A novel bicyclic hexapeptide, RA-XVIII, from Rubia cordifolia: Structure, semi-synthesis, and cytotoxicity

Article abstract of DOI:10.1016/j.bmcl.2007.11.030

A new bicyclic peptide of RA-series, RA-XVIII (3), was isolated from the roots of Rubia cordifolia L. Its structure was established to be a hydroxylated derivative of RA-VII by the semi-synthesis of 3 from deoxybouvardin, and its cytotoxicity against P-38

Full text of DOI:10.1016/j.bmcl.2007.11.030

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 808–811
A novel bicyclic hexapeptide, RA-XVIII, from Rubia cordifolia:
Structure, semi-synthesis, and cytotoxicity
Ji-Ean Lee, Yukio Hitotsuyanagi, Ik-Hwi Kim, Tomoyo Hasuda and Koichi Takeya*
School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received 17 September 2007; revised 2 November 2007; accepted 9 November 2007
Available online 17 November 2007
Abstract—A new bicyclic peptide of RA-series, RA-XVIII (3), was isolated from the roots of Rubia cordifolia L. Its structure was
established to be a hydroxylated derivative of RA-VII by the semi-synthesis of 3 from deoxybouvardin, and its cytotoxicity against
P-388 cells was 0.012 lg/mL.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Natural antitumor bicyclic hexapeptides, RA-VII (1)1
Tyr-3
and deoxybouvardin (2),2 and their congeners,3 charac-
Ala-2
O
O
OMe
Me
HN
terized by unique structural features of having a
14-membered strained cycloisodityrosine unit, have
attracted considerable attention recently (Fig. 1). They
are provided with unique biological actions including
inhibition of protein synthesis through interaction with
eukaryotic ribosomes,4,5 and peptide 1 was recently
shown to cause conformational changes of F-actin and
stabilization of actin filaments to induce G2 arrest.6 As
part of our series of studies on the RA-series peptides
from Rubia cordifolia L.,7 the present paper describes
the isolation and structure elucidation of a new peptide,
RA-XVIII (3). Semi-syntheses of 3 and its analogues
from 2 and 1, and the cytotoxicity of representative
analogues of this series against P-388 leukemia cells
are also described.
N
Me
O
HN
D
-Ala-1
Me
Me
Me
Ala-4
NH
O
O
N
α
α
Hb
Ha
Hb
Ha
δa
N
β
γ
β
δaO
Me
γ
δb
Tyr-6
Tyr-5
εa
δb
εb
εa
εb
O
ζ
R2
ζ
OR1
RA-VII (1): R1 = Me, R2 = H
deoxybouvardin (2): R1 = R2 = H
RA-XVIII (3): R1 = Me, R2 = OH
Figure 1.
Peptide 3 was prepared from a chloroform–methanol
(10:1)-soluble portion of a methanol extract of the dried
roots of R. cordifolia (55 kg), by a series of column chro-
matography using silica gel, alumina, and then amino-
propyl-bonded silica gel eluting with a chloroform–
methanol mixture. The resulting RAs-rich fraction ob-
tained after removal of the solvent was crystallized from
methanol to give a crude crystalline RA mixture, which
was subjected to separation by reversed-phase HPLC
(ODS) to afford RA-XVIII (3)8 [4.8 mg, 8.7 · 10ꢀ6%,
25
amorphous solid, ½aꢁ ꢀ222 (c 0.13, MeOH)]. The
D
molecular formula C41H50N6O10 of 3 was obtained from
the [M+H]+ ion, m/z 787.3677 (calcd for C41H51N6O10,
1
787.3667), in HR-ESI-MS. The H and 13C NMR spec-
tra of 3 in CDCl3 (Table 1) showed signals typical of an
RA-series peptide, demonstrating the presence of two
conformers in a ratio of 89:11.9,10 The structure of 3
was determined by using the resonances caused by the
major conformer, which included signals for three sec-
ondary methyl groups (dH/dC 1.11/18.5, 1.30/20.7, 1.35/
16.6), three N-methyl groups (dH/dC 2.69/29.3, 2.86/
39.8, 3.11/30.5), two O-methyl groups (dH/dC 3.79/55.3,
4.09/61.4), six N-substituted methines (dH/dC 3.58/68.4,
4.36/47.9, 4.51/57.3, 4.74/46.4, 4.84/44.6, 5.39/54.2), a
Keywords: RA-XVIII; Deoxybouvardin; Cytotoxicity; Rubia cordifo-
lia; Synthesis.
*
Corresponding author. Tel.: +81 42 676 3007; fax: +81 42 677
1436; e-mail: takeyak@ps.toyaku.ac.jp
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.030

J.-E. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 808–811
Table 1. NMR data for the major conformer of RA-XVIII (3) in CDCl3 at 300 K
809
a
b
a
b
Position
D-Ala-1
dH
dC
Position
dH
dC
a
b
4.36 (qd, 7.0, 6.6)
1.30 (d, 6.8, 3H)
47.9
20.7
ba
bb
c
da
db
ea
eb
f
C@O
NMe
a
ba
bb
c
da
db
ea
eb
2.63 (dd, 11.4, 3.1)
3.67 (t, 11.4)
37.0
C@O
NH
a
172.3
135.4
132.7
131.0
124.1
125.8
158.0
169.2
30.5
6.44 (d, 6.6)
4.84 (dq, 8.4, 7.0)
1.35 (d, 7.0, 3H)
7.25 (dd, 8.4, 2.2)
7.41 (dd, 8.4, 2.2)
6.85 (dd, 8.4, 2.5)
7.18 (dd, 8.4, 2.5)
Ala-2
Tyr-3
44.6
16.6
b
C@O
NH
a
ba
bb
c
d
e
f
C@O
NMe
OMe
a
172.6
6.35 (d, 8.4)
3.58 (dd, 10.6, 5.0)
3.38 (dd, 14.1, 5.0)
3.34 (dd, 14.1, 10.6)
68.4
32.7
3.11 (s, 3H)
Tyr-6
4.51 (dd, 11.9, 3.7)
3.01 (dd, 18.2, 11.9)
2.88 (dd, 18.2, 3.7)
57.3
35.9
130.7
130.2c
114.1c
158.4
168.0
39.8
7.05 (d-like, 8.6, 2H)
6.83 (d-like, 8.6, 2H)
131.2
107.5
106.0
149.4
155.9
133.9
170.6
29.3
6.24 (d, 1.9)
3.89 (d, 1.9)
2.86 (s, 3H)
3.79 (s, 3H)
55.3
46.4
Ala-4
Tyr-5
4.74 (dq, 7.7, 6.6)
1.11 (d, 6.6, 3H)
f
b
18.5
171.8
C@O
NMe
OMe
OH
C@O
NH
a
2.69 (s, 3H)
4.09 (s, 3H)
5.91 (br s)
6.71 (d, 7.7)
5.39 (dd, 11.4, 3.1)
61.4
54.2
a 1H spectrum recorded at 500 MHz referenced to residual CHCl3 (7.26 ppm); J-values given in Hz in parentheses.
b 13C spectrum recorded at 125 MHz referenced to CDCl3 (77.03 ppm).
c 2C.
1,4-substituted benzene ring (dH/dC 6.83/114.1 · 2, 7.05/
130.2 · 2), two aromatic rings, methylenes of the three
tyrosine residues (dC 32.7, 35.9, 37.0), six amide car-
bonyl groups (dC 168.0, 169.2, 170.6, 171.8, 172.3,
172.6), and three amide protons (dH 6.35, 6.44, 6.71).
a hydroxyl group at the ea-position of Tyr-6, which was
further confirmed by the semi-synthesis of 3 from 2.
Semi-synthesis of 3 from 2 was performed as shown in
Scheme 1. Thus, treatment of 2 with nitric acid and sul-
furic acid in a chloroform–acetic acid mixture afforded
nitro compound 4.11 The position of the nitro group
was confirmed by observation of the H-da of Tyr-6 as
a singlet signal (dH 7.46) which showed an NOE correla-
tion with H-ba. O-Methylation of 4 with (trimethyl-
silyl)diazomethane gave methyl ether 5, which gave, by
subsequent catalytic hydrogenation over Pd/C, amine
6. Amine 6 was then converted into a diazonium inter-
mediate, which, on treatment with copper(II) nitrate
1
The H NMR data of 3 were very similar to those of
110 except that the H-da signal of Tyr-6 in 3 was at dH
6.24 as a meta-coupled doublet (J = 1.9 Hz), whereas
that in 1 was at dH 6.57 as a doublet of doublets
(J = 8.3, 2.0 Hz). Consequently, 3 was suggested to be
an analogue of 1 with a modified aromatic ring of
Tyr-6. The molecular formula of 3 showed that it was
equivalent to that of 1 with one additional oxygen.
The additional oxygen atom in 3 indicated the presence
of a hydroxyl group in 3; the proton signal of which was
and copper(I) oxide in an aqueous media,12 produced
25
D
found to be identical to natural 3 by comparison of their
1
observed as a broad singlet at dH 5.91 in the H NMR
a compound, ½aꢁ ꢀ223 (c 0.12, MeOH), which was
spectrum. HMBC correlations from the hydroxyl pro-
ton to C-da, C-ea, and C-f (Fig. 2) placed this hydroxyl
group at the ea-position of Tyr-6 in 3, which was verified
by the chemical shift value of the C-ea signal, dC 149.4.
Therefore, 3 was concluded to be an analogue of 1 with
1
optical rotations, H and 13C NMR, and mass spectra.
Accordingly, the absolute structure of 3 was determined
to be as shown in Figure 1.
In this series of peptides, hydroxylation of the Tyr-6 aro-
matic ring apparently increases the electron density of
this ring and changes the polarity of the molecules,
which may affect their biological activities. To investi-
gate such a positional effect of the hydroxyl group on
the cytotoxic activity in the peptides of this series, an
isomeric d-hydroxide, 7, was prepared from 1 by using
a similar protocol employed in the synthesis of 3 from
2 (Scheme 2). Nitration of 1 gave nitro compound 8,
whose nitro group was confirmed to be at the da posi-
tion of Tyr-6 by observation of H-ea of Tyr-6 as a sin-
glet signal (dH 7.74) and an NOE correlation of this
signal with the methoxyl proton of Tyr-6 (dH 4.03).11
N
O
Me
Tyr-6
Tyr-5
δa
εa
O
HO
ζ
O
Me
HMBC
H
C
Figure 2. Key HMBC correlations for 3.

810
J.-E. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 808–811
O
O
O
O
O
OMe
OMe
OMe
Me
HN
Me
HN
Me
HN
N
Me
N
Me
N
Me
O
O
O
HN
O
HN
HN
Me
Me
Me
Me
Me
Me
Me
Me
Me
HNO3, H2SO4
CHCl3-AcOH
93%
TMSCHN2
MeCN-MeOH
93%
NH
O
NH
O
NH
O
O
N
O
N
O
N
H
H
N
Me
N
Me
N
Me
β
NOE
O
O
O
H
δa
O
O
O
OMe
O2N
O2N
OH
OH
4
2 (deoxybouvardin)
5
O
O
O
OMe
OMe
Me
HN
Me
N
Me
N
Me
O
O
HN
1) t-BuONO
aq. HBF4
THF
HN
O
HN
Me
Me
Me
Me
O
Me
H2, Pd/C, HCl
EtOH-dioxane
76%
NH
O
NH
O
O
N
N Me
2) Cu(NO3)2
Cu2O, H2O
N
N
O
O
Me
Me
40%
O
OMe
O
OMe
H2N
HO
6
3 (RA-XVIII)
Scheme 1.
O
O
N
O
O
O
O
OMe
OMe
OMe
OMe
Me
HN
Me
HN
Me
Me
HN
Me
N
Me
N
Me
N
Me
O
O
O
O
HN
Me
1) t-BuONO
aq. HBF4
THF
O
HN
O
HN
HN
HN
Me
O
Me
Me
Me
Me
O
Me
Me
Me
O
Me
Me
H2, Pd/C, HCl
EtOH-dioxane
92%
HNO3, H2SO4
NH
O
NH
O
NH
O
NH
O
2) Cu(NO3)2
Cu2O, H2O
N
Me CHCl3-AcOH
88%
O
N
N
N
N
Me
N
Me
N
Me
N
Me
O
O
O
O
46%
O2N
H2N
HO
δa
εa
O
OMe
O
OMe
O
OMe
O
OMe
H
NOE
1 (RA-VII)
9
8
7
Scheme 2.
Table 2. Cytotoxicity of RA-VII (1), deoxybouvardin (2), RA-XVIII
(3), and their analogues 4–9 against P-388 leukemia cells
Then, 8 was subjected to catalytic hydrogenation to
yield amine 9, which was then converted into phenol 7.
Compound
IC50 (lg/mL)
The present new peptide 3, its analogues 4–9, and as ref-
erences, 1 and 2, were evaluated for their cytotoxicity
against P-388 leukemia cells. The results are summarized
in Table 2. The IC50 value of 3 was 0.012 lg/mL, indi-
cating that its cytotoxicity was one-fourth of that of 1,
though slightly higher than that of 2. Analogues 4–9
were all less cytotoxic than peptide 1, thus suggesting
that introduction of a hydroxyl, a nitro, or an amino
group at the da or the ea position of Tyr-6 causes a de-
crease in the cytotoxic activity. As regards the effect of a
substituent at different positions of Tyr-6, introduction
of a substituent at the da position seemed to decrease
the activity more than that at the ea position: the activ-
ities of 3, 5, and 6 tended to be higher than those of the
corresponding analogues 7, 8, and 9, respectively.
1
2
3
4
5
6
7
8
9
0.0030
0.014
0.012
0.014
0.010
0.0071
0.093
0.056
0.70
RA-XVIII (3) is a natural peptide of the RA-series in
which the ea position of Tyr-6 is hydroxylated. Since
cytotoxic peptides 3 and 6, having an additional hydro-
xyl or amino group on the Tyr-6 aromatic ring, are read-
ily prepared from 2, the most accessible congener in

J.-E. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 808–811
811
7. Hitotsuyanagi, Y.; Ishikawa, H.; Hasuda, T.; Takeya, K.
Tetrahedron Lett. 2004, 45, 935.
nature, these peptides may be expected to be useful
starting materials for producing new modified RA-series
peptides.
8. IR mmax (film) cmꢀ1: 3297, 2926, 1660, 1635, 1513, 1259,
1094, 1034.
9. 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.
References and notes
10. Morita, H.; Kondo, K.; Hitotsuyanagi, Y.; Takeya, K.;
Itokawa, H.; Tomioka, N.; Itai, A.; Iitaka, Y. Tetrahedron
1991, 47, 2757.
1. Itokawa, H.; Takeya, K.; Mihara, K.; Mori, N.; Hama-
naka, T.; Sonobe, T.; Iitaka, Y. Chem. Pharm. Bull. 1983,
31, 1424.
2. 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.
3. Itokawa, H.; Takeya, K.; Hitotsuyanagi, Y.; Morita, H.
In The Alkaloids; Cordell, G. A., Ed.; Academic Press:
NY, 1997; Vol. 49, p 301.
11. The location of the nitro group in the resulting nitration
reaction product is apparently controlled by the nature of
the substituent at the f position of Tyr-6. In 2, the
hydroxyl group at f having a strong electron-donating
effect causes the nitro group to be introduced to ea to
produce an ortho-nitrated compound, 4, whereas, in the
case of 1, the methoxyl group at f sterically inhibits the
introduction of a nitro group to the corresponding
position, so that the nitro group is introduced to the
less-hindered da position, which is also activated by the
diphenyl ether oxygen, and the nitration product is 8.
12. Boger, D. L.; Zhou, J.; Borzilleri, R. M.; Nukui, S.; Castle,
S. L. J. Org. Chem. 1997, 62, 2054.
´
´ ´
4. Zalacaın, M.; Zaera, E.; Vazquez, D.; Jimenez, 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.