Identification and Synthesis of a Nitrophenyl Metabolite of Rinskor

Article pubs.acs.org/OPRD

Cite This: Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Identification and Synthesis of a Nitrophenyl Metabolite of Rinskor Active from Terrestrial Aerobic Soil Studies Belgin Canturk,*,† Pete Johnson,† Joshua Taylor,§ Jeremy Kister,‡ and Jesse Balcer§ †

Product Design & Process R&D, Corteva Agriscience, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States Discovery Chemistry, Corteva Agriscience, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States § Regulatory Sciences, Corteva Agriscience, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States

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ABSTRACT: To support the registration of crop protection products, over 100 regulatory studies are conducted for an active ingredient. These studies allow regulatory agencies and the agroscience industry to ensure that new crop protection products meet the requirements for users, consumers, and the environment prior to their use in agriculture. Rinskor active is a Corteva Agriscience auxinic herbicide, which was launched in 2018 for the control of broadleaf weeds, grass weeds, and sedges in rice fields. To support the registration of Rinskor, four terrestrial aerobic soil studies were conducted to determine the soil degradation profile and rate of the active ingredient. These studies were conducted using carbon-14-labeled active ingredient test systems. From these studies, three major metabolites were generated, and the structures of these metabolites had to be identified via the preparation of an authentic standard of each metabolite. This Article discusses the identification and synthesis of one of the major metabolites, a nitrophenyl metabolite, formed from terrestrial aerobic soil studies. KEYWORDS: crop protection products, auxinic herbicides, Rinskor, soil metabolism, nitration

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Rinskor active5 (2) (Figure 1) represents the latest addition to the auxinic herbicide family. A primary use of 2 is to control important broadleaf weeds, grass weeds, and sedges in rice fields, with an exceptionally low application rate of 7.5−30 g per hectare (g/ha) of land. This amount of material applied can be contrasted with the 280−2240 g/ha that is required for 1 depending on the type of application. In addition to a low use rate, 2 also exhibits favorable environmental and toxicological profiles, which were contributing factors in its earning an American Chemical Society 2018 Green Chemistry Challenge Award. Prior to registering a new crop protection product, over 100 core regulatory studies are required by different regulatory agencies throughout the world. A series of environmental fate and metabolism studies are conducted to determine the rate and route of degradation of active ingredients in the environment (e.g., air, soil, and water), crops, and animals. They utilize carbon-14-labeled active ingredient test material, which allows the tracking and quantification of the parent and metabolites formed during the studies. Metabolites generated from these studies via hydrolysis, reduction, photolysis, or oxidation (phase I metabolism) and conjugates (phase II metabolism)6 that exceed trigger values must be identified, and their structures must be confirmed. In some cases, metabolites must be further evaluated for risk assessment based on exposure and hazard data to ensure that there is no risk to the environment or off-target species.7 Aerobic soil metabolism is one of the key regulatory studies that provides route and rate end points for an active ingredient

INTRODUCTION Herbicides are valuable for farmers because their use can prevent yield loss caused by the presence of weeds that compete with crops for water and nutrients.1 Weeds can also reduce crop quality, act as reservoirs for pests and diseases, and interfere with crop management and handling by preventing the proper operation of agricultural machinery. The world population growth along with a decrease in arable land are a few main factors that emphasize the increased need for new, effective, and sustainable herbicides. Throughout the years, herbicides exerting their phytotoxic activity via different modes of action have been developed.2 The discovery and commercialization of the first auxinic herbicide, 2,4-D (1) (Figure 1),3,4 in the 1940s marked the

Figure 1. Structures of the auxinic herbicides 2,4-D (1) and Rinskor active (2).

start of modern weed management practices enabled by this selective herbicide, which, to date, remains as one of the most widely used herbicides. Even though 1 provides the control of several important weeds in different cropping systems, additional auxinic herbicides possessing different attributes (e.g., crop selectivity, weed spectrum, residual activity in the soil, and reduced use rates) have been developed to address other needs. © XXXX American Chemical Society

Special Issue: Corteva Agriscience Received: June 25, 2019

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DOI: 10.1021/acs.oprd.9b00290 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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Figure 2. Carbon-14-labeled standards of 2.

Figure 3. LC-radio chromatograms of 120DAT 3, loam soil from the USA: metabolites A−C, aerobic soil metabolites that exceeded trigger values, and parent 2.

test systems to be 15 days, whereas the average parent DT90 was 305 days. From these studies, three metabolites exceeded 5% of the applied mass at two or more consecutive time points and needed to be identified by LC-radio/UV-vis and LC-MS using an authentic standard (Figure 3). Note that metabolites A−C were generated in all of the soils listed above, except for metabolite B, which was not generated in loamy sand from the U.K. Metabolites A and C were quickly identified as hydrolyzed metabolite 6 and hydrolyzed/demethylated metabolite 7, respectively, as confirmed by authentic standards on hand (Figure 4).

in soil under aerobic conditions. The desired route of soil degradation leads to metabolites that are nontoxic or less active/deactivated compared with the parent. Rapid rates of degradation of active ingredients in soil are preferred, and this would generally be defined as a DT50 < 30 days and nonpersistent.8,9 To accurately reflect the different soil types of the globe and their biodiversity, at least four dissimilar soil types are studied. Common aerobic soil metabolites are derived via the hydrolysis, reduction, oxidation, and dealkylation of an active ingredient.6 Less common aerobic soil metabolites including nitration10 and dehalogenation11 have also been observed. Herein the identification and synthesis of a nitrophenyl metabolite of 2 that was formed in laboratory terrestrial aerobic soil metabolism studies will be discussed.

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RESULTS AND DISCUSSION Terrestrial Soil Metabolism Studies. The laboratory soil metabolism studies of 2 were conducted using four different aerobic soils, a loam soil from the USA, a loam soil from Germany, a silt loam soil from the U.K., and a loamy sand from the U.K.12 To fully elucidate the route of metabolic soil degradation, each soil was separately dosed with independently carbon-14-labeled test systems of 2 including Rinskor-PhUL-14C (3), Rinskor-Py-4-14C (4),13 and Rinskor-Bn-UL-14C (5) at a rate equal to 120 g active ingredient/hectare (a.i./ha) (Figure 2). The results of the laboratory aerobic soil metabolism studies of 2 showed the average parent DT50 for the four aerobic soil

Figure 4. Aerobic soil metabolites of 2, hydrolyzed metabolite 6 and hydrolyzed/demethylated metabolite 7

Structure Elucidation via Mass Spectral Analysis. To elucidate the structure of metabolite B, high resolution mass spectral analysis was performed. The preliminary mass spectral information for metabolite B is shown in Figure 5. The radiochemical peak at 23.93 min in the top chromatogram (5A) aligned with an observed mass spectral component at B



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Figure 5. Chromatographic and mass spectral information for metabolite B. (A) Radiochemical chromatogram of metabolite B, (B) extracted ion chromatogram (m/z 379.967 ± 0.025 Da), (C) full-scan mass spectrum (average 23.65−23.91 min), and (D) MS/MS product ion mass spectrum (m/z 380).

23.78 min (5B). The protonated molecule at m/z 379.966 ± 0.005 (5C) at 23.78 min peak produced a match to the molecular formula C12H5Cl2F2N2O3 for metabolite B. MS/MS product ions of the protonated molecule are shown in Figure 5D. The measured isotopic pattern in Figure 5C closely matched the predicted elemental composition of the proposed formula for the dosed material. On the basis of the mass spectral data, it was hypothesized that metabolite B was generated via nitration,9 as mentioned above, it is a less commonly observed soil metabolite. It was anticipated that nitration occurred at the six-position of the phenyl ring of 2, but it could not be ruled out that nitration could have occurred at the five-position of the phenyl ring. For definitive structure confirmation, both of the proposed metabolites, 8 and 9, were concurrently prepared (Figure 6). Note that for metabolite confirmation, submilligram quantities of material are needed for a UV retention time match with a test system.

Synthesis of the Proposed Metabolites. The six-step synthetic route leading to the proposed 5-nitrophenyl metabolite 8 is described in Scheme 1. The synthesis began with an ortho-chlorination of the commercially available 2fluoro-5-nitrophenol (10) using a mixture of sulfuryl chloride (SO2Cl2) and diisobutylamine (i-Bu)2NH in toluene at 70 °C, which led to the desired chlorinated product 11 in 30% yield. The phenol 11 was then methylated using standard alkylation conditions, methyl iodide (MeI) and potassium carbonate (K2CO3) in acetone, to provide 12 in 90% yield, which was subjected to an ortho-lithiation/borylation sequence to produce a 1.0:0.8 regioisomeric mixture of boronic esters 13 and 14, respectively. Without further purification, the crude mixture was converted to 6-arylpicolinates 16 via a Suzuki− Miyaura cross-coupling with methyl 4-amino-6-bromo-3chloro-5-fluoropicolinate 1510 using bis(triphenylphosphine)palladium(II)chloride [PdCl2(PPh3)2] as the catalyst and cesium fluoride (CsF) as the base in a mixture of acetonitrile (CH3CN)/water (3:1) at 120 °C under microwave irradiation to give coupled product 16 in 11% yield over two steps after the column chromatography purification. The treatment of 16 with boron tribromide (BBr3) in dichloromethane (CH2Cl2) led to an inseparable 3.4:1.0 mixture of the desired 5nitrophenyl 8 and the 3-bromo derivative 17. The two-step route to access the proposed 6-nitrophenyl metabolite 9 is described in Scheme 2. The treatment of 2 with BBr3 resulted in the cleavage of the methyl ether as well as the removal of the benzyl ester to give 7 in 72% yield. The nitration of 7 using sodium nitrate (NaNO3) in sulfuric acid

Figure 6. Proposed 5- and 6-nitrophenyl aeroboic soil metabolites, 8 and 9. C



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Scheme 1. Synthesis of Proposed 5-Nitrophenyl Metabolite 8

Scheme 2. Synthesis of Proposed 6-Nitrophenyl Metabolite 9

three major metabolites were generated above trigger values, which necessitated their identification. With authentic standards on hand, two of the metabolites were identified as hydrolyzed metabolite 6 and hydrolyzed/demethylated metabolite 7. On the basis of mass spectral analysis, the third metabolite was proposed to be generated via the nitration of the phenyl ring of 2. For definitive metabolite confirmation, both 5- and 6-nitrophenyl regioisomers 8 and 9 were prepared and compared with the test soil system. On the basis of a UV retention time match, 9 was confirmed to be the desired aerobic soil metabolite. To further evaluate the ecotoxicity of metabolite 9, a multigram scale batch of 9 was prepared along with a carbon-14-labeled radiotracer 20. Ecotoxicology studies conducted on the 6-nitrophenyl metabolite 9 showed low toxicity to the nontarget species.

(H2SO4) gave 6-nitrophenyl 9 in 29% yield. The modest yield was attributed to material loss because of the difficulty in purification of compound 9. With the proposed metabolite standards 8 and 9 in-hand, these standards were compared with the soil extract sample using LC-radio/UV−vis (Figure 7). As predicted, 6-nitrophenyl metabolite 9 matched the retention time of the observed aerobic soil metabolite B. Moreover, to determine the ecotoxicological risk for nontarget species in the environment associated with the aerobic soil metabolite 9, further ecotoxicology studies needed to be conducted. Toward this end, 50 g of 9 as well as a carbon-14 radiotracer of 9 were required. The route used to prepare the small quantities for the metabolite identification discussed above was not amenable to scale-up; therefore, an alternative safe route needed to be quickly identified to support the studies. After a brief screening of various nitration conditions, nitronium tetrafluoroborate (NO2BF4) in sulfolane and CH3CN as the solvent was identified as suitable conditions for scale-up. Using these conditions, 7 was converted to 9 in 72% yield (Scheme 3). It is worth noting that reverse-phase column chromatography was used for the purification owing to the difficult removal of sulfolane from the final product. In addition, a carbon-14 radiotracer 20 was prepared in two steps starting from 18. The treatment of carbon-14-labeled standard 18 with BBr3 provided intermediate acid 19 (Scheme 4). Without further purification, 19 was treated with NO2BF4/ sulfolane in CH3CN to afford the desired radiotracer 20 in 59% yield.15 Pleasingly, ecotoxicology studies conducted using standards 9 and 20 showed low toxicity to nontarget species.

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EXPERIMENTAL PROCEDURES General. All reagents were commercially available and used as purchased without further purification. Unless otherwise noted, all reactions were performed in round-bottomed flasks under nitrogen. Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker Avance III HD 500 MHz, a Bruker 400 MHz, or a Varian Gemini 300 MHz spectrometer. NMR data are reported in parts per million (δ) downfield from tetramethylsilane as an internal reference. Mass spectra were obtained using an LC-MS system (Waters Micromass ZQ). High-resolution mass spectra (HRMS) were obtained on an Agilent 6210 time-of-flight liquid chromatography−mass spectrometry (TOF LC-MS) apparatus. IR spectra were obtained on neat samples using attenuated total reflection (ATR) on a Fisher Scientific Nicolet 6700 Fourier-transform infrared (FT-IR) apparatus. Melting points were obtained using the OptiMelt automated melting point system (Sanford Research Systems), and melting points are uncorrected. The quantitation of radioactivity was performed using a Perki-

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CONCLUSIONS To support the registration studies of Rinskor active (2), four terrestrial aerobic soil studies were conducted using carbon-14radiolabeled active ingredient standards. From these studies, D



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Figure 7. (A) LC-radio chromatogram of 120DAT 3, loam soil from the USA. (B) UV detector chromatogram retention time of 9 at 24.83 min. (C) UV detector chromatogram retention time of 8 at 27 min.14

purity was determined using an IN/US β-RAM 2 radioflow detector. Radio-HPLC Conditions for Soil Samples. Samples were analyzed using an Agilent 1260 Infinity liquid chromatography system (Agilent Technologies, Santa Clara, CA) and a β-RAM model 5 apparatus (LabLogic Systems, Sheffield, U.K.). The βRAM model 5 was installed after the LC column, and the detector was operated using IN-FLOW 2:1 cocktail at a ratio of 2:1 with an efficiency of 94%. The measurement mode was fraction stop flow at a 30 s count time. The column used was a Synerg Hydro-RP column (4 μm, 80A, 150 × 4.6 mm) (Phenomenex, Torrance, CA). A flow rate of 1 mL/min and the mobile phases consisting of 0.1% formic acid in water (A) and 0.1% formic acid in (80/20) MeOH/CH3CN (B) were

Scheme 3. Synthesis of 6-Nitrophenyl Metabolite 9

nElmer Tri-Carb 2910 TR liquid scintillation analyzer with PerkinElmer Ultima-Gold liquid scintillation cocktail. Highperformance liquid chromatography was performed using an Agilent 1260 high-performance liquid chromatography (HPLC) system. Chemical purity was determined using an Agilent 1260 Infinity DAD UV detector, and radiochemical E



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Scheme 4. Synthesis of Carbon-14 Phenyl-Labeled 6-Nitrophenyl Metabolite 20

which was purified by column chromatography (TeledyneISCO CombiFlash Rf, 220 g RediSep silica gel column eluting with a gradient of hexanes/EtOAc 0−20%) to afford the desired product as a light-orange solid (1.87 g, 31% yield). Mp 104−107 °C; 1H NMR (400 MHz, chloroform-d) δ 7.60 (dd, J = 9.2, 4.9 Hz, 1H), 7.18 (t, J = 9.1 Hz, 1H), 5.95 (s, 1H); 13C NMR (126 MHz, chloroform-d) δ 154.38, 152.36, 144.20, 142.40, 142.28, 117.78, 117.72, 116.49, 116.46, 114.64, 114.48; ESIMS m/z 190 [(M − H)−]. 2-Chloro-4-fluoro-3-methoxy-1-nitrobenzene (12). A 25 mL round-bottomed flask was charged with 2-chloro-6-fluoro3-nitrophenol (757 mg, 3.95 mmol), acetone (8 mL), MeI (0.35 mL, 5.53 mmol), and powdered (∼325 mesh) potassium carbonate (819 mg, 5.93 mmol). The reaction mixture was allowed to stir at room temperature under an atmosphere of N2 overnight. The reaction mixture was then diluted with EtOAc (60 mL) and washed with H2O (2 × 15 mL) and saturated NaCl (15 mL). The organic phase was dried over Na2SO4, filtered, and concentrated in vacuo to give 0.78 g of a light-yellow oil, which was column-chromatographed (Teledyne-ISCO CombiFlash Rf, 40 g RediSep silica gel eluting with a gradient of hexanes/EtOAc, 0−50%) to afford the desired product as a light-yellow solid (0.729 g, 90% yield). Mp 42−44 °C; 1H NMR (400 MHz, chloroform-d) δ 7.64 (dd, J = 9.2, 4.8 Hz, 1H), 7.16 (t, J = 9.4 Hz, 1H), 4.04 (d, J = 1.7 Hz, 3H); 13C NMR (126 MHz, chloroform-d) δ 159.13, 157.08, 146.02, 145.91, 145.28, 123.64, 123.61, 120.58, 120.50, 115.45, 115.28, 62.07, 62.03; ESIMS m/z 205. 2-(4-Chloro-2-fluoro-3-methoxy-5-nitrophenyl)-4,4,5,5tetramethyl-1,3,2-dioxaborolane (13) and 2-(3-Chloro-5fluoro-4-methoxy-2-nitrophenyl)-4,4,5,5-tetramethyl-1,3,2dioxaborolane (14). To a −78 °C solution of diisopropylamine (0.697 mL, 4.89 mmol) in THF (14 mL) was added a 2.5 M solution of n-BuLi (2.13 mL, 5.34 mmol) in hexane. The mixture was stirred at 0 °C for 10 min, then cooled to −78 °C, and a solution of 2-chloro-4-fluoro-3-methoxy-1-nitrobenzene (0.91 g, 4.45 mmol) in THF (3 mL) was added dropwise. The resulting dark-brown solution was stirred at −78 °C for 1 h; then, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.18 mL, 5.78 mmol) was added, and the mixture was stirred at −78 °C for 1 h, then at 0 °C for 30 min. The reaction mixture was poured into a saturated NH4Cl solution and extracted with EtOAc (two times). The combined organic layers were dried over MgSO4, filtered, and concentrated. The residue contained a (1.7:1.0:0.8) mixture of 2-chloro-4-fluoro3-methoxy-1-nitrobenzene/2-(4-chloro-2-fluoro-3-methoxy-5nitrophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane/2-(3chloro-5-fluoro-4-methoxy-2-nitrophenyl)-4,4,5,5-tetramethyl1,3,2-dioxaborolane (1.47 g), which was used without further purification in the next step. Methyl 4-Amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxy-5-nitrophenyl)-5-fluoropicolinate (16). In a microwave

used. The gradient started at 5% B, with a 2 min hold, and was then linearly increased to 37.5% B over 17 min then linearly increased to 100% B by 35 min, followed by a 10 min hold, and then returned to 5% B. The equilibration time was 9 min, and the Agilent 1260 Infinity UV detector was set at 254 nm. Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry. Samples were analyzed using a Sciex 5600 TripleTOF mass spectrometer equipped with a DuoSpray ion source (Sciex, Concord, Canada) coupled to an Agilent 1290 Infinity liquid chromatography system (Agilent Technologies) and PAL HTC-xt autosampler (Leap Technologies, Carborro, NC). The mass spectrometer was operated in the positive electrospray ionization (ESI) mode. A Phenomenex Synergi Hydro-RP (4.6 × 150 mm with 4.0 μm particle size) column was used for the separation, and the mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid in (80/20) MeOH/CH3CN (B). The gradient started with 5% B, was held for 2 min, and was then linearly increased to 37.5% B within 17 min, then linearly increased to 100% B to 35 min and held for another 10 min, and then returned to 5% B. The 1.0 mL/min sample flow was split 4:1, with approximately four parts of the flow directed to a Berthold LB 509 radioflow detector (Berthold Technologies, Bad Wildbad, Germany) used to assist in the location of metabolite peaks and approximately one part of the sample flow directed to the mass spectrometer. These data were recorded using optimized parameters for the ESI source and MS as follows. The full-scan mode data were collected with a mass range of m/z 100−1000 with an accumulation time of 0.20 s. Targeted MS/MS experiments were collected with a mass range of m/z 60−1000 with an accumulation time of 0.15 s in the high-sensitivity mode. The MS2 fragments were simultaneously collected in high (50 eV), middle (35 eV), and low (20 eV) collision energy, respectively. The ion spray voltage of mass spectrometry was set at 5500 V in positive ion mode. The interface heater temperature was 500 °C, and curtain gas, ion source gas 1, and ion source gas 2 were set at 30, 50, and 50 psi, respectively. The declustering potential was optimized at 80 V. Sciex PeakView software was used to process the data from the mass spectrometer. 2-Chloro-6-fluoro-3-nitrophenol (11). A 250 mL, threenecked round-bottomed flask equipped with a magnetic stir bar, reflux condenser, and J-KEM temperature probe was charged with 2-fluoro-5-nitrophenol (5 g, 31.80 mmol), 200 mL of toluene, and i-Bu2NH (0.45 mL, 2.55 mmol). The resultant mixture was warmed to 70 °C, treated dropwise with SO2Cl2 (2.72 mL, 33.40 mmol), and stirred overnight. The reaction mixture was then treated with an additional 2 mL of SO2Cl2 and stirred at 70 °C for 4 h. Additional SO2Cl2 (4 mL) and i-Bu2NH (0.4 mL) were added, and the reaction mixture was stirred at 70 °C overnight. The mixture was then concentrated in vacuo to give 10.8 g of a dark-yellow oil, F



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off-white solid (72%, 76 mg). 1H NMR (500 MHz, DMSO-d6) δ 13.67 (s, 1H), 10.65 (s, 1H), 7.34 (dd, J = 8.5, 1.5 Hz, 1H), 7.09−6.92 (m, 3H); 19F NMR (471 MHz, DMSO-d6) δ −132.53 (dd, J = 27.7, 7.0 Hz), −138.78; 13C NMR (126 MHz, DMSO-d6) δ 166.53, 150.86, 148.90, 146.62, 146.59, 146.20, 144.16, 142.19, 142.06, 141.88, 141.76, 137.15, 137.05, 125.54, 123.61, 123.58, 122.51, 122.40, 121.19, 112.48. 4-Amino-3-chloro-6-(4-chloro-2-fluoro-3-hydroxy-6-nitrophenyl)-5-fluoropyridine-2-carboxylic Acid (9). A mixture of 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-hydroxyphenyl)-5-fluoropyridine-2-carboxylic acid (500 mg, 1.49 mmol) in 5 mL of concentrated H2SO4 was cooled in an ice−water bath and treated in one portion with NaNO3 (140 mg, 1.64 mmol). The reaction mixture turned orange in color and slowly became homogeneous. After stirring at room temperature for 60 min, an aliquot of the reaction mixture was diluted with H2O and analyzed by HPLC, which showed only a trace amount of the starting material remaining. After stirring for an additional 30 min, the reaction mixture was cautiously poured into ca. 30 mL of ice−water and then filtered. The filtrate was extracted with EtOAc (3 × 30 mL) and then washed with saturated NaCl (1 × 30 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give an orange oil, which was dissolved in isopropyl alcohol (IPA) and purified via a reverse-phase column chromatography (Teledyne-ISCO CombiFlash Rf, 86 g RediSep C18 column eluting with a gradient of CH3CN/ H2O with 0.1% formic acid, 0−100%). Fractions containing the desired product were combined and concentrated in vacuo and taken up in CH3CN and concentrated (three times) to give an orange oil, which was taken up in a 3:1 mixture of hexanes and Et2O (4 mL) and then allowed to stir overnight. After stirring overnight, the solid was removed by vacuum filtration and washed with hexanes/Et2O (v/v 2:1). The solid was dried at 50−60 °C to give the desired product as a yellow powder (29%, 162 mg). Mp 145−150 °C (decomp); 1H NMR (400 MHz, DMSO-d6) δ 13.68 (s, 1H), 8.22 (d, J = 1.7 Hz, 1H), 7.10 (s, 2H); 19F NMR (376 MHz, DMSO-d6) δ −130.04, −139.74; 13C NMR (126 MHz, DMSO-d6) δ 166.22, 150.02, 148.52, 148.39, 148.06, 146.59, 146.55, 146.27, 144.25, 141.82, 141.72, 138.49, 134.25, 134.14, 123.35, 123.02, 122.97, 118.63, 118.61, 118.48, 113.24, 113.22; IR 3473, 3372, 1623, 1526, 1328, 1249, 1215, 991, 969, 894 cm−1; HRMS-ESI (m/ z) [M + H]+ calcd for C12H5Cl2F2N3O5, 378.9574; found, 378.9575. NO2BF4/Sulfolane Procedure for 4-Amino-3-chloro-6-(4chloro-2-2-fluoro-3-hydroxy-6-nitrophenyl-5-fluoropicolinic Acid (9). To a 1000 mL three-necked round-bottomed flask fitted with a nitrogen inlet, a temperature probe, and an overhead stirring apparatus was charged CH3CN (375 mL) and 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-hydroxyphenyl)5-fluoropicolinic acid (10 g, 29.8 mmol). The reaction mixture was treated portion-wise with NO2BF4, (0.5 M in sulfolane, 65.7 mL, 4.36 g, 32.8 mmol), (three portions, 5 min of wait time between each addition). The reaction mixture was a cloudy yellow mixture upon initial stirring of starting material and CH3CN, but after the addition of two portions of NO2BF4, the reaction became homogeneous and yellow in color. (Note: The reaction mixture changed from yellow to a yellow/red color after the complete addition of nitronium tertafluoroborohydrate.) The reaction mixture was stirred at room temperature for 1 h, and an aliquot analyzed by HPLC indicated the consumption of the starting material. The reaction mixture was concentrated in vacuo to yield a yellow/

vessel was added a degassed (argon) suspension of methyl 4amino-6-bromo-3-chloro-5-fluoropicolinate (1.26 g, 4.43 mmol), a crude mixture of 2-(4-chloro-2-fluoro-3-methoxy-5nitrophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane and 2-(3chloro-5-fluoro-4-methoxy-2-nitrophenyl)-4,4,5,5-tetramethyl1,3,2-dioxaborolane (1.47 g, 4.43 mmol), CsF (2.02 g, 13.30 mmol), and bis(triphenylphosphine)palladium(II) chloride (0.31 g, 0.44 mmol) in CH3CN (11.08 mL), and water (3.69 mL). The reaction mixture was stirred under microwave irradiation at 120 °C over 30 min. The reaction mixture was poured into a saturated NaHCO3 solution and extracted with EtOAc (two times). The combined organic layers were dried over MgSO4, filtered, and concentrated. The crude mixture was column-chromatographed (Teledyne-ISCO CombiFlash Rf, 40 g RediSep silica gel column eluting with a gradient of hexane/ EtOAc 0−100%) to afford the desired product as a light-brown solid (205 mg, 11% yield over two steps). 1H NMR (500 MHz, DMSO-d6) δ 8.08 (d, J = 6.1 Hz, 1H), 7.23 (s, 2H), 4.03 (d, J = 1.1 Hz, 3H), 3.88 (s, 3H); 19F NMR (471 MHz, DMSO-d6) δ −120.92, −120.98, −137.14, −137.20; ESIMS m/z 408 [(M + H)+]. 4-Amino-3-chloro-6-(4-chloro-2-fluoro-3-hydroxy-5-nitrophenyl)-5-fluoropicolinic Acid (8) and 4-Amino-3-bromo-6(4-chloro-2-fluoro-3-hydroxy-5-nitrophenyl)-5-fluoropicolinic Acid (17). To a 0 °C suspension of methyl 4-amino-3chloro-6-(4-chloro-2-fluoro-3-methoxy-5-nitrophenyl)-5-fluoropicolinate (69 mg, 0.169 mmol) in CH2Cl2 (3.4 mL) was added a 1 M solution in CH2Cl2 of boron tribromide (0.845 mL, 0.845 mmol) dropwise. The reaction mixture was stirred at 0 °C for 30 min and then at room temperature overnight. The reaction mixture was diluted with CH2Cl2 (ca. 5 mL), and a 2 N HCl solution was added (ca. 5 mL), followed by MeOH (ca. 2 mL). Upon vigorous agitation, the product precipitated out as an off-white solid. The heterogeneous mixture was stirred for 1 h, then filtered, and the solid was washed with CH2Cl2 and dried in vacuo to afford a 3.4:1.0 inseparable mixture of 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-hydroxy-5nitrophenyl)-5-fluoropicolinic acid (22 mg, 34% yield) and 4amino-3-bromo-6-(4-chloro-2-fluoro-3-hydroxy-5-nitrophenyl)-5-fluoropicolinic acid (7 mg, 10% yield) as an off-white solid. 1H NMR (500 MHz, DMSO-d6) δ 13.70 (s, 1H), 11.88 (s, 1H), 7.74 (d, J = 6.1 Hz, 1H), 7.11 (s, 2H); 19F NMR (471 MHz, DMSO-d6) δ −124.28, −124.34, −137.99, −138.06; ESIMS m/z 380 [(M + H)+]. 17: 1H NMR (500 MHz, DMSO-d6) δ 13.70 (s, 1H), 11.88 (s, 1H), 7.73 (d, J = 6.1 Hz, 1H), 7.03 (s, 2H); 19F NMR (471 MHz, DMSO-d6) δ −124.23, −124.28, −137.71, −137.78; ESIMS m/z 426 [(M + H)+]. 4-Amino-3-chloro-6-(4-chloro-2-fluoro-3-hydroxyphenyl)5-fluoropyridine-2-carboxylic Acid (7). A solution of benzyl 4amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxyphenyl)-5-fluoropicolinate (138 mg, 0.31 mmol) in CH2Cl2 (3 mL) was cooled in an ice−water bath. To the solution was added a 1 M solution of BBr3 in CH2Cl2 (3.14 mL, 3.14 mmol) dropwise over 10 min. After the mixture was stirred for 5 min at 0 °C, the ice−water bath was removed, and the yellow slurry was stirred at room temperature for 2 h. An aliquot was taken out and dissolved in CH3CN/H2O. On the basis of HPLC analysis, all of the starting material was consumed. The reaction mixture was poured into a 5% solution of Na2CO3 and then extracted. The pH of the aqueous layer was ca. 11, and upon adjusting the pH to ca. 3 using 1 N HCl, the product precipitated out of solution. The product was filtered to give an G



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water bath) to give the desired product (38.1 mg, 2.4 mCi) as a yellow solid. The radiochemical purity was >99.05%, as determined by HPLC, and the specific activity was 24 mCi/ mmol.

orange oil, which was purified via reverse-phase column chromatography (Teledyne-ISCO CombiFlash Rf, 1.9 kg C18 column eluting with a gradient of 65% CH3CN/25% THF/ 15% H2O in 0.1% phosphoric acid and H2O in 0.1% formic acid). Fractions containing the desired product were collected and concentrated in vacuo to remove CH3CN and THF. The product, in water, was then extracted using EtOAc, dried over MgSO4, filtered, and concentrated in vacuo to yield a yellow oil. The oil was titrated using hot CH2Cl2 and agitated via sonication and stirred for 20 min to precipitate out the product. The precipitate was then filtered and dried in vacuo to give a yellow solid (72%, 8.13 g). 4-Amino-3-chloro-6-(4-chloro-2-fluoro-3-hydroxy-6-nitrophenyl-UL-14C)-5-fluoropyridine-2-carboxylic Acid (20). A stock solution of benzyl 4-amino-3-chloro-6-(4-chloro-2fluoro-3-methoxyphenyl)-5-fluoropicolinate (ca. 4 mCi) was transferred to a 50 mL round-bottomed flask; then, the solution was concentrated in vacuo. To the flask was added CH2Cl2 (3 mL), and the solution was cooled to 0 °C using an ice−water bath; then, a 1 M solution of BBr3 in CH2Cl2 (1.7 mL, 1.7 mmol) was added dropwise under N2. Upon the complete addition of BBr3, the resulting yellow slurry was stirred for 10 min at 0 °C, and then the ice-water bath was removed. The reaction mixture was allowed to stir at room temperature for 90 min. An aliquot was taken out and dissolved in CH3CN/H2O, and based on HPLC and β-RAM analyses, all of the starting material was consumed. The reaction mixture was cooled using an ice−water bath, and then H2O (ca. 2 mL) and 2 N NaOH (ca. 5 mL) were added dropwise. The aqueous layer was transferred to a 100 mL pear flask with a pipet. The pH of the aqueous layer was adjusted to ca. 3 using 1 N HCl (ca. 2.5 mL). The product precipitated out of solution, and then water was removed via a filter stick. The remaining solid was washed with water (1 mL, two times), and water was removed via a filter stick. The solid was taken up in CH3CN/MeOH and then transferred to a 50 mL roundbottomed flask, concentrated, and dried in vacuo to give the desired product (57 mg). This material was used in the next step without further purification. To a 50 mL one-necked round-bottomed flask containing 4amino-3-chloro-6-(4-chloro-2-fluoro-3-hydroxyphenyl)-5-fluoropicolinic acid (57 mg, 0.17 mmol) was added CH3CN (4 mL). To the off-white slurry solution was added NO2BF4 (0.5 M sulfolane, 0.36 mL, 0.18 mmol) dropwise. After all of the NO2BF4 was added, the yellow slurry solution became homogeneous. The progress of the reaction was monitored by HPLC and β-RAM. An aliquot was taken out after 4 h, and based on HPLC and β-RAM analyses, the reaction was incomplete; therefore, an additional NO2BF4 (ca. 0.070 mL) was added. The reaction mixture was stirred for an additional 1 h and then concentrated in vacuo. The recovered oil was directly loaded onto a reverse-phase Teledyne-ISCO CombiFlash Rf, 40 g C18 column eluting with a gradient of 65% CH3CN/25% THF/15% H2O in 0.1% phosphoric acid and H2O in 0.1% formic acid. The purity of the fractions was analyzed by HPLC and β-RAM. Fractions containing the product in >97% purity were combined and concentrated in vacuo to remove CH3CN and THF. The product was extracted out of water with EtOAc (ca. 30 mL). The aqueous layer was pipetted out and then back-extracted with EtOAc (ca. 20 mL). The combined organic layers were sparged with N2 to remove excess EtOAc and then transferred to a 25 mL pear-shaped round-bottomed flask and then concentrated in vacuo (35 °C

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Belgin Canturk: 0000-0002-9848-1054 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Laura Laughlin for her environment fate expertise and advisement during the analytical phase of the Rinskor regulatory studies. We thank James Knobelsdorf and Amanda Jevons for their help with the preparation of compound 9. We also thank Dr. Jeff Gilbert and Dr. Mingming Ma for their constructive suggestions during the preparation of this manuscript. Corteva Agriscience, Arylex, and Rinksor are trademarks of Dow AgroSciences, DuPont, or Pioneer and their affiliated companies or respective owners.

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REFERENCES

(1) Cobb, A. H.; Reade, J. P. H. Herbicides and Plant Physiology, 2nd ed.; Wiley-Blackwell: United Kingdom, 2010. (2) For a complete list of herbicidal modes-of-action classified by group, see: https://hracglobal.com/tools/classification-lookup (accessed May 14, 2019). (3) Walsh, T. A.; Schmitzer, P. R.; Masters, R. A.; Lo, W. C.; Gast, R. E.; Claus, J. S.; Finkelstein, B. L. Chapter 5: New Auxin Mimics and Herbicides. In Modern Crop Protection Compounds, 2nd ed.; WileyVCH: Weinheim, Gerrmany, 2012; Vol. 1, pp 277−304. (4) Grossmann, K. Auxin Herbicides: Current Status of Mechanism and Mode of Action. Pest Manage. Sci. 2009, 66, 113−120. (5) Epp, J. B.; Alexander, A. L.; Balko, T. W.; Buysse, A. M.; Brewster, W. K.; Bryan, K.; Daeuble, J. F.; Fields, S. C.; Gast, R. E.; Green, R. A.; Irvine, N. M.; Lo, W. C.; Lowe, C. T.; Renga, J. M.; Richburg, J. S.; Ruiz, J. M.; Satchivi, N. M.; Schmitzer, P. R.; Siddall, T. L.; Webster, J. D.; Weimer, M. R.; Whiteker, G. T.; Yerkes, C. N. The discovery of ArylexTM active and RinskorTM active: Two novel auxin herbicides. Bioorg. Med. Chem. 2016, 24, 362−371. (6) Van Eerd, L. L.; Hoagland, R. E.; Zablotowicz, R. M.; Hall, J. C. Pesticide metabolism in plants and microorganisms. Weed Sci. 2003, 51, 472−495. (7) Gehen, S.; Corvaro, M.; Jones, J.; Ma, M.; Yang, Q. Challenges and Opportunities in the Global Regulation of Crop Protection Products. Org. Process Res. Dev. 2019, submitted. (8) Oh, B.-Y. Pesticide Residues for Food Safety and Environment Protection. http://www.fftc.agnet.org/library.php?func=view&style= type&id=20110715225622 (accessed June 17, 2019). (9) Rao, P. S. C.; Mansell, R. S., Baldwin, L. B.; Laurent, M. F. Pesticides and Their Behavior in Soil and Water. http://agris.fao.org/ agris-search/search.do?recordID=US9317257 (accessed June 17, 2019). (10) (a) Kodaka, R.; Sugano, T.; Katagi, T.; Takimoto, Y. Claycatalyzed nitration of a Carbamate Fungicide Diethofencarb. J. Agric. Food Chem. 2003, 51, 7730−7737. (b) Ju, K.-S.; Parales, R. E. Nitroaromatic Compounds, from Synthesis to Biodegradation. Microbiol. Mol. Biol. Rev. 2010, 74, 250. (c) Budde, C. L.; Beyer, H



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A.; Munir, I. Z.; Dordick, J. S.; Khmelnitsky, Y. L. Enzymatic nitration of phenols. J. Mol. Catal. B: Enzym. 2001, 15, 55. (11) Kearney, P. C.; Helling, C. S. Reactions of Pesticides in Soils. In Residue Reviews: Residues of Pesticides and Other Foreign Chemicals in Foods and Feeds; Gunther, F. A., Ed.; Springer, New York; Vol. 25, 1969. (12) USDA Texture Classes: loam soil from USA: % sand/silt/clay (41:39:20), pH 7.2; loam soil from Germany: % sand/silt/clay (45:43:12), pH 6.2; silt loam soil from U.K.: % sand/silt/clay (36:55:9), pH 5.0; loamy sand from U.K.: % sand/silt/clay (78:17:5), pH 7.4. (13) Johnson, P. Development of a Novel Route for Incorporation of Carbon-14 into the Pyridine of Rinskor Active. Org. Process Res. Dev. 2019, submitted. (14) The radio-LC flow path is a UV detector, followed by a β-RAM flow cell detector. Because of this, there is a time delay between the UV and radio traces, and the UV retention time is slightly before the radio retention time for a chemical species. (15) Walker, M. D.; Andrews, B. I.; Burton, A. J.; Humphreys, L. D.; Kelly, G.; Schilling, M. B.; Scott, P. W. The Development of a New Manufacturing Route to the Novel Anticonvulsant, SB-406725A. Org. Process Res. Dev. 2010, 14 (1), 108−113.

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Identification and Synthesis of a Nitrophenyl Metabolite of Rinskor

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