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Nitrogen-containingheterocycliccompounds from the roots of Callerya speciosa

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Life ScienceS
|
Pharmacology
49
SEPTEMBER 2022
Volume 64 Number 3
Introduction
Callerya speciosa
(Champ.) Schot. [synonym:
Millettia speciosa
Champ.] is a valuable medicinal plant
belonging to the family Leguminosae, which is widely
distributed in Southeast Asia in the tropical and subtropical
forests of Hainan Island and southern mainland China [1].
C. speciosa
is also found in the north areas of Vietnam
such as Tuyen Quang, Bac Kan, Bac Giang, and Phu
Tho. In recent years,
C. speciosa
has been planted more
frequently in areas of Vietnam as its roots have been used
as a traditional medicine for the treatment of fever, cough,
headache, backache, rheumatism, chronic bronchitis,
and nephritis [2]. Previous phytochemical studies on
this species revealed the presence of phenolics, phenolic
glycosides, pterocarpans, flavonoids, isoflavonoids,
sterols, and chromones [3-9]. In a previous work, we
reported the isolation and structural characterization of a new
oleanane triterpenoid along with three known compounds
from the ethyl acetate extract of the
C. speciosa
roots
[10]. In a further investigation of chemical constituents
from the n-butanol extract of the roots of this species, we
isolated four nitrogen-containing compounds including
uridine (
1
), 2-(
β
-D-glucopyranosyl)-3-isoxazolin-5-
one (
2
), adenosine (
3
), and hypaphorine (
4
), and their
structures were fully chara
cterized.
Materials and methods
General experiment procedure
1D- and 2D-NMR spectra were acquired on a Bruker
Avance 500 Ultrashield NMR Spectrometer. ESI-MS was
measured on an Agilent LC-MSD-Trap SL. Thin layer
chromatography was carried on silica gel 60 F254 (0.25
mm, Merck) and reversed phase RP18 F254S (0.25 mm,
Merck) plates. Column chromatography was performed
using silica gel 60 (230-400 mesh, Merck), YMC RP-
18 resins (30-50 μm, Fuji Silysia Chemical Ltd), and
Sephadex LH-20 gel (Amersham Pharmacia Biotech).
Plant material
The plant material was collected in the Tan Yen
district, Bac Giang province, in March of 2018. The
sample identification was done by Dr. Nguyen The
Cuong, Institute of Ecology and Biological Resources,
Vietnam Academy of Science and Technology (VAST),
and a voucher specimen was preserved in the Laboratory
of Natural Products Research, Institute of Chemistry,
VAST.
Extraction and isolation
The powdered roots of
C. speciosa
(870 g) were
extracted with 95% methanol three times at room
temperature. The extracts were filtered, combined, and
Nitrogen-containing
heterocyclic
compounds
from the roots of
Callerya speciosa
Duc Thien Dao
1
, Quoc Thang
Le
2
, Thanh Tam Nguyen
1, 3*
1
Institute of Chemistry, Vietnam Academy of Science and Technology
2
Department of Chemistry, Hue University of Education
3
Graduate University of Science and Technology, Vietnam Academy of Science and Technology
Received 30 October 2021; accepted 29 November 2021
*
Corresponding author: E-mail: [email protected].
vn
Abstract:
The four nitrogen-containing heterocyclic compounds uridine (1), 2-(
β
-D-glucopyranosyl)-3-isoxazolin-5-
one (2), adenosine (3), and hypaphorine (4) were isolated from the n-butanol extract of the roots of
Callerya
speciosa
. Their structures were characterized on the basis of extensive nuclear magnetic resonane (NMR),
mass spectroscopic analyses,
and comparison with reported values. This is the first report on the isolation of
compounds 1-4 from
Callerya speciosa
.
Keywords:
Callerya speciosa
, Leguminosae, nitrogen-containing heterocyclic compounds.
Classification number:
3.3
DOI : 10.31276/VJSTE.64(3).49-52
Life ScienceS
|
Pharmacology
50
SEPTEMBER 2022
Volume 64 Number 3
concentrated under reduced pressure. The obta
ined
residue was successively dissolved with water and re-
extracted in turn with ethyl acetate and n-butanol. The
organic solvents were concentrated to give 4.6 g and 9.7
g of the corresponding extracts.
The n-butanol extract was fractionated by silica gel
column chromatography eluted with CH
2
Cl
2
:MeOH:H
2
O
(4:1:0.1 – 1.5:1:0.2, v/v) to give 7 fractions. Fraction 2
was re-purified by silica gel column (CH
2
Cl
2
:MeOH:H
2
O
= 4:0.8:0.1, v/v), then Sephadex LH-20 column (MeOH)
to afford compound
1
(4 mg). Fraction 4 was re-purified
on a silica gel column (CH
2
Cl
2
:MeOH:H
2
O = 3:1:0.1,
v/v), then Sephadex LH-20 column (MeOH) to give
compounds
2
(3 mg) and
3
(4 mg). Fraction 6 was
chromatographed on a reversed phase silica gel (RP-
18) column (MeOH:H
2
O = 1:1), then silica gel column
(CH
2
Cl
2
:MeOH:H
2
O = 2.5:1:0.1, v/v) to yield compound
4
(7 mg).
Uridine (1):
Yellowish solid. ESI-MS:
m/z
267.2 [M+Na]
+
.
1
H-NMR (500 MHz, CD
3
OD): δ
H
8.02 (1H, d,
J
=7.0 Hz,
H-6), 5.92 (1H, d,
J
=4.0 Hz, H-1′), 5.71 (1H, d,
J
=7.0 Hz,
H-5), 4.21-4.19 (1H, m, H-2′), 4.18-4.16 (1H, m, H-3′),
4.03-4.02 (1H, m, H-4′), 3.86 (1H, dd,
J
=10.0, 2.0 Hz,
H-5a′), 3.75 (1H, dd,
J
=10.0, 2.0 Hz, H-5b′).
13
C-NMR
(125 MHz, CD
3
OD): δ
C
166.19 (C-4), 152.47 (C-2),
142.73 (C-6), 102.66 (C-5), 90.75 (C-1′), 86.37 (C-4′),
75.72 (C-2′), 71.31 (C-3′), 62.28 (C-5′).
2-(
β
-D-glucopyranosyl)-3-isoxazolin-5-one (2):
Colourless solid. ESI-MS:
m/z
248.1 [M+H]
+
.
1
H-NMR (500 MHz, CD
3
OD): δ
H
8.44 (1H, d,
J
=3.0 Hz,
H-3), 5.33 (1H, d,
J
=3.0 Hz, H-4), 4.92 (1H, d,
J
=7.5
Hz, H-1′), 3.87-3.85 (1H, m, H- H-6a′), 3.69-3.67 (1H,
m, H-6b′), 3.61-3.60 (1H, m, H-2′), 3.46 (1H, t,
J
=7.5
Hz, H-3′), 3.41-3.40 (1H, m, H-5′), 3.36 (1H, d,
J
=7.5
Hz, H-4′).
13
C-NMR (125 MHz, CD
3
OD): δ
C
173.96 (C-
5), 154.73 (C-3), 90.86 (C-4), 90.43 (C-1′), 80.39 (C-5′),
78.67 (C-3′), 73.85 (C-2′), 70.93 (C-4′), 62.46 (C-6′).
Adenosine (3):
White solid. ESI-MS:
m/z
268.1 [M+H]
+
.
1
H-NMR
(500 MHz, CD
3
OD): δ
H
8.32 (1H, s, H-8), 8.20 (1H, s,
H-2), 5.99 (1H, d,
J
=6.5 Hz, H-1′), 4.76 (1H, dd,
J
=6.0,
5.5 Hz, H-2′), 4.35 (1H, dd,
J
=5.0, 3.0 Hz, H-3′), 4.19 (1H,
dd,
J
=3.5, 3.0 Hz, H-4′), 3.91 (1H, dd,
J
=12.5, 3.0 Hz,
H-5a′), 3.77 (1H, dd,
J
=12.5, 3.0 Hz, H-5b′).
13
C-NMR
(125 MHz, CD
3
OD): δ
C
157.41 (C-6), 153.52 (C-2),
150.02 (C-4), 142.01 (C-8), 121.24 (C-5), 91.26 (C-1′),
88.17 (C-4′), 75.48 (C-2′), 72.65 (C-3′), 63.47 (C-5′).
Hypaphorine (4):
Colourless solid. ESI-MS:
m/z
247.3 [M+H]
+
.
1
H-NMR (500 MHz, CD
3
OD): δ
H
7.67 (1H, dd,
J
=6.5,
1.0 Hz, H-5), 7.39 (1H, dd,
J
=6.5, 1.0 Hz, H-8), 7.23
(1H, s, H-2), 7.15 (1H, dt,
J
=6.0, 1.0 Hz, H-7), 7.09 (1H,
dt,
J
=6.0, 1.0 Hz, H-6), 3.91 (1H, t,
J
=6.0 Hz, H-11),
3.44 (2H, d,
J
=6.0 Hz, H-10), 3.29 [9H, s, -N
+
(CH
3
)
3
].
13
C-NMR (125 MHz, CD
3
OD): δ
C
171.34 (C-12), 137.98
(C-9), 128.37 (C-4), 125.16 (C-2), 122.63 (C-7), 120.07
(C-6), 119.22 (C-5), 112.55 (C-8), 109.21 (C-3), 80.58
(C-11), 52.73, 52.71, 52.69 [-N
+
(CH
3
)
3
], 24.62 (C-10).
Results and discussion
Compound
1
was obtained as yellowish solid. The
13
C-NMR and HSQC spectra of compound
1
revealed
resonances for nine carbons of which four carbons
C
166.19 (C-4), 152.47 (C-2), 142.73 (C-6), 102.66
(C-5)] were assigned to a uracil and five carbons [δ
C
90.75 (C-1′), 86.37 (C-4′), 75.72 (C-2′), 71.31 (C-3′),
62.28 (C-5′)] to a ribofuranosyl unit. The
1
H-NMR
spectrum showed signals of a pair of doublets at δ
H
8.02
(1H, d,
J
=7.0 Hz, H-6) and 5.71 (1H, d,
J
=7.0 Hz, H-5);
a ribofuranose with an anomeric proton signal at δ
H
5.92 (1H, d,
J
=4.0 Hz, H-1′); and other sugar protons in
regions δ
H
4.21-3.75 ppm. The HMBC spectrum showed
correlations from H-1′ (δ
H
5.92) to C-2 (δ
C
152.47) and
C-6 (δ
C
142.73) indicating uracil linked to a ribofuranose
via a β-N1-glycosidic bond. The molecular formula
of
1
was deduced to be C
9
H
12
N
2
O
6
based on NMR data
and an ESI-MS pseudo-molecular ion peak at
m
/z 267.2
[M+Na]
+
. The
1
H- and
13
C-NMR data from compound
1
were consistent with uridine in literature [11]. Therefore,
compound
1
was elucidated to be uridine.
Compound
2
was isolated as colourless solid. The
1
H-NMR spectrum of compound
2
shows typical signals
of isoxazolin-5-one with two doublets of H-3 and H-4
at δ
H
8.44 and 5.33, respectively (
3
J
3,4
=3.0 Hz). In
addition, the signals of a
β
-D-glucopyranosyl unit with
an anomeric proton signal at δ
H
4.92 (1H, d,
J
=7.5 Hz,
H-1′) and complex proton signals in the region of δ
H
3.87-
3.36 were also observed. Corresponding to the
1
H-NMR
spectrum, the
13
C-NMR of
2
showed one carbonyl (δ
C
173.96), two methine carbons (δ
C
154.73,90.86), and a
glucose unit (δ
C
90.43, 80.39, 78.67, 73.85, 70.93, and
62.46). The Heteronuclear Multiple Bond correlation
(HMBC) correlation between H-1′ (δ
H
4.92) and C-3 (δ
C
154.73) were detected. A molecular formula of C
9
H
13
NO
7
was determined for compound
2
on the basis of an ion
peak [M + H]
+
at
m/z
248.1 in ESI-MS and NMR data.

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