Synthesis and antifungal activities of 3-substituted phthalide derivatives

1 Introduction

Phytopathogenic fungi easily infect many crops and are often hard to control. Therefore, the development of new compounds that effectively inhibit these agricultural diseases is still highly desirable. Phthalide is an important heterocyclic scaffold found in several plants [1]. Molecules containing this pharmacophore exhibit a broad spectrum of biological activities [2], such as antioxidant [3], analgesic [4], antithrombotic [5], antiplatelet [6], insecticidal [7], [8], [9], bactericidal [10], antifungal [11], and anti-HIV [12], [13] activities. For example, 3-n-butylphthalide (NBP) (Fig. 1), an antiplatelet drug, which was approved in 2002 by the State Food and Drug Administration of China for the clinical treatment of acute ischemic stroke [14]. Mycophenolic acid (MPA) is in clinical trial for the prevention and reversal of transplant rejection and as an anticancer drug [15]. Corollosporine is a new phthalide derivative from the marine fungus Corollospora maritima which exhibits concentration-dependent antibacterial activity against Staphylococcus aureus and other microorganisms [16]. (±)-Chrycolide is isolated from the leaves and stem of a popular vegetable Chrysanthemum coronarium, which shows plant growth inhibiting activity [17].

Fig. 1:

Selected biologically important compounds containing a phthalide scaffold.

Encouraged by the numerous pharmacological activities of phthalide derivatives, and in continuation of our ongoing work on the discovery and development of compounds with superior antifungal activities [18], [19], we prepared two series of 3-substitued phthalide derivatives and evaluated their antifungal activities against nine phytopathogenic fungi in vitro.

2 Results and discussion

As shown in Scheme 1, isobenzofuran-1(3H)-one (1) was prepared by the reduction of phthalic anhydride with sodium borohydride (NaBH₄), followed by cyclization in 3 n hydrochloric acid (HCl) at room temperature. Compound 1 was then subjected to radical bromination in the presence of N-bromosuccinimide/azobisisobutyronitrile (NBS/AIBN) to give the key intermediate 2. Finally, two series of 3-substituted phthalide derivatives (3a–j and 4a–m) were synthesized from intermediate 2 by Williamson etherification reaction in the presence of potassium carbonate (K₂CO₃). To our surprise, 5-methyl-2-(3-oxo-1,3-dihydroisobenzofuran-1-yl)-3-(3-oxo-1,3-dihydroisobenzofuran-1-yloxy)is-oxazol bromide (3j) was obtained when compound 2 reacted with the nucleophile 5-methylisoxazol-3-ol (5) under the same conditions. Closer scrutiny of this reaction revealed that it was completely determined by the reaction time. If the reaction time was prolonged to 12 h, compound 3i was immediately and completely transformed into the unexpected quaternary salt 3j [20]. The structures of all target compounds were characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR and high-resolution mass spectrometry.

Scheme 1:

Synthetic route of the preparation of phthalide derivatives 3a–j and 4a–m.

Scheme 2:

The conversion of compound 3i–j.

The antifungal activities of 3-substituted phthalide derivatives 3a–j and 4a–m against nine phytopathogenic fungi [i.e. Fusarium solani (FS), Botryosphaeria berengriana f. sp. Piricola (BP), Curvularia lunata (CL), Fusarium oxysporum schlecht. f. sp. C. maxima (FM), Fusarium graminearum (FG), Alternaria alternata (AA), Pyricularia oryzae (PO), Fusarium oxysporum f. sp. Vasinfectum (FV), and Alternaria brassicae (AB)] were investigated at a concentration of 50 μg mL⁻¹in vitro by the poisoned food technique [21]. Hymexazol, a commercially available agricultural fungicide, was used as a positive control at the same concentration (50 μg mL⁻¹).

As is shown in Table 1, the results revealed that most of derivatives displayed certain inhibitory effects on the growth of the tested phytopathogenic fungi. Among all the synthesized compounds, ten derivatives 3b, 3c, 3e, 3f, 3g, 3i, 3j, 4a, 4c, and 4j (42, 48, 43, 53, 52, 45, 41, 44, 41, 50%, respectively) generally exhibited more pronounced antifungal activity against BP than hymexazol (31%). This finding indicated that the synthesized 3-substituted phthalide derivatives could represent a new structure skeleton for inhibiting BP. Compounds 3b, 3c, 3f, 3g, 3i, 4c, 4j, and 4k (45, 43, 58, 46, 53, 44, 52, 51%, respectively) showed relatively higher activity against FS than hymexazol (42%). Preliminary structure-activity relationships (SARs) presumed that introduction electron-donating groups -OH and -NH₂ on the benzene ring could enhance their antifungal activity against FS, probably because they form a hydrogen bond with the active site of the enzyme in the fungus leading to metabolic system disorder [22]. For FV strains, compounds 3b, 3j, 4c, 4j, 4k, and 4m (38, 43, 38, 37, 39, 39%, respectively) displayed slightly higher inhibition rates than hymexazol (37%). Toward FG strains, the introduction of two nitrogen dioxide (-NO₂) group or -OH group on the benzene ring give the compounds 3e (62%) and 3f (52%), which show better activities than hymexazol (52%). For CL strains, seven compounds 3c (48%), 3g (41%), 3j (43%), 4c (56%), 4j (55%), 4k (61%), and 4m (42%) display more potent antifungal activities than hymexazol (40%). In addition, all compounds showed poor fungicidal activities against the four tested fungi (FM, AA, PO, and AB) except compounds 3c, 3i, 4c, 4j, and 4k. Based on these results compounds 3c, 4c, 4j, and 4k provide broad-spectrum antifungal activities to the tested fungi and could be taken under consideration for further research.

3 Conclusion

In conclusion, 24 3-substituted phthalide derivatives were designed and synthesized via reduction, bromine substitution and Williams etherification, and evaluated in vitro for their antifungal activities against nine phytopathogenic fungi at the concentration of 50 μg mL⁻¹. Among these derivatives, four compounds 3c, 4c, 4j, and 4k generally exhibit broad-spectrum antifungal activities as compared with the commercially available agricultural fungicide hymexazol. This clearly demonstrates that the introduction of appropriate thioether and oxyether substituents on the 3-position of phthalide leads to more potent antifungal derivatives.

4 Experimental section