Design, Synthesis, Fungicidal Activity, and Unexpected Docking Model of the First Chiral Boscalid Analogues Containing Oxazolines

Article abstract of DOI:10.1021/acs.jafc.6b03464

Chirality greatly influences the biological and pharmacological properties of a pesticide and will contribute to unnecessary environmental loading and undesired ecological impact. No structure and activity relationship (SAR) of enantiopure succinate dehydrogenase inhibitors (SDHIs) was documented during the structure optimization of boscalids. On the basis of commercial SDHIs, oxazoline natural products, and versatile oxazoline ligands in organic synthesis, the first effort was devoted to explore the chiral SDHIs and the preliminary mechanism thereof. Fine-tuning furnished chiral nicotinamides 4ag as a more promising fungicidal candidate against Rhizoctonia solani, Botrytis cinerea, and Sclerotinia sclerotiorum, with EC₅₀ values of 0.58, 0.42, and 2.10 mg/L, respectively. In vivo bioassay and molecular docking were investigated to explore the potential in practical application and plausible novelty in action mechanism, respectively. The unexpected molecular docking model showed the different chiral effects on the binding site with the amino acid residues. This chiral nicotinamide also featured easy synthesis and cost-efficacy. It will provide a powerful complement to the commercial SDHI fungicides with the introduction of chirality.

Full text of DOI:10.1021/acs.jafc.6b03464

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Article
Design, Synthesis, Fungicidal Activity and Unexpected Docking
Model of the First Chiral Boscalid Analogues Containing Oxazolines
Shengkun Li, Dangdang Li, Taifeng Xiao, Shasha Zhang, Zehua Song, and Hongyu Ma
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03464 • Publication Date (Web): 28 Oct 2016
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Design, Synthesis, Fungicidal Activity and Unexpected Docking
Model of the First Chiral Boscalid Analogues Containing Oxazolines
Shengkun Li*, Dangdang Li, Taifeng Xiao, ShaSha Zhang, Zehua Song, Hongyu Ma,
Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural
University, Weigang 1, Xuanwu District, Nanjing 210095, People’s Republic of
China.
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Corresponding Author: Shengkun Li, Email SKL505@outlook.com
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ABSTRACT: Chirality greatly influences
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pesticide's biological and
pharmacological properties, and will contribute to unnecessary environment loading
and undesired ecological impact. No structure and activity relationship (SAR) of
enantiopure succinate dehydrogenase inhibitors (SDHIs) were documented during the
structure optimization of boscalids. Based on commercial SDHIs, oxazoline natural
products and versatile oxazoline ligands in organic synthesis, the first effort was
devoted to explore the chiral SDHIs and the preliminary mechanism thereof.
Fineꢀtuning furnished chiral nicotinamides 4ag as a more promising fungicidal
candidate against Rhizoctonia solani, Botrytis cinerea and Sclerotinia sclerotiorum,
with EC50 values of 0.58, 0.42 and 2.10 mg/L, respectively. In vivo bioassay and
molecular docking were investigated to explore the potential in practical application
and plausible novelty in action mechanism, respectively. The unexpected molecular
docking model showed the differently chiral effect on the binding site with the amino
acids residues. This chiral nicotinamides also featured easy synthesis and
costꢀefficacy. It will provide a powerful complement to the commercial SDHI
fungicides with the introduction of chirality.
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KEYWORDS: chiral pesticide, oxazoline, nicotinamide, spoxazomicin, fungicide
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INTRODUCTION
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With an increasing trend for introduction of enantioenriched novel ones, around
oneꢀthird of all agrochemicals are chiral compounds[1]. Chirality greatly influences a
drug's biological and pharmacological properties[2]. Chiral centers are crucial for
biological processes, because the response of an organism to them often depends on
how these molecules fits the particular sites on biological receptors. The application
of chiral pesticides will contribute to unnecessary environment loading and undesired
toxic and ecological impact.
Recent analysis of past and present synthetic methodologies on medicinal
chemistry demonstrates the popularity and significance of amide formation, which is
also frequently used in agrochemical discovery. Amide fungicides are classic but
vibrant among different commercial available agrochemicals, the most famous class
are the succinate dehydrogenase inhibitors (SDHIs). Since the first SDHI carboxin
launched, 18 SDHIs have been developed as agricultural fungicides[3]. Homology
models and docking simulations were also developed to explain binding behaviors
and the peculiarities of the resistance profiles[4]. Schematic overview of structure
showed the SDHIs consist of other three parts (polar core + linker + substituent rest),
keeping the amide bond as constant. Significant progress were made on the
hydrophobic rest optimization, which was speculated to be embedded in the groove on
the target surface. With boscalid as a model, aryl ether and heterocycles, etc. are
typically hydrophobic units modification[5]. The latest commercial generation was
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developed by Syngenta, including isopyrazam, sedaxane and benzovindiflupyr
(Figure 1), we envisage the “dearomatization” may be a tendency for the hydrophobic
rest optimization, while no SAR of enantiopure SDHIs were discussed.
Rethinking the aforementioned the importance of chirality on biological processes，
we envision that this “invaginated hydrophobic groove” on the target surface may be
sensitive to chirality. Herein, we report the first effort to explore the novel SDHIs
with chiral hydrophobic rest and the preliminary mechanism will also be discussed.
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MOLECULAR DESIGN
A literature survey confirmed the popularity of chiral oxazoline in organic
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synthesis[6], natural products and pesticide chemistry (Figure 2). Since the
development of commercially available oxazoline insecticide etoxazole [7], many
etoxazole annalogues with oxazoline unit were patented with acaricidal/insecticidal
activities[8]. DuPont Company explored the structure activity relationships and
evaluate the potential of 2,4ꢀdiaryloxazolines as insecticides and acaricides [9], the
Syngenta group showed novel oxazolines subꢀgroup with chiral Indanyl core as
insecticides[10]. Recently, Wang’s group documented many elegant works on this kind
of bioactive agrochemicals as acaricidal/insecticidal ingredients[11]( Figure 2). In 2011,
the Spoxazomicins AꢀC were isolated by Kazuro Shiomi [12], those alkaloids showed
antitrypanosomal activity without cytotoxicity or obvious antibacterial and antifungal
activities; in the same year, the new oxazoline compound, nocazoline A was isolated
by Zhu[13]. Three years later, the enantiomer of nocazoline A, Yanglingmycin, was
isolated by Wu, interestingly, it exhibited good and broad antibacterial activities.
The chiral difference in biological effect and the importance of oxazolines as
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“privileged ligands” in organic synthesis and medicinal chemistry intrigued us deeply.
We envision that this simple but amusing scaffold can also act as good “ligands” for
biological “acceptor” to facilitate novel pesticide discovery. A novel class of
nicotinamides containing chiral oxazoline was designed and synthesized, which
featured “easy synthesis, Cꢀring dearomatization and chirality formation” (Figure 3).
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MATERIALS AND METHODS
Instruments and Chemicals. All solvents and reagents were purchased from
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commercial sources (Energy, Meryer or Aladdin Chemicals etc.), they were
analytically pure and used as received. Anhydrous solvents were dried and distilled by
standard techniques before use; chlorobenzene was dried over 4A MS for 72hrs
before use, ZnCl2 was flameꢀdried under vacuum just before use. Silica gel GF254 and
column chromatography silica gel for isolation (200~300 mesh) were both purchased
from Qingdao Broadchem Industrial Co., Ltd. Reaction progress was monitored by
thinꢀlayer chromatography (TLC) on silica gel GF254 with ultraviolet (UV254nm)
detection or visualized with phosphomolybdic acid. Yields of all the tiltle compounds
were not optimized. Melting points (M.P.) were recorded on Shenguang WRSꢀ1B
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melting point apparatus and are uncorrected. HNMR and 13C NMR spectra were
carried out utilizing a Bruker AV400 spectrometer with CDCl3 as solvent and
tetramethylsilane as the internal standard and the chemical shifts (δ) were recorded in
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parts per million (ppm). Data for HꢀNMR are reported as follows: chemical shift (δ:
ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet),
coupling constant (Hz), integration and assignment (italic H). Data for 13C NMR are
reported in terms of chemical shift (δ: ppm). (C) stands for quaternary carbon, (CH)
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stands for tertiary carbon, (CH2) stands for secondary carbon, (CH3) stands for
primary carbon. Elemental analyses were performed on a CHNꢀOꢀRapid instrument.
Mass spectrometry (MS) data were obtained with Waters Xevo TQꢀS Micro
Spectrometer. QSAR Analyses and Molecular Docking were performed with Tripos
SYBYL X 2.0 program.
General Procedure for the Synthesis of Compound 2. A threeꢀneck roundꢀbottom
schlenk flask fitted with a magnetic stir bar, a reflux condenser and an addition funnel,
was charged with sodium borohydride (0.95g, 25mmol) and 50mL dry
tetrahydrofuran (THF) under N2 atmosphere; then 1.65g of LꢀPhenylalanine(10mmol)
was added in one portion and cooled to 0 °C with an ice bath. A solution of iodine
（2.54g, 10mmol）in dry THF (25 mL) was added slowly and dropwise with an
addition funnel under vigorous stirring. After completion of the iodine addition, the
whole system was put into a preheated oil bath (80 °C), the progress of the reaction
was monitored by TLC until the reaction was complete (~12h). The flask was then
cooled to room temperature, and cold water was added cautiously to quench the
reaction. The solvent was removed under vacuum, 20mL of 20% aqueous KOH was
added to the white paste and the solution was stirred for 1 h and extracted by
dichloromethane (DCM, 30mL X 4), The organic extracts were combined and dried
over sodium sulfate and concentrated in vacuum to afford a white semisolid 1.298g
(yield 86%) and was used for next step without further purification.
General Procedure for the synthesis of Compound 3. Method 1[14]; an oven dried
twoꢀnecked schlenk flask was purged with nitrogen and charged with freshly flame
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dried ZnCl2 (405 mg, 3 mmol), anhydrous chlorobenzene (6 mL), 2ꢀaminobenzonitrile
(118 mg, 1 mmol) and (R)ꢀ2ꢀaminoꢀ2ꢀphenylethanol (1.2 mmol) respectively. The
mixture was stirred at reflux and the reaction progress was monitored by TLC until
the reaction was complete (24 h~48h). The solvent was then removed under vacuum
and the residue was stirred with 30% NaOH (10mL) for 0.5 h. The product was
extracted with dichloromethane(15mL X 3) and purified by flash column
chromatography on 200~300 mesh silica gel (hexane/EtOAc, 2:1) to give the
(R)ꢀ2ꢀ(4ꢀphenylꢀ4,5ꢀdihydrooxazolꢀ2ꢀyl)aniline as white solid (Yield 60.1%).
Method 2: A tiny modification from the literature report procedure[15]. To an oven
dried tube under nitrogen atomosphere, was added 2ꢀaminobenzonitrile (118 mg, 1
mmol), (R)ꢀ2ꢀaminopropanꢀ1ꢀol, and freshly flame dried ZnCl2 (13mg, 10%mmol).
The mixture was sealed with teflon tape and stirred at 150℃，the reaction progress
was monitored by TLC until the consumption of aminobenzonitrile （6h~8h）. The
reaction mixture was quenched and suspended with ethyl acetate（50mL），30% NaOH
(5 mL) was added and the organic phase was washed with H2O (10mL X 2) and
saturated aqueous NaCl（10mL）respectively，then dried over anhydrous sodium
sulfate, filtered, and concentrated by evaporation under vacuum to give the crude
products，which was subject to flash chromatography purification on silica gel
(hexane/EtOAc, v/v = 2:1)to give (R)ꢀ2ꢀ(4ꢀmethylꢀ4,5ꢀdihydrooxazolꢀ2ꢀyl)aniline as a
light yellow oil(68.4%).
The other 2ꢀ(4, 5ꢀdihydrooxazolꢀ2ꢀyl)anilines were synthesized accordingly, with
yields ranging from 55.4% to 72.8%.
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General Procedure for the synthesis of Compound 4.
With Acyl Chloride: To a dried schlenk flask charged with the aforementioned
synthesized 2ꢀ(4,5ꢀdihydrooxazolꢀ2ꢀyl)aniline 3 (1mmol)，were successively added
anhydrous DCM (10mL) and distilled NEt3 under N2 atomosphere. The solution of
nicotinoyl chloride in anhydrous DCM was added slowly under iceꢀbath. The mixture
was allowed to warm gradually to ambient temperature and stirred for 24 h. Saturated
aqueous NH4Cl（15mL）was added to quench the reaction, separated and extracted
with dichloromethane (15mL X 3). Then the combined organic phase was washed
with water (10mL X 2) and saturated aqueous NaCl (10mL), dried over anhydrous
sodium sulfate, and concentrated to give the crude products, which was subject to
flash chromatography purification on silica gel (hexane/EtOAc, v/v =2:1) to give the
desired product.
Compounds 4ba−4bd were synthesized according to this procedure.
With Pyridyl acid: To a dried schlenk flask charged with the aforementioned
synthesized 2ꢀ(4,5ꢀdihydrooxazolꢀ2ꢀyl)aniline 3 (1mmol) and the pyridyl acid (1.05
mmol), were added anhydrous DCM (8 mL) and DiPEA (1.5mmol) and the mixture
was vigorously stirred for dissolution, Nꢀ(3ꢀDimethylaminopropyl)ꢀ N′ꢀ
ethylcarbodiimide hydrochloride (EDCIꢀHCl) (0.211 g, 1.1 mmol) and
4ꢀdimethylaminopyridine (DMAP, 0.012 g, 0.1 mmol), and then the mixture was
stirred overnight at room temperature until the full consumption of compound 3
detected by TLC. The mixture was quenched by the addition of a saturated aqueous
solution of NH4Cl (20 mL) and separated. The water phase was extracted with
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dichloromethane (15mL X 3), and the combined organic phase was washed with water
(10mL X 2) and saturated aqueous NaCl (10mL) successively, dried over anhydrous
sodium sulfate, concentrated under vacuum and purified by chromatography on silica
gel (hexane/EtOAc, v/v = 4:1 ~ 2:1) to give the desired product.
Unless otherwise mentioned, the other oxazoline nicotinamides were synthesized
according to this procedure.
Take compound 4be as an example for physicochemical detection and structure
elucidation: (R)ꢀNꢀ(2ꢀ(4ꢀmethylꢀ4,5ꢀdihydrooxazolꢀ2ꢀyl)phenyl)nicotinamide (4be),
white solid, m.p. 88.1~88.4℃, purified on silica chromatography (Hexane/ Ethyl
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acetate= 4:1), Yield 79%. HꢀNMR (CDCl3, 400 MHz) δ: 1.47(d, J = 5.08Hz, 3H,
CH3), 3.97(dd, J1 = 5.92Hz, J2 = 5.76 Hz, 1H, 1H in OCH2), 4.52~4.61(m, 2H, 1H in
OCH2 and 1H in CHNꢀCH3), 7.17 (ddd, 1H , J1 = 6.32Hz, J2= 5.88Hz, J3 = 0.84 Hz,
1H, aromatic H in phenyl ring) 7.53~7.57(m, 2H, aromatic H in phenyl ring and
Pyridyl ring ), 7.92(dd, J1 = 6.32Hz, J2 = 1.24 Hz, 1H, aromatic H in phenyl ring),
8.53(m, 1H, aromatic H in Pyridyl ring), 8.79(dd, J1 = 3.88Hz, J2 = 1.08 Hz, 1H,
aromatic H in phenyl ring), 8.90(dd, J1 = 6.20Hz, J2 = 0.56 Hz, 1H, aromatic H in
Pyridyl ring), 9.37(d, 1H, J = 1.40 Hz, aromatic H in Pyridyl ring), 13.41(s, 1H, NH).
13CꢀNMR and DEPT135 (CDCl3, 100 MHz) δ: 21.6(CH3), 61.9(CH), 72.9(OCH2),
113.8(C), 119.9(CH), 123.1(CH), 124.1(CH), 129.4(CH), 131.7(C), 132.8(CH),
137.2(CH), 139.7, 147.5(CH), 150.6(CH), 163.2(C), 163.9(C). Elemental anal. calcd
for C16H15N3O2: C, 68.31; H, 5.37; N, 14.94; Found: C, 68.42; H, 5.41; N, 14.96.
ESIꢀMS, Calcd for [M+H] 282.12, found 282.19.
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Biological Assay. The fungi were provided by the Department of Pesticide, College
of Plant Protection, Nanjing Agricultural University (Nanjing, China). The fungicidal
activity of the target compounds was tested in vitro against the three plant pathogenic
fungi using the mycelium growth rate test. All the tested compounds were dissolved
in DMSO at a concentration of 10 mg·mL−1. The media containing compounds at a
concentration of 50 ꢁg·mL−1 were then poured into Petri dishes for initial screening.
In the precision antifungal test, the 10 mg·mL−1 solution was diluted to 25, 12.5, 6.25,
3.125, 1.56 ꢁg·mL−1 and the above experiments were repeated three times, the
inhibition rates were calculated separately.
The in vivo fungicidal activity of the target compounds was carried out on tomato.
Appropriate amounts of all the test samples, including synthesized nicotinamides and
positive control boscalid, in 0.2 mL of dimethyl sulfoxide (DMSO) were suspended in
20mL distilled water with triton (0.1%) at a concentration of 100 mg/L. Each
suspension was sprayed onto the fruits of tomato (Lycopersicum esculentum), which
is washed and treated with water and 75% aqueous ethyl alcohol in advance. After
evaporation under ambient environment (~28℃), the epidermis (Ø 4.5 cm) on fruits
was punctured with inoculating needle, then each pathogen was inoculated. 1%
aqueous DMSO containing 0.1% triton was set up as blank control. All the treated
fruits were then placed into illumination incubator (25℃, 100% relative humidity) for
4 days. This experiments were repeated for three times.
The statistical analyses were performed by SPSS software (SPSS Statistic19.0).
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QSAR Analyses and Molecular Docking. The 3DꢀQuantitative StructureꢀActivity
Relationship (3DꢀQSAR), and Molecular Docking were performed with the SYBYL
X 2.0 program, using Topomer CoMFA and SurflexꢀDock respectively. Besides the
core of the molecule, we split the functional groups of synthesized compounds into
two Rꢀgroups that refer to the amine moiety and carboxylic acid moiety groups. The
synthesized oxazoline amides were used to create a data set in which the inhibition
rate and EC50 of all compounds was determined against Botrytis cinerea.
Threeꢀdimensional structures of the target compounds were built by the ChemBio 3D
Ultra software version 12.0. The Structures were imported to Tripos SYBYL X 2.0
and optimized for lowest energy geometry. Crystal structure of succinate
dehydrogenase (SDH) was downloaded from RCSB Protein Data Bank (PDB code:
2FBW). The dabigatran was docked in the corresponding binding site by an empirical
scoring function in SurflexꢀDock. Before the docking process, the ligand (P/CBE 202)
was extracted and all water molecules were removed from the crystal structure.
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RESULTS AND DISCUSSION
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Synthesis. The chiral amino alcohols were synthesized by the reduction of the
corresponding chiral amino acids with previously reported systems of NaBH4ꢀH2SO4
or NaBH4ꢀI2 in good to excellent yields[16]. In most case, the NaBH4ꢀI2 was used to
provide chiral amino alcohols. The chiral oxazoline units were established by
condensation of commercially available aromatic nitrile and different amino alcohols
based on the ZnCl2 catalyzed WitteꢀSeeliger reaction[14], It can also be synthesized
under microwave irradiation with higher temperature[15]. For the sake of easy workup,
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we carried out this transformation with the modified WitteꢀSeeliger reaction in a
sealed tube at 150℃ catalyzed by freshly flame dried ZnCl2 under solvent free
conditions. The amide bond formation was realized in good to excellent yields
either by direct condensation of acyl chloride with amine or DMAP catalyzed steglich
type reaction from organic acids activated and dehydrated by EDCI (Scheme 1).
Antifungal Activity and SAR Discussion. We began our investigation with the
simple and easily synthesized nicotinamide containing the unsubstituted oxazoline
unit (compound 4ba), the initial attempt for the bioactivity screening was not so
successful because of its poor physicochemical properties. This was readily solved by
the introduction of substituents on either the pyridinyl ring (4aa, 4ca and 4da) or the
oxazoline subuinit (4bb~4be). For the preliminary assessment of steric effect of the
substituents on the bioactivities, the smallest CH3 and bulky aromatic Phenyl ring
were designed and decorated on the 4ꢀposition of oxazoline, methyl effect showed
more advantages over the phenyl counterpart. The Hydroxyl group on the pyridinyl
ring is detrimental for the antifungicidal activity, as can be seen from the table 1, the
introduction of OH will lead sharp decrease in bioactivity, no matter what the
substituent and the configuration of the oxazoline ring is, especially for the
synthesized amides against the Botrytis cinerea and Sclerotinia sclerotiorum” (table1,
4ca~4ce). The importance of nicotinamide subunit on the bioactivity was confirmed
by the synthesis and bioassay of the corresponding nicotinic acid ester and benzoate
(Figure 4).
The electron donating groups (EDGs) are more beneficial for the antifungnal
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bioactivity of nicotinamide with unmodified oxazoline ring (MeO > Cl, 4aa vs 4da),
while the situation changed inversely when the oxazoline part was substituted and the
chirality was introduced (Cl > MeO, 4ab~4ae and 4db~4de). Replacement of
nicotinyl group by isonicotinyl gave comparable activities in most cases, while when
the 4ꢀposition of oxazoline units was decorated by phenyl ring with Rꢀconfiguration,
the inhibition against Rhizoctonia solani and Sclerotinia sclerotiorum dropped sharply
with the increment of EC50 from 5.34 and 9.75 to 17.57 and 18.02 mg/L, respectively
(compound 4be vs 4ee). Fineꢀtuning of the substituent on pyridyl ring protrude
2ꢀClꢀPyridinyl amide as good candidates for further optimization. The effect of the
substitution and the chiral properties or steric configuration in the 4ꢀposition of
oxazoline on the fungicidal activity is prominent. For most case, the Rꢀconfiguration
is preferred for both nicotamides and isonicotamides, with similar if not superior
inhibitory effect.
With this interesting and encouraging results, we envisage that fine tuning of the
size and configuration of substituents may be beneficial for the discovery of more
promising fungicidal agents. Keeping 2ꢀClꢀnicotamide as constant, compounds
4af~4am were synthesized and screened (Table 2). The antifungal activity was
enhanced when the bulkier aliphatic groups were introduced, including Et, iPr and
Butyl, with the R configuration as promising enantiomers, and it followed Ph < Me <
iBu < iPr < Et (4ae, 4ad, 4ak, 4ah, 4ag). The EC50 values of the aforementioned
nicotamides against Botrytis cinerea were 13.99; 3.14, 1.15, 0.51 and 0.42 mg/L
respectively. Through with same configuration, the isomeric formation of substituents
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(iBu, sꢀBu, tꢀBu) provided significant difference in bioactive outcomes (4aj vs 4al vs
4am).
It is noteworthy that 4ag, 4ai, 4ak and 4al demonstrated more potent bioactivity
than the commercial fungicide boscalid against Botrytis cinerea. Take all the factors
together and based on the preliminary SAR study, compound 4ag may be served as a
potent fungicidal ingredient. It featured chiral difference, easy synthesis, good activity
and costꢀefficiency than the commercial available boscalid.
The compound 4ag showed higher bioactivities against Rhizoctonia solani and
Botrytis cinerea than the positive control boscalid, with the EC50 values of 0.58, and
0.42 mg/L, respectively. The tiny weaker bioactivity against Sclerotinia sclerotiorum
than boscalid can be counterbalanced by the easy synthesis and costꢀefficiency. As
inherently physiochemical and structural properties (i.e., large size and permeability)
were crucial for pesticideꢀlikeness[17], the calculated octanolꢀwater partition
coefficients (ClogP) and Topological Polar Surface Area (TPSA) were extracted from
ChemBioOffice. Statistical analysis showed 90% of the ClogP values of fungicides
ranged from 1.0 to 6.0, and the mean value of ClogP for commercial fungicides was
3.3. The synthesized nicotamides were all distributed between 2.1 and 5.1. The
optimized candidates, with ClogP values of 3.2 to 3.6 (Table 2), correlated well with
the statistical analysis. The strong fungicidal activity and easy synthesis demonstrated
the potential in the discovery of novel chiral SDHIs.
In vivo Antifungal Activity and Discussion. With the “original model” boscalid as
positive control, in vivo bioassay was carried out for the compound 4ag against
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Botrytis cinerea. The Sꢀenantiomer (4af) of 4ag was also tested to document whether
in vivo chiral difference exsits. As can be seen in Figure 5, 4af demonstrated moderate
efficacy, while chiral difference in biological response was prominent. To our delight,
the chiral nicotinamide 4ag afford the good preventative effect against Botrytis
cinerea. With the preventative rate of 54.8%, 4ag showed no significant difference
from that of the positive control boscalid, which demonstrates the practically potential
of this novel chiral nicotinamides for crop protection.
QSAR Analyses and Molecular Docking. Topomer CoMFA is an
alignmentꢀindependent 3DꢀQSAR method that combines the topomer search method
with the conventional CoMFA method. The sterically favored and disfavored regions
are shown in green and yellow respectively. The positively charged favored regions
are shown in blue in the electrostatic field, and the opposite effect are shown in red.
The virtual model demonstrated the necessity of decoration of electron withdrawing
groups and bulky substituents on pyridine and oxazoline respectively.
Based on the aforementioned results, our effort was further devoted to explore the
possible and preliminary mechanism of the observed antifungal activity and chiral
difference of synthesized chiral nicotamides. The target protein (pdb code: 2FBW)[18]
was downloaded, modified and optimized by Tripos SYBYLꢀX 2.0, the ligand was
extracted (P/CBE202) and all the waters were removed from the protein(see SI for
more details), based on which surflexꢀdock was carried out. The threeꢀdimensional
schematic diagrams clearly explained the possible optimal combination between the
ligands and receptor protein. It is speculated that this kind of nicotamides and
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ananlogues were bound with Qp site of SDH[5b, 19]. To explore the plausible binding
models of the synthesized chiral nicotinamides, 4ag and its enantiomer 4af were
chosen again in surflexꢀdock, with boscalid and carboxin as positive control and
reference compound, respectively. The weaker interactions with the amino acid
residues of MET36, SER39, ILE40, AGR43, PRO169 were reported previously [5b], to
make docking model more clear and easier to read, only major interactions through
hydrogen bond were highlighted.
As can be seen from Figure 6, the synthesized chiral nicotinamides (4af and 4ag),
and the boscalid were embedded well in the same protein groove established by
extraction of carboxin from the original protein crystal (PDB 2FBW). Interestingly,
though our design of novel nicotinamides was based on the commercial boscalid, the
embedded subunits were different (structures shown in brown in Figure 6). The
substructure nicotinamide of boscalid is buried in the protein cavity, while this
situation changed to oxazoline for both 4af and 4ag, casting pyridyl ring out. This
may be due to the sensitivity of the protein cavity to the size of ligand. It is speculated
that the smaller fiveꢀmembered oxazoline ring will be much easier and more suitable
than the biphenyl ring for docking to the “groove” in the target protein. Two major
hydrogen bonds were formed on TRP58 and TRP173 amino acid residues for 4af and
boscalid, while only one was formed between the compound 4ag and TRP173. The
hydrogen bonding distance to TRP173 for 4af, 4ag and boscalid was 2.04 Å, 2.27 Å,
and 1.90 Å respectively.
In accordance with our original design and hypothesis, the docking model also
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differentiated the chirality of the enantiomenrs 4af and 4ag. As shown in Figure 6, the
enantiomers have entirely different bonding sites to the amino acid residues. The
hydrogen bond was constructed based on the “oxygen” of oxazoline in 4af, while the
one for 4ag is donated by “nitrogen” of oxazoline. To some extent, those interesting
differences may demonstrate novel mechanism for the chiral nicotinamides compared
with the commercial available SDHIs.
In summary, a series of novel Nꢀ(2ꢀ(4, 5ꢀdihydrooxazolꢀ2ꢀyl) phenyl) nicotinamides
were designed and synthesized, featured enantiomers of different oxazolines at ortho
position of the arylamine part. The results of the bioassay indicated that all of them
exhibited moderate to remarkable antifungal activities against the tested fungi.
Structureꢀactivity relationships (SAR) were investigated to demonstrate the pivotal
role of chirality of the oxazoline ring in various biological response. Fineꢀtuning of
the chiral substituents protrudes compound 4ag with better activity than the
commercial boscalid. In vivo bioassay and molecular docking were investigated to
explore the potential in practical application and plausible novelty in action
mechanism, respectively. This is the first exploration of chiral nicotinamides for novel
SDHIs fungicides discovery. The present results will provide a powerful complement
to the commercial SDHI fungicides and will lay solid foundation for novel fungicides
discovery. Further structural optimization and investigation of the action mechanism
of the chiral analogues are in progress and will be reported in due course.
386
387
AUTHOR CONTRIBUTIONS
S. Li conceived and designed this work; S. Li performed the skeleton construction and
388
analyzed the data. T. Xiao carried out derivative synthesis and the biological
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experiments. D. Li conducted the in vivo bioassay and analyzed NMR and MASS data.
D. Li, S. Zhang and H. Ma carried out MASS detection. S. Li wrote the paper.
391
392
ACKNOWLEDGMENT
This work was financially supported by National Natural Science Foundation of
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China (No. 31401777), Natural Science Foundation of Jiangsu Province
(No.BK20140684), and the Fundamental Research Funds for the Central Universities
(No. KJQN201510). This work was dedicated to Prof. Wenjun Wu on the occasion of
his retirement from Northwest A&F University.
SUPPORTING INFORMATION
Graphics in biological test together with molecular docking are available.
REFERENCES
400
401
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405
406
407
408
409
410
411
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413
414
415
416
417
418
419
420
421
422
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[8]. Patents N.O.s: EP0686345A1; EP1625792A1; JP2009292793A; US5633271A; WO0058291A1;
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Hillegass, K.; Jones, L.; Yatsko, C., Insecticidal Oxazolines. 2001, 800, 188ꢀ198.
[10].O’Sullivan, A. C.; Schaetzer, J. H.; Luethy, C.; Mathews, C. J.; Elliott, C.; Pitterna, T.; Pabba, J.;
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IndanylꢀOxazolines, Thiazolines and AlkoxyꢀAlkylꢀImidazolines. 2015, 1204, 411ꢀ430.
[11].(a) Li, Y.; Li, C.; Zheng, Y.; Wei, X.; Ma, Q.; Wei, P.; Liu, Y.; Qin, Y.; Yang, N.; Sun, Y.; Ling, Y.;
Yang, X.; Wang, Q., Design, Synthesis, Acaricidal Activity, and Mechanism of Oxazoline
Derivatives Containing an Oxime Ether Moiety. J. Agric. Food Chem. 2014; (b) Yu, X.; Liu, Y.; Li,
Y.; Wang, Q., Design, Synthesis, and Acaricidal/Insecticidal Activities of Oxazoline Derivatives
Containing a Sulfur Ether Moiety. J. Agric. Food Chem. 2015, 63 (44), 9690ꢀ5; (c) Yu, X.; Liu, Y.;
Li, Y.; Wang, Q., Design, Synthesis, Acaricidal/Insecticidal Activity, and StructureꢀActivity
Relationship Studies of Novel Oxazolines Containing Sulfone/Sulfoxide Groups Based on the
Sulfonylurea Receptor ProteinꢀBinding Site. J. Agric. Food Chem. 2016, 64 (15), 3034ꢀ40.
[12].Inahashi, Y.; Iwatsuki, M.; Ishiyama, A.; Namatame, M.; NishiharaꢀTsukashima, A.; Matsumoto,
A.; Hirose, T.; Sunazuka, T.; Yamada, H.; Otoguro, K.; Takahashi, Y.; Omura, S.; Shiomi, K.,
Spoxazomicins AꢀC, novel antitrypanosomal alkaloids produced by an endophytic actinomycete,
Streptosporangium oxazolinicum K07ꢀ0460(T). The Journal of antibiotics 2011, 64 (4), 303ꢀ7.
[13].Fu, P.; Liu, P.; Qu, H.; Wang, Y.; Chen, D.; Wang, H.; Li, J.; Zhu, W., αꢀPyrones and
Diketopiperazine Derivatives from the MarineꢀDerived Actinomycete Nocardiopsis dassonvillei
HR10ꢀ5. J. Nat. Prod., 2011, 74 (10), 2219ꢀ2223.
[14].Wolińska, E., Asymmetric Henry reactions catalyzed by copper(II) complexes of chiral
1,2,4ꢀtriazineꢀoxazoline ligands: the impact of substitution in the oxazoline ring on ligand activity.
Tetrahedron: Asymmetry 2014, 25 (15), 1122ꢀ1128.
[15].Hassani, R.; Requet, A.; Marque, S.; Gaucher, A.; Prim, D.; Kacem, Y.; Hassine, B. B., Efficacious
and rapid metalꢀ and solventꢀfree synthesis of enantiopure oxazolines. Tetrahedron: Asymmetry
2014, 25 (18ꢀ19), 1275ꢀ1279.
[16].(a) McKennon, M. J.; Meyers, A. I.; Drauz, K.; Schwarm, M., A convenient reduction of amino
acids and their derivatives. J. Org. Chem. 1993, 58 (13), 3568ꢀ3571; (b) Abiko, A.; Masamune, S.,
An improved, convenient procedure for reduction of amino acids to aminoalcohols: Use of
NaBH4ꢀH2SO4. Tetrahedron Letters 1992, 33 (38), 5517ꢀ5518.
[17].(a) Rao, H.; Huangfu, C.; Wang, Y.; Wang, X.; Tang, T.; Zeng, X.; Li, Z.; Chen, Y.,
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Discovery and Screening Library Development. Molecular Informatics 2015, 34 (5), 331ꢀ338; (b)
Hao, G.; Dong, Q.; Yang, G., A Comparative Study on the Constitutive Properties of Marketed
Pesticides. Molecular Informatics 2011, 30 (6ꢀ7), 614ꢀ622.
[18].2FBW,
The
Protein
with
PDB
code
2FBW
was
downloaded
from
http://www.rcsb.org/pdb/explore/explore.do?structureId=2FBW.
[19].Maklashina, E. C., G. , The quinoneꢀbinding and catalytic site of complex II. Biochim.Biophys.
Acta 2010, 12, 1877ꢀ1882.
461
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Figures
Figure 1. SDHIs model and typical fungicides
Oxazoline Scaffold as Privileged "Ligands" in both Biology and Chemistry
Commercial Pesticide and Bioactive Molecules
F
O
H
F
O
OC2H5
O
N
O
N
N
N
R
H
N
S
OH
F
H
F
Spoxazomicin A and B
Etoxazole
R=(Sulfur) Ether or Sulfone/Sulfoxide
R2
R1
Oxazoline Ligands for Asymmetric Catalysis
O
N
O
O
O
O
O
N
R1
R2
N
N
O
N
PAr2
N
N
N
R2
R1
Phosphinooxazolines
R1
R
R2
O
R
N
R2
PyBisoxazoline
R1
Bisoxazoline
R2
Trisoxazolines
R1
Figure 2. Popularity of chiral oxazolines in bioactive molecules and organic synthesis
Figure 3. Design of the first chiral SDHIs with oxazoline
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O
O
O
N
O
N
H
EC50 value
(ug/mL)
H
N
Cl
N
Cl
Cl
N
O
N
O
N
O
Rhizoctonia solani
Botrytis cinerea
44.39
>200
>200
>12.5
>12.5
>12.5
5.10
3.17
11.69
Sclerotinia sclerotiorum
Figure 4. Importance of nicotinamide unit on bioactivity
Figure 5. In vivo activity of 4af and 4ag
Figure 6. Docking model of 4af, 4ag and boscalid
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Scheme
Scheme 1. Synthesis of Chiral Nicotamides
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Tables
Table 1. Preliminary Synthesis and Antifungal activity of Chiral Nicotinamides
Structure
S.P. =
Bioactivity (EC50, mg/L)
Properties
ClogP
Compd
4aa
4ab
4ac
4ad
4ae
4ba
4bb
4bc
4bd
4be
4ca
4cb
4cc
4cd
4ce
4da
4db
4dc
4dd
4de
4ea
4eb
4ec
4ed
4ee
R=
H
R. S.
B. C.
S. S.
tPSA
63.05
63.05
63.05
63.05
63.05
63.05
63.05
63.05
63.05
63.05
83.23
83.23
83.23
83.23
83.23
72.28
72.28
72.28
72.28
72.28
63.05
63.05
63.05
63.05
63.05
41.46
2ꢀClꢀnicotinyl
2ꢀClꢀnicotinyl
2ꢀClꢀnicotinyl
2ꢀClꢀnicotinyl
2ꢀClꢀnicotinyl
nicotinyl
56.51±0.69 l
11.18±0.24 e
29.56±1.44 i
4.99±0.13 b
27.10±0.67 h
ꢀ
151.17±0.57 l
16.15±0.28 de
20.24± 1.86 f
3.14±0.10 a
13.99±0.15 cde
ꢀ
99.73± 0.53 m
25.75±0.51 h
30.28±1.67 j
11.29±0.31 c
21.33±0.42 g
ꢀ
2.160
2.679
3.718
2.679
3.718
2.198
2.717
3.756
2.717
3.756
3.486
4.005
5.044
4.005
5.044
2.943
3.453
4.492
3.453
4.492
2.198
2.717
3.756
2.717
3.756
3.437
SꢀCH3
SꢀPh
RꢀCH3
RꢀPh
H
nicotinyl
SꢀCH3
SꢀPh
RꢀCH3
RꢀPh
H
8.31±0.33 cd
21.61±1.16 g
8.21±0.07 cd
5.34±0.13 b
114.44±3.71 p
94.26±0.72 n
44.31±0.62 k
23.91±0.13 g
21.67±1.11 fg
11.78±0.29 bc
10.60±0.42 b
>200
19.73±0.15 f
25.58±0.46 h
20.92±0.45 fg
9.75±0.08 b
>200
nicotinyl
nicotinyl
nicotinyl
2ꢀOHꢀnicotinyl
2ꢀOHꢀnicotinyl
2ꢀOHꢀnicotinyl
2ꢀOHꢀnicotinyl
2ꢀOHꢀnicotinyl
2ꢀOCH3ꢀnicotinyl
SꢀCH3
SꢀPh
>200
>200
>200
>200
RꢀCH3 99.96±2.11 o
138.79±1.81 k
174.31±8.99 m
23.57±0.43 g
17.01±0.43 e
57.02±0.57 j
13.65±0.20 bcd
14.37±0.21 cde
28.18±0.20 h
12.31±0.12 bc
34.02±0.28 i
11.45±0.18 bc
16.32±0.34 de
1.66±0.12 a
>200
RꢀPh
86.96±0.45 m
9.41±0.04 d
16.34±0.13 f
134.66±0.52 r
12.77±0.12 e
127.99±0.45 q
9.57±0.32 d
7.34± 0.15 c
37.67±0.43 j
5.71±0.14 b
17.57±0.33 f
1.59±0.11 a
>200
H
28.62±0.16 i
31.35±0.11 j
147.88±2.01 n
54.53±0.32 l
40.33± 0.46 k
28.44±0.43 i
25.57±0.43 h
18.13±0.15 e
15.09±0.24 d
18.02±0.48 e
0.34±0.05 a
2ꢀOCH3ꢀnicotinyl SꢀCH3
2ꢀOCH3ꢀnicotinyl SꢀPh
2ꢀOCH3ꢀnicotinyl RꢀCH3
2ꢀOCH3ꢀnicotinyl RꢀPh
isonicotinyl
isonicotinyl
isonicotinyl
isonicotinyl
isonicotinyl
Boscalid
H
SꢀCH3
SꢀPh
RꢀCH3
RꢀPh
Note: R. S.: Rhizoctonia solani; B. C.: Botrytis cinerea; S. S.: Sclerotinia sclerotiorum. The EC50 values
are presented as mean ± standard deviation of triplicate experiments. Different small letters in the same
column showed significant difference at P < 0.05 level, through Duncan’s multiple range test in SPSS
statistics 19.0. The alphabetical order is consistent with the high to low order of the antifungal activity.
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Table 2. Further Optimization and Discovery of More Potent Chiral Nicotinamides
Structure
Bioactivity (EC50,mg/L)
Properties
Compd
4ab
4ad
4af
R=
R.S.
B.C.
S.S.
ClogP tPSA
SꢀCH3
Rꢀ CH3
SꢀEt
11.18±0.24 k 16.14±0.27 h 25.75±0.51f 2.679 63.05
4.99±0.13 e
3.58±0.11 d
0.58±0.09 a
7.36±0.11 h
2.95±0.21 c
9.89±0.20 j
8.17±0.08 i
6.16±0.09 f
6.63±0.05 g
3.14±0.10 f 11.29±0.31 e 2.679 63.05
2.30±0.12 e
0.42±0.04 a
3.29±0.11 f
0.51±0.03 a
3.32±0.03 f
1.15±0.08 c
0.70±0.02 b
3.83±0.15 g
1.66±0.12 d
4.58±0.12 c 3.208 63.05
2.10±0.03 b 3.208 63.05
4.55±0.07 c 3.607 63.05
2.25±0.04 b 3.607 63.05
4.87±0.08 c 4.136 63.05
0.63±0.05 a 4.136 63.05
2.11±0.03 b 4.136 63.05
6.36±0.12 d 4.006 63.05
0.34±0.05 a 3.437 41.46
4ag
4ah
4ai
RꢀEt
SꢀiPr
RꢀiPr
4aj
SꢀiBu
RꢀiBu
SꢀsꢀBu
SꢀtꢀBu
4ak
4al
4am
Boscalid 1.59±0.11 b
Note: R. S.: Rhizoctonia solani; B. C.: Botrytis cinerea; S. S.: Sclerotinia sclerotiorum. The EC50 values
are presented as mean ± standard deviation of triplicate experiments. Different small letters in the same
column showed significant difference at P < 0.05 level, through Duncan’s multiple range test in SPSS
statistics 19.0. The alphabetical order is consistent with the high to low order of the antifungal activity.
Graphic for table of contents
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