Asymmetric synthesis and bioselective activities of α-amino-phosphonates based on the dufulin motif

Article abstract of DOI:10.1021/acs.jafc.6b01256

The asymmetric synthesis of enantiomerically pure α-aminophosphonates with high and bioselective activities is a challenge. Here, we report that both enantiomers of α-aminophosphonates bearing the N-benzothiazole moiety can be prepared in high yields (up

Full text of DOI:10.1021/acs.jafc.6b01256

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Article
Asymmetric Synthesis and Bioselective Activities
of #-Amino-phosphonates Based on Dufulin Motif
Guoping Zhang, Ge-Fei Hao, Jianke Pan, Jian Zhang, Deyu Hu, and Bao-An Song
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01256 • Publication Date (Web): 10 May 2016
Downloaded from http://pubs.acs.org on May 18, 2016
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Asymmetric Synthesis and Bioselective Activities of αꢀAmino-
phosphonates Based on Dufulin Motif
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Guoping Zhang,†, ‡ Gefei Hao,§ Jianke Pan,† Jian Zhang,† Deyu Hu,† and Baoan
Song* †
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†State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering,
Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Guizhou University, Huaxi District, Guiyang 550025, China
‡ Department of Chemistry, Huaibei Normal University, Huaibei, Anhui 235000, China
§Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of
Chemistry, Central China Normal University, Wuhan430079, China
*Corresponding author (Tel.: 86ꢀ851ꢀ83620521; Fax: 86ꢀ851ꢀ83622211; Eꢀmail:
songbaoan22@yahoo.com)
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ABSTRACT: The asymmetric synthesis of enantiomerically pure αꢀaminophosphonates
with high and bioselective activities is a challenge. Here, we report that both enantiomers
of αꢀaminophosphonates bearing Nꢀbenzothiazole motief can be prepared in high yields
(up to 99%) and excellent enantioselectivities (up to 99% ee) by using chiral thiourea
organocatalysts. Evaluation of the antiviral activities of our reaction products against
cucumber mosaic virus (CMV) led to promising hits with high and selective biological
activities, wherein (R)ꢀenantiomers exhibit higher biological activities than the
corresponding (S)ꢀenantiomers. Especially, compound (R)-3b with excellent antiꢀCMV
activity (curative activity, 72.3%; protection activity, 56.9%; inactivation activity, 96.9%)
at 500 ꢁg/mL emerged as a potential inhibitor of the plant virus. The difference in the
selective bioactivity could be affected by the combination mode of the threeꢀdimensional
space between the enantiomers of αꢀaminophosphonate and cucumber mosaic virus coat
protein (CMVꢀCP) via florescence spectroscopy and molecular docking.
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KEYWORDS: asymmetric synthesis; αꢀaminophosphonate; bioselective activity;
cucumber mosaic virus coat protein; florescence spectroscopy; molecular docking
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INTRODUCTION
Chiral αꢀaminophosphonic derivatives show interesting bioactivities.1ꢀ4 Therefore, the
synthesis of enantiomerically enriched αꢀaminophosphonates has received considerable
attention, with a number of catalytic enantioselective methods reported.5ꢀ10 Among these
methods, the most direct approach involves the addition of phosphites to imines.
Unfortunately, in all these asymmetric methods, highly selectivities are generally
restricted to imine substrates without Nꢀheterocyclic moieties that are necessary for
bioactivity.11ꢀ14 In recent years, Dufulin (Figure 1) a pesticide product, which was
developed by our group has been widely used to prevent and control rice, vegetable, and
tobacco viral diseases in China.15ꢀ16 To exploit highly effective antiviral chiral agents,
asymmetric catalysis is seen as one of the economical strategies to satisfy the growing
demand for enantiomerically pure novel αꢀaminophosphonates based on “dufulin”
skeleton.
Chiral thioureas are privileged organic catalysts which were found effective in a large
set of reactions such as Michael additions,17ꢀ18 Mannich,19ꢀ21 and Aldol,22ꢀ23 DielsꢀAlder
reactions,24etc. Thiourea derivatives can bind to the nitrogen or oxygen atom serving as
catalysts. Notably, Nꢀbenzothiazole moiety in the design of synthetic compounds contains
the nitrogen atom which easily forms hydrogen bonding of chiral thiourea. In addition,
there are few studies in synthesizing highly enantiomerically pure αꢀaminphosphonates
with Nꢀbenzothiazole that exhibit high selective activities.
In this study, chiral αꢀaminophosphonates based on the structure of Dufulin were
synthesized by thiourea organic catalyst (Figure 1). The antiviral activities against
cucumber mosaic virus (CMV) of both enantiomers from our catalytic reactions were
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investigated. Meanwhile, The origins of bioselective activities were further investigated
via fluorescence spectroscopy analysis and molecular docking.
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MATERIALS AND METHODS
General Information. All reactions were carried out in oven dried glassware with
magnetic stirring. Unless otherwise stated, all reagents were purchased from Aladdin
Chemicals Co. (Aladdin, Shanghai, China) and used without further purification. All
solvents used in the reactions were distilled from appropriate drying agents prior to use.
Silica gel GF254ꢀcoated glass plates (Branch Qingdao Haiyang Chemical Co., Qingdao,
China) were used for thin layer chromatography under detection at 254 nm. Silica gel 200
~ 300 mesh (Branch Qingdao Haiyang Chemical Co., Qingdao, China) was applied to
column chromatography. NMR spectra were recorded on a JEOL ECXꢀ500 spectrometer
(JEOL, Tokyo, Japan). Infrared (IR) spectra were recorded on Bruker VECTOR 22
spectrometer (Bruker, Karlsruhe, Germany) using KBr disks. HRMS data were measured
on Thermo Scientific Q Exactive (Thermo, Missour, USA). HPLC analysis was
conducted by an Agilent Technologies 1200 Series system (Agilent, California, USA)
with a 250 mm × 4.6 mm i.d., 5 ꢁm, Chiralpak IA (Daicel) column. Optical rotation
values were measured by a Wzzꢀ2s polarimeter (Shanghai Yue Feng instrument and
Meter Co., Shanghai, China). Fluorescence spectra were performed on Horiba
FluoroMaxꢀ 4 spectrofluorometer (Horiba, Paris, France). The CMVꢀCP protein was
purified from the supernatant by an AKTA purifier protein liquid chromatography system
(GE Healthcare, Pittsburgh, USA) with the NiꢀNTA column.
General Procedure for Preparation of Thiourea-Quinine Catalyst, Q-1.25, 26
To a solution of 9ꢀaminoquinine 27 (1.60 g, 5 mmol) in CH2Cl2 (10 mL) was slowly
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added a solution of 3,5ꢀbis(trifluoromethyl)phenyl isothiocyanate (1.39g, 5.1 mmol) in 10
mL of THF at ambient temperature. The mixture was stirred overnight, and the solvent
was removed in vacuo. The residue was recrystallized with ethanol to afford the catalyst
Q-1 (1.5 g, 75% yield) as white solid (m.p. 121ꢀ123°C; lit.,26 m.p. 121.0ꢀ121.5 °C).
Catalyst Q-2 was prepared under the same reaction condition using 9ꢀaminoquinindium.
General Procedure for Preparation of Aldeimines, 1a~1r.28 A solution of 2ꢀaminoꢀ
4ꢀmethylbenzothiazole (10 mmol) and aldehyde (10 mmol) in toluene (40 mL) was
heated to reflux for 6 h in the presence of HOAc (25mol%). The mixture was cooled
down to room temperature and the solvent was removed in vacuo. The residue was
purified by column chromatography on silica gel with EtOAc/hexane (1:10, v/v) as eluant,
affording the imine, 1a~1r.
General Procedure for Asymmetric Synthesis of αꢀAminophosphonates
Containing N-benzothiazole, 3a~3r. Imine, 1 (0.1 mmol) was added to a dry tube
containing a solution of catalyst, Q (0.01mmol), dry solvent (1 mL), and 4A activating
molecular sieves (100 mg). The mixture was stirred at room temperature for 0.5 h. Then
diphenylphosphite, 2 (0.10 mmol) was added in the mixture. The resulting reaction
mixture was stirred at room temperature for 12 h. The reaction mixture was then purified
by column chromatography on silica gel with EtOAc/hexane (2/1, v/v) as eluant,
affording the product 3. Enantiomeric excess of the product was determined by chiral
stationary phase HPLC analysis. The representative data for 3a is shown below.
(R)ꢀDiphenylꢀ1ꢀ(4ꢀmethylbenzothiazoleꢀ2ꢀamino)ꢀ1ꢀ(4ꢀchlorophenyl)methylphosphonate
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(3a). White solid, m.p. 147–149 °C, [α]D + 80.30 (c 1.00, CHCl3), the opposite
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configuration: [α]D ꢀ78.64 (c 0.93, CHCl3); H NMR (500 MHz, DMSOꢀd6): δ 2.42 (s,
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3H), 6.17 (dd, J = 20.0, 10.0 Hz, 1H), 6.94 (t, J = 10.0 Hz, 1H), 6.98 (dd, J = 10.0 Hz,
2H), 7.04ꢀ7.06 (m, 3H), 7.14ꢀ7.18 (m, 2H), 7.28ꢀ7.33 (m, 4H), 7.48ꢀ7.50 (m, 3H), 7.66
(dd, J = 10.0, 5.0 Hz, 2H), 9.36 (dd, J = 10.0, 5.0 Hz, 1H); 13C NMR (125 MHz, DMSOꢀ
d6) δ 18.46 (s), 54.80 (d, J = 157.5 Hz), 119.11 (s), 120. 85 (d, J = 3.8 Hz), 120.92 (d, J =
3.8 Hz), 122.19 (s), 126.01 (s), 126.91 (s), 128.44 (s), 129.16 (s), 130.44 (s), 130.80 (d, J
= 6.3 Hz), 130.95(s), 134.25 (s), 150.35 (dd, J = 18.9, 10.1 Hz), 150.66 (s), 164.87 (d, J =
10.1 Hz); 31P NMR (202 MHz, DMSOꢀd6) δ 14.60 (s); IR (KBr, cmꢀ1): 3265 (s), 3053 (s),
1587 (s), 1543 (s), 1489 (s), 1256 (s), 1209 (s), 1026 (s), 949 (s), 937 (s); HRMS (ESI)
m/z for C27H22ClN2O3PS [M+H]+ cacld. 521.0856, found. 521.0848; HPLC analysis:
Chiralpak IA, hexane: iPrOH = 75:25, 1.0 mL/min, tR = 12.03 min (major, R), 17.03 min
(minor, S).
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Antiviral Biological Assay. The procedure of purifying CMV and the method to test
the antiꢀCMV activity of both enantiomers of αꢀaminophosphonates bearing Nꢀ
benzothiazole motief were the same as those reported previously in the literature.29, 30
Purification of CMV-CP.31, 32 Genetically engineered CMVꢀCP was expressed, into
which hexahistidine (His) tags were incorporated. To express the protein, we transformed
each plasmid into E. coli strain BL21 (DE3) RIL. The E. coli strain BL21 (DE3) RIL
harboring the recombinant plasmid was cultured in LB medium that contained 30 ꢁg/mL
kanamycin at 37 °C until the OD at 600 nm reached 0.65. Protein expression was induced
at 16 °C by adding IPTG at 1 mM for 16 h. Bacteria were harvested by centrifugation at
12,000 rpm for 10 min at 4 °C. The pellets were suspended in a buffer that contained 20
mM PBS, 150 mM NaCl, 20 mM imidazole, and 1 mM βꢀmercaptoethanol. The
suspension was sonicated for 35 min in an ice bath and then centrifuged at 12,000 rpm for
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30 min at 4 °C to remove cell debris. The protein was purified from the supernatant by an
AKTA purifier protein liquid chromatography system. The NiꢀNTA column was washed
with above buffer, and the proteins were eluted with buffer (50 mM PBS, 250 mM
imidazole, 150 mM NaCl, pH 7.4). The protein was desalinated into a storage buffer (20
mM PBS, and 100 mM NaCl, pH 7.4). The expressed protein was initially assayed by
coomassie brilliant blue method in a smallꢀscale experiment, in which the final
concentration was 10 ꢁM.
RESULTS AND DISCUSSION
Chemistry. Chiral αꢀaminophosphonates based on the structure of Dufulin were
prepared using our methodology.
Optimization
of
Asymmetric
Reaction
Conditions.
The
enantioselective
hydrophosphonylation reaction of Nꢀ(4ꢀmethylꢀbenzothiazolꢀ2ꢀyl)imine, 1a, with
diphenylphosphite, 2, was chosen as a model reaction to optimize reaction conditions
(Figure 2, Table 1). Without any catalyst, the desired product 3a could not be obtained
(entry 1) (Table 1). In initial studies, different catalysts were screened in toluene as the
solvent (1 mL) at room temperature for 12 h (entry 2 and 3) (Table 1). It was found that
the use of the catalyst Q-1 afforded the product 3a in good enantioselectivity with
moderate yield (entry 3) (Table 1). The effects of solvents were then evaluated by using
Q-1 as the catalyst (entry 3~8) (Table 1). CH2Cl2 (entry 7) (Table 1) was found to be an
optimal solvent, giving 3a in a good yield (up to 84%) and high enantioselectivity (up to
96%). When using two equivalents of diphenylphosphite (entry 9) (Table 1), the reaction
yield decreased from 84% to 66%, and ee value decreased from 96% to 88%. When 4A
molecular sieves (100 mg) was added to the reaction mixture, the reaction proceeded
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cleanly (97% yield) with 99% ee (entry 10) (Table 1). It should be noted that the other
enantiomer of the reaction could be obtained by using Q-2 as the catalyst (entry 11)
(Table 1). Diethyl phosphite (instead of diphenyl phosphite) could also be used, but with
a lower yield and ee under the current condition without further optimizations (entry 12)
(Table 1). Finally, the optimal condition was set as: 1a (1.0 equ.), diphenylphosphite (1.0
equ.), catalyst Q (0.1 equ.), 4A molecular sieves (100 mg), CH2Cl2 (1mL), room
temperature, 12 h.
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Asymmetric synthesis of αꢀaminophosphonates, 3a~3r. With the optimized reaction
conditions in hand, the scope of imines were examined to demonstrate the generality of
this enantioselective hydrophosphonylation reaction (Table 2). All substituted imines,
1a~1r, using the catalyst Q-1 or Q-2 underwent the reaction smoothly, leading to the
desired enantiomerically pure αꢀaminophosphonates, 3a~3r, in good to excellent isolated
yields of 93 – 99% and with high levels of enantioselectivity ranging from 91 – 99% ee.
Significantly, the electronic properties of substituted imines do not obviously influence
the reaction outcomes. Quantitative yields and high enantioselectivities were obtained for
aromatic imines bearing a substitutent at the paraꢀ, orthoꢀ, and metaꢀ positions of either
electronꢀdonating groups, such as CH3ꢀ (product 3b), CH3Oꢀ (product 3d, 3i, 3l) and
electronꢀwithdrawing groups, such as Fꢀ(product 3f), and CF3ꢀ (product 3h), and imines
with aromatic substituent also proved to be good substrates (product 3o~3r) with a high
yield and ee values.
Determination of absolute configuration of chiral αꢀaminophosphonates. To determine
the absolute configuration of our product, the crystal of the product 3f obtained by Q-1
catalyst was generated and analyzed by Xꢀray crystallography (Figure 3). The structure 3f
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is given in Figure 3. The absolute configuration of the molecule was determined to be R.
Accordingly the absolute configuration of the product 3f obtained by Q-2 catalyst was S
by analogy. The absolute configuration of all other products, 3, was assigned by analogy.
Antiviral Activity. The antiviral activities of the enantiomers of αꢀaminophosphonates,
3a~3r, against cucumber mosaic virus (CMV) were tested. Dufulin and Ningnanmycin33
were used as the positive controls respectively.
In Vivo AntiꢀTMV Activity. As shown in Table 3, (R)ꢀcompounds generally exhibited
higher antiviral activity than the corresponding (S)ꢀenantiomer. Among them, some
compounds display higher in vivo activity than controls. Compounds (R)ꢀ3b possesses the
best antiꢀCMV activity at 500 ꢁg/mL (curative activity, 72.3%; protection activity, 56.9%;
inactivation activity, 96.9%), which is significantly higher than that of Ningnanmycin
(curative activity, 45.3%; protection activity, 47.9%; inactivation activity, 71.3%) and
Dufulin (curative activity, 50.4%; protection activity, 54.1%; inactivation activity, 78.2%)
at 500 ꢁg/mL. Compound (S)-3b had lower in vivo activity (curative activity, 25.1%;
protection activity, 37.4%; inactivation activity, 57.2%) at 500 ꢁg/mL. In addition, the
phenyl substitution pattern greatly affected the in vivo antiꢀCMV activity of (R)ꢀαꢀ
aminophosphonates. The Fꢀand NO2ꢀ moieties at the 2 position of phenyl ring also
showed good antiꢀCMV activity in vivo (compounds (R)-3f and (R)-3j). Introduction of
the thiophene hetercycle at the position of phenyl ring also possessed good activity
(compound (R)-3p). In a short, the Rꢀconfiguration of title compounds is the preferred
antiviral configuration. Among them, compound (R)-3b could have great potential for
further development as chiral antivirus agents.
The Origins of Bioselective Acticity. CMVꢀCP is an important protein involved in the
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plant virus infections,34ꢀ37 and is being studied as potential protein target to develop
effective antivirus agents.38 To determine the cause of the better activity of the (R)ꢀ versus
the (S)ꢀ compounds, the interactions between chiral compounds 3b and cucumber mosaic
virus coat protein (CMVꢀCP) were performed via fluorescence spectroscopy (Figure 4).
Dufulin and Ningnanmycin were used as the positive controls respectively. The effects of
drugs on CMVꢀCP have been evaluated through binding constants. The binding constant
(Ka) between (R)-3b and CMVꢀCP is 2.19×105 L/mol, indicating a strong ligandꢀ protein
interactions. In contrast, the corresponding binding constants of (S)ꢀ3b (1.17×104 L/mol),
Dufulin (5.25×104 L/mol) and Ningnanmycin (2.40×104 L/mol) with CMVꢀCP are much
lower. Results of the binding studies via fluorescence spectroscopy are consistent with
our experimental observations that (R)-3b shows high antivirus activities. To further
understand the origins of the experimental results showing that (R)ꢀenantiomers exhibited
better activity than (S)ꢀenantiomers, molecular docking with AutoDock 4.039 between
chiral compound 3b and CMVꢀCP was performed (Figure 5). The Xꢀray crystal structure
of CMVꢀCP used for computation was downloaded from RCSB.40 Most of parameters for
the docking calculation were set to the default values. Each docked structure was scored
by the builtꢀin scoring function and was clustered by 1 Å of RMSD criteria. Finally, the
enzyme ligand complex structures were selected according to the criteria for Autodock
Score. The interactions between both enantiomers of 3b and CMVꢀCP can likely occur in
the binding pocket defined by five residues (Phe58, Thr57, Val218, His55 and Ala147).
The binding energy of (R)-3b with CMVꢀCP (ꢀ8.74 Kcal/mol) is lower than that of (S)-3b
(ꢀ6.57 Kcal/mol) and the interaction between (R)-3b and CMVꢀCP is therefore stronger
than that of (S)-3b. The results from molecular docking are in agreement with our in vivo
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experimental observations on the antiviral activities and the results from fluorescence
spectroscopy.
In summary, we have developed a highly efficient and selective catalytic method for
the synthesis of αꢀaminophosphonates with Nꢀbenzothiazole. The products from our
reaction showed potent antiviral activities against CMV, with R-enantiomer performing
much better than the Sꢀenantiomer. The difference in the selective bioactivity could be
affected by the combination mode of the threeꢀdimensional space between the
enantiomers of αꢀaminophosphonate and CMVꢀCP via fluorescence spectroscopy and
molecular docking. Given the promising bioactivity of this class of molecules and the
simplicity of the catalysts and substrates, our results are expected to find important
applications in developing future generations of plant protective agents.
ASSOCIATED CONTENT
Supporting Information
1
Physical and analytical data, H NMR, 13C NMR, HPLC spectra of intermediates, 1a~1r,
target compounds, 3a~3r, this material is available free of charge via the Internet at
http://pubs.acs.org; crystallographic data of compound 3f (CCDC1402656) for this paper
could be obtained free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif
ACKNOWLEDGMENTS
The authors gratefully acknowledge the National Natural Science Foundation of China
(Nos. 21132003, 21362004)
Notes
The authors declare no competing financial interest.
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ABRREVIATIONS
CMV, cucumber mosaic virus; CMV-CP, cucumber mosaic virus coat protein; PBS,
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phosphonic acid buffer solution; RCSB, research collaboratory for structural
bioinformatics; RMSD, root mean square deviation.
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FIGURE CAPTIONS
Figure 1 Synthesis of chiral αꢀaminophosphonates based on Dufulin motif.
Figure 2 Asymmetric synthetic route of condition optimization.
Figure 3 Absolute configuration of compound 3f obtained by thioureaꢀquinine catalyst Q-
1.
Figure 4 Fluorescence emission spectra of CMVꢀCP in the presence of testing compounds
with different concentrations (λex 280 nm). Inset: the linear relationship for quenching
CMVꢀCP.
Figure 5 Molecular docking models of CMVꢀCP in a complex with (R)-3b and (S)-3b
respectively.
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Table 1 Condition Optimization for The Enantioselective Addition of Phosphite to Imine
Entrya
Catalyst
ꢀ
Solvent
Toluene
Toluene
Toluene
THF
Yield (%)e
ee (%)f
1
2
0
0
Quinine
Qꢀ1
32
45
17
21
78
84
80
66
97
96
43
10 (S)
85 (R)
20 (R)
49 (R)
89 (R)
96 (R)
94 (R)
88 (R)
99 (R)
94 (S)
73 (R)
3
4
Qꢀ1
5
Qꢀ1
Et2O
6
Qꢀ1
Hexane
CH2Cl2
Xylene
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
7
Qꢀ1
8
Qꢀ1
9b
10c
11
12d
Qꢀ1
Qꢀ1
Qꢀ2
Qꢀ2
378
379
380
381
a Reaction Conditions:1a (0.10 mmol), diphenylphosphite (0.10 mmol), catalyst (0.01 mmol), solvent
b
c
(1 mL), r.t, 12 h; 2.0 equiv of diphenylphosphite was employed; 100 mg 4Å molecular sieve was
employed; dusing diethylphosphite; eisolated yields; fthe ee was determined by chiral HPLC analysis.
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Table 2 Enantioselective Hydrophosphonylation of Imine
Entry
1
R
Product
3a (R)/(S)
3b (R)/(S)
3c (R)/(S)
3d (R)/(S)
3e (R)/(S)
3f (R)/(S)
3g (R)/(S)
3h (R)/(S)
3i (R)/(S)
3j (R)/(S)
3k (R)/(S)
3l (R)/(S)
3m (R)/(S)
3n (R)/(S)
3o (R)/(S)
3p (R)/(S)
3q (R)/(S)
3r (R)/(S)
Yield (%)a
99/94
95/96
93/95
94/93
99/97
98/96
98/96
98/97
98/97
95/95
95/95
97/98
96/96
94/96
97/95
95/95
95/96
96/97
ee (%)b
97/96
97/96
91/94
96/95
97/96
97/95
97/93
98/94
98/96
95/94
97/93
97/97
98/97
94/97
98/97
98/99
92/94
98/96
pꢀClC6H4
2
pꢀCH3C6H4
pꢀBrC6H4
3
4
pꢀOMeC6H4
Ph
5
6
oꢀFC6H4
7
oꢀClC6H4
8
oꢀCF3C6H4
oꢀOMeC6H4
oꢀNO2C6H4
2,4ꢀCl2C6H4
mꢀOMeC6H4
mꢀBrC6H4
mꢀNO2C6H4
2ꢀfuranyl
9
10
11
12
13
14
15
16
17
18
2ꢀthienyl
2ꢀbenzofuranyl
2ꢀbenzothienyl
383
384
aIsolated yield after silica gel chromatography; bthe ee was determined by chiral HPLC analysis.
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Table 3 Inhibitory Effect of The Title Compounds against CMV in vivo at 500 µg/mL
Curative
Protective Inactivation
Curative
effect (%)a effect (%)a
Protective
Inactivation
effect (%)a
67.5±2.4
57.2±1.3
51.1±1.7
50.7±2.8
60.4±2.8
77.8±2.3
67.3±1.4
51.4±2.1
66.3±2.8
71.1±1.9
47.9±1.7
47.3±1.9
47.2±2.6
57.5±1.9
73.1±2.6
71.6±2.1
41.4±2.6
36.5±2.4
78.2±2.3
Compd.
Compd.
effect (%)a effect (%)a effect (%)a
(R)ꢀ3a
(R)ꢀ3b
(R)ꢀ3c
(R)ꢀ3d
(R)ꢀ3e
(R)ꢀ3f
(R)ꢀ3g
(R)ꢀ3h
(R)ꢀ3i
53.4±1.8
72.3±1.2
34.4±2.2
47.8±2.5
70.2±0.4
71.4±2.1
58.6±1.8
43.1±2.2
57.5±1.7
64.2±1.5
46.1±1.8
36.2±2.5
22.9±2.3
34.7±3.4
55.1±1.3
62.4±1.8
43.3±2.1
41.2±2.3
45.3±1.7
51.1±2.5
56.9±2.6
47.2±1.6
52.2±1.7
50.6±1.2
57.3±1.4
42.1±2.5
48.2±2.7
45.8±2.9
67.4±2.1
46.4±2.2
46.3±1.8
35.1±1.6
44.4±1.9
51.4±2.1
53.6±2.4
27.4±3.3
29.8±1.8
47.9±2.1
79.6±1.9
96.3±1.2
63.5±1.9
72.5±2.6
93.4±1.7
94.1±1.3
88.3±2.3
75.6±1.8
82.2±1.0
90.7±1.6
82.7±1.6
70.8±2.2
55.4±2.2
71.6±1.7
88.6±2.7
89.3±1.7
55.8±1.9
53.3±2.4
71.3±1.6
(S)ꢀ3a
(S)ꢀ3b
(R)ꢀ3c
(S)ꢀ3d
(S)ꢀ3e
(S)ꢀ3f
36.8±2.3
25.1±2.5
21.5±2.3
27.3±3.1
42.1±1.6
54.5±1.9
45.2±2.7
20.3±3.3
34.8±2.6
47.3±2.2
17.4±2.7
15.5±3.1
10.6±2.8
23.8±1.5
44.2±1.7
49.7±1.9
35.2±2.4
19.6±3.1
50.4±1.2
37.4±3.3
37.4±1.7
24.6±3.2
43.4±2.5
39.8±2.3
44.4±1.7
34.3±3.1
39.5±2.6
22.4±1.9
43.1±2.8
23.1±1.4
34.1±2.3
30.2±2.1
32.7±2.6
42.5±2.3
47.3±2.4
17.6±3.1
18.2±2.7
54.1±1.7
(S)ꢀ3g
(S)ꢀ3h
(S)ꢀ3i
(R)ꢀ3j
(R)ꢀ3k
(R)ꢀ3l
(S)ꢀ3j
(S)ꢀ3k
(S)ꢀ3l
(R)ꢀ3m
(R)ꢀ3n
(R)ꢀ3o
(R)ꢀ3p
(R)ꢀ3q
(R)ꢀ3r
Controlb
(S)ꢀ3m
(S)ꢀ3n
(S)ꢀ3o
(S)ꢀ3p
(S)ꢀ3q
(S)ꢀ3r
Dufulinc
386
387
a Average of three replicates; b Ningnanmycin was used as the control; c Dufulin was also used as the
control.
388
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Figure 1
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Figure 2
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Figure 3
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Figure 4
400
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Figure 5
403
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