Design, synthesis, and biological evaluation of biotin-labeled (-)-ternatin, a potent fat-accumulation inhibitor against 3T3-L1 adipocytes

Article abstract of DOI:10.1016/j.bmcl.2008.11.003

The design, synthesis, and biological activity of biotin-labeled (-)-ternatin are reported. Chemical modification, that is, biotinylation, was conducted using Click chemistry at the 6-position (NMe-d-ProGly moiety), which was a plausible location selected

Full text of DOI:10.1016/j.bmcl.2008.11.003

Bioorganic & Medicinal Chemistry Letters 19 (2009) 92–95
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological evaluation of biotin-labeled (À)-ternatin,
a potent fat-accumulation inhibitor against 3T3-L1 adipocytes
Kenichiro Shimokawa a, Kaoru Yamada a, Osamu Ohno a, Yuichi Oba b, Daisuke Uemura a,c,
*
a Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, Japan
b Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan
c Institute for Advanced Research, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis, and biological activity of biotin-labeled (À)-ternatin are reported. Chemical mod-
Received 8 October 2008
Revised 29 October 2008
Accepted 3 November 2008
Available online 6 November 2008
ification, that is, biotinylation, was conducted using Click chemistry at the 6-position (NMe-D-ProGly
moiety), which was a plausible location selected on the basis of our SAR studies. The compound displayed
sufficient fat-accumulation inhibitory effect against 3T3-L1 adipocytes for further bio-organic studies.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Ternatin
Cyclic peptide
Biotinylation
Click chemistry
Fat-accumulation inhibitory effect
Health problems caused by obesity have led to new crises in
diseases such as cancer, cardiovascular disease, hypertension,
hyperlipidemia and diabetes, and have shortened the life expec-
tancy.1 Considerable effort has been devoted to the discovery of
anti-obesity drugs worldwide. At the cellular level, obesity is char-
acterized by an increase in the number and size of adipocytes dif-
ferentiated from fibloblastic preadipocytes in adipose tissues.2 For
basic studies on obesity, 3T3-L1 murine preadipocytes, which dif-
ferentiate into mature adipocytes under appropriate conditions,
have been used as a model for adipose cells. For instance, tea chate-
chins2 and flavonoids3 have been reported to suppress adipocyte
differentiation (adipogenesis), which lead to the inhibition of fat
accumulation in 3T3-L1 adipocytes. In addition, capsaicin induces
apoptosis and suppresses adipogenesis in 3T3-L1 cells.4 However,
the effective concentrations of these compounds are rather high
Due to its potent and unique bioactive profile, synthetic studies
of 1 have been carried out, and these have enabled large-scale
preparation and useful derivatization.6 Continuing studies of the
structure–activity relationship (SAR) provided additional insights
regarding further chemical modifications.7
To clarify the detailed mechanism of the inhibitory effect of 1 on
fat accumulation, we began bio-organic studies on 1. In general,
small molecules are thought to exhibit bioactivity by interacting
with receptors in living cells, but this has not yet been clarified
for 1. Therefore, to identify the target bio-molecule that bind to
1, we planned to use biotin–streptavidin affinity purification which
is a classical but reliable solution.8 For this purpose, biotinylated
derivatives with considerable bioactivity must be synthesized.
(>1
l
M). In 2006, (À)-ternatin (1, Fig. 1), a highly N-methylated
cyclic heptapeptide from the mushroom Coriolus versicolor, was
isolated in the course of our continuing search for fat-accumula-
tion-inhibitors from natural sources.5
A biological evaluation
revealed that 1 potently inhibited fat accumulation against 3T3-
L1 adipocytes with an IC50 value of 27 nM. Recently, we also
reported its novel in vivo bioactivity6: 1 suppresses body-weight
gain and fat accumulation in diet-induced obese mice, which
may be promising lead in the development of anti-obesity drugs.
* Corresponding author. Tel./fax: +81 52 789 5869.
E-mail address: uemura@chem3.chem.nagoya-u.ac.jp (D. Uemura).
Figure 1. Structure of (À)-ternatin (1).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.003

K. Shimokawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 92–95
93
We describe here the design, synthesis, and fat-accumulation
inhibitory effects of [NMe-
-ProGly (NMe-propargylglycine)6]-
D
ternatin (2) and biotin-labeled ternatin 3 which would be useful
in further bio-organic studies.
Initially, we sought to install new functional groups at the 7-po-
sition, since the hydroxy group was pre-formatted in natural com-
pound 1. As described in our 1st-generation SAR study,7a the
unusual b-OH-D
-Leu7 amino acid moiety was a plausible candidate
that did not show a significant loss of bioactivity by replacement
with normal amino acids such as Ala, Leu, and Ser. However, ester-
ification between 1 and a tag (biotin-linker conjugate) bearing the
carboxyl group was not successful due to the lower reactivity of
the hydroxyl group. Similarly, neither of the sterically less-hin-
dered synthetic analogues [
D lM) and [D-
-Ser7]-ternatin (IC50 12
Thr7]-ternatin (IC50 3.1
l
M) gave adequate results. Recently, how-
ever, we clarified the essential and inessential amino acid moieties
in 1 for a potent fat-accumulation-inhibitory effect against 3T3-L1
adipocytes, in the 2nd-generation SAR study.7b Based on the SAR
profile, the 6-position was the best location, since potent bioactiv-
ity is believed to retain after chemical modification.
Thus, we planned a concise synthesis of biotin-labeled probe 3,
which is outlined inFigure 2. We envisaged that 3 would be assem-
bled from three key components; cyclic peptide 2, triethylene gly-
col-based linker 4, and (+)-
D-biotin. Use of the newly designed
analogue [NMe-
D
-ProGly6]-ternatin (2) enables the rapid installa-
tion of a biotin-linker conjugate via Click chemistry, that is,
[3 + 2] azide–alkyne cycloaddition, at the final stage.
Scheme 1. Synthesis of [NMe-
MeI (2.2 equiv), NaH (2.2 equiv), THF, rt, quant.; (b) HATU, DIPEA, CH2Cl2, rt, 65%;
(c) 50% TFA/CH2Cl2, 0 °C; (d) Boc-NMe- -Ala-OH, HATU, DIPEA, CH2Cl2, rt, 62%; (e)
1 M NaOH aq, 1,4-dioxane, 0 °C, 48%; (f) right fragment 11 (1.0 equiv), HATU, DIPEA,
CH2Cl2, 76%; (g) LiOH, H2O, t-BuOH, THF; (h) 50% TFA/CH2Cl2, 0 °C; (i) HATU, HOAt,
DIPEA, DMF, CH2Cl2 (1.5 mM), 2 days, 28% in 3 steps.
D
-ProGly6]-ternatin (2). Reagents and conditions: (a)
The synthesis of 2 began with the N-methylation of Boc-
Gly-OH (6) (Scheme 1). The use of an appropriate amount of NaH
(2.2 equiv) gave the desired Boc-NMe-
-ProGly-OH (7).9 Next, 7
was coupled with b-OH-
-Leu-OEt (5)6 in the presence of HATU
D-Pro-
L
D
D
to provide dipeptide 8. Removal of the Boc group of 8 with 50%
TFA/CH2Cl2 followed by a second HATU-mediated condensation
with Boc-NMe-L-Ala-OH gave tripeptide 9. After alkaline hydrolysis
Synthesis of the probe 3 is shown in Scheme 2. First, triethylene
glycol derivative 4 was prepared as a linker. We anticipated that 4
would be helpful for increasing the solubility of the target probes
in aqueous solution. The azide alcohol 14, readily prepared from
commercially available triethylene glycol (13),11 was subjected to
hydrogenation followed by Boc protection of the resulting amino
group to afford alcohol 15. Next, 15 was converted into azide 16
by sequential mesylation and azidation. The Boc group of 16 was
cleaved under acidic conditions to provide linker 4 as a TFA salt.
Next, the functional unit, that is, biotin, was incorporated. Bio-
tin-linker conjugate 17 was synthesized by HATU-mediated cou-
of the ester group, the left fragment 10 was obtained. Next, the for-
mation of an amide bond between the two fragments, the carbox-
ylic acid 10 and the amine 116 (a key common fragment),
generated heptapeptide 12 in medium yield. Methyl ester hydroly-
sis followed by Boc deprotection of 12 gave the cyclic precursor. Fi-
nally, the key macrolactamization between the amino group in the
NMe-L L
-Ala5 moiety and the carboxyl group in the -Leu4 moiety
was performed in the presence of HATU (2.0 equiv) and HOAt
(2.0 equiv) at low concentration (1.5 mM). After HPLC purification
of the reaction mixture, [NMe-
tained in 28% yield (from 12).
D
-ProGly6]-ternatin (2)10 was ob-
pling of linker 4 and (+)-D-biotin in quantitative yield after HPLC
Figure 2. Synthetic design of biotin-labeled ternatin 3.

94
K. Shimokawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 92–95
Based on the results, compound 2 showed 1.4-fold more potent
bioactivity than natural 1, which entirely agreed with our previous
SAR profile. On the other hand, it was found that 2 is considerably
less toxic compared to 1. The bioactivity of probe 3 (IC50 for fat
accumulation: 46 lM) was 1700-fold weaker than that of 1, how-
ever, it is still potent enough to be used in further applications as a
chemical probe. Consequently, the modification at the 6-position,
which was defined in our whole SAR study, provided generally po-
tent bioactivity. Meanwhile, 18 did not exhibit any bioactivity with
regard to fat-accumulation-inhibition or cytotoxicity (both
IC50: > 160 lM). This evidence shows the chemical structure
responsible for bioactivity is the peptide core.
In summary, we designed highly bioactive [NMe-D
-ProGly6]-
ternatin (2) bearing an alkyne group for chemical modification.
The use of ‘Click Chemistry’ enabled the rapid and reliable incorpo-
ration of a biotin unit. Bioactivity of bioin-labeled ternatin 3 was
potent enough for its application in bio-organic studies. Further
studies on this topic are now in progress.
Acknowledgment
This study was supported in part by Grants-in-Aid for Scientific
Research for Creative Scientific Research (Grant No. 16GS0206) and
the Global COE program in Chemistry at Nagoya University (Grant
No. B-021) from the Ministry of Education, Culture, Sports, Science
and Technology, Japan. We are indebted to Ono Pharmaceutical
Co., Ltd. and Banyu Pharmaceutical Co., Ltd. for their financial
support.
References and notes
Scheme 2. Synthesis of biotin-labeled ternatin 3. Reagents and conditions: (a) H2,
5% Pd/C, Boc2O, MeOH, quant.; (b) MsCl, NEt3, THF; (c) NaN3, DMF, 50 °C, 64% in 2
steps; (d) 50% TFA/CH2Cl2; (e) (+)-D-biotin, HATU, DIPEA, DMF, quant. in 2 steps; (f)
1. (a) Mann, C. C. Science 2005, 307, 1716; (b) Kordik, C. P.; Reitz, A. B. J. Med.
Chem. 1999, 42, 181; (c) Pi-Sunyer, X. Science 2003, 299, 859.
2. Furuyashiki, T.; Nagayasu, H.; Aoki, Y.; Bessho, H.; Hashimoto, T.; Kanazawa, K.;
Ashida, H. Biosci. Biotechnol. Biochem. 2004, 68, 2353.
CuSO4, sodium ascorbate, t-BuOH, H2O, 57%.
3. (a) Harmon, A. W.; Joyce, B. H. Am. J. Physiol. Cell Physiol. 2001, 280, 807; (b)
Hsu, C.-L.; Yen, G.-C. J. Agric. Food Chem. 2007, 55, 8404.
4. Hsu, C.-L.; Yen, G.-C. J. Agric. Food Chem. 2007, 55, 1730.
purification. Finally, the alkyne–azide cycloaddition reaction12 of 2
and 17 was performed by using CuSO4 and sodium ascorbate in
water, which provided desired 313 in moderate yield. In addition,
we prepared biotin-linker conjugate 1814 to examine the influence
of its chemical structure on bioactivity (as a negative control).
5. (a) Shimokawa, K.; Mashima, I.; Asai, A.; Yamada, K.; Kita, M.; Uemura, D.
Tetrahedron Lett. 2006, 47, 4445; (b) Shimokawa, K.; Mashima, I.; Asai, A.; Ohno,
T.; Yamada, K.; Kita, M.; Uemura, D. Chem. Asian J. 2008, 3, 438.
6. Shimokawa, K.; Yamada, K.; Kita, M.; Uemura, D. Bioorg. Med. Chem. Lett. 2007,
17, 4447.
7. (a) Shimokawa, K.; Iwase, Y.; Yamada, K.; Uemura, D. Org. Biomol. Chem. 2008,
6, 58; (b) Shimokawa, K.; Iwase, Y.; Miwa, R.; Yamada, K.; Uemura, D. J. Med.
Chem. 2008, 51, 5912.
The three synthetic derivatives, [NMe-D
-ProGly6]-ternatin (2),
biotin-labeled ternatin 3 and biotin-linker conjugate 18, were as-
sessed with regard to their fat-accumulation-inhibitory effect
against 3T3-L1 adipocytes (Table 1). The bioassay consisted of
the treatment of confluent 3T3-L1 preadipocytes with each sample
and further incubation for 7 days. After this period, control cells
were completely differentiated into mature adipocytes. Both the
rate of fat accumulation and the cell viability of adipocytes treated
with each sample were calculated. Among the samples tested, no
cytotoxicity was observed at the concentration that gave 50% fat-
accumulation inhibition (IC50).
8. Hübel, K.; Lebmann, T.; Waldmann, H. Chem. Soc. Rev. 2008, 37, 1361.
9. The use of excess NaH resulted in complex mixture of N-methylated and C,N-
dimethylated products (C-methylation was occurred at the terminal alkyne
group). For example, the use of 3.3 equiv NaH afforded the mixture in 1:1 ratio,
which cannot be separated by silica-gel column chromatography.
28
10. Spectroscopic data for 2: [
a
]
D À53.1° (c, 0.58, CHCl3); IR (CHCl3) 3346, 3308,
3008, 2963, 2870, 1635, 1507, 1411, 1078 cmÀ1
;
1H NMR (600 MHz, C6D6) d
7.92 (d, J = 9.5 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 6.21 (d, J = 7.0 Hz, 1H), 5.88 (d,
J = 3.7 Hz, 1H), 5.77 (dd, J = 11.3, 4.7 Hz, 1H), 5.67 (q, J = 6.6 Hz, 1H), 5.25–5.17
(m, 1H), 5.08 (t, J = 9.5 Hz, 1H), 4.48 (dd, J = 7.3, 3.0 Hz, 1H), 4.36 (q, J = 7.3 Hz,
1H), 4.29 (dd, J = 11.3, 4.0 Hz, 1H), 3.95–3.90 (m, 1H), 3.29 (s, 3H), 3.05 (ddd,
J = 17.6, 4.7, 2.9 Hz, 1H), 2.79 (s, 3H), 2.694 (s, 3H), 2.689 (s, 3H), 2.48 (ddd,
J = 17.6, 11.3, 2.9 Hz, 1H), 2.38–2.32 (m, 1H), 2.14 (quint., J = 6.6 Hz, 1H), 2.10–
2.04 (m, 1H), 1.83–1.73 (m, 2H), 1.67–1.60 (m, 1H), 1.60 (t, J = 2.9 Hz, 1H),
1.50–0.60 (m, 4H) 1.48 (d, J = 7.0 Hz, 3H), 1.43 (d, J = 6.6 Hz, 3H), 1.25 (d,
J = 6.6 Hz, 3H), 1.16 (d, J = 6.6 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H), 0.93 (d, J = 6.6 Hz,
3H), 0.90 (d, J = 7.0 Hz, 3H), 0.88 (d, J = 7.3 Hz, 3H), 0.60 (d, J = 7.0 Hz, 3H), 0.58
(t, J = 7.3 Hz, 3H); 13C NMR (150 MHz, C6D6) 175.3, 174.4 (2C), 174.3, 173.7,
168.8, 168.2, 76.1, 71.8, 69.9, 59.2, 56.2, 55.18, 55.11, 51.4, 49.84, 49.79, 40.5,
38.9, 37.9, 33.7, 30.7, 30.3, 29.7, 29.3, 26.7, 26.0, 25.3, 23.8, 23.3, 22.6, 21.34,
21.30, 18.5, 15.8, 14.8, 13.5, 13.2, 11.6; HRMS (FAB) calcd for C39H67N7O8Na
(M+Na)+ 784.4949, found 784.4954.
Table 1
Fat-accumulation inhibitory effect of synthetic compounds 2, 3, and 18 and cell
viability of 3T3-L1 adipocytes.a
Compound
Fat-accumulation inhibitory effect:
IC50 M)
Cell viability: IC50
M)
(
l
(l
(À)-Ternatin (1)
0.027 0.003
0.019 0.001
0.28 0.03
> 5.2b
11. Jeong, S. W.; O’Brien, D. F. J. Org. Chem. 2001, 66, 4799.
[NMe-D
-ProGly6]-
12. (a) Rostovsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int.
Ed. 2002, 41, 2596; For reviews on Click Chemistry, see: (b) Kolb, H. C.; Finn, M.
G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004; (c) Gil, M. V.; Arévalo,
M. J.; López, Ó. Synthesis 2007, 1589.
ternatin (2)
3
18
46 2.6
>160
>86
>160
13. Spectroscopic data for 3: 1H NMR (600 MHz, CD3OD) d 7.94 (d, J = 8.5 Hz, 1H),
7.77 (s, 1H), 7.72 (d, J = 9.2 Hz, 1H), 5.54 (dd, J = 11.7, 4.4 Hz, 1H), 5.52 (q,
J = 7.0 Hz, 1H), 5.94–5.86 (m, 3H), 4.62 (d, J = 3.7 Hz, 1H), 4.55 (s, 1H), 4.50 (q,
a
Values are means of quadruplicate determinations.
54% inhibition at 5.2 lM. Not tested at higher concentrations.
b

K. Shimokawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 92–95
95
J = 5.1 Hz, 2H), 4.47 (dd, J = 7.7, 4.0 Hz, 1H), 4.29 (dd, J = 7.7, 4.4 Hz, 1H), 4.10
(dd, J = 11.0, 4.0 Hz, 1 H), 3.85 (t, J = 5.1 Hz, 2H), 3.61–3.55 (m, 6H), 3.50 (t,
J = 5.5 Hz, 2H), 3.38–3.32 (m, 4H), 3.22–3.16 (m, 1H), 3.09 (s, 3H), 3.07 (s, 3H),
2.98 (s, 3H), 2.94 (s, 3H), 2.91 (dd, J = 12.8, 4.7 Hz, 1H), 2.69 (d, J = 12.8 Hz, 1H),
2.19 (t, J = 7.7 Hz, 2H), 2.09 (ddd, J = 15.8, 11.0, 4.7 Hz, 1H), 1.94–1.86 (m, 1H),
1.85–1.81 (m, 1H), 1.76–1.39 (m, 12H), 1.22–1.17 (m, 1H), 1.20 (d, J = 6.0 Hz,
3H), 1.15 (d, J = 7.3 Hz, 3H), 1.03 (d, J = 7.7 Hz, 3H), 1.02 (d, J = 7.0 Hz, 3H), 0.99
(d, J = 7.3 Hz, 3H), 0.98 (t, J = 7.3 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H), 0.93 (d,
J = 6.6 Hz, 3H), 0.91 (d, J = 5.8 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H), two amide
protons in biotin moiety and a hydroxy proton were not observed; 13C NMR
(150 MHz, CD3OD) 183.5, 177.6, 175.0, 174.2, 174.1, 171.3, 171.2, 170.3, 170.2,
151.9, 145.0, 124.6, 77.6, 76.7, 71.5, 71.3, 70.7, 70.5, 63.4, 61.6, 60.3, 57.9, 57.0,
56.8, 56.1, 53.0, 51.3, 51.2, 50.7, 41.0, 40.9, 40.4, 39.2, 36.8, 35.0, 31.9, 31.5,
30.4, 30.2, 29.8, 29.7, 29.5, 27.6, 26.8, 26.6, 26.2, 24.0, 23.7, 23.2, 21.22, 21.19,
15.5, 15.0, 14.3, 12.1; HRMS (FAB) calcd for C55H96N13O12S (M+H)+ 1162.7022,
found 1162.7008.
14. Compound 18 was synthesized as shown below.