Discovery of halogenated eurypamide B analogues as inhibitors of lipid droplet accumulation in macrophages

Article abstract of DOI:10.1016/j.bmcl.2005.06.070

Halogenated cyclic isodityrosine-tripeptides were synthesized as analogues of a marine natural product, eurypamide B. Although the original eurypamides showed no inhibitory activity, the new analogues were found to inhibit lipid droplet accumulation in macrophages with a low micromolar IC₅₀ value.

Full text of DOI:10.1016/j.bmcl.2005.06.070

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4189–4191
Discovery of halogenated eurypamide B analogues as inhibitors
of lipid droplet accumulation in macrophages
Rika Obata,a Taichi Ohshiro,b Hiroshi Tomodab and Shigeru Nishiyamaa,*
aDepartment of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1,
Kohoku-ku, Yokohama 223-8522, Japan
bLaboratory of Microbial Chemistry, School of Pharmaceutical Sciences, Kitasato University,
5-9-1, Shirokane, Minato-Ku, Tokyo 108-8641, Japan
Received 7 May 2005; revised 17 June 2005; accepted 27 June 2005
Available online 28 July 2005
Abstract—Halogenated cyclic isodityrosine–tripeptides were synthesized as analogues of a marine natural product, eurypamide B.
Although the original eurypamides showed no inhibitory activity, the new analogues were found to inhibit lipid droplet accumula-
tion in macrophages with a low micromolar IC50 value.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Marine natural product eurypamides1 represent a class
of isodityrosine-derived 17-membered cyclic peptides
including aminopeptidase B inhibitor OF4949I-IV,2
angiotensin-I converting enzyme inhibitor K-13,3 and
renieramide4 recently reported. The common scaffold
for OF4949 and K-13 has been taken as a new lead
for protease inhibitors (Fig. 1).5 More recently, the de-
sign and synthesis of macrocyclic structures having a
diaryl ether moiety or the related cyclic compounds
(Fig. 1) were also reported for drug candidates, such
as protease inhibitors and farnesyltransferase inhibi-
tors.6 We have recently completed a total synthesis of
Figure 1. Reported cyclic compounds.
eurypamides A, B, and D using thallium(III) trinitrate
(TTN)-oxidative coupling of an o, o0-dihalogenated
(Fig. 2).8 Based on these findings, several halogenated
eurypamide B derivatives were synthesized to examine
tyrosine derivative for cyclization.7 Although isodityro-
sine-derived 17-membered cyclic peptides showed a
the biological activity.
variety of biological activities, interestingly, euryp-
amides showed no antimicrobial, anti-inflammatory,
or cytotoxic activities.1 Among the molecular modeling
study of eurypamide derivatives including synthetic
2. Synthesis of o,o0-halogenated eurypamide analogues
intermediates, flexibility of the diaryl ether linkage
and peptide chain in the halogenated analogue 1 were
Halogenated eurypamide B derivatives (5a–c, 6a–c) were
prepared in good yield by following our total synthetic
different from that of halogen-free structure
2
route7 (Scheme 1). Coupling reaction of dibrominated
tyrosine derivative 37 with commercially available N-
Boc-threonine or N-Boc-O-benzyl-threonine, under the
conditions using BOP, provided the dipeptides. Subse-
quent deprotection of the N-Boc group and further con-
densation with a diiodinated tyrosine derivative,9 readily
Keywords: Cyclic isodityrosine; Eurypamide; Thallium(III) oxidation;
Lipid droplet accumulation inhibitor; Antiatherosclerotic agents.
*
Corresponding author. Tel.: +81 45 566 1599; fax: +81 45 566
1717; e-mail: nisiyama@chem.keio.ac.jp
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.070

4190
R. Obata et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4189–4191
Table 1. Inhibition of lipid droplet accumulation in macrophages
Compounds
Inhibitory activity (IC50, lM)a
Eurypamide A1
>18.8
>18.8
>22.6
>22.6
3.1
7
Eurypamide A0
Eurypamide B7
Eurypamide D7
5a
5b
5c
6a
6b
6c
3.2
nt
>12.1
>13.5
>12.1
a Values are the mean of three experiments (nt = not tested).
Figure 2. Model structures for conformational study. Lower left:
overlapped minimum conformers of compound 1. Lower right:
overlapped minimum conformers of compound 2.
In conclusion, we have developed low cytotoxic and bio-
logically active eurypamide analogues by simple modifi-
cation. Further investigation in these areas including
additional analogue synthesis with structure–activity
relationship study, and other biological assays will be
reported in due course.
prepared from tyrosine, yielded the linear tripeptides
4. Key ring closure of 4–5 via intramolecular phenolic
oxidation with TTN smoothly proceeded at 0 °C with
yield of 89% at best. Deprotection of the N-Boc group
of 5b and 5c gave 6a and 6b, respectively, and hydrolysis
of 5c gave 6c. The NMR data of 5 and 6 are shown in
Supplement data.
References and notes
1. Reddy, M. V. R.; Harper, M. K.; Faulkner, D. J.
Tetrahedron 1998, 54, 10649.
2. (a) Sano, S.; Ikai, K.; Katayama, K.; Takesako, K.;
Nakamura, T.; Obayashi, A.; Ezure, Y. J. Antibiot. 1986,
39, 1685; (b) Sano, S.; Ikai, K.; Yoshikawa, Y.; Nakam-
ura, T.; Obayashi, A. J. Antibiot. 1987, 40, 512.
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1987, 40, 455; Boger, D. L.; Yohannes, D. Bioorg. Med.
Chem. Lett. 1993, 13, 245.
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Debiyus, C.; Hooper, J.; Gomez-Paloma, L.; Riccio, R. J.
Nat. Prod. 2002, 65, 407.
3. Biological activities of eurypamides and their analogues
The obtained analogues 5a, 5b, 6a–6c, and parent euryp-
amides A (300R,400S), A0 (300S,400R), B, and D7 were
screened for their biological effects. Among biological
assessments, a cell-based assay of lipid droplet synthesis
using mouse peritoneal macrophages as a model of mac-
rophage-derived foam-cell formation10 showed positive
results.11 As shown in Table 1, compounds 5a and 5b
inhibited accumulation of lipid droplets with IC50 values
of 3.1 and 3.2 lM, respectively: these compounds reduce
the size and number of the cytosolic lipid droplets in mac-
rophages. Moreover, no morphological changes or cyto-
toxicity were observed at concentrations up to 10 lM.
These indicated that 5a and 5b possessed no effect against
cell viability, but specific inhibitory activity against accu-
mulation of lipid droplets. The other three analogues 6a–c
and parent eurypamides A–D showed no inhibitory activ-
ity and cytotoxicity up to 12 lM. From these results of 5a,
5b, and 6a–c, more hydrophobic characters, for example,
carrying protecting groups and halogen atoms than that
of parent eurypamide, are required to exhibit the biolog-
ical activity for this assay.
5. Janetka, J. W.; Raman, P.; Satyshur, K.; Flentke, G. R.;
Rich, D. H. J. Am. Chem. Soc. 1997, 119, 441.
6. (a) Dinsmore, C. J.; Bogusky, M. J.; Culberson, J. C.;
Bergman, J. M.; Homnick, C. F.; Zartman, C. B.; Mosser,
S. D.; Schaber, M. D.; Robinson, R. G.; Koblan, K. S.;
Huber, H. E.; Graham, S. L.; Hartman, G. D.; Huff, J. R.;
Williams, T. M. J. Am. Chem. Soc. 2001, 123, 2107; (b)
Venkatraman, S.; Njoroge, F. G.; Girijavallabhan, V.
Tetrahedron 2002, 58, 5453; (c) Dumez, E.; Snaith, J. S.;
Jackson, R. F. W. J. Org. Chem. 2002, 67, 4882.
7. (a) Ito, M.; Yamanaka, M.; Kutsumura, N.; Nishiyama,
S. Tetrahedron Lett. 2003, 44, 7949; (b) Ito, M.; Yama-
naka, M.; Kutsumura, N.; Nishiyama, S. Tetrahedron
2004, 60, 5623.
8. Calculations were done using SPRTANÕ02 for Macintosh
(Wavefunction Inc., CA, USA) on a Power Mac G5
Scheme 1. Reagents and conditions: (a) N-Boc-O-benzyl-Thr, BOP, Et3N/MeCN 94%; (b) TFA/CH2Cl2; (c) N-Z-o,o0-diiodo-Tyr, BOP, Et3N/MeCN
4a 99% in two steps, N-Boc-o,o0-diiodo-Tyr, BOP, Et3N/MeCN–dioxane 4b 94% in two steps, 4c;7 (d) TTN, MeOH–THF (1:4), 0 °C, 40–60 min, 5a
89%, 5b 69%, 5c;7 (e) TFA/CH2Cl2, 5b to 6a 78%, 5c to 6b quant.; (f) 1 N-NaOH/MeOH, 5c to 6c 95%.

R. Obata et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4189–4191
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(Apple Computer Inc., CA, USA) computer. Halogenated
1 and halogen-free 2 model structures of the tripeptide
bonds were generated as b-sheet conformation, then the
tyrosine moiety was cyclized to construct an isodityrosine
bond, and the models were pre-minimized. The stable
local minimum conformers were distributed based on
molecular mechanics calculations with MMFF94 force
field. The first 20 stable minimum conformers were aligned
at the residue of the DOPA moiety as shown in Figure 1.
Decrease of conformational heterogeneity of the diaryl
ether moiety and peptide chain linkage was observed in 1
compared to 2.
9. Nishiyama, S.; Kim, M. H.; Ymamamura, S. Tetrahedron
Lett. 1994, 35, 8397.
10. Namatame, I.; Tomoda, H.; Ishibashi, S.; Omura, S. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 737.
11. None of the tested compounds showed antimicrobial
activity. Antimicrobial activity was tested against Bacil-
lus subtilis, Escherichia coli, Candida albicans, Staphylo-
coccus aureus, Micrococcus luteus, Bacteroides fragilis,
Acholeplasma laidlawii, Pyricularia oryzae, Aspergillus
niger, Mucor racemosus, Saccharomyces cerevisiae, and
Pseudomonas aeruginosa at 10 lg/6 mm diameter paper
disks.