Discovery of novel obovatol-based phenazine analogs as potential antifungal agents: synthesis and biological evaluation in vitro

2.1 General information

The leaves of M. obovata were collected from Qingdao, Shandong Province, P. R. China and obovatol (1) was isolated from the leaves of M. obovata according to our previously reported procedures [10]. All chemical reagents and solvents were obtained from commercial sources and used without further purification unless otherwise stated in this work. Difenoconazole (>98%) and hymexazole (>99%) were purchased from Aladdin Reagents Co., Ltd. and used as the positive controls. Analytical thin-layer chromatography (TLC) and column chromatography (CC) were performed using silica gel 60 GF₂₅₄ and silica gel (200–300 mesh) (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), respectively. Melting points (m.p.) were determined on a SGWX-4B digital melting-point apparatus and are uncorrected (Shanghai INESA Physico optical Instrument Co., Ltd.). ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) were recorded on a Bruker Avance Ⅲ 500 magnetic resonance spectrometer (Bruker, Germany) in deuterochloroform (CDCl₃) or deuterated dimethyl sulfoxide (DMSO-d₆) with tetramethylsilane (TMS) as internal standard. High resolution electrospray ionization mass spectrometry (HR-ESI-MS) was measured on a Waters Xevo G2-XS QToF spectrometer (Waters Technologies, USA).

2.2 General procedure for the synthesis of phenazine derivatives of obovatol (8a–k, 8h′–8k′, and 8l)

To a stirred solution of obovatol (1, 200 mg, 0.71 mmol) in dry dichloromethane (10 mL) in an ice bath, a solution of concentrated nitric acid (1.42 mmol) in dry dichloromethane (5 mL) was added dropwise while the temperature was maintained below 0 °C. After that, the reaction mixture was allowed to warm up to room temperature naturally and the reaction progress was monitored by TLC analysis. When the reaction was complete after 5 h, the mixture was extracted with dichloromethane (3  × 50 mL) and the combined organic phase was dried over anhydrous Na₂SO₄ and subsequently concentrated in vacuo. The obtained residue was used directly for the next step. A solution of substituted ortho-Phenylenediamine or 2,3-diaminonaphthalene (1.07 mmol) in CHCl₃ (10 mL) was added to a solution of the residue from the previous step in CHCl₃ (10 mL) at room temperature. The reaction system was stirred overnight and the mixture was concentrated, and purified by silica gel column chromatography to afford compounds 8a–k, 8h′–8k′, and 8l in 11.1–24.4% yield (two steps from 1 to 8a–k, 8h′–8k′, and 8l).

2.3 Crystal structure determination of 8i′

A suitable crystal was selected and examined with a SuperNOVA single-crystal X-ray diffractometer from Agilent Technologies Inc., Santa Clara, CA (USA). Diffraction data were collected at T = 293(2) K using CuKα radiation (λ = 1.54184 Å). Using Olex2 [26], the structure was solved with Direct Methods using the program Shelxs [27] and refined with full matrix-least squares minimization using Shelxl [28]. Table 1 summarizes the crystal data and numbers pertinent to data collection and refinement.

CCDC 2026867 (8i′) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre viawww.ccdc.cam.ac.uk/data_request/cif.

2.4 Antifungal assay of compounds 8a–k, 8h′–8k′, and 8l

The tested plant pathogenic fungi Alternaria solani, F. solani, Botrytis cinerea, and Colletotrichum orbiculare were provided by the Key Laboratory of Plant Pathology, College of Plant Protection, Shanxi Agricultural University, Taigu, P. R. China. These fungi were grown on potato dextrose agar (PDA) plates at 25 ± 1 °C for 96 h before used for the biological assays and maintained at 4 °C with periodic subculturing. Four tested phytopathogenic fungal strains were cultured at 25 ± 1 °C on potato dextrose agar (PDA) until sporulation. Then, sterile distilled water was poured onto the Petri dishes and the conidia were scraped with a glass rod. The mixture was filtered by quadruple-layer cheesecloth to remove mycelial debris and the spores were obtained and suspended in sterile distilled water. Finally, the spore suspension was diluted to a suitable concentration (30–40 spores per field vision under 100-fold magnification) with sterile water for further testing and an additional 0.5% aqueous glucose solution was added.

All the samples were dissolved in dimethyl sulfoxide (DMSO final concentration ≤ 1%, v/v) and diluted to a series of desired concentrations with aseptic aqueous 0.1% Tween-80 (v/v) solution. Then, the sample solution was further mixed with the prepared spore suspension and 30 μL aliquots of the treated spore suspension were placed on separate glass slides in triplicate. The glass slides containing the spores were incubated in an incubator and kept at a suitable moisture. For different fungal spores, the culture conditions were different. A. solani, F. solani, and C. orbiculare were incubated at 25 ± 1 °C for 3–6 h and B. cinerea was incubated at 20 ± 1 °C for 7 h. The spores were considered to have germinated if the length of the germ tube was at least half the length of the spore. The number and percentage of the germinated spores were calculated in the compound-treated group when the percentage of germinated spores was more than 90% that in the blank control group. 1% aqueous DMSO solution was used as a blank control.

Corrected inhibition rate (in %) of spore germination: (R_c – R_t)/R_c × 100 where R_c is the percentage of germinated spores in the blank control test and R_t represents the percentage of germinated spores in the compound-treated test sample. The experimental data of the antifungal assay were analyzed by Excel 2010 and SPSS 20.0 software for Windows.

3.1 Synthesis and characterization

The isolated obovatol (1) was used as precursor for the further synthetic steps. As depicted in Scheme1, the intermediate 7 was obtained by oxidation of 1 in the presence of concentrated nitric acid. Subsequently, the unpurified 7 was used directly in the next step, and reacted with substituted ortho-Phenylenediamine or 2,3-diaminonaphthalene to produce the target products 8a–k, 8h′–8k′, and 8l via cyclization reaction. It should be noted that two different products 8h–k and 8h′–8k′ were obtained after cyclization of each monosubstituted ortho-Phenylenediamine and intermediate 7. Unexpectedly, none of the nitration products 5 and 6 was found when obovatol was treated with concentrated nitric acid. The structures of all the target compounds were characterized by ¹H and ¹³C nuclear magnetic resonance, high-resolution mass spectrometry (HR-MS) and melting points (m.p.). Furthermore, the configuration of compound 8i′ was unambiguously identified by X-ray crystallography which established the crystal and molecular structure (Figure 2, Table 1). This clearly demonstrated that the phenazine heterocycle was introduced into the obovatol skeleton.

Figure 2:

The molecular structure of 8i′ in the crystal and atom numbering scheme adopted. Non-H atoms are drawn as ellipsoids at the 50% probability level, hydrogen atoms as spheres with arbitrary radius. Important bond lengths (Å) and angles (°): Cl1–C1 1.732(4), O1–C5 1.368(4), O1–C16 1.406(5), N1–C3 1.347(5), N1–C4 1.343(5), N2–C9 1.338(5), N2–C10 1.341(5); C5–O1–C16 117.9(3), C4–N1–C3 116.2(3), C9–N2–C10 117.5(3), C2–C1–Cl1 120.5(3), C12–C1–Cl1 117.3(3), N1–C3–C2 119.1(3), N1–C3–C10 121.9(4), N1–C4–C5 120.2(3), N1–C4–C9 122.2(3), O1–C5–C4 113.9(3), C6–C5–O1 125.5(3), N2–C9–C4 120.9(3), N2–C9–C8 119.4(3), N2–C10–C3 121.3(4), N2–C10–C11 119.9(3), C17–C16–O1 119.9(4), C21–C16–O1 118.3(4).

3.2 Antifungal activity

The antifungal activity of derivatives 8a–k, 8h′–8k′, and 8l against four plant pathogenic fungi in vitro was tested using the spore germination method. As is shown in Table 2, the results revealed that most of compounds lost antifungal activity when compared with the precursor obovatol. Firstly, none of the tested compounds showed antifungal activity against A. solani. On the other hand, we found that eight of the 16 compounds (8b, 8g, 8h–k, 8i′, and 8k′) exhibited better inhibitory activity against F. solani than obovatol and two positive controls (difenoconazole and hymexazole). Specifically, compounds 8b and 8i′ displayed the highest inhibitory activity against F. solani with the IC₅₀ values of 64.61 and 79.97 μg mL⁻¹, respectively, whereas the IC₅₀ values of obovatol was 114.52 μg mL⁻¹ and the positive controls were greater than 500 μg mL⁻¹. These findings may indicate that obovatol phenazine derivatives possess specific inhibitory on F. solani spores. In addition, only compound 8b (IC₅₀ = 223.45 μg mL⁻¹) showed moderate inhibitory effects on B. cinerea spores. Derivatives 8b and 8h exhibited certain antifungal activity against C. orbiculare spores with the IC₅₀ values of 124.75 and 130.19 μg mL⁻¹, respectively. In summary, it can be found that compound 8b showed excellent and broad spectrum antifungal activity, which is worthy of further investigation.

The preliminary structure-activity relationship study showed that among the eight compounds with significant inhibitory effect on the spores of F. solani, six compounds (8h–k, 8i′, and 8k′) are monosubstituted derivatives, and all of them contain electron-withdrawing groups on the benzene ring (8h: 4-nitrophenyl; 8i/8i′: 3/4-chlorophenyl; 8j: 4-bromophenyl; 8k/8k′: 3/4-trifluoromethylphenyl). This may indicate that the introduction of a monosubstituted electron-withdrawing group containing a benzene ring into obovatol can give rise to further potentially antifungal compounds against F. solani. This finding provides a basis for the further development of effective antifungal agents based on obovatol.