Synthesis, Antifungal Activity and Structure-Activity Relationships of Novel 3-(Difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic Acid Amides

A series of novel 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid amides were synthesized and their activities were tested against seven phytopathogenic fungi by an in vitro mycelia growth inhibition assay. Most of them displayed moderate to excellent activities. Among them N-(2-(5-bromo-1H-indazol-1-yl)phenyl)-3-(difluoro-methyl)-1-methyl-1H-pyrazole-4-carboxamide (9m) exhibited higher antifungal activity against the seven phytopathogenic fungi than boscalid. Topomer CoMFA was employed to develop a three-dimensional quantitative structure-activity relationship model for the compounds. In molecular docking, the carbonyl oxygen atom of 9m could form hydrogen bonds towards the hydroxyl of TYR58 and TRP173 on SDH.


Introduction
Amide compounds are traditional fungicides. Their common target is mitochondrial respiratory chain enzyme complex II (succinate dehydrogenase, SDH) [1]. With the development of boscalid, many researchers paid attention to the amide fungicides with a renewed focus on this traditional fungicide class. A series of novel highly efficient amide fungicides have been used for crop protection. In particular, the 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-acyl group has been the most outstanding acyl moiety group in recent years, and a number of excellent commercial fungicides with this group were successfully developed, such as: isopyrazam (  In our previous work [2], we designed and synthesized a series of amide compounds based on bioisosterism by introducing an N atom instead the C atom in an ortho-aniline. Bioassays showed that some target molecules exhibited excellent antifungal activity against Pythium aphanidermatum and Rhizoctonia solani. By summarizing the structural characteristics of the recent commercial pyrazole amide fungicides and the previous experience in our group, we designed new some molecules according to the active fragments mosaic theory and introduced the 3-(difluoromethyl)-1-methyl-1H-pyrazole-4carboxylic acyl group into these substituted anilines. Three series of novel amides were thus synthesized as shown in Schemes 1-3. Some target molecules exhibited good antifungal activity. We report these results in this paper.

Synthesis of Compounds
The synthetic route to the target compounds 3a is shown in Scheme 1. The aldehyde in 2-nitrobenzaldehyde and amine in 2-aminobenzenethiol were condensed to form an imine, then the intermediate 1 was obtained via a intramolecular cyclization between the thiol and imine. The nitro group of 1 was reduced with hydrazine hydrate to provide the intermediates 2, which were subsequently acylated to produce the target amides 3a.
The synthetic route to the target compounds 6a-6g is shown in Scheme 2. First, the aminopyridine was protected as the corresponding benzaldehyde imine, so the formation could be accomplished in situ under the cross-coupling conditions. The intermediates 4 were prepared via a Suzuki coupling by reacting the imines and phenylboronic acid under palladium catalysis. Intermediates 5 were obtained after subsequent cleavage of the imines using HCl.
The synthetic route to the target compounds 9a-9n is shown in Scheme 3. The chlorine atom in 1-chloro-2-nitrobenzene was replaced by an amino group via an aromatic nucleophilic substitution reaction, giving the intermediates 7. Then the reduction of the nitro group and the acylation of the amino group were performed exactly as presented in Scheme 1.  The synthetic route to the carboxylic acid chloride 10 is shown in Scheme 4. The carboxylic acid was treated with thionyl chloride, then the carboxylic acid chloride was obtained.

In Vitro Antifungal Activity
The in vivo antifungal activity results of the title compounds against seven phytopathogenic fungi are listed in Table 1. The target molecules exhibited different levels of antifungal activity against these fungi. Their inhibitory activities to Colletotrichum orbiculare, Rhizoctonia solani, Pythium aphanide-rmatum and Botrytis cinerea were higher than against the other three fungi.
Compound 9h exhibited excellent activity against Fusarium moniliforme Sheld and Rhizoctonia solani, but little effect on the others. Among compounds 9a-9n, the compounds with an indazole group (9m and 9n) exhibited the highest antifungal activity against the tested fungi. The second group are the compounds with open chain tertiary amines (9k and 9l) and the third one are the amides with nitrogen-containing aliphatic rings (9a, 9b, etc.). Those compounds with imidazole or triazole rings (9d and 9h) exhibited little antifungal activity. The results show the compounds 9c and 9m exhibited higher activity against most of the seven fungi than boscalid and the series 3 and series 6 displayed poorer activities than the series 9.
In order to further study the activities of the best target compounds, we chose five compounds for precise virulence measurements against the seven fungi.

QSAR Analyses
The topomer CoMFA model was optimized. A cross-validation q 2 value of 0.636 and a non-cross-validation r 2 value of 0.995 with an optimized component of 6 were obtained, which suggested that the model has good predictive ability (q 2 > 0.5). The sterically favored and disfavored regions are shown in green and yellow. In the electrostatic field, the positively charged favored regions are shown in blue, and the negatively charge favored regions are shown in red.
We chose the molecule 9m with highest activity which makes it is easier to explain the contour map. The bromoindazole group in compound 9m, with an increasing steric hindrance with its larger substituent exhibits stronger bioactivities in the steric field map (Figure 2a). The compound 9h with a small triazole group exhibited poor activity. In the electrostatic region, the benzene ring of the indazole hovered in the blue blocks (Figure 2b), indicating that electropositivity was beneficial for the antifungal activity. Compound 9m has a more electropositive benzene ring which may be attributed to that the N atoms in the indazole which decrease the electron density of the benzene ring.

Molecular Docking
In an effort to elucidate the possible mechanism of the observed antifungal activity of these compounds, molecular docking of compounds 9m to the binding site of SDH (pdb code: 2FBW [3,4]) pdb was performed. The three-dimensional schematic diagrams clearly explained the possible optimal combination between the ligands and receptor protein ( Figure 3). Compound 9m is bound to the Qp [5] site of SDH. Two hydrogen bonds were formed between the carboxyl oxygen of compound 9m and amino acid residues. The hydrogen bonding distance between the amino hydrogen of TRP173 and the carboxyl oxygen of 9m was found to be 2.02 Å. Another hydrogen bond between the hydroxyl hydrogen of TYR58 and the carboxyl oxygen of 9m was found to be 1.91 Å (Figure 3a). The amino acid residues of MET36, SER39, ILE40, AGR43, PRO169, HIS216 and ILE218 interacted with the ligand, including weak interaction such as wan der Waals interactions and polar interactions. The results agreed well with the molecule docking of carboxin [6], boscalid and bixafen [7].
The compounds 9m (the light salmon color) and boscalid (the light blue color) were aligned in the active pocket (Figure 3b). The total docking scores were 6.59 and 5.26, respectively. The carboxylic acid building block moiety appears to be more harmonious than the amide building block. The results of this molecular docking study demonstrated the perfect combination between compound 9m and the key residues in the binding cavity of SDH [8]. Thus, the stable complex could support the postulation that our active compounds may act on the same enzyme target where SDH inhibitors act confirming the molecular design of the reported class of antifungal agents.

General Information
1 H (300 MHz) and 13 C-NMR (75 MHz) spectra were obtained using an Avance DPX300 spectrometer (Bruker, Billerica, MA, USA) in CDCl3 or DMSO-d6 solution with tetramethylsilane as the internal standard. Melting points were determined using an X-4 binocular microscope melting point apparatus (Beijing Tech Instruments Co., Beijing, China). High resolution mass spectrometry data were obtained with an Accurate-Mass-Q-TOF MS 6520 system equipped with an electrospray ionization (ESI) source (Agilent, Santa Clara, CA, USA).

General Procedure for the Preparation of 2-Aryl-3-aminopyridines 5a-5g
In a 250 mL flask, 2-chloropyridin-3-amine (5 g, 38.9 mmol), p-tolylboronic acid (5.5 g, 46.7 mmol), benzaldehyde (4.12 g, 38.9 mmol) and toluene (80 mL) were stirred at room temperature for 10 min, and trans-dichloro(triphenylphosphine) palladium [5] (0.136 g, 0.21 mmol) was added. The mixture was stirred for 15 min, and a solution of Na2CO3 (5.0 g, 46.7mmol) in water (80 mL) was added. The mixture was heated to reflux for 7 h. The suspension was filtered when the mixture was cooled to room temperature, and the layers were allowed to separate. The organic layer was treated with 3 N HCl (60 mL). The organic layer containing the benzaldehyde was discarded and the pH adjusted to 12 with 50% aqueous NaOH [12]. After extraction with ethyl acetate (3×60 mL) the organic layer was concentrated to an oil. Pure 2-(p-tolyl)pyridin-3-amine (5f) was obtained as a brown paste by column chromatography

Bioassays
The fungi were provided by the Laboratory of Institute of Plant Protection, Chinese Academy of Agricultural Sciences (Beijing, China). The fungicidal activity of the target compounds was tested in vitro against the seven plant pathogenic fungi using the mycelia growth inhibition method [15]. The tested compounds were dissolved in DMSO at a concentration of 10 mg·mL −1 . The media containing compounds at a concentration of 50 μg·mL −1 were then poured into Petri dishes for initial screening. In the precision antifungal test, the 10 mg·mL −1 solution was diluted to 100, 50, 25, 12.5, 6.25, 3.125, 1.56 μg·mL −1 and the above experiments were repeated three times, the inhibition rates were calculated separately. The statistical analyses were performed by SPSS software version 17.0.

QSAR Analyses
Topomer CoMFA (in the SYBYL X 2.0 program) was performed to analysis the relationship between structure and activity. Topomer CoMFA is an alignment-independent 3D-QSAR method that combines the topomer search method [16] with the conventional CoMFA method. Besides the core of the molecule, we split the functional of compound into two R-groups that refer to the R1 (amide moiety) and R2 (carboxylic acid moiety) groups. In total, 21 compounds obtained from synthesis were used to create a data set in which the inhibition rate of all compounds was determined (Table 1) against Colletotrichum orbiculare. Three-dimensional structures of the target compounds were built by the Chem3D software version 12.0.

Molecular Docking
Docking was performed by Surflex-Dock (in the SYBYL X2.0 program). The ligand structures were energetically minimized using MM2 energy minimizations in ChemBio 3D. All bound water and key ligands were eliminated from the protein, and the polar hydrogen atoms and the AMBER7 FF99 charges were added to the proteins.

Conclusions
Twenty-one novel 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid amides had been synthesized by introducing the 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-acyl group in these amide compounds. The bioassays showed that all of them exhibited moderate to remarkable antifungal activities against the seven tested fungi. The compounds containing indazole groups displayed stronger antifungal activities than the others. By comparing the activities and structures of the compounds 9c and 9m with the highly antifungal compounds which we found in previous work [2], it could be determined that the indazole group plays a significant role in improving the activities of the molecules. We found a novel lead compound 9m with higher antifungal activity and broader spectrum than the control boscalid. Topomer CoMFA was employed to develop a 3D QSAR model on the antifungal activity of target molecules. A molecular docking study showed the mode of action of these structures on SDH. The present findings provided a powerful complement to the SDHIs of fungicides, and warrant future investigation of the mechanism of action of these analogues. Further studies on biological behavior and structural optimization are in progress in our laboratory.