4-Disubstituted Pyrazolin-3-Ones—Novel Class of Fungicides against Phytopathogenic Fungi

: The search for fungicides of novel classes is the long-standing priority in crop protection due to the continuous development of fungal resistance against currently used types of active compounds. Recently, 4-nitropyrazolin-3-ones were discovered as highly potent fungicides, of which activity was believed to be strongly associated with the presence of a nitro group in the pyrazolone ring. In this paper, a series of 4-substituted pyrazolin-3-ones were synthesized and their fungicidal activity against an important species of phytopathogenic fungi ( Venturia inaequalis , Rhizoctonia solani , Fusarium oxysporum , Fusarium moniliforme , Bipolaris sorokiniana , and Sclerotinia sclerotiorum ) was tested in vitro. We discovered that 4-mono and 4,4-dihalogenated pyrazolin-3-ones demonstrate fungicidal activity comparable to that of 4-nitropyrazolin-3-ones and other modern fungicides (such as kresoxim methyl). This discovery indicates that NO 2 moiety can be replaced by other groups of comparable size and electronic properties without the loss of fungicidal activity and signiﬁcantly expands the scope of potent new fungicides based on a pyrazolin-3-one fragment.


Introduction
In today's world, the problem of microbial contamination of agricultural crops is critical to providing food for a growing human population [1]. Fungi represent one of the most harmful groups of phytopathogens, which account for up to 80% of crop losses [2][3][4][5]. Moreover, phytopathogenic fungi produce toxic metabolites, which represent a serious risk for public health as food contaminants [6][7][8][9]. Besides, fungal pathogens can cause opportunistic fungal infections in humans and animals [10,11].
Despite active research [12][13][14], the output of new fungicides has been relatively constant for the past 20 years [15]. To date, only a few classes of compounds dominate the market (77% of sales in 2018 [15]): Quinone outside Inhibitors (QoI, strobilurins, C3), De-Methylation Inhibitors (DMI, triazoles and imidazoles, G1) [16], Succinate-dehydrogenase inhibitors (SDHI, C2) [17], Dithiocarbamates (M03), Chloronitriles (M05), Carboxylic Acid Amides (CAA, H5) and Phenyl Amides (PA, A1), which has created the basis for the development of pest resistance [18]. In addition, the use of fungicides with the same mechanism of action both for agricultural needs and in medicine can create conditions for outbreaks of human diseases caused by ant. imycotic-resistant strains of fungi [19,20]. In this regard, the development of novel types of fungicides (First-in-Class compounds) is a necessary and urgent task [21].
The five-membered pyrazolin-3-one (pyrazolone) ring is a privileged structural motif in medicinal chemistry with a wide range of biological activities [22] (Figure 1). The regard, the development of novel types of fungicides (First-in-Class compounds) is a necessary and urgent task [21].
The five-membered pyrazolin-3-one (pyrazolone) ring is a privileged structural motif in medicinal chemistry with a wide range of biological activities [22] (Figure 1). The practical importance of pyrazolones attracts continuous interest to the development of their synthetic methodology and properties [23,24]. The history of the medical use of 4-unsubstituted pyrazolinones began as early as 1887 with the discovery of antipyrine (1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one), one of the first nonopioid analgesics and antipyretics. This discovery prompted the study of pyrazolone derivatives, including C4monosubstituted pyrazolones, pyrazolone-based Schiff bases, and their metal complexes, which possess anti-inflammatory, antipyretic and analgesic [22,[25][26][27], antitumor/cytotoxic [22,25,[28][29][30], antimicrobial [22,25,[31][32][33], antioxidant [22,26], and protein denaturation inhibiting [27] activities. The interest of medicinal chemists to pyrazolones has been maintained and is increasing at the present time [22]. Recently, C4-disubstituted pyrazolones, primarily spiropyrazolones, have also attracted increasing attention as biologically active compounds [34][35][36]. Due to the wide spectrum of the biological activity of disubstituted pyrazolones, new asymmetric methods for their synthesis are being actively developed [36][37][38][39][40][41]. C4-disubstituted pyrazolones are recognized as valuable antitumor agents [35,[42][43][44][45][46], antimicrobial substances [47], inhibitors of trypanosomal phosphodiesterase B1 [48], RalA inhibitors [49], and miticides [50]. However, their potential as effective fungicides has not been expected. In our previous reports, 4-nitropyrazoline-3-ones were discovered as the novel class of highly potent broad spectrum fungicides [51,52]. However, the understanding of the underlying modes of action was lacking. To shed light on this issue, it is desirable to know how the structure of pyrazolone derivatives affects their fungicidal activity. Our previous study [51] of the structure-activity relationship revealed the importance of the aromatic substituent at N2, a small alkyl substituents at the C4 and C5 atoms of the pyrazolone ring, and the C(sp 3 )-hybridized C4 atom for high fungicidal activity [51]. Since the In our previous reports, 4-nitropyrazoline-3-ones were discovered as the novel class of highly potent broad spectrum fungicides [51,52]. However, the understanding of the underlying modes of action was lacking. To shed light on this issue, it is desirable to know how the structure of pyrazolone derivatives affects their fungicidal activity. Our previous study [51] of the structure-activity relationship revealed the importance of the aromatic substituent at N2, a small alkyl substituents at the C4 and C5 atoms of the pyrazolone ring, and the C(sp 3 )-hybridized C4 atom for high fungicidal activity [51]. Since the unnitrated pyrazolone (1) did not exhibit significant activity [52] (Figure 1), fungicidal properties were believed to be associated with the presence of a nitro group in the pyrazolone ring. In the present work, we aimed to synthesize and study other pyrazolones with a C(sp 3 )-hybridized C4 atom containing no nitro-group to reveal the role of substituents at this position for the fungicidal activity.

The Synthesis of the 4,4-Disubstituted Pyrazolone Derivatives
We have synthesized and tested a series of pyrazolones with variable substituent at position 4 instead of the NO 2 group to reveal the role of this substituent. The structure of one of the most active nitropyrazolones, 4,5-dimethyl-4-nitro-2-phenyl-2,4-dihydro-3Hpyrazol-3-one (1a, Scheme 1), was used as the reference [51,52].
Agrochemicals 2023, 2, FOR PEER REVIEW 3 unnitrated pyrazolone (1) did not exhibit significant activity [52] (Figure 1), fungicidal properties were believed to be associated with the presence of a nitro group in the pyrazolone ring. In the present work, we aimed to synthesize and study other pyrazolones with a C(sp 3 )-hybridized C4 atom containing no nitro-group to reveal the role of substituents at this position for the fungicidal activity.

The Synthesis of the 4,4-Disubstituted Pyrazolone Derivatives
We have synthesized and tested a series of pyrazolones with variable substituent at position 4 instead of the NO2 group to reveal the role of this substituent. The structure of one of the most active nitropyrazolones, 4,5-dimethyl-4-nitro-2-phenyl-2,4-dihydro-3Hpyrazol-3-one (1a, Scheme 1), was used as the reference [51,52]. Scheme 1. Synthetic approaches to 4-substituted pyrazolin-3-ones used in the present study. Scheme 1. Synthetic approaches to 4-substituted pyrazolin-3-ones used in the present study.

Study of the Fungicidal Activity of 4,4-Disubstituted Pyrazolones
In the next step we tested the fungicidal activity of the synthesized 4-disubstituted 2-phenyl-5-methylpyrazolin-3-ones against six species of phytopathogenic fungi characterized by high impact on crop production: Venturia inaequalis, Rhizoctonia solani, Fusarium oxysporum, Fusarium moniliforme, Bipolaris sorokiniana, and Sclerotinia sclerotiorum. Tests were performed by using the mycelium radial growth inhibition method in Petri dishes at a concentration of 10 mg/L and 5 mg/L in the culture medium. Kresoxim-methyl and triadimefon were used as the reference compounds (Table 1). Synthetic approaches to the target compounds 1b-1j and 2d-g are shown in Scheme 1. A previously developed synthetic procedure using N2O4 as a nitrating agent [51] was used for the synthesis of 1a. The advantages of this method are the absence of metal-containing reagents in contrast to the metal salt/NaNO2 system [52], high selectivity, and scalability up to multigram quantities without the yield drop and without the need for chromatographic purification of the target product. 4-Methyl-4-hydroxypyrazolone (1b) was synthesized by aerobic oxidation of 4-methyl-2-phenyl-5-dimethyl-pyrazolin-3-one (1) under basic conditions (K2CO3) [53]. The further methylation step gave 4-methoxypyrazolone (1c). Monohalogenated fluoro-, chloro-and bromopyrazolones (1d-f) were synthesized by the halogenation of pyrazolone 1 with Selectfluor™, N-chlorosuccinimide (NCS), and N-bromosuccinimide (NBS), respectively. It should be noted that these procedures provided up to quantitative yields. 4,4-Dihalogenated pyrazolones (2d-f) were synthesized similarly from 2-phenyl-5-dimethyl-pyrazol-3-one (2) employing two equivalents of halogenating agents. 4-Azido-4,5-dimethyl-2-phenyl-2,4-dihydro-3H-pyrazol-3one (1g) was synthesized with nearly quantitative yield (99%) according to the modified literature procedure [54]. However, the same procedure was less effective for the synthesis of diazide (2g) from 2, where only 25% yield of 2g was obtained. Thiocyanate (1h) was synthesized according to the previously developed procedure for the thiocyanation of the CH-acidic substrates [55]. Dimers (1i and 1j) were synthesized by dehydrogenative dimerization of 1 employing mixed heterogeneous photocatalysis and homogeneous organocatalysis in photooxidative system N-hydroxyphthalimide (NHPI)/TiO2 [56]. The reaction proceeded under air as the terminal oxidant to obtain the mixture of diastereomeric dimers with a total yield of 84%.

Study of the Fungicidal Activity of 4,4-Disubstituted Pyrazolones
In the next step we tested the fungicidal activity of the synthesized 4-disubstituted 2phenyl-5-methylpyrazolin-3-ones against six species of phytopathogenic fungi characterized by high impact on crop production: Venturia inaequalis, Rhizoctonia solani, Fusarium oxysporum, Fusarium moniliforme, Bipolaris sorokiniana, and Sclerotinia sclerotiorum. Tests were performed by using the mycelium radial growth inhibition method in Petri dishes at a concentration of 10 mg/L and 5 mg/L in the culture medium. Kresoxim-methyl and triadimefon were used as the reference compounds (Table 1). Synthetic approaches to the target compounds 1b-1j and 2d-g are shown in Scheme 1. A previously developed synthetic procedure using N2O4 as a nitrating agent [51] was used for the synthesis of 1a. The advantages of this method are the absence of metal-containing reagents in contrast to the metal salt/NaNO2 system [52], high selectivity, and scalability up to multigram quantities without the yield drop and without the need for chromatographic purification of the target product. 4-Methyl-4-hydroxypyrazolone (1b) was synthesized by aerobic oxidation of 4-methyl-2-phenyl-5-dimethyl-pyrazolin-3-one (1) under basic conditions (K2CO3) [53]. The further methylation step gave 4-methoxypyrazolone (1c). Monohalogenated fluoro-, chloro-and bromopyrazolones (1d-f) were synthesized by the halogenation of pyrazolone 1 with Selectfluor™, N-chlorosuccinimide (NCS), and N-bromosuccinimide (NBS), respectively. It should be noted that these procedures provided up to quantitative yields. 4,4-Dihalogenated pyrazolones (2d-f) were synthesized similarly from 2-phenyl-5-dimethyl-pyrazol-3-one (2) employing two equivalents of halogenating agents. 4-Azido-4,5-dimethyl-2-phenyl-2,4-dihydro-3H-pyrazol-3one (1g) was synthesized with nearly quantitative yield (99%) according to the modified literature procedure [54]. However, the same procedure was less effective for the synthesis of diazide (2g) from 2, where only 25% yield of 2g was obtained. Thiocyanate (1h) was synthesized according to the previously developed procedure for the thiocyanation of the CH-acidic substrates [55]. Dimers (1i and 1j) were synthesized by dehydrogenative dimerization of 1 employing mixed heterogeneous photocatalysis and homogeneous organocatalysis in photooxidative system N-hydroxyphthalimide (NHPI)/TiO2 [56]. The reaction proceeded under air as the terminal oxidant to obtain the mixture of diastereomeric dimers with a total yield of 84%.

Study of the Fungicidal Activity of 4,4-Disubstituted Pyrazolones
In the next step we tested the fungicidal activity of the synthesized 4-disubstituted 2phenyl-5-methylpyrazolin-3-ones against six species of phytopathogenic fungi characterized by high impact on crop production: Venturia inaequalis, Rhizoctonia solani, Fusarium oxysporum, Fusarium moniliforme, Bipolaris sorokiniana, and Sclerotinia sclerotiorum. Tests were performed by using the mycelium radial growth inhibition method in Petri dishes at a concentration of 10 mg/L and 5 mg/L in the culture medium. Kresoxim-methyl and triadimefon were used as the reference compounds (Table 1). Synthetic approaches to the target compounds 1b-1j and 2d-g are shown in Scheme 1. A previously developed synthetic procedure using N2O4 as a nitrating agent [51] was used for the synthesis of 1a. The advantages of this method are the absence of metal-containing reagents in contrast to the metal salt/NaNO2 system [52], high selectivity, and scalability up to multigram quantities without the yield drop and without the need for chromatographic purification of the target product. 4-Methyl-4-hydroxypyrazolone (1b) was synthesized by aerobic oxidation of 4-methyl-2-phenyl-5-dimethyl-pyrazolin-3-one (1) under basic conditions (K2CO3) [53]. The further methylation step gave 4-methoxypyrazolone (1c). Monohalogenated fluoro-, chloro-and bromopyrazolones (1d-f) were synthesized by the halogenation of pyrazolone 1 with Selectfluor™, N-chlorosuccinimide (NCS), and N-bromosuccinimide (NBS), respectively. It should be noted that these procedures provided up to quantitative yields. 4,4-Dihalogenated pyrazolones (2d-f) were synthesized similarly from 2-phenyl-5-dimethyl-pyrazol-3-one (2) employing two equivalents of halogenating agents. 4-Azido-4,5-dimethyl-2-phenyl-2,4-dihydro-3H-pyrazol-3one (1g) was synthesized with nearly quantitative yield (99%) according to the modified literature procedure [54]. However, the same procedure was less effective for the synthesis of diazide (2g) from 2, where only 25% yield of 2g was obtained. Thiocyanate (1h) was synthesized according to the previously developed procedure for the thiocyanation of the CH-acidic substrates [55]. Dimers (1i and 1j) were synthesized by dehydrogenative dimerization of 1 employing mixed heterogeneous photocatalysis and homogeneous organocatalysis in photooxidative system N-hydroxyphthalimide (NHPI)/TiO2 [56]. The reaction proceeded under air as the terminal oxidant to obtain the mixture of diastereomeric dimers with a total yield of 84%.

Study of the Fungicidal Activity of 4,4-Disubstituted Pyrazolones
In the next step we tested the fungicidal activity of the synthesized 4-disubstituted 2phenyl-5-methylpyrazolin-3-ones against six species of phytopathogenic fungi characterized by high impact on crop production: Venturia inaequalis, Rhizoctonia solani, Fusarium oxysporum, Fusarium moniliforme, Bipolaris sorokiniana, and Sclerotinia sclerotiorum. Tests were performed by using the mycelium radial growth inhibition method in Petri dishes at a concentration of 10 mg/L and 5 mg/L in the culture medium. Kresoxim-methyl and triadimefon were used as the reference compounds (Table 1). Synthetic approaches to the target compounds 1b-1j and 2d-g are shown in Scheme 1. A previously developed synthetic procedure using N2O4 as a nitrating agent [51] was used for the synthesis of 1a. The advantages of this method are the absence of metal-containing reagents in contrast to the metal salt/NaNO2 system [52], high selectivity, and scalability up to multigram quantities without the yield drop and without the need for chromatographic purification of the target product. 4-Methyl-4-hydroxypyrazolone (1b) was synthesized by aerobic oxidation of 4-methyl-2-phenyl-5-dimethyl-pyrazolin-3-one (1) under basic conditions (K2CO3) [53]. The further methylation step gave 4-methoxypyrazolone (1c). Monohalogenated fluoro-, chloro-and bromopyrazolones (1d-f) were synthesized by the halogenation of pyrazolone 1 with Selectfluor™, N-chlorosuccinimide (NCS), and N-bromosuccinimide (NBS), respectively. It should be noted that these procedures provided up to quantitative yields. 4,4-Dihalogenated pyrazolones (2d-f) were synthesized similarly from 2-phenyl-5-dimethyl-pyrazol-3-one (2) employing two equivalents of halogenating agents. 4-Azido-4,5-dimethyl-2-phenyl-2,4-dihydro-3H-pyrazol-3one (1g) was synthesized with nearly quantitative yield (99%) according to the modified literature procedure [54]. However, the same procedure was less effective for the synthesis of diazide (2g) from 2, where only 25% yield of 2g was obtained. Thiocyanate (1h) was synthesized according to the previously developed procedure for the thiocyanation of the CH-acidic substrates [55]. Dimers (1i and 1j) were synthesized by dehydrogenative dimerization of 1 employing mixed heterogeneous photocatalysis and homogeneous organocatalysis in photooxidative system N-hydroxyphthalimide (NHPI)/TiO2 [56]. The reaction proceeded under air as the terminal oxidant to obtain the mixture of diastereomeric dimers with a total yield of 84%.
It should be noted that the synthesized C4-disubstituted pyrazolones have a different spectrum of activity than kresoxim-methyl . Pyrazolones 1a, 1d, 1f, 2e, 2f and 2g are more active against F. moniliforme, and 4,4-dibromopyrazolone 2f also showed outstanding activity against S. sclerotiorum, the least affected by kresoxim-methyl, even at concentrations as low as 5 mg/L . Pyrazolones 1a, 1d, 1e, 2e, 2f and 2g showed activity at or above the kresoxim-methyl level against B. sorokiniana, with nitropyrazolone 1a being the most active on the list . Pyrazolones 1a, 1e, 1f, 2e, 2f and 2g have excellent activity against V. inaequalis and R. solani: up to 100% mycelium growth inhibition at a concentration of 10 mg/L or 5 mg/L in the case of dihalogen pyrazolones 2e and 2f. Thus, the newly discovered fungicidal compounds can serve as a useful addition to the current range of agricultural fungicides for the control of fungi that are the least susceptible to existing fungicides.
Most of the previously known biologically active C4-disubstituted pyrazolones, with the exception of TELIN [43], belong to the class of spiro compounds bearing two C-C bonds at C4. Thus, the main structural feature that distinguishes the pyrazolones tested by us from other biologically active C4-disubstituted pyrazolones is the presence of a C4-heteroatom bond, which may be a key aspect for the manifestation of fungicidal activity.
In all experiments, see Supplementary File, RT stands for 22-25 • C. 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE II 300 and Bruker Fourier 300HD (300.13 and 75.47 MHz, respectively) spectrometers in CDCl 3 and DMSO-D 6 . FT-IR spectra were recorded on Bruker Alpha instrument. High-resolution mass spectra (HR-MS) were measured on a Bruker maXis instrument using electrospray ionization (ESI). The measurements were performed in a positive ion mode (interface capillary voltage-4500 V); mass range from m/z 50 to m/z 3000 Da; external calibration with Electrospray Calibrant Solution (Fluka). A syringe injection was used for all acetonitrile solutions (flow rate 3 µL/min). Nitrogen was applied as a dry gas; the interface temperature was set at 180 • C.
For investigation of fungicidal activity, aseptic polystyrene Petri dishes (90 × 17 mm) were used. All glassware used for addition and mixing of acetone solutions of the tested compounds with agar medium were sterilized before usage. Experiments were performed in a laminar flow cabinet.

Conclusions
Pronounced broad spectrum fungicidal activity was found to be characteristic for a wide range of C4-disubstituted pyrazolin-3-ones. This discovery shows that 4-nitropyrazolin-3ones, previously reported as a novel class of fungicides, are actually the subgroup of a more diverse type of fungicidal structures, which can be explored further. Currently, the most active compounds are 4,4-dichloro-, 4,4-dibromo pyrazolin-3-ones 2e and 2f. 4-Methyl-4-chloro-, 4-methyl-4-bromo-and 4,4-diazidopyrazolones showed somewhat lower activity. For the most active structures, efficient synthesis procedures have been proposed that allow for obtaining substances with high to quantitative yields. The ease of synthesis, the availability of reagents, and high fungicidal activity comparable to that of commercial fungicides, make the discovered 4-disubstituted pyrazolin-3-ones attractive candidates for the role of a new class of fungicidal compounds.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/agrochemicals2010004/s1, 1 H and 13 C NMR spectra of the synthesized compounds, FT-IR and HRMS data for new compounds.

Conflicts of Interest:
The authors declare no conflict of interest.