Design and synthesis of a bis(cycloisodityrosine) analogue of RA-VII, an antitumor bicyclic hexapeptide

Article abstract of DOI:10.1016/j.bmcl.2008.10.064

An analogue of an antitumor bicyclic hexapeptide RA-VII was prepared, in which the Ala-2 and Tyr-3 residues of RA-VII were replaced by a cycloisodityrosine unit. In the crystalline state, the peptide backbone structures and the side-chain conformations at Tyr-3, Tyr-5, and Tyr-6 of this analogue and of RA-II were very similar. This analogue, however, showed much weaker cytotoxicity against P-388 leukemia cells than parent RA-VII.

Full text of DOI:10.1016/j.bmcl.2008.10.064

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6458–6461
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of a bis(cycloisodityrosine) analogue of RA-VII, an
antitumor bicyclic hexapeptide
*
Ji-Ean Lee, Yukio Hitotsuyanagi, Yoshie Nakagawa, Saori Kato, Haruhiko Fukaya, Koichi Takeya
School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
An analogue of an antitumor bicyclic hexapeptide RA-VII was prepared, in which the Ala-2 and Tyr-3 res-
idues of RA-VII were replaced by a cycloisodityrosine unit. In the crystalline state, the peptide backbone
structures and the side-chain conformations at Tyr-3, Tyr-5, and Tyr-6 of this analogue and of RA-II were
very similar. This analogue, however, showed much weaker cytotoxicity against P-388 leukemia cells
than parent RA-VII.
Received 16 August 2008
Revised 13 September 2008
Accepted 15 October 2008
Available online 18 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
RA-VII
Bouvardin
Cycloisodityrosine
X-ray crystallography
Rubia cordifolia
Cytotoxicity
RA-VII (1),1,2 isolated from Rubia cordifolia L. and R. akane Nakai
(Rubiaceae) is a bicyclic hexapeptide with a potent antitumor
activity (Fig. 1). The antitumor activities of this compound and also
of bouvardin (NSC 259968, 2)3 from Bouvardia ternifolia (Cav.)
Schltdl. (Rubiaceae) which is structurally closely related to 1 are
considered to be due to inhibition of protein synthesis through
interaction with eukaryotic ribosomes.4,5 Recently, 1 was shown
to cause conformational changes in F-actin and stabilization of ac-
tin filaments to induce G2 arrest.6 Peptide 1 is known to exist as a
mixture of two or three stable conformers in solution,7,8 and the
most populated conformer, having trans, trans, trans, trans, cis,
and trans (t–t–t–t–c–t) configuration at the peptide bonds between
cytotoxicity. In the present study, to obtain information about the
effect of the side-chain conformation of Tyr-3 upon the activity, we
designed an analogue having a restricted Tyr-3 side-chain rotation,
synthesized it, performed its X-ray crystallographic studies, and
evaluated its cytotoxicity.
Tyr-3
Ala-2
O
OR1
Me
N
Me
HN
D-Ala-1/Ala-2, Ala-2/Tyr-3, Tyr-3/Ala-4, Ala-4/Tyr-5, Tyr-5/Tyr-6,
O
HN
and Tyr-6/D-Ala-1, respectively, has been identified as an active
D
-Ala-1
Tyr-6
O
conformer.9–11 Of the three tyrosines at positions-3, -5, and -6 in
Me
O
Me
Ala-4
peptide 1, Tyr-5 and Tyr-6 form a cycloisodityrosine unit by form-
NH
O
ing a linkage between the phenolic oxygen of Tyr-5 and the C of
N Me
e
Tyr-6. Due to a planar amide bond and 1,3-disubstituted- and
1,4-disubstituted-phenyl rings included in the 14-membered ring
of the cycloisodityrosine unit, the rotation of the side chains of
those residues is restricted. The remaining side chain at Tyr-3 ro-
R2
N
O
H
Me
Tyr-5
tates about the C –Cb (
v
1) and Cb–C
(v
2) bonds. Since the substi-
tuent at the zeta position of Tyr-3 is known to greatly relate to the
activity,12 the
1 and 2 angles of Tyr-3, defining the spatial orien-
O
a
c
OR3
v
v
RA-VII (1): R1 = R3 = Me, R2 = H
Bouvardin (2): R1 = Me, R2 = OH, R3 = H
RA-II (11): R1 = R2 = H, R3 = Me
tation of the Tyr-3 phenyl ring, appear to play a critical role in the
* Corresponding author. Tel.: +81 42 676 3007; fax: +81 42 677 1436.
E-mail address: takeyak@ps.toyaku.ac.jp (K. Takeya).
Figure 1. Structures of natural RA-series peptides.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.064

J.-E. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6458–6461
6459
MeO
MeO
O
cis/trans
isomerization
Tyr-3
Tyr-3
rotatation restricted
Tyr-2
γ
β
Ala-2
Me
χ2
χ1
O
O
O
rotatable
bonds
by a 14-membered
ring structure
α
N
N
H
O
O
Me
HN
HN
HN
preferential adoption of a
trans amide by deletion of
the N-methyl group
O
HN
O
D
-Ala-1
Ala-4
Me
O
Me
Me
Me
NH
O
NH
O
N Me
O
N Me
N
Me
N
Me
O
Tyr-5
Tyr-6
O
O
OMe
OMe
RA-VII (1)
bis(cycloisodityrosine) analogue 3
Figure 2. Design of conformationally restricted analogue of 1.
MeO
O
MeO
O
1) TFA
2) Cbz-D-Ala-OH
O
O
MeO
O
MeO
O
HOBt, EDC
CHCl3
98% (two steps)
Me
CbzHN
N
H
N
H
CO2Me
CO2R
HN
Boc
HN
Tyr-2
Tyr-3
O
4
O
O
N
H
N
H
MeI, NaOH
n-Bu4NHSO4
CH2Cl2
5: R = Me
6: R = H
O
O
LiOH, H2O2
MeOH/H2O
HN
HN
1) H2, Pd/C
2) HOBt
EDC
CHCl3
O
HN
O
HN
EtOH
Me
Me
Me
Me
Me
Me
85%
42%
2) DPPA, Et3N
DMF
CbzHN
BnO2C
O
O
NH
O
Boc
Me
N
O
N
91%
HN
N
Boc
(two steps)
45%
Me
Me
N
Me
Me
N
RO2C
N
Me
(two steps)
O
O
Me
BnO2C
N
1) TFA
2) Boc-Ala-OH
N
Tyr-6
Tyr-5
O
1) TFA
O
O
O
HOBt, EDC
CHCl3
OMe
OMe
O
OMe
3
10
87% (two steps)
O
OMe
7: R = Me
8: R = Bn
1) LiOH, H2O2, MeOH/H2O
2) DEAD, Ph3P, BnOH, THF
9
49% (two steps)
Scheme 1. Synthesis of analogue 3.
Our approach to the application of restriction to the Tyr-3 side-
chain rotation was to introduce a cycloisodityrosine unit in place of
the Ala-2/Tyr-3 moiety of peptide 1, that is, preparation of an ana-
logue having two cycloisodityrosine units in one molecule as
shown in 3 (Fig. 2). In this analogue 3, the rotations about the
lated under phase-transfer catalysis conditions to prepare the
other cycloisodityrosine unit, 7, corresponding to Tyr-5 and Tyr-6
of the analogue. Subsequent conversion of the methyl ester func-
tionality of 7 to a benzyl ester group afforded 8, which, after re-
moval of the Boc group, was coupled with Boc-Ala-OH to afford
tripeptide 9. After deprotection, tripeptide 9 was coupled with acid
6 to give hexapeptide 10. Removal of the N- and C-terminal pro-
tecting groups of 10 and subsequent formation of the macrocycle
with diphenylphosphoryl azide (DPPA) and triethylamine under
high-dilution conditions in DMF (0.001 M) furnished 3 in 45% yield
from 10.
C –Cb and Cb–C bonds of Tyr-3 are restricted simultaneously as
a
c
in the case of those in Tyr-6. However, Tyr-3 in the newly intro-
duced second cycloisodityrosine unit bears no N-methyl group,
so that, in contrast to the Tyr-5/Tyr-6 bond of the original cycloiso-
dityrosine unit, the Tyr-2/Tyr-3 bond adopts a trans configuration
as in the active conformer of 1.
The route of synthesis of 3 is illustrated in Scheme 1. The start-
ing material, cycloisodityrosine 4, corresponding to Tyr-2 and
The solution structure of thus obtained analogue 3 was ana-
lyzed by the NMR experiments. In CDCl3 at 300 K, analogue 3 gave
a single set of sharp peaks (Fig. 3),14 suggesting that only one single
conformer was present in this solution. The amide configuration of
this solution structure was determined by analysis of NOESY data.
Tyr-3 of 3, was prepared from 3-iodo-
procedure reported previously.13 Compound 4 was deprotected
and coupled with Cbz- -Ala-OH to afford tripeptide 5, which was
then converted to acid 6. Another portion of 4 was N,N0-dimethy-
L-tyrosine according to the
D
From the NOE correlations between D-Ala-1 H-a/Tyr-2 NH and be-

6460
J.-E. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6458–6461
Figure 3. Five hundred megahertz 1H NMR spectrum of analogue 3 in CDCl3.
tween
2 was determined to be trans, and from the correlations between
Tyr-2 H- /Tyr-3 NH, the amide bond between Tyr-2/Tyr-3 to be
trans. Analogously, from the correlations between Tyr-3 H- /Ala-
4 NH, Ala-4 H- /Tyr-5 NMe, Ala-4 Me/Tyr-5 NMe, Tyr-5 H- /Tyr-
6 H- , and Tyr-6 H- -Ala-1 NH, the amide bonds between Tyr-
3/Ala-4, Ala-4/Tyr-5, Tyr-5/Tyr-6, and Tyr-6/ -Ala-1 were deter-
D-Ala-1 Me/Tyr-2 NH, the amide bond between D-Ala-1/Tyr-
a
a
a
a
a
a/D
D
mined to be trans, trans, cis, and trans, respectively, to show the se-
quence of the amide configurations of 3 to be t–t–t–t–c–t as in the
active conformer of 1. In addition, an NOE correlation between
D-
Ala-1 H- /Ala-4 Me was observed. Such a transannular cross-peak
a
was also observed in the conformer of natural RAs having t–t–t–t–
c–t configuration.15 The structure in the solid state was obtained by
the crystallography of 3 (Fig. 4).16 The results showed that the crys-
tal structure of 3 was basically identical to the solution structure of
3 in CDCl3, derived from its NOESY experiments. The amide config-
Figure 4. ORTEP representation of analogue 3.
Table 1
X-ray calculated backbone dihedrals (degree) in analogue 3.
Residue
-Ala-1
Dihedral angle
136.3(3)
Residue
Ala-4
Dihedral angle
D
u
w
x
u
w
x
ꢀ164.5(3)
ꢀ129.2(3)
156.4(3)
176.3(3)
178.3(3)
Tyr-2
Tyr-3
u
w
x
ꢀ141.7(3)
116.6(3)
ꢀ162.7(3)
49.1(4)
49.3(4)
ꢀ174.8(3)
Tyr-5
Tyr-6
u
w
x
ꢀ115.5(3)
111.1(3)
ꢀ21.3(4)
ꢀ75.7(4)
ꢀ179.7(3)
176.8(3)
u
w
x
u
w
x
Figure 5. Superposition of the crystal structures of analogue 3 (red) and RA-II (11,
blue).

J.-E. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6458–6461
6461
d, J = 8.2 Hz, Tyr-6 H-
e
a), 6.77 (1H, d, J = 8.2 Hz, Tyr-3 H-
e
a), 6.65 (1H, dd,
uration of the crystal structure of 3 was also t–t–t–t–c–t (Table 1)
and the distance between -Ala-1 H- and the nearest methyl
hydrogen of Ala-4, responsible for the above mentioned significant
NOE correlation in 3, was 2.38 Å. This distance is reasonable for
producing an NOE cross-peak.
The similarity in the three-dimensional structural features of 1
and 3 was highlighted by superimposing the crystal structure of 3
over that of RA-II (11),17 whose conformational property is known
to be identical to that of 1 and whose crystallographic data are
available (Fig. 5).18 The spatial positions of the phenyl rings of
the three tyrosines and the peptide backbone conformation at res-
idues 2–6 of these two peptides 3 and 11 are almost superimpos-
able, which indicated that analogue 3 may effectively mimic one of
the lowest-energy conformations in peptide 1 including the side
chain of Tyr-3.
Analogue 3 and as reference, 1, were evaluated for their cyto-
toxicity against P-388 leukemia cells. Their IC50 values were 7.5
and 0.0015 lg/mL, respectively. The result apparently does not
agree with our hypothesis that the side-chain conformation at
Tyr-3 of peptide 1, as shown in the crystal structure of 11, is a ma-
jor factor which determines the cytotoxic activity of the com-
pounds of this series. The bulky phenoxy tether connecting the
J = 8.2, 2.0 Hz, Tyr-3 H-da), 6.62 (1H, dd, J = 8.2, 2.0 Hz, Tyr-6 H-da), 6.36 (1H, d,
J = 6.9 Hz, -Ala-1 NH), 6.08 (1H, d, J = 9.2 Hz, Tyr-2 NH), 5.88 (1H, d, J = 6.4 Hz,
Tyr-3 NH), 5.36 (1H, dd, J = 11.4, 3.3 Hz, Tyr-5 H- ), 4.73 (1H, m, Ala-4 H- ),
4.70 (1H, m, Tyr-2 H- ), 4.68 (1H, d, J = 2.0 Hz, Tyr-3 H-db), 4.46 (1H, dd,
J = 12.0, 3.6 Hz, Tyr-6 H- ), 4.35 (1H, d, J = 2.0 Hz, Tyr-6 H-db), 4.35 (1H,
quintet, J = 6.9 Hz, -Ala-1 H- ), 3.95 (3H, s, Tyr-6 OMe), 3.94 (3H, s, Tyr-3
OMe), 3.64 (1H, t, J = 11.4 Hz, Tyr-5 H-bb), 3.37 (1H, m, Tyr-3 H- ), 3.27 (1H,
D
a
D
a
a
a
a
D
a
a
dd, J = 12.8, 5.1 Hz, Tyr-2 H-ba), 3.16 (1H, m, Tyr-6 H-ba), 3.14 (3H, s, Tyr-5
NMe), 3.12–2.98 (2H, m, Tyr-3 H2-b), 3.00 (1H, m, Tyr-6 H-bb), 2.95 (1H, t,
J = 12.8 Hz, Tyr-2 H-bb), 2.69 (3H, s, Tyr-6 NMe), 2.61 (1H, dd, J = 11.4, 3.3 Hz,
Tyr-5 H-ba), 1.36 (3H, d, J = 6.9 Hz,
H3-b); 13C NMR (125 MHz, 300 K, CDCl3, d)
Tyr-2 (170.6 C@O, 158.3 f, 133.6 , 131.1 da, 131.0 db, 127.3
, 37.3 b), Tyr-3 (167.9 C@O, 152.0 b, 146.7 f, 130.7
111.9 a, 58.7 , 56.2 OMe, 32.4 b), Ala-4 (171.7 C@O, 46.3
(169.5 C@O, 158.2 f, 134.9 , 132.9 da, 130.7 db, 126.0 b, 124.4
b, 30.6 NMe), Tyr-6 (170.5 C@O, 153.1 b, 146.5 f, 128.1 , 120.9 da, 113.3 db,
D-Ala-1 H3-b), 1.14 (3H, d, J = 6.9 Hz, Ala-4
D-Ala-1 (171.8 C@O, 48.2
a
, 21.1 b),
b, 56.3
c
e
a, 125.0 e
a
e
c
, 121.9 da, 119.3 db,
, 19.0 b), Tyr-5
a, 53.9 , 36.9
e
a
a
c
e
e
a
e
c
112.3
e
a, 57.6
a, 56.2 OMe, 35.1 b, 29.2 NMe).
MeO
ζ
ζ
εb
O
εa
εa
δa
Tyr-2
Tyr-3
εb
δb
δb
γ
γ
β
δa
O
Ha
Hb
Ha
Hb
β
α
α
N
H
H
O
H
HN
O
HN
Cb of Ala-2 and the C of Tyr-3 in the present compound 3, how-
e
D
-Ala-1
Ala-4
Me
Me
ever, may be hampering its necessary close access to the relevant
binding site, resulting in giving low cytotoxicity. Synthesis of fur-
ther analogues and their analyses may give further information
to this problem.
NH
H
O
H
O
N Me
Hb
α
α
Hb
Ha
N
β
β
Ha
δaO
γ
Me
γ
References and notes
δb
εa
δa
εa
δb
εb
Tyr-6
Tyr-5
1. Itokawa, H.; Takeya, K.; Mihara, K.; Mori, N.; Hamanaka, T.; Sonobe, T.; Iitaka, Y.
Chem. Pharm. Bull. 1983, 31, 1424.
2. Itokawa, H.; Takeya, K.; Hitotsuyanagi, Y.; Morita, H.. In The Alkaloids; Cordell,
G. A., Ed.; Academic Press: NY, 1997; Vol. 49, p 301.
ζ
εb
O
ζ
OMe
15. Hitotsuyanagi, Y.; Ishikawa, H.; Hasuda, T.; Takeya, K. Tetrahedron Lett. 2004,
45, 935.
3. Jolad, S. D.; Hoffman, 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.
4. Zalacaín, M.; Zaera, E.; Vázquez, D.; Jiménez, A. FEBS Lett. 1982, 148, 95.
5. Sirdeshpande, B. V.; Toogood, P. L. Bioorg. Chem. 1995, 23, 460.
6. Fujiwara, H.; Saito, S.; Hitotsuyanagi, Y.; Takeya, K.; Ohizumi, Y. Cancer Lett.
2004, 209, 223.
7. 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.
8. Morita, H.; Kondo, K.; Hitotsuyanagi, Y.; Takeya, K.; Itokawa, H.; Tomioka, N.;
Itai, A.; Iitaka, Y. Tetrahedron 1991, 47, 2757.
16. Crystallography of 3: C46H49N6O10ꢁCH4Oꢁ3(O), M = 925.95, 0.19 ꢂ 0.12 ꢂ
0.09 mm, monoclinic, space group C2, a = 18.9568(18) Å, b = 14.9739(14) Å,
c = 16.4613(15) Å, b = 93.3980(10)°, V = 4664.4(8) Å3, Z = 4, DX = 1.319 Mg mꢀ3
,
l(Mo
K
a
) = 0.10 mmꢀ1
,
T = 100 K, 13,383 measured reflections, 9102
independent reflections, 7496 reflections with I > 2
r
(I), R[F2 > 2 (F2)] = 0.059,
r
wR(F2) = 0.155, S = 1.05. The structure was solved by direct methods using
SHELXS-97,19 and refined by full-matrix least-squares on F2 using SHELXL-
97.20 Crystallographic data for compound 3 reported in this paper have been
deposited with the Cambridge Crystallographic Data Centre under the
reference number CCDC 698521. These data can be obtained free of charge
9. Itokawa, H.; Kondo, K.; Hitotsuyanagi, Y.; Isomura, M.; Takeya, K. Chem. Pharm.
Bull. 1993, 41, 1402.
10. Itokawa, H.; Saitou, K.; Morita, H.; Takeya, K.; Yamada, K. Chem. Pharm. Bull.
1992, 40, 2984.
via
http://www.ccdc.cam.ac.uk/data_request/cif,
or
by
The
e-mailing
Cambridge
data_request@ccdc.cam.ac.uk,
or by contacting
Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax:
+44 1223 336033.
17. Itokawa, H.; Takeya, K.; Mori, N.; Sonobe, T.; Mihashi, S.; Hamanaka, T. Chem.
Pharm. Bull. 1986, 34, 3762.
18. Hitotsuyanagi, Y.; Sasaki, S.-i.; Matsumoto, Y.; Yamaguchi, K.; Itokawa, H.;
Takeya, K. J. Am. Chem. Soc. 2003, 125, 7284.
19. Sheldrick, G. M. SHELXS-97: Program for the Solution of Crystal Structures;
University of Göttingen: Göttingen, Germany, 1997.
11. Boger, D. L.; Zhou, J. J. Am. Chem. Soc. 1995, 117, 7364.
12. Itokawa, H.; Kondo, K.; Hitotsuyanagi, Y.; Nakamura, A.; Morita, H.; Takeya, K.
Chem. Pharm. Bull. 1993, 41, 1266.
13. Hitotsuyanagi, Y.; Ishikawa, H.; Naito, S.; Takeya, K. Tetrahedron Lett. 2003, 44, 5901.
14. Data for 3: colorless prisms, mp > 300 °C (MeOH–H2O); 1H NMR (500 MHz,
300 K, CDCl3) d 7.47 (1H, dd, J = 8.6, 1.8 Hz, Tyr-2 H-db), 7.37 (1H, dd, J = 8.4,
2.2 Hz, Tyr-5 H-db), 7.32 (1H, dd, J = 8.4, 1.8 Hz, Tyr-2 H-da), 7.30 (1H, dd,
J = 8.4, 2.0 Hz, Tyr-2 H-
dd, J = 8.4, 2.3 Hz, Tyr-5 H-
(1H, dd, J = 8.4, 2.3 Hz, Tyr-5 H-
e
a), 7.27 (1H, dd, J = 8.4, 2.2 Hz, Tyr-5 H-da), 7.21 (1H,
b), 7.10 (1H, dd, J = 8.6, 2.0 Hz, Tyr-2 H- b), 6.91
a), 6.84 (1H, d, J = 7.2 Hz, Ala-4 NH), 6.83 (1H,
20. Sheldrick, G. M. SHELXL-97: Program for the Refinement of Crystal Structures;
University of Göttingen: Göttingen, Germany, 1997.
e
e
e