Synthesis and biological evaluation of tamandarin B analogues

Article abstract of DOI:10.1021/ol0530023

The synthesis of two tamandarin B analogues in which the N,O-Me ₂Tyr⁵ unit was replaced by N-Me-phenylalanine (N-MePhe⁵) and (S)-2-(methylamino)-3-(naphthalen-2-yl)propanoic acid (N-MeNaphth⁵) is described. The

Full text of DOI:10.1021/ol0530023

ORGANIC
LETTERS
2006
Vol. 8, No. 3
511-514
Synthesis and Biological Evaluation of
Tamandarin B Analogues
Javier Adrio,† Carmen Cuevas,§ Ignacio Manzanares,§ and Madeleine M. Joullie´*,‡
Department of Chemistry, UniVersity of PennsylVania, Philadelphia, PennsylVania
19104-6323, Departamento de Quimica Organica, Faculdad de Ciencias, UniVersidad
Autonoma de Madrid, 28049 Madrid, Spain, and Pharma Mar S.A., Sociedad
Unipersonal, AVd. de los Reyes, 1.P.I. La Mina Norte 28770 Colmenar Viejo, Spain
mjoullie@sas.upenn.edu
Received December 12, 2005
ABSTRACT
The synthesis of two tamandarin B analogues in which the N,O-Me2Tyr5 unit was replaced by N-Me-phenylalanine (N-MePhe5) and (S)-2-
(methylamino)-3-(naphthalen-2-yl)propanoic acid (N-MeNaphth5) is described. The choice of the macrocyclization site was crucial to achieve
satisfactory macrolactamization. Coupling between norstatine (Nst1) and threonine (Thr6) afforded only a 15% yield, while lactamization between
proline (Pro4) and the aromatic moiety could be achieved in 65% yield.
Tamandarins A and B (1 and 2) are two cyclic depsipeptides
isolated from a Brazilian ascidian by Fenical and co-
workers.1 Their structures are similar to that of didemnin B
(3), an ascidian metabolite isolated by Rinehart et al. in 1981
(Figure 1).2 The two species that produce 1 and 3 originate
from remote geographic locations but use a common method
to defend themselves and their vulnerable larvae by produc-
ing these structurally similar but not identical depsipeptides.3
Some members of the family exhibited outstanding biological
activity, especially a manifest capacity to inhibit cell
proliferation, which has stimulated much effort into their
development for therapeutic use.4 Tamandarins A (1) and B
(2), recently synthesized in our laboratory,5 were reported
to exhibit much of the same biological activity as didemnin
B (3) and aplidine (4), maintaining similar levels of in vitro
antitumor activity as well as protein biosynthesis inhibition
properties,1,4 suggesting that the propionic acid unit results
only in a minor conformational modification and may not
(4) (a) Li, W.-R.; Ewing, W. R.; Harris, B. D.; Joullie´, M. M. J. Am.
Chem. Soc. 1990, 112, 7659. (b) Sakai, R.; Rinehart, K. L.; Kishore, V.;
Kundu, B.; Faircloth, G.; Gloer, J. B.; Carney, J. R.; Namikoshi, M.; Sun,
F.; Hughes, R. G., Jr.; Gravalos, G.; de Quesada, T. G.; Wilson, G. R.;
Heid, R. M. J. Med. Chem. 1996, 39, 2819. (c) Schmidt, U.; Kroner, M.;
Griesser, H. Tetrahedron Lett. 1988, 29, 4407. (d) Grubb, D. R.; Ly, J. D.;
Vaillant, F.; Johnson, K. L.; Lawen, A. Oncogene 2001, 20, 4085. (e) Ding,
X.; Vera, M. D.; Zhao, Y.; Leonard, M. S.; Joullie´, M. M. Bioorg. Med.
Chem. Lett. 2001, 11, 231.
‡ University of Pennsylvania.
† Universidad Auto´noma de Madrid.
§ Pharma Mar S.A.
(1) Vervoort, H.; Fenical, W.; de Epifanio, R. J. Org. Chem. 2000, 65,
782.
(2) Rinehart, K. L.; Gloer, J. B., Jr.; Hughes, R. G., Jr.; Renis, H. E.;
McGovren, J. P.; Swynenberg, E. B.; Stringfellow, D. A.; Kuentzel, S. L.;
Li, L. H. Science 1981, 212, 933.
(3) Joullie´, M. M.; Leonard, M. S.; Portonovo, P.; Liang, B.; Ding, X.;
La Clair, J. J. Bioconjugate Chem. 2003, 14, 30.
(5) (a) Liang, B.; Richard, D. J.; Portonovo, P. S.; Joullie´, M. M. J. Am.
Chem. Soc. 2001, 123, 4469. (b) Joullie´, M. M.; Portonovo, P. S.; Liang,
B.; Richard, D. J. Tetrahedron Lett. 2000, 41, 9373.
10.1021/ol0530023 CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/04/2006

Scheme 1
molecule in the solid state7 or in solution.8 Since comparable
cytotoxic activity was observed in didemnins when residue
5 was replaced with either aliphatic hydrophobic amino
acids,9 such as N-MeLeu,5 or aromatic residues, such as
N-MePhe,5,4e we decided to prepare two tamandarin B
analogues containing hydrophobic residues by replacing
N,O-Me2Tyr5 with N-Me-phenylalanine (N-MePhe5) and (S)-
2-(methylamino)-3-(naphthalen-2-yl)propanoic acid (N-
MeNaphth5). The synthesis previously described for taman-
darin B5b was used as the basis for the synthesis of our
N-MePhe5 analogue, differing only in the preparation of the
tetrapeptide moiety, which consists of Leu3, Pro4, Phe5, and
Thr6 residues linked in sequence. We began our synthesis
with commercially available Cbz-L-Phe, which was methy-
lated using dimethyl sulfate to afford Cbz-N-MePhe 5. The
coupling of the resulting acid with Boc-Thr-OSEM 6 formed
ester 7. Subsequent hydrogenolysis and coupling with acid
8 yielded the desired tetrapeptide 9 (Scheme 1).
Hydrogenolysis of tetrapeptide 9 provided the free amine
10, which was coupled with the activated pentafluorophenyl
PFP ester 11 to afford the linear precursor 12 in 57% yield
(Scheme 2). Selective cleavage of the SEM ester in the
presence of the Boc and TIPS protective groups was achieved
with MgBr2‚Et2O.10 The Cbz group of the resulting acid was
removed to obtain the corresponding amino acid by hydro-
genolysis using Pd(OH)2 as the catalyst. Macrocyclization
with HATU gave the Boc derivative 13. The macrocycle
(13) was treated with HCl (gas) in dioxane, which success-
fully removed both TIPS and Boc protective groups. The
macrocycle salt 14 was coupled with the corresponding side
chain 15 (prepared as previously described).4a
Figure 1. Structures of didemnins and tamandarins.
be required for bioactivity. Furthermore, the synthesis of
several side chain analogues of tamandarin A have shown
that structural modifications can afford compounds with
biological activity similar to that of didemnin B analogues.5
These observations support the belief that tamandarins are
simplified didemnin mimics. This concept was corroborated
by fluorescence studies of the predator-prey interactions of
1 and 3.3
There is a large amount of information on the structure-
activity relationships (SAR) of the side chains of didemnins.6
However, the length and complexity of the synthetic routes
for accessing the didemnins have hindered the development
of large numbers of macrocycle-modified compounds. How-
ever, a few such analogues are known. The role of the N,O-
Me2Tyr5 residue of didemnin B was reported to be important
for biological activity. To determine the importance of
aromaticity at this position, two analogues, N-MeLeu5 and
N-MePhe5, were synthesized and shown to retain antitumor
activity and the ability to inhibit protein synthesis.9c
The easier synthetic access to tamandarin derivatives could
accelerate the preparation of new analogues and the develop-
ment of structure-activity relationships for these natural
products. To the best our knowledge, no tamandarins with
modified macrocycles have been reported.
Unfortunately, the macrolactamization reaction took place
in only 15% yield. Several attempts to improve this yield
by changing the solvent, the base, and the coupling reagent
failed. Because we required a robust protocol for the
synthesis of different analogues, the difficulties found in the
macrocyclization step led us to modify the synthetic strategy.
Since the first total synthesis of didemnins reported by
Rinehart et al.11 in 1987, several different macrocyclization
strategies have been described. Among them, a protocol first
reported by Shioiri12 and then by Lloyd-Williams and Giralt13
The N,O-Me2Tyr5 residue at position 5 of the didemnins
was shown to be a potentially important pharmacophoric
element, which could interact with a hydrophobic pocket in
the receptor because it extends out of the main body of the
(6) Vera, M. D.; Joullie´ M. M. Med. Res. ReV. 2002, 22, 102.
(7) Hossain, M. B.; van der Helm, D.; Antel, J.; Sheldrick, G. M.;
Sanduja, S. K.; Weinheimcr, A. J. Proc. Natl. Acad. Sci. U.S.A. 1988, 85,
4118.
(8) Kessler, H.; Will, M.; Antel, J.; Beck, H.; Sheldrick, G. M. HelV.
Chim. Acta 1989, 72, 530.
(9) (a) Pfizenmayer, A. J.; Ramanjulu, J.; Vera, M. D.; Ding, X.; Xiao,
D.; Chen, W.-C.; Joullie´, M. M. Bioorg. Med. Chem. Lett. 1996, 6, 2713.
(b) Pfizenmayer, A. J.; Ramanjulu, J.; Vera, M. D.; Ding, X.; Xiao, D.;
Chen, W.-C.; Joullie´, M. M.; Tandon, D.; Toogood, P. L. Bioorg. Med.
Chem. Lett. 1998, 8, 3653. (c) Pfizenmayer, A. J.; Ramanjulu, J.; Vera, M.
D.; Ding, X.; Xiao, D.; Chen, W.-C.; Joullie´, M. M. Tetrahedron 1999, 55,
313.
(10) Chen, W.-C.; Vera, M. D.; Joullie´, M. M. Tetrahedron Lett. 1997,
38, 4025.
(11) Rinehart, K. L.; Kishore, V. N.; Nagarajan, S.; Lake, R. J.; Gloer,
J. B.; Bozich, F. A.; Li, K. M.; Maleczka, R. E., Jr.; Todsen, W. L.; Munro,
M. H. G.; Sullins, D. W.; Sakai, R. J. Am. Chem. Soc. 1987, 109, 6846.
512
Org. Lett., Vol. 8, No. 3, 2006

Scheme 3
A retrosynthetic analysis for the synthesis of N-MeNaphth5
(17) using this approach is shown in Figure 2. The formation
of the macrocyclic core is the key step, and the site between
Pro4 and N,O-Me2Tyr5 was selected as the point of macro-
cyclization. Linear precursor 19 was disconnected into units
20 and 21. The depsipeptide 21 was prepared by coupling
alcohol 22 and norstatine 23 (Figure 2).
Figure 2. Retrosynthetic analysis of compound 17.
The synthesis of the norstatine moiety was performed as
described in our previous work,5b but using Boc instead of
Cbz as the nitrogen protecting group and TBDMS instead
of TIPS as the oxygen protecting group (Scheme 3).
The coupling between Leu-Pro-OBn 24 and the hydroxy-
valeric acid 25 using DCC and diisopropyl ethylamine in
CH2Cl2 afforded alcohol 22 in good yield. Subsequently,
reaction with norstatine 23 employing DCC and DMAP gave
depsipeptide 26 (Scheme 4).
selected the site between Pro4 and N,O-Me2Tyr5 to afford
the macrolactam. We assumed that the same approach could
be applied to tamandarin analogues and changed the mac-
rolactamization site for the synthesis of other tamandarin B
analogues.
Scheme 2
Scheme 4
The coupling between threonine phenacyl ester 27 and
N-L-3-(2-naphthyl)alanine 28 using DCC/DMAP afforded the
desired intermediate 20 in 68% yield. The phenacyl ester
was removed using Zn in AcOH/H2O in 60% yield (Scheme
5).
Depsipeptide 21, obtained by acidic deprotection of
compound 26, was coupled with acid 20 using HBTU, HOBt,
(12) Hamada, Y.; Kondo, Y.; Shibata, M.; Shioiri, T. J. Am. Chem. Soc.
1989, 111, 669.
(13) Jou, G.; Gonzalea, I.; Albericio, F.; Lloyd-Williams, P.; Giralt, E.
J. Org. Chem. 1997, 62, 354.
Org. Lett., Vol. 8, No. 3, 2006
513

Scheme 5
Table 1. Data of in vitro Cytotoxicity (GI50, nM) of
Compounds 2, 16, and 17
tamandarin B 2 N-MePhe 16 N-MeNaphth 17
DU-145
LN-CaP
IGROV
IGROV-ET
SK-BR-3
MEL-28
A-549
K-562
PANC-1
HT-29
7.08
5.84
7.31
173
2.37
1.52
2.94
215
0.66
2.74
4.56
23.2
3.81
4.77
26.1
358
2.17
1.98
2.09
159
1.83
4.14
6.96
20.0
6.24
4.60
6.84
429
5.44
3.03
7.62
8.47
14.0
6.32
30.5
1250
3.90
59.7
and DIPEA to afford the linear precursor 19 in 54% yield.
The benzyl protecting groups were removed by hydrogena-
tion in 96% yield. Finally, the macrocyclization (HATU/
NMM/CH3CN) afforded the desired macrocycle 18 in 65%
yield (Scheme 6).
LOVO
LOVO-DOX
HELA
1.17
44.5
2.81
36.4
HELA-APL
Scheme 6
17: prostate carcinoma tumor cells (DU-145 and LN-CaP),
ovarian cells sensitive (IGROV) or resistant (IGROV-ET)
to ET-743, SK-BR-3 breast adenocarcinoma, MEL-28 ma-
lignant melanoma, A-549 lung carcinoma NSCL, K-562
chronic myelogenous leukemia, PANC-1 pancreatic epithe-
loid carcinoma, HT-29 colon carcinoma cells, LoVo lymph
node metathesis cells and the corresponding LoVo-Dox cells
resistant to Doxorubicin, and cervix epitheloid carcinoma
(HeLa) or resistant (HeLa-Apl) to aplidine.
A conventional colorimetric assay14 was set up to estimate
GI50 values, that is, the drug concentration that causes 50%
cell growth inhibition after 72 h continuous exposure to the
test molecules. Tamandarin B (2) is included in the test for
comparison. The results obtained are shown in Table 1.
The results revealed that the cytotoxic activity of analogues
16 and 17 is generally better than that of 2. This remarkable
activity suggests that analogues 16 and 17 might be used as
scaffolds for further development of new types of antitumor
compounds.
The coupling of the hydrochloride salt of macrocycle 18
with the lactyl side chain 15 gave the corresponding
tamandarin B analogue 17 (Scheme 7).
In conclusion, two tamandarin B analogues involving
structural changes in the macrocycle have been synthesized
and shown to have enhanced cytotoxic activity against many
human tumor cell lines.
Scheme 7
Acknowledgment. Financial support by NIH is gratefully
acknowledged. We thank Pharma Mar for a postdoctoral
Fellowship for J.A.
Supporting Information Available: Experimental pro-
cedures and characterization data for new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL0530023
(14) (a) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon,
J.; Vistica, D.; Waren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Nat.
Cancer Inst. 1990, 82, 1107. (b) Faircloth, G. T.; Stewart, D.; Clement, J.
J. J. Tissue Cult. Methods 1988, 11, 201.
A panel of 14 human tumor cell lines was used to evaluate
the cytotoxic potential of the tamandarin B analogues 16 and
514
Org. Lett., Vol. 8, No. 3, 2006