Isolation, structural elucidation, and synthesis of RA-XVII, a novel bicyclic hexapeptide from Rubia cordifolia, and the effect of side chain at residue 1 upon the conformation and cytotoxic activity

Article abstract of DOI:10.1016/j.tetlet.2003.11.112

A novel antitumor bicyclic hexapeptide RA-XVII was isolated from the roots of Rubia cordifolia. By spectral studies and synthetic approach, its structure was determined to be [D-2-aminobutyric acid-1]deoxybouvardin. Studies on the effect of side chain at

Full text of DOI:10.1016/j.tetlet.2003.11.112

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 935–938
Isolation, structural elucidation, and synthesis of RA-XVII,
a novel bicyclic hexapeptide from Rubia cordifolia, and the
effect of side chain at residue 1 upon the conformation
and cytotoxic activity
Yukio Hitotsuyanagi, Hiroshi Ishikawa, Tomoyo Hasuda and Koichi Takeya*
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received 16 October 2003; revised 21 November 2003; accepted 21 November 2003
Abstract—A novel antitumor bicyclic hexapeptide RA-XVII was isolated from the roots of Rubia cordifolia. By spectral studies and
synthetic approach, its structure was determined to be [D-2-aminobutyric acid-1]deoxybouvardin. Studies on the effect of side chain
at residue 1 on cytotoxic activity and conformation showed that although it had little effect on the conformation of the molecule, it
decreased the activity as it grew longer.
ꢀ 2003 Elsevier Ltd. All rights reserved.
RA-VII (1) is an antitumor bicyclic hexapeptide from
Rubiaceous plants. Seventeen congeners, RA-I–XVI1;2
and RA-dimer-A,3 have been isolated from Rubia akane
Nakai or R. cordifolia L., and two congeners, bouvardin
(NSC 259968) (2) and deoxybouvardin (RA-V) (3), from
another Rubiaceous plant, Bouvardia ternifolia (Cav.)
Schlecht.4 These peptides possess a promising antitumor
activity, and RA-VII (1) underwent phase I clinical trials
as an anticancer drug in Japan.5 In the present study, we
isolated a novel bicyclic hexapeptide RA-XVII (4) as a
minor peptide component from the roots of R. cordifolia
L., which had D-2-aminobutyric acid as residue 1. In the
present letter, we describe the isolation and structural
elucidation of this new peptide, and the studies of the
effect of the side chain at residue 1 upon the confor-
mation and cytotoxic activity of RA series peptides.
Tyr-3
Ala-2
O
OMe
Me
N
O
1
2
3
HN
Me
R
R
H
R
O
HN
O
D-Ala-1
(D-Aa-1)
Ala-4
1
Me
Me
Me RA-VII (1)
R
Me
Me
OH
H
H
(2)
deoxybouvardin(3)
RA-XVII (4)
NH
O
bouvardin
O
N
H
H
Me
Et
2
R
N
H
H
Me
H
Et
Me [D-2-aminobutyric acid-1]RA-VII (5)
Me
Tyr-6
Tyr-5
H
n-Pr
[D-norvaline-1]RA-VII (6)
O
3
OR
* Corresponding author. Tel.: +81-426-76-3007; fax: +81-426-77-1436; e-mail: takeyak@ps.toyaku.ac.jp
0040-4039/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.11.112

936
Y. Hitotsuyanagi et al. / Tetrahedron Letters 45 (2004) 935–938
Methanol extract obtained from the dried roots of R.
cordifolia (50 kg) was partitioned between chloroform
and water. The chloroform-soluble portion was sub-
jected to a series of column chromatographic studies
using silica gel, alumina, and then aminopropyl-bonded
silica gel, eluting with a chloroform/methanol mixture to
give an RAs-rich fraction. The residue obtained after
removal of the solvent of this fraction was crystallized
from methanol to afford crystals of crude RAs (8.7 g).
Separation of the crude RAs by reversed-phase HPLC
(ODS) afforded RA-XVII (4, 0.24 mg, 4.8 · 10ꢀ7% yield)
and Ala-4 (d 1.13, d, J ¼ 6.7 Hz) in 4 were almost
identical to those in 3.7 The major differences between
the spectra of 3 and 4 were that the spectrum of 4 had no
doublet methyl resonance for D-Ala-1 observed in 3 and
that, instead, it showed resonances of triplet methyl
protons (d 0.85, t, J ¼ 7.4 Hz) and methylene protons (d
1.77, m and 1.58, m), all coupled to each other, were
observed. Those methylene protons were also coupled to
the methine proton (d 4.33, m) assigned to the a-proton
of residue 1. A transannular NOESY correlation
between this a-proton and the methyl protons of Ala-4
residue indicated that the a-proton and the methyl
group are located in proximity, thus suggesting that the
as an amorphous powder, ½aꢁ24 )194ꢁ (c 0.01, CHCl3).
D
The molecular formula was determined to be
C41H50N6O9 by HR-ESIMS (m=z 771.3737 [M+H]þ, for
C41H51N6O9 D + 1.9 mmu). The quantity of the thus
obtained peptide was very small, but it was sufficient for
configuration of the amino acid at residue 1 was
D as in
other peptides of this series. Accordingly, on the basis of
those observations and the molecular formula derived
from HR-ESIMS, we assigned the structure of 4 to a
1
1
the 1D H NMR, H–1H COSY, and NOESY spectral
analysis.
deoxybouvardin analogue in which
replaced by -2-aminobutyric acid.
D-Ala-1 of 3 was
D
As in the case of other related peptides of this series,
peptide 4 was present in CDCl3 as a mixture of one
major (89% population) conformer and one minor
conformer having a trans and cis amide bond between
Confirmation of the assigned structure was performed
by a synthetic approach shown in Scheme 1. The tetra-
peptide fragment 12 consisting of residues 1–4 of 4 was
1
Ala-2 and Tyr-3 residues, respectively.6 The H NMR
synthesized by stepwise extension of Cbz-N-methyl-
L-
-ala-
spectrum of 4 indicated a close resemblance between the
structures of 4 and deoxybouvardin (3): characteristic
resonances of O-methyl at Tyr-3 (d 3.79, s), N-methyls
at Tyr-3 (d 2.85, s), Tyr-5 (d 3.11, s), and Tyr-6 (d 2.66,
s), and alanyl methyls at Ala-2 (d 1.36, d, J ¼ 6.9 Hz)
tyrosine (7).8 Compound 7 was first coupled to
L
nine methyl ester, and subsequent O-methylation of 8
with diazomethane afforded dipeptide 9. After removal
of the Cbz protecting group, the less reactive N-methyl-
dipeptide ester was coupled to Boc-
L
-alanine by using
OH
OR
OMe
d or e
c
a
O
H
N
H
N
H
N
Cbz
Cbz
CO2Me
CO2Me
Me
N
CO2H
N
Me
Boc
N
Me
O
Me
8: R = H
9: R = Me
Me Me
O
7
10
b
OMe
R1
O
H
N
H
N
Cbz
CO2R2
Me
N
H
N
O
O
O
O
OMe
OMe
Me
HN
Me
HN
O
Me Me
O
N
Me
N
Me
O
Me
N Me
O
11: R1 = Et, R2 = Me
12: R1 = Et, R2 = H
15: R1 = n-Pr, R2 = Me
16: R1 = n-Pr, R2 = H
f
f
HN
HN
R1
O
Me
R
h or i
NHCbzO
Me
NH
O
N Me
BnO2C
N
N
O
O
Me
Boc
Me
N
N Me
BnO2C
O
O
OR2
O
g
OMe
14: R = Et
5: R1 = Et, R2 = Me
4: R1 = Et, R2 = H
6: R1 = n-Pr, R2 = Me
j
O
OMe
17: R = n-Pr
13
Scheme 1. Reagents and conditions: (a) H-Ala-OMeꢂHCl, EDC, HOBt, Et3N, CH2Cl2, 71%; (b) CH2N2, Et2O–MeOH, 100%; (c) H2, Pd/C, HCl,
MeOH; Boc-Ala-OH, PyBOP, i-Pr2NEt, CH2Cl2, 88%; (d) TFA; Cbz- -2-aminobutyric acid, EDC, HOBt, CHCl3, 89% for 11; (e) TFA; Cbz-
D
D-
norvaline, EDC, HOBt, CHCl3, 99% for 15; (f) LiOH, THF–MeOH–H2O, 92% for 12 and 99% for 16; (g) TFA; 12 or 16, EDC, HOObt, THF, 90%
for both 14 and 17; (h) H2, Pd/C, EtOH; DPPA, Et3N, 0.001 M, rt, 72 h, 44% for 5; (i) H2, Pd/C, EtOH; DPPA, Et3N, 0.001 M, 0 ꢁC, 72 h, 49% for 6;
(j) AlCl3, CH2Cl2, 49%.

Y. Hitotsuyanagi et al. / Tetrahedron Letters 45 (2004) 935–938
trans
937
cis
Me
O
O
O
OMe
OMe
Me
HN
Me
HN
N
N
H
H
H
H
O
O
Me
HN
O
HN
H
R
Me
Me
R
O
Me
Me
H
H
NH
O
NH
O
trans
trans
O
N
N
H
H
H
H
N
N
O
O
Me
Me
cis
cis
O
O
OMe
major conformer (89%)
OMe
minor conformer (11%)
Figure 1. Key sequential NOESY correlations commonly observed in 1 (R ¼ Me), 5 (R ¼ Et), and 6 (R ¼ n-Pr) in CDCl3.
PyBOP (1H-benzotriazol-1-yloxytripyrrolidinophos-
phonium hexafluorophosphate) to afford tripeptide 10.
89:11 at 300 K. Close similarity of the chemical shift
1
values of the corresponding proton signals in their H
Further elongation of the peptide chain at the N-ter-
minus with Cbz-D-2-aminobutyric acid gave tetrapep-
tide 11, which, on treatment with lithium hydroxide,
provided acid 12.
NMR spectra and observation of the same sequential
NOESY correlations in their NOESY spectra suggested
that the respective structures of the two conformers of
peptides 1, 5, and 6 were practically identical (Fig. 1).
The cytotoxic activities (IC50) of RA-VII (1) and ana-
logues 5 and 6 on P-388 cells were 0.0023, 0.0076, and
0.026 lg/mL, respectively. Thus, the results indicated
that although the side chain at residue 1 had little effect
on their conformation in solution, the longer carbon
Acid 12 was then subjected to the coupling reaction with
N-deprotected 139 by using 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (EDC) and 3-
hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOObt)
to afford hexapeptide 14. Macrocyclization of the pep-
tide obtained by deprotection of 14 was performed by
using diphenylphosphoryl azide (DPPA, 2 equiv) and
triethylamine (10 equiv) in DMF (room temperature,
3 days, peptide concentration 0.001 M) to afford peptide
side chain at
D
-Ala-1 of peptide 1 decreased the activity.
5,10 i.e. [
D-2-aminobutyric acid-1]RA-VII. The yield of 5
References and notes
from 14 was 44%. Selective O-demethylation at Tyr-6
residue afforded a compound,11 which was shown to be
identical to 4 isolated from R. cordifolia, by the com-
1. (a) Itokawa, H.; Takeya, K.; Mori, N.; Hamanaka, T.;
Sonobe, T.; Mihara, K. Chem. Pharm. Bull. 1983, 31,
1424–1427; (b) Itokawa, H.; Takeya, K.; Mihara, K.;
Mori, N.; Hamanaka, T.; Sonobe, T.; Iitaka, Y. Chem.
Pharm. Bull. 1984, 32, 284–290.
1
parison of their H NMR and HR-ESI mass spectra, tR
of HPLC analysis, and optical rotations.
2. Itokawa, H.; Takeya, K.; Hitotsuyanagi, Y.; Morita, H. In
The Alkaloids; Cordell, G. A., Ed.; Academic: New York,
1997; Vol. 49, pp 301–387.
3. Hitotsuyanagi, Y.; Aihara, T.; Takeya, K. Tetrahedron
Lett. 2000, 41, 6127–6130.
The amino acid at residue 1 of all the RA-series peptides
isolated so far is D-alanine. RA-XVII (4) is the first
example of RA-series peptides whose residue 1 amino
acid is not
peptide 4 is
D
D
-alanine. The residue at 1 of the present
-2-aminobutyric acid, which is not known
4. 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.
5. (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, 67–78; (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,
199–207.
in cyclic peptides of plant origin, though occasionally
found in the cyclic peptides of microbial metabolites.12
Peptide 4 had an ethyl side chain at residue 1 and yet it
was biologically active and gave an IC50 value of
0.028 lg/mL when tested on P-388 murine leukemia
cells. Therefore, by a similar sequence of reactions
employed for the synthesis of 5 (Scheme 1), we synthe-
sized another peptide of this series, [D-norvaline-1]RA-
VII (6),13 in which the residue at 1 had an n-propyl
group, and studied the influence of the side chain of
residue 1 upon their conformation and cytotoxic activ-
ities. The NMR spectral features of 5 and 6 were very
similar to those of 1, showing that in CDCl3, these three
peptides were present in two conformers at the ratio of
6. Morita, H.; Kondo, K.; Hitotsuyanagi, Y.; Takeya, K.;
Itokawa, H.; Tomioka, N.; Itai, A.; Iitaka, Y. Tetrahedron
1991, 47, 2757–2772.

938
Y. Hitotsuyanagi et al. / Tetrahedron Letters 45 (2004) 935–938
7. Data for 4: 1H NMR (500 MHz, CDCl3, 300 K, major
52, 2957–2958; (b) Inoue, T.; Inaba, T.; Umezawa, I.;
Yuasa, M.; Itokawa, H.; Ogura, K.; Komatsu, K.; Hara,
H.; Hoshino, O. Chem. Pharm. Bull. 1995, 43, 1325–
1335.
conformer, J in Hz) d 7.42 (dd, 1H, J ¼ 8.4, 2.3, Tyr-5 H-
db), 7.28 (dd, 1H, J ¼ 8.5, 2.3, Tyr-5 H-da), 7.21 (dd, 1H,
J ¼ 8.4, 2.4, Tyr-5 H-eb), 7.05 (d-like, 2H, J ¼ 8.6, Tyr-3
H2-d), 6.84 (m, 1H, Tyr-5 H-ea), 6.84 (d-like, 2H, J ¼ 8.6,
Tyr-3 H2-e), 6.82 (d, 1H, J ¼ 8.2, Tyr-6 H-ea), 6.70 (d, 1H,
J ¼ 7.5, Ala-4 NH), 6.53 (dd, 1H, J ¼ 8.2, 2.0, Tyr-6 H-da),
9. Hitotsuyanagi, Y.; Hasuda, T.; Matsumoto, Y.; Yama-
guchi, K.; Itokawa, H.; Takeya, K. Chem. Commun. 2000,
1633–1634.
6.28 (d, 1H, J ¼ 7.2,
D
-Abu-1 NH), 6.22 (d, 1H, J ¼ 8.5,
10. Data for 5: white crystalline powder, mp 232–235 ꢁC,
Ala-2 NH), 5.66 (br s, 1H, Tyr-6 OH), 5.39 (dd, 1H,
J ¼ 11.4, 3.0, Tyr-5 H-a), 4.89 (dq, 1H, J ¼ 8.5, 6.9, Ala-2
H-a), 4.74 (dq, 1H, J ¼ 7.5, 6.7, Ala-4 H-a), 4.60 (1H, dd,
11.5, 4.6, Tyr-6 H-a), 4.37 (d, 1H, J ¼ 2.0, Tyr-6 H-db),
½aꢁ24 )169ꢁ (c 0.24, CHCl3); HR-ESIMS m=z 807.3750
D
[M+Na]þ, calcd for C42H52N6O9Na 807.3693.
11. Data for synthetic 4: ½aꢁ26 )237ꢁ (c 0.20, CHCl3); 13C
D
NMR (125 MHz, CDCl3, 300 K, major conformer, d)
D-
4.33 (m, 1H,
D
-Abu-1 H-a), 3.79 (s, 3H, Tyr-3 OCH3),
Abu-1 (171.3, 53.0, 27.8, 9.3), Ala-2 (172.5, 44.5, 16.8),
Tyr-3 (168.0, 158.4, 130.7, 130.2 · 2, 114.1 · 2, 68.4, 55.3,
39.7, 32.7), Ala-4 (171.7, 46.4, 18.5), Tyr-5 (169.3, 157.9,
135.7, 133.1, 131.0, 125.9, 124.2, 54.2, 36.9, 30.5), Tyr-6
(170.8, 151.1, 143.0, 127.6, 121.7, 115.7, 113.1, 57.7, 35.8,
29.3).
3.68 (dd, 1H, J ¼ 11.4, 11.4, Tyr-5 H-bb), 3.57 (dd, 1H,
J ¼ 10.6, 5.1, Tyr-3 H-a), 3.39 (dd, 1H, J ¼ 14.0, 5.1, Tyr-3
H-ba), 3.35 (dd, 1H, J ¼ 14.0, 10.6, Tyr-3 H-bb), 3.11 (s,
3H, Tyr-5 NCH3), 3.05 (dd, 1H, J ¼ 17.9, 11.5, Tyr-6 H-
ba), 3.00 (dd, 1H, J ¼ 17.9, 4.6, Tyr-6 H-bb), 2.85 (s, 3H,
Tyr-3 NCH3), 2.66 (s, 3H, Tyr-6 NCH3), 2.63 (dd, 1H,
12. (a) Vanderhaeghe, H.; Parmentier, G. Bull. Soc. Chim.
Belg. 1959, 68, 716–718; (b) Watanabe, K. J. Antibiot.
1961, 14, 14–17; (c) Sasse, F.; Steinmetz, H.; Schupp, T.;
Petersen, F.; Memmert, K.; Hofmann, H.; Heusser, C.;
Brinkmann, V.; Von Matt, P.; Ho¨fle, G.; Reichenbach, H.
J. Antibiot. 2002, 55, 543–551.
J ¼ 11.4, 3.0, Tyr-5 H-ba), 1.77 (m, 1H,
1.58 (m, 1H,
-Abu-1 H-bb), 1.36 (d, 3H, J ¼ 6.9, Ala-2
H3-b), 1.13 (d, 3H, J ¼ 6.7, Ala-4 H3-b), 0.85 (t, 3H,
-Abu-1 H3-c); HPLC tR 32.7 min (Inertsil ODS-
D
-Abu-1 H-ba),
D
J ¼ 7.4,
D
3, 4.6 · 250 mm, 5 lm, 37% H2O/MeOH, 0.6 mL/min, UV
254 nm).
8. (a) Inaba, T.; Umezawa, I.; Yuasa, M.; Inoue, T.;
Mihashi, S.; Itokawa, H.; Ogura, K. J. Org. Chem. 1987,
13. Data for 6: amorphous powder, ½aꢁ24 )237ꢁ (c 0.38,
D
CHCl3); HR-ESIMS m=z 821.3930 [M+Na]þ, calcd for
C43H54N6O9Na 821.3850.