Homosecoiridoid alkaloids with amino acid units from the flower buds of lonicera japonica

Article abstract of DOI:10.1021/np4005773

Nine new homosecoiridoid alkaloids, named lonijaposides O-W (1-9), along with 19 known compounds, were isolated from an aqueous extract of the flower buds of Lonicera japonica. Their structures and absolute configurations were determined by spectroscopic data analysis and chemical methods. Lonijaposides O-W have structural features that involve amino acid units sharing the N atom with a pyridinium (1-5) or nicotinic acid (6-9) moiety. The absolute configurations of the amino acid units were determined by oxidation of each pyridinium ring moiety with potassium ferricyanide, hydrolysis of the oxidation product, and Marfey's analysis of the hydrolysate. This procedure was validated by oxidizing and hydrolyzing synthetic model compounds. The phenylalanine units in compounds 4, 5, and 9 have the d-configuration, and the other amino acid units in 1-3 and 6-8 possess the l-configuration. Compounds 1, 4, 6, and 9 and the known compounds 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 5′-O-methyladenosine exhibited antiviral activity against the influenza virus A/Hanfang/359/95 (H3N2) with IC₅₀ values of 3.4-11.6 μM, and 4 inhibited Coxsackie virus B3 replication with an IC₅₀ value of 12.3 μM.

Full text of DOI:10.1021/np4005773

Article
pubs.acs.org/jnp
Homosecoiridoid Alkaloids with Amino Acid Units from the Flower
Buds of Lonicera japonica
Yang Yu,† Chenggen Zhu,† Sujuan Wang, Weixia Song, Yongchun Yang, and Jiangong Shi*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
S
* Supporting Information
ABSTRACT: Nine new homosecoiridoid alkaloids, named
lonijaposides O−W (1−9), along with 19 known compounds,
were isolated from an aqueous extract of the flower buds of
Lonicera japonica. Their structures and absolute configurations
were determined by spectroscopic data analysis and chemical
methods. Lonijaposides O−W have structural features that
involve amino acid units sharing the N atom with a pyridinium
(1−5) or nicotinic acid (6−9) moiety. The absolute configurations of the amino acid units were determined by oxidation of each
pyridinium ring moiety with potassium ferricyanide, hydrolysis of the oxidation product, and Marfey’s analysis of the hydrolysate.
This procedure was validated by oxidizing and hydrolyzing synthetic model compounds. The phenylalanine units in compounds
4, 5, and 9 have the D-configuration, and the other amino acid units in 1−3 and 6−8 possess the L-configuration. Compounds 1,
4, 6, and 9 and the known compounds 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 5′-O-methyladenosine
exhibited antiviral activity against the influenza virus A/Hanfang/359/95 (H3N2) with IC50 values of 3.4−11.6 μM, and 4
inhibited Coxsackie virus B3 replication with an IC50 value of 12.3 μM.
he flower buds of Lonicera japonica Thunb., known as “Jin
TYin Hua” in Chinese, provide some of the most common
ingredients for formulations used in traditional Chinese
medicine to treat influenza, cold, fever, and infections.1−5
The constituents of the extracts of L. japonica flower buds,
which exhibit differing structural features and biological
activities, have been characterized in chemical and pharmaco-
logical studies; some of the identified compound classes have
been caffeoyl quinic acids, secoiridoids, flavonoids, saponins,
cerebrosides, polyphenols, and nitrogen-containing iridoids.6−14
As part of a program to assess the chemical and biological
diversity of traditional Chinese medicines, a detailed chemical
study was conducted on an aqueous extract of L. japonica
flower buds, since their decoctions are used in a variety of
formulations. Our previous studies on the aqueous extract led
to the isolation of 18 homosecoiridoids with the following
structural characteristics: secoiridoid nuclei coupled with N-
substituted nicotinic acid or pyridine units (lonijaposides A−N)
and secoiridoid nuclei coupled with phenylpyruvic acid-derived
moieties (loniphenyruviridosides A−D).15,16 Additionally,
aqueous extractions were performed on the flower buds; then
the residue was further extracted with EtOH (95%), which
resulted in the characterization of six new aromatic glycosides
and 48 known compounds.17 The present report describes a
continuation of this investigation on the aqueous extract, and
nine new homosecoiridoid alkaloids containing amino acid
units (1−9) have been isolated in addition to 19 known
compounds. Reported herein are the isolation, structure
determination, and biological activity of these isolates.
RESULTS AND DISCUSSION
■
The IR spectrum of compound 1 indicated the presence of
hydroxy group, carboxylic acid, conjugated double bond, and
aromatic ring absorptions. Its molecular formula of
C24H29NO11 was determined by HRESIMS combined with
the NMR data (Table 1 and Experimental Section). The NMR
spectra of 1 showed resonances attributable to a terminal
monosubstituted double bond, a trans-disubstituted double
bond, and a trisubstituted double bond. They also displayed
resonances due to four methines (dioxygen-bearing and
nitrogen-bearing), a methyl group vicinal to a methine, and
two quaternary carboxyl groups; there were also diagnostic
resonances for pyridinium-3″-yl and β-glucopyranosyl moieties.
These data indicated that 1 is an analogue of a homosecoiridoid
possessing a N-substituted pyridinium moiety.15,16 The
presence of a β-glucopyranosyl unit was confirmed by
enzymatic hydrolysis of 1 with snailase, which produced
glucose as identified by TLC comparison using an authentic
sugar sample. The glucose isolated from the hydrolysate gave a
positive optical rotation, [α]20D +42.7 (c 0.09, H2O), indicating
it to be D-glucose.15,16 The structure of the aglycone moiety of
1 was established by 2D-NMR data analysis. The proton
resonances and protonated carbon resonances in the NMR
spectra were assigned on the basis of the gHSQC data. In the
1H−1H gCOSY spectrum of 1, homonuclear vicinal coupling
correlations between H-1/H-9/H-5/H-6/H-7 and H-9/H-8/
H2-10, combined with the chemical shifts and coupling
Received: July 16, 2013
Published: November 26, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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dichroism (CD) data of compound 1 and lonijaposides H and
K−M demonstrated that these compounds have the same
configuration in the secoiridoid moiety, which was determined
by a single-crystal X-ray crystallographic analysis of the co-
occurring secologanic acid using anomalous scattering of Cu
Kα radiation.16 To determine the absolute configuration of the
alanine unit, the pyridinium moiety in 1 was oxidized with
potassium ferricyanide [K3Fe(CN)6],18 then hydrolyzed using
6 N HCl to liberate the amino acid, and finally analyzed by
Marfey’s method.19 This resulted in the assignment of the L-
configuration to the alanine unit in 1 (Figure S12, Supporting
Information). Therefore, the structure of compound 1 was
determined as shown, with this isolate assigned as lonijaposide
O.
Compound 2 gave the molecular formula C25H31NO11, with
one more CH2 unit than 1, as indicated by HRESIMS.
Comparison of the NMR data between these compounds
(Tables 1 and 2) indicated the presence of an additional
methoxy group [δH 3.73 (s) and δC 54.8] in 2. Additionally, the
H-3 and C-3 resonances in 2 were deshielded by ΔδH +0.16
and ΔδC +2.3 ppm, respectively, whereas the C-4 and C-11
resonances were shielded, in turn, by ΔδC −2.7 and −3.1 ppm.
This revealed that 2 is the methyl 11-carboxylate of 1, which
was confirmed by 2D-NMR data analysis. In particular, a
HMBC correlation of OCH3/C-11 supported the location of
the methoxy group. The chemical shifts of the alanine unit in 2
were in good agreement with those of 1, which confirmed the
inner salt to be formed via the alanine carboxylate in both
compounds. The L-configuration of the alanine unit in 2 was
verified by the same protocol as described for 1. Thus, the
structure of compound 2 (lonijaposide P) was determined as
shown.
The spectroscopic data of compound 3, lonijaposide Q,
indicated that it is an analogue of 1, with the molecular formula
C26H32N2O12. Comparison of the NMR data between 3 and 1
(Tables 1 and 2) revealed that instead of containing the alanine
unit and OH-11 group of 1, 3 contains a glutamic acid unit and
an amino group at these respective positions. The 11-carbamide
constants of these protons, demonstrated the presence of a
secoiridoid moiety with a C-6−C-7 trans-double bond and a
vinyl group at C-9. This was confirmed in the gHMBC
spectrum by the two- and three-bond heteronuclear correla-
tions of H-1/C-3, C-5, and C-8; H-3/C-1, C-4, C-5, and C-11;
H-5/C-1, C-3, C-4, C-6, C-7, C-8, C-9, and C-11; H-6/C-4, C-
5, C-7, and C-9; H-7/C-5 and C-6; H-8/C-1, C-5, and C-9; H-
9/C-1, C-4, C-5, C-6, C-8, and C-10; and H2-10/C-8 and C-9.
Also, gCOSY correlations of H-1′/H-2′/H-3′/H-4′/H-5′/H2-
6′, combined with gHMBC correlations of H-1/C-1′ and H-1′/
C-1, confirmed that the β-D-glucopyranosyl unit is located at C-
1 in 1. The gCOSY correlations of H-5″ with H-4″ and H-6″,
together with gHMBC correlations of H-2″/C-3″, C-4″, C-6″,
and C-7; H-4″/C-2″, C-6″, and C-7; H-5″/C-3″, C-4″, and C-
6″; H-6″/C-2″, C-4″, and C-5″; H-6/C-3″; and H-7/C-2″, C-
3″, and C-4″, provided evidence for the occurrence of the
pyridinium-3″-yl moiety at C-7. In addition, a vicinal coupling
gCOSY correlation between H-2‴ and H3-3‴, gHMBC
correlations of H-2‴/C-1‴, C-3‴, C-2″, and C-6″ and H3-
3‴/C-1‴ and C-2‴, and their chemical shifts revealed an
alanine unit sharing the N atom with the pyridinium moiety.
Accordingly, the planar structure of 1 was elucidated as shown,
which is different from lonijaposides I and K−M16 with respect
to the substituent at the N atom of the pyridinium moiety.
Hence, the deshielded shift of C-3 and the shielded shifts of C-
4 and C-11 suggested that the inner salt is formed via an alanine
carboxylate in 1. Similarities between the NMR and circular
1
was indicated by the chemical shift of H-3 (δH 7.52) in the H
NMR spectrum and the undetected C-4 and C-11 resonances
in the 13C NMR spectrum of 3, which distinguished it from
those of 1, 2, and other derivatives with an 11-carboxylate and
11-carboxylic acid unit.15,16 The presence of the glutamic acid
unit was confirmed by two- and three-bond correlations
between H-2″ and H-6″/C-2‴; H-2‴/C-1‴, C-4‴, C-2″, and
C-6″; H2-3‴/C-1‴, C-2‴, C-4‴, and C-5‴; and H2-4‴/C-2‴,
C-3‴, and C-5‴ in the gHMBC spectrum of 3. The chemical
shifts of C-1‴ (δC 174.5) and C-5‴ (δC 179.9) indicated that
the inner salt is formed via the 1‴-carboxylate. Oxidation of 3
using K3Fe(CN)6 followed by acid hydrolysis and Marfey’s
analysis of the hydrolysate supported the L-configuration for the
glutamic acid unit in 3. Therefore, the structure of compound 3
was determined as shown.
Compound 4 exhibited spectroscopic data similar to those of
1. The molecular formula C30H33NO11 of 4 was determined
from its HRESIMS and NMR data. On comparison of their
NMR data, this indicated that instead of containing an alanine
unit as in 1, 4 contains a phenylalanine unit, as verified by 2D-
NMR analysis (Figures S40−S42, Supporting Information). A
gCOSY correlation of H-2‴/H2-3‴ and HMBC correlations of
H-2‴/C-1‴; H2-3‴/C-1⁗, C-2⁗, and C-6⁗; and H-2″ and H-
6″/C-2‴, in addition to their shifts, confirmed the presence of
the phenylalanine unit that shared the N atom with the
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a
1
Table 1. H NMR Spectroscopic Data (δ) for Lonijaposides O−W (1−9)
no.
1
2
3
4b
5b
6
7
8
9b
1
5.53 d (7.2)
7.60 s
5.57 d (7.8)
7.76 s
5.50 d (7.5)
7.52 s
5.50 d (7.5)
7.75 s
5.48 d (8.4)
7.26 s
5.57 d (7.2)
7.69 s
5.48 d (7.2)
7.41 s
5.60 d (7.5)
7.78 s
5.51 d (7.2)
7.71 s
3
5
3.70 dd (7.8,
7.2)
3.71 dd (7.8,
7.2)
3.72 dd (7.5,
7.0)
3.61 dd (8.0,
6.5)
3.11 dd
(12.0,5.4)
3.70 dd (7.2,
6.6)
3.70 dd (7.2,
6.6)
3.75 dd (7.5,
7.5)
3.64 dd (7.8,
7.8)
6
6.68 dd (15.6,
7.8)
6.67 dd (16.2,
7.8)
6.68 dd (15.5,
7.0)
6.23 dd (16.0,
8.0)
5.83 t (12.0)
6.53 d (12.0)
6.73 dd (15.6,
7.2)
6.71 dd (16.2,
7.2)
6.73 dd (16.0,
7.5)
6.28 dd (16.2,
7.8)
7
8
6.59 d (15.6)
6.59 d (16.2)
6.58 dd (15.5) 6.43 d (16.0)
6.63 d (15.6)
6.59 d (16.2)
6.67 d (16.0)
6.49 d (16.2)
5.81 ddd (17.4, 5.79 ddd (18.0, 6.81 ddd (17.5, 5.70 ddd (16.5, 5.68 ddd (16.8,
5.82 ddd (16.8, 5.82 ddd (17.4, 5.82 ddd (18.0, 5.73 ddd (17.4,
10.2, 7.8)
10.8, 7.8)
10.5, 8.5)
10.0, 7.5)
10.2, 7.2)
10.2, 7.8)
10.8, 7.8)
10.0, 7.5)
10.2, 7.8)
9
2.86 ddd (7.8,
7.2, 7.2)
2.85 ddd (7.8,
7.8, 7.2)
2.86 m
2.80 ddd (7.5,
7.5, 6.5)
2.36 ddd (8.4,
7.2, 5.4)
2.87 ddd (7.8,
7.2, 6.6)
2.85 ddd (7.8,
7.2, 6.6)
2.87 ddd (7.5,
7.5, 7.5)
2.82 ddd (7.8,
7.8, 7.2)
10a
10b
1′
5.38 d (17.4)
5.33 d (10.2)
4.90 d (8.0)
5.37 d (18.0)
5.33 d (10.8)
4.90 d (8.0)
5.37 d (17.5)
5.32 d (10.5)
4.90 d (8.0)
5.33 d (17.0)
5.31 d (10.5)
4.90 d (8.0)
5.13 d (10.2)
4.96 d (16.8)
4.85 d (7.8)
5.38 d (16.8)
5.34 d (10.2)
4.90 d (8.0)
5.37 d (17.4)
5.32 d (10.8)
4.88 d (7.8)
5.39 d (18.0)
5.35 d (10.0)
4.93 d (8.0)
5.34 d (17.4)
5.33 d (10.2)
4.91 d (7.8)
2′
3.35 dd (8.0,
9.0)
3.34 dd (9.0,
8.0)
3.34 dd (9.0,
8.0)
3.34 dd (9.0,
8.0)
3.30 dd (9.0, 7.8) 3.35 dd (9.0,
8.0)
3.34 dd (9.0,
7.8)
3.35 dd (9.0,
8.0)
3.35 dd (9.0,
7.8)
3′
4′
3.54 dd (9.0,
9.0)
3.53 dd (9.0,
9.0)
3.54 dd (9.0,
9.0)
3.54 dd (9.0,
9.0)
3.52 dd (9.0, 9.0) 3.54 dd (9.0,
9.0)
3.53 dd (9.0,
9.0)
3.55 dd (9.0,
9.0)
3.55 dd (9.0,
9.0)
3.41 dd (9.0,
9.0)
3.40 dd (9.0,
9.0)
3.41 dd (9.0,
9.0)
3.42 dd (9.0,
9.0)
3.38 dd (9.0, 9.0) 3.41 dd (9.0,
9.0)
3.41 dd (9.0,
9.0)
3.42 dd (9.0,
9.0)
3.43 dd (9.0,
9.0)
5′
3.50 m
3.49 m
3.49 m
3.50 m
3.48 m
3.49 m
3.48 m
3.50 m
3.51 m
6′a
6′b
3.92 d (12.0)
3.91 d (12.0)
3.91 d (12.5)
3.91 d (12.0)
3.92 d (12.0)
3.91 d (12.0)
3.91 d (12.0)
3.91 d (12.0)
3.92 d (12.0)
3.72 dd (12.0,
6.0)
3.70 dd (12.0,
6.0)
3.71 dd (12.5,
6.0)
3.72 dd (12.0,
6.0)
3.71 dd (12.0,
6.0)
3.72 dd (12.0,
6.0)
3.73 dd (12.0,
6.0)
3.71 dd (12.0,
6.0)
3.75 dd (12.0,
6.0)
2″
4″
5″
8.77 s
8.76 s
8.80 s
8.34 s
8.72 s
9.03 s
8.89 s
9.08 s
8.89 s
9.10 s
8.90 s
8.88 s
8.70 s
8.55 d (7.2)
8.53 d (7.8)
8.58 d (8.0)
8.35 d (8.0)
8.40 d (8.4)
7.98 dd (8.4, 6.0)
7.99 dd (7.2,
6.6)
7.99 dd (7.8,
6.0)
8.01 dd (8.0,
6.5)
7.87 dd (8.0,
6.0)
6″
2‴
8.68 d (6.6)
5.35 q (7.2)
8.69 d (6.0)
5.35 q (7.2)
8.70 d (6.5)
5.22 m
8.59 d (6.0)
5.49 m
8.74 d (6.0)
8.85 s
8.88 s
8.90 s
8.38 s
5.43 dd (12.0,
4.2)
5.32 m
4.89 d (8.4)
4.82 d (7.0)
5.58 dd (9.0,
4.2)
3‴
4‴
1.93 d (7.2)
1.92 d (7.2)
2.33 m; 2.38 m 3.75 m; 3.50 m 3.90 m; 3.51 m
2.56 m; 2.76 m
2.29 m; 2.22 m 2.45 m
2.65 m
3.72 m; 3.57 m
1.35 m
1.24 m; 1.08 m 1.15 d (7.0)
5‴
6‴
0.92 d (6.6)
0.97 d (6.6)
0.87 t (7.2)
1.11 d (7.2)
0.85 d (7.0)
OMe
3.73 s
3.75 s
a
NMR data (δ) were measured in D2O at 600 MHz for 1, 2, 5−7, and 9 and at 500 MHz for 3, 4, and 8. Coupling constants (J) in Hz are given in
parentheses. The assignments were based on DEPT, H−1H COSY, gHSQC, and HMBC experiments, and the data were presented as calculated
using the solvent peak (δ 4.80 ppm) as the reference. Data of the phenyl unit: δ 7.08 (2H, d, J = 7.0 Hz, H-2⁗ and H-6⁗), 7.25 (3H, m, H-3⁗, H-
4⁗, and H-5⁗) for 4; 7.11 (2H, d, J = 7.2 Hz, H-2⁗ and H-6⁗), 7.29 (2H, t, J = 7.2 Hz, H-3⁗ and H-5⁗), and 7.25 (H, t, J = 7.2 Hz, H-4⁗) for 5;
7.09 (2H, d, J = 7.2 Hz, H-2⁗ and H-6⁗) and 7.27 (3H, m, H-3⁗, H-4⁗, and H-5⁗) for 9.
1
pyridinium moiety in 4. The chemical shifts of C-3, C-4, C-11,
and C-1‴, as compared with those of lonijaposides I and K−
M,16 suggested that 4 possesses 11-carboxylic acid and 1‴-
carboxylate units. The phenylalanine unit was determined to
have the D-configuration using the same protocol as described
for 1. Therefore, the structure of compound 4 (lonijaposide R)
was determined as shown.
The spectroscopic data of compound 5 indicated it to be an
isomer of 4. Comparison of the NMR data of these two
compounds revealed a variation in the coupling constant
between H-6 and H-7 (J6,7), which was 16.0 Hz in 4 and 12.0
Hz in 5. This suggests that 5 is a geometrical isomer of 4 at the
C-6−C-7-cis double bond, which was corroborated by 2D-
NMR and NOE difference spectroscopic data analysis (Figures
S52−S55, Supporting Information). As observed in the NOE
difference spectrum of 5, irradiation of H-6 enhanced H-1 and
H-7, which verified that H-6 and H-7 are cis-oriented and that
the secoiridoid moiety has the same configuration as in 4. The
chemical shifts of the C-3, C-4, and C-11 resonances in 5 were
consistent with those of lonijaposides I and K−M.16 This
demonstrated that the inner salt is formed via the 11-
carboxylate. By applying Marfey’s method, the D-configuration
was assigned to the phenylalanine unit in 5 (lonijaposide S), for
which the structure was determined as shown.
Compound 6 gave the molecular formula C28H35NO13, as
indicated by its (+)-HRESIMS and NMR data. The NMR data
of 6 (Tables 1 and 2) revealed the presence of leucine and
3″,5″-disubstituted nicotinic acid units in place of the respective
alanine and pyridinium-3″-yl moieties in 1. This was verified by
the gCOSY cross-peaks of H-2‴/H2-3‴, H-4‴/H3-5‴, and H3-
6‴ and gHMBC correlations of H-2‴and H2-3‴/C-1‴; H3-5‴,
H3-6‴/C-3‴, and C-4‴; H-2″/ C-3″, C-4″, C-6″, and C-7″; H-
4″/C-2″, C-5″, C-6″, and C-7″; and H-6″/C-2″, C-4″, and C-
5″. gHMBC correlations of H-2″ and H-6″/C-2‴, in addition
to their chemical shifts, also confirmed that the leucine and
nicotinic acid units share the same N atom. The chemical shifts
of the C-3, C-4, and C-11 resonances indicated the formation
of the inner salt via the 11-carboxylate. Oxidation of 6 followed
by acid hydrolysis and Marfey’s analysis of the hydrolysate
revealed an L-configuration for the leucine unit. Thus, the
structure of compound 6 (lonijaposide T) was determined as
shown.
The spectroscopic data of compound 7 (Tables 1 and 2 and
Experimental Section) demonstrated that it is an isomer of 6
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Journal of Natural Products
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a
Table 2. 13C NMR Spectroscopic Data (δ) for Lonijaposides O−W (1−9)
b
b
b
no.
1
2
3
4
5
6
7
8
9
1
99.9
154.9
112.6
40.9
100.1
157.2
109.9
40.8
100.0
154.4
N.D.
41.3
100.2
157.5
110.1
40.6
98.9
151.5
116.1
37.2
99.8
151.8
116.0
41.3
99.7
151.7
115.9
41.1
100.2
157.3
109.9
40.9
100.0
156.6
111.0
40.6
3
4
5
6
140.4
128.3
136.3
47.5
139.9
128.6
136.0
47.4
140.9
128.2
1366
47.8
140.1
128.4
136.1
47.5
139.9
126.0
137.5
47.2
141.7
127.9
136.8
47.8
141.6
127.9
136.7
47.7
140.2
128.5
136.1
47.4
140.5
128.1
136.1
47.5
7
8
9
10
11
1′
2′
3′
4′
5′
6′
2″
3″
4″
5″
6″
7″
1‴
2‴
3‴
4‴
5‴
6‴
OMe
122.5
175.3
101.9
75.4
122.7
172.2
102.0
75.4
122.6
N.D.
102.0
75.5
122.9
173.3
102.1
75.5
122.5
177.5
101.9
75.4
122.4
177.9
101.9
75.5
122.4
177.7
101.8
75.4
122.7
172.2
102.1
75.4
122.8
174.2
102.0
75.4
c
78.4
78.5
78.6
78.6
78.5
78.5
78.4
78.5
78.5
72.3
72.3
72.5
72.4
72.4
72.5
72.4
72.3
72.3
79.1
79.2
79.3
79.3
79.2
79.2
79.1
79.2
79.2
63.5
63.5
63.5
63.5
63.5
63.5
63.5
63.5
63.5
144.4
140.5
144.5
130.2
144.4
144.5
140.4
144.6
130.2
144.3
144.9
141.1
145.2
130.5
144.9
145.0
140.1
145.1
130.4
144.7
145.8
140.6
148.7
130.0
145.0
145.3
139.4
144.3
140.7
145.5
170.9
176.1
77.5
145.4
139.3
145.2
140.5
144.3
170.8
175.0
84.3
145.5
139.3
144.3
140.2
145.2
170.5
175.3
88.9
145.6
139.2
144.6
139.8
145.4
c
170.9
c
c
176.5
74.0
20.7
176.5
74.0
20.7
174.5
78.5
174.5
79.7
42.0
174.6
79.9
41.6
172.3
79.9
41.9
34.4
43.9
40.4
34.5
31.1
27.9
27.3
21.5
179.9
24.9
12.8
20.5
23.2
17.8
54.8
54.8
a
Data were measured in D2O at 125 MHz for 1−5, 7, and 9 and at 150 MHz for 6 and 8. The assignments were based on DEPT, 1H−1H COSY,
HSQC, HMQC, and HMBC experiments, and the data were presented as calculated using C-6′ (δ 63.5 ppm) as the reference. “N.D.” means that the
b
carbon resonance was not displayed in the spectrum. Data of the phenyl unit: δ 138.4 (C-1⁗), 131.8 (C-2″″′ and H-6⁗), 132.1 (C-3⁗ and C-5⁗),
and 130.6 (C-4⁗) for 4; 138.3 (C-1⁗), 131.4 (C-2⁗ and H-6⁗), 132.1 (C-3⁗ and C-5⁗), and 130.4 (C-4⁗) for 5; 138.2 (C-1⁗), 131.8 (C-2⁗ and
c
H-6⁗), 132.0 (C-3⁗ and C-5⁗), and 130.6 (C-4⁗) for 9. The resonance data were obtained from the HMBC spectrum.
with an isoleucine unit in place of the leucine unit. This was
corroborated by gCOSY correlations of H-2‴/H-3‴/H2-4‴/
H3-5‴ and H-3‴/H3-6‴ and gHMBC correlations of H2-4‴/C-
2‴ and C-6‴; H3-5‴/C-3‴ and C-4‴; H3-6‴/C-2‴, C-3‴, and
C-4‴; and H-2″ and H-6″/C-2‴ (Figures S77−S79, Supporting
Information). The L-configuration of the isoleucine unit was
determined by applying Marfey’s analysis in combination with
the chemical shifts of the C-5‴ and C-6‴ resonances.20−22
Therefore, the structure of compound 7 (lonijaposide U) was
determined as shown.
Compound 8 was found to be a further isomer of 6, as
indicated from its spectroscopic data (Tables 1 and 2 and
Experimental Section). The NMR data of 8 suggested the
presence of valine and methoxy units in place of the leucine
moiety in 6. Although resonances for the carboxylic carbons
(C-7″ and C-1‴) were not observed in the 13C NMR spectrum
of 8 due to the limited amount of sample obtained, their
chemical shifts were assigned on the basis of the correlations
from H-2″ and H-4″ to the carbon resonance at δC 170.5 (C-
7″) and from H-2‴ to the carbon resonance at δC 175.3 (C-
1‴), respectively, in the HMBC spectrum (Figure S90,
Supporting Information). Additionally, the gCOSY spectrum
of 8 displayed vicinal coupling correlations between H-3‴ and
H-2‴, H3-4‴, and H3-5‴, and the gHMBC spectrum exhibited
long-range correlations from H-3 and OCH3 to C-11 and from
H-2″, H-6″, H3-4‴, and H3-5‴ to C-2‴. These correlations
provided evidence that the OCH3 group is located at C-11 and
that the valine unit shares the N atom with the nicotinic acid
moiety in 8. Similarities between the chemical shifts of the
nicotinic acid moieties of 8 with 7 suggested that the inner salt
is formed via the 1‴-carboxylate. Therefore, the L-configuration
was assigned to the valine unit based on Marfey’s analysis.
Accordingly, the structure of compound 8 (lonijaposide V) was
determined as shown.
The spectroscopic data of compound 9 (Tables 1 and 2 and
Experimental Section) indicated that it is an analogue of 6 with
a phenylalanine unit in place of the leucine unit. This was
corroborated by 2D-NMR data analysis (Figures S100−S102,
Supporting Information). As compared to those of 6, the shifts
of the C-3, C-4, C-11, and C-1‴ resonances suggested that the
inner salt is formed via the 1‴-carboxylate in 9. By application
of the aforementioned Marfey’s method, the phenylalanine unit
in 9 was determined to have the D-configuration. Therefore, the
structure of compound 9 (lonijaposide W) was determined as
shown.
Since the sample amounts of the isolated compounds 1−9
were limited, the protocol used to determine the absolute
configurations of the amino acid units was first established by
the oxidation and hydrolysis of synthetic model compounds.
Alkylation of nicotinic acid (10) and pyridine (10′) with 1-
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iodopropane yielded 3-carboxy-1-propylpyridin-1-ium iodide
(11) and 1-propylpyridin-1-ium iodide (11′), respectively,
which were oxidized by K3Fe(CN)6 to produce 6-oxo-1-propyl-
1,6-dihydropyridine-3-carboxylic acid (12) and 1-propylpyr-
idin-2(1H)-one (12′) (Scheme 1).18 This demonstrated that
comparing the NMR and [α]20 data with those of the
D
authentic samples. This confirmed that the amide derivatives
could be hydrolyzed to release the corresponding amino acids,
and their configurations could then be determined by Marfey’s
method.19
The known compounds were identified by comparing their
spectroscopic data with reported data as adinoside A,24
stryspinoside,25 dimethyl secoxyloganoside,26 ketologanin,27
chlorogenic acid, methyl chlorogenate, 3,5-dicaffeoylquinic
acid, 3,4-dicaffeoylquinic acid, caffeic acid, protocatechuic
acid,28 5-caffeoylquinic acid,29 4-caffeoylquinic acid,30 syrin-
gin,31 caffeic acid 4-O-β-D-glucopyranoside,30 vanillic acid 4-O-
β-D-(6-O-benzoyl)glucopyranoside,28 gentisic acid 5-O-β-D-
glucopyranoside,32 benzyl 2-O-β-D-glucopyranosyl-2,6-dihy-
droxybenzoate,33 adenosine, and 5′-O-methyladenosine.34
In in vitro assays, compounds 1, 4, 6, and 9 and the known
compounds 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic
acid, and 5′-O-methyladenosine showed antiviral activity
against the influenza virus A/Hanfang/359/95 (H3N2),35
with respective IC50 values of 11.6, 6.8, 10.3, 8.2, 10.2, 4.9,
and 3.4 μM and respective SI values of 23.0, 41.5, 16.2, 32.3,
120.3, 88.7, and 48.3. (The positive control, oseltamivir, gave an
IC50 value of 1.3 μM and a SI value of 1164.2.) Compound 4
inhibited Coxsackie virus B3 replication with an IC50 of 12.3
μM (the positive control, pleconaril, gave an IC50 value of 0.3
μM).35 Compounds 1, 2, and 4 and caffeic acid, at 10 μM,
showed inhibitory activity against the release of glucuronidase
in rat polymorphonuclear leukocytes (PMN) induced by PAF
with 63.2 3.6%, 72.3 2.8%, 53.8 5.6%, and 40.3 3.3%
inhibition rates, respectively, while the other isolates exhibited
inhibition rates lower than 30%. The positive control
(ginkgolide B) gave an inhibition rate of 78.0 4.3% at the
same concentration.15,36 These results, combined with our
previous studies,15,16 show that diverse compounds contribute
toward pharmacological efficacy, which supports the traditional
uses of L. japonica flower buds. In addition, these compounds
were also assessed for their inhibitory activity against HIV-1
replication37 and several human cancer cell lines,38 but all were
inactive at a concentration of 10 μM.
Scheme 1. Synthesis and Oxidation of 3-Carboxy-1-
propylpyridin-1-ium Iodide (11) and 1-Propylpyridin-1-ium
Iodide (11′)
the pyridinium moiety in compounds 1−9 could be oxidized
readily by K3Fe(CN)6 to produce the hydrolyzable amide
derivatives. In addition, (S)- and (R)-2-[2-oxopyridin-1(2H)-
yl]propanoic acid (14 and 14′) were prepared by alkylation of
pyridine with (S)- and (R)-2-bromopropanoic acid, respec-
tively, and oxidation of the alkylated products (13 and 13′)
with K3Fe(CN)6 (Scheme 2). (S)- and (R)-Methyl 1-(1-
Scheme 2. Synthesis and Hydrolysis of (S)- and (R)-2-[2-
Oxopyridin-1(2H)-yl]propanoic Acid (14 and 14′)
methoxy-1-oxo-3-phenylpropan-2-yl)-6-oxo-1,6-dihydropyri-
dine-3-carboxylate (17 and 17′) were synthesized from
commercially available coumalic acid (2-oxo-2H-pyran-5-
carboxylic acid, 15) and L- or D-methyl 2-amino-3-phenyl-
propanoate hydrochloride,23 respectively (Scheme 3). Hydrol-
ysis of the two pairs of stereoisomers 14 and 14′ and 17 and
17′ with 6 N HCl at 110 °C for 16 h produced L- and D-alanine
and L- and D-phenylalanine, respectively, as identified by
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a model 343 polarimeter (Perkin-Elmer). UV and CD
spectra were measured on a JASCO J-810 spectropolarimeter. IR
spectra were recorded on a Nicolet 5700 FT-IR microscope
spectrometer (FT-IR microscope transmission). 1D- and 2D-NMR
1
spectra were obtained at 300, 400, 500, or 600 MHz for H and 75,
100, 125, or 150 MHz for 13C, respectively, on INOVA 300 MHz, 400
MHz, 500 MHz, or SYS 600 MHz spectrometers (Varian), with
solvent peaks serving as references (unless otherwise noted). ESIMS
data were measured with a Q-Trap LC/MS/MS (Turbo Ionspray
source) spectrometer. HRESIMS data were, in turn, measured on an
AccuTOF-CS JMS-T100CS spectrometer (JEOL). Column chroma-
tography was performed with silica gel (200−300 mesh, Qingdao
Marine Chemical Inc., Qingdao, People’s Republic of China) and
Pharmadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). HPLC
separation was performed on an instrument with a Waters 600
controller, a Waters 600 pump, and a Waters 2487 dual λ absorbance
detector (detecting wavelength: 250 nm) on a Prevail (250 × 10 mm
i.d.) semipreparative column packed with C18 (5 μm) (flow rate: 2
mL/min). Glass, precoated silica gel GF254 plates were used for TLC.
Spots were visualized under UV light or by spraying with 7% H2SO4 in
95% EtOH followed by heating.
Scheme 3. Synthesis and Hydrolysis of (S)- and (R)-Methyl
1-(1-Methoxy-1-oxo-3-phenylpropan-2-yl)-6-oxo-1,6-
dihydropyridine-3-carboxylate (17 and 17′)
Plant Material. See refs 15 and 16.
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Extraction and Isolation. For the extraction and preliminary
fractionation of the extract, see ref 15. Fraction B3-10 (22.1 g) was
separated by MPLC over RP silica gel and eluted with a gradient of
EtOH (0−50%) in H2O to give subfractions B3-10-1−B3-10-13.
Subfraction B3-10-7 (1.6 g) was separated further by flash
chromatography over RP silica gel, eluted with a gradient of
CH3CN (0−30%) in H2O, to obtain subfractions B3-10-7-1−B3-10-
7-5. Of these, subfraction B3-10-7-2 (102 mg) was subjected to RP-
HPLC using CH3CN−H2O (7:93) containing 0.1% HOAc as the
mobile phase to afford 1 (33 mg, 0.00027%, tR = 16.3) and 2 (2 mg,
0.000016%, tR = 20.1). Subfraction B3-10-10 (2.1 g) was separated by
flash chromatography over RP silica gel and eluted with a gradient of
EtOH (0−50%) in H2O to give subfractions B3-10-10-1−B3-10-10-4.
Subfraction B3-10-10-2 (58 mg) was subjected to RP-HPLC using
CH3CN−H2O (7:93) containing 0.1% HOAc as mobile phase to yield
6 (6 mg, 0.00005%, tR = 17.4), 7 (5 mg, 0.000042%, tR = 17.9), and 8
(1.5 mg, 0.000012%, tR = 21.1).
Lonijaposide S (5): beige, amorphous solid; [α]20 −120.0 (c 0.05,
D
H2O); UV (H2O) λmax (log ε) 202 (4.35, sh), 231 (4.02, sh), 285
(3.51, sh) nm; CD (H2O) 204 (Δε −7.04), 210 (Δε −6.69), 222 (Δε
−9.07), 247 (Δε +3.62), 278 (Δε −0.05), 291 (Δε +0.09); IR (Nujol)
νmax 3375, 2954, 2918, 2851, 1735, 1641, 1575, 1543, 1500, 1466,
1
1396, 1185, 1164, 1078, 961, 927, 831, 797, 722, 702, 662 cm−1; H
NMR (D2O, 600 MHz) data, see Table 1; 13C NMR (D2O, 125 MHz)
data, see Table 2; (+)-ESIMS m/z 584 [M + H]+, 606 [M + Na]+, 622
[M + K]+; (−)-ESIMS m/z 582 [M − H]−; HRESIMS m/z 584.2129
[M + H]+ (calcd for C30H34NO11, 584.2126).
Lonijaposide T (6): beige, amorphous solid; [α]20 −185.8 (c 0.12,
D
H2O); UV (H2O) λmax (log ε) 209 (4.35, sh), 233 (4.26, sh), 262
(4.09, sh), 309 (3.60) nm; CD (H2O) 197 (Δε −6.29), 216 (Δε
+0.65), 232 (Δε −1.55), 244 (Δε −2.75), 268 (Δε −6.78); IR
(Nujol) νmax 3347, 2959, 2925, 1633, 1605, 1390, 1278, 1199, 1165,
1074, 938, 786, 755 cm−1; 1H NMR (D2O, 600 MHz) data, see Table
1; 13C NMR (D2O, 150 MHz) data, see Table 2; (+)-ESIMS m/z 594
[M + H]+, 632 [M + K]+; (−)-ESIMS m/z 592 [M − H]−; HRESIMS
m/z 594.2194 [M + H]+ (calcd for C28H36NO13, 594.2181), 616.2006
[M + Na]+ (calcd for C28H35NO13Na, 616.2001).
Fraction B3-14 (4.3 g) was separated by flash chromatography over
RP silica gel and eluted with a gradient of EtOH (0−50%) in H2O to
give subfractions B3-14-1−B3-14-5. Subfraction B3-14-2 (36 mg) was
subjected to RP-HPLC using CH3CN−H2O (7:93), containing 0.1%
HOAc as the mobile phase, to afford 3 (3 mg, 0.000024%, tR = 19.6).
Subfraction B4 (86 g) was separated over a RP silica gel column and
eluted with a gradient of EtOH (0−100%) in H2O to yield
subfractions B4-1−B4-7. Of these, subfraction B4-6 (9.8 g) was
separated further by flash chromatography over RP silica gel and
eluted with a gradient of MeOH (0−50%) in H2O to give subfractions
B4-6-1−B4-6-4. Subfractions B4-6-1 (176 mg) and B4-6-2 (90 mg)
were separately subjected to RP-HPLC, using for B4-6-1 CH3CN−
H2O (13:87) containing 0.1% HOAc as the mobile phase, to afford 4
(120 mg, 0.001%, tR = 23.1) and 9 (1 mg, 0.000 008 3%, tR = 19.6),
and using for B4-6-2 CH3CN−H2O (15:85) containing 0.1% HOAc as
the mobile phase, to afford 5 (2 mg, 0.000 016%, tR = 23.7).
Lonijaposide O (1): beige, amorphous solid; [α]20D −160.0 (c 0.18,
H2O); UV (H2O) λmax (log ε) 230 (4.39), 256 (4.21, sh), 304 (3.71)
nm; CD (H2O) 195 (Δε −3.45), 214 (Δε +0.19), 237 (Δε −2.88),
243 (Δε −2.72), 261 (Δε −5.63); IR (Nujol) νmax 3352, 2926, 1630,
1504, 1453, 1394, 1360, 1283, 1195, 1161, 1074, 1042, 755, 689, 629
cm−1; 1H NMR (D2O, 600 MHz) data, see Table 1; 13C NMR (D2O,
125 MHz) data, see Table 2; (+)-ESIMS m/z 508 [M + H]+, 530 [M
+ Na]+, 546 [M + K]+; HRESIMS m/z 508.1827 [M + H]+ (calcd for
C24H30NO11, 508.1813), 530.1641 [M + Na]+ (calcd for
C24H29NO11Na, 530.1633).
Lonijaposide U (7): beige, amorphous solid; [α]20D −169.3 (c 0.15,
H2O); UV (H2O) λmax (log ε) 232 (4.28, sh), 264 (4.07, sh), 310
(3.61) nm; CD (H2O) 198 (Δε −4.95), 218 (Δε +0.19), 227 (Δε
−0.55), 235 (Δε −0.34), 246 (Δε −0.84), 270 (Δε −2.17); IR
(Nujol) νmax 3399, 2958, 2919, 2851, 1640, 1597, 1543, 1466, 1392,
1
1288, 1163, 1075, 947, 791, 720, 680 cm−1; H NMR (D2O, 600
MHz) data, see Table 1; 13C NMR (D2O, 125 MHz) data, see Table
2; (−)-ESIMS m/z 592 [M − H]−; HRESIMS at m/z 594.2194 [M +
H]+ (calcd for C28H36NO13, 594.2181), 616.2006 [M + Na]+ (calcd
for C28H35NO13Na, 616.2001).
Lonijaposide V (8): beige, amorphous solid; [α]20D −117.5 (c 0.04,
H2O); UV (H2O) λmax (log ε) 212 (4.32, sh), 233 (4.25, sh), 264
(4.07, sh), 308 (3.64) nm; CD (H2O) 199 (Δε −6.13), 216 (Δε
+2.19), 238 (Δε −0.23), 266 (Δε −4.29); IR (Nujol) νmax 3375, 2961,
2920, 2851, 1728, 1697, 1627, 1599, 1390, 1306, 1261, 1165, 1076,
1047, 940, 867, 789, 770, 660 cm−1; 1H NMR (D2O, 500 MHz) data,
see Table 1; 13C NMR (D2O, 150 MHz) data, see Table 2;
(−)-ESIMS m/z 592 [M − H]−; HRESIMS m/z 594.2175 [M + H]+
(calcd for C28H36NO13, 594.2181).
Lonijaposide W (9): beige, amorphous solid; [α]20D −112.0 (c 0.05,
H2O); UV (H2O) λmax (log ε) 203 (4.15, sh), 232 (3.83, sh), 266
(3.64, sh), 312 (3.34) nm; CD (H2O) 197 (Δε −4.20), 203 (Δε
−3.48), 217 (Δε −1.23), 227 (Δε −1.05), 244 (Δε −2.49), 268 (Δε
−4.94); IR (Nujol) νmax 3375, 2923, 2853, 1728, 1640, 1402, 1243,
1164, 1074, 932, 706, 596 cm−1; 1H NMR (D2O, 600 MHz) data, see
Table 1; 13C NMR (D2O, 125 MHz) data, see Table 2; (−)-ESIMS
m/z 627 [M]−; HRESIMS m/z 628.2034 [M + H]+ (calcd for
C31H34NO13, 628.2025), 650.1846 [M + Na]+ (calcd for
C31H33NO13Na, 650.1844).
Lonijaposide P (2): beige, amorphous solid; [α]20 −129.5 (c 0.10,
D
H2O); UV (H2O) λmax (log ε) 232 (4.07), 253 (3.95, sh), 300 (3.43)
nm; CD (H2O) 198 (Δε −1.42), 207 (Δε −0.39), 221 (Δε −0.29),
240 (Δε −1.62), 262 (Δε −4.31); IR (Nujol) νmax 3330, 2906, 1695,
1627, 1505, 1436, 1394, 1360, 1302, 1252, 1196, 1162, 1072, 948, 794,
1
770, 687 cm−1; H NMR (D2O, 600 MHz) data, see Table 1; 13C
NMR (D2O, 125 MHz) data, see Table 2; (+)-ESIMS m/z 522 [M +
H]+, 544 [M + Na]+, 560 [M + K]+; HRESIMS m/z 522.1973 [M +
H]+ (calcd for C25H32NO11, 522.1970), 544.1788 [M + Na]+ (calcd
for C25H31NO11Na, 544.1789).
Enzymatic Hydrolysis of Compounds 1 and 4. A solution of
each compound (5 mg) in H2O (1 mL) was treated with snailase (20
mg, LJ0427B2011Z, Shanghai Sangon Biotech Co., Ltd., Shanghai,
People’s Republic of China) at 37 °C for 12 h. The reaction mixture
was extracted with EtOAc (2 × 3 mL). The H2O phases of the
hydrolysates of 1 and 4 were separately concentrated to dryness and
then eluted with CH3CN−H2O (6:1) on a silica gel column to yield
glucose with an [α]20D value of +42.7 (c 0.09, H2O) and +43.6 (c 0.16,
H2O), respectively. The solvent system CH3CN−H2O (4:1) was used
for the TLC identification of glucose (Rf = 0.38).
Synthesis and Oxidation of 3-Carboxy-1-propylpyridin-1-
ium Iodide (11) and 1-Propylpyridin-1-ium Iodide (11′). A
solution of either nicotinic acid (50 mg) or pyridine (0.1 mL) and 1-
iodopropane (70 mg) in N,N-dimethylformamide (DMF, 2 mL) was
stirred at room temperature for 12 h, then evaporated under reduced
pressure to give a residue. The residue was separated over silica gel and
eluted with CHCl3−MeOH (8:1) to yield 3-carboxy-1-propylpyridin-
1-ium iodide (11, 63 mg) from nicotinic acid, and 1-propylpyridin-1-
ium iodide (11′, 86 mg) from pyridine, which were identified by
ESIMS and NMR analysis (Figures S106−S110, Supporting
Information). K3Fe(CN)6 (100 mg) and KOH (40 mg) were
Lonijaposide Q (3): beige, amorphous solid; [α]20D −129.0 (c 0.08,
H2O); UV (H2O) λmax (log ε) 229 (4.14), 258 (3.98), 300 (3.47) nm;
IR (Nujol) νmax 3317, 2920, 1727, 1665, 1636, 1548, 1394, 1276, 1239,
1188, 1153, 1072, 1042, 796, 683, 614, 582 cm−1; 1H NMR (D2O, 500
MHz) data, see Table 1; 13C NMR (D2O, 125 MHz) data, see Table
2; (+)-ESIMS m/z 565 [M + H]+, 587 [M + Na]+; HRESIMS m/z
565.2034 [M + H]+ (calcd 565.2035 for C26H33N2O12).
Lonijaposide R (4): beige, amorphous solid; [α]20D −195.0 (c 0.30,
H2O); UV (H2O) λmax (log ε) 204 (4.44, sh), 230 (4.37), 257 (4.23,
sh), 305 (3.75) nm; CD (H2O) 194 (Δε −1.92), 207 (Δε −0.85), 202
(Δε −0.90), 223 (Δε + 0.95), 242 (Δε −3.17), 263 (Δε −8.70), 299
(Δε −1.08); IR (Nujol) νmax 3348, 2921, 2851, 1681, 1630, 1501,
1
1366, 1277, 1199, 1164, 1075, 1045, 950, 928, 754, 703 cm−1; H
NMR (D2O, 500 MHz) data, see Table 1; 13C NMR (D2O, 125 MHz)
data, see Table 2; (+)-ESIMS m/z 584 [M + H]+, 606 [M + Na]+;
HRESIMS m/z 584.2141 [M + H]+ (calcd for C30H34NO11,
584.2126), 606.1951 [M + Na]+ (calcd for C30H33NO11Na, 606.1946).
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Journal of Natural Products
Article
successively added into a solution of 11 (30 mg) or 11′ (40 mg) in
H2O−benzene (1:1, 2 mL). The mixture was stirred at room
temperature for 2 h, then partitioned with EtOAc (2 × 2 mL). The
EtOAc phase was separated over silica gel and eluted with petroleum
ether−EtOAc (4:1) to give 6-oxo-1-propyl-1,6-dihydropyridine-3-
carboxylic acid (12) from 11, and 1-propylpyridin- 2(1H)-one (12′)
from 11′, as identified by ESIMS and NMR spectroscopic analysis
(Figures S111−S116, Supporting Information).
acetone (1%, 115 μL) and 6% aqueous triethylamine (60 μL) were
added. The mixture was kept at 40 °C for 2 h, then diluted with H2O
(150 μL) and filtered. The standard FDAA-amino acids were prepared
in the same manner using the corresponding L- and D-amino acids
(0.8−1.3 mg). The FDAA-amino acid derivatives from the hydrolysate
were compared with standard FDAA-amino acids using HPLC
analysis: Previl C18 (250 × 4.6, 5 μm); flow rate, 1 mL/min; UV
detection at 340 nm; linear gradient elution increasing CH3CN from
25% to 45% in 0.1% aqueous TFA buffer over a period of 45 min. The
absolute configuration of each amino acid was determined by
comparing the retention time of the hydrolysate FDAA derivatives
with those of the standard D- and L-amino acid FDAA derivatives.
Anti-influenza Virus, Coxsackie Virus, and Herpes Simplex
Virus Assay. See ref 35.
Synthesis and Hydrolysis of (S)- and (R)-2-[2-Oxopyridin-
1(2H)-yl]propanoic Acid (14 and 14′). A solution of pyridine (1.24
g) and either (S)- or (R)-2-bromopropanoic acid (2 g) in CH2Cl2 (10
mL) was refluxed for 4 h. The solvent was removed under reduced
pressure to give a residue. The residue was separated over silica gel and
eluted with CHCl3−MeOH (5:1) to yield (S)-1-(1-carboxyethyl)-
pyridin-1-ium bromide {13, 2.6 g, [α]20 −5.1 (c 2.3, MeOH)} from
Anti-inflammatory Activity Assay. See refs 15 and 36.
HIV-1 Replication Inhibition Assay. See ref 37.
Cells, Culture Conditions, and Cell Proliferation Assay. See
ref 38.
D
(S)-2-bromopropanoic acid, and 13′ [the (R)-isomer, 2.4 g, [α]20
D
+5.2 (c 1.8, MeOH)] from (R)-2-bromopropanoic acid. K3Fe(CN)6
(700 mg) and KOH (40 mg) were successively added into a solution
of 13 or 13′ (200 mg) in H2O−benzene (1:1, 5 mL). The mixture was
stirred at room temperature for 8 h, then acidified with 6 N HCl to pH
5 and filtered. The filtrate was evaporated under reduced pressure to
yield a residue, which was chromatographed over silica gel, eluting
with CHCl3−MeOH (6:1), to afford (S)-2-[2-oxopyridin-1(2H)-
ASSOCIATED CONTENT
* Supporting Information
■
S
Isolation of the known compounds, IR, MS, 1D/2D NMR, UV,
and CD spectra of compounds 1−9, HPLC chromatograms of
FDAA derivatives of the standard amino acids, and hydrolysates
of the oxidation products of compounds 1−9. This material is
available free of charge via the Internet at http://pubs.acs.org.
yl]propanoic acid {14, 123 mg, [α]20 +4.4 (c 1.0, MeOH)} from
D
13, and the (R)-isomer {14′, 144 mg, [α]20 −4.2 (c 1.3, MeOH)}
D
from 13′. The synthesized compounds 13, 13′, 14, and 14′ were
characterized by ESIMS, NMR, and CD spectroscopic techniques
(Figures S117−S129, Supporting Information). The enantiomers 14
and 14′ (100 mg each) were separately hydrolyzed with 6 N HCl (4
mL) to liberate the corresponding L-alanine {24.3 mg, [α]20D +14.3 (c
AUTHOR INFORMATION
Corresponding Author
■
1.3, H2O)} from 14 and D-alanine {21.2 mg, [α]20 −14.5 (c 1.1,
D
*Tel: 86-10-83154789. Fax: 86-10-63017757. E-mail: shijg@
imm.ac.cn (J.S).
H2O)} from 14′, as identified by ESIMS and NMR spectroscopic
analyses (Figures S130−S135, Supporting Information) and their
[α]20 values.
SyDnthesis and Hydrolysis of (S)- and (R)-Methyl 1-(1-
methoxy-1-oxo-3-phenylpropan-2-yl)-6-oxo-1,6-dihydropyri-
dine-3-carboxylate (17 and 17′). A solution of coumalic acid (15,
500 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 1
g), and 4-dimethylaminopyridine (DMAP, 800 g) in anhydrous
MeOH (20 mL) was stirred at room temperature for 4 h. The solvent
was removed under reduced pressure to give a residue, which was then
partitioned between H2O (20 mL) and CH2Cl2 (2 × 20 mL). The
CH2Cl2 phase was concentrated and purified by column chromatog-
raphy over silica gel to afford methyl coumalate (16, 412 mg), which
was identified by ESIMS and NMR spectroscopic analysis (Figures
S136−S138, Supporting Information). Methyl coumalate (50 mg) and
either L-phenylalanine or D-phenylalanine (80 mg) in anhydrous
MeOH (5 mL) were stirred at room temperature for 2 h, then
evaporated under reduced pressure. The residue was separated over
silica gel and eluted with petroleum ether−EtOAc (4:1) to yield 17
(59 mg) from L-phenylalanine and 17′ [the (R)-isomer, 63 mg] from
D-phenylalanine, which were characterized using the ESIMS,
HRESIMS, NMR, and CD spectroscopic techniques (Figures S139−
S147, Supporting Information). These enantiomers (17 and 17′; 30
mg each) were separately hydrolyzed with 6 N HCl (2 mL) to liberate
Author Contributions
†Y. Yu and C. Zhu contributed equally to this study.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the National Natural Sciences
Foundation of China (NNSFC; grant nos. 20772156,
30825044, and 20932007), the Program for Changjiang
Scholars and Innovative Research Team in University
(PCSIRT, grant no. IRT1007), and the National Science and
Technology Project of China (nos. 2012ZX09301002-002 and
2011ZX0 9307-002-01) is acknowledged.
REFERENCES
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