Isolation, structure determination, and synthesis of allo-RA-V and neo-RA-V, RA-series bicyclic peptides from Rubia cordifolia L.

Article abstract of DOI:10.1002/chem.201103185

Two bicyclic hexapeptides, allo-RA-V (4) and neo-RA-V (5), and one cyclic hexapeptide, O-seco-RA-V (6), were isolated from the roots of Rubia cordifolia L. Their gross structures were elucidated on the basis of spectroscopic analysis and X-ray crystallography of compound 5. The absolute stereochemistry of compounds 4 and 5 were established by their total syntheses, and the absolute stereochemistry of compound 6 by chemical correlation with deoxybouvardin (3). Comparison of the 3D structures of highly active RA-VII (1) with less-active compounds 4 and 5 suggests that the orientation of the Tyr-5 and/or Tyr-6 phenyl rings plays a significant role in their biological activity. The isolation of peptides 4-6, along with compound 3, and the comparison of their structures seem to indicate that peptide 6 may be the common precursor to bicyclic peptides 3-5 in the plant. Allo allo: Two cycloisodityrosine-modified congeners of deoxybouvardin (RA-V) have been isolated from the roots of Rubia cordifolia. Comparison of their structures with that of RA-VII revealed that the orientation of one or both of the Tyr-5 and Tyr-6 phenyl rings plays an essential role in expressing the cytotoxic activity. Copyright

Full text of DOI:10.1002/chem.201103185

FULL PAPER
DOI: 10.1002/chem.201103185
Isolation, Structure Determination, and Synthesis of Allo-RA-V and
Neo-RA-V, RA-Series Bicyclic Peptides from Rubia cordifolia L.
Yukio Hitotsuyanagi, Masumi Odagiri, Saori Kato, Jun-ichi Kusano, Tomoyo Hasuda,
Haruhiko Fukaya, and Koichi Takeya*[a]
Abstract: Two bicyclic hexapeptides,
allo-RA-V (4) and neo-RA-V (5), and
one cyclic hexapeptide, O-seco-RA-V
(6), were isolated from the roots of
Rubia cordifolia L. Their gross struc-
tures were elucidated on the basis of
spectroscopic analysis and X-ray crys-
tallography of compound 5. The abso-
lute stereochemistry of compounds 4
and 5 were established by their total
syntheses, and the absolute stereo-
chemistry of compound 6 by chemical
correlation with deoxybouvardin (3).
Comparison of the 3D structures of
highly active RA-VII (1) with less-
active compounds 4 and 5 suggests that
the orientation of the Tyr-5 and/or
Tyr-6 phenyl rings plays a significant
role in their biological activity. The iso-
lation of peptides 4–6, along with com-
pound 3, and the comparison of their
structures seem to indicate that peptide
6 may be the common precursor to bi-
Keywords: configuration determi-
nation · cytotoxicity · peptides ·
structure elucidation · total synthe-
sis
AHCTUNGTRENGNcUN yclic peptides 3–5 in the plant.
Introduction
RA-VII (1), isolated from Rubia cordifolia L. and R. akane
Nakai (Rubiaceae),[1,2] bouvardin (NSC 259968; 2), isolated
from Bouvardia ternifolia (Cav.) Schltdl. (Rubiaceae),[3] and
deoxybouvardin (RA-V; 3),[1–3] isolated from those three
plants are bicyclic hexapeptides that are characterized by
the presence of a unique strained 14-membered cycloisodi-
tyrosine unit in their structures (Scheme 1). These peptides
possess promising antitumor activity, and from the late
1980s until the early 1990s, peptide 1 underwent phase I
clinical trials as an anticancer drug in Japan.[4] The antitu-
mor action of these peptides are believed to be due to inhi-
ACHTUNGTRENNUNGbition of protein synthesis through interaction with eukary-
otic ribosomes.[5,6] In addition, peptide 1 also causes confor-
mational changes in F-actin, which stabilizes the actin fila-
ments and induces G2 arrest.[7]
Herein, we report the isolation and structural elucidation
of two bicyclic hexapeptides, allo-RA-V (4) and neo-RA-V
(5), and one cyclic hexapeptide, O-seco-RA-V (6), along
with the known RA-series peptides from the roots of Rubia
cordifolia L. The absolute stereochemistry of compounds 4
and 5 were established by their total syntheses, and the ste-
reochemistry of compound 6 was established by chemical
[a] Dr. Y. Hitotsuyanagi, M. Odagiri, S. Kato, J.-i. Kusano, T. Hasuda,
H. Fukaya, Prof. Dr. K. Takeya
School of Pharmacy, Tokyo University of Pharmacy
and Life Sciences,
1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan)
Fax : (+81)42-677-1436
E-mail: takeyak@ps.toyaku.ac.jp
Supporting information for this article, including full compound char-
acterization, is available on the WWW under http://dx.doi.org/
10.1002/chem.201103185.
Scheme 1. Structures of RA-series peptides and bouvardins.
Chem. Eur. J. 2012, 18, 2839 – 2846
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2839

correlation with deoxybouvardin (3). These peptides were
also assayed for their cytotoxic activity against HL-60 and
HCT-116 cell lines.
1,2,4-trisubstituted phenyl ring (Table 1, Scheme 2). Analysis
of the HMBC spectrum revealed that peptides 3 and 4 have
the same peptide sequence at residues 1–4, with a difference
noted at residues 5 and 6. In peptide 3, Tyr-5 has a para-
AHCTUNGTREGsNNUN ubstituted phenyl ring and Tyr-6 has a trisubstituted phenyl
Results and Discussion
ring, whereas in peptide 4, Tyr-5 has a trisubstituted phenyl
ring and Tyr-6 a para-substituted phenyl ring. The chemical
shift (dC =150.8 ppm) of the Ceb atom in Tyr-5 indicates that
the oxygen atom is located on this carbon. The Hdb atom of
Tyr-5 in compound 4 showed a very upfield resonance in the
1H NMR spectrum (dH =3.96 ppm), as did the Hdb atom of
Isolation: A methanol extract obtained from dried roots of
R. cordifolia (50 kg) gave four fractions by HP-20 column
chromatography (H2O/MeOH, 1:0, then 1:1, then 0:1, then
acetone). Sequential column chromatography of the MeOH
eluate on silica gel, charcoal,
and aminopropyl-bonded silica
gel with a series of solvent mix-
tures gave a fraction rich in RA
compounds. The residue of this
fraction, obtained after removal
of the solvent, gave crystals of
crude RAs on crystallization
from MeOH/iPr2O. Repeated
reversed-phase HPLC (ODS)
purifications of the mother
liquor from the crystallization
gave allo-RA-V (4) (2.2 mg),
Table 1. NMR data of the major conformers of allo-RA-V (4) and neo-RA-V (5).[a]
4[b]
5[c]
Residue
dH [ppm]
dC [ppm]
dH [ppm]
dC [ppm]
d-Ala-1
a
4.36 (quintet, J=6.4 Hz)
1.44 (d, J=6.9 Hz, 3H)
--
7.05 (d, J=6.1 Hz)
4.65 (quintet, J=6.8 Hz)
1.40 (d, J=6.8 Hz, 3H)
--
48.0
20.0
173.1
–
45.7
16.0
172.3
–
4.95[d]
48.1
20.9
173.2
–
45.3
16.4
173.3
–
b
1.66 (d, J=6.9 Hz, 3H)
--
C=O
NH
8.13 (d, J=6.6 Hz)
Ala-2
Tyr-3
a
4.96[d]
b
1.47 (d, J=6.6 Hz, 3H)
--
10.16 (d, J=7.4 Hz)
4.07 (dd, J=10.7, 4.7 Hz)
3.86 (dd, J=13.8, 10.7 Hz)
3.80 (dd, J=13.8, 4.7 Hz)
C=O
NH
6.31 (d, J=6.8 Hz)
a
3.55 (dd, J=11.0, 4.8 Hz)
68.2
32.8
–
68.4
33.6
–
ba
bb
3.33[d]
neo-RA-V
(5)
(6.3 mg),
3.29 (dd, J=14.1, 11.0 Hz)
O-seco-RA-V (6) (42.7 mg),
and six other known RA-series
peptides, including deoxybou-
vardin.
g
--
130.7
130.3[e]
114.0[e]
158.4
168.6
39.9
55.3
46.4
18.4
170.7
–
--
131.8
130.8[e]
114.4[e]
158.8
169.0
39.9
55.1
46.5
18.9
171.4
–
d
7.04 (d-like, J=8.6 Hz, 2H)
6.84 (d-like, J=8.6 Hz, 2H)
--
--
2.87 (s, 3H)
3.79 (s, 3H)
4.80 (dq, J=8.7, 6.8 Hz)
0.97 (d, J=6.8 Hz, 3H)
7.27 (d-like, J=8.6 Hz, 2H)
7.01 (d-like, J=8.6 Hz, 2H)
--
--
3.13 (s, 3H)
3.70 (s, 3H)
5.22 (dq, J=8.5, 6.6 Hz)
1.27 (d, J=6.6 Hz, 3H)
e
z
C=O
NMe
OMe
a
Structures of allo-RA-V (4)
and neo-RA-V (5): Allo-RA-V
Ala-4
Tyr-5
[aŠ2D5 =À234,
c=0.13,
b
(4;
C=O
NH
--
--
MeOH), was obtained as an
amorphous solid. Its molecular
formula was determined to be
C40H48N6O9 from the [M+Na]+
peak found at 779.3346 (calcd
for C40H48N6O9Na: 779.3380) in
the high-resolution mass spec-
6.66 (d, J=8.7 Hz)
7.29 (d, J=8.5 Hz)
a
5.23 (dd, J=10.6, 1.5 Hz)
2.20 (d, J=16.0 Hz)
3.71 (dd, J=16.0, 10.6 Hz)
50.4
33.2
–
6.16 (d, J=9.9 Hz)
2.36 (d, J=13.7 Hz)
4.31 (dd, J=13.7, 9.9 Hz)
54.6
34.7
–
b (pro-R)
b (pro-S)
g
--
129.4
121.9
113.6
115.5
150.8
142.2
169.5
30.4
–
--
129.2
128.8
140.8
115.7
129.3
154.9
171.8
30.4
–
da
db
ea
eb
6.52 (dd, J=8.3, 1.8 Hz)
3.96 (d, J=1.8 Hz)
6.76 (d, J=8.3 Hz)
--
--
--
7.17 (dd, J=8.2, 2.5 Hz)
7.06 (d, J=2.5 Hz)
7.07 (d, J=8.2 Hz)
--
--
--
1
trum (ESI). The H NMR spec-
z
trum of compound 4 in CDCl3
indicated the presence of three
stable conformers in the ratio
86:7:7; the structure of the
most-populated conformer was
determined on the basis of
NMR data. The 1H and
13C NMR spectra of compound
4 were similar to those of com-
pound 3, and 1H-1H COSY,
HSQC, and HMBC analysis
showed that peptide 4 consists
of three alanine groups, one
C=O
NMe
OH
a
2.79 (s, 3H)
5.50 (br s)
2.94 (s, 3H)
11.26 (br s)[f]
Tyr-6
4.43 (dd, J=11.5, 2.2 Hz)
64.1
38.6
–
5.61 (dd, J=11.5, 4.1 Hz)
3.30 (dd, J=16.2, 11.5 Hz)
3.50 (dd, J=16.2, 4.1 Hz)
--
7.11 (dd, J=8.5, 1.9 Hz)
6.77 (d, J=1.9 Hz)
7.06 (d, J=8.5 Hz)
--
60.6
35.8
–
b (pro-R)
b (pro-S)
g
3.33[d]
3.09 (dd, J=14.1, 2.2 Hz)
--
7.17 (dd, J=8.3, 2.2 Hz)
7.54 (dd, J=8.3, 2.2 Hz)
7.07 (dd, J=8.3, 2.4 Hz)
7.11 (dd, J=8.3, 2.4 Hz)
--
--
135.6
130.6
130.8
124.9
126.7
158.8
169.6
32.2
–
125.5
129.2
141.7
116.0
128.6
154.4
170.8
31.1
–
da
db
ea
eb
z
--
--
C=O
NMe
OH
3.22 (s, 3H)
--
3.21 (s, 3H)
11.35 (br s)[f]
N,O-dimethyltyrosine
and two N-methyltyrosine
group,
[a] 1H NMR spectra recorded at 600 MHz and 13C NMR spectra at 150 MHz. [b] In CDCl3. [c] In
[D5]pyridine. [d] Multiplicity could not be determined owing to signal overlapping. [e] Two carbon atoms.
groups, one of which contains a [f] Assignments may be reversed.
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Chem. Eur. J. 2012, 18, 2839 – 2846

RA-series Bicyclic Peptides from Rubia Cordifolia L
FULL PAPER
[D5]pyridine displayed two sets of signals in an 81:19 ratio,
thereby implying that it consisted of two conformers. The
1H and 13C NMR spectra of compound 5 generally resem-
bled those of compound 3, thereby indicating that it consists
of three alanine groups and three modified N-methyltyro-
sine groups (Table 1), and the HMBC spectrum revealed
that its amino acid sequence is also the same as in com-
pound 3 (Scheme 2). However, the phenyl rings of Tyr-5 and
Tyr-6 in compound 5 were both 1,2,4-trisubstituted, whereas
in compound 3, only Tyr-6 was trisubstituted. The chemical
shift of the Ceb signals in Tyr-5 and Tyr-6 (dC =129.3 and
128.6 ppm, respectively) indicated that no oxygen function-
ality was connected to those carbon atoms. A cross-peak
was observed between the Tyr-5 Ceb and Tyr-6 Hdb atoms in
the HMBC spectrum (Scheme 2). Those NMR spectroscopic
features, and the fact that compounds 5 and 3 had the same
molecular formula, suggested that the Tyr-5 and Tyr-6
groups of compound 5 were linked to each other at their
e positions to form a biphenyl bond. The proximity of the
Tyr-5 Hdb and Tyr-6 Hdb atoms was indicated by the cross-
peak between those protons in the NOESY spectrum, which
confirmed the presence of a biphenyl bond at this position.
Accordingly, peptide 5 was determined to be a deoxybouvar-
din analogue in which the aromatic rings of Tyr-5 and Tyr-6
are connected at their e positions to form a 12-membered
cyclodityrosine unit. The relative stereochemistry of com-
pound 5 was determined by X-ray crystallography to be as
shown in Figure 1.[10] These data show that peptide 5 has the
same relative configuration as compounds 1–3 at all the
amino acid chiral centers.
Scheme 2. 1H-1H COSY and selected HMBC correlations for allo-RA-V
(4) and neo-RA-V (5).
Tyr-6 in compound 1 (dH =4.34 ppm)[8] and the Hdb atom in
compound 3 (dH =4.35 ppm).[9] This unusual upfield shift of
an aromatic proton in compounds 1 and 3 adjacent to the di-
phenyl ether bond is thought to be due to their location just
above the phenyl ring of the other tyrosine in the 3D struc-
ture of the cycloisodityrosine.[8] From these observations, we
concluded that peptide 4 has a cycloisodityrosine structure,
as shown in Scheme 1, in which the phenolic oxygen of
Tyr-6 is connected to the Ce atom of Tyr-5 to form a diphen-
yl ether bond. Inequivalent chemical shifts of the Hda/Cda
and Hdb/Cdb atoms, and of the Hea/Cea and Heb/Ceb atoms of
1
Tyr-6 in the H and 13C NMR spectra also indicated that this
phenyl ring was involved in the formation of the cyclic di-
phenyl ether structure. The relative stereochemistry of com-
pound 4 was deduced from the NMR data. The chemical
shift of the proton and carbon signals of residues 1–4 were
very similar to those of compounds 1 and 3. In the NOESY
spectrum, NOE correlations were observed between d-Ala-
1 Ha/Ala-2 NH, Ala-2 Ha/Tyr-3 NMe, Tyr-3 NMe/Tyr-3 Ha,
Tyr-3 Ha/Ala-4 NH, Ala-4 Ha/Tyr-5 NMe, Ala-4 H3b/Tyr-
5 NMe, Tyr-5 Ha/Tyr-6 Ha, Tyr-6 Ha/d-Ala-1 NH, and d-Ala-
1 Ha/Ala-4 H3b. These correlations are characteristic of the
major conformer of peptides 1 and 3 in solution, where the
molecule takes a type II b-turn at residues 1–4 and a
type VI b-turn at residues 1 and 4–6.[8] Thus, we concluded
that peptide 4 has the same relative configuration as pep-
tides 1–3 at the amino acid chiral centers and that it adopts
a very similar peptide backbone conformation to those of
compounds 1–3 in solution.
Figure 1. ORTEP of neo-RA-V (5).
Neo-RA-V (5), [aŠ2D5 =À290 (c=0.14, 1,4-dioxane), was
obtained as colorless prisms, m.p. >3008C. Its quasi-molecu-
lar ion peak at m/z 757.3596 ([M+H]+; calcd for
C40H49N6O9: 757.3561) in the HRMS (ESI) spectrum indicat-
ed that peptides 5 and 3 share a common molecular formula,
C40H48N6O9. The 1H NMR spectrum of compound 5 in
Synthesis of allo-RA-V (4) and neo-RA-V (5): The absolute
structures of allo-RA-V (4) and neo-RA-V (5) were estab-
lished by their total synthesis. In cyclopeptide synthesis,
ring-closing at the correct position on the linear peptide is
Chem. Eur. J. 2012, 18, 2839 – 2846
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2841

K. Takeya et al.
essential for successful macrocyclization. Following the pre-
vious syntheses of RA-series peptides,[11–14] we attempted
our ring formation by linking the Tyr-6 and d-Ala-1 groups
to form an 18-membered macrocycle, because the NMR
spectroscopic data and the crystal structure data of peptides
4 and 5 suggested that they have very similar conformation-
al features to those of the previously synthesized compounds
1–3.[8,9] Accordingly, the cycloisodityrosine unit (for com-
pound 4) and the cyclodityrosine unit (for compound 5)
were prepared first, to which the tetrapeptide segments, cor-
responding to residues 1–4, were connected to form hexa-
peptides that were then subjected to macrocyclization to
construct the 18-membered cyclopeptide rings.
In the synthesis of allo-RA-V (4; Scheme 3), the forma-
tion of the 14-membered cycloisodityrosine unit was the
major task. Several methods are known for its preparation,
including: intramolecular phenolic oxidative coupling reac-
tions with thalliumACHTUNGTRENNUNG
(III) nitrate,[11,15] intramolecular Ullmann
reactions,[12,16] and intramolecular SNAr reactions.[13,17,18] We
chose a copper-mediated etherification reaction with an
arylboronic acid and a phenol.[19,20] Though low yields had
been reported for the synthesis of cycloisodityrosines,[18d] we
found that the use of 4-dimethylaminopyridine as a base in-
stead of pyridine or triethylamine significantly improved the
yield of the cyclic ether.[21] This method is attractive and is
of value for the synthesis of cycloisodityrosines as it uses
commercially available chiral amino acids and involves short
reaction sequences.
Iodotyrosine derivative 8, obtained by O-benzylation of
N-Boc-3-iodo-l-tyrosine methyl ester (7), was converted
into boronic acid 9, which, after subsequent hydrolysis, gave
carboxylic acid 10. Acid 10 was linked to l-tyrosine methyl
ester to give dipeptide 11, which was treated with copper(II)
acetate and 4-dimethylaminopyridine in the presence of 4 ꢁ
molecular sieves to afford the desired cycloisodityrosine
(12) in 47% yield. N-Methylation of compound 12 under
phase-transfer catalysis conditions[22] gave the fully function-
alized cycloisodityrosine (13), which was usable for the syn-
thesis of compound 4. The Boc group in compound 13 was
removed, and the resulting amine was coupled with tetra-
peptide 14 provided hexapeptide 15. Ester hydrolysis of
compound 15 with in situ generated lithium peroxide gave
acid 16. Deprotection of the N-terminus of compound 16
and subsequent cyclization with EDC·HCl (8 equiv) and
HOOBt (8 equiv) in DMF under dilute conditions
(0.0013m) gave bicyclic peptide 17 in 50% yield. Removal
of the benzyl group of peptide 17 by hydrogenolysis gave
the product ([aŠ2D5 =À221, c=0.097, MeOH), which was
shown to be identical to natural product 4 by comparison of
Scheme 3. Synthesis of allo-RA-V (4). Reagents and conditions:
a) BnOH, diethyl azodicarboxylate, PPh3, THF/toluene 8:3, 08C, 1 h,
then RT, 3 h, 97%; b) 1) bis(pinacolato)diboron, [PdCl2ACHTUNRGTNEUNG(dppf)]·CH2Cl2,
KOAc, DMSO, 808C, 20 h, 2) NaIO4, NH4OAc, acetone/H2O 1:1, RT,
17 h, 86%; c) LiOH, THF/MeOH/H2O 3:3:1, 08C, 1 h, then RT, 1 h,
88%; d) l-Tyr-OMe·HCl, EDC·HCl, HOAt, Et3N, CHCl3, 08C, 1 h, then
RT, 24 h, 95 %; e) CuACHTNUTRGNEG(UN OAc)2, DMAP, 4 ꢁ molecular sieves, CH2Cl2, RT,
2 days, 47%; f) MeI, nBu4NHSO4, KOH, K2CO3, CH2Cl2, RT, 1 h, 72%;
g) 1) TFA, 08C, 1 h, 2) compound 14, EDC·HCl, HOOBt, THF, RT,
2 days, 71%; h) LiOOH, MeOH/H2O 6:1, 08C, 1 h, then RT, 22 h, 92%;
i) 1) TFA, CHCl3, 08C, 2 h, 2) EDC·HCl, HOOBt, DMF, 08C, 1 h, then
RT, 4 days, 50%; j) H2, Pd(OH)2/C, EtOH, RT, 3 h, 88%. For a list of ab-
breviations see Ref. [30].
the functionalized amino acid components used for the syn-
thesis of compound 4 were exploited.
Iodotyrosine derivative 8 was N-methylated under phase-
transfer catalysis conditions to give the N-methyltyrosine de-
rivative 18, which, after removal of the Boc group, was cou-
pled with boronic acid derivative 10 to give dipeptide 19.
1
their H and 13C NMR spectra, IR spectra, mass spectra, and
optical rotations. Thus, the absolute structure of allo-RA-V
(4) was determined to be as shown in Scheme 1.
For the synthesis of neo-RA-V (5; Scheme 4), the prepa-
ration of the cyclodityrosine unit was the major problem be-
cause synthesis of this unit had not been reported before.
We chose the intramolecular Suzuki–Miyaura cross-coupling
reaction[23] of a modified tyrosyl–tyrosine dipeptide, where
The [PdACTHNUGTRENNU(G PPh3)4]-catalyzed intramolecular Suzuki–Miyaura
coupling reaction of compound 19 efficiently gave cyclodi-
tyrosines 20 and 20’ in 50% and 14% yields, respectively.[24]
1
Although the H, 13C, and 2D NMR data could not distin-
guish the structural differences between compounds 20 and
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Chem. Eur. J. 2012, 18, 2839 – 2846

RA-series Bicyclic Peptides from Rubia Cordifolia L
FULL PAPER
mainder of the synthesis. N-Methylation of compound 20
gave compound 21. After Boc deprotection, compound 21
was coupled with tetrapeptide 14 to afford hexapeptide 22,
which was then treated with lithium peroxide to give
carboxylic acid 23. After removal of the Boc group, com-
pound 23 was subjected to macrocyclization at a concentra-
tion of 0.0013m in DMF with EDC·HCl and HOOBt to
afford bicyclic peptide 24 in 60% yield. However, debenzy-
lation of compound 24 under standard catalytic hydrogenol-
ysis conditions (H2, Pd(OH)2 or Pd/C, EtOH) did not afford
compound 5 at all: it gave just a complex mixture of prod-
ucts. When compound 24 was treated with palladium on
charcoal and 1,4-cyclohexadiene in EtOH,[26] it gave the de-
sired deprotected compound in 80% yield, though the reac-
25
D
tion was very sluggish. The thus obtained compound ([aŠ
=
À274, c=0.09, 1,4-dioxane), was shown to be identical to
natural product 5, by comparison of their H and 13C NMR
1
spectra, IR spectra, mass spectra, and optical rotations. Ac-
cordingly, the absolute structure of neo-RA-V (5) was deter-
mined to be as shown in Scheme 1.
25
D
Structure of O-seco-RA-V (6): O-seco-RA-V (6), [aŠ
=
À82 (c=0.42, MeOH), was obtained as an amorphous solid
and afforded a quasi-molecular ion peak at m/z 759.3685
[M+H]+ in the high-resolution mass spectrum (ESI), which
corresponded to a molecular formula of C40H50N6O9. The
1H NMR spectra of compound 6 in CD3OD showed that it
was a mixture of four conformers in the ratio 54:42:3:1. 2D
NMR spectroscopy revealed that peptide 6 was composed
of three alanine groups and three N-methyltyrosine groups,
of which one was O-methylated, and that the sequence of
those alanine and N-methyltyrosine groups was the same as
that in deoxybouvardin (RA-V, 3), with the N,O-dimethyl-
tyrosine as residue 3.[27] Thus, peptide 6 was shown to be a
deoxybouvardin analogue with no diphenyl ether bond be-
tween Tyr-5 and Tyr-6. Because peptide 6 was considered to
have the same configuration as compounds 3–5 at all of the
amino acid residues, we tried to establish its absolute stereo-
chemistry by chemical correlation with deoxybouvardin (3),
whose absolute stereochemistry had been established by
degradation studies[1] and by the total synthesis.[11,12a,c] As
shown in Scheme 5, hydrogenolysis of compound 3 gave
O-seco-derivative 25, which was converted into ditriflate 26.
The palladium-mediated reduction[28] of compound 26 af-
forded N-methylphenylalanine derivative 27. Analogously,
peptide 6 was converted into ditriflate 28, which gave com-
pound 27 by subsequent reduction. Because the spectroscop-
ic data (1H and 13C NMR, IR, and MS) and optical rotation
([aŠ2D5 =À85, c=0.14, MeOH) of compound 27 that was de-
rived from compound 6 were the same as those ([aŠ2D5 =À86,
c=0.12, MeOH) of compound 27 that was derived from de-
oxybouvardin (3), the structure of compound 6 was deter-
Scheme 4. Synthesis of neo-RA-V (5). Reagents and conditions: a) MeI,
nBu4NBr, NaOH, CH2Cl2, RT, 1 h, 88%; b) 1) TFA, CHCl3, 08C, 1 h,
2) compound 10, EDC·HCl, HOAt, Et3N, DMF, 08C, 1 h, then RT,
4 days, 84%; c) [PdACHTUNGTRENNUNG(PPh3)4], Na2CO3, toluene/MeOH/H2O 8:3:1, 908C,
2 h, 50% yield of compound 20 and 14% yield of compound 20’; d) tolu-
ene, 808C, 20 h, 74% yield of compound 20 and 20% yield of compound
20’; e) MeI, nBu4NHSO4, NaOH, CH2Cl2, RT, 2 h, 84%; f) 1) TFA,
CHCl3, 08C, 1 h, 2) compound 14, EDC·HCl, HOOBt, THF, RT, 3 days,
44%, g) LiOOH, MeOH/H2O 3:1, 08C, 1 h, then RT, 22 h, quant.;
h) 1) TFA, CHCl3, 08C, 2 h, 2) EDC·HCl, HOOBt, DMF, 08C, 1 h, then
RT, 4 days, 60%; i) Pd/C, 1,4-cyclohexadiene, EtOH, RT, 7 days, 80%.
For a list of abbreviations see Ref. [30].
20’, owing to line broadening of the signals and their com-
plexity caused by cis/trans isomerization of the tertiary
amide bond, we tentatively assigned compounds 20 and 20’
to be atropisomers. Although a biphenyl axis usually re-
quires three or four ortho substituents for its conformers to
become atropisomers,[25] their 12-membered ring structure
and the two bulky benzyloxy groups might provide sufficient
À
steric hindrance to the rotation about the C C biphenyl
bond. After heating the minor isomer (20’) in toluene at
808C for 20 h, it yielded a 74% yield of compound 20 with
20% of starting isomer 20’, thereby indicating that the
major isomer (20) is thermodynamically more stable than
the minor isomer (20’). Major isomer 20 was used in the re-
mined to be cyclo-(d-Ala-Ala-N-Me-TyrACTHNGUETRNNU(G OCH3)-Ala-N-Me-
Tyr-N-Me-Tyr), that is, O-seco-RA-V (Scheme 1). Although
peptide 6 has been previously reported as a synthesized
monocyclic analogue of deoxybouvardin,[29] this is the first
report on the isolation of compound 6 from nature.[30]
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2843

K. Takeya et al.
and their IC50 values are summarized in Table 2. The results
indicated that only peptides 1 and 3 had potent cytotoxic ac-
tivities. Superposition of the crystal structures of compounds
Table 2. Cytotoxic activity.[a]
Compound
HL-60
0.0049
0.0060
5.0
>100
80
HCT-116
0.0063
0.0083
5.0
>100
>100
1
3
4
5
6
[a] IC50 [mgmLÀ1].
1 and 5, and the energy-minimized structure of compound 4
(obtained by a Monte Carlo conformational search) is
shown in Figure 2. This picture indicates that both the cyclo-
Scheme 5. Chemical correlation of O-seco-RA-V (6) with deoxybouvar-
din (3). Reagents and conditions: a) H2, Pd/C, EtOH, 508C, 24 h, 78%;
b) Tf2NPh, Et3N, CH2Cl2, RT, 44 h, 91%; c) Pd
HCO2H, DMF, 608C, 42 h, 75%; d)Tf2NPh, Et3N, CH2Cl2, RT, 44 h,
98%; e) Pd(OAc)2, dppf, Et3N, HCO2H, DMF, 608C, 24 h, 75%. For a
ACHTUNGTRENN(NUG OAc)2, dppf, Et3N,
ACHTUNGTRENNUNG
list of abbreviations see Ref. [30].
Deoxybouvardin (RA-V, 3), commonly detected in Rubia
akane, R. cordifolia, and Bouvardia ternifolia, is composed
of one d-alanine group, two l-alanine groups, and three
modified N-methyl-l-tyrosine groups. One of those
N-methyl-l-tyrosine groups is O-methylated, and the other
two are connected to form a cycloisodityrosine. Cycloisodi-
tyrosines are tyrosyl–tyrosine dipeptide derivatives in which
one of the two phenoxy groups forms an ether linkage with
the aromatic carbon at the e position of the other tyrosine.
In peptides 1–3, an ether linkage is formed between the
hydroxy oxygen atom at the z position of Tyr-5 and the
carbon atom at the e position of Tyr-6, whereas in allo-RA-
V (4), the ether linkage is formed between the carbon atom
at the e position of Tyr-5 and the hydroxy oxygen atom at
the z position of Tyr-6. In neo-RA-V (5), those two tyrosines
Figure 2. Superposition of the crystal structures of RA-VII (1, blue) and
neo-RA-V (5, green), and the energy-minimized structure of allo-RA-V
(4, red).
isodityrosine rings in compounds 1 and 4, and the cyclodi-
AHCTUNGTREGtNNNU yrosine ring in compound 5 have an apparently equal effect
À
are linked by a C C bond between the two e positions to
on the 18-membered cyclohexapeptide backbone, so that
the 18-membered backbones of the three are almost super-
imposable. RA-series peptides adopt between two and three
stable conformations in solution.[8] The-most populated con-
former, represented by the crystal structure of compound 1
(Figure 2), has been identified as the active conforma-
tion,[12d,e,31] with the 4-methoxyphenyl ring of Tyr-3 also im-
portant for expressing the activity.[32] The phenyl rings of
Tyr-5 and Tyr-6 in compounds 4 and 5 were not superimpos-
able on those in the active compound 1, thus suggesting
that, in addition to the 4-methoxybenzyl side-chain of Tyr-3,
a certain orientation of both or either of the phenyl rings of
Tyr-5 and Tyr-6 is also required for expressing the activity.
form an unusual 12-membered cyclodityrosine structure.
The key question in the biosynthesis of RA-series pep-
tides is whether the formation of the diphenyl ether linkage
in the cycloisodityrosine moiety occurs prior to, or after, the
formation of the cyclohexapeptide chain. Our isolation of
O-seco-RA-V (6) along with peptides 3–5 from Rubia cordi-
folia may suggest that the cyclohexapeptide ring is formed
first to produce compound 6, which, by subsequent phenolic
oxidative coupling reactions, generates bicyclic peptides 3–5
in the plant body.
Cytotoxic activities: Peptides 1 and 3–6 were evaluated for
their cytotoxic activity against human promyelocytic leuke-
mia HL-60 cells and human colon carcinoma HCT-116 cells,
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Chem. Eur. J. 2012, 18, 2839 – 2846

RA-series Bicyclic Peptides from Rubia Cordifolia L
FULL PAPER
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Conclusion
Two bicyclic hexapeptides, allo-RA-V (4) and neo-RA-V
(5), and
a
previously known cyclic hexapeptide,
O-seco-RA-V (6), were isolated along with known RA-
series peptides from the roots of Rubia cordifolia. The total
synthesis of compounds 4 and 5 were performed by con-
structing the 14-membered cycloisodityrosine unit of com-
pound 4 through an intramolecular phenol/arylboronic-acid
coupling reaction mediated by copper(II) acetate, and the
12-membered cyclodityrosine unit of compound 5 through
an intramolecular Suzuki–Miyaura cross-coupling reaction;
these reactions unambiguously determined the absolute
structures of compounds 4 and 5. The absolute structure of
compound 6 was established by chemical correlation with
compound 3. The fact that the 3D structures of the 18-mem-
bered cyclopeptide backbones of highly cytotoxic compound
1 and less-active compounds 4 and 5 are nearly superimpos-
able and yet they show obviously different activities may
suggest that, in addition to the proper alignment of the 4-
methoxyphenyl ring of Tyr-3, the orientation of one or both
of the Tyr-5 and Tyr-6 phenyl rings plays an essential role in
expressing the activity. This information should be useful for
designing structurally more simplified RA analogues. In this
study, peptides 3–6 were isolated from the same plant
source, R. cordifolia. This fact and the comparison of their
structures may indicate that compound 6 is biosynthesized
first, with subsequent oxidative coupling reactions producing
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2845

K. Takeya et al.
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Received: October 9, 2011
Published online: February 1, 2012
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Chem. Eur. J. 2012, 18, 2839 – 2846