High value-added application of natural forest product α-pinene: design, synthesis and 3D-QSAR study of novel α-campholenic aldehyde-based 4-methyl-1,2,4-triazole-thioether compounds with significant herbicidal activity

Abstract For exploring novel natural product-derived herbicides, 16 novel α-campholenic aldehyde-based 4-methyl-1,2,4-triazole-thioether compounds were designed, synthesized, and characterized by FT-IR, 1H NMR, 13C NMR, ESI-MS and elemental analysis. The preliminary bioassay showed that, at 100 µg/mL, most of the target compounds displayed significant inhibition activity against root-growth of rape(Brassica campestris L.), with inhibition rates of 85.0%～98.2%(A-class activity level), much better than that of the positive control flumioxazin. In addition, an effective and reasonable 3D-QSAR model was established by CoMFA method in SYBYL-X 2.1.1 software. It was found that, the steric field was the major factor towards the herbicidal activity of the target compounds against B. campestris L., and the introduction of bulky groups into m- and p-position of the benzene ring was favourable to increase the herbicidal activity. This kind of title compounds deserved further study as potential leading compounds for the discovery and development of novel herbicidal agents.


Introduction
Turpentine oil is an abundant natural forest biomass resource from living pine trees, and its dominant component a-pinene is a bicyclic monoterpene with a C ¼ C and a four-membered ring, which provides a great potential for its chemical modification with the aim of discovering novel herbicides to overcome the developed resistance of currently commercial herbicides to weeds (Lin et al. 2019), an essential issue in agriculture.As one of the key intermediates prepared by the epoxidation and catalytic isomerization of a-pinene (Sidorenko et al. 2018, Stekrova et al. 2015), a-campholenic aldehyde is also a natural product occurring in some essential oils, such as Eucalyptus globulus Labill (Li et al. 2007), and containing a five-membered ring along with a C¼C.It was found that, further structural modification of a-campholenic aldehyde could improve its biological activities, including cockroach-repellent (Han et al. 2012), antiinflammatory (Borisova et al. 2021, Borisova et al. 2018), and antifungal activity (Huang et al. 2016, Ma et al. 2016, Yin et al. 2016).
Recently, it has been found that 1,2,4-triazole-thioether compounds showed broadspectrum bioactivities, such as herbicidal activity (Li et al. 2021, Wang et al. 2021, Yu et al. 2020), antifungal (Huang et al. 2021, Lin et al. 2017), anticancer (Patel et al. 2021), antioxidant (Gultekin et al. 2018), and antiviral (Shao et al. 2021) activities.Herein, in continuation of our interest in natural product-derived bioactive derivatives (Huang et al. 2021, Lin et al. 2017, Lin et al. 2019, Li et al. 2022, Li et al. 2021, Li et al. 2021, Ma et al. 2016), a series of novel a-campholenic aldehyde-based 4-methyl-1,2,4triazole-thioether compounds were rationally designed.As shown in Figure 1, 1,2,4-triazole-thioether moiety has been proved to be a promising pharmacophore for the discovery of novel bioactive compounds, especially for that of leading compounds with herbicidal activity, and thus was integrated into the naturally bioactive skeleton of a-campholenic aldehyde.Then, the target compounds were synthesized by multi-step reaction and characterized by FT-IR, 1 H NMR, 13 C NMR, ESI-MS and elemental analysis.In addition, the herbicidal activity evaluation and three-dimensional quantitative structure-activity relationship(3D-QSAR) study of the target compounds were carried out as well.

Chemistry
As shown in Scheme 1, the target compounds 6a-p were synthesized by multi-step reaction.Firstly, 2,3-epoxypinane 2 was prepared by the oxidation of a-pinene in the presence of peracetic acid as oxidative agent.Then, intermediate 2 was converted into a-campholenic aldehyde 3 by catalytic isomerization.Subsequently, intermediate 4 was obtained from CrO 3 -oxidation of a-campholenic aldehyde 3, followed by the condensation of intermediate 4 with 4-methylthiosemicarbazide to give key intermediate 5. Finally, the target compounds 6a-p were synthesized via the cyclization of intermediate 5 and the nucleophilic substitution of the resulting heterocycle with various chlorides or bromides.All the target compounds were characterized by FT-IR, 1 H NMR, 13 C NMR, ESI-MS and elemental analysis.The related spectra can be found in Supplemental material.The FT-IR spectra of the target compounds showed the stretching vibrations of the C-H bonds related to C¼C-H and Ar-H in the range of 3075 $ 3031 cm À1 .The absorption bands at 1669 $ 1626 cm À1 were attributed to the C ¼ N stretching vibration.The stretching vibrations of Ar-C¼C exhibited at around 1600 and 1500 cm À1 .The 1 H NMR spectra of compounds 6a-p displayed characteristic signals at 6.96 $ 8.12, 5.21 $ 5.24 and 3.04 $ 3.52 ppm, which were assigned to Ar-H, C¼C-H and N-CH 3 , respectively.The 13 C NMR spectra of the target compounds showed peaks for C¼C at 121.53 $ 121.70 ppm, 1,2,4-triazole ring at 148.62 $ 156.16 ppm and benzene ring at d 147.93 $ 122.63 ppm.Their molecular weights and element contents of C, H, and N were confirmed by ESI-MS and elemental analysis, respectively.

Herbicidal activity
The herbicidal activities of title compounds 6a-p against B. campestris L. and E. crusgalli L. were evaluated by the rape petri dish method and the barnyard grass beaker method at the concentrations of 10 and 100 mg/mL, respectively.Flumioxazin, a commonly used and commercially available herbicide, was employed as positive control.The results were listed in Table S1.
It was found that, at the concentration of 100 mg/mL, all the target compounds except 6j(R ¼ p-CH 3 Bn) and 6k(R ¼ o-Cl Bn) displayed significant inhibition activity against root-growth of rape(B.campestris L.), with inhibition rates of 85.0% $ 98.2% (Aclass activity level), much better than that of the positive control flumioxazin.Among them, compounds 6b(R ¼ n-C 3 H 7 ) and 6g(R ¼ Bn) showed the best inhibition rates of 98.2%.Especially, even at the low concentration of 10 mg/mL, compounds 6b(R ¼ n-C 3 H 7 ), 6d(R ¼ n-C 4 H 9 ) and 6e(R ¼ i-C 4 H 9 ), exhibited significant inhibition rates of 88.3%, 90.6%, and 82.8%, respectively, against B. campestris L., implying that the three compounds deserved further study as potential leading compounds of novel herbicidal agents.In addition, six aliphatic compounds 6a-f and ten aromatic compounds 6g-p held average growth-inhibition rates of 94.1% and 87.8% against B. campestris L. at 100 mg/mL, respectively, indicating that introduction of aliphatic groups into target compounds would be more beneficial to improve activity than that of aromatic groups.

3 D-QSAR analysis
In order to further investigate the structure-activity relationship of target compounds bearing different R groups, 3D-QSAR analysis was carried out by CoMFA method in SYBYL-X 2.1.1 software.According to the literatures (Li et al. 2022, Li et al. 2021), the herbicidal activities of target compounds against B. campestris L.was selected for sample data in the built model, and the effectiveness and reasonability of the built model were also confirmed.
The steric and electrostatic contribution maps of this 3D-QSAR model were shown in Figure S73.The contribution rate of the steric field was 77.3% while that of the electrostatic field was 22.7%, revealing that the steric field was the major factor towards the herbicidal activity of the target compounds against B. campestris L. In Figure S73(A), the yellow region was located around the o-position of the benzene ring, indicating that the introduction of small groups at this position was beneficial to enhance the herbicidal activity, and the green regions located around the mand pposition of the benzene ring, represented that the bulky groups at the positions were favourable to increase the herbicidal activity.For example, compound 6h(R ¼ o-CH 3 Bn) displayed higher herbicidal activity than that of 6k(R ¼ o-Cl Bn), and 6m(R ¼ p-Cl Bn) and 6p(R ¼ p-NO 2 Bn) exhibited better herbicidal activities than those of 6j(R ¼ p-CH 3 Bn).In Figure S73(B), the electrostatic field contours were displayed in two distinguishing colours.The blue area indicated that the introduction of electron-donating groups was beneficial to improve activity, and the red area indicated that the introduction of electron-withdrawing groups was favourable to increase activity.Therefore, the introduction of electron-donating groups at the m-position of benzene rings was favourable for herbicidal activity.For instance, compound 6i(R ¼ m-CH 3 Bn) exhibited better herbicidal activity than that of compound 6l(R ¼ m-Cl Bn).

Conclusion
In summary, sixteen novel a-campholenic aldehyde-based 4-methyl-1,2,4-triazole-thioether compounds were rationally designed and synthesized by multi-step reaction.All the target compounds were characterized by means of FT-IR, 1 H NMR, 13 C NMR, ESI-MS and elemental analysis.It was found that, at the concentration of 100 mg/mL, all the target compounds except 6j(R ¼ p-CH 3 Bn) and 6k(R ¼ o-Cl Bn) displayed significant inhibition activity against root-growth of rape(B.campestris L.), with inhibition rates of 85.0%～98.2%(A-classactivity level), much better than that of the positive control flumioxazin.Compounds 6b(R ¼ n-C 3 H 7 ), 6d(R ¼ n-C 4 H 9 ) and 6e(R ¼ i-C 4 H 9 ) deserved further study as potential leading compounds of novel herbicidal agents.In addition, an effective and reasonable 3D-QSAR model was established by CoMFA method for the optimization of the title compounds.