Oleanane-Type Triterpene Conjugates with 1H-1,2,3-Triazole Possessing of Fungicidal Activity

The triazole pesticide is an organic nitrogen-containing heterocyclic compound with a 1,2,3-Triazole ring. In order to develop a potential glucosamine-6-phosphate synthase (GlmS) inhibitor bactericide, 18 triazole-derivative compounds were synthesized efficiently. In addition, these compounds have not been reported in the literature. The structure was confirmed by high-resolution mass spectrometry (HRMS), 1H NMR and 13C NMR. The potential use of the most promising derivatives has been investigated by testing their antifungal activity and enzyme inhibitory activity, revealing inhibitory activities in the low micromolar range. Among them, the antifungal effects of compounds 1e, 1f, 1g, 2e, 2f, and 2g on Sclerotinia sclerotiorum were particularly significant, all of which were above 83%. These compounds will be further investigated as potential antifungal lead compounds. Their structure–activity relationships are discussed based on the effects of substituted phenyl groups on compounds.


Introduction
The development of lead compounds of new pesticides based on natural products plays an important role in the field of pesticide research [1,2]. Many natural compound derivatives have shown strong vitality as pesticides. There are a wide range of natural compounds that are biologically diverse and unique in their role. Moreover, they are easily degradable and compatible with the environment [3]. The newly developed fungicide based on natural products has the advantages of safety, high efficiency, low residue and low resistance to chemicals. It also ensures the high yield of agricultural products such as vegetables in addition to solving the problem of pesticide residues. At present, 1,2,3-Triazole and its derivatives have not been discovered in nature and can only be synthesized by artificial means [4]. The 1,2,3-Triazole structure in which three nitrogen atoms are arranged adjacently to eachother in the five-membered ring was first reported by Michael in 1893 [5]. For a long time, the synthesis and application of triazole rings have received great attention from scientific researchers in various fields at home and abroad. An abundance of research has shown that this class of compounds bears significant anti-inflammatory effects [6]. Further, studies have shown bacteriostatic [7], anti-tumor [8,9], and anti-HIV effects [10,11] in medical applications; and this type of compound is widely used in fungicides [12], herbicides [13], and insecticides [14] in agriculture. In addition, such compounds have shown very important applications in the fields of organic synthesis, medicinal chemistry, organometallic catalysis, and materials science [15][16][17]. Rational design based on specific biological targets is one of the important means of developing new pesticides internationally. According to the literature, entagenic acid (EA, Figure 1) has good inhibitory activity on GlmS [18], which has been verified by molecular docking experiments. The researchers used oleanolic acid which has high structural similarity to entagenic acid as raw material, the oleanolic acid oxime ester and oleanolic acid oxime ether compounds were synthesized and their biological activities were determined [19][20][21][22]. Subsequently, some target compounds exhibited certain inhibitory activity and good antifungal activity against GlmS. This experiment will continue to develop new glucosamine-6-phosphate synthase inhibitors. Based on the results accumulated in the early stages of these experiments, various important biological activities possessed by the triazole group were considered. In this paper, a novel hexosaminidase (GlmS) inhibitor oleanane-type conjugate with 1,2,3-Triazole fragment was designed and synthesized followed by the determination of biological activity of the target compound and inhibition of Candida albicans GlcN-6-P synthase. We hope to report the synthesis (Scheme 1) and biological activity in more detail, as well as the structure-activity relationship studies. We report the preliminary results of the study here.

Results and Discussion
As shown in Scheme 1, we envisioned that the target compounds, namely triazole derivatives 1 and 2 could be synthesized from the intermediates 3, 4, 5 and 6. According to known methods, the first three steps can be prepared using oleanolic acid as a starting material [23,24]. Tf 2 O was added to compounds 3 and 4 under N 2 protection for 15 min; and then reacted with sodium azide to form intermediate compounds 5 and 6. Then cyclization of 5 and 6 with an alkyne provided the desired triazole derivatives 1 and 2 under the conditions of copper sulfate and sodium ascorbate in high yields, respectively.
All the derivatives were synthesized according to the procedures described in Scheme 1 with good overall yields of 70-89%. The synthesized compounds were characterized by 1 H-NMR, 13 C-NMR, and HRMS (Supporting information). We used CDCl 3 as a solvent to confirm the structure of compounds by NMR. The physical data of the target compounds are given in Table 1 Fungicidal activities of the target compounds 1 and 2 against six fungal species were evaluated as previously reported [25] and compared with the commercial fungicide chlorothalonil. As shown in Table 2, the resulting data revealed that most of the tested compounds displayed a certain degree of fungicidal activity against the six species. The determination results of antifungal activity exhibited that the oleanane-type conjugates with 1,2,3-Triazole fragment showed some antifungal activity against six kinds of pathogenic fungi at a concentration of 50 µg/mL. In general, the following structure-activity relationships (SAR) are observed in series of compounds 1 and 2: (1) As a whole, the target compound had a certain inhibitory effect on the tested strains, all compounds inhibited Sclerotinia sclerotiorum, Botrytis cinerea Pers and Rhizoctonia solani Kuhn at 50 µg/mL, and the inhibition rate was above 50%.

Bioassay of Enzyme Inhibitory Activities
Inhibitory activities of all the synthesized compounds towards Candida albicans GlcN-6-P synthase were evaluated using the optimized Elson-Morgan method as previously reported [25]. The absorption value of the solution was measured at 585 nm, and then the concentration was measured using the specification curve which was determined by the relation between the absorption value and the concentration of glucosamine-6-phosphate. The inhibition rates are given in Table 3 at 0.35 mm.
The enzyme activity of compounds with a methyl substituent was slightly better than those possessing a benzyl substituent. However, this observation was not particularly obvious. When R 2 was a halogen-substituted aryl (1e, 1g), the inhibitory activity of the compound was better than that of others in the same series. Benzyl substituted compounds (2) also showed relatively good inhibitory activity.

Chemical Synthesis
General procedure for the synthesis of compounds 3 and 4. Slight modifications of the synthesis methods referring to the literature [26][27][28] have been made. We use a onepot method to synthesize 3 and 4 instead of the step-by-step method. The yield of the intermediate product 3 or 4 prepared by this method is desirably more than 90%. This method is innovative and reduces the cumbersome post-processing steps.
General procedure for the synthesis of compounds 5 and 6. Under the protection of nitrogen, place the reaction device in a low-temperature tank, add dry pyridine (20 mmol, 1.6 mL) to a three-neck round bottom flask, and add trifluoromethanesulfonic anhydride (7.5 mmol, 1.3 mL). The raw materials 3 and 4 (5 mmol) dissolved in anhydrous CH 2 Cl 2 in a constantpressure dropping funnel were slowly released. Reaction at low temperature lasted 1 h. The reaction was monitored by thin-layer chromatography (TLC) [V (ethyl acetate): V (petroleum ether) = 1 :12]. After the reaction had completed, dichloromethane was added for extraction (3 × 50 mL), washed with water, dried, and concentrated. The product was dissolved in anhydrous DMF, sodium azide (20 mmol, 1.49 g) was added, and the reaction was carried out at 60 • C for 4 h. The reaction was monitored by thin-layer chromatography (TLC) [V (ethyl acetate): V (petroleum ether) = 1: 12] until the reaction was complete, and extracted with dichloromethane (3 × 50 mL), washed with water, dried and concentrated. Purification by silica gel chromatography with 20:1 petroleum ether-EtOAc as the eluent afforded 5 or 6.
General procedure for the synthesis of title compounds 1 and 2. Intermediate 5 or 6 (1 mmol) was placed in a 250 mL round-bottom flask, a mixture of 10 mL of methanol and 10 mL of water was added, then catalytic amount of CuSO 4 ·5H 2 O and sodium ascorbate were added, and finally substituted phenylacetylene or alkyl alkyne (1.2 mmol) was added. The reaction was monitored by thin-layer chromatography (TLC) [V (ethyl acetate): V (petroleum ether) = 1:15] and the reaction was stopped after 10 h. After being filtered, extracted with CH 2 Cl 2 (3 × 50 mL), dried (Na 2 SO 4 anhydrous), and taking [V (ethyl acetate): V (petroleum ether) = 1:30] as an eluent, the target compound 1a∼1i/2a∼2i was obtained by silica gel column chromatography.
The results of the antifungal activity assay showed that the oleanane-type conjugates with 1,2,3-Triazole fragment had certain antifungal activity against the six pathogenic fungi at a concentration of 50 µg/mL. The target compounds demonstrated conspicuous inhibitory effects on Sclerotinia sclerotiorum, Botrytis cinerea Pers and Rhizoctonia solani Kuhn, with an inhibition rate higher than 50%. Compared with the raw material oleanolic acid OA, the antifungal activity of the target was significantly improved. The antifungal effects of compounds 1e, 1f, 1g, 2e, 2f and 2g on Sclerotinia sclerotiorum were particularly prominent, 85.6%, 83.1%, 87.6%, 86.8%, 87.7%, and 89.6%, respectively. Structural analysis of the compound revealed that the benzene ring (e.g., fluorine, chlorine, nitro) of the electron-withdrawing substituent increases the activity. Subsequent work will further explore the development of new lead compounds through the study of structure-activity relationships, laying the experimental foundation for the research of novel glucosamine-6phosphate synthase inhibitors.