Design and synthesis of novel n -butyphthalide derivatives as promising botanical fungicides

: In order to obtain novel botanical fungicides, three series of novel 6-substituted n -butyphthalide derivatives have been designed and synthesized via nucleophilic addition, reduction, nitri ﬁ cation, amination, sulfonation, Sandmeyer and Suzuki reaction. The mycelium growth rate method was used to evaluate the inhibition activity against eight phytopathogenic fungi in vitro . Preliminary bioassay tests showed that compounds 6f , 6n , 6p , 6r and 7a exhibited better activity for some fungi at 50 μ g/mL than the positive drug hymexazol and lead compound n -butyphthalide (NBP). The preliminary structure – activity relationships indicated that the antifungal activity is signi ﬁ cantly affected by the substituents on the benzene ring.


Introduction
Plant disease is a natural disaster caused by plant pathogenic fungi, which not only tremendously influence the yield and quality of grain, vegetables and fruits, but also some pathogenic fungi can produce carcinogenic, neurotoxic or teratogenic secondary metabolites (such as aflatoxin and zearalenone) in the process of growth and metabolism in the host body. Therefore, the development of novel compounds that effectively inhibit these agricultural diseases is still highly desirable [1]. Phthalides are a relatively small group of natural compounds found in several plant and fungal genera, which usually possess a broad scope of pharmacological and biological activities [2,3], such as the three compounds (Z)-butylidenephthalide (1), n-butylphthalide (2) and (Z)-ligustilide (3) (Figure 1). Identified in the essential oil of Lingusticum chuanxiong, these were shown to exhibit antifungal [4], antiplatelet [5], neuroprotection [6], anticancer [7], antiinflammatory [8] and insecticidal effects [9,10]. However, to the best of our knowledge, phthalide compounds were rarely reported on their structural modifications based on agricultural bioactivities. Thus, in continuation of our aim to search for novel bioactive molecules with antiphytopathogenic effects [11][12][13][14][15][16], we selected n-butylphthalide as the lead compound. Three series of 6-substitued phthalide derivatives were designed, synthesized and evaluated for their antifungal activities against eight phytopathogenic fungi in vitro.

Results and discussion
As shown in Scheme 1, the lead compound n-butylphthalide (2) was synthesized according to our previous method. (Z)-butylidenephthalide (1) was prepared by the nucleophilic addition of phthalic anhydride with n-BuLi, followed by dehydration in toluene with p-toluenesulfonic acid (p-TsOH) as catalyst. Then compound 2 was obtained by the treatment of compound 1 with Pd/C in 94.5% yield [17]. Afterward, compound 2 was nitrated using potassium nitrate in H 2 SO 4 to obtain the corresponding nitro compound 3, which was reduced with iron powder and NH 4 Cl in THF/H 2 O to give amino compound 4 [18]. Compound 4 was subjected to standard Sandmeyer reactions to give the 6-bromobutylphthalide (5). Finally, the target compounds 6a-s were prepared by suzuki reaction of compound 5, and compounds 7a-f and 8a-c were synthesized by sulfonylation and acylation reaction of compound 4 (Scheme 2). All the target compounds are new compounds and their structures were characterized by spectrometric methods including 1 H NMR, 13 C NMR and high-resolution mass spectra (HRMS).

Compounds
Average inhibition rate ± SD (%) (n = ) Although it is difficult to extract clear structure-activity relationships (SARs) from the presented biological data, the conclusion still can be summarized is that the spectrum of antifungal activity is significantly impacted by the presence of substituents. Firstly, compared with the lead compound NBP, introduction of benzene ring can enhance the activity against BC and AS (6a vs NBP), and introduction of pyridine ring can increase the activity against BC, VM, SS and AS but decrease the activity against other fungi (6r vs NBP). Meanwhile, bringing chlorine atom to the compound 6r afforded the moderate potent compound 6s. Secondly, introduction of 4-OH on the benzene ring of compound 6a only significantly enhances the activity against BC strains (6e vs NBP). Thirdly, for the halogenated compounds, introduction of 2-F afforded the slightly more potent compound than that of 2-Cl (6f vs 6l), and monohalogenated compounds display better antifungal activity than polyhalogenated compounds against all phytopathogenic fungi at the concentration of 50 μg/mL (6f vs 6i and 6j, 6m vs 6k and 6l). Furthermore, it is noticeable that bearing a thiophene group at R position exhibited more pronounced antifungal effects than that of benzothiophene group (6p vs 6q ). Finally, compounds with sulfonylamine substitutions (7a-f ) were found to have varying degree of antifungal activity. Amongst them, compound 7a with para-fluorine substitution was found to be more active than that of others. But unfortunately, compounds having the amide substitutions (8a-c) showed worse activity than aryl (6a-s) and sulfonamide substitutions (7a-f ).
Meanwhile, the effects of compounds on the growth of VM and FS at the concentration of 50 μg/mL were shown in Figure 2. It can be seen clearly that the mycelium diameter was significantly smaller than that of the positive control hymexazol after treated of VM stains with tested compounds 6r, 6h, 6s, 6n and 6b. Similarly, the mycelium growth also reflected that the compound 6r had better inhibitory effects on FS strains than that of 6n, 6p, 6h, 6f and hymexazol.

Conclusion
In summary, 28 novel n-butyphthalide derivatives were synthesized and evaluated for their antifungal activities against eight phytopathogenic fungi in vitro at the concentration of 50 μg/mL. Among all the derivatives, compounds 6a, 6f, 6n, 6r, 6s and 7a generally exhibited the promising and broad-spectrum antifungal activities, especially compound 6r displayed the more pronounced antifungal activity than the lead compound NBP and hymexazol against VM, SS and AS strains. It clearly demonstrated that introduction of appropriate substituents on the 6-position of NBP would lead to more potent derivatives. It also implied that 6r might be considered as new promising lead candidates for further design and synthesis of agricultural fungicides.

Experimental section 4.1 General information
All reagents and solvents were of reagent grade or purified according to standard methods before use. Thin-layer chromatography (TLC) and preparative thin-layer chromatography (PTLC) were used with silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd., China). Melting points (m. p.) were determined on a digital m.p. apparatus and were uncorrected. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance NEO 400 and 100 MHz instruments, respectively, using TMS as the internal standard and CDCl 3 or DMSO-d 6 as the solvent. HRMS were carried out with an APEX II Bruker 4.7T AS instrument.
To a mixture of phthalic anhydride (40.0 g, 270 mmol) in anhydrous THF (50 mL), n-butyllithium (100 mL, 1.0 eq, 2.7 M n-hexane solution) was added dropwise and reacted at −78°C under N 2 . When the reaction was complete according to TLC analysis, the reaction was quenched with water. Subsequently, the reaction mixture was adjusted to pH 1-2 with 10% HCl and extracted with EtOAc (3 × 150 mL). The combined organic phase was washed with brine (200 mL), dried over anhydrous Na 2 SO 4 and concentrated to give 40.0 g of a brown oily liquid. Then anhydrous toluene (150 mL) and p-toluenesulfonic acid (4.6 g, 27.0 mmol) were added and the reaction mixture was refluxed for 6 h. When the reaction was complete (TLC control), the organic solvent was removed. The crude material was purified by silica gel column chromatography to give compound 1 (16.8 g, 33.1%) as an oily liquid. 1

3-Butyl-6-phenylisobenzofuran-1(3H)-one (6a)
Yield: 95%, colorless oily liquid. 1    were used for the assays. Potato dextrose agar (PDA) medium was prepared in the flasks and sterilized. The synthesized compounds were dissolved in acetone before mixing with PDA, and the concentration of test compounds in the medium was fixed at 50 μg/mL. The medium was then poured into sterilized Petri dishes. All types of fungi were incubated in PDA at 27 ± 0.5°C for 5 days to get new mycelium for the antifungal assays, and a mycelia disk of approximately 4 mm diameter cut from culture medium was picked up with a sterilized inoculation needle and inoculated in the center of the PDA Petri dishes. The inoculated Petri dishes were incubated at 27 ± 0.5°C for 4 days. Acetone without any compounds mixed with PDA served as a control, while hymexazol, a commercial agricultural fungicide, was used as positive control. For each treatment, three replicates were conducted. The radial growths of the fungal colonies were measured and the data were statistically analyzed. The inhibitory effects of the test compounds on these fungi in vitro were calculated by the formula: Inhibition rate (%) = (C−T) × 100/(C−4 mm), where C represents the diameter of fungi growth on untreated PDA, and T represents the diameter of fungi on treated PDA. Statistical analysis was processed by the SPSS 21.0 (SPSS Inc., Chicago, USA) software.