Three new amino acid derivatives from edible mushroom Pleurotus ostreatus

Article abstract of DOI:10.1080/10286020.2017.1311870

Three new amino acid derivatives, oxalamido-L-phenylalanine methyl ester (1), oxalamido-L-leucine methyl ester (2), and lumichrome hydrolyzate (3), together with nine known compounds (4–12), were isolated from the solid culture of edible mushroom Pleurotu

Full text of DOI:10.1080/10286020.2017.1311870

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Three new amino acid derivatives from edible
mushroom Pleurotus ostreatus
Xiao-Jie Lu, Bao-Min Feng, Shao-Fei Chen, Dan Zhao, Gang Chen, Hai-Feng
Wang & Yue-Hu Pei
To cite this article: Xiao-Jie Lu, Bao-Min Feng, Shao-Fei Chen, Dan Zhao, Gang Chen, Hai-Feng
Wang & Yue-Hu Pei (2017): Three new amino acid derivatives from edible mushroom Pleurotus
ostreatus, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2017.1311870
To link to this article: http://dx.doi.org/10.1080/10286020.2017.1311870
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Date: 16 April 2017, At: 07:49

Journal of asian natural Products research, 2017
http://dx.doi.org/10.1080/10286020.2017.1311870
Three new amino acid derivatives from edible mushroom
Pleurotus ostreatus
Xiao-Jie Lua,b, Bao-Min Fengc, Shao-Fei Chena,b, Dan Zhaoa,b, Gang Chena,b
,
Hai-Feng Wanga,b and Yue-Hu Peia,b
aKey laboratory of structure-Based drug design and discovery, Ministry of education, shenyang
Pharmaceutical university, shenyang 110016, china; bschool oftraditional chinese Materia Medica, shenyang
Pharmaceutical university, shenyang 110016, china; cschool of life sciences and Biotechnology, dalian
university, dalian 116622, china
ABSTRACT
ARTICLE HISTORY
received 24 october 2016
accepted 21 March 2017
Three new amino acid derivatives, oxalamido-L-phenylalanine methyl
ester (1), oxalamido-L-leucine methyl ester (2), and lumichrome
hydrolyzate (3), together with nine known compounds (4–12),
were isolated from the solid culture of edible mushroom Pleurotus
ostreatus. Their structures were elucidated on the basis of extensive
spectroscopic analysis. The absolute configurations of 1 and 2
were established by the chiral synthesis and confirmed by circular
dichroism (CD) analysis of their total synthesis products and natural
isolates. All new compounds were evaluated for their antioxidant
effects, antimicrobial activities, and cytotoxic activity. Compounds
1–3 showed weak antifungal activities against Candida albicans with
minimum inhibitory concentration (MIC) value of 500 μg/ml.
KEYWORDS
edible mushroom; Pleurotus
ostreatus; antioxidation;
antimicrobial activity
CONTACT hai-feng Wang
wanghaifeng0310@163.com; Yue-hu Pei
peiyueh@vip.163.com
supplemental data for this article can be accessed http://dx.doi.org/10.1080/10.1080/10286020.2017.1311870.
© 2017 informa uK limited, trading as taylor & francis Group

2
X.-J. LU ET AL.
1. Introduction
Mushrooms have become increasingly attractive as a functional food due to their bioac-
tive metabolites in the development of drugs and nutraceuticals [1]. Mushroom mycelia
have been considered as alternatives and replacement of dried fruiting bodies for their
rich bioactive metabolites. e technique of solid-stated fermentation refers to micro-or-
ganism growing on moist solid substrates without free flowing water. Recently, fermented
foods produced by inoculating the edible and medicinal mushrooms on the cooked rice
medium have attracted wide attention due to enhancement of diversity of pharmacological
bioactivities and surprising metabolites [2,3]. Examples include sesquiterpenoids from
Pleurotus cystidiosus with inhibitory activity against α-glucosidase and protein tyrosine
phosphatase–1B (PTP1B) [4], terpenoids from Astraeus odoratus with antibacterial and
cytotoxic activities [5], lectin from Agaricus bisporus with immunomodulatory effect [6],
β-D-glucans from Cookeina tricholoma with antinociceptive property [7]. In this work,
we tried to grow the edible mushroom on the cooked rice medium for their bioactive
secondary metabolites.
Pleurotus ostreatus, also known as the oyster mushroom, is a fresh edible mushroom
frequently consumed in Southeast Asia and belongs to the phyla Basidiomycete, order
Agaricales, family Pleurotaceae, genus Plearotus. Compounds with medicinal properties
have been isolated from the fruiting body and mycelia culture of this mushroom, including
benzofuranone monoterpenoids with nitric oxide production (NO) inhibitory activity [8],
bisablolene sesquiterpenes with PTP1B inhibitory activity [9], pleurospiroketal sesquiter-
penes with NO inhibitory activity [10], cyclododecane skeleton diterpenoid and tetrahyd-
robenzofuran sesquiterpenoid with cytotoxicity against Hela and HepG2 cells [8], protein
hydrolysates with antioxidant activity [11], protein with antitumor activity [12], polysac-
charides with antitumor activity [13], mine elements with antioxidant and immunomodu-
latory activity [14], and sterol with osteomalacia- and rickets-prevented activity [15]. In the
present research, we reported the isolation and structure elucidation of three new amino
acid derivatives (1–3), along with nine known compounds (4–12) from the EtOAc extract of
the solid culture of P. ostreatus (Figure 1). e new structures were elucidated by extensive
spectroscopic methods. Finally, the absolute configurations of 1 and 2 were established by
the chiral synthesis. All new compounds were evaluated for their antioxidant effects by
2,2-diphenyl-1-picryldrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzothiazolin-6-sulfonic
acid) (ABTS) free radical scavenging activities, antimicrobial activities against Bacillus sub-
tilis, Staphylococcus aureus and Escherichia coli, and cytotoxicity against HL-60 cell line.
Unfortunately, these compounds were found to be inactive.
Figure 1. the structures of compounds 1–3.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
2. Results and discussion
Compound 1 was obtained as a white amorphous powder. Its HRESIMS exhibited a
[M + Na]+ at m/z 273.0858, corresponding to a molecular formula of C12H14N2O4. e
infared absorption (IR) spectrum indicated the presence of an amino group at 3385, 3304
cm−1, an ester carbonyl at 1744 cm−1, and amide carbonyl at 1662 cm−1. e 1H NMR (nuclear
magnetic resonance) and 13C NMR spectra of 1 exhibited the presence of a mono-substituted
benzene ring with signals at δH 7.18–7.29 (5H, m) and δC 137.3, 129.0 (×2), 128.3 (×2),
126.6, which were unambiguously confirmed by the 2D-NMR. And a methyl ester group
with the signals at δH 3.65 (3H, s) and δC 52.1, 171.1 was observed. e 13C NMR (100 MHz,
DMSO-d6) and heteronuclear single quantum coherence (HSQC) (Table 1) spectra showed
12 carbon signals, and the multiplicity of carbon signals was classified into nine sp2 carbons
(δ 171.1, 161.4, 160.2, 137.3, 129.0, 129.0, 128.3, 128.3, 126.6, including three carbonyls, five
methines, one quaternary), three sp3 carbons (53.6, 52.1, 35.7, including one methylene,
one methine, and one methoxy) by analysis of HSQC data.
e HMBC (heteronuclear multiple bond correlation) correlations of structure 1 between
H-3 (δH 3.10, 3.14) and C-1 (δC 171.1), C-2 (δC 53.6), C-4 (δC 137.3), C-5 (δC 129.0),
between H-2 (δH 4.56, td) and C-1 (δC 171.1), C-3 (δC 35.7), C-4 (δC 137.3), C-1′ (δC 160.2),
between OCH3 (δH 3.65, s) and C-1 (δC 171.1), between NH (δH 8.86, d) and C-1′ (δC
160.2), as well as between NH2 (δH 7.79, s) and C-1′ (δC 160.2) (Figure 2) permitted the
identification of the phenylalanine structure unit. Meanwhile, these data indicated that the
compound was an amino acid derivative with carboxylate group and 2-amino-2-oxacetyl
group. us, the planar structure of 1 was deduced as shown in Figure 1. Despite its relative
structural simplicity, difficulties were encountered during the determination of its absolute
configuration using amide hydrolysis and comparing optical data method due to the limited
yield. A straightforward and efficient method was developed to assign the stereochemical
configuration. Optical pure L-amino acid derivatives were synthesized using the specific
L-amino acid as initial materials. e final assignment of oxalamido-L-phenylalanine methyl
a
Table 1 1h and 13c nMr spectral data for 1 and 2 in dMso-d6
1
2
Position
δH, muti (J in Hz)
δH, muti (J in Hz)
Phe
1
2
171.1, c
53.6, ch
35.7, ch2
−
172.0, c
50.5, ch
38.7, ch2
−
4.56, td (8.7, 5.7)
3.10, dd (13.9, 9.5);
3.14, dd (14.5, 5.9)
−
7.19–7.21, m
7.25–7.27, m
7.19–7.21, m
7.25–7.27, m
7.19–7.21, m
−
4.33, td (8.4, 3.6)
1.51–1.53, m
1.76–1.86, m
1.53–1.55, m
0.84, d (6.0)
3
4
5
6
7
8
9
1′
2′
137.3, c
129.0, ch
128.3, ch
126.6, ch
128.3, ch
129.0, ch
160.2, c
24.3, ch
21.0, ch3
22.8, ch3
0.88, d (6.0)
160.5, c
161.6, c
52.0, ch3
−
−
161.4, c
52.1, ch3
−
1-och3
2-nh
2′-nh2
3.65, s
8.86, d (8.4)
8.03, s
3.63, s
8.92, d (8.0)
8.10, s
7.85, s
7.79, s
a400 Mhz for 1h nMr and 100 Mhz for 13c nMr. data were assigned based on the hsQc, hMBc, 1h-1h cosY, and noesY
experiments.

4
X.-J. LU ET AL.
Figure 2. Key hMBc (blue) and noesY (red) correlations of 1–3.
ester (1) was made by comparison of the circular dichroism (CD) spectrum of 1 with that
of L-chiral synthetic product.
Compound 2 was obtained as a white amorphous powder. Its HRESIMS exhibited a
[M + H]+ at m/z 217.1150, [M + Na]+ at m/z 239.1059, corresponding to a molecular for-
mula of C9H16N2O4. e IR spectrum indicated the presence of an amino group at 3381,
3313 cm−1, ester carbonyl at 1743 cm−1, and amide carbonyl at 1667 cm−1. Comparison of
NMR data of 2 with that of 1 suggested both of them shared the same basic structure except
for the amino acid residue (Table 1). e structure of 2 was elucidated by the analysis of
2D NMR data including the HSQC, HMBC and COSY spectra (Figure 2). In the HMBC
spectrum, correlations from H-3 (δH 1.52, m and δH 1.81, m) to C-1 (δC 172.0), C-2 (δC
50.5), C-4 (δC 24.3), C-5 (δC 21.0), from H-2 (δH 4.33, td) to C-1 (δC 172.0), C-3 (δC 38.7)
and C-4 (δC 24.3), from H-5 (δH 0.84, 3H, d) to C-2 (δC 50.5), C-3 (δC 38.7), C-4 (δC 24.3)
and C-6 (δC 22.8), from –OCH3 (δH 3.63, s) to C-1 (δC 172.0), from NH (δH 8.92, d) to
C-1′ (δC 160.5), and from NH2 (δH 7.85, s) to C-1′ (δC 160.5) afforded the leucine structure
unit. Furthermore, the tert-butyl methine was confirmed by 1H-1H COSY correlation. e
residues of compound 2 were the same as that of 1. Consequently, the planar structure of
2 was identified as shown in Figure 1. Using the same synthetic strategy in 1, the absolute
configuration of 2 was identified as oxalamido-L-leucine methyl ester.
Compound 3 was obtained as yellow amorphous powder. Its HRESIMS exhibited [M−
H2O + H]+ at m/z 243.0875 and [M−H2O + Na]+ at m/z 265.0684, corresponding to a
molecular formula of C12H12N4O3, with 9° of unsaturation. e IR spectrum indicated the
presence of an amino group at 3388, 3180 cm−1, and the presence of ester carbonyl at 1707
cm−1 and amide carbonyl at 1580 cm−1. Analysis of 1H and 13C NMR data of compound 3
showed the existence of two methyl singlets at δH 2.47, 2.49 and δC 19.6, 20.2, which were
unambiguously confirmed by the HSQC spectrum. e HSQC spectrum of compound
3 showed two aromatic proton signals at δH 7.71, 7.92 and its carbon signals at δC 125.8,
128.7. Meanwhile, four amino group protons were deduced according to the HSQC spec-
trum (Table 2). Careful comparison of the 1H and 13C NMR data of 3 (Table 2) with those
of known lumichrome [16] revealed that compound 3 is the hydrolyzate of lumichrome
(Figure 1), which was confirmed by its two primary amine acute IR peaks at 3388 and 3180
cm−1. Nitrogen double-bond configuration was determined as E relationship based on the
NOESY (nuclear overhauser effect spectroscopy) crosspeaks. On the basis of the above
evidence, the structure of 3 was established as (E)-6-((2-amino-4,5-dimethylphenyl)imino)
dihydropyrimidine-2,4,5(3H)-trione.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 2. 1h nMr (400 Mhz) and 13c nMr (100 Mhz) spectral data of 3 in dMso-d6.
No.
δH
δC
HMBC (1H→13C)
2
4
5
6
1′
2′
3′
4′
5′
6′
1-nh
3-nh
2′-nh
–
–
–
–
–
150.1, c
160.7, c
162.5, c
146.5, c
141.6, c
138.3, c
128.7, ch
144.7, c
138.9, c
125.8, ch
–
–
–
–
–
–
–
–
7.92, s
–
–
7.71, s
11.7, s
11.8, s
7.98, s
7.69, s
2.47, s
2.49, s
1′, 4′, 5′, 4′-ch3
–
–
2′
–
–
-
–
3′
1′, 6′
–
–
4′-ch3
5′-ch3
19.6, ch3
20.2, ch3
Scheme 1. retrosynthetic analysis.
To verify absolute configurations of 1 and 2, the total synthesis of 1 and 2 was carried out.
Our retrosynthetic analysis of 1 and 2 was shown in Scheme 1. Compounds 1 and 2 were
composed of 2-amino-2-oxacetyl chloride (3) and corresponding amino acid methyl ester
(4). Amino acid methyl ester (4) could be prepared by the corresponding amino acid (6).
Ethyl 2-chloro-2-oxoacetate (5), instead of 2-amino-2-oxacetyl chloride (3), was afforded
to form a peptide bond because 2-amino-2-oxacetyl chloride (3), itself is very unstable.
To synthesize compound 1, we firstly performed the condensation of L-phenylalamine
methyl ester with ethyl 2-chloro-2-oxoacetate. And the target compound 1 was afforded
by aminolysis of ethyl ester of C [17]. Similarly, we also have synthesized compound 2 by
the same procedures (Scheme 2). e spectroscopic data of synthetic 1 and 2, including 1H
NMR, 13C NMR, circular dichroism, and some ESI mass spectra, almost matched the data
originated for natural products 1 and 2, respectively. erefore, the absolute configurations
of compounds 1 and 2 were established as oxalamido-L-phenylalanine methyl ester (D1),
oxalamido-L-leucine methyl ester (D2), respectively (Figure 3).
e known compounds (4−12) were identified as uracil (4) [18], nicotinamide (5) [19],
1H-indole-2,3-dione (6) [20], 1H-indole-3-carboxaldehyde (7) [21], 1H-indole-3-carboxylic

6
X.-J. LU ET AL.
Scheme 2. synthesis of amino acid derivatives.
acid (8) [18], cyclo (Leu-Pro) (9) [22], uridine (10) [23], 2′-O-methyluridine (11) [18],
and adenosine (12) [24] by comparing its NMR spectral data with those reported in the
literatures.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a Perkin-Elmer 241 polarimeter (Perkin-Elmer Co.,
Jena, Germany). Ultraviolet Visible (UV) spectra were measured on Shimadzu UV-1601
(Shimadzu, Tokyo, Japan). IR spectra were measured on a Bruker IFS-55 infrared spec-
trophotometer (Bruker Co., Zurich, Switzerland). e NMR spectral data were recorded
on Bruker AV-400 (400 MHz for 1H and 100 MHz for 13C) with tetramethylsilane (TMS)
as the internal standard (Bruker Co.). e HR-ESI-MS data were obtained on the micro
TOF-Q mass instrument (Bruker Co.). CD spectra were measured on Bio-logic MOS
450 (Bio-Logic, Paris, France). Chromatography was carried out on silica gel (200–300
mesh; Qingdao Haiyang Chemical Factory, Qingdao, China), Sephadex LH-20 (Pharmacia,
Piscataway, NJ, USA), HPLC (high-performance liquid chromatography) (Shimadzu
LC-8A vp, Kyoto, Japan), and Varioskan Flash Multimode Reader (ermo Scientific,
Waltham, MA, USA).
2,2-Diphenyl-1-picrylhudrazyl (DPPH·), 2,2′-azinobis (3-ethylbenzothiazoline-6-sul-
fonic acid) (ABTS), ascorbic acid, and L-amino acid were purchased from Sigma-Alddrich
(St. Louis, MO, USA). All chemicals used for synthesis were analytical reagent grade with
no further purification.
3.2. Fungal material
e strain of Pleurotus ostreatus used in this work was purchased from Institute of Edible
Fungi, Shenyang Agricultural University, Liaoning, China, and deposited in School of
Traditional Chinese Material Medica, Shenyang Pharmaceutical University, China. P.
ostreatus strain was cultured on the Petri dish of potato dextrose agar at 25 °C for 10 days.
Agar plugs were inoculated in 500-ml Erlenmeyer flasks containing 120 ml of medium
(0.4% glucose, 1% malt extract, and 0.4% yeast extract), and the final pH of the medium

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Figure 3. isolated and synthetic ecd spectra of oxalamido-L-phenylalanine methyl ester and oxalamido-
L-leucine methyl ester.
was adjusted to 6.5 before sterilization, and incubated at 25 °C on a rotary shaker at 170
rpm for 7 days. Large-scale fermentation was carried out in 263 of 500 ml Fernbach flasks,
each containing 80 g of rice and 120 ml of distilled H2O. Each flask was inoculated with
15.0–20.0 ml culture medium and incubated at 25 °C for 25 days.

8
X.-J. LU ET AL.
3.3. Extraction and isolation
e fermented rice substrate in 263 flasks was soaked with exhaustive ethyl acetate (EtOAc)
and extracted five times under ultrasonic. e combined organic solvent was evaporated
to dryness under vacuum to afford the crude extracts (70.7 g). Similarly, n-BuOH extracts
(137.7 g) were obtained using the same method. EtOAc extracts were subjected to sillica
gel column chromatography using a stepwise solvent gradient method with CH2Cl2/MeOH
(100:0→0:100, v/v) to give 12 fractions (Fr.1−Fr.12). Fr.1 (5 g) was subjected to CC on silica
gel column (6 × 55 cm) using a stepwise solvent gradient method with petroleum ether (PE)/
EtOAc (100:0–0:100) to give six subfractions (Fr.1.1–Fr.1.6). Fr.1.4 (400 mg) was chromato-
graphed over Sephadex LH-20 (2.5 × 40 cm) eluted with MeOH, followed by semi-prepara-
tive HPLC (45% CH3OH/H2O, a flow rate at 3 ml/min) to yield compound 6 (50 mg, tR 7.5
min). Fr.2 (15 g) was separated over silica gel CC (7.5 × 120 cm) eluting with CH2Cl2/MeOH
(100:0–0:100) to yield eight subfractions (Fr.2.1–Fr.2.2). Fr.2.2 (1.7 g) was chromatographed
over Sephadex LH-20 (4.5 × 45 cm) eluting with MeOH, followed by semi-preparative HPLC
(30% CH3OH/H2O, a flow rate at 3 ml/min) to yield compound 7 (15 mg, tR 31 min). Fr.2.3
(1.6 g) was also chromatographed over Sephadex LH-20 (4.5 × 45 cm) eluting with MeOH
to yield six subfractions (Fr.2.3.1–Fr.2.3.6). Fr.2.3.4 (300 mg) was purified by HPLC (27%
CH3OH/H2O, a flow rate at 3 ml/min) to give compound 8. Fr.2.3.5 (200 mg) was separated
over semi-preparative HPLC (33% CH3OH/H2O, a flow rate at 3 ml/min) to yield compound
1 (2.0 mg, tR 63.4 min). Fr.3 (2.5 g) was also purified by Sephadex LH-20 (MeOH), followed
by semi-preparative HPLC (36% CH3OH/H2O, a flow rate 3 ml/min) to yield compound
2 (4.5 mg, tR 29.5 min). Compound 10 (6.4 mg) was obtained by recrystallization afer
Sephadex LH-20 (MeOH). e n-BuOH extracts were also subjected to silica gel column
chromatography eluted with a solvent gradient of CH2Cl2/MeOH (100:0→100:100, v/v) to
give six fractions (Fr.1–Fr.6). Fr.1 was subjected to silica gel column eluting with PE/EtOAc
(100:0–0:100) to yield six subfractions (Fr.1–Fr.6). Fr.6 was chromatographed over Sephadex
LH-20 (CH2Cl2/MeOH, 1:1), followed by semi-preparative HPLC (24% MeOH/H2O, a
flow rate 3 ml/min) to give compound 9 (5.1 mg, tR 63 min). Fr.2 (4.9 g) was separated on
silica gel CC, eluted with petroleum ether/acetone (100:2–100:50) to give four subfractions
(Fr.2.1–Fr.2.4). Subfraction Fr.2.3 was subjected to Sephadex LH-20 eluting with MeOH to
give four subfractions (Fr.2.3.1–Fr.2.3.4). Compound 3 (3.0 mg) was obtained from sub-
fraction Fr.2.3.4 by recrystallization. Fr.3 (10 g) was subjected to CC on silica gel column
(6 × 60 cm) eluting with CH2Cl2/MeOH (100:0–0:100) to yield seven subfractions (Fr.3.1–
Fr.3.7). Fr.3.4 (1.6 g) was chromatographed over Sephadex LH-20 (4.5 × 45 cm) eluting
with MeOH to give four subfractions (Fr.3.4.1–Fr.3.4.4). Fr.3.4.2 (247 mg) was purified by
semi-preparative HPLC (10% CH3OH/H2O, a flow rate at 3 ml/min) to yield compounds
5 (53 mg, tR 20 min) and 11 (5.7 mg, tR 26.5 min). Compound 4 (6.0 mg) was obtained by
recrystallization from Fr.3.4.4. Fr.4 (30 g) was applied to silica gel CC eluting with CH2Cl2/
MeOH (100:0–0:100) to yield six subfractions (Fr.3.1–Fr.3.6). Fr.3.5 (5 g) was subjected to
Sephadex LH-20 eluting with MeOH to yield six subfractions (Fr.3.5.1–Fr.3.5.6). Compound
12 (100 mg) was obtained by recrystallization afer gel permeation chromatography on
Sephadex LH-20 eluted with MeOH.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
3.3.1. Oxalamido-L-Phenylalamine methyl ester (1)
White amorphous powder; [ꢀ]2D5 +50 (c 0.02, MeOH). UV (MeOH) λmax (log ε) 205 (3.53)
nm; IR (KBr) νmax (cm−1): 3385, 3304, 2956, 2925, 1744, 1662, 1605, 1541; 1H and 13C NMR
spectral data are shown in Table 1; HR-ESI-MS (positive) m/z: 273.0858 [M + Na]+ (calcd
for C12H14N2O4Na, 273.0846).
3.3.2. Oxalamido-L-leucine methyl ester (2)
White amorphous powder; [ꢀ]2D5 +22.2 (c 0.045, MeOH); UV (methanol) λmax (log ε) 209
(3.44) nm; IR (KBr) νmax(cm−1): 3381, 3313, 2956, 2927, 2855, 1743, 1667, 1208, 1025, 802;
1H and 13C NMR and 2D NMR spectral data were shown in Table 1; HR-ESI-MS (positive)
m/z: 217.1150 [M + H]+ (calcd for C9H17N2O4, 217.1183), 239.1059 [M + Na]+ (calcd for
C9H16N2O4Na, 239.1002).
3.3.3. (E)-6-((2-Amino-4,5-dimethylphenyl)imino)dihydropyrimidine-2,4,5(3H)-trione
(3)
Yellow amorphous powder; UV (MeOH) λmax (log ε) 206 (2.97), 246 (2.57), 258 (2.56) nm;
IR (KBr) νmax(cm−1): 3388, 3180, 3000, 2923, 2854, 2805, 1707, 1580, 1362, 1345, 1005;
1H and 13C NMR and 2D NMR data were shown in Table 2, HR-ESI-MS (positive) m/z:
243.0875 [M−H2O + H]+ (calcd for C12H13N2O2, 243.0877), 265.0684 [M−H2O + Na]+ (calcd
for C12H12N4O3Na, 265.0696).
3.4. Synthetic experiment
3.4.1. General procedure for the synthesis of intermediates B1–B2
At the room temperature, 10-ml acetyl chloride was slowly added dropwise to 40 ml of
methanol. Afer the mixture was cooled to r.t., L-amino acid hydrochloride was added.
e reaction was stirred for 30 min, and then solutions were concentrated under reduced
pressure to give desired L-amino acid methyl ester hydrochloride as white solid in almost
quantitative yields. No further purification was required unless indicated as below.
3.4.2. General procedure for the synthesis of intermediates C1–C2
To a slurry of L-amino acid methyl ester hydrochloride substrate in 20–30 volumes of
dry dichloromethane at room temperature, three equivalents of anhydrous potassium car-
bonate was added followed by a solution of 1.1 equivalents of commercially available ethyl
2-chloro-2-oxoacetate by dropwise. Reaction mixture was slowly stirred at room tempera-
ture for 2 h. Afer completion of the reaction monitored by TLC, the residue was quenched
with water and extracted twice with CH2Cl2. e merged organic layer was washed three
times with saturated brine and dried over anhydrous MgSO4 for 30 min, then filtered and
concentrated under reduced pressure to afford intermediate C as yellow oil with good yields.
No further purification was required unless indicated as below.
Methyl (2-ethoxy-2-oxoacetyl) -L-phenylalaninate (C1): light yellow oil; 1H-NMR (400
MHz, DMSO-d6): δ 9.26 (1H, d, J = 8.0 Hz, 2-NH), 7.29–7.19 (5H, m, H-5–H-9), 4.56 (1H,
ddd, J = 9.8, 8.0, 5.1 Hz, H-2), 4.21 (2H, qd, J = 7.1, 1.6 Hz, 2′-OCH2CH3), 3.64 (3H, s,
1-OCH3), 3.14 (1H, dd, J = 13.9, 5.1 Hz, H-3a), 3.06 (1H, dd, J = 13.9, 9.8 Hz, H-3b), 1.24
(3H, t, J = 7.1 Hz, 2′-OCH2CH3).

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X.-J. LU ET AL.
1
Methyl (2-ethoxy-2-oxoacetyl) -L-leucinate (C2): light yellow oil; H-NMR (400 MHz,
DMSO-d6): δ 9.24 (1H, d, J = 8.0 Hz, 2-NH), 4.35 (2H, ddd, J = 10.7, 8.0, 4.0 Hz, H-2),
4.25 (2H, q, J = 7.1 Hz, 2′-OCH2CH3), 3.64 (3H, s, 1-OCH3), 1.76 (1H, td, J = 11.0, 4.0 Hz,
H-3a), 1.61–1.52 (2H, m, H-3b, H-4), 1.28 (3H, t, J = 7.1 Hz, 2′-OCH2CH3), 0.89 (3H, d,
J = 6.4 Hz, H-5 or 4-CH3), 0.85 (3H, d, J = 6.2 Hz, 4-CH3 or H-5).
To a solution of intermediate C in five volumes of ethanol cooled at 0 °C, 2–10 equivalents
of aq. ammonia (14.8 N) were added. Reaction mixture was slowly warmed up to room
temperature overlight. Concentration under reduced pressure gave target product as white
solid in good yields. Furthermore, the residue was purified by column chromatography on
silica gel (3:1 PE/EA) and HPLC (35% Methol-H2O) to give desired product as white powder.
Oxalamido-L-phenylalanine methyl ester (D1): white powder; CD (c 2.00 mM, MeOH)
Δε196 nm − 2.45, Δε219 nm + 7.43, Δε258 nm + 0.394; e 1H-NMR spectral data were same as
compound 1: δ 8.87 (1H, d, J = 8.5 Hz, 2-NH), 8.03 (1H, s, 2′-NH2), 7.80 (1H, s, 2′-NH2),
7.29–7.18 (5H, H-5–H-9), 4.56 (1H, dt, J = 8.5, 5.4 Hz, H-2). 3.65 (3H, s, 1-OCH3), 3.15
(1H, dd, J = 13.7, 5.4 Hz, H-3a), 3.11 (1H, dd, J = 13.7, 5.4 Hz, H-3b).
Oxalamido-L-leucine methyl ester (D2): white powder; CD (c 2.30 mM, MeOH) Δε196 nm
−
0.95, Δε209 nm + 1.31, Δε234 nm − 1.35; e 1H-NMR spectral data were same as natural isolates:
δ 8.91 (1H, d, J = 8.4 Hz, 2-NH), 8.10 (1H, s, 2′-NH2), 7.85 (1H, s, 2′-NH2), 4.33 (1H, ddd,
J = 8.4, 3.9, 3.0 Hz, H-2), 3.63 (3H, s, 1-OCH3), 1.84–1.78 (1H, m, H-3a), 1.59–1.51 (2H, m,
H-3a, H-4), 0.88 (3H, d, J = 6.2 Hz, H-5 or 4-CH3), 0.84 (3H, d, J = 6.0 Hz, 4-CH3 or H-5).
3.5. 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH·) scavenging assay
e DPPH· scavenging activity was assessed according to the described method with minor
modifications [25], using lower DPPH concentration (0.2 mM) and different sample to
DPPH ratio (1:1). To a 100 μl aliquot of the sample with different concentrations, 100 μl
of DPPH solution (0.2 mM) was added in a 96-well microplate. e mixture was shaken
vigorously and incubated in darkness for 30 min. e absorbance of the reaction solution at
517 nm was recorded using a varioskan flash multimode reader. Ascorbic acid was used as a
positive control. e percentage of scavenging DPPH versus concentration was plotted using
the following equation: DPPH scavenging activity (%) = [1 – (S – SB)/(C – CB)] × 100%,
where S, SB, C, and CB are the absorption of the sample, the blank sample, the control and
the blank control, respectively. All experiments were tested in triplicate.
3.6. 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) free radical (ABTS+)
scavenging assay
e determination of ABTS+ scavenging was carried out on the basis of the previous method
with minor modifications [25]. ABTS radical cations (ABTS+) were obtained by reacting
7 mM stock solution of ABTS+ with 2.45 mM potassium persulfate and allowing the mix-
ture to stand in the dark at room temperature for 14 h before use. e ABTS+ solution was
diluted with ethanol to the absorbance of 0.70 0.05 at 734 nm. To a 100 μl aliquot of the
sample with different concentrations, 150 μl ABTS+ solution in a 96-well microplate was
added. Afer reacting at room temperature for 30 min, the absorbance was recorded using
varioskan flash multimode reader at 734 nm. e percentage of ABTS+ scavenging activity

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
was calculated using the same equation in DPPH assay. Similarly, all experiments were
carried out in triplicate, and ascorbic acid was used as positive control.
3.7. Antimicrobial bioassay
e minimum inhibitory concentration (MIC) values for all compounds were determined by
the dilution method. For sample preparation, each of the test compounds was dissolved in
DMSO and then diluted with sterile broth to a concentration of 500 μg/ml. Further dilutions
of the compounds in the test medium were prepared at the required quantities of 250, 125,
etc. down to 3.9 μg/ml. Chloramphenicol and fluconazole were used as positive controls
for bacteria and fungus, respectively. e in vitro antimicrobial activity of the compounds
was tested by tube-dilution technique using individually packaged, flat bottomed, 96-well
microtitre plates (NCCLS. 2000). Bacterial strains were maintained on LB medium for
48 h at 37 °C and fungal strains were on PDA medium for 48 h at 28 °C. e cell density
for bacteria was 2–4 × 107 CFU/ml and 2–4 × 105 CFU/ml for fungus. A serial dilution of
compounds were performed in the microplates and incubated for 12 h. e last tube with
no growth of micro-organism was recorded to represent the MIC value expressed in μg/ml.
3.8. Cytotoxicity assay
Antitumor activity of 1–3 was evaluated by the MTT assay against HL-60 cell lines. HL-60
cell (American Type Culture Collection) was maintained in RPMI-1640 medium (Gibco)
with 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 1 mmol/L
ʟ-glutamine, and 10% (v/v) heat-inactivated FBS in a humidified atmosphere of 5% CO2 at
37.0 °C. Logarithmic phase cells were used for experiments. Appropriate dilutions of the
test samples were added to the cultures. e growth inhibition was calculated comparing
with control cells. 5-Fluorouracil was used as a positive control.
3.9. Antioxidation of compounds 1–3
Antioxidant activities of compounds 1–3 were evaluated by their ability to scavenge DPPH
and ABTS radicals. All compounds with IC50 (50% inhibitory concentration) values of above
200 μM were inactive in the DPPH radical scavenging assay and ABTS assay, comparable
with that of the standard compound ascorbic acid with an IC50 value of 10.2 μM.
3.10. Antibacterial and antifungal activity of compounds 1–3
e antimicrobial activities of compounds 1–3 were assessed for B. subtilis, S. aureus,
E. coli and Candida albicans. None of these compounds exhibited antibacterial
activities against B. subtilis, S. aureus and E. coli. Compounds 1–3 showed weak antifungal
activities against C. albicans with MIC value of 500 μg/ml. Fluconazole was used as positive
control against C. albicans (SC5314) with MIC value of 500 μg/ml.

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X.-J. LU ET AL.
Acknowledgments
We thank Mrs. Wen Li and Mr. Yi Sha, Department of Analytical Testing Centre, Shenyang
Pharmaceutical University, for measurements of the NMR data.
Disclosure statement
e authors declare no competing financial interest.
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