Synthesis and biological evaluation of MS-347a derivatives against plant pathogenic fungi based on a strategy of laeA gene introduction

Abstract

Our group previously identified MS-347a (1) as a new fungicide candidate from the culture broth of the mutant strain, Aspergillus sp. KTF-0058, which had the laeA gene inserted. This mutant strain was able to produce a sufficient supply of 1, allowing for its use to investigate the structure–activity relationship. To this end, we synthesized 11 derivatives and evaluated their antifungal activity. Among these derivatives, the aldehyde derivative exhibited superior antifungal potency as compared to 1, and some acyl derivatives were able to maintain the antifungal activity of 1 despite significant structural changes. From these results, it is found that the aldehyde derivative is one of the most promising fungicidal candidates, and the introduction of acyl groups could be utilized to create chemical probes for target-identification experiments.

Introduction

As the world’s population continues to grow, global food security becomes ever more important. Whether through climate change, conflicts, or infectious disease, the global food supply is under great strain and is vulnerable to collapse. As such, our group has been aiming to tackle fungal plant pathogens with fungicidal natural products to ensure and maintain the food supply. In fact, pathogenic plant fungi have already caused significant damage to humanity’s essential food crops, and that damage has been increasing.^1,2) The rapid emergence of drug-resistant pathogenic plant fungi to several conventional fungicides is a major contributing factor to the increasing damage to crops, and this resistance could be attributed to the highly plastic genomes of fungi.³⁾ Considering this, it is likely preferable to use multiple fungicides with unique mechanisms of action against these pathogens to minimize the emergence of drug resistances. Therefore, continuous screening for new fungicide candidates is in high demand for efficient control of pathogenic plant fungal disease. Accordingly, we conducted screening of microbial secondary metabolites and found that the culture broth of Aspergillus sp. FKI-5362 strain demonstrated antifungal activity. However, we were not able to initially isolate and identify the active compound due to its extremely low production. To settle this challenge, we introduced the putative methyltransferase laeA, known as a global regulator for expressions of secondary metabolite biosynthetic genes, into the strain.^4,5) The laeA gene introduced mutant (KTF-0058) strain produced the target compound in ten-folds greater quantity compared to the wild strain, enabling the identification of MS-347a (1) as a fungicidal compound (Fig. 1). Ultimately, we revealed that 1 showed potent antifungal activity against broad plant pathogenic fungal species. In this study, we synthesized new derivatives from 1 which was produced under the laeA gene introduction strategy to elucidate the structure–activity relationship of 1 for antifungal activity against pathogenic plant fungi, as well as to develop more potent fungicide candidates.

Fig. 1. Structure of MS-347a (1)

Materials and methods

1. General experimental procedures

High- and low-resolution mass spectra were obtained using an AB Sciex QSTAR Hybrid LC/MS/MS Systems (AB Sciex, Framingham, MA, USA) and JEOL JMS-T100LP (JEOL, Tokyo, Japan). NMR spectra were measured using AVANCE III HD-600 (Bruker, CA, USA), and Varian-INOVA600 with ¹H NMR and ¹³C NMR at 600 MHz and 150 MHz, respectively, and JEOL JNM-ECA-500 (JEOL, Tokyo, Japan), with ¹H NMR and ¹³C NMR at 500 MHz and 125 MHz in DMSO-d₆ and CDCl₃. The chemical shifts are reported in ppm and referenced to DMSO-d₆ (2.50 ppm) and CDCl₃ (7.26 ppm) in the ¹H NMR spectra and DMSO-d₆ (39.50 ppm) and CDCl₃ (77.16 ppm) in the ¹³C NMR.

2. Fermentations of Aspergillus sp. KTF-0058 strain

One loop of Aspergillus sp. FKI-5362 and KTF-0058 strains grown on an LcA slant (0.1% glycerol, 0.08% KH₂PO₄, 0.02% K₂HPO₄, 0.02% MgSO₄·7H₂O, 0.02% KCl, 0.2% NaNO₃, 0.02% yeast extract, and 1.5% agar, pH 6.0) was inoculated into 10 mL of a seed culture medium (2% glucose, 0.2% yeast extract, 0.05% MgSO₄·7H₂O, 0.5% polypepton (Nihon Pharmaceutical Co., Tokyo, Japan), 0.1% KH₂PO₄, and 0.1% agar, pH 6.0) and incubated on a rotary shaker (210 rpm) at 27°C for 3 days. One liter of the seed culture was inoculated into a production medium (10 kg of water-sodden rice). Static fermentation was continued at 27°C for 14 days.

3. Isolations of MS-347a (1)

MS-347a (1): The culture of KTF-0058 strain was extracted with 10 L of methanol. After filtration, the filtrate was concentrated in vacuo to remove the methanol. The concentrated extract (2 L) was applied on an ODS column (500 mL of resin, YMC Co. Ltd., Kyoto, Japan), which was eluted stepwise with 0, 20, 40, 60, 80, and 100% of MeOH aqueous solutions (each 1 L). The 40% MeOH fraction was concentrated to dryness to afford a crude extract. The extract was chromatographed on a silica gel column and eluted stepwise with a mixture of CHCl₃/MeOH (100/0, 100/1, 100/3, 100/5, 100/10, 100/100, and 0/100). After concentrating 100/0 and 100/1 fractions in vacuo, the compound which was insoluble in MeOH was washed 5 times with 100 µL of methanol by centrifugation at 10,000 × g for 1 min, isolating compound 1. HR-ESIMS m/z 317.0662 [M+H] ⁺ (calcd m/z 317.0661 [M+H] ⁺, molecular formula C₁₆H₁₂O₇).

4. Synthesis of MS-347a derivatives

General procedure for derivatives 2, 6, 7 and 12

To a stirred solution of 1 in pyridine was added acetic anhydride, propionic anhydride, butyric anhydride, or BzCl at rt. After 24 hr, the reaction solution was evaporated by nitrogen blowdown. The residual oil was purified by PTLC (hexane : acetone=3 : 2 or n-hexane : AcOEt=1 : 2) to afford acylated MS-347a.

5. Acetyl derivative 2

A reaction of 1 (20.0 mg, 63.2 µmol) with acetic anhydride (149 µL, 1.54 mmol) in pyridine (200 µL, 2.43 mmol), followed by PTLC purification gave acetyl derivative 2 (6.6 mg, 26%) as a yellow solid. ¹H-NMR (500 MHz, DMSO-d₆) δ 7.57 (d, J=1.8 Hz, 1H), 7.20 (d, J=1.8 Hz, 1H), 7.17 (dd, J=12.3, 4.9 Hz, 1H), 6.91(dd, J=12.3, 4.9 Hz, 1H), 5.22 (s, 2H), 4.28 (dd, J=4.9, 2.0 Hz, 1H), 3.66 (s, 3H), 2.31 (s, 3H), 2.13 (s, 3H); and ¹³C-NMR (125 MHz, DMSO-d₆) δ 173.5, 170.3, 169.1, 166.8, 158.1, 155.7, 148.7, 144.4, 137.7, 125.6, 118.5, 115.2, 114.8, 114.6, 63.9, 56.9, 56.1, 52.5, 20.9, 20.7. HRMS (ESI+) m/z calcd for C₂₀H₁₇O₉ (M+H⁺): 401.0873, found: 401.0872.

6. Aromatized derivative 3

To a stirred solution of 1 (20.0 mg, 63.2 µmol) in MeOH (632 µL, 0.1 M) was added Pd/C (2.0 mg, 10% wt of SM) at room temperature. The reaction mixture was evacuated and backfilled with H₂, which was reaerated a total ten times. After being stirred for 1 hr, the reaction mixture filtered through a pad of celite, and the filtrate was concentrated under reduced pressure. The residue was purified by PTLC (hexane : AcOEt=6 : 1) to give aromatized derivative 3 (8.2 mg, 43%) as a yellow solid. ¹H-NMR (500 MHz, DMSO-d₆) δ 12.05 (s, 1H), 7.94 (dd, J=10.8, 9.0 Hz, 1H), 7.76 (dd, J=10.3, 1.3 Hz, 1H), 7.45 (dd, J=10.3, 1.3 Hz, 1H), 7.03 (d, J=1.6 Hz, 1H), 6.78 (dd, J=1.6 Hz, 1H), 5.59 (t, J=7.3 Hz, 1H), 4.60 (d, J=6.9 Hz, 2H), 3.89 (s, 3H); and ¹³C-NMR (125 MHz, DMSO-d₆) δ 180.3, 168.8, 160.6, 155.7, 155.5, 154.6, 136.1, 133.0, 123.0, 119.8, 116.7, 107.8, 107.1, 104.3, 62.4, 52.8. HRMS (ESI+) m/z calcd for C₁₆H₁₃O₆ (M+H⁺): 301.0712, found: 301.0718.

7. Methyl derivative 4

To a stirred solution of 1 (20.0 mg, 63.2 µmol) in DMF (632 µL, 0.1 M) were added K₂CO₃ (52.4 mg, 379.2 µmol) and MeI (19.6 µL, 316.0 µmol) at room temperature. After being stirred for 12 hr, the reaction mixture was quenched with sat. NH₄Cl aq. and diluted with AcOEt. The resulting mixture was extracted with AcOEt and washed with brine. The organic phase was dried over Na₂SO₄, concentrated under the reduced pressure. The residue was purified by PTLC (AcOEt) to give methyl derivative 4 (9.1 mg, 44%) as a yellow solid. ¹H-NMR (500 MHz, DMSO-d₆) δ 7.08 (dd, J=4.8, 11.9 Hz, 1H), 7.07 (d, J=1.8 Hz, 1H), 6.97 (d, J=1.8 Hz, 1H), 6.85 (dd, J=11.9, 2.1 Hz, 1H), 5.58 (t, J=7.3 Hz, 1H), 4.61 (d, J=7.3 Hz, 2H) 4.23 (dd, J=4.8, 1.8 Hz, 1H), 3.86 (s, 3H), 3.69 (s, 3H); and ¹³C-NMR (125 MHz, DMSO-d₆) δ 173.8, 167.1, 159.1, 156.8, 156.6, 151.2, 136.4, 125.8, 115.0, 111.6, 106.6, 105.3, 62.3, 57.1, 56.3, 53.2, 52.5. HRMS (ESI+) m/z calcd for C₁₇H₁₅O₇ (M+H⁺): 3310818, found: 331.0818.

8. Aldehyde derivative 5

To a stirred solution of 1 (20.0 mg, 63.2 µmol) in DCM (632 µL, 0.1 M) was added MnO₂ (54.9 mg, 632 µmol) at room temperature. After being stirred for 3 hr, the reaction mixture filtered through a pad of celite, and the filtrate was concentrated under reduced pressure. The residue was purified by PTLC (hexane : AcOEt=1 : 2) to give aldehyde derivative 5 (7.0 mg, 34%) as a yellow solid. ¹H-NMR (600 MHz, CDCl₃) δ 12.33 (s, 1H) 10.02 (s, 1H), 7.42 (d, J=1.3 Hz, 1H), 7.31 (d, J=1.3 Hz, 1H), 7.08 (dd, J=9.9, 4.2 Hz, 1H), 6.70 (dd, J=9.9, 1.3 Hz, 1H), 4.14 (dd, J=4.2, 1.9 Hz, 1H), 3.90 (s, 3H); and ¹³C-NMR (150 MHz, DMSO-d₆) δ 192.7, 180.8, 166.9, 161.2, 160.7, 155.8, 141.9, 140.0, 126.3, 113.4, 111.5, 109.4, 57.1, 56.5(2C), 29.5. HRMS (ESI+) m/z calcd for C₁₆H₁₁O₇ (M+H⁺): 315.0505, found: 315.0509.

9. Propionyl derivative 6

A reaction of 1 (19.7 mg, 62.2 µmol) with propionic anhydride (200 µL, 1.54 mmol) in pyridine (200 µL, 2.43 mmol), followed by PTLC purification gave propionyl derivative 6 (5.8 mg, 22%) as a white solid. ¹H-NMR (600 MHz, CDCl₃) δ 7.34 (d, J=1.8 Hz, 1H), 7.01 (d, J=1.8 Hz, 1H), 6.94 (dd, J=9.9, 4.2 Hz, 1H), 6.71 (dd, J=9.9, 1.8 Hz, 1H), 5.19 (s, 2H), 4.07 (dd, J=3.6, 1.8 Hz, 1H), 3.82 (s, 3H), 2.76 (dd, J=17.1, 7.2 Hz, 1H), 3.74 (dd, J=17.1, 7.2 Hz, 1H), 2.44 (q, J=7.2 Hz, 2H), 1.28 (t, J=7.2 Hz, 3H), 1.19 (t, J=7.2 Hz, 3H); and ¹³C-NMR (150 MHz, CDCl₃) δ 174.0, 173.8, 173.0, 167.4, 157.7, 156.4, 149.8, 143.3, 135.7, 126.2, 118.7, 116.3, 115.8, 114.6, 64.4, 57.4, 56.6, 53.0, 27.7, 27.6, 9.18, 8.88. HRMS (ESI+) m/z calcd for C₂₂H₂₀O₉Na (M+Na⁺): 451.1011, found: 451.1005.

10. Butyryl derivative 7

A reaction of 1 (20.0 mg, 63.2 µmol) with butyric anhydride (200 µL, 1.20 mmol) in pyridine (200 µL, 2.43 mmol), followed by PTLC purification gave butyryl derivative 7 (6.3 mg, 22%) as a white solid. ¹H-NMR (600 MHz, CDCl₃) δ 7.34 (d, J=1.2 Hz, 1H), 6.99 (d, J=1.8 Hz, 1H), 6.94 (dd, J=9.6, 3.6 Hz, 1H), 6.70 (dd, J=9.6, 1.2 Hz, 1H), 5.18 (s, 2H), 4.07 (dd, J=3.6, 1.8 Hz, 1H), 3.81 (s, 3H), 2.72 (dd, J=16.5, 7.5 Hz, 1H), 2.67 (dd, J=16.5, 7.5 Hz, 1H), 2.40 (t, J=7.2 Hz, 2H), 1.84–1.78 (m, 2H), 1.73–1.67 (m, 2H), 1.06 (t, J=7.2 Hz, 3H), 0.97 (t, J=7.2 Hz, 3H); and ¹³C-NMR (150 MHz, CDCl₃) δ 173.8, 173.2, 172.2, 167.4, 157.7, 156.4, 149.7, 143.3, 135.7, 126.3, 118.8, 116.3, 115.8, 114.6, 64.3, 57.5, 56.6, 53.0, 36.1 (2C), 18.5, 18.1, 13.9, 13.8. HRMS (ESI+) m/z calcd for C₂₄H₂₄O₉Na (M+Na⁺): 479.1324, found: 479.1319.

11. Cyclopropanecarbonyl derivative 8

To a stirred solution of 1 (20.0 mg, 63.2 µmol) in DCM (632 µL, 0.1 M) were added Et₃N (29.1 µL, 208.6 µmol) and cyclopropanecarbonyl chloride (18.9 µL, 208.6 µmol) at room temperature. After being stirred for 12 hr, the reaction mixture was quenched with sat. NH₄Cl aq. and diluted with AcOEt. The resulting mixture was extracted with AcOEt and washed with brine. The organic phase was dried over Na₂SO₄, concentrated under the reduced pressure. The residue was purified by PTLC (hexane : AcOEt=1 : 2) to give cyclopropanecarbonyl derivative 8 (28.0 mg, 95%) as a yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 7.34 (d, J=1.7 Hz, 1H), 7.03 (d, J=1.7 Hz, 1H), 6.93 (dd, J=9.9, 3.8 Hz, 1H), 6.69 (dd, J=9.9, 1.7 Hz, 1H), 5.18 (s, 2H), 4.07 (dd, J=5.4, 1.6 Hz, 1H), 3.83 (s, 3H), 1.99–1.94 (m, 1H), 1.74–1.59 (m, 1H), 1.30–1.2.0 (m, 2H), 1.09–1.05 (m, 4H), 0.96–0.91 (m, 2H); and ¹³C-NMR (150 MHz, CDCl₃) δ 174.6, 173.8, 173.2, 167.4, 157.7, 156.3, 149.7, 143.3, 135.7, 126.3, 118.8, 116.5, 115.8, 114.6, 64.5, 57.5, 56.6, 53.0, 13.4, 12.9, 9.3(2C), 9.1(2C). HRMS (ESI+) m/z calcd for C₂₄H₂₁O₉ (M+H⁺): 453.1186, found: 453.1188.

12. Cyclobutanecarbonyl derivative 9

To a stirred solution of 1 (20.0 mg, 63.2 µmol) in DCM (632 µL, 0.1 M) were added Et₃N (29.1 µL, 208.6 µmol) and cyclobutanecarbonyl chloride (23.8 µL, 208.6 µmol) at room temperature. After being stirred for 12 hr, the reaction mixture was quenched with sat. NH₄Cl aq. and diluted with AcOEt. The resulting mixture was extracted with AcOEt and washed with brine. The organic phase was dried over Na₂SO₄, concentrated under the reduced pressure. The residue was purified by PTLC (hexane : AcOEt=1 : 2) to give cyclobutanecarbonyl derivative 9 (5.2 mg, 17%) as a yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 7.32 (d, J=1.6 Hz, 1H), 6.98 (d, J=1.6 Hz, 1H), 6.94 (dd, J=9.9, 3.9 Hz, 1H), 6.70 (dd, J=9.9, 1.6 Hz, 1H), 5.17 (s, 2H), 4.07 (dd, J=3.9, 1.6 Hz, 1H), 3.81 (s, 3H), 3.56–3.49 (m, 1H), 3.27–3.21 (m, 1H), 2.65–2.54 (m, 2H), 2.38–2.21 (m, 6H), 2.10–1.89 (m, 4H); and ¹³C-NMR (125 MHz, CDCl₃) δ 175.0, 173.8, 173.7, 167.4, 157.7, 156.4, 143.4, 135.7 (2C), 126.3 (2C), 118.7, 114.6 (2C), 64.4, 57.5, 56.6, 53.0, 38.3, 38.0, 29.8, 25.4 (2C), 25.2, 18.6 (2C). HRMS (ESI+) m/z calcd for C₂₆H₂₅O₉ (M+H⁺): 481.1499, found: 481.1488.

13. Cyclopentanecarbonyl derivative 10

To a stirred solution of 1 (20.0 mg, 63.2 µmol) in DCM (632 µL, 0.1 M) were added Et₃N (29.1 µL, 208.6 µmol) and cyclobutanecarbonyl chloride (25.4 µL, 208.6 µmol) at room temperature. After being stirred for 12 hr, the reaction mixture was quenched with sat. NH₄Cl aq. and diluted with AcOEt. The resulting mixture was extracted with AcOEt and washed with brine. The organic phase was dried over Na₂SO₄, concentrated under the reduced pressure. The residue was purified by PTLC (hexane : AcOEt=1 : 2) to give cyclobutanecarbonyl derivative 10 (12.0 mg, 37%) as a yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 7.32 (s, 1H), 7.00 (s, 1H), 6.94 (dd, J=9.9, 1.3 Hz, 1H), 6.71 (dd, J=9.9, 1.3 Hz, 1H), 5.16 (s, 2H), 4.07 (dd, J=3.6, 1.8 Hz, 1H), 3.80 (s, 3H), 3.14 (quin, J=7.9 Hz, 1H), 2.84 (quin, J=7.9 Hz, 1H), 2.14–2.04 (m, 4H), 1.98–1.91 (m, 3H), 1.85–1.58 (m, 9H); and ¹³C-NMR (125 MHz, CDCl₃) δ 176.4, 175.1, 173.8, 167.4, 157.7, 156.4, 149.9, 143.5, 135.7, 126.3, 118.7, 116.5, 115.8, 114.5, 64.4, 57.5, 56.6, 53.0, 44.0, 43.8, 30.2, 30.1, 29.8 (2C), 26.0 (2C), 25.9 (2C). HRMS (ESI-) m/z calcd for C₂₈H₂₇O₉ (M−H)⁻: 507.1655, found: 507.1633.

14. Benzoyl derivative 11

A reaction of 1 (20.0 mg, 63.2 µmol) with BzCl (32 µL, 270 µmol) in pyridine (200 µL, 2.43 mmol), followed by PTLC purification gave benzoyl derivative 12 (5.1 mg, 15%) as a white solid. ¹H-NMR (600 MHz, CDCl₃) δ 8.22 (dd, J=7.9, 1.2 Hz, 2H), 8.12–8.11 (m, 2H), 7.65–7.63 (m, 1H), 7.62–7.59 (m, 1H), 7.51 (t, J=7.9 Hz, 2H), 7.50–7.47 (m, 3H), 7.24 (d, J=1.2 Hz, 1H), 6.93 (dd, J=9.6, 3.6 Hz, 1H), 6.72 (dd, J=9.6, 1.2 Hz, 1H), 5.48 (s, 2H), 4.05 (dd, J=3.6, 1.2 Hz, 1H), 3.65 (s, 3H); and ¹³C-NMR (150 MHz, CDCl₃) δ 173.7, 167.3, 166.2, 165.3, 157.7, 156.4, 149.9, 143.3, 135.7, 133.7 (2C), 130.5 (2C), 129.9 (3C), 128.7 (2C), 128.6 (3C), 126.2, 118.8, 116.5, 116.0, 114.8, 65.0, 57.3, 56.6, 53.0. HRMS (ESI+) m/z calcd for C₃₀H₂₀O₉Na (M+Na⁺): 547.1011, found: 547.1004.

15. Cyclohexanecarbonyl derivative 12

To a stirred solution of 1 (20.0 mg, 63.2 µmol) in DCM (632 µL, 0.1 M) were added Et₃N (29.1 µL, 208.6 µmol) and cyclobutanecarbonyl chloride (27.9 µL, 208.6 µmol) at room temperature. After being stirred for 12 hr, the reaction mixture was quenched with sat. NH₄Cl aq. and diluted with AcOEt. The resulting mixture was extracted with AcOEt and washed with brine. The organic phase was dried over Na₂SO₄, concentrated under the reduced pressure. The residue was purified by PTLC (hexane : AcOEt=1 : 2) to give cyclobutanecarbonyl derivative 11 (24.0 mg, 89%) as a yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 12.18 (s, 1H), 7.01 (dd, J=9.9, 3.8 Hz, 1H), 6.87 (s, 1H), 6.79 (s, 1H), 6.74 (dd, J=9.9, 1.2 Hz, 1H), 5.13 (s, 2H), 4.11 (dd, J=3.8, 1.2 Hz, 1H), 3.88 (s, 3H), 2.43–2.37 (m, 1H), 1.98–1.21 (m, 10H); and ¹³C-NMR (125 MHz, CDCl₃) δ 180.4, 175.6, 167.2, 160.9, 159.6, 155.4, 145.8, 136.7, 126.3, 113.5, 110.6, 109.8, 105.6, 64.7, 57.3, 56.6, 53.3, 43.2, 29.1 (2C), 25.8, 25.5 (2C). HRMS (ESI+) m/z calcd for C₂₃H₂₃O₈ (M+H⁺): 427.1393.

16. Evaluation of antifungal activity

The method was performed according to the protocols in a previous study.⁶⁾ The following microorganisms were used for the antifungal activity evaluation on a paper disk method: Pyricularia oryzae APU15-60A (quinone outside inhibitors-sensitive [QoIS]), P. oryzae APU15-63A (quinone outside inhibitors-resistant [QoIR]), Botrytis cinerea MAFF-306820, Leptosphaeria maculans MAFF-726728, Rizoctonia solani MAFF-237699, and Colletotrichum gloeosporioide MAFF-237219 strains.^7–11) The QoIS and QoIR strains were collected from the paddy field of Akita Prefecture in 2015. Compound 1 was prepared as 10 mg/mL DMSO solution. Each paper disk (diameter 6 mm, thin type, Advantec, Tokyo, Japan) impregnated with 10, 3, 1, 0.3, 0.1, 0.03, and 0.01 µg of compounds were put into an agar plate. The B. cinerea MAFF-306820, L. maculans MAFF-726728, C. gloeosporioide MAFF-237219, R. solani MAFF-237699, QoIS, and QoIR strains were each grown on PSA solid medium (0.5% potato peptone, 2% sucrose, 0.05% L-glutamic acid Na, and 2% agar). Sterile filter disks impregnated with each compound solution were placed on the agar plate, and the plates were incubated at 25°C for 3 days. After incubation, the diameter (mm) of the inhibition zone was measured.

Results and discussion

Our investigation commenced with the acquisition of 1 by fermentation of the laeA gene introduced mutant strain, KTF-0058, which was established by us.⁵⁾ This laeA gene introduction strategy enabled us to obtain 1 in significantly greater quantities (180 mg/kg). With sufficient quantities of 1 for derivatization in hand, we tackled the synthesis of derivatives as shown in Scheme 1 and Scheme 2. As a first step, acylation of 1 with acetic anhydride and pyridine provided acetyl derivative 2 in 26% yield. In this transformation, undesired side reactions such as aromatization occurred simultaneously, resulting in the low yield of 2. This result made us realize the instability of the epoxy moiety even under relatively mild conditions, which would be attributed to an energetically less favored dearomatized xanthone structure. Therefore, we tried hydrogenation of the olefin moiety with Pd/C and H₂ to prevent the undesired aromatization reactions. However, unexpected deoxygenative aromatization proceeded to give 3 in 43% yield, suggesting that the epoxy cyclohexadiene ring would be unstable also under the hydrogenation conditions. From this result, we decided to derivatize the 3-hydroxybenzyl alcohol moiety. Selective methylation of the phenol moiety under K₂CO₃ and MeI-used conditions furnished methyl derivative 4 in 44% yield. Subsequently, oxidation of the primary alcohol utilizing MnO₂ gave aldehyde derivative 5 in 34% yield.

Scheme 1. Synthesis of derivatives-2–5.

Scheme 2. Synthesis of derivatives-6–12.

6; R₂=Ethyl (22% yield), 7; R₂=Propyl (22% yield), 8; R₂=Cyclopropyl (95% yield), 9; R₂=Cyclobutyl (17% yield), 10; R₂=Cyclopentanyl (34.0% yield), 11; R₂=Phenyl (32.0% yield), 12; 89% yield.

In order to verify the structure–activity relationship of 1, we started by evaluating the antifungal activity of four derivatives against pathogenic plant fungi (Table 1). Compound 3 and 4 lost the activity, whereas 2 retained the efficacy against the six different fungal strains. It is noteworthy that 5 showed more potent fungicidal activity compared to the parent compound, approximately 2–30 times higher potency. Considering the antifungal activity of 1 and 3, the epoxy moiety of 1 appears to be a very important functional group. Considering the labile epoxy moiety and the activity of those derivatives, the oxirane might bind covalently to the molecular target(s), resulting in showing corresponding fungicidal activity. In contrast to 1, methyl derivative 4 turned out to be not active against all 6 fungal strains due to the extremely low solubility of the compound in water and organic solvents such as DMSO. On the other hand, since bisacetylated derivative 2 showed a respectable level of the activity, the electron deficient chromone ring somewhat increase the activity. This is probably due to the electron density of the chromone moiety affects the electrophilicity of the oxirane. In these regards, further investigations are needed to identify the molecular target(s) and prove our hypothesis.

Table 1. Antifungal activity of 2–5 against phytopathogenic fungi

Strain	Compound	Inhibition zone diameter (mm)
		µg/disk
		10	3	1	0.3	0.1	0.03
Pyricularia oryzae APU15-60A (QoIS)	1	24.19	16.62	12.46	8.80	—	—
	2	23.13	17.62	12.11	8.73	—	—
	3	10.68	—	—	—	—	—
	4	—	—	—	—	—	—
	5	19.37	16.43	12.81	10.43	8.38	—
	AMPH	N.T.	N.T.	12.46	N.T.	10.40	N.T.
	KXM	N.T.	N.T.	29.14	N.T.	N.T.	N.T.
Colletotrichum gloeosporioides MAFF-237219	1	8.64	7.73	—	—	—	—
	2	7.98	—	—	—	—	—
	3	—	—	—	—	—	—
	4	—	—	—	—	—	—
	5	10.52	8.99	7.48	7.06	—	—
	AMPH	N.T.	N.T.	12.28	N.T.	N.T.	N.T.
Leptosphaeria maculans MAFF-726728	1	18.70	13.89	9.67	—	—	—
	2	12.05	8.87	—	—	—	—
	3	—	—	—	—	—	—
	4	—	—	—	—	—	—
	5	14.03	12.35	9.57	8.49	—	—
	AMPH	N.T.	N.T.	12.45	N.T.	N.T.	N.T.
Pyricularia oryzae APU15-63A (QoIR)	1	20.54	14.97	12.29	8.17	—	—
	2	27.82	20.63	13.27	9.43	—	—
	3	—	—	—	—	—	—
	4	—	—	—	—	—	—
	5	19.50	18.90	13.28	11.49	9.29	—
	AMPH	N.T.	N.T.	14.81	N.T.	N.T.	N.T.
	KXM	N.T.	N.T.	—	N.T.	N.T.	N.T.
Botrytis cinerea MAFF-306820	1	15.58	—	—	—	—	—
	2	15.29	2.55	—	—	—	—
	3	—	—	—	—	—	—
	4	—	—	—	—	—	—
	5	14.01	11.52	10.40	9.28	—	—
	AMPH	N.T.	N.T.	10.25	N.T.	N.T.	N.T.
Rizoctonia solani MAFF-237699	1	11.06	8.56	—	—	—	—
	2	12.11	—	—	—	—	—
	3	—	—	—	—	—	—
	4	—	—	—	—	—	—
	5	10.13	9.92	7.67	—	—	—
	AMPH	N.T.	N.T.	7.73	N.T.	N.T.	N.T.

AMPH, Amphotericin B; KXM, Kresoxim-methyl. N.T., Not tested; —, no inhibition. Pyricularia oryzae APU15-60A, Susceptible to QoI; Pyricularia oryzae APU15-63A, Resistance to QoI.

Next, we sought to synthesize more derivatives based on 2 and 5 to gain more insight into the structure–activity relationship. Considering the instability of the epoxy moiety in 5, we chose the Wittig reaction using isolated ylid reagents (Scheme 2A), which do not need any additives such as bases. Unfortunately, aromatized products were obtained instead of the desired homologated products, suggesting that the epoxy moiety is more reactive than the aldehyde moiety even under the mild nucleophilic conditions. With the purpose of increasing the reactivity of the aldehyde moiety, we raised reaction temperature, which was also unfruitful. This situation prompted us to conduct acylation using several acid chloride or anhydride reagents for the synthesis of bisacyl derivatives such as 2. We synthesized six kinds of acyclic and cyclic bisacyl derivatives (Scheme 2B). In the case of cyclohexanecarbonyl chloride-used reaction, the mono acylated product 12 was obtained even with excess amount of the reagent. We evaluated the antifungal activity of 6–12 (Table 2). For QoI-sensitive (QoIS) (P. oryzae APU15-60A), QoI-resistant (QoIR) (P. oryza APU15-63A), and Colletotrichum gloeosporioide MAFF-237219, all compounds except 10 demonstrated antifungal activities comparable to the parent compound, which suggested that several acyl groups are tolerated for the chemical modifications. Intriguingly, 7 exhibited great antifungal activity against QoIS, QoIR, and Colletotrichum gloeosporioide but not against the other strains, Botrytis cinerea MAFF-306820, Leptosphaeria maculans MAFF-726728, and Rizoctonia solani MAFF-237699, which implies that the one or two carbons length of the alkyl chain in the acyclic acyl type would be appropriate for broad antifungal activity. In case of the efficacy against Botrytis cinerea MAFF-306820, Leptosphaeria maculans MAFF-726728 strains, 6, 8, and 12 displayed satisfactory biological activities. Compounds 6, 8, and 12 demonstrate broad antifungal activity against all six strains, whereases 9 did not show antifungal activity against Botrytis cinerea MAFF-306820 and Leptosphaeria maculans MAFF-726728, suggesting that the suitable ring size of the acyl group would be three and six. However, cyclohexanecarbonyl derivative 12 is a mono acyl type compound and the number of acyl groups might influence its antifungal activity. Additionally, cyclopentenoyl derivative 10 did not show any antifungal activities against the 6 strains. This anomalous result does not fit in with the tendency of the other results.

Table 2. Antifungal activity of 6–12 against phytopathogenic fungi

Strain	Compound	Inhibition zone diameter (mm)
		µg/disk
		10	3	1	0.3	0.1	0.03
Pyricularia oryzae APU15-60A (QoIS)	1	23.12	17.40	12.04	10.29	7.12	—
	6	17.24	13.20	10.51	8.46	—	—
	7	7.95	11.33	11.20	7.79	—	—
	8	26.30	17.50	14.86	14.39	11.28	—
	9	16.45	16.16	11.03	10.84	—	—
	10	—	—	—	—	—	—
	11	11.45	9.07	7.58	7.01	—	—
	12	20.75	14.72	12.31	11.46	—	—
	AMPH	N.T.	N.T.	17.82	N.T.	N.T.	N.T.
	KXM	N.T.	N.T.	20.32	N.T.	N.T.	N.T.
Colletotrichum gloeosporioides MAFF-237219	1	9.40	8.57	7.92	—	—	—
	6	7.98	6.90	6.75	—	—	—
	7	6.96	7.18	6.94	6.76	—	—
	8	10.92	8.31	8.18	—	—	—
	9	8.83	8.54	6.94	—	—	—
	10	—	—	—	—	—	—
	11	6.99	6.52	6.09	—	—	—
	12	7.47	7.58	7.30	6.92	—	—
	AMPH	N.T.	N.T.	9.90	N.T.	N.T.	N.T.
Leptosphaeria maculans MAFF-726728	1	18.50	13.47	8.40	—	—	—
	6	8.87	7.82	7.30	—	—	—
	7	—	—	—	—	—	—
	8	9.76	8.60	8.10	—	—	—
	9	—	—	—	—	—	—
	10	—	—	—	—	—	—
	11	7.32	—	—	—	—	—
	12	8.44	7.40	6.76	—	—	—
	AMPH	N.T.	N.T.	13.77	N.T.	N.T.	N.T.
Pyricularia oryzae APU15-63A (QoIR)	1	14.49	8.39	7.37	6.99	—	—
	6	11.72	10.52	7.56	6.94	—	—
	7	6.92	7.82	7.59	6.46	—	—
	8	14.22	11.32	7.53	—	—	—
	9	7.30	7.04	—	—	—	—
	10	—	—	—	—	—	—
	11	8.05	7.33	6.58	6.12	—	—
	12	8.02	8.02	7.71	6.88	8.40	—
	AMPH	N.T.	N.T.	9.87	N.T.	N.T.	N.T.
	KXM	N.T.	N.T.	—	N.T.	N.T.	N.T.
Botrytis cinerea MAFF-306820	1	12.36	10.80	9.63	—	—	—
	6	9.32	8.39	—	—	—	—
	7	—	—	—	—	—	—
	8	10.88	9.58	8.40	—	—	—
	9	—	—	—	—	—	—
	10	—	—	—	—	—	—
	11	7.75	—	—	—	—	—
	12	9.49	9.46	7.69	7.22	—	—
	AMPH	N.T.	N.T.	9.30	N.T.	N.T.	N.T.
Rizoctonia solani MAFF-237699	1	18.30	11.36	—	—	—	—
	6	12.11	8.90	7.05	—	—	—
	7	—	—	—	—	—	—
	8	13.77	11.36	—	—	—	—
	9	12.36	9.01	—	—	—	—
	10	—	—	—	—	—	—
	11	8.71	—	—	—	—	—
	12	8.98	7.71	—	—	—	—
	AMPH	N.T.	N.T.	11.47	N.T.	N.T.	N.T.

AMPH, Amphotericin B; KXM, Kresoxim-methyl. N.T., Not tested; —, no inhibition. Pyricularia oryzae APU15-60A, Susceptible to QoI; Pyricularia oryzae APU15-63A, Resistance to QoI.

In conclusion, we have previously identified 1 as a potent antifungal compound against broad pathogenic plant fungal species after utilizing the laeA gene introduction strategy which allowed for sufficient supply of 1 for the structure–activity relationship study. To this end, we synthesized 11 derivatives and evaluated their antifungal activity. Among them, 5 showed more potent activity compared to 1 and acyl derivatives 2, 6, 8, 12 maintained comparable activity. From this investigation, it is found that 5 would be one of the most promising derivatives and the derivatization with acyl groups would be a useful approach to create chemical probes for target identification experiments. We are planning to conduct in vivo tests using 1 and 5 against pathogenic plant fungi and synthesize chemical probes from 1 via acylation for the target identification.

Acknowledgements

We are grateful to Distinguished Emeritus Professor S. Ōmura (Kitasato University, Tokyo, Japan) for his helpful support and valuable suggestions. Further, we are grateful to Dr. K. Nagai, Ms. R. Seki, and Ms. N. Sato, School of Pharmacy, Kitasato University for the measurements of mass and NMR spectra.

Conflicts interests

The authors declare no competing financial interest.

Author’s contributions

Aoi. K., S. H., Atsushi. K., P.W., and Y. A. conceptualized and directed the project. H. K., T. T., A. S., Y. A., T. T., and K. I. constructed and maintained Aspergillus sp. S. H., H. K., T. T., and A. S., isolated MS-347a. Aoi. K. and Atsushi. K. synthesized derivatives. S. H., Aoi. K., A. N., M. H., S. K., T. T., T. C., T. U., and S. F. performed the biological experiments. Aoi. K., S. H., Atsushi K., P. W., and Y. A. drafted the manuscript with the assistance from all co-authors. All authors approved the final manuscript. Aoi. K., S. H., and Atsushi K. contributed equally.

Data availability statement

The data underlying this article are available in the article and in its online supplementary material.

Funding

This study was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) and the Japan Agency for Medical Research & Development (AMED) under grant number JP21am0101096.

Electronic supplementary materials

The online version of this article contains supplementary material, which is available at https://www.jstage.jst.go.jp/browse/jpestics/.

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