Rubiyunnanins C-H, cytotoxic cyclic hexapeptides from Rubia yunnanensis inhibiting nitric oxide production and NF-κB activation

Article abstract of DOI:10.1016/j.bmc.2010.10.019

Six new (rubiyunnanins C-H, 1-6) and five known (7-11) cyclic hexapeptides were isolated from the roots of Rubia yunnanensis (Franch.) Diels. The structures and stereochemistry of 1-6 were established by extensive spectroscopic analyses and chemical metho

Full text of DOI:10.1016/j.bmc.2010.10.019

Bioorganic & Medicinal Chemistry 18 (2010) 8226–8234
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Rubiyunnanins C–H, cytotoxic cyclic hexapeptides from Rubia yunnanensis
inhibiting nitric oxide production and NF-
jB activation
Jun-Ting Fan a,b, Jia Su a,b, Yan-Min Peng , Yan Li , Jia Li , Yu-Bo Zhou , Guang-Zhi Zeng ,
He Yan , Ning-Hua Tan
a
b,c
a
c
c
a
a,b
a,
⇑
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, PR China
Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China
National Centre for Drug Screen, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Six new (rubiyunnanins C–H, 1–6) and five known (7–11) cyclic hexapeptides were isolated from the
roots of Rubia yunnanensis (Franch.) Diels. The structures and stereochemistry of 1–6 were established
by extensive spectroscopic analyses and chemical methods. All compounds (1–11) not only exhibited
cytotoxic activities against a panel of eleven cancer cell lines with IC50 values ranging from 0.001
Received 11 August 2010
Revised 5 October 2010
Accepted 5 October 2010
Available online 31 October 2010
to 56.24
IFN- -induced RAW 264.7 murine macrophages with IC50 values ranging from 0.05 to 12.68
Furthermore, this is the first time it is being reported that compounds 2 and 7–10 significantly inhibited
l
M, but also exerted inhibitory activities against nitric oxide (NO) production in LPS and
c
l
M.
Keywords:
Rubia yunnanensis
Rubiyunnanins C–H
Cyclopeptides
TNF-
a
-induced NF-
j
B activation in HEK-293-NF-
M, respectively.
j
B luciferase stable cells with IC50 values of 35.07, 0.03,
1.69, 12.64 and 1.18
l
Cytotoxicity
Ó 2010 Elsevier Ltd. All rights reserved.
Inhibition of NO production
NF-jB pathway inhibitors
1
. Introduction
first two RAs, isolated from the stems, leaves and flowers of
1
8
Bouvardia ternifolia (Rubiaceae) in 1977. Subsequently, other 28
RAs have been reported from three Rubia plants, that is, Rubia
Rubia yunnanensis (Franch.) Diels (Rubiaceae, Chinese name
Xiao-Hong-Shen’), is a perennial herb native to Yunnan province,
1
7,19–21
‘
cordifolia, Rubia akane and Rubia yunnanensis.
Recently,
China. Its roots have a long history of use in Chinese traditional
medicine for the treatment of tuberculosis, menoxenia, rheuma-
tism, contusion, hematemesis, anemia and lipoma.1 Previous
studies have shown that rubiaceae-type bicyclic hexapeptides
two novel cyclic peptides named rubiyunnanins A and B from
R. yunnanensis, were reported from our laboratory. Preliminary
studies on antitumor mechanism of RAs indicated that bouvardin
2
2
and RA-VII inhibited protein synthesis through interaction with
2–4
5–8
23–25
(
RA-series cyclopeptides, RAs),
anthraquinones
and arbor-
eukaryotic ribosomes.
Furthermore, RA-V and RA-XII have
9
–14,8
inane-type triterpenoids
are the major types of chemical
been reported to significantly inhibit overproduction of NO and
8
constituents of this plant. Within these chemical constituents,
RAs have attracted great interest in terms of their distinctive
bicyclic structural feature and significant antitumor activities
induction of inducible nitric oxide synthase (iNOS). Recently,
RA-VII has been reported to change the conformational structure
of F-actin to induce G
both in vivo and vitro.
2
arrest and exhibit anti-angiogenic activity
1
5,16
26,27
in vivo and in vitro.
formed with one
N-methyl- -tyrosines and an additional L-a
RAs are homodicyclohexapeptides mainly
-alanine, one -alanine, three modified
-amino acid. The
Moreover, a possible biosynthetic path-
D
-
a
L
-a
way for RAs was proposed, predicting that RAs could be synthe-
sized via a non-ribosomal peptide biosynthetic mechanism.1
In our investigation on structurally interesting cyclopeptides from
R. yunnanensis and further antitumor mechanism of RAs, six new
(rubiyunnanins C–H, 1–6) and five known (7–11) RAs were iso-
lated from the titled plant roots. Herein, their isolation, structural
elucidation, cytotoxic activities and inhibitory activities against
7,28
L
-a
most unusual feature is a 14-membered ring formed by a phenolic
oxygen linkage between two adjacent tyrosines with a cis peptide
bond, and the 14-membered ring which is fused to a 18-membered
1
7
cyclic hexapeptide ring. Bouvardin and deoxybouvardin were the
NO production in LPS and IFN-
macrophages were described. Mostly importantly, it was the first
time to report that RAs potently inhibit TNF- -induced NF-
activation in HEK-293-NF- B luciferase stable cells.
c-induced RAW 264.7 murine
⇑
Corresponding author. Tel.: +86 871 5223800; fax: +86 871 5223800.
E-mail address: nhtan@mail.kib.ac.cn (N.-H. Tan).
a
jB
j
0
968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.019

J.-T. Fan et al. / Bioorg. Med. Chem. 18 (2010) 8226–8234
8227
2
2
2
. Results and discussion
acid
c-methyl ester unit as AA (Fig. 2). In addition, the upfield shifts
1
3
of the C NMR signals of C-2
a, C-2b, C-2c and C-2d compared with
.1. Structure determination
those of 9, together with the MS information also corroborated 1
was an analog of 9 whose glutamine was replaced by glutamic acid
The methanol extract of the air-dried and powdered roots of
c-methyl ester. Moreover, the sequence of the amino acid residues
R. yunnanensis (100 kg) was suspended in water and then parti-
tioned successively with EtOAc and n-BuOH. The EtOAc layer was
repeatedly chromatographed over a series of silica gel, RP-18 silica
gel, Sephadex LH-20 and ODS HPLC to afford eleven RAs (Fig. 1).
These included six new ones (rubiyunnanins C–H, 1–6) and five
known ones, RA-V (7), RA-I (8), RA-XXIV (9), RA-XII (10), RY-II (11).
Rubiyunnanin C (1) was obtained as a white crystal. Its molecular
in 1 was confirmed from the HMBC correlations of Glu -methyl es-
c
2
1
3
2
4
3
ter -NH/Ala -CO, Tyr -NMe/Glu c-methyl ester -CO, Ala -NH/Tyr -
5
4
6
5
1
6
CO, Tyr -NMe/Ala -CO, Tyr -NMe/Tyr -CO and Ala -NH/Tyr -CO
(Fig. 2). In the ROESY spectrum, NOE correlations were observed
3
5
between Tyr -NMe/H-2
a
, H-3
a
, Tyr -NMe/H-4
a
and H-5
-methyl es-
ter /Tyr , Ala /Tyr and Tyr /Tyr were trans, trans and cis, respec-
tively. Furthermore, crucial NOE correlations of H-1 /H-4b, H-6
H-6db, H-6db/H-5 a were also observed, revealing the similar
relative configuration to that of RAs (Fig. 3). The absolute configura-
a/H-6a,
indicating the N-methyl peptide bonds between Glu
c
2
3
4
5
5
6
formula was established as C43
H
52
N
6
O
11 by its positive HRFABMS
a
a/
+
(m/z 828.3713, [M] ), indicating 21 degrees of unsaturation. Its UV
e
spectrum showed the existence of phenyl groups based on the
absorptions at 203 and 277 nm. The IR spectrum exhibited absorp-
1
4
tions of Ala and Ala were identified as D (R) and L (S), respectively,
by application of the Marfey’s method. The remaining configura-
tions of Glu c-methyl ester , Tyr , Tyr and Tyr in 1 were deter-
ꢀ1
31
tion bands at 3414 and 1660 cm ascribable to OH, NH and CO
1
13
2
3
5
6
groups. The H and C NMR spectra of 1 in C
5
D
5
N (Table 1) displayed
characteristics of typical RAs and demonstrated the presence of two
mined to be L (S), L (S), L (S) and L (S), respectively, by analyzing
information from the similar NOE correlations, carbon and proton
chemical shifts and proton coupling constants, compared with those
of 9. Accordingly, the structure of 1 was determined as shown in Fig-
ure 1.
conformers in a ratio of 74:26.2
NMR spectral data of 1 displayed the major conformer’s signals for
two methyls (d /d 1.35/19.2, 1.54/21.8), three amide N-methyl
signals (d /d 3.00/30.4, 3.02/29.7, 3.26/40.1), two O-methyl signals
/d 3.51/51.5, 3.66/55.2), five methylenes (d /d 2.36/27.0; 2.56/
0.3; 2.61, 3.66/ 36.8; 3.32, 3.56/36.3; 3.85, 3.92/33.9), six -amino
methines(d /d 4.13/68.9, 5.01/58.0, 5.09/46.9, 5.16/48.3, 5.32/48.4,
.75/54.5), two 1,4-disubstituted benzene rings (d 131.9, 158.9 and
/d 136.1, 158.9 and d /d 6.90/
7.00/114.5 ꢁ 2, 7.32/130.8 ꢁ 2; d
24.7, 6.95/126.6, 7.19/130.9, 7.44/133.5), one 1,2,4-trisubstituted
128.1, 145.5, 152.8 and d /d 4.64/115.1, 6.75/
22.1, 7.21/117.9), seven carbonyl signals (d 168.6, 169.9, 171.3,
72.1, 172.6, 173.0, 173.2) and three amide protons (d 7.36, 8.81,
.91). An extensive comparison of 1D and 2D NMR spectra data of
with those of RA-XXIV (9)21 in C
N indicated that both com-
9,30
Further analysis of 1D and 2D
H
C
H
C
(d
H
C
H
C
Rubiyunnanin D (2) and rubiyunnanin E (3) were obtained
as white powders. Compound 2 had the molecular formula
3
a
41 48 6 11
C H N O , established from the positive HRESIMS (m/z
H
C
+
5
C
823.3259, [M+Na] ), indicating 21 degrees of unsaturation. Com-
pound 3 had the molecular formula C42 12 by its positive
HRESIMS (m/z 853.3391, [M+Na] ), corresponding to 21 degrees of
d
H
C
C
H
C
50 6
H N O
+
1
1
13
benzene ring (d
C
H
C
5 5
unsaturation. The H and C NMR spectra of 2 and 3 in C D N (Table
1
1
9
1
C
1) also displayed characteristics of typical RAs and demonstrated the
presence of two conformers in a ratio of 78:22 and 77:23, respec-
tively. Comparison of the 1D and 2D NMR spectra of 2 in C D N with
5 5
H
5 5
D
those of 1 showed their structural similarities. The main difference
pounds were very similar, except for the resonances due to those
between 2 and 1 was the absence of two O-methyl groups. In the
2
1
1
13
of the second amino acid residue (AA ). In the H– H COSY spectrum,
cross-peaks of H-2 /H-2b, NH-2 and H-2b/H-2 suggestedthat C-2
was further extended by two methylene carbons at C-2b and C-2
The observed HMBC correlations of H-2b, H-2 and the methoxyl
173.2) indicated that 1 had a glutamic
C NMR spectra of 1 and 2, the C-2
48.4, 27.0, 30.3, 173.2 in 1 to d
27.4, 31.0, 175.4 in 2, which revealed the replacement of the meth-
oxyl group (d 51.5) in 1 by a hydroxyl group in 2 at the C-2d position.
a, C-2b, C-2
c
and C-2d resonances
a
c
a
were shifted downfield from d
C
C
48.8,
c
.
c
C
group (d
H
3.51) with C-2d (d
C
C
Moreover, the upfield shifts of C-3cand C-3f from d 131.9 and 158.9
δ
ε
β
δ
ε
R
α
HN
1
O
O
OR
2
β
γ
ξ
β
N
α
O
O
R1
α
HN
OR2
2
1
3
β
γ
ξ
O
N
α
2
3
HN
4
HN
4
O
R3
β
α
α
β
NH
O
O
ξ
1
β
β
α
α
O
N
NH
O
O
ξ
6
5
α
N
H
b
R
H
δa
εa
3
α
β
O
N
6
5
α
β
N
b
δa
H
a
Hb
γ
Ha
Hb
δa
εa
α
β
δb
εa
γ
β
Ha
γ
δa
δb
δb
εa
γ
6'
εb
ξ
O
O
εb
HO
5'
O
δb
HO
4'
εb
ξ
OH
O
εb
HO
2
'
1'
3'
OH
R
1
R
CH
H
CH
CH
CH
CH
2
R
H
H
OH
H
3
R1
R2
CH3
H
CH3 OH
CH3
CH3
R3
H
H
1
2
3
7
8
9
CH
CH
CH
CH
CH
CH
2
2
2
3
2
2
CH
CH
CH
2
2
2
COOCH
COOH
COOH
3
3
4
5
6
10
11
CH CH CONH
CH3
CH3
CH3
2
CH OH
2
2
2
3
3
3
3
H
H
OH
CH
H
H
2
CONH
2
Figure 1. Chemical structures for compounds 1–11.

8
228
J.-T. Fan et al. / Bioorg. Med. Chem. 18 (2010) 8226–8234
Table 1
1
H NMR and 13C NMR data for compounds 1–3 in C
5
D
5
N (d in ppm, J in Hz)
1
2
3
a
b
a
b
a
b
d
H
d
C
d
H
d
C
d
H
d
C
-Ala1
a
5.16 (q, 7.0)
1.54 (d, 7.0)
48.3 d
5.17 (m)
48.4 d
5.17 (m)
48.3 d
D
b
21.8 q
1.55 (d, 6.5)
21.9 q
1.55 (d, 7.0)
21.9 q
C@O
NH
173.0 s
173.2 s
173.3 s
8.81 (d, 8.0)
8.80 (d, 8.0)
8.82 (d, 8.0)
AA2
a
b
c
5.32 (dd, 15.3, 8.0)
2.36 (m)
2.56 (m)
48.4 d
27.0 t
30.3 t
5.44 (dd, 15.3, 8.0)
2.48 (m)
2.75 (m)
48.8 d
27.4 t
31.0 t
5.45 (dd, 15.3, 8.0)
2.46 (m), 2.53 (m)
2.75 (m)
48.8 d
27.3 t
31.0 t
d
173.2 s
172.6 s
175.4 s
172.9 s
175.4 s
172.9 s
C@O
NH
OMe
9.91 (d, 8.5)
3.51 (s)
9.92 (d, 8.5)
9.94 (d, 8.0)
51.5 q
Tyr3
a
4.13 (dd, 10.5, 4.5)
3.85 (dd, 13.8, 4.5)
3.92 (dd, 13.8, 10.5)
68.9 d
33.9 t
4.11 (dd, 10.5, 4.5)
3.89 (m)
69.1 d
34.0 t
4.16 (dd, 9.3, 5.8)
3.89 (m)
68.9 d
34.4 t
ba
bb
c
da
db
131.9 s
130.8 d
130.8 d
114.5 d
114.5 d
158.9 s
168.6 s
40.1 q
130.2 s
131.1 d
131.1 d
116.6 d
116.6 d
157.7 s
168.9 s
40.2 q
133.2 s
117.7 d
120.7 d
148.4 s
112.8 d
147.5 s
169.0 s
40.2 q
7.32 (d, 8.5)
7.32 (d, 8.5)
7.00 (d, 8.5)
7.00 (d, 8.5)
7.34 (overlap)
7.34 (overlap)
7.22 (overlap)
7.22 (overlap)
7.31 (d, 1.5)
6.90 (overlap)
e
e
f
a
b
6.97 (d, 8.5)
C@O
NMe
OMe
3.26 (s)
3.66 (s)
3.30 (s)
3.31 (s)
3.68 (s)
55.2 q
56.0 q
Ala4
Tyr5
a
b
C@O
NH
5.09 (m)
1.35 (d, 7.0)
46.9 d
19.2 q
172.1 s
5.09 (m)
1.36 (d, 6.5)
47.0 d
19.3 q
172.2 s
5.09 (m)
1.36 (d, 7.0)
47.0 d
19.2 q
172.3 s
7.36 (d, 7.5)
7.37 (d, 8.0)
7.37 (d, 8.0)
a
5.75 (dd, 11.5, 3.0)
2.61 (dd, 11.5, 3.0)
3.66 (overlap)
54.5 d
36.8 t
5.75 (d, 11.0)
2.62 (d, 11.0)
3.63 (t, 11.0)
54.6 d
36.9 t
5.75 (dd, 11.5, 3.0)
2.62 (dd, 11.5, 3.0)
3.63 (t, 11.5)
54.6 d
36.9 t
ba
bb
c
da
db
136.1 s
133.5 d
130.9 d
124.7 d
126.6 d
158.9 s
169.9 s
30.4 q
136.1 s
133.6 d
131.1 d
124.7 d
126.7 d
159.0 s
170.0 s
30.5 q
136.1 s
133.6 d
131.1 d
124.7 d
126.7 d
159.1 s
170.0 s
30.5 q
7.44 (dd, 8.5, 2.0)
7.19 (overlap)
6.90 (overlap)
7.43 (d, 8.0)
7.43 (dd, 8.5, 2.0)
7.25 (overlap)
6.90 (overlap)
7.22 (overlap)
6.89 (br d, 8.0)
6.98 (br d, 8.0)
e
e
f
a
b
6.95 (dd, 8.3, 2.5)
7.00 (dd, 8.3, 2.0)
C@O
NMe
3.00 (s)
3.00 (s)
3.00 (s)
Tyr6
a
5.01 (dd, 12.3, 3.0)
3.56 (dd, 17.5, 3.0)
3.32 (m)
58.0 d
36.3 t
5.01 (br d, 12.0)
3.55 (br d, 17.5)
3.30 (overlap)
58.1 d
36.4 t
5.02 (dd, 11.8, 3.0)
3.56 (dd, 18.5, 3.0)
3.31 (overlap)
58.1 d
36.4 t
ba
bb
c
da
db
128.1 s
122.1 d
115.1 d
117.9 d
152.8 s
145.5 s
171.3 s
29.7 q
128.2 s
122.2 d
115.2 d
117.9 d
152.8 s
145.5 s
171.4 s
29.8 q
128.2 s
122.2 d
115.3 d
117.9 d
152.8 s
145.5 s
171.4 s
29.8 q
6.75 (br d, 8.5)
4.64 (br s)
7.21 (d, 8.5)
6.74 (br d, 8.0)
4.66 (br s)
7.22 (overlap)
6.74 (br d, 8.0)
4.69 (br s)
7.19 (overlap)
e
e
f
a
b
C@O
NMe
3.02 (s)
3.00 (s)
3.00 (s)
a
Data were measured at 500 MHz.
Data were measured at 125 MHz.
b
in 1 to d
C
130.2 and 157.7 in 2, as well as the downfield shifts of C-3d
from d 130.8 and 114.5 in 1 to d 131.1 and 116.6 in 2 fur-
55.2) in 1
by a hydroxyl group in 2 at the C-3f position. But for compound 3, its
D and 2D NMR spectra were closely related to those of 2, except for
the presence of an O-methyl group (d /d 3.68/56.0) and a hydroxyl
deduced to be L (S), L (S) and L (S) respectively, from the similarities
observed in NOE correlations, carbon and proton chemical shifts and
proton coupling constants as compared with those of 1. Therefore,
the structures of 2 and 3 were defined as shown in Figure 1.
Rubiyunnanin F (4) was obtained as a white powder and gave
and C-3
e
C
C
ther confirmed the replacement of the methoxyl group (d
C
1
H
C
61 7
the molecular formula C48H N O15 from its positive HRESIMS
3
+
1
group attached to aromatic ring in Tyr . The HMBC correlation ob-
served between methoxyl proton (d 3.68) and C-3f (d 147.5 s) indi-
cated that the methoxyl group was positioned at C-3f. In addition,
the hydroxyl group was deduced to be located at the C-3 a position,
which was verified by the apparent downfield shift of the C-3 a sig-
nal at d 148.4 together with the key HMBC correlation of H-3 b/C-
a (Fig. 2). The absolute configurations of Ala , Glu and Ala in 2
and 3 were identified as D (R), L (S) and L (S), respectively. The
(976.4334 [M+H] ), with 21 degrees of unsaturation. Its H and
1
3
H
C
3
C NMR spectra in CD OD (Table 2) displayed characteristics of
typical RAs and demonstrated the presence of two conformers in
a ratio of 72:28. Further comparison of the 1D and 2D NMR spectra
of 4 with those of RA-XII (10), which was a RAs glucoside isolated
e
e
3
2,2
C
e
from R. cordifolia and R. yunnanensis,
except for the resonances due to those of AA . The H– H COSY
correlations of H-2 /H-2b/H-2 and HMBC correlations of H-2b
and H-2 with the carbonyl carbon at C-2d (d 177.3), together
showed many similarities
1
2
4
2
1
1
3e
a
c
remaining configurations of Tyr , Tyr and Tyr6 in 2 and 3 were
3
5
c
C

J.-T. Fan et al. / Bioorg. Med. Chem. 18 (2010) 8226–8234
8229
OCH3
OH
O
O
O
O
O
O
OCH3
OH
OCH3
N
N
HN
O
O
HN
O
HN
HN
NH
O
NH
O
N
O
N
N
N
O
O
O
O
OH
OH
1
3
NH2
O
O
OCH3
O
O
OCH
3
N
N
HN
O
HN
O
O
HN
HN
NH
O
NH
O
O
N
O
N
N
O
N
O
HO
O
O
HO
HO
HO
1
1
H- H COSY
HMBC(H C)
O
O
O
O
HO
HO
HO
OH
OH
4
6
Figure 2. Selected 1H– H COSY and HMBC correlations of compounds 1, 3, 4 and 6.
1
as L (S), L (S), L (S) and L (S), respectively, from the comparison of
the NOE correlations, carbon and proton chemical shifts and proton
coupling constants with those of 10. Therefore, the structure of 4
was determined as shown in Figure 1.
Rubiyunnanin G (5) and rubiyunnanin H (6) were obtained as
white powders. Compound 5 possessed the molecular formula
+
C
45
56
H N
6
O
14 by its positive HRESIMS (m/z 927.3734, [M+Na] ),
corresponding to 21 degrees of unsaturation. Compound 6 gave
the molecular formula C46
H
58
N
6
+
O
15 on the basis of its positive
HRESIMS (m/z 957.3882, [M+Na] ), indicating 21 degrees of unsat-
1
13
3
uration. The H and C NMR spectra of 5 and 6 in CD OD (Table 2)
displayed characteristics of typical RAs and demonstrated the pres-
ence of two conformers in a ratio of 73:27 and 77:23, respectively.
The similarities of the 1D and 2D NMR spectra of 5, 6 and 10 showed
that 5 and 6 were also RAs glucosides. The major difference be-
tween 5 and 10 was that the hydroxyl group in 5 was substituted
with the methoxyl group in 10 at the C-3f position. In addition,
the upfield shifts of C-3
to d 129.6 and 158.4 in 5, together with the downfield shift of
C-3 from d 115.0 in 10 to d 116.8 in 5 further corroborated this
substitution. But for compound 6, it was also structurally similar
to 10 except for the presence of an oxygenated methine (d /d
.66/74.3) and the absence of a methylene at d 36.6 (C-6b). Based
on MS information, a hydroxyl group was assigned to 6 at C-6b,
C
c and C-3f from d 132.2 and 159.9 in 10
C
e
C
C
H
C
Figure 3. Selected NOE correlations of compounds 1 and 6.
4
C
1
1
with the MS information implied that 4 was an analog of 10 whose
which was supported by the H– H COSY correlation of H-6b/H-
and HMBC correlations of H-6b/C-6da, C-6db and Tyr6 C@O
(Fig. 2). The hydroxyl group at C-6b was determined to be -ori-
ented on the basis of the coupling constant of H-6b/H-6 (d,
J = 8.8 Hz) and NOE correlations of H-6b/Tyr -NMe, H-6da, which
2
2
Ala was replaced by Gln . As for the glucopyranosyl moiety of 4,
HMBC correlation between the anomeric proton (d 4.98, d,
J = 7.5 Hz) (b-form) and C-6f indicated the linkage of the sugar
group and C-6f (Fig. 2). The acidic hydrolysis of 4 gave -glucose
as a sugar residue, which was determined by GC analysis of its
corresponding trimethylsilylated -cysteine adduct. The absolute
configurations of Ala and Ala were assigned as D (R) and L (S),
6a
H
a
a
6
D
3
3,34
is the same configuration as that of RA-IV and RA-XVI (Fig. 3).
1
2
4
L
The absolute configurations of Ala , Ala and Ala in 5 and 6 were
identified as D (R), L (S) and L (S), respectively. The remaining con-
figurations of Tyr , Tyr and Tyr in 5 and 6 were deduced to be L (S),
1
4
2
3
5
6
3
5
6
respectively, while that of Gln , Tyr , Tyr and Tyr were deduced

8
230
J.-T. Fan et al. / Bioorg. Med. Chem. 18 (2010) 8226–8234
Table 2
1
H NMR and 13C NMR data for compounds 4–6 in CD
3
OD (d in ppm, J in Hz)
4
5
6
a
c
a
c
b
c
d
H
d
C
d
H
d
C
d
H
d
C
-Ala1
a
4.49 (q, 7.0)
1.26 (d, 6.5)
49.0 d
4.45 (m)
48.8 d
4.48 (overlap)
1.27 (overlap)
48.9 d
D
b
21.4 q
1.23 (d, 7.0)
21.0 q
20.8 q
C@O
173.7 s
173.4 s
173.8 s
AA2
a
b
4.80 (m)
1.96 (m)
49.5 d
27.4 t
4.74 (overlap)
1.29 (d, 7.0)
45.6 d
16.4 q
4.73 (overlap)
1.27 (overlap)
45.5 d
16.6 q
c
2.24 (t, 7.0)
31.9 t
d
177.3 s
173.7 s
C@O
174.6 s
174.4 s
Tyr3
a
3.87 (overlap)
3.29 (m)
68.8 d
34.0 t
3.84 (dd, 11.0, 5.0)
3.18 (dd, 13.5, 5.0)
3.25 (dd, 13.5, 11.0)
68.8 d
33.7 t
3.84 (overlap)
3.20 (m)
68.6 d
33.6 t
ba
bb
c
132.3 s
131.5 d
115.1 d
160.0 s
170.9 s
40.5 q
129.6 s
131.4 d
116.8 d
158.4 s
171.1 s
40.3 q
131.6 s
131.5 d
115.0 d
160.0 s
171.0 s
40.3 q
d ꢂ 2
7.11 (d, 8.5)
6.87 (d, 8.5)
6.98 (d, 8.5)
6.73 (d, 8.5)
7.07 (d, 8.4)
6.85 (d, 8.4)
e
f
ꢂ 2
C@O
NMe
OMe
2.94 (3H, s)
3.75 (3H, s)
2.92 (s)
2.88 (s)
3.75 (s)
55.7 q
55.7 q
Ala4
Tyr5
a
b
C@O
4.73 (m)
1.10 (d, 6.5)
47.8 d
18.9 q
173.0 s
4.74 (overlap)
1.08 (d, 6.5)
47.7 d
18.8 q
173.0 s
4.73 (overlap)
1.12 (d, 6.4)
47.8 d
18.8 q
172.6 s
a
5.43 (br d, 11.5)
2.65 (m)
3.60 (overlap)
55.6 d
37.4 t
5.46 (dd, 11.5, 3.0)
2.68 (dd, 11.5, 3.0)
3.57 (overlap)
55.7 d
37.4 t
5.35 (dd, 11.6, 3.2)
2.69 (dd, 11.6, 3.2)
3.56 (overlap)
56.0 d
37.5 t
ba
bb
c
da
db
136.9 s
134.1 d
132.0 d
125.2 d
127.4 d
159.7 s
171.4 s
31.1 q
137.0 s
134.1 d
131.8 d
125.1 d
127.2 d
159.8 s
171.3 s
31.1 q
136.9 s
134.8 d
131.1 d
124.9 d
127.6 d
160.5 s
171.8 s
31.1 q
7.20 (dd, 8.5, 2.0)
7.46 (br d, 8.5)
6.79 (dd, 8.5, 2.0)
7.28 (dd, 8.5, 2.0)
7.21 (dd, 8.5, 2.0)
7.47 (dd, 8.5, 2.0)
6.81 (dd, 8.5, 2.5)
7.28 (dd, 8.5, 2.5)
7.14 (overlap)
7.43 (dd, 8.4, 2.0)
6.68 (dd, 8.8, 2.4)
7.38 (dd, 8.4, 2.4)
e
e
f
a
b
C@O
NMe
3.06 (s)
3.06 (s)
3.05 (s)
Tyr6
a
4.68 (dd, 12.0, 3.5)
3.13 (m)
3.00 (m)
58.7 d
36.5 t
4.70 (dd, 11.5. 4.0)
3.08 (overlap)
3.02 (m)
58.6 d
36.6 t
4.48 (overlap)
4.66 (d, 8.8)
64.4 d
74.3 d
ba
bb
c
da
db
131.6 s
122.7 d
115.8 d
118.6 d
154.6 s
145.4 s
172.1 s
30.2 q
132.3 s
122.6 d
115.8 d
118.7 d
154.6 s
145.4 s
172.3 s
30.1 q
135.8 s
124.3 d
117.4 d
118.2 d
154.5 s
147.3 s
170.9 s
32.0 q
6.65 (br d, 8.0)
4.59 (br s)
7.08 (d, 8.0)
6.66 (br d, 8.5)
4.62 (d, 1.5)
7.09 (d, 8.5)
6.82 (dd, 8.4, 2.0)
5.03 (d, 2.0)
7.11 (d, 8.4)
e
e
f
a
b
C@O
NMe
2.59 (s)
2.61 (s)
2.31 (3H, s)
0
0
0
0
0
0
Glc
1
2
3
4
5
6
4.98 (d, 7.5)
3.60 (overlap)
3.51 (m)
3.44 (overlap)
3.44 (overlap)
3.70 (dd, 12.0, 4.5)
102.8 d
74.9 d
77.9 d
71.4 d
78.2 d
62.5 t
4.98 (d, 7.5)
3.57 (overlap)
3.49 (m)
3.42 (overlap)
3.42 (overlap)
3.70 (m)
102.8 d
74.9 d
77.9 d
71.3 d
78.2 d
62.5 t
5.01 (d, 7.6)
3.56 (overlap)
3.48 (m)
3.42 (overlap)
3.42 (overlap)
3.69 (dd, 12.0, 4.8)
3.87 (overlap)
102.6 d
74.8 d
77.8 d
71.3 d
78.2 d
62.5 t
3
.87 (overlap)
3.88 (d, 12.0)
a
b
c
Data were measured at 500 MHz.
Data were measured at 400 MHz.
Data were measured at 100 MHz.
L (S) and L (S), respectively, from the similarity in their NOE corre-
lations, carbon and proton chemical shifts and proton coupling con-
stants, compared with those of 10. Consequently, the structures of 5
and 6 were defined as shown in Figure 1.
R. yunnanensis were tested for their cytotoxicities against five can-
cer cell lines BEL-7402, A549, BGC-823, U251 and B16. Results
indicated that the EtOAc fraction was most active against cancer
cell lines (Table 3). Furthermore, all eleven RAs (1–11) isolated
from the active EtOAc fraction were evaluated for their cytotoxic-
ities against eleven cancer cell lines, HEPG2, BEL-7402, SMMC-
7721, MDA-MB-231, DU-145, PC-3, A549, BGC-823, Hela, U251
and B16 (Table 4). Compounds 1, 7–11 exhibited significant
cytotoxicities, whereas 2–6 showed weak activities. From these
active constituents, RA-V (7) showed the best activity with nano-
molar IC50 values. Further analysis suggested that the replacement
Moreover, by comparing the NMR spectroscopic data, MS data
and physical characteristics with the reported data in the litera-
35
33
tures, five known RAs were identified as RA-V (7), RA-I (8),
RA-XXIV (9), RA-XII (10), RY-II (11).3
2
1
32
2
.2. Bioactivity
2
2
2
In the present study, the methanol extract, EtOAc-soluble
fraction, n-BuOH-soluble fraction and H O-soluble fraction of
of Ala in 7 by Glu
c-methyl ester in 1, Glu in 2 and 3, Gln in 4 and
2
9 or Ser in 8 and 11 caused an apparent decrease in the

J.-T. Fan et al. / Bioorg. Med. Chem. 18 (2010) 8226–8234
8231
Table 3
The IC50 values of the methanol extract and its different solvent fractions on five
Table 5
Inhibition activities of NO production and NF-jB activation by
cancer cell lines by SRB method (
l
g/mL)
compounds 1–11
Extract
BEL-7402 A549
BGC-823 U251 B16
Compd
IC50 (lM)
Methanol extract
EtOAc-soluble fraction
n-Butanol-soluble fraction ND
NDa
14.32
ND
ND
2.18
ND
ND
ND
5.32
ND
ND
0.02
ND
NOa
5.16
7.22
11.25
1.77
12.64
12.68
0.05
0.25
2.22
0.32
1.99
NF-j
Bb
NDe
35.07
ND
ND
ND
ND
0.03
1.69
12.64
1.18
ND
8.16
16.17
ND
3.66
26.75
ND
1
2
3
4
5
6
7
8
9
H
2
O-soluble fraction
ND
0.58
Taxol
0.0008 0.02
0.11
a
ND: no detected (>50 lg/mL).
cytotoxicity. The introduction of a hydroxyl group at the C-3ea or
1
1
0
1
C-6b position, or the replacement of OMe by OH at the C-3f posi-
tion also seems to decrease the activity. Moreover, RAs glycosides
exhibited much weaker activities than their aglycons.
c
MG-132
0.35
PS-341d
0.014
In a previous report, RA-V (7) and RA-XII (10) were found to
show inhibitory effects on NO production and iNOS induction. In
this study, compounds 1–11 were also evaluated for their effects
a
Effect on NO production induced by LPS and IFN-
RAW 264.7 macrophages.
c in
8
b
Effect on TNF-
B luciferase stable cells.
Positive control for LPS and IFN-
production.
a induced NF-jB activation in HEK-293-
NK-
j
on NO production in LPS and IFN-
phages. All compounds showed inhibitory activities with IC50 val-
ues ranging from 0.05 to 12.68 M (Table 5). Free radical NO is
produced by nitric oxide synthase (NOS) as a by-product during
conversion of -arginine to -citrulline. In the family of NOS, the
inducible NOS (iNOS) is the direct target of NF- B signaling path-
way. Moreover, NF- B is a family of dimeric transcription factors
c-induced RAW 264.7 macro-
c
c
stimulated NO
l
d
Positive control for NK-
jB luciferase assay.
e
ND: no detected (>25
lg/mL).
L
L
j
j
3. Experimental
3.1. General experimental procedures
which plays vital roles in regulation of proliferation, inflammation,
immune responses and apoptosis in various human patholo-
3
6,37
gies.
hibit NO production by regulating the NF-
Therefore, the effects of compounds 1–11 on NF-
way were examined using HEK-293-NF- B luciferase stable cell
lines. Compounds2 and 7–10 exhibited potent inhibitory activities
with IC50 values of 35.07, 0.03, 1.69, 12.64 and 1.18 M, respec-
tively (Table 5). Compound 7 has the best activity against NO pro-
duction and NF- B signaling pathway, while compound 2 has the
worst activity against them. There is good correlation between
the activities of these RAs against NO production and NF- B activa-
tion (Table 5), implying that the inhibition of NO production was
due to the inhibition of NF- B signaling pathway. This is the first
report demonstrating NF- B inhibitory activity of RAs. Further-
more, compared the results in Table 4 with Table 5, it was deduced
that the activities against cancer cell proliferation and NF- B inhi-
bition of RAs have good correlation, suggesting that the down-reg-
ulation of the NF- B signaling pathway might play an important
role in the mechanism of RAs against tumor cell growth. The more
Hence, the above results suggested that these RAs may in-
B signaling pathway.
B signaling path-
j
Melting points were obtained on an X-4 micromelting point
apparatus. Optical rotations were measured with a Horiba SEPA-
300 polarimeter. UV spectra were obtained using a Shimadzu
UV-2401A spectrophotometer. IR spectra were obtained by a Tenor
27 spectrophotometer using KBr pellets. 1D and 2D NMR spectra
were recorded on Bruker AM-400, DRX-500 or AV-600 spectrome-
ters with TMS as internal standard. Coupling constants were ex-
pressed in hertz, and chemical shifts (d) were expressed in ppm
with reference to the solvent signals. Mass spectra were performed
on a VG Autospec-3000 spectrometer or an API Qstar pulsa TOF
spectrometer. The GC analysis was performed on an HP5890 gas
chromatograph (Agilent, American) equipped with a quartz capil-
j
j
l
j
j
j
j
lary column (30 mm ꢁ 0.32 mm, 0.25
lm); detection, FID. Analyti-
j
cal or semipreparative HPLC was performed on an Agilent 1100
liquid chromatograph with
150 mm; 9.4 mm ꢁ 250 mm).
a
Zorbax Eclipse-C18 (4.6 mm ꢁ
j
Column chromatography was performed with silica-gel (200–
300 mesh, Qingdao Yu-Ming-Yuan Chemical Co. Ltd., Qingdao,
China), Sephadex LH-20 (Pharmacia Fine Chemical Co., Uppsala,
detail research on RAs and NF-
progress.
jB signaling pathway is still in
Table 4
The IC50 values of compounds 1–11 on eleven cancer cell lines (
l
M)
Compd
HEPG2
BEL-7402
SMMC-7721
MDA-MB-231
DU-145
PC-3
A549
BGC-823
Hela
U251
B16
1
2
3
4
5
6
7
8
9
7.84
1.97
10.69
21.28
ND
14.32
ND
35.97
0.13
2.41
9.09
8.82
12.97
ND
1.52
15.95
37.58
6.32
39.32
ND
0.01
0.16
1.44
5.97
8.57
ND
0.67
1.00
0.94
0.87
30.34
ND
12.47
36.41
ND
0.01
0.39
3.26
3.71
6.91
0.02
5.97
0.76
1.12
15.78
29.97
38.90
33.66
41.35
0.14
1.01
8.01
8.68
12.76
0.11
45.99
15.83
43.73
41.62
36.29
ND
0.01
0.59
2.03
10.23
54.76
22.31
36.82
ND
0.05
0.80
3.66
6.55
14.40
ND
37.17
45.74
8.18
25.95
32.82
0.06
0.42
2.85
1.82
5.02
15.09
33.16
19.15
32.01
48.17
0.04
0.91
6.16
5.18
3.52
26.25
56.24
14.99
48.61
42.31
0.001
1.96
10.81
10.03
8.20
15.28
22.17
3.44
25.94
29.24
0.001
0.22
1.01
1.40
2.89
0.04
a
ND
20.99
ND
ND
0.08
2.02
8.45
7.49
11.45
1.07
1
1
0
1
2.88
10.65
0.01
Taxol
CPT
0.001
0.07
0.03
0.02
0.33
16.24
a
ND: no detected (>50 lg/mL).

8
232
J.-T. Fan et al. / Bioorg. Med. Chem. 18 (2010) 8226–8234
Sweden), Lichroprep RP-18 gel (40–63
lM, Merck, Darmstadt,
3
Sephadex LH-20 (CHCl –MeOH, 1:1) and then purified over re-
Germany). Fractions were monitored by TLC (GF254, Qingdao
Yu-Ming-Yuan Chemical Co. Ltd., Qingdao, China), and spots were
peated silica gel H eluting with petroleum ether–acetone (6:4) to
afford RA-V (7) (4 g, 0.004%).
visualized by heating Si gel plates spray with 5% H
2
SO
4
in EtOH
3
Fr.2 (257 g) was chromatographed over silica gel using CHCl –
or 2% ninhydrin reagent. The plate was handed in a sealed glass
vessel with about 1 mL of concentrated HCl and hydrolyzed in a
drying incubator (110 °C) for 1 h, and orange spots were visualized
by spraying with 2% ninhydrin reagent.38
MeOH gradient system (95:5–9:1) to give seven subfractions
(Fr.2-1 to Fr.2-7). Fr.2-4 (104 g) was subjected to a reversed-phase
column (RP-18) eluting with MeOH–H
subfractions (Fr.2-4-1 to Fr.2-4-6). Fr.2-4-2 (18 g) were separated
by Sephadex LH-20 (CHCl –MeOH, 1:1) and then subjected to
RP-18 column (MeOH–H O, 30–50%) to afford RA-I (8) (145 mg,
.00015%). Then Fr.2-4-5 was also passed through Sephadex LH-
2
O (20–90%) to afford six
3
3
.2. Plant material
2
0
The roots of R. yunnanensis were commercially purchased from
20 (CHCl
3
2
–MeOH, 1:1), RP-18 column (MeOH–H O, 20–40%), and
the Yunnan Lv-Sheng Pharmaceutical Co. Ltd., Kunming, China. The
material was identified by Prof. Su-Gong Wu at Kunming Institute
of Botany. A voucher specimen (No. Wu20070905) has been depos-
ited in the Herbarium of Kunming Institute of Botany, Chinese
Academy of Sciences.
repeated silica gel H column (CHCl –MeOH, 20:1–10:1) to yield
rubiyunnanin C (1) (34 mg, 0.000034%).
3
3
Fr.3 (94 g) was subjected to a silica gel column using CHCl –
MeOH (70:1–9:1) to furnish five subfractions (Fr.3-1 to Fr.3-5).
Fr.3-3 (12 g) was chromatographed over a silica gel column using
petroleum ether–acetone (1:1–0:1), followed by a Sephdex LH-20
3
.3. Cell lines and reagents
3
column (CHCl –MeOH, 1:1) and then further purified by repeated
3
silica gel H eluting with CHCl –MeOH (20:1) to give RA-XXIV (9)
Eleven cancer cell lines, HEPG2 (human hepatocellular carci-
(290 mg, 0.00029%).
noma), BEL-7402 (human hepatocellular carcinoma), SMMC-7721
human hepatocellular carcinoma), MDA-MB-231 (human breast
Fr.4 (365 g) was separated by a silica gel column using EtOAc–
MeOH (20:1–8:2) to yield four subfractions (Fr.4-1 to Fr.4-4). Fr.4-
(
carcinoma), DU-145 (human prostate carcinoma), PC-3 (human
prostate carcinoma), A549 (human non-small cell lung carcinoma),
BGC-823 (human gastric carcinoma), Hela (human cervical carci-
noma), U251 (human golima) and B16 (murine melanoma) were
used in our experiments. All cancer cell lines were cultured in RPMI
3 (50 g) was separated by Sephdex LH-20 (CHCl
followed by silica gel (CHCl –MeOH, 95:5–0:1) to afford seven sub-
fractions (Fr.4-3-1 to Fr.4-3-7). Fr.4-3-5 (21 g) was further sepa-
rated by repeated RP-18 (MeOH–H O, 40–60%) and led to the
isolation of RA-XII (10) (6 g, 0.006%). Fr. 4-3-7 (46 mg) was sepa-
rated by ODS HPLC (33% ACN, 4‰ TFA) to obtain rubiyunnanin D
(2) (9 mg, 0.000009%) and rubiyunnanin E (3) (29 mg, 0.000029%).
Fr.5 (87 g) was divided into four subfractions (Fr.5-1 to Fr.5-4)
3
–MeOH, 1:1) and
3
2
1
640 medium supplemented with 10% FBS under a humidified
atmosphere of 5% CO at 37 °C. The murine monocytic macrophage
cell lines RAW 264.7 were cultured in RPMI 1640 medium
Hyclone, USA) with 10% FBS under a humidified atmosphere of
% CO at 37 °C. pNF- B-Luc vector (Clontech Laboratories, Inc.)
2
(
5
3
by CC over silica gel eluting with gradient CHCl –MeOH (95:5–
2
j
0:1). Fr. 5-3 (35 g) was subjected to CC over silica gel using
EtOAc–MeOH (20:1–8:2) to give four subfractions (Fr.5-3-1 to
Fr.5-3-4). Then Fr. 5-3-2 (4.5 g) and Fr. 5-3-4 (6 g) were separated
and pCDNA3.1(ꢀ) (Invitrogen, USA) were cotransfected into hu-
man embryonic kidney (HEK)-293 cells with a 10:1 ratio, and
HEK-293-NF-
G418 selection and cultured in HG-DMEM medium (Gibco, USA)
with 10% FBS and supplemented with 700 g/mL G418 under a
humidified atmosphere of 5% CO at 37 °C.
Roswell Park Memorial Institute 1640 (RPMI 1640) (Sigma–Al-
drich, Milan, Italy), fetal bovine serum (FBS), sulforhodamine B
j
B luciferase stable cell lines were established with
by Sephadex LH-20 (CHCl
3
–MeOH, 1:1) and subjected to RP-18
colum (MeOH–H O, 20–50%) to afford cyclopeptides-rich fractions,
2
l
respectively. RY-II (11) (57 mg, 0.000057%) was finally purified by
ODS HPLC (23% ACN, 4‰ TFA) from subfraction Fr. 5-3-2.
Rubiyunnanin G (5) (6 mg, 0.000006%) and rubiyunnanin H (6)
(8 mg, 0.000008%) were purified by ODS HPLC (40% ACN, 4‰
TFA) from subfraction Fr. 5-3-4.
2
(
SRB) (Sigma–Aldrich), trichloroacetic acid (TCA) and Tris solution
were used for the cytotoxic assay. Griess reagent (1:1, v/v, 1%
Fr.6 (151 g) was subjected to a silica gel column using CHCl
3
/
sulfanilamide and 0.1% N-(1-naphthyl) ethylenediamine dihydro-
MeOH/H O (9:1:0.1–8:2:0.2) to furnish four subfractions (Fr.6-1
2
chloride in 2.5% H
diphenyltetrazoliun bromide) (Sigma–Aldrich), lipopolysaccharide
LPS), interferon-gamma (IFN- ) were used for measurement of
NO. The single luciferase reporter assay system (Promega, USA)
and TNF- were used for NF- B luciferase assay.
3
PO
4
), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
to Fr.6-4). Fr.6-3 (27 g) was then applied to a silica gel column
using EtOAc–MeOH (20:1–8:2) to afford cyclopeptides-rich
(
c
fraction (Fr.6-3-2), then separated by RP-18 colum (MeOH–H
10–40%) to afford six subfractions (Fr.6-3-2-1 Fr.6-3-2-6).
Rubiyunnanin F (4) (22 mg, 0.000022%) was purified by ODS HPLC
2
O,
-
a
j
(
28% ACN, 4‰ TFA) from subfraction Fr. 6-3-2-1.
2
D
7
3
.4. Extraction and isolation
Rubiyunnanin C (1): White crystal; mp 253–254 °C; ½ ꢃ
a
ꢀ
3
221.0 (c 0.11, CHCl /MeOH = 1:1); UV (MeOH) kmax (log e) 203
The air-dried and powdered roots of R. yunnanensis (100 kg)
(4.80), 277 (3.78) nm; IR (KBr)
mmax 3414, 2937, 1737, 1660,
ꢀ1
1
13
were extracted three times with methanol (3 ꢁ 100 L). After re-
moval of the solvent under vacuum, the methanol extract (21 kg)
was suspended in water and partitioned successively with ethyl
acetate and n-butanol, respectively. The EtOAc part (6.4 kg) was
subjected to silica gel column chromatography eluted with
1514, 1445, 1239, 1209, 1096, 838, 803 cm ; for H and
NMR data, see Table 1; positive FABMS m/z 829 (100) [M+H] ; po-
sitive HRFABMS m/z 828.3713 [M] , cacld for
C
+
+
43 52 6 11
C H N O ,
828.3694.
2
D
3
Rubiyunnanin D (2): Amorphous powder; ½
MeOH); UV (MeOH) kmax (log ) 203 (4.69), 276 (3.70) nm; IR
(KBr) max 3421, 2938, 1659, 1516, 1448, 1203, 1138, 838,
722 cm ; for H and C NMR data, see Table 1; positive FABMS
a
ꢃ
ꢀ149.7 (c 0.57,
CHCl
3
–MeOH (1:0, 95:5, 9:1, 8:2, 0:1) to afford five fractions.
–MeOH,
5:5, 9:1, 8:2) were combined and again rechromatographed on
e
Fractions which contained cyclopeptides (1.2 kg, CHCl
9
3
m
ꢀ
1
1
13
+
+
a silica gel column eluting with a CHCl
3
–MeOH gradient system
m/z 801 (10) [M+H] ; positive HRESIMS m/z 823.3259 [M+Na] ,
cacld for C41 11Na, 823.3278.
Rubiyunnanin E (3): Amorphous powder; ½
MeOH); UV (MeOH) kmax (log ) 204 (4.81), 280 (3.87) nm; IR
(KBr) max 3426, 2938, 1664, 1515, 1446, 1204, 1134, 839, 801,
(
70:1–8:2) to yield six fractions (Fr.1–Fr.6).
48 6
H N O
2
3
Fr.1 (139 g) was applied to silica gel column chromatography
CC) using petroleum ether–acetone (7:3–0:1) to furnish five
a
ꢃ
ꢀ124.5 (c 0.58,
D
(
e
subfractions (Fr.1-1 to Fr.1-5). Fr.1-4 (30 g) was separated using
m

J.-T. Fan et al. / Bioorg. Med. Chem. 18 (2010) 8226–8234
8233
ꢀ
13
7
22 cm 1; for 1H and C NMR data, see Table 1; positive FABMS
monosaccharide mixture. The dry powder was dissolved in pyri-
dine (2 mL), -cysteine methyl ester hydrochloride (1.5 mg) was
+
+
m/z 831 (50) [M+H] ; positive HRESIMS m/z 853.3391 [M+Na] ,
cacld for C42 12Na, 853.3384.
Rubiyunnanin F (4): Amorphous powder; ½
MeOH); UV (MeOH) kmax (log ) 203 (4.87), 276 (3.80) nm; IR
max 3423, 2932, 1663, 1512, 1447, 1414, 1248, 1208, 1075,
L
H
50
N
6
O
added, and the mixture was heated at 60 °C for 1 h. Thereafter,
trimethylsilylimidazole (1.5 mL) was added to the reaction mix-
ture in ice water and kept at 60 °C for 30 min. The mixture was di-
rectly subjected to GC analysis under the following conditions:
column temperature 180–280 °C; programmed increase, at 3 °C/
2
D
5
aꢃ
ꢀ182.5 (c 0.37,
e
(
KBr) m
ꢀ1
1
13
8
39, 803 cm ; for H and C NMR data, see Table 2; positive FAB-
+
+
MS m/z 976 (92) [M+H] , 814 (13) [M+2HꢀGlc] ; positive HRESIMS
min; carrier gas, N
250 °C; injection volume, 4
tion of -glucose for compound 4 was determined by comparing
2
(1 mL/min); injector and detector temperature,
+
m/z 976.4334 [M+H] , cacld for C48
Rubiyunnanin G (5): Amorphous powder; ½
MeOH); UV (MeOH) kmax (log ) 203 (4.57), 277 (3.53) nm; IR
KBr) max 3440, 2936, 1658, 1640, 1517, 1449, 1207, 838,
10 cm 1; for 1H and C NMR data, see Table 2; positive FABMS
H
62
N
7
O
15, 976.4303.
lL; and split ratio, 1:50. The configura-
28
aꢃ
ꢀ127.3 (c 0.11,
D
D
e
the retention time with the derivative of authentic sample (
cose: 18.074 min).
D-glu-
(
8
m
ꢀ
13
+
+
m/z 905 (24) [M+H] ; positive HRESIMS m/z 927.3734 [M+Na] ,
cacld for C45 14Na, 927.3752.
Rubiyunnanin H (6): Amorphous powder; ½
MeOH); UV (MeOH) kmax (log ) 203 (4.81), 276 (3.83) nm; IR
KBr) max 3424, 2933, 1657, 1640, 1512, 1248, 1075, 826 cm ;
Acknowledgements
56 6
H N O
2
D
7
aꢃ
ꢀ244.0 (c 0.12,
This work was supported by the National Natural Science Foun-
dation of China (30725048), National Basic Research Program of
China (2009 CB522300), the Fund of Chinese Academy of Sci-
ences (KSCX2-YW-R-177), the Innovative Group Program from
the Science and Technology Department of Yunnan Province
e
ꢀ1
(
m
1
13
for H and C NMR data, see Table 2; positive FABMS m/z 935
95) [M+H] ; positive HRESIMS m/z 957.3882 [M+Na] , cacld for
+
+
(
C
46
H
58
N
6
O
15Na, 957.3857.
(
2008OC011) and the Fund of State Key Laboratory of Phytochem-
istry and Plant Resources in West China, Kunming Institute of Bot-
any, Chinese Academy of Sciences. We thank Mrs. Ogunlana
Olubanke and Dr. Abiodun Humphrey Adebayo for proof reading
the manuscript.
3
.5. Marfey’s analysis of the absolute configuration of amino
acids
Hydrolysis of compounds 1–6 (1 mg each) were achieved in sep-
arate experiments, by the addition of 1 mL of 6 N HCl at 115 °C for
8 h. The hydrolyzate was evaporated to dryness under a stream of
to remove traces of HCl. Then, the resultant hydrolyzate was
redissolved in 900 L of acetone, thereafter, 1 M NaHCO (20 L)
and a 1% solution of N -(2,4-dinitro-5-fluorophenyl)- -alaninamide
-FDAA, Marfey’s reagent, Sigma, 100 L) in acetone were added,
and the mixture was heated at 40 °C for 1 h. The reaction mixture
was cooled to RT and quenched by addition of 2 N HCl (10 L), dried,
anddissolvedin50%aqueousCH CN(600 L). Fivemicrolitersofthe
FDAA derivative was analyzed by HPLC using a C18 column (Agilent,
.6 mm ꢁ 150 mm, 5 m, Zorbax Eclipse-C18) maintained at 30 °C.
Aqueous CH CN containing 4‰ TFA was used as the mobile phase
with linear gradient elution (20–35%, 40 min) at a flow rate of
mL/min. FDAA-derivatived amino acids were detected by absorp-
tion at 340 nm.
To 50 L of a 50 mM aqueous solution of
of Ala (no hydrolysis), Glu (no hydrolysis) and Glu (hydrolysis with
N HCl, 115 °C, 18 h) were added 1 M NaHCO (20 L) and a 1%
solution of -FDAA (100 L) in acetone, respectively. Each mixture
was heated at 40 °C for 1 h. The reaction mixture was cooled to RT
and quenched by the addition of 2 N HCl (10 L). It was dried, dis-
solved in 50% aqueous CH CN (600 L). Then 5 L of the FDAA
Supplementary data
1
N
2
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.10.019. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
l
3
l
a
L
(L
l
l
References and notes
3
l
1.
Dian Nan Ben Cao; Lan, M., Ed.; Yunnan People’s Publishing House: Kunming,
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4
l
2.
3.
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D and L-configurations
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l
l
derivative was analyzed by HPLC.
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6
6, 638.
L
-FDAA derivatives of standard
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D
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L
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1
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2
L
derivatives of acid hydrolysates of compounds 1–6 were summa-
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-Glu (18.218), -Ala (19.522); 3, -Ala (14.388), -Glu
-Ala (14.248), -Ala (19.578); 5, -Ala
-Ala (14.372), -Ala (19.794).
1
L
D
L
2
2
0. Lee, J. E.; Hitotsuyanagi, Y.; Takeya, K. Tetrahedron 2008, 64, 4117.
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(18.212),
(14.395),
L
D
D
D
L
L
-Ala (19.512); 4,
-Ala (19.710); 6,
L
D
L
2008, 56, 730.
L
D
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