Astins K-P, six new chlorinated cyclopentapeptides from Aster tataricus

Article abstract of DOI:10.1016/j.tet.2013.07.006

During further investigation on the methanol extract of roots and rhizomes of Aster tataricus, six new chlorinated cyclopentapeptides, designated as astins K-P (9-14), together with eight known derivatives, astins A-H (1-8), were isolated. Structures of the new cyclopeptides were established using extensive spectroscopic methods, and the absolute configurations were determined by the advanced Marfey's method. Astin P (14) represents a unique cyclopentapeptide linking as cyclo-(l-Pro (Cl₂)¹-l-allo-Thr ²-l-Ser³-l-β-Phe⁴-l-Ava⁵). Astin B (2) showed cytotoxicity against BGC-823 cell with IC₅₀ value of 19.2 μg/ml. Astin C (3) exhibited cytotoxicity on HCT-116 and BGC-823 cells with IC₅₀ values of 13.4 and 3.3 μg/ml, respectively.

Full text of DOI:10.1016/j.tet.2013.07.006

Tetrahedron 69 (2013) 7964e7969
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Astins KeP, six new chlorinated cyclopentapeptides from Aster
tataricus
,
b
,
,
,
,
*
Hui-Min Xu a y, Guang-Zhi Zeng a y, Wen-Bing Zhou a b, Wen-Jun He a, Ning-Hua Tan a
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming 650201, PR China
b University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 May 2013
Received in revised form 19 June 2013
Accepted 3 July 2013
Available online 8 July 2013
During further investigation on the methanol extract of roots and rhizomes of Aster tataricus, six new
chlorinated cyclopentapeptides, designated as astins KeP (9e14), together with eight known derivatives,
astins AeH (1e8), were isolated. Structures of the new cyclopeptides were established using extensive
spectroscopic methods, and the absolute configurations were determined by the advanced Marfey’s
method. Astin P (14) represents a unique cyclopentapeptide linking as cyclo-(
L L L-
-Pro (Cl2)1- -allo-Thr2-
Ser3- -Phe4- -Ava5). Astin B (2) showed cytotoxicity against BGC-823 cell with IC50 value of 19.2
Astin C (3) exhibited cytotoxicity on HCT-116 and BGC-823 cells with IC50 values of 13.4 and 3.3
L-
b
L
m
m
g/ml.
g/ml,
Keywords:
Aster tataricus
Cyclopentapeptides
Compositae
respectively.
Ó 2013 Elsevier Ltd. All rights reserved.
Astins KeP
Cytotoxicity
1. Introduction
by inducing activated T-cell apoptosis.9 Intrigued by the distinctive
chlorinated proline feature and their potential antitumor and im-
munosuppressive activities,9,10 six new chlorinated cyclo-
pentapeptides, designated as astins KeP (9e14), together with eight
known derivatives, astins AeH (1e8), were isolated from the
methanol extract of roots and rhizomes of the titled plant (Fig. 1). In
this paper, we reported their structure elucidation, absolute con-
figuration assignments, and biological activities (cytotoxic, anti-
HBV and immunosuppressive activities).
In the frame of our long-term research program directed at
discovery of structurally unique and potentially useful cyclopeptide
drugs from traditional Chinese medicines (TCMs),1 we had the op-
portunity to investigate the TCM Aster tataricus Linn. f. (Compositae,
Chinese name ‘zi-wan’). A. tataricus, widespread in China, has been
used for relieving cough and eliminating phlegm, and documented
in Chinese Pharmacopoeia.2 It has been described to produce many
natural compounds of biological interest, such as triterpenes, ste-
rols and cyclopeptides.3 Previous chemical investigation on this
plant has led to the isolation of nine cyclopentapeptides (astins
AeI),4 and two cyclotetrapeptides (tataricins A and B).5 These
cyclopeptides belong to a family of Compositae-type cyclopeptides
or astins, and share remarkable structural similarity, which are
characterized by the presence of four nonproteinogenic amino acids
2. Results and discussion
2.1. Structure determination
The methanol extract of the air-dried and powdered roots and
rhizomes of A. tataricus was partitioned between EtOAc and n-bu-
tanol. The EtOAc portion was subjected to a series of silica gel,
Sephadex LH-20, RP-18 column chromatography (CC) and HPLC to
afford six new cyclopentapeptides, astins KeP (9e14), together
with eight known ones, astins AeH (1e8). The known compounds
were identified by comparing to literature data.4 Astin C (3) was
further confirmed by an X-ray experiment (CCDC 933250, see S8,
Supplementary data (SM)).
(L-b
-Phe,
L
-Abu,
L
-allo-Thr, chlorinated
L-Pro derivatives) and one
proteinogenic amino acid
(L
-Ser).6,7 Besides their interesting
structures, these cyclopeptides also show potential biological ac-
tivities. Astins AeC (1e3) have shown the potent antitumor activity
by using Sarcoma 180A in mice.4a,8 Moreover, it is noteworthy that
astin C (3) was recently found to have immunosuppressive activity
Astin K (9), obtained as an amorphous powder, was a cyclo-
peptide as evidenced by its positive reaction with ninhydrin using
a thin layer chromatography (TLC) protosite acidic hydrolysis.11 Its
* Corresponding author. Tel./fax: þ86 871 65223800; e-mail address: nhtan@
mail.kib.ac.cn (N.-H. Tan).
y
These authors contributed equally to this paper.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.07.006

H.-M. Xu et al. / Tetrahedron 69 (2013) 7964e7969
7965
was established to cis like that of 3, which was confirmed by the
similar coupling constants in Pro (Cl2) of the astins, the NOE cor-
relation observed between Hb and Hg of Pro (Cl2) in the ROESY
spectrum, and X-ray crystallographic analysis (Figs. 2 and 3, see S8,
SM). In the HMBC spectrum the correlations were showed from
allo-Thr5-NH to -Phe4-CO, -Phe4-NH to Ser3-CO, Ser3-NH to allo-
b b
Thr2-CO, allo-Thr2-NH to Pro (Cl2)1eCO, and Pro (Cl2)1-Ha and Pro
(Cl2)1-Hd to allo-Thr5-CO, which established the sequence of cyclo-
(Pro (Cl2)1-allo-Thr2-Ser3- -Phe4-allo-Thr5).
b
R1
H
OH
H
H
OH
H
H
R2
R3 R4 R5 R6
OH
Cl Cl Cl OH
Cl Cl OH
X-Y
1
2
3
4
5
6
7
8
9
OH Cl Cl
H
H
H
H
H
H
OH
H
CH-CH
CH-CH
CH-CH
C=C
H
H
H
Cl
H
H
H
H
H
H
H
Cl OH
Cl OH
C=C
H
H
OH
OH
CH-CH
CH-CH
C=C
CH-CH
C=C
H
Cl OH
OH
Cl OH
OH OH Cl Cl
H
10 OH OH
H
H
H
H
Cl
Cl
11
12
13
H
H
H
H
H
H
H
H
H
OH
OH
CH-CH
C=C
Cl Cl
OAc CH-CH
Fig. 3. X-ray structure of astin C (3).
Fig. 1. Structures of compounds 1e14.
The absolute configuration of 9 was assigned using advanced
Marfey’s method12 and LCeMS analysis. The results indicated that
molecular formula, C25H33N5O8Cl2, was deduced from the HRESIMS
at m/z 602.1788 [MþH]þ, indicating 11 degrees of unsaturation. Its
UV spectrum peaks at 203 and 266 nm suggested the existence of
a phenyl group. The IR absorption bands at 3404 and 1649 cmꢀ1
indicated the presence of OH, NH, and C]O groups. In the 1D NMR
the absolute configurations at the
tion for Ser, Thr, -Phe residues (see Table S1, SM). In the ROESY
spectrum, the presence of a strong cross peak between Ha in Pro
(Cl2)1 and Ha in Thr5 indicated the configuration of Pro (Cl2)1 and
a cis peptide bond like that of astin AeH.4,6 Because the threonine
residue has two stereogenic centers, the -allo-Thr and -Thr stan-
dards were carried out and subjected to the advanced Marfey’s
analysis using HPLC. The identical retention times of the -Marfey
derivatized -allo-Thr and Thr in the hydrolyzates of 9 suggested an
-configuration of the allo-Thr residue. Therefore, 9 was established
as depicted in Fig. 1.
Astin (10) was obtained as amorphous powder. Its molecular
a-carbons were the L configura-
b
L
spectra, four amide protons at
d 8.21, 9.56, 10.13 and 10.21 ppm and
L
L
five amide carbonyls at 168.7, 171.4, 171.8, 172.7 and 175.2 ppm
d
were clearly observed, which supported 9 could be a cyclo-
pentapeptide with a proline residue. Detail analysis of the 1D NMR
L
L
spectra, two methyls [dH C
Thr5-
)], two methylenes [dH/C 2.63 and 3.27/43.9 (
and 5.12/52.2 (Pro (Cl2)1-
)], one O-methylene [dH C
61.5 (Ser3- 4.73/59.4 (Ser3-
)], seven methines [dH C
(Pro (Cl2)1- ), 5.30/58.6 (allo-Thr2- ), 5.53/60.4 (allo-Thr5-
66.8 (Pro (Cl2)1- -Phe4- ), 6.42/66.8 (Pro (Cl2)1-
), 5.73/53.0 (
two O-methines [dH C
4.57/69.0 (allo-Thr5- ), 4.95/67.6 (allo-Thr2-
)] and one monosubstituted benzene ring [dH/C 7.10/127.4 (
-Phe4-
), 7.14/128.9 ( ), dC 143.2 ( )]
-Phe4-ε), 7.40/126.4 ( -Phe4- -Phe4-
were showed. The five amino acid residues were ascribed to one
serine (Ser), two threonines (Thr), one -phenylalanine ( -Phe) and
/
1.73/23.2 (allo-Thr2-
g
), 1.77/22.6 (allo-
-Phe4-
); 3.94
4.21 and 4.40/
), 4.78/56.1
), 5.55/
)],
L
g
b
a
d
/
L
b
g
/
a
a
formula was determined as C25H32N5O8Cl according to the
[MþNa]þ peak at 588.1847 in the HRESIMS spectrum. Analysis of
the 1D NMR spectra revealed that all proton and carbon signals of
10 closely resembled to those of 9 except for the signals in the Pro
residue. In the Pro residue of 10, the presence of a trisubstituted
a
b
b
b
a
/
b
b
b
z
b
b
d
b
g
double bond was established by the chemical shifts of dH/C 5.95/
124.7 (Pro1- ) and dC 127.8 (Pro1- ). The 1He1H COSY correlations
g s
b
b
of Ha/Hb (2H)/Hg and HMBC correlation of Hb with allo-Thr2-CO
one dichloroproline (Pro (Cl2)) on the basis of the observations
above together with the 2D NMR spectra (1He1H COSY, HSQC and
HMBC, Fig. 2). In addition, the Pro residue containing two chlorine
atoms was disclosed to be 2,3-dichloroproline by the 1He1H COSY
correlations of Ha/Hb/Hg/Hs (2H) in Pro (Cl2)1 residue as shown in
Fig. 2. The relative configuration of the two chlorines in Pro (Cl2)
indicated that the double bond was located at Cg and Cs, and the
only chlorine atom was attached to Cs (see S3, SM). Thus, the planar
O4 (5)
structure of 10 was determined as cyclo-(
Pro (Cl)1-allo-Thr2-
Ser3- -Phe4-allo-Thr5).
b
Astin M (11) showed a pseudomolecular ion peak at m/z
558.2089 [MþNa]þ in the HRESIMS spectrum, indicating a molec-
ular formula of C25H34N5O6Cl. Compared the 1D NMR data and
molecular formula of 11 with those of 3,4a it showed that the only
difference between them was also happened in the Pro1 residue, in
which the chlorine atom in Cb of 3 was displaced with one hy-
drogen atom in 11. The change was further supported by the 1He1H
COSY correlations of Ha/Hb (2H)/Hg/Hs (2H) in the Pro residue. The
configuration of Hg was
correlation of Hg with Ha (see S4, SM). Thus, 11 was established as
cyclo-(Pro (Cl)1-Abu2-Ser3- -Phe4-Abu5).
a-oriented as evidenced by the ROESY
b
Astin N (12) had the molecular formula C25H32N5O6Cl based on
the HRESIMS spectrum ([MþNa]þ m/z 556.1955); 2 mass units less
than that of 11. Comparison of the 1D NMR data of 12 with those of 11
strongly suggested a similar structure of both compounds. One ap-
Fig. 2. a. Selected 1He1H COSY, HMBC, ROESY correlations of 9. b. Selected coupling
(Hz) and ROESY correlations in Pro (Cl2)1 of 9.
parent change in 12 was one more double bond (dH 6.41 (Pro-
s) and
dC 130.0 (Pro- ),124.5 (Pro- )) appearance in the Pro residue. It was
g
s

7966
H.-M. Xu et al. / Tetrahedron 69 (2013) 7964e7969
located between the Cg and Cs based on the HMBC correlation of Hs
indicated that the cis dichlorinated proline residue and the number
of hydroxyl groups in the side chains play important roles. These
findings are beneficial to further synthesize new astin analogues
and discover drug candidates.
with Pro (Cl)1eCO (see S5, SM). Thus the planar structure of 12 was
O4 (5)
elucidated as cyclo-(
Pro (Cl)1-Abu2-Ser3- -Phe4-Abu5).
b
The molecular formula (C27H35N5O7Cl2) of Astin O (13) was
deduced from the HRESIMS analysis of the pseudomolecular ion
peak at m/z 634.1802 [MþNa]þ. Its 1D NMR spectra were quite
consistent with those of 3 except for the resonance of Ser3 resi-
due.4a In the 13C NMR spectrum of 13, a characteristic acetyl group
4. Materials and methods
4.1. General experimental procedures
[
dC 20.9 (CH3COe) and 171.0 (CH3COe)] was observed. It was at-
tached to the hydroxyl group of Ser3 residue as evidenced by the
HMBC resonances from Hb1 4.97 (dd,11.4, 4.2) and Hb2 5.12 (dd,11.4,
9.0) to [dC 171.0 (CH3eCOeOe)] (see S6, SM). Accordingly, 13 was
elucidated to be an analogue of 3 in which Ser3 was substituted by
acetoxylation (Fig. 1). So, the planar structure of 13 was cyclo-(Pro
Optical rotations were measured on a Horiba SEPA-300 polar-
imeter. IR spectra were obtained on a Tensor 27 spectrometer with
KBr pellets. UV spectra were recorded using a Shimadzu UV-2401A
spectrophotometer. 1D and 2D NMR spectra were performed on
a Bruker AVANCE III-600 spectrometer with TMS as the internal
standard. Mass spectra were measured on a VG Auto Spec-3000 or
API-Qstar-Pulsar instrument. Analytical or semi-preparative HPLC
was performed on Agilent 1100 with Zorbax Eclipse-C18
(Cl2)1-Abu2- (Ac-Ser)3- -Phe4-Abu5).
b
Astin P (14) was isolated as an amorphous powder. It showed
a [MꢀH]ꢀ peak at m/z 598.1820 in the HRESIMS spectrum, which
was consistent with the molecular formula of C26H35N5O7Cl2 ac-
counting for 11 degrees of unsaturation. Comparison of the 1D NMR
(4.6 mmꢁ150 mm, 1 mL/min; 9.4 mmꢁ250 mm, 3 mL/min; 5
mm).
Column chromatography was performed using silica gel (100e200
and 200e300 mesh, Qingdao Yu-Ming-Yuan Chemical Co. Ltd.,
Qingdao, P. R. China), Sephadex LH-20 (Pharmacia Fine Chemical
spectra of 9 and 14 revealed that a methine [dH C
Thr5-
)] in 9 was replaced with two methylene signals [dH C
20.1 (Ava5- ), 1.86/32.9 (Ava5-
)] in 14, indicating that the allo-Thr
/
4.57/69.0 (allo-
b
/
1.65/
g
b
Co., Uppsala, Sweden) or Lichroprep RP-18 gel (40e63 mm, Merck,
residue in 9 was replaced by the Ava residue in 14. This change was
further supported by the 1He1H COSY data of Ha/Hb (2H)/Hg (2H)/
Hs (3H) in Ava5 residue (see S7, SM) and the acidic hydrolysis ex-
periment. It is should be noteworthy that this is the first time for
having found an Ava residue in the Compositae-type cyclo-
pentapeptides.4,6,7 Thus, 14 was identified as a unique cyclo-
Darmstadt, Germany). Fractions were monitored by TLC (GF254,
Qingdao Yu-Ming-Yuan Chemical Co. Ltd., Qingdao, P. R. China), and
spots were visualized by the chemical detection of cyclopeptides.11
4.2. Plant material
pentapeptide linking as cyclo-(Pro (Cl2)1-allo-Thr2-Ser3-
b
-Phe4-
Roots and rhizomes of A. tataricus were commercially purchased
from the Yunnan Lv-Sheng Pharmaceutical Co. Ltd., P. R. China, and
identified by Professor Xi-Wen Li at the Kunming Institute of Botany,
Chinese Academy of Sciences. Avoucher specimen (No. 200704) was
deposited in the Herbarium of Kunming Institute of Botany.
Ava5) (Fig. 1).
Except the variable proline derivatives, 9 and 10 have the same
residues and 11e13 also have the same residues. Since small
amounts of 10, 12 and 13 were obtained, only 9, 11, 14 were sub-
jected to establish their absolute configuration of the amino acid
residues by the advanced Marfey’s method described above. The
results were depicted in the SM (Table S1) and suggested that all
4.3. Extraction and isolation
the amino acid residues were
L
configurations. Furthermore, the
The air dried and powdered roots and rhizomes of A. tataricus
(50 kg) were extracted three times with methanol (3ꢁ50 L). The
extract was concentrated to 13 kg, and then was suspended in
water and partitioned successively with ethyl acetate (EtOAc) and
n-butanol, respectively. The EtOAc fraction (2 kg) was subjected to
silica gel CC and eluted with gradient CHCl3/MeOH (20:1, 10:1,
85:15, 8:2, and 0:1) to yield six fractions (Fr.1eFr.6).
strong cross peaks between Ha of Pro1 residue and Ha of the fifth
residue in the ROESY spectrum of 11 and 14 revealed the Pro resi-
due was also
L
configuration and cis peptide bond like that of 9. The
configuration of the Thr residues in 10 and 14 was indicated as
allo-Thr by using HPLC method as described in 9. Therefore, the
structures of 9e14 were finally established and shown in Fig. 1.
L-
Fr.1 (320 g) was chromatographed over silica gel using gradient
petroleum ether/acetone (10:1e1:1) to furnish three subfractions
(Fr.1-1eFr.1-3). Fr.1-2 (50 g) was subjected to silica gel column us-
ing gradient CHCl3/acetone (15:1e5:1) to afford subfractions (Fr.1-
2-1eFr.1-2-3). Fr.1-2-1 (2 g) was applied to Sephadex LH-20 using
CHCl3/MeOH (1:1), and then silica gel CC (CHCl3/EtOAc, 10:1e5:1)
to yield two subfractions (Fr.1-2-1-1eFr.1-2-1-2). Astin L (10)
(5 mg) was afforded from subfraction Fr.1-2-1-1 (34 mg) by a fur-
ther purification using semi-HPLC (20% CH3CN, 0.5% TFA). Addi-
tionally, Fr.1-2-2 (4 g) was separated by a silica gel column eluting
with CHCl3/EtOAc (1:3) and then purified by repeated silica gel CC
eluting with CHCl3/MeOH (9:1) to obtain astin K (9) (30 mg).
Fr.2 (75 g) was subjected to silica gel CC and eluted with gradient
petroleum ether/acetone (2:1e1:2) to yield six subfractions (Fr.2-
1eFr.2-6). Fr.2-2 (14 g) was separated into four subfractions (Fr.2-2-
1eFr.2-2-4) on a silica gel column using CHCl3/MeOH (7:1). Fr.2-2-1
(282 mg) was firstly purified by Sephadex LH-20 (CHCl3/MeOH,
1:1), and then by semi-HPLC (35% CH3OH, 0.5% TFA) to yield astin F
(6) (10 mg), astin H (8) (20.9 mg), and astin M (11) (7 mg). Fr.2-3
(21 g) was separated by Sephadex LH-20 (CHCl3/MeOH, 1:1) and
followed by silica gel CC (CHCl3/MeOH, 20:1e5:1) to afford five
subfractions (Fr.2-3-1eFr.2-3-5). Fr.2-3-4 (3 g) was purified by
semi-HPLC to obtain astin N (12) (5 mg). Fr.2-5 (4 g) was separated
2.2. Biological assays
In the present study, compounds 1e14 were tested for cytotoxic
activity against BEL-7402, BGC-823, A549, HeLa, and HCT-116 cell
lines by SRB or MTT method.13 Only 2 showed cytotoxicity against
BGC-823 cell with IC50 value of 19.2
icity on HCT-116 and BGC-823 cells with IC50 values of 13.4 and 3.3
ml, respectively. In addition,1e14 were tested foranti-HBV activityon
Hep G 2.2.15 cell;14 but they exhibited no inhibitory activity against
the secretion of HBsAg and HBeAg. Moreover, 2, 4, 9 were also ex-
amined forimmunosuppressiveactivityagainstactivatedlymphnode
cells from mice by the MTT method.9 Unfortunately, none of them
exhibited obvious immunosuppressive activity.
m
g/ml, and 3 exhibited cytotox-
mg/
3. Conclusions
To the best of our knowledge, astins are the only chlorinated
cyclopeptides in higher plants and obtained only from A. tataricus.
Therefore, discovery of these six new chlorinated cyclopeptides
astins KeP (9e14) expands the cyclopeptide diversity in A. tataricus
and could be considered as one of characteristic constituents for
this TCM quality control. Furthermore, cytotoxic activity results

H.-M. Xu et al. / Tetrahedron 69 (2013) 7964e7969
7967
Table 1
by a silica gel column (CHCl3/MeOH, 20:1e5:1) to get astin D (4)
1H (600 MHz) and 13C (150 MHz) NMR data for compounds 9 and 10 in pyridine-d5
in ppm, J in Hz)
(d
(1 g). Fr.2-6 (1.2 g) was subjected to Sephadex LH-20 eluted with
CHCl3/MeOH (1:1) to give two subfractions (Fr.2-6-1eFr.2-6-2).
Fr.2-6-1 (88.7 mg) was further fractionated by silica gel CC eluting
with EtOAc/MeOH (25:1) to afford astin A (1) (13.6 mg). Fr.2-6-2
(535 mg) was repeated submitted to CC (CHCl3/MeOH, 20:1),
then purified by silica gel CC (CHCl3/acetone) to afford astin O (13)
(3 mg).
Fr.3 (55 g) was separated into five subfractions (Fr.3-1eFr.3-5)
by silica gel CC using elution with EtOAc/CHCl3 (3:1-5:1). Fr.3-2
(40 g) was further purified by Sephadex LH-20 (CHCl3/MeOH,
1:1), and then by recrystallization (CHCl3/MeOH) to yield astin C (3)
(8 g).
Fr.4 (85 g) was divided into two subfractions (Fr.4-1eFr.4-2).
Then Fr.4-2 (60 g) was separated by repeated RP-18 (MeOH/H2O,
25%e70%) and led to obtain three subfractions (Fr.4-2-1eFr.4-2-3).
Fr.4-2-2 (14 g) was passed through Sephadex LH-20 (CHCl3/MeOH,
1:1) and repeated silica gel CC (CHCl3/MeOH, 20:1-5:1) to afford
four subfractions (Fr.4-2-2-1eFr.4-2-2-4). Astin P (14) (15.1 mg)
was purified by semi-HPLC (45% CH3OH, 0.5% TFA) from subfraction
Fr.4-2-2-1 (300 mg). Fr.4-2-2-2 (4 g) was submitted to repeated
silica gel CC, and purified by Sephadex LH-20 to afford astin E (5)
(1.1 g). Astin G (7) (20 mg) was given from Fr.4-2-2-4 (292 mg) by
semi-HPLC (15% CH3OH, 0.5% TFA).
9
10
dC
dH, mult
dC
dH, mult
Pro1
a
66.8
6.42 (d, 4.8)
70.4
6.37 (m)
b1
b2
66.8
5.55 (overlapped)
53.3
4.41 (overlapped)
4.75 (m)
g
d1
d2
56.1
52.2
4.78 (m)
3.94 (t, 11.0)
5.12 (dd, 11.0, 6.9)
124.7
127.8
5.95 (m)
C]O
allo-Thr2
a
b
g
168.7
169.3
58.6
67.6
23.2
5.30 (t, 9.8)
4.95 (m)
1.73 (d, 6.0)
59.8
69.3
22.2
5.36 (overlapped)
4.65 (overlapped)
1.63 (d, 6.6)
C]O
NH
Ser3
a
b1
b2
171.8
172.4
9.56 (d, 9.8)
8.77 (d, 9.0)
59.4
61.5
4.73 (m)
4.21 (dd, 11.1, 3.3)
4.40 (dd, 11.1, 5.1)
59.8
61.1
4.61 (m)
4.47 (dd, 10.5, 4.5)
4.65 (overlapped)
C]O
NH
b
a1
a2
b
g
d
ε
171.4
43.9
170.4
42.1
10.13 (d, 4.2)
10.47 (d, 6.0)
-Phe4
2.63 (t, 12.8)
3.27 (dd, 12.8, 5.1)
5.73 (m)
2.75 (dd, 13.5, 7.2)
3.09 (dd, 13.5, 5.4)
5.47 (overlapped)
Fr.5 (146 g) was subjected to a silica gel CC using CHCl3/MeOH
(15:1e5:1) to furnish two subfractions (Fr.5-1eFr.5-2). Fr.5-2 (50 g)
was purified by Sephadex LH-20 (CHCl3/MeOH, 1:1) to yield astin B
(2) (15 g).
53.0
143.2
126.4
128.9
127.4
172.7
52.8
143.6
127.3
129.2
127.5
172.2
7.40 (d, 7.2)
7.14 (t, 7.2)
7.10 (t, 7.2)
7.48 (d, 7.6)
7.28 (t, 7.6)
7.17 (t, 7.6)
z
C]O
4.3.1. Astin K (9). Amorphous powder; ½a D17:3
ꢀ88.7 (c 0.14, MeOH);
ꢂ
NH
8.21 (d, 6.0)
8.21 (d, 6.0)
allo-Thr5
UV (MeOH) lmax (log ε) 203 (3.11), 266 (4.40) nm; IR (KBr) nmax
a
b
g
60.4
69.0
22.6
5.53 (overlapped)
4.57 (m)
1.77 (d, 6.0)
59.6
69.0
22.2
5.36 (overlapped)
4.43 (overlapped)
1.55 (d, 6.0)
3404, 2935, 1649, 1533, 1423, 1312, 1080, 758, 700, 588 cmꢀ1 1H
;
(600 MHz) and 13C (150 MHz) NMR, see Table 1; positive ESIMS m/z
602 [MþH]þ; HRESIMS m/z 602.1788 [MþH]þ (calcd for
C25H34N5O8Cl2, 602.1784).
C]O
NH
175.2
172.9
10.21 (d, 3.6)
10.09 (d, 6.6)
4.3.2. Astin L (10). Amorphous powder; ½a D24:6
ꢀ357.6 (c 0.05,
ꢂ
Table 2
1H (600 MHz) and 13C (150 MHz) NMR data for compounds 11 and 12 in pyridine-d5
MeOH); UV (MeOH) lmax (log ε) 208 (4.70), 284 (2.97) nm; IR (KBr)
nmax 3421, 2930, 1646, 1434, 1204, 1080, 703, 527 cmꢀ1 1H
;
(d
in ppm, J in Hz)
(600 MHz) and 13C (150 MHz) NMR, see Table 1; negative ESIMS m/
z 564 [MꢀH]ꢀ, 1129 [2MꢀH]ꢀ; HRESIMS m/z 588.1847 [MþNa]þ
(calcd for C25H32N5O8ClNa, 588.1837).
11
12
dC
dH, mult
dC
dH, mult
Pro1
4.3.3. Astin M (11). Amorphous powder; ½a D24:2
ꢂ
ꢀ52.2 (c 0.20,
a
b1
60.9
41.2
5.10 (overlapped)
2.78 (m)
69.0
56.9
5.71 (m)
4.58 (m)
MeOH); UV (MeOH) lmax (log ε) 207 (4.01) nm; IR (KBr) nmax 3430,
b2
3.29 (d, 8.4)
4.78 (d, 16.2)
2934, 1636, 1549, 1451, 1288, 1204, 1132, 702, 517 cmꢀ1 1H
;
g
d1
d2
57.1
58.3
4.73 (t, 5.1)
4.13 (d, 14.3)
4.33 (dd, 14.3, 5.1)
130.0
124.5
(600 MHz) and 13C (150 MHz) NMR, see Table 2; positive ESIMS m/z
536 [MþH]þ; 1093 [2MþNa]þ; HRESIMS m/z 558.2089 [MþNa]þ
(calcd for C25H34N5O6ClNa, 558.2095).
6.41 (br s)
C]O
Abu2
a
b1
b2
172.3
170.1
55.9
24.7
5.15 (m)
2.26 (m)
56.1
25.7
5.10 (m)
2.04 (m)
2.14 (m)
0.98 (t, 7.6)
4.3.4. Astin N (12). Amorphous powder; ½a D24:4
ꢀ105.8 (c 0.05,
ꢂ
MeOH); UV (MeOH) lmax (log ε) 207 (4.12) nm; IR (KBr) nmax 3406,
2972, 1639, 1533, 1431, 1319, 1207, 1140, 1079, 700 cmꢀ1 1H
;
g
11.8
172.8
1.11 (t, 7.2)
9.30 (d, 9.0)
11.3
172.8
(600 MHz) and 13C (150 MHz) NMR, see Table 2; positive ESIMS m/z
556 [MþNa]þ; 1089 [2MþNa]þ; HRESIMS m/z 556.1955 [MþNa]þ
(calcd for C25H32N5O6ClNa, 556.1938).
C]O
NH
Ser3
a
b1
b2
8.91 (d, 8.4)
60.4
61.5
4.76 (m)
4.29 (dd, 11.1, 3.9)
4.46 (dd, 11.1, 5.1)
60.3
61.2
4.70 (m)
4.44 (dd, 11.1, 4.5)
4.54 (m)
4.3.5. Astin O (13). Amorphous powder; ½a D23:7
ꢀ113.3 (c 0.05,
ꢂ
C]O
NH
b
a1
a2
b
171.2
43.8
170.8
42.8
MeOH); UV (MeOH) lmax (log ε) 207 (4.10), 262 (3.10) nm; IR (KBr)
8.49 (d, 5.4)
9.19 (d, 6.0)
nmax 3422, 2923, 2853, 1646, 1530, 1461, 1239, 701 cmꢀ1 1H
;
-Phe4
(600 MHz) and 13C (150 MHz) NMR, see Table 3; positive ESIMS m/z
634 [MþNa]þ, 1247 [2MþNa]þ; HRESIMS m/z 634.1802 [MþNa]þ
(calcd for C27H35N5O7Cl2Na, 634.1811).
2.65 (t, 12.6)
3.22 (dd, 12.6, 4.8)
5.67 (m)
2.80 (dd, 13.4, 9.6)
3.10 (dd, 13.4, 5.1)
5.60 (m)
53.3
143.6
126.7
129.1
52.8
143.5
127.1
129.2
g
d
7.39 (d, 7.2)
7.14 (t, 7.2)
7.45 (d, 7.2)
7.24 (overlapped)
4.3.6. Astin P (14). Amorphous powder; ½a D24:7
ꢀ21.7 (c 0.03,
ꢂ
ε
MeOH); UV (MeOH) lmax (log ε) 205 (3.81), 254 (3.73) nm; IR (KBr)
(continued on next page)

7968
H.-M. Xu et al. / Tetrahedron 69 (2013) 7964e7969
Table 2 (continued )
acetone. To each a half portion (50
m
L) were added 20
-leucinamide or 1-
fluoro-2,4-dinitrophenyl-5-D-leucinamide (L-FDLA or D-FDLA, 1% in
mL NaHCO3
(1 M) and 100 L 1-fluoro-2,4-dinitrophenyl-5-
m
L
11
12
dC
dH, mult
dC
dH, mult
acetone), and the mixture was heated at 45 ꢃC for 1.5 h. Reaction
was cooled to room temperature, and then acidified with 2 N HCl
z
127.6
172.8
7.09 (m)
127.6
172.4
7.16 (t, 7.2)
C]O
NH
Abu5
a
(10
each solution of FDLA derivatives were analyzed by LC/MS.
The analysis of the - and D, -FDLA (mixture of - and
derivatives was performed using an Agilent Eclipse XDB-C18 col-
umn (4.6ꢁ150 mm, 5
m) maintained at 40 ꢃC. Acetonitrile/0.1%
mL), dried and dissolved in 50% aqueous CH3CN. About 5 mL of
8.40 (d, 6.0)
9.05 (d, 6.6)
55.2
24.6
5.10 (overlapped)
1.81 (m)
1.87 (m)
53.9
24.9
5.03 (m)
1.77 (m)
1.90 (m)
1.77 (d, 6.0)
L
L
D
L-FDLA)
b1
b2
m
g
10.9
1.01 (t, 7.5)
10.9
172.1
HCOOH/H2O was used as the mobile phase under a linear gradient
elution mode (acetonitrile, 30%e50%, 50 min) at a flow rate of 1 mL/
min. A Waters Xevo TQ-S mass spectrometer was used for detection
in ESI (negative) mode. The capillary voltage was kept at 2.5 KV, and
the ion source at 350 ꢃC. Nitrogen gas was used as a sheath gas at
400 L/h. A mass range of m/z 100e1000 was scanned in 0.2 s. The
retention times (tR, min) of the Marfey-derivatized amino acids are
summarized in Table S1 (see SM). However, this was not conclusive
since the allo-Thr standard was not used. The Thr residues in
compounds were subsequently confirmed on the basis of chemical
manipulation using the advanced Marfey’s method as follows.
C]O
NH
172.9
10.12 (d, 3.6)
9.94 (d, 4.8)
Table 3
1H (600 MHz) and 13C (150 MHz) NMR data for compounds 13 and 14 in pyridine-d5
(d
in ppm, J in Hz)
13
14
dC
dH, mult
dC
dH, mult
Pro1
Pro1
66.9
66.4
55.8
52.3
a
66.2
5.47 (d, 6.6)
5.63 (dd, 6.6, 4.8)
5.06 (m)
4.14 (dd, 12.2, 7.5)
4.69 (dd, 12.2, 6.0)
5.76 (overlapped)
5.69 (d, 5.4)
5.13 (m)
3.99 (t, 10.5)
5.04 (m)
b
g
d1
63.6
57.5
53.2
4.5. Advanced Marfey’s analysis of the configuration of Thr
L
-Thr or D,L-allo-Thr standard was derivatized with L,D-FDLA or
d2
C]O
Abu2
a
b1
b2
167.3
168.2
allo-Thr2
58.5
L
-FDLA with the advanced Marfey’s method to produce four ste-
reoisomers of Thr, which were analyzed using the HPLC Waters
1525-2707-2998 system [column, Eclipse XDB-C18 column
55.8
26.4
5.29 (m)
2.19 (m)
2.31 (m)
1.15 (t, 7.5)
5.26 (t, 9.8)
4.99 (m)
67.4
(4.6ꢁ150 mm, 5
flow rate, 1 mL/min using a linear gradient (30e50% CH3CN over
50 min)]. The retention times of the -FDLA-derivatized amino
acids were as follows: -Thr- -FDLA (10.470), -Thr- -Thr-
-FDLA (¼
-FDLA, 17.537), -allo-Thr- -FDLA (11.940), -allo-Thr-
-FDLA (¼
allo-Thr- -FDLA, 14.268). The tR of the Thr residue in the hydroly-
mm); mobile phase, CH3CN in 0.1% HCOOH/H2O;
g
10.6
172.9
1.74 (d, 6.0)
9.38 (d, 9.8)
C]O
NH
Ser3
a
b1
b2
23.3
171.7
Ser3
59.6
61.5
L/D
8.43 (d, 7.2)
L
L
L
D
D
L
L
L
D
L
L-
56.7
63.1
4.64 (m)
3.79 (m)
4.23 (dd, 10.8, 3.6)
4.41 (dd, 10.8, 5.4)
d
D
4.97 (dd, 11.4, 4.2)
5.12 (dd, 11.4, 9.0)
1.98 (s)
zates of 9, 10, and 14 was similar to the L-allo-Thr-L-FDLA standard
at 11.930, 11.913, 11.902 min.
OCOCH3
OCOCH3
C]O
NH
20.9
171.0
168.4
d
d
d
171.3
Acknowledgements
10.16 (d, 5.4)
10.13 (d, 4.2)
b
-Phe4
b
-Phe4
a1
a2
b
g
d
41.6
2.86 (dd, 13.5, 6.9)
3.03 (dd, 13.5, 5.7)
5.38 (m)
44.0
2.65 (t, 12.5)
3.35 (dd, 12.5, 4.8)
5.76 (overlapped)
This work was supported by the National Basic Research Pro-
gram of China (2013CB127505 and 2009CB522300), the National
Natural Science Foundation of China (U1032602, 91013002,
21102152, 30725048), the National New Drug Innovation Major
Project of China (2011ZX09307-002-02), the Foundation of Chinese
Academy of Sciences (Hundred Talents Program), and the Natural
Science Foundation of Yunnan Province (2012GA003 and
2011FZ206).
52.6
143.1
127.4
129.2
127.5
171.9
53.1
143.5
126.4
128.9
127.3
172.9
7.47 (d, 7.7)
7.33 (t, 7.7)
7.23 (overlapped)
7.46 (d, 7.4)
7.17 (t, 7.4)
7.12 (t, 7.4)
ε
z
C]O
NH
Abu5
a
b1
b2
g
9.44 (d, 6.0)
8.21 (d, 6.0)
Ava5
53.6
32.9
53.2
25.6
5.16 (m)
1.62 (m)
1.77 (m)
0.89 (t, 7.2)
d
5.35 (m)
1.86 (m)
d
Supplementary data
10.9
d
20.1
13.8
174.1
1.65 (m)
0.73 (t, 7.2)
Detailed 1D and 2D NMR spectra, MS, IR, UV, ½a D
ꢂ
of compounds
d
9e14, X-ray crystallographic data of 3 and bioassay protocols used
are supplied. Supplementary data associated with this article can
be found in the online version, at http://dx.doi.org/10.1016/
j.tet.2013.07.006. These data include MOL files and InChiKeys of the
most important compounds described in this article.
C]O
NH
173.4
9.90 (d, 7.2)
10.21 (d, 3.6)
nmax 3426, 2932, 1680, 1385, 1289, 1206, 1136, 801, 723 cmꢀ1 1H
;
(600 MHz) and 13C (150 MHz) NMR, see Table 3; negative ESIMS m/
z 598 [MꢀH]ꢀ, 634 [MþCl]ꢀ; HRESIMS m/z 598.1820 [MꢀH]ꢀ (calcd
for C26H34N5O7Cl2, 598.1835).
References and notes
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