1,3,4-Oxadiazole Contained Sesquiterpene Derivatives: Synthesis and Microbiocidal Activity for Plant Disease

Abstract

A series of 1,3,4-oxadiazole contained sesquiterpene derivatives were synthesized, and the activity of the target compounds against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas axonopodis pv. citri (Xac), and tobacco mosaic virus (TMV) were evaluated. The biological activity results showed that the EC₅₀ values of compounds H4, H8, H11, H12, H14, H16, and H19 for Xac inhibitory activity were 33.3, 42.7, 56.1, 74.5, 37.8, 43.8, and 38.4 μg/ml, respectively. Compounds H4, H8, H15, H19, H22, and H23 had inhibitory effects on Xoo, with EC₅₀ values of 51.0, 43.3, 43.4, 50.5, 74.6, and 51.4 μg/ml, respectively. In particular, the curative and protective activities of compound H8 against Xoo in vivo were 51.9 and 49.3%, respectively. In addition, the EC₅₀ values of the inactivation activity of compounds H4, H5, H9, H10, and H16 against TMV were 69.6, 58.9, 69.4, 43.9, and 60.5 μg/ml, respectively. The results of molecular docking indicated that compound H10 exhibited a strong affinity for TMV-coat protein, with a binding energy of −8.88 kcal/mol. It may inhibit the self-assembly and replication of TMV particles and have an anti-TMV effect, which supports its potential usefulness as an antiviral agent.

Introduction

Most plant diseases are caused by biological agents such as bacteria, fungi, viruses, and nematodes, which have adverse impact on the growth and development of plants (Das et al., 2016). Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) seriously threatens the growth and production of rice by affecting the tillering stage of rice (Wang et al., 2021). Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri (Xac) reduces the quality and yield of fruits (Graham et al., 2004). Tobacco mosaic virus (TMV) can survive in dry plant debris for up to 100 years, and the associated plant diseases cause economic losses of more than USD 30 billion each year (Wang et al., 2018; Guo et al., 2021). At present, pesticides are the main means of controlling crop diseases and insect pests (Kemmitt et al., 2018). For plant disease, such as Xoo, Xac or TMV, although there are traditional medicines (such as Bismerthiazol, Thiodiazole copper, Ningnanmycin and Ribavirin), their effectiveness is limited various forms of disease and insect resistance (Buttimer et al., 2017; Liu et al., 2021). Natural products have special structural characteristics and unique biological activity mechanisms, and they are an important source for discovery of highly effective, safe, and environmentally compatible drugs (Zhang et al., 2018; Zheng and Hua, 2020; Li and Wang, 2021).

Sesquiterpenes are the most common type of terpenoids in terms of the number of compounds and the type of structural skeleton. They have thousands of representative structures and more than 300 different skeletons (Sacchettini and Poulter, 1997; Arroo, 2007). Sesquiterpenes are natural products of terpenoids found in plants, fungi, marine organisms, insects, and microorganisms. They are widely used in agriculture, medicine, perfume, cosmetics, and biofuels (Liu C.-L. et al., 2021; Liu T. et al., 2021; Mai et al., 2021). Sesquiterpenes have a variety of biological activities due to their complex three-dimensional structure, such as antiviral (Shang et al., 2016; Zhao et al., 2017), antibacterial (Duan et al., 2020; Wang et al., 2020), antifungal (Aricu et al., 2016), insecticidal and antifeedant activities (Inocente et al., 2019). In addition, at least some have excellent pharmacological activity, such as artemisinin for anti-malaria (Platon et al., 2021). There may also have anti-inflammatory (Gao et al., 2015), anti-HIV (Liu Y.-P. et al., 2021), and cytotoxic activity (Ryu et al., 2015). Collectively, sesquiterpenes offer a wide potential for research and commercial applications.

Heterocyclic compounds often combine good activity, high selectivity, and low dosage, thus features attractive to new pesticide research (Jin and Zhang_, 2010; Wu et al., 2011; Wu et al., 2013). The presence of nitrogen in the molecule is usually accompanied by the emergence of new compound activities or the enhancement of the original activity characteristics of natural terpenoids (Lungu., 2015). Among them, 1,3,4-oxadiazole is a kind of heterocyclic compound with a variety of biological activities, and its derivatives show antiviral (Gan et al., 2017; He et al., 2021), antibacterial (Vasantha et al., 2019; Yu et al., 2021; Wang S. et al., 2021), antifungal (Wen et al., 2019; Wang X. et al., 2021) and insecticidal activity (Yang et al., 2020) in agricultural applications. Some also proved to be attractive anti-cancer (Kumar et al., 2009), anti-depressant (Ergun et al., 2010), anti-HIV (Parizadeh et al., 2018), and anti-inflammatory (Naseer et al., 2019) medicines. Additionally, the presence of alkyl groups on the oxadiazole nucleus increases their ability to penetrate active sites and enhance their biological activity (Vasantha et al., 2019).

In view of the above findings, as one of the most active research fields in natural product chemistry, sesquiterpenes can be derived from their skeletons to obtain active different compounds. In this study, using the principle of active substructure splicing, sclareolide was used as the lead compound and the active fragment of oxadiazole was introduced (Figure 1). A series of 1,3,4-oxadiazole contained sesquiterpene derivatives were synthesized and their biological activities were evaluated.

FIGURE 1

Results and Discussion

Antibacterial Activity in Vitro

The in vitro antibacterial activity of synthetic compounds H1–H23 against Xoo and Xac was tested by the turbidity method (Zhang et al., 2021). The preliminary biological activity results are shown in Table 1. The inhibitory activities of compounds H4, H8, H15, H19, and H22 on Xoo were 64.5, 70.2, 69.5, 65.7, and 60.1% at 100 μg/ml, respectively, which were higher than that of thiodiazole copper (56.7%). The inhibitory effects of compounds H4, H8, H14, and H19 on Xac at 100 μg/ml were 73.3, 65.1, 70.5, and 66.6%, respectively, which were better than that of bismerthiazol (64.6%).

The concentration values for 50% of maximal effect (EC₅₀) of some compounds are shown in Table 2. The EC₅₀ values of compounds H8 and H15 against Xoo were 43.3 and 43.4 μg/ml, respectively, which were close to bismerthiazol (41.8 μg/ml) and superior to that of thiodiazole copper (61.4 μg/ml). Compounds H4 and H14 had an inhibitory effect on Xac, with their EC₅₀ values being 33.3 and 37.8 μg/ml, respectively, thus better than for bismerthiazol (38.2 μg/ml).

Antibacterial Activity in Vivo

To further verify the control effect of the compound on rice bacterial leaf blight, the in vivo antibacterial activity of compound H8 was determined by the leaf-cutting method at 200 μg/ml (Zhang et al., 2021). The results are shown in Table 3, Table 4; Figure 2. The curative activity of compound H8 was 51.9%, which was better than that of bismerthiazol (47.1%) and thiodiazole copper (46.1%). Concomitantly, the compound H8 showed good protective activity of 49.3% compared to bismerthiazol (45.8%) and thiodiazole copper (43.7%).

FIGURE 2

Anti-TMV Activity in Vivo

According to the classic literature method (Ren et al., 2020), the activity of the target compound H1–H23 on TMV was tested. Preliminary bioactivity showed that most of the compounds exhibited a good inhibitory effect on TMV at 500 μg/ml. The results are shown in Table 5. Compared with ribavirin, most compounds had moderate to good activity. The curative activities of compounds H8, H12, H16, and H19 were 68.3, 63.5, 67.5, and 63.3%, respectively, which were significantly higher than ribavirin (45.4%). Notably, the curative activity of compound H9 was 77.5%, which was better than ningnanmycin (70.0%). The inactivation potency of compounds H3, H4, H5, H9, and H16 were 81.7, 82.0, 87.5, 82.0, and 87.3%, respectively, which were higher than that of ribavirin (72.3%). It was worth noting that the inactivation potency of compound H10 was 90.5%, which was slightly better than that of ningnanmycin (90.0%)

The EC₅₀ values of some compounds were further tested, as shown in Table 6. The results indicated that the EC₅₀ value of compound H10 was 43.9 μg/ml, which was better than ningnanmycin (44.8 μg/ml).

Molecular Docking and MD Simulation

TMV coat protein (TMV-CP) plays an important role in the replication and assembly of plant viruses. Our goal was to investigate the interaction between active target compounds and TMV-CP. The binding method of ligand molecules (compound H10 and ningnanmycin) and TMV-CP (PDB 97 code: 1EI7) was explored through molecular docking, and the results are shown in Figures 3A,B. Compound H10 had a strong affinity for TMV-CP, with a binding energy of -8.88 kcal/mol, while that of ningnanmycin was 6.35 kcal/mol. The hydroxyl oxygen atom of compound H10 formed a strong hydrogen bond with ASN73 and ELU131 (the bond length is 3.1Å and 2.8Å, respectively), and the residue ELU131 can also be seen in ningnanmycin. Compound H10 had two hydrophobic interactions with amino acid residues TYR139 and THR136 in addition to interacted with VAL260 via hydrophobic bonds like ningnanmycin.

FIGURE 3

The stability and interaction mode of the ligand molecule and TMV-CP under the simulated conditions were further studied through molecular dynamics (MD) simulation, and the root-mean-square deviation (RMSD) of the atom and its initial position was measured (Figures 3C,D). Due to the significant interaction between the ligand and the binding site, the difference in energy characteristics results in a stable conformation and strong binding. Therefore, the biological activity can be influenced by optimizing the structure of the compound, and the properties of inhibiting TMV can be explored.

Structure-Activity Relationship Analysis

The preliminary structure-activity relationship showed that the different substituents R of sesquiterpene derivatives had a great influence on Xoo, Xac, and TMV. According to Table 1, when there are electron-withdrawing F, Cl or F, Br atoms on the benzene ring at the same time, the activity of the compound against Xoo is reduced: H4 (R = Ph) > H21 (R = 4-Br-2-F-Ph) > H1 (R = 2-Cl-5-F-Ph) > H9 (R = 2-Br-5-F-Ph) > H16 (R = 2-Br-4-F-Ph) > H17 (R = 3-Cl-2-F-Ph) > H6 (R = 2-Cl-4-F-Ph). The position of difluoro substitution on the aromatic ring also had an effect on the activity of Xac: H8 (R = 2,4-di-F-Ph) > H12 (R = 2,3-di-F-Ph) > H5 (R = 2,6-di-F-Ph) > H13 (R = 3,5-di-F-Ph) > H2 (R = 2,5-di-F-Ph). As shown in Table 5, introduction of different groups at the 4-position of the aromatic ring, altered the compounds’ curative activities against TMV, with the electron-donating group having improved activity over the electron-withdrawing group: H19 (R = 4-OCH₃-Ph) > H23 (R = 4-NO₂-Ph) > H3 (R = 4-CF₃-Ph) > H22 (R = 4-OCF₃-Ph) > H14 (R = 4-Cl-Ph) > H18 (R = 4-Br-2-F-Ph). The type and position of a single halogen atom on the benzene ring and heterocyclic ring may affect the inactivation potency of the compound: H10 (R = 3-Br-Ph) > H11 (R = 4-Cl-Py) > H14 (R = 4-Cl-Ph) > H15 (R = 2-Cl-Ph) > H18 (R = 4-Br-2-F-Ph) > H20 (R = 5-Cl-thiazol).

Materials and Methods

General Information

Melting points (uncorrected) of the synthetic compounds were determined using the XT-4 micro melting point instrument (Beijing Tech Instrument Co., China). All of the reactions were performed using a magnetic stir bar, followed by thin-layer chromatography (TLC) on silica gel GF254 and identified by UV. The ¹H, ¹³C, and ¹⁹F nuclear magnetic resonance (NMR) spectra were obtained with AVANCE III HD 400 MHz or 500 MHz (Bruker Corporation, Switzerland) system in CDCl₃, and used TMS as an internal standard at room temperature. High-resolution mass spectrometer (HRMS) data was conducted using an Orbitrap LC-MS instrument (Q-Exative, Thermo Scientific™, United States). All reagents and solvents were purchased from commercial suppliers and were not subjected to further purification and drying.

Chemistry

According to the synthetic route shown in Scheme 1, the target compounds H1–H23 were obtained. The natural product sclareolide was used as raw material to produce hydrazide intermediate 1 by hydrazinolysis reaction with hydrazine hydrate under weakly alkaline conditions. Intermediate 1 continues to form a closed loop with carbon disulfide under reflux to obtain oxadiazole intermediate 2. Then, under the alkaline condition in the presence of anhydrous potassium carbonate, intermediate 2 reacts with different substituted benzyl halides to synthesize the target compounds H1–H23.

SCHEME 1

Synthesis

General Procedure for the Preparation of the Intermediates 1 and 2

As shown in Scheme 1, the previously published methods were used (Zhang et al., 2013; Mishra et al., 2017). The raw material sclareolide (500 mg, 1 mol) was dissolved in a round bottom flask with EtOH, and hydrazine hydrate (1 ml, 11 mol) was added and stirred at room temperature for 2 h. After the reaction was completed, an appropriate amount of water was added to the system, and the precipitate was collected by filtration to obtain Intermediate 1. Subsequently, Intermediate 1 (300 mg, 1 mol) was dissolved in DMF and stirred for 30 min, carbon disulfide (743 mg, 5 mol) was slowly added and refluxed for 6–8 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over NaSO₄ and concentrated under vacuum. The residue was purified by silica gel chromatography with petroleum ether/ethyl acetate (8:1) concentrated eluent to obtain Intermediate 2.

General Procedures for the Preparation of Target Compounds H1-H23

According to the published method (Wang et al., 2019), Intermediate 2 (200mg, 1 mol) and potassium carbonate (107mg, 1.2 mol) were dissolved in a round bottom flask with DMF and stirred for 30 min. Different substituted benzyl halides were added and reacted at room temperature for 6–7 h. An appropriate amount of water was added to the reaction mixture to filter the residue. The crude product was subjected to column chromatography with petroleum ether/ethyl acetate (5:1) to extract target compounds H1-H23.

The structures of synthesized compounds H1-H23 were confirmed by ¹H NMR, ¹³C NMR, ¹⁹F NMR, and HRMS.

(1R,2R,8aS)-1-((5-((2-fluoro-5-(trifluoromethyl)benzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H1). Yield 95%; White solid; m. p.75–76°C. ¹H NMR (400 MHz, CDCl₃) δ 7.81 (dd, J = 6.8, 2.1 Hz, 1H), 7.61–7.54 (m, 1H), 7.19 (t, J = 8.9 Hz, 1H), 4.47 (s, 2H), 2.90 (ddd, J = 76.1, 16.3, 5.7 Hz, 2H), 1.95–1.89 (m, 2H), 1.74–1.51 (m, 4H), 1.46 (d, J = 3.7 Hz, 1H), 1.40–1.30 (m, 4H), 1.21 (s, 3H), 1.00 (dd, J = 12.1, 2.2 Hz, 1H), 0.88 (s, 3H), 0.87 (s, 3H), 0.80 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.5, 162.7 (d, J = 254.3 Hz), 162.3, 128.8 (d, J = 8.0 Hz), 127.4 (d, J = 3.7 Hz), 127.3 (d, J = 3.7 Hz), 124.6 (d, J = 15.6 Hz), 123.5 (d, J = 272.0 Hz), 116.2 (d, J = 22.7 Hz), 73.2, 59.0, 55.7, 44.5, 41.5, 39.3, 38.8, 33.3, 33.2, 29.5, 23.3, 21.4, 21.1, 20.4, 18.3, 15.1.¹⁹F NMR (376 MHz, CDCl₃) δ -61.91, -110.96. HRMS (ESI+) m/z Calcd for C₂₅H₃₃F₄SN₂O₂ [M + H]⁺ 501.21934; Found 501.21936.

(1R,2R,8aS)-1-((5-((2,5-difluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H2). Yield 63%; White solid; m. p.84–86°C. ¹H NMR (400 MHz, CDCl₃) δ 7.26–7.22 (m, 1H), 7.04–7.00 (m, 1H), 6.98–6.95 (m, 1H), 4.40 (s, 2H), 2.91 (ddd, J = 76.7, 16.3, 5.6 Hz, 2H), 1.96–1.91 (m, 2H), 1.75–1.67 (m, 2H), 1.54–1.49 (m, 2H), 1.47–1.43 (m, 1H), 1.40–1.30 (m, 4H), 1.21 (s, 3H), 1.00 (dd, J = 12.1, 2.2 Hz, 1H), 0.88 (s, 3H), 0.87 (s, 3H), 0.80 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.5, 162.6, 158.3 (d, J = 245.5 Hz), 156.8 (d, J = 246.6 Hz), 125.0 (dd, J = 17.1, 8.1 Hz), 117.7 (dd, J = 24.7, 3.6 Hz), 116.6 (dd, J = 21.2, 5.5 Hz), 116.3 (dd, J = 20.9, 5.4 Hz), 73.2, 59.0, 55.7, 44.5, 41.5, 39.3, 38.8, 33.4, 33.2, 29.7, 23.3, 21.4, 21.1, 20.4, 18.3, 15.1.¹⁹F NMR (376 MHz, CDCl₃) δ -118.08, -122.78. HRMS (ESI+) m/z Calcd for C₂₄H₃₃F₂SN₂O₂ [M + H]⁺ 451.22253; Found 451.22229.

(1R,2R,8aS)-2,5,5,8a-tetramethyl-1-((5-((4-(trifluoromethyl)benzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)decahydronaphthalen-2-ol (H3). Yield 64%; White solid; m. p.81–83°C. ¹H NMR (400 MHz, CDCl₃) δ 7.58 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 4.44 (s, 2H), 2.89 (ddd, J = 75.7, 16.2, 5.7 Hz, 2H), 1.93–1.87 (m, 1H), 1.73–1.58 (m, 4H), 1.44 (ddt, J = 10.0, 6.8, 5.2 Hz, 4H), 1.33 (ddd, J = 13.5, 6.2, 1.3 Hz, 2H), 1.21 (s, 3H), 0.99 (dd, J = 12.1, 2.2 Hz, 1H), 0.88 (s, 3H), 0.86 (s, 3H), 0.80 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.4, 162.5, 140.1 (d, J = 1.3 Hz), 130.1 (d, J = 32.6 Hz), 129.4, 129.2, 125.6 (d, J = 3.8 Hz), 125.6 (d, J = 11.2 Hz), 123.9 (d, J = 272.3 Hz), 73.2, 59.1, 55.8, 44.5, 41.5, 39.4, 38.8, 36.0, 33.3, 33.2, 23.3, 21.4, 21.1, 20.4, 18.3, 15.1. HRMS (ESI+) m/z Calcd for C₂₅H₃₄F₃SN₂O₂ [M + H]⁺ 483.22876; Found 483.22870.

(1R,2R,8aS)-1-((5-(benzylthio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H4). Yield 91%; Pink solid; m. p.78–80°C. ¹H NMR (400 MHz, CDCl₃) δ 7.41 (dd, J = 8.0, 1.4 Hz, 2H), 7.35–7.30 (m, 3H), 4.42 (s, 2H), 2.90 (ddd, J = 77.6, 16.2, 5.6 Hz, 2H), 1.91 (ddd, J = 15.1, 9.0, 4.4 Hz, 2H), 1.73–1.64 (m, 1H), 1.59–1.47 (m, 2H), 1.43–1.34 (m, 4H), 1.32–1.23 (m, 2H), 1.20 (s, 3H), 0.99 (dd, J = 12.1, 2.2 Hz, 1H), 0.88 (s, 3H), 0.87 (s, 3H), 0.80 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.1, 163.1, 135.7, 129.1, 128.7, 128.0, 73.2, 59.1, 55.7, 44.5, 41.5, 39.3, 38.8, 36.8, 33.3, 33.2, 23.3, 21.4, 21.1, 20.4, 18.3, 15.1. HRMS (ESI+) m/z Calcd for C₂₄H₃₅SN₂O₂ [M + H]⁺ 415.24138; Found 415.24130.

(1R,2R,8aS)-1-((5-((2,6-difluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H5). Yield 84%; Pink solid; m. p.87–88°C. ¹H NMR (400 MHz, CDCl₃) δ 7.21–7.16 (m, 1H), 6.88–6.83 (m, 2H), 4.24 (s, 2H), 2.49–2.40 (m, 2H), 2.08–2.04 (m, 1H), 1.89–1.76 (m, 2H), 1.67–1.59 (m, 2H), 1.48–1.39 (m, 4H), 1.38–1.28 (m, 2H), 1.25 (s, 3H), 1.02 (d, J = 2.6 Hz, 1H), 0.88 (s, 3H), 0.87 (s, 3H), 0.82 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 168.3, 161.5 (d, J = 250.1 Hz), 161.4 (d, J = 250.1 Hz), 154.5, 128.9 (d, J = 10.5 Hz), 113.9 (d, J = 19.4 Hz), 111.2 (d, J = 25.1 Hz), 111.2 (d, J = 12.8 Hz), 73.2, 59.1, 56.6, 42.1, 39.4, 38.6, 36.2, 33.3, 33.1, 26.3, 21.9, 20.9, 20.7, 18.1, 15.1.¹⁹F NMR (376 MHz, CDCl₃) δ -113.32, -113.49. HRMS (ESI+) m/z Calcd for C₂₄H₃₃F₂SN₂O₂ [M + H]⁺ 421.22253; Found 421.22253.

(1R,2R,8aS)-1-((5-((2-chloro-4-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H6). Yield 84%; White solid; m. p.86–87°C. ¹H NMR (400 MHz, CDCl₃) δ 7.51 (dd, J = 8.3, 6.3 Hz, 1H), 7.09 (dd, J = 8.5, 2.6 Hz, 1H), 6.90 (td, J = 8.4, 2.6 Hz, 1H), 4.21 (s, 2H), 2.45 (t, J = 9.3 Hz, 2H), 2.08–2.04 (m, 1H), 1.83 (ddd, J = 30.2, 10.4, 5.2 Hz, 2H), 1.67–1.58 (m, 2H), 1.47–1.39 (m, 4H), 1.32 (m, 2H), 1.24 (s, 3H), 1.01 (d, J = 2.3 Hz, 2H), 0.88 (s, 3H), 0.87 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.4, 162.9, 162.2 (d, J = 250.8 Hz), 135.0 (d, J = 10.4 Hz), 132.6 (d, J = 8.9 Hz), 129.9 (d, J = 3.7 Hz), 117.1 (d, J = 24.9 Hz), 114.3 (d, J = 21.1 Hz), 73.2, 59.0, 55.7, 44.4, 41.5, 39.3, 38.8, 33.8, 33.3, 33.2, 23.3, 21.4, 21.1, 20.4, 18.3, 15.1.¹⁹F NMR (376 MHz, CDCl₃) δ -111.21. HRMS (ESI+) m/z Calcd for C₂₄H₃₃FClSN₂O₂ [M + H]⁺ 467.19298; Found 467.19293.

(1R,2R,8aS)-2,5,5,8a-tetramethyl-1-((5-((2-(trifluoromethyl)benzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)decahydronaphthalen-2-ol (H7). Yield 98%; Pink solid; m. p.115–117°C. ¹H NMR (400 MHz, CDCl₃) δ 7.67 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.45 (dd, J = 14.3, 6.6 Hz, 1H), 7.37–7.29 (m, 1H), 4.35 (s, 2H), 2.49–2.40 (m, 2H), 2.08–2.03 (m, 1H), 1.95–1.84 (m, 2H), 1.67–1.61 (m, 2H), 1.47–1.39 (m, 4H), 1.37–1.29 (m, 2H), 1.25 (s, 3H), 1.00 (dd, J = 12.5, 2.7 Hz, 1H), 0.88 (s, 3H), 0.87 (s, 3H), 0.82 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 168.6, 154.6, 137.4, 132.2 (d, J = 24.9 Hz), 131.9 (d, J = 5.0 Hz), 128.2, 127.1, 125.7 (d, J = 5.8 Hz), 124.3 (d, J = 274.0 Hz), 73.2, 59.1, 56.6, 44.5, 42.1, 39.4, 38.6, 36.2, 33.3, 33.1, 26.3, 21.9, 20.9, 20.7, 18.1, 15.1.¹⁹F NMR (376 MHz, CDCl₃) δ -59.24. HRMS (ESI+) m/z Calcd for C₂₅H₃₄F₃SN₂O₂ [M + H]⁺ 483.22876; Found 483.22855.

(1R,2R,8aS)-1-((5-((2,4-difluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H8). Yield 72%; White solid; m. p.93–95°C. ¹H NMR (400 MHz, CDCl₃) δ 7.44 (dd, J = 15.4, 8.5 Hz, 1H), 6.83–6.78 (m, 1H), 6.77–6.72 (m, 1H), 4.12 (s, 2H), 2.50–2.40 (m, 4H), 2.10–1.73 (m, 1H), 1.49–1.40 (m, 4H), 1.38–1.28 (m, 2H), 1.25 (s, 3H), 1.02 (d, J = 2.4 Hz, 1H), 0.88 (s, 3H), 0.87 (s, 3H), 0.83 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.5, 162.8 (d, J = 249.6 Hz), 162.8, 161.1 (d, J = 250.7 Hz), 132.3 (dd, J = 9.6, 4.8 Hz), 119.4 (dd, J = 14.5, 3.5 Hz), 111.5 (dd, J = 21.0, 3.5 Hz), 104.2 (d, J = 25.4 Hz), 73.3, 59.1, 55.8, 44.6, 41.6, 39.4, 38.8, 33.4, 33.3, 29.5, 23.3, 21.5, 21.2, 20.4, 18.4, 15.2.¹⁹F NMR (376 MHz, CDCl₃) δ -109.30, -112.11. HRMS (ESI+) m/z Calcd for C₂₄H₃₂F₂SN₂O₂Na [M + Na]⁺ 473.20448; Found 473.20499.

(1R,2R,8aS)-1-((5-((2-bromo-5-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H9). Yield 84%; White solid; m. p.120–122°C. ¹H NMR (400 MHz, CDCl₃) δ 7.47 (dd, J = 8.8, 5.3 Hz, 1H), 7.30 (dd, J = 9.3, 3.0 Hz, 1H), 6.82 (td, J = 8.4, 3.1 Hz, 1H), 4.22 (s, 2H), 2.56–2.39 (m, 2H), 2.10–1.74 (m, 4H), 1.61 (dd, J = 11.6, 4.2 Hz, 1H), 1.44 (ddd, J = 20.8, 12.2, 5.6 Hz, 4H), 1.32 (ddd, J = 16.5, 10.4, 3.4 Hz, 2H), 1.25 (s, 3H), 1.02 (d, J = 2.5 Hz, 1H), 0.88 (s, 3H), 0.87 (s, 3H), 0.83 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 168.3, 161.8 (d, J = 246.9 Hz), 154.7, 140.3 (d, J = 8.0 Hz), 133.6 (d, J = 8.1 Hz), 118.6 (d, J = 3.6 Hz), 118.2 (d, J = 23.4 Hz), 115.8 (d, J = 23.9 Hz), 74.1, 59.1, 56.6, 42.1, 39.4, 38.6, 36.2, 33.3, 33.1, 26.3, 21.9, 20.9, 20.7, 18.1, 15.1.¹⁹F NMR (376 MHz, CDCl₃) δ -114.65. HRMS (ESI+) m/z Calcd for C₂₄H₃₂FBrSN₂O₂Na [M + Na]⁺ 533.12441; Found 533.12457.

(1R,2R,8aS)-1-((5-((3-bromobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H10). Yield 83%; White solid; m. p.68–70°C.

¹H NMR (400 MHz, CDCl₃) δ 7.57 (t, J = 1.7 Hz, 1H), 7.44–7.40 (m, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 4.37 (s, 2H), 3.05–2.72 (m, 2H), 1.96–1.79 (m, 4H), 1.73–1.66 (m, 1H), 1.56–1.42 (m, 4H), 1.28 (ddd, J = 13.4, 6.6, 3.5 Hz, 2H), 1.20 (s, 3H), 1.01 (d, J = 2.2 Hz, 1H), 0.88 (s, 3H), 0.86 (s, 3H), 0.80 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.3, 162.6, 138.1, 132.0, 131.1, 130.3, 127.8, 122.6, 73.2, 59.0, 55.7, 44.5, 41.5, 39.3, 38.8, 36.0, 33.4, 33.2, 23.3, 21.4, 21.1, 20.4, 18.3, 15.1. HRMS (ESI+) m/z Calcd for C₂₄H₃₄BrSN₂O₂ [M + H]⁺ 493.15189; Found 493.15204.

(1R,2R,8aS)-1-((5-(((6-chloropyridin-3-yl)methyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H11). Yield 72%; White solid; m. p.102–104°C. ¹H NMR (400 MHz, CDCl₃) δ 8.44–8.36 (m, 2H), 7.69 (dd, J = 8.2, 2.3 Hz, 1H), 4.07 (s, 2H), 2.55–2.39 (m, 2H), 2.06 (dt, J = 11.6, 3.1 Hz, 1H), 1.88–1.74 (m, 2H), 1.68–1.60 (m, 2H), 1.44 (dt, J = 20.2, 5.8 Hz, 4H), 1.32 (ddd, J = 16.5, 11.1, 3.4 Hz, 2H), 1.26 (s, 3H), 1.02 (d, J = 2.7 Hz, 1H), 0.88 (s, 6H), 0.83 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 168.0, 154.9, 149.9, 139.5, 133.8, 123.9, 89.0, 59.1, 56.6, 42.1, 39.4, 38.6, 36.2, 33.3, 33.0, 29.4, 26.4, 21.9, 20.9, 20.7, 18.1, 15.1. HRMS (ESI+) m/z Calcd for C₂₃H₃₁ClSN₃O₂ [M-H]^- 448.18200; Found 448.18344.

(1R,2R,8aS)-1-((5-((2,3-difluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-olH-12 (H12). Yield 67%; White solid; m. p.99–100°C. ¹H NMR (400 MHz, CDCl₃) δ 7.19 (ddd, J = 8.8, 5.8, 3.1 Hz, 1H), 6.95 (td, J = 9.0, 4.5 Hz, 1H), 6.92–6.84 (m, 1H), 4.12 (s, 2H), 2.53–2.40 (m, 2H), 2.06 (dt, J = 11.6, 3.2 Hz, 1H), 1.87 (ddd, J = 14.0, 6.8, 3.2 Hz, 2H), 1.78 (dd, J = 13.5, 7.2 Hz, 2H), 1.69–1.59 (m, 2H), 1.49–1.32 (m, 4H), 1.25 (s, 3H), 1.02 (d, J = 2.5 Hz, 1H), 0.88 (s, 3H), 0.88 (s, 3H), 0.83 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 168.3, 159.6, 156.8 (d, J = 243.0 Hz), 155.9 (d, J = 255.4 Hz), δ 127.6 (dd, J = 17.6, 8.1 Hz), 117.5 (dd, J = 24.4, 3.4 Hz), 116.0 (dd, J = 24.6, 8.7 Hz), 115.0 (dd, J = 23.9, 8.5 Hz), 88.8, 59.1, 56.6, 42.1, 39.4, 38.6, 36.2, 33.3, 33.1, 26.3, 21.9, 20.9, 20.7, 18.1, 15.1.¹⁹F NMR (376 MHz, CDCl₃) δ -119.09, -123.36. HRMS (ESI+) m/z Calcd for C₂₄H₃₃F₂SN₂O₂ [M + H]⁺ 451.22253; Found 451.22275.

(1R,2R,8aS)-1-((5-((3,5-difluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H13). Yield 60%; White solid; m. p.86–88°C. ¹H NMR (400 MHz, CDCl₃) δ 6.90 (dd, J = 8.2, 2.2 Hz, 2H), 6.65 (tt, J = 9.0, 2.3 Hz, 1H), 4.08 (s, 2H), 2.54–2.41 (m, 2H), 2.07 (dt, J = 11.8, 3.2 Hz, 1H), 1.91–1.79 (m, 2H), 1.65–1.58 (m, 2H), 1.49–1.38 (m, 4H), 1.39–1.27 (m, 2H), 1.26 (s, 3H), 1.02 (d, J = 2.7 Hz, 1H), 0.88 (s, 6H), 0.83 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 168.1, 162.9 (d, J = 248.2 Hz), 162.7 (d, J = 248.1 Hz), 154.8, 142.6 (d, J = 9.0 Hz), 111.8 (d, J = 11.7 Hz), 111.8 (d, J = 25.4 Hz), 102.3 (d, J = 25.3 Hz), 88.9, 59.1, 56.6, 42.1, 39.4, 38.6, 36.2, 33.3, 33.1, 26.4, 21.9, 20.9, 20.7, 18.1, 15.1.¹⁹F NMR (376 MHz, CDCl₃) δ -110.18, -110.18. HRMS (ESI+) m/z Calcd for C₂₄H₃₃F₂SN₂O₂ [M + H]⁺ 451.22253; Found 451.22287.

(1R,2R,8aS)-1-((5-((4-chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H14). Yield 65%; White solid; m. p.95–96°C.

¹H NMR (500 MHz, CDCl₃) δ 7.38–7.34 (m, 2H), 7.32–7.28 (m, 2H), 4.37 (s, 2H), 2.89 (ddd, J = 21.7, 16.3, 5.7 Hz, 2H), 1.99–1.84 (m, 2H), 1.74–1.66 (m, 2H), 1.56–1.48 (m, 2H), 1.46–1.34 (m, 4H), 1.26 (dd, J = 13.3, 3.2 Hz, 1H), 1.20 (s, 3H), 0.99 (dd, J = 12.2, 2.1 Hz, 1H), 0.88 (s, 3H), 0.86 (s, 3H), 0.80 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.4, 162.8, 134.5, 133.9, 130.5, 128.9, 73.3, 59.1, 55.8, 44.6, 41.6, 39.4, 38.8, 36.1, 33.4, 33.3, 23.4, 21.5, 21.2, 20.4, 18.4, 15.2. HRMS (ESI+) m/z Calcd for C₂₄H₃₄ClSN₂O₂ [M + H]⁺ 449.20240; Found 449.20154.

(1R,2R,8aS)-1-((5-((2-chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H15). Yield 74%; White solid; m. p.87–89°C.

¹H NMR (500 MHz, CDCl₃) δ 7.56 (dd, J = 7.3, 1.9 Hz, 1H), 7.39 (dd, J = 7.7, 1.3 Hz, 1H), 7.26–7.18 (m, 2H), 4.53 (s, 2H), 2.89 (ddd, J = 96.9, 16.3, 5.5 Hz, 2H), 1.92 (ddd, J = 14.8, 8.8, 4.3 Hz, 2H), 1.75–1.67 (m, 2H), 1.55–1.47 (m, 2H), 1.44–1.33 (m, 4H), 1.26 (dd, J = 13.5, 3.5 Hz, 1H), 1.20 (s, 3H), 0.99 (dd, J = 12.1, 2.0 Hz, 1H), 0.88 (s, 3H), 0.86 (s, 3H), 0.80 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.4, 163.1, 134.3, 133.9, 131.5, 129.8, 129.6, 127.1, 73.3, 59.1, 55.8, 44.6, 41.6, 39.4, 38.9, 34.61, 33.4, 33.3, 23.3, 21.5, 21.2, 20.4, 18.4, 15.2. HRMS (ESI+) m/z Calcd for C₂₄H₃₄ClSN₂O₂ [M + H]⁺ 449.20240; Found 449.20117.

(1R,2R,8aS)-1-((5-((2-bromo-4-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H16). Yield 70%; White solid; m. p.92–94°C. ¹H NMR (500 MHz, CDCl₃) δ 7.60 (dd, J = 8.6, 5.9 Hz, 1H), 7.32 (dd, J = 8.1, 2.6 Hz, 1H), 6.97 (td, J = 8.3, 2.7 Hz, 1H), 4.50 (s, 2H), 2.89 (ddd, J = 95.7, 16.2, 5.6 Hz, 2H), 1.95–1.87 (m, 2H), 1.68 (dd, J = 9.9, 6.5 Hz, 2H), 1.58–1.48 (m, 2H), 1.46–1.34 (m, 4H), 1.30–1.25 (m, 1H), 1.20 (s, 3H), 0.99 (dd, J = 12.2, 2.1 Hz, 1H), 0.87 (s, 3H), 0.86 (s, 3H), 0.79 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.4, 162.9, 162.0 (d, J = 251.6 Hz), 132.6 (d, J = 8.5 Hz), 131.7 (d, J = 3.5 Hz), 124.8 (d, J = 9.7 Hz), 120.3 (d, J = 24.8 Hz), 114.9 (d, J = 21.1 Hz), 73.3, 59.1, 55.8, 44.6, 41.6, 39.4, 38.9, 36.4, 33.4, 33.3, 23.3, 21.5, 21.2, 20.4, 18.4, 15.2.¹⁹F NMR (376 MHz, CDCl₃) δ -111.25. HRMS (ESI+) m/z Calcd for C₂₄H₃₃FBrSN₂O₂ [M + H]⁺ 511.14247; Found 511.14197.

(1R,2R,8aS)-1-((5-((3-chloro-2-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H17). Yield 61%; White solid; m. p.79–81°C. ¹H NMR (500 MHz, CDCl₃) δ 7.44–7.39 (m, 1H), 7.36–7.32 (m, 1H), 7.03 (dt, J = 8.2, 4.2 Hz, 1H), 4.44 (s, 2H), 2.90 (ddd, J = 96.4, 16.3, 5.7 Hz, 2H), 1.92 (ddd, J = 14.8, 8.9, 4.4 Hz, 2H), 1.76–1.66 (m, 2H), 1.56–1.47 (m, 2H), 1.45–1.32 (m, 4H), 1.31–1.26 (m, 1H), 1.20 (s, 3H), 1.00 (dd, J = 12.2, 2.1 Hz, 1H), 0.88 (s, 3H), 0.86 (s, 3H), 0.80 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.5, 162.6, 156.4 (d, J = 250.3 Hz), 130.5, 129.7, 125.3 (d, J = 14.4 Hz), 124.7 (d, J = 4.7 Hz), 121.3 (d, J = 17.8 Hz), 73.3, 59.1, 55.8, 44.6, 41.6, 39.4, 38.8, 33.4, 33.3, 30.0, 23.3, 21.5, 21.2, 20.4, 18.4, 15.2.¹⁹F NMR (376 MHz, CDCl₃) δ -118.45. HRMS (ESI+) m/z Calcd for C₂₄H₃₃FClSN₂O₂ [M + H]⁺ 467.19298; Found 467.19138.

(1R,2R,8aS)-1-((5-((4-bromobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H18). Yield 62%; White solid; m. p.99–101°C. ¹H NMR (400 MHz, CDCl₃) δ 7.46 (s, 1H), 7.44 (s, 1H), 7.31 (s, 1H), 7.29 (s, 1H), 4.36 (s, 2H), 2.89 (ddd, J = 77.1, 16.3, 5.7 Hz, 2H), 1.96–1.85 (m, 2H), 1.63–1.42 (m, 4H), 1.41–1.29 (m, 4H), 1.28–1.24 (m, 1H), 1.20 (s, 3H), 0.99 (dd, J = 12.1, 2.2 Hz, 1H), 0.88 (s, 3H), 0.86 (s, 3H), 0.80 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.3, 162.7, 135.0, 131.8, 131.8, 130.8, 122.0, 122.0, 73.2, 59.0, 55.8, 44.5, 41.5, 39.4, 38.8, 36.1, 33.4, 33.2, 23.3, 21.4, 21.1, 20.4, 18.3, 15.1. HRMS (ESI+) m/z Calcd for C₂₄H₃₄BrSN₂O₂ [M + H]⁺ 493.15189; Found 493.15070.

(1R,2R,8aS)-1-((5-((4-methoxybenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H19). Yield 70%; White solid; m. p.86–88°C. ¹H NMR (400 MHz, CDCl₃) δ 7.34 (s, 1H), 7.32 (s, 1H), 6.86 (s, 1H), 6.84 (s, 1H), 4.39 (s, 2H), 3.79 (s, 3H), 2.90 (ddd, J = 78.2, 16.3, 5.6 Hz, 2H), 1.92 (ddd, J = 16.1, 8.8, 4.4 Hz, 2H), 1.77–1.65 (m, 2H), 1.60–1.49 (m, 2H), 1.46–1.33 (m, 4H), 1.29–1.23 (m, 1H), 1.20 (s, 3H), 1.00 (dd, J = 12.1, 2.2 Hz, 1H), 0.88 (s, 3H), 0.87 (s, 3H), 0.80 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.1, 163.2, 159.3, 130.3, 127.5, 114.1, 73.2, 59.0, 55.7, 55.2, 44.4, 41.5, 39.3, 38.8, 36.4, 33.3, 33.2, 23.2, 21.4, 21.1, 20.3, 18.3, 15.1. HRMS (ESI+) m/z Calcd for C₂₅H₃₇SN₂O₃ [M + H]⁺ 445.25194; Found (H20). Yield 75%; White solid; m. p.98–100°C. ¹H NMR (400 MHz, CDCl₃) δ 7.52 (s, 1H), 4.55 (s, 2H), 2.92 (ddd, J = 75.5, 16.2, 5.7 Hz, 2H), 1.96–1.89 (m, 2H), 1.75–1.69 (m, 2H), 1.55–1.50 (m, 2H), 1.45–1.34 (m, 4H), 1.28–1.25 (m, 1H), 1.22 (s, 3H), 1.01 (dd, J = 12.2, 2.2 Hz, 1H), 0.88 (s, 3H), 0.87 (s, 3H), 0.80 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.8, 161.9, 152.3, 140.9, 135.9, 73.2, 59.0, 55.7, 44.5, 41.5, 39.4, 38.7, 33.3, 33.2, 28.5, 23.3, 21.4, 21.1, 20.3, 18.3, 15.1. HRMS (ESI+) m/z Calcd for C₂₁H₃₀ClS₂N₃O₂Na [M + Na]⁺ 478.13602; Found 478.13550.

(1R,2R,8aS)-1-((5-((4-bromo-2-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (H21). Yield 68%; White solid; m. p.120–122°C. ¹H NMR (400 MHz, CDCl₃) δ 7.40 (t, J = 8.3 Hz, 1H), 7.23 (dd, J = 7.1, 2.0 Hz, 2H), 4.38 (s, 2H), 2.89 (ddd, J = 77.6, 16.3, 5.6 Hz, 2H), 1.92 (ddd, J = 13.8, 8.7, 4.4 Hz, 2H), 1.63–1.42 (m, 4H), 1.41–1.30 (m, 4H), 1.28–1.24 (m, 1H), 1.20 (s, 3H), 1.00 (dd, J = 12.1, 2.2 Hz, 1H), 0.88 (s, 3H), 0.86 (s, 3H), 0.80 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.4, 162.6, 160.6 (d, J = 253.0 Hz), 132.4 (d, J = 4.0 Hz), 127.6 (d, J = 3.8 Hz), 122.7 (d, J = 14.6 Hz), 122.3 (d, J = 9.5 Hz), 119.2 (d, J = 24.4 Hz), 73.2, 59.0, 55.7, 44.5, 41.5, 39.3, 38.7, 33.3, 33.2, 29.5, 23.3, 21.4, 21.1, 20.3, 18.3, 15.1.¹⁹F NMR (376 MHz, CDCl3) δ -113.84. HRMS (ESI+) m/z Calcd for C₂₄H₃₃BrFSN₂O₂ [M + H]⁺ 511.14247; Found 511.14252.

(1R,2R,8aS)-2,5,5,8a-tetramethyl-1-((5-((4-(trifluoromethoxy)benzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)decahydronaphthalen-2-ol (H22). Yield 70%; White solid; m. p.83–85°C. ¹H NMR (500 MHz, CDCl₃) δ 7.45 (s, 1H), 7.43 (s, 1H), 7.16 (s, 1H), 7.14 (s, 1H), 4.39 (s, 2H), 2.88 (ddd, J = 21.7, 16.4, 5.8 Hz, 2H), 1.90 (ddd, J = 15.5, 7.8, 4.4 Hz, 2H), 1.60–1.39 (m, 4H), 1.38–1.22 (m, 4H), 1.19 (s, 3H), 1.14–1.07 (m, 1H), 0.98 (dd, J = 12.1, 2.2 Hz, 1H), 0.86 (s, 3H), 0.85 (s, 3H), 0.78 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.4, 162.8, 148.9, 134.7, 130.7, 121.2, 120.4 (d, J = 257.8 Hz), 73.3, 59.1, 55.8, 44.6, 41.6, 39.4, 38.8, 35.9, 33.4, 33.3, 23.4, 21.5, 21.2, 20.4, 18.4, 15.2.¹⁹F NMR (376 MHz, CDCl₃) δ -57.72. HRMS (ESI+) m/z Calcd for C₂₅H₃₃F₃SN₂O₃ [M + H]⁺ 521.20562; Found 521.20575.

(1R,2R,8aS)-2,5,5,8a-tetramethyl-1-((5-((4-nitrobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)decahydronaphthalen-2-ol (H23). Yield 53%; White solid; m. p.105–107°C. ¹H NMR (500 MHz, CDCl₃) δ 8.17 (s, 1H), 8.15 (s, 1H), 7.61 (s, 1H), 7.59 (s, 1H), 4.45 (s, 2H), 2.87 (ddd, J = 21.6, 16.3, 5.7 Hz, 2H), 1.94–1.83 (m, 2H), 1.60–1.39 (m, 4H), 1.38–1.29 (m, 4H), 1.18 (s, 3H), 1.13–1.06 (m, 1H), 0.96 (dd, J = 12.2, 2.1 Hz, 1H), 0.86 (s, 3H), 0.84 (s, 3H), 0.78 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.7, 162.2, 147.5, 143.7, 130.1, 124.0, 73.3, 59.1, 55.8, 44.6, 41.6, 39.4, 38.8, 35.7, 33.4, 33.3, 23.4, 21.5, 21.2, 20.4, 18.4, 15.2. HRMS (ESI+) m/z Calcd for C₂₄H₃₄SN₃O₄ [M + H]⁺ 460.22645; Found 460.22681.

Biological Activity Test Method

The in vitro antibacterial activities of target compounds H1-H23 against Xoo and Xac was evaluated by the turbidity method (Zhang et al., 2021). According to Schaad’s method (Zhang et al., 2021), the curative and protective activities of compound H8 against rice bacterial blight were determined in vivo. Based on the previous work (Wang et al., 2019; Luo et al., 2020), TMV was extracted and purified, and the interaction mode of active molecules with TMV-CP was explored by molecular docking. Detailed methods for bacterial bioactivity testing, as well as specific steps for TMV extraction and purification can be found in the Supplementary Datasheet S1.

Conclusion

In conclusion, a series of 1,3,4-oxadiazole contained sesquiterpene derivatives were synthesized, and the biological activity of title compounds was evaluated. The results exhibited that the synthetic compounds had good antibacterial activity against Xoo and Xac. The EC₅₀ values of compounds H4, H8, H11, H12, H14, H16, and H19 for Xac inhibitory activity were 33.3, 42.7, 56.1, 74.5, 37.8, 43.8, and 38.4 μg/ml, respectively. Compounds H4, H8, H15, H19, H22, and H23 had inhibitory effects on Xoo, with EC₅₀ values of 51.0, 43.3, 43.4, 50.5, 74.6, and 51.4 μg/ml, respectively. In particular, the curative and protective activities of compound H8 were 51.9 and 49.3%, respectively, showing good antibacterial activity against Xoo in vitro. In addition, the EC₅₀ values of the inactivation activities of the compounds H4, H5, H9, H10, and H16 against TMV were 69.6, 58.9, 69.4, 43.9, and 60.5 μg/ml, respectively. It is worth noting that the molecular docking results indicated that compound H10 binds to the active site of TMV-CP through amino acid residues ASN73, VAL260, TYR139, ELU131, and THR136. And it existed a strong affinity for TMV-CP, with a binding energy of -8.88 kcal/mol. Thus, the process of self-assembly and replication of TMV particles is inhibited and the anti-TMV effect is played.

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