Society Awards 2017
Development of the novel fungicide fenpyrazamine
2017 Volume 42 Issue 3 Pages 137-143
Details
2017 Volume 42 Issue 3 Pages 137-143
Fenpyrazamine is a novel fungicide with an aminopyrazolinone structure developed by Sumitomo Chemical Co., Ltd. Fenpyrazamine has good fungicidal properties, such as high antifungal activity, preventive activity, translaminar activity, inhibition activity in lesion development, and long lasting activity. The target enzyme of fenpyrazamine is 3-keto reductase in the ergosterol biosynthetic pathway. Fenpyrazamine shows high efficacy against gray mold, stem rot, and brown rot in field trials. Formulated products, PROLECTUS® and PIXIO®DF, have been registered since 2012. PROLECTUS® was first launched in Italy in 2012, and PIXIO®DF was launched in Japan in 2014.
In the agricultural industry, gray mold (Botrytis cinerea) and stem rot (Sclerotinia sclerotiorum), which attack the fruits of fruit trees and vegetables, greatly reduce the yield and productivity of crops and are therefore important diseases to control. Many fungicides, such as benomyl, have been developed to control gray mold. However, the lifecycle of gray mold in particular is short and many spores are produced; therefore, it is widely known as a phytopathological fungus that easily acquires fungicide resistance.1) Novel fungicides are much anticipated for the control of gray mold. Fenpyrazamine is a novel fungicide that was discovered and developed by Sumitomo Chemical Co., Ltd. This fungicide exhibits an aminopyrazolinone structure (Fig. 1), for which there are no previous examples among existing agricultural chemicals. Fenpyrazamine has a particularly high efficacy against gray mold, stem rot, and brown rot in fruits and vegetables. Sumitomo Chemical Co., Ltd. obtained Korean registration for fenpyrazamine fungicide, and thereafter obtained EU registration. They launched “PROLECTUS®” in Italy in October 2012, followed by “Pixio® DF” in Japan in January 2014. Sumitomo Chemical Co., Ltd. then obtained registration for fenpyrazamine and launched this fungicide in various EU countries and Australia. To date, fenpyrazamine has been registered in more than 30 countries.
As a method for discovering new lead compounds, we not only evaluated the original compounds in our chemical library, but also evaluated the biological activities of compounds that had unique chemical structures and were introduced from external libraries. In our evaluation, a compound introduced from an external library was found to show disease controlling efficacy on powdery mildew on wheat. As the compound had a unique chemical structure, we chose it as a lead compound and commenced screening. On careful investigation of the effects of benzene substituents, we discovered that we were able to confer on the compound disease controlling efficacy against gray mold by introducing a chlorine atom at the ortho position of the benzene ring (compound A). This discovery was an extremely important one for us because we were searching for the next generation fungicide to control gray mold at the time. We decided upon compound A as a lead compound for discovering fungicides to control gray mold, and as a result of careful investigation, the following outline of the structure–activity relationships for gray mold that control efficacy became clear (Fig. 2).
As a result of intensive research into the synthesis and biological evaluation of several hundred compounds, we discovered compound B (Fig. 3), which had high activity against target diseases.2)
Through optimization of the structure of these compounds, we found that conversion of the branched hydrocarbon group R3 (Fig. 2) to a –C(=O)OCH3 group, especially a –C(=O)SCH3 group, drastically improved the efficacy against gray mold.3) After we re-evaluated a variety of combinations of the substituents (R2, R3) bonded to the nitrogen atoms in the first and second positions of the pyrazolinone ring and to substituent R1 in benzene, we selected compound C (Fig. 3), which showed excellent activity against gray mold disease. Since the activity of compound C appeared to be the same or higher than that of benchmarked products, we decided to proceed with the development of compound C from the standpoint of biological activity. However, it might be difficult to obtain registration for compound C in the EU because of its physicochemical properties. So, improvements were required.
1.3. Discovery of fenpyrazamineIn addition to further modifying a series of compounds to improve their persistence in soil, we carried out simple soil residue evaluations in parallel. As a result of these investigations, we discovered fenpyrazamine. Fenpyrazamine has a thiol ester group terminally substituted by an allyl group with an unsaturated hydrocarbon at the nitrogen atom at the first position in the pyrazolinone ring and a methyl group at the ortho position in the benzene ring. Although fenpyrazamine showed excellent activity against gray mold disease, equivalent to that of compound C, it is rapidly degraded in soil. Following careful consideration of various development criteria, we finally selected fenpyrazamine as the compound for development (Fig. 3). The physical and chemical properties of fenpyrazamine are shown in Table 1.
| General Name (ISO) | Fenpyrazamine |
| Chemical Name (IUPAC) | S-Allyl 5-amino-2, 3-dihydro-2-isopropyl-3-oxo-4-(o-tolyl)pyrazole-1-carbothioate |
| CAS RN | 473798-59-3 |
| Molecular Formula | C17H21N3O2S |
| Molecular Weight | 331.43 |
| Physical Form | Solid (Ta) |
| Color | White (Ta) |
| Odor | Faint aromatic odor (Ta) |
| Density | 1.262 g/mL (20°C) |
| Melting Point | 116.4°C |
| Vapor Pressure | 2.89×10−8 Pa (25°C) |
| Solubility | Water: 20.4 mg/L (20°C) |
| Hexane: 902 mg/L (20°C) | |
| Toluene: 112978 mg/L (20°C) | |
| Ethyl acetate:>250 g/L (20°C) | |
| Acetone:>250 g/L (20°C) | |
| Methanol:>250 g/L (20°C) | |
| Octanol-water partition coefficient (log Pow) | 3.52 |
In plate tests using potato dextrose agar (PDA), fenpyrazamine showed high antifungal activity against members of the Sclerotiniaceae such as Botrytis spp. (e.g. gray mold), Sclerotinia spp. (e.g. stem rot), and Monilinia spp. (e.g. blossom blight). EC50 values (mg/L) of fenpyrazamine against Botrytis cinerea, Sclerotinia scleotiorum and Monilinia laxa were approximately 0.02, 0.1, and 0.02, respectively (Table 2).4)
| Fungal species | EC50 (mg/L) | EC90 (mg/L) |
|---|---|---|
| Botrytis cinerea | 0.020 | 0.14 |
| Botrytis allii | 0.030 | 0.67 |
| Botrytis tulipae | 0.030 | 0.67 |
| Sclerotinia sclerotiorum | 0.11 | 0.47 |
| Sclerotinia minor | 0.049 | 0.25 |
| Sclerotinia triformis | 0.012 | 0.041 |
| Monilinia laxa | 0.020 | 0.15 |
| Monilinia fructigena | 0.048 | 0.31 |
| Monilinia fructicola | 0.079 | 0.58 |
| Magnaporthe grisea | >5.0 | >5.0 |
| Septoria tritici | >5.0 | >5.0 |
| Pythium aphanidermatum | >5.0 | >5.0 |
| Rhizoctonia solani | >5.0 | >5.0 |
Conidia of B. cinerea were incubated in potato dextrose broth (PDB) containing fenpyrazamine at 5 mg/L for a predetermined time. After incubation, the conidia were washed in sterile distilled water to remove fenpyrazamine. After washing, the conidia were inoculated onto PDA without any fungicides and incubated at 18°C for 18 hr. Then living conidia were counted. After 24 hr incubation in PDB containing fenpyrazamine, no living conidia were detected indicating that fenpyrazamine had fungicidal action against B. cinerea (Fig. 4).4)
In preventive activity tests (fungicides were applied one day before inoculation of B. cinerea), fenpyrazamine showed a high preventive activity (% control was 100) against gray mold at a low concentration of 1/16 of the concentration (250 mg a.i./L) registered in Japan for gray mold on cucumbers (Fig. 5).
In translaminar activity tests (fungicides were applied on the abaxial side of leaves and B. cinerea was inoculated on the adaxial side of leaves one day after application), fenpyrazamine showed high efficacy (% control was more than 80) against gray mold even at a low concentration of 1/16 of the concentration (250 mg a.i./L) registered in Japan (Fig. 5). This result indicated that fenpyrazamine was rapidly absorbed into the plant after treatment and was transferred to untreated areas. In terms of translaminar activity, fenpyrazamine would be expected to show good efficacy in controlling gray mold in cases where application in the field was a little uneven.
2.5. Inhibition activity in lesion developmentIn inhibition of lesion development tests (B. cinerea was inoculated one day before application), fenpyrazamine showed high efficacy (% control was more than 80) against gray mold at a low concentration of 1/16 of the concentration (250 mg a.i./L) registered in Japan (Fig. 6). This result indicated that fenpyrazamine can suppress disease development during the early stages after infection and would therefore be expected to show good efficacy in controlling gray mold on vegetables even when applied immediately after the first symptoms of disease appeared.
In 14-day long-lasting preventive activity tests, fenpyrazamine showed high efficacy (% control was more than 90) against gray mold at a low concentration of 1/4 of the concentration (250 mg a.i./L) registered in Japan for gray mold on cucumbers (Fig. 7).
In rainfastness tests, fenpyrazamine showed high efficacy against gray mold on grape (% control was more than 75) under conditions in which artificial rainfall was dispersed, 50 mm in total within two hours of application (25 mm/hour) (Fig. 8).
In summary, the results of the biological property tests predicted that fenpyrazamine would provide high efficacy against disease in the field.5)
Conidia of B. cinerea were inoculated onto PDA containing 0.2 mg/L of fenpyrazamine, and incubated for 18 hr at 18°C. After incubation, growth of the germ tubes was observed under a microscope. Fenpyrazamine did not inhibit spore germination, but remarkably inhibited germ tube elongation after germination and caused swelling of the germ tubes (Fig. 9). Moreover, the swelling observed was similar to the morphological changes associated with epoxiconazole, an ergosterol biosynthesis inhibitor (SBI). From these results, we deduced that the mode of action of fenpyrazamine involves changes in biological membrane structures similar to those induced by SBI fungicides.6)
The morphological changes in the germ tubes and metabolomic analysis (data not shown) suggested that fenpyrazamine affected the biosynthesis of ergosterol. Thus, we compared accumulation patterns for sterols in gray mold treated with fenpyrazamine and epoxiconazole (an SBI fungicide). An abnormal accumulation of intermediate sterols other than ergosterol was detected in the unsaponifiable lipids from B. cinerea treated with fenpyrazamine by gas chromatography. The pattern of abnormal accumulation of intermediate sterols was different from that for epoxiconazole (Fig. 10). These results suggested that fenpyrazamine affected the biosynthesis of ergosterol, but the target enzyme is different from that of epoxiconazole.
We examined the identity of the abnormally accumulated sterols detected by gas chromatography (uk1, uk2, and uk3) using CG/MS. We were unable to identify uk1, but uk2 was identified as fecosterone and uk3 was identified as 4-methylfecosterone (Fig. 10). The intermediates possessed a ketone at the third position. Therefore, these result suggested strongly that the target enzyme of fenpyrazamine was 3-keto reductase in the ergosterol biosynthetic pathway.6)
3.4. Inhibitory activity of 3-keto reductase on gray moldIn collaboration with Sumitomo Dainippon Pharma Co., Ltd., Sumitomo Chemical Co., Ltd. established a purification system for 3-keto reductase of B. cinerea from recombinant yeast and an in vitro evaluation system for the inhibition of 3-keto reductase.6) The 3-keto reductase inhibitory activity of fenpyrazamine and Ref. 3, known to inhibit 3-keto reductase, could be evaluated using this system. Both compounds clearly inhibited 3-keto reductase directly. The IC50 value for fenpyrazamine was 0.15 µM, compared with 0.60 µM for Ref. 3 (Fig. 11). These results confirmed that the target enzyme of fenpyrazamine was 3-keto reductase in the ergosterol biosynthetic pathway (Fig. 12).6) In terms of FRAC classification, fenpyrazamine is classified in FRAC code 17.7)
The cross-resistance between fenpyrazamine and other fungicides, whose FRAC codes were different from fenpyrazamine, was evaluated using a plate test (medium: PDA). Resistance factors were calculated based on the EC50 values. Five types of resistant isolates (benzimidazole, dicarboximide, QoI, SDHI, or fluazinam) were evaluated for their resistance to fenpyrazamine and a resistance factor of approximately 1.0 was obtained for each (Table 3). These results indicated that there was no cross-resistance between fenpyrazamine and the five groups of fungicides.
| Phenotype | Resistance factorc) |
|---|---|
| Benzimidazole resistant | 1.1 |
| Dicarboximide resistant | 0.67 |
| QoIa) resistant | 1.1 |
| SDHIb) resistant | 1.0 |
| fluazinam resistant | 0.67 |
a) Electron transfer inhibitors at the quinone outside site of the bc1 complex. b) Succinate dehydrogenase inhibitors. c) EC50 value of tested isolate/EC50 value of sensitive isolate.
Recently, multidrug resistant (MDR) B. cinerea isolates have been detected in the EU. The mechanism of multidrug resistance has been found to be associated with drug efflux. The antifungal activity of fenpyrazamine to two MDR isolates was evaluated using a plate test (medium: PDA) and resistance factors were calculated based on the EC50 values. MDR1 isolate overexpressed the ABC transporter gene atrB and MDR2 isolate possessed a rearrangement in the MFS transporter gene mfsM2 and overexpressed this gene. Kretschmer et al. reported that the resistance factors of MDR1 and MDR2 isolates to Ref. 4 were approximately 8 and 3, respectively.8) In this study, the resistance factors of MDR1 and MDR2 isolates to Ref. 4 were found to be 7.0 and 4.4, respectively. Whereas, the resistance factors of MDR1 and MDR2 isolates to fenpyrazamine were 1.8. These results indicated that both MDR1 and MDR2 did not significantly affect the antifungal activity of fenpyrazamine (Table 4). We also evaluated the preventive activity of fenpyrazamine to MDR1 and MDR2 isolates and found it to be almost equal to that of sensitive isolates (data not shown).
| Phenotype | Resistance factorc) | |
|---|---|---|
| Fenpyrazamine | Reference 4 | |
| MDR 1a) | 1.8 | 7.0 |
| MDR 2b) | 1.8 | 4.4 |
a) Overexpression of the ABC transporter gene atrB. b) Promoter rearrangement in the MFS transporter gene mfsM2 and overexpression the gene. c) EC50 value of tested isolate/EC50 value of sensitive isolate.
Fenpyrazamine showed high efficacy (>90% disease control) against gray mold on grapes in two field tests in Italy (Fig. 13). Fenpyrazamine can therefore be considered effective practically in the control of gray mold on grapes.9)
Fenpyrazamine showed high efficacy (>80% disease control) against gray mold on eggplants in a field trial under curative conditions and severe disease incidence (% of diseased fruit in the untreated control (UTC) was greater than 70%) (Fig. 14). Fenpyrazamine can therefore be considered effective practically in the control of gray mold on eggplants.5)
Fenpyrazamine showed high efficacy (>80% disease control) against brown rot on nectarines in a field trial in Italy under conditions of severe disease (>90% of diseased flower clusters). (Fig. 15). Fenpyrazamine can therefore be considered effective practically in the control of brown rot on nectarines.
Fenpyrazamine has a unique aminopyrazolinone structure. Fenpyrazamine also shows good fungicidal properties, such as fungicidal action, high antifungal activity against the Sclerotiniaceae including multidrug-resistant isolates of B. cinerea, preventive efficacy, translaminar activity, inhibition activity in lesion development, long lasting activity, and rainfastness. The target enzyme of fenpyrazamine is 3-keto reductase in the ergosterol biosynthetic pathway. Fenpyrazamine also showed high efficacy against disease-causing Sclerotiniaceae, such as Botrytis spp., Sclerotinia spp., and Monilinia spp., at field trials indicating its practical applicability.
The authors would like to thank everyone involved in the practical evaluation tests and those who offered advice regarding the development of fenpyrazamine from the Japan Plant Protection Association, the national institutes and plant protection associations of various prefectures and research organizations, including universities. The authors would also like to thank everyone involved in the development of fenpyrazamine at Sumitomo Chemical. Co., Ltd.
