2021 Volume 46 Issue 2 Pages 229-234
To investigate the effects of bioisosteric replacement of the phenyl group with the thienyl group, N-phenylcarboxamide and three regioisomers of N-(substituted-thienyl)carboxamide were synthesized. The inhibitory activity on the succinate dehydrogenase prepared from the gray mold Botrytis cinerea as well as the fungicidal activity against B. cinerea were evaluated. Two isomers, N-(2-substituted-3-thienyl)carboxamide and N-(4-substituted-3-thienyl)carboxamide exhibited the same level of activity as the phenyl derivative, whereas N-(3-substituted-2-thienyl)carboxamide exhibited lower activity than the phenyl derivative, suggesting that the 2-substituted-3-thienyl and 4-substituted-3-thienyl groups functioned as bioisosteres of the phenyl group in N-phenylcarboxamide, but the other did not.
Penthiopyrad (1; Fig. 1) is a heterocyclic carboxamide fungicide that was developed by Mitsui Chemicals Agro, Inc., and it exhibits high activity against various plant pathogens that belong to the Ascomycota and Basidiomycota phyla.1,2) Penthiopyrad is classified as a succinate dehydrogenase inhibitor (SDHI), which inhibits mitochondrial respiration in plant pathogenic fungi by binding to succinate dehydrogenase (SDH).3,4) Penthiopyrad consists of a pyrazole carbonyl moiety and a thiophene amine moiety. With regard to the pyrazole carbonyl moiety, there is a large difference in the activity against gray mold between positional isomers, and this difference has been attributed to the difference in binding affinities to SDH in a docking study.5)
With regard to the thiophene amine moiety, penthiopyrad (1) and the corresponding N-phenyl compound, N-[2-(1,3-dimethylbutyl)]phenyl-1-methyl-3-(trifluoromethyl)pyrazole-4-carboxamide (2), exhibit the same level of activity against various diseases, suggesting that the 2-substituted-3-thienyl group can function as a bioisostere of the 2-substituted-phenyl group.1) As a thienyl group with a substituent in the ortho position of the amino group, three possible regioisomers are permitted (i.e., the 2-substituted-3-thienyl, 4-substituted-3-thienyl, and 3-substituted-2-thienyl groups; Fig. 2). Thus, it is an interesting question whether these isomers can be bioisosteres of the phenyl group similar to the 2-substituted-3-thienyl group.
Several studies that compared the biological activities of the two positional isomers of thiophene have been reported.6–9) However, few studies have compared the biological activities of the three positional isomers of thiophene with two or more substituents at the thiophene ring,10) and it has been reported that one of the three isomers could not be synthesized due to the instability of its intermediate.11) Because bioisosterism has the potential to play an important role in the rational modification of lead compounds into safer and more effective agents, it is scientifically significant to investigate the biological activities of the three thiophene regioisomers in detail.
In this study, we used N-[2-(substituted)phenyl-3-thienyl]-1-methyl-3-(trifluoromethyl)pyrazole-4-carboxamides, which have the same level of activity as penthiopyrad against gray mold,1,2) as model compounds, and two other thiophene regioisomers were synthesized to evaluate the differences in their fungicidal activities. Additionally, to determine the factors that cause the difference in fungicidal activity, in vivo and in vitro antifungal activities against Botrytis cinerea (which is a pathogen of gray mold), SDH inhibitory activity, and the stability of each regioisomer in water were examined.
Melting points were measured using a Mettler FP62 melting-point apparatus (Mettler-Toledo, Greifensee, Switzerland). IR spectra were obtained using a JASCO FT-IR-7300 spectrometer (JASCO, Tokyo, Japan). 1H-NMR spectra were obtained using a JEOL JNM-Λ400 FT-NMR system at 400 MHz (JEOL Ltd., Tokyo, Japan) with tetramethylsilane as the internal standard. Melting-point, IR spectral, and 1H-NMR spectral data of each compound are provided in Supplemental Table S1.
1.1. 3-Amino-2-substituted thiophene derivativesPenthiopyrad (1), N-(2-phenyl-3-thienyl)-1-methyl-3-(trifluoromethyl)pyrazole-4-carboxamide (3), and N-[2-(4-chlorophenyl)-3-thienyl]-1-methyl-3-(trifluoromethyl)pyrazole-4-carboxamide (4) were synthesized according to the method in the literature.1)
1.2. 4-Amino-3-substituted thiophene derivatives (Scheme 1)1.2.1. N-[3-(4-Chlorophenyl-4-thienyl)]-1-methyl-3-(trifluoromethyl)pyrazole-4-carboxamide (6)
1-Methyl-3-(trifluoromethyl)pyrazole-4-carbonyl chloride (7), which was prepared from 1-methyl-3-(trifluoromethyl)pyrazole-4-carboxylic acid (0.38 g, 1.96 mmol) and thionyl chloride (1 mL), was added to a solution of 3-amino-4-(4-chlorophenyl)thiophene12) (6a; 0.41 g, 1.96 mmol) in pyridine (3 mL) at room temperature. The reaction mixture was stirred for 1 hr at room temperature and then poured into a 5% hydrochloric acid solution (20 mL) and extracted with ethyl acetate (20 mL). The organic layer was washed successively with saturated sodium hydrogen carbonate solution and brine, then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and finally, compound 6 (0.53 g, 70%) was obtained as a white solid via silica gel purification (hexane : ethyl acetate=7 : 3).
1.2.2. N-(3-Phenyl-4-thienyl)-1-methyl-3-(trifluoromethyl)pyrazole-4-carboxamide (5)Compound 5 was synthesized from 3-amino-4-phenylthiophene (5a)12) using a method similar to that described in Section 1.2.1.
1.3. 2-Amino-3-substituted thiophene derivatives (Scheme 2)1.3.1. N-[3-(4-Chlorophenyl)-2-thienyl]-1-methyl-3-(trifluoromethyl)pyrazole-4-carboxamide (10)1.3.1.1. 2-Nitro-3-(4-chlorophenyl) thiophene (10b)
Tetrakis(triphenylphosphine)palladium (0.64 g, 0.56 mmol) was added under nitrogen to a solution of 3-bromo-2-nitorothiophene (8; 2.0 g, 9.61 mmol), 4-chlorophenylboronic acid (10c, 1.50 g, 9.61 mmol), ethanol (5 mL), and 2 M potassium carbonate solution (10 mL) in toluene (60 mL). The mixture was stirred under reflux for 7 hr under nitrogen. The cooled mixture was washed with water (40 mL), and the organic layer was dried over anhydrous sodium sulfate. The solvent was removed in vacuo, and the residue was purified using silica gel (hexane : ethyl acetate=10 : 1) to produce the compound 10b (2.1 g, 91%) as a yellow solid.
1.3.1.2. N-[3-(4-Chlorophenyl)-2-thienyl]-1-methyl-3-(trifluoromethyl)pyrazole-4-carboxamide (10)To convert 2-nitro-3-(4-chlorophenyl)thiophene (10b) to 2-amino-3-(4-chlorophenyl)phenylthiophene, we first tried nitro reduction with iron under acidic and heating conditions (Bechamp reduction) used for synthesizing 3-amino-2-phenylthiophene in high yield.1) However, only a complicated reaction mixture was obtained as determined via TLC analysis, and the desired amino compound could not be obtained under those conditions. This fact suggests that 2-amino-3-(4-chlorophenyl)thiophene is unstable upon Bechamp reduction. Thus, we performed a catalytic hydrogenation reaction using Pd/C, which is a milder reaction condition as compared to Bechamp reduction, and carried out the next amidation without isolating the obtained 2-amino-3-(4-chlorophenyl)thiophene. We describe the detailed procedure below. 2-Nitro-3-(4-chlorophenyl)thiophene (10b; 0.5 g, 2.09 mmol) and 1.0 g of 5% Pd/C (50% wet) were added to 1,4-dioxane (20 mL) and stirred under a hydrogen atmosphere at room temperature for 11 hr, and the reaction mixture was filtrated to remove the Pd/C catalyst. Pyridine (1.65 g, 20.9 mmol) and 1-methyl-3-(trifluoromethyl)pyrazole-4-carbonyl chloride (7), which was prepared from 1-methyl-3-(trifluoromethyl)pyrazole-4-carboxylic acid (0.49 g, 2.53 mmol) and thionyl chloride (2 mL), were added to the filtrate at room temperature, and the resulting mixture was stirred at room temperature for 4 hr. Ethyl acetate (50 mL) was added to the reaction mixture and washed successively with 5% hydrochloric acid solution (30 mL), saturated with sodium hydrogen carbonate solution (30 mL) and brine (30 mL), and then dried over anhydrous sodium sulfate. The solvent was removed in vacuo, and the residue was purified using silica gel (hexane : ethyl acetate=7 : 3) to produce the compound 10 (0.36 g, 45%) as a white solid.
1.3.2. N-(3-Phenyl-2-thienyl)-1-methyl-3-(trifluoromethyl)pyrazole-4-carboxamide (9)Compound 9 was synthesized from 3-bromo-2-nitorothiophene (8) and phenylboronic acid (9c) by methods similar to those described in Sections 1.3.1.1. and 1.3.1.2.
1.4. 2-Substituted aniline derivativesCompound 2, N-(2-phenylphenyl)-1-methyl-3-(trifluoromethyl)pyrazole-4-carboxamide (11), and N-[2-(4-chlorophenyl)phenyl]-1-methyl-3-(trifluoromethyl)pyrazole-4-carboxamide (12) were synthesized according to the method in the literature.1,13)
2. Biological evaluation of kidney bean gray mold (in vivo assay)Each compound was dissolved in acetone, diluted with water, and sprayed onto kidney beans (Phaseolus vulgaris, variety: Green Top from Tokita Seed Co. Ltd., Japan) at the cotyledonous stage until a runoff of the test solution was observed. Then, after the dried leaves were cut and placed in plastic cups, they were covered with wet paper discs to maintain the humidity. Spores of gray mold (Botrytis cinerea, MCAG stock culture No. 40212) were collected in potato sucrose broth medium, and paper discs were inoculated by soaking them in the spore suspension (1×105 spores/mL). The plastic cups were kept in the dark at 20°C. Four days after inoculation, the gray mold lesions were measured, and the protective value was calculated using the following formula:
 |
Botrytis cinerea (MCAG stock culture No. 40226) was precultured on PDA (potato dextrose agar: Difco, Detroit, MI) medium at 24°C for 5 days under dark conditions. A 5 mm2 disk from precultured mycelia was transferred to a PDA plate containing a test compound at 0.01, 0.1, 1, and 10 ppm, and mycelia were grown at 24°C for 6 days under dark conditions. Each colony was measured, and the inhibition rate of each test compound was calculated and compared with an untreated control.
4. Inhibition of SDH in the cell-free system4.1. Mitochondria preparationMitochondria were extracted from the B. cinerea protoplast, which was prepared via the following procedure. Botrytis cinerea (MCAG stock culture No. 40226) was grown on PDA (Difco) medium under BLB light, and conidial suspensions were collected. The conidial suspension of B. cinerea was incubated with 150 mL of modified liquid medium (KH2PO4, 2 g; MgSO4·7H2O, 0.5 g; glucose, 10 g; starch, 10 g; K2HPO4, 1.5 g; (NH4)2SO4, 1 g; yeast extract (Difco), 2 g; distilled water, 1,000 mL) at 22°C for 23 hr while shaking (115 rpm min−1).14) The resulting mycelia were harvested by filtration through a nylon mesh (0.63 mm), rinsed with distilled water, and then suspended in 10 mL/g of an enzyme solution (2% Driselase 20 (ASKA Animal Health Co., Ltd.), 0.5% Cellulase Onozuka RS (Yakult HONSYA Co., Ltd.,), 0.25% Zymolyase 20-T (NACALAI TESQUE, Inc.), 2% Meicelase (SERVICETEC JAPAN Corp.), and 0.6 M KCl). After incubation at 37°C for 1 hr, the resulting protoplasts were obtained by centrifugation at 1,500×g for 10 min.15) The protoplast pellet was resuspended in 3 mL of extraction buffer (0.5 M sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% bovine serum albumin, 5 mM cysteine, and 1/15 M sodium phosphate buffer; pH 7.4) and disrupted using a sonicator at 0–4°C. The total mitochondria pellet was collected by centrifugation at 3,000×g for 10 min and resuspended in 5 mL of wash buffer (0.5 M sucrose, 1 mM EDTA, and 1/15 M sodium phosphate buffer ; pH 7.4).16)
4.2. SDH activity in the cell-free systemThe mitochondrial suspension used for the SDH assay was adjusted to 1 µg/mL by measuring the amount of protein using the Bradford reagent (595 nm). SDH inhibitory activity was measured using the MitoCheck Complex II Activity Assay Kit as per the manufacturer’s protocol (Cayman Chemical Company, Ann Arbor, MI, USA).
5. Stability in waterEach test compound was dissolved in acetonitrile to prepare a 2,500 ppm solution, and the solution (1 µL) was added to 0.5 mL of buffer solution in a glass vial. The buffer solution (pH 4, 7) was prepared following OECD TG111 (ANNEX3)17) except for the pH 11 solution, which was prepared by mixing 0.1 N NaOH solution and 0.2 M borax solution.18) The glass vial was sealed with a vial cap and left to stand in a thermostatic chamber at 50°C under dark conditions. After 14 days, the vial was collected from the chamber, and 0.5 mL of acetonitrile was added to the vial to obtain a sample for measurement. The sample (20 µL) was injected into a high-performance liquid chromatography column (Chemicals Evaluation and Research Institute, L-column ODS 5 µm, 4.6×250 mm) maintained at 40°C under isocratic conditions (water : acetonitrile=45 : 55, v/v) with a combined flow rate of 1.0 mL/min. The area of the peak was determined, and the attenuation rate was calculated with the value of day 0 as 100%.
6. Data analysisStatistical analysis was performed using the GraphPad Prism 6.07 software (GraphPad Prism, San Diego, CA, USA). The IC50 and IC90 values were determined using a four-parameter logistic Hill equation with an upper constraint of 100% extension and a lower constraint of 0% extension. Data from at least three independent experiments were used with two technical repeats for each condition. One-way analysis of variance followed by Tukey’s post hoc test was used to assess the significant differences (p<0.05) between inhibitors.
The in vivo fungicidal activities of various positional isomers against gray mold with regard to N-(substituted-thienyl)carboxamides and the corresponding N-(2-substituted-phenyl)carboxamides are shown in Table 1. We selected the phenyl or 4-chlorophenyl group as the substituent because these substituents are expected to show high activity against gray mold.1,2)
 |  |  |  | |
|---|---|---|---|---|
| R | Fungicidal activity against gray mold | |||
| Type A | Type B | Type C | Type D | |
 | 3 (1) | NDb) | NDb) | 3 (2) |
 | 3 (3) | 3 (5) | 1 (9) | 3 (11) |
 | 3 (4) | 3 (6) | 0 (10) | 3 (12) |
a) Numbers in parentheses represent compound No. b) ND: No Data.
Regarding the two substituents of the thiophene ring, N-(2-substituted-3-thienyl)carboxamides (Type A) and N-(3-substituted-4-thienyl)carboxamides (Type B) showed high activity against gray mold at a level similar to that of N-(2-substitituted-phenyl)carboxamides (Type D). Their activities are on the same level as penthiopyrad (1) and the corresponding phenyl compound (2). These results indicated that Types A and B can be bioisosteres of the corresponding phenyl compound (Type D). However, Type C compounds (9 and 10) exhibited lower activity than the other compounds (Types A, B, and D). The low fungicidal activity observed for Type C compounds was very interesting because the three-dimensional (3D) structure of Type C was presumed to be almost the same as those of Types A and B. Therefore, we conducted a series of studies to find the cause of the lower in vivo fungicidal activity of Type C compounds.
2. Difference in fungicidal activity in vitro against B. cinereaThe in vitro antifungal activity of each isomer was examined to confirm the direct effect of the compounds against the pathogen without the influence of the plants. The IC50 and IC90 values of each isomer against B. cinerea, which causes gray mold disease, are listed in Table 2.
| No. | Type | IC50 (ppm)a) | IC90 (ppm)a) |
|---|---|---|---|
| 4 | A | 0.271±0.0907a | 1.53±0.602a |
| 6 | B | 0.514±0.122a | 1.62±0.402a |
| 10 | C | 8.63±0.131b | 84.6±78.6b |
| 12 | D | 0.418±0.100a | 1.63±0.501a |
a): Letters (a, b) devote homogenous subsets at p<0.05. Data are expressed as mean±S.D.
Compounds 4 (Type A), 6 (Type B), and 12 (Type D), which exhibited high fungicidal activity in vivo, had almost the same in vitro IC50 and IC90 values, and no significant differences in the IC50 and IC90 values (P<0.05) in Tukey's post hoc test were observed. However, compound 10 (Type C), which exhibited lower activity in vivo, also exhibited low activity in vitro; i.e., its IC50 and IC90 values were approximately 21-fold and 52-fold higher than those of the phenyl derivative (Type D), respectively. These results indicated that one of the reasons for the difference in activity against gray mold in vivo is the difference in direct antifungal activity against B. cinerea.
3. Difference in SDH inhibitory activityWe examined whether the difference in antifungal activity against B. cinerea was due to the difference in inhibitory activity against SDH, which is the action site of the test compounds. The inhibitory activities of the isomers on SDH are listed in Table 3.
| No. | Type | IC50 (ppm)a) |
|---|---|---|
| 4 | A | 2.57±0.851a,b |
| 6 | B | 1.01±0.715a |
| 10 | C | 7.41±2.36b |
| 12 | D | 0.978±0.180a |
a): Letters (a, b) devote homogenous subsets at p<0.05. Data are expressed as mean±S.D.
The inhibitory activity of SDH was high in the order of compounds 12, 6, 4, and 10. Among them, compounds 4, 6, and 12 showed no significant difference in IC50 values (p<0.05) in Tukey’s post hoc test. The SDH inhibitory activity of compound 10, which exhibited lower activity in both the in vivo and in vitro tests, was significantly lower than that of compounds 6 and 12 in Tukey’s post hoc test at the 95% confidence level. These results agree with the in vivo and in vitro test results. However, the difference in the IC50 of SDH between compounds 10 and 4 is relatively small (approximately 3 times) and not significant in Tukey’s post hoc test at the 95% confidence level, whereas the difference in in vitro activity between compounds 10 and 4 is approximately 32 times. Thus, the SDH inhibitory activity seems insufficient to explain the difference in fungicidal activity between the two compounds.
4. Difference in chemical stabilityThe difference in activity between compound 10 and compounds 4, 6, and 12 in the in vivo and in vitro tests was likely influenced by differences in metabolic detoxification or chemical stability of each compound as well as the difference in activity at the action site. Therefore, the differences in chemical stability, i.e., the stabilities of compounds 4, 6, 10, and 12 in water, were compared. Because the in vivo test was conducted under dark conditions, this stability experiment was conducted under dark conditions as well. The results are provided in Table 4.
| No. | Type | Recovery rate (%)a) | ||
|---|---|---|---|---|
| pH 4 | pH 7 | pH 11 | ||
| 4 | A | 101.8±2.6 | 101.8±3.7 | 94.8±6.2 |
| 6 | B | 102.3±3.1 | 100.5±0.4 | 101.9±3.2 |
| 10 | C | 98.7±1.2 | 99.4±0.1 | 56.0±2.1 |
| 12 | D | 103.4±9.4 | 102.4±2.9 | 100.1±8.5 |
a): Incubation temperature; 50°C, Incubation period; 14days. Data are expressed as mean±S.D. from two experiments, each done in duplicate.
Compounds 4, 6, and 12, which exhibited high activity both in vivo and in vitro, showed a high recovery rate at each pH, indicating that the chemical stability of these compounds was relatively high. However, compound 10, whose in vivo and in vitro activities were considerably lower, showed a lower recovery rate under alkaline conditions of pH 11, though a high water stability at pH 4 and 7 was observed. These results indicated that the chemical stability of compound 10 was relatively low. It is uncertain whether this instability under alkaline conditions is directly related to the lower activity in vivo, but compound 10 may be more susceptible to some chemical reactions, including metabolic detoxification, than the other isomers. Further information could be obtained by examining the metabolites and degradation products of each isomer.
We described in Section 1.3.1.2 that 2-amino-3-(4-chlorophenyl)thiophene, an intermediate of compound 10, is unstable under Bechamp reduction conditions. Although compound 10 seems to have been stabilized to some extent by covalent binding of an amine to the pyrazolyl carbonyl group, which is an electron-withdrawing group, the low stability of 2-amino-3-phenylthiophene as a partial structure might be one factor that lowers the stability of the entire compound.
In this study, we synthesized three types of positional isomers of the thiophene ring that were expected to be the bioisosteres of the phenyl ring and investigated their biological activities in detail. The in vivo fungicidal activity of each positional isomer was evaluated. N-(2-substitututed-3-thienyl)carboxamides (Type A) and N-(4-substituted-3-thienyl)carboxamides (Type B) exhibited the same activity levels as the corresponding N-(2-substituted)-phenylcarboxamides (Type D). However, the N-(3-substituted-2-thienyl)carboxamides (Type C) showed significantly lower activity as compared to the other isomers. Therefore, the in vitro fungicidal activity of each positional isomer was evaluated. The Type C isomer, which showed lower activity in vivo, exhibited a lower activity in vitro than the other isomers, which is consistent with the in vivo fungicidal activity. The Type C isomer showed significantly lower inhibitory activity on SDH as compared to the other isomers. These results were qualitatively consistent with the in vivo and in vitro data.
The Type C isomer was significantly less stable in water than the other isomers under alkaline conditions. The Type C isomer exhibited lower fungicidal activity both in vivo and in vitro than other isomers, likely owing to multiple factors, such as inhibitory activity at the action site and compound stability.
In summary, we investigated whether a thiophene ring can become a bioisostere for a benzene ring for fungicidal activity against plant pathogens in vivo and in vitro. We also evaluated the inhibitory activity at the action site. Two of the thiophene regioisomers could act as bioisosteres for the benzene ring, whereas one isomer could not. Future studies, such as examining the metabolites of each isomer in detail, may clarify the factors that cause the difference in fungicidal activity between each regioisomer.
We would like to thank T. Sato at Mitsui Chemicals Agro, Inc. (MCAG) for preparation of the compounds and M. Endo at MCAG for providing data of chemical stability.
The online version of this article contains supplementary materials (Supplemental Table S1), which are available at http://www.jstage.jst.go.jp/browse/jpestics/.
