Semisynthesis of an analogue of antitumor bicyclic hexapeptide RA-VII by fixing the Ala-2/Tyr-3 bond to cis by incorporating a triazole cis-amide bond surrogate

Article abstract of DOI:10.1021/ol040005r

We prepared an analogue of an antitumor bicyclic hexapeptide RA-VII whose amide configuration between residues 2 and 3 was fixed to cis by incorporating a triazole cis-amide bond surrogate. This analogue was shown, by NMR studies, to take almost the same

Full text of DOI:10.1021/ol040005r

ORGANIC
LETTERS
2004
Vol. 6, No. 7
1111-1114
Semisynthesis of an Analogue of
Antitumor Bicyclic Hexapeptide RA-VII
by Fixing the Ala-2/Tyr-3 Bond to Cis by
Incorporating a Triazole cis-Amide Bond
Surrogate
Yukio Hitotsuyanagi, Shuichi Motegi, Tomoyo Hasuda, and Koichi Takeya*
School of Pharmacy, Tokyo UniVersity of Pharmacy and Life Science,
1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
takeyak@ps.toyaku.ac.jp
Received January 5, 2004
ABSTRACT
We prepared an analogue of an antitumor bicyclic hexapeptide RA-VII whose amide configuration between residues 2 and 3 was fixed to cis
by incorporating a triazole cis-amide bond surrogate. This analogue was shown, by NMR studies, to take almost the same conformation as
that of the minor conformer of RA-VII. It showed no cytotoxic activity.
RA-VII (1)1,2 and bouvardin (2)3 are plant-origin antitumor
bicyclic hexapeptides having a unique 14-membered cyclo-
isodityrosine unit. Their antitumor action is considered to
be due to inhibition of protein synthesis through interaction
with eukaryotic 80 S ribosomes,4,5 and accordingly, for
effective interaction a particular molecular conformation is
considered to be essential. In solution, due to the cis/trans
isomerization at the three tertiary amides, cyclic peptides of
this series can take several conformations. Peptide 1 takes
two, and sometimes three, stable conformations. Their ratios
vary, but of those three conformers, one conformer in which
the amide bond between Ala-2/Tyr-3 is trans (major) and
another one in which it is cis (minor) are always observed
in solution (Figure 1). The third minor one observed
occasionally involves isomerization at the amide bonds
between Ala-2/Tyr-3 and between Ala-4/Tyr-5. The amide
(1) (a) Itokawa, H.; Takeya, K.; Mihara, K.; Mori, N.; Hamanaka, T.;
Sonobe, T.; Iitaka, Y. Chem. Pharm. Bull. 1983, 31, 1424-1427. (b)
Itokawa, H.; Takeya, K.; Mori, N.; Hamanaka, T.; Sonobe, T.; Mihara, K.
Chem. Pharm. Bull. 1984, 32, 284-290.
(2) Itokawa, H.; Takeya, K.; Hitotsuyanagi, Y.; Morita, H. In The
Alkaloids; Cordell, G. A., Ed.; Academic Press: New York, 1997; Vol.
49, pp 301-387.
(3) Jolad, S. D.; Hoffmann, J. J.; Torrance, S. J.; Wiedhopf, R. M.; Cole,
J. R.; Arora, S. K.; Bates, R. B.; Gargiulo, R. L.; Kriek, G. R. J. Am. Chem.
Soc. 1977, 99, 8040-8044.
(4) Zalaca´ın, M.; Zaera, E.; Va´zquez, D.; Jime´nez, A. FEBS Lett. 1982,
148, 95-97.
(5) Sirdeshpande, B. V.; Toogood, P. L. Bioorg. Chem. 1995, 23, 460-
470.
10.1021/ol040005r CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/06/2004

fixing the amide bond between Ala-2 and Tyr-3 residues to
cis by replacing the relevant amide bond with a triazole cis-
amide bond surrogate, because the configuration at the other
two tertiary amides, between Ala-4/Tyr-5 and between Tyr-
5/Tyr-6, are less flexible and, apparently, usually are trans
and cis, respectively, in solutions. This approach of fixing
an amide bond by incorporating this surrogate has an
advantage that the desired product is prepared by reacting
readily available chiral thionopeptides with the reagents under
mild conditions without affecting the side chains and other
chiral centers.12 Thus, by a sequence of reactions as shown
in Scheme 1, first, the tetrapeptide fragment 12, incorporating
Scheme 1a
Figure 1. Population of the two conformers of 1 in solution.
configuration in the major conformer is thus trans-trans-
trans-trans-cis-trans (t-t-t-t-c-t) whereas that of the minor
conformer is t-c-t-t-c-t at D-Ala-1/Ala-2, Ala-2/Tyr-3, Tyr-
3/Ala-4, Ala-4/Tyr-5, Tyr-5/Tyr-6 and Tyr-6/D-Ala-1, re-
spectively, which has been verified by X-ray crystallography,
NOESY experiments, and computational methods.6-8 The
major conformer has been identified as an active conformer
on the grounds that [Pro-2]RA-VII (3)9 and [N-desmethyl-
Tyr-3]RA-VII (4),10,11 both taking a single conformer of t-t-
t-t-c-t amide configuration in solution, expressed a significant
cytotoxic activity. However, it is not known whether the
minor conformer characterized by having a cis amide bond
between Ala-2/Tyr-3 or t-c-t-t-c-t amide configuration shows
any activity or not. In the present study, we designed and
prepared an analogue of RA-VII whose conformer in solution
should be exclusively of the t-c-t-t-c-t amide configuration
and examined its solution conformation and cytotoxicity.
a Reagents and conditions: (a) NaH, MeI, DMF, 82%; (b) 4 M
HCl-dioxane; 8, Et3N, THF, 87%; (c) H2NNHCHO, Hg(OAc)2,
MeCN; p-TsOH, powdered 4 Å MS, CHCl3, 79%; (d) 4 M HCl-
dioxane; Boc-D-Ala-OH, EDC, HOBt, Et3N, CHCl3, 89%; (e) LiOH,
THF-MeOH-H2O, 95%.
a cis-amide bond surrogate (residues 1-4), was prepared,
which was linked to the cycloisodityrosine unit (residues 5
and 6) 13 and then subjected to macrocyclization (Scheme
2). For macrocyclization, a critical step in the synthesis of
cyclopeptides, the carboxyl group of Tyr-6 and the amino
(7) Morita, H.; Kondo, K.; Hitotsuyanagi, Y.; Takeya, K.; Itokawa, H.;
Tomioka, N.; Itai, A.; Iitaka, Y. Tetrahedron 1991, 47, 2757-2772.
(8) Boger, D. L.; Patane, M. A.; Zhou, J. J. Am. Chem. Soc. 1995, 117,
7357-7363.
(9) Itokawa, H.; Kondo, K.; Hitotsuyanagi, Y.; Isomura, M.; Takeya,
K. Chem. Pharm. Bull. 1993, 41, 1402-1410.
The major feature of our present preparation of a peptide
1 analogue having t-c-t-t-c-t amide configuration (5) included
(10) Itokawa, H.; Saitou, K.; Morita, H.; Takeya, K.; Yamada, K. Chem.
Pharm. Bull. 1992, 40, 2984-2989.
(11) Boger, D. L.; Zhou, J. J. Am. Chem. Soc. 1995, 117, 7364-7378.
(12) Hitotsuyanagi, Y.; Motegi, S.; Fukaya, H.; Takeya, K. J. Org. Chem.
2002, 67, 3266-3271.
(6) Bates, R. B.; Cole, J. R.; Hoffmann, J. J.; Kriek, G. R.; Linz, G. S.;
Torrance, S. J. J. Am. Chem. Soc. 1983, 105, 1343-1347.
1112
Org. Lett., Vol. 6, No. 7, 2004

for macrocyclization. In another experiment using diphenyl-
phosphoryl azide (DPPA, 4 equiv) and NaHCO3 (8 equiv),
this macrocyclization was even less efficient (yield 6.5%),
though DPPA was reportedly successfully employed in the
synthesis of RA-VII (1).13b,c
Scheme 2a
The solution conformation of analogue 5 was studied by
NMR experiments. In CD3OD, analogue 5 was shown to
exist in a single conformation. The key NOESY correlations
are shown in Figure 2. The correlation between Ala-2 HR/
a Reagents and conditions: (a) TFA; 12, EDC, HOObt, THF,
73%; (b) H2, Pd/C, EtOH, 94%; (c) 4 M HCl-dioxane; (d) FDPP,
N,N-diisopropylethylamine, DMF, 9.5% from 15.
group of D-Ala-1 were chosen as the cyclization site, as was
the case for the RA-VII syntheses.13
Figure 2. Key sequential and transannular NOESY correlations
for 5 in CD3OD.
Preparation of tetrapeptide fragment 12 proceeded as
shown in Scheme 1, starting with O-methylation of the
N-protected tyrosyl-alanine methyl ester 6 to afford methyl
ether 7 in 82% yield. After removal of the Boc group of 7,
the resultant amine was reacted with benzotriazole thio-
acylating agent 814 to provide thionotripeptide 9 in 87% yield.
Treatment of 9 with formic hydrazide and mercury(II) acetate
and subsequent dehydration using p-toluenesulfonic acid and
powdered 4 Å molecular sieves afforded triazolotripeptide
10 in 79% yield. Tripeptide 10 was converted into tetrapep-
tide 11 by the standard procedure (89%), which, after the
subsequent hydrolysis of the methyl ester group, afforded
carboxylic acid 12 in 95% yield (Scheme 1).
After removal of the Boc group, cycloisodityrosine 13,
prepared by partial degradation of natural RA-VII (1),15 was
coupled to acid 12 to provide hexapeptide 14 in 73% yield.
Debenzylation of 14 afforded 15 in 94% yield, and subse-
quent removal of the N-Boc group by treatment of 15 with
4 M HCl in dioxane afforded free hexapeptide 16. Then, 16
was subjected to macrocyclization by treating it with
pentafluorophenyl diphenylphosphinate (FDPP, 4 equiv) and
N,N-diisopropylethylamine (6 equiv) in DMF (concentration
0.002 M, 0 °C, 3 d, then room temperature, 2 d) to link the
Tyr-6 and D-Ala-1 residues together to afford cyclopeptide
5 (9.5% yield from 15). The yield of macrocyclization of
16 to 5 was very low: the incorporated cis-amide bond
surrogate may interfere with 16 taking suitable conformations
Tyr-3 HR indicated that the configuration of the peptide bond
between Ala-2/Tyr-3 residues was exclusively cis as ex-
pected. The correlations between Ala-4 Me/Tyr-5 NMe and
between Ala-4 HR/Tyr-5 NMe indicated that the amide bond
between Ala-4/Tyr-5 was trans, and the correlation between
Tyr-5 HR/Tyr-6 HR indicated that the amide bond between
Tyr-5/Tyr-6 was cis. Thus, analogue 5 was determined to
take the same t-c-t-t-c-t amide configuration in solution as
that of the minor conformer of peptide 1. The chemical shift
and coupling constant values of analogue 5 and the minor
conformer of peptide 1, having t-c-t-t-c-t amide configuration,
were generally quite similar, indicating that their solution
structures were approximately identical. In addition to those
observations, a transannular NOESY correlation observed
between D-Ala-1 HR/Ala-4 Me further demonstrated that the
structures of the backbone conformation of analogue 5 and
the minor conformer of peptide 1 were almost identical.7
Analogue 5 and peptide 1 were evaluated for the cytotoxic
activity using P-388 murine leukemia cells. Their IC50 values
were >10 and 0.0027 µg/mL, respectively. We may con-
clude, therefore, that the minor conformer of 1 having the
t-c-t-t-c-t configuration, accompanying the major conformer
in solution in all solvents tested, does not possess significant
activity.
The present and the previous results,9-11 demonstrating
that the major conformer with the t-t-t-t-c-t configuration is
responsible for the activity, whereas the minor one with the
t-c-t-t-c-t configuration takes little, if any, part in expressing
the activity, provide further knowledge about the conforma-
tion-activity relationships of the peptides of this series,
(13) (a) Inaba, T.; Umezawa, I.; Yuasa, M.; Inoue, T.; Mihashi, S.;
Itokawa, H.; Ogura, K. J. Org. Chem. 1987, 52, 2957-2958. (b) Boger, D.
L.; Yohannes, D. J. Am. Chem. Soc. 1991, 113, 1427-1429. (c) Bigot, A.;
Dau, M. E. T. H.; Zhu, J. J. Org. Chem. 1999, 64, 6283-6296.
(14) Shalaby, M. A.; Grote, C. W.; Rapoport, H. J. Org. Chem. 1996,
61, 9045-9048.
(15) Hitotsuyanagi, Y.; Hasuda, T.; Matsumoto, Y.; Yamaguchi, K.;
Itokawa, H.; Takeya, K. Chem. Commun. 2000, 1633-1634.
Org. Lett., Vol. 6, No. 7, 2004
1113

regarding the mechanisms of interactions between the
peptides and the ribosomes. The results also emphasize the
importance of the conformation in the expression of biologi-
cal activities and suggest that more active analogues or
analogues having new functions such as resistance against
enzymatic hydrolysis may be designed and synthesized, e.g.,
by replacement of the amide bonds of peptide 1, having t-t-
t-t-c-t amide configuration, with cis- or trans-amide bond
isosteres.
Supporting Information Available: Experimental details
and 1H NMR spectra of 5, 7, 9-12, 14, and 15. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL040005R
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