[Lys3]didemnins as potential affinity ligands

Article abstract of DOI:10.1016/S0960-894X(00)00585-0

The synthesis and biological activity of N(ε)-Z-[Lys³]didemnin B are reported. This novel analogue retains antiproliferative, cytotoxic, and protein biosynthesis inhibition activities, but at reduced levels. This result suggests the use of [Lys

Full text of DOI:10.1016/S0960-894X(00)00585-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 13±16
[Lys3]Didemnins as Potential Anity Ligands
Matthew D. Vera, Amy J. P®zenmayer, Xiaobin Ding, Dong Xiao
and Madeleine M. Joullie*
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA
Received 20 September 2000; accepted 5 October 2000
AbstractÐThe synthesis and biological activity of Ne-Z-[Lys3]didemnin B are reported. This novel analogue retains anti-
proliferative, cytotoxic, and protein biosynthesis inhibition activities, but at reduced levels. This result suggests the use of [Lys3]di-
demnin derivatives as potential anity probes for studying the molecular target(s) of the didemnin class of natural products. # 2000
Elsevier Science Ltd. All rights reserved.
Introduction
The didemnin family of cyclic depsipeptide natural pro-
ducts (Fig. 1) exhibits antitumor, antiviral, and immu-
nosuppressive activities,1 3 as well as the ability to
induce very rapid apoptosis4 and to inhibit eukaryotic
protein biosynthesis.5 Aplidine, a congener having a
pyruvate residue at position 9 and enhanced antitumor
activity, is undergoing clinical evaluation against can-
cer.6 The mechanistic basis of these biological activities
is incompletely understood. Two didemnin binding
proteins, eukaryotic elongation factor 1a (EF-1a) and
palmitoyl protein thioesterase 1 (PPT1), have been
identi®ed using an anity chromatography ligand.7,8
A
mechanistic model for the EF-1a-mediated inhibition of
protein biosynthesis in vitro by didemnins has been
proposed9 and recently re®ned10 in which didemnins are
believed to inhibit the EF-2-catalyzed translocation step
of the elongation cycle. Additional work demonstrated
that inhibition of protein biosynthesis is not sucient to
induce apoptosis in MCF7 cells,11 indicating that other
antiproliferative or cytotoxic mechanisms may be oper-
ating. Recent studies with PPT1, the other didemnin
binding protein, showed that inhibition of PPT1 activity
by a series of didemnin congeners is independent of both
antiproliferative potency and side-chain structure.12
These results imply that an unidenti®ed molecular target
may play a role in mediating the biochemical e€ects of
this group of natural products.
*Corresponding author Tel.: +1-215-898-3158; fax: +1-215-898-
5129; e-mail: mjoullie@sas.upenn.edu
Figure 1. Structures of didemnins A, B, and analogue 3.
0960-894X/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00585-0

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M. D. Vera et al. / Bioorg. Med. Chem. Lett. 11 (2001) 13±16
Rationale and Chemical Synthesis
In order to clarify the mechanistic basis for the e€ects of
didemnins on cells, we have focused on synthetic
didemnin analogues designed for use as probe mole-
cules. Biological activity is highly sensitive to the struc-
ture of the side-chain peptide anchored to Thr6 of the
didemnin macrocycle.13 16 For example, didemnin A (1)
shows consistently lower levels of activity than didem-
nin B (2).2,15 The anity ligand used to identify EF-1a
and PPT1 was a didemnin A derivative in which the N-
Me-d-Leu7 residue was acylated with a biotinylated lin-
ker.7 Since the two binding proteins identi®ed using this
ligand do not seem sucient to explain the anti-
proliferative e€ects of didemnins, we believed it would
be fruitful to synthesize additional anity ligand candi-
dates. Our ®rst target is compound 3, in which the
macrocyclic Leu3 residue is replaced by a protected Lys3
residue. This analogue provides a protected nucleophilic
amino group for eventual derivatization to yield an
anity probe that leaves intact the side-chain peptide of
didemnin B, an important structural determinant of
biological activity.
Scheme 2. (a) 2,2,2-Trichloroethyl chloroformate, Na2CO3, H2O,
dioxane, 87%; (b) proline methyl ester hydrochloride, PyBroP, DIEA,
CH2Cl2, 77%; (c) LiOH, H2O, THF, 74%; (d) BOP-Cl, NMM,
CH2Cl2, then amine 6 (Scheme 1).
The synthesis of 3 follows the overall approach used in
our laboratory to obtain several congeners and analo-
gues of the didemnins.14,17 N-Boc-threonine (4) was
sequentially alkylated with SEM-Cl (Scheme 1) and
then acylated with a derivative of N-Z-tyrosine pre-
activated with isopropenyl chloroformate to yield dep-
sipeptide 5 in one operation. Catalytic hydrogenolysis
a€orded secondary amine 6.
Scheme 1. (a) Et3N, SEM-Cl, THF, then (b) Z-Me2TyrOH, IPCF,
Et3N, DMAP (cat), CH2Cl2, 85% (2 steps); (c) 10% Pd/C, H2, EtOAc/
MeOH (1:1), quant.
.
Scheme 3. (a) MgBr2 Et2O, CH2Cl2, 96%; (b) HATU, DIEA,
CH2Cl2, amine 11, 61%.
The tetrapeptide fragment was elaborated as shown in
Scheme 2. Monoprotected lysine derivative 7 was con-
verted to its Troc carbamate and then coupled to pro-
line methyl ester using the PyBroP reagent, followed by
saponi®cation, to a€ord dipeptide 9. Preactivation of 9
with BOP-Cl, followed by reaction with secondary
amine 6 (Scheme 1) furnished the tetrapeptide SEM
ester 10.
Hydrolysis of the silyl ether (Scheme 4) followed by
stepwise oxidation using Dess±Martin20 and Masa-
mune21 conditions delivered the linear carboxylic acid.
Reductive fragmentation with acid-washed zinc dust
followed by treatment with HATU induced
macrocyclization in 55% yield.
The protected linear precursor was synthesized as out-
lined in Scheme 3. Treatment of 10 with MgBr2 ether-
ate18 caused smooth and selective cleavage of the SEM
ester, in the presence of three carbamates and an alkyl
ester.19 Coupling to the previously-reported17 HIP-iso-
statine amine fragment (11) using HATU a€orded linear
silyl ether 12.
The cyclic MOM ether (13) was cleaved using dime-
thylboron bromide (Scheme 5) and the resulting alcohol
was oxidized under Dess±Martin conditions. The Boc
and TIPS protecting groups were removed selectively
using anhydrous HCl to obtain a macrocyclic amine salt.
BOP-mediated coupling of this salt with the previously
reported17 side-chain acid (14) a€orded analogue 3.22

M. D. Vera et al. / Bioorg. Med. Chem. Lett. 11 (2001) 13±16
15
intact for the purpose of obtaining a benchmark for
biological activity.
The biological activities of the protected analogue (3)
were less than those of didemnin B. The anti-
proliferative activity, as measured by the 50% growth
inhibition parameter (GI50) was substantially reduced.
Interestingly, cytotoxic activity, as measured by the
LC50, was only modestly reduced. This result is in
agreement with reports of intact cytotoxic activity in
natural congeners having modi®cations at HIP2 24,25 or
inversion of con®guration at Pro4,26 indicating a toler-
ance for modi®cation in this region. The reduction in
protein biosynthesis inhibition potency was of an inter-
mediate magnitude, as measured in this cell-free trans-
lation assay. The observation that the leucine-to-lysine
modi®cation exerts such disparate e€ects on these types
of activity underscores the complex and multifaceted
nature of the biological actions of didemnins.
Scheme 4. (a) HOAc, H2O, THF, 84%; (b) Dess±Martin periodinane,
96%; (c) KMnO4, tBuOH, NaH2PO4, quant.; (d) Zn, HOAc, H2O,
quant.; (e) HATU, DIEA, CH2Cl2, 55%.
In spite of the reduction in potency, the residual anti-
proliferative and cytotoxic activities of 3 suggest its
potential use as a competent but unoptimized anity
chromatography ligand for studying the molecular tar-
get(s) of the didemnins. Previous studies have indicated
that the potency of didemnins can be attenuated, in
some cases by several orders of magnitude, by mod-
i®cation of the side-chain peptide structure. This sug-
gests that second-generation anity probes based on
the [Lys3]didemnin sca€old with modi®ed side chains
might be feasible. Studies with 3 and similar analogues
are currently underway.
Acknowledgements
We gratefully acknowledge Dr. Deepika Ahuja and
Prof. Peter Toogood (University of Michigan) for eval-
uating protein biosynthesis inhibition activity. We
thank the Developmental Therapeutics Program of the
National Cancer Institute for evaluating compound 3 in
the NCI-60 tumor cell screen. Additionally, we grate-
fully acknowledge ®nancial support of this work from
the NIH (CA-40081) and NSF (CHE-01449).
Scheme 5. (a) Me2BBr, CH2Cl2, 30ꢀC, quant.; (b) Dess±Martin
periodinane, 95%; (c) HCl, EtOAc, 30 to 0ꢀC, quant.; (d) BOP,
NMM, CH2Cl2, side-chain acid 14, 81%.
References and Notes
Biological Activity and Conclusions
Analogue 3 was tested in both the NCI-60 human
tumor cell screen23 and also a cell-free assay for protein
biosynthesis inhibition activity,9 as shown in the Table 1.
Since the e-amino group of lysine would be acylated by
a linker during anity chromatography, the benzyl
carbamate protecting group at this position was left
1. Li, W.-R.; Joullie, M. M. The didemnins: biological proper-
ties, chemistry, and total synthesis. In Studies in Natural Pro-
ducts Chemistry, Vol. 10, Stereoselective Synthesis (Part F);
Atta-ur-Rahman, Ed.; Elsevier: Amsterdam, 1992; pp 241±302.
2. Rinehart, K. L., Jr.; Gloer, J. B.; 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. Rinehart, K. L. J. Didemnin and Its Biological Properties;
ESCOM: St. Louis, MO, 1988.
Table 1. Comparison of biological activities of 2 and 3
4. Grubb, D. R.; Wolvetang, E. J.; Lawen, A. Biochem. Bio-
phys. Res. Commun. 1995, 215, 1130.
5. SirDeshpande, B. V.; Toogood, P. L. FASEB J. 1993, 7,
A1083(abstr.).
6. Rinehart, K. L. Med. Res. Rev. 2000, 20, 1.
Biological activity
2
3
Growth inhibition (GI50
)
13 nM
66 nM
3.8 mM
3 mM
6.4 mM
22 mM
25 mM
154 mM
Total growth inhibition (TGI)
Cytotoxicity (LC50
Cell-free protein biosynthesis
inhibition (IC50
)
7. Crews, C. M.; Collins, J. L.; Lane, W. S.; Snapper, M. L.;
Schreiber, S. L. J. Biol. Chem. 1994, 269, 15411.
)

16
M. D. Vera et al. / Bioorg. Med. Chem. Lett. 11 (2001) 13±16
8. Crews, C. M.; Lane, W. S.; Schreiber, S. L. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 4316.
9. SirDeshpande, B. V.; Toogood, P. L. Biochemistry 1995,
34, 9177.
10. Ahuja, D.; Vera, M. D.; SirDeshpande, B. V.; Morimoto,
H.; Williams, P. G.; Joullie, M. M.; Toogood, P. L. Biochem-
istry 2000, 39, 4339.
11. Beidler, D. R.; Ahuja, D.; Wicha, M. S.; Toogood, P. L.
Biochem. Pharmacol. 1999, 58, 1067.
21. Abiko, A.; Roberts, J. C.; Takemasa, T.; Masamune, S.
Tetrahedron Lett. 1986, 27, 4537.
22. Compound 3: Rf 0.26 (5% MeOH/CHCl3); 1H NMR (500
MHz, CDCl3) d 0.71±1.05 (m, 18H), 1.16±1.42 (m, 13H), 1.43±
1.80 (m, 6H), 1.82±2.20 (m, 10H), 2.26±2.66 (m, 6H), 2.85±
2.99 (m, 2H), 3.12±3.74 (m, 11H), 3.79 (s, 3H), 3.99±4.13 (m,
2H), 4.24±4.39 (m, 2H), 4.50 (brs, 1H), 4.59±5.31 (m, 7H),
5.43±5.45 (m, 1H), 6.15 (m, 1H), 6.82 (d, J=8.3 Hz, 2H), 7.03
(d, J=8.2 Hz, 2H), 7.20 (d, J=10.2 Hz, 1H), 7.28±7.39 (m,
6H), 7.66±7.67 (m, 1H), 7.72 (d, J=9.3 Hz, 1H); 13C NMR
(125 MHz, CDCl3) d 11.6, 14.1, 14.5, 15.0, 15.4, 16.3, 16.9,
17.7, 18.6, 20.2, 21.2, 22.7, 23.2, 23.3, 24.5, 25.1, 25.9, 27.1,
27.8, 28.4, 29.3, 29.7, 29.9, 30.3, 31.3, 31.4, 31.9, 32.5, 33.8,
33.9, 36.2, 38.8, 41.0, 45.3, 46.8, 47.2, 49.1, 50.8, 55.2, 55.3,
56.4, 57.3, 57.8, 66.1, 66.3, 66.4, 67.8, 70.2, 75.6, 81.5, 114.1,
128.0, 128.4, 130.0, 130.3, 137.0, 156.5, 158.6, 168.6, 169.3,
170.4, 170.7, 171.7, 172.7, 173.4, 174.2, 204.6; IR (neat) 3330
(m), 2957 (m), 2931 (m), 2359 (w), 1730 (m), 1635 (s), 1539 (m),
1514 (m), 1456 (m), 1247 (m), 1168 (m), 731 (w) cm 1; LRMS
m/z calcd for C65H96N8O17Na (M+Na): 1283.7, found 1283.6.
23. Boyd, M. R.; Paull, K. D. Drug Dev. Res. 1995, 34, 91.
24. Banaigs, B.; Abou Mansour, E.; Bonnard, I.; Boulanger,
A.; Francisco, C. Tetrahedron 1999, 55, 9559.
12. Meng, L.; Sin, N. M. C. C. Biochemistry 1998, 37, 10488.
13. Jouin, P.; Poncet, J.; Dufour, M.-N.; Aumelas, A.; Pan-
taloni, A. J. Med. Chem. 1991, 34, 486.
14. Mayer, S. C.; Ramanjulu, J.; Vera, M. D.; P®zenmayer,
A.; Joullie, M. M. J. Org. Chem. 1994, 59, 5192.
15. Sakai, R.; Rinehart, K. L.; Kishore, V.; Kundu, B.; Fair-
cloth, 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.
16. Schmidt, U.; Griesser, H.; Haas, G.; Kroner, M.; Riedl,
B.; Schumacher, A.; Sutoris, F.; Haupt, A.; Emling, F. J.
Peptide Res. 1999, 54, 146.
17. Li, W.-R.; Ewing, W. R.; Harris, B. D.; Joullie, M. M. J.
Am. Chem. Soc. 1990, 112, 7659.
18. Kim, S.; Park, Y. H.; Kee, I. S. Tetrahedron Lett. 1991,
32, 3099.
25. Vervoort, H.; Fenical, W.; Epifanio, R. D. J. Org. Chem.
2000, 65, 782.
19. Chen, W.-C.; Vera, M. D.; Joullie, M. M. Tetrahedron
Lett. 1997, 38, 4025.
20. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277.
26. Abou-Mansour, E.; Boulanger, A.; Badre, A.; Bonnard,
I.; Banaigs, B.; Combaut, G.; Francisco, C. Tetrahedron 1995,
51, 12591.