Studies on scavenger receptor inhibitors. Part 1: Synthesis and structure-activity relationships of novel derivatives of sulfatides

Article abstract of DOI:10.1016/S0968-0896(02)00120-7

Scavenger receptors have been proven to be implicated in the formation of atherosclerotic lesions. A series of novel derivatives of sulfatides were synthesized, and their inhibitory activities against incorporation of DiI-acetyl-LDL into macrophages were

Full text of DOI:10.1016/S0968-0896(02)00120-7

Bioorganic & Medicinal Chemistry 10 (2002) 2445–2460
Studies on Scavenger Receptor Inhibitors.
Part 1: Synthesis and Structure–Activity Relationships of
Novel Derivatives of Sulfatides
Kazuya Yoshiizumi,* Fumio Nakajima, Rika Dobashi, Noriyasu Nishimura and
Shoji Ikeda
R & D Laboratories, Nippon Organon K.K., 1-5-90, Tomobuchi-cho, Miyakojima-ku, Osaka 534-0016, Japan
Received 26 October 2001; accepted 8 April 2002
Abstract—Scavenger receptors have been proven to be implicated in the formation of atherosclerotic lesions. A series of novel
derivatives of sulfatides were synthesized, and their inhibitory activities against incorporation of DiI-acetyl-LDL into macrophages
were evaluated in order to clarify the structure–activity relationships of sulfatides as a scavenger receptor inhibitor and find out
novel inhibitors with synthetic easiness. The chemical modification of the substructures of sulfatides led to the establishment of the
following structure–activity relationships; (1) the ceramide moiety can be replaced with another structure bearing two long chains, (2) the
galactose moiety can be replaced with another structure or be deleted without a large decrease in the inhibitory activity, (3) the sulfate
moiety was crucial, and it was the most preferable functional group for a potent inhibitory activity. The inhibitory activity of (S)-2-octa-
decanoylamino-2-tetradecylcarbamoyl)ethyl sulfate sodium salt (3a) against incorporation of DiI-acetyl-LDL into macrophages was
proven to be based on the inhibition against the binding of acetyl-LDL to the surface of macrophages. We discovered novel scavenger
receptor inhibitors with synthetic easiness, such as (S)-2-octadecanoylamino-2-(tetradecylcarbamoyl)ethyl sulfate sodium salt (3a) and 2-
octadecanoylamino-1-(octadecanoylaminomethyl)ethyl sulfate sodium salt (13q). # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
of cholesterol ester in the macrophages. Modified LDLs
are recognized by special molecules, scavenger recep-
tors, expressed in the surface of the macrophages. After
the binding of modified LDLs to scavenger receptors,
the formed complexes are delivered to lysosomes by
endocytosis. In lysosomes, modified LDLs are degra-
dated to their protein portion and cholesterol ester por-
tion, and the latter is further hydrolyzed to free
cholesterol by a lysosomal cholesterol esterase. Free
cholesterol generated in lysosomes is then transported
into cytoplasm, where it is re-esterified to cholesterol
ester by acyl-coenzyme A/cholesterol acyltransferase
(ACAT), leading to cholesterol ester-rich lipid droplets
(Fig. 1).
Atherosclerosis in early stage is characterized by the
presence of foam cells in the arterial intima. Foam cells,
which are filled with cholesterol ester-rich lipid droplets,
lead to atherosclerotic plaques. It is generally accepted
that the formation of the foam cells begins with the
attachment of circulating monocytes to the lumenal
surface of endothelial cells. Then, the monocytes
migrate into the subendothelial space, where they dif-
ferentiate into macrophages. The resulting macrophages
uptake lipoproteins, and accumulate a large amount of
cholesterol ester derived from the lipoproteins, which
results in the formation of the foam cells.1ꢀ4
Various evidences have demonstrated that modified low
density lipoproteins (modified LDLs), not native low
density lipoprotein, are responsible for the accumulation
The presence of scavenger receptors which can incor-
porate acetyl-LDL, one of modified LDLs, was pre-
dicted by Brown and Goldstein 23 years ago.5 Then,
Kodama etal. succeeded in hte cloning of com-
plementary DNA of scavenger receptor for acetyl-LDL
in 1990, which led to the clarification of the sub-
structures of the scavenger receptor.6,7 After that, novel
scavenger receptors which can recognize modified LDLs
have been identified by several research groups,8ꢀ15 and
*Corresponding author at current address: Medicinal Chemistry
Department, Organon Laboratories Ltd., Newhouse, Motherwell,
Lanarkshire ML1 5SH, UK. Tel.: +44-1698-736000; fax: +44-1698-
736001; e-mail: k.yoshiizumi@organon.nhe.akzonobel.nl
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00120-7

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K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
now the scavenger receptors cloned by Kodama et al.
are called class A scavenger receptor.
because sulfatides is a mixture of structurally related
compounds. Chemical synthesis enables us to obtain a
certain component of sulfatides, but there have been
some problems in the chemical synthesis of sulfatides.
One of them was the difficulty of the synthesis of the
ceramide moiety which has two asymmetric carbons and
one olefin group. Although many synthetic routes for
ceramide have been reported,19ꢀ21 all of them still have
drawbacks such as many steps, low yield, and/or
formation of isomer.
If the binding of modified LDLs to scavenger receptors
is blocked, it could be thought that the accumulation of
cholesterol ester and the subsequent formation of foam
cells do nothappen, because modified LDLs are not
incorporated into the macrophages. Therefore, com-
pounds with an inhibitory activity against the binding
of modified LDLs to scavenger receptors (referred to as
scavenger receptor inhibitor) should be effective in the
treatment of atherosclerosis. Recently, Suzuki et al.
reported that class A scavenger receptor/apolipoprotein
E double-knockoutmice developed 60% smaller aht er-
osclerotic lesions than single-apolipoprotein E knockout
littermates.16 This was the first in vivo evidence that
class A scavenger receptors are implicated in formation
of atherosclerotic lesions, meaning that class A sca-
venger receptor inhibitors would prevent the progress of
atherosclerosis.
By the way, the structure–activity relationships (SARs)
of sulfatides as scavenger receptor inhibitor have not
been investigated yet, so it was unclear which sub-
structures of sulfatides are important for its inhibitory
activity. Herein, at first, we report the synthesis of novel
derivatives of sulfatides. In addition, we describe the
SARs of sulfatides as a scavenger receptor inhibitor.
Chemistry
Before the cloning of the scavenger receptors by
Kodama etal., Brown and Goldstein had already found
that negatively charged macromolecules, such as poly
vinyl sulfate, polyG, and maleyl-LDL, inhibited the
binding of 125I-acetyl-LDL to the surface of macro-
phages.17 Moreover, they discovered that sulfatides, a
mixture of negatively charged glycolipids having two
long chains (Fig. 2), also exhibited the inhibitory activ-
ity.17 Therefore, sulfatides can be one of scavenger
receptor inhibitors with low molecular weight.18 How-
ever, we considered that the use of sulfatides itself for
the treatment of atherosclerosis would be impractical,
The synthetic route for the peptide derivative 1a was
outlined in Scheme 1. 2,3,4,6-Tetra-O-acetyl-a-d-galac-
topyranosyl bromide 15 was treated with N-(tert-
butoxycarbonyl)-l-serine benzyl ester 16 in the presence
of silver carbonate, silver perchlorate, and molecular
sieves to give b-glycoside 17. Removal of tert-butoxy-
carbonyl (Boc) group of compound 17 with tri-
fluoroacetic acid (TFA) afforded amine 18, which was
acylated with octadecanoyl chloride. The obtained N-
octadecanoyl serine benzyl ester 19 was hydrogenated in
the presence of palladium–carbon (Pd–C), followed by
condensation with tetradecylamine by use of 1-ethyl-3-
[3-(dimethylamino)propyl]carbodiimide hydrochloride
(EDCI) and 1-hydroxybenzotriazole hydrate (HOBt) to
yield peptide 21. Removal of the four acetyl groups of
compound 21 was achieved with sodium methoxide in
methanol. According to the procedure reported by
22
Guilbertetal.,
the obtained tetraol 22 was treated
with di-n-butyltin oxide, followed by sulfation with sul-
fur trioxide-trimethylamine complex to afford the target
compound 1a, regioselectively.
Scheme 2 shows the synthetic route for the galactose-
deleted derivatives of compound 1a. To begin with,
condensation of N-Boc-O-benzyl-l-serine 23 with alkyl-
amine R2NH2 gave N-Boc-serinamides 24. The Boc
group of compounds 24 was removed by use of TFA to
give amines 25. Acylation of 25 with acyl chloride
R1COCl to yield N-acyl-O-benzyl-serinamides 26.
Hydrogenolysis of compounds 26 over Pd/C afforded
Figure 1. Schematic progress of atherosclerosis.
Figure 2. Structure of sulfatides.

K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
2447
alcohols 8. Finally, compounds 8 were sulfated with
chlorosulfonic acid in pyridine, followed by treatment
with sodium carbonate to give the target compounds 3a
and 3c–j. The enantiomer of 3a, compound 3b, was
prepared from N-Boc-O-benzyl-d-serine in the same
manner as compound 3a (scheme notshown).
sulfate 4a. Removal of the benzyl group of compound
27a by hydrogenolysis over Pd–C gave carboxylic acid
6a. Sulfoxide 10a was oxidized with 3-chloroperoxy-
benzoic acid (m-CPBA) to give sulfone 11a.
Scheme 5 shows the synthetic route for thiol 9a and
ethylthiopropyl sulfate 5a. Atfirst, N-Boc-l-cystine 30n
was converted to cystinamide 30a in three steps. The
disulfide bond of 30a was cleaved reductively with tri-
butylphosphine to yield N-octadecanoylcysteinamide
9a. Treatment of 9a with 3-bromopropanol in the pre-
sence of Na2CO3, followed by sulfation of the resulting
alcohol 31a to afford 3-ethylthiopropyl sulfate 5a.
Three peptides 10a, 27a, and 28a were prepared from
the corresponding N-Boc-amino acids (10n, 27n, and
28n) in the same manner as compound 26, as shown in
Scheme 3. Then, O-benzyl-N-octadecanoyltyrosinamide
28a was hydrogenated, and then sulfated in a harder
condition to afford compound 2a (Scheme 4). The ben-
zyl ester of compound 27a was reduced with sodium
borohydride in methanol–tetrahydrofuran, followed by
sulfation of the resulting alcohol 29a to afford butyl
Compounds 12j and 13q–s were synthesized according
to the routes shown in Scheme 6. 2-Aminoethanol 32 or
Scheme 1. Reagents: (a) N-(tert-butoxycarbonyl)-l-serine benzyl ester (16), Ag2CO3, AgClO4, molecular sieves; (b) TFA; (c) ClCO(CH2)16CH3,
Et3N; (d) Pd/C (10%), H2; (e) H2N(CH2)13CH3, EDCI, HOBt; (f) NaOMe; (g) (i) n-Bu2SnO; (ii) SO3–Et3N.
Scheme 2. Reagents: (a) H2NR2, EDCI, HOBt; (b) TFA; (c) ClCOR1, iPr2EtN; (d) Pd/C (10%), H2; (e) (i) ClSO3H, pyridine; (ii) Na2CO3.

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K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
Scheme 3. Reagents: (a) H2N(CH2)13CH3, EDCI, HOBt; (b) TFA; (c) ClCO(CH2)16CH3, iPr2EtN.
Scheme 4. Reagents: (a) Pd/C (10%), H2; (b) (i) ClSO3H, pyridine; (ii) Na2CO3; (c) NaBH4, MeOH; (d) mCPBA.
Scheme 5. Reagents: (a) H2N(CH2)13CH3, EDCI, HOBt; (b) TFA; (c) ClCO(CH2)16CH3, iPr2EtN; (d) n-Bu3P; (e) K2CO3, BrCH2CH2CH2OH; (f)
(i) ClSO3H, pyridine; (ii) Na2CO3.

K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
2449
1,3-diamino-2-propanol 34 was acylated, and then
followed by sulfation to give the target compounds.
bearing two long chains (Table 1, compound 1a). We
chose octadecanoyl group [CO(CH2)16CH3] and tetra-
decyl group [(CH2)13CH3] as two long chains of 1a. The
peptide derivative 1a was slightly less potent than sul-
fatides, but it showed a moderate inhibitory activity. We
concluded that the ceramide moiety can be replaced
with the peptide structure.
Results and Discussion
For the purpose of monitoring the inhibitory activity of
the novel derivatives of sulfatides against acetyl-LDL
binding to scavenger receptors, all of them were
tested for inhibitory activity against incorporation of
1,10 -dioctadecyl-3,3,30,30 -tetramethylindocarbocyanine
perchlorate-labeled acetyl-LDL (DiI-acetyl-LDL) into
macrophages at the concentration of 100 mg/mL or
100 mM at 37 ꢁC. When a tested compound showed
more than 40% inhibitory activity, the compound was
also tested in lower concentrations. The obtained results
are summarized in Tables 1–3.
Next, compound 2a which has a phenylene moiety
instead of the galactose moiety was prepared (Table 2).
It exhibited almost the same potent activity as 1a, which
means that the galactose moiety is not necessary for a
potent inhibitory activity, and it can be replaced with
another structure like the phenylene moiety. The effect
of the 4-ethylphenyl moiety of 2a on the inhibitory
activity was investigated next (Table 2). Ethyl sulfate 3a
and butyl sulfate 4a showed a potent inhibitory activity
comparable to that of 2a. The potency of 3-ethylthio-
propyl sulfate 5a was weak compared to that of 2a.
These results indicate that the ethylphenyl group of 2a
can be replaced with another group which is not so long
as 3-ethylthiopropyl group.
Sulfatides consists of three substructures: sulfate moi-
ety, galactose moiety, and ceramide moiety (Fig. 2). At
first, we synthesized a novel derivative in which the cer-
amide moiety was replaced with a peptide structure
Scheme 6. Reagents: (a) ClCOCH[(CH2)13CH3]2, iPr2EtN; (b) (i) ClSO3H, (ii) Na2CO3; (c) ClCOR3, Et3N.
Table 1. Effects of sulfatides and its related compounds on incorporation of DiI-acetyl-LDL into macrophages
Compound
Structure
Inhibitory activity against incorporation of DiI-acetyl-LDL (%)a
100 mg/mL
30 mg/mL
10 mg/mL
3 mg/mL
Sulfatides
83.2(3)
77.4(3)
56.2(3)
<20(3)
1a
99.7(3)
63.1(2)
NT
<20(3)
NTb
NT
Galactosylceramide
<20(1)
NT
aThe number on the left side of parentheses represents% inhibition at each concentration. The number inside parentheses represents the number of
experiment.
bNT, not tested.

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K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
Our next interest was focussed on the effect of the sul-
fate group of 3a on the inhibitory activity. Interestingly,
the carboxylic acid derivative 6a did notshow a poetnt
inhibitory activity at 100 mM (Table 2), even though
maleyl-LDL bearing carboxylic acids as negatively
charged functional groups have been known to be a
scavenger receptor inhibitor.17 Galactosylceramide
(Table 1), the phenol derivative 7a, and the hydroxy
derivative 8a (Table 2) exhibited a weak inhibitory
activity of less than 30%. Conversion of the sulfate
group of 3a into another sulfur-containing functional
group did not lead to the discovery of compounds with
more potent inhibitory activity (9a, 10a, and 11a). We
can summarize these results as follows; among the
functional groups investigated here, sulfate group was
the most preferable for a potent inhibitory activity
against incorporation of DiI-acetyl-LDL into macro-
phages.
Then, in order to elucidate whether the stereochemistry
of the asymmetric carbon of 3a is critical for the inhibi-
tory activity, the enantiomer of 3a (compound 3b) was
evaluated for the inhibitory activity. As shown in Table 3,
3b showed almost the same inhibitory activity as that of
3a, meaning that the stereochemistry of the asymmetric
carbon does not affect the inhibitory activity.
Next, we investigated the modification of the two long
chains of 3a. The replacement of the octadecanoyl
group with a shorter group, such as acetyl or decanoyl,
weakened the potency (3a vs 3c, 3d). The conversion of
the tetradecyl group into propyl group also caused a
Table 2. Effects of compound 1a and its related compounds on incorporation of DiI-acetyl-LDL into macrophages
Compd
X
Inhibitory activity against incorporation of DiI-acetyl-LDL(%)a
100 mM
30 mM
10 mM
1a
99.7(3)b (124 mM)
69.5(2)
63.1(2)c (37 mM)
<20(3)d (12.4 mM)
<20(1)
2a
3a
4a
5a
55.2(2)
43.8(4)
36.3(2)
NTe
61.4(5)
20.5(2)
62.7(3)
<20(1)
32.0(1)
NTe
6a
7a
HO2CCH2—
<20(1)
<20(1)
NTe
NTe
NTe
NTe
8a
9a
HO—
HS—
27.3(1)
39.1(1)
NTe
NTe
NTe
NTe
10a
11a
20.4(1)
25.2(1)
NTe
NTe
NTe
NTe
aThe number on the left side of parentheses represents % inhibition at each concentration. The number inside parentheses represents the number of
experiment.
b% inhibition at 124 mM (100 mg/mL).
c% inhibition at 37 mM (30 mg/mL).
d% inhibition at 12.4 mM (10 mg/mL).
eNT, not tested.

K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
2451
Table 3. Effects of compound 3a and its related compounds on incorporation of DiI-acetyl-LDL into macrophages
Compound
Structure
Inhibitory activity against incorporation of DiI-acetyl-LDL(%)a
100 mM
3 mM
10 mM
3a
61.4(5)
43.8(4)
20.5(2)
3b
3c
57.1(4)
49.5(2)
NTb
28.7(1)
NTb
NTb
NTb
NTb
NTb
NTb
23.6(1)
NTb
NTb
NTb
NTb
<20(1)
3d
3e
31.4(2)
65.2(1)
NTb
NTb
3f
<20(1)
NTb
3g
3h
3i
<20(1)
NTb
45.4(1)
NTb
57.4(2)
25.5(1)
63.1(2)
NTb
3j
12j
13q
13r
<20(1)
NTb
60.7(3)
42.8(2)
NTb
<20(1)
13s
<20(1)
NTb
NTb
aThe number on the left side of parentheses represents % inhibition at each concentration. The number inside parentheses represents the number of
experiment.
bNT, not tested.

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K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
reduction in the inhibitory activity (3a vs 3g). The doc-
osanoyl derivative 3e and the nonadecyl derivative 3i
were as potent as 3a, while the hexacosanoyl deriva-
tive 3f was less potent than 3a. These results indicate
that the existence of two long chains which consist of
14–22 carbons is necessary for a potent inhibitory
activity.
rophages by inhibiting the binding of DiI-acetyl-LDL to
scavenger receptors.
Kodama etal. found outhtatType I and II class A
scavenger receptors are comprised of six domains;6,7 N-
terminal cytoplasmic domain, transmembrane domain,
spacer domain, coiled-coil domain, collagen-like
domain, and cysteine-rich domain. As a result of step-
wise deletion of Type I and II class A scavenger recep-
tors, Doi et al. found that, among the six domains,
acetyl-LDL binds to the collagen-like domain.23 Col-
lagen-like domain in human Type I and II class A sca-
venger receptors consist of 69 residue sequence bearing
glycine (Gly) as every third residue, that is, (Gly-X-Y)23.
Lysine residues which are located in the Y positions of
Gly-X-Y triplets near the C-terminus have been repor-
ted to be implicated in the ligand binding. We specu-
lated that the sulfate moiety of 3a or 13q would interact
withe-amino group of one of such lysine residues, and
inhibit the binding of acetyl-LDL.
It was noteworthy that both compounds 3j and 12j
having two long chains (tetradecyl and hexadecanoyl)
were weak inhibitors, while compound 13q with another
type of diamide structure was a potent inhibitor. These
results means that the existence of two long chains like
tetradecyl is necessary, but not enough for a potent
inhibitory activity, and another unknown factor is also
important. The replacement of methylene group in the
two octadecanoyl groups of 13q with oxygen atom
resulted in a reduction in the inhibitory activity (13r and
13s). This result means that the lipophilic property of
the two long chains is important for the potent inhibi-
tory activity. The SARs of sulfatides as a scavenger
receptor inhibitor are summarized in Figure 3.
Recently, class A scavenger receptor/apolipoprotein E
double-knockoutmice were generaetd by Suzuki et
al.,16 and they reported that, (1) degradation of acetyl-
LDL in the macrophages from double-knockout mice
was reduced to less than a third compared to that in
wild-type mice macrophages, (2) oxidized-LDL degra-
dation activity of the macrophages from double-knock-
out mice was 50% of that of wild-type mice
macrophages, (3) the size of atherosclerotic lesions in
double-knockout mice were 60% smaller than that in
apolipoprotein E knockout mice.16 These results are
direct evidence that class A scavenger receptors plays an
important role in the formation of atherosclerotic
lesions. Therefore, 3a and 13q with class A scavenger
receptor inhibitory activity are expected to prevent the
formation of atherosclerosis lesions and become a
medicine for the treatment of atherosclerosis.
Compounds which can inhibit the endocytosis of the
formed receptor–ligand complex or inhibit the degrada-
tion of acetyl-LDL in the lisosomes are thought to be
able to exhibit the inhibitory activity against incorpora-
tion of DiI-acetyl-LDL into macrophages. In order to
confirm that the inhibitory activity of 3a againstincor-
poration of DiI-acetyl-LDL into macrophages is based
on the inhibition of the binding of DiI-acetyl-LDL to
the surface of macrophages, 3a was examined for the
inhibitory activity against the binding of 125I-acetyl-
LDL to macrophages at 4 ꢁC (the binding of acetyl-
LDLs to the surface of macrophages can occur at 4 ꢁC,
but the incorporation of acetyl-LDLs into macrophages
can notdo at4 ꢁC). Compound 3a and sulfatides
exhibited the inhibitory activity of 42% at 10 mM
(6.7 mg/mL) and 54% at3 mg/mL, respectively (Table 4).
This resultmeans ht at(1) 3a has the inhibitory activity
againstht e binding of 125I-acetyl-LDL to macrophage,
(2) the inhibitory activity of 3a againstDiI-aceyt l-LDL
incorporation is based on the inhibition of binding of
acetyl-LDL to the surface of macrophages in the same
manner as sulfatides. Acetyl-LDL is known to bind to
scavenger receptors expressed on the surface of macro-
phages, so it was thought that 3a showed its inhibitory
activity of incorporation of DiI-acetyl-LDL into mac-
The reports on the profile of the double-knockout mice
by Suzuki etal. shows ht atclass A scavenger recepot rs
are one of promising drug targets for atherosclerosis.
However, in spite of the disruption of class A scavenger
receptor gene, the atherosclerotic lesions did not dis-
appear completely. This indicates that other scavenger
receptors, such as CD36,9 SR-B1,11 and LOX-1,14 may
Table 4. Effects of compound 3a and sulfatides on binding of
125I-acetyl-LDL to macrophages
Compound
Concentration
Inhibitory activity
againstbinding of
125I-acetyl-LDL(%)a
3a
30 mM (20 mg/mL)
10 mM (6.7 mg/mL)
3 mM (2.0 mg/mL)
10 mg/mL
81.1(2)
41.8(2)
6.6(2)
95.1(3)
54.3(3)
5.9(3)
Sulfatides
3 mg/mL
1 mg/mL
aThe number on the left side of parentheses represents % inhibition at
each concentration. The number inside parentheses represents the
number of experiment.
Figure 3. SARs of sulfatides.

K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
2453
also participate in the progress of atherosclerosis. The
inhibitory activities of 3a and 13q against these receptors
will be evaluated.
Benzyl
(S)-2-Amino-3-(2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyra-
nosyl)oxypropionate (18). A solution of benzyl (S)-2-
tert-butoxycarbonylamino-3-(2,3,4,6-tetra-O-acetyl-b-d-
galactopyranosyl)oxypropionate (17) (2.88 g, 4.60 mmol)
in TFA (10 m) was stirred for 3 h under ice-bath cool-
ing. The reaction mixture was poured into a mixture of
ethyl acetate (100 mL) and an aqueous solution of
sodium hydrogen carbonate (100 mL) under ice-bath
cooling. The resulting aqueous layer was extracted with
ethyl acetate, and then the organic layers were com-
bined, washed with an aqueous solution of sodium
chloride, dried over magnesium sulfate, and con-
centrated in vacuo to give a colorless oil. Purification of
the oil by column chromatography (eluent chloroform/
methanol=50:1) gave the title compound as a colorless
oil (1.28 g, yield=53%).
Conclusion
In conclusion, we clarified the structure–activity rela-
tionships of sulfatides as a scavenger receptor inhibitor.
The obtained information would be very useful for
design of effective scavenger receptor inhibitors. In
addition, we succeeded in the discovery of novel sca-
venger receptor inhibitors, such as 3a and 13q, which
were synthesized easily compared to sulfatides. Further
pharmacological evaluation about compounds 3a and
13q is in progress, and the results will be reported in due
course.
1H NMR (CDCl3) d : 1.72 (br s, 2H), 1.99 (s, 3H), 2.04
(s, 3H), 2.07 (s, 3H), 2.15 (s, 3H), 3.66 (t, J=4.4 Hz,
1H), 3.75–3.95 (m, 2H), 4.10–4.25 (m, 3H), 4.54 (d,
J=7.9 Hz, 1H), 5.00 (dd, J=3.4, 10.5 Hz, 1H), 5.18 (s,
2H), 5.20 (dd, J=7.9, 10.5 Hz, 1H), 5.38 (d, J=3.4 Hz,
1H), 7.38 (s, 5H). Anal. calcd for C24H31NO12: C, 54.85;
H, 5.95; N, 2.67; found: C, 55.01; H, 5.79; N, 2.77.
Experimental
Melting points were determined on a capillary melting
point apparatus (Yamato MR-21) and are uncorrected.
1H NMR spectra of all the compounds synthesized here
were determined with Brucker DPX-250 at 250 MHz
(tetramethylsilane as an internal standard). In NMR
description, s, d, t, dd, ddd, td, m, br, and br s mean
singlet, doublet, triplet, double doublet, double double
doublet, triple doublet, multiplet, broad peak, and
broad singlet, respectively. Elemental analysis data were
obtained by use of Yanagimoto CHNcorder MT-5).
Column chromatography was performed using silica gel
(YMC-GEL SIL-60A) under medium pressure. No
attempt was made to maximize the yields.
Benzyl
(S)-2-Hexadecanoylamino-3-(2,3,4,6-tetra-O-acetyl-ꢀ-D-
A solution of
galactopyranosyl)oxypropionate (19).
octadecanoyl chloride (0.72 g, 2.36 mmol) in N,N-dime-
thylformamide (DMF, 10 mL) was added at 0 ꢁC slowly
to a solution of benzyl (S)-2-amino-3-(2,3,4,6-tetra-O-
acetyl-b-d-galactopyranosyl)oxypropionate (18) (1.18 g,
2.25 mmol) and triethylamine (0.46 g, 4.54 mmol) in
DMF (10 mL). The resulting mixture was stirred at 0 ꢁC
for 2 h. The reaction mixture was diluted with water
(200 mL), and then the whole was extracted with diethyl
ether. The organic layer was dried over magnesium sul-
fate and concentrated in vacuo to give a colorless oil.
The oil was purified with column chromatography (elu-
entfirs,t n-hexane/ethyl acetate=3:1, then 1:1) to give
Benzyl
(S)-2-tert-Butoxycarbonylamino-3-(2,3,4,6-tetra-O-ace-
tyl-ꢀ-D-galactopyranosyl)oxypropionate (17). A mixture
of N-tert-butoxycarbony-l-serine benzyl ester (16)
(0.72 g, 2.44 mmol), silver carbonate (0.34 g, 1.22 mmol),
silver perchlorate (0.26 g, 1.22 mmol) and molecular
sieves 4A (1.00 g), dry dichloromethane (5 mL) was stir-
red at room temperature for 5 h. A mixture of 2,3,4,6-
the title compound as
yield=85%).
a
colorless oil (1.52 g,
tetra-O-acetyl-a-d-galactopyranosyl
bromide
(15)
(0.50 g, 1.22 mmol), molecular sieves 4A (1.00 g), and
dry dichloromethane (3 mL), which had been stirred for
5 h, was added to the above mixture, and then the
resulting mixture was stirred at room temperature for
18 h. Insoluble matter in the reaction mixture was
removed by Celite-filtration, and then the obtained fil-
trate was concentrated in vacuo. The obtained oil was
purified by column chromatography (eluent n-hexane/
ethyl acetate=2:1) to give the title compound as a
colorless oil (0.16 g, yield=21%).
1H NMR (CDCl3) d: 0.88 (t, J=6.6 Hz, 3H), 1.20–1.40
(m, 28H), 1.55–1.75 (m, 2H), 1.99 (s, 3H), 2.05 (s, 3H),
2.07 (s, 3H), 2.16 (s, 3H), 2.25 (t, J=7.6 Hz, 2H), 3.82 (t,
J=7.4 Hz, 1H), 3.91 (dd, J=3.5, 10.5 Hz, 1H), 4.05–
4.20 (m, 2H), 4.29 (dd, J=3.1, 10.5 Hz, 1H), 4.44 (d,
J=7.9 Hz, 1H), 4.65–4.90 (m, 1H), 4.97 (dd, J=3.4,
10.5 Hz, 1H), 5.16 (dd, J=7.9, 10.5 Hz, 1H), 5.20 (s,
2H), 5.37 (dd, J=1.0, 3.4 Hz, 1H), 6.30 (d, J=7.6 Hz,
1H), 7.25–7.45 (m, 5H). Anal. calcd for C42H65NO13: C,
63.70; H, 8.27; N, 1.77; found: C, 63.99; H, 8.35; N,
1.79.
1H NMR (CDCl3) d: 1.42 (s, 9H), 1.95 (s, 3H), 2.00 (s,
3H), 2.05 (s, 3H), 2.11 (s, 3H), 3.75–3.90 (m, 2H), 4.00–
4.15 (m, 2H), 4.20–4.35 (m, 1H), 4.35–4.50 (m, 2H), 4.96
(dd, J=3.4, 10.5 Hz, 1H), 5.05–5.25 (m, 3H), 5.25–5.40
(m, 2H), 7.33 (s, 5H). Anal. calcd for C29H39NO14: C,
55.67; H, 6.28; N, 2.24; found: C, 55.87; H, 6.25; N,
2.29.
(S)-2-Hexadecanoylamino-3-(2,3,4,6-tetra-O-acetyl-ꢀ-D-
galactopyranosyl)oxypropionic acid (20). A mixture of
Pd–C (10%, 0.50 g) and dioxane (1 mL) was added to a
solution of benzyl (S)-2-hexadecanoylamino-3-(2,3,4,6-
tetra-O-acetyl-b-d-galactopyranosyl)oxypropionate (19)

2454
K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
(1.40 g, 1.77 mmol) in tetrahydrofuran (THF, 10 mL),
and then the resulting mixture was stirred at room tem-
perature for 2 h under hydrogen atmosphere. Pd/C was
removed by filtration, and then the obtained filtrate was
concentrated in vacuo to give a brown oil. Purification
of the oil by column chromatography (eluent n-hexane/
ethyl acetate=1:1) gave the title compound as a colorless
solid (1.19 g, yield=96%).
1H NMR (CD3OD) d: 0.90 (t, J=6.6 Hz, 6H), 1.20–1.40
(m, 50H), 1.40–1.75 (m, 4H), 2.23 (t, J=7.3 Hz, 2H),
3.15–3.30 (m, 2H), 3.40–4.60 (m, 10H), 4.57 (br s, 6H).
Anal. calcd for C41H80N2O8: C, 67.54; H, 11.06; N,
3.84; found: C, 67.55; H, 10.94; N, 3.55.
N-[(S)-1-Tetradecylcarbamoyl-2-(3-sulfo-ꢀ-D-galactopyra-
nosyl)oxyethyl]hexadecanamide (1a).
A
mixture of
N-[(S)-1-tetradecylcarbamoyl-2-(b-d-galactopyranosyl)o-
xyethyl]hexadecanamide (22) (110 mg, 0.151 mmol), di-
n-butyltin oxide 38 mg (38 mg, 0.151 mmol), methanol
(20 mL) was refluxed for 3 h. The solventwas removed,
and then a mixture of sulfur trioxide–trimethylamine
complex (42 mg, 0.302 mmol) and dioxane (20 mL) was
added to the obtained residue. The resulting mixture
was stirred at room temperature for 65 h. Methanol
(20 mL) was added to the reaction mixture, and then
insoluble matter was collected by filtration to give a
colorless powder. Purification of this crude product of
the title compound by recrystallization from acetonitrile
and the subsequent preparative TLC (developing sol-
1H NMR (CDCl3) d: 0.89 (t, J=6.6 Hz, 3H), 1.20–1.40
(m, 28H), 1.55–1.75 (m, 2H), 2.00 (s, 3H), 2.07 (s, 3H),
2.09 (s, 3H), 2.17 (s, 3H), 2.28 (t, J=7.6 Hz, 2H), 3.91
(td, J=1.0, 6.5 Hz, 1H), 4.00 (dd, J=4.4, 10.9 Hz, 1H),
4.05–4.30 (m, 3H), 4.53 (d, J=7.9 Hz, 1H), 4.65–4.75
(m, 1H), 5.01 (dd, J=3.4, 10.4 Hz, 1H), 5.18 (dd,
J=7.8, 10.4 Hz, 1H), 5.40 (dd, J=1.0, 3.4 Hz, 1H), 6.43
(d, J=7.1 Hz, 1H). Anal. calcd for C35H59NO13: C,
59.90; H, 8.47; N, 2.00; found: C, 60.06; H, 8.44; N, 2.15.
N-[(S)-1-Tetradecylcarbamoyl-2-(2,3,4,6-tetra-O-acetyl-
ꢀ-D-galactopyranosyl)oxyethyl]hexadecanamide
(21).
EDCI (75 mg, 0.39 mmol) and tetradecylamine (77 mg,
0.36 mmol) were added at0 ꢁC to a solution of (S)-2-
hexadecanoylamino-3-(2,3,4,6-tetra-O-acetyl-b-d-galac-
topyranosyl)oxypropionic acid (20) (0.25 g, 0.36 mmol)
and HOBt(53 mg, 0.39 mmol) in DMF (5 mL), and then
the resulting mixture was stirred at room temperature
for 19 h. The reaction mixture was diluted with water
(30 mL), and then the whole was extracted with ethyl
acetate. Combined organic layer was washed with water
and brine, and dried over magnesium sulfate. Con-
centration of the solution under reduced pressure gave a
colorless oil. Purification of the oil by column chroma-
tography (eluent n-hexane/ethyl acetate=1:1) gave the
vent/chloroform:methanol=4:1)
gave
the
compound as a colorless solid (7 mg, yield=5.7%).
title
1H NMR (CD3OD) d: 0.91 (t, J=6.6 Hz, 6H), 1.20–1.45
(m, 50H), 1.45–1.80 (m, 4H), 2.20–2.40 (m, 2H), 3.10–
3.30 (m, 2H), 3.55–4.65 (m, 10H), 4.84 (br s, 6H). Anal.
.
calcd for C41H80N2O11S 1.0H2O: C, 59.53; H, 9.99; N,
3.39; found: C, 59.31; H, 10.20; N, 3.59.
tert-Butyl N-[(S)-2-benzyloxy-1-(tetradecylcarbamoyl)-
ethyl]carbamate (24a). EDCI (9.34 g, 48.7 mmol) and
HOBt (6.58 g, 48.7 mmol) were added to a mixture of N-
Boc-O-benzyl-l-serine (12.0 g, 40.6 mmol), tetra-
decylamine (10.4 g, 48.7 mmol), and DMF under ice-
cooling, and the resulting mixture was stirred at room
temperature for 20 h. Chloroform (400 mL) was added
to the reaction mixture, and then the resulting mixture
was washed with water, a saturated aqueous solution of
sodium hydrogen carbonate, and a saturated aqueous
solution of sodium chloride, dried over magnesium sul-
fate, and concentrated in vacuo. The obtained residue
was purified with column chromatography (eluent;
chloroform) to give the title compound as a colorless
powder (19.3 g, yield=97%). Recrystallization from a
mixture of methanol and water gave an analytical
sample having the following physical chemical proper-
ties.
title compound as
yield=16%).
a
colorless powder (53 mg,
1H NMR (CDCl3) d: 0.89 (t, J=6.6 Hz, 6H), 1.20–1.40
(m, 50H), 1.40–1.80 (m, 4H), 2.00 (s, 3H), 2.06 (s, 3H),
2.09 (s, 3H), 2.16 (s, 3H), 2.25 (t, J=7.5 Hz, 2H), 3.15–
3.40 (m, 2H), 3.81 (dd, J=7.9, 9.5 Hz, 1H), 3.92 (td,
J=1.0, 6.7 Hz, 1H), 3.99 (dd, J=3.6, 9.5 Hz, 1H), 4.05–
4.20 (m, 2H), 4.30–4.45 (m, 1H), 4.59 (d, J=7.5 Hz,
1H), 5.05 (dd, J=3.3, 10.6 Hz, 1H), 5.15 (dd, J=7.5,
10.6 Hz, 2H), 5.42 (dd, J=1.0, 3.3 Hz, 1H), 6.43 (t,
J=5.5 Hz, 1H), 6.51 (d, J=6.6 Hz, 1H). Anal. calcd for
C49H88N2O12: C, 65.59; H, 9.89; N, 3.12; found: C,
65.77; H, 10.01; N, 3.11.
N-[(S)-1-Tetradecylcarbamoyl-2-(ꢀ-D-galactopyranosyl)-
oxyethyl]hexadecanamide (22). A solution of sodium
methoxide in methanol (28%, 165 mg, 0.854 mmol) was
Mp 57.0–57.5 ꢁC. 1H NMR (CDCl3) d: 0.88 (t,
J=6.6 Hz, 3H), 1.10–1.35 (m, 22H), 1.45 (s, 9H), 1.35–
1.65 (m, 2H), 3.20–3.35 (m, 2H), 3.56 (dd, J=6.7,
9.2 Hz, 1H), 3.91 (dd, J=4.0, 9.2 Hz, 1H), 4.10–4.30 (m,
1H), 4.50 (d, J=11.8 Hz, 1H), 4.59 (d, J=11.8 Hz, 1H),
5.25–5.50 (br, 1H), 6.30–6.50 (br, 1H), 7.20–7.40 (m,
5H). Anal. calcd for C29H50N2O4: C, 70.98; H, 10.27; N,
5.71; found: C, 71.00; H, 10.31; N, 5.67.
added at0 ꢁC to
a
solution of N-[1(S)-tetra-
decylcarbamoyl-2-(2,3,4,6-tetra-O-acetyl-b-d-galacto-
pyranosyl)oxyethyl] hexadecanamide (21) (174 mg,
0.194 mmol) in THF (10 mL) and methanol (1 mL), and
then the resulting mixture was stirred at room tempera-
ture for 3 h. Diluted hydrochloric acid was added to the
reaction mixture until the pH of the mixture showed 7.
The resulting mixture was diluted with water (30 mL),
and the precipitated solid was collected by filtration to
give the title compound as a colorless powder (90 mg,
yield=64%).
(S)-2-Amino-3-benzyloxy-N-tetradecylpropionamide (25a).
A mixture of tert-butyl N-[(S)-2-benzyloxy-1-(tetra-
decylcarbamoyl)ethyl]carbamate (24) (19.0 g, 38.7 mmol)
and TFA (70 mL) was stirred 1 h for under ice-cooling.
The reaction mixture was poured into a mixture of a

K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
2455
saturated aqueous solution of sodium hydrogen car-
bonate and ethyl acetate. The organic layer was washed
with a saturated aqueous solution of sodium hydrogen
carbonate and a saturated aqueous solution of sodium
chloride, dried over magnesium sulfate, and con-
centrated in vacuo. The obtained residue was purified
with column chromatography (eluent n-hexane/ethyl
acetate=1:1) to give the title compound as a colorless
powder (11.3 g, yield=75%). Recrystallization from
ethyl acetate gave an analytical sample having the
following physical chemical properties.
N-[(R)-2-Benzyloxy-1-(tetradecylcarbamoyl)ethyl]octade-
canamide (26b). Mp 87.0–89.0 ꢁC. H NMR (CDCl3) d:
1
0.88 (t, J=6.6 Hz, 6H), 1.20–1.40 (m, 50H), 1.40–1.70
(m, 4H), 2.21 (t, J=7.6 Hz, 2H), 3.15–3.30 (m, 2H), 3.48
(dd, J=8.0, 9.2 Hz, 1H), 3.89 (dd, J=4.2, 9.2 Hz, 1H),
4.45–4.60 (m, 1H), 4.53 (d, J=11.7 Hz, 1H), 4.62 (d,
J=11.7 Hz, 1H), 6.30–6.45 (br, 2H), 7.20–7.45 (m, 5H).
Anal. calcd for C42H76N2O3: C, 76.77; H, 11.66; N,
4.26; found: C, 76.99; H, 11.65; N, 4.26.
Benzyl (S)-4-tetradecylcarbamoyl-4-octadecanoylamino-
butanoate (27a). Mp 93.5–94.5 ꢁC. H NMR (CDCl3) d:
1
Mp 60.5–62.5 ꢁC. 1H NMR (CDCl3) d: 0.88 (t,
J=6.6 Hz, 3H), 1.15–1.40 (m, 22H), 1.40–1.60 (m, 2H),
1.64 (br s, 2H), 3.15–3.30 (m, 2H), 3.58 (dd, J=3.8,
6.6 Hz, 1H), 3.64 (dd, J=6.6, 8.8 Hz, 1H), 3.75 (dd,
J=3.8, 8.8 Hz, 1H), 4.54 (s, 2H), 7.20–7.45 (m, 6H).
Anal. calcd for C24H42N2O2: C, 73.80; H, 10.84; N,
7.17; found: C, 73.86; H, 10.83; N, 7.25.
0.88 (t, J=6.5 Hz, 6H), 1.15–1.40 (m, 50H), 1.40–1.75
(m, 4H), 1.85–2.20 (m, 4H), 2.30–2.70 (m, 2H), 3.10–
3.30 (m, 2H), 4.35–4.50 (m, 1H), 5.10 (d, J=12.3 Hz,
1H), 5.16 (d, J=12.3 Hz, 1H), 6.25–6.45 (br, 2H), 7.25–
7.45 (m, 5H). Anal. calcd for C44H78N2O4: C, 75.59; H,
11.25; N, 4.01; found: C, 75.57; H, 11.,25; N, 3.78.
N-[(S)-2-(4-Benzyloxy)phenyl-1-(tetradecylcarbamoyl)-
ethyl]octadecanamide (28a). Mp 123.5–125.5 ꢁC. 1H
NMR (CDCl3) d: 0.88 (t, J=6.6 Hz, 6H), 1.10–1.40 (m,
50H), 1.40–1.80 (m, 4H), 2.16 (t, J=7.6 Hz, 2H), 2.90
(dd, J=8.6, 13.7 Hz, 1H), 2.95–3.20 (m, 3H), 4.40–4.60
(m, 1H), 5.03 (s, 2H), 5.55 (br, 1H), 6.14 (d, J=7.8 Hz,
1H), 6.90 (d, J=8.7 Hz, 2H), 7.13 (d, J=8.7 Hz, 2H),
7.30–7.45 (m, 5H). Anal. calcd for C48H80N2O3: C, 78.63;
H, 11.00; N, 3.82; found: C, 78.60; H, 10.98; N, 3.85.
N-[(S)-2-Benzyloxy-1-(tetradecylcarbamoyl)ethyl]octade-
canamide (26a). A solution of octadecanoyl chloride
(1.71 g, 5.64 mmol) in THF (5 mL) was added dropwise
to
a
mixture of (S)-2-amino-3-benzyloxy-N-tetra-
decylpropionamide (25) (2.00 g, 5.12 mmol), diisopro-
pylethylamine (660 mg, 5.11 mmol), and THF (15 mL)
under ice-cooling, and then the resulting mixture was
stirred at room temperature for 18 h. After the reaction
mixture had been concentrated in vacuo, chloroform
(50 mL) was added to the obtained residue. The result-
ing mixture was washed with a saturated aqueous solu-
tion of sodium chloride, dried over magnesium sulfate
and concentrated in vacuo. The obtained residue was
purified with column chromatography (eluent n-hexane/
ethyl acetate=2:1) to give the title compound as a
brown powder (2.38 g, yield=71%). Recrystallization
from ethyl acetate gave an analytical sample having the
following physical chemical properties.
N-[(R)-2-[(R)-2-Tetradecylcarbamoyl-2-octadecanoyl-
aminoethyl]dithio- 1 - (tetradecylcarbamoyl)ethyl]octadec-
1
anamide (30a). H NMR (CDCl3) d: 0.88 (t, J=6.6 Hz,
12H), 1.20–1.40 (m, 100H), 1.40–1.70 (m, 8H), 2.25 (t,
J=7.5 Hz, 4H), 2.86 (dd, J=10.0, 14.7 Hz, 2H), 2.97
(dd, J=4.5, 14.7 Hz, 2H), 3.15–3.40 (m, 4H), 5.33 (ddd,
J=4.5, 9.2, 10.0 Hz, 2H), 6.44 (d, J=9.2 Hz, 2H), 8.17
(t, J=4.8 Hz, 2H). Anal. calcd for C70H138N4O4S2: C,
72.23; H, 11.95; N, 4.81; found: C, 72.21; H, 12.02; N, 4.79.
Mp 87.0–89.0 ꢁC. 1H NMR (CDCl3) d: 0.88 (t,
J=6.6 Hz, 6H), 1.20–1.40 (m, 50H), 1.40–1.70 (m, 4H),
2.21 (t, J=7.6 Hz, 2H), 3.15–3.30 (m, 2H), 3.48 (dd,
J=8.0, 9.2 Hz, 1H), 3.89 (dd, J=4.2, 9.2 Hz, 1H), 4.45–
4.60 (m, 1H), 4.53 (d, J=11.7 Hz, 1H), 4.62 (d,
J=11.7 Hz, 1H), 6.30–6.45 (br, 2H), 7.20–7.45 (m, 5H).
Anal. calcd for C42H76N2O3: C, 76.77; H, 11.66; N,
4.26; found: C, 76.78; H, 11.63; N, 4.30.
N-[(S)-2-Hydroxy-1-(tetradecylcarbamoyl)ethyl]octadec-
anamide (8a). A mixture of N-[(S)-2-benzyloxy-1-(tetra-
decylcarbamoyl)ethyl]octadecanamide (26) (1.40 g,
2.13 mmol), Pd/C (10%, 1.13 g), dioxane (120 mL), and
methanol (200 mL) was stirred at room temperature
under hydrogen atmosphere for 46 h. After addition of
chloroform (100 mL) to the reaction mixture, Pd/C was
removed by filtration. The obtained filtrate was con-
centrated in vacuo to dryness. Ethyl acetate (20 mL) was
added to the obtained reside, and then the resulting
mixture was stirred. Finally, the insoluble crystalline
solid was collected by filtration to give the title com-
The following compounds 10a, 26b, 27a, 28a, and 30a
were synthesized from N-Boc-O-benzyl-d-serine, N-
Boc-l-methionine sulfoxide (10n), N-Boc-l-glutamic
acid 5-benzyl ester (27n), N-Boc-O-benzyl-l-tyrosine
(28n), and N-Boc-l-cystine (30n), respectively, in the
same manner as the synthesis of 26a.
pound as
a colorless crystalline powder (1.05 g,
yield=87%). Recrystallization from chloroform gave
an analytical sample having the following physical
chemical properties.
N-[(S)-3-Methanesulfinyl-1-(tetradecylcarbamoyl)propyl]-
octadecanamide (10a). Mp 106.5–108.5 ꢁC. 1H NMR
(CDCl3) d: 0.88 (t, J=6.6 Hz, 6H), 1.10–1.40 (m, 50H),
1.40–1.70 (m, 4H), 2.10–2.55 (m, 4H), 2.58 (s), 2.67 (s),
2.70–3.00 (m, 2H), 3.15–3.30 (m, 2H), 4.50–4.70 (m,
1H), 6.75–7.15 (br, 2H). Anal. calcd for C37H74N2O3S:
C, 70.87; H, 11.89; N, 4.47; found: C, 70.88; H, 11.93;
N, 4.23.
Mp 119.0–121.0 ꢁC. 1H NMR (CDCl3) d: 0.88 (t,
J=6.6 Hz, 6H), 1.15–1.40 (m, 50H), 1.40–1.75 (m, 4H),
1.90 (br s, 1H), 2.20–2.35 (m, 2H), 3.10–3.35 (m, 2H),
3.60 (dd, J=4.1, 11.6 Hz, 1H), 4.18 (dd, J=2.5, 11.6 Hz,
1H), 4.30–4.45 (m, 1H), 6.55–6.75 (br, 1H), 6.75–6.95
(br, 1H). Anal. calcd for C35H70N2O3: C, 74.15; H,
12.44; N, 4.94; found: C, 74.08; H, 12.48; N, 4.89.

2456
K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
The following compounds 8c–j were synthesized from
N-Boc-O-benzyl-l-serine and the corresponding alkyl-
amine and acyl chloride via the corresponding N-[(S)-2-
benzyloxy-1-(alkylcarbamoyl)ethyl]alkanamide in the
same manner as 8a.
J=4.1, 11.5 Hz, 1H), 4.17 (dd, J=2.3, 11.5 Hz, 1H),
4.30–4.45 (m, 1H), 6.55–6.70 (br, 1H), 6.75–6.90 (br,
1H). Anal. calcd for C40H80N2O3: C, 75.41; H, 12.66; N,
4.40; found: C, 75.31; H, 12.62; N, 4.27.
N-[(S)-2-Hydroxy-1-(propylcarbamoyl)ethyl]-2-tetrade-
1
N-[(S)-2-Hydroxy-1-(tetradecylcarbamoyl)ethyl]acetamide
(8c). 1H NMR (CDCl3) d: 0.88 (t, J=6.6 Hz, 3H),
1.20–1.40 (m, 22H), 1.40–1.60 (m, 2H), 2.05 (s, 3H),
3.23 (td, J=6.7, 6.7 Hz, 2H), 3.40–3.70 (m, 2H), 4.12
(dd, J=2.9, 10.9 Hz, 2H), 4.30–4.45 (m, 1H), 6.67 (d,
J=6.9 Hz, 1H), 6.79 (br, 1H). Anal. calcd for
C19H38N2O3: C, 66.63; H, 11.18; N, 8.18; found: C,
66.35; H, 11.18; N, 8.47.
cylhexadecanamide (8j). H NMR (CDCl3) d: 0.88 (t,
J=6.6 Hz, 6H), 0.91 (t, J=7.4 Hz, 3H), 1.10–1.40 (m,
48H), 1.40–1.70 (m, 6H), 2.00–2.20 (m, 1H), 3.10–3.30
(m, 2H), 3.35–3.65 (m, 2H), 4.18 (d, J=2.6, 11.9 Hz,
1H), 4.30–4.45 (m, 1H), 6.57 (d, J=7.1 Hz, 1H),
6.80–7.00 (m, 1H). Anal. calcd for C36H72N2O3: C,
74.42; H, 12.49; N, 4.82; found: C, 74.70; H, 12.55;
N, 4.93.
N-[(S)-2-Hydroxy-1-(tetradecylcarbamoyl)ethyl]decan-
The following compounds 8b and 7a were synthesized
from the corresponding benzyloxy derivatives 26b and
28a, respectively, in the same manner as 8a.
amide (8d). Mp 127.0–128.0 ꢁC. H NMR (CDCl3) d:
1
0.88 (t, J=6.6 Hz, 6H), 1.15–1.40 (m, 34H), 1.40–1.75
(m, 4H), 2.15–2.30 (m, 3H), 3.15–3.30 (m, 2H), 3.50–
3.70 (m, 2H), 4.10–4.20 (m, 1H), 4.30–4.45 (m, 1H), 6.64
(d, J=7.0 Hz, 1H), 6.83 (br, 1H). Anal. calcd for
C27H54N2O3: C, 71.31; H, 11.97; N, 6.16; found: C,
71.48; H, 11.89; N, 6.11.
N-[(R)-2-Hydroxy-1-(tetradecylcarbamoyl)ethyl]octade-
canamide (8b). Mp 119.5–120.5 ꢁC. H NMR (CDCl3)
1
d: 0.88 (t, J=6.6 Hz, 6H), 1.15–1.40 (m, 50H), 1.40–1.70
(m, 5H), 2.20–2.30 (m, 2H), 3.15–3.30 (m, 2H), 3.50–
3.65 (m, 1H), 4.10–4.25 (m, 1H), 4.30–4.40 (m, 1H),
6.50–6.65 (m, 1H), 6.70–6.85 (br, 1H). Anal. calcd for
N-[(S)-2-Hydroxy-1-(tetradecylcarbamoyl)ethyl]docosa-
ꢁ
1
.
namide (8e). Mp 115.0–117.0 C. H NMR (CDCl3) d:
0.88 (t, J=6.6 Hz, 6H), 1.15–1.40 (m, 58H), 1.40–1.75
(m, 4H), 2.10 (br s, 1H), 2.15–2.30 (m, 2H), 3.15–3.30
(m, 2H), 3.50–3.70 (m, 1H), 4.10–4.25 (m, 1H), 4.30–
4.40 (m, 1H), 6.62 (d, J=7.3 Hz, 1H), 6.75–6.90 (m,
1H). Anal. calcd for C39H78N2O3: C, 75.18; H, 12.62; N,
4.50; found: C, 75.21; H, 12.63; N, 4.49.
C35H70N2O3 0.25H2O: C, 73.56; H, 12.43; N, 4.90;
found: C, 73.57; H, 12.39; N, 4.77.
N-[(R)-2-(4-Hydroxyphenyl)-1-(tetradecylcarbamoyl)-
ethyl]octadecanamide (7a). Mp 134.5–136.5 ꢁC. 1H
NMR (DMSO-d6) d: 0.85 (t, J=6.6 Hz, 6H), 1.10–1.60
(m, 54H), 2.02 (t, J=7.3 Hz, 2H), 2.61 (dd, J=8.9,
13.7 Hz, 1H), 2.80 (dd, J=5.7, 13.7 Hz, 1H), 2.90–3.10
(m, 2H), 4.25–4.45 (m, 1H), 6.61 (d, J=8.5 Hz, 2H),
6.98 (d, J=8.5 Hz, 2H), 7.70–7.80 (m, 1H), 7.86 (d,
J=8.3 Hz, 1H), 9.08 (s, 1H). Anal. calcd for
C41H74N2O3: C, 76.58; H, 11.60; N, 4.36; found: C,
76.41; H, 11.58; N, 4.50.
N-[(S)-2-Hydroxy-1-(tetradecylcarbamoyl)ethyl]hexaco-
1
sanamide (8f). H NMR (CDCl3) d: 0.88 (t, J=6.6 Hz,
6H), 1.15–1.40 (m, 66H), 1.40–1.75 (m, 4H), 2.20–2.30
(m, 2H), 3.15–3.30 (m, 2H), 3.30–3.45 (m, 1H), 3.50–
3.65 (m, 1H), 4.10–4.20 (m, 1H), 4.30–4.40 (m, 1H), 6.57
(d, J=6.5 Hz, 1H), 6.65–6.85 (m, 1H). Anal. calcd for
C43H86N2O3: C, 76.04; H, 12.76; N, 4.12; found: C,
76.06; H, 12.91; N, 3.98.
(S)-2-Octadecanoylamino-2-(tetradecylcarbamoyl)ethyl
sulfate sodium salt (3a). Chlorosulfonic acid (70 mL,
1.06 mmol) was added dropwise to pyridine (2 mL) at
room temperature with attention, and then the resulting
mixture was stirred at 70–75 ꢁC for 30 min. After the
reaction mixture had been cooled to room temperature,
N-[(S)-2-Hydroxy-1-(propylcarbamoyl)ethyl]octadecan-
amide (8g). 1H NMR (CDCl3) d: 0.80–1.00 (m, 6H),
1.10–1.40 (m, 26H), 1.40–1.75 (m, 4H), 2.24 (t,
J=7.5 Hz, 2H), 3.21 (td, J=6.6, 6.6 Hz, 2H), 3.50–3.70
(m, 1H), 3.81 (br s, 1H), 4.07 (dd, J=3.0, 11.0 Hz, 1H),
4.35–4.50 (m, 1H), 6.73 (d, J=6.9 Hz, 1H), 6.99 (br,
1H). Anal. calcd for C24H48N2O3: C, 69.86; H, 11.72; N,
6.79; found: C, 70.05; H, 11.99; N, 6.78.
a
solution of N-[(S)-2-hydroxy-1-(tetradecylcarb-
amoyl)ethyl]octadecanamide (0.20 g, 0.353 mmol) in
pyridine (5 mL) was added to the reaction mixture, and
then the resulting mixture was stirred at 70–75 ꢁC for
1 h. After evaporation of pyridine from the reaction
mixture in vacuo, methanol (10 mL) was added to the
obtained residue. A saturated aqueous solution of
sodium carbonate was added to the resulting mixture till
pH of the mixture showed 9. Water (10 mL) was added
to the alkaline mixture, and then the precipitating solid
was collected by filtration, and dried in vacuo. After the
obtained solid had been stirred with hot chloroform
(40 mL), insoluble matter was removed by filtration.
The obtained filtrate was concentrated in vacuo.
Finally, the residue was stirred with ethyl acetate
(10 mL), and then the precipitating crystals were col-
lected by filtration to give the title compound as a
colorless powder (150 mg, Yield=64%).
N-[(S)-2-Hydroxy-1-(octylcarbamoyl)ethyl]octadecan-
amide (8h). Mp 114.0–115.0 ꢁC. H NMR (DMSO-d6)
1
d: 0.85 (t, J=6.6 Hz, 6H), 1.10–1.60 (m, 42H), 2.05–2.20
(m, 2H), 2.90–3.10 (m, 2H), 3.40–3.60 (m, 2H), 4.15–
4.30 (m, 1H), 4.77 (t, J=5.6 Hz, 1H), 7.60–7.80 (m, 2H).
Anal. calcd for C29H58N2O3: C, 72.15; H, 12.11; N,
5.80; found: C, 72.21; H, 11.98; N, 5.80.
N-[(S)-2-Hydroxy-1-(nonadecylcarbamoyl)ethyl]octade-
canamide (8i). Mp 122.0–124.5 ꢁC. H NMR (CDCl3)
1
d: 0.88 (t, J=6.6 Hz, 6H), 1.15–1.40 (m, 60H), 1.40–1.80
(m, 5H), 2.20–2.35 (m, 2H), 3.15–3.30 (m, 2H), 3.58 (dd,

K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
2457
Mp 139.0–144.0 ꢁC (dec). H NMR (CDCl3) d: 0.88 (t,
(S)-2-Octadecanoylamino-2-(octylcarbamoyl)ethyl sulfate
sodium salt (3h). Mp 120.0–122.0 ꢁC (dec). 1H NMR
(DMSO-d6) d: 0.85 (t, J=6.6 Hz, 6H), 1.10–1.60 (m,
42H), 2.05–2.15 (m, 2H), 2.95–3.10 (m, 2H), 3.84 (d,
J=5.9 Hz, 2H), 4.25–4.40 (m, 1H), 7.71 (t, J=5.4 Hz,
1H), 7.88 (d, J=8.0 Hz, 1H). Anal. calcd for
1
J=6.5 Hz, 6H), 1.10–1.40 (m, 50H), 1.40–1.80 (m, 4H),
2.15–2.30 (m, 2H), 3.00–3.30 (m, 2H), 4.10–4.30 (m,
2H), 4.65–4.80 (m, 1H), 7.20–7.60 (br, 2H). Anal. calcd
for C35H69N2NaO6S 1.5H2O: C, 60.40; H, 10.43; N,
4.02; found: C, 60.57; H, 10.51; N, 4.09.
.
.
C29H57N2NaO6S 1.5H2O: C, 56.93; H, 9.88; N, 4.58;
found: C, 56.90; H, 9.65; N, 4.50.
The following compounds 3b–j were synthesized from
the corresponding hydroxyl derivatives 8b–j in the same
manner as 3a.
(S)-2-Nonadecylcarbamoyl-2-(octadecanoylamino)ethyl
sulfate sodium salt (3i). Mp 137.0–139.0 ꢁC (dec). 1H
NMR (DMSO-d6) d: 0.85 (t, J=6.6 Hz, 6H), 1.10–1.60
(m, 64H), 2.05–2.20 (m, 2H), 2.95–3.10 (m, 2H), 3.84 (d,
J=6.4 Hz, 2H), 4.25–4.40 (m, 1H), 7.60–7.75 (m, 1H),
(R)-2-Octadecanoylamino-2-(tetradecylcarbamoyl)ethyl
ꢁ
1
sulfate sodium salt (3b). Mp 139.0–144.0 C (dec). H
NMR (CDCl3) d: 0.88 (t, J=6.5 Hz, 6H), 1.10–1.40 (m,
50H), 1.40–1.80 (m, 4H), 2.15–2.30 (m, 2H), 3.00–3.30
(m, 2H), 4.10–4.30 (m, 2H), 4.65–4.80 (m, 1H), 7.20–
7.60 (br, 2H). Anal. calcd for C35H69N2NaO6S 1.5H2O:
C, 60.40; H, 10.43; N, 4.02; found: C, 60.11; H, 10.14;
N, 3.70.
7.80–7.95
(m,
.
1H).
Anal.
calcd
for
C40H79N2NaO6S 1.0H2O: C, 63.46; H, 10.78; N, 3.70;
found: C, 63.43; H, 10.65; N, 3.79.
.
(S)-2-Propylcarbamoyl-2-(2-tetradecylhexadecanoylamino)-
ethyl sulfate sodium salt (3j). Mp 135.0–138.0 ꢁC (dec).
1H NMR (DMSO-d6) d: 0.80–0.95 (m, 6H), 1.10–1.35
(m, 48H), 1.35–1.65 (m, 6H), 2.10–2.50 (m, 1H), 3.00–
3.30 (m,2H), 4.05–4.40 (m, 2H), 4.65–4.85 (m, 1H),
(S)-2-Acetyamino-2-(tetradecylcarbamoyl)ethyl sulfate
sodium salt (3c). Mp 140.0–143.0 ꢁC (dec). 1H NMR
(CDCl3) d: 0.88 (t, J=6.6 Hz, 3H), 1.15–1.40 (m, 22H),
1.40–1.60 (m, 2H), 2.02 (s, 3H), 3.00–3.35 (m, 2H),
4.10–4.40 (m, 2H), 4.70–4.90 (m, 1H), 7.30–7.90 (br,
6.90–7.30
(br,
.
2H).
Anal.
calcd
for
C36H71N2NaO6S 1.5H2O: C, 60.90; H, 10.50; N, 3.94;
found: C, 60.52; H, 10.26; N, 3.67.
.
2H). Anal. calcd for C19H37N2NaO6S 1.5H2O: C, 48.39;
H, 8.55; N, 5.94; found: C, 48.35; H, 8.35; N, 5.71.
4-[(S) -2-Octadecanoylamino - 2- (tetradecylcarbamoyl)-
ethyl]phenyl sulfate sodium salt (2a). Chlorosulfonic
acid (0.10 mL, 1.50 mmol) was added dropwise to pyri-
dine (2 mL) at room temperature, and then the resulting
mixture was stirred at 70–75 ꢁC for 30 min. After the
reaction mixture was cooled to room temperature, a
solution of (S)-N-[2-(4-hydroxyphenyl)-1-(tetradecyl-
carbamoyl)ethyl] octadecanamide (0.33 g, mmol) in
pyridine (5 mL) was added to the reaction mixture, and
then the resulting mixture was stirred at 80–85 ꢁC for
38 h. After evaporation of pyridine from the reaction
mixture in vacuo, methanol (10 mL) was added to the
obtained residue. A saturated aqueous solution of
sodium carbonate was added to the resulting mixture till
pH of the mixture showed 9. Water (10 mL) was added
to the alkaline mixture, and then the precipitating solid
was collected by filtration, and dried in vacuo. After the
obtained solid was stirred with a mixture of chloroform
and methanol (1:1, 50 mL), insoluble matter was
removed by filtration. The obtained filtrate was con-
centrated in vacuo to dryness. Finally, the obtained
residue was stirred with ethyl acetate (10 mL), and then
the precipitating crystals were collected by filtration to
give the title compound as a colorless powder (220 mg,
Yield=58%).
(S)-2-Decanoylamino-2-(tetradecylcarbamoyl)ethyl sulfate
sodium salt (3d). Mp 140.0–145.0 ꢁC (dec). 1H NMR
(CDCl3) d: 0.88 (t, J=6.6 Hz, 6H), 1.15–1.40 (m, 34H),
1.40–1.65 (m, 4H), 2.25 (t, J=7.5 Hz, 2H), 3.00–3.30
(m, 2H), 4.10–4.40 (m, 2H), 4.65–4.85 (m, 1H), 7.20–
7.70
(br,
2H).
Anal.
calcd
for
.
C27H53N2NaO6S 0.75H2O: C, 56.87; H, 9.63; N, 4.91;
found: C, 56.92; H, 9.42; N, 4.51.
(S)-2-Docosanoylamino-2-(tetradecylcarbamoyl)ethyl sul-
fate sodium salt (3e). Mp 137.0–140.0 ꢁC (dec). 1H
NMR (CDCl3) d: 0.88 (t, J=6.6 Hz, 6H), 1.10–1.40 (m,
58H), 1.40–1.70 (m, 4H), 2.10–2.35 (m, 2H), 3.00–3.30
(m, 2H), 4.10–4.40 (m, 2H), 4.65–4.85 (m, 1H), 7.20–
.
7.70 (br, 2H). Anal. calcd for C39H77N2NaO6S 1.5H2O:
C, 62.28; H, 10.72; N, 3.72; found: C, 62.33; H, 10.75;
N, 3.68.
(S)-2-Hexacosanoylamino-2-(tetradecylcarbamoyl)ethyl
sulfate sodium salt (3f). Mp 133.0–134.0 ꢁC (dec). 1H
NMR (CDCl3) d: 0.88 (t, J=6.6 Hz, 6H), 1.10–1.40 (m,
66H), 1.40–1.70 (m, 4H), 2.15–2.35 (m, 2H), 3.00–3.30
(m, 2H), 4.10–4.40 (m, 2H), 4.60–4.85 (m, 1H), 7.20–
.
7.70 (br, 2H). Anal. calcd for C43H85N2NaO6S 2.0H2O:
C, 63.20; H, 10.98; N, 3.43; found: C, 63.21; H, 10.90;
N, 3.14.
Mp 148.0 ꢁC (dec). H NMR (DMSO-d6) d: 0.80–0.95
1
(m, 6H), 1.10–1.50 (m, 54H), 1.95–2.10 (m, 2H), 2.60–
3.10 (m, 4H), 4.30–4.50 (m, 1H), 6.95–7.15 (m, 4H),
7.80–8.00 (m, 2H). Anal. calcd for C41H73N2
NaO6S 1.5H2O:C, 63.78; H, 9.92; N, 3.63; found: C,
63.49; H, 9.56; N, 3.61.
(S)-2-Octadecanoylamino-2-(propylcarbamoyl)ethyl sulfate
sodium salt (3g). Mp 91.5–92.5 ꢁC (dec). 1H NMR
(DMSO-d6) d: 0.81 (t, J=7.4 Hz, 3H), 0.85 (t,
J=6.7 Hz, 3H), 1.10–1.60 (m, 32H), 2.11 (t, J=7.3 Hz,
2H), 2.90–3.05 (m, 2H), 3.84 (d, J=5.8 Hz, 2H), 4.25–
4.40 (m, 1H), 7.74 (t, J=5.7 Hz, 1H), 7.87 (d, J=7.9 Hz,
.
N-[(S)-4-Hydroxy-1-(tetradecylcarbamoyl)butyl]octade-
canamide (29a). Sodium borohydride (97 mg, 2.56 mmol)
was added at room temperature to a solution of benzyl
.
1H). Anal. calcd for C24H47N2NaO6S 2.0H2O: C, 52.35;
H, 9.33; N, 5.08; found: C, 52.64; H, 8.98; N, 5.22.

2458
K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
(S)-4-(octadecanoylamino)-4-(tetradecylcarbamoyl)but-
anoate (1.00 g, 1.43 mmol) in THF (15 mL), and then
the resulting mixture was heated to 70 ꢁC. After addi-
tion of methanol (0.50 mL) to the mixture at the same
temperature in 30 min, the reaction mixture was cooled
to room temperature. After the solvent of the mixture
had been evaporated in vacuo, the obtained residue was
diluted with water (50 mL). The resulting mixture was
extracted with chloroform, and then the chloroform
layer was washed with a saturated solution of sodium
chloride, dried over magnesium sulfate, and con-
centrated in vacuo to dryness. The obtained solid was
purified by column chromatography (eluent chloroform:
methanol=20:1) to give the title compound as a colorless
powder (140 mg, yield=16%).
to a solution of N-[(S)-3-methanesulfinyl-1-(tetra-
decylcarbamoyl)propyl]octadecanamide (10a) (100 mg,
0.159 mmol) in dichloromethane (5 mL), and then the
resulting mixture was stirred at room temperature for
16 h. After addition of a saturated aqueous solution of
sodium hydrogen carbonate (15 mL), the resulting mix-
ture was stirred at room temperature for 5 min.
Chloroform (20 mL) was added to the mixture, and then
the organic layer was separated. The aqueous layer was
extracted with chloroform twice, and then the organic
layers were combined. The obtained solution was
washed with a saturated aqueous solution of sodium
chloride, dried over magnesium sulfate, and con-
centrated in vacuo. The obtained colorless powder was
recrystallized from ethyl acetate to yield the target
compound as
Yield=21%).
a
colorless powder (21.8 mg,
Mp 121.0–122.5 ꢁC. 1H NMR (CDCl3) d: 0.88 (t,
J=6.6 Hz, 6H), 1.15–1.40 (m, 50H), 1.40–2.00 (m, 8H),
2.20 (t, J=7.6 Hz, 2H), 2.42 (br s, 1H), 3.10–3.35 (m,
2H), 3.60–3.80 (m, 2H), 4.40–4.60 (m, 1H), 6.37 (d,
J=8.1 Hz, 1H), 6.49 (br, 1H). Anal. calcd for
C37H74N2O3: C, 74.69; H, 12.54; N, 4.71; found: C,
74.55; H, 12.51; N, 4.66.
Mp 130.0–132.0 ꢁC. 1H NMR (CDCl3) d: 0.88 (t,
J=6.6 Hz, 6H), 1.15–1.40 (m, 50H), 1.40–1.70 (m, 4H),
2.10–2.35 (m, 4H), 2.99 (s, 3H), 2.90–3.40 (m, 4H), 4.61
(dd, J=6.8, 6.8 Hz, 1H), 6.30–6.50 (m, 2H). Anal. calcd
for C37H74N2O4S 0.5H2O: C, 68.15; H, 11.59; N, 4.29;
found: C, 68.07; H, 11.24; N, 4.10.
.
(S)-4-Octadecanoylamino-4-(tetradecylcarbamoyl)butyl
sulfate sodium salt (4a). The title compound was syn-
thesized from the hydroxyl derivative (29a) in the same
manner as 3a.
N-[(R)-2-Mercapto-1-(tetradecylcarbamoyl)ethyl]octade-
canamide (9a).
A
mixture of (30a) (333 mg,
0.286 mmol), tributylphosphine (116 mg, 0.572 mmol),
methanol (20 mL), and water (0.5 mL) was stirred at
room temperature for 19 h. Chloroform (100 mL) was
added to the reaction mixture, and then the resulting
mixture was washed with water, and then dried over
magnesium sulfate. The solution was concentrated in
vacuo, and then the obtained colorless oil was purified
by column chromatography (eluent first, chloroform,
then chloroform/methanol=20:1) to give the title com-
pound as a colorless powder (168 mg, Yield=50%).
Mp 164.0–166.0 ꢁC (dec). H NMR (DMSO-d6) d: 0.84
1
(t, J=6.5 Hz, 6H), 1.10–1.80 (m, 58H), 2.09 (t,
J=7.3 Hz, 2H), 2.90–3.10 (m, 2H), 3.65–3.80 (m, 2H),
4.10–4.25 (m, 1H), 7.60–7.80 (m, 2H). Anal. calcd for
.
C37H73N2NaO6S 1.0H2O: C, 62.15; H, 10.57; N, 3.92;
found: C, 62.29; H, 10.39; N, 3.90.
(S)-4-Tetradecylcarbamoyl-4-octadecanoylaminobutanoic
acid (6a). Benzyl (S)-4-tetradecylcarbamoyl-4-octadeca-
noylaminobutanoate (27a) (1.00 g, 1.43 mmol) was dis-
solved in a mixture of methanol (5 mL) and dioxane
(20 mL) with heating. A mixture of Pd/C (10%, 1.52 g)
and dioxane (10 mL) was added to the above solution,
and then resulting mixture was stirred at room tem-
perature under hydrogen atmosphere for 2 h. Pd–C was
removed by filtration to give a filtrate. The obtained
Pd–C was washed with chloroform, and the obtained
wash was combined with the filtrate. The resulting
solution was concentrated in vacuo to afford the title
compound as a colorless powder (750 mg, Yield=86%).
Recrystallization of the obtained powder from ethyl
acetate gave an analytical sample.
1H NMR (CDCl3) d: 0.88 (t, J=6.6 Hz, 6H), 1.20–1.40
(m, 50H), 1.40–1.75 (m, 4H), 1.67 (dd, J=7.7, 10.7 Hz,
1H), 2.25 (t, J=7.5 Hz, 2H), 2.70 (ddd, J=7.0, 10.1,
13.8 Hz, 1H), 3.04 (ddd, J=4.3, 7.7, 13.8 Hz, 1H), 3.20–
3.40 (m, 2H), 4.52 (ddd, J=4.3, 7.0, 8.1 Hz, 1H), 6.30
(br, 1H), 6.39 (d, J=8.1 Hz, 1H). Anal. calcd for
C35H70N2O2S: C, 72.10; H, 12.10; N, 4.80; found: C,
72.11; H, 12.38; N, 5.01.
N-[(R)-2-(3-Hydroxypropylthio)-1-(tetradecylcarbamoy-
l)ethyl]octadecanamide (31a). Potassium carbonate
(238 mg, 1.72 mmol) was added to a solution of N-[(R)-2
-mercapto-1-(tetradecylcarbamoyl)ethyl]octadecanamide
(9a) (500 mg, 0.858 mmol) and 3-bromopropanol
(144 mg, 1.03 mmol) in DMF (6 mL) and THF (3 mL),
and then the resulting mixture was stirred at room
temperature for 64 h. After addition of water (100 mL)
to the reaction mixture, the diluted mixture was
extracted with ethyl acetate. The organic layer was
washed with water, and dried over magnesium sulfate.
The solution was concentrated in vacuo, and finally the
obtained residue was purified by column chroma-
tography (eluent n-hexane/ethyl acetate=1:1) to give
the title compound as a colorless powder (237 mg,
yield=50%).
Mp 112.5–115.5 ꢁC. H NMR (DMSO-d6) d: 0.75–0.95
1
(m, 6H), 1.10–1.60 (m, 54H), 1.60–2.00 (m, 2H), 2.10 (t,
J=7.3 Hz, 2H), 2.18 (t, J=7.4 Hz, 2H), 2.90–3.15 (m,
2H), 4.10–4.30 (m, 1H), 7.65–7.90 (m, 2H), 12.01 (br s,
1H). Anal. calcd for C37H72N2O4: C, 72.97; H, 11.92; N,
4.60; found: C, 72.81; H, 11.91; N, 4.54.
N-[(S)-3-Methanesulfonyl-1-(tetradecylcarbamoyl)propy-
l]octadecanamide (11a).
perbenzoic acid (mCPBA, 70%, 43 mg, 0.175 mmol) in
A
solution of m-chloro-
dichloromethane (5 mL) was added at room temperature

K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
2459
1H NMR (CDCl3) d: 0.88 (t, J=6.6 Hz, 6H), 1.10–1.40
(m, 50H), 1.40–1.80 (m, 5H), 1.80–1.95 (m, 2H), 2.22 (t,
J=7.5 Hz, 2H), 2.60–3.05 (m, 4H), 3.15–3.35 (m, 2H),
3.65–3.80 (m, 2H), 4.40–4.55 (m, 1H), 6.40–6.55 (m,
2H). Anal. calcd for C38H76N2O3S: C, 71.19; H, 11.95;
N, 4.37; found: C, 71.20; H, 11.96; N, 4.66.
2.22 (t, J=7.6 Hz, 4H), 3.15–3.50 (m, 4H), 3.70–3.85
(m, 1H), 3.95–4.10 (br, 1H), 6.15–6.35 (br, 2H). Anal.
calcd for C39H78N2O3: C, 75.18; H, 12.62; N, 4.50;
found: C, 74.87; H, 12.53; N, 4.63.
2-Octadecanoylamino-1-(octadecanoylaminomethyl)ethyl
sulfate sodium salt (13q). The title compound was syn-
thesized from 35q in the same manner as compound 3a.
3-[(R)-2-Octadecanoylamino-2-(tetradecylcarbamoyl)-
ethyl]thiopropyl sulfate sodium salt (5a). The title com-
pound was synthesized from 31a in the same manner as
3a.
ꢁ
1
Mp 144.0–147.0 C (dec). H NMR (CDCl3) d: 0.88 (t,
J=6.6 Hz, 6H), 1.15–1.40 (m, 56H), 1.40–1.70 (m, 4H),
2.19 (t, J=7.5 Hz, 4H), 3.15–3.40 (m, 2H), 3.40–3.65
(m, 2H), 4.35–4.50 (m, 1H), 7.10–7.50 (br, 2H). Anal.
ꢁ
1
Mp 183.0–185.0 C (dec). H NMR (CDCl3) d: 0.88 (t,
J=6.6 Hz, 6H), 1.20–1.50 (m, 50H), 1.50–1.80 (m, 4H),
1.95–2.15 (m, 2H), 2.21 (t, J=7.5 Hz, 2H), 2.65–3.10
(m, 4H), 3.15–3.45 (m, 2H), 4.15–4.35 (m, 2H), 4.60–
4.80 (m, 1H), 6.85–7.00 (m, 1H), 7.30–7.45 (m, 1H).
.
calcd for C39H77N2NaO6S 1.25H2O: C, 62.66; H, 10.72;
N, 3.75; found: C, 62.62; H, 10.59; N, 3.47.
The following compounds 13r and 13s were synthesized
from 1,3-diamino-2-propanol and the corresponding
acyl chloride via the corresponding hydroxyl derivative
in the same manner as compound 13q.
.
Anal. calcd for C38H75N2NaO6S2 1.0H2O: C, 59.96; H,
10.20; N, 3.68; found: C, 59.66; H, 9.99; N, 3.97.
N-(2-Hydroxyethyl)-2-tetradecylhexadecanamide (33j).
The title compound was synthesized from 2-aminoetha-
nol and 2-tetradecylhexadecanoyl chloride in the same
manner as compound 26.
2-(3-Tetradecyloxypropanoyl)amino-1-[(3-tetradecyloxy-
propanoyl)amino methyl]ethyl sulfate sodium salt (13r).
Mp 143.5–146.0 ꢁC (dec). H NMR (CDCl3) d: 0.88 (t,
1
J=6.6 Hz, 6H), 1.10–1.45 (m, 44H), 1.45–1.65 (m, 4H),
2.35–2.60 (m, 4H), 3.20–3.80 (m, 12H), 4.40–4.55 (m,
1H), 7.30–7.50 (br, 2H). Anal. calcd for
C37H73N2NaO8S 2.0H2O: C, 58.09; H, 10.14; N, 3.66;
found: C, 57.88; H, 9.83; N, 3.60.
Mp 116.0–117.5 ꢁC. 1H NMR (CDCl3) d: 0.88 (t,
J=6.6 Hz, 6H), 1.10–1.70 (m, 52H), 1.95–2.10 (m, 1H),
2.59 (br s, 1H), 3.35–3.50 (m, 2H), 3.65–3.80 (m, 2H),
5.75–5.90 (m, 1H). Anal. calcd for C32H65NO2: C,
77.51; H, 13.21; N, 2.82; found: C, 77.56; H, 13.17; N,
2.69.
.
2-(15-Ethoxypentadecanoyl)amino-1-[(15-ethoxypentade-
canoyl)aminomethyl] ethyl sulfate sodium salt (13s). Mp
110.0–112.0 ꢁC (dec). 1H NMR (CDCl3) d: 1.19 (t,
J=7.0 Hz, 6H), 1.00–1.45 (m, 40H), 1.45–1.75 (m, 8H),
2.05–2.35 (m, 4H), 3.15–3.70 (m, 4H), 3.39 (t,
J=6.6 Hz, 4H), 3.46 (q, J=7.0 Hz, 4H), 4.35–4.55 (m,
1H), 7.10–7.45 (br, 2H). Anal. calcd for
2-(2-Tetradecylhexadecanoyl)aminoethyl sulfate sodium
salt (12j). The title compound was synthesized from 33j
in the same manner as compound 3a. Mp 125.0–
ꢁ
1
126.0 C. H NMR (CDCl3) d: 0.88 (t, J=6.5 Hz, 6H),
1.10–1.70 (m, 52H), 2.00–2.20 (m, 1H), 3.40–3.70 (m,
2H), 4.00–4.25 (m, 2H), 6.93 (br s, 1H). Anal. calcd for
.
C37H73N2NaO8S 1.3H2O: C, 59.06; H, 10.13; N, 3.72;
found: C, 59.09; H, 9.89; N, 3.67.
.
C32H64NNaO5S 1.0H2O: C, 62.40; H, 10.80; N, 2.27;
found: C, 62.47; H, 10.50; N, 2.21.
Inhibitory activity against incorporation of DiI-acetyl-
LDL into macrophages
N-(2-Hydroxy-3-octadecanoylaminopropyl)octadecana-
mide (35q). A solution of octadecanoyl chloride (2.76 g,
9.10 mmol) in THF (30 mL) was added dropwise to a
mixture of 1,3-diamino-2-propanol (34) (400 mg,
4.44 mmol), triethylamine (990 mg, 9.77 mmol), and
THF (200 mL) under ice-cooling, and then the resulting
mixture was stirred at room temperature for 20 h. After
the reaction mixture had been diluted with water
(100 mL) to the reaction mixture, the resulting mixture
was extracted with chloroform. Combined organic layer
was washed with a diluted hydrochloric acid, a satu-
rated aqueous solution of sodium hydrogen carbonate,
and a saturated aqueous solution of sodium chloride
succesively, dried over magnesium sulfate, and con-
centrated in vacuo. The obtained solid was recrys-
tallized from ethanol to give the title compound as a
colorless crystalline powder (2.25 g, Yield=81%).
Recrystallization from ethyl acetate gave an analytical
sample having the following physical chemical properties.
Dulbecco’s modified Eagle medium (DMEM) and fetal
calf serum (FCS) were purchased from GIBCO BRL
(Grand Island, NY, USA). Lipoprotein-deficient serum
(LPDS) was purchased from Sigma Chemical Co. (St.
Louis, MO, USA). Na125I was purchased from Du Pont
New England Nuclear (Boston, MA, USA). Female
ddY mice were obtained from Nihon SLC Co. (Shi-
zuoka, Japan). BCA Protein Assay Reagent Kit was
purchased from PIERCE (Rockford, IL, USA). All
other reagents were purchased from Sigma Chemical
Co. (St. Louis, MO, USA).
LDL was isolated from normolipidemic human plasma
containing 0.1% EDTA, 0.02% sodium azide and
0.5 mg/mL benzamidine ata densiyt of 1.019–1.063 g/
mL by sequential ultracentrifugation.24 LDL was
acetylated by repeated additions of acetic anhydride as
described.25 DiI-acetyl-LDL was prepared by co-incu-
bation of acetyl-LDL with DiI solution as described.26
Acetyl-LDL was radioiodinated at a specific activity of
Mp 127.0–128.5 ꢁC. 1H NMR (CDCl3) d: 0.88 (t,
J=6.5 Hz, 6H), 1.15–1.40 (m, 56H), 1.40–1.75 (m, 4H),

2460
K. Yoshiizumi et al. / Bioorg. Med. Chem. 10 (2002) 2445–2460
100–300 cpm/ng protein with Na125I using the iodine
monochloride method.27
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Mouse peritoneal macrophages were prepared accord-
ing to the method of Cohn and Morse.28 Peritoneal
macrophages were harvested in phosphate-buffered sal-
ine without Ca2+ and Mg2+ (PBS[ꢀ]) from non-stimu-
lated female ddY mice (25–30 g). The cells were
centrifuged at 800g for 5 min, washed once with PBS[ꢀ],
and suspended at2 ꢂ106 cells/mL in DMEM containing
10% FCS, 100 U/mL penicillin and 100 mg/mL strepto-
mycin. The cells suspension (0.4 mL) was plated in 24-
well plastic dishes for uptake assay, and the suspension
(1 mL) was plated in 12-well plastic dishes for binding
assay. After an incubation at 37 ꢁC for 2 h, non-adher-
ent cells were removed by washing twice with PBS[ꢀ].
Adherent cell monolayers were cultured with 1 mL of
DMEM containing 10% FCS, and were used for the
experiments next day.
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The cells were incubated for 4 h at 37 ꢁC in 0.3 mL of
DMEM containing 10% LPDS, 10 mg/mL DiI-Ac-
LDL, and a drug. After the incubation, the cells were
washed once rapidly and twice with ice-cold 50 mM
Tris–HCl (pH 7.4), 150 mM NaCl, 2 mg/mL BSA, and
were washed once with ice-cold 50 mM Tris–HCl (pH
7.4), 150 mM NaCl. The cells were dissolved in 2 mM
SDS, and the fluorescence was measured at the excita-
tion wavelength of 530 nm and at the emission wave-
length of 590 nm. The concentration of cell protein was
determined by BCA Protein Assay Reagent Kit.
14. Sawamura, T.; Kume, N.; Aoyama, T.; Moriwaki, H.;
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H.; Yazaki, Y.; Horiuchi, S.; Takahashi, K.; Kruijt, J. K.; van
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125I-Acetyl-LDL binding assay
The cells were pre-incubated in DMEM containing
10 mM HEPES-Na (pH 7.4) and 10% LPDS for 30 min
at4 ꢁC. The medium was then changed to 1 mL of
DMEM containing 10 mM HEPES-Na (pH 7.4), 10%
LPDS, 10 mg/mL 125I-Ac-LDL, and a drug, and the cell
were further incubated for 2 h at 4 ꢁC. Non-specific
binding was estimated in the presence of a 40-fold
excess unlabeled Ac-LDL. After the incubation, the
cells were washed once rapidly and twice for 10 min with
ice-cold 50 mM Tris–HCl (pH 7.4), 150 mM NaCl,
2 mg/mL BSA, and were washed once with ice-cold
50 mM Tris–HCl(pH 7.4), 150 mM NaCl. The cells were
dissolved in 0.1 N NaOH, and the amount of 125I-Ac-
LDL bound to cells was counted in a gamma counter.
The concentration of cell protein was determined by the
method of Lowry et al.29
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References and Notes
1. Stary, H. C.; Chandler, A. B.; Glagov, S.; Guyton, J. R.;
Insull, W.; Rosenfeld, M. E.; Schaffer, S. A.; Schwartz, C. J.;
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840.
27. Goldstein, J. L.; Basu, S. K.; Brown, M. S. Methods
Enzymol. 1983, 98, 241.
28. Cohn, Z. A.; Morse, S. I. J. Exp. Med. 1959, 110, 419.
29. Lowry, O. H.; Rosebrough, H. J.; Farr, A. L.; Randall,
R. J. J. Biol. Chem. 1951, 193, 265.
2. Gerrity, R. G. Am. J. Pathol. 1981, 103, 181.