Synthesis and antibacterial activities of Yanglingmycin analogues

Article abstract of DOI:10.1248/cpb.c14-00578

The synthesis of Yanglingmycin and its enantiomer, along with eighteen Yanglingmycin analogues is reported. The structures were confirmed mainly by analyses of NMR spectral data. Antibacterial activity assays showed that Yanglingmycin and some of its analogues exhibited significant antibacterial activities against two important agricultural pathogenic bacteria, Ralstonia solanacearum and Pseudomonas syringae pv. actinidiae, with minimum inhibitory concentration (MIC) values ranging from 3.91 to 15.62 μg/mL. The antibacterial activities exhibited by Yanglingmycin and its analogues are promising, suggesting potential in the development of compounds for novel bactericides.

Full text of DOI:10.1248/cpb.c14-00578

Vol. 63, No. 1
Chem. Pharm. Bull. 63, 33–37 (2015)
33
Regular Article
Synthesis and Antibacterial Activities of Yanglingmycin Analogues
Long-Bo Li,a,b Wen-Jia Dan,a,b Fang-Fang Tan,a,b Li-Hui Cui,a,b Zhi-Peng Yuan,a,b
,a,b
Wen-Jun Wu,b,c and Ji-Wen Zhang*
b
a College of Sciences, Northwest A&F University; Yangling, Shaanxi Province 712100, P.R. China: Shaanxi
Province Key Laboratory of Research and Development of Botanic Pesticide, Northwest A&F University; Yangling,
c
Shaanxi Province 712100, P.R. China: and State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest
A&F University; Yangling, Shaanxi Province 712100, P.R. China.
Received August 10, 2014; accepted October 8, 2014; advance publication released online October 30, 2014
The synthesis of Yanglingmycin and its enantiomer, along with eighteen Yanglingmycin analogues is
reported. The structures were confirmed mainly by analyses of NMR spectral data. Antibacterial activity
assays showed that Yanglingmycin and some of its analogues exhibited significant antibacterial activities
against two important agricultural pathogenic bacteria, Ralstonia solanacearum and Pseudomonas syringae
pv. actinidiae, with minimum inhibitory concentration (MIC) values ranging from 3.91 to 15.62µg/mL. The
antibacterial activities exhibited by Yanglingmycin and its analogues are promising, suggesting potential in
the development of compounds for novel bactericides.
Key words Yanglingmycin; structural analogue; substituted cyclic azole; antibacterial activity
Bacterial diseases pose a major threat to agricultural pro- Pinner reaction to obtain the corresponding methyl benzimi-
duction worldwide. For example, Ralstonia solanacearum, date hydrochlorides,8) which were then reacted with each the
which causes plant bacterial wilt, is one of the most destruc- following compounds: L-serine methyl ester hydrochloride,
tive plant pathogens, and affects many regions of the world.1) D-serine methyl ester hydrochloride, L-cysteine methyl ester
It is estimated that R. solanacearum is responsible for US$ hydrochloride, D-cysteine methyl ester hydrochloride, L-2,3-
1 billion in losses each year.2) Pseudomonas syringae pv. diaminopropionic acid methyl ester hydrochloride, and D-2,3-
actinidiae, which causes kiwifruit canker, is another devastat- diaminopropionic acid methyl ester hydrochloride.9,10) Amino
ing plant pathogen and has received significant attention in acid methyl ester hydrochlorides were obtained by reacting the
recent years.3–6) Thus, there has been considerable interest corresponding amino acids with AcCl in MeOH.11) The prod-
in development of bactericides for Ralstonia solanacearum ucts were reduced by LiAlH4 to obtain Yanglingmycin and its
and Pseudomonas syringae pv. actinidiae. Unfortunately, analogues.
there have been few reports of effective bactericides against
Antimicrobial Assay for Antimicrobial Activity of Yan-
these pathogens. In the process of screening new agricultural glingmycin and Its Analogues All synthesized compounds
antibiotics, we report the synthesis a new broad spectrum were evaluated for their in vitro antibacterial activities against
antibiotic, Yanglingmycin (Fig. 1), which was found to be ef- Ralstonia solanacearum and Pseudomonas syringae pv. ac-
fective against the previously mentioned pathogens.7) Although tinidiae. The corresponding inhibition zone diameters and
Yanglingmycin can be isolated from the fermentation broth of minimum inhibitory concentrations (MICs) were determined
Streptomyces djakartensis, the yield is too low to conduct ex- using the filter paper method and the micro-broth dilution
periments to determine the mechanism of bactericidal action. method, respectively, and are presented in Tables 1 and 2.
In order to investigate the bactericidal activities of Yangling- Yanglingmycin, its enantiomer, and eighteen Yanglingmycin
mycin and its analogues, Yanglingmycin and its analogues analogues synthesized by systematically varying the hetero-
were designed and synthesized. In this paper, Yanglingmycin atom and substituent configuration on the heterocycle and
and its enantiomer and eighteen Yanglingmycin analogues substituents on the phenyl group have been characterized.
were synthesized and their antibacterial activities against Some of the synthesized compounds showed more potent
Ralstonia solanacearum and Pseudomonas syringae pv. acti- antibacterial activities against Ralstonia solanacearum and
nidiae are reported.
Pseudomonas syringae pv. actinidiae than that of Ampicillin.
In particular, compounds 4.2a and 4.2a′ exhibited MIC values
of 3.91µg/mL for Ralstonia solanacearum and 7.81µg/mL
Results
Chemistry Chart 1 describes the synthesis of all target for Pseudomonas syringae pv. actinidiae. Compounds 4.1a
compounds. Benzonitrile, 2-hydroxybenzonitrile, and 4-hy- and 4.1a′ exhibited MIC values of 15.62µg/mL for Ralstonia
droxybenzonitrile were used as starting material for the solanacearum and 7.81µg/mL for Pseudomonas syringae pv.
actinidiae. In comparison, compound 3.1a exhibited MIC val-
ues greater than 125µg/mL for both pathogen species. None of
the other compounds characterized exhibited significant anti-
bacterial activity.
Discussion
A comparison among the compounds screened shows that
Fig. 1. Structure of Yanglingmycin
*To whom correspondence should be addressed. e-mail: nwzjw@nwsuaf.edu.cn
© 2015 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 63, No. 1 (2015)
Reagents and conditions: (I) AcCl, MeOH, 16h; (II) AcCl, MeOH, reflux, 2h; (III) NEt3, CH2Cl2, reflux, 24h; (IV) LiAlH4, Et2O, 1h.
Chart 1. Synthesis of Yanglingmycin and Its Analogues
Table 1. Diameter of Inhibition Zone of Yanglingmycin Analogues
against the Tested Bacteria
a significant change in antibacterial activity. Focusing on the
heteroatom substitution, compounds with oxygen and sulfur
heteroatoms on the heterocycle presented better antibacterial
activities than those with a nitrogen heteroatom on the het-
erocycle. Since the oxazole and thiazole are isoelectronic,
they can be expected to share similar chemical properties
and activities. On the other hand, the imidazole has an extra
proton on the nitrogen heteroatom. Proton transfer between
the two nitrogen atoms in the imidazole could cause enough
of a structural change to disrupt antibacterial activity, which
is consistent with lower bactericidal activity of Yanglingmy-
cin analogues with nitrogen heteroatom substitution on the
heterocycle. According to the MIC data, compounds 4.2a and
4.2a′ were approximately 4 times more active than 4.1a and
4.1a′ although the inhibition zone diameters of 4.2a and 4.2a′
ranged from 16 to 37% lower than the inhibition diameters of
4.1a and 4.1a′. Since the MICs of compounds 4.2a and 4.2a′
suggest significantly higher activity than those of 4.1a and
4.1a′, but the inhibition zone diameters of these (4.1a, 4.1a′,
4.2a, 4.2a′) compounds are somewhat similar, it is likely that
the diffusivity of the compounds in the inoculated culture
medium during the antimicrobial activity screening process,
carried out with the filter paper method, is the cause for this
discrepancy. Further variation of substituents on the phenyl
group in Yanglingmycin analogues may further improve upon
the selectivity of antibacterial activity.
Diameter of inhibition zone (mm)
in 1µL/disk (mean S.D.)
Compounds
Pseudomonas syringae
Ralstonia solanacearum
pv. actinidiae
3.1a
3.1a′
3.1b
9.2 0.77 (++)
—
—
—
—
—
3.1b′
3.1c
—
—
—
—
3.1c′
3.2a
—
—
—
—
3.2a′
3.3a
—
—
—
—
3.3a′
4.1a
—
—
11.7 0.58 (++)
12.6 0.58 (++)
—
17.2 0.34 (+++)
17.3 0.63 (+++)
4.1a′
4.1b
—
4.1b′
4.1c
—
—
—
—
4.1c′
4.2a
—
—
14.5 0.71 (+++)
13.5 0.71 (+++)
13.6 0.21 (+++)
8.7 0.46 (+)
8.0 0.62 (+)
14.6 0.58 (++)
4.2a′
Ampicillin
All values were means of three replicates, “+++” means transparent; “++”
means clear; “+” means visible; “—” means no inhibitory effect.
Conclusion
holding all other structural components constant, 2-hydroxy
Yanglingmycin and its structural analogues have been
substitution at the phenyl group produced the most active synthesized. A number of the compounds displayed higher
compounds. Yanglingmycin analogues with a 4-hydroxy sub- bactericidal activity than ampicillin towards Ralstonia sola-
stituted phenyl group and or an unsubstituted phenyl group nacearum and Pseudomonas syringae pv. actinidiae. Different
did not exhibit significant bactericidal activities. The data substitutions for the heteroatom, on the phenyl group, and on
suggest that the 2-substitution on the phenyl group elicits a the heterocycle suggest that 2-substitution of the phenyl group,
geometric effect, since the same functional group at a dif- a more polar functional group on the heterocycle yield, and
ferent position on the phenyl group has dramatically lower oxygen or sulfur heteroatom substitution improved bacteri-
activity. Similarly, if we consider the effect of the functional cidal activity.
group at the chiral center on the heterocycle of Yanglingmy-
cin analogues, we find that the hydroxymethyl substitution
Experimental
yields considerably improved bactericidal activities compared
General Solvents were of analytical reagent (AR) grade
to methoxycarbonyl substition. Enantiomeric configuration of unless otherwise mentioned. TLC was performed on E. Merck
the substituents on the heterocycle was not observed to elicit 60F254 silica gel plates. Column chromatography was car-

Vol. 63, No. 1 (2015)
Chem. Pharm. Bull.
35
Table 2. MIC of Yanglingmycin Analogues against the Tested Bacteria
Tested bacteria
Minimum inhibitory concentration (µg/mL)
4.1a
4.1a′
4.2a
4.2a′
3.1a
Ampicillin
Ralstonia solanacearum
15.62
7.81
15.62
7.81
3.91
7.81
3.91
7.81
>125
>125
>125
>125
Pseudomonas syringae pv. actinidiae
ried out with silica gel (Qingdaohaiyang Co., Ltd., Qingdao, uct was chromatographed on silica gel to obtain the products
Shandong, China); compounds were eluted with petroleum (4.1a–4.3a′). The yields of 4.1a, a′, b, b′, c, c′, 4.2a, a′, 4.3a,
ether and ethyl acetate in sequence. Melting point (mp) was a′ were 86%, 87%, 84%, 84%, 88%, 86%, 87%, 86%, 76%,
1
measured using Yanagimoto apparatus (uncorrected). H- and 75%, respectively.
13C-NMR were obtained on a Bruker-Avance-500 spectrom-
eter with DMSO-d6 or CDCl3 as solvent and SiMe4 as internal oxazole (3.1a) Colorless oil. H-NMR (CDCl3, 500MHz) δ:
standard. MS were recorded under electrospray ionization 11.65 (1H, brs), 7.67 (1H, dd, J=1.5, 7.0Hz), 7.40 (1H, m), 7.02
(ESI) conditions using a Thermo LCQ Fleet instrument.
(R)-2-(2-Hydroxyphenyl)-4-methoxycarbonyl-4,5-dihydro-
1
(1H, dd, J=0.5, 8.5Hz), 6.89 (1H, m), 4.99 (1H, dd, J=7.5,
General Procedure for the Synthesis of 2a–c Acetyl 10.5Hz), 4.68 (1H, dd, J=7.5, 9.0Hz), 4.58 (1H, dd, J=9.0,
chloride (11.4mL, 160mmol) was added dropwise to a stirred 10.5Hz), 3.81 (3H, s). ESI-MS m/z: 222 [M+H]+. [α]D20 −35.0
solution of corresponding substituted benzonitrile (1a–c) (c=0.1, MeOH).
(20mmol) in anhydrous methanol (9.7mL, 240mmol) at room
(S)-2-(2-Hydroxyphenyl)-4-methoxycarbonyl-4,5-dihydro-
1
temperature. TLC analysis showed complete consumption of oxazole (3.1a′) Colorless oil. H-NMR (CDCl3, 500MHz) δ:
the substituted benzonitrile starting material. The product was 11.65 (1H, brs), 7.66 (1H, dd, J=1.5, 7.0Hz), 7.40 (1H, m), 7.02
observed to precipitate out from the mixture as a white solid. (1H, dd, J=0.5, 8.5Hz), 6.88 (1H, m), 4.99 (1H, dd, J=7.5,
Then the mixture was filtered and washed by ethyl acetate, 10.5Hz), 4.68 (1H, dd, J=7.5, 9.0Hz), 4.58 (1H, dd, J=9.0,
and the product was dried under low pressure to give methyl 10.5Hz), 3.81 (3 H, s). 13C-NMR (CDCl3, 125MHz) δ: 170.98,
benzimidate hydrochloride (2a–c) as a white powder. The 167.58, 159.94, 134.05, 128.38, 118.83, 116.95, 110.05, 68.92,
yields of 2a, b and c were 90%, 89%, 92%, respectively.
67.19, 52.83. ESI-MS m/z: 222 [M+H]+. [α]D20 +32.1 (c=0.1,
General Procedure for the Synthesis of 2d–f and 2d′–f′ MeOH).
Acetyl chloride (1.6mL, 22mmol) was added to MeOH
(R)-2-(4-Hydroxyphenyl)-4-methoxycarbonyl-4,5-dihydro-
(100mL), and the solution was cooled to 0°C. The solution oxazole (3.1b) White solid. mp: 128–130°C. 1H-NMR
was stirred for 5min. Amino acid (L-serine, D-serine, L- (CDCl3, 500MHz) δ: 7.76 (2H, d, J=8.5Hz), 6.77 (2H, d,
cysteine, D-cysteine, L-2,3-diaminopropionic acid, and D-2,3- J=8.5Hz), 4.94 (1H, dd, J=7.5, 10.5Hz), 4.71 (1H, dd, J=7.5,
diaminopropionic acid) (20mmol) was then added to the 8.5Hz), 4.61 (1H, dd, J=8.5, 10.5Hz), 3.73 (3H, s). ESI-MS
acetyl chloride solution in methanol (MeOH), and the solution m/z: 222 [M+H]+. [α]D20 −139.8 (c=0.1, MeOH).
was heated to reflux for 2h, then cooled to room temperature.
(S)-2-(4-Hydroxyphenyl)-4-methoxycarbonyl-4,5-dihydro-
The reaction was evaporated under reduced pressure and gave oxazole (3.1b′) White solid. mp: 128–130°C, 1H-NMR
a colorless solid. The solid was washed with CH2Cl2 (20mL) (CDCl3, 500MHz) δ: 7.76 (2H, d, J=8.5Hz), 6.77 (2H, d,
to give amino acid methyl ester hydrochloride (2d–f, 2d′–f′) J=8.5Hz), 4.94 (1H, dd, J=7.5, 10.5Hz), 4.71 (1H, dd, J=7.5,
as a white solid. The yields of 2d, d′, e, e′, f and f′ were 95%, 8.5Hz), 4.61 (1H, dd, J=8.5, 10.5Hz), 3.74 (3H, s). 13C-NMR
96%, 94%, 96%, 95%, 95%, respectively.
(CDCl3, 125MHz) δ: 171.56, 167.57, 160.77, 130.71, 117.37,
General Procedure for the Synthesis of 3.1a–3.3a′ To a 115.67, 69.72, 67.53, 52.76. ESI-MS m/z: 222 [M+H]+. [α]D20
mixture of the hydrochloride salt of each substituted methyl +138.8 (c=0.1, MeOH).
benzimidate (2a–c) (1mmol) and corresponding amino acid
(R)-2-Phenyl-4-methoxycarbonyl-4,5-dihydrooxazole (3.1c)
1
methyl ester hydrochloride (1mmol) in CH2Cl2 (20mL) was Colorless oil. H-NMR (CDCl3, 500MHz) δ: 7.98 (2H, m), 7.50
added Et3N (0.14mL, 1mmol). The reaction solution was (1H, m), 7.41 (2H, m), 4.96 (1H, dd, J=8.0, 10.5Hz), 4.70 (1H,
stirred 24h at room temperature, and the reaction was checked dd, J=8.0, 8.5Hz), 4.60 (1H, dd, J=8.5, 10.5Hz), 3.82 (3H, s).
for completion by TLC. The insoluble salts were filtered off ESI-MS m/z: 206 [M+H]+. [α]D20 −130.4 (c=0.1, MeOH).
and the filtrate was washed with an NaHCO3 saturated aque-
(S)-2-Phenyl-4-methoxycarbonyl-4,5-dihydrooxazole (3.1c′)
ous solution, dried over Na2SO4. The organic layer was con- Colorless oil. 1H-NMR (CDCl3, 500MHz) δ: 7.98 (2H, m), 7.50
centrated under vacuum, and the crude product was chromato- (1H, m), 7.41 (2H, m), 4.96 (1H, dd, J=8.0, 10.5Hz), 4.70 (1H,
graphed on silica gel to obtain the products (3.1a–3.3a′). The dd, J=8.0, 8.5Hz), 4.60 (1H, dd, J=8.5, 10.5Hz), 3.82 (3H, s).
yields of 3.1a, a′, b, b′, c, c′, 3.2a, a′, 3.3a, a′ were 83%, 82%, 13C-NMR (CDCl3, 125MHz) δ: 171.66, 166.35, 131.90, 128.62,
84%, 85%, 85%, 85%, 83%, 82%, 84%, 83%, respectively.
General Procedure for the Synthesis of 4.1a–4.2a′ [α]D20 +128.2 (c=0.1, MeOH).
LiAlH4 (52.8mg, 1.1mmol) was added slowly in parts to a (S)-2-(2-Hydroxyphenyl)-4-methoxycarbonyl-4,5-dihydro-
128.38, 126.94, 69.57, 68.63, 52.74. ESI-MS m/z: 206 [M+H]+.
1
stirred solution of 3.1a–3.2a′ (1mmol) in Et2O (10mL). After thiazole (3.2a) Colorless oil. H-NMR (CDCl3, 500MHz) δ:
1h, the reaction was completed (checked by TLC). Ethyl 12.30 (1H, brs), 7.39 (2H, m), 7.02 (1H, dd, J=1.0, 8.0Hz),
acetate was added, and then the mixture was washed with 6.88 (1H, m), 5.35 (1H, dd, J=8.0, 9.5Hz), 3.82 (3H, s), 3.68
saturated aq. NaHCO3 (2×5mL), H2O (2×5mL), saturated (1H, dd, J=8.0, 11.0Hz), 3.59 (1H, dd, J=9.5, 11.0Hz). ESI-
aq. NaCl (2×5mL), and dried with anhydrous Na2SO4. The MS m/z: 238 [M+H]+. [α]D20 +13.7 (c=0.1, MeOH).
mixture was concentrated under vacuum, and the crude prod-
(R)-2-(2-Hydroxyphenyl)-4-methoxycarbonyl-4,5-dihydro-

36
Chem. Pharm. Bull.
Vol. 63, No. 1 (2015)
thiazole (3.2a′) Colorless oil. 1H-NMR (CDCl3, 500MHz) (CDCl3, 125MHz) δ: 165.59, 131.54, 128.35, 128.30, 69.27,
δ: 12.30 (1H, brs), 7.39 (2H, m), 7.01 (1H, dd, J=1.0, 8.0Hz), 68.06, 63.92. ESI-MS m/z: 178 [M+H]+. [α]D20 +47.6 (c=0.1,
6.88 (1H, m), 5.35 (1H, dd, J=8.0, 9.5Hz), 3.82 (3H, s), MeOH).
3.68 (1H, dd, J=8.0, 11.0Hz), 3.59 (1H, dd, J=9.5, 11.0Hz).
(S)-2-(2-Hydroxyphenyl)-4-hydroxymethyl-4,5-dihydrothi-
1
13C-NMR (CDCl3, 125MHz) δ: 174.33, 170.61, 159.16, 133.59, azole (4.2a) White solid. mp: 43–45°C. H-NMR (CDCl3,
130.80, 118.97, 117.28, 115.95, 76.68, 52.91, 33.67. ESI-MS m/z: 500MHz) δ: 7.41 (1H, dd, J=1.5, 7.5Hz), 7.35 (1H, m), 6.99
238 [M+H]+. [α]D20 −14.1 (c=0.1, MeOH).
(1H, dd, J=1.0, 8.0Hz), 6.88 (1H, td, J=1.0, 7.5Hz), 4.87 (1H,
(R)-2-(2-Hydroxyphenyl)-4-methoxycarbonyl-4,5-dihydro- m), 3.97 (1H, dd, J=5.0, 11.0Hz), 3.83 (1H, dd, J=5.0, 11.0Hz),
imidazole (3.3a) White solid. mp: 154–156°C. 1H-NMR 3.42 (1H, dd, J=7.5, 11.0Hz), 3.34 (1H, dd, J=7.5, 11.0Hz).
(500MHz, CDCl3) δ: 7.35 (2H, m), 7.01 (1H, dd, J=1.0, ESI-MS m/z: 210 [M+H]+. [α]D20 +15.8 (c=0.1, MeOH).
8.0Hz), 6.84 (1H, m), 4.54 (1H, t, J=8.0Hz), 4.15 (2H, d,
J=8.0Hz), 3.80 (3H, s). ESI-MS m/z: 221 [M+H]+. [α]D20 −11.7 azole (4.2a′) White solid. mp: 44–46°C. H-NMR (CDCl3,
(c=0.1, MeOH). 500MHz) δ: 7.41 (1H, dd, J=1.5, 7.5Hz), 7.35 (1H, m), 6.99
(R)-2-(2-Hydroxyphenyl)-4-hydroxymethyl-4,5-dihydrothi-
1
(S)-2-(2-Hydroxyphenyl)-4-methoxycarbonyl-4,5-dihydro- (1H, dd, J=1.0, 8.0Hz), 6.88 (1H, td, J=1.0, 7.5Hz), 4.87
imidazole (3.3a′) White solid. mp: 152–154°C. 1H-NMR (1H, m), 3.97 (1H, dd, J=5.0, 11.5Hz), 3.83 (1H, dd, J=5.0,
(CDCl3, 500MHz) δ: 7.34 (2H, m), 7.01 (1H, dd, J=1.0, 11.5Hz), 3.42 (1H, dd, J=7.5, 11.0Hz), 3.34 (1H, dd, J=7.5,
8.0Hz), 6.84 (1H, m), 4.54 (1H, t, J=8.0Hz), 4.15 (2H, d, 11.0Hz). 13C-NMR (CDCl3, 125MHz) δ: 173.48, 158.96,
J=8.0Hz), 3.80 (3H, s). 13C-NMR (CDCl3, 125MHz) δ: 173.02, 133.23, 130.66, 118.99, 117.06, 116.18, 77.69, 64.08, 32.69. ESI-
165.67, 160.64, 133.03, 126.41, 118.24, 117.63, 111.42, 52.75. MS m/z: 210 [M+H]+. [α]D20 −16.3 (c=0.1, MeOH).
ESI-MS m/z: 221 [M+H]+. [α]D20 +13.7 (c=0.1, MeOH).
(S)-2-(2-Hydroxyphenyl)-4-hydroxymethyl-4,5-dihydro-
1
(S)-2-(2-Hydroxyphenyl)-4-hydroxymethyl-4,5-dihydrooxa- imidazole (4.3a) Yellow oil. H-NMR (CDCl3, 500MHz) δ:
zole (Yanglingmycin) (4.1a) Colorless needle-shaped crystal. 7.80(1H, brs), 7.53 (1H, dd, J=1.5, 8.0Hz), 7.21 (1H, m), 6.71
1
mp: 78–80°C. H-NMR (CDCl3, 500MHz) δ: 7.66 (1H, dd, (1H, dd, J=0.5, 8.5Hz), 6.62 (1H, m), 4.03 (1H, m), 3.73 (1H,
J=1.5, 7.5Hz), 7.39 (1H, m), 7.01 (1H, d, J=8.0Hz), 6.89 (1H, m), 3.49 (1H, m), 3.44 (1H, m), 3.39 (1H, m). ESI-MS m/z: 193
m), 4.50 (2H, m), 4.36 (1H, t, J=6.0Hz), 3.89 (1H, dd, J=3.5, [M+H]+. [α]D20 +25.7 (c=0.1, MeOH).
11.5Hz), 3.71 (1H, dd, J=3.5, 11.5Hz). 13C-NMR (CDCl3,
(R)-2-(2-Hydroxyphenyl)-4-hydroxymethyl-4,5-dihydro-
125MHz) δ: 166.93, 159.78, 133.68, 128.22, 118.81, 116.75, imidazole (4.3a′) Yellow oil. 1H-NMR (CDCl3, 500MHz)
110.40, 68.58, 66.87, 63.95. ESI-MS m/z: 194 [M+H]+. [α]D20 δ: 7.80 (1H, brs), 7.53 (1H, dd, J=1.5, 8.0Hz), 7.21 (1H, m),
−16.4 (c=0.1, MeOH).
6.71 (1H, dd, J=0.5, 8.5Hz), 6.62 (1H, m), 4.03 (1H, m), 3.73
(R)-2-(2-Hydroxyphenyl)-4-hydroxymethyl-4,5-dihydroox- (1H, m), 3.49 (1H, m), 3.44 (1H, m), 3.39 (1H, m). 13C-NMR
1
azole (4.1a′) White solid. mp: 76–78°C. H-NMR (CDCl3, (CDCl3, 125MHz) δ: 174.50, 170.62, 137.91, 132.76, 123.51,
500MHz) δ: 7.66 (1H, dd, J=1.5, 7.5Hz), 7.38 (1H, m), 7.00 120.77, 115.53, 68.93, 65.22, 46.76. ESI-MS m/z: 193 [M+H]+.
(1H, d, J=8.0Hz), 6.88 (1H, m), 4.52 (2H, m), 4.38 (1H, m), [α]D20 −25.3 (c=0.1, MeOH).
3.90 (1H, dd, J=3.5, 11.5Hz), 3.71 (1H, dd, J=3.5, 11.5Hz).
Filter Paper Assay The antibacterial activities of com-
pounds against two strains of bacteria were evaluated by the
ESI-MS m/z: 194 [M+H]+. [α]D20 +17.3 (c=0.1, MeOH).
(S)-2-(4-Hydroxyphenyl)-4-hydroxymethyl-4,5-dihydrooxa- filter paper assay. The standard bacterial strains were obtained
zole (4.1b) White solid. mp: 192–194°C. H-NMR ((CD3)2SO, from College of Plant Protection, Northwest A&F University.
1
500MHz) δ: 10.04 (1H, s), 7.69 (2H, d, J=9.0Hz), 6.81 (2H, d, Ampicillin (Sigma, Shanghai, China) was used as a positive
J=9.0Hz), 4.79 (1H, t, J=5.5Hz), 4.39 (1H, m), 4.24 (2H, m), control. Mueller–Hinton (Hangzhou Microbial Reagent Co.,
3.59 (1H, m), 3.42 (1H, m). 13C-NMR ((CD3)2SO, 125MHz) δ: Ltd., Hangzhou, China) agar was used as an assay medium.
163.25, 160.66, 130.17, 118.83, 115.65, 69.95, 68.43, 63.74. ESI- The medium at 45°C was mixed with a suspension containing
MS m/z: 194 [M+H]+. [α]D20 −51.6 (c=0.1, MeOH).
the bacterial pathogen at approximately 108 colony forming
(R)-2-(4-Hydroxyphenyl)-4-hydroxymethyl-4,5-dihydro- units (CFU)·mL−1. Next, the mixture was poured on 9cm
oxazole (4.1b′) White solid. mp: 192–194°C. 1H-NMR Petri dishes. The tested compounds were dissolved in acetone
((CD3)2SO, 500MHz) δ: 10.04 (1H, s), 7.69 (2H, d, J=9.0Hz), at the concentration of 1000ppm, The filter papers (6mm in
6.81 (2H, d, J=9.0Hz), 4.78 (1H, t, J=5.5Hz), 4.38 (1H, m), diameter) were impregnated with 1µL/disc of each compound,
4.24 (2H, m), 3.58 (1H, m), 3.42(1H, m). 13C-NMR ((CD3)2SO, then were completely dried and placed on the inoculated agar.
125MHz) δ: 163.27, 160.67, 130.18, 118.80, 115.65, 69.96, The inoculated plates were incubated at 37°C for 24h. Anti-
68.41, 63.72. ESI-MS m/z: 194 [M+H]+. [α]D20 +50.2 (c=0.1, bacterial activity was evaluated by measuring the zone of
MeOH).
(S)-2-Phenyl-4-hydroxymethyl-4,5-dihydrooxazole
White solid. mp: 81–83°C. 1H-NMR (CDCl3, 500MHz) δ:
inhibition against the test organism. Experiments were run in
(4.1c) triplicate.
Minimum Inhibitory Concentration (MIC) Anti-
7.88 (2H, m), 7.45 (1H, m), 7.37 (2H, m), 4.48 (1H, m), 4.42 bacterial activities were also measured by the micro-broth
(1H, m), 4.34 (1H, m), 3.97 (1H, m), 3.67 (1H, m). 13C-NMR dilution method in 96-well culture plates using the Mueller–
(CDCl3, 125MHz) δ: 165.58, 131.54, 128.35, 128.30, 69.27, Hinton broth, according to the National Committee for Clini-
68.08, 63.94. ESI-MS m/z: 178 [M+H]+. [α]D20 −51.1 (c=0.1, cal Laboratory Standards.12) The tested bacteria were incu-
MeOH).
bated in the Mueller–Hinton broth for 12h at 30°C at 190rpm,
(R)-2-Phenyl-4-hydroxymethyl-4,5-dihydrooxazole (4.1c′) and the spore concentration was diluted to approximately
White solid. mp: 82–84°C. 1H-NMR (CDCl3, 500MHz) δ: 1×105–1×106 CFU/mL with Mueller–Hinton broth. After incu-
7.88 (2H, m), 7.46 (1H, m), 7.37 (2H, m), 4.48 (1H, m), 4.43 bation for 24h at 30°C, the MICs were examined.
(1H, m), 4.34 (1H, m), 3.97 (1H, m), 3.67 (1H, m). 13C-NMR

Vol. 63, No. 1 (2015)
Chem. Pharm. Bull.
37
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Acknowledgments This work was supported by the
National Key S&T Research Foundation of China
(2010CB126105) and the National Natural Science Foundation
of China (31371958, 21372185), as well as the Funds of Cen-
tral Colleges Basic Scientific Research Operating Expenses
(QN2011117).
Conflict of Interest The authors declare no conflict of
interest.
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