Design, Catalyst-Free Synthesis of New Novel α-Trifluoromethylated Tertiary Alcohols Bearing Coumarins as Potential Antifungal Agents

A new method for the synthesis of α-trifluoromethylated tertiary alcohols bearing coumarins is described. The reaction of 3-(trifluoroacetyl)coumarin and pyrrole provided the target compounds with high yields under catalyst-free, mild conditions. The crystal structure of compound 3fa was investigated by X-ray diffraction analysis. The biological activities, such as in vitro antifungal activity of the α-trifluoromethylated tertiary alcohols against Fusarium graminearum, Fusarium oxysporum, Fusarium moniliforme, Rhizoctonia solani Kuhn, and Phytophthora parasitica var nicotianae, were investigated. The bioassay results indicated that compounds 3ad, 3gd, and 3hd showed broad-spectrum antifungal activity in vitro. Compound 3cd exhibited excellent fungicidal activity against Rhizoctonia solani Kuhn, with an EC50 value of 10.9 μg/mL, which was comparable to that of commercial fungicidal triadimefon (EC50 = 6.1 μg/mL). Furthermore, molecular docking study suggested that 3cd had high binding affinities with 1W9U, like argifin.

In this paper, we describe the first synthetic method for the preparation of α-trifluoromethylated tertiary alcohols bearing coumarins. Our approach is based on the two-component reaction of 3-(trifluoroacetyl)coumarin and pyrrole, and this method required neither catalyst/additive nor special conditions. In addition, the in vitro antifungal activity of the title compounds was also studied.

Results and Discussion
We readily prepared 3-(trifluoroacetyl)coumarin 1a in two steps and directly used it as an electrophile for the Friedel-Crafts alkylation of 2-methylpyrrole 2a. During the process of exploring reaction conditions, we were initially pleased to find that the reaction proceeded smoothly in CH2Cl2 at room temperature to provide the desired product 3aa with 46% yield in the presence of 5 mol % of AlCl3 (Table 1, entry 1). The success indicated that the reaction could be promoted by Lewis acid. Subsequently, a series of Lewis acids were screened, and the reactions could proceed (Table 1, entries 2-6). However, unfortunately, only low yields of tertiary alcohol product were formed. To our surprise, 97% yield was observed by performing the reaction in the absence of catalyst (Table 1, entry 7). Finally, the reaction media were screened. Solvent evaluation indicated that the solvents Coumarin is an important heterocyclic skeleton frequently found in numerous natural products, pharmaceutical molecules, fluorescent probes, and materials [35][36][37][38][39][40][41][42]. The combination of some privileged structures, a benzopyrone ring, a trifluoromethyl moiety, and a pyrrole ring for the synthesis of quaternary carbon organic molecules could be of significant importance, especially for new drugs and materials.
In this paper, we describe the first synthetic method for the preparation of αtrifluoromethylated tertiary alcohols bearing coumarins. Our approach is based on the twocomponent reaction of 3-(trifluoroacetyl)coumarin and pyrrole, and this method required neither catalyst/additive nor special conditions. In addition, the in vitro antifungal activity of the title compounds was also studied.

Results and Discussion
We readily prepared 3-(trifluoroacetyl)coumarin 1a in two steps and directly used it as an electrophile for the Friedel-Crafts alkylation of 2-methylpyrrole 2a. During the process of exploring reaction conditions, we were initially pleased to find that the reaction proceeded smoothly in CH 2 Cl 2 at room temperature to provide the desired product 3aa with 46% yield in the presence of 5 mol % of AlCl 3 (Table 1, entry 1). The success indicated that the reaction could be promoted by Lewis acid. Subsequently, a series of Lewis acids were screened, and the reactions could proceed (Table 1, entries 2-6). However, unfortunately, only low yields of tertiary alcohol product were formed. To our surprise, 97% yield was observed by performing the reaction in the absence of catalyst ( have a remarkable influence on the yields (Table 1, entries 7-15). The yield sharply decreased when chloroform, ethyl acetate, acetonitrile, toluene, and tetrahydrofuran were used as the solvents (Table 1, entries [8][9][10][11][12]. Methylene chloride was the best solvent, giving the highest yield of 97%. Unfortunately, the yield sharply decreased when reducing the reaction temperature to 0 ℃ (Table 1, entry 16). Having established the preferred reaction conditions, we next examined the substrate scope of the Friedel-Crafts reaction ( Table 2). A range of 3-(trifluoroacetyl)coumarins with different substituents at the 6-, 7-, or 8-positions proceeded smoothly to afford the corresponding α-trifluoromethyl tertiary alcohols 3aa-ia with excellent yields. Subsequently, pyrroles 2b-f with different groups were also tested for this synthesis. They proceeded well, furnishing the desired products 3ab-af with moderate to excellent yields. The position and electronic property of substituents had a remarkable influence on the yields. The pyrrole ring with electron-donating substituents at the 2-, 3-, or 4-positions proceeded smoothly to afford the corresponding α-trifluoromethyl tertiary alcohols with excellent yields, while the presence of electron-donating substituents in the 2-and 5-positions had a detrimental effect on the yield, due to the activity of the 2-position pyrrole higher than the 3-positon. It was gratifying that excellent yields could be achieved by adding Sc(OTf)3 and increasing the reaction temperature. Unfortunately, the pyrrole ring with an electrondeficient group was an infeasible substrate, presumably because electron withdrawal affected the activity of the pyrrole. Notably, when the pyrrole and N-methylpyrrole were employed, a comparable yield was obtained. Having established the preferred reaction conditions, we next examined the substrate scope of the Friedel-Crafts reaction ( Table 2). A range of 3-(trifluoroacetyl)coumarins with different substituents at the 6-, 7-, or 8-positions proceeded smoothly to afford the corresponding α-trifluoromethyl tertiary alcohols 3aa-ia with excellent yields. Subsequently, pyrroles 2b-f with different groups were also tested for this synthesis. They proceeded well, furnishing the desired products 3ab-af with moderate to excellent yields. The position and electronic property of substituents had a remarkable influence on the yields. The pyrrole ring with electron-donating substituents at the 2-, 3-, or 4-positions proceeded smoothly to afford the corresponding α-trifluoromethyl tertiary alcohols with excellent yields, while the presence of electron-donating substituents in the 2-and 5-positions had a detrimental effect on the yield, due to the activity of the 2-position pyrrole higher than the 3-positon. It was gratifying that excellent yields could be achieved by adding Sc(OTf) 3 and increasing the reaction temperature. Unfortunately, the pyrrole ring with an electron-deficient group was an infeasible substrate, presumably because electron withdrawal affected the activity of the pyrrole. Notably, when the pyrrole and N-methylpyrrole were employed, a comparable yield was obtained.
To test the feasibility of potential large-scale application of the process, the reaction of 3-(trifluoroacetyl)coumarin 1a with 2-methylpyrrole 2a was carried out at the 10 mmol scale under the standard conditions (Scheme 1). The reaction furnished a 95% yield of the product 3aa, which is quite comparable to the yield obtained in the small scale reaction (97% yield).
To determine the structures of the products, a single crystal of compound 3fa was obtained ( Figure 3). The structures of other products can therefore be determined by analogy.
The in vitro antifungal activity of target compounds 3aa-3hd against six representative phytopathogens is summarized in Table 3. Triadimefon was used as a positive control at a concentration of 10 µg/mL. In general, the title compounds exhibited a certain degree of fungicidal activity at a concentration of 500 µg/mL. Among them, compounds 3ad, 3bd, and 3cd showed over 90% inhibition against F. graminearum (from corn). Compounds 3ad, 3bd, 3gd, and 3hd showed over 80% inhibition against F. oxysporum. Compounds 3ad and 3bd showed over 80% inhibition against F. graminearum (from wheat). In particular, 3ad, 3gd, and 3hd exhibited excellent activity (>98%) against R. solani Kuhn. Compounds 3ac, 3ad, 3bd, 3gd, and 3hd exhibited higher antifungal activity (> 80%) against P. parasitica var nicotianae. Furthermore, it was also worth noting that 3ad, 3gd, and 3hd exhibited broad-spectrum antifungal activity and could be considered as new fungicidal leads for further optimization.  To test the feasibility of potential large-scale application of the process, the reaction of 3-(trifluoroacetyl)coumarin 1a with 2-methylpyrrole 2a was carried out at the 10 mmol scale under the standard conditions (Scheme 1). The reaction furnished a 95% yield of the product 3aa, which is quite comparable to the yield obtained in the small scale reaction (97% yield). To determine the structures of the products, a single crystal of compound 3fa was obtained (Figure 3). The structures of other products can therefore be determined by analogy. To determine the structures of the products, a single crystal of compound 3fa was obtained (Figure 3). The structures of other products can therefore be determined by analogy. The in vitro antifungal activity of target compounds 3aa-3hd against six representative phytopathogens is summarized in Table 3. Triadimefon was used as a positive control at a concentration of 10 ug/mL. In general, the title compounds exhibited a certain degree of fungicidal activity at a concentration of 500 ug/mL. Among them, compounds 3ad, 3bd, and 3cd showed over 90% inhibition against F. graminearum (from corn). Compounds 3ad, 3bd, 3gd, and 3hd showed over 80% inhibition against F. oxysporum. Compounds 3ad and 3bd showed over 80% inhibition against F. graminearum (from wheat). In particular, 3ad, 3gd, and 3hd exhibited excellent activity (>98%) against R. solani Kuhn. Compounds 3ac, 3ad, 3bd, 3gd, and 3hd exhibited higher antifungal activity (> 80%) against P. parasitica var nicotianae. Furthermore, it was also worth noting that 3ad, 3gd, and 3hd exhibited broad-spectrum antifungal activity and could be considered as new fungicidal leads for further optimization.
The in vitro fungicidal activity of the title compounds against six phytopathogens showed that the substituents have a great impact on the activity. Overall, the fungicidal activity of 3-ethyl-2,4-dimethyl-1H-pyrrole derivatives (3ad, 3bd, 3cd, 3gd, and 3hd) showed generally higher inhibition against the tested fungi than other substituted pyrrole derivatives.   The in vitro fungicidal activity of the title compounds against six phytopathogens showed that the substituents have a great impact on the activity. Overall, the fungicidal activity of 3-ethyl-2,4-dimethyl-1H-pyrrole derivatives (3ad, 3bd, 3cd, 3gd, and 3hd) showed generally higher inhibition against the tested fungi than other substituted pyrrole derivatives.
Several compounds with higher preliminary antifungal activities at a concentration of 500 µg/mL were selected for determination of the median effective concentration (EC 50 ) values. As shown in Table 4, four compounds displayed good fungicidal activity against R. solani Kuhn. Among them, compound 3cd was the most potent and had the EC 50 values of 10.9 µg/mL. The results indicated that most of the coumarin derivatives containing α-trifluoromethylated tertiary alcohols exhibited good fungicidal activity. Studies have reported that coumarin compounds can bind with the groove of chitinese and revealed better fungal inhibitory activity [43]. The molecular docking of the compound 3cd with 1W9U was conducted to explore the probable interaction with chitinese, and argifin was used as the standard of comparison. As can be seen from Figure 4, compared with argifin, the coumarin derivative 3cd shows some similar aminoacid residues interacting with the receptor. For instance, TYR 245 formed a strong hydrogen bond with the fluorine that emerged as a hydrogen bond donor in the title compound. Then, another hydrogen bond appeared between the hydrogen in the pyrrole ring and ASP 246. It is worth noting that the target compound bonds to GLU 177 by a strong hydrogen bond, while argifin bonds to GLU 177 by the Van der Waals force. Although 3cd does not bind to GLU 178 by hydrogen bond as argifin does, it also binds to GLU 178 by Pi-Pi stacked interaction. Concurrently, the title compound 3cd connected with the TRP52, PHE76, GLY136, TRP137, PHE251, and TRP384 residues, which also formed weak interactions with the chitinese inhibitor argifin. As can be seen from the above results, our synthesized coumarin compounds are expected to be novel chitinese inhibitors, and the relative work is on the way. Studies have reported that coumarin compounds can bind with the groove of chitinese and revealed better fungal inhibitory activity [43]. The molecular docking of the compound 3cd with 1W9U was conducted to explore the probable interaction with chitinese, and argifin was used as the standard of comparison. As can be seen from Figure 4, compared with argifin, the coumarin derivative 3cd shows some similar aminoacid residues interacting with the receptor. For instance, TYR 245 formed a strong hydrogen bond with the fluorine that emerged as a hydrogen bond donor in the title compound. Then, another hydrogen bond appeared between the hydrogen in the pyrrole ring and ASP 246. It is worth noting that the target compound bonds to GLU 177 by a strong hydrogen bond, while argifin bonds to GLU 177 by the Van der Waals force. Although 3cd does not bind to GLU 178 by hydrogen bond as argifin does, it also binds to GLU 178 by Pi-Pi stacked interaction. Concurrently, the title compound 3cd connected with the TRP52, PHE76, GLY136, TRP137, PHE251, and TRP384 residues, which also formed weak interactions with the chitinese inhibitor argifin. As can be seen from the above results, our synthesized coumarin compounds are expected to be novel chitinese inhibitors, and the relative work is on the way. (A)

Chemicals and Instruments
All chemicals, except 3-(trifluoroacetyl)coumarin 1, which was synthesized according our reported procedure, were purchased from commercial sources and used without further purification. 1 H NMR, 19 F NMR, and 13 C NMR spectra were obtained using a Bruker DPX-400 spectrometer (Brucker Technologies Co., Karlsruhe, Germany) in CDCl3 or DMSO solution with TMS as an internal standard. HR-MS(APCI) spectra were performed using a Waters Q-Tof MicroTM instrument (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and X-rays were measured at 293 K on a Rigaku RAXIAS-IV type diffractometer (Brucker Technologies Co., Karlsruhe, Germany). Most reaction yields, except compound 3fa, were not optimized. CCDC 2,167,896 contains the supplementary crystallographic data for this paper (Table S1 in the Supplement Materials). These data may be obtained free of charge via http://www.ccdc.cam.ac.uk; access on 15 November 2022.

General Procedure for the Preparation of Compounds 3aa-3af
A mixture of 3-(trifluoroacetyl)coumarin (1.0 mmol) and pyrrole (1.0 mmol) was stirred in methylene chloride (10 mL) at room temperature. After completion of the reaction, the mixture was concentrated under vacuum to yield the crude product, which was further purified by column chromatography.
To a mixture of 3-(trifluoroacetyl)coumarin (1.0 mmol) and pyrrole (1.0 mmol) in methylene chloride (10 mL) was added Sc(OTf)3 (5%), and the resulting mixture was heated under reflux. After completion of the reaction, the mixture was concentrated under vacuum to yield the crude product, which was further purified by column chromatography.

Chemicals and Instruments
All chemicals, except 3-(trifluoroacetyl)coumarin 1, which was synthesized according our reported procedure, were purchased from commercial sources and used without further purification. 1 H NMR, 19 F NMR, and 13 C NMR spectra were obtained using a Bruker DPX-400 spectrometer (Brucker Technologies Co., Karlsruhe, Germany) in CDCl 3 or DMSO solution with TMS as an internal standard. HR-MS(APCI) spectra were performed using a Waters Q-Tof MicroTM instrument (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and X-rays were measured at 293 K on a Rigaku RAXIAS-IV type diffractometer (Brucker Technologies Co., Karlsruhe, Germany). Most reaction yields, except compound 3fa, were not optimized. CCDC 2,167,896 contains the supplementary crystallographic data for this paper (Table S1 in the Supplement Materials). These data may be obtained free of charge via http://www.ccdc.cam.ac.uk; access on 15 November 2022.

General Procedure for the Preparation of Compounds 3aa-3af
A mixture of 3-(trifluoroacetyl)coumarin (1.0 mmol) and pyrrole (1.0 mmol) was stirred in methylene chloride (10 mL) at room temperature. After completion of the reaction, the mixture was concentrated under vacuum to yield the crude product, which was further purified by column chromatography.
To a mixture of 3-(trifluoroacetyl)coumarin (1.0 mmol) and pyrrole (1.0 mmol) in methylene chloride (10 mL) was added Sc(OTf) 3 (5%), and the resulting mixture was heated under reflux. After completion of the reaction, the mixture was concentrated under vacuum to yield the crude product, which was further purified by column chromatography.  13 C NMR (101 MHz, DMSO-d 6 ) δ mycelium of the fungi reached the edges of the control plate (without sample), the inhibitory index was calculated as follows: Inhibitory index (%) = (Db − Da)/(Db − Dc) × 100%, where Da is the colony diameter of the growth zone in the test plate, Db is the colony diameter of the growth zone in the control plate, and Dc is the diameter of the mycelial disc. The median effective concentration (EC 50 ) of each compound with a significant fungicidal activity was further evaluated in three independent experiments. The statistical analyses were performed using SPSS software (IBM SPSS Statistic 26).

Molecular Docking
The molecular docking studies of compound 3cd and triadimefon were performed with the assistance of Discovery Studio 2016 software and pymol. The crystal structure of Tre was acquired from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB code 2JF4). The ligand validoxylamine was extracted, and all water molecules were eliminated from this crystal complex. Libdock was applied for simulating and evaluating the interactions between the compounds and the target protein by an empirical scoring function.

Conclusions
In conclusion, we have developed a new and practical catalyst-free method for novel α-trifluoromethylated tertiary alcohols bearing coumarins at ambient temperature. This procedure means a promising approach for attaining these new heterocycles, since it applies mild and easily operational conditions (no special reagents, catalysts, or additives). Altogether, 29 new α-trifluoromethylated tertiary alcohols were synthesized in high to excellent yields. The crystal structure of compound 3fa was studied by single-crystal XRD analysis. The biological activity of the α-trifluoromethylated tertiary alcohols was also tested in in vitro antifungal assays. The bioassay results indicated that the compounds showed broad-spectrum fungicidal activity in vitro.