Semisynthesis, anti-oomycete and anti-fungal activities of ursolic acid ester derivatives

Abstract Using ursolic acid (UA) as the lead compound, thirteen UA ester derivatives (3 and 7a-l) were synthesized by modifying their C-3 and C-28 positions, respectively, and their structures were well characterized by 1H NMR, 13C NMR, HRMS and melting points. Furthermore, we evaluated the anti-oomycete and anti-fungal activities of these compounds against Phytophthora capsici and Fusarium graminearum in vitro. The results showed that compound 7h exhibited prominent anti-oomycete and anti-fungal activities, and the median effective concentration (EC50) values of 7h against P. capsici and F. graminearum were 70.49 and 113.21 mg/L, respectively. This study suggested that the anti-oomycete and anti-fungal activities of esters synthesized by introducing acyloxy group at C-3 position of UA was more conspicuous than that of esters synthesized by introducing benzyloxy group at C-28 position. This result will pave the way for further modification of UA to develop potential new fungicides. Graphical Abstract


Introduction
The Irish famine that shocked the world was caused by the plant pathogenic oomycetes, which can cause the potato tuber to rot, and then lead to the potato harvest failure (McGowan and Fitzpatrick 2020).Phytophthora capsici Leonian is a kind of oomycetes.The pepper blight caused by P. capsici is a soil borne disease with strong epidemic and wide occurrence range (Wang et al. 2019).It can infect the whole pepper plant, causing the root and stem base of the plant to rot and mildew.The production may be reduced by about 30% in light cases, or even cut off in serious cases (Hausbeck and Lamour 2004;Barchenger et al. 2018;Wang et al. 2019).Similarly, it can also infect tomato, cucumber, grape etc., seriously threatening the production of vegetables and fruits (Lamour et al. 2012).At present, phenylamide fungicides, mainly metalaxyl, are commonly used to control pepper blight (Wang et al. 2021).However, long-term use of the same kind of fungicides can change the structure of soil flora, increase the number of drug-resistant strains, and lead to the pesticide efficacy decrease or even ineffective (Pennisi et al. 1998;Parra and Ristaino 2001;Hawkins et al. 2019;dann and McLeod 2021).In view of this, it is extremely urgent to research and develop a new low-toxicity fungicide to control P. capsici (Fawke et al. 2015).
Wheat, as the second largest food crop in the world, has been infected by Fusarium for many years (Figueroa et al. 2018).Fusarium graminearum Schw. is the main pathogen causing wheat scab, which is easy to spread in warm and humid conditions (Wegulo et al. 2015).It can cause the wheat seedlings to wither, the stem base and wheat ears to rot, and then lead to the reduction of 1000-grain weight of wheat and poor flour quality (Wang and Miller 1988;Goswami and Kistler 2004).In serious cases, deoxynivalenol (doN) toxin can be produced, which can affect the digestive system and immune system of humans and animals (Trail 2009).Tebuconazole, carbendazim etc. currently available in the market have good control effect on F. graminearum, but excessive use can also decrease the efficacy and make the pathogen resistant to pesticide (Schisler et al. 2002;Anand et al. 2003;Javad et al. 2006).In addition to breeding resistant varieties, the best way is to develop plant-based pesticides with high efficiency and low toxicity based on natural products (Alabouvette et al. 2006;Palazzini et al. 2007;Cantrell et al. 2012).
In recent years, pentacyclic triterpenoids have been used as potential substance for the development of potent pesticide due to their high natural abundance and relatively non-toxic property (Mallavadhani et al. 2003).Ursolic acid (UA, 1, Figure 1), 3β-hydroxy-ursane-12-ene-28-carboxylic acid is a typical ursane pentacyclic triterpenoids compound, which can be extracted from Ligustrum lucidum, Prunella vulgaris and other plants (Lopez-Hortas et al. 2018).At present, UA has been widely used in medicine, food, cosmetics and other industries because of its low toxicity and safety.For example, UA has anti-neoplastic (Khwaza et al. 2020), anti-hepatitis (Liang et al. 2021), anti-bacterial (Wolska et al. 2010), anti-inflammatory (Wolska et al. 2010), anti-diabetes (oboh et al. 2021(oboh et al. ), anti-viral (Xiao et al. 2018)), anti-HIV (Baglin et al. 2003) and other activities, and has attracted more and more attention in the pharmaceutical community.Because of its various biological activities, UA is highly sought after by people.However, few studies have been reported on the effects of UA on plant oomycetes and fungal diseases.
Esters play an important role in pesticides, such as carbamate, methoxyacrylate, pyrethroid, etc.In recent years, we have focused on the synthesis of ester compounds (Chen et al. 2021a;2022;Che et al. 2022a).Previous studies have shown that the introduction of acyloxy groups into the skeleton of natural products can significantly improve their anti-plant pathogenic activity (Tian et al. 2020;2021;Chen et al. 2021a;2021b;2022;Xing et al. 2022;Che et al. 2022a;2022b;2022c;2022d).Encouraged by the above results, a series of UA esters were synthesized by modifying the hydroxyl group at position C-3 and the carboxyl group at position C-28 with UA as the lead compound, and their inhibitory activities against P. capsici and F. graminearum were determined.

Chemistry
It has been shown that the C-28 carboxyl group of UA can esterify the phenolic hydroxy group of resveratrol (Li et al. 2021).Therefore, we studied the reaction of UA with phenol under the action of EdC .HCl or EdC and dMAP, and compound 2 was not obtained when the reaction time was extended to 48 h, as shown in Scheme 1.The results showed that UA could not react with simple phenol with strong acidity.
As depicted in Scheme 2, phenol was replaced with benzyl alcohol, and compound 3 was obtained by the same esterification method, but even if the reaction time was extended to 48 h, the yield was only 9.3%.We changed the synthesis strategy.First, under the action of PBr 3 , benzyl alcohol was preferentially converted into benzyl bromide.Then, under the action of dMF and K 2 Co 3 , UA reacted with benzyl bromide for 8 h, and compound 3 was synthesized with 85% high yield.The previous research of our research group has shown that sulfonate derivatives have potential anti-oomycete activity (Tian et al. 2020;2021;Chen et al. 2021a;2021b;2022;Xing et al. 2022;Che et al. 2022a;2022b;2022c;2022d).In view of this, we plan to introduce sulfonyloxy active group at C-3 hydroxy position of UA.As shown in Scheme 3, Et 3 N was used as catalyst, and compounds 5a-e were not obtained even if dMAP was added in 1 equivalent ratio and the reaction time was extended to 48 h.
Studies have shown that acyloxy derivatives also exhibit potential anti-oomycete activity.As depicted in Scheme 4, the target compounds 7a-l were first prepared by using Method 1, namely traditional EdC .HCl condensation and dMAP catalysis.Secondly, in order to make better use of UA and further optimize the reaction conditions, Method 2 was adopted, that is, first brominate the carboxylic acid 6a-l, and then react with UA under the catalysis of Et 3 N to prepare the target compounds 7a-l.As shown in Table S1 (Supplementary material), Method 2 was superior to Method Scheme 1. the synthetic route of 28-benzyloxy ua derivatives (2).Scheme 2. the synthetic route of 28-benzyloxy ua derivatives (3).
1 in UA utilization.The yields of target compounds 7a-l synthesized by Method 2 were 40-75%, while the yields of compounds 7a-l synthesized by Method 1 were only 20-51%.For example, the yield of compound 7 l prepared by method 2 was 2.3 times that of method 1.

Anti-oomycete activity
The inhibitory activities of the target compounds 3 and 7a-l against P. capsici were determined at concentrations of 50 and 100 mg/L with metalaxyl as positive control Scheme 3. the synthetic route of 3-sulfonyloxy ua derivatives (5a-e).
in Table S2 (Supplementary material).The results suggested that most of the target compounds exhibit good anti-oomycete activity.
As shown in Table S2, it was found that at the concentration of 50 or 100 mg/L, the 3-acyloxy UA derivatives (7a-l) had higher inhibitory activity against P. capsici than the UA, which indicated that the activity of UA modified at C-3 position has been enhanced.However, compared with the 28-benzyloxy UA derivative (3), the modified compound has less active than the UA, so no further synthesis was required.At the same time, the result also indicates that C-3 modification has higher inhibitory activity against P. capsici than C-28 modification, which can be used as a future research direction.
At the concentration of 100 mg/L, 7a-l showed moderate inhibitory activity against P. capsici.Compound 7h had the highest inhibitory activity (55.49%), while 7b had the lowest inhibitory (41.21%).Compounds 7 g and 7i had outstanding activity, and the inhibition rate was more than 50%.In addition, some interesting information was found through the comparative analysis of structure-activity relationship (SAR).(1) When R 2 was methyl, the position of methyl was different, and the anti-oomycete activity was also different (meta > para > ortho, e.g.7c > 7d > 7b).For example, the inhibition rate of 7c was 50.00%, that of 7b was 41.21% and 7d was 47.25%.(2) When R 2 was Cl or Br, the anti-oomycete activity was different with different positions on benzene ring (para > ortho, e.g.7h > 7 g, 7j > 7i).For example, the inhibitory activities of 7h, 7 g, 7j and 7i on oomycete were 55.49%, 50.55%, 53.05% and 49.25% respectively.(3) When R 2 was No 2 or contains both No 2 and Cl, the presence of Cl can enhance the anti-oomycete activity.For example, the inhibition rate of 7k against P. capsici was 46.90%, while the inhibition rate of 7 l reached 50.80%, so 7 l > 7k.
To better explore the SAR of these target compounds, we selected some representative target compounds and performed EC 50 determination.The EC 50 values for these compounds, including the commercial fungicide metalaxyl, against P. capsici was shown in Table S3 (Supplementary material).When R 2 on the benzene ring was a chlorine substituent in compound 7h, excellent anti-oomycete activity against P. capsici was exhibited.

Anti-fungal activity
With triadimefon as the positive control, the inhibitory activity of the target compounds 3 and 7a-l against F. graminearum at concentrations of 100 and 200 mg/L was determined respectively in Table S4 (Supplementary material), and one compound with good activity was screened out.The anti-fungal activity at five concentration gradients was determined by mycelial growth rate method, which is described in Table S5 (Supplementary material).
As shown in Table S4, it was found that the inhibitory activities of the target compounds against F. graminearum were generally low, but the anti-fungal activities of 3-acyloxy UA derivatives (7a-l) and 28-benzyloxy UA derivative (3) were higher than those of the UA in the conditions of 100 or 200 mg/L.And the inhibitory activities of 7a-l were higher than that of 3, which was consistent with the conclusion drawn above.
When the concentration was 200 mg/L, only compound 7h has the highest inhibitory activity against F. graminearum, the inhibitory rate was 60.71%, and other compounds generally have low inhibitory activity.Therefore, compound was selected for 7h, and their antifungal activity was determined at five concentration gradients by mycelial growth rate method.The results showed that the antifungal activity of the compound can be improved when the para position substituent was Cl.

General
See supplementary material for general procedures.

Synthesis of 28-benzyloxy derivatives of ursolic acid (3)
To a solution of C 6 H 5 CH 2 oH (9.25 mmol) in the toluene (12 mL) at 40 °C for 20 min to dissolve.Then PBr 3 (3.20 mmol) was slowly dripped inwardly.After the dripping, the temperature was continued to 100 °C and the reaction lasted for 1 h before cooling down to room temperature.The organic phase was washed with water (15 mL), saturated aq.brine (10 mL × 3), dried over anhydrous Na 2 So 4 , concentrated under reduced pressure and was C 6 H 5 CH 2 Br obtained (Yang et al. 2021;Fan et al. 2022).
UA (1, 0.50 mmol) was dissolved in dMF (5 mL), anhydrous K 2 Co 3 (3.60 mmol) was added and stirred for 0.5 h at room temperature, then C 6 H 5 CH 2 Br (2.20 mmol) was added inward.Continue the reaction at room temperature and monitor by TLC until the end of the reaction (8 h).Then the mixture was extracted with ethyl acetate (10 mL × 3).Subsequently, the organic phase was combined, saturated aqueous aq.brine (10 mL × 3), dried over anhydrous Na 2 So 4 , concentrated under reduced pressure, and purified by CC to obtain 28-benzyloxy UA derivative (3) in 85% yield.The structure of compound 3 was confirmed to be correct by 1 H NMR, 13 C NMR, HRMS, and m.p., and the data of compound 3 can be found in the supplementary material.

General procedure for the synthesis of 3-acyloxy derivatives of ursolic acid (7a-l)
To a solution of R 2 CooH (6a-l, 0.80 mmol), PPh 3 (0.60 mmol) and Br 3 CSo 2 Ph (0.50 mmol) in dry dichloromethane (10 mL), the mixture was stirred at room temperature for 1-2 h, and the reaction process was checked by TLC analysis.Then, UA (1, 0.40 mmol) and Et 3 N (1.50 mmol) were added to the reaction.After the addition, the solution was continually stirred at room temperature for 10 h.The reaction was detected by TLC, H 2 o (15 mL) was added to the reaction, and extracted with CH 2 Cl 2 (10 mL × 3).Subsequently, the organic phase was combined, washed by saturated aq.brine (10 mL × 3), dried over anhydrous Na 2 So 4 , concentrated under reduced pressure, and purified by CC to obtain 3-acyloxy UA derivatives in 40-75% yields.The structures of compounds 7a-l were confirmed to be correct by 1 H NMR, 13 C NMR, HRMS, and m.p., and the data of compounds 7a-l can be found in the supplementary material.

Anti-oomycete and anti-fungal activities of 3 and 7a-l against P. capsici and F. graminearum
detailed methods for determining anti-oomycete and anti-fungal activities are included in the supplementary material.

Statistical analysis
Statistical analyses of the data were performed with the SPSS software (SPSS Inc., Chicago, IL, USA).The EC 50 value for each compound was estimated by linear regression of the probit-transformed relative inhibition value on log 10 -transformed compound concentration.

Conclusion
In summary, thirteen ursolic acid ester derivatives of ursolic acid (3 and 7a-l) were synthesized and their structures were well characterized by 1 H NMR, 13 C NMR, HRMS and m.p.The anti-oomycete and anti-fungal activities of thirteen compounds were determined in this study.Among the thirteen ursolic acid esters tested, compound 7h has significantly higher activity than that of UA in both anti-oomycete and anti-fungal activities, which can be used as a key research object for further study.The result of this study shows that the anti-oomycete and anti-fungal activities of esters synthesized by introducing acyloxy group at C-3 position of UA are higher than that of esters synthesized by introducing benzyloxy group at C-28 position.The study provides an idea for further exploring the biological activities of UA esters, and develops the application of UA esters in agriculture.