Synthesis, Structural Determination, and Antifungal Activity of Novel Fluorinated Quinoline Analogs

A series of new fluorinated quinoline analogs were synthesized using Tebufloquin as the lead compound, 2-fluoroaniline, ethyl 2-methylacetoacetate, and substituted benzoic acid as raw materials. Their structures were confirmed by 1H NMR, 13C NMR, and HRMS. The compound 8-fluoro-2,3-dimethylquinolin-4-yl 4-(tert-butyl)benzoate (2b) was further determined by X-ray single-crystal diffraction. The antifungal activity was tested at 50 μg/mL, and the bioassay results showed that these quinoline derivatives had good antifungal activity. Among them, compounds 2b, 2e, 2f, 2k, and 2n exhibited good activity (>80%) against S. sclerotiorum, and compound 2g displayed good activity (80.8%) against R. solani.

Among the more than 1200 commercialized pesticides, 424 pesticides contain at least one fluorine atom [21]. After organic compounds are fluorinated, their physical and chemical properties such as lipophilicity, water solubility, and metabolic stability will undergo significant changes [22], due to that they [23,24] have the characteristics of small radius and large electronegativity. Therefore, many commercial medicines or pesticides heterocycles containing fluorine atoms were commercialized. For example (Figure 1), the fluoroquinoline fungicide Ipflufenoquin [25] was recently developed by Japan's Soda Company. The Meiji Fruit Industry in Japan has developed the fungicide Tebufloquin [26], which has excellent control effects on rice blast, and the commercial antifungal Ciprofloxacin.
In our previous work [27][28][29][30][31], many compounds based on the natural quinoline structure were synthesized and showed good activity. In recent work [32], we found that in the 8-position quinoline ring added p-chlorophenoxy group, some compounds showed certain activity. As we continued our effort to develop high-efficiency and low-toxicity new quinoline-based fungicides, the natural quinoline structure was selected as key core, 8-position of quinoline ring was replaced by fluorine atom, and the 4-position of hydroxyl on the quinoline ring was esterified ( Figure 2 [32]). All new quinoline compounds were characterized by 1 H NMR, 13 C NMR, and HRMS, and their antifungal activity was tested.

Synthesis and Spectra Analysis
The synthetic route of title fluorinated quinoline analogs is shown in Scheme 1. First, 2-fluorophenol was directly reacted with ethyl 2-methylacetoacetate under polyphosphoric acid (PPA) to give 2,3-dimethyl-4-hydroxyquinoline intermediate 1. The PPA was used as both a solvent and an acidic catalyst, and the reactants are directly one-step synthesis of quinoline rings. Then, intermediate 1 undergoes esterification with various substituted benzoic acids to generate the target compound. When EDC•HCl and DMAP are used as condensation agent, the solvent has a great influence on the yield of this reaction. It was found that using DMF as solvent can give higher yield than using dichloromethane (DCM) as solvent (Table 1). The two CH 3 protons are at 2.3 and 2.8 ppm, respectively. The protons of the benzene ring are assigned at 7~9 ppm. Taking compound 2b as an example, the protons of two methyl groups can be found at δ 2.32 ppm and δ 2.80 ppm as singlets in the 1 H NMR, while in the 13 C NMR data, they are found at 12.87 ppm and 24.37 ppm, respectively. The C=O was found at 163.73 ppm in the 13 C NMR.

Structure Determination
A colorless crystal of 8-fluoro-2,3-dimethylquinolin-4-yl 4-(tert-butyl)benzoate, 2b, suitable for X-ray diffraction study, was cultivated in the test tube from EtOH by selfvolatilization. A crystal with dimensions of 0.36 mm × 0.28 mm × 0.18 mm was mounted on a Bruker APEX-II CCD diffractometer equipped with graphite-monochromatic MoKα radiation (λ = 0.71073 Å). The crystal structure was solved by direct methods with SHELXS-97 [33] and refined by full-matrix least-squares refinements based on F 2 with SHELXL-97. All nonhydrogen atoms were refined anisotropically, and all hydrogen atoms were located in the calculated positions and refined with a riding model. The detailed crystal data are listed in Table 2.

Fungicidal Activity and SAR
The antifungal activities of compounds 2a-2p against ten phytopathogenic fungi are listed in Table 3. Generally, these compounds showed good antifungal activity. For example, compounds 2b(4-Bu), 2e(4-F), 2g(4-OMe) and 2p displayed moderate activity (40~60%) against A. solani. For the P. oryae, compounds 2b, 2d, 2f, and 2p also exhibited moderate activity (40~60%), which is the same as the A. solani. The inhibition rate of compound 2f against P. capsicum reached 58.1%, which was the same as that of the positive control Tebufloquin (58.1%). Compounds 2e and 2j were both 45% inhibitory to F. oxysporum, which is a little better than the positive control Tebufloquin (42.9%). Among these fungi, compounds 2b, 2e, 2f, 2k, and 2n possessed good activity (>80%) against S. sclerotiorum, which were higher than the positive control Tebufloquin (75.0%). Compounds 2b and 2d also had good activity (53.8%) against B. cinerea, which was a little lower than that of the positive control Tebufloquin (56.7%), but the activity of compound 2n (57.7%) was a little higher than that of the positive control Tebufloquin (56.7%). For the R. solani, compounds 2g (80.8%) and 2p (76.9%) exhibited good activity, which were higher than the positive control Tebufloquin (69.7%). The inhibition rates of compounds 2b, 2f, and 2n against C. arachidicola were 46.7%, 46.7%, and 60%, respectively, which were higher than the positive control Tebufloquin (37.5%). The inhibitory rates of compounds 2e, 2f, 2k, and 2n, against P. piricola were 72.0%, 76.0%, 76.0%, and 76.0%, respectively, which were also higher than the positive control Tebufloquin (65.4%). Notably, all the title compounds had lower inhibition rates against G. zeae; only compounds 2b and 2n exceeded 30%. From Table 3, it can be concluded that substitution on the benzene ring can influence the activity. The 4-position of the benzene ring had an electron-given group with better activity, such as compounds 2b(4-Bu), 2g(4-MeO), 2j(4-Me), and 2n(4-i-Pr), while the 4-position of the benzene ring had an electron-withdraw group, which had low activity, except for compound 2e(4-F), which may be the influence of the fluorine atom. When the R was an alkyl group, such as compound 2p (cyclopropyl group), it also exhibited good activity.

Instruments
Melting points were determined using an X-4 apparatus and uncorrected. 1 H NMR and 13 C NMR spectra were measured on a Bruker AC-P500 and AC-P400 instrument using TMS as an internal standard and deuterated chloroform, CDCl 3 , as the solvent. HR-ESI-MS was tested using an Agilent 1100 HPLC-JEOL AccuTOF instrument. All reagents were of analytical grade or were freshly prepared before use.

Synthesis of Intermediate 1
The synthetic route is shown in Scheme 1. In a 250 mL three-necked flask, 2-fluoroaniline (11.11 g, 100.00 mmol), ethyl 2-methylacetoacetate (14.42 g, 100.00 mmol), and polyphosphoric acid (50.69 g, 150.00 mmol) were added, and the mixture was heated at 150 • C. After the reaction was completed, the mixture was cooled to room temperature. The three-necked flask was placed in an ice bath, and the pH was adjusted to 7-8 by 10% aqueous sodium hydroxide solution. Then, it was filtered and dried to give 8-fluoro-2,3-dimethylquinolin-4-

Fungicide Bioassays
The fungicidal activities of title chiral niacinamide derivatives 2a-2p were tested in vitro against A. solani (AS), G. zeae (GZ), P. oryae (PO), P. capsici (PC), S. sclerotiorum (SS), B. cinerea (BC), R. solani (RS), F. oxysporum (FO), C. arachidicola (CA), and P. piricola (PP), and their relative percent inhibition (%) was determined using the mycelium growth rate method according to the previous work [34,35]. Tebufloquin was used as the positive control. Each compound was dissolved in DMSO with 1% Tween to prepare the 500 µg/mL stock solution. The ten fungi were inoculated into a Petri dish containing 50 µg/mL stock solution and incubated in a 27 • C incubator. A DMSO solvent containing 1% Tween was used as a blank assay. The fungicidal effect was determined 48-72 h later. The inhibition rate of the compound compared to the blank assay was calculated by the following equation: where CK is the average diameter of the mycelium in the blank test, and AI is the average value of the mycelium in the presence of these compounds.

Conclusions
In summary, sixteen new fluorinated quinoline compounds were synthesized, and their structures were characterized by 1 H NMR, 13 C NMR, X-ray diffraction, and HRMS. The antifungal activity results showed that some of the compounds exhibited good antifungal activity at 50 µg/mL. Among them, compounds 2b, 2e, 2f, 2k, and 2n exhibited good antifungal activity (>80%) against S. sclerotiorum. The antifungal activity of compound 2g against R. solani was 80.8%. This can give new clues for designing new highly active quinoline compounds in the next study.