Design, synthesis and evaluation of herbicidal activity of novel carboxylic acid inhibitors containing 1,3,4 oxadiazole fragments

Abstract

Carboxylic acid and 1,3,4-oxadiazole moieties are key structural components in many synthetic bioactive agents. A series of novel 2-[(5-phenyl-1,3,4-oxadiazol-2-yl)thio]propanoate derivatives (5aa–5bb) were designed and synthesized, and their structures were confirmed by ¹H NMR, ¹³C NMR, and HRMS. The spatial configuration of 2-[(5-phenyl-1,3,4-oxadiazol-2-yl)thio]propanoate (5aa) was further determined by X-ray diffraction analysis. The synthesized target compounds were evaluated for in vitro herbicidal activity, from which compound 5ap was identified as the most potent (ED₅₀ = 96.89 g/ha; field trial dosage: 750 g/ha). Moreover, 5ap exhibited high selectivity toward rice and maize, along with low toxicity to honeybees. Transmission electron microscopy (TEM) analysis suggested that 5ap likely disrupts normal metabolic processes and photosynthesis in plants, ultimately leading to plant death. In summary, compound 5ap demonstrates promising herbicidal activity along with favorable biosafety profiles, indicating its potential as a candidate for further development as a herbicide.

1 Introduction

Food security is a paramount national priority. However, crop production has long been severely impacted by weed infestation, resulting in direct annual economic losses amounting to USD 32 billion due to weed-related damage (Sreekanth et al., 2025; Zheng et al., 2026). Herbicides can effectively reduce weed damage when used properly. However, long-term use of a single herbicide often leads to pesticide residues and the development of resistant weeds (Cheng et al., 2025; Palma-Bautista et al., 2026; Zhang et al., 2026). According to statistics, 273 weed species have developed resistance to existing herbicide mechanisms and target sites (https://www.weedscience.org/Pages/Species.aspx). Therefore, developing new, green, and low-toxicity herbicides has become an urgent priority.

The development and utilization of heterocyclic compounds have long been a research focus in the pharmaceutical field (Atmaram and Roopan, 2022). 1,3,4-Oxadiazole is an important nitrogen and oxygen-containing heterocyclic scaffold that has attracted considerable attention in the design of bioactive molecules (Chen et al., 2025; Yin et al., 2025; Devadiga et al., 2026). More importantly, this structural motif has also been successfully introduced into agrochemicals, and some 1,3,4-oxadiazole-containing herbicides, such as flusulfinam and oxadiargyl, have been commercialized. These examples demonstrate that 1,3,4-oxadiazole is a valuable heterocyclic core for the discovery of new herbicidal compounds. Therefore, introducing a 1,3,4-oxadiazole moiety into candidate structures may provide a feasible strategy for the development of novel herbicides (Jiang et al., 2010; Sanyal et al., 2015; Li et al., 2025; Liu et al., 2025; Wang et al., 2025).

Carboxylic acid compounds have made indelible contributions to the field of pesticide innovation, particularly in the development of herbicides (Lei et al., 2019; Liu et al., 2024). Carboxylic acid herbicides, more accurately referred to as synthetic auxin herbicides, serve as the cornerstone of the modern era of selective chemical weed control. Since their introduction in the 1940s, they have fundamentally transformed the management of broadleaf weeds, marking the dawn of a new epoch in chemical weed control. To this day, they remain one of the most widely used classes of herbicides globally (Schulz et al., 2025; Ung et al., 2025; Zhang et al., 2026). Their mechanism of action involves inhibiting photosynthesis, disrupting the synthesis of nucleic acids and proteins, interfering with plant hormone balance, and impeding the uptake of water and inorganic salts by roots. This cascade of physiological disruptions ultimately leads to the death of target weeds (Grossmann, 2010; Cai et al., 2023). Currently, common commercially available carboxylic acid herbicides include 2,4-D, MCPA, and dicamba, among others (Blachnio et al., 2023; Pernak et al., 2024; Qi et al., 2025).

In this study, a series of 2-[(5-phenyl-1,3,4-oxadiazol-2-yl) thio] propanoate derivatives were synthesized via the active fragment fusion strategy by linking 1,3,4-oxadiazole with carboxylic acid structural moieties (Figure 1). Herbicidal activity assays were conducted on the target compounds, leading to the identification of the most representative bioactive compound within the series. Building on this, the median effective dosage (ED₅₀) and weed control spectrum of the compound were evaluated, along with its safety assessment on crops and honeybees. The efficacy was further validated through field plot trials. Finally, transmission electron microscopy (TEM) was employed to observe the ultrastructural damage characteristics in leaf cells, aiming to preliminarily explore its potential herbicidal mechanism of action.

Figure 1

2 Materials and methods

2.1 Test reagents and instruments

All reagents and chemicals were purchased from commercial sources. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Avance-400 spectrometer (Bruker BioSpin AG, Fällanden, Switzerland). Using dimethyl sulfoxide-d6 and chloroform-d6 (DMSO-d6, CD₃Cl) as solvents, and tetramethylsilane (TMS) as the internal standard. High-resolution mass spectrometry analysis was conducted using the FTICR-MS instrument from Varian 7.0 T (Varian IonSpec, Lake Forest, CA, USA). Single crystal X-ray diffraction analysis was performed on the Bruker SMART APEX II X-ray single crystal diffractometer (Bruker AXS, Karlsruhe, BW, Germany). 3WP-2000 Self-Propelled Bioassay Spray Tower: Travel distance of 1340 mm, spray volume of 30 mL, and spray pressure of 0.3 MPa (Nanjing Institute of Agricultural Mechanization, Ministry of Agriculture and Rural Affairs, Nanjing, China). Linong HD400 Manual Knapsack Sprayer: Equipped with a herbicide-specific flat fan nozzle, operating at a spray pressure of 450 kPa and an application water volume of 600 L/ha (manufactured by Linong (Singapore) Pte. Ltd.),. Ultra-thin sectioning machine (Leica UC7), Leica; Transmission electron microscope (HT7700), Corporation Hitachi Manufacturing Co., Ltd. 150-mesh Fanghua membrane copper mesh (AZH150), Beijing Zhongjingkeyi Technology Co., Ltd. (Beijing, China).

2.2 Test plants

The test plant seeds were all provided by the Seed Resource Library of the Biotechnology Institute of the Hunan Academy of Agricultural Sciences. They included Echinochloa crus-galli, Amaranthus retroflexus, Portulaca oleracea, Digitaria sanguinalis, Rumex acetosa, Monochoria vaginalis, Lactuca sativa, Oryza, Zea mays, Nicotiana tabacum and Glycine max.

2.3 Synthetic procedures

2.3.1 Synthesis of compounds 2aa–2bb

Synthesis of compound 2aa as an example. In a 100 mL round-bottom flask, benzoic acid (1.22 g, 10 mmol) was dissolved in methanol (25 mL). H₂SO₄ (36.8 mmol, 2 mL) was slowly added dropwise at room temperature, and the resulting mixture was heated to 90 °C under reflux. The reaction was monitored by TLC. Upon completion, the reaction mixture was extracted with ethyl acetate (2×50 mL). The combined organic layers were washed with saturated aqueous NaHCO₃ (1×50 mL) and brine (2×50 mL), then dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The desired product (2aa) was obtained as a yellow oil (1.02 g, 83% yield).

2.3.2 Synthesis of compounds 3aa–3bb

Synthesis of compound 3aa as an example. In a 100 mL round-bottom flask, compound 2aa (0.95 g, 7 mmol) was dissolved in methanol (25 mL). Hydrazine hydrate (50%, 20.4 mmol, 2 mL) was added at room temperature, and the resulting mixture was heated to 90 °C under reflux. The reaction was monitored by TLC. Upon completion, part of the methanol was removed under reduced pressure, and the concentrated solution was stored in a refrigerator at 4 °C overnight. A white solid precipitated, which was collected by filtration and dried to give compound 3aa (0.8 g, 84% yield).

2.3.3 Synthesis of compounds 4aa–4bb

Synthesis of compound 4aa as an example. In a 100 mL round-bottom flask, compound 3aa (0.8 g, 5.9 mmol) was dissolved in methanol (25 mL). Potassium hydroxide (12 mmol, 0.76 g) and carbon disulfide (66 mmol, 4 mL) were added at room temperature, and the resulting mixture was heated to 90 °C under reflux. The reaction was monitored by TLC. Upon completion, the excess methanol was removed under reduced pressure. The residue was extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with saturated aqueous brine (2 × 50 mL), then dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The desired product (4aa) was obtained as a white solid (0.64 g, 62% yield).

2.3.4 Synthesis of target compounds 5aa–5bb

Synthesis of compound 5aa as an example. In a 100 mL round-bottom flask, compound 4aa (0.36 g, 2 mmol) was dissolved in DMF (25 mL). Add K₂CO₃ (0.42 g, 3 mmol) and methyl 2-bromopropionate (0.37 g, 2.2 mmol) sequentially, and heat to 60 °C. The reaction was monitored by TLC. Upon completion, the reaction mixture was extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with saturated brine (2 × 50 mL), then dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The desired product (5aa) was obtained as a yellow solid (0.43 g, 81% yield).

2.4 X-ray diffraction of compound 5aa

The temperature for the diffraction experiment was 50.0(10) K. Using Cu Kα as the incident light (λ = 1.54184), the maximum value of the maximum 2θ angle for data collection was 146.188. A total of 6680 diffraction points were collected, and 2235 independent diffraction points were obtained (Rint = 0.01177). Transparent crystals of appropriate size of 0.14 mm × 0.11 mm × 0.09 mm were obtained by recrystallization from a 1:10 mixture of dichloromethane and ethanol. The crystallographic data of the target compound has been uploaded to the Cambridge Crystallographic Data Centre (CCDC), with the CCDC number being 2481325. The X-ray single crystal diffraction data of the target compound 5aa can be accessed through https://www.ccdc.cam.ac.uk (Figure 2).

Figure 2

2.5 Biological assays

2.5.1 Greenhouse weeding activity

At a dosage of 375 g/ha, the herbicidal activity of the target compounds 5aa-5bb against E.c. and A.r. was determined. The test weeds were treated with the post-emergence method by stem and leaf spraying. Dissolve the target compounds 5aa-5bb in an appropriate amount of DMF, then dilute to 20 mL with 0.6% Tween 80 aqueous solution. The single-leaf weeds were treated with the measured at the 2–3 leaf stage, and the double-leaf weeds were treated at the 2–4 leaf stage. The untreated weeds were mixed with an equal amount of DMF and 0.6% Tween 80 solution for a blank control. Each treatment was repeated three times. Quinclorac and 2,4-D were used as positive controls. After 7 days, the aboveground parts of the plants were cut and weighed to calculate the fresh weight inhibition rate; The term half maximal effective dosage (ED₅₀) was calculated by the inhibition rate of A.r. under the application dosage of 0, 18.75, 37.5, 75, 93.75, 112.5, 150, 187.5, 225 and 375 g/ha were sprayed on amaranth. All tests were repeated in three times.

E: Inhibition rate

C: Plant height or fresh weight of control group plants

T: Plant height or fresh weight of treatment group plants

2.5.2 Crop safety and herbicidal spectrum

To determine the safety of highly active compounds on common crops and their herbicidal spectra, the stem and leaf spraying method was used at a dosage of 1125 g/ha to assess the safety of the highly active compounds on four crops (rice, tobacco, corn, and soybean); At a dosage of 375 g/ha, the herbicidal activity against several weeds (ragweed, wild buckwheat, duckweed, purslane, and watercress) was evaluated, and 2,4-D was used as a positive control. The herbicidal effect was observed 7 days after application, and the aboveground parts were cut off to calculate the fresh weight inhibition rate.

2.5.3 Field plot experiment

To further assess the herbicidal potential of highly active compounds, a field plot experiment was conducted using paddy fields as the test subjects. The rice was sown by direct seeding and the experiment was carried out when the plants reached the 3–5 leaf stage. At a dosage of 750 g/ha, the herbicidal activity was determined by foliar spraying. 2,4-D was used as the positive control. There were a total of 3 treatments in the experiment, with 2 replicates for each treatment, arranged in a randomized block design. The plot area was 2.5 m² (1 × 2.5 m²). The conditions of each treatment were observed by visual inspection 7 days and 14 days after application.

2.6 Bees safety test

According to the international standard OECD NO.245, the acute contact toxicity of highly active compounds to bees was determined using the oral feeding method, with 2,4-D as the positive control. Adult worker bees of uniform size, lively and healthy were selected as the test bees. They were acclimated for 24 hours before the experiment, and the mortality rate during the acclimation period was 0. The test bees for the oral test were stopped from feeding 2 hours before the experiment. The dose for the oral toxicity test of bees was set at 200 μg per bee, and a blank control was also set. There were 15 bees in each repetition, and 4 repetitions were set for each treatment. After the bees finished drinking the liquid medicine, they were fed with sucrose water. 96 h after the administration of the medicine, the mortality rate of each treatment was counted.

2.7 Characterization by transmission electron microscopy

To preliminarily explore the potential herbicidal mechanism of highly active compounds, the damage characteristics of chloroplasts after treatment with the compounds were characterized and observed by electron microscopy. The ultrastructure of chloroplasts in the leaves of pigweed was observed by spraying the ED₅₀ (96.89 g/ha) dosage of the compound on the leaves, and 2,4-D was selected as the positive control. The specific operation of the transmission electron microscope follows the methods described in the literature (Mo et al., 2025).

3 Results and discussion

3.1 Synthesis

The synthetic route of the target compounds is illustrated in Figure 3. Compounds 2aa–2bb were obtained as benzoate esters (yields 90–97%) by refluxing differently substituted benzoic acids with sulfuric acid in methanol at 90 °C. Compounds 3aa–3bb were prepared by reacting the corresponding substituted benzoate esters with hydrazine hydrate in methanol at 90 °C (yields 53–67%). Compounds 4aa–4bb were synthesized from compounds 3aa–3bb with potassium hydroxide, carbon disulfide, and methanol at 90 °C (yields 85–93%). Compounds 5aa–5bb were formed by the reaction of 5-phenyl-1,3,4-oxadiazole-2-thiol with methyl 2-bromopropionate in the presence of potassium carbonate in DMF at 60 °C (yields 62–83%). The structures of all target compounds were confirmed by ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry (HRMS). In addition, the structure of compound 5aa was unequivocally verified by X-ray single-crystal diffraction.

Figure 3

Methyl 2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)propanoate(5aa). yellow solid, yield 81%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 8.02-8.00 (m, 2H, Ar-H), 7.56-7.48 (m, 3H, Ar-H), 4.55-4.49 (m, 1H, SCHCH₃), 3.79 (s, 3H, OCH₃), 1.75 (d, J=7.6 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.37, 166.14, 162.44, 131.86, 129.10, 126.76, 123.47, 53.15, 44.55, 18.21. HRMS (ESI) C₁₂H₁₂N₂O₃S [M+H]⁺: calcd. 265.0642, found 265.0636.

Methyl 2-((5-(o-tolyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ab). Yellow solid, yield 87%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.87 (d, J=6.4 Hz, 1H, Ar-H), 7.42-7.39 (m, 1H, Ar-H), 7.34-7.29 (m, 2H, Ar-H), 4.54-4.49 (m, 1H, SCHCH₃), 3.79 (s, 3H, OCH₃), 2.69 (s, 3H, CH₃), 1.75 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.37, 166.31, 162.20, 138.35, 131.76, 131.33, 128.75, 126.19, 122.48, 53.13, 44.47, 22.09, 18.20. HRMS (ESI) C₁₃H₁₄N₂O₃S [M+H]⁺: calcd. 279.0798, found 279.0797.

Methyl 2-((5-(m-tolyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ac). Yellow solid, yield 83%. ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 7.79-7.75 (m, 2H, Ar-H), 7.51-7.44 (m, 2H, Ar-H), 4.61-4.56 (m, 1H, SCHCH₃), 3.70 (s, 3H, OCH₃), 2.40 (s, 3H, CH₃), 1.62 (d, J=7.6 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆, δ ppm): 171.35, 166.15, 161.95, 139.47, 133.32, 129.84, 127.22, 124.11, 123.28, 53.54, 44.32, 21.27, 18.24. HRMS (ESI) C₁₃H₁₄N₂O₃S [M+H]⁺: calcd. 279.0798, found 279.0797.

Methyl 2-((5-(p-tolyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ad). Yellow solid, yield 84%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.89 (d, J=8.4 Hz, 2H, Ar-H), 7.30 (d, J=8.0 Hz, 2H, Ar-H), 4.53-4.47 (m, 1H, SCHCH₃), 3.79 (s, 3H, OCH₃), 2.42 (s, 3H, CH₃), 1.74 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.38, 166.30, 161.99, 142.43, 129.79, 126.70, 120.68, 53.11, 45.51, 21.65, 18.19. HRMS (ESI) C₁₃H₁₄N₂O₃S [M+H]⁺: calcd. 279.0798, found 279.0797.

Methyl 2-((5-(2,3-dimethylphenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ae). Yellow solid, yield 83%. ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 7.62 (d, J=8.0 Hz, 1H, Ar-H), 7.42 (d, J=7.2 Hz, 1H, Ar-H), 7.29 (t, J=7.6 Hz, 1H, Ar-H), 4.60-4.54 (m, 1H, SCHCH₃), 3.70 (s, 3H, OCH₃), 2.48 (s, 3H, CH₃), 2.34 (s, 3H, CH₃), 1.63 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆, δ ppm): 171.38, 166.72, 161.71, 138.76, 136.71, 133.49, 127.45, 126.52, 122.98, 53.33, 44.19, 20.64, 18.23, 17.16. HRMS (ESI) C₁₄H₁₆N₂O₃S [M+H]⁺: calcd. 293.0956, found 293.0955.

Methyl 2-((5-(2,4-dimethylphenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5af). Yellow solid, yield 81%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.77 (d, J=7.6 Hz, 1H, Ar-H), 7.15 (s, 1H, Ar-H), 7.12 (d, J=7.6 Hz, 1H, Ar-H), 4.53-4.48 (m, 1H, SCHCH₃), 3.79 (s, 3H, OCH₃), 2.65 (s, 3H, CH₃), 2.38 (s, 3H, CH₃), 1.75 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.74, 166.52, 161.64, 141.79, 138.23, 132.54, 128.76, 126.97, 119.74, 53.13, 44.46, 22.02, 21.41, 18.21. HRMS (ESI) C₁₄H₁₆N₂O₃S [M+H]⁺: calcd. 293.0956, found 293.0955.

Methyl 2-((5-(2,5-dimethylphenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ag). Yellow solid, yield 80%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.69 (s, 1H, Ar-H), 7.21 (s, 2H, Ar-H), 4.54-4.49 (m, 1H, SCHCH₃), 3.79 (s, 3H, OCH₃), 2.63 (s, 3H, CH₃), 2.37 (s, 3H, CH₃), 1.75 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.41, 166.52, 161.90, 135.80, 135.26, 132.18, 131.71, 129.20, 122.24, 53.13, 44.49, 21.56, 20.82, 18.21. HRMS (ESI) C₁₄H₁₆N₂O₃S [M+H]⁺: calcd. 293.0956, found 293.0955.

Methyl 2-((5-(3,4-dimethylphenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ah). Yellow solid, yield 79%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.79 (s, J=7.6 Hz, 1H, Ar-H), 7.25 (d, J=7.6 Hz, 1H, Ar-H), 4.53-4.47 (m, 1H, SCHCH₃), 3.79 (s, 3H, OCH₃), 2.33 (s, 6H, CH₃), 1.74 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.41, 166.44, 161.88, 141.19, 137.60, 130.31, 122.72, 124.28, 120.95, 53.10, 44.52, 20.00, 19.73, 18.20. HRMS (ESI) C₁₄H₁₆N₂O₃S [M+H]⁺: calcd. 293.0956, found 293.0955.

Methyl 2-((5-(3,5-dimethylphenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ai). Yellow solid, yield 81%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.62 (s, 2H, Ar-H), 7.16 (s, 1H, Ar-H), 4.54-4.48 (m, 1H, SCHCH₃), 3.79 (s, 3H, OCH₃), 2.38 (s, 6H, CH₃), 1.75 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.41, 166.48, 162.13, 138.86, 133.60, 124.48, 123.20, 53.12, 44.53, 21.22, 18.22. HRMS (ESI) C₁₄H₁₆N₂O₃S [M+H]⁺: calcd. 293.0956, found 293.0955.

Methyl 2-((5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5aj). Yellow solid, yield 81%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.96-7.94 (m, 1H, Ar-H), 7.56-7.54 (m, 1H, Ar-H), 7.49-7.45 (m, 1H, SCHCH₃), 7.43-7.38 (m, 1H, Ar-H), 4.56-4.50 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.76 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.29, 164.43, 163.18, 133.08, 132.52, 131.30, 131.00, 127.13, 122.76, 53.18, 44.59, 18.25. HRMS (ESI) C₁₂H₁₁ClN₂O₃S [M+H]⁺: calcd 299.0252, found 299.0251.

Methyl 2-((5-(3-chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ak). Yellow solid, yield 83%.¹H NMR (400 MHz, CDCl₃, δ ppm): 8.00 (s, 1H, Ar-H), 7.90 (d, J=6.0 Hz, 1H, Ar-H), 7.52-7.50 (m, 1H, Ar-H), 7.45 (t, J=8.0 Hz, 1H, Ar-H), 4.56-4.51 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.76 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.28, 164.94, 135.26, 131.89, 130.49, 126.73, 125.07, 124.83, 53.20, 44.61, 18.20. HRMS (ESI) C₁₂H₁₁ClN₂O₃S [M+H]⁺: calcd 299.0252, found 299.0251.

Methyl 2-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate. (5al) Yellow solid, yield 81%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.96-7.93 (m, 2H, Ar-H), 7.50-7.47 (m, 2H, Ar-H), 4.55-4.50 (m, 1H, SCHCH₃), 3.79 (s, 3H, OCH₃), 1.75 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.26, 165.26, 138.10, 129.49, 128.00, 121.92, 53.16, 44.58, 18.18. HRMS (ESI) C₁₂H₁₁ClN₂O₃S [M+H]⁺: calcd 299.0252, found 299.0251.

Methyl 2-((5-(2,3-dichlorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5am). Yellow solid, yield 84%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.86-7.83 (m, 1H, Ar-H), 7.67-7.64 (m, 1H, Ar-H), 7.46 (t, J=8.0 Hz, 1H, Ar-H), 4.57-4.51 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.77 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.19, 163.90, 163.59, 135.09, 133.31, 131.60, 129.37, 127.66, 124.89, 53.21, 44.62, 36.51, 18.23. HRMS (ESI) C₁₂H₁₀Cl₂N₂O₃S [M+H]⁺: calcd 332.9862, found 332.9860.

Methyl 2-((5-(2,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5an). Yellow solid, yield 83%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.91 (d, J=8.4 Hz, 1H, Ar-H), 7.57 (d, J=2.0 Hz, 1H, Ar-H), 7.41-7.38 (m, 1H, Ar-H), 4.56-4.50 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.76 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.21, 163.67, 163.42, 138.22, 133.81, 131.64, 131.47, 131.23, 131.08, 127.67, 121.57, 121.24, 53.22, 44.64, 18.22. HRMS (ESI) C₁₂H₁₀Cl₂N₂O₃S [M+H]⁺: calcd 332.9862, found 332.9860.

Methyl 2-((5-(2,5-dichlorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ao). Yellow solid, yield 83%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.95 (d, J=2.4 Hz, 1H, Ar-H), 7.49 (d, J=8.8 Hz, 1H, Ar-H), 7.45-7.42 (m, 1H, Ar-H), 4.57-4.52 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.77 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.17, 163.66, 163.27, 133.20, 132.51, 132.40, 131.28, 130.49, 123.93, 53.21, 44.63, 18.22. HRMS (ESI) C₁₂H₁₀Cl₂N₂O₃S [M+H]⁺: calcd 332.9862, found 332.9860.

Methyl 2-((5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ap). Yellow solid, yield 81%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 8.09 (d, J=6.0 Hz, 1H, Ar-H), 7.86-7.83 (m, 1H, Ar-H), 7.59 (d, J=8.4 Hz, 1H, Ar-H), 4.57-4.51 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.76 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.19, 164.22, 163.30, 136.31, 133.74, 131.32, 128.41, 125.72, 123.22, 53.21, 44.65, 18.19. HRMS (ESI) C₁₂H₁₀Cl₂N₂O₃S [M+H]⁺: calcd 332.9862, found 332.9860.

Methyl 2-((5-(3,5-dichlorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5aq). Yellow solid, yield 88%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.90 (d, J=1.6 Hz, 2H, Ar-H), 7.52 (t, J=2.0 Hz, 1H, Ar-H), 4.58-4.52 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.77 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.18, 163.88, 163.66, 136.07, 135.37, 131.69, 128.27, 126.03, 124.97, 53.23, 44.67, 18.19. HRMS (ESI) C₁₂H₁₀Cl₂N₂O₃S [M+H]⁺: calcd 332.9862, found 332.9860.

Methyl 2-((5-(2-fluorophenyl)-1,3,4-oxadiazol-2-yl)thio) propanoate(5ar). Yellow solid, yield 88%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 8.04-8.00 (m, 1H, Ar-H), 7.57-7.51 (m, 1H, Ar-H), 7.31-7.22 (m, 2H, Ar-H), 4.55-4.49 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.75 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.29, 163.01, 163.00, 162.89, 162.84, 161.16, 158.59, 133.69, 133.61, 129.55, 129.54, 124.72, 124.68, 117.12, 116.92, 112.04, 111.93, 53.15, 44.55, 18.19. HRMS (ESI) C₁₂H₁₁FN₂O₃S [M+H]⁺: calcd. 283.0546, found 283.0548.

Methyl 2-((5-(3-fluorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5as). Yellow solid, yield 74%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.80 (d, J=8.0 Hz, 1H, Ar-H), 7.72-7.68 (m, 1H, Ar-H), 7.52-7.46 (m, 1H, Ar-H), 7.26-7.21 (m, 1H, Ar-H), 4.56-4.51 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.76 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.24, 165.07, 165.03, 164.02, 162.97, 161.55, 131.02, 130.94, 125.33, 125.24, 122.51, 122.48, 119.01, 118.80, 113.87, 113.63, 53.16, 44.58, 18.17. HRMS (ESI) C₁₂H₁₁FN₂O₃S [M+H]⁺: calcd. 283.0546, found 283.0548.

Methyl 2-((5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5at). Yellow solid, yield 74%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 8.03-8.00 (m, 2H, Ar-H), 7.22-7.18 (m, 2H, Ar-H), 4.54-4.49 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.75 (d, J=7.6 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.32, 166.09, 165.31, 163.57, 162.51, 129.11, 129.03, 119.84, 119.80, 116.59, 116.37, 53.16, 44.58, 18.19. HRMS (ESI) C₁₂H₁₁FN₂O₃S [M+H]⁺: calcd. 283.0546, found 283.0548.

Methyl 2-((5-(2,3-difluorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5au). Yellow solid, yield 71%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.81-7.77 (m, 1H, Ar-H), 7.40-7.34 (m, 1H, Ar-H), 7.29-7.22 (m, 1H, Ar-H), 4.56-4.51 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.76 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.19, 165.53, 161.98, 161.94, 152.44, 152.33, 149.96, 149.85, 149.70, 147.11, 146.97, 124.93, 124.88, 124.86, 124.81, 124.14, 124.10, 120.65, 120.48, 113.99, 113.91, 53.18, 44.59, 18.16. HRMS (ESI) C₁₂H₁₀F₂N₂O₃S [M+H]⁺: calcd. 300.1038, found 300.1034.

Methyl 2-((5-(2,4-difluorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5av). Yellow solid, yield 73%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.74-7.70 (m, 1H, Ar-H), 7.28-7.21 (m, 2H, Ar-H), 4.56-4.51 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.76 (d, J=7.6 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.20, 163.57, 161.93, 159.63, 159.61, 157.20, 157.18, 157.15, 154.64, 154.62, 120.49, 120.41, 120.25, 120.16, 118.66, 118.58, 118.43, 118.34, 115.78, 115.75, 115.51, 115.49, 113.05, 112.96, 112.91, 112.82, 53.18, 44.60, 18.17. HRMS (ESI) C₁₂H₁₀F₂N₂O₃S [M+H]⁺: calcd. 300.1038, found 300.1034.

Methyl 2-((5-(3,4-difluorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5aw). Yellow solid, yield 74%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.86-7.77 (m, 2H, Ar-H), 7.35-7.29 (m, 1H, Ar-H), 4.56-4.50 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.76 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.19, 164.30, 163.06, 153.93, 153.80, 151.91, 151.77, 151.38, 151.26, 149.41, 149.28, 123.58, 123.54, 123.51, 123.47, 120.49, 120.45, 120.42, 120.38, 118.54, 118.36, 116.23, 116.03, 53.16, 44.60, 18.14. HRMS (ESI) C₁₂H₁₀F₂N₂O₃S [M+H]⁺: calcd. 300.1038, found 300.1034.

Methyl 2-((5-(3,5-difluorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ax). Yellow solid, yield 74%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.57-7.53 (m, 2H, Ar-H), 7.02-6.97 (m, 1H, Ar-H), 4.58-4.52 (m, 1H, SCHCH₃), 3.81 (s, 3H, OCH₃), 1.76 (d, J=6.4 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.16, 164.54, 164.41, 164.21, 163.57, 162.05, 161.92, 126.20, 126.10, 125.99, 110.05, 109.97, 109.85, 109.77, 107.53, 107.28, 107.03, 53.21, 44.64, 18.17. HRMS (ESI) C₁₂H₁₀F₂N₂O₃S [M+H]⁺: calcd. 300.1038, found 300.1034.

Methyl 2-((5-(2-bromophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ay). Yellow solid, yield 74%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.90-7.88 (m, 1H, Ar-H), 7.76-7.74 (m, 1H, Ar-H), 7.47-7.43 (m, 1H, Ar-H), 7.41-7.36 (m, 1H, Ar-H), 4.56-4.51 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.76 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.24, 164.91, 163.20, 134.61, 132.61, 131.44, 127.65, 124.82, 121.50, 53.18, 44.60, 18.27. HRMS (ESI) C₁₂H₁₁BrN₂O₃S [M+H]⁺: calcd. 342.9747, found 342.9750.

Methyl 2-((5-(4-bromo-2-chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5az). Yellow solid, yield 82%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.84 (d, J=8.0 Hz, 1H, Ar-H), 7.73 (d, J=2.0 Hz, 1H, Ar-H), 7.56-7.54 (m, 1H, Ar-H), 4.56-4.51 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.76 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.18, 163.75, 163.44, 134.03, 133.82, 131.71, 130.58, 126.28, 121.67, 53.21, 44.63, 18.22. HRMS (ESI) C₁₂H₁₀BrN₂O₃S [M+H]⁺: calcd. 376.9357, found 376.9360.

Methyl 2-((5-(4-bromo-3-chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5ba). Yellow solid, yield 84%. ¹H NMR (400 MHz, CDCl₃, δ ppm): 8.08 (s, 1H, Ar-H), 7.76 (s, 2H, Ar-H), 4.57-4.52 (m, 1H, SCHCH₃), 3.80 (s, 3H, OCH₃), 1.76 (d, J=7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 171.20, 164.32, 163.34, 135.73, 134.63, 128.14, 126.54, 125.71, 123.88, 53.22, 44.65, 18.19. HRMS (ESI) C₁₂H₁₀BrN₂O₃S [M+H]⁺: calcd. 376.9357, found 376.9360.

Methyl 2-((5-(2-methyl-4-chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)propanoate(5bb). Yellow solid, yield 77%. ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 7.84 (d, J=8.4 Hz, 1H, Ar-H), 7.54 (s, 1H, Ar-H), 7.46 (d, J=6.0 Hz, 2H, Ar-H), 4.61-4.56 (m, 1H, SCHCH₃), 3.79 (s, 3H, OCH₃), 2.58 (s, 3H, CH₃), 1.63 (d, J=7.6 Hz, 3H, CH₃-H). ¹³C NMR (100 MHz, DMSO-d₆, δ ppm): 171.32, 165.49, 161.90, 140.42, 136.63, 131.83, 130.75, 127.04, 121.43, 53.35, 44.25, 21.60, 18.26. HRMS (ESI) C₁₃H₁₃ClN₂O₃S [M+H]⁺: calcd. 313.0409, found 313.0408.

3.2 Herbicidal activity

The inhibitory rates of the target compounds 5aa-5bb against the two weed species are shown in Table 1 and Supplementary Figures 1, 2. At the initial screening dosage of 375 g/ha, compounds 5ab, 5ai, 5am, 5ap, 5aq, 5au, 5ax, 5ay, and 5bb exhibited good herbicidal activity against A.r., comparable to the reference herbicide 2,4-D and superior to quinclorac. In terms of control efficacy against E.c., compounds 5ap and 5ba showed the best performance, both achieving over 80% inhibition, outperforming 2,4-D.

A follow-up screening was conducted for the compounds that demonstrated better efficacy against A.r. The inhibitory rates from the compound re-screening are shown in Table 1 and Supplementary Figure 3. At a dosage of 225 g/ha, compound 5ap exhibited significantly superior efficacy compared to the other compounds, with performance comparable to that of 2,4-D.

The herbicidal activity of the target compounds was significantly influenced by both the electronic nature and substitution pattern of the aromatic substituents. In general, halogen-substituted derivatives, especially chlorinated analogues, showed better herbicidal activity than methyl-substituted compounds. However, the activity pattern also depended strongly on the substitution position and the weed species tested. For example, among the monochloro-substituted derivatives, 5ak (3-Cl) exhibited stronger activity than 5aj (2-Cl) and 5al (4-Cl) against A.r., while 5al showed slightly higher activity than 5ak and 5aj against E.c. Furthermore, dichloro-substituted derivatives generally performed better than monochloro-substituted analogues, with 5ap (3,4-Cl) showing the most favorable overall herbicidal activity. These results suggest that introduction of chlorine atoms, particularly at the 3- and 4-positions of the phenyl ring, is beneficial for herbicidal activity in this compound series.

Through preliminary and follow-up screenings of active compounds, it was determined that compound 5ap is the most active compound in this series. Using growth inhibition rate as the y-axis and dosage value as the x-axis, the ED₅₀ value was calculated with GraphPad Prism 8.0.2. The determined ED₅₀ value for compound 5ap is 96.89 g/ha (Figure 4).

Figure 4

3.3 Crop safety and herbicidal spectrum of compound 5ap

At a dosage of 1125 g/ha, the safety of compound 5ap was evaluated on four crops (corn, rice, soybean, tobacco). Compound 5ap caused a certain degree of phytotoxicity to soybean and tobacco, with limited recovery in later stages, consistent with the reference herbicide 2,4-D. However, compound 5ap showed lower phytotoxicity to rice and corn, significantly less than that caused by 2,4-D, with no adverse effects observed in later growth stages, indicating relatively high safety. Therefore, compound 5ap shows potential for application in corn and rice fields (Supplementary Figure 4.).

To further evaluate the herbicidal potential of compound 5ap and understand its broad-spectrum activity, the herbicidal efficacy of compound 5ap was tested against five weed species. The results showed that compound 5ap did not exhibit significant herbicidal activity against M.v., P. p., with its effectiveness being lower than that of the reference herbicide 2,4-D. In contrast, compound 5ap demonstrated highly effective control against P.o., D.s., and L.s., achieving an inhibition rate of 100%. Notably, its efficacy against Digitaria sanguinalis was significantly superior to that of 2,4-D (Supplementary Figure 5). Therefore, compound 5ap shows potential for controlling weeds such as D.s. and P. o.

3.4 Compound 5ap herbicidal activity in rice fields

At 14 days after treatment, visual observation showed that at a dosage of 750 g/ha, compound 5ap achieved a comprehensive control efficacy of over 85% against weeds in rice fields, with no apparent phytotoxicity symptoms observed on rice plants, indicating relatively high safety. In contrast, the positive control 2,4-D exhibited significant phytotoxicity damage. In summary, compound 5ap demonstrates good herbicidal activity against weeds in rice fields and warrants further in-depth investigation (Supplementary Figure 6).

3.5 Bee safety assessment

The safety of the target compound 5ap was evaluated on honeybees at a dose of 200 μg/bee. The mortality rate in the acute oral toxicity test was 5%, which is superior to the reference herbicide 2,4-D, and no obvious poisoning symptoms were observed. This test was conducted as a limit test for acute oral contact of compound 5ap with honeybees. According to the classification standard for acute toxicity of pesticides to honeybees: extremely toxic (LD₅₀ ≤ 0.001 μg/bee); highly toxic (0.001 μg/bee< LD₅₀ ≤ 2.00 μg/bee); moderately toxic (2.00 μg/bee< LD₅₀ ≤ 11.00 μg/bee); and low toxicity (LD₅₀ > 11.00 μg/bee). Therefore, it can be preliminarily determined that compound 5ap exhibits low acute oral toxicity to honeybees (Supplementary Figure 7).

3.6 Transmission electron microscopy experiment

The high-activity compound 5ap and 2,4-D (positive control) were applied as foliar sprays at ED₅₀ dosage to A.r. leaves for observation of chloroplast ultrastructure. The TEM results showed that in the blank control group, the chloroplasts of A.r. cells appeared elongated and spindle-shaped, distributed at the cell periphery. The thylakoid membranes formed by grana stacking and the stroma were neatly arranged within the double membrane of chloroplasts, parallel to the long axis of the chloroplasts. After treatment with 5ap, the chloroplast structure of A.r. was significantly deformed, with cells undergoing plasmolysis. Chloroplasts were fragmented and dispersed, with most chloroplast envelopes changing from elongated to oval shapes. Osmiophilic granules appeared within the chloroplasts, the grana thylakoid lamellae disappeared, and the stroma stacking became disorganized. Following 2,4-D treatment, the chloroplast structure of A.r. was severely damaged, similar to that observed after 5ap treatment, with the appearance of more osmiophilic granules. Comparison between the blank control and treatment groups revealed that 5ap treatment caused substantial damage to the chloroplast structure of A.r., ultimately leading to abnormal growth of the weed (Figure 5). These ultrastructural changes suggest that compound 5ap may interfere with chloroplast-related physiological functions, particularly photosynthesis-associated metabolism, thereby resulting in abnormal growth and eventual death of the weed. However, the precise molecular target of 5ap remains to be clarified.

Figure 5

4 Conclusion

This study employed an active fragment splicing strategy to design and synthesize a series of methyl 2-[(5-phenyl-1,3,4-oxadiazol-2-yl)thio]propanoate derivatives. The structures were characterized using ¹H NMR, ¹³C NMR, and HRMS spectra. Among these compounds, compound 5ap demonstrated excellent herbicidal activity and good crop safety to rice, results that were further validated in field trials. In bee safety tests, compound 5ap exhibited low toxicity to honeybees. Transmission electron microscopy analysis revealed that 72 hours after treatment with compound 5ap, the chloroplast structure in leaf cells was damaged, cells underwent plasmolysis, and the cell membrane was severely deformed. Both osmiophilic granules and starch grains within the chloroplasts increased. These observations suggest that compound 5ap likely interferes with normal metabolic processes and photosynthesis in plants, ultimately leading to plant death. In conclusion, compound 5ap demonstrates promising herbicidal activity with high biological safety, indicating significant potential for further development.

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