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Article

Study on Design, Synthesis and Herbicidal Activity of Novel 6-Indazolyl-2-picolinic Acids

by
Qing Liu
1,2,
Rong-Chuan Shi
1,2,
Hui-Ting Li
1,
Wei Wei
1,
Xiao Yuan
1,
Shang-Zhong Liu
1,2 and
Yi-Ming Cao
1,2,*
1
Innovation Center of Pesticide Research, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, China
2
Key Laboratory of National Forestry and Grassland Administration on Pest Chemical Control, China Agricultural University, Beijing 100193, China
*
Author to whom correspondence should be addressed.
Molecules 2024, 29(2), 332; https://doi.org/10.3390/molecules29020332
Submission received: 12 December 2023 / Revised: 29 December 2023 / Accepted: 5 January 2024 / Published: 9 January 2024
Browse Figures
Versions Notes

Abstract

:
Thirty-eight new 4-amino-3,5-dicholo-6-(1H-indazolyl)-2-picolinic acids and 4-amino-3,5-dicholo-6-(2H-indazolyl)-2-picolinic acids were designed by scaffold hopping and synthesized to discover potential herbicidal molecules. All the new compounds were tested to determine their inhibitory activities against Arabidopsis thaliana and the root growth of five weeds. In general, the synthesized compounds exhibited excellent inhibition properties and showed good inhibitory effects on weed root growth. In particular, compound 5a showed significantly greater root inhibitory activity than picloram in Brassica napus and Abutilon theophrasti Medicus at the concentration of 10 µM. The majority of compounds exhibited a 100% post-emergence herbicidal effect at 250 g/ha against Amaranthus retroflexus and Chenopodium album. We also found that 6-indazolyl-2-picolinic acids could induce the up-regulation of auxin genes ACS7 and NCED3, while auxin influx, efflux and auxin response factor were down-regulated, indicating that 6-indazolyl-2-picolinic acids promoted ethylene release and ABA production to cause plant death in a short period, which is different in mode from other picolinic acids.

Graphical Abstract

1. Introduction

The production loss caused by diseases, pests, and weeds in different crops and regions ranges from 30% to more than 90% [1]. At present, about 250 plant species classified as weeds in arable land influence the normal growth of crops through competing limited land, water, and other nutrients. Application of synthetic herbicides is the most widely used and effective method to control weeds [2], and has been used for more than 70 years. Currently, herbicides account for more than 40% of the global pesticide market; however, weed damage still results in 8–13% global crop losses every year [3,4]. Furthermore, the long-term and extensive application of herbicides has triggered the exponential increase in some herbicide-resistant weeds, seriously threatening the productivity and profitability of the farms or companies. Until now, at least 60 countries have reported herbicide-resistant weed biotypes, including more than 500 herbicide combinations, and the annual cost of treating herbicide-resistant weeds is about USD 4 billion worldwide [5,6,7,8].
Synthetic auxinic herbicides mimic the natural plant hormone indole-3-acetic acid (IAA) and some have already been used for decades, but the weeds generate corresponding resistance against them much slower than others, which can be attributed to the potential multiple sites of actions and complex action mechanism of this kind of herbicides [6,7,9,10]. Synthetic auxinic herbicides contain several structural skeletons, in particular 2-picolinic acid. During the 1940s, Corteva (former Dow AgroSciences) discovered a series of herbicides containing structural skeleton 2-picolinic acid, such as picloram, clopyralid, aminopyralid, halauxifen-methyl (ArylexTM active) and florpyrauxifen-benzyl (RinskorTM active). In recent years, the last two were launched following intensive research on the structure–activity relationship and soil metabolites (Figure 1) [11,12,13,14].
Many of the researchers also tended to introduce heterocycles or bicyclic heterocycles at the 6 position of 2-picolinic acid to develop highly active and environment-friendly herbicides. For instance, Bayer Crop Science, Corteva Agriscience, etc., introduced benzothiazole, benzofuran, indole, isoxazoline and other heterocycles at the 6 position of 2-picolinic acid [15,16,17]. In our group, we used the 1H-pyrazole group to replace the chlorine atom at the 6 position of clopyralid and picloram to obtain new chemotype compounds (Figure 2), and some of the resulted compounds displayed a wider herbicidal spectrum and good crop safety [18,19].
Indazole is a 10π electron aromatic heterocyclic ring with a unique electronic structure and chemical properties [20,21] and is a potential fragment in herbicidal compounds [22,23,24]. In our previous study, some 6-(5-substitued phenylpyrazolyl)-2-picolinic acids were found to have herbicidal activities. In addition, modifying 2-picolinic acid could alter the binding mode of lead compounds at the auxinic herbicide binding pocket. To discover potential herbicidal molecules with low resistance, we further modified 6-(5-substitued phenylpyrazolyl)-2-picolinic acids by replacing the pyrazolyl group with an indazolyl group (Figure 3).
The method of exploring the expression of auxin-related genes gave rise to researchers’ extensive attention to study active compounds’ action mechanism. In 2000, BASF [25] reported that the 1-aminocyclopropane-1-carboxylic acid synthase (ACS) activity, level of 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene significant increase in Galium spurium within 2 h after the application of high concentrations of IAA and picloram. The treatment also aroused the up-regulation of 9-cis-epoxy urea dioxygenase (NCED), which triggered abscisic acid (ABA) to increase 24 times compared to the control group after 24 h. Jiaqi Xu et al. [26] also found that halauxifen-methyl induced over expressions of ACS and NCED genes. Upregulated genes destroyed the homeostasis of IAA and stimulated the excessive production of ethylene and ABA, eventually leading to plant death. In 2019, Lei et al. [27] reported that genes IAA5 (auxin-induce gene), GH3.3 (auxin-regulate gene) and AUX1 (auxin-influx gene) were up-regulated after Arabidopsis thaliana (A. thaliana) was treated by a synthetic compound [28,29,30,31]. Therefore, the response of auxin-related genes was used for the initial investigation of the mechanism of action of 6-indazolyl-2-picolinic in this study.

2. Results and Discussion

2.1. Chemistry

A series of new 6-indazolyl-2-picolinic acids 1A–7d (Table 1) were provided via the route illustrated in Scheme 1 (some of them are mixtures). All the obtained compounds were characterized via 1H NMR, 13C NMR, and HRMS.
The substituted indazoles as intermediates were synthesized in two steps: (1) substituted o-fluorobenzaldehyde reacts with hydrazine hydrate in tetrahydrofuran to generate corresponding crude substituted benzylidenehydrazine; (2) intermediate indazole forms in the presence of two equivalents of sodium bicarbonate. At the beginning, substituted salicylaldehydes were used as starting materials, and intermediate benzylidenehydrazine could be easily obtained. However, the subsequent cyclization reaction for forming indazole could not proceed. Instead, when o-fluorobenzaldehyde was used as the starting material, the cyclization reaction could proceed smoothly by using 100% hydrazine hydrate as a solvent [32]. 1H-Indazole and 2H-indazole are tautomeric isomers, and 1H-indazole is the most significant one, since it is thermodynamically more stable than 2H-indazole. In the coupling reaction of 1H-indazole and 4-amino-3,5,6-chloro-2-picolinonitrile, sodium salt of 1H-indazole would inevitably transform to sodium salt of 2H-indazole at room temperature or above; thus, the isomer 4-amino-3,5-dichloro-6-(2H-indazolyl)-2-picolinonitrile could form alongside the designed product 4-amino-3,5-dichloro-6-(1H-indazolyl)-2-picolinonitrile.
When the substituent is at the 7 position of the indazole ring, only compound V containing 2H-indazolyl formed probably due to the steric effect. And when the R substituents CH3, F, Cl and Br were at the 6 position of 1H-indazole, the obtained product isomers were difficult to separate by column chromatography, and their biological activities were tested with their mixtures.
The preliminary results of the subsequent biological assay to inhibit A. thaliana root growth showed that the biological activity of the new compounds containing the 1H-indazolyl fragment was better than those with the 2H-indazolyl fragment. Thus, we attempted to optimize the conditions of the coupling reaction for the synthesis of 6-(1H-indazolyl)-2-picolinonitrile (r1) (Scheme 2).
The results in Table 2 showed that inorganic bases potassium carbonate, potassium hydroxide and sodium hydroxide were uncapable of converting 1H-indazole to its salt due to the weak alkalinity or low solubility. Cesium carbonate with its better solubility could make the reaction proceed despite the fact that the starting materials could not be fully converted. We also found that the reaction temperature of salt formation influences the ratio of r1 and r2. For instance, the proportion of compound r1 can be significantly increased with the decreasing in T1, but the proportion of r1 cannot be increased by reducing T2. In order to further increase the proportion of r1, the solvent was changed to acetonitrile, of which the melting point is much lower than that of 1,4-dioxane. However, the lower boiling point acetonitrile limited the temperature of the reaction and thus resulted in lower conversion. The ratio of r1 was increased to 82.9% when 1,2-dimethoxyethane, which has a higher boiling point, was employed as solvent and cesium carbonate was used as the base, but 4-amino-3,5,6-trichloro-2-picolinonitrile could still not be fully consumed. When changing the solvent to 1,2-diethoxyethane with a much higher boiling point, more r2 was generated and the reaction was still incomplete, albeit less amounts of 5-bromo-1H-indazole and 4-amino-3,5,6-trichloro-2-picolinonitrile were left compared to other conditions.
In the above-mentioned experiments, it was unable to obtain a single isomer as the product, and the reaction could also not be completed. Finally, sodium hydride was employed as the base, and the obtained r1 and r2 were separated by column chromatography. For cases in which r1 and r2 cannot be easily separated, the mixture of r1 and r2 was used in biological activity investigation.

2.2. Phenotypic Study of Arabidopsis thaliana and SAR Analysis

All new compounds were tested against A. thaliana root growth at concentrations from 200 µmol/L to 3 µmol/L. When the inhibition at a certain concentration (µmol/L) is greater than 80%, the test was continued at half the concentration used. The inhibition effect of some new compounds, picloram and DMSO (solvent), at different concentrations against A. thaliana root growth is shown in Figure 4.
The results from Figure 4a show that compounds 5a, 6Cc, and 7Cc displayed significant inhibitory activity against A. thaliana root growth and were better than the commercial herbicide picloram at the concentrations 50 and 25 µM. Figure 4b shows that 7Cc at 3 µM had the same inhibition effect with picloram at 12.5 µM. Structure–activity relationship analysis revealed that the electron-donating substituents amino and methoxy in indazolyl decreased the inhibition activity of new compounds; when the substituents are proton, methyl, and halide atoms, the inhibition activity of compound IV was better than that of compound V; when the substituents are at positions 4, 6 and 7 on the indazole ring, the new compounds have similar inhibition activity, and their inhibition activity was better than those at positions 5; when the indazole ring was substituted by electron-withdrawing groups, the inhibition activity was significantly improved. The influence of halide atoms on inhibition activity was related to their electronegativity and weaker electronegativity resulted in higher inhibition activity. Overall, the substituents in indazole ring improve the inhibition activity of the compounds in the following order: bromine > chlorine > fluorine ≈ methyl > amino > methoxy.

2.3. Evaluation of Herbicidal Activity

2.3.1. Root Growth Inhibition of Weeds in Petri Dishes

The herbicidal activity of new compounds was evaluated according to a reported procedure [18], in which picloram was used as the control, and each experiment had three replicates. Compounds 1A–7d were tested to evaluate their effect to control the root growth of five grass seeds including Echinochloa crusgalli (EC), Amaranthus retroflexus (AR), Chenopodium album (CA), Abutilon theophrasti Medicus (AM) and Brassica napus (BN) at concentrations of 500 µM (Figure 5) and 250 µM (Figure 6).
The results showed that most compounds have a certain inhibitory effect on the roots of weeds but their inhibitory activity on EC was generally weak. The relationship of structure–activity showed that the positions of substituents are related with the root inhibitory activity of weeds. The inhibitory effect is much better with substituents on the 4 position of the indazole ring, while the 5 position substitution results in poor inhibitory activity. In addition, compounds with electron-withdrawing substituents on the indazole ring showed better activity. There is no significant difference in inhibitory activities between 1H- and 2H-indazolyl isomers.
Moreover, compound 5a had a similar inhibitory effect compared to picloram, and their root growth inhibition on four dicotyledonous grasses at 10 µM/L was tested (Figure 7).
Compound 5a had a higher inhibitory activity on the root growth of BN (like A. thaliana, a member of the Cruciferae family) compared to picloram when the concentration was decreased to 10 μM, and the inhibitory activity of compound 5a on the roots of AM was also significantly higher than picloram. For CA and AR, two species of weeds in the Amaranthaceae family, the inhibitory effect of compound 5a was not as good as that of picloram.

2.3.2. Herbicidal Activity

In addition, the herbicidal activities of the 38 new compounds against the four dicotyledonous weeds and one monocotyledonous weed were tested in a glasshouse. The test was carried out at a range of concentrations (from high to low) until the visual injury effect was less than 60%. Some results are displayed in Table 3 and Figure 8, and the full results can be found in Table S1.
Most compounds exhibit excellent herbicidal activity at 1000 g/ha, and some of them (as shown in Table 3) could completely control BN, CA and AR at a dosage of 250 g/ha. The herbicidal activities of new compounds containing a 4 position substituent were superior to those containing the 7 position substituent and to those with substituent at the 5 and 6 position. The electronic properties of the substituent did not have a significant effect on the activity.

2.4. The Response of Auxin Relative Genes

Compound 7Cc had superior inhibitory activity against the root growth of A. thaliana compared to picloram, and the evaluation of auxin-related gene response was carried out by treating A. thaliana with compound 7Cc (Figure 9a). The results showed that compound 7Cc did not upregulate AUX1 (auxin-influx gene), PIN2 (auxin-efflux gene) [33], GH3.3 (auxin-regulate gene), or ARF2 (auxin response factor) as picloram did (Figure 9b), but upregulated the expression of ACS7 and NCED3 genes and promoted the production of ethylene and ABA, which affect the physiological processes of plant growth. In order to figure out the impact of each isomer of 7Cc on the auxin gene response (Figure 9b), compounds 5A and 5a were employed to explore the auxin-related gene response, respectively.
The AUX1, PIN2, and ARF2 genes’ expression of A. thaliana treated with compounds 5A and 5a (Figure 9c,d) remained down-regulated, implying that they are unable to be transported via carrier proteins as traditional commercial picolinic herbicides, and they are unable to bind auxin receptor proteins TIR1 and AFBs to release ARFs [34,35]. Compound 5a induced a higher level of ACS7 and NCED3 expression compared to 5A, explaining why it inhibited A. thaliana root growth better than compound 5A.
The new synthesized compounds also contain the 2-picolinic acid skeleton, promote ethylene release and ABA production by causing up-regulation of ACS and NCED genes, leading to the death of the treated plant in a short time. Such an action mechanism is different from those of other 2-picolinic acid herbicides, which might mitigate the potential growth of resistance. However, the exact mechanism of action remains unclear.

3. Materials and Methods

3.1. Chemicals, Experimental Instruments and Plant Materials

Solvents and reagents were provided by Bide Pharmatech (Beijing, China). The commercial herbicide picloram was provided by Nutrichem Company Ltd. (Beijing, China). Arabidopsis thaliana (A. thaliana ecotype Columbia-0, Col-0) and weed seeds were provided by the Laboratory of National Forestry and Grassland Administration on Pest Chemical Control, China Agricultural University, Beijing, China. 1H NMR and 13C NMR spectra were obtained at 500 MHz using a Bruker AVANCE NOE500 spectrometer (Billerica, MA, USA) in DMSO-d6 solution. HRMS was performed using an Agilent 6540 Q-TOF instrument (Santa Clara, CA, USA) instrument. The A. thaliana and weed root growth data were obtained using IMAGEJ software (https://imagej.nih.gov/ij/).

3.2. Synthesis

3.2.1. General Synthetic Procedure of Intermediates II

Compound I (100 mmol) and tetrahydrofuran (250 mL) were added into a 500 mL three-mouth round-bottom flask at 25 °C, and 80% hydrazine hydrate (110 mmol) was added drop-wise to the reaction solution under stirring. Subsequently, the reaction mixture was heated to 65 °C and maintained at this temperature for 2 h. Then, the mixture was cooled to 25 °C, and concentrated under a vacuum to obtain the crude-substituted benzylidenehydrazine. This crude intermediate was dissolved in 100% hydrazine hydrate (100 mL), Na2CO3 (200 mmol) was then added under stirring at 25 °C, and the reaction mixture was heated to 100 °C and maintained at this temperature for 4 h. After the reaction was completed, the mixture was cooled to 25 °C, quenched and acidified to pH 5–6 with an aqueous hydrochloric acid solution, and extracted using ethyl acetate (3 × 15 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under a vacuum. The residue was purified via flash column chromatography (n-hexane/ethyl acetate = 15:1) to afford intermediate II (yields 78.5–90.3%).

3.2.2. General Synthetic Procedure of Intermediate III

In a 50 mL three-mouth round-bottom flask, sodium hydride (60%, 8.96 mmol) was added to extra dry 1,4-dioxane (15 mL), and compound II (5.60 mmol) in extra dry 1,4-dioxane (10 mL) was added drop-wise under stirring at 25 °C. The reaction mixture was heated to 50 °C and maintained at this temperature for 3 h. Then, 4-amino-3,5,6-trichloropicolinonitrile (5.60 mmol) was added under stirring and heated to 100 °C for 12 h. The reaction solution was cooled to 25 °C, and was quenched with water. The solid was filtered to provide a mixture of compound III 1 and compound III 2, which were separated via flash column chromatography (n-hexane/ethyl acetate = 10:1) to obtain compounds III 1 (yields 37.6–42.4%) and compounds III 2 (yields 35.2–45.0%).

3.2.3. General Synthetic Procedure of Product

In a 25 mL round-bottom flask, compound III (1.067 mmol) was dissolved in 80% aqueous sulfuric acid (10 mL), and was heated to 100 °C and maintained at this temperature for 2 h. The reaction solution was cooled to 25 °C, and quenched with water. A white solid was collected through filtration to obtain the product (yields 90.1–99.0%).
Compound 1A 4-amino-3,5-dichloro-6-(1H-indazol-1-yl)-2-picolinic acid. White solid; 181.9–182.7 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.86 (s, 1H), 8.40 (s, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.58 (d, J = 8.1 Hz, 1H), 7.50–7.45 (m, 1H), 7.30–7.26 (m, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.09, 151.20, 146.84, 146.40, 139.92, 136.57, 127.93, 124.58, 122.49, 121.62, 111.93, 111.48, 110.45. HRMS calcd. For C13H7Cl2N4O2 ([M – H]−), 320.9946; found, 320.9945.
Compound 1a 4-amino-3,5-dichloro-6-(2H-indazol-2-yl)-2-picolinic acid. White solid; 185.1–185.6 °C; H NMR (500 MHz, DMSO-d6) δ 13.88 (s, 1H), 8.77 (s, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.41 (s, 2H), 7.34 (dd, J = 6.9, 1.6 Hz, 1H), 7.13 (dd, J = 6.9, 1.6 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.90, 151.00, 149.08, 147.30, 146.75, 127.52, 125.98, 122.75, 121.69, 118.07, 112.44, 110.58. HRMS calcd. For C13H7Cl2N4O2 ([M – H]−), 320.9946; found, 320.9947.
Compound 2A 4-amino-3,5-dichloro-6-(4-methyl-1H-indazol-1-yl)-2-picolinic acid. White solid; 195.2–196.4 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.95 (s, 1H), 8.81 (s, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.42 (s, 2H), 7.24 (dd, J = 8.8, 6.7 Hz, 1H), 6.88 (d, J = 6.7 Hz, 1H), 2.53 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.09, 151.15, 146.87, 146.50, 139.89, 135.54, 131.68, 128.03, 124.90, 122.34, 111.44, 110.53, 109.25, 18.63. HRMS calcd. For C14H9Cl2N4O2 ([M – H]−), 335.0103; found, 335.0103.
Compound 2a 4-amino-3,5-dichloro-6-(4-methyl-2H-indazol-2-yl)-2-picolinic acid. White solid; 198.9–201.1 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.31 (s, 1H), 8.81 (s, 1H), 7.57 (d, J = 7.0 Hz, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.41 (s, 2H), 7.23 (dd, J = 8.8, 6.7 Hz, 1H), 6.91–6.85 (m, 1H). 13C NMR (126 MHz, DMSO-d6) δ 168.24, 153.85, 146.47, 143.92, 138.24, 134.72, 130.70, 129.35, 126.83, 120.32, 114.89, 110.58, 105.08, 20.43. HRMS calcd. For C14H9Cl2N4O2 ([M − H]−), 335.0103; found, 335.0104.
Compound 2B 4-amino-3,5-dichloro-6-(5-methyl-1H-indazol-1-yl)-2-picolinic acid. White solid; 190.8–192.2 °C; 1H NMR (500 MHz, DMSO-d6) δ 12.74 (s, 1H), 8.30 (d, J = 0.7 Hz, 1H), 7.65–7.62 (m, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.31 (dd, J = 8.7, 1.3 Hz, 1H), 7.28 (s, 2H), 2.44 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.10, 151.17, 146.79, 146.48, 138.60, 136.05, 131.59, 129.77, 124.99, 120.49, 111.74, 111.32, 110.19, 21.29. HRMS calcd. For C14H9Cl2N4O2 ([M − H]−), 335.0103; found, 335.0103.
Compound 2b 4-amino-3,5-dichloro-6-(5-methyl-2H-indazol-2-yl)-2-picolinic acid. White solid; 193.6–192.2 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.11 (s, 1H), 8.63 (s, 1H), 7.61 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.39 (s, 2H), 7.18 (dd, J = 8.9, 1.4 Hz, 1H), 2.39 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.91, 150.98, 148.15, 147.29, 146.72, 131.65, 130.47, 124.91, 121.96, 119.36, 117.84, 112.28, 110.41, 21.84. HRMS calcd. For C14H9Cl2N4O2 ([M − H]−), 335.0103; found, 335.0101.
Compound 2Cc (mixture) 4-amino-3,5-dichloro-6-(6-methyl-1H-indazol-1-yl)-2-picolinic acid and 4-amino-3,5-dichloro-6-(6-methyl-2H-indazol-2-yl)-2-picolinic acid (1: 1.27). Yellow solid; 149.5–160.2 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.27 (s, 1H), 8.34 (s, 1H), 7.93 (s, 1H), 7.83 (s, 1H), 7.30 (s, 2H), 2.45 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.83, 151.00, 146.53, 145.97, 136.24, 131.04, 125.24, 124.92, 124.26, 122.30, 115.36, 111.65, 110.16, 23.06; 1H NMR (500 MHz, DMSO-d6) δ 12.76 (s, 1H), 8.72 (s, 1H), 8.04 (s, 1H), 7.77 (s, 1H), 7.41 (s, 2H), 2.42 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.01, 151.27, 148.74, 146.95, 146.65, 139.18, 131.11, 125.88, 121.80, 121.10, 120.79, 112.61, 110.52, 23.48. HRMS calcd. For C14H9Cl2N4O2 ([M − H]−), 335.0103; found, 335.0105.
Compound 2d 4-amino-3,5-dichloro-6-(7-methyl-2H-indazol-2-yl)-2-picolinic acid. White solid; 190.8–191.6 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.94 (s, 1H), 8.73 (s, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.41 (s, 2H), 7.10 (d, J = 6.6 Hz, 1H), 7.03 (dd, J = 8.4, 6.7 Hz, 1H), 2.53 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.92, 150.95, 149.55, 147.48, 146.78, 127.66, 126.15, 126.12, 123.01, 121.46, 118.99, 112.41, 110.76, 17.29. HRMS calcd. For C14H9Cl2N4O2 ([M − H]−), 335.0103; found, 335.0101.
Compound 3A 4-amino-3,5-dichloro-6-(4-amino-1H-indazol-1-yl)-2-picolinic acid. Pale yellow solid; 170.5–171.4 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.34 (s, 1H), 8.39 (s, 1H), 7.24 (s, 2H), 7.08 (dd, J = 8.0, 7.5 Hz, 1H), 6.58 (d, J = 8.2 Hz, 1H), 6.28 (d, J = 7.5 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 166.17, 151.01, 146.90, 143.01, 141.58, 135.01, 129.44, 114.30, 111.13, 110.55, 103.73, 98.37. HRMS calcd. For C13H8Cl2N5O2 ([M − H]−), 336.0055; found, 336.0056.
Compound 3a 4-amino-3,5-dichloro-6-(4-amino-2H-indazol-2-yl)-2-picolinic acid. Pale yellow solid; 179.3–180.2 °C; 1H NMR (500 MHz, DMSO-d6) δ 14.03 (s, 0H), 11.14 (s, 1H), 8.98 (s, 2H), 7.82 (dd, J = 8.2, 0.9 Hz, 1H), 7.07 (dd, J = 8.2, 8.1 Hz, 1H), 6.60 (s, 2H), 6.35 (dd, J = 8.1, 0.8 Hz, 1H). 13C NMR (126 MHz, DMSO-d6)13C NMR (126 MHz, DMSO-d6) δ 171.14, 166.86, 151.82, 149.35, 148.38, 145.27, 143.65, 133.20, 109.89, 106.39, 105.05, 100.59, 99.87. HRMS calcd. For C13H8Cl2N5O2 ([M − H]−) 336.0055; found, 336.0057.
Compound 3C 4-amino-3,5-dichloro-6-(6-amino-1H-indazol-1-yl)-2-picolinic acid. Pale yellow solid; 172.3–173.3 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.42 (s, 1H), 8.02 (s, 1H), 7.46 (d, J = 8.6 Hz, 1H), 7.23 (s, 2H), 6.59 (dd, J = 8.6, 1.5 Hz, 1H), 6.44 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ 166.18, 150.93, 149.54, 147.06, 142.29, 136.33, 121.69, 116.36, 113.50, 111.06, 110.82, 92.11. HRMS calcd. For C13H8Cl2N5O2 ([M − H]−), 336.0055; found, 336.0060.
Compound 3c 4-amino-3,5-dichloro-6-(6-amino-2H-indazol-2-yl)-2-picolinic acid. Pale yellow solid; 158.9–160.0 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.35 (s, 1H), 8.92 (s, 1H), 7.99 (d, J = 8.9 Hz, 1H), 7.73 (s, 1H), 7.46 (s, 2H), 7.10 (dd, J = 8.9, 1.4 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.83, 151.03, 148.01, 146.90, 146.75, 131.20, 127.30, 124.22, 120.75, 118.79, 112.72, 111.92, 110.63. HRMS calcd. For C13H8Cl2N5O2 ([M − H]−), 335.0055; found, 336.0062.
Compound 3d 4-amino-3,5-dichloro-6-(7-amino-2H-indazol-2-yl)-2-picolinic acid. Yellow solid; 214.2–214.9 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.23 (s, 1H), 8.55 (s, 1H), 7.39 (s, 2H), 6.91 (d, J = 8.0 Hz, 1H), 6.85 (dd, J = 8.3, 7.1 Hz, 1H), 6.33 (d, J = 6.9 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 166.00, 150.87, 147.53, 146.88, 142.90, 138.72, 125.29, 124.53, 122.48, 112.21, 110.66, 107.65, 104.43. HRMS calcd. For C13H8Cl2N5O2 ([M − H]−), 335.0055; found, 336.0057.
Compound 4A 4-amino-3,5-dichloro-6-(4-methoxy-1H-indazol-1-yl)-2-picolinic acid. White solid; 199.1–201.6 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.36 (s, 1H), 9.16 (s, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.38 (dd, J = 8.9, 8.4 Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H), 6.72 (s, 2H), 3.81 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 169.31, 166.67, 158.44, 148.88, 148.62, 145.36, 140.35, 132.17, 112.99, 111.66, 105.79, 105.48, 99.33, 56.46. HRMS calcd. For C14H9Cl2N4O3 ([M − H]−), 351.0052; found, 351.0052.
Compound 4a 4-amino-3,5-dichloro-6-(4-methoxy-2H-indazol-2-yl)-2-picolinic acid. White solid; 221.3–222.5 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.56 (s, 1H), 7.95 (s, 1H), 7.18 (dd, J = 8.0, 7.9 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 6.47 (s, 1H), 6.46 (s, 2H), 3.84 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 169.12, 157.35, 150.28, 145.45, 142.77, 135.39, 131.83, 128.28, 124.79, 118.32, 112.67, 105.39, 101.68, 56.92. HRMS calcd. For C14H9Cl2N4O3 ([M − H]−), 351.0052; found, 351.0050.
Compound 4B 4-amino-3,5-dichloro-6-(5-methoxy-1H-indazol-1-yl)-2-picolinic acid. White solid; 179.8–180.5 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.72 (s, 1H), 8.28 (s, 1H), 7.53 (d, J = 9.1 Hz, 1H), 7.31 (d, J = 2.3 Hz, 1H), 7.28 (s, 2H), 7.13 (dd, J = 9.1, 2.4 Hz, 1H), 3.83 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.12, 155.49, 151.19, 146.89, 146.42, 135.98, 135.62, 125.25, 119.32, 113.20, 111.16, 109.80, 101.16, 55.96. HRMS calcd. For C14H9Cl2N4O3 ([M − H]−), 351.0052; found, 351.0051.
Compound 4b 4-amino-3,5-dichloro-6-(5-methoxy-2H-indazol-2-yl)-2-picolinic acid. White solid; 223.0–224.3 °C; 1H NMR (500 MHz, DMSO-d6) δ13.67 (s, 1H), 8.58 (d, J = 0.9 Hz, 1H), 7.62 (d, J = 9.3 Hz, 1H), 7.38 (s, 2H), 7.05 (d, J = 2.4 Hz, 1H), 7.02 (dd, J = 9.3, 2.4 Hz, 1H), 3.80 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 168.10, 157.32, 150.76, 148.85, 142.45, 130.63, 129.46, 125.82, 120.97, 115.23, 110.35, 104.74, 100.38, 55.52. HRMS calcd. For C14H9Cl2N4O3 ([M − H]−), 351.0052; found, 351.0056.
Compound 4C 4-amino-3,5-dichloro-6-(6-methoxy-1H-indazol-1-yl)-2-picolinic acid. White solid; 193.1–194.4 °C; 1H NMR (500 MHz, DMSO-d6) δ 12.98 (s, 1H), 11.51 (s, 1H), 8.77 (d, J = 2.5 Hz, 1H), 7.94 (d, J = 8.9 Hz, 1H), 6.76 (s, 2H), 6.57 (dd, J = 8.9, 2.6 Hz, 1H), 3.82 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 170.35, 166.52, 164.08, 148.87, 148.77, 145.82, 144.15, 133.37, 107.49, 107.27, 106.68, 102.96, 100.25, 55.63. HRMS calcd. For C14H9Cl2N4O3 ([M − H]−), 351.0052; found, 351.0056.
Compound 4c 4-amino-3,5-dichloro-6-(6-methoxy-2H-indazol-2-yl)-2-picolinic acid. White solid; 244.6–245.0 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.05 (s, 1H), 8.59 (s, 1H), 7.65 (d, J = 9.1 Hz, 1H), 7.07 (s, 2H), 6.99 (s, 1H), 6.77 (dd, J = 9.1, 2.1 Hz, 1H), 3.83 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.95, 159.22, 150.34, 150.09, 146.92, 125.71, 122.50, 117.79, 117.45, 110.77, 108.33, 94.92, 55.60. HRMS calcd. For C14H9Cl2N4O3 ([M − H]−), 351.0052; found, 351.0054.
Compound 4d 4-amino-3,5-dichloro-6-(7-methoxy-2H-indazol-2-yl)-2-picolinic acid. White solid; 203.0–203.7 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.97 (s, 1H), 8.71 (s, 1H), 7.41 (s, 2H), 7.31 (d, J = 8.4 Hz, 1H), 7.03 (dd, J = 8.3, 7.5 Hz, 1H), 6.68 (d, J = 7.3 Hz, 1H), 3.93 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.90, 150.97, 150.50, 147.32, 146.76, 142.78, 125.92, 123.51, 123.42, 113.20, 112.37, 110.60, 104.23, 55.70. HRMS calcd. For C14H9Cl2N4O3 ([M − H]−), 351.0052; found, 351.0060.
Compound 5A 4-amino-3,5-dichloro-6-(4-fluoro-1H-indazol-1-yl)-2-picolinic acid. White solid; 161.2–162.3 °C; 1H NMR (500 MHz, DMSO-d6) δ 12.80 (s, 1H), 8.55 (d, J = 0.8 Hz, 1H), 7.50 (td, J = 8.0, 5.1 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.35 (s, 2H), 7.09 (dd, J = 10.2, 7.6 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.80, 156.63, 154.62, 151.19, 151.15, 151.02, 146.96, 146.77, 127.88, 127.83, 123.63, 123.59, 114.64, 114.60, 113.42, 113.26, 112.79, 110.85, 105.48, 105.35. HRMS calcd. For C13H6Cl2FN4O2 ([M − H]−) 338.9852; found, 338.9859.
Compound 5a 4-amino-3,5-dichloro-6-(4-fluoro-2H-indazol-2-yl)-2-picolinic acid. White solid; 177.9–178.5 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.78 (s, 1H), δ 8.99 (d, J = 0.9 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.46 (s, 2H), 7.34 (ddd, J = 8.8, 7.5, 5.4 Hz, 1H), 6.91 (dd, J = 10.7, 7.4 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.98, 156.14, 154.15, 151.24, 146.87, 145.92, 142.43, 142.36, 132.50, 129.47, 129.41, 114.41, 114.23, 111.98, 110.82, 108.43, 108.40, 107.01, 106.86. HRMS calcd. For C13H6Cl2FN4O2 ([M − H]−) 338.9852; found, 338.9858.
Compound 5B 4-amino-3,5-dichloro-6-(5-fluoro-1H-indazol-1-yl)-2-picolinic acid. White solid; 197.8–198.4 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.23 (s, 1H), 8.40 (d, J = 0.9 Hz, 1H), 7.69 (dd, J = 8.9, 2.5 Hz, 1H), 7.66 (dd, J = 9.2, 4.4 Hz, 1H), 7.39 (ddd, J = 9.1, 2.5, 2.3 Hz, 1H), 7.33 (s, 2H). 13C NMR (126 MHz, DMSO-d6) δ 166.01, 159.20, 157.32, 151.28, 146.74, 146.13, 136.95, 136.43, 136.38, 124.86, 124.78, 117.29, 117.08, 113.73, 113.66, 111.61, 110.25, 105.98, 105.79. HRMS calcd. For C13H6Cl2FN4O2 ([M − H]−), 338.9852; found, 338.9850.
Compound 5b 4-amino-3,5-dichloro-6-(5-fluoro-2H-indazol-2-yl)-2-picolinic acid. White solid; 193.5–194.8 °C; 1H NMR (500 MHz, DMSO-d6) δ 14.02 (s, 1H), 8.77 (s, 1H), 7.81 (dd, J = 9.4, 4.7 Hz, 1H), 7.54 (dd, J = 9.4, 2.2 Hz, 1H), 7.44 (s, 2H), 7.28 (ddd, J = 9.3, 2.4, 2.2 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.83, 159.17, 157.27, 150.98, 146.93, 146.61, 146.55, 126.48, 126.41, 120.88, 120.78, 120.64, 120.56, 119.35, 119.12, 112.60, 110.49, 104.08, 103.89. HRMS calcd. For C13H6Cl2FN4O2 ([M − H]−), 338.9852; found, 338.9850.
Compound 5Cc (mixture) 4-amino-3,5-dichloro-6-(6-fluoro-1H-indazol-1-yl)-2-picolinic acid and 4-amino-3,5-dichloro-6-(6-fluoro-2H-indazol-2-yl)-2-picolinic acid (1: 0.47). Yellow solid; 160.1–167.9 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.94 (s, 1H), 8.77 (s, 1H), 7.80 (dd, J = 9.4, 4.7 Hz, 1H), 7.53 (dd, J = 9.4, 2.4 Hz, 1H), 7.43 (s, 2H), 7.28 (ddd, J = 9.4, 2.5, 2.2 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 166.00, 157.33, 151.30, 146.75, 146.15, 136.40, 124.87, 124.78, 120.78, 120.70, 117.28, 117.06, 113.70, 111.61, 111.23, 105.96, 105.77; 1H NMR (500 MHz, DMSO-d6) δ 13.11 (s, 1H), 8.40 (s, 1H), 7.68 (dd, J = 8.9, 2.5 Hz, 1H, 7.65 (dd, J = 8.9, 2.5 Hz, 1H), 7.39 (td, J = 9.1, 2.5 Hz, 1H), 7.33 (s, 2H). 13C NMR (126 MHz, DMSO-d6) δ 165.85, 159.22, 151.04, 147.08, 146.61, 136.97, 126.43, 126.36, 120.89, 120.80, 119.24, 119.01, 113.77, 112.53, 110.50, 104.04, 103.84. HRMS calcd. For C13H6Cl2FN4O2 ([M − H]−), 338.9852; found, 338.9852.
Compound 5d 4-amino-3,5-dichloro-6-(5-fluoro-2H-indazol-2-yl)-2-picolinic acid. White solid; 203.6–204.2 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.62 (s, 1H), 8.24–8.15 (m, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.18 (dd, J = 11.4, 7.6 Hz, 1H), 7.09 (ddd, J = 7.8, 4.5, 4.4 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.84, 153.62, 151.61, 151.04, 146.97, 146.77, 140.21, 140.08, 127.33, 124.89, 124.84, 122.78, 122.73, 118.18, 118.15, 112.77, 110.78, 110.40, 110.27. HRMS calcd. For C13H6Cl2FN4O2 ([M − H]−), 338.9852; found, 338.9851.
Compound 6A 4-amino-3,5-dichloro-6-(4-chloro-1H-indazol-1-yl)-2-picolinic acid. White solid; 195.3–195.8 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.28 (s, 1H), 8.50 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.49 (dd, J = 7.5, 0.9 Hz, 1H), 7.38 (d, J = 7.3 Hz, 2H), 7.38 (s, 1H). 133C NMR (126 MHz, DMSO-d6) δ 165.99, 151.27, 146.85, 145.89, 140.99, 134.46, 129.09, 125.58, 123.34, 122.15, 112.08, 111.03, 110.87. HRMS calcd. For C13H6Cl3N4O3 ([M − H]−), 354.9556; found, 354.9559.
Compound 6a 4-amino-3,5-dichloro-6-(4-chloro-2H-indazol-2-yl)-2-picolinic acid. White solid; 206.2–207.3 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.98 (s, 1H), 8.93 (s, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.48 (s, 2H), 7.36 (dd, J = 8.7, 7.2 Hz, 1H), 7.25 (d, J = 7.1 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.78, 151.03, 149.42, 146.93, 146.84, 128.13, 125.62, 125.25, 122.11, 121.65, 117.34, 112.77, 110.74. HRMS calcd. For C13H6Cl3N4O3 ([M − H]−), 354.9556; found, 354.9532.
Compound 6B 4-amino-3,5-dichloro-6-(5-chloro-1H-indazol-1-yl)-2-picolinic acid. White solid; 198.5–199.1 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.17 (s, 1H), 8.41 (d, J = 0.7 Hz, 1H), 8.00 (d, J = 1.7 Hz, 1H), 7.64 (d, J = 8.9 Hz, 1H), 7.51 (dd, J = 8.9, 2.0 Hz, 1H), 7.36 (s, 2H). 13C NMR (126 MHz, DMSO-d6) δ 165.83, 151.05, 147.36, 146.92, 146.71, 128.59, 127.20, 126.18, 122.01, 120.45, 120.24, 112.67, 110.53. HRMS calcd. For C13H6Cl3N4O3 ([M − H]−), 354.9556; found, 354.9555.
Compound 6b 4-amino-3,5-dichloro-6-(5-chloro-2H-indazol-2-yl)-2-picolinic acid. White solid; 204.2–205.3 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.40 (s, 1H), 8.80 (s, 1H), 7.92 (d, J = 1.3 Hz, 2H), 7.78 (d, J = 9.2 Hz, 1H), 7.46 (s, 2H), 7.34 (dd, J = 9.2, 2.0 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.83, 151.05, 147.36, 146.94, 146.74, 128.58, 127.18, 126.20, 122.01, 120.46, 120.27, 112.63, 110.52. HRMS calcd. For C13H6Cl3N4O3 ([M − H]−), 354.9556; found, 354.9554.
Compound 6Cc (mixture) 4-amino-3,5-dichloro-6-(6-chloro-1H-indazol-1-yl)-2-picolinic acid and 4-amino-3,5-dichloro-6-(6-chloro-2H-indazol-2-yl)-2-picolinic acid (0.48: 1). Yellow solid; 158.0–171.4 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.27 (s, 1H), 8.91 (s, 1H), 7.92 (d, J = 9.0 Hz, 1H), 7.88 (s, 1H), 7.48 (s, 2H), 7.18 (dd, J = 8.9, 1.6 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.96, 151.34, 146.67, 145.92, 140.33, 133.40, 132.33, 123.38, 123.26, 123.21, 112.63, 111.85, 110.59; 1H NMR (500 MHz, DMSO-d6) δ 13.27 (s, 1H), 8.49 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.79 (s, 1H), 7.37(s, 2H), 7.36 (dd, J = 8.5, 1.7 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.83, 151.05, 149.05, 147.00, 146.78, 136.73, 127.06, 123.87, 123.86, 120.21, 116.87, 111.82, 110.54. HRMS calcd. For C13H6Cl3N4O2 ([M − H]−), 338.9556; found, 338.9555.
Compound 6d 4-amino-3,5-dichloro-6-(7-chloro-2H-indazol-2-yl)-2-picolinic acid. White solid; 211.5–212.1 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.89 (s, 1H), 8.94 (s, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.48 (dd, J = 6.8, 0.4 Hz, 1H), 7.46 (s, 2H), 7.13 (dd, J = 8.4, 7.2 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.84, 151.02, 147.06, 146.82, 146.41, 127.88, 126.94, 123.24, 122.98, 122.28, 121.11, 112.79, 110.89. HRMS calcd. For C13H6Cl3N4O3 ([M − H]−), 354.9556; found, 354.9555.
Compound 7A 4-amino-3,5-dichloro-6-(4-bromo-1H-indazol-1-yl)-2-picolinic acid. Yellow solid;181.5–182.3 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.51 (s, 1H), 8.39 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 7.4 Hz, 1H), 7.42 (dd, J = 7.8, 0.4 Hz, 1H), 7.37 (s, 2H). 13C NMR (126 MHz, DMSO-d6) δ 165.95, 151.29, 146.93, 145.95, 140.68, 135.85, 129.39, 125.36, 125.18, 113.85, 111.98, 111.53, 110.83. HRMS calcd. For C13H6BrCl2N4O3 ([M − H]−), 398.9051; found, 398.9055.
Compound 7a 4-amino-3,5-dichloro-6-(4-bromo-2H-indazol-2-yl)-2-picolinic acid. Yellow solid; 206.4–207.2 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.60 (s, 1H), 8.83 (s, 1H), 7.76 (d, J = 8.7 Hz, 1H), 7.45 (s, 2H), 7.41 (d, J = 7.1 Hz, 1H), 7.30 (dd, J = 8.6, 7.2 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.77, 151.03, 148.90, 146.92, 146.69, 128.56, 126.92, 125.51, 123.40, 117.74, 113.30, 112.79, 110.76. HRMS calcd. For C13H6BrCl2N4O3 ([M − H]−), 398.9051; found, 398.9054.
Compound 7B 4-amino-3,5-dichloro-6-(5-bromo-1H-indazol-1-yl)-2-picolinic acid. Yellow solid; 207.5–208.6 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.45 (s, 1H), 8.41 (s, 1H), 8.15 (s, 1H), 7.66–7.57 (m, 2H), 7.35 (s, 2H). 13C NMR (126 MHz, DMSO-d6) δ 165.98, 151.29, 146.81, 145.97, 138.76, 135.95, 130.68, 126.27, 124.03, 114.75, 114.04, 111.76, 110.43. HRMS calcd. For C13H6BrCl2N4O3 ([M − H]−), 398.9051; found, 398.9056.
Compound 7b 4-amino-3,5-dichloro-6-(5-bromo-2H-indazol-2-yl)-2-picolinic acid. Yellow solid; 209.9–210.2 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.39 (s, 1H), 8.79 (s, 1H), 8.08 (s, 1H), 7.71 (d, J = 9.2 Hz, 1H), 7.44–7.42 (m, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.81, 151.06, 147.39, 146.95, 146.77, 130.78, 126.02, 123.85, 122.87, 120.48, 115.36, 112.60, 110.52. HRMS calcd. For C13H6BrCl2N4O3 ([M − H]−), 398.9051; found, 398.9053.
Compound 7Cc (mixture) 4-amino-3,5-dichloro-6-(6-bromo-1H-indazol-1-yl)-2-picolinic acid and 4-amino-3,5-dichloro-6-(6-bromo-2H-indazol-2-yl)-2-picolinic acid (1: 1.03). Yellow solid; 143.7–158.9 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.73 (s, 1H), 8.86 (s, 1H), 8.01 (s, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.43 (s, 2H), 7.43 (dd, J = 8.5, 1.5 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.83, 149.59, 146.99, 146.80. 146.78, 140.68, 127.12, 123.60, 120.90, 120.32, 112.62, 110.58; 1H NMR (500 MHz, DMSO-d6) δ 13.11 (s, 1H), 8.44 (s, 1H), 7.90 (s, 1H), 7.82 (d, J = 8.9 Hz, 1H), 7.33 (s, 2H), 7.25 (d, J = 9.0 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.96, 151.34, 151.05, 146.68, 145.91, 136.78, 126.14, 125.73, 123.98, 123.46, 121.53, 120.19, 114.80, 111.85. HRMS calcd. For C13H6BrCl2N4O3 ([M − H]−), 398.9051; found, 398.9050.
Compound 7d 4-amino-3,5-dichloro-6-(7-bromo-2H-indazol-2-yl)-2-picolinic acid. Yellow solid; 212.3–213.2 °C; 1H NMR (500 MHz, DMSO-d6) δ 13.21 (s, 1H), 8.97 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 7.1 Hz, 1H), 7.46 (s, 2H), 7.07 (dd, J = 7.0, 1.0 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.85, 151.01, 147.47, 147.07, 146.83, 130.35, 128.01, 123.67, 122.52, 121.63, 112.78, 110.99, 110.92. HRMS calcd. For C13H6BrCl2N4O3 ([M − H]−), 398.9051; found, 398.9053.

3.3. Determination of the Biological Activities

3.3.1. Phenotypic Study for the Inhibition of A. thaliana Root Growth

Seeds of Col-0 were surface-sterilized using 1% sodium hypochlorite solution, and were sown onto 1/2 Modified Medium (with vitamins, sucrose and phytagel) with compounds at 200, 100, 50, 25, 12, 6 and 3 µM in Petri dishes. Subsequently, seeds were incubated at 4 °C for two days under darkness, and were cultured on vertically oriented Petri dishes at 22 °C for 7 d under light/dark (16 h/8 h) cycling in a plant incubator. The taproot length of 7-day-old seedlings was measured using IMAGEJ software. The inhibition percentage of A. thaliana root growth was calculated based on the following equation:
P 0 = L a 0 − L a L a 0 × 100 %
where P0 is the inhibition percentage, and La and La0 are the average lengths of the roots if A. thaliana in the presence of compounds and untreated controls, respectively.

3.3.2. Evaluation of the Herbicidal Activity

The herbicidal activity of synthetic compounds was evaluated according to a reported procedure, and picloram was used as the positive control and the results represent the bioactivity triplicate [18]. Preliminary herbicidal activities of synthetic compounds against BN, AM, EC, AR and CA were screened at concentrations of 500 and 250 μM in Petri dish tests. Emulsions of new compounds and picloram were prepared by dissolving them in the mixture of DMSO (0.1 mL) and Tween-80 (0.1 mL) followed by dispersing in deionized water (10 mL). A mixture of the same amount of water, DMSO and Tween-80 was used as the untreated control. The seeds were soaked in warm water (25 °C) for 15 h before use, and about 10 seeds of BN, AM, AR, CA and EC were placed onto germinating paper (6 cm) wetted with 5 mL emulsions in a 6 cm Petri dish. The plates were placed in a dark room and allowed to germinate for 36 h at 25 ± 1 °C, then transferred to 22 °C for four to seven days under light/dark (16 h/8 h) cycling. The lengths of five BN, AM, AR, CA and EC root radicles randomly selected from each plate were measured, respectively. The average of the root lengths was calculated, and the inhibition percentage was calculated using the following equation:
P 1 = L w 0 − L w L w 0 × 100 %
where P1 is the inhibition percentage, and Lw and Lw0 are the average lengths of the five weed’s roots in the presence of compounds and untreated controls, respectively.
Furthermore, the vivo post-emergence herbicidal activity of new compounds against four dicotyledonous weeds, BN, AM, AR and CA, and one monocotyledonous weed, EC, was tested at a dosage of 1000, 500 and 250 g ha−1 in a glasshouse (Xian Zheng Fei Greenhouse, Science and Technology Park, China Agricultural University, Beijing, China). The plant growth medium was obtained by mixing peat soil, flower soil and vermiculite at a mass ratio of 1:3:2. Preparation of the emulsions of new compounds and picloram was the same for tests in Petri dishes. The emulsions were sprayed using a spray bottle at a dosage of 1000, 500 and 250 g ha−1 after the plants reached the two-leaf stage. Subsequently, the seedings grew in the greenhouse (natural environment, no additional lighting, 25–35 °C). Weed growth and toxic symptoms were observed regularly after treatment and the growth inhibitory activities of each compound were visually evaluated 14 d after treatment based on the following index: all dead: 100%; stems atrophy and dead leaves: 80%; stems atrophy and partially dead leaves: 60%, normal stems and partially dead leaves: 40%; normal stems and partially curled leaves: 20%, and normal stems and leaves = 0.

3.4. Quantitative Real-Time PCR

A. thaliana was cultured on 1/2 Modified Medium (with vitamins, sucrose, and phytagel) in Petri dishes (10 × 10 cm), and placed in a plant incubator after the seeds were incubated at 4 °C for two days under darkness. The seeds were cultured on vertically oriented Petri dishes at 22 °C for two weeks under light/dark (16 h/8 h) cycling. Seedlings then were carefully placed on filter paper wetted with the solution of compound 7Cc, 5A, 5a, picloram at a concentration of 100 μM and deionized water in a Petri dish (10 × 10 cm), respectively. After 12 h, 24 h and 72 h, the whole seedlings were rapid frozen with liquid nitrogen, collected and stored at −80 °C. Detailed steps are described in the Supporting Information.

4. Conclusions

In this study, based on scaffold hopping, 38 4-amino-3,5-dicholor-6-(1H-indazol-1-yl)-2-picolinic acid and 4-amino-3,5-dicholor-6-(2H-indazol-1-yl)-2-picolinic acid compounds were designed and synthesized via a four-step synthetic route with good yields. The results of primary bioassay on the root growth inhibition of A. thaliana demonstrated that most compounds had an excellent inhibitory effect, especially with substituents at the 4, 6, 7 position and electron-withdrawing groups on the indazole ring. Compound 5a had a comparable performance against the commercial herbicide picloram, while 7Cc was superior to picloram. The herbicidal activity in Petri dishes showed that most of the compounds were able to inhibit root growth in five weeds, while compound 5a showed significantly better activity than picloram in the root inhibition assay of BN and AM at a concentration of 10 µM. Most of the compounds exhibited excellent herbicidal activity in post-emergence at 1000 and 500 g/ha, while compound 5a also had an injurious effect on AR and CA at 250 g/ha. Overall, 6-indazolyl-2-picolinic acids with an electron-withdrawing substituent on the indazole ring and substituent at the 4 position exhibited excellent root inhibitory and herbicidal activities. Compounds 7Cc, 5A, and 5a did not arouse the up-regulation of auxin-related gene as picloram did, but they promoted ethylene release and ABA production through the up-regulation of ACS and NCED genes to cause plant death in a short period of time. These results provide new perspectives and insights for the future design of compounds with similar structures.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules29020332/s1, Figure S1: The root inhibition of compounds on EC at 500 µM and 250 µM; Figure S2: The root inhibition of compounds on AR at 500 µM and 250 µM; Figure S3: The root inhibition of compounds on CA at 500 µM and 250 µM; Figure S4: The root inhibit ion of compounds on AM at 500 µM and 250 µM; Figure S5: The root inhibition of compounds on BN at 500 µM and 250 µM; Figure S6: Summary of the visual injury percentages resulting from treatment of the five dicotyledonous weeds 14 days with the compounds at concentrations 1000 g/ha; Table S1: Herbicidal activities of some compounds against five weeds (reflected in terms of the visual injury effect %); Table S2: Primers for real-time PCR used in this study; Additional Experimental Details for qPCR; The1H and 13C NMR spectra of compounds 1A–7d.

Author Contributions

Conceptualization, Y.-M.C. and S.-Z.L.; methodology, Q.L.; software, Q.L.; bioassay, Q.L., R.-C.S. and W.W.; validation, H.-T.L. and X.Y. All authors have read and agreed to the published version of H.-T.L. the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key R&D Program, grant number 2022YFD1700403.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Molecules 29 00332 g001
Figure 1. Commercial 2-picolinic acid and 2-picolinate herbicides from 1940s.
Figure 1. Commercial 2-picolinic acid and 2-picolinate herbicides from 1940s.
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Molecules 29 00332 g002
Figure 2. Structures of herbicidal compounds 6-phenylpyrazolyl-2-picolinic acids [18,19].
Figure 2. Structures of herbicidal compounds 6-phenylpyrazolyl-2-picolinic acids [18,19].
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Molecules 29 00332 g003
Figure 3. Design strategy of new 6-indazolyl-2-picolinic acid target compounds.
Figure 3. Design strategy of new 6-indazolyl-2-picolinic acid target compounds.
Molecules 29 00332 g003
Molecules 29 00332 sch001
Scheme 1. Synthetic route to 6-indazolyl-2-picolinic acids. Reagent and conditions: (a) NH2-NH2·H2O, THF, 65 °C, 2 h; (b) NaHCO3, NH2-NH2·H2O, 100 °C, 4 h; (c) NaH, extra dry 1,4-dioxane, 100 °C, 12 h; (d) 80% H2SO4, H2O, 100 °C, 3 h.
Scheme 1. Synthetic route to 6-indazolyl-2-picolinic acids. Reagent and conditions: (a) NH2-NH2·H2O, THF, 65 °C, 2 h; (b) NaHCO3, NH2-NH2·H2O, 100 °C, 4 h; (c) NaH, extra dry 1,4-dioxane, 100 °C, 12 h; (d) 80% H2SO4, H2O, 100 °C, 3 h.
Molecules 29 00332 sch001
Molecules 29 00332 sch002
Scheme 2. Reaction of 5-bromo-1H-indazole and 4-amino-3,5,6-trichloro-2-picolinonitrile.
Scheme 2. Reaction of 5-bromo-1H-indazole and 4-amino-3,5,6-trichloro-2-picolinonitrile.
Molecules 29 00332 sch002
Molecules 29 00332 g004
Figure 4. The inhibition activity of A. thaliana root growth for some compounds. (a): the assay concentrations were 50 µM and 25 µM; (b): the assay concentrations were 12.5 µM, 6 µM and 3 µM.
Figure 4. The inhibition activity of A. thaliana root growth for some compounds. (a): the assay concentrations were 50 µM and 25 µM; (b): the assay concentrations were 12.5 µM, 6 µM and 3 µM.
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Molecules 29 00332 g005
Figure 5. The root inhibition activity of new compounds on five grasses at 500 µM.
Figure 5. The root inhibition activity of new compounds on five grasses at 500 µM.
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Figure 6. The root inhibition activity of new compounds on five grasses at 250 µM.
Figure 6. The root inhibition activity of new compounds on five grasses at 250 µM.
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Figure 7. The weed root growth inhibition activity. ((a): 5a; (b): picloram; (c): water).
Figure 7. The weed root growth inhibition activity. ((a): 5a; (b): picloram; (c): water).
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Molecules 29 00332 g008
Figure 8. Summary of the visual injury percentages resulting from treatment of the four dicotyledonous weeds by the compounds over 14 days at the concentration of 500 g/ha.
Figure 8. Summary of the visual injury percentages resulting from treatment of the four dicotyledonous weeds by the compounds over 14 days at the concentration of 500 g/ha.
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Molecules 29 00332 g009
Figure 9. Auxin-related genes response. ((a): compound 7Cc; (b): picloram; (c): compound 5A; (d): compound 5a).
Figure 9. Auxin-related genes response. ((a): compound 7Cc; (b): picloram; (c): compound 5A; (d): compound 5a).
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Table 1. The structures of new compounds.
Table 1. The structures of new compounds.
No.Compd.RNo.Compd.R
1AIVH3cV6-NH2
2AIV4-CH3 3dV7-NH2
2BIV5-CH34aV4-OCH3
3AIV4-NH24bV5-OCH3
3CIV6-NH24cV6-OCH3
4AIV4-OCH34dV7-OCH3
4BIV5-OCH35aV4-F
4CIV6-OCH35bV5-F
5AIV4-F5dV7-F
5BIV5-F6aV4-Cl
6AIV4-Cl6bV5-Cl
6BIV5-Cl6dV7-Cl
7AIV4-Br7aV4-Br
7BIV5-Br7bV5-Br
1aVH7dV7-Br
2aV4-CH32CcIV+V6-CH3
2bV5-CH35CcIV+V6-F
2dV7-CH36CcIV+V6-Cl
3aV4-NH27CcIV+V6-Br
Table 2. Reaction condition optimization for increasing the percentage of r1 in the product.
Table 2. Reaction condition optimization for increasing the percentage of r1 in the product.
Solvent Base Temperature
(T1) a		Temperature
(T2) b		Reaction Progress
Conversion/Ratio of r1:r2
Extra dry dioxaneNaH50 °C100 °CComplete/r1:r2 = 1:1
Extra dry dioxaneNaH25 °C100 °CIncomplete (5%) c/r1:r2 = 1:1
Extra dry dioxaneK2CO350 °C100 °CNo reaction
Extra dry dioxaneKOH50 °C100 °CNo reaction
Extra dry dioxaneNaOH50 °C100 °CNo reaction
Extra dry dioxaneCsCO350 °C100 °CIncomplete (10%) c/r1:r2 = 2.5:1
Extra dry dioxaneCsCO325 °C100 °CIncomplete (15%) c/r1:r2 = 3.4:1
Extra dry dioxaneCsCO325 °C50 °CIncomplete (20%) c/r1:r2 = 1.3:1
AcetonitrileCsCO3−13 °C80 °CIncomplete (25%) c/r1:r2 = 3.9:1
1,2-DimethoxyethaneCsCO3−10 °C85 °CIncomplete (15%) c/r1:r2 = 4.9:1
1,2-DiethoxyethaneCsCO3−10 °C110 °CIncomplete (5%) c/r1:r2 = 2.25:1
a 3-h reaction time at T1 temperature. b 12-h reaction time at T2 temperature. c Percentage of unreacted 4-amino-3,5,6-trichloropicolinonitrile.
Table 3. Herbicidal activities of some compounds against five weeds.
Table 3. Herbicidal activities of some compounds against five weeds.
CompoundDosage
(g ha−1)		BN (%)AM (%)CA (%)AR (%)EC (%)
1a1000100301001000
500100/1001000
250100/151000
2b10001001001001000
50010045701000
250100/451000
2d10001001001001000
50080401001000
25060/1001000
3d100010001001000
500100/1001000
250100/30800
4A10001001001001000
5001000701000
250100/101000
4d1000100801001000
50010070651000
2501001030950
5A10001001001001000
50010051001000
250100/751000
5a10001001001001000
50065101001000
25015/40750
6A1000100851001000
500100101001000
250100/45850
6a1000100801001000
5002051001000
250//45900
6b100010010801000
500100/601000
250100/101000
6d1000100701001000
50050251001000
250//65900
7a1000100701001000
50010001001000
250100/50800
7d1000100751001000
5004051001000
250//30850
Picloram100010010010010010
500100651001000
2501004080850
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MDPI and ACS Style

Liu, Q.; Shi, R.-C.; Li, H.-T.; Wei, W.; Yuan, X.; Liu, S.-Z.; Cao, Y.-M. Study on Design, Synthesis and Herbicidal Activity of Novel 6-Indazolyl-2-picolinic Acids. Molecules 2024, 29, 332. https://doi.org/10.3390/molecules29020332

AMA Style

Liu Q, Shi R-C, Li H-T, Wei W, Yuan X, Liu S-Z, Cao Y-M. Study on Design, Synthesis and Herbicidal Activity of Novel 6-Indazolyl-2-picolinic Acids. Molecules. 2024; 29(2):332. https://doi.org/10.3390/molecules29020332

Chicago/Turabian Style

Liu, Qing, Rong-Chuan Shi, Hui-Ting Li, Wei Wei, Xiao Yuan, Shang-Zhong Liu, and Yi-Ming Cao. 2024. "Study on Design, Synthesis and Herbicidal Activity of Novel 6-Indazolyl-2-picolinic Acids" Molecules 29, no. 2: 332. https://doi.org/10.3390/molecules29020332

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