2,4-Bis(octadecanoylamino)benzenesulfonic acid sodium salt as a novel scavenger receptor inhibitor with low molecular weight

Article abstract of DOI:10.1016/j.bmcl.2004.03.082

In order to investigate the effect of the fixation of the orientations of the two long chains, three types of novel derivatives of scavenger receptor inhibitor 1 were synthesized, and their biological activities were evaluated. Among the novel derivatives

Full text of DOI:10.1016/j.bmcl.2004.03.082

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2791–2795
2,4-Bis(octadecanoylamino)benzenesulfonic acid sodium salt as a
novel scavenger receptor inhibitor with low molecular weight
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 20 February 2004;accepted 22 March 2004
Abstract—In order to investigate the effect of the fixation of the orientations of the two long chains, three types of novel derivatives
of scavenger receptor inhibitor 1 were synthesized, and their biological activities were evaluated. Among the novel derivatives, 2,4-
bis(octadecanoylamino)benzenesulfonic acid sodium salt (4d) showed the most potent inhibitory activity against the incorporation
of 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate-labeled acetyl-LDL (DiI-acetyl-LDL) into macrophages. 2,5-
Bis(octadecanoylamino)benzenesulfonic acid sodium salt (4c), a regioisomer of 4d, did not exhibit as potent an inhibitory activity as
4d, meaning that the substitution pattern of two long chains on the benzene ring must be important. Compound 4d exhibited 10
times more potent inhibitory activity against the binding of 125I-labeled acetyl-LDL to the surface of macrophages than compound 1.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
receptors, which can recognize modified LDLs, and now
the scavenger receptors discovered by Kodama and co-
workers are called scavenger receptor class A.
The presence of form cells is one of characteristic fea-
tures in the early stages of atherosclerosis. Numerous
studies have demonstrated that the formation of foam
cells begins with the attachment of circulating mono-
cytes to the lumenal surface of endothelial cells. Then,
the monocytes migrate into the subendothelial space,
where they differentiate into macrophages. The resulting
macrophages uptake lipoproteins, and then accumulate
a large amount of cholesterol ester derived from the
lipoproteins, which leads to the deposition of lipid
droplets observed in form cells.1–4
Compounds which possess an inhibitory activity against
the binding of modified LDLs to scavenger receptors
(referred to as scavenger receptor inhibitors) should in-
hibit the accumulation of cholesterol ester, which should
lead to the inhibition of the formation of foam cells.
Therefore, such scavenger receptor inhibitors are ex-
pected to prevent the progress of atherosclerosis. Re-
cently, Suzuki et al. generated class A scavenger
receptor/apolipoprotein E double knockout mouse, and
compared it with apolipoprotein E single knockout lit-
termate.15 As a result, the double knockout mouse
developed 60% smaller atherosclerotic lesions than the
single knockout mouse. This result supports our work-
ing hypothesis strongly.
It is generally accepted that the accumulation of cho-
lesterol ester is caused by the uptake of modified low
density lipoproteins (modified LDLs), not native low
density lipoproteins (native LDLs). Scavenger receptors
expressed on the surface of the macrophages recognize
modified LDLs, and then incorporate them by endocy-
tosis. The first scavenger receptors were identified by
Kodama and co-workers in 1990 as receptors for acetyl-
LDLs.5;6 After that, several research groups7–14 suc-
ceeded in the cloning of several types of scavenger
Sulfatides, a mixture of glycolipids having two long
chains (Fig. 1), is known to be an inhibitor against the
binding of acetyl-LDLs to scavenger receptors.16 In the
previous paper, we reported the structure–activity rela-
tionships (SAR) of sulfatides.17 In addition, we
described novel scavenger receptor inhibitors, such as
compound 1, with synthetic facility.17 In this paper, we
report the synthesis of novel derivatives with two long
alkyl chains whose orientations are fixed, and then
describe their biological activities.
* Corresponding author. Tel.: +81-6-6347-9718;fax: +81-6-6347-9716;
e-mail: zumikazu@kd6.so-net.ne.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.082

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K. Yoshiizumi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2791–2795
O
NH2
NH2
NH2
a
HO
OH
O
HN
R
HO2C
CH3O2C
NaO3SO
O
(CH2)12CH3
NH2
OH
15
14
OH
b
R = -(CH2)14CH3, -(CH2)16CH3, -(CH2)22CH3, -CHOH(CH2)21CH3,
-(CH2)13CH=CH(CH2)7CH3, -CHOH(CH2)12CH=CH(CH2)7CH3, etc.
Sulfatides
NHCO(CH2)16CH3
NHCO(CH2)16CH3
c
HO
CH3O2C
O
NHCO(CH2)16CH3
NHCO(CH2)16CH3
(CH2)16CH3
HN
17
16
NaO3SO
NH(CH2)13CH3
O
d, e
1
NHCO(CH2)16CH3
Figure 1. Structures of sulfatides and compound 1.
NaO3SO
NHCO(CH2)16CH3
2. Chemistry
3a-c
The synthetic route for the trans-4-hydroxy-L-proloine
derivative 2 is shown in Scheme 1. To begin with, con-
densation of trans-N-Boc-4-hydroxy- -proline 10 with
CH3(CH2)16CONH
NaO3SO
NHCO(CH2)16CH3
L
tetradecylamine gave N-Boc-4-hydroxyprolinamide 11.
The Boc group of compound 11 was removed by use of
trifluoroacetic acid (TFA) to give amine 12. Acylation of
12 with octadecanoylchloride yielded N-octadecanoyl-4-
hydroxyprolinamide 13. Finally, compound 13 was
sulfated with chlorosulfuric acid in pyridine, followed by
treatment with sodium carbonate to give the target
compound 2.
3d
Scheme 2. Reagents: (a) concd H2SO4/CH3OH;(b) ClCO(CH 2)16CH3,
i-Pr2EtN;(c) NaBH 4, MeOH;(d) ClSO 3H, pyridine;(e) Na 2CO3.
pyridine and subsequent treatment with sodium car-
bonate yielded sulfate ester sodium salts 3a,b, and 3c. The
attempt to synthesize the 2,4-disubstituted derivative 3d
failed because of the unstability of the final compound.
Benzyl sulfate-type derivatives (3a–c) were prepared as
outlined in Scheme 2. First of all, diaminobenzoic acids
14 were esterified under an acidic condition to give the
corresponding methyl diaminobenzoates 15. Acylation
of the two amino groups of compounds 15 afforded
compounds 16. The ester moieties of compounds 16 were
reduced by sodium borohydride in the presence of
methanol in THF to give benzyl alcohols 17. Finally,
sulfation of compounds 17 with chlorosulfuric acid in
Scheme 3 shows the synthetic routes for benzenesulfonic
acid-type derivatives and a related compound. Diami-
NHCO(CH2)mCH3
NHCO(CH2)mCH3
NH2
NH2
a, b
NaO3S
HO3S
4b-d, 5d, 6d, 7d
18
Boc
Boc
a
N
N
HO
HO
NHCO(CH2)16CH3
NH2
c,b
d
CO2H
CONH(CH2)13CH3
NaO3S
HO3S
10
11
19
8
b
H2N
NO2
CO(CH2)16CH3
H2N
NH2
N
NH
CONH(CH2)13CH3
c
HO
HO
HO2C
HO2C
CONH(CH2)13CH3
20
21
13
12
c
d, e
CH3(CH2)16NHCO
HO2C
NHCO(CH2)16CH3
CO(CH2)16CH3
N
NaO3SO
CONH(CH2)13CH3
2
9d
Scheme 1. Reagents: (a) H2N(CH2)13CH3, EDCI, HOBt;(b) TFA;(c)
ClCO(CH2)16CH3, i-Pr2EtN;(d) ClSO 3H, pyridine;(e) Na 2CO3.
Scheme 3. Reagents: (a) ClCO(CH2)mCH3, i-Pr2EtN;(b) Na 2CO3;
(c) ClCO(CH2)16CH3, i-Pr2EtN;(d) Pd–C, NaBH 4, H2.

K. Yoshiizumi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2791–2795
2793
nobenzenesulfonic acids 18 or 4-aminobenzenesulfonic
acid 19 was acylated with appropriate acid chloride,
then treated with sodium carbonate to afford bis(acyl-
amino)benzenesulfonic acid sodium salts 4b–d, 5d, 6d,
7d, or mono(acylamino)benzenesulfonic acid sodium
salt 8. In the case of benzoic acid 9d, first of all, 2-amino-
4-nitrobenzoic acid 20 was hydrogenated in the presence
of palladium–carbon and sodium borohydride, then the
obtained 2,4-diaminobenzoic acid 21 was converted into
the target compound 9d.
tested in lower concentrations. The obtained results are
summarized in Tables 1 and 2.
The 4-hydroxyproline derivative 2 was a weaker inhib-
itor than compound 1, though compound 2 has the same
two long chains as compound 1. Among the benzyl
sulfate derivatives 3a–c, the 3,5-disubstituted benzyl
derivative 3b showed the most potent inhibitory activity
against the DiI-acetyl-LDL incorporation. The 2,5-
disubstituted benzyl derivative 3c was slightly less potent
than 3b. The inhibitory activity of the 3,4-disubstituted
benzyl derivative 3a was less than 10% at 100 lM. These
results might mean that the substitution pattern of two
octadecanoylamino moieties on the ring is related to the
potency. That is to say, when the substitution pattern of
two octadecanoylamino moieties is so-called meta, a
comparatively strong inhibitory activity might be ob-
tained, and when the pattern is ortho, a comparatively
weak inhibitory activity might be obtained. The two
long chains of compound 2, as well as those of com-
pound 3a, are substituted next to each other on the ring.
The reason why compound 2 was a weak inhibitor could
be explained by the undesirable substitution pattern of
the two long chains.
3. Results and discussion
In the previous paper, we reported that we had synthe-
sized novel derivatives of sulfatides and established the
following SAR: (1) sulfate group was the most favorable
among investigated functional groups, (2) the galactose
moiety can be deleted or replaced with another structure
without a large decrease in the inhibitory activity, (3) b-
(N-acylamino)alcohol moiety of sulfatides can be re-
placed with another structure, (4) the existence of two
long alkyl chains is essential for potent inhibitory
activity. In addition, the synthesis of novel derivatives of
sulfatides led to the discovery of novel scavenger
receptor inhibitors, such as compound 1.
Next, we were interested in the inhibitory activities of
the benzenesulfonic acid-type derivatives, which were
thought to be more stable than the benzyl sulfate-type
derivatives. The potency of the 3,5-disubstituted deriv-
ative 4b was almost equal to that of the corresponding
benzyl sulfate derivative 3b. Interestingly, also in the
benzenesulfonic acid series, the para-substitution deriv-
ative 4c was not as potent as the meta-substitution
derivative 4b, which was consistent with the result ob-
served in the benzyl sulfate series. The 2,4-disubstituted
derivative 4d19 with a meta-substitution pattern exhib-
ited the strongest activity in the benzenesulfonic acid
series.
Compound 1 has two long alkyl chains, which can be
located in all directions. We were interested in fixing the
orientations of the two long chains, and we designed
compounds 2, 3a–d, and 4b–d (see Fig. 2), and synthe-
sized them. In order to monitor the inhibitory activities
of the synthesized compounds against acetyl-LDL
binding to scavenger receptors, all the compounds were
tested for an inhibitory activity against the incorpora-
tion of 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbo-
cyanine perchlorate-labeled acetyl-LDL (DiI-acetyl-
LDL) into macrophages at the concentration of 100 lM
at 37 °C.18 When a tested compound showed an inhibi-
tory activity of more than 50%, the compound was also
Then, substitution pattern was fixed to 2,4-disubstitu-
tion, and the effect of the length of two acyl moieties was
investigated. The tetradecanoyl derivative 6d and the
docosadecanoyl derivative 7d were slightly less potent
than 4d, and the decanoyl derivative 5d was inactive at
100 lM. In order to clarify whether both of the two
octadecanoylamino moieties of 4d are required for the
excellent potency, the inhibitory activity of the 4-
monosubstituted derivative 8 was tested. Compound 8
exhibited a very weak activity of less than 10% at
100 lM. This result indicates that both of the two long
chains are necessary for the potent inhibitory activity.
The carboxylic acid derivative 9d was much less potent
than the sulfonic acid derivative 4d, meaning that sul-
fonic acid is a more preferable functional group than
carboxylic acid.
O
(CH2)16CH3
HN
NaO3SO
NH(CH2)13CH3
O
1
NHCO(CH2)16CH3
NHCO(CH2)16CH3
CO(CH2)16CH3
N
NaO3SO
NaO3SO
CONH(CH2)13CH3
2
3a-d
Finally, the inhibitory activity of compound 4d against
the binding of 125I-labeled acetyl-LDL to scavenger
receptor was tested,20 and compared with those of
compound 1 and sulfatides. As apparent from Table 3,
compound 4d showed 10 times stronger 125I-acetyl-LDL
binding inhibitory activity (IC50 ¼ 2.0 lM) than
compound 1. The potency of compound 4d was thought
NHCO(CH2)16CH3
NaO3S
NHCO(CH2)16CH3
4b-d
Figure 2. Design of novel derivatives of compound 1.

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K. Yoshiizumi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2791–2795
Table 1. Effects of the 4-hydroxylproline derivative 2 and benzyl sulfates 3a–c on the incorporation of DiI-acetyl-LDL into macrophages
Compound
Structure
Inhibitory activity against the incorporation of DiI-acetyl-LDL (%)a
100 lM
30 lM
10 lM
CO(CH2)16CH3
N
2
23.7(1)
NTb
NTb
NaO3SO
CONH(CH2)13CH3
NHCO(CH2)16CH3
NHCO(CH2)16CH3
3a
<10(1)
NTb
NTb
NaO3SO
NaO3SO
NHCO(CH2)16CH3
3b
3c
1
63.0(3)
40.8(1)
61.4(5)
40.4(2)
NTb
NTb
NHCO(CH2)16CH3
CH3(CH2)16CONH
NaO3SO
NTb
NHCO(CH2)16CH3
NHCO(CH2)16CH3
CONH(CH2)13CH3
43.8(4)
20.5(2)
NaO3SO
a The number on the left side of parentheses represents %inhibition at each concentration. The number inside parentheses represents the number of
experiment.
b NT: not tested.
Table 2. Effects of benzenesulfonic acids and related compounds on the incorporation of DiI-acetyl-LDL into macrophages
Compound
Structure
Inhibitory activity against the incorporation of DiI-acetyl-LDL (%)a
100 lM
30 lM
10 lM
NHCO(CH2)16CH3
4b
71.2(3)
46.2(3)
18.5(2)
NaO3S
NHCO(CH2)16CH3
CH3(CH2)16CONH
NaO3S
4c
4d
5d
6d
7d
8
32.1(1)
84.6(3)
<10(1)
66.6(1)
85.7(1)
<10(1)
NTb
NTb
57.0(2)
NTb
NTb
NTb
NTb
NHCO(CH2)16CH3
CH3(CH2)16CONH
NaO3S
NHCO(CH2)16CH3
72.5(3)
NTb
CH3(CH2)8CONH
NaO3S
NHCO(CH2)8CH3
NHCO(CH2)12CH3
NHCO(CH2)20CH3
CH3(CH2)12CONH
NaO3S
48.0(1)
48.0(1)
NTb
CH3(CH2)20CONH
NaO3S
NHCO(CH2)16CH3
NaO3S

K. Yoshiizumi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2791–2795
2795
Table 2 (continued)
Compound
Structure
Inhibitory activity against the incorporation of DiI-acetyl-LDL (%)a
100 lM
30 lM
10 lM
CH3(CH2)16CONH
HO2C
NHCO(CH2)16CH3
9d
1
<10(1)
NTb
NTb
NHCO(CH2)16CH3
CONH(CH2)13CH3
61.4(5)
43.8(4)
20.5(2)
NaO3SO
a The number on the left side of parentheses represents %inhibition at each concentration. The number inside parentheses represents the number of
experiment.
b NT: not tested.
Table 3. Effects of compounds 4d, 1, and sulfatides on the binding of
125I-acetyl-LDL to macrophages
5. Kodama, T.;Freeman, M.;Rohrer, L.;Zabrecky, J.;
Matsudaira, P.;Krieger, M. Nature 1990, 343, 531.
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Krieger, M. Nature 1990, 343, 570.
Compound
Inhibitory activity against the binding of
125I-acetyl-LDL, IC50
a
7. Gough, P. J.;Greaves, D. R.;Gordon, S. J. Lipid Res.
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4d
1
Sulfatides
2.0 lM
41.8% at 30 lM (2)b;c
2.6 lg/mL
a IC50 represents the concentration required 50% inhibition against the
binding of 125I-acetyl-LDL.
b The number inside parentheses represents the number of experiment
at each concentration.
c 81.1% at 100 lM (2), and 6.6% at 10 lM (2).
11. Ramprasad, M. P.;Fischer, W.;Witztum, J. L.;Sambr-
ano, G. R.;Quehenberger, O.;Steinberg, D. Proc. Natl.
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to be almost the same as that of sulfatides (IC50 ¼ 2.6 lg/
mL). Compound 4d is being evaluated in an in vivo
model of atherosclerosis. The results will be presented in
a subsequent paper.
4. Conclusion
14. Adachi, H.;Tsujimoto, M.;Arai, H.;Inoue, K.
Chem. 1997, 272, 31217.
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15. Suzuki, H.;Kurihara, Y.;Takeya, M.;Kumada, N.;
Kataoka, M.;Jishage, K.;Ueda, O.;Sakaguchi, H.;
Higashi, T.;Suzuki, T.;Takashima, Y.;Kawabe, Y.;
Cynshi, O.;Wada, Y.;Honda, M.;Kurihara, H.;Abura-
tani, H.;Doi, T.;Matsumoto, A.;Azuma, S.;Noda, T.;
Toyoda, Y.;Itakura, H.;Yazaki, Y.;Horiuchi, S.;
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Steinbrecher, U. P.;Ishibashi, S.;Maeda, N.;Gordon,
S.;Kodama, T. Nature 1997, 386, 292.
In conclusion, we succeeded in the discovery of novel
scavenger receptor inhibitor 4d whose inhibitory activity
against 125I-acetyl-LDL binding was 10 times more po-
tent than that of compound 1. The substitution pattern
of two long acylamino chains on the benzene ring was
considered to be associated with the inhibitory activity.
The obtained information would be very useful for the
design of effective scavenger receptor inhibitors.
16. Brown, M. S.;Basu, S. K.;Falck, J. R.;Ho, Y. K.;
Goldstein, J. L. J. Supramol. Struct. 1980, 13, 67.
17. Yoshiizumi, K.;Nakajima, F.;Dobashi, R.;Nishimura,
N.;Ikeda, S. Bioorg. Med. Chem. 2002, 10, 2445.
18. The details of the method are described in Ref. 17.
19. 1H NMR (250 MHz, DMSO-d6) d: 0.84 (t, J ¼ 6:5 Hz,
6H), 1.24 (s, 56H), 1.40–1.70 (m, 4H), 2.24 (t, J ¼ 6:3 Hz,
2H), 2.27 (t, J ¼ 6:2 Hz, 2H), 7.44 (dd, J ¼ 1:8, 8.7 Hz,
1H), 7.52 (d, J ¼ 8:7 Hz, 1H), 8.39 (d, J ¼ 1:8 Hz, 1H),
9.94 (s, 1H), 10.42 (s, 1H). Anal. Calcd for
C42H75N2NaO5SÆ0.5H2O: C, 67.07;H, 10.18;N, 3.72;
found: C, 67.02;H, 10.09;N, 3.84.
References and notes
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Insull, W.;Rosenfeld, M. E.;Schaffer, S. A.;Schwartz, C.
J.;Wagner, W. D.;Wissler, R. W. Arterioscler. Thromb.
1994, 14, 840.
2. Gerrity, R. G. Am. J. Pathol. 1981, 103, 181.
3. Stary, H. C. Atherosclerosis 1987, 64, 91.
4. Schwartz, C. J.;Valente, A. J.;Sprague, E. A.;Kelley, J.
L.;Cayatte, A. J.;Mowery, J. Circulation 1992, 86(Suppl
III), 117.
20. The details of the method are described in Ref. 17.