Synthesis and inhibitory effect on fat accumulation of (-)-ternatin derivatives modified in the β-OH-d-Leu7 moiety

Article abstract of DOI:10.1039/b714710d

An efficient synthesis of (-)-ternatin derivatives directed toward their SAR at the β-OH-d-Leu⁷ moiety and their biological activities against 3T3-L1 murine adipocytes are described. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b714710d

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Synthesis and inhibitory effect on fat accumulation of (−)-ternatin derivatives
modified in the b-OH-D-Leu7 moiety†
Kenichiro Shimokawa,a Yoshiaki Iwase,a Kaoru Yamadaa and Daisuke Uemura*a,b
Received 24th September 2007, Accepted 13th November 2007
First published as an Advance Article on the web 22nd November 2007
DOI: 10.1039/b714710d
An efficient synthesis of (−)-ternatin derivatives directed
toward their SAR at the b-OH-D-Leu7 moiety and their
biological activities against 3T3-L1 murine adipocytes are
described.
(−)-Ternatin (1) is a highly N-methylated cyclic heptapeptide
that was isolated from the mushroom Coriolus versicolor during
our continuing search for potential anti-obesity agents from
natural resources such as mushrooms. In our previous paper, we
described the isolation, structure elucidation and synthesis of 1,
which potently inhibited fat accumulation against 3T3-L1 murine
adipocytes.1 Additionally, we also reported a concise synthesis of
Fig. 1 (A) Stereostructure of 1 fixed by intramolecular H-bond networks
(indicated with dotted lines); (B) H–D exchange experiment evaluated in
1H NMR spectrum (600 MHz) in C6D6 with addition of D2O.
1 in solution and its in vivo biological activity.2 Treatment with 1
at 5 mg kg−1 per day was found to suppress the increase in body
weight and fat accumulation in diet-induced obese mice.
To further evaluate 1 as a new lead compound for therapeutic
development, we commenced on new research to clarify the
detailed mode of action of 1 with regard to fat-accumulation in-
hibition in adipocytes. Moreover, a structure–activity relationship
(SAR) study of 1 was first investigated in parallel, aimed at the
recognition of the importance of side chain functionalities as well
as a suitable site for advanced functionalization in its structure,
e.g., biotinylation and introduction of a fluorescent unit.
OH group in the b-OH-D-Leu7 moiety is supposed to contribute
toward the stabilization of the macrocyclic conformation of 1 by
forming a H-bond between the OH proton (HC) in that moiety and
1
=
the C O in the Ile moiety, whereas two H-bonds of NH protons
(HA, HB) act strongly on stabilizing b-turn structure. Presumably,
these intramolecular H-bonds are key interactions in 1, since they
build the b-turn structure, which is one of the major motifs of
peptide and protein secondary structure playing a key role in many
biological processes.4,5
In order to demonstrate the intramolecular H-bonds of com-
pound 1 in solution, we evaluated hydrogen–deuterium (H–D)
exchange properties of NH and OH protons (Fig. 1B). The
experiment was conducted by adding 20 lL of D2O in a C6D6
solution. As a result, NH protons HA and HB remained over 24 h,
expectedly. Meanwhile, a OH proton HC and a NH proton HD
smoothly exchanged within 24 h. These results strongly suggested
the existence of intramolecular H-bonds of two NH protons, HA
and HB. However, the possibility of a H-bond of HC was unclear
due to the flexible nature of the OH proton.
To confirm directly whether the b-OH-D-Leu7 moiety is im-
portant for the bioactivity of 1, chemical modification at this
position was first investigated. We describe here the first and
efficient synthesis of ternatin derivatives and the SAR at the b-
OH-D-Leu7 moiety with regard to the inhibitory activity of fat
accumulation against 3T3-L1 adipocytes.
Structurally, the existence of non-coded (D- and N-methylated)
amino acids is a novel feature in 1. Of particular interest is the
effect on biological activity of modifying the unusual b-OH-D-Leu
[(2R,3R)-3-hydroxyleucine], which is a key constituent of 1. Based
on an analysis of the X-ray crystal structure of (−)-ternatin (1),3
it is proposed that 1 adopts type II b-turn structure in the region
of L-Leu4 to b-OH-D-Leu7 moieties with the assistance of a novel
intramolecular H-bond network (Fig. 1A). In this network, the
For this purpose, we designed two types of derivatives, a non-
OH series [D-Ala7-1 (1a), D-Leu7-1 (1b), D-Ser(OBn)7-1 (1c)] and
a OH series [D-Thr7-1 (1d) and D-Ser7-1 (1e)], as final targets. The
latter compounds were used to realize whether b-OH-D-Leu7 could
be replaced with normal b-hydroxy-a-amino acids such as serine
and threonine.
aDepartment of Chemistry, Graduate School of Science, Nagoya University,
Furo-cho, Chikusa, Nagoya 464-8602, Japan. E-mail: uemura@chem3.
chem.nagoya-u.ac.jp; Fax: 81-52-789-3654; Tel: 81-52-789-3654
bInstitute for Advanced Research, Nagoya University, Furo-cho, Chikusa,
Nagoya 464-8602, Japan
Synthesis of the derivatives 1a–e was performed in solution by
exploiting our efficient synthetic route to 1, which is amenable to
† Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/b714710d
58 | Org. Biomol. Chem., 2008, 6, 58–60
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large-scale synthesis for extensive biological evaluations. Begin-
ning with four D-amino acid methyl esters 2a–d, we assembled
tripeptides 5a–d in the C to N direction (Scheme 1). Couplings
of 2a–d with Boc-NMe-D-Ala-OH provided dipeptides 3a–d.
The Boc deprotections followed by second HATU-mediated
condensations with Boc-NMe-L-Ala-OH gave tripeptides 4a–d.
Scheme 1 Synthesis of the carboxylic acid fragments 4a–d.
The final conversions to the desired 1a–d were elaborated by
key fragment couplings and cyclization reactions (Scheme 2).
The alanine methyl esters of 4a–d were cleaved by treatment
with LiOH to yield the carboxylic acid fragments 5a–d as key
intermediates. Attempts to couple 5a–d with amine 7, prepared
by the Boc deprotection of tetrapeptide 6, proceeded smoothly
to provide the linear peptides 8a–d in 65–100% yields from 5a–d.
The leucine methyl esters were then saponified, and the resulting
carboxylic acids were subjected to Boc deprotection. Finally, the
key macrolactamizations of the linear peptides were accomplished
in the presence of HATU, HOAt and DIPEA under dilute
conditions to generate the cyclized products 1a–d in successful
yields (56–62% yields from 8a–d). Furthermore, modification of
compound 1c was performed. The Bn protecting group of 1c was
removed by hydrogenolysis to give 1e in 74% yield.
The bioactivities of synthetic derivatives were then evaluated
with regard to their inhibitory effect on fat accumulation and cy-
totoxicity against 3T3-L1 murine adipocytes. Table 1 summarizes
the biological activities of derivatives 1a–e with two controls, (−)-
ternatin (1) and (−)-noradrenaline (+)-bitartrate salt, respectively.
Based on the results, all synthetic derivatives were found to
exhibit an inhibitory effect. The potency of inhibition tended to
decrease in the order 1b > 1d > 1c > 1a > 1e. Interestingly,
Scheme 2 Synthesis of ternatin derivatives 1a–e.
the simplified D-Leu7-derivative 1b displayed 8-fold lower activity
compared to compound 1 which differs only in the presence
of the hydroxy group in the b-position of the Leu7 moiety. In
contrast, other derivatives showed less activity (120–520 fold) than
1, though they still remained active. It is interesting to note that
synthetic derivatives possessed almost the same IC50 values of
fat-accumulation inhibition independent of the presence of the
hydroxy group in their structures [a non-OH series (1a and 1c) vs.
a OH series (1d and 1e)].
With regard to the amino acid in the 7-position in 1, the results
indicated that the specific side chain, i.e., isobutyl group, is strictly
required for potent bioactivity. On the other hand, the presence of
the hydroxy group in the b-position of the amino acid residue is
not absolutely important. Therefore, it may be possible to install
Table 1 Fat-accumulation inhibitory effect of derivatives 1a–e and cell viability of 3T3-L1 murine adipocytesa
Compound
Fat-accumulation inhibitory effect: IC50/lM
Cell viability b: IC50/lM
[D-Ala7]ternatin (1a)
[D-Leu7]ternatin (1b)
[D-Ser(OBn)7]ternatin (1c)
[D-Thr7]ternatin (1d)
[D-Ser7]ternatin (1e)
(−)-Ternatin (1)
14
0.22 0.02
5.9
3.1 0.4
12
0.027 0.003
260
4
>150
3.3 0.2
89 10
1
51
9
3
>150
0.28 0.03
>300
(−)-Noradrenaline
(+)-bitartrate salt
6
a Values are means of quadruplicate determinations. b Cell viability was calculated independently to exclude undesired fat-accumulation inhibition arising
from the toxicity of tested compounds. At the concentration of 50% fat-accumulation inhibition (IC50), no cell toxicity was observed for any of the
compounds.
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new functional groups at this position, since all derivatives still
possess considerable activity.
and Technology, Japan. We are indebted to Ono Pharmaceutical
Co., Ltd. and Banyu Pharmaceutical Co., Ltd. for their financial
support.
At 10–15 fold higher concentrations compared to those of
IC50 values of the fat-accumulation inhibitory effect, cytotoxicity
was observed for all derivatives. This may suggest that fat-
accumulation inhibition is the direct cause of toxicity at higher
concentration. In addition, the fat-accumulation inhibitory effect
of compounds may be caused by the prevention of adipogenesis.
Further investigation on the mechanism of inhibition is urgently
needed.
In conclusion, we have constructed a series of ternatin deriva-
tives modified in the b-OH-D-Leu7 moiety based on our convergent
synthetic route. Actually, unusual b-OH-leucine is a novel class of
amino acid that is commonly found in pharmacologically impor-
tant cyclic peptides such as lysobactin,6 YM-254890,7 GE3,8 and
cyclosporin A9,10 (as MeBmt; (4R)-4-[(E)-butenyl]-4,N-dimethyl-
L-threonine). On the other hand, we found that the naturally
occurring b-OH-D-Leu7 in 1 is not absolutely required but is
important for the potent inhibitory effect on fat accumulation
against 3T3-L1 adipocytes. Further studies on this bioactive
molecule are ongoing.
Notes and references
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This study was supported in part by Grants-in-Aid for Scientific
Research for Creative Scientific Research (16GS0206) and the
21st century COE program (Establishment of COE on Materials
Science) from the Ministry of Education, Culture, Sports, Science
10 D. H. Rich, M. K. Dhaon, B. Dunlap and S. P. F. Miller, J. Med. Chem.,
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60 | Org. Biomol. Chem., 2008, 6, 58–60
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