for the treatment of hypercholester-
olemia avoiding potential toxic effects of inhibiting
cholesterol synthesis.
The transcription rate of the LDL receptor gene
appears to be the main mechanism by which the level
of receptor expression is controlled. Either cholesterol
itself, possibly in a regulatory pool, or an oxysterol
derivative regulates transcription of the LDL receptor,
HMG CoA reductase, and synthase genes. Cholesterol
The choice of where to target a new drug therapy was
directed by an understanding of the regulatory process
for controlling cellular cholesterol levels. Putative
pathways, involving the SREBP, for the regulation of
LDL receptors and cholesterol biosynthesis are shown
in Figure 1. Several groups have shown interest in
X Abstract published in Advance ACS Abstracts, J uly 1, 1996.
S0022-2623(96)00153-7 CCC: $12.00 © 1996 American Chemical Society

3344 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Ashton et al.
Sch em e 1a
a
Reagents: (i) cyclohexylpropionyl chloride, Et3N, CH2Cl2; (ii) H2-Pd/C, EtOH; (iii) RCOCl, Et3N, CH2Cl2; (iv) NaOH (1 M), EtOH,
room temperature; (v) LiBH4, THF, room temperature; (vi) mCPBA, CHCl3, room temperature.
controlling HMG CoA reductase gene expression using
oxysterols or oxysterol mimetics.9 Indeed Larsen et al.9j
have recently shown that a series of oxysterol mimetics
may suppress HMG CoA reductase gene expression
without concomitant suppression of the LDL receptor
gene. Studies by Kandutsch10 demonstrated a correla-
tion between the binding affinity of oxysterols for an
“oxysterol-binding protein” and repression of HMG CoA
reductase activity. This suggested that compounds may
be discovered which act as antagonists to selectively
enhance LDL receptor gene expression by inhibition of
oxysterol binding to a related SREBP. A recent publi-
cation showed indeed that some sterol derivatives are
capable of upregulating the LDL receptors in CHO cells,
in the presence of 25-hydroxycholesterol.11
Steroidal mimetics seemed more suitable for screen-
ing than oxysterols for the principal reason that they
amine (Schemes 1 and 2). The carbamate 10n and urea
10h were prepared by treating in situ the isocyanate
would have no steroid-related side effects. A series of
derived from the aniline 8a with cyclohexylmethanol
benzamides (e.g., RP 64477, 2) were shown to be
and cyclohexylmethylamine, respectively (Scheme 2).
effective inhibitors of acyl coenzyme A:cholesterol acyl-
The benzamide 12 was prepared in two steps from 1,3-
transferase (ACAT). It was proposed that these com-
phenylenediamine, by successive acylation with 3-cy-
pounds mimic the structure of cholesteryl ester and
clohexylpropionyl chloride (to give 11) and 4-acetoxy-
thereby compete for the binding site of the natural
benzoyl chloride (Scheme 1).
enzyme substrate.12
The retroamide 14 was prepared by coupling 4-ac-
etoxyaniline with 2-methyl-5-nitrobenzoyl chloride fol-
lowed by hydrogenation and acylation of the nitro group
and deprotection of the phenol (Scheme 3). The N-
benzylaniline 15 was prepared by reductive alkylation
of the aniline 5 with 4-acetoxybenzaldehyde using
sodium borohydride (Scheme 4).
Compounds with prototype structure 3 were therefore
chosen from a 2D database search of registry com-
pounds. It was assumed that the phenol group of 3
would substitute for the C-3 hydroxyl or ketone of a
typical oxysterol, such as 25-hydroxycholesterol, and
that the benzanilide would have some topographical
identity with the steroid nucleus. Screening compounds
in a HepG2 whole cell assay and measuring the number
of LDL receptors expressed on the cell surface led to
the discovery of compound 1 (RPR102359) which was
found to increase the expression of the LDL receptors
in HepG2 cells by 80% when tested at 3 µM (the
standard, mevinolin, increased LDL receptor expression
by 70% at 3 µM). The functional activity of the LDL
receptors was confirmed by their ability to internalize
human [125I]LDL.13
The 1,2-diphenylpropenonitriles 20-22 were prepared
by Knoevenagel condensation of the appropriate aryl-
acetonitrile with the benzaldehyde 19 (Scheme 5). The
Z configuration assigned to the product is consistent
1
with NMR spectroscopy. In the H NMR, irradiation
of the vinylic proton of compound 20 gave an NOE effect
on the protons at positions 2 and 4 of the pyridine ring,
indicating a cis relationship between the pyridine ring
and the vinylic proton. In the coupled 13C NMR
spectrum, a coupling constant of 14.5 Hz between the
nitrile carbon and the vinylic proton indicated a trans
relationship between these two groups.
The 2-phenylbenzimidazoles 25-27 were prepared by
treating the appropriate 1,2-benzenediamine with the
benzoyl chloride derived from 24 in the presence of
Ch em istr y
The benzamides 1, 6a -m , 9a ,g,l, and 10b-f,i-k ,m
were prepared in a regiospecific manner by acylation
of the appropriate 3-nitroaniline followed by hydrogena-
tion of the nitro group and acylation of the resulting

New LDL Receptor Upregulators
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3345
Sch em e 2a
a
Reagents: (i) 4-acetoxybenzoyl chloride, Et3N, CH2Cl2; (ii) H2, Pd/C, EtOH; (iii) RCOCl, Et3N, CH2Cl2; (iv) triphosgene, THF, Et3N,
then cyclohexylmethylamine; (v) triphosgene, THF, Et3N, then cyclohexylmethanol; (vi) aqueous NaOH, EtOH; (vii) acetic anhydride.
Sch em e 3a
a
Reagents: (i) SOCl2, CH2Cl2; (ii) 4-acetoxyaniline, Et3N, CH2Cl2; (iii) H2, Pd/C, EtOH; (iv) 3-cyclohexylpropionyl chloride, Et3N, CH2Cl2;
(v) aqueous NaOH, EtOH.
Sch em e 4a
odology starting from 4-methyl-3-nitrobenzoyl chloride
(Scheme 7). The ketones 34 and 35 were prepared in
four steps from toluene via a Friedel-Crafts acylation
followed by nitration, hydrogenation, and acylation
(Scheme 8).
Resu lts a n d Discu ssion
A chemistry program was initiated to increase the in
vitro activity of the lead compound 1. Chemical modi-
fications to 1 have been examined in five main areas of
the molecule as indicated in Figure 2. Each part of the
molecule was systematically modified and the activity
compared to that of 1 as shown in Tables 1-5. Mevi-
nolin was used in the first assays as a standard for the
upregulation of LDL receptors (see Table 1) and was
later replaced by compound 20.
Changes to the 4-hydroxyphenyl group (region 1)
included replacement by aromatic and aliphatic groups,
selections of which are listed in Table 1. The first two
entries (compounds 1 and 6a ) show similar activities,
more likely due to the fact that the acetoxy group is
hydrolyzed in the biological test, presumably by me-
a
Reagents: (i) toluene, PTSA, reflux, then NaBH4, EtOH.
triethylamine at -60 °C to give the N-(2-aminophenyl)-
carboxamide (higher temperatures led to the formation
of the bis-carboxamide). Cyclization to the benzimida-
zole was achieved by heating to 200-220 °C (Scheme
6).
The formation of the retroamide 30 in the right-hand
part of the molecule was achieved by standard meth-

3346 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Ashton et al.
Sch em e 5a
a
Reagents: (i) B2H6, THF; (ii) H2, Pd/C, EtOAc; (iii) 3-cyclohexylpropionyl chloride, Et3N, CH2Cl2; (iv) MnO2, THF, reflux; (v) ArCH2CN,
MeOH, K2CO3, reflux.
Sch em e 6a
a
Reagents: (i) H2, Pd/C, EtOH; (ii) 3-cyclohexylpropionyl chloride, Et3N, CH2Cl2; (iii) SOCl2, toluene, reflux, then 1,2-NH2-Ar, Et3N,
CH2Cl2, -60 °C, then 200-220 °C, neat, 0.5-2 h.
Sch em e 7a
a
Reagents: (i) 2-(1-cyclohexenyl)ethylamine, Et3N, CH2Cl2; (ii) H2, Pd/C, EtOAc; (iii) 4-acetoxybenzoyl chloride, Et3N, CH2Cl2, then
aqueous NaOH, EtOH.
Sch em e 8a
a
Reagents: (i) 4-cyclohexylbutyryl chloride, AlCl3, reflux; (ii) H2SO4, HNO3, -7 °C; (iii) H2, Pd/C, EtOH; (iv) ArCOCl, CH2Cl2, Et3N.
tabolism occurring in the whole cell assay during the
24 h incubation time. (This is also applicable to all
acetoxyphenyl compounds in subsequent tables.) Re-
placement by aliphatic moieties resulted in loss of
activity as indicated for the cyclohexyl derivative 6c.
The phenyl compound 6b was equiactive with 1, and
aromatic groups substituted meta and para to the amide
bond are tolerated with limited functionality (entries
6a ,d ,g,h ,j). In general when the meta and para posi-
tions have electron-donating groups, activity is retained;

New LDL Receptor Upregulators
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3347
Ta ble 2. Effect of Modifications to R (Region 2) in Compound
1 in Vitro in HepG2 Cellsa
upregulation
of the LDL
receptors (%)
F igu r e 2. Five areas for chemical modifications of compound
1.
EC50
(µM)
compd
R
(at 3 µM)
1
9a
cyclohexylethyl
cyclohexylmethylb
cyclohexylpropyl
octyl
80
3
32
-10
-20
-2
25
1.7
Ta ble 1. Effect of Modification of R (Region 1) in Compound 1
in Vitro in HepG2 Cellsa
10b
10c
10d
10e
10f
9g
4
phenethyl
(1-piperidinyl)ethyl
cyclopentylethyl
(cyclohexyloxy)methylb
17
a
b
See footnote a of Table 1. Prepared as the acetate of the
phenol residue.
upregulation
of the LDL
receptors (%) EC50
Ta ble 3. Effect of Modification of R (Region 3) in Compound 1
in Vitro in HepG2 Cellsa
b
compdc
R
(at 3 µM)
(µM)
1
4-hydroxyphenyl
4-acetoxyphenyl
phenyl
80
82
50
0
59
0
1.7
2.5
2
6a
6b
6c
6d
6e
6f
6g
6h
6i
cyclohexyl
3-hydroxyphenyl
2-hydroxyphenyl
4-(methoxycarbonyl)phenyl
4-(hydroxymethyl)phenyl
3,4-(methylenedioxy)phenyl
4-nitrophenyl
4-aminophenyl
3-pyridyl
4-pyridyl
2
3
44
111
11
58
83
-5
7
7.6
0.6
upregulation
of the LDL
receptors (%)
EC50
6j
6k
6l
3.8
1.2
compd
R
(at 3 µM)
(µM)
1
12
Me
80
6
1.7
Hb
6m
4-pyridyl N-oxide
10i
10j
10k
9l
Br
Cl
F
6
8
12
-13
-3
a
Each assay was performed in five replicates, and the results
correspond to a mean value (see the Experimental Section for
b
OMeb
OH
details on the assay). The EC50, here, is the concentration at
which one-half the maximum upregulation is observed and is
measured for compounds increasing the number of LDL receptors
by more than 40% at 3 µM. It was observed in repeat assays that
the EC50 did not vary despite differences between assays in the
degree of maximum upregulation of receptors achieved. c In the
same assay mevinolin gave 70% upregulation at 3 µM with an
EC50 of 0.2 µM.
10m
a
b
See footnote a of Table 1. Prepared as the acetate of the
phenol residue.
size, the cyclopentylethyl derivative 10f only being
weakly active. Replacement of the cyclohexyl group by
a straight alkyl chain (10c) and phenyl (10d ) abolished
activity. Introduction of heteroatoms, like in the (1-
piperidinyl)ethyl 10e and the (cyclohexyloxy)methyl 9g,
led to a loss of activity, indicating a hydrophobic
interaction with the receptor. In this region the cyclo-
hexylethyl moiety appears optimal for activity. This
group has the requisite size and is able to attain a
suitable conformation for hydrophobic interactions.
Substitutions for the methyl group in the central
phenyl ring in region 3 were examined, and selected
examples are collected in Table 3. Removal of the
methyl group gave the unsubstituted analogue 12,
which was inactive. Replacement of the methyl group
by bromine, chlorine, and fluorine abolished activity
(entries 10i-k ). Substitution of the methyl group by
methoxy and hydroxy also resulted in loss of activity
as shown for compounds 9l and 10m . These results
indicate that the methyl group not only is important
for conformational reasons but also has a hydrophobic
interaction. The syntheses of different substituents at
when the positions are substituted with electron-
withdrawing groups, the activity is abolished, as seen
for compounds 6f,i. Substitution at the ortho position
is disfavored (compound 6e) probably due to this posi-
tion having the greatest conformational influence with
respect to the orientation of the aromatic ring relative
to the amide bond. The 3-pyridyl compound 6k was
surprisingly active, while compounds 6l,m were devoid
of activity. The 3,4-(methylenedioxy)phenyl derivative
6h was the most potent compound in this series, being
approximately 3-fold more potent than the lead com-
pound 1.
Modifications to the ethylcyclohexyl group in region
2 are listed in Table 2. Minor changes in this region
have dramatic effects on activity. Altering the alkyl
chain length by one carbon unit severely reduced the
activity as indicated for the cyclohexylmethyl and
cyclohexylpropyl derivatives 9a and 10b. A similar
detrimental effect was observed when changing the ring

3348 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Ashton et al.
Ta ble 4
1. Effect of Modification of the Central Amide (Region 4) in Compound 1 in Vitro in HepG2 Cellsa
compd
X
upregulation of the LDL receptors (%) (at 3 µM)
EC50 (µM)
1
14
15
-CONH-
-NHCO-
-CH2NH-
80
60
-11
1.7
7
2. Effect of Modification of Ar in R,â-Unsaturated Nitrile Compounds Related to 1 in Vitro in HepG2 Cellsa
compd
Ar
upregulation of the LDL receptors (%) (at 3 µM)
EC50 (µM)
20
21
22
3-pyridyl
4-hydroxyphenyl
3,4-(methylenedioxy)phenyl
126
21
108
1
0.7
3. Effect of Modification of R in Benzimidazole Compounds Related to 1 in Vitro in HepG2 Cellsa
compd
R
upregulation of the LDL receptors (%) (at 3 µM)
EC50 (µM)
25
26
27
H
Me
OH
45
91
134
1.3
1.3
0.7
a
See footnote a of Table 1.
that position proved to be lengthy and difficult, and the
priority was given to the investigation of the other parts
of the molecule.
series show comparable activities to the benzamide
series, with compounds 22 and 27 being as potent as
6h .
Modifications to the amide bond in region 4 are shown
in Table 4-1. The reverse amide 14 had slightly reduced
activity compared to 1. Reduction of the amide carbonyl
gave the benzylamine 15 which was devoid of activity.
The amide bonds in both 1 and 14 have the transoid
geometry, which is a conformation unlikely to be favored
by the nonrigid benzylamine 15. The position of the
carbonyl function in the bond linking the two aromatic
rings will also exert a profound electronic effect in this
region, and the directional influence of this electronic
effect is likely to play a role in the activity of a
compound. Other replacements for the amide bond are
depicted in Tables 4-2 and 4-3. The R,â-unsaturated
nitriles 20-22 and the benzimidazoles 25-27 are rigid
families of compounds that both fit well when super-
imposed with the preferred low-energy conformation of
1 (Figure 3). Both the R,â-unsaturated nitriles and the
benzimidazoles would exert a directional electronic
effect similar to that of a carbonyl of an amide moiety,
and activities similar to that of the parent compound 1
are observed. Analogues from these different chemical
Changes to the amide bond in region 5 are listed in
Table 5. Reversal of the amide bond gave compound
30 which was equipotent with 1. Replacement of the
amide bond by urea (10h ) or carbamate (10n ) moieties
abolished activity. The ketomethylene group was a poor
substitute for the amide bond in 1 as indicated for
compound 34. Replacement of the 4-hydroxyphenyl
moiety of 34 with 3-pyridyl gave 35 with improved
efficacy. The same effect was already observed in the
R,â-unsaturated nitriles series (Table 4-2).
Con clu sion
A new series of compounds possessing potent in vitro
activity for upregulating LDL receptors of HepG2 cells
has been discovered, and three new series related to
compound 1 with good activities (exemplified by com-
pounds 20, 27, and 35) have been identified. Further
testing confirmed that compound 1 does not inhibit
cholesterol biosynthesis: neither by direct enzyme
inhibition of cholesterol biosynthesis nor by attenuating
HMG CoA reductase gene transcription.14 Figure 4

New LDL Receptor Upregulators
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3349
F igu r e 3. Stereoplot of an overlay of compounds 1 (yellow), 20 (red), and 27 (blue), showing the good superposition of the amide
linker with the R,â-unsaturated nitriles and the imidazole moieties. The cyclohexylmethyl group is omitted for clarity.
Ta ble 5. Effect of Modification of the Top Amide (Region 5) in
Compound 1 in Vitro in HepG2 Cellsa
upregulation
of the LDL
receptors (%) EC50
compd
Ar
X
(at 3 µM)
(µM)
1
4-hydroxyphenyl -NHCOCH2-
4-hydroxyphenyl -NHCONH-
4-hydroxyphenyl -NHCO2-
4-hydroxyphenyl -CONHCH2-
4-hydroxyphenyl -COCH2CH2-
80
1.7
10h
10n
30
34
35
10 (@ 1 µM)
16 (@ 1 µM)
75
10
2
2
3-pyridyl
-COCH2CH2- 111
a
See footnote a of Table 1.
F igu r e 5. Effect of 25-hydroxycholesterol (2.5 µM) on LDL
receptor expression in HepG2 cells and in the presence of
compound 1 at different concentrations.
ure 5) supports the hypothesis that these compounds
may be antagonists of a SREBP. According to our
knowledge, this is the first example of nonsteroidal
compounds with these properties.
Exp er im en ta l Section
Ch em ica l Meth od s. Melting points were taken on an
Electrothermal melting point apparatus and are uncorrected.
Infrared spectra were recorded in potassium bromide on a
Nicolet 205XB FI spectrometer. Proton NMR spectra were
recorded using a Varian VXR 400 (or a Varian XL 200 when
specified) spectrometer; peak positions are reported in parts
per million relative to internal tetramethylsilane on the δ
scale. Mass spectra were recorded on
a VG 7070E/250
spectrometer. Microanalyses were performed on a Carlo-Erba
1106 microanalyzer. All the reactions were performed at room
temperature unless otherwise stated. All organic solutions
were dried with magnesium sulfate. Yields were not opti-
mized. The acyl chlorides used in the following syntheses were
either purchased or prepared by treating the appropriate acid
with thionyl chloride (1.2 equiv).
F igu r e 4. Effect of mevinolin (1 µM) on LDL receptor
expression in HepG2 cells and in the presence of compound 1
at different concentrations.
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
4-h yd r oxyben za m id e, 1. To a solution of 6a (21 g, 0.05 mol)
in ethanol (210 mL) was added dropwise sodium hydroxide
solution (2 M, 50 mL). The mixture was stirred at room
temperature for 30 min and then poured into water (1 L)
containing 12 mL of concentrated HCl. The resultant white
precipitate was collected by filtration, washed with water, and
dried to give 1 (13.5 g, 71%) as a white solid: mp 171-173 °C
shows that compound 1 is able to produce an additional
degree of upregulation of receptors, in the presence of
a maximally upregulating concentration of mevinolin,
again suggesting that its mechanism of action is not
through inhibition of sterol formation. Compound 1 was
also shown not to stimulate or inhibit ACAT activity in
HepG2 cells. The observation that the increase in LDL
receptor expression still occurs in the presence of an
inhibitory concentration of 25-hydroxycholesterol (Fig-
1
(ethanol); H NMR (CDCl3) 0.85-0.96 (2H, m, C6H11), 1.18-
1.31 (4H, m, C6H11), 1.53-1.76 (7H, m, CH2C6H11), 2.27 (3H,
s, Ar′H), 2.32 (2H, t, J ) 8 Hz, COCH2), 6.92 (2H, d, J o ) 8
Hz, ArH), 7.13 (1H, d, J o ) 8 Hz, Ar′H), 7.55 (1H, dd, J o ) 8

3350 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Ashton et al.
Hz, J m ) 2 Hz, Ar′H), 7.79 (2H, d, J o ) 8 Hz, ArH), 7.84 (1H,
d, J m ) 2 Hz, Ar′H), 8.18 (1H, s, ArCONHAr′), 8.65 (1H, s,
Ar′NHCOR). Anal. (C23H28N2O3) C,H,N.
7.15 (1H, d, J o ) 8 Hz, Ar′H), 7.27-7.46 (3H, m, ArH), 7.66
(1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.92 (1H, d, J m ) 2 Hz,
Ar′H), 8.03 (1H, s, Ar′NHCOR), 8.29 (1H, s, ArCONHAr′).
Anal. (C23H28N2O3) C,H,N.
3-Cycloh e xyl-N -(4-m e t h yl-3-n it r op h e n yl)p r op ion a -
m id e, 4. To a solution of 4-methyl-3-nitroaniline (18.8 g, 0.124
mol) in dichloromethane (250 mL) was added dropwise 3-cy-
clohexylpropionyl chloride (24 g, 0.124 mol) followed by tri-
ethylamine (19 mL, 0.136 mol). After stirring for 1 h at room
temperature, the solution was concentrated in vacuo. The
residue was suspended in 5% sodium bicarbonate solution and
then collected by filtration. The solid was dissolved in dichlo-
romethane and then purified by filtration through silica gel.
Concentration of the filtrate in vacuo gave a solid which was
recrystallized from tert-butyl methyl ether to give 4 (23 g, 64%)
as an off-white solid: mp 133-135 °C; 1H NMR (CDCl3) 0.85-
0.97 (2H, m, C6H11), 1.17-1.32 (4H, m, C6H11), 1.57-1.75 (7H,
m, CH2C6H11), 2.31-2.43 (2H, t, J ) 8 Hz, -COCH2-), 2.54 (3H,
s, CH3Ar), 7.25 (1H, d, J o ) 12 Hz, ArH), 7.76 (1H, dd, J o ) 12
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
2-h yd r oxyben za m id e, 6e. Compound 6e was obtained in
99% yield as the acetate and then hydrolyzed following the
method described for 1 to give a white solid: mp 185-186 °C;
1H NMR (CDCl3) 0.85-0.97 (2H, m, C6H11), 1.18-1.34 (4H,
m, C6H11), 1.56-1.78 (7H, m, CH2C6H11), 2.28 (3H, s, CH3Ar′),
2.35 (2H, t, J ) 8 Hz, COCH2), 6.94 (1H, td, J o ) 8 Hz, J m
)
1 Hz, ArH), 6.99 (1H, dd, J o ) 8 Hz, J m ) 1 Hz, ArH), 7.16
(1H, d, J o ) 8 Hz, Ar′H), 7.40 (1H, td, J o ) 8 Hz, J m ) 1 Hz,
ArH), 7.57 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.90 (1H, d,
J m ) 2 Hz, Ar′H), 7.96 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, ArH),
8.71 (1H, s, Ar′NHCOR), 9.56 (1H, s, ArCONHAr′). Anal.
(C23H28N2O3) C,H,N.
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
4-(m eth oxyca r bon yl)ben za m id e, 6f. Compound 6f was
obtained in 77% yield: mp 220-222 °C (ethoxyethanol); 1H
NMR (DMSO-d6) 0.82-0.94 (2H, m, C6H11), 1.08-1.27 (4H,
m, C6H11), 1.48 (2H, q, J ) 8 Hz, CH2-cyclohexyl), 1.56-1.74
(5H, m, C6H11), 2.17 (3H, s, CH3Ar′), 2.30 (2H, t, J ) 8 Hz,
COCH2), 3.90 (3H, s, CH3CO2Ar), 7.17 (1H, d, J o ) 8 Hz, Ar′H),
Hz, J m ) 3 Hz, ArH), 7.86 (1H, s, ArNHCO), 8.13 (1H, d, J m
3 Hz, ArH). Anal. (C16H22N2O3) C,H,N.
)
3-Cycloh exyl-N-(4-m et h yl-3-a m in op h en yl)p r op ion a -
m id e, 5. A solution of 4 (100 g, 0.35 mol) in ethyl acetate
(1100 mL) was hydrogenated under atmospheric pressure with
10% Pd/C (5 g) for 16 h. The suspension was filtered through
Celite, and the filter was washed with hot ethyl acetate (1000
mL). The combined solution was concentrated in vacuo, and
the residue was triturated with diethyl ether. The resultant
white solid was collected by filtration and then dried in vacuo
to give 5 (82 g, 91%) as a white solid: mp 134-136 °C; 1H
NMR (CDCl3) 0.85-0.98 (2H, m, C6H11), 1.18-1.33 (4H, m,
C6H11), 1.50-1.71 (7H, m, CH2C6H11), 2.11 (3H, s, CH3Ar), 2.32
(2H, t, J ) 8 Hz, COCH2), 3.30 (2H, s, NH2Ar), 6.62 (1H, dd,
J o ) 8 Hz, J m ) 2 Hz, ArH), 6.94 (1H, d, J o ) 8 Hz, ArH), 7.13
(1H, s, ArNHCO), 7.24 (1H, d, J m ) 2 Hz, ArH). Anal.
(C16H24N2O) C,H,N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
6a -f,h ,i,k ,l. To a solution of 5 in dichloromethane was added
the appropriate acyl chloride (1 equiv) followed by triethyl-
amine (1.2 equiv). The solution was stirred at room temper-
ature and the reaction monitored by TLC. Upon evaporation
of the solvent, the residue was washed with water, filtered,
dried, and recrystallized when needed.
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
4-a cetoxyben za m id e, 6a . Compound 6a was obtained in
67% yield: mp 188-191 °C (2-propanol); 1H NMR (200 MHz,
CDCl3) 0.82-1.02 (2H, m, C6H11), 1.08-1.36 (4H, m, C6H11),
1.54-1.82 (7H, m, CH2C6H11), 2.24 (3H, s, CH3Ar′), 2.30 (2H,
t, J ) 8 Hz, COCH2), 2.36 (3H, s, CH3CO2Ar), 7.12 (1H, d, J o
) 8 Hz, Ar′H), 7.18 (2H, d, J o ) 8 Hz, ArH), 7.50 (1H, dd, J o
) 8 Hz, J m ) 2 Hz, Ar′H), 7.72 (1H, d, J m ) 2 Hz, Ar′H), 7.79
(2H, d, J o ) 8 Hz, ArH), 9.04 (1H, s, Ar′NHCO), 9.12 (1H, s,
ArNHCOAr′). Anal. (C25H30N2O4) C,H,N.
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
ben za m id e, 6b. Compound 6b was obtained in 72% yield:
mp 189-191 °C (ethanol); 1H NMR (CDCl3) 0.85-0.92 (2H,
m, C6H11), 1.08-1.32 (4H, m, C6H11), 1.54-1.77 (7H, m,
CH2C6H11), 2.27 (3H, s, CH3Ar′), 2.33 (2H, t, J ) 8 Hz, COCH2),
7.18 (1H, d, J o ) 8 Hz, Ar′H), 7.45-7.58 (4H, m, Ar′H, ArH),
7.83 (1H, d, J m ) 2 Hz, ArH), 7.95 (2H, m, Ar′H), 8.70 (1H, s,
Ar′NHCOR), 8.97 (1H, s, ArNHCOAr′). Anal. (C23H28N2O2)
C,H,N.
7.39 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.66 (1H, d, J m
)
2 Hz, Ar′H), 8.09 (4H, s, ArH), 9.84 (1H, s, Ar′CONHR), 10.82
(1H, s, ArCONHAr′). Anal. (C25H30N2O4) C,H,N.
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
4-(h yd r oxym eth yl)ben za m id e, 6g. To a suspension of 6f
(0.25 g, 0.59 mmol) in THF (5 mL) was added a solution of
LiBH4 (2 M in THF, 1 mL, 2 mmol) under argon. The mixture
was stirred for 7 h and allowed to stand for 16 h. The reaction
was then quenched with a saturated solution of ammonium
chloride (5 mL) at 0 °C. The aqueous solution was extracted
with diethyl ether (20 mL). The organic layer was washed
with water, dried, filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography (eluant
CH2Cl2:MeOH, 19:1) to give 6g (0.15 g, 63%) as a white solid:
mp 165 °C; 1H NMR (DMSO-d6) 0.82-0.94 (2H, m, C6H11),
1.06-1.28 (4H, m, C6H11), 1.48 (2H, q, J ) 8 Hz, CH2C6H11),
1.56-1.74 (5H, m, C6H11), 2.16 (3H, s, CH3Ar′), 2.29 (2H, t, J
) 8 Hz, COCH2), 4.58 (2H, d, J ) 6 Hz, HOCH2Ar), 5.35 (1H,
t, J ) 6 Hz, HOCH2Ar), 7.05 (1H, d, J o ) 8 Hz, Ar′H), 7.38
(1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.45 (2H, d, J o ) 8 Hz,
ArH), 7.55 (1H, d, J m
ArH), 9.82 (1H, s, Ar′NHCOR), 9.85 (1H, s, ArCONHAr′).
Anal. (C24H30N2O3) C,H,N.
) 2 Hz, Ar′H), 7.94 (2H, d, J o ) 8 Hz,
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
3,4-(m eth ylen ed ioxy)ben za m id e, 6h . Compound 6h was
1
obtained in 86% yield: mp 202-203 °C; H NMR (DMSO-d6)
0.82-0.94 (2H, m, C6H11), 1.06-1.27 (4H, m, C6H11), 1.46 (2H,
q, J ) 8 Hz, CH2C6H11), 1.56-1.74 (5H, m, C6H11), 2.15 (3H,
s, CH3Ar′), 2.29 (2H, t, J ) 8 Hz, COCH2), 6.13 (2H, s, OCH2O),
7.04 (1H, d, J o ) 8 Hz, ArH), 7.14 (1H, d, J o ) 8 Hz, Ar′H),
7.37 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.50 (1H, d, J m
)
2 Hz, Ar′H), 7.58 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, ArH), 7.62
(1H, d, J o ) 8 Hz, ArH), 9.70 (1H, s, ArCONHAr′), 9.83 (1H,
s, Ar′NHCOR). Anal. (C24H28N2O4) C,H,N.
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
4-n itr oben za m id e, 6i. Compound 6i was obtained in 65%
1
yield as a white solid: mp 198-200 °C; H NMR (DMSO-d6)
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
cycloh exa n a m id e, 6c. Compound 6c was obtained in 71%
0.82-0.94 (2H, m, C6H11), 1.06-1.28 (4H, m, C6H11), 1.49 (2H,
q, J ) 8 Hz, CH2C6H11), 1.58-1.76 (5H, m, C6H11), 2.18 (3H,
s, CH3Ar′), 2.30 (2H, t, J ) 8 Hz, COCH2), 7.18 (1H, d, J o ) 8
Hz, Ar′H), 7.49 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.69
(1H, d, J m ) 2 Hz, Ar′H), 8.20 (2H, d, J o ) 8 Hz, ArH), 8.38
(2H, d, J o ) 8 Hz, ArH), 9.82 (1H, s, Ar′NHCOR), 10.22 (1H,
s, ArCONHAr′). Anal. (C23H27N3O4) C,H,N.
1
yield: mp 207-208 °C; H NMR (DMSO-d6) 0.82-1.84 (23H,
m, CH2C6H11, C6H10), 1.93 (3H, s, CH3Ar), 2.27 (2H, t, J ) 8
Hz, COCH2), 2.38 (1H, tt, J a ) 12 Hz, J e ) 4 Hz, CHCO), 7.07
(1H, d, J o ) 8 Hz, ArH), 7.35 (1H, dd, J o ) 8 Hz, J m ) 2 Hz,
ArH), 7.58 (1H, d, J m ) 2 Hz, ArH), 9.11 (1H, s, ArNHCOR),
9.77 (1H, s, CyCONHAr). Anal. (C23H34N2O3) C,H,N.
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
4-a m in oben za m id e, 6j. Compound 6i (2 g, 4.9 mmol) was
hydrogenated at atmospheric pressure in ethanol (250 mL)
with 5% Pd/C (0.1 g) until uptake of hydrogen was complete.
The mixture was filtered through Celite and then concentrated
in vacuo. The residue was recrystallized from ethyl acetate
to give 6j (2 g, 63%) as a white solid: mp 195-197 °C; 1H NMR
(DMSO-d6) 0.82-0.94 (2H, m, C6H11), 1.06-1.27 (4H, m,
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
3-h yd r oxyben za m id e, 6d . Compound 6d was obtained in
76% yield as the acetate and then hydrolyzed following the
method described for 1 to give a white solid: mp 213-215 °C;
1H NMR (CDCl3) 0.85-0.98 (2H, m, C6H11), 1.08-1.34 (4H,
m, C6H11), 1.56-1.74 (7H, m, CH2C6H11), 2.27 (3H, s, CH3Ar),
2.34 (2H, t, J ) 8 Hz, COCH2), 7.01-7.06 (2H, m, ArH, ArOH),

New LDL Receptor Upregulators
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3351
C6H11), 1.49 (2H, q, J ) 8 Hz, CH2C6H11), 1.56-1.74 (5H, m,
C6H11), 2.14 (3H, s, CH3Ar′), 2.29 (2H, t, J ) 8 Hz, COCH2),
5.71 (2H, s, NH2Ar), 6.59 (2H, d, J o ) 8 Hz, ArH), 7.11 (1H, d,
J o ) 8 Hz, Ar′H), 7.36 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H),
7.68 (1H, d, J o ) 2 Hz, Ar′H), 7.71 (2H, d, J o ) 8 Hz, ArH),
9.44 (1H, s, ArCONHAr′), 9.80 (1H, s, Ar′NHCOR). Anal.
(C23H29N3O2) C,H,N.
(4.8 g, 82%): 1H NMR (CDCl3) 2.36 (3H, s, CH3CO2Ar), 4.06
(3H, s, CH3OAr′), 6.99 (1H, d, J o ) 8 Hz, Ar′H), 7.26 (2H, d, J o
) 8 Hz, ArH), 7.93 (2H, d, J o ) 8 Hz, ArH), 8.05 (1H, dd, J o
)
8 Hz, J m ) 2 Hz, Ar′H), 8.49 (1H, s, ArCONHAr′), 9.45 (1H, d,
J m ) 2 Hz, Ar′H6).
N-(2-Hyd r oxy-5-n itr op h en yl)-4-a cetoxyben za m id e, 7d .
To a solution of 2-amino-4-nitrophenol (5 g, 32.5 mmol) in
dichloromethane (150 mL) were added 4-acetoxybenzoyl chlo-
ride (13.5 g, 68 mmol) and triethylamine (13 mL, 97 mmol).
The mixture was stirred for 2 h and left to stand for 16 h. The
resultant precipitate was collected by filtration, washed with
water, and dried to give 7d (7.9 g, 77%) as a yellow solid: 1H
NMR (DMSO-d6) 2.31 (3H, s, CH3CO2Ar), 7.09 (1H, d, J o ) 8
Hz, Ar′H), 7.31 (2H, d, J o ) 8 Hz, ArH), 8.01 (1H, dd, J o ) 8
Hz, J m ) 2 Hz, Ar′H), 8.04 (2H, d, J o ) 8 Hz, ArH), 8.75 (1H,
d, J m ) 2 Hz, Ar′H), 9.69 (1H, s, ArCONHAr′).
N-(2-Acetyl-5-n itr op h en yl)-4-a cetoxyben za m id e, 7e. A
suspension of 7d (5 g, 15.8 mmol) in acetic anhydride (250 mL)
was stirred for 6 h and then warmed gently until dissolution
of the remaining solid. The solution was then poured onto ice/
water (2 L) and stirred for 2 h. The resultant precipitate was
collected by filtration, washed with water, and dried to give
7e (5.4 g, 96%) as a white solid: 1H NMR (DMSO-d6) 2.10 (3H,
s, CH3CO2Ar′), 2.34 (3H, s, CH3CO2Ar), 7.41 (2H, d, J o ) 8
Hz, ArH), 6.52 (1H, d, J o ) 8 Hz, Ar′H), 8.05 (1H, dd, J o ) 8
Hz, J m ) 2 Hz, Ar′H), 8.23 (2H, d, J o ) 8 Hz, ArH), 8.96 (1H,
d, J m ) 2 Hz, Ar′H), 9.98 (1H, s, ArCONHAr′).
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
n icotin a m id e, 6k . Compound 6k was obtained as a white
solid in 74% yield: mp 179-180 °C (toluene); 1H NMR (DMSO-
d6) 0.82-0.94 (2H, m, C6H11), 1.15-1.28 (4H, m, C6H11), 1.49
(2H, q, J ) 8 Hz, CH2C6H11), 1.56-1.74 (5H, m, C6H11), 2.18
(3H, s, CH3Ar′), 2.30 (2H, t, J ) 8 Hz, COCH2), 7.17 (1H, d, J o
) 8 Hz, Ar′H), 7.39 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.57
(1H, m, PyH), 7.69 (1H, d, J m ) 2 Hz, Ar′H), 8.31 (1H, m, PyH),
8.76 (1H, m, PyH), 9.13 (1H, d, PyH), 9.87 (1H, s, Ar′NHCOR),
10.08 (1H, s, PyCONHAr′). Anal. (C22H23N3O2) C,H,N.
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
ison icotin a m id e, 6l. Compound 6l was obtained in 79% yield
as a white solid: mp 158-160 °C (toluene); 1H NMR (DMSO-
d6) 0.82-0.94 (2H, m, C6H11), 1.16-1.38 (4H, m, C6H11), 1.49
(2H, q, J ) 8 Hz, CH2C6H11), 1.57-1.74 (5H, m, C6H11), 2.18
(3H, s, CH3Ar′), 2.30 (2H, t, J ) 8 Hz, COCH2), 7.18 (1H, d, J o
) 8 Hz, Ar′H), 7.40 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.68
(1H, d, J m ) 2 Hz, ArH), 7.87 (2H, m, PyH), 8.79 (2H, m, PyH),
9.88 (1H, s, Ar′NHCOR), 10.17 (1H, s, PyCONHAr′). Anal.
(C22H27N3O2) C,H,N.
N-[5-[(3-Cycloh exylpr opion yl)am in o]-2-m eth ylph en yl]-
ison icotin a m id e N-Oxid e, 6m . Compound 6l (0.39 g, 1.07
mmol) was stirred with m-chloroperoxybenzoic acid (0.54 g,
1.6 mmol) in chloroform (40 mL) for 6 h. The organic solution
was washed with sodium hydroxide solution (1 M, 25 mL) and
water and then dried, filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography (eluant
CH2Cl2:MeOH, 9:1) to give 6m (0.2 g, 53%) as a white solid:
mp 216-218 °C; 1H NMR (DMSO-d6) 0.82-0.94 (2H, m,
C6H11), 1.05-1.28 (4H, m, C6H11), 1.48 (2H, q, J ) 8 Hz, -CH2-
cyclohexyl), 1.51-1.74 (5H, m, C6H11), 2.15 (3H, s, CH3Ar′),
2.30 (2H, t, J ) 8 Hz, COCH2), 7.18 (1H, d, J o ) 8 Hz, Ar′H),
N-(5-Am in o-2-m eth ylp h en yl)-4-a cetoxyben za m id e, 8a .
Compound 7a (51 g, 0.162 mol) was hydrogenated at atmo-
spheric pressure in ethyl acetate (1 L) with 5% Pd/C (5 g). The
mixture was shaken for 24 h and then filtered through Celite
and concentrated in vacuo. The residue was triturated with
petroleum ether (500 mL, bp 40-60 °C), collected by filtration,
and dried to give 8a (44 g, 95%) as a white solid: mp 124-
126 °C; 1H NMR (CDCl3) 2.20 (3H, s, CH3Ar′), 2.33 (3H, s, CH3-
CO2Ar), 3.37 (2H, s, NH2Ar′), 6.46 (1H, dd, J o ) 8 Hz, J m ) 2
Hz, Ar′H), 6.98 (1H, d, J o ) 8 Hz, Ar′H), 7.20 (2H, d, J o ) 8
Hz, ArH), 7.46 (1H, d, J m ) 2 Hz, Ar′H), 7.67 (ArCONHAr′),
7.88 (2H, d, J o ) 8 Hz, ArH). Anal. (C16H16N2O3) C,H,N.
N-(5-Am in o-2-br om op h en yl)-4-a cetoxyben za m id e, 8b.
A solution of 7b (8.3 g, 22 mmol) in toluene (100 mL) was
hydrogenated at atmospheric pressure with 5% Pd/C (0.6 g)
for 16 h. The reaction mixture was heated to 100 °C to dissolve
the solid precipitate and then filtered through Celite while hot.
The solvent was concentrated in vacuo and the residue
recrystallized from ethyl acetate to give 8b (3 g, 41%) as a
white solid: 1H NMR (CDCl3) 2.34 (3H, s, CH3CO2Ar), 3.80
(2H, br s, NH2Ar′), 6.36 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H),
7.25 (2 H, d, J o ) 8 Hz, ArH), 7.29 (1H, d, J o ) 8 Hz, Ar′H),
7.95 (2H, d, J o ) 8 Hz, ArH), 8.00 (1H, d, J m ) 2 Hz, Ar′H),
8.36 (1H, s, ArCONHAr′).
N-(5-Am in o-2-m eth oxyph en yl)-4-acetoxyben zam ide, 8c.
Compound 7c (4.5 g, 13.6 mmol) was hydrogenated at atmo-
spheric pressure in ethyl acetate (200 mL) with 5% Pd/C (0.34
g) for 16 h. The reaction mixture was then filtered through
Celite and concentrated in vacuo to give 8c (3.9 g, 95%) as a
brown solid: 1H NMR (CDCl3) 2.34 (3H, s, CH3CO2Ar), 3.85
(3H, s, CH3OAr′), 6.45 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H),
6.75 (1H, d, J o ) 8 Hz, Ar′H), 7.23 (2H, d, J o ) 8 Hz, ArH),
7.91 (2H, d, J o ) 8 Hz, ArH), 8.01 (1H, d, J m ) 2 Hz, Ar′H),
8.52 (1H, s, ArCONHAr′).
N-(2-Acetoxy-5-am in oph en yl)-4-acetoxyben zam ide, 8d.
A suspension of 7e (5.4 g, 14.2 mmol) in ethanol (1 L) was
hydrogenated at atmospheric pressure with 5% Pd/C (1.75 g)
for 16 h. The reaction mixture was then filtered through Celite
and concentrated in vacuo to give 8d (4.9 g, 100%) as a foamy
solid: 1H NMR (DMSO-d6) 1.94 (3H, s, CH3CO2Ar′), 2.32 (3H,
s, CH3CO2Ar), 5.12 (2H, br s, NH2Ar′), 6.35 (1H, dd, J o ) 8
Hz, J m ) 2 Hz, Ar′H), 6.85 (1H, d, J o ) 8 Hz, Ar′H), 7.07 (1H,
d, J m ) 2 Hz, Ar′H), 7.36 (2H, d, J o ) 8 Hz, ArH), 8.15 (2H, d,
J o ) 8 Hz, ArH), 9.25 (1H, s, ArCONHAr′).
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
9a ,g,l. To a solution of 8a or 8c in dichloromethane was added
the appropriate acyl chloride (1 equiv) followed by triethyl-
amine (1.2 equiv). The solution was stirred at room temper-
ature for 3 h and left to stand for 16 h. The solution was
7.48 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.67 (1H, d, J m
)
2 Hz, Ar′H), 7.95 (2H, m, PyH), 8.37 (2H, m, PyH), 9.88 (1H,
s, Ar′NHCOR), 10.11 (1H, s, PyCONHAr′). Anal. (C22H27N3O3)
C,H,N.
N-(2-Meth yl-5-n itr op h en yl)-4-a cetoxyben za m id e, 7a .
To a solution of 2-methyl-5-nitroaniline (37.5 g, 0.246 mol) in
dichloromethane (800 mL) was added 4-acetoxybenzoyl chlo-
ride (50 g, 0.29 mol) in dichloromethane (300 mL) followed by
triethylamine (103 mL, 0.73 mol). The mixture was stirred
for 4 h and left to stand for 16 h. The solution was concen-
trated in vacuo and the residue washed with sodium bicarbon-
ate solution. The solid was collected by filtration, washed with
water, and dried to give 7a (24 g, 33%) as a white solid: mp
1
196-200 °C (ethyl acetate); H NMR (DMSO-d6) 2.32 (3H, s,
CH3Ar′), 2.41 (3H, s, CH3CO2Ar), 7.33 (2H, d, J o ) 8 Hz, ArH),
7.58 (1H, d, J o ) 8 Hz, ArH), 8.1-8.7 (3H, m, ArH, Ar′H), 8.38
(1H, d, J m ) 2 Hz, Ar′H), 10.16 (1H, s, ArCONHAr′).
N-(2-Br om o-5-n itr op h en yl)-4-a cetoxyben za m id e, 7b.
To a solution of 2-bromo-5-nitro aniline (10 g, 46 mmol) in
dichloromethane (250 mL) were added 4-acetoxybenzoyl chlo-
ride (10.1 g, 50 mmol) and triethylamine (7 mL, 50 mmol).
The solution was stirred for 2 h, left to stand for 72 h, and
then washed with saturated sodium bicarbonate solution (100
mL), hydrochloric acid (1 M, 100 mL), and water (100 mL).
The organic solution was dried, filtered, and concentrated in
vacuo to give a solid. This was recrystallized from ethyl
acetate to give 7b (8.3 g, 48%) as a yellow solid: 1H NMR
(DMSO-d6) 2.32 (3H, s, CH3CO2Ar), 7.34 (2H, d, J o ) 8 Hz,
ArH), 8.03-8.1 (4H, m, ArH, Ar′H), 8.49 (1H, m, Ar′H), 10.36
(1H, s, ArCONHAr′).
N-(2-Meth oxy-5-n itr op h en yl)-4-a cetoxyben za m id e, 7c.
To a solution of 2-methoxy-5-nitroaniline (3 g, 18 mmol) in
dichloromethane (80 mL) were added 4-acetoxybenzoyl chlo-
ride (3.9 g, 19.6 mmol) and triethylamine (2.7 mL, 19.6 mmol).
The mixture was stirred for 4 h and left to stand for 16 h. The
reaction mixture was washed with 1 M hydrochloric acid and
water and then dried and concentrated in vacuo to give 7c

3352 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Ashton et al.
concentrated in vacuo; then the residue was washed with
water, filtered, and dried.
N -[2-Me t h yl-5-[(3-p ip e r id in -1-ylp r op ion yl)a m in o]-
p h en yl]-4-h yd r oxyben za m id e, 10e. Compound 8a was
treated with 3-bromopropionyl chloride as described in the
general procedure, using 2.2 equiv of triethylamine. The
resultant amide was dissolved in neat piperidine at room
temperature and then stirred for 2 h. The reaction mixture
was concentrated in vacuo, washed with water, filtered, and
N-[5-[(2-Cycloh exyla cetyl)a m in o]-2-m eth ylp h en yl]-4-
a cetoxyben za m id e, 9a . Compound 9a was obtained in 78%
1
yield as a white solid: mp 179-180 °C; H NMR (DMSO-d6)
0.90-1.03 (2H, m, C6H11), 1.06-1.29 (4H, m, C6H11), 1.57-
1.74 (5H, m, C6H11), 2.17 (5H, m, COCH2, CH3Ar′), 2.31 (3H,
1
dried to give 10e (15%) as a white solid: mp 206-208 °C; H
s, CH3CO2Ar), 7.16 (1H, d, J o ) 8 Hz, Ar′H), 7.29 (2H, d, J o
)
NMR (DMSO-d6) 1.33-1.42 (2H, m, C5H10N), 1.46-1.60 (4H,
m, C5H10N), 2.14 (3H, s, CH3Ar′), 2.30-2.40 (4H, m, C5H10N),
2.43 (2H, t, J ) 8 Hz, CH2C5H10), 2.58 (2H, t, COCH2), 6.86
(2H, d, J o ) 8 Hz, ArH), 7.15 (1H, d, J o ) 8 Hz, Ar′H), 7.35
(1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.61 (1H, d, J m ) 2 Hz,
Ar′H), 7.85 (2H, d, J o ) 8 Hz, ArH), 9.60 (1H, s, Ar′NHCOR),
10.13 (1H, s, ArCONHAr′). Anal. (C22H27N3O3‚1/4H2O) C,H,N.
N-[5-[(3-Cyclopen tylpr opion yl)am in o]-2-m eth ylph en yl]-
4-h yd r oxyben za m id e, 10f. Compound 8a was acylated with
cyclopentylpropionyl chloride and the intermediate acetoxy
hydrolyzed to give 10f (22%) as a white solid: mp 185-188
8 Hz, ArH), 7.39 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.65
(1H, d, J m ) 2 Hz, Ar′H), 8.02 (2H, d, J o ) 8 Hz, ArH), 9.83
(Ar′NHCOR), 9.90 (1H, s, ArCONHAr′). Anal. (C24H28N2O4)
C,H,N.
N -[5-[[2-(C y c lo h e x y lo x y )a c e t y l]a m i n o ]-2-m e t h -
ylp h en yl]-4-a cetoxyben za m id e, 9g. Compound 9g was
1
obtained in 94% yield as a white solid: mp 159-161 °C; H
NMR (CDCl3) 1.22-1.46 (5H, m, C6H10), 1.53-1.60 (1H, m,
C6H10), 1.74-1.84 (2H, m, C6H10), 1.91-2.00 (2H, m, C6H10),
2.30 (3H, s, CH3-Ar′), 2.35 (3H, s, CH3CO2Ar), 3.40 (1H, m,
OCH), 4.06 (2H, s, COCH2O), 7.29 (1H, d, J o ) 8 Hz, Ar′H),
7.39 (2H, d, J o ) 8 Hz, ArH), 7.63 (1H, dd, J o ) 8 Hz, J m ) 2
Hz, Ar′H), 7.70 (Ar′NHCOR), 7.71 (2H, d, J o ) 8 Hz, ArH),
7.79 (1H, d, J m ) 2 Hz, Ar′H), 8.40 (1H, s, ArCONHAr′). Anal.
(C24H28N2O5) C,H,N.
1
°C; H NMR (DMSO-d6) 1.04-1.16 (2H, m, C5H9), 1.42-1.64
(6H, m, CH2C5H9), 1.69-1.81 (3H, m, C5H9), 2.15 (3H, s, CH3-
Ar′), 2.30 (2H, t, J ) 8 Hz, COCH2), 6.86 (2H, d, J o ) 8 Hz,
ArH), 7.13 (1H, d, J o ) 8 Hz, Ar′H), 7.38 (1H, dd, J o ) 8 Hz,
J m ) 2 Hz, Ar′H), 7.62 (1H, d, J m ) 2 Hz, Ar′H), 7.85 (2H, d,
J o ) 8 Hz, ArH), 9.59 (1H, s, Ar′NHCOR), 9.83 (1H, s,
ArCONHAr′), 10.06 (1H, s, HOAr). Anal. (C22H26N2O3) C,H,N.
1-(Cycloh exylm et h yl)-3-[3-(4-h yd r oxyb en za m id o)-4-
m eth ylp h en yl]u r ea , 10h . To a solution of triphosgene (0.99
g, 3.33 mmol) in dichloromethane (150 mL) were added
compound 8a (2.84 g, 10 mmol) and triethylamine (2.8 mL,
20 mmol). The mixture was heated under reflux for 7 h before
addition of cyclohexylmethylamine (2.26 g, 20 mmol). The
reaction mixture was left to stand at room temperature for 16
h and then concentrated in vacuo. The solid residue was
washed with water and hydrolyzed (following the procedure
described for compound 1) to give 10h (3 g, 70%) as a white
solid: mp 210-212 °C; 1H NMR (DMSO-d6) 0.83-0.95 (2H,
m, C6H11), 1.06-1.26 (3H, m, C6H11), 1.32-1.44 (1H, m, C6H11),
1.58-1.72 (5H, m, C6H11), 2.22 (3H, s, CH3Ar), 2.92 (2H, t, J
) 6 Hz, CONHCH2C6H11), 6.08 (1H, t, J ) 6 Hz, ArNH-
CONHR), 6.85 (2H, d, J o ) 8 Hz, Ar′H), 7.06 (1H, d, J o ) 8
Hz, ArH), 7.14 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, ArH), 7.43 (1H,
d, J m ) 2 Hz, ArH), 7.85 (2H, d, J o ) 8 Hz, Ar′H), 8.31 (1H, s,
HOAr′), 9.52 (1H, s, ArNHCONHR), 10.03 (1H, s, Ar′CON-
HAr). Anal. (C22H27N3O3‚1/3H2O) C,H,N.
N-[2-Br om o-5-[(3-cycloh exylp r op ion yl)a m in o]p h en yl]-
4-h yd r oxyben za m id e, 10i. To a solution of 8b (1.4 g, 4
mmol) in dichloromethane (125 mL) were added 3-cyclohexyl-
propionyl chloride (0.77 g, 4.4 mmol) and triethylamine (0.62
mL, 0.44 mmol). The reaction mixture was stirred for 3 h and
then washed with saturated sodium bicarbonate solution (50
mL), hydrochloric acid (1 M, 50 mL), and water (50 mL). The
organic solution was dried, filtered, and concentrated in vacuo
to give a gum. This was dissolved in ethanol (10 mL) and
hydrolyzed (following the method described for the preparation
of compound 1) to give 10i (0.4 g, 22% overall yield) as a white
solid: mp 140-143 °C; 1H NMR (CDCl3) 0.84-0.97 (2H, m,
C6H11), 1.08-1.32 (4H, m, C6H11), 1.54-1.77 (7H, m, CH2C6H11),
2.33 (2H, t, J ) 8 Hz, COCH2), 6.96 (2H, d, J o ) 8 Hz, ArH),
7.47 (1H, d, J o ) 8 Hz, Ar′H), 7.76-7.84 (3H, m, ArH, Ar′H),
8.38 (1H, s, Ar′NHCOR), 8.41 (1H, d, J m ) 2 Hz, Ar′H6), 8.77
(1H, s, ArCONHAr′). Anal. (C22H25BrN2O3) C,H,N.
N -[5-[(3-C y c lo h e x y lp r o p io n y l)a m in o ]-2-m e t h o x y -
p h en yl]-4-a cetoxyben za m id e, 9l. To a solution of 8c (3.7
g, 12.3 mmol) in dichloromethane (90 mL) were added 3-cy-
clohexylpropionyl chloride (2.37 g, 13.5 mmol) and triethyl-
amine (1.88 mL, 13.5 mmol). The mixture was stirred for 4 h
and then washed with 1 M hydrochloric acid and water. The
organic solution was dried, filtered, and concentrated in vacuo
to give 9l (3.4 g, 63%) as a white solid mp 195-196 °C
(ethanol); 1H NMR (DMSO-d6) 0.82-0.95 (2H, m, C6H11), 1.09-
1.26 (4H, m, C6H11), 1.49 (2H, q, J ) 8 Hz, CH2C6H11), 1.57-
1.75 (5H, m, C6H11), 2.29 (2H, t, J ) 8 Hz, COCH2), 2.32 (3H,
s, CH3CO2Ar), 3.80 (3H, s, CH3OAr′), 7.00 (1H, d, J o ) 8 Hz,
Ar′H), 7.29 (2H, d, J o ) 8 Hz, ArH), 7.50 (1H, dd, J o ) 8 Hz,
J m ) 2 Hz, Ar′H), 7.97-8.03 (3H, m, ArH, Ar′H), 9.45 (1H, s,
Ar′NHCOR), 9.79 (1H, s, ArCONHAr′). Anal. (C25H30N2O5)
C,H,N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
10b-f. To a solution of 9 (acetoxy derivative) in ethanol was
added 2 equiv of sodium hydroxide (1 M). The mixture was
stirred at room temperature for 2 h and then concentrated in
vacuo. The residue was diluted with water and acidified to
pH 1 with concentrated HCl. The precipitate was collected
by filtration, washed with water, and dried.
N-[5-[(4-Cycloh exylbu tyr yl)a m in o]-2-m eth ylp h en yl]-4-
h yd r oxyben za m id e, 10b. Compound 10b was obtained as
a white solid: mp 146-147 °C; 1H NMR (DMSO-d6) 0.80-0.91
(2H, m, C6H11), 1.04-1.27 (6H, m, CH2C6H11), 1.53-1.72 (7H,
m, CH2CH2C6H11), 2.15 (3H, s, CH3Ar′), 2.25 (2H, t, J ) 8 Hz,
COCH2), 6.85 (2H, d, J o ) 8 Hz, ArH), 7.13 (1H, d, J o ) 8 Hz,
Ar′H), 7.37 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.62 (1H, d,
J m ) 2 Hz, Ar′H), 7.85 (2H, d, J o ) 8 Hz, ArH), 9.58 (1H, s,
Ar′NHCOR), 9.80 (1H, s, ArCONHAr′), 10.04 (1H, s, HOAr);
MS m/z 395 (MH+), 274 (MH+ - CH2CH2CH2C6H11), 242 (MH+
- HOC6H4CO). Anal. (C24H30N2O3‚3/2H2O) C,H,N.
N-[5-(Non an oylam in o)-2-m eth ylph en yl]-4-h ydr oxyben -
za m id e, 10c. Compound 10c was obtained in 80% yield as a
white solid: mp 160-161 °C; 1H NMR (DMSO-d6) 0.85 (3H, t,
J ) 6 Hz, RCH3) 1.20-1.34 (10H, m, C5H10CH3), 1.53-1.62
(2H, m, COCH2CH2), 2.15 (3H, s, CH3Ar′), 2.28 (2H, t, J ) 8
Hz, COCH2), 6.85 (2H, d, J o ) 8 Hz, ArH), 7.14 (1H, d, J o ) 8
Hz, Ar′H), 7.37 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.62
(1H, d, J m ) 2 Hz, Ar′H), 7.75 (2H, d, J o ) 8 Hz, ArH), 9.60
N-[2-Ch lor o-5-[(3-cycloh exylp r op ion yl)a m in o]p h en yl]-
4-h yd r oxyben za m id e, 10j. To a solution of 4-chloro-3-
nitroaniline (2.07 g, 12 mmol) in toluene (30 mL) were added
3-cyclohexylpropionyl chloride (2.3 g, 13 mmol) and triethyl-
amine (2.5 mL, 18 mmol). The mixture was stirred for 2 h
and heated to 100 °C for a further 2 h. The solvent was
concentrated in vacuo and the residue partitioned between
diethyl ether (100 mL) and hydrochloric acid (1 M, 50 mL).
The organic solution was washed with water, dried, filtered,
and concentrated in vacuo. The solid residue was recrystal-
lized from tert-butyl methyl ether to give beige needles (1.72
g, 46%). This compound (1.6 g, 5.2 mmol) was hydrogenated
in toluene (50 mL) at atmospheric pressure with 5% Pd/C (0.25
g) for 3 h. Ethanol (50 mL) was then added to dissolve the
(1H, s, Ar′NHCOR), 9.83 (1H, s, ArCONHAr′). Anal. (C23H30
-
N2O3) C,H,N.
N-[5-[(3-P h en ylp r op ion yl)a m in o]-2-m et h ylp h en yl]-4-
h yd r oxyben za m id e, 10d . Compound 10d was obtained in
55% yield as a white solid: mp 215-217 °C; 1H NMR (DMSO-
d6) 2.15 (3H, s, CH3Ar′), 2.61 (2H, t, J ) 8 Hz, CH2Ph), 2.90
(2H, t, J ) 8 Hz, COCH2), 6.85 (2H, d, J o ) 8 Hz, ArH), 7.15
(1H, d, J o ) 8 Hz, Ar′H), 7.17-7.31 (5H, m, Ph), 7.37 (1H, dd,
J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.61 (1H, d, J m ) 2 Hz, Ar′H),
7.85 (2H, d, J o ) 8 Hz, ArH), 9.60 (1H, s, Ar′NHCOR), 9.88
(1H, s, ArCONHAr′). Anal. (C23H22N2O3) C,H,N.

New LDL Receptor Upregulators
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3353
solid precipitate, and the mixture was filtered through Celite.
The filtrate was concentrated in vacuo to give a light brown
solid (1.4 g, 97%). To a solution of this solid (1.3 g, 4.6 mmol)
in dichloromethane (30 mL) were added 4-acetoxybenzoyl
chloride (1 g, 5.1 mmol) and triethylamine (0.8 mL, 5.8 mmol).
The solution was stirred for 2 h, left to stand for 24 h, and
then washed with hydrochloric acid (1 M, 100 mL) and water
(100 mL), dried, and filtered. Concentration in vacuo gave a
brown viscous oil which was dissolved in ethanol (10 mL) and
hydrolyzed (following the method described for the preparation
of compound 1) to give 10j (0.6 g, 36%) as a white solid: mp
8 Hz, ArH), 6.67 (1H, d, J o ) 8 Hz, ArH), 7.06 (1H, t, J o ) 8
Hz, ArH), 7.15-7.27 (2H, m, ArH, ArNHCOR).
N-[3-[(3-Cycloh exylp r op ion yl)a m in o]p h en yl]-4-a ce-
toxyben za m id e, 12. To a solution of 11 (2 g, 8.1 mmol) in
dichloromethane (100 mL) and triethylamine (1.16 mL, 11
mmol) was added 4-acetoxybenzoyl chloride (1.8 g, 9 mmol).
The mixture was stirred for 5 h and then treated with
saturated sodium bicarbonate solution (75 mL). The phases
were separated, and the organic layer was washed with water
and dried. Concentration of the organic solution left a gum,
which, after trituration with cyclohexane, gave 12 (2.7 g, 82%)
as a white solid: mp 149-150 °C; 1H NMR (CDCl3) 0.84-0.96
(2H, m, C6H11), 1.07-1.33 (4H, m, C6H11), 1.54-1.76 (7H, m,
CH2C6H11), 2.30 (2H, t, J ) 8 Hz, COCH2), 2.35 (3H, s, CH3-
CO2Ar), 7.15 (2H, d, J o ) 8 Hz, ArH), 7.23-7.30 (1H, m, Ar′H),
1
147-149 °C; H NMR (DMSO-d6) 0.82-0.94 (2H, m, C6H11),
1.16-1.29 (4H, m, C6H11), 1.50 (2H, q, J ) 8 Hz, CH2C6H11),
1.57-1.75 (5H, m, C6H11), 2.32 (2H, t, J ) 8 Hz, COCH2), 6.87
(2H, d, J o ) 8 Hz, ArH), 7.42 (1H, d, J o ) 8 Hz, Ar′H), 7.52
(1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.86 (2H, d, J o ) 8 Hz,
ArH), 7.91 (1H, d, J m ) 2 Hz, Ar′H), 9.70 (1H, s, Ar′NHCOR),
10.06 (1H, s, ArCONHAr′). Anal. (C22H25ClN2O3) C,H.N.
N-[5-[(3-Cycloh exylp r op ion yl)a m in o]-2-flu or op h en yl]-
4-h yd r oxyben za m id e, 10k . Compound 10k was prepared
using a similar route to compound 10j and obtained as a white
solid in 51% overall yield: mp 103-105 °C; 1H NMR (DMSO-
d6) 0.82-0.94 (2H, m, C6H11), 1.08-1.29 (4H, m, C6H11), 1.49
(2H, q, J ) 8 Hz, CH2C6H11), 1.56-1.75 (5H, m, C6H11), 2.30
(2H, t, J ) 8 Hz, COCH2), 6.86 (2H, d, J o ) 8 Hz, ArH), 7.19
(1H, t, J ) 8Hz, Ar′H), 7.40-7.48 (1H, m, Ar′H), 7.82-7.90
(3H, m, ArH, Ar′H), 9.80 (1H, s, Ar′NHCOR), 9.95 (1H, s,
ArCONHAr′). Anal. (C22H25FN2O3) C,H,N.
7.38-7.44 (2H, m, Ar′H), 7.52 (1H, s, Ar′H), 7.82 (2H, d, J o
)
8 Hz, ArH), 7.94 (1H, s, Ar′NHCOR), 8.13 (1H, s, ArCONHAr′).
Anal. (C24H28N2O4) C,H,N.
2-Meth yl-5-n itr o-N-(4-a cetoxyp h en yl)ben za m id e, 13.
To a solution of 4-acetoxyaniline (4.14 g, 27.4 mmol), in
dichloromethane (200 mL), were added 2-methyl-5-nitroben-
zoyl chloride (6.6 g, 33 mmol) and triethylamine (3 mL, 41
mmol). The reaction mixture was stirred for 5 h. The
resultant solid was filtered and dried to give 13 (6 g, 71%) as
a white solid: 1H NMR (CDCl3) 2.30 (3H, s, CH3CO2Ar), 2.60
(3H, s, CH3Ar), 7.07 (2H, d, J o ) 8 Hz, Ar′H), 7.45 (1H, d, J o
) 8 Hz, ArH), 7.80 (2H, d, J ) 8 Hz, Ar′H), 8.19 (1H, dd, J o
)
8 Hz, J m ) 2 Hz, ArH), 8.38 (1H, d, J m ) 2 Hz, ArH), 9.98
(1H, s, Ar′NHCOAr).
N-[2-Hydr oxy-5-[(3-cycloh exylpr opion yl)am in o]ph en yl]-
4-h yd r oxyben za m id e, 10m . To a solution of 8d (2.4 g, 7.3
mmol) in dichloromethane (75 mL) were added 3-cyclohexyl-
propionyl chloride (1.5 g, 8.7 mmol) and triethylamine (1.5 mL,
11 mmol). The mixture was stirred for 6 h and left to stand
for 16 h. The reaction mixture was washed with hydrochloric
acid (1 M, 50 mL) and water (50 mL) and then dried, filtered,
and concentrated in vacuo to give an off-white solid. This was
dissolved in ethanol (85 mL) and hydrolyzed (following the
method described for the preparation of compound 1) to give
10m (0.7 g, 30% overall yield) as a white solid: mp 209-216
°C; 1H NMR (DMSO-d6) 0.82-0.94 (2H, m, C6H11), 1.09-1.28
(4H, m, C6H11), 1.48 (2H, q, J ) 8 Hz, CH2C6H11), 1.57-1.75
(5H, m, C6H11), 2.27 (2H, t, J ) 8 Hz, COCH2), 6.81 (1H, d, J o
) 8 Hz, Ar′H3), 6.88 (2H, d, J o ) 8 Hz, ArH), 7.28 (1H, dd, J o
) 8 Hz, J m ) 2 Hz, Ar′H), 7.84 (2H, d, J o ) 8 Hz, ArH), 7.95
(1H, d, J m ) 2 Hz, Ar′H), 9.35 (1H, s, Ar′NHCOR), 9.70 (1H,
s, ArCONHAr′). Anal. (C22H26N2O4) C,H,N.
Cycloh exylm eth yl N-[3-(4-Hydr oxyben zam ido)-4-m eth -
ylp h en yl]ca r ba m a te, 10n . To a solution of triphosgene (0.99
g, 3.33 mmol) in THF (150 mL) were added compound 8a (2.84
g, 10 mmol) and triethylamine (2.8 mL, 20 mmol). The
reaction mixture was heated under reflux for 5 h and then
cooled to room temperature. Upon addition of cyclohexyl-
methanol (2.3 g, 20 mmol), the mixture was heated under
reflux for 2 h and then left to stand at room temperature for
16 h. The solution was concentrated in vacuo, and the residue
was triturated with diethyl ether to give a solid. This was
hydrolyzed (following the procedure described for compound
1) to give 10n (1.2 g, 30%) as a white solid: mp 112-115 °C;
1H NMR (CDCl3) 0.91-1.03 (2H, m, C6H11), 1.10-1.31 (4H,
m, C6H11), 1.59-1.80 (5H, m, C6H11), 2.24 (3H, s, CH3Ar), 3.95
(2H, d, J ) 6 Hz, OCH2C6H11), 6.08 (1H, t, J ) 6 Hz, OCH2CH),
6.79 (1H, s, HOAr′), 6.86 (2H, d, J o ) 8 Hz, Ar′H), 7.12 (1H, d,
J o ) 8 Hz, ArH), 7.20 (1H, br s, ArNHCO2R), 7.33 (1H, s,
Ar′CONHAr), 7.64-7.73 (3H, m, ArH, Ar′H), 7.36 (1H, d, J m
) 2 Hz, ArH). Anal. (C22H26N2O4‚1/5H2O) C,H,N.
2-Met h yl-5-[(3-cycloh exylp r op ion yl)a m in o]-N-(4-h y-
d r oxyp h en yl)ben za m id e, 14. A solution of 13 (5.5 g, 17.5
mmol) was hydrogenated at atmospheric pressure in ethyl
acetate (200 mL) with 5% Pd/C (0.5 g) for 24 h. The reaction
mixture was then filtered through Celite and concentrated in
vacuo to give a solid which was dissolved in dichloromethane
(80 mL). To this solution were added triethylamine (2.5 mL,
18 mmol) and 3-cyclohexylpropionyl chloride (3 g, 17.5 mmol).
The mixture was stirred for 3 h, washed with 1 M hydrochloric
acid, sodium bicarbonate solution, and water, and then dried
and concentrated in vacuo to give a brown gum. This was
hydrolyzed (following the method described for the preparation
of compound 1) to give 14 (1.65 g, 25% overall yield) as a white
solid: mp 215 °C; 1H NMR (DMSO-d6) 0.83-0.92 (2H, m,
C6H11), 1.06-1.28 (4H, m, C6H11), 1.49 (2H, q, J ) 8 Hz,
CH2C6H11), 1.56-1.74 (5H, m, C6H11), 2.26-2.35 (5H, m, CH3-
Ar, COCH2), 6.72 (2H, d, J o ) 8 Hz, Ar′H), 7.18 (1H, d, J o ) 8
Hz, ArH), 7.50 (2H, d, J o ) 8 Hz, Ar′H), 7.58 (1H, dd, J o ) 8
Hz, J m ) 2 Hz, ArH), 7.64 (1H, d, J m ) 2 Hz, ArH), 9.22 (1H,
s, Ar′OH), 9.92 (1H, s, Ar′CONHAr), 10.03 (1H, s, ArNHCOR).
Anal. (C23H28N2O3‚1/3H2O) C,H,N.
3-Cycloh exyl-N-[3-[(4-h yd r oxyben zyl)a m in o]-4-m eth -
ylp h en yl]p r op ion a m id e, 15. To compound 5 (4.5 g, 17.3
mmol) in toluene (210 mL) were added 4-acetoxybenzaldehyde
(5.67 g, 34.5 mmol) and p-toluenesulfonic acid (50 mg). The
mixture was heated under reflux with a Dean-Stark ap-
paratus for 3 h and then concentrated in vacuo. The oily,
yellow residue was dissolved in ethanol (400 mL); then sodium
borohydride (1.5 g, 39 mmol) was added portionwise. The
reaction mixture was stirred for 2 h, left to stand for 16 h,
and then concentrated in vacuo to give a solid. This was
triturated with ethyl acetate, collected by filtration, and dried
1
to give 15 (3.5 g, 50%) as a white solid: mp 171-172 °C; H
NMR (DMSO-d6) 0.80-0.92 (2H, m, C6H11), 1.08-1.25 (4H,
m, C6H11), 1.43 (2H, q, J ) 8 Hz, CH2C6H11), 1.56-1.72 (5H,
m, C6H11), 2.05 (3H, s, CH3Ar), 2.21 (2H, t, J ) 8 Hz, COCH2),
4.16 (2H, br d, Ar′CH2NHAr), 5.31 (1H, br t, Ar′CH2NHAr)
6.55-6.84 (5H, m, ArH), 7.15 (2H, d, J o ) 8 Hz, Ar′H), 9.20
(1H, s, Ar′OH), 9.44 (1H, s, ArNHCOR). Anal. (C23H30N2O2‚1/
2H2O) C,H,N.
3-(3-Cycloh exylp r op ion a m id o)a n ilin e, 11. To a stirred
solution of 1,3-phenylenediamine (3.25 g, 30 mmol) in dichlo-
romethane (100 mL) was added 3-cyclohexylpropionyl chloride
(1.75 g, 10 mmol). The mixture was stirred for 5 h and left to
stand for 16 h. A solution of saturated sodium bicarbonate
(150 mL) was added to the mixture, and the phases were
separated. The organic phase was washed with water, dried,
and concentrated in vacuo to give 11 (2.1 g, 85%) as an off-
white solid: 1H NMR (CDCl3) 0.82-0.98 (2H, m, C6H11), 1.06-
1.34 (4H, m, C6H11), 1.49-1.78 (7H, m, CH2C6H11), 2.33 (2H,
2-(Hyd r oxym eth yl)-4-n itr otolu en e, 16. A solution of
diborane in THF (800 mL, 1 M diborane in THF, 0.8 mol) was
added over 1 h to a solution of 2-methyl-5-nitrobenzoic acid
(100 g, 0.55 mol) in THF (400 mL), cooled to 0 °C under
nitrogen. The stirred reaction mixture was allowed to reach
room temperature overnight. The mixture was then cooled
to 0 °C again, and water (500 mL) was added over 30 min,
t, J ) 8 Hz, COCH2), 3.72 (2H, br s, ArNH2), 6.42 (1H, d, J o
)

3354 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Ashton et al.
followed by sodium hydroxide solution (400 mL, 1 M). The
resultant solid was collected by filtration, washed with water,
and dried to give 16 (86 g, 92%); 1H NMR (CDCl3) 2.00 (1H, t,
J ) 6 Hz, CH2OH), 2.24 (3H, s, CH3Ar), 4.78 (2H, d, CH2OH),
7.21 (1H, d, J o ) 8 Hz, ArH), 8.15 (1H, dd, J o ) 8 Hz, J m ) 2
Hz, ArH), 8.30 (1H, d, J m ) 2 Hz, ArH).
3-(Hyd r oxym eth yl)-4-m eth yla n ilin e, 17. Compound 16
(76 g, 0.45 mol) was hydrogenated at atmospheric pressure in
ethyl acetate (750 mL) with 5% Pd/C (9.5 g) for 24 h. The
resulting mixture was boiled and then filtered through Celite
while hot. The filtrate was concentrated in vacuo and the
residue triturated with diethyl ether to give a solid. This was
collected by filtration and dried to give 17 (62 g, 87%) as a
white solid: mp 106-111 °C; 1H NMR (CDCl3) 2.22 (3H, s,
CH3Ar), 3.58 (2H, br s, ArNH2), 4.61 (2H, s, ArCH2OH), 6.56
(1H, dd, J o ) 8 Hz, J m ) 2 Hz, ArH), 6.74 (1H, d, J m ) 2 Hz,
ArH), 6.86 (1H, d, J o ) 8 Hz, ArH).
22 was obtained in 76% yield as an off-white solid: mp 179-
181 °C; 1H NMR (DMSO-d6) 0.82-0.95 (2H, m, C6H11), 1.06-
1.29 (4H, m, C6H11), 1.50 (2H, q, J ) 8 Hz, CH2C6H11), 1.56-
1.75 (5H, m, C6H11), 2.26-2.36 (5H, m, CH3Ar, COCH2), 6.12
(2H, s, OCH2O), 7.06 (1H, d, J o ) 8 Hz, ArH), 7.20-7.27 (2H,
m, ArH, Ar′H), 7.46 (1H, d, J m ) 2 Hz, Ar′H), 7.62 (1H, dd, J o
) 8 Hz, J m ) 2 Hz, Ar′H), 7.96 (1H, d, J m ) 2 Hz, ArH), 8.01
(1H, s, olefinic-H), 9.96 (1H, s, ArNHCOR). Anal. (C26H28N2O4)
C,H,N.
5-Am in o-2-m eth ylben zoic Acid , 23. 2-Methyl-5-nitroben-
zoic acid (1.8 g, 10 mmol) in ethanol (30 mL) was hydrogenated
at atmospheric pressure with 5% Pd/C (0.2 g). The mixture
was stirred for 1 h and then filtered through Celite. Concen-
tration of the filtrate in vacuo gave 2315 (1.5 g, 100%) as a
white solid: mp 193-197 °C.
5-[(3-Cycloh exylp r op ion yl)a m in o]-2-m et h ylb en zoic
Acid , 24. To a solution of 23 (1.5 g, 10 mmol) in dichlo-
romethane (35 mL) were added 3-cyclohexylpropionyl chloride
(1.75 g, 10 mmol) and triethylamine (3 mL, 22 mmol). The
reaction mixture was stirred for 1 h and left to stand for 16 h.
The mixture was washed with water, dried, and concentrated
in vacuo to give a solid. This was recrystallized from aqueous
ethanol to give 24 (2.65 g, 91%) as a white solid: mp 221-
223 °C; 1H NMR (DMSO-d6) 0.71-0.95 (2H, m, C6H11), 1.06-
1.29 (4H, m, C6H11), 1.49 (2H, q, J ) 8 Hz, CH2C6H11), 1.56-
1.74 (5H, m, C6H11), 2.30 (2H, t, J ) 8 Hz, COCH2), 2.45 (3H,
s, CH3-Ar), 7.20 (1H, d, J o ) 8 Hz, ArH), 7.68 (1H, dd, J o ) 8
Hz, J m ) 2 Hz, ArH), 8.08 (1H, d, J m ) 2 Hz, ArH), 9.94 (1H,
s, ArNHCOR). Anal. (C17H23NO3‚1/5H2O) C,H,N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
25-27. To a solution of the appropriate 1,2-arylenediamine
in dichloromethane, at -60 °C, were added 5-(3-cyclohexyl-
propionamido)-2-methylbenzoylchloride (0.5 equiv, prepared
by reacting 24 with 1 equiv of thionyl chloride in toluene under
reflux for 8 h) and triethylamine (2 equiv). The mixture was
stirred for 15 min and allowed to reach room temperature. The
solution was then washed with water, dried over magnesium
sulfate, and filtered. The filtrate was concentrated in vacuo
and the residue triturated with ethyl acetate to give an off-
white solid. This was heated to 200 °C neat for 30 min. The
residue was boiled in acetonitrile and then treated with
charcoal and filtered through Celite while hot. The filtrate
was concentrated in vacuo to give a solid which was collected
by filtration and dried.
N-[3-(1H-Ben zim id a zol-2-yl)-4-m eth ylp h en yl]-3-cyclo-
h exylp r op ion a m id e, 25. Compound 25 was obtained in 20%
yield as a light brown solid: mp 197-200 °C; 1H NMR (CDCl3)
0.83-0.96 (2H, m, C6H11), 1.07-1.32 (4H, m, C6H11), 1.52-
1.76 (7H, m, CH2C6H11), 2.30-2.41 (5H, m, COCH2, CH3Ar),
6.98 (1H, d, J o ) 8 Hz, ArH), 7.29-7.37 (3H, m, ArH), 7.63
(1H, d, ArH), 7.61-7.70 (2H, m, ArH), 8.55 (1H, s, ArNHCOR).
Anal. (C23H27N3O) C,H,N.
N-[3-(5-Meth yl-1H-ben zim idazol-2-yl)-4-m eth ylph en yl]-
3-cycloh exylp r op ion a m id e, 26. Compound 26 was obtained
in 22% overall yield as an off-white solid: mp 157-159 °C; 1H
NMR (CDCl3) 0.80-0.93 (2H, m, C6H11), 1.05-1.28 (4H, m,
C6H11), 1.54 (2H, q, J ) 8 Hz, -CH2C6H11), 1.59-1.73 (5H, m,
C6H11), 2.25 (3H, s, CH3Ar), 2.31 (2H, t, J ) 8 Hz, COCH2),
2.47 (3H, s, CH3-benzimidazole), 6.90 (1H, d, J o ) 8 Hz, ArH),
7.09 (1H, d, J ) 8 Hz, ArH), 7.29-7.38 (3H, m, ArH), 7.49
(1H, d, J ) 8 Hz, ArH), 8.56 (1H, s, ArNHCOR). Anal.
(C24H29N3O) C,H,N.
N-[3-(Hyd r oxym eth yl)-4-m eth ylp h en yl]-3-cycloh exyl-
p r op ion a m id e, 18. To a solution of 17 (49 g, 0.36 mol) in
dichloromethane (1 L) was added 3-cyclohexylpropionyl chlo-
ride (62.6 g, 0.36 mol) and triethylamine (51 mL, 0.36 mol).
The reaction mixture was stirred for 1 h and then washed with
water (940 mL). The organic layer was separated, dried,
filtered, and concentrated in vacuo to give 18 (86 g, 87%) as a
white solid: mp 121-125 °C; 1H NMR (CDCl3) 0.85-0.98 (2H,
m, C6H11), 1.08-1.34 (4H, m, C6H11), 1.55-1.81 (8H, m,
CH2C6H11, ArCH2OH), 2.28 (3H, s, CH3Ar), 2.33 (2H, t, J ) 8
Hz, COCH2), 4.63 (2H, s, ArCH2OH), 7.10 (1H, d, J o ) 8 Hz,
ArH), 7.32 (1H, s, ArNHCOR), 7.37 (1H, dd, J o ) 8 Hz, J m
2 Hz, ArH), 7.46 (1H, d, J m ) 2 Hz, ArH).
)
3-Cycloh exyl-N-(3-for m yl-4-m et h ylp h en yl)p r op ion a -
m id e, 19. To a solution of 18 (83.7 g, 0.3 mol) in THF (1 L)
was added activated manganese dioxide (85 g, 0.97 mol)
portionwise. The reaction mixture was stirred for 4 h at room
temperature and then heated to 60 °C for 2 h, left to stand at
room temperature for 16 h, and filtered through Celite. The
solvent was concentrated in vacuo and the residue purified
by flash column chromatography on silica gel (eluant CH2Cl2:
MeOH, 98:2). Concentration of the solvent in vacuo yielded a
residue which was triturated with diethyl ether to give 19 (59
1
g, 71%) as a white solid: mp 134-137 °C; H NMR (CDCl3)
0.86-0.99 (2H, m, C6H11), 1.08-1.35 (4H, m, C6H11), 1.59-
1.78 (7H, m, CH2C6H11), 2.40 (2H, t, J ) 8 Hz, COCH2), 2.62
(3H, s, CH3Ar), 7.21 (1H, d, J o ) 8 Hz, ArH), 7.51 (1H, s,
ArNHCOR), 7.78 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, ArH), 7.87
(1H, d, J m ) 2 Hz, ArH2), 10.22 (1H, s, ArCHO).
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
20-22. To a solution of 19 in methanol (10 mL) were added
the appropriate arylacetonitrile (1 equiv) and potassium
carbonate (2 equiv). The mixture was heated under reflux for
2 h and then cooled to room temperature and diluted with
ethyl acetate. The mixture was washed with water, dried,
filtered, and concentrated in vacuo to give a yellow solid which
was triturated with diethyl ether.
N-[3-[2-Cya n o-2-(3-p yr id yl)vin yl]-4-m et h ylp h en yl]-3-
cycloh exylp r op ion a m id e, 20. Compound 20 was obtained
in 51% yield as a white solid: mp 140-141 °C; 1H NMR
(CDCl3) 0.86-0.99 (2H, m, C6H11), 1.08-1.35 (4H, m, C6H11),
1.59-1.78 (7H, m, CH2C6H11), 2.30 (3H, s, CH3Ar), 2.42 (2H,
t, J ) 8 Hz, COCH2), 7.22 (1H, d, J o ) 8 Hz, ArH), 7.43 (1H,
m, PyH), 7.55 (1H, br s, PyH), 7.75-7.88 (2H, s, olefinic-H,
ArH), 7.91 (1H, d, J m ) 2 Hz, ArH), 7.98 (1H, m, PyH), 8.66
(1H, m, PyH), 8.93 (1H, s, ArNHCOR). Anal. (C24H27N3O)
C,H,N.
N -[3-(5-H y d r o x y -1H -b e n z i m i d a z o l-2-y l)-4-m e t h -
ylp h en yl]-3-cycloh exylp r op ion a m id e, 27. Starting from
4-hydroxy-1,2-phenylenediamine (freshly prepared by hydro-
genation of 3-nitro-4-aminophenol at atmospheric pressure, in
ethanol with 5% Pd/C), compound 27 was obtained in 12%
N -[3-[2-Cya n o-2-(4-h yd r oxyp h e n yl)vin yl]-4-m e t h yl-
p h en yl]-3-cycloh exylp r op ion a m id e, 21. Compound 21 was
1
obtained in 37% yield as a cream solid: mp 186-187 °C; H
1
NMR (DMSO-d6) 0.82-0.95 (2H, m, C6H11), 1.06-1.29 (4H,
m, C6H11), 1.49 (2H, q, J ) 8 Hz, CH2C6H11), 1.56-1.75 (5H,
m, C6H11), 2.29 (3H, s, CH3-Ar), 2.32 (2H, t, J ) 8 Hz, COCH2),
6.89 (2H, d, J o ) 8 Hz, Ar′H), 7.21 (1H, d, J o ) 8 Hz, ArH),
7.57-7.65 (3H, m, ArH, Ar′H), 7.89 (1H, s, olefinic-H), 7.93
(1H, d, J m ) 2 Hz, ArH), 9.95 (1H, s, Ar′OH), 9.96 (1H, s,
ArNHCOR). Anal. (C25H28N2O2‚1/5H2O) C,H,N.
overall yield as an off-white solid: mp 167-175 °C; H NMR
(DMSO-d6) 0.83-0.96 (2H, m, C6H11), 1.06-1.30 (4H, m,
C6H11), 1.50 (2H, q, J ) 8 Hz, -CH2-cyclohexyl), 1.57-1.76
(5H, m, C6H11), 2.33 (2H, t, J ) 8 Hz, COCH2), 2.47 (3H, s,
CH3Ar), 6.78 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, benzimidazole-
H), 7.30 (1H, d, J o ) 8 Hz, ArH), 7.46 (1H, d, J o ) 8 Hz,
benzimidazole-H), 7.56 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, ArH),
8.02 (1H, d, J m ) 2 Hz, ArH), 9.36 (1H, s, benzimidazole-H),
10.02 (1H, s, ArNHCOR). Anal. (C23H27N3O2‚H2O) C,H,N.
N-[3-[2-Cya n o-2-[3,4-(m eth ylen ed ioxy)p h en yl]vin yl]-4-
m eth ylph en yl]-3-cycloh exylpr opion am ide, 22. Compound

New LDL Receptor Upregulators
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3355
4-Me t h yl-3-n it r o-3-[(cycloh e xe n -1-yle t h yl)a m in o]-
ben za m id e, 28. To a solution of 4-methyl-3-nitrobenzoyl
chloride (6 g, 30 mmol; prepared from 4-methyl-3-nitrobenzoic
acid and 3 equiv of thionyl chloride in toluene under reflux
for 2 h) were added 2-(cyclohexen-1-yl)ethylamine (3.75 g, 30
mmol) and triethylamine (6 g, 60 mmol). The mixture was
stirred for 1 h and then left to stand for 16 h. The reaction
mixture was washed with hydrochloric acid (1 M, 100 mL),
saturated sodium bicarbonate (200 mL), and water and then
dried, filtered, and concentrated in vacuo. The residue was
triturated with diethyl ether and collected by filtration to give
28 (6.8 g, 79%) as a white solid: 1H NMR (CDCl3) 1.53-1.69
(4H, m, C6H9), 1.94-2.08 (4H, m, C6H9), 2.27 (2H, t, J ) 8 Hz,
CH2C6H9), 2.96 (3H, s, CH3Ar), 3.54 (2H, m, CONHCH2R), 5.55
(1H, m, olefinic-H), 6.21 (1H, br s, ArCONHR), 7.43 (1H, d, J o
) 8 Hz, ArH), 7.91 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, ArH), 8.30
(1H, d, J m ) 2 Hz, ArH).
3-Am in o-4-m et h yl-N-[(cycloh exylet h yl)a m in o]ben za -
m id e, 29. Compound 28 (6.8 g, 23.6 mmol) was hydrogenated
at atmospheric pressure in ethyl acetate (175 mL) with 5%
Pd/C (0.7 g) for 16 h. The reaction mixture was then warmed
and filtered through Celite while hot. The filtrate was
concentrated in vacuo, and the residue was triturated with
diethyl ether to give 29 (5.1 g, 83%) as a yellow solid: 1H NMR
(CDCl3) 0.86-1.01 (2H, m, C6H11), 1.09-1.27 (4H, m, C6H11),
1.45-1.78 (7H, m, CH2C6H11), 2.19 (3H, s, CH3-Ar), 3.45 (2H,
m, CONHCH2R), 6.05 (1H, br, s, ArCONHR), 6.94 (1H, dd, J o
) 8 Hz, J m ) 2 Hz, ArH), 7.08 (1H, d, J o ) 8 Hz, ArH), 7.14
(1H, d, J m ) 2 Hz, ArH).
COCH2), 7.46 (1H, d, J o ) 8 Hz, ArH), 8.09 (1H, dd, J o ) 8
Hz, J m ) 2 Hz, ArH), 8.52 (1H, d, J m ) 2 Hz, ArH); MS m/z
190 (MH+).
4-Cycloh e xyl-1-(3-a m in o-4-m e t h ylp h e n yl)b u t a n -1-
on e, 33. Compound 32 was hydrogenated at atmospheric
pressure in ethanol (75 mL) with 5% Pd/C (0.43 g) for 16 h.
The mixture was then filtered through Celite and concentrated
in vacuo to give a brown solid. This was purified by flash
column chromatography on silica gel (eluant dichloromethane)
to give 33 (1.86 g, 48%) as a yellow solid: 1H NMR (CDCl3)
0.80-0.95 (2H, m, C6H11), 1.07-1.32 (6H, m, COCH2CH2-
CH2C6H11), 1.58-1.78 (7H, m, CH2C6H11), 2.21 (3H, s, CH3-
Ar), 2.87 (2H, t, J ) 8 Hz, COCH2), 3.75 (2H, br s, ArNH2),
7.11 (1H, d, J o ) 8 Hz, ArH), 7.5-7.31 (2H, m, ArH).
N -[5-(4-Cycloh e xylb u t yr yl)-2-m e t h ylp h e n yl]-4-h y-
d r oxyben za m id e, 34. To a solution of compound 33 (4 g, 15
mmol) in dichloromethane (400 mL) were added 4-acetoxy-
benzoyl chloride (3.37 g, 17 mmol) and triethylamine (5.6 mL,
40 mmol). The solution was left to stand for 24 h and then
washed with water, dried, filtered, and concentrated in vacuo
to give a yellow solid. This was triturated with diethyl ether
to give a white solid which was collected by filtration and
hydrolyzed (following the procedure described for compound
1) to give 34 (1.6 g, 30% overall yield) as a white solid: mp
177 °C; 1H NMR (DMSO-d6) 0.72-0.99 (2H, m, C6H11), 1.08-
1.29 (6H, m, COCH2CH2CH2C6H11), 1.56-1.72 (7H, m, CH2-
C6H11), 2.29 (3H, s, CH3Ar), 2.95 (2H, t, J ) 8 Hz, COCH2),
6.86 (2H, d, J o ) 8 Hz, ArH), 7.40 (1H, d, J o ) 8 Hz, Ar′H),
7.74 (1H, dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 7.88 (2H, d, J o ) 8
Hz, ArH), 7.91 (1H, d, J m ) 2 Hz, Ar′H), 9.71 (1H, s,
ArCONHAr′). Anal. (C24H29NO3) C,H,N.
N-[5-(4-Cycloh exylbu tyr yl)-2-m eth ylp h en yl]p yr id in e-
3-ca r boxa m id e, 35. To a solution of compound 33 (1.86 g,
7.2 mmol) in dichloromethane (300 mL) were added nicotinoyl
chloride hydrochloride (1.28 g, 7.9 mmol) and triethylamine
(2.6 mL, 19 mmol). The solution was stirred for 5 h and then
washed with water, dried, filtered, and concentrated in vacuo.
The residue was triturated with pentane to give a white solid
which was recrystallized (toluene:cyclohexane, 3:1) to give 35
(0.95 g, 36%) as a white solid: mp 93-94 °C; 1H NMR (CDCl3)
0.82-0.94 (2H, m, C6H11), 1.11-1.31 (6H, m, COCH2CH2-
CH2C6H11), 1.60-1.81 (7H, m, CH2C6H11), 2.41 (3H, s, CH3-
Ar), 2.95 (2H, t, J ) 8 Hz, COCH2), 7.35 (1H, d, J o ) 8 Hz,
Ar′H), 7.50 (1H, m, PyH), 7.35 (1H, s, PyCONHAr′), 7.78 (1H,
dd, J o ) 8 Hz, J m ) 2 Hz, Ar′H), 8.26 (1H, m, PyH), 8.47 (1H,
br s, Ar′H), 8.83 (1H, m, PyH), 9.14 (1H, br s, PyH). Anal.
(C23H28N2O2) C,H,N.
Biologica l Meth od s. Ma ter ia ls. HepG2 cells were ob-
tained from ECCAC at Porton Down. All cell culture reagents
were obtained from Gibco BRL. MAC-C7, a mouse anti-human
LDL receptor monoclonal, was produced by biofermentation
of the C7 cell clone. All other antibodies were obtained from
Boehringer Mannheim. 25-Hydroxycholesterol was obtained
from Sigma Ltd. ATTAPHOS was obtained from Millipore.
Cell Cu ltu r e. Cells were maintained in Dulbecco’s modi-
fied Eagle’s medium supplemented with fetal calf serum in
75 cm2 culture flasks in a humidified 5% CO2 atmosphere at
37 °C and subcultured every 3-4 days. Cells beyond passage
10 were not used in assays.
LDL Recep tor Assa y. LDL receptors were quantitated
by a fluorescence method using the monoclonal antibody MAC-
C7. HepG2 cells were plated in 96-well culture plates at
10 000 cells/well and grown for 3 days to 80% confluence;
compounds were added in DMEM containing 1% bovine serum
albumin (BSA) and incubated for 24 h. The culture medium
was aspirated, and the cell monolayers were washed with
phosphate-buffered saline (PBS) and fixed with 3% formalde-
hyde at 4 °C. The formaldehyde solution was removed, and
the cells were washed with ammonium sulfate solution fol-
lowed by PBS. MAC-C7 solution, 0.5 µg/mL in PBS containing
10% FCS, was added and incubated for 60 min at 4 °C.
Following removal of the MAC-C7 solution and washing with
PBS, the monolayers were incubated with an anti-mouse IgG
monoclonal antibody conjugated to alkaline phosphatase and
incubated for 60 min at 4 °C. The second antibody solution
was removed, and the alkaline phosphatase substrate AT-
3-(4-H yd r oxyb en za m id o)-4-m et h yl-N-[(2-cycloh exyl-
eth yl)a m in o]ben za m id e, 30. To a solution of 4-acetoxyben-
zoyl chloride (0.76 g, 3.87 mmol) in dichloromethane (100 mL)
were added compound 29 (1 g, 3.87 mmol) and triethylamine
(0.86 g, 8.5 mmol). The mixture was stirred for 3 h, left to
stand for 16 h, and then washed with hydrochloric acid (1 M,
75 mL), saturated sodium bicarbonate (75 mL), and water (75
mL). The organic solution was filtered and then concentrated
in vacuo to give a solid. This was hydrolyzed (following the
method described for compound 1) to give 30 (0.3 g, 20%
1
overall) as a white solid: mp 211-212 °C; H NMR (DMSO-
d6) 0.83-0.96 (2H, m, C6H11), 1.09-1.27 (4H, m, C6H11), 1.42
(2H, q, J ) 8 Hz, CH2C6H11), 1.57-1.77 (5H, m, C6H11), 2.25
(3H, s, CH3-Ar), 3.28 (2H, m, CONHCH2R), 6.87 (2H, d, J o
8 Hz, Ar′H), 7.33 (1H, d, J o ) 8 Hz, ArH), 7.64 (1H, dd, J o
)
)
8 Hz, J m ) 2 Hz, ArH), 7.70 (1H, d, J m ) 2 Hz, ArH), 7.78
(2H, d, J o ) 8 Hz, Ar′H), 8.38 (1H, br t, ArCONHR), 9.75 (1H,
s, Ar′CONHAr). Anal. (C23H28N2O3‚2/3H2O), C,H,N.
4-Cycloh exyl-1-(4-m eth ylp h en yl)bu ta n -1-on e, 31. To a
suspension of aluminum chloride (27.87 g, 0.2 mol) in dry
toluene (425 mL) was added 4-cyclohexylbutyryl chloride (38.6
g, 0.2 mol; prepared from 4-cyclohexylbutyric acid and 1.5
equiv of thionyl chloride at room temperature for 56 h) in dry
toluene (270 mL). The mixture was heated under reflux for
1.5 h, cooled to room temperature, and then washed with water
(5 × 250 mL), dried, filtered, and concentrated in vacuo to give
a yellow oil. This was purified by distillation under reduced
pressure (bp 142-146 °C, 0.2 mbar) to give 31 (42.5 g, 85%)
as a colorless oil which solidified on standing: 1H NMR (CDCl3)
0.82-0.96 (2H, m, C6H11), 1.07-1.31 (6H, m, COCH2CH2-
CH2C6H11), 1.49-1.79 (7H, m, CH2C6H11), 2.40 (3H, s, CH3-
Ar), 2.91 (2H, t, J ) 8 Hz, COCH2), 7.25 (2H, d, J o ) 8 Hz,
ArH), 7.85 (2H, d, J o ) 8 Hz, ArH).
4-Cycloh exyl-1-(4-m eth yl-3-n itr op h en yl)bu ta n -1-on e,
32. Compound 31 (38 g, 0.16 mol) was added portionwise to
a stirred solution of concentrated sulfuric acid (86 mL) and
concentrated nitric acid (70 mL), maintained at -7 °C. After
addition, the mixture was stirred for 20 min at -7 °C and then
poured onto ice. The resultant yellow oil was extracted with
diethyl ether (3 × 200 mL). The combined ether extracts were
washed with water, dried, filtered, and concentrated in vacuo
to give an oil. This was purified by filtration through silica
gel (eluant cyclohexane:ethylacetate, 3:1) to give 32 (42.3 g,
93%) as a yellow oil: 1H NMR (CDCl3) 0.82-0.97 (2H, m,
C6H11), 1.08-1.31 (6H, m, COCH2CH2CH2C6H11), 1.60-1.81
(7H, m, CH2C6H11), 2.68 (3H, s, CH3Ar), 2.96 (2H, t, J ) 8 Hz,

3356 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Ashton et al.
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added with propidium iodide at 20 µg/mL. The culture plates
were incubated at 4 °C for 1 h and the reaction terminated by
addition of 6 M NaOH. Fluorescence readings at 436 and 590
nm were taken in a Cytofluor 2400 fluorescence plate reader;
an increase in fluorescence reading denoted an increase in
MAC-C7 binding and hence LDL receptor numbers. Cell
numbers per well were determined by propidium iodide
fluorescence at 530 and 640 nm.
Ack n ow led gm en t. We are grateful to Barry Wy-
man, Imtiaz Ahmed, and Clare Howells for technical
assistance, Iain McLay for modeling studies, Michael
Podmore, Mark Vine, and Don Daley for spectroscopy,
and Anne Stevens for microanalysis. We are also
grateful to Anne White, J ulia Lloyd, and Melanie Wong
for the in vitro assays on HepG2 cells.
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