Synthesis and antitumor activity of cyclodepsipeptide zygosporamide and its analogues

Article abstract of DOI:10.1016/j.bmcl.2008.06.066

The synthesis and structure-activity relationships of zygosporamide, a known potent and selective cytotoxic natural product against SF-268 and RXF 393 cell lines, are described. The potencies of the synthetic zygosporamide are similar to those reported fo

Full text of DOI:10.1016/j.bmcl.2008.06.066

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4385–4387
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antitumor activity of cyclodepsipeptide zygosporamide
and its analogues
c,
You Wang a, Feiran Zhang b, Yihua Zhang a, Jun O. Liu b, , Dawei Ma
*
*
a Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, China
b Department of Pharmacology and Department of Oncology, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205, USA
c State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and structure–activity relationships of zygosporamide, a known potent and selective cyto-
toxic natural product against SF-268 and RXF 393 cell lines, are described. The potencies of the synthetic
zygosporamide are similar to those reported for the natural product toward all cancer cell lines examined
with the exception of SF-268, the underlying cause of which remains to be elucidated.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 30 April 2008
Revised 17 June 2008
Accepted 18 June 2008
Available online 21 June 2008
Keywords:
Anti-tumor
Cyclopeptide
Zygosporamide (1) is a cyclic depsipeptide that was isolated
from the seawater-based fermentation broth of a marine-derived
fungus identified as Zygosporium masonii (Fig. 1).1 It is composed
esterification
Ph
O
O
NH
O
of four hydrophobic amino acids (D-Leu, L-Leu, and two L-Phe)
O
and one hydrophobic hydroxy acid ((S)-2-hydroxy-4-methylpenta-
noicacid; O-Leu). In the National Cancer Institute 60 cancer cell
lines screen, this compound displayed highly selective cytotoxicity
against CNS (central nervous system) cancer cell line SF-268 and
renal cancer cell line RXF 393, with GI50 values of 6.5 nM and less
than 5.0 nM, respectively. These values are at least 1000ꢀ lower
than most of the 54 (out of 60) cancer cell lines tested. The unusual
cell type selectivity displayed by this structurally simple com-
pound prompted us to initiate a synthetic and SAR program toward
zygosporamide. It is our long-term goal to explore the mode of ac-
tion by this natural product, particularly its selectivity toward dif-
ferent cancer cell lines.2
NH
HN
H
N
O
O
Ph
macrocyclization
zygosporamide (1)
Figure 1. Structure and bond-disconnections of zygosporamide.
In a parallel procedure, acid 5 was condensed with the p-tolu-
enesulfonic acid salt of L-Phe-OBn to produce amide 6 in 90% yield.
Esterification of 4a with 6 under Yamaguchi conditions4 gave rise
to ester 7. After hydrogenolysis of 7, the macrocyclization precur-
sor was obtained. As we anticipated, the HATU-mediated macro-
Since zygosporamide contains a
with
D-leucine that is connected
L
-phenylalanine, we decided to carry out the macrocyclization
at this site (Fig. 1).3 The cyclization precursor could be assembled
via ester bond formation. Based on this analysis, we started the to-
tal synthesis by preparing tripeptide 4b as depicted in Scheme 1.
lactamization of this amino acid proceeded smoothly in
a
mixture of DMF and methylene chloride to furnish the cyclic dep-
sipeptide 15 in 50% yield. Its analytical data were identical to those
previously reported.1
Alanine-scan technique is a classical tool to study the struc-
ture–activity relationship of proteins as well as smaller polypep-
tides.6 Because the chirality of Ala is identical to that of the
corresponding residue in parent peptides, substitution of other
amino acids with Ala is not expected to cause significant confor-
mational changes. Following the above procedure, we synthesized
four Ala-substituted analogues of zygosporamide (8–11, Fig. 2) by
Condensation of N-Cbz-D-leucine with L-leucine methyl ester under
the action of EDC, HOBt, and DIPEA provided dipeptide 3 in 92%
yield. After hydrolysis of 3 with aqueous LiOH in THF and MeOH,
the resultant acid was condensed with L-phenylalanine methyl es-
ter to afford amide 4a, which was then hydrolyzed with LiOH to
give the tripeptide 4b.
* Corresponding authors.
E-mail address: madw@mail.sioc.ac.cn (D. Ma).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.066

4386
Y. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4385–4387
replacing each amino acid residue with Ala, in order to determine
which residue(s) plays an important role in the interaction with
the putative cellular target. In addition, a -lactic acid substituted
cyclodepsipeptide 12 and a cyclic pentapeptide 13 were elabo-
rated to see if the amide unit is important for antitumor activity.
Noteworthy is that the similar modification to cyclic depsipep-
tide, sansalvamide A, has resulted in some new potent antitumor
agents.7
Before testing the synthetic zygosporamide and its analogues
in cancer cell lines, we determined the IC50 values of paclitaxel
as a reference compound in our cell proliferation assay. As shown
in Table 1, the cytotoxicity of paclitaxel in five tested cancer cell
lines (SF-268, SF-295, A549, MDA-MB-231, and HCT-116) is com-
parable to that previously reported, which validated our assay
conditions.
Using the same assay, we next determined the cytotoxicity of
the synthetic zygosporamide and its analogues. The results for
inhibition of the growth of two CNS cancer cell lines (SF-268 and
SF-295), a lung cancer cell line (A549), a breast cancer cell line
(MDA-MB-231), and a colon cancer cell line (HCT-116) are summa-
rized in Table 2. The IC50 values for the synthetic zygosporamide
(1) against most cell lines including SF-295, A549, MDA-MB-231,
and HCT-116 are similar to those previously reported for the natu-
ral product.1 A notable exception, however, is the IC50 value of the
CO2Me
NH
HO2C
NHCbz
L
L-Leu-OMe
HCl
NHCbz
EDC, HOBt, DIPEA, CH2Cl2
92%
O
2
3
Ph
H
O
N
CO2R
1. aq LiOH, THF, MeOH
NH
2.
L-Phe-OMe
EDC,HOBt, DIPEA
HCl
NHCbz
O
95%
4a: R = Me
4b: R = H
aq LiOH, THF, MeOH
OH
H
N
L-Phe-OBn
BnO2C
HO
BOP, DIPEA, MeCN
90%
O
O
Ph
HO
5
6
Ph
O
H
O
N
synthetic zygosporamide (1) against SF-268 (4.6 0.7 lM), which
is about 700-fold higher than that previously reported. The under-
lying cause of the discrepancy in potencies against SF-268 cell line
between our synthetic sample and the natural product remains un-
known.8 It is possible that the SF-268 cells we received from ATCC
have undergone further changes upon passage in cell culture, los-
ing the unique genetic or epigenetic characteristics that conferred
its unusually high sensitivity to zygosporamide in the previous
study.
O
TCBC, DIPEA, DMAP
toluene, 80%
O
NH
4b + 6
NHCbz NH
O
BnO2C
Ph
7
1. Pd/C, H2, MeOH
1
2. HATU, DIPEA, DMF/CH2Cl2 (1:4), 0.002 M
50%
Taking advantage of our new synthetic route to zygospora-
mide, we made several analogues for a preliminary structure–
activity relationship study. Most synthetic analogues (8–13)
showed lower cytotoxicity toward SF-268, SF-295, and A549 in
comparison with the synthetic zygosporamide (1). However, ana-
logue 11 exhibited slightly higher cytotoxicity and analogues 8–9
showed similar cytotoxicity toward MDA-MB-231. In addition,
the cytotoxicity of analogues 9 and 11–13 was considerably high-
er than that of synthetic zygosporamide toward HCT-116. Thus,
Ala2-substituted analogues10 did not show significant cytotoxic-
Scheme 1.
Ph
O
Ph
O
O
O
NH
NH
O
NH
O
O
O
HN
NH
HN
H
H
N
N
ity at the highest concentration tested (50
stitued analogues 8 only had limited cytotoxicity. This indicates
that
-Leu2 and -Phe5 side-chain functional groups are indispens-
able for the cellular activity. The potencies of the -lactic acid–
l
M), while Ala5-sub-
O
O
O
O
Ph
L
L
8
9
L
Ph
O
O
O
substituted cyclodepsipeptide 12 and the cyclic pentapeptide 13
were more than 8-fold and more than 4-fold lower than the com-
pound 1 (except for HCT-116), respectively, which suggests that
O-Leu side-chain and the ester linkage are preferred at this posi-
tion at least for cytotoxicity toward certain cancer cell lines. In
addition, substituted analogues 9 and 11 generally maintained
O
NH
O
NH
O
O
O
NH
HN
NH
HN
H
N
H
N
O
O
O
Ph
O
Ph
10
11
Table 1
In vitro cytotoxicity data for paclitaxel toward SF-268, SF-295, A549, MDA-MB-231,
and HCT-116 cell lines
Ph
O
O
Ph
O
O
Cell line
IC50 (nM) Paclitaxel
IC50 (nM)* NSC125973**
NH HN
NH
O
O
O
SF-268
SF-295
A549
MDA-MB-231
HCT-116
15.3 2.7
290 220
23.1 1.8
40.5 7.6
13.6 2.4
25
63
25
50
6.3
NH
HN
NH
HN
H
N
H
N
O
O
O
Ph
O
Ph
*
13
12
IC50 values in this column are from the data base of DTP NCI/NIH (http://
www.dtp.nci.nih.gov/).
**
Figure 2. Structures of synthetic analogues of zygosporamide.
In DTP data base, compound NSC125973 is paclitaxel.

Y. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4385–4387
4387
Table 2
In vitro cytotoxicity data for synthetic zygosporamide and its analogues toward SF-268, SF-295, A549, MDA-MB-231 and HCT-116 cell lines
compound
IC50
(
lM) SF-268
IC50
(l
M) SF-295
IC50
(l
M) A549
IC50
(l
M) MDA-MB-231
IC50
(l
M) HCT-116
1
8
9
10
4.6 0.7
ꢃ35*
4.2 1.5
19.7 8.8
8.7 0.9
>50
2.5 0.5
ꢃ31*
>50
5.7 2.1
7.5 6.3
5.0 1.8
>50
ꢃ22*
>50
10.4 1.5
>50
2.1 0.4
>50
>50
11
12
13
8.8 1.5
ꢃ6*
>50
>50
21.0 5.9
7.4
2.8 2.1
>50
>50
2.7 0.6
7.8 6.0
1.9 1.3
11.5
ꢃ31*
ꢃ32*
20.5 2.0
0.0065
14.4
15
Zygosporamide**
8.5
*
Those dose–response curves are not sigmoid curves.
Values from Ref. 1.
**
moderate activities. These data suggest that the hydrophobic
group of
-Leu1 and the phenyl group of -Phe3 are not absolutely
References and notes
D
L
1. Oh, D. C.; Jensen, P. R.; Fenical, W.. Tetrahedron Lett. 2006, 47, 8625.
2. For our previous studies in this direction, see Ma, D.; Zou, B.; Cai, G.; Hu, X.; Liu,
J. O. Chem. Eur. J. 2006, 12, 7615.
3. For the discussion on selection of macrocyclization site in cyclopeptide
synthesis, see: (a) Wipf, P. Chem. Rev. 1995, 95, 2115; (b) Humphrey, J. M.;
Chamberlin, A. R. Chem. Rev. 1997, 97, 2243; (c) Hamada, Y.; Shioiri, T. Chem. Rev.
2005, 105, 4441.
required for activity. Hence, the preliminary analysis of structure–
activity relationships reveals that the hydrophobic group of
D
-Leu1 may be a good target for further rational design, and the
phenyl group of L L
-Phe3 or -Phe5 also could be further optimized
to generate new compounds with higher potency against prolifer-
ation of certain cancer cells.
4. (a) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull. Chem. Soc.
Jpn. 1979, 52, 1989; (b) Hikota, M.; Sakurai, Y.; Horita, K.; Yonemitsu, O.
Tetrahedron Lett. 1990, 31, 6367.
In summary, we have achieved the first total synthesis of zygos-
poramide and found that the synthetic compound possessed
similar cytotoxicity toward SF-295, A549 and MDA-MB-231 and
HCT-116, but a significantly lower potency against SF-268 in com-
parison with that reported for natural zygosporamide. The preli-
minary SAR studies reported here should be helpful for further
development of more potent antitumor compounds. Investigation
in this direction is being actively pursued in this group and will
be reported elsewhere in due course.
5. Selected data for synthetic 1: ½a D25
ꢁ
ꢂ 117 (c 0.26, CH3CN); 1H NMR (500 MHz,
CD3CN) d 7.52 (d, J = 9.6 Hz, 1H), 7.33–7.22 (m, 10H), 7.12 (d, J = 6.2 Hz, 1H), 7.07
(d, J = 8.2 Hz, 1H), 6.85 (d, J = 9.3 Hz, 1H), 4.85–4.81 (m, 1H), 4.74 (dd, J = 9.7,
5.2 Hz, 1H), 4.59–4.55 (m, 1H), 4.13–4.06 (m, 2H), 3.34 (dd, J = 14.0, 9.0 Hz, 1H),
3.11 (dd, J = 13.9, 11.0 Hz, 1H), 3.03 (dd, J = 13.5, 6.0 Hz, 1H), 2.88 (dd, J = 13.5,
8.9 Hz, 1H), 1.75–1.71 (m, 1H), 1.57–1.48 (m, 4H), 1.44–1.38 (m, 2H), 1.29–1.23
(m, 2H), 0.92–0.90 (m, 6H), 0.89–0.84 (m, 9H), 0.77 (d, J = 6.6 Hz, 3H); 13C NMR
(125 MHz, CD3CN) d 173.6, 173.0, 172.7, 171.3, 170.9, 138.6, 138.2, 130.4, 129.2,
127.6, 127.4, 76.6, 54.1, 53.8, 53.5, 40.8, 40.7, 39.8, 39.4, 37.3, 25.5, 25.4, 25.2,
23.3, 23.0, 22.7, 22.2, 21.1; ESI-HRMS: calcd for
657.3623, found 657.3629.
C
36H50N4O6Na (M+Na)+
6. Marshall, G. R. Tetrahedron 1993, 49, 3547.
Acknowledgments
7. (a) Liu, S.; Gu, W.; Lo, D.; Ding, X.-Z.; Ujiki, M.; Adrian, T. E.; Soff, G. A.; Silverman,
R. B. J. Med. Chem. 2005, 48, 3630; (b) Gu, W.; Liu, S.; Silverman, R. B. Org. Lett.
2002 2002, 4, 4171.
8. Recently, several reports have appeared regarding the different biological
activity displayed by synthetic and natural cyclopeptides, see: (a) Napolitano,
A.; Bruno, I.; Riccio, R.; Gomez-Paloma, L. Tetrahedron 2005, 61, 6808; (b) Pettit,
G. R.; Lippert, J. W., III; Taylor, S. R.; Tan, R.; Williams, M. D. J. Nat. Prod. 2001, 64,
883.
The authors are grateful to the Chinese Academy of Sciences
and the National Natural Science Foundation of China (Grant
20632050 and 20621062) for their financial support. We are grate-
ful to Dr. William Fenical for providing an authentic sample of nat-
ural zygosporamide.