Total synthesis and cytotoxicity of bisebromoamide and its analogues

Article abstract of DOI:10.1016/j.tetlet.2010.11.058

A highly convergent route for assembling bisebromoamide, its stereoisomers and a simplified analogue has been accomplished, which features connecting its left part and right part via thiazoline ring formation at the final stage. Preliminary biological stu

Full text of DOI:10.1016/j.tetlet.2010.11.058

Tetrahedron Letters 52 (2011) 2124–2127
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Total synthesis and cytotoxicity of bisebromoamide and its analogues
⇑
Wenhua Li, Shouyun Yu, Mingzhi Jin, Hongguang Xia, Dawei Ma
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Lu, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 1 December 2010
A highly convergent route for assembling bisebromoamide, its stereoisomers and a simplified analogue
has been accomplished, which features connecting its left part and right part via thiazoline ring formation
at the final stage. Preliminary biological studies revealed that compounds with both proposed and revised
structures, and a simplified analogue have similar potency against the proliferation of HeLa S3 cell, indi-
cating that the stereochemistry of methylthiazoline part, and methyl group at the 4-methylproline res-
idue in bisebromoamide, have limited influence on its cytotoxicity.
Ó 2010 Elsevier Ltd. All rights reserved.
Bisebromoamide (1, Fig. 1) is a potent cytotoxic linear peptide
that was isolated from the marine cyanobacterium Lyngbya sp.
by Suenaga and co-workers last year.1 Biological studies revealed
that bisebromoamide could selectively inhibit the phosphorylation
of ERK in NRK cells by PDGF (platet-derived growth factor)-stimu-
AKT, PKD, PLC
tion. Furthermore, this peptide exhibited cytotoxicity against HeLa
S3 cells with an IC50 value of 0.04 g/mL, as well as a panel of 39
c1 or S6 ribosomal protein at the same concentra-
l
human cancer cell lines at the Japanese Foundation for Cancer Re-
search with an average GI50 value of 40 nM.1
lation at 10 to 0.1
l
M, without an effect on the phosphorylation of
Structurally, bisebromoamide contains seven amino acid resi-
dues, namely, N-pivaloyl-alanine, N-methyl-3-bromo-tyrosine (N-
Me-Br-Tyr), 4-methylproline (Me-Pro), 2-methylcystine (Me-Cys),
leucine (Leu), N-methylphenyl-alanine (N-Me-Phe) and 2-(1-oxo-
propyl)pyrrolidine (Opp). Among them, 4-methylproline is con-
nected with 2-methylcystine through methylthiazoline (Me-Tzn).
The stereochemistry of bisebromoamide was assigned by chemical
degradation and treatment with Marfey’s reagent followed by
comparison of HPLC retention times with the authentic samples.
A foreshadowing of the problems regarding the structure was that
the configuration of the methylthiazoline in the initial Letter was S,
which was inconsistent with that observed in other methylthiazo-
line-containing natural products, such as thiangazole,2 halipep-
tins3,5a and largazole.4
Me-Pro
Leu
Me-Tzn
O
Ala
O
N
O
N
O
N
H
O
N
S
N
N
N
H
O
O
Opp
N-Me-Br-Tyr
OH
N-Me-Phe
Br
Proposed structure of bisebromoamide (1)
As a continuing effort on the total synthesis and structure–
activity relationship (SAR) studies of complex natural peptides,5
we wish to report here our synthesis and preliminary SAR results
of bisebromoamide. Our studies confirm the structural revision of
this natural peptide, which was made by Ye and co-workers in
their recently disclosed total synthesis.6
Our synthetic strategy is outlined in Figure 1. Given that the
methylthiazoline is sensitive to both acid and base,7 we planned
to construct this unit at the late stage of the synthesis. Accordingly,
bisebromoamide was degraded to two units with similar complex-
ity, cyanide 3 and protected aminothiol 4. Further amide bond dis-
connection led to smaller amino acid fragments. This highly
convergent and flexible route would allow the preparation of its
analogues, thereby facilitating the subsequent studies on struc-
ture–activity relationship.
O
CN
O
N
O
N
BocHN
O
O
N
N
H
+
N
H
O
N
BocS
O
OH
4
3
Br
Figure 1. Proposed structure of bisebromoamide and its retrosynthetic analysis.
⇑
Corresponding author.
E-mail address: madw@mail.sioc.ac.cn (D. Ma).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.11.058

W. Li et al. / Tetrahedron Letters 52 (2011) 2124–2127
2125
OH
OMe
O
O
BocN
BocN
O
TMSCHN2
NBS, DMF
98%
FmocN
O
OAllyl
1. Et2NH, CH3CN
FmocHN
O
CH2Cl2, MeOH
quant.
N
2. D-Fmoc-Leu-OH, HATU,
DIPEA, CH2Cl2
95%
OAllyl
OBn
OBn
6
5
17
OMe
O
18
OMe
O
N
BocN
1. TFA, CH2Cl2
2. HOAt, HATU,
BocHN
1. Pd(PPh3)4, NMA, THF
O
19
2. , HATU, HOAt, DIPEA
O
O
DIPEA, Boc-Ala-OH
97%
FmocHN
O
41% for 2 steps
OBn
N
OBn
N
Br
Br
8
7
N
H
xTFA
O
19
20
O
OR
O
N
1. TFA, CH2Cl2
N
H
1. Et2NH, CH3CN
21
2. , HOAt, HATU,
2. DIPEA, CH2Cl2,
pivaloyl chloride,
quant. for 2 steps
O
O
DIPEA, CH2Cl2, DMF
54% for 2 steps
O
N
BocHN
BocS
O
N
H
O
OBn
9
O
: R=Me
10
N
aq. LiOH, THF
quant.
Br
BocHN
: R=H
OH
Scheme 1.
BocS
21
4
Scheme 3.
Our synthesis commenced with preparation of N-methyl-3-bro-
motyrosine (Scheme 1). Methylation of commercially available
tyrosine derivative 5 followed by bromination with NBS provided
fully protected N-methyl-3-bromo-tyrosine 7. After removal of
Boc group with TFA, the liberated amine was coupled with Boc-
Ala-OH under the action of HATU and HOAt to give dipeptide 8.
Switching the protecting group from Boc to Piv delivered amide
9, which was hydrolysed to give acid 10.
material). Noteworthy is that hydrogenation in protic solvents
led to formation of a debromination product.
We next turned our attention to the synthesis of the aminothiol
fragment 4. As depicted in Scheme 3, after Fmoc removal of phen-
ylalanine derivative 17, coupling with D-Fmoc-Leu-OH was con-
ducted in the presence of HATU to produce dipeptide 18. After
deallylation via Pd chemistry, the liberated carboxylic acid was
condensed with TFA salt 19 that was prepared from its correspond-
ing Boc-protected precursor,12 to provide tripeptide 20 in a moder-
ate yield. The free amine liberated from tripeptide 20 with Et2NH
was condensed with the free acid (S)-21,13 to generate the desired
protected aminothiol 4 in 54% yield.
Although the protected aminothiol 4 was obtained successfully,
the yields for last two condensation steps were not satisfactory.
We believed that this problem should result from reactive ketone
moiety. Therefore, an alternative route to 4 was developed, as
shown in Scheme 4. At this stage we employed amino alcohol 22
The assembly of cyanide 3 is illustrated in Scheme 2. Methyla-
tion of pyroglutamic acid derivative 118 gave lactam 12 with syn
configuration.9 The lactam 12 was then reduced to cyclic amine
13 via treatment with LiBEt3H and Et3SiH successively.10 The
tert-butyl ester 13 was converted to cyanide 15 via deprotection,
primary amide formation and subsequent dehydration.11 The syn
configuration was secured by the X-ray analysis of a sulfonylamide
derivative of 15. Hydrogenation of 15 followed by condensation of
the liberated amine with acid 10 afforded tripeptide 16, which was
hydrogenated carefully in ethyl acetate to generate the desired
cyanide 3 with 39% yield (74% yield based on recovered starting
LiHMDS
1. LiBEt3H
then MeOTf
1. Pd(PPh3)4, NMA;
CO2t-Bu
O
N
O
CO t-Bu
2
toluene, 60%
N
2. Et3SiH
2. 22, HATU, HOAt,
Cbz
Cbz
BF3•Et2O
DIPEA, CH2Cl2
94% for 2 steps
HO
O
11
12
87%
FmocHN
O
1. Et2NH, CH3CN
18
N
2. 21, HOAt,
N
1. TFA, CH2Cl2
TFAA, Et3N
HATU, DIPEA
N
H
xTFA
76% for 2 steps
CO2t-Bu
99% for 3 steps
CONH2
N
2. NH4OH, HOBt
EDCI, DMF
N
OH
Cbz
Cbz
22
23
13
14
CN
N
O
O
HO
O
1. Pd/C, H2, THF
BocHN
BocS
DMP, NaHCO3
CH2Cl2, 100%
N
N
H
O
N
O
4
2. 10, HATU,
CN
N
H
N
HOAt, DIPEA
Cbz
O
N
82%
15
OR
Pd/C, H2, EtOAc
16: R = Bn
3: R = H
Br
24
39% (74% b.r.s.m.)
Scheme 2.
Scheme 4.

2126
W. Li et al. / Tetrahedron Letters 52 (2011) 2124–2127
O
O
N
O
N
O
N
H
O
O
N
O
N
BocHN
O
N
H
O
1. TFA, CH2Cl2
S
N
N
1
N
H
O
N
.
2. 3, NaHCO3, MeOH,
BocS
O
pH=6 buffer, 70 °C
61%
OH
Br
29
4
O
O
N
O
N
O
N
H
O
O
N
O
N
BocHN
BocS
O
N
H
O
1. TFA, CH2Cl2
S
N
N
N
N
H
O
2. 3, NaHCO , MeOH,
.
pH=6 buff3er, 70 °C
63%
O
OH
30
28
Br
O
O
N
O
N
O
N
H
O
N
O
N
O
O
N
N
H
O
O
N
S
N
N
S
N
N
H
O
N
N
H
O
O
O
OH
OH
Br
Br
revised structure of bisebromoamide (2)
31
Scheme 5.
Scheme 6.
(prepared from 19 via NaBH4 reduction) as a coupling partner. The
same coupling sequence was carried out to produce tetrapeptide
24 with a good yield. The tetrapeptide 24 was then oxidized with
Dess–Martin reagent to give 4 in quantitative yield.
Table 1
In vitro cytotoxicity data for synthetic bisebromoamide and its analogues towards
HeLa S3 cell lines
Compound
IC50 (lg/mL)
With two key fragments in hand, we completed the synthesis of
bisebromoamide as outlined in Scheme 5. Treatment of the pro-
tected aminothiol 4 with TFA gave free aminothiol, which was con-
densed with cyanide 3 in the presence of NaHCO3 in a mixture of
methanol and buffer to deliver the target molecule 1.14 However,
spectroscopic data for 1 did not match the characterization data re-
ported for natural bisebromoamide. After careful comparison of
our data with those reported, we found disturbing discrepancies
in regard to the positions of 4-methylproline, 2-methylcystine
and leucine residues. The different configuration in the methyl-
thiazoline part between 1 and other methylthiazoline-containing
natural products2–4 suggested that the proposed stereochemistry
at this position for bisebromoamide might be wrong. Thus, peptide
28 was prepared from (R)-21 following the same procedure, and
reacted with the cyanide 3, after deprotection, to afford 2. To our
delight, the spectroscopic data for 2 showed unambiguous agree-
ment with those of nature one, which indicated that the structure
of bisebromoamide should be revised to 2.
In order to check if the stereochemistry of other amino acid res-
idues, and the methyl group at the 4-methylproline residue play
some roles for maintaining the cytotoxicity of bisebromoamide.
Two other isomers 29 and 30, and a simplified analogue 31 of bise-
bromoamide were assembled from suitable intermediates follow-
ing a similar procedure as described above (Scheme 6). All our
synthetic compounds were evaluated as inhibitors against the pro-
liferation of HeLa S3 cell, and the results were summarized in Table
1. Surprisingly, peptide 1 was even more potent than bisebromoa-
mide 2 in our assay. This result demonstrated that the stereochem-
istry at the methylthiazoline part of bisebromoamide has limited
influence to its cytotoxicity. Similar trend was seen by changing
1
2
29
30
31
0.038
0.165
0.296
—
0.134
a
a
Inactive at 8 lg/mL.
the stereochemistry of the N-Me–Br-Tyr residue, as evident from
compound 29 that has an IC50 value of 0.296 g/mL. However,
changing the configuration of the alanine residue led to losing
cytotoxicity because compound 30 was found inactive even at
the concentration of 8 lg/mL, indicating that the stereochemistry
of this residue plays a key role for maintaining the cytotoxicity.
More interestingly, simplified analogue 31 was found as potent
as bisebromoamide, and, thereby delivering a valuable lead for
further SAR studies because its synthesis is easier than
bisebromoamide.
In conclusion, we have achieved the total synthesis and struc-
tural revision of bisebromoamide by developing a highly conver-
gent route. Accompanying the total synthesis its SAR was
preliminarily explored. These results should be of benefit to the
further SAR studies of this relatively simple but potent antitumor
agent.
l
Acknowledgements
The authors are grateful to National Basic Research Program of
China (973 Program, grant 2010CB833200), Chinese Academy of

W. Li et al. / Tetrahedron Letters 52 (2011) 2124–2127
2127
2005, 7, 4237; (d) Yu, S.; Pan, X.; Ma, D. Chem. Eur. J. 2006, 12, 6572; (e) Ma, D.;
Zou, B.; Cai, G.; Hu, X.; Liu, J. O. Chem. Eur. J. 2006, 12, 7615; (f) Xie, W.; Ding, D.;
Zi, W.; Li, G.; Ma, D. Angew. Chem., Int. Ed. 2008, 47, 2844; (g) Tan, L.; Ma, D.
Angew. Chem., Int. Ed. 2008, 47, 3614; (h) Li, W.; Gan, J.; Ma, D. Angew. Chem.,
Int. Ed. 2009, 48, 8891; (i) Li, W.; Gan, J.; Ma, D. Org. Lett. 2009, 11, 5694; (j) Li,
W.; Schlecker, A.; Ma, D. Chem. Commun. 2010, 5403.
Sciences and the National Natural Science Foundation of China
(grant 20632050 and 20921091) for their financial support.
Supplementary data
6. Gao, X.; Liu, Y.; Kwong, S.; Xu, Z.; Ye, T. Org. Lett. 2010, 12, 3018.
7. (a) Wipf, P.; Fritch, P. C. J. Am. Chem. Soc. 1996, 118, 12358; (b) Wipf, P.; Fritch,
P. C.; Geib, S. J.; Sefler, A. M. J. Am. Chem. Soc. 1998, 120, 4105; (c) McKeever, B.;
Pattenden, G. Tetrahedron 2003, 59, 2713. And see also Refs. 5a,d.
8. Deguest, G.; Bischoff, L.; Fruit, C.; Marsais, F. Tetrahedron: Asymmetry 2006, 17,
2120.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.11.058.
References and notes
9. Charier, J.-D.; Duffy, J. E. S.; Hitchcock, P. B.; Young, D. W. J. Chem. Soc., Perkin
Trans. 1 2001, 2367.
10. Zlatopolskiy, B. D.; Loscha, K.; Alvermann, P.; Kozhushkov, S. I.; Nikolaev, S. V.;
Zeeck, A.; de Meijere, A. Chem. Eur. J. 2004, 10, 4708.
11. Deiters, A.; Chen, K.; Eary, C. T.; Martin, S. F. J. Am. Chem. Soc. 2003, 125, 4541.
12. Conrow, R.; Portoghese, P. S. J. Org. Chem. 1986, 51, 938.
13. (a) Seebach, V. D.; Aebi, J. D.; Gander-Coquoz, M.; Naef, R. Helv. Chim. Acta 1987,
70, 1194; (b) Pattenden, G.; Thom, S. M.; Jones, M. F. Tetrahedron 1993, 49,
2131; (c) Boyce, R. J.; Mulqueen, G. C.; Pattenden, G. Tetrahedron 1995, 51,
7321.
1. Teruya, T.; Sasaki, H.; Fukazawa, H.; Suenaga, K. Org. Lett. 2009, 11, 5062.
2. (a) Jansen, R.; Kunze, B.; Reichenback, H.; Jurkiewicz, E.; Hunsmann, G.; Hofle,
G. Liebigs Ann. Chem. 1992, 357; (b) Jansen, R.; Schomburg, D.; Hofle, G. Liebigs
Ann. Chem. 1993, 701.
3. (a) Randazzo, A.; Bifulco, G.; Giannini, C.; Bucci, M.; Debitus, C.; Cirino, G.;
Gomez-Paloma, L. J. Am. Chem. Soc. 2001, 123, 10870; (b) Monica, C. D.;
Randazzo, A.; Bifulco, G.; Cimino, P.; Aquino, M.; Izzo, I.; De Riccardis, F.;
Gomez-Paloma, L. Tetrahedron Lett. 2002, 43, 5707; (c) Nicolaou, K. C.; Schlawe,
D.; Kim, D. W.; Longbottom, D. A.; de Noronha, R. G.; Lizos, D. E.; Manam, R. R.;
Faulkner, D. J. Chem. Eur. J. 2005, 11, 6197.
14. (a) Bergeron, R. J.; Wiegand, J.; McManis, J. S.; McCosar, B. H.; Weimar, W. R.;
Brittenham, G. M.; Smith, R. E. J. Med. Chem. 1999, 42, 2432; For a recent
comprehensive review about chemistry of thiazoline, see (b) Gaumont, A.-C.;
Gulea, M.; Levillain, J. Chem. Rev. 2009, 109, 1371.
4. Kanchan, T.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008, 130, 1806.
5. For our recent studies on the synthesis of cyclodepsipeptides, see: (a) Yu, S.;
Pan, X.; Lin, X.; Ma, D. Angew. Chem., Int. Ed. 2005, 44, 135; (b) Xie, W.; Zou, B.;
Pei, D.; Ma, D. Org. Lett. 2005, 7, 2775; (c) Zou, B.; Long, K.; Ma, D. Org. Lett.