Design and Discovery of Novel Chiral Antifungal Amides with 2-(2-Oxazolinyl)aniline as a Promising Pharmacophore

Article abstract of DOI:10.1021/acs.jafc.8b02778

Inspired by established succinate dehydrogenase inhibitors (SDHIs), our continuing efforts toward the discovery of chiral antifungal amides turned to the optimization of their polar regions with 2-(2-oxazolinyl)aniline as a known pharmacophore. Scaffold hopping and bioactivity-guided convergent synthesis enabled the identification of promising antifungal categories. Fine tuning of the substituents and chirality furnished seven amides (1s, 1t, 2d, 2h, 2j, 3k, and 2l) as antifungal candidates, with EC₅₀ values lower than 5 mg/L. The first investigation of chiral amides of acyclic acids as SDHIs was conducted, and compound 2d was selected as a promising candidate against Botrytis cinerea, with a preventative efficacy of up to 93.9% at 50 mg/L, which is better than that of boscalid. The different binding models between compounds with different configurations were simulated for compound 2d and its diastereoisomers. The benefits of synthetic accessibility and cost-effectiveness highlight the practical potential for compound 2d as a good alternative to known SDHI fungicides.

Full text of DOI:10.1021/acs.jafc.8b02778

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Agricultural and Environmental Chemistry
Design and Discovery of Novel Chiral Antifungal Amides
with 2-(2-Oxazolinyl)aniline as a Promising Pharmacophore
Lu Zhang, Wei Li, Taifeng Xiao, Zehua Song, René Csuk, and Shengkun Li
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02778 • Publication Date (Web): 09 Aug 2018
Downloaded from http://pubs.acs.org on August 14, 2018
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Design and Discovery of Novel Chiral Antifungal Amides with 2-(2-
Oxazolinyl)aniline as a Promising Pharmacophore
†
†
‡
†
#
,†
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Lu Zhang, Wei Li, Taifeng Xiao, Zehua Song, René Csuk, Shengkun Li *
Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural
†
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University, Weigang 1, Xuanwu District, Nanjing 210095, People’s Republic of China.
‡
Ningbo Customs District, Ningbo 315012, Zhejiang Province, People’s Republic of China
#
Organic Chemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 2, D-
06120 Halle (Saale), Germany
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*Corresponding Author (Email: SKL505@outlook.com)
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ABSTRACT: Inspired by established succinate dehydrogenase inhibitors (SDHIs), our
continuing efforts toward the discovery of chiral antifungal amides turned to the
optimization of their polar regions with 2-(2-oxazolinyl)aniline as a known
pharmacophore. Scaffold hopping and bioactivity-guided convergent synthesis enabled
the identification of promising antifungal categories. Fine tuning of the substituents and
chirality furnished 7 amides (1s, 1t, 2d, 2h, 2j, 3k, and 2l) as antifungal candidates,
with EC50 values lower than 5 mg/L. The first investigation of chiral amides of acyclic
acids as SDHIs was conducted, and 2d was selected as a promising candidate against
B. cinerea, with a preventative efficacy of up to 93.9% at 50 mg/L, which is better than
that of boscalid. The different binding models between compounds with different
configuration were simulated for 2d and its diastereoisomers. The benefits of synthetic
accessibility and cost-effectiveness highlight the practical potential for 2d as a good
alternative to known SDHIs fungicides.
KEYWORDS: chiral pesticide, oxazoline alkaloids, respiratory chain, fungicide,
structure-activity relationship
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INTRODUCTION
The respiratory electron transport chain (ETC) can create an electrochemical
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proton gradient across the membrane via a series of redox reactions, which transfer
electrons from NADH to O with concomitant production of adenosine triphosphate
2
(ATP). Many fungicides have been developed and launched based on this crucial
biological process, mainly targeting mitochondrial redox carrier protein complexes I to
IV. The respiratory inhibitors are ranked first for disease control in modern plant
protection, in terms of either commercial species or sales volume, mainly addressing
the protein targets SDH (complex II) and cytochrome bc1 (Complex III).2, 3 Among
different respiratory chain inhibitors, SDHIs have been attracting increasing attention
especially after the commercialization of boscalid in 2003. Significant advances have
been achieved for this kind of fungicides as exemplified by the more recent researches4-
9. Succinate dehydrogenase was also chosen as a potential target for exploring natural
products as probes for the discovery of novel antibiotics.10
The importance of the ubiquinone-binding pocket was clearly established during
the discovery of new SDHIs. 11 The continuing interest in the agrochemicals targeting
the respiratory chain prompted us to consider the possibility of chiral ligands for the
ubiquinone binding pocket. The rationality was confirmed by a survey of the chiral
SDHIs launched since 2010, including sedaxane,12 benzovindiflupyr,13 and
1
4
pydiflumetofen. We envisioned that the binding pocket was sensitive to the innate
chirality of the small ligands, and our efforts were therefore devoted to chiral drimane
terpenoids15-17 and chiral carboxamides which simulated the natural ubiquinone and
commercial respiratory inhibitors, respectively. With the hypothesis that
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dearomatization of the “substituent” in the SDHIs model can be used to optimize the
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system (Figure 1), novel chiral antifungal nicotinamides were synthesized, and 2-(2-
oxazolinyl)aniline was validated as a promising pharmacophore.
As shown in Figure 1, 23 SDHIs have been released as commercial fungicides .2
The core moiety (the polar part) is essential for both binding and in vivo potency. The
core is speculated to be inserted deeply into the cavity of SDH, and mainly interact with
11
the Arg87 and His267 in SDHc and SDHb, respectively (Figure 1). We envision that
the cavity and the interactions of amino acid residues with small molecules are
enantioselective.
Structure analysis demonstrated that with the exception of oxathiin carboxamides
(carboxin and oxycarboxin), aromatic acids are common in the cyclic fragments
2
attached to the amide functionality. 2-(2-Oxazolinyl)aniline is embodied in numerous
bioactive compounds of relevance to crop protection and medicinal chemistry.18, 19 Our
previous investigation with this motif for the dearomatization of boscalid culminated in
the discovery of (R)-LE001 as an antifungal candidate (Figure 1). With 2-(2-
oxazolinyl)aniline as a pharmacophore (inserted into the groove formed by SDHb and
SDHc), novel molecules were designed to diversify and optimize the polar cores. All
of the prepared compounds can potentially form hydrogen bonds and π-π interaction
with the amino acid residues in the cavity. Considering the privileged subunits,
synthetic accessibility, aforementioned potential interactions and cost-effectiveness,
our study featured the introduction of versatile polar parts for the establishment of novel
amides of 2-(2-oxazolinyl)aniline (Figures 1 and 2).
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MATERIALS AND METHODS
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Instruments, Chemicals and Related Materials. Unless otherwise mentioned, all
solvents and reagents were analytically pure and were purchased from commercial
sources (Energy, Shanghai, China). Anhydrous solvents were dried and distilled by
standard techniques before use. Silica gel GF254 and silica gelfor column
chromatography (200-300 mesh) were both purchased from Qingdao Broadchem
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Industrial Co., Ltd. (Qingdao, Shandong, China). The H NMR and C NMR spectra
were recorded on an AV 400 spectrometer (Bruker, Switzerland) with CDCl or d -
3
6
DMSO as the solvent and tetramethylsilane (TMS) as the internal standard. Elemental
analyses were performed on a Vario EL instrument (Elementar, Hesse, Germany).
Melting points (m.p.) were recorded on a WRS-1B melting point apparatus (Shenguang,
Shanghai, China) and are uncorrected. Electrospray ionization mass spectrometry (ESI-
MS) data were obtained with aXevo TQ-S Micro-Spectrometer (Waters, Milford, MA).
The single-crystal diffraction was carried out on an AXS Smart APEX CCD
diffractometer (Bruker, Switzerland).
General Procedures for the Condensation of Acids and Amines. Method 1A: To
a dried Schlenk flask charged with 4-(difluoromethyl)-1-methyl-1H-pyrazole-3-
carboxylic acid (1 mmol, 176 mg) and the synthesized (R)-2-(4-methyl-4,5-
dihydrooxazol-2-yl)aniline (1.2 mmol, 211 mg) was added anhydrous CH
2
Cl (5 mL)
2
for dissolution. 4-Dimethylaminopyridine(DMAP) (0.06 g, 0.5 mmol) and N-(3-
(dimethyl amino)propyl)-N′-ethylcarbodiimide hydrochloride (EDCI−HCl) (0.268 g,
1.4 mmol) were then added while the reaction flask was in an ice bath. The mixture was
allowed to gradually warm to room temperature, and it was stirred overnight until full
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consumption of the carboxylic acid was detected by thin layer chromatography (TLC).
The mixture was quenched by the addition of a saturated aqueous solution of NaHCO
3
(20 mL) and separated. The water phase was extracted with dichloromethane (10 mL ×
3), and the combined organic phase was sequentially washed with water (10 mL × 2)
and saturated aqueous NaCl (10 mL), dried over anhydrous sodium sulfate, and
concentrated under vacuum. Purification by silica gel column chromatography (254
mm x 17 mm i.d.) on silica gel with hexane/EtOAc (5:1, v/v) as the eluent gave (R)-4-
(difluoromethyl)-1-methyl-N-(2-(4-methyl-4,5-dihydrooxazol-2-yl)phenyl)-1H-
pyrazole-3-carboxamide (1a).
Data for 1a: yield 66%; white solid; m.p. 146.8-147.7 ℃; 1H NMR (400 MHz,
CDCl
3.82 (dd, J
CH), 6.47 (m, br, CHF
ring), 7.91 (s, 1H, H in pyrazole), 8.67 (d, J = 8.80 Hz, 1H, H in phenyl ring), 11.77 (s,
3
) δ 1.41 (d, J = 6.7 Hz, 3H, CH
= 11.2 Hz, J = 4.4 Hz, 1H, OCH
), 7.11 (m, 1H, H in phenyl ring), 7.48-7.54 (m, 2H, H in phenyl
3
), 3.69 (dd, J
1
= 11.2 Hz, J
2
= 3.6 Hz, 1H, OCH
2
),
1
2
2
), 4.02 (s, 3H, N-CH
3
), 4.55 (m, 1H, N-
2
br, 1H, NH). Elemental anal. calcd for C16
H
16
F N
2 4
O
2
: C, 57.48; H, 4.82; N, 16.76.
+
Found: C, 57.51; H, 4.86; N, 16.63. ESI-MS: calcd for C16
found: 335.23.
H
17
F
2
N
4
O
2
[M+ H] : 335.13,
Compounds 1b, 1c, 1d, 1e, 1f, 1q, 1r were prepared similarly by the variation of
either acid parts or chiral amine moieties.
Method 1B: To a dried Schlenk flask charged with (R)-mandelic acid (0.152 g, 1
mmol) and the synthesized (R)-2-(4-methyl-4,5-dihydrooxazol-2-yl)aniline (0.176 g, 1
mmol) was added anhydrous CH
2
Cl (5 mL) for dissolution. This mixture was chilled
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with an ice-bath, and DMAP (0.024 g, 0.2 mmol) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU) ( 0.493 g, 1.3 mmol) was added
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sequentially under a N
the full conversion of (R)-2-(4-methyl-4,5-dihydrooxazol-2-yl)aniline was observed.
The mixture was quenched by the addition of a saturated aqueous solution of NH Cl
2
atmosphere. The reaction progress was monitored by TLC until
4
(10 mL) and separated. The aqueous phase was extracted with dichloromethane (10 mL
× 3), and the combined organic phase was sequentially washed with water (10 mL × 2)
and saturated aqueous NaCl (10 mL), dried over anhydrous sodium sulfate, and
concentrated under vacuum. Purification by column chromatography (254 mm x 17 mm
i.d.) on silica gel with hexane/EtOAc ( 2:1, v/v) gave the desired product (R)-2-
hydroxy-N-(2-((R)-4-methyl-4,5-dihydrooxazol-2-yl)phenyl)-2-phenylacetamide (2b).
1
Data for 2b: yield 46%; pale yellow solid; m.p. 115.4-116.9 ℃; H NMR (400
MHz, CDCl
Hz, 1H, OH), 4.39-4.49 (m, 2H, NCH and OCH
7.09 (m, 1H, H in phenyl ring), 7.30-7.41 (m, 4H, H in phenyl ring), 7.46 (m, 1H, H in
phenyl ring), 7.50-7.57 (m, 2H, H in phenyl ring), 7.81 (dd, J = 7.9 Hz, J = 1.7 Hz,
1H, H in phenyl ring), 8.72 (dd, J = 8.5 Hz, J = 1.1 Hz, 1H, H in phenyl ring), 12.56
(s, br, 1H, NH). C NMR (100 MHz, CDCl ) δ 21.5 (CH ), 61.8 (CH), 72.7 (CH ),
3
) δ 1.26 (d, J = 6.24 Hz, 3H, CH
3
), 3.87 (m, 1H, OCH
2
), 4.25 (d, J = 4.6
2
), 5.20 (d, J = 4.6 Hz, 1H, Ph-CH-OH),
1
2
1
2
1
3
3
3
2
74.8 (CH), 113.7 (C), 119.8 (CH), 122.9 (CH), 127.4 (2 × CH), 128.6 (CH), 128.6 (2 ×
CH), 129.1 (CH), 132.5 (CH), 139.1 (C), 139.4 (C), 163.0 (C), 171.8 (C). Elemental
anal. calcd for C18
H
18
F N
2 2
O
3
: C, 69.66; H, 5.85; N, 9.03. Found: C, 69.71; H, 5.90; N,
+
9.11. ESI-MS calcd for C18
H
19
N
2
O
3
[M+H] : 311.14, found: 311.17. The structure of
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2b was confirmed by single-crystal diffraction, and the data was given the CCDC
number 1838207.
The compounds listed in Figure 2B were prepared similarly by variation of the
mandelic acid or chiral amines.
Compounds 4a, 4b, 4c, and 4d were prepared according to a similar manipulation.
As an example, the spectroscopic data of compound 4d are as follows: (R)-N-(2-(4-
benzyl-4,5-dihydrooxazol-2-yl)phenyl)-2-oxo-2-phenylacetamide: yield 61%, white
1
solid, m.p. 129.8-30.9 ℃; H NMR (400 MHz, CDCl
3
) δ 2.83 (dd, J
1
= 13.9 Hz, J
2
=
=
8.2 Hz, 1H, Ph-CH
2
), 3.27 (dd, J
1
= 13.9 Hz, J
2
= 5.4 Hz, 1H, Ph-CH
2
), 4.12 (dd, J
1
8.5 Hz, J = 7.6 Hz, 1H, OCH
2
2
), 4.35 (dd, J
1
= 8.5 Hz, J
2
= 8.4 Hz, 1H, OCH ), 4.75
2
(m, 1H, =NCH), 7.19 (m, 1H, H in phenyl ring), 7.23-7.29 (m, 4H, H in phenyl ring),
7.50-7.57 (m, 3H, H in phenyl ring), 7.66 (m, 1H, H in phenyl ring), 7.88 (dd, J = 7.9
Hz, J = 1.6 Hz, 1H, H in phenyl ring), 8.31-8.36 (m, 2H, H in phenyl ring), 8.89 (dd,
= 8.4 Hz, J
MHz, CDCl ) δ 41.6 (CH
1
2
1
3
J
1
2
= 1.1 Hz, 1H, H in phenyl ring), 13.46 (s, br, 1H, NH). C NMR (100
3
2
), 67.8 (CH ), 70.6 (CH), 114.6 (C), 120.1 (CH), 123.6 (CH),
2
126.6 (CH), 128.5 (2 × CH), 128.6 (2 × CH), 129.3 (2 × CH), 129.5 (CH), 131.1
(2 × CH), 132.6 (CH), 133.4 (C), 134.3 (CH), 137.5 (C), 138.7 (C), 161.3 (C), 163.5
1
1
1
1
1
86
87
88
89
90
(C), 188.0 (C). Elemental anal. calcd for C24
H
20
N
2
O
3
: C, 74.98; H, 5.24; N, 7.29. Found:
+
C, 74.95; H, 5.33; N, 7.31. ESI-MS calcd for C24
385.20.
H
21
N
2
O
3
[M+H] : 385.16, found:
General Procedures for the Synthesis of Amides from Acids with an NH group
(Method 2). The synthesis of 1h is presented as an example (Figure 4).
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Step 1: To the stirred solution of L-proline(2.0 g, 17.39 mmol) in tetrahydrofuran
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2
2
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2
2
2
(THF) (30 mL) and water (15 mL) was added NaHCO
3
(2.8 g, 33.3 mmol) slowly in
O) (4.06 g, 18.62
portions while in an ice-bath, and then di-tert-butyl dicarbonate((Boc)
2
mmol) was added. The mixture was allowed to gradually warm to room temperature
and was stirred overnight until full consumption of the carboxylic acid was observed.
The volatile solvent was evaporated under reduced pressure, and the inorganic phase
was extracted with dichloromethane (20 mL × 3). The combined organic phase was
washed sequentially with water (15 mL × 2) and saturated aqueous NaCl (15 mL), dried
over anhydrous sodium sulfate, and concentrated under vacuum to furnish the crude
Boc-L-Proline as a colorless oil in 96% yield. This intermediate was used for the next
step without further purification.
Step 2: Boc-1h was successfully synthesized in 72% yield following Method 1.
Step 3: To a solution of Boc-1h (373 mg, 1 mmol) in CH
2
Cl (5 mL) in an ice bath
2
was added trifluoroacetic acid (TFA) (1.5 mL, 20 mmol), and the mixture was stirred
until full conversion was reached as judged by TLC (~ 30 min). Saturated aqueous
NaHCO (15 mL) was added while the mixture was in an ice bath, and the organic
3
phase was separated. The aqueous layer was extracted with dichloromethane (10 mL ×
3), and the organic extracts were combined and washed with saturated aqueous NaCl
(10 mL), dried over Na
2
SO , filtered, and concentrated in vacuo. The crude residue was
4
subjected to flash chromatography (254 mm x 17 mm i.d.) on silica gel with petroleum
ether/EtOAc (1:1, v/v) as the eluent to give (R)-N-(2-((S)-4-methyl-4,5-dihydrooxazol-
2-yl)phenyl)pyrrolidine-3-carboxamide (1h).
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Data for 1h: yield 64%; white solid; m.p.: 104.5-105.1 ℃; 1H NMR (400 MHz,
CDCl
(m, 1H, H in pyrrolidine ring), 2.15-2.31 (m, 2H, H in pyrrolidine ring), 3.07-3.11 (m,
2H, H in pyrrolidine ring), 3.88-3.96 (m, 2H, 1H in OCH and 1H in CH-CO), 4.44-
4.50 (m, 2H, 1H in OCH and 1H in CH-N), 7.08 (dd, J = 7.9 Hz, J
phenyl ring), 7.45 (m, 1H, H in phenyl ring), 7.84 (dd, J = 7.9 Hz, J
3
) δ 1.39 (d, J = 6.3 Hz, 3H, CH
3
), 1.72-1.86 (m, 2H, H in pyrrolidine ring), 2.02
2
2
1
2
= 7.3 Hz, H in
= 1.7 Hz, H in
1
2
phenyl ring), 8.81 (d, J = 8.4 Hz, H in phenyl ring), 12.65 (s, br, 1H, H in phenyl ring).
Elemental anal. calcd for C15
H
19
N
3
O
2
: C, 65.91; H, 7.01; N, 15.37. Found: C, 65.89; H,
+
7.05; N, 15.43. ESI-MS calcd for C15
H
20
N
3
O
2
[M+ H] : 274.16, found: 274.26.
Compounds 1g, 1i, 1j, 1k, 1l, 1m, 1n, 1s, 1t, 1u, and 1v were prepared similarly
by variation of either the acid or chiral amine moieties.
Synthesis of Amides from Isoquinoline-3-Carboxylic Acid (Method 3). The
synthesis of 1o is selected as an example (Figure 5).
Step 1: To a stirred suspension of (S)-phenylalanine (10 g, 60.6 mmol) and 37%
formalin (23 mL) was added concentrated HCl (77 mL). The reaction mixture was
immersed in a preheated oil bath (95 ℃) for 1 h. Second portions of formalin (10 mL)
and concentrated HCl (20 mL) were added, and the reaction mixture was stirred until
the full conversion of the phenylalanine. The resulting mixture was chilled with an ice
bath and filtered. The precipitate was dissolved in hot water, concentrated ammonium
hydroxide was added to neutralize the system and then the mixture was chilled with an
ice-bath. The precipitate was filtered, washed with cold water, collected and dried under
vacuum. This crude (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, I (75% yield)
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was used for the next step without further purification.
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Step 2: Distilled thionyl chloride (SOCl ) (1.1mL, 15 mmol) was added dropwise
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to a stirred solution of compound I (1.77 g, 10 mmol) in anhydrous methanol (15 mL)
in an ice-bath under a nitrogen atmosphere. The reaction mixture was heated in a
preheated oil bath (80 ℃) for 6 h. The solvent was evaporated under reduced pressure
to give the crude residue, which was partitioned into dichloromethane (50 mL) and a
saturated aqueous solution of NaHCO
dried over anhydrous Na SO , filtered and concentrated. The crude residue was
subjected to flash column chromatography (305 mm x 32 mm i.d.) on silica gel (200-
300 m) with 1% NEt in petroleum ether/dichloromethane (1:5, v/v) as the eluent to
3
(30 mL). The organic phase was separated and
2
4
3
give (S)-methyl 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (II) in 81% yield.
Step 3: To a stirred solution of compound II (1.11 g, 5.8 mmol) in N,N-
dimethylformamide (DMF) ( 5 mL) and xylene (20 mL) was added Pd/C (0.9 g), and
the reaction mixture was immersed in a preheated oil bath (150 ℃) and refluxed until
full conversion was achieved as judged by TLC. The heterogeneous system was filtered
and concentrated under vacuum. The crude residue was purified by flash
chromatography (305 mm x 32 mm i.d.) on silica gel with 1% NEt in petroleum
3
ether/dichloromethane = 1:5 (v/v) as the eluent to provide methyl isoquinoline-3-
carboxylate (III) in 68% yield.
Step 4: To a stirred solution of compound III (0.374 g, 2 mmol) in methanol (2
mL) was added 2 M aqueous NaOH solution (2 mL), and the mixture was immersed in
a preheated oil-bath (100 ℃) and refluxed until full hydrolysis was reached as detected
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by TLC. The volatile solvent was removed through rotary evaporation and the system
was adjusted to pH 6 by the addition of 0.5 N aqueous HCl. The resulting inorganic
phase was extracted with dichloromethane (15 mL × 3), and the combined organic
phase was dried over anhydrous Na
2
SO and concentrated to give crude isoquinoline-
4
3-carboxylic acid (IV) in 82% yield, which was directly used in the next step.
Step 5: The synthesis of (R)-N-(2-(4-methyl-4,5-dihydrooxazol-2-yl)phenyl)-
isoquinoline-3-carboxamide (1o, 73%) was conducted according to Method 1 as
described for 1b.
1
Data for compound 1o: yield 73 %; pale yellow solid; m.p. 105.9-107.1 ℃; H
NMR (400 MHz, CDCl
7.8 Hz, 1H, OCH ), 4.55 (dd, J
7.15 (m, 1H, H in phenyl ring), 7.55 (m, 1H, H in phenyl ring), 7.72 (m, 1H, H in phenyl
ring), 7.77 (m, 1H, H in phenyl ring), 7.90 (dd, J = 7.84 Hz, J = 1.72 Hz, 1H, H in
phenyl ring), 8.02 (d, J = 7.6 Hz, 1H, H in phenyl ring), 8.07 (d, J = 8.0 Hz, 1H, H in
phenyl ring), 8.72 (s, 1H, H in pyridine ring), 9.09 (dd, J = 8.5 Hz, J = 1.2 Hz, 1H, H
in phenyl ring), 9.30 (s, 1H, H in pyridine ring), 13.93 (s, br, 1H, NH). Elemental anal.
3
) δ 1.55 (d, J = 6.5 Hz, 3H, CH
3
), 3.96 (dd, J
1
= 7.8 Hz, J
2
=
2
1
= 7.8 Hz, J = 7.8 Hz, 1H, OCH
2
2
), 4.64 (m, 1H, N-CH),
1
2
1
2
calcd for C20H
17
N
3
O
2
: C, 72.49; H, 5.17; N, 12.68; Found: C, 72.53; H, 5.21; N, 12.71.
+
ESI-MS calcd for C20
H
18
N
3
O
2
[M+H] : 332.14, found: 332.23.
(S)-N-(2-(4-methyl-4,5-dihydrooxazol-2-yl)phenyl)isoquinoline-3-carboxamide
(1p) was successfully prepared in 68% yield.
Synthesis of Amides from β-Carboline-3-Carboxylic Acid (Method 4). The
synthesis of 1x is presented as an example (Figure 6).
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Step 1: To a mixture of L-tryptophan (10.2 g, 50 mmol) and acetaldehyde (8.5 mL,
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150 mmol) in water (80 mL) was added 0.5 M H
2
SO (1.5 mL), and the mixture was
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stirred at room temperature. The precipitate was isolated by filtration, washed with cold
water and dried under vacuum. The crude β-carboline acid, V (82% yield) was directly
used in the next step without further purification.
Step 2: (3S)-Methyl 1-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-
carboxylate (VI) was prepared in 89% yield from precursor V following Method 3.
Step 3: To the stirred solution of compound VI (1.8 g, 7.4 mmol) in DMF (20 mL)
was added potassium permanganate (KMnO ) (1.58 g, 10 mmol) in portions while the
4
mixture was in an ice bath. The reaction mixture was stirred vigorously and allowed to
gradually warm to room temperature until the full consumption of the starting material
was detected by TLC. The heterogeneous mixture was filtered and rinsed with methanol.
The combined organic phase was concentrated under vacuum. The crude residue was
subjected to column chromatography (305 mm x 32 mm i.d.) with 1% NEt in
3
petroleum ether/dichloromethane (1:8, v/v) as the eluent to provide methyl 1-methyl-
9H-pyrido[3,4-b]indole-3-carboxylate (VII) in 62% yield.
Step 4: Compound VII was hydrolyzed under basic conditions to produce acid
VIII, which was then condensed with (R)-2-(4-methyl-4,5-dihydrooxazol-2-yl)aniline
or (S)-2-(4-methyl-4,5-dihydrooxazol-2-yl)aniline according to Method 3, to give
desired amides 1w and 1x, respectively.
Data for 1x: (S)-1-methyl-N-(2-(4-methyl-4,5-dihydrooxazol-2-yl)phenyl)-9H-
1
pyrido[3,4-b]indole-3-carboxamide; yield 63%; yellow solid; m.p. 288.3-288.9°C; H
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NMR (400 MHz, d
CH ), 3.99 (dd, J = 7.5 Hz, J
1H, OCH ), 4.63 (m, 1H, N-CH), 7.18 (dd, J
ring), 7.31 (dd, J = 7.4 Hz, J = 7.3 Hz, 1H, H in phenyl ring), 7.56-7.63 (m, 2H, H in
phenyl ring), 7.66 (d, J = 8.1 Hz, 1H, H in phenyl ring), 7.88 (dd, J = 7.8 Hz, J = 1.6
6
-DMSO) δ 1.45 (d, J = 6.4 Hz, 3H, CH
= 7.5 Hz, 1H, OCH ), 4.56 (dd, J
= 7.8 Hz, J = 7.3 Hz, 1H, H in phenyl
3
), 2.94 (s, 3H, pyridine-
3
1
2
2
1
= 9.3 Hz, J = 7.5 Hz,
2
2
1
2
1
2
1
2
Hz, 1H, H in phenyl ring), 8.39 (d, J = 7.8 Hz, 1H, H in phenyl ring), 8.84 (s, 1H, H in
pyridine), 9.04 (d, J = 8.3 Hz, 1H, H in phenyl ring), 12.06 (s, 1H, NH), 13.77 (s, 1H,
NH). Elemental anal. calcd for C23
H
20
N
4
O
2
: C, 71.86; H, 5.24; N, 14.57. Found: C,
+
71.89; H, 5.21; N, 14.61. ESI-MS calcd for C23
H
21
N
4
O
2
[M+H] : 385.17, found:385.24.
In vitro and in vivo antifungal activity. All the plant pathogens were provided
by the Department of Pesticide, College of Plant Protection, Nanjing Agricultural
University (Nanjing, China). The in vitro antifungal activities of the target compounds
9
were tested using the mycelium growth rate test as in our previous report. The in vivo
antifungal activities of the prepared compounds against Botrytis cinerea and Sclerotinia
sclerotiorum were determined on strawberry fruits of and oilseed rape leaves,15
respectively. The statistical analyses were performed by SPSS software (SPSS Statistic
22.0).
9
Molecular Docking. The molecular docking studies of all the synthesized
compounds were performed with the assistance of Tripos SYBYL X 2.0 software. The
crystal structure of succinate dehydrogenase (SDH, respiratory complex II) was
2
0
acquired from the RCSB Protein Data Bank (PDB code 2FBW). The ligand carboxin
(P/CBE 202) was extracted and all water molecules were eliminated from this crystal
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complex. The 3D structures of the synthesized chiral amides were built in ChemBio 3D
Ultra software, Version 12.0, before being imported to Tripos SYBYL-X 2.0 and
optimized by the Powell method to determine lowest energy geometry. Surflex-Dock
was applied for simulating and evaluating the interactions between the amides and the
target protein by an empirical scoring function.4
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RESULTS AND DISCUSSIONS
Design and Synthesis. A scaffold-hopping tactic was applied for the discovery of
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novel antifungal amides. As depicted in Figure 2, the optimization of the polar regions
described herein can be categorized into cyclic and acyclic acids. The 2-(2-
oxazolinyl)aniline core and chiral analogues were successfully synthesized following
9
the procedure described in our previous report. As shown in Figure 3, either EDCI or
HBTU can be used in Steglich-type amidation. Heterocyclic acids, mandelic acid, and
related mimics were selected for preparation of chiral amides in moderate to good yields.
The common subunits in fungicides, including pyrazolyl carboxylic acid, pyrazine-2-
carboxylic acid, indazolyl and quinolyl carboxylic acids, and mandelic acid, are
commercially available.
The free NH groups of L-proline and homoproline were protected with (Boc) O in
2
excellent yield (> 95%) before condensation with 2-(2-oxazolinyl)aniline. Deprotection
of Boc-group was efficiently conducted with superstiochiometric trifluoroacetic acid
(TFA). The related chiral amides can be prepared in fair to good yields (Figure 4).
Isoquinoline-3-carboxylic acids and β-carboline-3-carboxylic acids were
synthesized via Pictet-Spengler cyclization. Tiny modifications of pH were necessary
to cyclize different amino acids. L-phenylalanine was suspended in excess formalin and
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treated by refluxing concentrated HCl to furnish tetrahydro-3-isoquinoline carboxylic
21
acid (I, Figure 5). The Pictet-Spengler cyclization could be much milder for the
construction of the β-carboline ring with L-tryptophan and acetaldehyde (V, Figure 6).
The ester of isoquinoline-3-carboxylic acids (III) was successfully prepared through
oxidation of the precursor II with Pd/C in refluxing xylene. Hydrolysis of III furnished
isoquinoline-3-carboxylic acid (IV) for the preparation of 1o and 1p. Notably, the
chirality of tetrahydro-β-carboline-3-carboxylic acid V can be controlled through
2
2
simple treatment with aqueous sulfuric acid. Intermediate VI underwent oxidation,
hydrolysis, and condensation to smoothly provide desired chiral amides 1w and 1x.
Structure and Activity Relationship (SAR). A bioactivity-guided mixed
1
6
synthesis was applied for the discovery of antifungal candidates against Magnaporthe
oryzae and Botrytis cinerea, the top two fungal plant pathogens based on scientific and
23
economic importance. The new chiral amides of 2-(2-oxazolinyl)aniline with a range
of acids can be divided into five groups: (1) heterocyclic acids that are commercially
available and ubiquitous in SDHIs, (2) biologically important isoquinoline and β-
carboline analogues, (3) inexpensive aliphatic acids, (4) benzoic acids, and (5) mandelic
acid and its analogues. The initial screening indicated the amides of heterocyclic or
mandelic acids were promising candidates. These compounds have the potential to
interact with amino acid residues via H-bonds and π-π interaction, which is consistent
with initial design. Typical antifungal profiles are provided in Table 1 and Table 2.
Amides of cyclic carboxylic acids (Figure 2) were initially screened at 10 mg/L
against up to eight kinds of plant pathogens (Table 1). The introduction of a pyrazole
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subunit (embedded in bixafen, penthiopyrad, benzovindiflupyr, sedaxane, etc.), only
provided modest activities (1a and 1b). This unexpected result may demonstrate the
unique properties of chiral oxazolines as the replacement for “substituent” in SDHIs.
The benzopyrazole counterparts of 1a and 1b (1e and 1f) gave improved bioactivities.
Neither dearomatization of the pyridine (1i and 1j) nor ring-contraction (1g and 1h)
improved the antifungal activities. The tetrahydroisoquinoline counterparts (1k and 1l,
1m and 1n) possessed enhanced activity against M. oryzae, showing that the presence
of fused rings may be beneficial for the bioactivity. Notably, ring-fusing position is
crucial for the antifungal effect (1o vs 1q, and 1p vs 1r). Interestingly, further
enhancement of the activities against R. solani and G. graminis was driven by the
introduction of a carboline ring, which was inspired by the harmine natural products.
The bioactivity against G. graminis is dependent on the chirality of either the oxazoline
or the carboline ring. Compound 1s was a prominent candidate against G. graminis
(EC50 = 3.53 mg/L) and showed superior efficacy in vitro to boscalid (Figure 7).
The amides from mandelic acid demonstrated good effects against both M. oryzae
and B. cinerea at 50 mg/L. Detailed and specific information is listed in Table 2.
Interestingly, the chirality of both mandelic and 2-(2-oxazolinyl)aniline subunits
showed pronounced effects on the biological results. An ethyl substituent in the R-
configuration on the oxazoline ring gave amides 2d and 3d broad-spectrum antifungal
activities. Synergistic effects against all the fungi except for P. capsica were detected
when using (R)-mandelic acid. This may be attributed to the special characteristics of
oomycetes. The antifungal effects of the four diastereoisomers of 2d against R. solani,
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S. scleotiorum, and B. cinerea were in the following order: (R,R)-isomer (2d) > (R,S)-
isomer (2e) > (S, S)-isomer (3e) > (S, R)-isomer (3d). We speculated that the chirality
of mandelic acid on bioactivity may be more remarkable since the (R,R)-isomer (2d)
was >10-fold more potent than the (S,R)-isomer (3d). Fine-tuning of the oxazoline gave
notable enhancements in the antifungal activities. Chiral amides 2h, 2l, and 2j were
identified as promising candidates against R. solani, B. cinerea and M. oryzae, with the
EC50 values of 2.98, 2.95 and 3.01 mg/L, respectively.
As shown in Figure 8, the hydroxyl group in mandelic acid is crucial for the
antifungal activities. The inhibitory effect against B. cinerea and S. scleotiorum dropped
sharply or were even lost when the hydroxyl group was formally reduced to a methylene
group (4a and 4b) or oxidized to a ketone group (4c and 4d).
In vivo Antifungal Activity and Discussion. The in vivo antifungal efficacies of
promising candidates 2d and 2l were evaluated against B. cinerea, with boscalid as a
positive control. As shown in Figure 9, both 2d and 2l showed good preventative
efficacies against B. cinerea. Gratifyingly, 2d displayed a stronger effect in vivo than
boscalid; the preventative efficacy of 2d was up to 93.9% at 50 mg/L. The disparity
with the in vitro results may be caused by their innate physicochemical characteristics.
Hydrophilicity was speculated to be a key factor, and the calculated ClogP values of 2d
and 2l were 3.3506 and 4.3896, respectively. Compound 3k possessed good
preventative efficacy (72.7%) against S. scleotiorum at 200 mg/L. Considering its
synthetic accessibility, cost-effectiveness, and tunability of the chirality, compound 2d
demonstrates practical potential as a novel antifungal ingredient for crop protection.
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Molecular Docking Study. To determine the plausible binding model and
elucidate the chiral deviation thereof, the docking of 2d, its stereoisomers (2e, 3d, and
2e) and analogue (2l) with the potential target protein (PDB code 2FBW) was conducted.
Major hydrogen bonds between the chiral amides and amino acid residues are shown
in Figure 10. Notably, the chirality of both the mandelic acid and the oxazoline ring had
distinct effects on the mode and length of the hydrogen bonds. The substituents on the
oxazoline showed a significant effect on the “composition” of the hydrogen bonds (2d
vs 2l). The sensitivity of the protein cavity to the size of the ligand is also noticeable.
The presence of a sterically bulky pendant on the oxazoline ring may force the attached
phenyl ring partially out of the binding pocket (2l). This effect could be mitigated by
docking the 4-benzyl-4,5-dihydrooxazole substructure properly into the active “groove”
of SDH. The order of total scores of 2d and isomers was well correlated with their
antifungal bioactivities against B. cinerea.
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In summary, novel chiral amides were conceived and synthesized with 2-(2-
oxazolinyl)aniline as a promising pharmacophore. Heterocyclic acids and mandelic
acids were demonstrated as building blocks for the optimization of the polar region in
the SDHIs. The chiral amides of acyclic acids have been discussed in the discovery of
novel SDHIs for the first time. Intentional fine-tuning of polar and hydrophobic regions
was accomplished. The resulting novel amides 1s, 1t, 2d, 2h, 2j, 3k and 2l demonstrated
significant in vitro antifungal activities. In vivo test confirmed our hypothesis and
showed the practical potential of 2d as a promising treatment for destructive B. cinerea.
Molecular docking showed the binding difference between 2d and its stereoisomers in
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the interaction with the potential SDH (PDB code: 2FBW). Synthesis of new chiral
amides and explorations of related biological mechanisms are underway, to discover
novel antifungal ingredients with practical potentials.
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AUTHOR CONTRIBUTIONS
S. Li conceived this work; L. Zhang, W. Li, and T. Xiao performed the chemical
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synthesis, biological tests, and molecular docking; Z. Song conducted structure
detection; S. Li wrote the paper; R. Csuk checked the manuscript.
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ACKNOWLEDGMENT
This work was financially supported by the National Natural Science Foundation
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of China (No. 21772094), the Fundamental Research Funds for the Central Universities
(KYZ201706, Y0201700151) and Postgraduate Research & Practice Innovation
Program of Jiangsu Province (SJCX17_0183). We are grateful to the editors and
reviewers for their kind advice.
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SUPPORTING INFORMATION
X-ray single-crystal diffraction for compound 2b (deposited in the Cambridge
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Structural Database with the CCDC number of 1838207), physical data of the target
compounds, molecular docking of compound 2d and its diastereoisomers, and in vivo
antifungal profiles. The Supporting Information is available free of charge via the
Internet at http://pubs.acs.org.
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19. Wan, L.; Qiao, K.; Yuan, X.; Zheng, M.-W.; Fan, B.-B.; Di, Z. C.; Zhang, D.; Fang, Z.; Guo, K.,
Nickel-catalyzed regioselective C-H bond mono- and bis-nitration of aryl oxazolines with tert-butyl
nitrite as nitro source. Adv. Synth. Catal. 2017, 359, 2596-2604.
20. Avian respiratory complex II with carboxin bound, https://www.rcsb.org/structure/2FBW,
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Lazo, J. S.; Ball, R. G., Synthesis, molecular modeling, 2-D NMR, and biological evaluation of ILV
mimics as potential modulators of protein kinase C. J. Am. Chem. Soc. 1993, 115, 3957-65.
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FIGURE CAPTIONS
Figure 1. Overview of SDHIs and design of novel chiral antifungal ingredients.
Figure 2. New chiral amides of 2-(2-oxazolinyl)aniline with different polar moieties.
Figure 3. General synthesis of target amides.
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Figure 4. Synthesis of 1h and related amides.
Figure 5. Synthesis of amides 1o and 1p.
Figure 6. Synthesis of amides 1w and 1x.
Figure 7. Antifungal activity of cyclic amides against G. graminsis.
Figure 8. Importance of hydroxyl group on antifungal activities
Figure 9. In vivo antifungal activities of 2d and 2l against B. cinerea (1% DMSO as CK)
Figure 10. Molecular docking of 2d and its isomers and analogue
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TABLE CAPTIONS
Table 1, Antifungal Activities of Chiral Amides from Heterocyclic Acids
Table 2, Antifungal Activities of Chiral Mandelic Amides in vitro (EC50, mg/L)
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Table 1, Antifungal Activities of Chiral Amides from Heterocyclic Acids (inhibitory rate at 10 mg/L)
Compd.
R. S.
B. C.
S. S.
A. S.
F. F.
G. G.
P. C.
M. O.
1
a
8.8
< 5.0
37.4
29.2
45.9
45.8
< 5.0
11.9
< 5.0
13.9
10.0
< 5.0
8.6
9.0
7.9
13.6
8.2
8.6
6.4
10.1
10.1
20.7
9.0
34.0
27.1
5.9
5.0
6.4
7.4
35.1
19.1
10.5
8.5
38.3
34.0
8.0
9.0
6.4
12.8
6.9
6.9
24.3
30.7
12.4
13.9
61.1
57.9
17.9
23.4
20.7
14.3
21.9
12.4
36.3
35.5
39.7
27.6
10.5
10.4
72.2
65.8
52.1
49.1
14.5
12.4
5.6
< 5.0
11.3
11.3
< 5.0
< 5.0
< 5.0
< 5.0
3.2
< 5.0
14.5
10.5
7.7
< 5.0
< 5.0
5.1
12.8
19.5
17.7
8.5
6.0
10.4
5.1
35.4
39.6
59.1
50.0
31.7
36.6
< 5.0
< 5.0
12.8
19.5
10.4
23.2
10.0
8.5
1
b
1
c
21.7
31.8
44.4
42.9
< 5.0
< 5.0
< 5.0
< 5.0
7.1
< 5.0
8.7
6.7
32.3
35.9
< 5.0
< 5.0
16.7
30.4
5.6
24.6
20.3
17.5
24.1
9.6
33.6
28.2
48.2
30.9
8.2
9.5
8.2
9.1
35.0
27.3
27.3
15.5
41.8
32.7
9.1
1
d
1
1
1
e
f
g
1
h
9.6
1
1
i
j
< 5.0
< 5.0
7.7
12.7
14.1
8.5
42.9
37.5
< 5.0
5.8
10.6
9.9
1
k
1
l
1
m
1
n
6.3
< 5.0
8.5
1
1
1
o
p
q
38.2
37.0
< 5.0
< 5.0
53.3
46.8
55.2
39.2
3.7
14.5
< 5.0
< 5.0
12.3
12.3
5.3
< 5.0
< 5.0
< 5.0
1
1
1
r
s
t
6.4
< 5.0
28.2
27.3
27.3
< 5.0
5.4
1
u
14.1
27.8
< 5.0
< 5.0
1
v
23.6
5.2
< 5.0
1
w
8.0
6.4
1
x
21.9
5
5
5
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Data are given as the mean of triplicate experiments, promising candidates with inhibitory rate＞ 40% are showed with a green
background. R.s. : Rhizoctonia solani, B.c. : Botrytis cinerea, S.s. : Sclerotinia scleotiorum, A.s. : Alternaria solani, F.f.: Fusarium
fujikuroi, G.g. : Gaeumanomyces graminis, P.c. : Phytophthora capsici, M.o. : Magnaporthe oryzae
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Table 2, Antifungal Activities of Chiral Mandelic Amides in vitro (EC50, mg/L)
Compd.
R. solani
S.sclerotiorum
B.cinerea
P. capsici
M. oryzae
2
3
2
3
2
3
a
a
b
b
c
c
14.92
˃50
13.76
˃50
˃50
˃50
˃50
32.96
˃50
40.71
12.41
42.56
16.97
18.74
˃50
15.59
˃50
˃50
˃50
˃50
˃50
˃50
˃50
13.75
˃50
3.95
9.07
31.76
35.58
˃50
˃50
9.21
˃50
˃50
˃50
˃50
3.83
˃50
9.73
12.73
7.32
8.09
13.35
5.38
˃50
˃50
˃50
˃50
6.57
5.98
˃50
˃50
2.95
˃50
˃50
˃50
1.53
˃50
˃50
˃50
˃50
˃50
17.25
15.52
13.31
9.76
9.93
˃50
12.27
˃50
19.97
˃50
˃50
˃50
18.27
˃50
16.52
˃50
10.33
˃50
˃50
˃50
˃50
˃50
˃50
˃50
˃50
18.85
˃50
10.54
16.62
18.93
˃50
˃50
˃50
˃50
˃50
˃50
˃50
˃50
˃50
3.01
9.73
5.75
12.98
5.56
15.52
6.91
14.38
1.12
5.32
8.74
5.76
15.76
8.94
9.98
8.12
7.76
7.23
8.14
2.98
9.76
˃50
2
3
d
d
2
3
2
3
e
e
f
f
2g
3g
2h
3h
2
3
2
3
i
i
j
j
˃50
6.47
9.37
7.71
7.98
˃50
7.23
˃50
6.77
1.59
2
3
k
k
2
3
l
l
2
3
m
m
Boscalid
0.29
2.53
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5
90
91
Data are given as the means of triplicate experiments, promising candidates with EC50 values ＜5 mg/L are highlighted in bold red
with a green background.
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Figure 1.
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Figure 2.
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Figure 3.
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