Structures of cytotoxic bicyclic hexapeptides, RA-XIX, -XX, -XXI, and -XXII, from Rubia cordifolia L.

Article abstract of DOI:10.1016/j.tet.2008.01.094

Novel bicyclic hexapeptides, RA-XIX, -XX, -XXI, and -XXII, were isolated from the roots of Rubia cordifolia L. The structures of RA-XIX and RA-XX were established by semisynthesis from a cycloisodityrosine, derived from previously reported RA-VII, and those of RA-XXI and RA-XXII by chemical correlation with RA-XX and previously reported RA-VIII, respectively. The IC₅₀ values of these new peptides against P-388 leukemia cells were 0.013-0.63 μg/mL.

Full text of DOI:10.1016/j.tet.2008.01.094

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 4117e4125
www.elsevier.com/locate/tet
Structures of cytotoxic bicyclic hexapeptides, RA-XIX, -XX, -XXI,
and -XXII, from Rubia cordifolia L.
*
Ji-Ean Lee, Yukio Hitotsuyanagi, Koichi Takeya
School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received 24 November 2007; received in revised form 18 January 2008; accepted 22 January 2008
Available online 31 January 2008
Abstract
Novel bicyclic hexapeptides, RA-XIX, -XX, -XXI, and -XXII, were isolated from the roots of Rubia cordifolia L. The structures of RA-XIX
and RA-XX were established by semisynthesis from a cycloisodityrosine, derived from previously reported RA-VII, and those of RA-XXI and
RA-XXII by chemical correlation with RA-XX and previously reported RA-VIII, respectively. The IC50 values of these new peptides against
P-388 leukemia cells were 0.013e0.63 mg/mL.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: RA-XIX; RA-XX; RA-XXI; RA-XXII; Rubia cordifolia; Bouvardin; Cytotoxicity
1. Introduction
RA-XIX (3), -XX (4), -XXI (5), and -XXII (6), were isolated.
This paper describes their isolation, structure determination,
and cytotoxicity against P-388 cells.
RA-VII (1)1,2 and bouvardin (NSC 259968, 2)3 are tumor
cell growth inhibitors isolated from the plants, Rubia cordifo-
lia L. (Rubiaceae) and Bouvardia ternifolia (Cav.) Schltdl.
(Rubiaceae), respectively. Their structures are characterized
by the presence of a 14-membered strained ring, an unusual
cycloisodityrosine unit, formed by an ether linkage between
the two aromatic rings of Tyr-5 and Tyr-6. Their antitumor ac-
tion is considered to be due to inhibition of protein synthesis
through interaction with eukaryotic ribosomes.4,5 Recently,
RA-VII (1) was shown to cause conformational changes in
F-actin and stabilization of actin filaments to induce G2 ar-
rest.6 In our further studies on the cyclopeptide constituents
in the roots of R. cordifolia, four new RA-series peptides,
2. Results and discussion
A methanol extract obtained from dried roots of R. cordifo-
lia (50 kg) was partitioned between chloroform and water. The
chloroform-soluble portion was subjected to a series of col-
umn chromatography using silica gel, alumina, and then ami-
nopropyl-bonded silica gel eluting with a series of chloroform/
methanol mixtures to give a fraction rich in RAs. The residue
of this fraction, obtained after removal of the solvent, was
crystallized from methanol to give crystals of crude RAs,
which, after reversed-phase HPLC (ODS), afforded RA-XIX
(3, 2.6 mg, 5.2ꢀ10ꢁ6%) and RA-XX (4, 4.6 mg, 9.2ꢀ
10ꢁ6%). The mother liquor from the crystallization gave
RA-XXI (5, 5.5 mg, 1.1ꢀ10ꢁ5%) and RA-XXII (6, 34.5 mg,
6.9ꢀ10ꢁ5%).
Abbreviations: Boc, tert-butyloxycarbonyl; Cbz, benzyloxycarbonyl;
DIEA, diisopropylethylamine; DMF, N,N-dimethylformamide; DPPA,
diphenylphosphoryl azide; EDC$HCl, N-ethyl-N0-(3-dimethylaminopropyl)-
carbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole; HOObt, 3,4-di-
hydro-3-hydroxy-4-oxo-1,2,3-benzotriazine; PyBOP, (benzotriazol-1-yl-
oxy)tripyrrolidinophosphonium hexafluorophosphate; TFA, trifluoroacetic acid;
THF, tetrahydrofuran.
RA-XIX (3) was obtained as an amorphous solid. Its
molecular formula was determined to be C44H56N6O9 from
the [MþH]þ peak at m/z 813.4187 (calcd for C44H57N6O9,
1
813.4187) in the HRESIMS. The H and 13C NMR spectra
* Corresponding author. Tel.: þ81 42 676 3007; fax: þ81 42 677 1436.
E-mail address: takeyak@ps.toyaku.ac.jp (K. Takeya).
of 3 in CDCl3 (Tables 1 and 2) showed signals typical of an
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.01.094

4118
J.-E. Lee et al. / Tetrahedron 64 (2008) 4117e4125
Table 1
1H NMR data for the major conformers of RA-XIX (3), -XX (4), -XXI (5), and -XXII (6) in CDCl3 at 300 Ka
Position
D-Ala-1
RA-XIX (3)
RA-XX (4)
RA-XXI (5)
RA-XXII (6)
dH
dH
dH
dH
a
4.34 (qd, 6.9, 6.8)
1.30 (d, 6.9, 3H)
6.39 (d, 6.8)
4.35 (qd, 7.0, 6.8)
1.31 (d, 7.0, 3H)
6.40 (d, 6.8)
4.37 (qd, 7.0, 6.9)
1.31 (d, 7.0, 3H)
6.40 (d, 6.9)
4.47 (septet, 7.0)
1.35 (d, 7.0, 3H)
6.38 (d, 7.0)
b
NH
a
AA-2
4.85 (m)
1.52e1.66 (m, 2H)
1.61 (m)
4.71 (m)
1.67e1.84 (m, 2H)
0.96 (t, 7.4, 3H)
4.70 (m)
1.67e1.84 (m, 2H)
0.96 (t, 7.4, 3H)
4.44 (dd, 7.9, 1.2)
4.09 (qd, 6.3, 1.2)
1.21 (d, 6.3, 3H)
b
g
da
db
NH
a
0.94 (d, 6.1, 3H)
0.93 (d, 6.1, 3H)
6.04 (d, 8.8)
6.15 (d, 8.9)
6.16 (d, 8.7)
6.60 (d, 7.9)
Tyr-3
3.60 (dd, 11.1, 4.2)
3.44 (dd, 14.2, 4.2)
3.32 (dd, 14.2, 11.1)
7.07 (d-like, 8.6, 2H)
6.83 (d-like, 8.6, 2H)
2.92 (s, 3H)
3.62 (dd, 11.1, 4.2)
3.46 (dd, 14.2, 4.2)
3.31 (dd, 14.2, 11.1)
7.08 (d-like, 8.6, 2H)
6.83 (d-like, 8.6, 2H)
2.92 (s, 3H)
3.62 (dd, 11.1, 4.2)
3.46 (dd, 14.2, 4.2)
3.31 (dd, 14.2, 11.1)
7.08 (d-like, 8.6, 2H)
6.83 (d-like, 8.6, 2H)
2.93 (s, 3H)
3.64 (dd, 10.7, 5.0)
3.39 (dd, 14.1, 5.0)
3.35 (dd, 14.1, 10.7)
7.07 (d-like, 8.6, 2H)
6.86 (d-like, 8.6, 2H)
2.98 (s, 3H)
ba
bb
d
3
NMe
OMe
a
3.80 (s, 3H)
3.79 (s, 3H)
3.79 (s, 3H)
3.80 (s, 3H)
Ala-4
Tyr-5
4.72 (dq, 7.5, 6.7)
1.14 (d, 6.7, 3H)
6.68 (d, 7.5)
4.73 (dq, 7.4, 6.7)
1.14 (d, 6.7, 3H)
6.69 (d, 7.4)
4.74 (dq, 7.5, 6.7)
1.13 (d, 6.7, 3H)
6.68 (d, 7.5)
4.75 (dq, 7.6, 6.7)
1.10 (d, 6.7, 3H)
6.71 (d, 7.6)
b
NH
a
5.40 (dd, 11.3, 3.0)
2.63 (dd, 11.3, 3.0)
3.67 (t, 11.3)
5.40 (dd, 11.4, 3.1)
2.63 (dd, 11.4, 3.1)
3.67 (t, 11.4)
5.39 (dd, 11.4, 3.0)
2.63 (dd, 11.4, 3.0)
3.68 (t, 11.4)
5.40 (dd, 11.3, 3.0)
2.63 (dd, 11.3, 3.0)
3.70 (t, 11.3)
ba
bb
da
db
3a
7.27 (dd, 8.4, 2.2)
7.42 (dd, 8.4, 2.2)
6.88 (dd, 8.4, 2.4)
7.21 (dd, 8.4, 2.4)
3.13 (s, 3H)
7.27 (dd, 8.4, 2.2)
7.42 (dd, 8.4, 2.2)
6.88 (dd, 8.4, 2.4)
7.21 (dd, 8.4, 2.4)
3.13 (s, 3H)
7.28 (dd, 8.5, 2.2)
7.43 (dd, 8.4, 2.2)
6.84 (dd, 8.5, 2.4)
7.21 (dd, 8.4, 2.4)
3.12 (s, 3H)
7.28 (dd, 8.4, 2.2)
7.41 (dd, 8.4, 2.2)
6.84 (dd, 8.4, 2.4)
7.20 (dd, 8.4, 2.4)
3.11 (s, 3H)
3b
NMe
a
Tyr-6
4.55 (dd, 12.0, 3.8)
3.10 (dd, 18.0, 12.0)
2.96 (dd, 18.0, 3.8)
6.58 (dd, 8.3, 2.0)
4.35 (d, 2.0)
4.54 (dd, 12.1, 3.9)
3.09 (dd, 18.1, 12.1)
2.96 (dd, 18.1, 3.9)
6.58 (dd, 8.3, 2.0)
4.34 (d, 2.0)
4.54 (dd, 12.0, 3.9)
3.05 (dd, 17.8, 12.0)
2.94 (dd, 17.8, 3.9)
6.52 (dd, 8.2, 2.0)
4.35 (d, 2.0)
4.57 (dd, 12.0, 3.9)
3.06 (dd, 18.1, 12.0)
2.94 (dd, 18.1, 3.9)
6.53 (dd, 8.2, 1.9)
4.35 (d, 1.9)
ba
bb
da
db
3a
6.80 (d, 8.3)
2.68 (s, 3H)
3.94 (s, 3H)
6.80 (d, 8.3)
2.68 (s, 3H)
3.94 (s, 3H)
6.82 (d, 8.2)
2.67 (s, 3H)
6.82 (d, 8.2)
2.69 (s, 3H)
NMe
OMe
OH
5.66 (s)
5.66 (s)
a
Recorded at 500 MHz, chemical shifts referenced to residual CHCl3 (7.26 ppm); J-values given in Hz are in parentheses.
RA-series peptide, and also demonstrated the presence of two
conformers in a ratio of 87:13.7,8 The structure of 3 was deter-
mined using the resonances caused by the major conformer,
which included signals for four secondary methyl groups
(dH/dC 0.93/22.9, 0.94/22.5, 1.14/18.6, 1.30/20.9), three N-
methyl groups (dH/dC 2.68/29.2, 2.92/39.8, 3.13/30.5), two
O-methyl groups (dH/dC 3.80/55.3, 3.94/56.2), four methyl-
enes (dH/dC 1.52e1.66/40.3; 2.63, 3.67/37.1; 2.96, 3.10/35.4;
3.32, 3.44/32.9), one methine (dH/dC 1.61/24.8), six N-
substituted methines (dH/dC 3.60/68.6, 4.34/48.0, 4.55/57.5,
4.72/46.4, 4.85/47.4, 5.40/54.2), two 1,4-disubstituted benzene
rings (dH/dC 6.83/114.0ꢀ2, 7.07/130.3ꢀ2, dC 130.8, 158.4; dH/
dC 6.88/124.3, 7.21/125.9, 7.27/132.8, 7.42/131.1, dC 135.1,
158.3), one 1,2,4-trisubstituted benzene ring (dH/dC 4.35/
113.4, 6.58/120.9, 6.80/112.3, dC 128.2, 146.5, 153.1), six am-
ide carbonyl groups (dC 167.8, 169.5, 170.6, 171.8, 172.1,
172.2), and three amide protons (dH 6.04, 6.39, 6.68). The
analysis of its 1He1H COSY and HMBC spectra revealed
the features of the component amino acid units and the amino
acid sequence of 3 (Fig. 2). In 3 the C-a (dC 47.4) of amino
acid at position-2 (AA-2) was connected to the methylene car-
bon (C-b, dC 40.3), which was further connected to the me-
thine (C-g, dC 24.8) of an isopropyl unit, thus indicating that
1
peptide 3 had a leucine unit as AA-2. The profiles of the H
and 13C NMR chemical shifts due to the peptide backbone
of 1 and 3 were very similar, indicating that they shared the
same relative configuration. These observations implied that
peptide 3 was an analogue of 1 whose Ala-2 was replaced
by leucine, which was confirmed by the semisynthesis of 3
as shown in Scheme 1. After N-deprotection by catalytic hy-
drogenolysis, dipeptide 79 was coupled with Boc-Leu-OH us-
ing PyBOP to afford tripeptide 8a. Peptide 8a was further
treated with Cbz-D-Ala-OH to produce tetrapeptide 9a, which
on treatment with lithium hydroperoxide gave tetrapeptide
acid 10a. After deprotection of the N-terminus of cycloisodi-
tyrosine 11, prepared by degradation of natural 1,9,10 11 was
coupled with acid 10a to afford hexapeptide 12a. Deprotection
and subsequent macrocyclization of 12a using DPPA gave
a product that was shown to be identical to natural 3 by com-
parison of their spectroscopic data and optical rotations. Thus,

J.-E. Lee et al. / Tetrahedron 64 (2008) 4117e4125
4119
Table 2
13C NMR data for the major conformers of RA-XIX (3), -XX (4), -XXI (5),
and -XXII (6) in CDCl3 at 300 Ka
2-aminobutyric acid, which was verified by semisynthesis of
4 through a similar protocol employed in the synthesis of 3
(Scheme 1). Thus, tetrapeptide acid 10b with a 2-aminobutyric
acid was prepared from dipeptide 79 and then converted into
hexapeptide 12b, which, after deprotection, was subjected to
macrocyclization to give a product that was shown to be iden-
tical to natural 4 by comparison of their spectroscopic data and
optical rotations. Thus, the absolute structure of RA-XX was
determined to be as shown in structure 4 (Fig. 1).
Position
RA-XIX (3) RA-XX (4) RA-XXI (5) RA-XXII (6)
dC dC dC dC
48.0 48.1 48.0 47.8
20.9
D-Ala-1
a
b
20.9
172.3
50.3
20.9
172.4
50.4
21.0
172.9
51.2
C]O 172.2
AA-2
Tyr-3
a
47.4
40.3
24.8
22.5
22.9
b
24.7
10.1
24.6
10.2
66.1
19.3
RA-XXI (5) was obtained as a white crystalline powder. Its
molecular formula was determined to be C41H50N6O9 from the
[MþNa]þ peak at m/z 793.3495 (calcd for C41H50N6O9Na,
g
da
db
1
793.3537) in the HRESIMS. The H and 13C NMR spectra
C]O 172.1
172.0
68.7
33.0
172.1
68.7
33.0
172.3
68.5
32.8
a
b
g
d
3
68.6
32.9
1
of 5 were very similar to those of 4, except that in the H
NMR spectrum of 5 one methoxyl signal was missing and in-
stead had a phenolic hydroxyl signal at dH 5.66. This phenolic
hydroxyl signal showed cross-peaks with C-3a, C-3b, and C-z
in the HMBC spectrum, thus indicating that in 5 a hydroxyl
group substituted for the methoxyl group at the z-position of
Tyr-6 in 4. A downfield shift of the 13C NMR signal for C-
3a in Tyr-6 from dC 112.3 for 4 to dC 115.7 for 5 also explains
the replacement of the methoxyl group by a hydroxyl group.
Treatment of 5 with (trimethylsilyl)diazomethane afforded
a product that was shown to be identical to natural 4 from their
spectroscopic data and optical rotations. Thus, the absolute
structure of RA-XXI was determined to be as shown in struc-
ture 5 (Fig. 1).
130.8
130.3b
114.0b
158.4
130.9
130.2b
114.1b
158.4
167.8
39.8
130.8
130.2b
114.1b
158.4
167.9
39.8
130.4
130.1b
114.2b
158.5
167.6
40.3
z
C]O 167.8
NMe
OMe
a
39.8
55.3
46.4
18.6
55.3
46.4
55.3
46.4
55.3
46.2
Ala-4
Tyr-5
b
18.6
18.6
18.6
C]O 171.8
171.8
54.2
37.0
171.8
54.2
37.0
171.6
54.4
36.9
a
54.2
37.1
b
g
135.1
132.8
131.1
124.3
125.9
158.3
135.1
132.8
131.0
124.3
125.9
158.3
169.4
30.5
135.6
133.0
131.1
124.2
125.9
157.9
169.2
30.5
135.7
133.0
131.0
124.2
125.9
157.9
169.1
30.5
da
db
3a
3b
z
RA-XXII (6) was obtained as an amorphous solid. Its mo-
lecular formula was determined to be C41H50N6O10 from the
[MþNa]þ peak at m/z 809.3461 (calcd for C41H50N6O10Na,
809.3486) in the HRESIMS. Its 1H NMR spectrum bore a close
resemblance to that of RA-VIII (13),11 but the signal for the
O-methyl group at Tyr-6 was missing. The presence of a phe-
C]O 169.5
NMe
a
30.5
57.5
Tyr-6
57.4
35.4
57.5
35.5
57.5
35.6
b
35.4
g
128.2
120.9
113.4
112.3
153.1
146.5
128.1
120.9
113.4
112.3
153.1
146.5
170.6
29.2
127.6
121.6
113.0
115.7
151.1
143.0
170.5
29.3
127.6
121.7
113.0
115.7
151.0
143.0
170.6
29.4
nolic hydroxyl signal at dH 5.66 and close similarity of the 13
C
da
db
3a
3b
z
NMR chemical shifts for the aromatic carbons of Tyr-6 in 6
with those in 5 indicated that the methoxyl group at Tyr-6 in
13 was replaced by a hydroxyl group in 6. Treatment of 6
with (trimethylsilyl)diazomethane afforded a product that
was shown to be identical to natural 13 from their spectro-
scopic data and optical rotations. Since the absolute structure
of 13 has already been established,11 that of RA-XXII was
determined to be as shown in structure 6 (Fig. 1).
C]O 170.6
NMe
OMe
29.2
56.2
56.2
a
Recorded at 125 MHz, chemical shifts referenced to CDCl3 (77.03 ppm).
Two carbons.
b
Peptides 3e6 were evaluated for their cytotoxicity against
P-388 murine leukemia cells, with peptide 1 as reference.
The results are summarized in Table 3. For the peptides having
a methoxyl group in Tyr-6, the order of cytotoxicity was
1>4>3. Peptide 6, having a hydroxyl group in its Thr-2,
was less cytotoxic than 5. This is consistent with our earlier
observations that the cytotoxicity decreases with increase in
the length of the carbon side chain or introduction of a polar
functionality at this location.12
The representative RA-series peptides, RA-VII (1) and
bouvardin (2), are composed of one D-alanine (D-Ala-1), two
L-alanines (Ala-2, Ala-4), one N,O-dimethyl-L-tyrosine (Tyr-3),
and one modified N,N0-dimethyl-L,L-cycloisodityrosine (Tyr-5,
Tyr-6). In some RA-series peptides, Ala-2 of 1 was replaced
by L-serine (RA-III),13 L-threonine (RA-VIII, 13),11
the absolute structure of RA-XIX was determined to be as
shown in structure 3 (Fig. 1).
RA-XX (4) was obtained as an amorphous solid. Its molec-
ular formula was determined to be C42H52N6O9 from the
[MþH]þ peak at m/z 785.3879 (calcd for C42H53N6O9,
785.3874) in the HRESIMS. Its 1H and 13C NMR spectra
were very similar to those of RA-XIX (3) except for the reso-
1
nances due to those of AA-2. In the H NMR spectrum of 4,
a characteristic triplet methyl signal (dH 0.96, t, J¼7.4 Hz)
1
was observed, and analysis of its He1H COSY and HMBC
spectra revealed the presence of a CH3eCH2eCH(NHe
C]O)eC]O unit (Fig. 3). Accordingly, peptide 4 was con-
sidered to be an analogue of 3 whose Leu-2 was replaced by

4120
J.-E. Lee et al. / Tetrahedron 64 (2008) 4117e4125
OMe
OMe
OMe
1. H2, Pd/C
HCl, MeOH
1. TFA
LiOH, H2O2
Me
O
O
H
N
H
N
H
N
H
H
N
Cbz
CO2Me
N
CO2Me
Cbz
CO2Me
2. Cbz-D-Ala-OH
EDC HCl
HOBt, CH2Cl2
2. Boc-Leu-OH
or Boc-Abu-OH
PyBOP, DIEA
THF-MeOH-H2O
N
H
N
Me
Boc
N
Me
N
.
O
O
Me
O
Me
Me
O
Me
R
R
CH2Cl2
9a (88%)
9b (85%)
7
8a (72%)
8b (72%) b, R = Me
a, R = i-Pr
OMe
R
R
O
O
O
O
OMe
OMe
Me
O
H
N
H
N
Me
N
Me
Cbz
N
CO2H
Me
O
O
N
H
N
Me
HN
HN
Me
O
O
HN
HN
1. H2, Pd/C
EtOH
R
Me
CbzHN
BnO2C
Me
Me
.
2. EDC HCl
10a (100%)
10b (99%)
O
O
NH
O
2. DPPA
Et3N
HOObt
THF
N Me
O
N Me
Boc
N Me
N
Me
N
Me
DMF
O
BnO2C
O
N
1. TFA
Me
O
O
OMe
OMe
O
OMe
3 (51%, R = i-Pr)
4 (49%, R = Me)
12a (66%)
12b (77%)
11
Scheme 1. Synthesis of RA-XIX (3) and RA-XX (4).
Leu-2
Me Me
Abu-2
Me
Thr-2
Me
Tyr-3
Ala-2
O
O
O
O
O
O
O
OMe
OMe
OMe
OMe
Me
HN
HO
N
Me
N
Me
N
Me
N
Me
O
O
O
O
HN
HN
HN
D-Ala-1
HN
HN
O
HN
HN
Me
O
Me
Me
Me
Me
Me
Me
Me
Ala-4
NH
O
NH
O
NH
O
NH
O
N Me
O
N Me
R1
H
O
N Me
O
N Me
α
α
H
b
N
N
Me
H
b
N
N
Me
β
β
O
O
O
H
O
Me
H
γ
δa
εa
Me
a
γ
a
δb
δa
εa
Tyr-6
Tyr-5
δb
εb
εb
O
O
O
O
ζ
ζ
OR2
OMe
OR
OR
RA-VII (1): R1 = H, R2 = Me
Bouvardin (2): R1 = OH, R2 = H
RA-XX (4): R = Me
RA-XXI (5): R = H
RA-XIX (3)
RA-XXII (6): R = H
RA-VIII (13): R = Me
Figure 1.
L-pyroglutamic acid (RA-IX), or L-glutamic acid (RA-X).14
RA-XIX (3) is the first example of RA-series peptides incor-
porating L-leucine, whereas RA-XX (4) and RA-XXI (5) are
also the first examples of this series of peptides incorporating
L-2-aminobutyric acid, and RA-XXII (6) is the second exam-
ple having L-threonine.
δa
δb
Tyr-3
γ
β
O
O
OMe
Leu-2
α
N
O
HN
HN
Ala-4
D-Ala-1
NH
O
O
N
N
O
Abu-2
O
N
Tyr-5
Tyr-6
HN
O
O
OMe
1H-1H COSY
HMBC
1H-1H COSY
HMBC
C
H
C
H
Figure 2. Key 1He1H COSY and HMBC correlations for determination of the
peptide sequence of 3.
Figure 3. Partial structure and selected 1He1H COSY and HMBC correlations
for 4.

J.-E. Lee et al. / Tetrahedron 64 (2008) 4117e4125
4121
Table 3
Cytotoxicity of RA-VII (1), RA-XIX (3), RA-XX (4), RA-XXI (5), and RA-
XXII (6) against P-388 leukemia cells
of crude RAs (8.5 g) and mother liquor (ML). The crystals
were then subjected to ODS HPLC using MeOH/H2O (60:40,
then 100:0) to give four fractions, C1 (4.42 g, mostly deoxybou-
vardin), C2 (0.32 g), C3 (3.44 g, mostly RA-VII), and C4
(0.23 g, MeOH eluate). Fraction C4 was separated by repeated
ODS HPLC using MeCN/H2O (37:63 and 45:55) to afford
RA-XIX (3, 2.6 mg, 5.2ꢀ10ꢁ6%) and RA-XX (4, 4.6 mg,
9.2ꢀ10ꢁ6%).
Compound
IC50 (mg/mL)
1
3
4
5
6
0.0023
0.024
0.013
0.041
0.63
After removal of the solvent, ML (10.2 g) was subjected to
ODS HPLC using MeOH/H2O (60:40, then 100:0) to give five
fractions, M1 (0.42 g), M2 (2.44 g, mostly deoxybouvardin),
M3 (0.33 g), M4 (0.11 g, mostly RA-VII), and M5 (1.12 g).
Fraction M1 was separated by repeated ODS HPLC using
MeCN/H2O (33:67) and then MeOH/H2O (55:45) to afford
RA-XXII (6, 34.5 mg, 6.9ꢀ10ꢁ5%). Separation of fraction
M3 by ODS HPLC using MeCN/H2O (30:70) yielded RA-
XXI (5, 5.5 mg, 1.1ꢀ10ꢁ5%).
3. Experimental
3.1. General
Melting points were determined on a Yanaco MP-3 appara-
tus and are recorded uncorrected. Optical rotations were mea-
sured on a JASCO P-1030 digital polarimeter, IR spectra on
a JASCO FT/IR 620 spectrophotometer, and UV spectra on
a JASCO V-530 spectrophotometer. NMR spectra were mea-
sured on a Bruker DRX-500 spectrometer at 300 K. The H
chemical shifts in CDCl3 or CD3OD were referenced to the re-
sidual CHCl3 (7.26 ppm) or CD2HOD (3.31 ppm), and the 13
chemical shifts were referenced to the solvent (CDCl3,
77.03 ppm; CD3OD, 49.0 ppm). Mass spectra were obtained
with a Micromass LCT spectrometer. Preparative HPLC was
carried out on a Shimadzu LC-6AD pump unit equipped
with a SPD-10A UV detector (l 254 nm) and a pre-packed
ODS column (5 mm, 20ꢀ250 mm), using a MeOH/H2O or
a MeCN/H2O solvent system at a flow rate of 10 mL/min.
3.4. Characteristics of each peptide
1
3.4.1. RA-XIX (3)
Amorphous solid; [a]D26 ꢁ224.4 (c 0.13, CHCl3); IR (film)
nmax 3390, 2954, 2932, 1655, 1635, 1513, 1413, 1265, 1128,
1032, 802 cmꢁ1; UV (MeOH) lmax (log 3) 205 (4.81), 225sh
(4.54), 277 (3.74) nm; 1H and 13C NMR, a mixture of two con-
formers in a ratio of 87:13 in CDCl3 at 300 K. For the data of
the major conformer, refer to Tables 1 and 2; HRESIMS m/z
813.4187 ([MþH]þ, calcd for C44H57N6O9, 813.4187).
C
3.4.2. RA-XX (4)
Amorphous solid; [a]D26 ꢁ218.4 (c 0.22, CHCl3); IR (film)
nmax 3388, 2934, 1654, 1635, 1513, 1414, 1265, 1128, 1031,
752 cmꢁ1; UV (MeOH) lmax (log 3) 205 (4.77), 225sh
(4.48), 277 (3.61) nm; 1H and 13C NMR, a mixture of two con-
formers in a ratio of 89:11 in CDCl3 at 300 K. For the data of
the major conformer, refer to Tables 1 and 2; HRESIMS m/z
785.3879 ([MþH]þ, calcd for C42H53N6O9, 785.3874).
3.2. Plant material
The roots of R. cordifolia L. were obtained from a market in
Tokyo in March 2004. The material was identified by Prof.
Koichi Takeya, and a voucher specimen (Tko-0403e01) has
been deposited at the Herbarium of Tokyo University of
Pharmacy and Life Sciences.
3.4.3. RA-XXI (5)
3.3. Extraction and isolation
White crystalline powder, mp>300 ꢂC; [a]D26 ꢁ230.1 (c
0.28, CHCl3); IR (film) nmax 3386, 2930, 1655, 1633, 1513,
1413, 1247, 1094, 1035, 754 cmꢁ1; UV (MeOH) lmax (log 3)
The dried roots (50 kg) of R. cordifolia were extracted with
MeOH (3ꢀ175 L). After removal of MeOH under reduced pres-
sure, the residue (3.6 kg) was partitioned between chloroform
and water. The chloroform-soluble portion (993 g) was placed
on a column of silica gel (Merck, 70e230 mesh, 3.6 kg) and
eluted with CHCl3 (9 L), EtOAc (18 L), and CHCl3/MeOH
(9:1, 27 L), sequentially to give three fractions. After removal
of the solvent, the residue of the CHCl3/MeOH (9:1) fraction
(152 g) was subjected to alumina (Merck, 3 kg) column chroma-
tography (CC) eluting sequentially with CHCl3 (2 L) and
CHCl3/MeOH (9:1, 12 L). After evaporation, the CHCl3/
MeOH (9:1) fraction (41.9 g) was subjected to aminopropyl-
bonded silica gel (Chromatorex, 200e350 mesh, 300 g) CC
eluting sequentially with CHCl3 (6 L) and CHCl3/MeOH (9:1,
1 L). The residue obtained after removal of the solvent of the
CHCl3 eluate was crystallized from methanol to give crystals
1
205 (4.77), 226sh (4.49), 278 (3.61) nm; H and 13C NMR,
a mixture of two conformers in a ratio of 87:13 in CDCl3 at
300 K. For the data of the major conformer, refer to Tables
1 and 2; HRESIMS m/z 793.3495 ([MþNa]þ, calcd for
C41H50N6O9Na, 793.3537).
3.4.4. RA-XXII (6)
Amorphous solid; [a]D26 ꢁ186.7 (c 0.34, CHCl3); IR (film)
nmax 3387, 2934, 1658, 1629, 1513, 1412, 1247, 1094, 1034,
752 cmꢁ1; UV (MeOH) lmax (log 3) 205 (4.76), 225sh
1
(4.47), 277 (3.61) nm; H and 13C NMR, a mixture of three
conformers in a ratio of 73:25:2 in CDCl3 at 300 K. For the
data of the most populated conformer, refer to Tables 1 and
2; HRESIMS m/z 809.3461 ([MþNa]þ, calcd for
C41H50N6O10Na, 809.3486).

4122
J.-E. Lee et al. / Tetrahedron 64 (2008) 4117e4125
3.5. Semisynthesis of RA-XIX (3)
amorphous solid. [a]D28 ꢁ82.0 (c 0.26, CHCl3); IR (film)
;
753 cmꢁ1 1H NMR (500 MHz, CDCl3, major conformer)
nmax 3288, 2955, 1724, 1627, 1514, 1455, 1248, 1035,
3.5.1. Boc-Leu-N,O-dimethyl-Tyr-Ala-OMe (8a)
Palladium (10%) on charcoal catalyst (5 mg) and hydro-
chloric acid (0.1 mL) were added to a solution of Cbz-N,O-di-
methyl-Tyr-Ala-OMe (7)9 (31.8 mg, 0.0742 mmol) in MeOH
(2 mL), and the mixture was stirred at room temperature under
an atmosphere of hydrogen for 2 h. The catalyst was filtered
off and the filtrate was concentrated to dryness. The residue
was dissolved in CH2Cl2 (2 mL) together with Boc-Leu-OH
(34.3 mg, 0.148 mmol) and PyBOP (77.2 mg, 0.148 mmol),
to which DIEA (52.9 mL, 0.304 mmol) was slowly added at
ꢁ20 ꢂC under an atmosphere of argon. The mixture was
stirred at this temperature for 1 h, and then at room tempera-
ture for 5 days. Aqueous citric acid (10%, 2 mL) was added
to the mixture, and the whole was extracted with CHCl3
(3ꢀ7 mL). The combined CHCl3 extracts were washed se-
quentially with saturated aqueous NaHCO3 (2 mL) and brine
(2 mL), dried over Na2SO4 and filtered, and the solvent
removed in vacuo. The residue was subjected to HPLC
(MeCN/H2O 70:30) to afford 8a (27.2 mg, 72%) as an amor-
phous solid. [a]D27 ꢁ102.7 (c 0.34, CHCl3); IR (film) nmax
3299, 2956, 1747, 1681, 1636, 1513, 1456, 1366, 1259,
d 8.15 (br d, 1H, J¼6.0 Hz), 7.36e7.28 (m, 5H), 7.10 (br d,
1H, J¼6.2 Hz), 7.01 (d-like, 2H, J¼8.6 Hz), 6.82 (d-like,
2H, J¼8.6 Hz), 5.74 (d, 1H, J¼7.8 Hz), 5.05 (s, 2H), 4.86
(m, 1H), 4.48 (quintet, 1H, J¼7.1 Hz), 4.39 (m, 1H), 4.32
(m, 1H), 3.75 (s, 3H), 3.66 (s, 3H), 3.10 (dd, 1H, J¼14.7,
3.5 Hz), 3.00 (dd, 1H, J¼14.7, 11.1 Hz), 2.89 (s, 3H), 1.37
(m, 1H), 1.34 (d, 3H, J¼7.1 Hz), 1.33 (m, 1H), 1.31 (d, 3H,
J¼7.2 Hz), 0.66 (br d, 3H, J¼5.7 Hz), 0.58 (d, 3H,
J¼5.6 Hz), ꢁ0.09 (br t, 1H, J¼11.0 Hz); 13C NMR
(125 MHz, CDCl3, major conformer) d 173.5 (s), 173.3 (s),
173.1 (s), 168.7 (s), 158.6 (s), 155.7 (s), 136.2 (s), 130.5 (d,
2C), 129.4 (s), 128.5 (d, 2C), 128.2 (d), 128.1 (d, 2C), 114.5
(d, 2C), 67.0 (t), 62.4 (d), 55.1 (q), 52.3 (q), 50.2 (d), 48.5
(d), 46.9 (d), 38.2 (t), 33.2 (t), 29.3 (q), 24.3 (d), 23.0 (q),
20.3 (q), 19.0 (q), 17.5 (q); HRESIMS m/z 613.3210
([MþH]þ, calcd for C32H45N4O8, 613.3237).
3.5.3. Cbz-D-Ala-Leu-N,O-dimethyl-Tyr-Ala-OH (10a)
A mixture of a LiOH solution [LiOH$H2O (4.6 mg,
0.11 mmol) in H2O (0.2 mL)] and aqueous H2O2 (35%,
0.1 mL) was slowly added to a cooled (0 ꢂC) solution of 9a
(22.3 mg, 0.0364 mmol) in a mixture of THF/MeOH (1:1,
2 mL). The solution was stirred at 0 ꢂC for 30 min and then
at room temperature for 4 h. Aqueous NaHSO3 (5%,
0.4 mL) and aqueous citric acid (10%, 0.8 mL) were added
to the solution at 0 ꢂC. After stirring for 20 min, the mixture
was extracted with CHCl3 (3ꢀ10 mL). The combined CHCl3
extracts were washed with brine (5 mL), dried over Na2SO4
and filtered, and the solvent removed in vacuo. The residue
was subjected to column chromatography (silica gel, CHCl3/
MeOH 5:1) to afford 10a (21.7 mg, 100%) as colorless nee-
dles. Mp 98e102 ꢂC (MeOH); [a]D28 ꢁ99.0 (c 0.16, CHCl3);
IR (film) nmax 3287, 2958, 1720, 1628, 1514, 1248, 1035,
1
1173, 1036, 772 cmꢁ1; H NMR (500 MHz, CDCl3, major
conformer) d 8.25 (d, 1H, J¼7.0 Hz), 7.02 (d-like, 2H,
J¼8.5 Hz), 6.82 (d-like, 2H, J¼8.5 Hz), 4.90 (d, 1H,
J¼7.4 Hz), 4.66 (dd, 1H, J¼11.0, 3.5 Hz), 4.52 (m, 1H),
4.16 (m, 1H), 3.75 (s, 3H), 3.73 (s, 3H), 3.18 (dd, 1H,
J¼14.7, 3.5 Hz), 2.98 (dd, 1H, J¼14.7, 11.0 Hz), 2.90 (s,
3H), 1.46 (m, 1H), 1.40 (d, 3H, J¼7.2 Hz), 1.39 (m, 1H),
1.37 (s, 9H), 1.16 (ddd, 1H, J¼14.2, 11.9, 4.1 Hz), 0.66 (d,
3H, J¼7.2 Hz), 0.64 (d, 3H, J¼7.0 Hz), ꢁ0.22 (ddd, 1H,
J¼14.2, 11.0, 3.2 Hz); 13C NMR (125 MHz, CDCl3, major
conformer) d 174.1 (s), 173.1 (s), 169.1 (s), 158.6 (s), 156.6
(s), 130.5 (d, 2C), 129.6 (s), 114.5 (d, 2C), 80.4 (s), 62.5
(d), 55.1 (q), 52.2 (q), 48.6 (d), 47.9 (d), 38.4 (t), 33.0 (t),
29.3 (q), 28.2 (q, 3C), 24.0 (d), 22.9 (q), 20.2 (q), 17.4 (q);
HRESIMS m/z 508.2992 ([MþH]þ, calcd for C26H42N3O7,
508.3023).
1
755 cmꢁ1; H NMR (500 MHz, CD3OD, major conformer)
d 7.37e7.27 (m, 5H), 7.11 (d-like, 2H, J¼8.6 Hz), 6.87 (d-
like, 2H, J¼8.6 Hz), 5.06 (d, 1H, J¼12.5 Hz), 5.03 (d, 1H, J¼
12.5 Hz), 4.93 (br d, 1H, J¼11 Hz), 4.46 (dd, 1H, J¼11.6,
2.8 Hz), 4.31 (q, 1H, J¼7.2 Hz), 4.16 (q, 1H, J¼7.2 Hz),
3.76 (s, 3H), 3.11 (dd, 1H, J¼14.4, 2.5 Hz), 3.00 (dd, 1H,
J¼14.4, 11.3 Hz), 2.89 (s, 3H), 1.38 (d, 3H, J¼7.2 Hz),
1.38 (m, 1H), 1.28 (m, 1H), 1.27 (d, 3H, J¼7.2 Hz), 0.68
(d, 3H, J¼6.5 Hz), 0.64 (d, 3H, J¼6.4 Hz), ꢁ0.05 (m,
1H); 13C NMR (125 MHz, CD3OD, major conformer)
d 178.0 (s), 175.5 (s), 175.3 (s), 171.0 (s), 160.2 (s), 157.9
(s), 138.1 (s), 131.7 (d, 2C), 130.8 (s), 129.5 (d, 2C),
129.1 (d, 2C), 128.9 (d), 115.5 (d, 2C), 67.6 (t), 63.9 (d),
55.6 (q), 51.6 (d), 50.7 (d), 48.3 (d), 39.5 (t), 34.2 (t),
30.2 (q), 25.4 (t), 23.5 (q), 20.9 (q), 18.8 (q), 17.9 (q); HRE-
SIMS m/z 599.3079 ([MþH]þ, calcd for C31H43N4O8,
599.3081).
3.5.2. Cbz-D-Ala-Leu-N,O-dimethyl-Tyr-Ala-OMe (9a)
A solution of 8a (24.6 mg, 0.0484 mmol) in TFA (0.6 mL)
was stirred at room temperature for 2 h. TFA was removed in
vacuo and the residue was dissolved in CHCl3 (3 mL). The so-
lution was washed sequentially with saturated aqueous
NaHCO3 (3 mL) and brine (3 mL), dried over Na2SO4 and fil-
tered, and the solvent removed in vacuo. The residue, Cbz-D-
Ala-OH (16.2 mg, 0.0725 mmol), and HOBt (9.8 mg,
0.073 mmol) were dissolved in CH2Cl2 (1 mL), to which
EDC$HCl (13.9 mg, 0.0725 mmol) was added at 0 ꢂC. The
mixture was stirred at 0 ꢂC for 1 h, and then at room temper-
ature for 3 days. Saturated aqueous NaHCO3 (3 mL) was
added to the solution and the whole was extracted with
CHCl3 (3ꢀ5 mL). The combined CHCl3 extracts were washed
with brine (3 mL), dried over Na2SO4 and filtered, and the sol-
vent removed in vacuo. The residue was subjected to HPLC
(MeCN/H2O 44:56) to afford 9a (26.2 mg, 88%) as an
3.5.4. Hexapeptide 12a
A solution of 11 (11.4 mg, 0.0198 mmol) in TFA (0.7 mL)
was stirred at room temperature for 2 h. TFA was removed in

J.-E. Lee et al. / Tetrahedron 64 (2008) 4117e4125
4123
vacuo. CHCl3 (5 mL) and saturated aqueous NaHCO3 (5 mL)
3.6. Semisynthesis of RA-XX (4)
were added to the residue, and the whole was extracted with
CHCl3 (3ꢀ5 mL). The combined CHCl3 extracts were washed
with brine (3 mL), dried over Na2SO4 and filtered, and the
solvent removed in vacuo. The residue was dissolved in
THF (1 mL) together with 10a (14.2 mg, 0.0237 mmol) and
HOObt (6.5 mg, 0.040 mmol), to which EDC$HCl (7.6 mg,
0.040 mmol) was added at 0 ꢂC. The mixture was stirred at
this temperature for 1 h and then at room temperature for
48 h. Saturated aqueous NaHCO3 (3 mL) was added to the
residue and the whole was extracted with CHCl3 (3ꢀ5 mL).
The combined CHCl3 extracts were washed with brine
(3 mL), dried over Na2SO4 and filtered, and the solvent
removed in vacuo. The residue was subjected to HPLC
(MeCN/H2O 60:40) to afford 12a (13.8 mg, 66%) as an
amorphous solid. [a]D26 ꢁ241.7 (c 0.13, CHCl3); IR (film)
1H NMR (500 MHz, CDCl3, mixture of rotamers) d 7.89 (br
m), 7.43e7.05 (m), 6.89 (dd, J¼8.4, 2.5 Hz), 6.83 (d,
J¼8.6 Hz), 6.80 (d, J¼8.7 Hz), 6.784 (d, J¼8.4 Hz), 6.775
(d, J¼8.4 Hz), 6.61 (dd, J¼8.2, 2.1 Hz), 6.54 (m), 6.25
(br m), 5.86 (br m), 5.52 (br m), 5.37 (br m), 5.32 (dd,
J¼11.4, 3.3 Hz), 5.15e4.68 (m), 4.64 (dd, J¼12.1, 3.6 Hz),
4.39 (m), 4.37 (d, J¼2.0 Hz), 4.13 (br m), 3.96 (s), 3.931
(s), 3.926 (s), 3.76 (s), 3.75 (s), 3.74 (s), 3.64 (t,
J¼11.6 Hz), 3.61 (t, J¼115 Hz), 3.31e3.14 (m), 3.27 (s),
3.18 (s), 3.05 (s), 3.01 (s), 2.98 (s), 3.00e2.93 (m), 2.96 (s),
2.90 (s), 2.88 (s), 2.85 (dd, J¼9.4, 3.6 Hz), 2.79 (dd,
J¼11.4, 3.1 Hz), 2.72 (dd, J¼11.4, 3.1 Hz), 2.66 (br s),
2.553 (s), 2.551 (s), 1.46 (br m), 1.34 (d, J¼7.0 Hz), 1.30
(d, J¼7.0 Hz), 1.26 (d, J¼6.8 Hz), 1.25 (d, J¼6.7 Hz), 1.18
(d, J¼7.0 Hz), 0.93 (d, J¼6.5 Hz), 0.87 (d, J¼6.4 Hz),
0.79 (d, J¼6.4 Hz), 0.67 (d, J¼6.4 Hz), 0.58 (d, J¼5.9 Hz);
HRESIMS m/z 1055.5127 ([MþH]þ, calcd for C59H71N6O12,
1055.5130).
3.6.1. Boc-Abu-N,O-dimethyl-Tyr-Ala-OMe (8b)
Palladium (10%) on charcoal catalyst (5 mg) and hydro-
chloric acid (0.1 mL) were added to a solution of Cbz-N,O-di-
methyl-Tyr-Ala-OMe (7)9 (32.3 mg, 0.0754 mmol) in MeOH
(2 mL), and the mixture was stirred at room temperature under
an atmosphere of hydrogen for 2 h. The catalyst was filtered
off and the filtrate was concentrated to dryness. The residue,
Boc-Abu-OH (30.6 mg, 0.151 mmol), and PyBOP (78.5 mg,
0.150 mmol) were dissolved in CH2Cl2 (2 mL), to which
DIEA (53.8 mL, 0.309 mmol) was slowly added at ꢁ20 ꢂC un-
der an atmosphere of argon. The mixture was stirred at ꢁ20 ꢂC
for 1 h and then at room temperature for 5 days. Aqueous cit-
ric acid (10%, 2 mL) was added to the mixture and the whole
was extracted with CHCl3 (3ꢀ7 mL). The combined CHCl3
extracts were washed sequentially with saturated aqueous
NaHCO3 (2 mL) and brine (2 mL), dried over Na2SO4 and fil-
tered, and the solvent removed in vacuo. The residue was sub-
jected to HPLC (MeCN/H2O 70:30) to afford 8b (26.2 mg,
72%) as an amorphous solid. [a]2D8 ꢁ105.8 (c 0.37, CHCl3),
IR (film) nmax 3425, 2978, 1642, 1515, 1249, 1174,
nmax 3292, 2955, 1739, 1635, 1514, 1249, 1030, 752 cmꢁ1
;
753 cmꢁ1 1H NMR (500 MHz, CDCl3, major conformer)
;
d 8.17 (d, 1H, J¼7.2 Hz), 7.04 (d-like, 2H, J¼8.5 Hz), 6.82
(d-like, 2H, J¼8.5 Hz), 4.88 (d, 1H, J¼7.2 Hz), 4.80 (dd,
1H, J¼10.9, 3.7 Hz), 4.55 (quintet, 1H, J¼7.3 Hz), 3.95 (m,
1H), 3.75 (s, 3H), 3.73 (s, 3H), 3.16 (dd, 1H, J¼14.6,
3.7 Hz), 2.97 (dd, 1H, J¼14.6, 10.9 Hz), 2.88 (s, 3H), 1.38
(s, 9H), 1.38 (d, 3H, J¼7.1 Hz), 1.03 (m, 1H), 0.62 (t, 3H,
J¼7.4 Hz), 0.12 (m, 1H); 13C NMR (125 MHz, CDCl3)
d 173.7 (s), 173.0 (s), 169.0 (s), 158.7 (s), 156.7 (s), 130.3
(d, 2C), 129.7 (s), 114.3 (d, 2C), 80.5 (s), 62.5 (d), 55.3 (q),
52.2 (q), 51.2 (d), 48.4 (d), 33.3 (t), 29.0 (q), 28.2 (q, 3C),
24.1 (t), 17.6 (q), 10.7 (q); HRESIMS m/z 502.2545
([MþNa]þ, calcd for C24H37N3O7Na, 502.2529).
3.6.2. Cbz-D-Ala-Abu-N,O-dimethyl-Tyr-Ala-OMe (9b)
3.5.5. RA-XIX (3)
A solution of 8b (30.0 mg, 0.0626 mmol) in TFA (0.6 mL)
was stirred at room temperature for 2 h. TFA was removed in
vacuo and the residue was dissolved in CHCl3 (3 mL). The so-
lution was washed sequentially with saturated aqueous
NaHCO3 (3 mL) and brine (3 mL), dried over Na2SO4 and fil-
tered, and the solvent removed in vacuo. The residue, Cbz-D-
Ala-OH (21.0 mg, 0.0941 mmol), and HOBt (12.7 mg,
0.0940 mmol) were dissolved in CH2Cl2 (1 mL), to which
EDC$HCl (18.0 mg, 0.0939 mmol) was added at 0 ꢂC. The
mixture was stirred at 0 ꢂC for 1 h and then at room tempera-
ture for 3 days. Saturated aqueous NaHCO3 (3 mL) was added
to the solution, and the whole was extracted with CHCl3
(3ꢀ5 mL). The combined CHCl3 extracts were washed with
brine (3 mL), dried over Na2SO4 and filtered, and the solvent
removed in vacuo. The residue was subjected to HPLC
(MeCN/H2O 45:55) to afford 9b (31.2 mg, 85%) as an amor-
phous solid. [a]D28 ꢁ82.1 (c 0.21, CHCl3); IR (film) nmax 3288,
Palladium (10%) on charcoal catalyst (24 mg) was added
to a solution of 12a (12.0 mg, 0.0114 mmol) in EtOH
(2 mL). Then, the reaction mixture was stirred at room tem-
perature under an atmosphere of hydrogen for 2.5 h. The cat-
alyst was filtered off and the filtrate was concentrated to
dryness. The residue was dissolved in DMF (11.4 mL). To
this solution were added triethylamine (15.9 mL,
0.114 mmol) and DPPA (4.9 mL, 0.023 mmol) at 0 ꢂC, and
after stirring at room temperature for 3 days, the solvent
was removed under reduced pressure. Saturated aqueous
NaHCO3 (10 mL) was added to the residue and the whole
was extracted with CHCl3 (3ꢀ10 mL). The combined
CHCl3 extracts were washed with brine (10 mL), dried
over Na2SO4 and filtered, and the solvent removed in vacuo.
The residue was separated by HPLC (MeOH/H2O 45:55) to
give a compound [4.7 mg, 51%, [a]2D6 ꢁ212.8 (c 0.22,
CHCl3)], which was shown to be identical to natural 3 by
2937, 1724, 1651, 1632, 1514, 1455, 1248, 1034, 754 cmꢁ1
;
comparison of their H and 13C NMR spectra, mass spectra,
and optical rotations.
1H NMR (500 MHz, CDCl3, major conformer) d 8.06 (br d,
1H, J¼6.0 Hz), 7.36e7.27 (m, 5H), 7.06 (m, 1H), 7.04 (d,
1

4124
J.-E. Lee et al. / Tetrahedron 64 (2008) 4117e4125
2H, J¼8.6 Hz), 6.82 (d, 2H, J¼8.6 Hz), 5.79 (d, 1H,
J¼7.6 Hz), 5.05 (s, 2H), 4.99 (br d, 1H, J¼9.1 Hz), 4.48 (quin-
tet, 1H, J¼7.1 Hz), 4.31 (m, 1H), 4.12 (m, 1H), 3.75 (s, 3H),
3.65 (br s, 3H), 3.12 (dd, 1H, J¼14.6, 3.6 Hz), 2.96 (dd, 1H,
J¼14.6, 11.1 Hz), 2.86 (s, 3H), 1.34 (br d, 3H, J¼8.9 Hz),
1.31 (d, 3H, J¼7.1 Hz), 1.20 (br m, 1H), 0.56 (br t, 3H,
J¼6.2 Hz), 0.26 (br m, 1H); 13C NMR (125 MHz, CDCl3, ma-
jor conformer) d 173.8 (s), 173.2 (s), 172.9 (s), 168.7 (s), 158.7
(s), 155.8 (s), 136.2 (s), 130.3 (d, 2C), 129.5 (d), 128.5 (d, 2C),
128.2 (d), 128.1 (d, 2C), 114.3 (d, 2C), 67.0 (t), 62.3 (d), 55.3
(q), 52.3 (q), 50.5 (d), 50.2 (d), 48.5 (d), 33.4 (t), 29.0 (q), 24.0
(t), 19.0 (q), 17.6 (q), 10.5 (q); HRESIMS m/z 585.2914
([MþH]þ, calcd for C30H41N4O8, 585.2924).
0 ꢂC for 1 h and then at room temperature for 48 h. Saturated
aqueous NaHCO3 (3 mL) was added to the residue and the
whole was extracted with CHCl3 (3ꢀ5 mL). The combined
CHCl3 extracts were washed with brine (3 mL), dried over
Na2SO4 and filtered, and the solvent removed in vacuo. The
residue was subjected to HPLC (MeCN/H2O 60:40) to afford
12b (17.9 mg, 77%) as an amorphous solid. [a]2D7 ꢁ190.6 (c
0.16, CHCl3); IR (film) nmax 3293, 2935, 1739, 1639, 1514,
1249, 1218, 1029, 752 cmꢁ1 1H NMR (500 MHz, CDCl3,
;
mixture of rotamers) d 7.76 (br m), 7.44e7.07 (m), 6.90
(dd, J¼8.4, 2.5 Hz), 6.87e6.83 (m), 6.84 (d, J¼8.6 Hz),
6.81 (d, J¼8.6 Hz), 6.781 (d, J¼8.3 Hz), 6.776 (d,
J¼8.3 Hz), 6.63e6.47 (m), 6.37 (br m), 5.87 (dd, J¼12.1,
4.8 Hz), 5.83 (m), 5.67 (br m), 5.35 (br m), 5.31 (dd,
J¼11.4, 3.2 Hz), 5.21 (t, J¼7.9 Hz), 5.15e4.68 (m), 4.64
(dd, J¼12.2, 3.6 Hz), 4.42e4.36 (m), 4.37 (d, J¼1.8 Hz),
4.13 (m), 3.96 (s), 3.931 (s), 3.926 (s), 3.76 (s), 3.75 (s),
3.74 (s), 3.63 (t, J¼11.2 Hz), 3.61 (t, J¼11.1 Hz), 3.32e3.14
(m), 3.26 (s), 3.18 (s), 3.03 (s), 3.01 (s), 2.98 (s), 2.98e2.81
(m), 2.96 (s), 2.88 (s), 2.86 (s), 2.77 (dd, J¼11.4, 3.1 Hz),
2.71 (dd, J¼11.4, 3.0 Hz), 2.550 (s), 2.547 (s), 1.68 (s), 1.39
(m), 1.35 (d, J¼7.0 Hz), 1.29 (d, J¼6.9 Hz), 1.25 (d,
J¼6.9 Hz), 1.23 (d, J¼7.1 Hz), 1.17 (m), 0.88 (t, J¼6.8 Hz),
0.77 (t, J¼7.0 Hz), 0.52 (t, J¼7.3 Hz), 0.49 (t, J¼7.3 Hz),
0.43 (br m); HRESIMS m/z 1049.4634 ([MþNa]þ, calcd for
C57H66N6O12Na, 1049.4636).
3.6.3. Cbz-D-Ala-Abu-N,O-dimethyl-Tyr-Ala-OH (10b)
A mixture of a LiOH solution [LiOH$H2O (5.9 mg,
0.14 mmol) in H2O (0.2 mL)] and aqueous H2O2 (35%,
0.1 mL) was slowly added to a cooled (0 ꢂC) solution of 9b
(27.2 mg, 0.0465 mmol) in a mixture of THF/MeOH (1:1,
2 mL). The solution was stirred at 0 ꢂC for 30 min and then
at room temperature for 4 h. Aqueous NaHSO3 (5%, 0.4 mL)
and aqueous citric acid (10%, 0.8 mL) were added to the
solution at 0 ꢂC. After stirring for 20 min, the mixture was
extracted with CHCl3 (3ꢀ10 mL). The combined CHCl3 ex-
tracts were washed with brine (5 mL), dried over Na2SO4
and filtered, and the solvent removed in vacuo. The residue
was subjected to column chromatography (silica gel, CHCl3/
MeOH 5:1) to afford 10b (26.3 mg, 99%) as colorless needles.
Mp 89e91 ꢂC (MeOH); [a]D28 ꢁ102.6 (c 0.15, CHCl3); IR
(film) nmax 3291, 2935, 1719, 1631, 1514, 1249, 1034,
3.6.5. RA-XX (4)
Palladium (10%) on charcoal catalyst (30 mg) was added to
a solution of 12b (15.6 mg, 0.0152 mmol) in EtOH (2 mL).
Then, the reaction mixture was stirred at room temperature un-
der an atmosphere of hydrogen for 2.5 h. The catalyst was fil-
tered off, the filtrate concentrated to dryness, and the residue
was dissolved in DMF (15.2 mL). To this solution were added
triethylamine (21.2 mL, 0.152 mmol) and DPPA (6.6 mL,
0.031 mmol) at 0 ꢂC, and after stirring at room temperature
for 3 days, the solvent was removed under reduced pressure.
Saturated aqueous NaHCO3 (10 mL) was added to the residue
and the whole was extracted with CHCl3 (3ꢀ10 mL). The
combined CHCl3 extracts were washed with brine (10 mL),
dried over Na2SO4 and filtered, and the solvent removed in va-
cuo. The residue was separated by HPLC (MeOH/H2O 40:60)
to give a compound [5.8 mg, 49%, [a]2D6 ꢁ213.7 (c 0.17,
CHCl3)], which was shown to be identical to natural 4 by com-
1
755 cmꢁ1; H NMR (500 MHz, CD3OD, major conformer)
d 7.37e7.27 (m, 5H), 7.15 (d-like, 2H, J¼8.6 Hz), 6.88
(d-like, 2H, J¼8.6 Hz), 5.12 (d, 1H, J¼12.4 Hz), 5.07 (d,
1H, J¼12.4 Hz), 5.04 (m, 1H), 4.35 (m, 1H), 4.20 (m, 1H),
4.17 (quintet, 1H, J¼7.1 Hz), 3.76 (s, 3H), 3.12 (dd, 1H,
J¼14.4, 3.4 Hz), 2.99 (dd, 1H, J¼14.4, 11.1 Hz), 2.87 (s,
3H), 1.36 (d, 3H, J¼7.3 Hz), 1.29 (d, 3H, J¼7.1 Hz), 1.17
(m, 1H), 0.57 (t, 3H, J¼7.3 Hz), 0.29 (m, 1H); 13C NMR
(125 MHz, CD3OD, major conformer) d 175.7 (s), 175.1 (s),
174.2 (s), 171.2 (s), 160.4 (s), 158.0 (s), 138.2 (s), 131.5 (d,
2C), 130.8 (s), 129.5 (d, 2C), 129.0 (d, 2C), 128.9 (d), 115.4
(d, 2C), 67.6 (t), 63.7 (d), 55.8 (q), 51.9 (d), 51.6 (d), 49.9
(d), 34.4 (t), 29.9 (q), 24.8 (t), 18.8 (q), 17.6 (q), 10.9 (q);
HRESIMS m/z 571.2751 ([MþH]þ, calcd for C29H39N4O8,
571.2768).
1
parison of their H and 13C NMR spectra, mass spectra, and
optical rotations.
3.6.4. Hexapeptide 12b
A solution of 11 (13.0 mg, 0.0226 mmol) in TFA (0.8 mL)
was stirred at room temperature for 2 h. TFA was removed in
vacuo. The residue was treated with CHCl3 (5 mL) and satu-
rated aqueous NaHCO3 (5 mL), and the whole was extracted
with CHCl3 (3ꢀ5 mL). The combined CHCl3 extracts were
washed with brine (3 mL), dried over Na2SO4 and filtered,
and the solvent removed in vacuo. The residue, 10b
(15.5 mg, 0.0272 mmol), and HOObt (7.4 mg, 0.045 mmol)
were dissolved in THF (1 mL), to which EDC$HCl (8.7 mg,
0.045 mmol) was added at 0 ꢂC. The mixture was stirred at
3.7. O-Methylation of RA-XXI (5)
(Trimethylsilyl)diazomethane (16.9 mL of a 2.0 M solution
in diethyl ether, 0.0338 mmol) was added to a stirred solution
of 5 (1.3 mg, 0.0017 mmol) in MeCN/MeOH (9:1, 0.5 mL) at
room temperature. After stirring at room temperature for 3
days, the mixture was concentrated in vacuo. The residue
was subjected to column chromatography (silica gel, CHCl3/
MeOH 20:1) to afford a compound [1.2 mg, 87%, [a]D25
ꢁ266.3 (c 0.06, CHCl3)], which was shown to be identical

J.-E. Lee et al. / Tetrahedron 64 (2008) 4117e4125
4125
1
to natural 4 by comparison of their H and 13C NMR spectra,
mass spectra, and optical rotations.
the formazan crystals formed in each well were dissolved by
stirring with a pipette. Optical density was recorded on a mi-
croplate reader (Tosoh MPR-A4i) at 550 nm. The cytotoxic
activities in Table 3 represent the average of three replicate
measurements for each test.
3.8. O-Methylation of RA-XXII (6)
(Trimethylsilyl)diazomethane (11.7 mL of a 2.0 M solution
in diethyl ether, 0.0234 mmol) was added to a stirred solution
of 6 (9.2 mg, 0.012 mmol) in MeCN/MeOH (9:1, 1 mL) at
room temperature. The mixture was stirred at room tempera-
ture for 10 h and concentrated in vacuo. The residue was sub-
jected to column chromatography (silica gel, CHCl3/MeOH
20:1) to give a compound [8.7 mg, 93%, [a]2D5 ꢁ151.7 (c
0.11, CHCl3)], which was shown to be identical to natural
References and notes
1. Itokawa, H.; Takeya, K.; Mihara, K.; Mori, N.; Hamanaka, T.; Sonobe, T.;
Iitaka, Y. Chem. Pharm. Bull. 1983, 31, 1424e1427.
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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, 8040e8044.
1
1311 by comparison of their H and 13C NMR spectra, mass
spectra, and optical rotations.
´
4. Zalacaın, M.; Zaera, E.; Vazquez, D.; Jimenez, A. FEBS Lett. 1982, 148,
´
´
95e97.
3.9. Assay for cytotoxic activity
5. Sirdeshpande, B. V.; Toogood, P. L. Bioorg. Chem. 1995, 23, 460e470.
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a 96-well plate. Murine P-388 leukemia cells (3ꢀ103 cells)
in 100 mL of RPMI-1640 medium (Nissui Pharmaceutical
Co. Ltd., Tokyo, Japan) supplemented with 5% fetal calf
serum (Mitsubishi Chemical Industry Co. Ltd., Tokyo, Japan)
and kanamycin (100 mg/mL) were placed into each well and
incubated at 37 ꢂC in a humidified atmosphere of 7% CO2.
After 24 h incubation, samples of test compounds at various
concentrations (10 mL) were added to the cultures, and the
mixtures were incubated for 48 h at 37 ꢂC. Then, 20 mL of
an MTT solution (5 mg/mL) was added to each well. After
a further incubation for 4 h, 100 mL of 10% sodium dodecyl
sulfatee0.01 M HCl solution was added to each well and
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, 1343e1347.
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Tomioka, N.; Itai, A.; Iitaka, Y. Tetrahedron 1991, 47, 2757e2772.
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Tetrahedron 1991, 47, 7007e7020.
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Chem. Pharm. Bull. 1993, 41, 1402e1410.
13. Itokawa, H.; Takeya, K.; Mori, N.; Sonobe, T.; Mihashi, S.; Hamanaka, T.
Chem. Pharm. Bull. 1986, 34, 3762e3768.
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