Rhein–Amino Acid Ester Conjugates as Potential Antifungal Agents: Synthesis and Biological Evaluation

In the search for crop protectants, amino acid ester conjugates have been widely investigated as potential antifungal agents. In this study, a series of rhein–amino acid ester conjugates were designed and synthesized in good yields, and their structures were confirmed by 1H-NMR, 13C-NMR and HRMS. The bioassay results revealed that most of the conjugates exhibited potent inhibitory activity against R. solani and S. sclerotiorum. In particular, conjugate 3c had the highest antifungal activity against R. solani with an EC50 value of 0.125 mM. For S. sclerotiorum, conjugate 3m showed the highest antifungal activity with an EC50 value of 0.114 mM. Satisfactorily, conjugate 3c exhibited better protective effects than that of the positive control, physcion, against powdery mildew in wheat. This research supports the role of rhein–amino acid ester conjugates as potential antifungal agents for plant fungal diseases.


Introduction
Fungicides play an irreplaceable role in protecting crops from pathogenic fungal infections and ensuring crop yield and quality [1,2]. Due to the long-term use of traditional fungicides, the emergence of drug-resistant strains and problems caused by the residue of these fungicides in the environment have become increasingly prominent [3,4]. Therefore, new antifungal agents with high efficiency, low toxicity and low environmental pollution must urgently be developed to address these challenges.
Natural products (NPs) have high chemical structure diversity, a wide range of biological activities and can easily be degraded by the environment, meaning that they play an important role in the development of crop protection agents [5,6]. The tuber of Rheum palmatum L. (Figure 1) is rich in 1,8-dihydroxy anthraquinones, and it is often used as a traditional folk medicine to treat rheumatoid arthritis, inflammation, cancer and cardiovascular diseases [7][8][9][10]. The active 1,8-dihydroxy anthraquinones in the tuber of Rheum palmatum L. are mainly rhein (1) and its analogs, which include emodin, aloeemodin, physcion ( Figure 1) and so on [11][12][13]. This research mainly focused on the modification of the chemical structure and pharmacological activities of rhein. Our previous studies have shown that various simple derivatives of rhein exhibit certain insecticidal and antifungal activities, especially against some plant pathogens [14]. These findings suggest that rhein could be used as a lead structure for the discovery of new antifungal agents. Amino acids are a class of active small molecules in organisms with important physiological and structural functions [15,16]. In addition, a significant number of amino acid or amino acid ester conjugates have been found in plants, in which a carboxylic group is usually conjugated with biologically active molecules via the amide bonds, such as hormones, flavonoids, vitamins, steroids and several heterocyclic compounds [17][18][19][20]. For this reason, many researchers have tried to conjugate active lead structures with amino acid (esters) moieties to find structures with better pharmacological or antifungal activities.
Herein, in order to find higher antifungal rhein derivatives, a series of rhein-amino acid ester conjugates were designed and synthesized by conjugating rhein with L-or D-amino acid esters via an amide bond. The antifungal activities of all the synthesized compounds were tested in vitro and in vivo, and the primary structure-activity relationship was discussed.

Chemistry
The synthetic route of rhein-amino acid ester conjugates 3a-3t is shown in Figure 2. Rhein was treated with SOCl 2 at the reflux temperature in CH 2 Cl 2 solution for 8 h, and intermediate 2 was afforded after the evaporation of the solvent. Then, intermediate 2 was allowed to react with the corresponding L-or D-amino acid esters in CH 2 Cl 2 at 0 • C for 2 h to yield target compounds 3a-3t. The corresponding reactant (amino acid ester) was used in the synthesis of rhein-amino acid ester conjugates, and the yields are listed in Figure 2, indicating that the rhein-amino acid ester conjugates were obtained in good yields and varied from 74% to 94%.
The structures of all the synthesized rhein-amino acid ester conjugates 3a-3t were effectively characterized by 1 H NMR, 13 C NMR and HRMS analyses (the corresponding spectra are available in the Supplementary Materials).

In Vitro Antifungal Activity
The in vitro antifungal activities of these rhein-amino acid ester conjugates 3a-3t were initially screened at 0.2 mM and 0.5 mM against four phytopathogenic fungi (Rhizoctonia solani, Sclerotinia sclerotiorum, Bipolaris maydis and Phytophthora capsici) using the mycelial growth rate method. The natural lead compound, rhein, was used as reference control. A commercial biofungicide, phenazine-1-carboxylic acid (PCA), was used as the positive control. The preliminary bioassay results of these conjugates against four phytopathogenic fungi are summarized in Table S1 and Figures 3     As seen in Figure 3, all of the conjugates exhibited moderate-to-good antifungal activities against R. solani at the concentration of 0.2 mM (this concentration of rhein was equal to 56.8 mg/L). Among all 20 conjugates, 11 conjugates, 3a, 3c, 3d, 3f, 3h, 3i, 3m, 3n, 3q, 3r and 3t, showed an efficacy over 50% against R. solani at 0.2 mM. In particular, at 0.5 mM (this concentration of rhein was equal to 142.1 mg/L), most of the conjugates showed great antifungal activities against R. solani, and compound 3c (rhein-L-Ala-OEt) exhibited excellent antifungal activities against R. solani with an inhibition rate of more than 90%. The data in Figure 4 indicated that seven conjugates (3c, 3f, 3h, 3i, 3m, 3q and 3r) out of the twenty tested conjugates showed moderate-to-strong antifungal activities against S. sclerotiorum with an efficacy over 50% at 0.2 mM. In particular, compound 3m (rhein-D-Met-OMe) exhibited excellent antifungal activities against S. sclerotiorum with an inhibition rate of more than 90% at 0.5 mM. As seen in Figures 5 and 6, almost all the conjugates presented poor antifungal activities against B. maydis and P. capsici, among which only compounds 3f, 3m and 3r showed an efficacy over 50% against P. capsici at 0.2 mM. Through this analysis of the preliminary antifungal activities results, it was inferred that these rhein-amino acid ester conjugates can be used as antifungal lead structures against R. solani and S. sclerotiorum.
In order to understand the antifungal activity of the more active conjugates against R. solani and S. sclerotiorum more clearly, the conjugates with inhibition rates of >50% at a concentration of 0.2 mM against these two fungi (shown in Figures 3 and 4) were further assayed to determine their median effective concentrations (EC 50 ). The results are presented in Table 1. The results showed that 11 conjugates (3a, 3c, 3d, 3f, 3h, 3i, 3m, 3n, 3q, 3r and 3t) exhibited potent antifungal activity against R. solani with EC 50 values between 0.125 and 0.197 mM, but these values were lower than that of PCA (EC 50 = 0.083 mM). Additionally, conjugate 3c had the highest antifungal activity with an EC 50 value of 0.125 mM. For S. sclerotiorum, conjugate 3m (rhein-D-Met-OMe) showed the highest antifungal activity with an EC 50 value of 0.114 mM, but this value was lower than PCA (EC 50 = 0.088 mM).

In Vivo Antifungal Activity against Powdery Mildew in Wheat
The antifungal activity of conjugate 3c against powdery mildew in wheat was evaluated at 0.4 mM and 0.2 mM. As shown in Table 2, in terms of its curative activity, conjugate 3c exhibited potent antifungal activity against powdery mildew in wheat with control efficiencies of 84.4% and 61.2% at 0.4 mM and 0.2 mM, respectively, which were similar values to those of the control agent, physcion (87.4% at 0.4 mM, and 67.2% at 0.2 mM). In terms of its protective activity, conjugate 3c had antifungal activity of 66.7% and 53.8% against powdery mildew in wheat at 0.4 mM and 0.2 mM, respectively, which were higher values than those of the control agent, physcion (54.2% at 0.4 mM, and 38.4% at 0.2 mM). Presently, extensive studies regarding the biological activities of amino acid ester conjugates are being carried out based on the conjugation of amino acid esters with an active lead structure. Zhang designed and synthesized a series of novel amino acid estercoupled caffeoylquinic acid derivatives, and the biological evaluation suggested that some amino acid ester-coupled derivatives exhibited varying degrees of lipid-lowering effects on oleic acid-elicited lipid accumulation in HepG2 liver cells [18]. Studies have shown that some curcumin-amino acid conjugates exhibit enhanced anti-inflammatory properties with potency higher than that of standard NSAID references (non-steroidal anti-inflammatory drugs, indomethacin, and ibuprofen) [21]. In our previous research, to improve the bioactivities of PCA (a biofungicide), a series of PCA-amino acid ester conjugates have been successfully prepared, some of which were shown to be more effective against Rhizoctonia solani Kuhn than PCA [22]. In this study, using natural rhein as a lead structure, 20 rhein-amino acid ester conjugates were successfully prepared, and the antifungal activities of these conjugates were initially determined. The current results confirmed that some rhein-amino acid ester conjugates have potent antifungal activities against R. solani and S. sclerotiorum, and they can be used as lead structures for the development of antifungal agents.

Chemicals and Instruments
All chemicals and reagents were commercially purchased and used directly without further purification. Flash column chromatography and analytical thin layer chromatography (TLC) were performed using silica gel 60 (200-300 mesh) and silica gel aluminum sheets F254 (Qingdao Marine Chemical Ltd., Qingdao, China), respectively. The melting points of all the compounds were determined using a WRR melting point apparatus (Shanghai Jingke Industrial Co. Ltd., Shanghai, China) and were uncorrected. Then, 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 or DMSO-d 6 solution at 400 MHz for 1 H and 101 MHz for 13 C on a Bruker Avance III HD 500 MHz NMR Spectrometer (Bruker (Beijing) Scientific Technology Co. Ltd., Beijing, China) using tetramethylsilane (TMS) as an internal standard. High-resolution mass spectra were obtained using a Bruker APEX IV Fourier-transform mass spectrometer (Bruker (Beijing) Scientific Technology Co. Ltd., Beijing, China).

Test Fungus
Plant pathogenic fungi, including Rhizoctonia solani, Sclerotinia sclerotiorum, Bipolaris maydis, and Phytophthora capsici, were provided by the Institute of Pesticide Research, Yangtze University, China. These four fungi were grown on potato dextrose agar (PDA) plates at 25 • C and maintained at 4 • C with periodic subculturing. The susceptible wheat variety, Chanceller and the tested pathogenic fungi, Blumeria graminis, isolated from a wheat plant infected with powdery mildew, were provided by the Institute for Plant Protection and Soil Sciences, Hubei Academy of Agricultural Sciences.
3.3. Preparation of 4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carbonyl Chloride (2) According to our previous literature report [23], rhein 1 (10 mmol), 30 mL of anhydrous dichloromethane, and 2~3 drops of DMF as a catalyst were added into a single-port bottle for stirring. Then, 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 a rotary evaporator to obtain 2 for the next step.
The target compounds with higher initial activities were further assayed to determine the EC 50 values according to the method described above. Based on the screening results, a series of test concentrations of the compound was set and evaluated to determine its inhibitory rate against the fungi. The log dose-response curves allowed for the determination of the EC 50 value for the bioassay using probit analysis.

In Vivo Protective and Curative Antifungal Bioassay
The control efficacy (protective and curative activity) of compound 3c against powdery mildew in wheat was assessed with pot experiments according to the method described in the literature with some modifications [26]. First, wheat seeds were grown in plastic pots (20 cm diameter) with about 15 plants per pot. After three weeks, plants in the three-leaf stage were used in antifungal activity experiments. For the protection assay, compound 3c solutions as well as the positive control physcion with different concentrations (0.4 and 0.2 mM) (containing 0.1% Tween 80 as the surfactant) were sprayed on the leaves of wheat on the first day. After 24 h, powdery mildew spores were inoculated into the leaves of wheat. Wheat seedlings sprayed with water were used as negative controls. Then, the plants were placed in a greenhouse at 25 • C with 100% relative humidity. After 7 days of greenhouse culture, the disease index of the wheat seedlings was measured. The grading standard of powdery mildew in wheat was used according to the method presented in the literature: Disease index (CK or PT) = ∑ (the number of leaves at each grade × the corresponding grade)/(the total number of leaves × the superlative grade). The protective efficacy of the tested compound was calculated according to the following formula: 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. For the curative assay, the powdery mildew spores were inoculated into the leaves for 24 h before the leaves were sprayed with the solutions under examination. The rest of the procedures were the same as the above.

Statistical Analysis
The collected data were analyzed using GraphPad Prism (Version 8.3.0) software.

Conclusions
In summary, a series of rhein-amino acid ester conjugates were obtained in good yields by conjugating rhein with L-or D-amino acid esters via an amide bond, and their structures were confirmed by 1 H-NMR, 13 C-NMR and HRMS. The bioassay results revealed that most of the conjugates exhibited potent inhibitory activity against R. solani and S. sclerotiorum. In particular, conjugate 3c had the highest antifungal activity against R. solani, with an EC 50 value of 0.125 mM. For S. sclerotiorum, conjugate 3m showed the highest antifungal activity, with an EC 50 value of 0.114 mM. Satisfactorily, conjugate 3c exhibited better protective effects than those of the positive control physcion against wheat powdery mildew. This research provides support for rhein-amino acid esters conjugates as potential antifungal agents for plant fungal diseases.