Synthesis of [Tyr-5-Ψ(CH2NMe)-Tyr-6]RA-VII, a reduced peptide bond analogue of RA-VII, an antitumor bicyclic hexapeptide

Article abstract of DOI:10.1016/j.bmcl.2012.02.093

A reduced peptide bond analogue of RA-VII, [Tyr-5-Ψ(CH ₂NMe)-Tyr-6]RA-VII (3), was designed and synthesized. The key reduced cycloisodityrosine unit was prepared by reduction of the cycloisodityrosine derived from natural RA-VII, followed by co

Full text of DOI:10.1016/j.bmcl.2012.02.093

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2757–2759
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Bioorganic & Medicinal Chemistry Letters
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Synthesis of [Tyr-5-W(CH2NMe)-Tyr-6]RA-VII, a reduced peptide bond
analogue of RA-VII, an antitumor bicyclic hexapeptide
⇑
Tomoyo Hasuda, Yukio Hitotsuyanagi, Mitsuyuki Shinada, 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:
A reduced peptide bond analogue of RA-VII, [Tyr-5-
W
(CH2NMe)-Tyr-6]RA-VII (3), was designed and syn-
Received 2 February 2012
Revised 24 February 2012
Accepted 27 February 2012
Available online 3 March 2012
thesized. The key reduced cycloisodityrosine unit was prepared by reduction of the cycloisodityrosine
derived from natural RA-VII, followed by connection with the tetrapeptide segment to afford a hexapep-
tide. Subsequent macrocyclization of the hexapeptide with FDPP under dilute conditions gave 3.
Analogue 3 showed cytotoxic activity against P-388 cells, but its activity was much weaker than that
of parent peptide RA-VII.
Keywords:
RA-VII
Ó 2012 Elsevier Ltd. All rights reserved.
[Tyr-5-W(CH2NMe)-Tyr-6]RA-VII
Bouvardin
Cytotoxicity
Rubia cordifolia
RA-VII (1), which was isolated from Rubia cordifolia L. and
in vivo use. We considered that the replacement of the peptide
bond between the Tyr-5 and Tyr-6 residues of RA-VII (1) with a
reduced peptide bond, –CH2NMe–, as in analogue 3, would
little affect the gross conformation of the molecule because this
peptide bond would be shared by both the 14-membered and
18-membered rings and the rotation about this bond would be
R. akane Nakai (Rubiaceae),1,2 and bouvardin (NSC 259968, 2),
which was isolated from Bouvardia ternifolia (Cav.) Schltdl. (Rubia-
ceae),3 are bicyclic hexapeptides that are structurally characterized
by the presence of a unique and strained 14-membered cycloisodi-
tyrosine unit in the molecule (Fig. 1). These peptides have promis-
ing antitumor activity, and peptide 1 had undergone phase I
clinical trials as an anticancer drug in Japan.4 The antitumor activ-
ity of these peptides is believed to be due to the inhibition of
protein synthesis through the interaction with eukaryotic ribo-
somes.5,6 Peptide 1 has been shown also to induce conformational
changes in F-actin, stabilizing actin filaments and inducing G2
arrest.7 During the phase I clinical trials of 1, we encountered
formulation problems associated with its poor solubility in water.
Therefore, the key consideration in our design of new peptide 1
analogues was to improve the solubility of 1. In this Letter,
we describe the synthesis of a novel analogue of RA-VII, [Tyr-5-
OR1
OMe
Ala-2
Me
O
O
O
O
Tyr-3
Me
HN
N
Me
N
Me
O
O
HN
Me
HN
HN
Ala-4
Me
Me
Me
Me
NH
O
NH
O
H
D
-Ala-1
O
N
Me
R2
H
O
N
W
(CH2NMe)-Tyr-6]RA-VII (3), and the evaluation of its
cytotoxicity.
N
Me
N
R4
O
H
Tyr-6
Modifications of the peptide backbone are often conducted for
the synthesis of bioactive peptide analogues.8 One example is the
replacement of the amide bond with a reduced peptide bond to
introduce a basic nitrogen into the peptide backbone.9 Compared
with the original peptide, such a reduced peptide bond analogue
is expected to have improved aqueous solubility through the for-
mation of an acid salt, and thus may simplify formulation and
Tyr-5
O
O
OMe
OR3
R1 R2 R3 R4
3
Me
RA-VII (1):
H
Me Me
bouvardin (2): Me
4: Me
OH
H
H
Me
Me
H
H
RA-II (5):
H
Me Me
⇑
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 RA-series peptides and bouvardins.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.093

2758
T. Hasuda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2757–2759
Figure 4. 1H NMR (500 MHz) spectrum of analogue 3.
in tetrahydrofuran (THF) at room temperature for 3 days. Aqueous
workup and subsequent treatment of the reaction mixture with
Boc2O gave desired reduced cycloisodityrosine 6 in 49% yield.
The tetrapeptide segment corresponding to residues 1–4 was
prepared from previously synthesized tripeptide 8.12b Extension
Figure 2. Energy-minimized structures of 3 with a cis (orange) or a trans (green)
amide bond between residues 2 and 3. The latter is superimposed with the crystal
structure of RA-II (5, blue).
at the N-terminus of tripeptide 8 by Boc-D-Ala-OH gave tetrapep-
restricted by the structural nature of the cycloisodityrosine unit
that would incorporate a meta-substituted phenyl ring and a
para-substituted phenyl ring. Indeed, N29-desmethyl-RA-VII (4),
which lacks an N-methyl group at the Tyr-6 residue, is known to
still adopt an unusual cis configuration at this amide bond, thus
retaining the conformational features of 1 and expressing potent
cytotoxicity.10 In analogue 3, the Monte Carlo conformational
search gave two distinctive conformers characterized by a cis or
a trans amide bond between the Ala-2 and Tyr-3 residues, and
tide 9, and subsequent ester hydrolysis of it afforded acid 10. After
removal of the Boc group, compound 6 was coupled with tetrapep-
tide carboxylic acid 10 in the presence of 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride (EDCÁHCl) and
3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt) to pro-
vide hexapeptide 11 in 68% yield. The benzyl-protecting group at
the C-terminus and the Boc group at the N-terminus of peptide
11 were removed sequentially by catalytic hydrogenolysis,
followed by treatment with HCl. The resulting deprotected hexa-
peptide was then subjected to macrocyclization under dilute con-
ditions (0.0027 M) in N,N-dimethylformamide (DMF) at 0 °C for
4 days with pentafluorophenyl diphenylphosphinate (FDPP) and
N,N-diisopropylethylamine (DIEA) to afford analogue 3 in 47% yield
from 11.
the former was slightly more stable (
D
E = À1.1 kcal/mol) than
the latter (Fig. 2).11 Comparison of the structure of the latter con-
former with the X-ray crystal structure of RA-II (5) indicated that
their 3D structures were similar (Fig. 2). Thus, it was thought that
analogue 3 might retain potent antitumor activity.
Analogue 3 was synthesized by the following procedure (Fig. 3).
Key reduced cycloisodityrosine unit 6, corresponding to Tyr-5 and
Tyr-6 residues, was prepared by selective reduction of the amide
bond of cycloisodityrosine 7, which was obtained by the degrada-
tion of natural RA-VII (1).12 The Boc group of 7 was removed by
treatment with trifluoroacetic acid (TFA) and the product was then
treated with 1.2 molar equiv of borane–tetrahydrofuran complex
The solution structure of analogue 3 was analyzed by NMR
spectroscopy.13 The 1H NMR spectrum of analogue 3 in pyridine-
d5 at 300 K showed the presence of three conformers in 54:44:2 ra-
tio (Fig. 4). In the NOESY spectrum, the most populated conformer
with population 54% (conformer-I, Fig. 5) showed NOE correlations
between Ala-2 H /Tyr-3 NMe, Tyr-3 NMe/Tyr-3 H , Ala-4 H /Tyr-5
a
a
a
OMe
OMe
O
O
O
OMe
OMe
Me
Me
HN
N
Me
N
Me
O
O
BocHN
1) TFA
2) Boc-
EDC•HCl
HOBt
CH2Cl2
91%
HN
HN
D
-Ala-OH
O
O
O
O
Me
CO2Me
Me
Me
CO2R
Me
HN
Me
HN
NHBoc
N
Me
N
Me
1) H2, Pd/C
EtOH
2) HCl
8
O
LiOH, THF
/MeOH/H2O
100%
9 R = Me
O
HN
HN
2) EDC
•HCl
10 R = H
Me
Me
Me
Me
Me
Me
dioxane
Boc
Me
Boc
Me
NHBocO
Me
NH
O
H
1) TFA
2) BH3•THF
THF
3) FDPP
DIEA
DMF
HOOBt
THF
68%
Me
N
Me
N
BnO2C
N
BnO2C
N
BnO2C
N
O
N
N
N
Me
1) TFA
H
H
H
H
H
O
47%
3) Boc2O
49%
O
O
OMe
O
OMe
11
O
OMe
OMe
7
6
3
Figure 3. Synthesis of analogue 3.

T. Hasuda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2757–2759
OMe
2759
OMe
References and notes
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.
Ala-2
O
O
Tyr-3
O
H
Me
Me
HN
Me
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. (a) Majima, H.; Tsukagoshi, S.; Furue, H.; Suminaga, M.; Sakamoto, K.;
Wakabayashi, R.; Kishino, S.; Niitani, H.; Murata, A.; Genma, A.; Nukariya, N.;
Uematsu, K.; Furuta, T.; Kurihara, M.; Yoshida, F.; Isomura, S.; Takemoto, T.;
Hirashima, M.; Izumi, T.; Nakao, I.; Ohashi, Y.; Ito, K.; Asai, R. Jpn. J. Cancer
Chemother. 1993, 20, 67; (b) Yoshida, F.; Asai, R.; Majima, H.; Tsukagoshi, S.;
Furue, H.; Suminaga, M.; Sakamoto, K.; Niitani, H.; Murata, A.; Kurihara, M.;
Izumi, T.; Nakao, I.; Ohashi, Y.; Ito, K. Jpn. J. Cancer Chemother. 1994, 21, 199.
5. Zalacaín, M.; Zaera, E.; Vázquez, D.; Jiménez, A. FEBS Lett. 1982, 148, 95.
6. Sirdeshpande, B. V.; Toogood, P. L. Bioorg. Chem. 1995, 23, 460.
7. Fujiwara, H.; Saito, S.; Hitotsuyanagi, Y.; Takeya, K.; Ohizumi, Y. Cancer Lett.
2004, 209, 223.
N
N
Me
H
O
H
O
H
HN
Ala-4
HN
O
HN
Me
H
Me
H
Me
H
Me
H
D
-Ala-1
NH
O
NH
O
O
N
Me
O
N
Me
H
H
H
H
N
N
Me
Me
Tyr-6
Tyr-5
8. (a) Ahn, J.-M.; Boyle, N. A.; MacDonald, M. T.; Janda, K. D. Mini Rev. Med. Chem.
2002, 2, 463; (b) Deska, J.; Kazmaier, U. Curr. Org. Chem. 2008, 12, 355.
9. Harbeson, S. L.; Shatzer, S. A.; Le, T. B.; Buck, S. H. J. Med. Chem. 1992, 35, 3949.
10. (a) Boger, D. L.; Yohannes, D.; Zhou, J.; Patane, M. A. J. Am. Chem. Soc. 1993, 115,
3420; (b) Boger, D. L.; Zhou, J. J. Am. Chem. Soc. 1995, 117, 7364.
11. The software used for this calculation was MacroModel ver. 7.0 (Schrödinger
Inc.). The Monte Carlo (MC) search was configured using the ‘automatic setup’
routine in MacroModel with all amide configurations in the macrocycle fixed
as trans except for the amide bond between Ala-2 and Tyr-3. The calculation
consisted of 50,000 MC steps with 500 iterations per step using the AMBER⁄
force field and the PR conjugate gradient (PRCG) with no solvation.
12. (a) Hitotsuyanagi, Y.; Hasuda, T.; Matsumoto, Y.; Yamaguchi, K.; Itokawa, H.;
Takeya, K. Chem. Commun. 2000, 1633; (b) Hitotsuyanagi, Y.; Hasuda, T.;
Aihara, T.; Ishikawa, H.; Yamaguchi, K.; Itokawa, H.; Takeya, K. J. Org. Chem.
2004, 69, 1481.
O
O
OMe
OMe
conformer-I
conformer-II
NOE H
H
Figure 5. Key NOE correlations of analogue 3.
NMe, and Ala-4 H3b/Tyr-5 NMe, indicating that the amide configu-
rations between Ala-2 and Tyr-3 and between Ala-4 and Tyr-5 are
both trans. The correlation between Tyr-5 H /Tyr-6 H demon-
strated the proximity of these protons, suggesting that the two
carbon atoms, Tyr-5 C and Tyr-6 C , are in a gauche or nearly an
eclipse arrangement as expected from the Monte Carlo conforma-
tional search of 3 (Fig. 2). Further, an NOE correlation was observed
between D-Ala-1 H /Ala-4 H3b. This correlation is usually observed
when this series of natural peptides adopt the conformation as de-
picted in the crystal structure of RA-II (5) in Figure 2. Accordingly,
this conformer seemed to have very similar structural features to
the calculated structure of 3 shown in green in Figure 2.
The conformer with population 44% (conformer-II, Fig. 5) dif-
fered from conformer-I in that it adopted a cis amide configuration
between Ala-2 and Tyr-3 (Fig. 5). The structure of the third con-
former with population 2% could not be determined due to weak
signal intensity.
13. NMR data of analogue 3 in pyridine-d5 at 300 K. Conformer-I:
4.81/dC 50.4; b, dH 1.56 (3H)/dC 20.3; C@O, dC 173.1; NH, dH 8.11), Ala-2 (
dH 4.76/dC 46.2; b, dH 1.47 (3H)/dC 16.6; C@O, dC 173.2; NH, dH 9.68), Tyr-3
, dH 4.15/dC 69.0; b, dH 3.88–3.94 (2H)/dC 33.7; , dC 131.9; d, dH 7.27 (2H)/
dC 130.9 (2C); , dH 7.00 (2H)/dC 114.4 (2C); f, dC 158.8; C@O, dC 169.5; NMe,
dH 3.09 (3H)/dC 40.3; OMe, dH 3.70 (3H)/dC 55.2), Ala-4 ( , dH 5.28/dC 47.2; b,
dH 1.46 (3H)/dC 18.6; C@O, dC 173.3; NH, dH 7.77), Tyr-5 ( , dH 5.00/dC 56.4;
b, dH 2.61, 2.85/dC 36.7; , dC 138.5; da, dH 7.30–7.38/dC 131.2; db, dH 7.53/dC
133.1; a, dH 7.30–7.38/dC 126.0; b, dH 6.83/dC 126.0; f, dC 158.6; CH2, dH
2.17, 2.35/dC 53.9; NMe, dH 2.76 (3H)/dC 28.8), Tyr-6 ( , dH 3.05/dC 67.8; b,
dH 2.41, 3.50/dC 30.1; , dC 134.2; da, dH 6.88/dC 121.9; db, dH 4.73/dC 118.8;
a, dH 6.83/dC 113.0; b, dC 154.0; f, dC 146.9; C@O, dC 171.5; NMe, dH 2.13
(3H)/dC 38.9; OMe, dH 3.84 (3H)/dC 56.3). Conformer-II: -Ala-1 ( , dH 5.00/dC
50.9; b, dH 1.50 (3H)/dC 20.3; C@O, dC 173.1; NH, dH 8.66), Ala-2 ( , dH 5.15/
dC 43.6; b, dH 0.96 (3H)/dC 17.8; C@O, dC 173.3; NH, dH 10.10), Tyr-3 ( , dH
5.78/dC 62.2; b, dH 3.21, 3.43/dC 34.2; , dC 130.1; d, dH 7.26 (2H)/dC 130.8
(2C); , dH 6.94 (2H)/dC 114.6 (2C); f, dC 159.0; C@O, dC 168.2; NMe, dH 3.40
(3H)/dC 29.5; OMe, dH 3.64 (3H)/dC 55.2), Ala-4 ( , dH 5.22/dC 47.7; b, dH
1.26/dC 19.1; C@O, dC 172.9; NH, dH 9.13), Tyr-5 (
, dH 5.05/dC ⁄; b, dH 2.85,
⁄/dC 36.2; , dC 138.6; da, dH 7.30–7.38/dC 129.7; db, dH 7.49/dC 133.2; a, dH
7.30–7.38/dC 126.3;
dH 2.81 (3H)/dC ⁄), Tyr-6 (
134.5; da, dH 6.98/dC 122.1; db, dH 4.65/dC 119.0;
D-Ala-1 (a, dH
a
a
a
,
a
a
(a
c
e
a
a
a
c
e
e
a
c
e
e
D
a
a
a
c
e
a
a
c
e
Analogue 3 and, as reference, peptide 1, were evaluated for their
cytotoxicity to P-388 leukemia cells, and their IC50 values were 5.0
e
b, dH 6.76/dC 126.1; f, dC 158.7; CH2, dH ⁄/dC ⁄; NMe,
a
, dH 3.09/dC 68.6; b, dH 2.32, 3.62/dC 29.7;
c
, dC
and 0.0043 l
g/mL, respectively.14 In spite of the resemblance of
ea, dH 6.87/dC 113.1;
eb, dC
154.1; f, dC 146.9; C@O, dC 171.4; NMe, dH 2.05 (3H)/dC 39.4; OMe, dH 3.86
(3H)/dC 56.3). Remarks: ⁄ not assigned in the present experiment.
14. The procedure for the cytotoxicity assay has previously been described, see:
Kim, I. H.; Takashima, S.; Hitotsuyanagi, Y.; Hasuda, T.; Takeya, K. J. Nat. Prod.
2004, 67, 863.
the 3D structure of the most populated conformer of analogue 3
in pyridine-d5 solution to that of the active conformation of this
series of peptides as represented by the crystal structure of RA-II
(5),15 analogue 3 showed only 1/1200 of the activity of peptide 1.
This unexpectedly weak activity of analogue 3 may be due to sub-
tle differences in the peptide backbone conformation and/or the
lack of amide carbonyl oxygen at Tyr-5 in analogue 3.
15. Itokawa, H.; Kondo, K.; Hitotsuyanagi, Y.; Isomura, M.; Takeya, K. Chem. Pharm.
Bull. 1993, 41, 1402.