Synthesis and Herbicidal Evaluation of Novel Ammonium Phenoxyacetates Based on Camphene‐Derived Primary Amine

ABSTRACT

To increase solubility, amines, including dimethylamine (DMA) and isopropylamine, are included in commercial formulations of phenoxyacetic acid herbicides, such as 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 2‐methyl‐4‐chlorophenoxyacetic acid (MCPA), but amine volatilization during production and use poses challenges for the social environment and living organisms. To mitigate the problem by replacing currently applied volatilized amines and developing high‐efficacy and environment‐friendly herbicides, three camphene‐derived ammonium phenoxyacetates and one glyphosate were synthesized and characterized. The preliminary herbicidal activity tests showed that several compounds displayed higher herbicidal performance against Lolium multiflorum Lam. and Brassica campestris than their corresponding herbicide‐amine salts. Compared to DMA salts of 2,4‐D and MCPA, compounds 5b–5c containing one or two chlorine atoms presented similar or higher herbicidal activity against B. campestris even at a lower concentration (0.0006 mmol/L). Besides, compound 5b with the half maximal inhibitory concentration (IC₅₀) value of 0.000281 mmol/L against B. campestris shoot growth showed 611.7% higher herbicidal activity than that of DMA salt of 2,4‐D, whereas compound 5c with an IC₅₀ value of 0.026 mmol/L against L. multiflorum Lam. shoot growth presented 38.5% higher herbicidal activity than DMA salt of MCPA. This study indicated that compounds 5b–5c could be promising herbicidal candidates.

To mitigate amine‐induced problems and find high‐efficacy herbicidal alternatives with low toxicity, four water‐soluble camphene‐derived ammonium salts were synthesized using camphene‐derived primary amine and different commercial herbicides. Most compounds displayed superior herbicidal activity against Lolium multiflorum Lam. and Brassica campestris than their herbicide‐amine salts. Particularly, compound 5b with an IC₅₀ value of 0.000281 mmol/L against Brassica campestris shoot growth showed over 7‐fold higher herbicidal activity than 2,4‐D dimethylamine salt, indicating its potential for practical application in sustainable agriculture.

1. Introduction

Herbicides have consistently played an irreplaceable role in the agricultural sector by controlling weeds and increasing crop productivity [1]. It is estimated that over 1.8 million tons of herbicides are consumed globally [2]. Despite the beneficial effects of herbicides, concerns about environmental and health issues associated with herbicide overuse and the serious occurrence of resistance weeds are emerging [3, 4, 5]. Particularly, the production and application of such commercial herbicidal formulations with an amine counterion may trigger substantial side effects on human health and the environment [3, 5]. For example, commonly used herbicides containing the phenoxy acid group, like 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 2‐methyl‐4‐chlorophenoxyacetic acid (MCPA), are applied in the form of ammonium salts with the use of an amine (i.e., dimethylamine [DMA] and isopropylamine [IPA]) to increase solubility, but the high volatility of amines tends to jeopardize bio‐efficacy and agro‐ecological security [5, 6, 7]. Volatilized amines are a hazard to production personnel, spraying workers, animals, and surrounding environment. Moreover, the volatilization of amine from those commercial forms is likely to promote herbicide volatilization, leading to heighted off‐target drift damage [5, 8]. In addition, the negative environmental effects may be further enhanced by amine associated aerosol particles [9, 10]. Therefore, the need for novel eco‐friendly water‐soluble herbicidal formulations with high activity and low toxicity is increasingly urgent.

Recently, botanical herbicides, mainly extracted or derived from plants, have become favorable substitutes to the currently used agrochemicals in the fight against weeds [11, 12]. Monoterpene is an essential group of secondary metabolites in various plants, which have been used as an important source in botanical herbicide preparation due to their unique molecular structure and modifiability [13, 14, 15]. As a monoterpene mixture of forest products, turpentine oil has shown good performance and great potential in the field of agriculture [16, 17, 18, 19]. It has been demonstrated that turpentine‐derived compounds by the utilization of the major components of turpentine, α‐pinene and β‐pinene, have the advantages of high biocompatibility and low toxicity [20, 21], displayed significant biological activities [22, 23], and thereby served as promising natural‐based herbicidal actives [17]. Camphene, a minor constituent of turpentine oil, can be produced by the isomerization reaction of α‐pinene [24], possessing a wide range of pharmacological activities, including antimicrobial, antitumor, antihyperlipidemia, and insecticidal activities [25, 26], which has been extensively explored for agricultural applications; however, very few studies on this compound have been performed as bioherbicides for weed management.

In our previous work, a number of compounds synthesized via derivation of volatile camphene are reported to exhibit high bioactive functions, such as ω‐acetoxymethyl camphene (compound 2), ω‐chloromethyl camphene (compound 3), and camphene‐based quaternary ammonium salts [27, 28]. Among them, camphene‐based quaternary ammonium salts presented highly effective and broad‐spectrum antimicrobial activity against common bacterial and fungal species [28]. Similarly, by taking advantage of the main component of heavy turpentine, longifolene, (E)‐2‐(4,8,8‐trimethyldecahydro‐1,4‐methanoazulen‐9‐ylidene)ethan‐1‐amine (ω‐aminomethyl longifolene), and longifolene‐derived primary amine carboxylates were developed by structural modification for weed control [29, 30]. All the prepared carboxylates had greater inhibitory effects against Lolium multiflorum Lam. and Brassica campestris than the commercial herbicide glyphosate, and some of them displayed robust efficacy with 100% inhibition in controlling these weeds at low concentrations (0.039–0.313 mmol/L) [30]. Recently, two novel phenoxyacetic herbicides synthesized from longifolene‐derived primary amine and two phenoxyacetic herbicides (i.e., 2,4‐D and MCPA) both have shown remarkable herbicidal performance against L. multiflorum Lam. root growth and B. campestris shoot growth with inhibition rates reaching 100% at concentrations of 0.039 and 0.156 mmol/L, respectively. Besides, the latter ammonium salt possessed a broad spectrum against six weeds (Setaria viridis, Eleusine indica, rice, Portulaca oleracea, Medicago sativa L., and clover) at 0.039 mmol/L [31]. It should be noted that when compared to longifolene, camphene is more available, and its chemical formula has five fewer carbon atoms, implying camphene‐derived amine compounds have higher hydrophilicity and lower lipophilicity than longifolene‐derived amine compounds. In that case, those camphene derivatives may potentially show better cell membrane permeability and stronger herbicidal activity, but this has not been reported yet. Herein, in this work, camphene‐derived primary amine (compound 5) was synthesized and then converted into two types of novel ammonium salts with the presence of different well‐known herbicides, including phenoxyacetic acid (PA), 2,4‐D, MCPA, and glyphosate. Relative to older DMA or IPA formulations, the introduction of compound 5 to the prepared formulations is anticipated to increase herbicide solubility, prevent off‐target damage, and improve the herbicidal efficacy. Besides, the preliminary herbicidal activities of compound 5 and the obtained derivatives against L. multiflorum Lam. and B. campestris were also evaluated and reported for the first time. This study is useful for the advancement of new herbicides and the high‐value‐added application of camphene.

2. Results and Discussion

2.1. Synthesis and Characterization

According to Scheme 1, target compounds 5a–5d were synthesized from the sustainable biomass resource camphene with yields of 80.4%–92.4%. As mentioned in our previous report, the synthesis of the key intermediate 5 utilized the modified synthetic method on the basis of Gabriel synthesis [29, 32]. Compound 2 was synthesized by adding camphene (1) and paraformaldehyde in glacial acetic acid at 110°C; compound 3 was obtained by refluxing at 30°C for 5 min, with acetyl chloride and anhydrous ethanol as the solvent [27]. Compound 3 was then reacted with potassium phthalimide in N,N‐dimethylformamide (DMF) at 110°C for 2 h to form compound 4. Compound 5 was synthesized from compound 4 and 80% N₂H₄·H₂O in the presence of 10% aqueous NaOH solution. Compounds 5a–5d were prepared from the reaction between compound 5 and the corresponding commercial herbicides, namely, PA, 2,4‐D, MCPA, and glyphosate. The target compounds were characterized by Fourier transform infrared spectroscopy (FTIR), ¹H NMR and ¹³C NMR spectra, and high‐resolution mass spectrometry (HRMS). Detailed spectra results for all compounds are provided in the Supporting Information.

SCHEME 1.

Synthetic route of target compounds 5a–5d. Reagents and conditions: (i) CH₃COOH, (HCHO) _n , reflux, 24 h; (ii) CH₃COCl, CH₃CH₂OH, reflux, 5 min; (iii) potassium phthalimide, DMF, 2 h; (iv) N₂H₄·H₂O, NaOH, reflux, 6 h; (v) absolute alcohol, RT, 15 min; (vi) glyphosate, deionized water, RT, 30 min.

Analysis of compounds 1–3 was presented in the Supporting Information, and the IR spectra of compounds 1–3 were shown in Figures S1–S3. In the IR spectrum of compound 4, the peak at 3070 cm⁻¹ was attributed to the C ═ C─H stretching vibration outside the main ring of camphene skeleton and on the benzene ring. The peaks at 2958 and 2867 cm⁻¹ were attributed to the stretching vibration bands of C─H alkane groups. The peaks at 1772 and 1689 cm⁻¹ represented the stretching vibration band of C ═ O groups in the heterocyclic ring and the stretching vibration band of the C ═ C bond outside the main ring of the camphene skeleton, respectively. The peaks at 1614, 1589, and 1450 cm⁻¹ were characteristic of the benzene ring. Other characteristic features for compound 4 were absorption of the imide C─N─C stretching vibration at 1390 cm⁻¹, and the absorption band of ortho‐substituted benzene ring at 741 cm⁻¹ (Figure S4). For compound 5, the absorption peaks of the ─NH₂ group located at 3376 and 3273 cm⁻¹. The absorption peak at 3060 cm⁻¹ was assigned to the ─C ═ C─H group outside the main ring of camphene skeleton. The peaks that appeared at 2961 and 2837 cm⁻¹ corresponded to the C─H stretching vibration bands of methyl or methylene groups. The absorption peaks at approximately 1648 and 1578 cm⁻¹ were ascribed to the C ═ C bond and the stretching vibration of the N─H bond, respectively (Figure S5). According to the IR spectrum of compound 5a shown in Figure S6, peaks observed at 3200–2250 and 2110 cm⁻¹ indicated the presence of primary ammonium cation. The peak at 3042 cm⁻¹ was attributed to the stretching vibration of the ═ C─H bond, whereas peaks at 2941 and 2872 cm⁻¹ were assigned to the stretching vibration band of the C─H alkane groups. The C ═ C stretching vibration band outside the main ring of camphene skeleton was observed at 1638 cm⁻¹. The stretching vibration band of the C ═ O group from the carboxylic acid at 1760 cm⁻¹ almost disappeared, but the antisymmetric stretching vibration band of the ─COO⁻ group could be found at 1620 and 1398 cm⁻¹. Besides, the absorption peaks at 1610, 1578, and 1488 cm⁻¹ were attributed to the benzene ring; peaks at 1052, 760, and 712 cm⁻¹ represented the stretching vibration band of the C─O band and benzene ring monosubstitution, respectively. For compound 5b, the characteristic absorption band of the primary ammonium cation was at 3200–2250 and 2114 cm⁻¹. The peaks at 3146, 2961, and 2865 cm⁻¹ were attributed to the ═ C─H stretching vibration band and C─H stretching vibration band, respectively. The peaks at 1640, 1625, and 1412 cm⁻¹ were assigned to the stretching vibration band of the C ═ C group and the antisymmetric stretching vibration band of the ─COO⁻ group. The peaks at 1585 and 1476 cm⁻¹ were related to the C ═ C bond, and the peak at 1065 cm⁻¹ corresponded to the C─O bond. Moreover, other characteristic features for compound 5b were the absorption band of trisubstituted benzene ring at 886 and 817 cm⁻¹, and the C─Cl stretching vibration band at 786 cm⁻¹ (Figure S7). As shown in Figure S8, similar to compounds 5a–5b, the characteristic absorption band of the primary ammonium cation for compound 5c was at 3200–2250 and 2112 cm⁻¹. The peaks at 3051, 2961, and 2858 cm⁻¹ were attributed to the ═ C─H stretching vibration band and C─H stretching vibration band, respectively. The peaks at 1624, 1405, 1578, and 1447 cm⁻¹ were assigned to the antisymmetric stretching vibration band of the ─COO⁻ group and the stretching vibration band of the C ═ C group, respectively. The peaks at 1052 and 770 cm⁻¹ represented the stretching vibration band of the C─O group and the stretching vibration band of the C═Cl group, respectively. The absorption peaks of a hydrogen atom in the benzene ring and the bending vibration of two adjacent hydrogen atoms on the benzene ring located at 880 and 815 cm⁻¹, respectively, reflected that compound 5c was benzene ring trisubstitution. In terms of compound 5d, the absorption peak of hydroxyl groups appeared at 3404 cm⁻¹. The characteristic absorption band of the primary ammonium cation for this compound was at 3200–2250 cm⁻¹, which was significantly enhanced when compared with compounds 5a–5c. This should be attributed to the absorption peak of the P─OH group at 2700–2600 cm⁻¹. The peaks at 1654, 1620, 1395, 1165, and 1045 cm⁻¹ were assigned to the stretching vibration band of the C ═ C group, the antisymmetric stretching vibration band of the ─COO⁻ group, the stretching vibration band of the R₃P ═ O group, and stretching vibration band of the C─O group, respectively (Figure S9).

The structures of all the synthesized compounds were also confirmed by NMR spectra (Figures S10–S27). In the ¹H NMR spectra of compound 4, four hydrogen atoms on the benzene ring show a distinct split double peak in the low field due to the binary substitution at the ortho position of the benzene ring. Influenced by two carbonyl groups on the phthalimide, the chemical shift of H‐1′ moves towards the lower field. The ¹³C NMR data indicate that compound 4 has 19 carbon atoms, which is consistent with the theoretical value. Because of the combined influence of the benzene ring and dicarboximide groups on the phthalimide, the chemical shifts of C‐1″ and C‐3″ move towards the lower field, which are higher than that of six carbon atoms on the benzene ring. In the ¹H NMR spectra of compound 5, the characteristic peak of the proton on the C ═ C bond is at a low field and is split into a triplet due to the effect of the adjacent methyl proton. Its ¹³C NMR data indicate that compound 5 has 11 carbon atoms, which is consistent with the theoretical value. The characteristic peak of C‐2′ is in the low field due to the C ═ C bond. The characteristic peak of C‐1′ also appeared at the low field, and its chemical shift is higher than that of other methyl and methylene carbons due to the effects of the amino group and the carbon‐carbon double bond. For compounds 5a–5d, triple peaks appeared at 4.95–5.00 ppm, corresponding to the proton chemical shift of methylene at the allylic position outside the camphene ring in their ¹H NMR spectra. The protons of the benzene ring for compounds 5a–5c were in the region of 6.76–7.41 ppm but cannot be observed for compounds 5 and 5d. The methylene protons in the phenoxyacetate group of compounds 5a–5c displayed signals at 4.38 or 4.45 ppm. The primary ammonium proton is reactive hydrogen, which can be lost by heavy water exchange. The ¹H NMR spectra of compounds 5a–5d showed that two characteristic signals at 0.91 and 0.94 ppm, respectively, were assigned to the methyl proton outside the camphene ring. The ¹³C NMR spectra of compounds 5a–5d exhibited peaks for the carbon signals of the carboxylate carbonyl group at about 174 or 170 ppm and the carbons of the C ═ C bond outside the camphene ring at 164 and 106 ppm. Other carbon atoms of the benzene ring displayed signals in the region of 114–166 ppm in the ¹³C NMR spectra of compounds 5a–5c, but those were absent in the ¹³C NMR spectra of compounds 5 and 5d. Besides, HRMS spectra of all compounds, gas chromatograms of compounds 1–3 and 5, and high performance liquid chromatograms of compounds 4 and 5a–5d were shown in Figures S28–S43.

It is well known that the boiling point serves as an indicator of the substance's volatility. Generally, a higher boiling point indicates lower volatility. Compounds 1 and 5 had relatively higher boiling points with 159°–160°C and 238°–240°C, respectively, when compared with DMA and IPA (6.9°C and 33°–34°C, respectively), which suggested that the synthesized compounds 5a–5d exhibited lower volatility. Moreover, the thermal stability of compounds 5a–5d was also evaluated by differential scanning calorimetry and thermogravimetry (DSC–TG), and their DSC–TG curves can be seen in Figures S44–S47. It is clear that three camphene‐derived ammonium phenoxyacetates 5a–5c were stable when the temperature was maintained between 0°C and 170°C, except for compound 5d. The thermal behaviors of compounds 5a–5c were similar, and their first recorded degradation temperatures were between 170°C and 180°C. The subsequent major weight losses of those three compounds were above 200°C. On the other hand, the first degradation stage of compound 5d in the range of 100°–150°C was attributed to the evaporation of water, and the other weight‐loss stages were attributed to the structural decomposition of compound 5d. On the basis of these results, different from older DMA or IPA formulations, compounds 5a–5d are odorless, and amine losses may not occur from these newly prepared herbicide‐amine salts at ambient temperatures, demonstrating their potential for practical applications.

2.2. Herbicidal Activities

The newly prepared compounds 5a–5d were assessed for preliminary herbicidal activity against L. multiflorum Lam. and B. campestris, and their corresponding DMA salts, namely, DMA salt of PA, DMA salt of 2,4‐D, DMA salt of MCPA, and glyphosate‐isopropylammonium (GLYP‐IPAM) salt, were chosen as positive controls. The inhibition rates of those tested compounds are listed in Tables 1, 2, 3, 4, and the toxicity regression equations and IC₅₀ values are shown in Tables 5 and 6. It can be found that several compounds showed remarkable herbicidal activity against target weeds. The negative inhibition rates of some tested compounds appeared in relatively lower concentrations, indicating their plant growth promotion. For L. multiflorum Lam., compounds 5 and 5a presented excellent herbicidal activity with 100% inhibition against the root and shoot growth of L. multiflorum Lam. at the concentrations of 3.13 and 6.25 mmol/L, respectively. Moreover, compounds 5b–5c containing one or two chlorine atoms exhibited relatively stronger activity: The inhibition rates for root growth exceeded 94% when treated at the concentration ranging from 0.078 to 0.625 mmol/L, and their inhibition rates on shoot growth were more than 67% at the same concentration (Figures 1 and 2). Furthermore, compound 5a displayed higher herbicidal activity against both the root and shoot growth of L. multiflorum Lam. than DMA salt of PA, whereas compounds 5c and 5d showed higher inhibition efficacy for shoot growth than their corresponding controls, respectively, but lower inhibition efficacy for the root growth (Figures S48–S51). Unfortunately, the activity of compound 5b against L. multiflorum Lam. root and shoot growth was weaker than that of DMA salt of 2,4‐D.

TABLE 1.

Inhibition rates of target compounds 5a–5d against root growth of Lolium multiflorum Lam.

Compounds	Concentrations (mmol/L)
	0.625 a (6.25 b )	0.313 a (3.13 b )	0.156 a (1.56 b )	0.078 a (0.781 b )	0.039 a (0.391 b )	0.020 a (0.200 b )	0.010 a (0.100 b )	0.005 a (0.050 b )	0.002 a (0.020 b )	0.001 a (0.010 b )
5	100 c	100	99.5 ± 0.84a	91.0 ± 1.06a	29.9 ± 6.12b	−6.34 ± 2.72cd	5.05 ± 3.94c	13.4 ± 16.33c	−16.0 ± 3.64d	d
5a	100	100	99.0 ± 1.74a	96.5 ± 4.13a	49.1 ± 7.07b	33.7 ± 12.61c	10.0 ± 3.45d	8.1 ± 7.71d	33.7 ± 6.32c	−6.7 ± 2.64e
5b	98.0 ± 2.58a	96.0 ± 0.29a	95.7 ± 0.94a	94.4 ± 1.03a	86.4 ± 2.08b	65.4 ± 5.38c	27.7 ± 5.25d	13.7 ± 9.21e	−5.93 ± 1.87f	0.30 ± 1.8f
5c	97.7 ± 0.59a	96.8 ± 0.24a	95.6 ± 1.36ab	94.1 ± 0.18ab	94.2 ± 0.39ab	88.3 ± 4.39b	88.9 ± 1.35b	58.3 ± 8.28c	26.2 ± 8.21d	3.21 ± 1.55e
5d	85.8 ± 3.9a	72.7 ± 2.95b	54.3 ± 3.48c	45.4 ± 8.67c	18.1 ± 10.97d	9.79 ± 5.2d	−14.2 ± 4.92f	−2.75 ± 2.5e	−1.13 ± 2.67e	d
DMA salt of PA	46.7 ± 13.9a	16.2 ± 9.9b	13.8 ± 8.9bc	4.1 ± 3.3bcd	1.8 ± 3.48cd	0.1 ± 8.66cd	−1.1 ± 8.84d	−5.3 ± 1.18d	−5.1 ± 5.3d	−9.3 ± 3.18d
DMA salt of 2,4‐D	100	100	98.2 ± 1.09a	97.2 ± 0.56a	83.9 ± 4.59b	70.5 ± 1.32c	36.1 ± 11.86d	6.0 ± 3.18e	2.1 ± 2.72e	1.2 ± 0.93e
DMA salt of MCPA	99.9 ± 0.16a	99.8 ± 0.28a	99.3 ± 0.51a	97.6 ± 0.59ab	98.4 ± 0.55ab	96.0 ± 2b	90.8 ± 1.18c	77.5 ± 4.85d	87.8 ± 0.87c	33.6 ± 0.67e
GLYP‐IPAM salt	88.2 ± 1.45a	87.9 ± 2.52a	84.5 ± 0.79a	72.4 ± 5.47ab	60.9 ± 8.14bc	51.9 ± 10.78cd	50.7 ± 11.54cd	43.9 ± 18.62d	27.3 ± 2.33e	d

Abbreviations: 2,4‐D, 2,4‐dichlorophenoxyacetic acid; GLYP‐IPAM, glyphosate‐isopropylammonium; MCPA, 2‐methyl‐4‐chlorophenoxyacetic acid; PA, phenoxyacetic acid.

^aThe concentrations of different camphene‐derived compounds and four ammonium salt solutions.

^bThe concentration of compounds 5 and 5a solutions.

^cThe inhibition rate (%). Values of inhibition rates are expressed as mean values ± the standard deviation (SD). Values followed by different letters are significantly different according to Duncan's multiple range test at p < 0.05.

^dThe data at this concentration were not determined.

TABLE 2.

Inhibition rates of target compounds 5a–5d against shoot growth of Lolium multiflorum Lam.

Compounds	Concentrations (mmol/L)
	0.625 a (6.25 b )	0.313 a (3.13 b )	0.156 a (1.56 b )	0.078 a (0.781 b )	0.039 a (0.391 b )	0.020 a (0.200 b )	0.010 a (0.100 b )	0.005 a (0.050 b )	0.002 a (0.020 b )	0.001 a (0.010 b )
5	100 c	100	74.9 ± 7.76a	31.9 ± 8.25b	9.85 ± 3.93c	−18.2 ± 1.54e	−6.36 ± 1.63d	−11.8 ± 1.15de	−33.3 ± 1.34f	d
5a	100	100	89.8 ± 17.74a	72.0 ± 9.42b	20.2 ± 3.38d	16.0 ± 2.68d	11.7 ± 4.98d	35.7 ± 6.59c	35.2 ± 7.58c	7.6 ± 3.92d
5b	85.8 ± 18.06a	73.6 ± 11.55b	72.4 ± 3.76b	66.9 ± 2.66b	41.0 ± 2.92c	35.0 ± 7.07c	30.5 ± 5.47c	13.9 ± 3.63d	2.83 ± 0.99d	7.28 ± 2.62d
5c	86.2 ± 3.2a	74.5 ± 3.66b	67.6 ± 2.09c	66.8 ± 3.71c	62.4 ± 4.9c	35.3 ± 2.65d	32.8 ± 5.87d	11.5 ± 4.17e	−5.16 ± 1.27f	−18.3 ± 5.4g
5d	65.3 ± 5.46a	34.2 ± 8.81b	10.0 ± 4.14c	0.27 ± 3.47cde	−12.3 ± 9.59f	−12.0 ± 4.47f	−7.94 ± 5.38ef	−2.05 ± 4.72def	5.68 ± 1.61cd	d
DMA salt of PA	16.6 ± 2.56a	12.4 ± 6.47a	10.0 ± 5.27ab	1.0 ± 5.26bc	1.0 ± 8.3bc	−4.3 ± 1.75cd	−9.3 ± 4.42cd	−13.1 ± 8.63d	−10.5 ± 5.53d	−6.4 ± 5.8cd
DMA salt of 2,4‐D	95.9 ± 1.2a	91.5 ± 1.19a	70.0 ± 4.42b	68.7 ± 7.1b	53.8 ± 4.96c	49.4 ± 2.9c	35.7 ± 14.12d	13.6 ± 3.89e	10.8 ± 2.42e	8.6 ± 3.48e
DMA salt of MCPA	63.1 ± 5ab	63.0 ± 2.04a	61.5 ± 7.32ab	57.9 ± 2.7ab	57.3 ± 3.17ab	56.6 ± 10.64ab	54.1 ± 1.82ab	48.7 ± 11b	35.8 ± 11.27c	8.6 ± 3.75d
GLYP‐IPAM salt	58.0 ± 6.36a	55.2 ± 9.48a	42.7 ± 8.43b	42.3 ± 10.56b	35.1 ± 0.49bc	26.0 ± 1.79cd	22.0 ± 2.79e	18.6 ± 2.56e	14.0 ± 7.04e	d

Abbreviations: 2,4‐D, 2,4‐dichlorophenoxyacetic acid; GLYP‐IPAM, glyphosate‐isopropylammonium; MCPA, 2‐methyl‐4‐chlorophenoxyacetic acid; PA, phenoxyacetic acid.

^aThe concentration of different camphene‐derived compounds and four ammonium salt solutions.

^bThe concentration of compounds 5 and 5a solutions.

^cThe inhibition rate (%). Values of inhibition rates are expressed as mean values ± SDs. Values followed by different letters are significantly different according to Duncan's multiple range test at p < 0.05.

^dThe data at this concentration were not determined.

TABLE 3.

Inhibition rates of target compounds 5a–5d against root growth of Brassica campestris.

Compounds	Concentrations (mmol/L)
	0.625 a (6.25 b )	0.313 a (3.13 b )	0.156 a (1.56 b )	0.078 a (0.781 b )	0.039 a (0.391 b )	0.020 a (0.200 b ) (0.0006 e )	0.010 a (0.100 b ) (0.0003 e )	0.005 a (0.050 b ) (0.00015 e )	0.002 a (0.020 b ) (0.00004 e )	0.001 a (0.010 b ) (0.000004 e )
5	100 c	100	96.6 ± 0.83a	91.3 ± 2.96b	41.3 ± 0.58c	9.38 ± 1.95e	12.0 ± 0.18e	7.44 ± 1.36e	21.4 ± 6.9d	d
5a	100	100	100	98.1 ± 2.64a	90.7 ± 6.86a	61.0 ± 13.43b	34.8 ± 12.77c	12.5 ± 4.28d	−17.6 ± 10.56e	−23.1 ± 12.64e
5b	97.1 ± 0.41a	96.9 ± 0.72a	96.9 ± 0.39a	95.7 ± 0.36a	95.6 ± 0.56a	95.4 ± 0.27a	95.2 ± 0.58a	95.3 ± 3.15a	94.0 ± 0.63	92.8 ± 0.6A
						84.6 ± 0.62b f	77.7 ± 0.67c f	56.1 ± 7.1d f	47.1 ± 1.78e f	45.3 ± 7.08e f
5c	97.8 ± 0.41a	97.2 ± 0.72a	97.2 ± 0.39a	95.3 ± 0.36a	95.5 ± 0.56a	95.6 ± 0.82a	91.8 ± 0.63b	91.7 ± 0.39b	88.9 ± 0.59bc	87.7 ± 0.88c
						83.3 ± 4.66d f	79.5 ± 3.76e f	60.3 ± 1.89f f	6.19 ± 3.37g f	4.66 ± 1.14g f
5d	93.6 ± 1.39a	79.1 ± 5.57b	66.0 ± 4.38c	52.2 ± 15.35d	40.4 ± 6.63e	25.4 ± 4.73f	−8.20 ± 1.74g	−23.3 ± 0.76h	−24.8 ± 3.94h	d
DMA salt of PA	89.0 ± 0.97a	82.2 ± 2.96a	67.6 ± 3.33b	41.7 ± 5.52c	40.3 ± 3.78c	27.1 ± 8.13d	18.5 ± 15.95de	4.5 ± 8.36f	−5.5 ± 7.56f	7.1 ± 5.58ef
DMA salt of 2,4‐D	92.8 ± 5.34a	90 ± 12a	88.1 ± 11.8a	86.3 ± 10.49a	85.9 ± 10.1a	83.8 ± 4.21a	82.8 ± 7.9ab	82.1 ± 5.59ab	81.8 ± 11.17ab	67.1 ± 4.87b
DMA salt of MCPA	100	100	100	100	100	100	100	100	100	98.0 ± 0.26
GLYP‐IPAM salt	79.5 ± 10a	71.2 ± 1.8ab	64.5 ± 2ab	63.6 ± 12.02b	48.2 ± 14.35c	42.5 ± 6.37c	24.1 ± 7.99d	15.7 ± 7.8de	6.63 ± 2.69e	d

Abbreviations: 2,4‐D, 2,4‐dichlorophenoxyacetic acid; GLYP‐IPAM, glyphosate‐isopropylammonium; MCPA, 2‐methyl‐4‐chlorophenoxyacetic acid; PA, phenoxyacetic acid.

^aThe concentrations of different camphene‐derived compounds and four ammonium salt solutions.

^bThe concentration of compounds 5 and 5a solutions.

^cThe inhibition rate (%). Values of inhibition rates are expressed as mean values ± SDs. Values followed by different letters are significantly different according to Duncan's multiple range test at p < 0.05.

^dThe data at this concentration were not determined.

^eThe diluted concentrations of compounds 5b and 5c solutions.

^fThe inhibition rate of compounds 5b–5c solutions at different diluted concentrations (%).

TABLE 4.

Inhibition rates of target compounds 5a–5d against shoot growth of Brassica campestris.

Compounds	Concentrations (mmol/L)
	0.625 a (6.25 b )	0.313 a (3.13 b )	0.156 a (1.56 b )	0.078 a (0.781 b )	0.039 a (0.391 b )	0.020 a (0.200 b ) (0.0006 e )	0.010 a (0.100 b ) (0.0003 e )	0.005 a (0.050 b ) (0.00015 e )	0.002 a (0.020 b ) (0.00004 e )	0.001 a (0.010 b ) (0.000004 e )
5	100c	100	90.9 ± 1.65a	82.3 ± 7.92a	30.5 ± 9.37b	10.6 ± 7.03d	23.0 ± 3.05bc	15.8 ± 4.73cd	10.1 ± 3.38d	d
5a	100	100	100	80 ± 2.64a	47 ± 9.45b	29.6 ± 3.51c	10.8 ± 6.64d	−8.6 ± 6.04e	−12.9 ± 3.57e	−10.8 ± 7.47e
5b	89.5 ± 1.92a	85.8 ± 1.6b	84.3 ± 1.69b	83.5 ± 0.54b	82.8 ± 0.45b	82.2 ± 0.4b	82.7 ± 0.19b	84.2 ± 0.81b	83.5 ± 0.79b	81.6 ± 2.19b
						72.2 ± 3.83c f	54.4 ± 6.58d f	24.1 ± 2.4f f	16.0 ± 5.31g f	33.8 ± 3.6e f
5c	91.8 ± 3.79a	84.9 ± 1.69b	81.4 ± 1.69bc	80.9 ± 0.54bc	81.2 ± 0.45bcd	81.5 ± 0.39bcd	79.0 ± 1.28cd	80.9 ± 1.15bcd	80.5 ± 2.08bcd	79.0 ± 1.64cd
						77.3 ± 1.17d f	55.9 ± 2.61e f	42.4 ± 5.8f f	7.99 ± 5.15g f	8.72 ± 5.63g f
5d	79.2 ± 6.23a	44.0 ± 4.75b	30.2 ± 8.6c	20.1 ± 2.69cd	17.7 ± 1.85d	14.7 ± 4.3d	−8.36 ± 1.66e	−21.7 ± 0.68f	−33.7 ± 15.11g	d
DMA salt of PA	63.9 ± 2.06a	50.2 ± 8.88b	32.9 ± 6.37c	12.3 ± 1.76de	12.9 ± 1.64de	7.4 ± 6.98ef	5.1 ± 8.71ef	−4.5 ± 2.09f	3.0 ± 7.7ef	23.3 ± 13.17cd
DMA salt of 2,4‐D	76.9 ± 7.93a	57.5 ± 5.03b	53.8 ± 7.38b	55.3 ± 0.45b	57.7 ± 10.04b	55.3 ± 9.99b	55.6 ± 2.6b	56.9 ± 8.03b	51.3 ± 0.94b	48.0 ± 0.81b
DMA salt of MCPA	87.4 ± 0.59a	85.4 ± 1.07ab	85.4 ± 1.24ab	85.1 ± 0.88ab	85.3 ± 1.21ab	86.1 ± 1.05ab	86.8 ± 0.27a	81.9 ± 1.61c	83.4 ± 2.04bc	82.6 ± 2.69c
GLYP‐IPAM salt	57.9 ± 16.7a	35.7 ± 5.34b	32.2 ± 15.89bc	23.3 ± 6.56bcd	23.0 ± 9.47bcd	22.5 ± 2.88bcd	15.6 ± 4.31cd	12.4 ± 1.34d	8.20 ± 3.88d	d

Abbreviations: 2,4‐D, 2,4‐dichlorophenoxyacetic acid; GLYP‐IPAM, glyphosate‐isopropylammonium; MCPA, 2‐methyl‐4‐chlorophenoxyacetic acid; PA, phenoxyacetic acid.

^aThe concentrations of different camphene‐derived compounds and four ammonium salt solutions.

^bThe concentration of compounds 5 and 5a solutions.

^cThe inhibition rate (%). Values of inhibition rates are expressed as mean values ± SDs. Values followed by different letters are significantly different according to Duncan's multiple range test at p < 0.05.

^dThe data at this concentration were not determined.

^eThe diluted concentrations of compounds 5b and 5c solutions.

^fThe inhibition rate of compounds 5b–5c solutions at different diluted concentrations (%).

TABLE 5.

Results of bioassay calculation of target compounds 5a–5d against Lolium multiflorum Lam.

Compounds	Root		Shoot
	Toxicity regression equation	IC50 (mmol/L)	Toxicity regression equation	IC50 (mmol/L)
5	Y = 1.384 + 3.606x R 2 = 0.936	0.413	Y = 0.054 + 3.825x R 2 = 0.991	0.968
5a	Y = 2.067 + 5.008x R 2 = 0.905	0.387	Y = 0.786 + 3.728x R 2 = 0.961	0.615
5b	Y = 3.247 + 1.79x R 2 = 0.899	0.015	Y = 1.359 + 1.043x R 2 = 0.941	0.050
5c	Y = 7.744 + 3.244x R 2 = 0.995	0.004	Y = 2.567 + 1.621x R 2 = 0.951	0.026
5d	Y = 1.415 + 1.563x R 2 = 0.985	0.124	Y = 1.047 + 2.974x R 2 = 0.975	0.445
DMA salt of PA	Y = 0.152 + 1.704x R 2 = 0.96	0.814	Y = −0.641 + 2.389x R 2 = 0.929	3.157
DMA salt of 2,4‐D	Y = 4.617 + 2.508x R 2 = 0.976	0.014	Y = 1.73 + 1.113x R 2 = 0.969	0.028
DMA salt of MCPA	Y = 3.928 + 1.336x R 2 = 0.908	0.001	Y = 0.654 + 0.452x R 2 = 0.662	0.036
GLYP‐IPAM salt	Y = 1.455 + 0.755x R 2 = 0.966	0.012	Y = 0.388 + 0.516x R 2 = 0.962	0.177

Abbreviations: 2,4‐D, 2,4‐dichlorophenoxyacetic acid; GLYP‐IPAM, glyphosate‐isopropylammonium; MCPA, 2‐methyl‐4‐chlorophenoxyacetic acid; PA, phenoxyacetic acid.

TABLE 6.

Results of bioassay calculation of target compounds 5a–5d against Brassica campestris.

Compounds	Root		Shoot
	Toxicity regression equation	IC50 (mmol/L)	Toxicity regression equation	IC50 (mmol/L)
5	Y = 1.212 + 2.35x R 2 = 0.938	0.305	Y = 0.89 + 1.584x R 2 = 0.873	0.274
5a	Y = 1.919 + 2.74x R 2 = 0.981	0.199	Y = 1.295 + 2.866x R 2 = 0.965	0.353
5b	Y = 1.386 + 1.432x R 2 = 0.989	0.000108	Y = 0.779 + 1.411x R 2 = 0.941	0.000281
5c	Y = 1.58 + 1.709x R 2 = 0.858	0.000119	Y = 1.121 + 1.745x R 2 = 0.988	0.000228
5d	Y = 1.56 + 1.32x R 2 = 0.978	0.066	Y = 0.969 + 1.769x R 2 = 0.923	0.283
DMA salt of PA	Y = 1.457 + 1.258x R 2 = 0.972	0.069	Y = 0.458 + 1.085x R 2 = 0.917	0.379
DMA salt of 2,4‐D	Y = 0.916 + 0.169x R 2 = 0.745	0.000034	Y = 0.532 + 0.197x R 2 = 0.685	0.002
DMA salt of MCPA	Y = 6.355 + 1.509x R 2 = 0.873	0.000061	Y = 1.139 + 0.099x R 2 = 0.923	0.000147
GLYP‐IPAM salt	Y = 1.226 + 0.96x R 2 = 0.967	0.053	Y = 0.107 + 0.589x R 2 = 0.941	0.657

Abbreviations: 2,4‐D, 2,4‐dichlorophenoxyacetic acid; GLYP‐IPAM, glyphosate‐isopropylammonium; MCPA, 2‐methyl‐4‐chlorophenoxyacetic acid; PA, phenoxyacetic acid.

FIGURE 1.

Herbicidal effect of compound 5b against Lolium multiflorum Lam. when the solution concentration was 0 mmol/L (A); 0.002 mmol/L (B); 0.005 mmol/L (C); 0.010 mmol/L (D); 0.020 mmol/L (E); 0.039 mmol/L (F); 0.078 mmol/L (G); and 0.156 mmol/L (H), respectively.

FIGURE 2.

Herbicidal effect of compound 5c against Lolium multiflorum Lam. when the solution concentration was 0 mmol/L (A); 0.002 mmol/L (B); 0.005 mmol/L (C); 0.010 mmol/L (D); 0.020 mmol/L (E); 0.039 mmol/L (F); 0.078 mmol/L (G); and 0.156 mmol/L (H), respectively.

As shown in Tables 3 and 4, Figures 3 and 4, and Figures S52–S55, all the target compounds also exhibited good inhibitory effect against B. campestris, and the inhibition rates to B. campestris root growth were higher than that of shoot growth. Similarly, compounds 5b–5c also had higher inhibition rates against the root growth of B. campestris within a certain concentration range and the shoot growth at the concentration of 0.625 mmol/L, respectively. Especially, compound 5c with a chlorine atom was found to be the most active among all the synthesized compounds when treated at 0.625 mmol/L, which possessed the most potent inhibition efficacy for both the B. campestris root and shoot growth. It is worth noting that even when the treatment concentration was reduced to 0.001 mmol/L, the inhibition rates of compounds 5b and 5c against the root growth of B. campestris were over 92% and 87%, respectively. Those two compounds even showed over 83% control against the root growth of B. campestris at a relative low concentration (0.0006 mmol/L). Meanwhile, their inhibition rates against shoot growth of B. campestris could reach over 72% at the same dosage. In most cases, compounds 5a–5d were similar or more effective inhibiting B. campestris than their corresponding DMA salts and GLYP‐IPAM salt.

FIGURE 3.

Herbicidal effect of compound 5b against Brassica campestris when the solution concentration was 0 mmol/L (A); 0.002 mmol/L (B); 0.005 mmol/L (C); 0.010 mmol/L (D); 0.020 mmol/L (E); 0.039 mmol/L (F); 0.078 mmol/L (G); and 0.156 mmol/L (H), respectively.

FIGURE 4.

Herbicidal effect of compound 5c against Brassica campestris when the solution concentration was 0 mmol/L (A); 0.002 mmol/L (B); 0.005 mmol/L (C); 0.010 mmol/L (D); 0.020 mmol/L (E); 0.039 mmol/L (F); 0.078 mmol/L (G); and 0.156 mmol/L (H), respectively.

The half maximal inhibitory concentration (IC₅₀) values of target compounds with excellent inhibitory effects against two different weeds at assay concentrations were further determined. As shown in Table 5, the IC₅₀ values of compounds 5a–5d against L. multiflorum Lam. root and shoot growth were 0.004–0.387 and 0.026–0.615 mmol/L, respectively, much lower than that of compound 5 (IC₅₀ values of root and shoot growth were 0.413 and 0.968 mmol/L, respectively). The IC₅₀ values of compounds 5a and 5c against L. multiflorum Lam. shoot growth were lower than that of their corresponding DMA salts, whereas the IC₅₀ values of compounds 5c–5d against L. multiflorum Lam. root growth were higher than that of DMA salt of MCPA and GLYP‐IPAM salt, respectively. According to Figure 5, the herbicidal activity of compound 5a against root growth of L. multiflorum Lam. was 110.3% and 13.2% higher than that of DMA salt of PA and compound 5, respectively. Although the herbicidal activity of compound 5b against root growth of L. multiflorum Lam. was over 27 times higher than that of compound 5, the herbicidal activity of this compound against L. multiflorum Lam. root growth was 6.7% lower than that of DMA salt of 2,4‐D. Similarly, compound 5b had higher herbicidal activity against L. multiflorum Lam. shoot growth than compound 5, but lower activity than DMA salt of 2,4‐D. Moreover, compounds 5a and 5c exhibited 413.3% and 38.5% higher herbicidal activities against shoot growth of L. multiflorum Lam. than their corresponding tested salts, respectively, and 187.2% and 134.8% higher than that of compound 5 against L. multiflorum Lam. shoot growth.

FIGURE 5.

Herbicidal activities of target compounds against the root growth (A) and shoot growth (B) of Lolium multiflorum Lam. compared to that of their corresponding salts. 2,4‐D, 2,4‐dichlorophenoxyacetic acid; GLYP‐IPAM, glyphosate‐isopropylammonium; MCPA, 2‐methyl‐4‐chlorophenoxyacetic acid; PA, phenoxyacetic acid.

It can be seen from Table 6 that the IC₅₀ values of compounds 5a–5d against B. campestris root and shoot growth were 0.000108–0.199 and 0.000228–0.353 mmol/L, respectively. In particular, compounds 5b–5c with the lowest IC₅₀ values were more favorable to herbicidal activity than compounds 5, 5a, and 5d. Moreover, compounds 5a–5b and 5d presented superior herbicidal activity than their controls except for the root growth of B. campestris (Figure 6). It is important to note that compound 5b was 611.7% and 711% higher than that of DMA salt of 2,4‐D and compound 5 against shoot growth of B. campestris, respectively. Despite compounds 5a and 5d displayed weaker herbicidal activity against B. campestris shoot growth than compound 5, their herbicidal activities against B. campestris shoot growth were 7.4% and 132.2% higher than that of DMA salt of PA and GLYP‐IPAM salt, respectively.

FIGURE 6.

Herbicidal activities of target compounds against the root growth (A) and shoot growth (B) of Brassica campestris compared to that of their corresponding salts. 2,4‐D, 2,4‐dichlorophenoxyacetic acid; GLYP‐IPAM, glyphosate‐isopropylammonium; MCPA, 2‐methyl‐4‐chlorophenoxyacetic acid; PA, phenoxyacetic acid.

Compounds 5a–5c were synthesized by reacting compound 5 with different herbicides, namely PA, 2,4‐D and MCPA, respectively, through the simple neutralization reaction. On the basis of the above results, compounds 5b–5c containing one or two chlorine atoms displayed the most significant herbicidal performance, and the reason is mainly due to higher activity of 2,4‐D and MCPA in comparison to that of PA. Further comparison of IC₅₀ values for those well‐performed compounds and their corresponding DMA salts are summarized in Table 7. It can be seen that compound 5b had much higher IC₅₀ values against the shoot growth of L. multiflorum Lam. and the root growth of B. campestris, but a lower IC₅₀ value against the shoot growth of B. campestris than DMA salt of 2,4‐D. Conversely, compound 5c possessed a lower IC₅₀ value against the shoot growth of L. multiflorum Lam., but relatively higher IC₅₀ values against the root and shoot growth of B. campestris than DMA salt of MCPA. Significantly, the IC₅₀ value of compound 5b against the shoot growth of B. campestris was 0.000281 mmol/L, and the IC₅₀ value of compound 5c against the shoot growth of L. multiflorum Lam. was 0.026 mmol/L. Besides, IC₅₀ values of compounds 5b–5c were almost comparable to that of their corresponding DMA salts against the root growth of L. multiflorum Lam. In addition, when compared to older DMA/IPA formulations, the prepared compounds 5a–5d without unpleasant and harmful odor exhibited enhanced herbicidal activity against L. multiflorum Lam. and B. campestris under certain circumstances, which is probably due to their stronger lipophilicity, leading to relatively better cell membrane permeability. Further studies, including cytotoxicity analysis, field trials, and herbicidal mode of action around the above compounds, are in progress.

TABLE 7.

Comparison of IC₅₀ values for compounds with relatively higher herbicidal activity and their corresponding salts.

Compounds	IC50 value (mmol/L)
	Lolium multiflorum Lam.		Brassica campestris
	Root	Shoot	Root	Shoot
5b	0.015	0.050	0.000108	0.000281
5c	0.004	0.026	0.000119	0.000228
DMA salt of 2,4‐D	0.014	0.028	0.000034	0.002
DMA salt of MCPA	0.001	0.036	0.000061	0.000147

Abbreviations: 2,4‐D, 2,4‐dichlorophenoxyacetic acid; MCPA, 2‐methyl‐4‐chlorophenoxyacetic acid.

3. Conclusions

In summary, a series of novel ammonium phenoxyacetates and glyphosate based on camphene‐derived primary amine were synthesized and characterized. Their herbicidal activities against the root and shoot growth of L. multiflorum Lam. and B. campestris were evaluated. The preliminary herbicidal activity tests indicated that some of the synthesized compounds exhibited better herbicidal activities than their corresponding herbicide‐amine salts. More interestingly, compound 5a displayed potent herbicidal efficacy for L. multiflorum Lam. and B. campestris; the root and shoot growth were completely inhibited when the concentrations of compound 5a were 3.13 and 1.56 mmol/L, respectively. The IC₅₀ values of compound 5a against root and shoot of L. multiflorum Lam. and the shoot growth of B. campestris were much lower than that of DMA salt of PA. Moreover, compounds 5b–5c also showed promising herbicidal activity against L. multiflorum Lam. and B. campestris within a certain concentration range. Those two compounds containing one or two chlorine atoms exhibited higher inhibition rates and lower IC₅₀ values than compounds 5, 5a, and 5d, especially for the root growth. In comparison to their corresponding DMA salts, compounds 5b–5c presented comparable to or higher inhibition efficacy for B. campestris even when the treatment concentration was as low as 0.0006 mmol/L. Those findings suggested that the prepared camphene‐derived ammonium salts, such as compounds 5b–5c, might be potential candidates with reduced ecological impact that can be used at low doses for effective weed control. Further research will be continued to explore the possibility of more high‐performance camphene derivatives for agricultural purposes.

4. Experimental Section

4.1. Materials

Camphene with a boiling point of 159–160°C was provided by Wuzhou Huangpu Chemical Pharmaceutical Co. Ltd., Guangxi, China (84.3%, gas chromatography [GC] analysis). PA, 2,4‐D, MCPA, glyphosate, DMA, and IPA were purchased from Shanghai Aladdin Biochemical Technology Co. Ltd. (Shanghai, China). Commercially available analytically pure solvents, including DMF, acetonitrile, deuterated trichloromethane (CDCl₃), deuterated water (D₂O), petroleum ether, acetic acid, and absolute alcohol, were used without further treatment or purification. Other chemical reagents, such as potassium phthalimide, sodium hydroxide, and anhydrous sodium sulfate, were purchased from Tianjin Damao Chemical Reagent Factory (Tianjin, China). Seeds of L. multiflorum Lam. and B. campestris were acquired from Barenbrug International Co. Ltd. (Tianjin, China) and Shenzhen Aoxin Libao Industrial Co. Ltd. (Shenzhen, China), respectively. Different formulations of DMA salts and GLYP‐IPAM salt were prepared through the neutralization reaction in our labs according to the procedure described in patents [33, 34].

4.2. Synthesis of Compound 4

(Z)‐2‐(2‐(3,3‐dimethylbicyclo[2.2.1]heptan‐2‐ylidene)ethyl)isoindoline‐1,3‐dione (4). As illustrated in Scheme 1, the starting material, camphene (compound 1), was commercially sourced and directly employed in the synthesis. Compounds 2–3 were synthesized in accordance with the methods in our previous studies [27]. Compound 3, with a yield of 95.2%, was obtained by the Prins and halogenation reaction from compound 1. Compound 3 (25.22 g, 0.1 mol) and potassium phthalimide (18.52 g, 0.1 mol) were dissolved in DMF (50 mL). The mixture was heated to 110°C and stirred for 2 h. When the reaction mixture was cooled to room temperature, distilled water (50 mL) was added to the above mixture for filtration. The crude product was collected after extracting with 20 mL of petroleum ether three times and drying in an oven at 50°C for 3 h. The crude product was then recrystallized with petroleum ether two times and dried to afford white crystal powder in a yield of 65.1%. The final product was obtained as a colorless crystalline solid by using absolute alcohol after solvent evaporation. Melting point (m.p.) 99.0°–99.5°C. IR (KBr): 3070, 2958, 2867, 1772, 1689, 1614, 1589, 1450, 1390, 741 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 7.84 (m, 2H, H‐4″, H‐7″), 7.70 (m, 2H, H‐5″, H‐6″), 5.04 (t, J = 8.0 Hz, 1H, H‐2′), 4.26–4.28 (d, J = 8.0 Hz, 2H, H‐1′), 3.25–3.26 (d, J = 4.0 Hz, 1H, H‐1), 1.89 (s, 1H, H‐4), 1.58–1.72 (m, 4H, H‐6eq, H‐7eq, H‐5eq, H‐5ax), 1.35–1.39 (m, 1H, H‐6ax), 1.26–1.28 (m, 1H, H‐7ax), 0.98 (s, 3H, 3‐CH₃), 0.96 ppm (s, 3H, 3‐CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 168.1 (C‐1″, C‐3″), 161.3 (C‐2), 133.8 (C‐6″, C‐5″), 132.4 (C‐3″a, C‐7″a), 123.1 (C‐7″, C‐4″), 108.93 (C‐2′), 47.8 (C‐4), 41.5 (C‐1), 37.4 (C‐1′), 37.0 (C‐7), 29.1 (C‐3), 28.1 (C‐6), 25.9 (3‐C(CH₃)₂), 23.8 ppm (C‐5). HRMS (ESI): m/z calcd for C₁₉H₂₂NO₂: 296.1651 [M + 1]⁺; found 296.1658.

4.3. Synthesis of Compound 5

(Z)‐2‐(3,3‐dimethylbicyclo[2.2.1]heptan‐2‐ylidene)ethan‐1‐amine (5). Compound 4 (2.18 g, 6 mmol), 80% N₂H₄·H₂O (8.64 g, 138 mmol), and 10% aqueous sodium hydroxide (5 g) were placed in a round‐bottom flask and stirred at 120°C for 6 h. After the temperature was lowered to room temperature, the reaction mixture was extracted with 10 mL of petroleum ether three times. The combined organic layer was washed with 10 mL of distilled water three times and dried over anhydrous sodium sulfate, then concentrated under reduced pressure. The solvent was evaporated, and the light yellow oily product with a yield of 91.8% and a GC purity of 91.8% was obtained by vacuum drying. IR (KBr): 3376, 3273, 3060, 2961, 2837, 1648, 1578 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ = 4.96–5.00 (t, J = 8.0 Hz, 1H, H‐2′), 3.17–3.23 (m, 2H, H‐1′), 2.88–2.90 (m, 1H, H‐2), 1.85 (s, 1H, H‐4), 1.54–1.62 (m, 3H, H‐5eq, H‐6eq, H‐7eq), 1.49 (s, 2H, NH₂), 1.31–1.39 (m, 1H, H‐5ax), 1.09–1.18 (m, 2H, H‐6ax, H‐7ax), 0.98 (s, 3H, 3‐CH₃), 0.95 ppm (s, 3H, 3‐CH₃). ¹³C NMR (100 MHz, CDCl₃) δ = 157.2 (C‐2), 116.3 (C‐2′), 47.6 (C‐4), 41.5 (C‐1), 41.0 (C‐1′), 40.5 (C‐7), 37.1 (C‐3), 29.1 (C‐6), 26.0 (3‐C(CH₃)₂), 23.6 ppm (C‐5). HRMS (ESI): m/z calcd for C₁₁H₁₇: 149.1330 [M─NH₂]⁺; found: 149.1330. The boiling point of compound 5 was 238°–240°C.

4.4. General Synthetic Procedure for Compounds 5a–5c

The preparation of compounds 5b–5c followed the typical procedure described below for the preparation of compound 5a.

(Z)‐2‐(3,3‐dimethylbicyclo[2.2.1]heptan‐2‐ylidene)ethan‐1‐aminium 2‐phenoxyacetate (5a). Compound 5 (0.83 g, 5 mmol) and absolute alcohol (8 mL) were mixed and transferred to a pressure‐equalizing dropping funnel. The mixture was added dropwise to the system containing PA (0.76 g, 5 mmol) and absolute alcohol (10 mL). After stirring the mixture at room temperature for 15 min, the resulting solid was collected by filtration, recrystallization with absolute alcohol and dried in vacuum. Compound 5a was obtained as a crystalline solid; yield 82.8%; m.p. 170.6°–172.5°C. IR (KBr): 3200, 3042, 2941, 2872, 2110, 1638, 1620, 1610, 1578, 1488, 1052, 760, 712 cm⁻¹. ¹H NMR (400 MHz, D₂O) δ = 7.24–7.26 (m, 2H, H‐3″, H‐5″), 6.84–6.95 (m, 3H, H‐4″, H‐6″, H‐8″), 4.95–4.99 (t, J = 8.0 Hz, 1H, H‐2′), 4.38 (s, 2H, H‐2″), 3.43–3.54 (m, 2H, H‐1′), 2.90–2.91 (d, J = 4.0 Hz, 1H, H‐1), 1.85 (m, 1H, H‐4), 1.53–1.61 (m, 3H, H‐5eq, H‐6eq, H‐7eq), 1.27–1.36 (m, 1H, H‐1), 1.18–1.20 (m, 1H, H‐6ax), 0.99–1.06 (1H, H‐7ax), 0.93 (s, 3H, 3‐CH₃), 0.91 ppm (s, 3H, 3‐CH₃). ¹³C NMR (100 MHz, D₂O) δ = 176.9 (C‐1″), 166.6 (C‐1‴), 157.6 (C‐2), 129.7 (C‐3‴, C‐5‴), 121.3 (C‐4‴), 114.4 (C‐2′), 105.4 (C‐2‴, C‐6‴), 66.5 (C‐2″), 47.3 (C‐4), 41.3 (C‐1), 38.2 (C‐1′, C‐7), 36.8 (C‐3), 28.5 (3‐CH₃), 28.2 (3‐CH₃), 24.9 (C‐6), 23.1 ppm (C‐5). HRMS (ESI): m/z calcd for C₁₁H₁₇: 1449.1330: [M─C₈H₇O₃─NH₂]⁺; found 149.1328.

(Z)‐2‐(3,3‐dimethylbicyclo[2.2.1]heptan‐2‐ylidene)ethan‐1‐aminium 2‐(2,4‐dichlorophenoxy)acetate (5b). The product was obtained as a white crystalline solid; yield 92.4%; m.p. 173.7°–176.8°C. IR (KBr): 3200, 3146, 2961, 2865, 2114, 1640, 1625, 1585, 1476, 1065, 886, 817, 786 cm⁻¹. ¹H NMR (400 MHz, D₂O) δ = 7.41 (s, 1H, H‐5″), 7.18–7.20 (d, J = 8.0 Hz, 1H, H‐3‴), 6.76–6.79 (d, J = 8.0 Hz, 1H, H‐2‴), 4.95–4.99 (t, J = 8.0 Hz, 1H, H‐2′), 4.45 (s, 2H, H‐2″), 3.44–3.55 (m, 2H, H‐1′), 2.91 (s, 1H, H‐1), 1.85–1.86 (s, 1H, H‐4), 1.54–1.58 (m, 3H, H‐5eq, H‐6eq, H‐eq), 1.27–1.36 (m, 1H, H‐5ax), 1.18–1.21 (m, 1H, H‐6ax), 1.01–1.03 (m, 1H, H‐7ax), 0.94 (s, 3H, 3‐CH₃), 0.91 ppm (s, 3H, 3‐CH₃). ¹³C NMR (100 MHz, CD₃OD) δ = 174.2 (C‐1″), 164.7 (C‐2), 153.6 (C‐1‴), 129.2 (C‐5‴), 127.2 (C‐3‴), 125.1 (C‐4‴), 123.2 (C‐6‴), 114.5 (C‐2‴), 106.5 (C‐2′), 68.1 (C‐2″), 42.0 (C‐4), 41.5 (C‐1), 38.1 (C‐1′, C‐7), 36.8 (C‐3), 28.0 (3‐CH₃), 27.5 (3‐CH₃), 24.7 (C‐6), 23.2 ppm (C‐5). HRMS (ESI): m/z calcd for C₁₁H₁₇: 149.1330: [M─C₈H₅Cl₂O₃─NH₂]⁺; found 149.1331; calcd for C₈H₅Cl₂O₃: 218.9621: [M─C₁₁H₂₀N]⁻; found 218.9621.

(Z)‐2‐(3,3‐dimethylbicyclo[2.2.1]heptan‐2‐ylidene)ethan‐1‐aminium 2‐(4‐chloro‐2‐methylphenoxy)acetate (5c). The product was obtained as a white crystalline solid; yield, 80.4%; m.p. 167.6°–169.6°C. IR (KBr): 3200, 3051, 2961, 2858, 2112, 1624, 1578, 1447, 1052, 880, 815, 770 cm⁻¹. ¹H NMR (400 MHz, D₂O) δ = 7.14 (s, 1H, H‐3‴), 7.06–7.09 (dd, J = 4.0 Hz, 1H, H‐5‴), 6.63–6.65 (d, J = 8.0 Hz, 1H, H‐6‴), 4.95–4.99 (t, J = 8.0 Hz, 1H, H‐2′), 4.38 (s, 2H, H‐2″), 3.47–3.52 (m, 2H, H‐1′), 2.91–2.92 (d, J = 4.0 Hz, 1H, H‐1), 2.13 (s, 3H, 6‴‐CH₃), 1.85–1.86 (dd, J = 4.0 Hz, 1H, H‐4), 1.53–1.63 (m, 3H, H‐5eq, H‐6eq, H‐7eq), 1.27–1.36 (m, 1H, H‐5ax), 1.18–1.21 (m, 1H, H‐6ax), 0.99–1.06 (m, 1H, H‐7ax), 0.94 (s, 3H, 3‐CH₃), 0.91 ppm (s, 3H, 3‐CH₃). ¹³C NMR (100 MHz, CD₃OD) δ = 175.2 (C‐1″), 164.8 (C‐2), 155.8 (C‐1‴), 129.7 (C‐5‴), 128.8 (C‐4‴), 125.8 (C‐4‴), 124.5 (C‐6‴), 112.1 (C‐2‴), 106.5 (C‐2′), 67.5 (C‐2″), 42.0 (C‐4), 41.4 (C‐1), 38.1 (C‐1′, C‐7), 36.8 (C‐3), 28.0 (3‐CH₃), 27.5 (3‐CH₃), 24.7 (C‐6), 23.2 (C‐5), 15.1 ppm (3‴‐CH₃). HRMS (ESI): m/z calcd for C₁₁H₁₇: 149.1330: [M─C₉H₈ClO₃─NH₂]⁺; found 149.1331; calcd for C₉H₈ClO₃ ⁻: 199.0167: [M─C₁₁H₂₀N]⁻; found 199.0161.

4.5. Synthesis of Compound 5d

(Z)‐2‐(3,3‐dimethylbicyclo[2.2.1]heptan‐2‐ylidene)ethan‐1‐aminium (phosphonomethyl)glycinate (5d). Compound 5 (0.17 g, 1 mmol) and glyphosate (0.17 g, 1 mmol) were dissolved in 10 mL of sterile deionized water. After evenly mixing at room temperature for 30 min, 100 mL of acetonitrile was added dropwise to the solution system for inducing the phase separation. When the milky white turbid solution color appeared, the reactant was filtered, and the filtrate was collected and further dried under vacuum to afford compound 5d in a yield of 83.8%. White crystalline solid; m.p. 187.8°–212.9°C. IR (KBr): 3404, 3200, 2961, 2865, 2640, 1654, 1620, 1165, 1045 cm⁻¹. ¹H NMR (400 MHz, D₂O) δ = 4.96–5.00 (t, J = 8.0 Hz, 1H, H‐2′), 3.71–3.75 (s, 2H, H‐2″), 3.45–3.66 (m, 2H, H‐1′), 3.12–3.15 (d, J = 12 Hz, 2H, H‐4″), 2.91–2.93 (d, J = 8.0 Hz, 1H, H‐1), 1.00–1.09 (m, 1H, H‐6ax), 0.94 (s, 3H, 3‐CH₃), 0.91 ppm (s, 3H, 3‐CH₃). ¹³C NMR (100 MHz, D₂O) δ = 170.3 (C‐1″), 166.6 (C‐2), 105.5 (C‐2′), 50.0 (C‐4″), 47.3 (C‐2″), 44.5 (C‐4), 43.2 (C‐1), 41.3 (C‐1′, C‐7), 36.8 (C‐3), 28.2 (3‐CH₃), 27.5 (3‐CH₃), 25.0 (C‐6), 23.1 ppm (C‐5). HRMS (ESI) m/z calcd for C₁₁H₁₇: 268.0067: [M─C₃H₇NO₅P─NH₂]⁺; found 168.006; calcd for C₃H₇NO₅P⁻: 168.0067: [M─C₁₁H₂₀N]⁻; found 168.0059.

4.6. Characterization

The structures of target compounds were identified by their spectral data. ¹H NMR and ¹³C NMR spectra were measured on a Bruker Ascend 400 MHz spectrometer with tetramethylsilane (TMS) as the internal standard. IR spectra were measured as KBr pellets on a Magna‐IR 550 FTIR spectrometer (Nicolet Co. Ltd., USA) in the 400–4000 cm⁻¹ region. The melting point was performed with Beijing Taike point apparatus (X‐4) and was uncorrected. The boiling point was determined through a simple distillation method. HRMS analysis was performed on an Agilent mass spectrometer (Agilent Technologies Inc., Palo Alto, USA) under electron spray ionization. The thermal behaviors of all the synthesized compounds were investigated using DSC–TG analysis with an STA 449 F3 Jupiter instrument (NETZSCH‐Gerätebau GmbH, Germany). All the samples were heated from 0°C to 300°C at a heating rate of 10°C/min under the nitrogen atmosphere. An Agilent 8860‐5977B GC–mass spectrometry equipped with a DB‐5 quartz capillary column (30 m, 0.25 mm, 0.25 µm) and FID was applied for GC analysis. The injection temperature and detector temperature were both at 200°C. The carrier gas was helium, and the injection volume was 1 µL. The temperature was programmed to increase from 100°C to 180°C at the rate of 5°C/min and continue to keep 0.5 min. The high‐performance liquid chromatography (HPLC) analysis of compounds 4 and 5a–5d was performed on an Agilent 1200 Series instrument equipped with an Inertsil ODS‐SP C18 column (4.6 × 250 mm²) and DAD detector. The injection volume of all samples was 25 µL, and the flow rate was set to 1 mL/min. The elution system was composed of methanol and water at different ratios varying by time, and the temperature was set to 30°C.

4.7. Preliminary Herbicidal Activity Evaluation

The preliminary herbicidal activities of the prepared compounds 5a–5d were determined on the inhibition tests of the root and shoot growth of L. multiflorum Lam. and B. campestris according to the reported methods [12, 35]. First, 0.0625 mmol of each tested compound, including compound 5, 5a–5c, PA, 2,4‐D, MCPA, DMA, IPA, and glyphosate was dissolved in 0.5 mL methanol in a 100 mL volumetric flask, respectively; then, 5 mL of 1% aqueous solution of Tween‐80 was added and diluted with deionized water to 0.625 mmol/L. The concentration was then diluted with the control solution (methanol and 1% aqueous solution of Tween‐80 in deionized water) to achieve the desired concentration following the established methods in the literature [30, 36]. The prepared stock solution of compounds 5 and 5a to be tested (0.625 mmol) was diluted to solutions of 6.25, 3.13, 1.56, 0.781, 00.391, 0.200, 0.100, 0.050 and 0.020  mmol/L, respectively. The solution of compound 5a was even diluted to the concentration of 0.010 mmol/L. Next, the seeds were soaked in 2% NaClO solution for 5 min, then rinsed with deionized water three times, and soaked with deionized water for 15 h. The treated seeds were placed in a 9 cm diameter Petri dish lined with two layers of filter paper and cultivated in dark for 24 h at 25°C. Afterwards, 10 seeds were added to each Petri dish containing 10 mL of the test solution or the blank control solution and cultivated in dark at 25°C for 3 days. Three replicates were used for each concentration and control. The inhibition percentage of seed germination was the mean value obtained through three independent experiments, and the standard deviation (±SDs) values were analyzed according to Duncan's multiple range test at p < 0.05. On the basis of the series inhibition rate, values of half maximal inhibitory concentration (IC₅₀) were calculated from probit analysis using SPSS software. Values of inhibition rates are expressed as mean values ± SDs. The root or shoot length of each seed was measured, and the inhibition rate (%) of the root or shoot growth was calculated according to the equation listed as follows:

$$y=\frac{x_0-x_1}{x_0}$$

where $y$ is the inhibition rate of the root or shoot growth, $x_0$ is the average root or shoot length of the blank control group in each test, and $x_1$ is the average root or shoot length of the group treated by the test compounds.

4.8. Statistical Analysis

SPSS software version 20.0 was used for statistical analysis. The data of the herbicidal activity evaluation in three replicas were calculated according to Duncan's multiple range test (p < 0.05). The herbicidal activity was expressed as the average percentage of germinated seeds (%), and results were presented as mean ± SD.

Author Contributions

This work was supervised by Hongyun Lan and Daozhan Huang. The synthesis and characterization of target compounds were performed by Ziqiang Zhao, Yanqun Huang, Hongyun Lan, and Daozhan Huang. Herbicidal activity evaluation was conducted by Rimei Chen, Libing Xu, Zhenfang Li, and Zhiqing Ning. Ziqiang Zhao wrote a preliminary draft of the article, and Yanqun Huang wrote the most of the article. Hongyun Lan, Daozhan Huang, and Yu Feng reviewed and edited.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv70826‐sup‐0001‐SuppMat.pdf

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.