Novel Fluorinated 7-Hydroxycoumarin Derivatives Containing an Oxime Ether Moiety: Design, Synthesis, Crystal Structure and Biological Evaluation

A series of fluorinated 7-hydroxycoumarin derivatives containing an oxime ether moiety have been designed, synthesized and evaluated for their antifungal activity. All the target compounds were determined by 1H-NMR, 13C-NMR, FTIR and HR-MS spectra. The single-crystal structures of compounds 4e, 4h, 5h and 6c were further confirmed using X-ray diffraction. The antifungal activities against Botrytis cinerea (B. cinerea), Alternaria solani (A. solani), Gibberella zeae (G. zeae), Rhizoctorzia solani (R. solani), Colletotrichum orbiculare (C. orbiculare) and Alternaria alternata (A. alternata) were evaluated in vitro. The preliminary bioassays showed that some of the designed compounds displayed the promising antifungal activities against the above tested fungi. Strikingly, the target compounds 5f and 6h exhibited outstanding antifungal activity against B. cinerea at 100 μg/mL, with the corresponding inhibition rates reached 90.1 and 85.0%, which were better than the positive control Osthole (83.6%) and Azoxystrobin (46.5%). The compound 5f was identified as the promising fungicide candidate against B. cinerea with the EC50 values of 5.75 μg/mL, which was obviously better than Osthole (33.20 μg/mL) and Azoxystrobin (64.95 μg/mL). Meanwhile, the compound 5f showed remarkable antifungal activities against R. solani with the EC50 values of 28.96 μg/mL, which was better than Osthole (67.18 μg/mL) and equivalent to Azoxystrobin (21.34 μg/mL). The results provide a significant foundation for the search of novel fluorinated 7-hydroxycoumarin derivatives with good antifungal activity.


Introduction
The rapid growth of the world population will be a great challenge to obtain the enough food produced by limited arable lands [1]. In addition, the phytopathogenic fungi have emerged as the destructive parasites that results inevitably in large reductions in the yields and quality of agricultural products [2], thus seriously hindering the sustainable development of agriculture [3,4]. Under these circumstances, the rational application of agricultural fungicides has been identified as a most effective way to protect crops and to elevate agriculture yields [5]. However, fungicide-resistance problems have appeared in recent years due to the unreasonable utilization of existing antifungal agents for a long time, which leads to a series of side-effects including environmental pollution and food safety threats [6,7]. Therefore, it is of great significance to develop novel high-efficiency and broad-spectrum fungicides.
Coumarin derivatives, the typical natural products containing benzopyrone structures, have widely served as secondary metabolites in plants [8,9]. Interestingly, the natural coumarins exhibit the noticeable bioactivities including antitumor, anticoagulant, anti-inflammatory, antioxidant, antifungal and antiviral effects [10][11][12]. In view of this, coumarin derivatives have been widely applied to agricultural and medicinal chemistries. The corresponding synthesis methods and pharmacological activities were also well reported, such as Osthole, Coumoxystrobin and Warfarin (Figure 1). Employing Osthole as a lead structure, our group synthesized a series of coumarin derivatives against the phytopathogenic fungi effectively (Figure 2), including coumarin [8,7-e][1,3]oxazine [13], furo [3,2-c]coumarin [14], pyrano [3,2-c]chromene-2,5-dione [14], coumarin-3-carboxamide derivatives [15], coumarin ring-opening derivatives [16] and pyrrole/pyrazole-substituted coumarin derivatives [17]. Due to the pharmacological diversity of coumarin derivatives, it has been described as one of "privileged scaffolds" [18,19]. Obviously, the diversified derivatization of the coumarin parent ring is an important strategy for the development of highly effective compounds, making coumarin a candidate for the development of more efficient compounds. anti-inflammatory, antioxidant, antifungal and antiviral effects [10][11][12]. In view of this, coumarin derivatives have been widely applied to agricultural and medicinal chemistries. The corresponding synthesis methods and pharmacological activities were also well reported, such as Osthole, Coumoxystrobin and Warfarin (Figure 1). Employing Osthole as a lead structure, our group synthesized a series of coumarin derivatives against the phytopathogenic fungi effectively (Figure 2), including coumarin [8,7-e][1,3]oxazine [13], furo [3,2-c]coumarin [14], pyrano [3,2-c]chromene-2,5-dione [14], coumarin-3-carboxamide derivatives [15], coumarin ring-opening derivatives [16] and pyrrole/pyrazole-substituted coumarin derivatives [17]. Due to the pharmacological diversity of coumarin derivatives, it has been described as one of "privileged scaffolds" [18,19]. Obviously, the diversified derivatization of the coumarin parent ring is an important strategy for the development of highly effective compounds, making coumarin a candidate for the development of more efficient compounds.    anti-inflammatory, antioxidant, antifungal and antiviral effects [10][11][12]. In view of this, coumarin derivatives have been widely applied to agricultural and medicinal chemistries. The corresponding synthesis methods and pharmacological activities were also well reported, such as Osthole, Coumoxystrobin and Warfarin (Figure 1). Employing Osthole as a lead structure, our group synthesized a series of coumarin derivatives against the phytopathogenic fungi effectively (Figure 2), including coumarin [8,7- [14], pyrano[3,2-c]chromene-2,5-dione [14], coumarin-3-carboxamide derivatives [15], coumarin ring-opening derivatives [16] and pyrrole/pyrazole-substituted coumarin derivatives [17]. Due to the pharmacological diversity of coumarin derivatives, it has been described as one of "privileged scaffolds" [18,19]. Obviously, the diversified derivatization of the coumarin parent ring is an important strategy for the development of highly effective compounds, making coumarin a candidate for the development of more efficient compounds.   As an active group, oxime ether has been extensively utilized in the modification of agrochemical-related compounds [20][21][22][23], which displays a ground-breaking prospect, such as Cymoxanil, Orysastrobin and Fenpyroximate ( Figure 1). Specifically, Cymoxanil marketed in the mid-1970s is a successful oxime ether fungicide [24]. It is reported that fluorine atom can effectively enhance the stability and biological activity of compounds in many ways owing to its small atomic radius and strong electronegativity, which thereby improves the hydrophobicity and liposolubility of compounds [25][26][27][28]. In this regard, fluorinated coumarin derivatives have been considered as a promising strategy in the application of medicinal and agricultural chemistry [29]. To the best of our knowledge, there were few researches regarding the investigation of the antifungal activity of fluorinated coumarin oxime ether derivatives.

Construction for Target Compounds
Three kinds of fluorinated 7-hydroxycoumarins 1a, 1b and 1c were synthesized as the parent compounds via Pechmann condensation reaction, as depicted in Scheme 1. Based on the parent structure 7-hydroxycoumarins, formyl group (-CHO) was then introduced at 8-position via Duff reaction using formylating agent hexamethylenetetramine [30,31]. The Duff reaction was the key step in the reaction route. In the literature procedure, the molar ratio of 7-hydroxycoumarin and hexamethylenetetramine was 1:2, and the reaction proceeded at reflux for 1.5 h. Then aqueous hydrochloric acid was added to the mentioned-above reaction solution to perform acid hydrolysis. Subsequently, the mixture was cooled to 70 • C and stirred for additional 45 min. As an active group, oxime ether has been extensively utilized in the modification of agrochemical-related compounds [20][21][22][23], which displays a ground-breaking prospect, such as Cymoxanil, Orysastrobin and Fenpyroximate ( Figure 1). Specifically, Cymoxanil marketed in the mid-1970s is a successful oxime ether fungicide [24]. It is reported that fluorine atom can effectively enhance the stability and biological activity of compounds in many ways owing to its small atomic radius and strong electronegativity, which thereby improves the hydrophobicity and liposolubility of compounds [25][26][27][28]. In this regard, fluorinated coumarin derivatives have been considered as a promising strategy in the application of medicinal and agricultural chemistry [29]. To the best of our knowledge, there were few researches regarding the investigation of the antifungal activity of fluorinated coumarin oxime ether derivatives.

Construction for Target Compounds
Three kinds of fluorinated 7-hydroxycoumarins 1a, 1b and 1c were synthesized as the parent compounds via Pechmann condensation reaction, as depicted in Scheme 1. Based on the parent structure 7-hydroxycoumarins, formyl group (-CHO) was then introduced at 8-position via Duff reaction using formylating agent hexamethylenetetramine [30,31]. The Duff reaction was the key step in the reaction route. In the literature procedure, the molar ratio of 7-hydroxycoumarin and hexamethylenetetramine was 1:2, and the reaction proceeded at reflux for 1.5 h. Then aqueous hydrochloric acid was added to the mentioned-above reaction solution to perform acid hydrolysis. Subsequently, the mixture was cooled to 70 °C and stirred for additional 45 min. To synthesize a series of fluorinated 7-hydroxycoumarin oxime ether derivatives, seven O-substituted hydroxylamines 3d-3j were synthesized according to the reported methods with the use of cheap raw materials (Scheme 1) [32]. Compounds 3a (hydroxylamine), 3b (N-methylhydroxyamine) and 3c (N-ethylhydroxylamine) in the form of the hydrochloride salt were commercially available.
Finally, the thirty target compounds 4a-4j, 5a-5j and 6a-6j were easily synthesized by the reaction between compounds 2a-2c and 3a-3j under a mild condition for 1 h, as To synthesize a series of fluorinated 7-hydroxycoumarin oxime ether derivatives, seven O-substituted hydroxylamines 3d-3j were synthesized according to the reported methods with the use of cheap raw materials (Scheme 1) [32]. Compounds 3a (hydroxylamine), 3b (N-methylhydroxyamine) and 3c (N-ethylhydroxylamine) in the form of the hydrochloride salt were commercially available.
Finally, the thirty target compounds 4a-4j, 5a-5j and 6a-6j were easily synthesized by the reaction between compounds 2a-2c and 3a-3j under a mild condition for 1 h, as shown in Scheme 1. The reaction progress was monitored using TLC (V ethyl acetate /V petroleum ether = 1:2). The desired target compounds were obtained by column chromatography from a mixed solution of ethyl acetate/petroleum ether (V petroleum ether :V ethyl acetate = 1:20), with yields of 17% to 83%. To the best of our knowledge, there have been no report on the production of oxime ether derivatives of 7-hydroxycoumarin at 8-position [33,34]. The structures of all the synthetic coumarin derivatives were confirmed by 1 H-NMR, 13 C-NMR, FTIR and HRMS spectral data. The data of target compound 5h was analyzed as a representative example. The 1 H-NMR spectrum of compound 5h showed two singlets at 11.53 and 8.88 ppm, which meant the molecular structure of compound 5h had a -OH group and -CH=N-group. The doublet at 4.82 ppm and the triplet at 2.59 ppm were attributed to the presence of propynyl. In the 13 C-NMR spectrum, a signal peak at 157.46 ppm indicated that target compound 5h had a carbonyl group. The quadruple peaks at 141.04 and 113.18 ppm were assigned to -CF 3 group. Meanwhile, three singles at 77.65, 76.40 and 62.81 ppm confirmed the existence of propynyl group in the structure of compound 5h. In the FTIR spectrum of compound 5h, the absorption peak of alkynyl group appears at 2131.6 cm −1 and the strong peak at 1736. 3

Single Crystal Structures of Compounds 4e, 4h, 5h and 6c
To further understand the actual structure of synthesized compounds, the structures of compound 4e, 4h, 5h and 6c were determined as representative examples by X-ray diffraction analysis. The tested single crystals were crystallized from the methanol containing the title compounds under room temperature until the size of the crystals was enough to test. The corresponding crystal structure diagram and crystal packing diagram were presented in Figure 3.   As shown in Figure 3D, C9-O4···N1 was an important hydrogen bond that combined with 7-hydroxy-2H-chromen-2-one and oxime ether fragment which construct the main scaffold of 6c. From this figure we could also see that these four compounds had trans structure, which might be owing to the low energy and stability of the trans structure.
As shown in Figure 3H, the intermolecular π-π conjugation force and hydrogen bonds between neighboring molecules were primary force for establishing the three-dimensional structure of compound 6c. The crystallographic data of compound 6c was deposited at the Cambridge Crystallographic Data Centre with an assigned number of CCDC 2043279, the crystallographic data were listed in Table 1. The CCDC numbers of 4e, 4h and 5h were 2043278, 2043275 and 2043276, respectively. The corresponding crystallographic data were listed in Tables S1-S3 (Supplementary Materials).

In Vitro Antifungal Activity of Target Compounds
The in vitro antifungal activity was evaluated by a mycelium growth rate method and the corresponding results were listed in Table 2. Osthole and Azoxystrobin were used as the positive control fungicides throughout the experiment. Noteworthy, the antifungal activities of target compounds against B. cinerea and R. solani were generally better than that of A. solani, G. zeae, C. orbiculare and A. alternata. Especially, the target compounds 5f and 6h exhibited outstanding antifungal activity against B. cinerea at 100 µg/mL with the corresponding inhibition rates of 90.1 and 85.0%, which were superior to the positive control fungicides Osthole (83.6%) and Azoxystrobin (46.5%). Meanwhile, the intermediate compounds 2a, 2b and target compounds 4f, 5a, 5f and 6f showed remarkable antifungal activities against R. solani at 100 µg/mL with the corresponding inhibition rates of 86.2, 64.6, 71.7, 64.6, 72.6 and 70.1%, which were equivalent to the positive control fungicides Osthole (82.7%) and Azoxystrobin (72.3%). Unfortunately, the partial target compounds were not sensitive to A. solani and G. zeae.  To better investigate the inhibitory performance of target compounds against phytopathogenic fungi, the compounds that exhibited the inhibition rates exceeding 60% at 100 µg/mL were further tested for their EC 50 values. Osthole and Azoxystrobin were served as the positive control. As exhibited in Table 3, the EC 50 values of target compounds 5f and 6h were notably as low as 5.75 µg/mL and 13.75 µg/mL, proving that they were much more effective than Osthole (33.20 µg/mL) and Azoxystrobin (64.95 µg/mL) against B. cinerea. Meanwhile, as listed in Table 4, the EC 50 values of all the detected compounds were lower than Osthole (67.18 µg/mL) against R. solani. Specifically, the EC 50 values of the target compounds 2b, 4f, 5a, 5f and 6f were 17.72, 7.48, 33.10, 28.96 and 28.70 µg/mL, respectively. The corresponding in vitro antifungal performances of compound 5f, Osthole and Azoxystrobin against B. cinerea were presented in Figure 4, which visually exhibited the corresponding in vitro inhibition of B. cinerea.   Figure 4, which visually exhibited the corresponding in vitro inhibition of B. cinerea.  The EC50 value was the average value of three replications.

Structure-Activity Relationships
The bioassay results in Tables 2-4 indicated that the antifungal activities of most synthesized 7-hydroxycoumarin oxime derivatives were certain poor, which made it difficult to extract a clear structure-activity relationship analysis. Nevertheless, some broad conclusions could still be drawn. First, the most target compounds were noticeably more efficient against B. cinerea and R. solani than A. solani, G. zeae, C. orbiculare and A. alternata. Second, compounds 4a, 5a, 5f, 6a and 6h displayed the preferable activity than the positive control Osthole against C. orbiculare. Meanwhile, compound 6a also possessed more effective activity against A. alternata than Osthole. Third, the antifungal activities generally could be improved if R 3 was Cl and R 4 was H atom, allyl or propargyl, highlighted by 4f, 5a, 5f, 6a, 6f and 6h, all of which displayed a broad spectrum of antifungal activity against B. cinerea and R. solani. Forth, we still could find that the intermediate compounds 2a and 2b showed a broad antifungal spectrum, which could be served as the active candidates for structure optimization.

Chemicals and Instruments
Resorcin, 4-chlororesorcinol, ethyl 4,4,4-trifluoroacetoacetate and ethyl 2-fluoroacetoacetate were obtained from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. Hydroxylamine hydrochloride, hexamethylenetetramine and trifluoroacetic acid were purchased from Aladdin Reagent Co. Ltd., Shanghai, China. All other chemicals were commercially available and used without further purification. The progress of reactions and the purity of products were monitored by TLC using silica gel GF/UV 254. The melting points of coumarin compounds were measured on an X-4 apparatus (uncorrected). Fourier transform infrared (FTIR) spectra were recorded on a Bruker Tensor 27 spectrometer (Billerica, MA, USA), and all samples were prepared as KBr plates. 1 H-NMR and 13 C-NMR spectra were detected on a Bruker Avance 400 MHz spectrometer with TMS as an internal standard. HR-MS (ESI) spectra were operated using a Thermo Exactive spectrometer (Waltham, MA, USA). X-rays were measured at 296 K on a Bruker SMART APEX2 CCD area detector diffractometer.
After stirring 45 min under 70 • C, the resulting mixture was quenched with ice water and extracted with ethyl acetate. The obtained organic layer was neutralized by sodium bicarbonate solution, washed with water, dried by anhydrous sodium sulfate and filtered. After concentrating under vacuum, the residue containing an intermediate 2 was purified by the column chromatography using ethyl acetate/petroleum ether (V ethyl acetate /V petroleum ether = 1:2) as eluent.

General Procedure for Synthesis of O-substituted hydroxylamines 3d-3j
The water (5 mL) mixing hydroxylamine hydrochloride (0.10 mol), ethyl acetate (0.18 mol) and 28% sodium hydroxide aqueous solution (25 mL) were stirred at 0 • C. The resultant mixture was then stirred at room temperature for 1 h. After that, halogenated hydrocarbon (0.11 mol) was dropwise added and the reaction was stirred at 60 • C for further 6 h. Subsequently, the reaction mixture was extracted with petroleum ether and ethyl acetate. The organic layer was combined and dried by anhydrous sodium sulfate, then the solvent was removed under reduced pressure and oily product was obtained. The obtained oily product was dissolved in ethyl alcohol, and 36% aqueous hydrochloric acid was added to the solution. The mixture was stirred at 50 • C for 4 h. Finally, the reaction was neutralized to pH 7.0 by saturated sodium bicarbonate aqueous solution, and then extracted with dichloromethane. The extracts were dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the title compounds 3d-3j.   fungicide candidate against B. cinerea with the EC 50 values of 5.75 µg/mL, which was obviously better than Osthole (33.20 µg/mL) and Azoxystrobin (64.95 µg/mL). Additionally, the compound 5f possessed outstanding antifungal activities against R. solani with the EC 50 values of 28.96 µg/mL, which was superior to Osthole (67.18 µg/mL) and equivalent to Azoxystrobin (21.34 µg/mL). The present work provides a practical foundation for further structural optimization of fluorinated coumarin oxime ether derivatives with the aim to improve the antifungal activity.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.