Natural Product-Based Fungicides: Design, Synthesis, and Antifungal Activity of Rhein Derivatives Against Phytopathogenic Fungi

Abstract

With the long-term use of certain types of traditional chemical fungicides, phytopathogen resistance and environmental pollution have made the application of these fungicides face unprecedented challenges. Therefore, using the low toxicity and structural diversity of natural product analogs to develop alternatives has become an important tactic to improve control efficiency and reduce pathogen resistance, as well as environmental risks. In this study, thirty-eight rhein derivatives were synthesized after our continuous efforts aiming to discover new anthraquinone-based antifungal agents. Their structures were characterized by ¹H-NMR, ¹³C-NMR and high-resolution mass spectrometry. The antifungal activities of rhein derivatives were first evaluated against four phytopathogenic fungi. The bioassay results indicated that most derivatives exhibited good antifungal activity against Rhizoctonia solani at 0.5 mM in vitro. Compounds 3e, 3j, 4a, 9d and 10f showed potent activities against R. solani, with inhibition rates over 50% at a low concentration of 0.2 mM in vitro. In particular, compound 10a strongly inhibited the growth of Sclerotinia sclerotiorum, Fusarium graminearum and P. capsica, with EC₅₀ values of 0.079 mM, 0.082 mM and 0.134 mM, respectively, which are comparable to the commercial biofungicide phenazine-1-carboxylic acid (PCA). An in vivo study showed that 10a presented excellent curative and protective activities (92.1% and 91.1%, 0.2 mM) against wheat powdery mildew. The phytotoxicity results indicated that rhein amino acid derivatives could significantly eliminate phytotoxicity to rice and rape and could be safely used in these two crops. The resistance development assay indicated that these rhein derivatives could effectively avoid the risk of resistance development in these two strains of fungi, R. solani and S. sclerotiorum. In conclusion, rhein derivatives can be used for the development of potential agricultural fungicides.

1. Introduction

Phytopathogenic fungal infections of crops can lead to significant declines in food production and have become a serious constraint to ensuring global crop production and food security [1,2]. The application of chemical fungicides for the control of phytopathogenic fungi remains the most extensively utilized and effective method to date [3,4]. With the long-term use of certain types of traditional chemical fungicides, phytopathogen resistance and environmental pollution have made the application of these fungicides face unprecedented challenges [5,6,7]. Therefore, using the low toxicity and structural diversity of natural product analogs to develop alternatives has become an important tactic to improve control efficiency and reduce pathogen resistance, as well as environmental risks [8,9,10].

Rhein (Figure 1), possessing a typical anthraquinone scaffold, is an important natural product, often isolated from Rheum palmatum, Rheum tanguticum and other traditional medicinal plants [11,12]. Studies have shown that rhein and its derivatives have a wide range of biological properties, such as anti-tumorigenic [13,14], antiviral [15], anti-oxidative [16], anti-inflammatory [17], antibacterial and insecticidal activities [18,19]. Currently, many clinical drugs and highly active molecules are modified from natural structures. For example, diacerein (Figure 1), a 1,8-dihydroxyacetylated derivative of rhein, has been developed as a drug for the treatment of osteoarthritis patients [20]. However, there have been few published studies on the antifungal activities of rhein and its derivatives against plant pathogens. Therefore, the development of new anthraquinone-based antifungal agents by the modification of rhein attracts our great interest, and it is considered a promising alternative to alleviate the pathogen resistance problems caused by the extensive use of other traditional fungicides.

More recently, we have prepared a series of rhein derivatives by structural modification of rhein at its 1- and 8-position hydroxyl groups and carboxyl group and evaluated their antifungal activities against six common plant pathogenic fungi: Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum, Fusarium pseudograminearum, Bipolaris maydis and Phytophthora capsica [21,22]. The results of the bioassay showed that rhein derivatives (Figure 1) could exhibit remarkably higher antifungal activity against R. solani compared to the parent compound rhein. In continuation of our efforts aimed at discovering new anthraquinone-based antifungal agents, in this paper, thirty-eight rhein derivatives were synthesized and evaluated for their antifungal activities against four phytopathogenic fungi (R. solani, S. sclerotiorum, F. graminearum and P. capsici) in vitro. An in vivo study against wheat powdery mildew was performed to identify the prospective application of highly active compounds as agricultural fungicides. Meanwhile, the phytotoxicity of the effective compounds was evaluated with rice Oryza sativa L. and Brassica napus L. In addition, the preliminary resistance developments of these rhein derivatives were explored to investigate their potential resistance risk to pathogenic fungi.

2. Materials and Methods

2.1. Chemicals and Instruments

The reagents and chemicals used are all commercially available and analytical or chemical grade. The melting points of all synthesized compounds were determined on a WRR melting point apparatus (Shanghai Jingke Industrial Co., Ltd., China) and are uncorrected. ¹H NMR (400 MHz) and ¹³C NMR spectra (101 MHz) were recorded on a Bruker Avance III HD 500 MHz NMR Spectrometer (Bruker Co., Switzerland) with CDCl₃ or DMSO-d₆ as a solution, as well as tetramethylsilane (TMS) as an internal standard. High-resolution mass spectrometry was performed on a Bruker APEX IV Fourier-transform mass spectrometer (Bruker Co., Switzerland). All reactions were monitored by TCL with silica gel aluminum sheets F254 (Qingdao Marine Chemical Ltd., China) and viewed under UV light at 254 nm.

2.2. Chemical Synthesis

The synthetic experiments were carried out from June 2022 to July 2023 at the Institute of Pesticide Research of Yangtze University. All anhydrous solvents mentioned are dried by standard technology before use. The synthesis of rhein ester and amide derivatives, compounds 3a–3l and 4a–4l, was the same as the method we reported previously [21]. Rhein ester and amide derivatives were prepared by the reaction of rhein chloride 2 with corresponding alcohols or amines, as shown in Scheme 1. The rhein amino acid derivatives 8a–8h and 9a–9f were obtained by the reaction formulas and conditions in Scheme 2. The specific synthesis method and experimental operation are described as follows.

2.2.1. Synthesis of 4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carbonyl Chloride (2)

Rhein 1 (10 mmol), 30 mL of anhydrous dichloromethane, and 2~3 drops of DMF as a catalyst were added in order into a single-port bottle for stirring. Then, the thionyl chloride (15 mmol) was slowly added to the flask and heated to reflux until the solid disappeared completely. The reflux reaction continued for 8 h, and the solvent was removed on the rotary evaporator to obtain compound 2 for the next step.

2.2.2. General Procedures for the Synthesis of Compounds 3a–3l

Compound 2 (10 mmol) dissolved in 30 mL of dry CH₂Cl₂ was added dropwise to a solution of the corresponding hydroxyl compounds (11 mmol) in 50 mL of CH₂Cl₂. The mixture was stirred at room temperature until the reaction was completed in 2~3 h (monitored by TLC). Then, the organic layer was washed with 0.1 mol/L aq. HCl (20 mL) and dried with anhydrous Na₂SO₄, and the solvent was removed under vacuum. The residue was purified by recrystallizing from a solution of CH₃OH to obtain pure compounds 3a–3l as yellow solids. Spectral data for representative target compounds 3a and 3b are given below; compounds 3c–3l are available in the Supporting Information.

Propyl 4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylate (3a)

Yellow solid; yield: 95.8%; m.p, 151~154 °C. ¹H NMR (400 MHz, CDCl₃) δ 12.05 (s, 1H), 11.99 (s, 1H), 8.43 (d, J = 1.6 Hz, 1H), 7.95 (d, J = 1.6 Hz, 1H), 7.88 (dd, J = 7.6, J = 1.2 Hz, 1H), 7.77–7.71 (m, 1H), 7.34 (dd, J = 8.4, J = 1.2 Hz, 1H), 4.35 (t, J = 6.8 Hz, 2H), 1.90–1.78 (m, 2H), 1.06 (t, J = 7.6 Hz, 3H). ¹³C-NMR (101 MHz, CDCl₃) δ 192.87, 181.03, 164.48, 162.82, 162.43, 138.29, 137.76, 133.90, 133.53, 125.33, 124.90, 120.41, 120.28, 118.21, 115.87, 67.67, 22.01, 10.48. HRMS calcd for C₁₈H₁₄O₆ [M + H]⁻: 325.0717, found 325.0711.

Isopropyl 4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylate (3b)

Yellow solid; yield: 96.1%; m.p, 177~179 °C. ¹H NMR (400 MHz, CDCl₃) δ 12.05 (s, 1H), 11.99 (s, 1H), 8.43 (d, J = 1.6 Hz, 1H), 7.95 (d, J = 1.6 Hz, 1H), 7.88 (dd, J = 7.6, J = 1.2 Hz, 1H), 7.77–7.71 (m, 1H), 7.34 (dd, J = 8.4, J = 1.2 Hz, 1H), 4.35 (t, J = 6.8 Hz, 2H), 1.90–1.78 (m, 2H), 1.06 (t, J = 7.6 Hz, 3H). ¹³C-NMR (101 MHz, CDCl₃) δ 192.88, 181.11, 163.89, 162.81, 16–2.41, 138.73, 137.73, 133.84, 133.56, 125.35, 124.89, 120.40, 120.29, 118.13, 115.89, 69.95, 21.87. HRMS calcd for C₁₈H₁₄O₆ [M + H]⁻: 325.0718, found 325.0710.

2.2.3. General Procedures for the Synthesis of Compounds 4a–4l

The preparation of compounds 4a–4l was similar to that of compounds 3a–3l. Compound 2 (10 mmol) dissolved in 30 mL of dry CH₂Cl₂ was added dropwise to a solution of the corresponding amine-group compounds (22 mmol) in 50 mL of dry CH₂Cl₂. The reaction was stirred for 2~3 h until the reaction was completed. Then, the organic phase was extracted with 0.1 mol/L aq. HCl (20 mL), dried with anhydrous Na₂SO₄ and concentrated under vacuum. The crude product was purified by recrystallization from the solution in CH₃OH to obtain pure compounds 4a–4l as yellow solids. Spectral data for representative target compounds 4a and 4b are given below; compounds 4c–4l are available in the Supporting Information.

N-Ethyl-4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (4a)

Yellow solid; yield: 97.5%; m.p, 283~284 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.90 (s, 2H), 8.92 (t, J = 5.2 Hz, 1H), 8.13 (d, J = 1.6 Hz, 1H), 7.83 (t, J = 8.0 Hz, 1H), 7.76 (d, J = 1.6 Hz, 2H), 7.41 (d, J = 7.6 Hz, 1H), 3.32 (d, J = 1.6 Hz, 2H), 1.16 (t, J = 7.2 Hz, 3H). ¹³C-NMR (101 MHz, DMSO-d₆) δ 192.00, 181.64, 164.15, 161.86, 161.64, 142.32, 138.04, 134.06, 133.81, 125.01, 122.84, 119.93, 118.03, 117.99, 116.58, 34.86, 14.99. HRMS calcd for C₁₇H₁₃NO₅ [M + H]⁻: 310.0721, found 310.0726.

4,5-Dihydroxy-N-isopropyl-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (4b)

Yellow solid; yield: 95.0%; m.p, >280 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.90 (s, 2H), 8.70 (d, J = 7.6 Hz, 1H), 8.13 (s, 1H), 7.83 (t, J = 8.0 Hz, 1H), 7.80–7.71 (m, 2H), 7.41 (d, J = 8.4 Hz, 1H), 4.14–4.08 (m, 1H), 1.20 (d, J = 6.4 Hz, 6H). ¹³C-NMR (101 MHz, DMSO-d₆) δ 192.02, 181.66, 163.56, 161.86, 161.61, 142.53, 138.05, 134.00, 133.82, 125.01, 122.96, 119.93, 118.21, 117.94, 116.58, 41.95, 22.61. HRMS calcd for C₁₈H₁₅NO₅ [M + H]⁺: 326.1023, found 326.1022.

2.2.4. Synthesis of Methyl 4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylate (5)

The preparation of compound 5 followed the procedure for compounds 3a–3l described above. Yellow solid; yield: 95.3%; m.p, 177~178 °C. ¹H NMR (400 MHz, CDCl₃) δ 12.03 (s, 1H), 11.97 (s, 1H), 8.42 (d, J = 1.6 Hz, 1H), 7.94 (d, J = 1.6 Hz, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 4.00 (s, 3H). ¹³C-NMR (101 MHz, CDCl₃) δ 192.84, 180.98, 164.91, 162.82, 162.42, 137.84, 137.78, 133.91, 133.48, 125.37, 124.93, 120.43, 120.29, 118.27, 115.85, 52.96. HRMS calcd for C₁₆H₁₀O₆ [M + H]⁻: 297.0405, found 297.0406.

2.2.5. Synthesis of Methyl 4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylate (6)

The solution of compound 5 (10 mmol) and NaH (30 mmol) in 100 mL DMF was stirred at 0 °C for 20 min. Then, the CH₃I (40 mmol) was added dropwise. The mixture was refluxed for 3 h. The DMF was evaporated under vacuum, and the remaining crude product was purified by column chromatography to obtain pure compound 6. Yellow solid; yield: 83.7%; m.p, 207~208 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.46 (d, J = 1.2 Hz, 1H), 7.94 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 4.07 (s, 3H), 4.02 (s, 3H), 4.00 (s, 3H). ¹³C-NMR (101 MHz, DMSO-d₆) δ 183.31, 182.43, 165.55, 159.58, 134.96, 134.79, 134.67, 134.29, 126.79, 123.93, 119.91, 119.12, 118.23, 118.21, 56.84, 56.58, 52.81, 29.71. HRMS calcd for C₁₈H₁₄NO₆ [M + H]⁺: 327.0863, found 327.0867.

2.2.6. Synthesis of 4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic Acid (7)

NaOH (12 mmol) dissolved in 30 mL of H₂O was added dropwise to a solution of compound 6 in 30 mL of CH₃OH and stirred for 2 h at room temperature. Then, 1N HCl (15 mL) was added to obtain a solid. The solid was filtered and then purified by recrystallizing from the solution of CH₃OH to obtain pure compound 7. Yellow solid; yield: 86.0%; m.p, 277~278 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (d, J = 1.2 Hz, 1H), 7.91 (d, J = 1.2 Hz, 1H), 7.82–7.76 (m, 1H), 7.72 (dd, J = 7.6, 1.1 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 3.98 (s, 3H), 3.93 (s, 3H). ¹³C-NMR (400 MHz, DMSO-d₆) δ 183.13, 181.33, 166.37, 159.29, 159.24, 136.24, 134.97, 134.75, 134.45, 126.52, 123.84, 119.49, 119.00, 118.68, 118.60, 56.91, 56.80. HRMS calcd for C₁₇H₁₂NO₆ [M + H]⁺: 313.0707, found 313.0709.

2.2.7. Synthesis of 4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracene-2-carbonyl Chloride (8)

Thionyl chloride (15 mL) was added to compound 7 (10 mmol), and the mixture was refluxed for 1 h. Then, excess thionyl chloride was removed under vacuum. Crude product 8 was obtained and used in the subsequent reaction without further purification.

2.2.8. General Procedures for the Synthesis of Compounds 9a–9h

Compound 8 (10 mmol) dissolved in 30 mL of CH₂Cl₂ was added dropwise to a solution of the corresponding L-AA ester (10 mmol), with triethylamine (22 mmol) as the attaching acid agent, in 50 mL of CH₂Cl₂ at 0 °C. The mixture was refluxed for 2 h until the reaction was complete. Then, the solvent was evaporated under vacuum, and the crude product was purified by column chromatography to obtain pure compounds 9a–9h. Spectral data for representative target compounds 9a and 9b are given below; compounds 9c–9h are available in the Supporting Information.

(R)-Methyl 2-(4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxamido)propanoate (9a)

Yellow solid; yield: 88.5%; m.p, 264~265 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.09 (d, J = 1.6 Hz, 1H), 7.88 (d, J = 1.6 Hz, 1H), 7.85 (dd, J = 7.6, J = 1.2 Hz, 1H), 7.70–7.64 (m, 1H), 7.33 (d, J = 7.6 Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H), 4.82 (t, J = 7.2 Hz, 1H), 4.07 (s, 3H), 4.02 (s, 3H), 3.82 (s, 3H), 1.58 (d, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 183.51, 182.19, 173.16, 164.87, 160.02, 159.64, 138.40, 134.90, 134.56, 134.23, 125.95, 123.86, 119.10, 118.39, 117.35, 115.71, 56.83, 56.57, 52.73, 48.84, 18.44. HRMS calcd for C₂₁H₁₉NO₇ [M + H]⁺: 398.1234, found 398.1228.

(R)-Methyl 2-(1,8-Dimethoxy-9,10-dioxo-9,10-dihydroanthracene-6-carboxamido)-3-methylbutanoate (9b)

Yellow solid; yield: 85.1%; m.p, 205~207 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.09 (d, J = 1.6 Hz, 1H), 7.91–7.84 (m, 2H), 7.71–7.64 (m, 1H), 7.34 (d, J = 7.6 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 4.79 (dd, J = 8.4, J = 5.2 Hz, 1H), 4.07 (s, 3H), 4.02 (s, 3H), 3.81 (s, 3H), 2.37–2.26 (m, 1H), 1.04 (dd, J = 6.8, J = 4.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 183.51, 182.17, 172.21, 165.35, 160.03, 159.64, 138.58, 134.92, 134.57, 134.24, 125.96, 123.85, 119.09, 118.38, 117.45, 115.55, 57.90, 56.82, 56.58, 52.42, 31.63, 19.08, 18.12. HRMS calcd for C₂₃H₂₃NO₇ [M + H]⁺: 426.1547, found 426.1553.

2.2.9. General Procedures for the Synthesis of Compounds 10a–10f

The preparation of compounds 10a–10f followed the procedure for compounds 9a–9h described above. Spectral data for representative target compounds 10a and 10b are given below; compounds 10c–10f are available in the Supporting Information.

(S)-Ethyl 2-(4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxamido)propanoate (10a)

Yellow solid; yield: 92.5%; m.p, 240~241 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.12 (d, J = 1.6 Hz, 1H), 7.90 (d, J = 1.6 Hz, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 6.8 Hz, 1H), 4.82 (t, J = 7.2 Hz, 1H), 4.29 (q, J = 7.2 Hz, 2H), 4.08 (s, 3H), 4.04 (s, 3H), 1.59 (d, J = 7.2 Hz, 3H), 1.35 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 183.51, 182.26, 172.77, 164.87, 159.99, 159.62, 138.49, 134.89, 134.55, 134.24, 125.90, 123.85, 119.10, 118.35, 117.31, 115.75, 61.84, 56.84, 56.58, 48.92, 18.49, 14.20. HRMS calcd for C₂₂H₂₁NO₇ [M + H]⁺: 412.1391, found 412.1397.

(S)-Methyl 2-(4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxamido)-3-methylbutanoate (10b)

Yellow solid; yield: 88.3%; m.p, 221~222 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J = 1.6 Hz, 1H), 7.90 (d, J = 1.6 Hz, 1H), 7.87 (dd, J = 7.6, J = 0.8 Hz, 1H), 7.73–7.66 (m, 1H), 7.35 (d, J = 8.0 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 4.81 (dd, J = 8.4, J = 5.2 Hz, 1H), 4.08 (s, 3H), 4.04 (s, 3H), 3.83 (s, 3H), 2.39–2.29 (m, 1H), 1.06 (dd, J = 6.8, J = 3.2 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 192.83, 181.01, 164.40, 162.79, 162.40, 138.24, 137.75, 133.85, 133.49, 125.35, 124.90, 120.41, 120.27, 118.17, 115.85, 62.14, 14.26. HRMS calcd for C₂₃H₂₃NO₇ [M + H]⁺: 426.1547, found 426.1548.

2.3. In Vitro Antifungal Assay

The in vitro antifungal assay was carried out from July 2023 to September 2023 at the Plant Pathology Laboratory of Yangtze University. The in vitro antifungal activities of compounds against four phytopathogenic fungi (Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum and Phytophthora capsici) were investigated using the mycelium linear growth rate method. The methods follow those previously described [23,24]. First, each tested compound was dissolved in 1 mL DMSO, then diluted to 50 mL with 0.1% Tween 80 aqueous solution and uniformly mixed with sterile molten potato dextrose agar (PDA) to obtain final concentrations of 0.2 mM and 0.5 mM. The aforementioned molten PDA (15 mL) was placed into 7 cm diameter Petri plates, allowed to solidify and then inoculated with 6 mm diameter hyphal plugs taken from the growing margin of each test fungus on a PDA plate. The inoculated culture plates were incubated in a sterile biochemical incubator at 28 °C. Each treatment was repeated three times, and PDA containing a corresponding concentration of DMSO in 0.1% Tween aqueous solution served as the solvent control. Hyphal growth was observed every day, and the diameter of each culture was measured when the hyphae of the solvent control group grew to 3/4 of the area of the Petri plate. Based on the above results of in vitro antifungal activity, the more active compounds were selected to determine their median effective concentration (EC₅₀) according to the same method described above. The relative inhibition rate of the tested compounds against pathogenic fungi relative to the solvent control was calculated via the following formula: inhibition rate (%) = (CK − PT)/(CK − 6 mm) × 100, where CK and PT are average diameters of the mycelium of the solvent control and treatment, respectively.

2.4. In Vivo Bioassay Against Wheat Powdery Mildew

The in vivo antifungal assay was performed from September 2023 to November 2023 at the Plant Pathology Laboratory of Yangtze University. The curative and protective activities of compound 10a against wheat powdery mildew were determined by the reported method with some modifications [25,26]. The susceptible wheat variety, Chanceller, and the tested pathogenic fungi, Blumeria graminis isolated from a powdery mildew-infected wheat plant, were provided by the Institute for Plant Protection and Soil Sciences, Hubei Academy of Agricultural Sciences. The experimental concentration was 0.2 mM, and sterile water was used as a control. Physcion, a commercial biofungicide registered for wheat powdery mildew, served as the positive control. Under greenhouse conditions, wheat seeds were planted in plastic pots, with about 15 plants in each pot. After three weeks of growth, when the wheat grew to 2–3 leaves, they were used for antifungal activity experiments.

In the curative activity experiment, wheat seedlings were inoculated with powdery mildew pathogen first and then sprayed with test compounds after 24 h. In the protective activity experiment, the wheat seedlings were first sprayed with the tested compounds, respectively, and then inoculated with powdery mildew of wheat 24 h later. Each treatment was repeated 5 times. After 7 days of greenhouse culture, the disease index of the wheat seedlings was measured. The grading standard of wheat powdery mildew was carried out according to the method in the literature [27]. The following formula was used to calculate the disease index (CK or PT) and control efficiency (CK or PT): Disease index (CK or PT) = ∑(the number of leaves at each Grade × the corresponding Grade)/(the total number of leaves × the superlative Grade). The relative control efficacy I (%) for the curative and protection activities is calculated by the following equation: Relative control efficacy I (%) = (CK − PT)/CK × 100, where CK is the disease index of the negative control and PT is the disease index of the treatment group.

2.5. Phytotoxic Bioassay

The phytotoxic bioassay was performed from September 2023 to October 2023 at the Weed Science Laboratory of Yangtze University. The effective compounds were selected to evaluate their phytotoxicity to their corresponding host crops, rice (Oryza sativa L.) and rape (Brassica napus L.), because of their potent antifungal activities against R. solani and S. sclerotiorum. Seeds were sterilized with ethanol for 5 min, washed with running tap water for 2 h and then germinated in moist dishes at 22 °C in the dark. All tested compounds, weighed out in the appropriate amounts, were dissolved in acetone (1 mL) and diluted with aqueous 0.1% Tween 80 to a final concentration of 0.2 mM. After two days of germination, seeds were transferred into a 6 cm Petri plate, to which 5 mL of the above inhibitor solution had been added in advance. Then, the plates were sealed with parafilm and incubated with a photoperiod of 16 h light/8 h dark at 22 °C for 2 days. The root lengths and the plant height of rice and rape selected from each plate were measured, and the means were calculated. A corresponding concentration of acetone in 0.1% Tween aqueous solution served as a negative control. Each treatment was carried out with five replicates.

2.6. Resistance Development Assay

The resistance development assay was performed from October 2023 to November 2023 at the Plant Pathology Laboratory of Yangtze University. The antifungal resistance development assay of the active compounds 3e, 4a, 9d, 10a and 10f (inhibition rates >80.0% at 0.2 mM in

Table 1. Preliminary antifungal activity of the tested compounds against four phytopathogenic fungi at concentrations of 0.2 mM and 0.5 mM at 72 h.

Compd.	Average Inhibition Rate ±SD (%) (n = 3)
	R. solani		S. sclerotiorum		F. graminearum		P. capsici
	0.2 (mM)	0.5 (mM)	0.2 (mM)	0.5 (mM)	0.2 (mM)	0.5 (mM)	0.2 (mM)	0.5 (mM)
3a	41.3 ± 3.9	61.4 ± 2.6	14.9 ± 0.7	38.2 ± 0.6	15.5 ± 2.1	31.2 ± 1.2	<10	20.7 ± 0.5
3b	38.1 ± 1.1	56.5 ± 1.5	18.1 ± 0.8	35.6 ± 1.1	<10	11.7 ± 0.5	<10	21.6 ± 0.3
3c	34.8 ± 1.5	55.1 ± 2.1	<10	21.9 ± 0.7	<10	<10	<10	<10
3d	24.1 ± 1.6	38.7 ± 1.5	36.4 ± 1.9	59.7 ± 2.1	14.8 ± 3.8	31.7 ± 0.8	12.4 ± 1.9	34.4 ± 2.5
3e	66.9 ± 1.7	82.7 ± 2.3	50.3 ± 2.1	76.4 ± 3.1	35.2 ± 1.3	56.8 ± 1.7	41.6 ± 2.1	71.6 ± 3.8
3f	43.0 ± 2.2	63.4 ± 2.5	31.1 ± 1.0	55.3 ± 1.1	13.9 ± 1.6	36.6 ± 0.8	29.1 ± 2.3	54.1 ± 2.6
3g	13.6 ± 1.9	31.3 ± 1.2	35.7 ± 2.2	55.9 ± 1.1	<10	21.4 ± 1.1	29.8 ± 2.1	54.6 ± 3.8
3h	47.5 ± 1.7	69.7 ± 2.4	23.9 ± 0.7	47.1 ± 0.9	28.8 ± 1.3	46.9 ± 1.5	11.8 ± 1.5	37.8 ± 2.6
3i	37.4 ± 1.9	55.4 ± 1.9	19.5 ± 2.1	45.1 ± 1.7	10.1 ± 0.9	29.8 ± 1.3	19.2 ± 1.8	41.5 ± 3.5
3j	51.5 ± 4.9	73.9 ± 3.5	18.6 ± 1.6	34.9 ± 1.2	<10	19.5 ± 0.4	13.1 ± 0.8	21.5 ± 1.5
3k	42.1 ± 0.8	61.5 ± 1.3	<10	22.9 ± 1.1	<10	22.3 ± 0.7	<10	<10
3l	39.7 ± 2.5	58.2 ± 3.1	14.3 ± 2.0	29.1 ± 2.1	<10	19.9 ± 0.3	11.6 ± 1.8	25.1 ± 2.5
4a	58.4 ± 1.1	75.7 ± 1.3	62.4 ± 0.6	87.9 ± 1.2	12.8 ± 1.1	33.9 ± 1.7	56.4 ± 1.5	83.4 ± 3.7
4b	44.7 ± 1.6	61.8 ± 1.7	<10	18.3 ± 0.6	<10	27.0 ± 0.8	<10	<10
4c	32.7 ± 1.7	46.9 ± 1.8	21.4 ± 0.6	37.1 ± 1.1	16.9 ± 2.1	34.27%	11.3 ± 2.7	29.5 ± 2.1
4d	35.5 ± 0.8	51.5 ± 1.1	12.1 ± 1.0	32.4 ± 0.8	<10	20.8 ± 0.6	<10	10.9 ± 1.8
4e	40.7 ± 1.7	57.9 ± 2.2	<10	24.7 ± 0.4	<10	20.3 ± 0.8	<10	14.8 ± 2.1
4f	36.7 ± 2.0	53.7 ± 3.2	<10	28.4 ± 0.7	12.9 ± 1.2	28.9 ± 1.5	<10	17.7 ± 2.1
4g	37.7 ± 3.3	53.8 ± 2.1	<10	15.7 ± 0.8	<10	15.8 ± 0.7	<10	<10
4h	43.5 ± 2.7	60.5 ± 1.6	<10	25.1 ± 0.8	<10	<10	<10	<10
4i	37.3 ± 2.4	52.5 ± 1.7	<10	13.0 ± 0.2	13.3 ± 1.0	29.8 ± 1.6	<10	10.7 ± 0.9
4j	48.6 ± 0.6	64.3 ± 1.3	<10	25.2 ± 0.5	<10	14.3 ± 0.5	<10	<10
4k	32.0 ± 3.8	46.9 ± 4.1	11.7 ± 0.7	35.1 ± 1.2	<10	25.3 ± 0.6	<10	12.6 ± 1.5
4l	35.3 ± 2.1	53.6 ± 1.7	10.4 ± 1.3	31.3 ± 1.3	<10	17.4 ± 0.5	<10	10.2 ± 1.8
9a	<10	29.4 ± 1.8	<10	19.2 ± 0.8	<10	<10	<10	15.7 ± 2.5
9b	<10	31.5 ± 1.5	<10	19.4 ± 0.5	<10	<10	12.1 ± 0.7	37.7 ± 2.7
9c	20.2 ± 3.0	51.6 ± 3.5	<10	24.5 ± 0.8	<10	16.0 ± 1.2	11.8 ± 1.2	28.8 ± 1.7
9d	55.4 ± 2.2	83.2 ± 3.5	13.2 ± 0.5	41.5 ± 1.2	12.5 ± 0.6	37.6 ± 1.3	15.6 ± 1.1	35.3 ± 1.7
9e	19.8 ± 0.8	46.3 ± 2.5	34.6 ± 1.4	60.5 ± 1.5	11.1 ± 0.3	32.2 ± 1.1	22.7 ± 1.3	46.4 ± 1.9
9f	28.2 ± 2.1	57.5 ± 2.5	24.4 ± 0.5	47.4 ± 1.0	<10	22.1 ± 1.0	11.3 ± 3.0	31.3 ± 2.1
9g	24.8 ± 1.8	50.4 ± 2.3	16.7 ± 1.2	39.49 ± 1.5	<10	22.6 ± 0.8	<10	29.0 ± 1.7
9h	23.9 ± 0.9	52.0 ± 2.8	<10	28.6 ± 1.3	<10	13.23 ± 0.3	11.2 ± 0.6	30.3 ± 1.5
10a	38.8 ± 1.2	62.1 ± 2.3	86.5 ± 0.8	100 ± 0.0	84.2 ± 1.2	100 ± 0.0	62.7 ± 1.7	85.6 ± 2.3
10b	24.9 ± 0.5	51.8 ± 1.9	19.1 ± 1.0	45.1 ± 2.3	12.1 ± 0.5	31.26 ± 0.8	17.6 ± 1.1	36.1 ± 1.6
10c	13.5 ± 1.5	34.4 ± 1.1	<10	29.2 ± 1.1	<10	<10	<10	21.7 ± 1.8
10d	<10	28.7 ± 1.8	<10	16.1 ± 0.4	<10	15.2 ± 0.3	<10	15.6 ± 0.7
10e	27.1 ± 1.3	47.3 ± 1.8	<10	29.9 ± 1.1	12.1 ± 0.4	36.68 ± 0.6	14.7 ± 0.8	35.7 ± 2.2
10f	54.6 ± 2.1	80.0 ± 3.2	25.6 ± 0.5	55.2 ± 1.2	24.5 ± 0.9	57.5 ± 1.8	12.8 ± 0.4	31.7 ± 1.6
Rhein	28.6 ± 1.3	47.8 ± 1.8	27.0 ± 1.2	45.4 ± 3.1	16.1 ± 0.5	35.8 ± 1.5	35.4 ± 2.4	52.6 ± 3.4
PCA	86.3 ± 0.9	100 ± 0.0	84.6 ± 1.2	100 ± 0.0	83.2 ± 0.8	100 ± 0.0	79.8 ± 1.6	97.5 ± 1.3

) against two plant pathogenic fungi (R. solani, S. sclerotiorum) was investigated using the mycelium linear growth rate method [28]. Each fungus was cultured continuously for three generations, and the DMSO control without any test compound was mixed with PDA as the blank control of the first generation. In addition, the second-generation fungal strains were cultured with the first-generation fungal strains. By parity of reasoning, the next generation can be operated in the same way.

3. Results and Discussion

3.1. Chemistry

The general synthetic routes of target compounds are depicted in Scheme 1 and Scheme 2. Firstly, commercially available rhein (1) as a starting material was reacted with thionyl chloride (SOCl₂) and DMF in CH₂Cl₂ to give intermediate 2. Then, compound 2 was reacted with corresponding hydroxyl or amine-group compounds to afford the target compounds 3a–3l and 4a–4l. On the other hand, compound 5 was synthesized by reacting starting material 1 with methanol in the presence of SOCl₂. Afterwards, the methoxylation of compound 5 with methyl iodide and NaH afforded intermediate 6. The key intermediate, methyl ether rhein 7, was then obtained by the hydrolysis of compound 6 with lithium hydroxide. Subsequently, compound 7 was reacted with SOCl₂ and DMF to obtain its corresponding acyl chloride 8. Finally, the corresponding target compounds 9a–9h and 10a–10f were obtained by reacting intermediate 8 with the corresponding L-AA ester or D-AA ester. The structures and melting points of all target compounds are shown in

Table 2. Structure of rhein derivatives 3a–3l and 4a–4l.

Compd.	R1	Compd.	R2
3a		4a
3b		4b
3c		4c
3d		4d
3e		4e
3f		4f
3g		4g
3h		4h
3i		4i
3j		4j
3k		4k
3l		4l

and

Table 3. Structure of rhein derivatives 9a–9h and 10a–10f.

Compd.	R3	R4	Compd.	R5	R6
9a	CH3		10a	CH2CH3
9b	CH3		10b	CH3
9c	CH2CH3		10c	CH3
9d	CH2CH3		10d	CH2CH3
9e	CH3		10e	CH3
9f	CH3		10f	CH3
9g	CH2CH3
9h	CH3

. In addition, the structures of all synthesized target compounds, including the partial intermediates, were well characterized by ¹H NMR, ¹³C NMR and HRMS analyses and gave satisfactory spectroscopic data that were in full accordance with their depicted structures.

3.2. Antifungal Activities In Vitro

All target compounds 3a–3l, 4a–4l, 9a–9h and 10a–10f were preliminarily screened for antifungal activity in vitro at 0.2 mM and 0.5 mM against four plant pathogenic fungi (Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum and Phytophthora capsici). Rhein and a commercial biofungicide, phenazine-1-carboxylic acid (PCA), were used as positive controls. The results of the preliminary antifungal activity of all rhein derivatives are listed in Table 1. The results of the preliminary bioassay showed that all the compounds exhibited various degrees of inhibitory activities against each of the tested fungi. Most compounds exhibited good antifungal activity against R. solani at 0.5 mM in vitro. In particular, compounds 3e, 3j, 4a, 9d and 10f showed potent activities against R. solani, with inhibition over 50% at 0.2 mM in vitro. Although most of these compounds exhibited weak activity against the other three fungal strains, fortunately, compound 10a displayed outstanding antifungal activities (100%) against S. sclerotiorum, F. graminearum and P. capsici, which was higher than that of the positive control PCA.

Based on the results of antifungal activities in vitro, the compounds with inhibitory rates over 50% against these strains of fungi at 0.2 mM were chosen to determine their median effective concentration (EC₅₀) values. As illustrated in Figure 2, compound 3e exhibited the best activity among all the target compounds against R. solani, with an EC₅₀ value of 0.144 mM, which is 3- to 4-fold lower than the EC₅₀ value of the parent molecule rhein. Particularly, compound 10a strongly inhibited the growth of S. sclerotiorum, F. graminearum and P. capsici, with EC₅₀ values of 0.079 mM, 0.082 mM and 0.134 mM, respectively, which is comparable to the commercial biofungicide PCA with corresponding EC₅₀ values of 0.088 mM, 0.091 mM and 0.113 mM against S. sclerotiorum, F. graminearum and P. capsici, respectively. These findings indicate that compound 10a exhibited a broad spectrum of antifungal activity and has the potential to be developed as an agricultural fungicide.

3.3. Antifungal Activities In Vivo

In vivo trials against wheat powdery mildew were performed to identify the prospective application of compound 10a as an agricultural fungicide. Rhein and a commercial biofungicide registered for wheat powdery mildew, physcion, were used as positive controls. As illustrated in

Table 4. In vivo antifungal activities of tested compounds (0.2 mM, 7 days after spraying) against wheat powdery mildew under greenhouse conditions.

Compd.	Curative Activity		Protective Activity
	Disease Index (%)	Control Efficiency (%)	Disease Index (%)	Control Efficiency (%)
10a	7.9	92.1 a	7.3	91.1 a
Rhein	87.9	12.1 c	70.0	15.4 c
Physcion	61.6	38.4 b	27.1	67.2 b
CK	100	-	82.7	-

Note: The data in the table was the average of 5 groups; data in a column with different normal letters means a significant difference at p = 0.05 level.

and Figure 3 and Figure S1, the highly active compound 10a exhibited excellent in vivo protective activity against wheat powdery mildew, providing control efficiency of 91.1% at 0.2 mM, which was significantly superior to the protective fungicide physcion (67.2%). It is worth mentioning that the curative activity of compound 10a was also remarkable and provided a relevant control efficiency of 91.1%. It can also be clearly seen from Figure 3A that when compound 10a was applied to the wheat leaves infected by powdery mildew for 7 days, the powdery mildew spores on the leaves gradually turned black and necrotic and were finally eradicated. This outcome indicates that rhein derivatives can be used for development as potential fungicides.

3.4. Phytotoxicity

The phytotoxicity of effective compounds 3e, 3j, 4a, 9d, 10a and 10f was evaluated at concentrations of 0.2 mM and 0.5 mM with rice (Oryza sativa L.) and rape (Brassica napus L.) because these compounds showed potent antifungal activities against rice sheath blight and rape sclerotinia stem rot caused by R. solani and S. sclerotiorum, respectively. The results are shown in Figure 4. From Figure 4, it is clearly seen that rhein had a negative effect on the growth of rice. Especially at a higher concentration of 0.5 mM, rhein significantly retarded the seedling growth of rice both on stems and roots of rice by over 58.4% (Figure 4A) and 52.8% (Figure 4B), respectively. Compared with the parent compound rhein, the phytotoxicity of rhein esters (3e, 3j) and amide (4a) derivative on the stems and roots of rice at a low concentration of 0.2 mM slightly decreased but still maintained a high level of phytotoxicity at a higher concentration of 0.5 mM. It is worth noting that once the rhein was coupled with amino acid esters, the phytotoxicity decreased substantially. In particular, compound 9d had a promotion effect on the stem length of rice.

For B. napus, a strong inhibition effect on the root length appeared after treatment with the parent compound rhein, 3e, 3j and 4a at a concentration of 0.5 mM (Figure 4D). Compared with the control, rhein amino acid ester derivatives (9d, 10a and 10f) had no effect on the root growth of rape at the two concentrations tested. On the whole, these chosen rhein derivatives gave no effect on stem length of rape except that rhein exhibited a high inhibition effect on the root of rape at 0.5 mM (Figure 4C). The results in Figure 4 indicate that the rhein amino acid derivatives could significantly eliminate their phytotoxicity to rice and rape and could be safely used in these two crops.

3.5. Preliminary Resistance Development

In order to evaluate the resistance risk of the effective compounds, compounds with inhibitory rates > 80.0% at 0.2 mM in Table 1 were chosen to determine the antifungal resistance against two plant pathogenic fungi (R. solani, S. sclerotiorum). It can be clearly seen from

Table 5. Preliminary resistance development of the effective compounds against two fungi at 0.5 mM at 72 h ^a.

Compd.	Average Inhibition Rate ± SD (%) (n = 3)
	R. solani			S. sclerotiorum
	Generation I	Generation II	Generation III	Generation I	Generation II	Generation III
3e	84.3 ± 1.3	82.3 ± 2.2	83.8 ± 1.7	77.4 ± 2.3	76.5 ± 1.8	76.1 ± 2.5
4a	74.8 ± 0.9	75.6 ± 1.2	74.4 ± 1.1	89.2 ± 1.7	88.5 ± 2.1	88.1 ± 1.5
9d	82.7 ± 2.4	81.3 ± 1.8	81.7 ± 2.1	-	-	-
10a	-	-	-	100 ± 0.0	100 ± 0.0	100 ± 0.0
10f	81.0 ± 3.1	79.6 ± 2.3	80.2 ± 2.4	-	-	-

^a Preliminary resistance development with effective compounds (antifungal activity in Table 1 >80% at 0.5 mM), “-” means not tested.

that all five selected rhein derivatives still showed potent antifungal activity in vitro after cultivating three generations, and the inhibition rate did not change. The current results suggest that these rhein derivatives could effectively avoid the risk of resistance to these two strains of fungi.

4. Conclusions

In summary, thirty-eight rhein-derived compounds were efficiently synthesized, and their structures were well characterized. Antifungal activity results indicated that most derivatives exhibited good antifungal activity against R. solani at 0.5 mM in vitro. Compounds 3e, 3j, 4a, 9d and 10f showed potent activities against R. solani, with inhibition over 50% at a low concentration of 0.2 mM in vitro. In particular, compound 10a strongly inhibited the growth of S. sclerotiorum, F. graminearum and P. capsici, with EC₅₀ values of 0.079 mM, 0.082 mM and 0.134 mM, respectively, which is comparable to the commercial biofungicide PCA. The in vivo study showed that compound 10a presented excellent curative and protective activities (92.1% and 91.1%, 0.2 mM) against wheat powdery mildew. The phytotoxicity results indicated that rhein amino acid derivatives could significantly eliminate their phytotoxicity to rice and rape and could be safely used in these two crops. The resistance development assay indicated that these rhein derivatives could effectively avoid the risk of resistance to these two strains of fungi. In conclusion, rhein derivatives can be used for the development of potential agricultural fungicides.