Structure-based hybridization of the bioactive natural products rhizonin A and ternatin leading to a selective fat-accumulation inhibitor against 3T3-L1 adipocytes

Article abstract of DOI:10.1016/j.bmcl.2008.11.109

Based on the structural similarity between the naturally occurring cyclic heptapeptides rhizonin A and ternatin, two novel analogues were designed. The synthetic analogues were assessed with regard to their fat-accumulation inhibitory effect against 3T3-L

Full text of DOI:10.1016/j.bmcl.2008.11.109

Bioorganic & Medicinal Chemistry Letters 19 (2009) 867–869
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based hybridization of the bioactive natural products rhizonin A
and ternatin leading to a selective fat-accumulation inhibitor against
3T3-L1 adipocytes
Kenichiro Shimokawa a, Kaoru Yamada a, Daisuke Uemura a,b,
*
a Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, Japan
b 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:
Based on the structural similarity between the naturally occurring cyclic heptapeptides rhizonin A and
ternatin, two novel analogues were designed. The synthetic analogues were assessed with regard to their
fat-accumulation inhibitory effect against 3T3-L1 adipocytes, and this led to the discovery of a potent and
selective fat-accumulation inhibitor compared to the parent compound rhizonin A.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 4 November 2008
Revised 26 November 2008
Accepted 28 November 2008
Available online 6 December 2008
Keywords:
Rhizonin A
Cyclic peptide
Structure–activity relationships
Fat-accumulation inhibitor
Rhizonin A (1) and ternatin (2) are novel bioactive cyclic hepta-
peptides that were isolated from the fungus Rhizopus microsporus
van Tieghem1 and the mushroom Coliorus versicolor,2,3 respec-
tively, (Fig. 1). They share common structural features in that they
the same concentration. To obtain a better understanding of struc-
ture–activity relationships (SAR) of 1 as well as of the relation be-
tween 1 and 2, we developed a synthesis of novel rhizonin A
analogues, which led to the discovery of a selective fat-accumula-
tion inhibitor against 3T3-L1 adipocytes.
First, we focused on the two NMe-FurAla moieties in 1, which
do not exist in 2. Uniquely, rhizonin A (1) (and its sister compound
rhizonin B1) is the only natural product that contains an unusual
FurAla moiety (including N-methylated variant) as a structural
component. Thus, we designed the di-phenylalanine-substituted
contain
acids, for example, NMe-FurAla [NMe-3-(3-furyl)alanine] or b-
OH- -Leu [(2R,3R)-3-hydroxy-Leu]. Interestingly, they show extre-
D-, N-methylated amino acids as well as unusual amino
D
mely high structural homology: (i) identical sequence of DDDLLLL
amino-acid configurations, (ii) positions of N-methylation, (iii)
contiguous L L
-Leu4-NMe- -Ala5 moieties. An analysis of the X-ray
crystal structures of 1 and 2 demonstrated that the solid-state con-
formation of 1 resembles that of 2, in particular with regard to a b-
turn structure in the region of positions 4 ? 7, which might be
mainly due to (i).4 Therefore, these two compounds could be clas-
sified as closely related cyclic peptides. However, their reported
biological activities are quite different. Recently, we discovered
that 2 significantly inhibited fat-accumulation both in vitro2,3
and in vivo.5 Meanwhile, 1 has only been investigated in vivo
and is known to exhibit a potent hepatotoxic effect in rat which
leads to death in rat and ducklings.6,7 To further examine the bio-
logical activity of 1, we recently achieved the chemical synthesis of
1 and evaluated its fat-accumulation inhibitory activity against
3T3-L1 adipocytes.8 While 1 exhibited a weak inhibitory effect at
high concentration (IC50 55 lM), it was also quite cytotoxic at
* Corresponding author. Tel.: +81 52 789 3654; fax: +81 52 789 5248.
E-mail address: uemura@chem3.chem.nagoya-u.ac.jp (D. Uemura).
Figure 1. Structures of rhizonin A (1) and ternatin (2).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.109

868
K. Shimokawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 867–869
Figure 2. Design of rhizonin A analogues 3 and 4.
analogue [NMe-L D
-Phe3, NMe- -Phe6]rhizonin A (3) to examine the
influence of unusual NMe-FurAla moieties on bioactivity (Fig. 2).
Moreover, with the concept of hybridization in mind, we sought
to synthesize other analogue [NMe-L D
-Leu3, NMe- -Phe6]rhizonin
A (4), which has three amino-acid sequences as in 2 on the right
side of the chemical structure.
The syntheses of analogues 3 and 4 were slightly modified from
the synthetic route toward 1. First, we prepared the right frag-
ments 8a and 8b, which are shown in Scheme 1. The starting mate-
Scheme 2. Synthesis of rhizonin A analogues 3 and 4. Reagents and conditions: (a)
50% TFA/CH2Cl2; (b) Boc-NMe- -Phe-OH, HATU, DIPEA, CH2Cl2; (c) 8a (1.0 equiv) for
3, 8b (1.0 equiv) for 4, HATU, DIPEA, CH2Cl2 (d) LiOH, t-BuOH, THF, H2O; (e) HATU,
D
HOAt, DIPEA, DMF, CH2Cl2 (1.5 mM).
rial NMe-L-Ala-OMe hydrochloride (5) was coupled with Boc-L-
Leu-OH in the presence of PyBroP to give dipeptide 6.
Removal of the Boc group in 50% TFA/CH2Cl2 followed by
amide-bond formation with the corresponding Boc-amino acids
provided tripeptides 7a and 7b, respectively. Finally, sequential
Boc deprotection, coupling, and methyl ester hydrolysis afforded
the desired 8a and 8b.
11. Fragment couplings of 11 with 8a and 8b were conducted be-
tween the carboxyl group in the
L
-Leu4 moiety and the amino
group in the NMe-
L
-Ala5 moiety to give heptapeptides 12a and
12b in moderate yield. Sequential methyl ester hydrolysis and
Boc deprotection of 12a and 12b provided the corresponding cyclic
precursors. Finally, the key macrolactamizations between the car-
Next, the left fragment 11 was prepared from the known dipep-
tide 99 in the same manner (Scheme 2). Removal of the Boc group
boxyl group in the D L
-Val1 moiety and the amino group in the -Val2
in 9 followed by coupling with Boc-NMe-D-Phe-OH afforded tri-
peptide 10, which was then subjected to Boc deprotection to give
moiety were performed in the presence of HATU (2.0 equiv) and
HOAt (2.0 equiv) at low concentration (1.5 mM). After HPLC purifi-
cation of the crude materials, rhizonin A analogues 310 and 411
were obtained in 13% yield from 12a and 8% yield from 12b (in
three steps), respectively.
The results of the in vitro fat-accumulation-inhibition assay for
synthetic rhizonin A analogues 3 and 4, as well as rhizonin A (1)
and ternatin (2), are shown in Table 1. Cell viability was calculated
independently to exclude undesired fat-accumulation inhibition
arising from the toxicity of the tested compounds.
Based on the results, both 3 and 4 more strongly inhibited fat-
accumulation than the parent compound 1, though 2 was the most
bioactive among the compounds tested. Hybrid compound 4 was
Table 1
Inhibitory effects of synthetic analogues on fat-accumulation against 3T3-L1 adipo-
cytes and their cell viabilitya
Compound
Fat-accumulation inhibitory
Cell viability:
IC50 M)
effect: IC50
(l
M)
(l
Rhizonin A (1)
3
4
55 3.7
42 1.1
5.6 2.1
70% at 62b
>240
>240
(lM)
Scheme 1. Synthesis of the right fragments 8a and 8b. Reagents and conditions: (a)
Ternatin (2)
0.027 0.003
0.28 0.03
Boc-
for 7a, Boc-NMe-
DIPEA, CH2Cl2; (e) LiOH, t-BuOH, THF, H2O.
L-Leu-OH, PyBroP, DIPEA, CH2Cl2; (b) 50% TFA/CH2Cl2; (c) Boc-NMe-
L-Phe-OH
a
Values are means of quadruplicate determinations.
Not tested at higher concentrations.
L
-Leu-OH for 7b, HATU, DIPEA, CH2Cl2; (d) Boc- -Val-OH, HATU,
L
b

K. Shimokawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 867–869
869
10-fold more potent than 1. The overall potency of inhibitory effect
is in the order (2>) 4 > 3 > 1. Thus, modifications on the right half of
that imitates the sequence of ternatin (2) should greatly
strengthen the bioactivity.
On the other hand, the toxic profiles of the synthetic analogues
showed an opposite tendency. Interestingly, 3 and 4 were found
References and notes
1. Steyn, P. S.; Tuinman, A. A.; van Heerden, F. R.; van Rooyen, P. H.; Wessels, P. L.;
Rabie, C. J. J. Chem. Soc. Chem. Commun. 1983, 47.
2. Shimokawa, K.; Mashima, I.; Asai, A.; Yamada, K.; Kita, M.; Uemura, D.
Tetrahedron Lett. 2006, 47, 4445.
3. Shimokawa, K.; Mashima, I.; Asai, A.; Ohno, T.; Yamada, K.; Kita, M.; Uemura, D.
Chem. Asian J. 2008, 3, 438.
4. Miller, R.; Galitsky, N. M.; Duax, W. L.; Langs, D. A.; Pletnev, V. Z.; Ivanov, V. T.
Int. J. Pept. Protein Res. 1993, 42, 539.
5. Shimokawa, K.; Yamada, K.; Kita, M.; Uemura, D. Bioorg. Med. Chem. Lett. 2007,
17, 4447.
6. Wilson, T.; Rabie, C. J.; Fincham, J. E.; Steyn, P. S.; Schipper, M. A. A. Fd. Chem.
Toxicol. 1984, 22, 275.
7. Rabie, C. J.; Lübben, A.; Schipper, M. A. A.; van Heerden, F. R.; Fincham, J. E. Int. J.
Food Microbiol. 1985, 1, 263.
8. Nakatsuka, H.; Shimokawa, K.; Miwa, R.; Yamada, K.; Uemura, D. Tetrahedron
Lett. 2009, 50, 186.
1
to be less toxic (IC50 > 240
toxicity (the expected IC50 value for cell viability is about 62–
176 M) at IC50 value for fat-accumulation inhibition. The selectiv-
lM), while 1 showed considerable cyto-
l
ity indices [SI; IC50 value for cytotoxicity/that for fat-accumulation]
of 3 and 4 were >12.3 and >43, and therefore higher than that of 1
(the expected SI is about 1–3). This result strongly suggests that the
two FurAla moieties are responsible for the potent cytotoxicity of 1
against 3T3-L1 adipocytes. Moreover, hybrid analogue 4 had a
greater SI than 2 (SI = 10). Therefore, 4 may be a plausible candidate
that possesses a selective and/or effective fat-accumulation inhibi-
tory effect against 3T3-L1 murine adipocytes.
9. Hernández, D.; Riego, E.; Francesch, A.; Cuevas, C.; Albericio, F.; Álvarez, M.
Tetrahedron 2007, 63, 9862.
23
10. Spectroscopic data for 3: [
a
]
À15.4° (c = 0.15, CHCl3); IR (CHCl3) 3326, 2933,
D
2337, 1650, 1506 cmÀ1
;
1H NMR (400 MHz, C6D6) d 7.85 (d, J = 9.6 Hz, 1H), 7.77
In summary, two novel analogues of rhizonin A (1) were de-
signed on the basis of structural hybridization with the potent
fat-accumulation inhibitor ternatin (2). The modification of the
two NMe-FurAla moieties in the structure of 1 led to analogues
with more potent and selective fat-accumulation inhibitory activ-
ities. Further chemical and biological studies on 1, including its
SAR, are now underway.
(d, J = 9.2 Hz, 1H), 7.41 (d, J = 7.2 Hz, 1H), 7.35 (s, 1H), 7.22–6.93 (m, 10H), 5.93
(dd, J = 12.4, 5.2 Hz, 1H), 5.89 (d, J = 10.0 Hz, 1H), 5.70–5.62 (m, 1H), 4.90 (t,
J = 9.2 Hz, 1H), 4.71 (t, J = 9.2 Hz, 1H), 4.38 (q, J = 6.8 Hz, 1H), 4.00–3.96 (m, 1H),
3.89 (dd, J = 15.2, 5.2 Hz, 1H), 3.66–3.58 (m, 1H), 3.40 (dd, J = 8.4, 4.8 Hz, 1H),
2.80 (dd, J = 15.2, 12.4 Hz, 1H), 2.71 (s, 3H), 2.69 (s, 3H), 2.63 (s, 3H), 2.33–2.25
(m, 1H), 2.24–2.12 (m, 1H), 1.90–1.75 (m, 2H), 1.55–1.47 (m, 1H), 1.37–1.27
(m, 2H), 1.25 (d, J = 6.8 Hz, 6H), 1.18–1.13 (m, 1H), 1.05 (d, J = 6.8 Hz, 3H), 1.02
(d, J = 6.8 Hz, 3H), 0.96 (t, J = 7.2 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 0.91 (d,
J = 6.4 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H), 0.47 (d, J = 7.2 Hz, 3H); HRMS (FAB) calcd
for C46H69N7O7Na (M+Na)+ 854.5196, found 854.5175.
23
11. Spectroscopic data for 4: [
a
]
–2.5° (c = 0.15, CHCl3); IR (CHCl3) 3300, 2966,
D
2881, 2363, 1644, 1499 cmÀ1
;
1H NMR (400 MHz, C6D6) d 7.87 (d, J = 9.2 Hz,
Acknowledgments
1H), 7.62 (d, J = 9.2 Hz, 1H), 7.36 (s, 1H), 7.18–6.90 (m, 5H), 6.85 (d, J = 6.4 Hz,
1H), 6.09–5.90 (m, 2H), 5.71–5.62 (m, 1H), 5.43–5.26 (m, 1H), 4.98 (t, J = 9.2 Hz,
1H), 4.79 (t, J = 9.2 Hz, 1H), 4.39 (q, J = 7.2 Hz, 1H), 3.48–3.35 (m, 2H), 3.06 (s,
3H), 2.65 (s, 3H), 2.58 (s, 3H), 2.54–2.45 (m, 2H), 2.33–2.25 (m, 1H), 2.05–1.77
(m, 4H), 1.32 (d, J = 6.8 Hz, 3H), 1.23 (d, J = 6.8 Hz, 3H), 1.06–0.80 (m, 4H), 1.05
(d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H), 0.99 (d, J = 6.8 Hz, 3H), 0.96 (t,
J = 4.8 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H), 0.87 (d, J = 6.4 Hz, 3H), 0.84 (d, J = 6.4 Hz,
3H), 0.82 (d, J = 6.4 Hz, 3H), 0.75 (d, J = 6.4 Hz, 3H); HRMS (FAB) calcd for
C43H71N7O7Na (M+Na)+ 820.5313, found 820.5291.
This study was supported in part by Grants-in-Aid for Creative
Scientific Research (Grant No. 16GS0206) and the Global COE pro-
gram in Chemistry at Nagoya University (Grant No. B-021) from
the Ministry of Education, Culture, Sports, Science, and Technology,
Japan. We are indebted to Banyu Pharmaceutical Co., Ltd. for their
financial support.