Convergent synthesis and in vivo inhibitory effect on fat accumulation of (-)-ternatin, a highly N-methylated cyclic peptide

Article abstract of DOI:10.1016/j.bmcl.2007.06.015

(-)-Ternatin (1), a highly N-methylated cyclic heptapeptide, is a potent inhibitor of fat accumulation against 3T3-L1 murine adipocytes (EC₅₀ = 0.14 μg/mL) [Shimokawa, K.; Mashima, I.; Asai, A.; Yamada, K.; Kita, M.; Uemura, D. Tetrahedron Lett. 2006, 47, 4445]. Compound 1 was synthesized from Boc-protected amino acids in solution. Upon treatment with 1 at 5 mg/kg/day, increases in body weight and fat accumulation in high-fat-fed mice were both significantly suppressed.

Full text of DOI:10.1016/j.bmcl.2007.06.015

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4447–4449
Convergent synthesis and in vivo inhibitory effect
on fat accumulation of (À)-ternatin, a highly
N-methylated cyclic peptide
a
a
b
a,c,
*
Kenichiro Shimokawa, Kaoru Yamada, Masaki Kita and Daisuke Uemura
a
Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, Japan
b
Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, Japan
c
Institute for Advanced Research, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, Japan
Received 11 May 2007; accepted 2 June 2007
Available online 8 June 2007
Abstract—(À)-Ternatin (1), a highly N-methylated cyclic heptapeptide, is a potent inhibitor of fat accumulation against 3T3-L1
murine adipocytes (EC50 = 0.14 lg/mL) [Shimokawa, K.; Mashima, I.; Asai, A.; Yamada, K.; Kita, M.; Uemura, D. Tetrahedron
Lett. 2006, 47, 4445]. Compound 1 was synthesized from Boc-protected amino acids in solution. Upon treatment with 1 at 5 mg/kg/
day, increases in body weight and fat accumulation in high-fat-fed mice were both significantly suppressed.
Ó 2007 Elsevier Ltd. All rights reserved.
Obesity is a serious problem that affects nearly 30% of
the adult population in developed countries, and may
cause serious lifestyle-related diseases such as cancer,
cardiovascular disease, hypertension, hyperlipidemia,
However, due to the limitation of the natural source and
the high cost of the SPPS method, a more effective large-
scale synthesis of 1 is required to further evaluate its bio-
logical and physiological activities. We describe here the
solution-phase synthesis of 1 and its in vivo inhibitory
effect on fat accumulation in diet-induced obese mice.
2
and diabetes. In general, obesity is associated with each
individual’s appetite, relative food preference, storage of
energy, thermogenesis, and metabolism. This can make
it difficult to develop effective anti-obesity drugs. How-
Figure 1 illustrates the two key fragments 2 and 3 that
are involved in our synthetic route. We thought amide
bond formation could be achieved between the amino
3
ever, there may be other approaches to solve this prob-
lem. Recently, several natural materials that may
prevent diet-induced increases in body weight have been
1
group in the D-allo-Ile moiety of 3 and the carboxylic
4
7
a focus of attention worldwide.
acid group in the b-OH-D-Leu moiety of 2. Cyclization
of the linear peptide would then occur between the ami-
5
no group in the L-(NMe)Ala moiety and the carboxylic
In a previous work, we described the isolation of a
highly N-methylated cyclic heptapeptide (À)-ternatin
4
acid group in the L-Leu moiety, which worked well as a
5
(
1) from the bracket fungus Coriolus versicolor, which
reactive coupling site in our previous synthesis.
significantly suppressed the fat accumulation against
T3-L1 murine adipocytes (EC50 = 0.14 lg/mL). The
structure of 1 was confirmed to be a cyclo [D-allo-
3
For effective coupling reactions, we selected phospho-
nium and uronium salt-based coupling reagents such
as PyBOP, PyBroP, and HATU, which have advantages
for fast and high-yield coupling reactions with low
1
2
3
4
5
Ile -L-(NMe)Ala -L-(NMe)Leu -L-Leu -L-(NMe)Ala -D-
6
NMe) Ala -(2R,3R)-3-hydroxy-Leu ] by spectroscopic
7
(
analyses and solid-phase peptide synthesis (SPPS) of 1.
1
6
racemization.
The left fragment 2 was synthesized by stepwise cou-
pling of Boc-NMe-D-Ala-OH and Boc-NMe-L-Ala-OH
to b-OH-D-leucine ethyl ester (5), which was easily pre-
pared from the known azide 4 by hydrogenolysis
(Scheme 1). First, 5 was coupled with Boc-NMe-D-
Ala-OH in the presence of PyBroP to yield dipeptide
Keywords: (À)-Ternatin; Cyclic peptide; Obesity; Inhibitory effect on
7
fat accumulation; Synthesis.
*
Corresponding author. Tel.: +81 52 789 3654; fax: +81 52 789
248; e-mail: uemura@chem3.chem.nagoya-u.ac.jp
5
0
960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.015

4448
K. Shimokawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4447–4449
Figure 1. Retrosynthetic analysis of (À)-ternatin (1).
6
. Removal of the Boc group of 6 followed by coupling
with Boc-NMe-L-Ala-OH and alkaline hydrolysis of
ethyl ester gave 2.
The right fragment 3 was prepared in the same fashion
(
Scheme 2). L-Leucine methyl ester (8) was coupled with
Boc-NMe-L-Leu-OH to give dipeptide 9. Removal of
the Boc group of 9 followed by coupling with Boc-
NMe-L-Ala-OH in the presence of HATU afforded tri-
peptide 10. Sequential removal of the Boc group of 10
followed by coupling with Boc-D-allo-Ile-OH gave tetra-
peptide 11. Finally, treatment of 11 with 50% TFA/
CH Cl gave 3 as a TFA salt. HATU-mediated frag-
2
2
ment coupling between a TFA salt of 3 and the left frag-
ment 2 gave heptapeptide 12 in good yield (88%).
Methyl ester hydrolysis of 12 and deprotection of the
Boc group provided linear peptide 13.
Scheme 2. Synthesis of the right fragment 3 and completion of the
synthesis of (À)-ternatin (1).
The final conversion of 13 to 1 was achieved by HATU
(2.0 equiv)/HOAt (2.0 equiv)-mediated macrolactamiza-
tion at low concentration (1.5 mM), which gave the
product in quantitative yield from 12. This successful
2
2
2
2
2
2
2
2
2
2
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Normal diet (group 1)
HF (group 2)
HF + 1 (group 3)
0
1
2
3
4
5
Weeks
Figure 2. Changes in body weight of C57BL/6J mice fed normal diet
(group 1; n = 8), high-fat diet (group 2; n = 8) or high-fat diet with 1
(group 3; n = 4). All values are means ± SE.
Scheme 1. Synthesis of the left fragment 2.

K. Shimokawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4447–4449
Table 1. Body and organ weights of mice after the experiment
4449
Tissues
Group 1
Group 2 (HF)
Group 3 (HF+1)
Body weight (g)
Liver (g)
24.5 ± 0.30
1.13 ± 0.02
27.0 ± 0.80
0.91 ± 0.04
25.8 ± 0.50*
0.93 ± 0.03
Fat tissues of internal organs
Perirenal (g)
Around genitals (g)
0.10 ± 0.01
0.30 ± 0.02
0.21 ± 0.01
0.81 ± 0.06
0.16 ± 0.03
0.50 ± 0.10
Subcutaneous fat tissues
Inguinal (g)
Dorsal (g)
0.11 ± 0.02
0.13 ± 0.03
0.25 ± 0.01
0.29 ± 0.03
0.16 ± 0.03
0.23 ± 0.04*
All values represent means ± SE, n = 8 for groups 1 and 2, n = 4 for group 3. Values without asterisk are significantly different at P < 0.05.
macrolactamization is notable compared to previous
examples of cyclization in the synthesis of cyclic pep-
tides. In conclusion, 1 was synthesized in 10 steps (lon-
gest sequence) in high yield (49%) from compound 8.
Synthetic 1 inhibited fat accumulation against 3T3-L1
murine adipocytes at a concentration similar to that of
the natural compound.
Research for Creative Scientific Research (16GS0206)
and the 21st century COE program (Establishment of
COE on Materials Science) from the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan.
We are also indebted to Ono Pharmaceutical Co., Ltd
and Banyu Pharmaceutical Co., Ltd for their financial
support.
8
The fat accumulation-inhibitory effect of (À)-ternatin
(1) in C57BL/6J mice (in vivo) has been examined
extensively.
References and notes
9
1
. Shimokawa, K.; Mashima, I.; Asai, A.; Yamada, K.; Kita,
M.; Uemura, D. Tetrahedron Lett. 2006, 47, 4445.
. Mann, C. C. Science 2005, 307, 1716.
. (a) Kordik, C. P.; Reitz, A. B. J. Med. Chem. 1999, 42, 181;
b) Pi-Sunyer, X. Science 2003, 299, 859.
. (a) Jayaprakasam, B.; Olson, L. K.; Schutzki, R. E.; Tai,
M.-H.; Nair, M. G. J. Agric. Food. Chem. 2006, 54, 243; (b)
Honda, S.; Aoki, F.; Tanaka, H.; Kishida, H.; Nishiyama,
T.; Okada, S.; Matsumoto, I.; Abe, K.; Mae, T. J. Agric.
Food. Chem. 2006, 54, 9055.
Mice were fed normal diet (group 1; n = 8), high-fat diet
2
3
(
(
HF) (group 2; n = 8) or HF with 1 (5 mg/kg/day)
group 3; n = 4) for 5 weeks. After this period, the body
(
weights of group 1 and group 2 mice were significantly
different, with means of 24.5 and 27.0 g, respectively
4
(
Fig. 2). Meanwhile, the increase in body weight in
group 3 was drastically suppressed (25.8 g) compared
to that in group 2. Interestingly, food consumption in
group 3 mice (2.2 g/day/head) was similar to that in
group 2 mice (2.2 g/day/head). This suggested that 1
suppressed the increase in body weight but did not affect
food consumption or appetite.
5. In our previous synthesis, cyclization was performed using
.77 mM linear peptide in CH Cl , HATU (2 equiv), HOAt
2 equiv), DIPEA (4.5 equiv) (73% yield). However, the
0
(
2
2
7
hydroxyl group in the b-OH-D-Leu moiety was protected
with the Bn group.
6
. Selected papers for the synthesis of N-methyl cyclic-
peptides and depsipeptides using phosphonium and uroni-
um-based salts: (a) Jou, G.; Gonzalez, I.; Albericio, F.;
Lloyd-Williams, P.; Giralt, E. J. Org. Chem. 1997, 62, 354;
(b) Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997,
97, 2243.
Furthermore, after these experiments, average weights
of the liver and the fat tissues of internal organs (perire-
nal and around the genitals) and subcutaneous parts
(
inguinal and dorsal tissue) in each group of mice were
measured (Table 1). In all cases, the weight of fat tissue
in group 3 was greatly reduced compared to that in
group 2, and was close to that in normal (group 1) mice.
Thus, 1 significantly suppressed fat accumulation in
both subcutaneous fat tissues and in those around inter-
nal organs.
7. Hale, K. J.; Manaviazar, S.; Delisser, M. Tetrahedron 1994,
0, 9181.
5
. Davies, J. S. J. Pept. Sci. 2003, 9, 471.
. Animals and diets; 4-week-old male C57BL/6J mice were
purchased from Charles River Japan Co. Ltd (Kanagawa,
Japan). The diets, MF (standard), and high-fat diet
8
9
(
4
3
composed of 40% beef suet, 10% cornstarch, 9% glucose,
TM
In summary, (À)-ternatin (1) was synthesized from
Boc-protected amino acids via a convergent route in
solution. Compound 1 significantly inhibited both the
increase in body weight and fat accumulation in high-
fat-fed mice. This work will enable further studies on
the derivatization of 1 and the evaluation of its struc-
ture–activity relationships.
TM
% AIN-76 with minerals, AIN-76 with vitamins, and
6% casein) were purchased from Oriental Yeast Co., Ltd
(Tokyo, Japan). The mice were individually housed under a
controlled temperature (24 ± 3 °C) and a 12-h light–dark
cycle. The mice had free access to water and standard
rodent diet for 8 days before the experiment. The mice
were then randomly divided into three groups for the study.
(
À)-Ternatin (1) (5 mg/kg/day) was administered by oral
injection in group 3. Food was changed twice a week. Daily
food intake and weekly body weight for each mouse were
measured throughout the study. After the 5-week feeding
period, mice were anesthetized with ether and killed by
cardiac puncture. Blood samples were obtained using
heparinized syringes. Livers and fat tissues were collected
and immediately weighed.
Acknowledgments
The diet-control experiments were performed by Mer-
cian Cleantec Co., Ltd (Kanagawa, Japan). This study
was supported in part by Grants-in-Aid for Scientific