Scaffold hopping approaches for the exploration of herbicidally active compounds inhibiting Acyl‐ACP Thioesterase

Abstract

BACKGROUND

The sustainable control of weed populations is a significant challenge facing farmers around the world. Although various methods for the control of weeds exist, the use of small molecule herbicides remains the most effective and versatile approach. Striving to find novel herbicides that combat resistant weeds via the targeting of plant specific modes of action (MoAs), we further investigated the bicyclic class of acyl‐acyl carrier protein (ACP) thioesterase (FAT) inhibitors in an effort to find safe and efficacious lead candidates.

RESULTS

Utilizing scaffold hopping and bioisosteric replacements strategies, we explored new bicyclic inhibitors of FAT. Amongst the investigated compounds we identified new structural motifs that showed promising target affinity coupled with good in vivo efficacy against commercially important weed species. We further studied the structure–activity relationship (SAR) of the novel dihydropyranopyridine structural class which showed promise as a new type of FAT inhibiting herbicides.

CONCLUSION

The current work presents how scaffold hopping approaches can be implemented to successfully find novel and efficacious herbicidal structures that can be further optimized for potential use in sustainable agricultural practices. The identified dihydropyranopyridine bicyclic class of herbicides were demonstrated to have in vitro inhibitory activity against the plant specific MoA FAT as well as showing promising control of a variety of weed species, particularly grass weeds in greenhouse trials on levels competitive with commercial standards. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The work presented herein details the scaffold hopping efforts to find novel herbicidal lead structures that target acyl‐ACP thioesterase. This resulted in the discovery of the new dihydropyranopyridine structural class that exhibits good in vitro inhibition of acyl‐ACP coupled with strong herbicidal activity in greenhouse trials.

ABBREVIATIONS

Ac

acetyl

ACP

acyl carrier protein

aq.

aqueous

br

broad

calcd.

calculated

d

doublet

dd

doublet of doublets

DMAP

4‐dimethylaminopyridine

DMF

N,N‐dimethylformamide

DMSO

dimethylsulfoxide

dppf

1,1′‐bis(diphenylphosphino)ferrocene

dt

doublet of triplets

dtbpf

[1,1‐bis(di‐tert‐butylphosphino)ferrocene]

EC

emulsifiable concentrates

ESI

electrospray ionization

Et

ethyl

FAT

acyl‐ACP thioesterase

FP

fluorescence polarization

h

hour

H‐bonds

hydrogen bonds

ha

hectare

HPLC

high performance liquid chromatography

HRMS

high resolution mass spectrometry

Hz

hertz

J

coupling constant

Lp

Lemna paucicostata

m

multiplet

m‐CPBA

meta‐chloroperoxybenzoic acid

Me

methyl

MHz

megahertz

mins

minutes

MoA

mode of action

MS

mass spectrometry

NBS

N‐bromosuccinimide

NMR

nuclear magnetic resonance

No.

number

Pd/C

palladium on carbon

pI₅₀

negative logarithm of the molar concentration inhibiting enzyme activity by 50%

q

quartet

rpm

revolutions per minute

rt

room temperature

s

singlet

SAR

structure–activity relationship

SFC

supercritical fluid chromatography

S_NAr

nucleophilic aromatic substitution

t

triplet

td

triplet of doublets

tt

triplet of triplets

THF

tetrahydrofuran

v/v

volume/volume percentage

1. INTRODUCTION

Global agriculture is currently facing unprecedented pressure from the effects of climate change, ¹ as well as having to contend with the continued burden exhibited by invasive pests and disease. ² Amongst the invasive pest classes, weed infestations apply a substantial strain on worldwide food production by preventing the growth of healthy crops via a variety of mechanisms. ³ With the ever‐increasing world population, it is critical that weed populations are effectively controlled to maximize crop yields. Herbicides represent a vital tool for farmers to control weeds, but the current solutions are facing a high level of scrutiny as agricultural practices are examined in order to achieve optimum levels of resource efficiency, safety and sustainability. ⁴ The crop protection industry aims to contribute to these goals by researching and developing the next generation of herbicides that set new safety and sustainability standards. ⁵ One way that this goal can be realized is by focusing on herbicidal modes of action (MoAs) that are plant specific, thus significantly reducing the risk of unwanted off‐target effects. ⁶ However, it is extremely challenging to discover a new plant specific MoA that is commercially viable on a sensible timeframe. As a result, researchers in the industry often take inspiration from competitor activities to stimulate their own research projects, particularly when it comes to compounds that could exhibit a new MoA. ⁷

One such area that has recently generated a high level of interest within the community is that of acyl‐acyl carrier protein (ACP) thioesterase (FAT) inhibitors. These activities were kick‐started by a research team at BASF, who elucidated that the herbicidal effect of cinmethylin (1) is caused by the inhibition of FAT (Fig. 1). ⁸ Although cinmethylin (1) was discovered in 1981 and later commercialized in 1989 for use in rice, it is the subject of renewed interest for its pre‐emergent control of problematic grass weeds in cereal crops. ⁹ Several years after the report of BASF, the herbicide methiozolin (2) which was commercialized in 2010, was also demonstrated to control weeds via the inhibition of FAT. ¹⁰ More recently, the team at Qingdao KingAgroot disclosed the new molecule cinflubrolin (3), which can be assumed inhibits FAT due to its high structural similarity with cinmethylin (1). The team at BASF also built on their report of cinmethylin (1) by assigning FAT as the herbicidal MoA of the previously unassigned herbicides cumyluron (4), oxaziclomefone (5) and bromobutide (6). ¹¹ These three compounds all share a similar gem‐dimethylbenzylamide structural motif in contrast to cinmethylin (1), methiozolin (2) and cinflubrolin (3), which exhibit a diether motif that is flanked by a substituted benzyl ring.

Figure 1.

Molecular structures of known herbicides that inhibit FAT with the recently reported bicyclic structures 7, 9, 10 and 11 and the spirocyclic compound 8.

We serendipitously started investigating FAT inhibitors by examining 1,8‐naphthyridine scaffolds that were reported by BASF in 1998. ¹² Our research activities resulted in the discovery of the strong pre‐emergent herbicide thiazolopyridine 7, ¹³ , ¹⁴ , ¹⁵ as well as the herbicidal spiro‐lactam 8 that arose from a study inspired by the structure of methiozolin (2). ¹⁶ We could successfully assign thiazolopyridine 7 as an inhibitor of FAT via an X‐ray co‐crystal structure and via its in vitro inhibition of FAT A (pI₅₀ = 6.9). The team at Syngenta has been active in this area recently, with the disclosure of isothiazoloyridine 9 and pyrazolopyridazine 10 which could have originated from their own scaffold hopping approaches. ¹⁷ , ¹⁸ In contrast to the other FAT inhibiting herbicide classes, the key necessity for this class of FAT inhibitors is the presence of the aromatic bicyclic system containing two nitrogen atoms separated by another atom that bridges between the two rings. Inspired by our observation that strong herbicidal activity could be maintained with the 2,3‐dihydro[1,3]thiazolo[4,5‐b]pyridine scaffold 11, ¹⁹ , ²⁰ we wanted to expand the scope of this class of FAT inhibitors by exploring new saturated ring systems in place of the dihydrothiazolo ring system using scaffold hopping strategies. It has been frequently shown that scaffold hopping is a useful tool for the optimization of crop protection agents, as it offers the opportunity to overcome certain drawbacks of the lead molecules such as insufficient efficacy, adverse toxicological effects, metabolic stability or narrow intellectual property space. ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ , ²⁷ The tuning of the compounds properties is accomplished by exploring isofunctional molecules via the replacement of a section of the compound or the entire scaffold. Herein, we report our scaffold hopping research activities on bicyclic FAT inhibitors that enabled us to discover the dihydropyranopyridine compound class and evaluate its potential as a new herbicide candidate.

2. MATERIALS AND METHODS

2.1. General synthesis remarks

All commercially purchased reagents and solvents were used without further purification. All reactions were conducted using dried glassware under an inert atmosphere with magnetic stirring unless otherwise stated. Low temperature reactions were carried out in a Dewar vessel filled with the appropriate cooling agent e.g., H₂O/ice (0 °C). Reactions using temperatures above room temperature (rt) were conducted using a heated oil bath or a Heat‐On block. Yields refer to spectroscopically pure compounds unless otherwise stated. Flash column chromatography was carried out with a Biotage Isolera™ using CHROMABOND® Flash SiOH (Macherey‐Nagel) columns in sizes ranging from 15 g to 120 g. NMR spectra were recorded using a Bruker AVII spectrometer with a Bruker TBI‐probe. The ¹H and ¹³C NMR shifts are reported in ppm related to the chemical shift of tetramethylsilane. ¹H NMR shifts were calibrated to residual solvent resonances (e.g., CHCl₃ = 7.26 ppm). ¹³C NMR shifts were calibrated to the center of the multiplet signal of the residual solvent resonance (e.g., CHCl₃ = 77.16 ppm). The ¹⁹F NMR shifts are reported in ppm related to the chemical shift of CFCl₃. High resolution mass spectrometry (HRMS) was conducted using a Waters Xevo G2‐XS QToF spectrometer using electrospray ionization (ESI). Preparative HPLC was performed using either Column 1 [YMC Triart C18 (250 × 20 mm, 5 μm)], Column 2 [Phenomenex Genimi NX C18 (150 × 40 mm, 5 μm)], or Column 3 [Phenomenex Gemini‐NX C18 (75 × 30 mm, 3 μm)]. Supercritical Fluid Chromatography (SFC) was performed using Column 4 [Daicel Chiralpak AD (250 × 30 mm, 10 μm)]. The ¹H, ¹³C and ¹⁹F NMR spectra for the characterized compounds can be found in the supporting information. Compound 12 was prepared as previously described in the literature. ²⁸ Samples of cinmethylin (1) and methiozolin (2) were purchased from commercial suppliers and used as received.

2.2. Synthetic procedures and characterization data

2.2.1. 6‐(2‐Fluorophenyl)‐5‐methyl‐2,3‐dihydro‐1H ‐pyrrolo[2,3‐b]pyridine (18)

To a stirred solution of 6‐chloro‐1H‐pyrrolo[2,3‐b]pyridine (13, 1.00 g, 6.55 mmol), (2‐fluorophenyl)boronic acid (14, 1.19 g, 8.52 mmol), and sodium carbonate (2.09 g, 19.7 mmol) in dimethyl sulfoxide (10 mL) and water (1.0 mL) was added Pd(dppf)Cl₂ (480 mg, 655 μmol) under a nitrogen atmosphere at rt. The reaction mixture was then heated to 90 °C. After 4 h, the reaction was cooled to rt and diluted with water (30 mL). The mixture was then extracted with ethyl acetate (3 × 30 mL) and the combined organic phases were washed with brine (30 mL) before concentrating in vacuo. The crude residue was purified by silica gel column chromatography (2% methanol/dichloromethane v/v) to yield 6‐(2‐fluorophenyl)‐1H‐pyrrolo[2,3‐b]pyridine (15, 1.35 g, 96%) as a pale yellow solid. To a stirred solution of 6‐(2‐fluorophenyl)‐1H‐pyrrolo[2,3‐b]pyridine (15, 500 mg, 2.36 mmol) in ethanol (50 mL) was added hydrochloric acid (12 M, 5.0 mL) and palladium on carbon (10%, 500 mg). The reaction mixture was then heated to 60 °C under a hydrogen atmosphere. After 16 h, the reaction mixture was filtered, and the filtrate was collected. The pH of the filtrate was adjusted to pH 8 with a saturated aqueous solution of sodium carbonate before extracting with ethyl acetate (3 × 50 mL). The combined organic phases were washed with brine (50 mL) then concentrated in vacuo. The crude residue was purified by silica gel column chromatography (5% methanol/dichloromethane v/v) to yield 6‐(2‐fluorophenyl)‐2,3‐dihydro‐1H‐pyrrolo[2,3‐b]pyridine (16, 401 mg, 80%) as a pale white solid. To a stirred solution of yield 6‐(2‐fluorophenyl)‐2,3‐dihydro‐1H‐pyrrolo[2,3‐b]pyridine (16, 50 mg, 0.23 mmol) in N,N‐dimethylformamide (5 mL) was added N‐bromosuccinimide (50 mg, 0.28 mmol) under a nitrogen atmosphere at rt. After 16 h, the reaction mixture was extracted with ethyl acetate (3 × 10 mL) and the combined organic phases were washed with brine (3 × 4.0 mL) and concentrated in vacuo. The crude residue was purified by silica gel column chromatography (5% methanol/dichloromethane v/v) to yield 5‐bromo‐6‐(2‐fluorophenyl)‐2,3‐dihydro‐1H‐pyrrolo[2,3‐b]pyridine (17, 1.35 g, 96%) as a pale yellow solid. Palladium diacetate (38 mg, 0.17 mmol), butyldi‐1‐adamantylphosphine (74 mg, 0.21 mmol), and trimethylboroxine (210 mg, 1.7 mmol) were sequentially added to a stirred suspension of 5‐bromo‐6‐(2‐fluorophenyl)‐2,3‐dihydro‐1H‐pyrrolo[2,3‐b]pyridine (17, 500 mg, 1.71 mmol) and potassium carbonate (520 mg, 3.76 mmol) in toluene (30 mL) and water (1.0 mL) at rt under a nitrogen atmosphere. The reaction mixture was then heated to 100 °C. After 16 h, the reaction mixture was cooled to rt and the solvent was removed in vacuo. The crude residue was purified by silica gel column chromatography (3% methanol/dichloromethane v/v) to yield 6‐(2‐fluorophenyl)‐5‐methyl‐2,3‐dihydro‐1H‐pyrrolo[2,3‐b]pyridine (18, 260 mg, 60%) as a pale yellow solid. ¹H NMR (600 MHz, CDCl₃) δ: 7.38–7.30 (m, 2 H), 7.21–7.18 (m, 2 H), 7.10 (ddd, J = 9.7, 8.5, 1.1 Hz, 1 H), 4.43 (br s, 1 H), 3.63 (t, J = 8.2 Hz, 2 H), 3.11–3.06 (m, 2 H), 2.06 (d, J = 1.7 Hz, 3 H). ¹³C NMR (150 MHz, CDCl₃) δ: 162.9 (C), 159.8 (d, J = 246.3 Hz, C), 149.0 (C), 134.2 (CH), 131.5 (CH), 129.5 (CH), 129.1 (d, J = 16.3 Hz, C), 124.3 (d, J = 4.4 Hz, CH), 121.9 (C), 121.1 (C), 115.7 (d, J = 22.9 Hz, CH), 44.8 (CH₂), 27.9 (CH₂), 18.3 (d, J = 4.4 Hz, CH₃). ¹⁹F NMR (565 MHz, CDCl₃) δ: −115.48 – −115.49 (m, F). HRMS (ESI, m/z): calcd. for C₁₄H₁₄FN₂, 229.1136 [M + H]⁺; found 229.1131.

2.2.2. 7‐(2‐Fluorophenyl)‐6‐methyl‐1,2,3,4‐tetrahydro‐1,8‐naphthyridine (24)

To a stirred solution of 2‐aminonicotinaldehyde (19, 2.93 g, 24.0 mmol) and (1‐(2‐fluorophenyl)ethan‐1‐one (20, 3.97 g, 28.8 mmol) in N,N‐dimethylformamide (30 mL) was added an aqueous solution of sodium hydroxide (2.5 M, 1.0 mL) at rt. The reaction mixture was then heated to 100 °C. After 12 h, the solvent was removed in vacuo and the crude residue was purified by silica gel column chromatography (20% methanol/dichloromethane v/v) to yield 2‐(2‐fluorophenyl)‐1,8‐naphthyridine (21, 3.20 g, 60%) as a pale yellow solid. To a stirred solution of 2‐(2‐fluorophenyl)‐1,8‐naphthyridine (21, 3.20 g, 14.3 mmol) in methanol (20 mL) was added palladium on carbon (10%, 320 mg). The reaction mixture was stirred at rt overnight under a hydrogen atmosphere. The mixture was then filtered and the filtrate was collected. Following concentration of the filtrate in vacuo, the crude residue was purified by silica gel column chromatography (20% ethyl acetate/petroleum ether v/v) to yield 7‐(2‐fluorophenyl)‐1,2,3,4‐tetrahydro‐1,8‐naphthyridine (22, 2.74 g, 84%) as a white solid. To a stirred solution of 7‐(2‐fluorophenyl)‐1,2,3,4‐tetrahydro‐1,8‐naphthyridine (22, 2.69 g, 11.8 mmol) in acetonitrile (20 mL) was added N‐bromosuccinimide (3.02 g, 17.0 mmol) at rt. After 16 h, the solvent was removed in vacuo and the crude residue was purified by silica gel column chromatography (17% ethyl acetate/petroleum ether v/v) to yield 6‐bromo‐7‐(2‐fluorophenyl)‐1,2,3,4‐tetrahydro‐1,8‐naphthyridine (23, 3.01 g, 83%) as a pale yellow solid. To a stirred solution of 6‐bromo‐7‐(2‐fluorophenyl)‐1,2,3,4‐tetrahydro‐1,8‐naphthyridine (23, 0.98 g, 3.2 mmol) and trimethylboroxine (0.40 g, 3.2 mmol) in N,N‐dimethylformamide (10 mL) was added Pd(PPh₃)₄ (370 mg, 0.32 mmol) and potassium carbonate (1.3 g, 9.6 mmol) at rt under an argon atmosphere. The reaction mixture was then heated to 80 °C. After 20 h, the reaction was cooled to rt, diluted with water (100 mL), and extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed with brine (100 mL) and subsequently dried over sodium sulfate before filtering and concentrating in vacuo. The crude residue was purified by silica gel column chromatography (20% ethyl acetate/petroleum ether v/v) to yield product 7‐(2‐fluorophenyl)‐6‐methyl‐1,2,3,4‐tetrahydro‐1,8‐naphthyridine (24, 500 mg, 64%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ: 7.39–7.29 (m, 2 H), 7.21–7.16 (m, 1 H), 7.13–7.05 (m, 2 H), 4.92 (br s, 1 H), 3.35 (br s, 2 H), 2.75 (br t, J = 6.1 Hz, 2 H), 2.02 (s, 3 H), 1.97–1.87 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 159.8 (d, J = 246.3 Hz, C), 154.3 (C), 149.3 (C), 138.7 (CH), 131.4 (d, J = 4.4 Hz, CH), 129.5 (d, J = 8.7 Hz, CH), 129.0 (d, J = 16.3 Hz, C), 124.3 (CH), 120.6 (C), 115.9 (C), 115.7 (d, J = 21.8 Hz, CH), 41.7 (CH₂), 26.5 (CH₂), 21.7 (CH₂), 17.7 (d, J = 3.3 Hz, CH₃). ¹⁹F NMR (565 MHz, CDCl₃) δ: −115.46 – −115.63 (m, F). HRMS (ESI, m/z): calcd. for C₁₅H₁₆FN₂, 243.1292 [M + H]⁺; found 243.1313.

2.2.3. 7‐(2‐Fluorophenyl)‐3,4‐dihydro‐2H ‐pyrano[2,3‐b]pyridine (31)

To a stirred solution of 1‐(2‐fluorophenyl)ethanone (20, 3.00 g, 21.7 mmol) and selenium dioxide (9.64 g, 86.9 mmol) in 1,4‐dioxane (20 mL) was added water (10 mL) dropwise at rt under a nitrogen atmosphere. The reaction mixture was then heated to 90 °C. After 1.5 days, the reaction was concentrated in vacuo and then dissolved in ethyl acetate (10 mL). The reaction was subsequently quenched by the addition of saturated aqueous solution of sodium thiosulfate (10 mL) at rt. The precipitated solids were filtered and washed with ethyl acetate (3 × 10 mL). The resulting solution was concentrated in vacuo to yield 2‐(2‐fluorophenyl)‐2‐oxoacetaldehyde (25, 2.20 g, 67%) as a white solid. To a stirred solution of 2‐(2‐fluorophenyl)‐2‐oxoacetaldehyde (25, 1.50 g, 9.86 mmol) and 3‐amino‐2‐methyl‐isothiourea hydroiodide (26, 2.76 g, 11.8 mmol) in ethanol (9 mL) was added sodium bicarbonate (1.66 g, 19.7 mmol) in water (6 mL) dropwise at rt under a nitrogen atmosphere. The reaction mixture was then cooled to 0 °C and stirred for 1 h before being quenched with a saturated aqueous solution of ammonium chloride. The aqueous layer was extracted with ethyl acetate (3 × 10 mL) and concentrated in vacuo to yield 5‐(2‐fluorophenyl)‐3‐methylsulfanyl‐1,2,4‐triazine (27, 1.10 g, 50%) as a white solid. To a stirred solution of 5‐(2‐fluorophenyl)‐3‐methylsulfanyl‐1,2,4‐triazine (27, 1.00 g, 4.52 mmol) in dichloromethane (12 mL) was added m‐chloroperoxybenzoic acid (2.75 g, 13.6 mmol, 85%) at rt under a nitrogen atmosphere. After 1 h, the reaction mixture was diluted with dichloromethane and washed sequentially with a saturated aqueous solution of sodium bicarbonate and brine. The organic phase was then dried over sodium sulfate and concentrated in vacuo to yield 5‐(2‐fluorophenyl)‐3‐methylsulfonyl‐1,2,4‐triazine (28, 1.02 g, 89%) as a light yellow solid. To a stirred solution of 5‐(2‐fluorophenyl)‐3‐methylsulfonyl‐1,2,4‐triazine (28, 300 mg, 1.19 mmol) and pent‐4‐yn‐1‐ol (29, 125 mg, 1.78 mmol) in tetrahydrofuran (9 mL) was added sodium hydride (57 mg, 1.42 mmol, 60% dispersion in mineral oil) at rt under a nitrogen atmosphere. After stirring overnight, the reaction was extracted with ethyl acetate, and the combined organic layers were washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel column chromatography (50% ethyl acetate/petroleum ether v/v) to yield 5‐(2‐fluorophenyl)‐3‐pent‐4‐ynoxy‐1,2,4‐triazine (30, 102 mg, 33%) as a white solid. To 5‐(2‐fluorophenyl)‐3‐pent‐4‐ynoxy‐1,2,4‐triazine (30, 400 mg, 1.64 mmol) was added chlorobenzene (6.0 mL, 56.1 mmol) at rt under a nitrogen atmosphere. The reaction mixture was then heated to 130 °C. After 20 h, the reaction was concentrated in vacuo and dissolved in methanol (3 mL). The crude material was purified by reverse phase column chromatography (0–53% acetonitrile/water v/v) to yield 7‐(2‐fluorophenyl)‐3,4‐dihydro‐2H‐pyrano[2,3‐b]pyridine (31, 0.32 g, 85%) as a light yellow oil. ¹H NMR (600 MHz, CDCl₃) δ: 8.05 (td, J = 8.0, 1.7 Hz, 1 H), 7.49–7.38 (m, 2 H), 7.35–7.29 (m, 1 H), 7.24–7.17 (m, 1 H), 7.15–7.07 (m, 1 H), 4.47–4.35 (m, 2 H), 2.84 (t, J = 6.4 Hz, 2 H), 2.12–2.00 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 161.0 (C), 160.8 (d, J = 249.2 Hz, C), 150.0 (C), 139.3 (CH), 131.2 (d, J = 2.2 Hz, CH), 130.1 (d, J = 8.7 Hz, CH), 126.9 (d, J = 12.0 Hz, C), 124.4 (d, J = 3.3 Hz, CH), 118.1 (d, J = 10.9 Hz, CH), 116.4 (C), 116.1 (d, J = 22.9 Hz, CH), 67.4 (CH₂), 24.8 (CH₂), 22.0 (CH₂). ¹⁹F NMR (565 MHz, CDCl₃) δ: −116.48 – −116.50 (m, F). HRMS (ESI, m/z): calcd. for C₁₄H₁₃FNO, 230.0976 [M + H]⁺; found 230.0981.

2.2.4. 6‐Bromo‐7‐(2‐fluorophenyl)‐3,4‐dihydro‐2H ‐pyrano[2,3‐b]pyridine (32)

To a stirred solution of 7‐(2‐fluorophenyl)‐3,4‐dihydro‐2H‐pyrano[2,3‐b]pyridine (31, 532 mg, 2.32 mmol) in N,N‐dimethylformamide (7 mL) was added N‐bromosuccinimide (620 mg, 3.49 mmol) at rt under a nitrogen atmosphere. The reaction mixture was then heated to 80 °C. After 1 h, the reaction mixture was extracted with ethyl acetate and the combined organic phases were washed with brine and concentrated in vacuo. The crude material was then purified by reverse phase column chromatography (0–70% acetonitrile/water v/v) to yield 6‐bromo‐7‐(2‐fluorophenyl)‐3,4‐dihydro‐2H‐pyrano[2,3‐b]pyridine (32, 500 mg, 70%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ: 7.65 (t, J = 1.0 Hz, 1 H), 7.42–7.36 (m, 2 H), 7.20 (td, J = 7.5, 1.1 Hz, 1 H), 7.11 (td, J = 9.1, 0.8 Hz, 1 H), 4.39–4.36 (m, 2 H), 2.86 (t, J = 6.4 Hz, 2 H), 2.07–2.02 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 160.0 (C), 159.7 (d, J = 247.6 Hz, C), 150.8 (C), 142.6 (CH), 131.3 (d, J = 3.3 Hz, CH), 131.0–130.1 (m, CH), 127.7 (br d, J = 16.3 Hz, C), 124.0 (d, J = 4.4 Hz, CH), 118.8 (C), 115.7 (d, J = 21.8 Hz, CH), 112.3 (C), 67.6 (CH₂), 24.7 (CH₂), 21.5 (CH₂). ¹⁹F NMR (565 MHz, CDCl₃) δ: −113.77 – −113.81 (m, F). HRMS (ESI, m/z): calcd. for C₁₄H₁₂BrFNO, 308.0081 [M + H]⁺; found 308.0076.

2.2.5. 7‐(2‐Fluorophenyl)‐6‐methyl‐3,4‐dihydro‐2H ‐pyrano[2,3‐b]pyridine (33a)

To a stirred solution of 6‐bromo‐7‐(2‐fluorophenyl)‐3,4‐dihydro‐2H‐pyrano[2,3‐b]pyridine (32, 200 mg, 0.68 mmol) and methylboronic acid (81 mg, 1.4 mmol) in 1,4‐dioxane (3 mL) was added cesium carbonate (440 mg, 1.4 mmol), water (1 mL), and Pd(PPh₃)₄ (79 mg, 68 μmol) under a nitrogen atmosphere. The reaction mixture was then heated at 80 °C. After 5 h, the reaction was cooled to rt and quenched with a saturated aqueous solution of ammonium chloride, then extracted with ethyl acetate and concentrated in vacuo. The crude material was dissolved in acetonitrile (5 mL) and purified by reverse phase column chromatography (0–70% acetonitrile/water v/v) to afford 7‐(2‐fluorophenyl)‐6‐methyl‐3,4‐dihydro‐2H‐pyrano[2,3‐b]pyridine (33a, 55 mg, 33%) as a yellow solid. ¹H NMR (600 MHz, CDCl₃) δ: 7.41 (td, J = 7.5, 1.7 Hz, 1 H), 7.36–7.31 (m, 1 H), 7.28 (s, 1 H), 7.19 (td, J = 7.5, 1.0 Hz, 1 H), 7.08 (td, J = 9.1, 0.9 Hz, 1 H), 4.36–4.32 (m, 2 H), 2.82 (t, J = 6.4 Hz, 2 H), 2.13 (d, J = 1.9 Hz, 3 H), 2.07–1.98 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 159.9 (br d, J = 218.0 Hz, C), 159.0 (C), 150.1 (C), 140.8 (CH), 131.7 (d, J = 4.4 Hz, CH), 129.8 (d, J = 7.6 Hz, CH), 128.2 (br d, J = 15.3 Hz, C), 125.5 (C), 124.2 (d, J = 3.3 Hz, CH), 116.5 (C), 115.5 (d, J = 22.9 Hz, CH), 67.2 (CH₂), 24.7 (CH₂), 22.1 (CH₂), 17.8 (d, J = 4.4 Hz, CH₃). ¹⁹F NMR (565 MHz, CDCl₃) δ: −115.34 – −115.39 (m, F). HRMS (ESI, m/z): calcd. for C₁₅H₁₅FNO, 244.1132 [M + H]⁺; found 244.1152.

2.2.6. 7‐(3‐Fluorophenyl)‐6‐methyl‐3,4‐dihydro‐2H ‐pyrano[2,3‐b]pyridine (33b)

Compound 33b (2.1% over seven steps) was prepared in an analogous manner to the preparation of compound 33a starting from 1‐(3‐fluorophenyl)ethenone. ¹H NMR (600 MHz, CDCl₃) δ: 7.35–7.30 (m, 1 H), 7.30–7.27 (m, 1 H), 7.26 (s, 1 H), 7.25–7.22 (m, 1 H), 7.04–6.98 (m, 1 H), 4.35–4.29 (m, 2 H), 2.79 (t, J = 6.4 Hz, 2 H), 2.24 (s, 3 H), 2.03–1.98 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 162.6 (br d, J = 245.2 Hz, C), 159.1 (C), 153.3 (br d, J = 2.2 Hz, C), 142.5 (br d, J = 7.6 Hz, C), 141.8 (CH), 129.5 (d, J = 8.7 Hz, CH), 125.0 (d, J = 3.3 Hz, CH), 123.7 (C), 116.3 (d, J = 21.8 Hz, CH), 116.3 (C), 114.7 (d, J = 20.7 Hz, CH), 67.3 (CH₂), 24.6 (CH₂), 22.1 (CH₂), 19.0 (CH₃). ¹⁹F NMR (565 MHz, CDCl₃) δ: −113.88 – −113.92 (m, F). HRMS (ESI, m/z): calcd. for C₁₅H₁₅FNO, 244.1132 [M + H]⁺; found 244.1120.

2.2.7. 7‐(2,6‐Difluorophenyl)‐6‐methyl‐3,4‐dihydro‐2H ‐pyrano[2,3‐b]pyridine (33c)

Compound 33c (5.4% over seven steps) was prepared in an analogous manner to the preparation of compound 33a starting from 1‐(3,5‐difluorophenyl)ethenone. ¹H NMR (600 MHz, CDCl₃) δ: 7.34–7.28 (m, 2 H), 6.97–6.92 (m, 2 H), 4.37–4.33 (m, 2 H), 2.83 (t, J = 6.5 Hz, 2 H), 2.09 (s, 3 H), 2.07–2.00 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 160.6 (br dd, J = 248.5, 7.6 Hz, C), 159.3 (C), 144.7 (C), 140.8 (CH), 129.8 (t, J = 10.4 Hz, CH), 126.5 (C), 117.3 (C), 112.7–109.9 (CH), 67.3 (CH₂), 24.8 (CH₂), 22.0 (CH₂), 17.5 (CH₃). ¹⁹F NMR (565 MHz, CDCl₃) δ: −113.14 – −113.18 (m, F). HRMS (ESI, m/z): calcd. for C₁₅H₁₄F₂NO, 262.1038 [M + H]⁺; found 262.1052.

2.2.8. 6‐Methyl‐7‐phenyl‐3,4‐dihydro‐2H ‐pyrano[2,3‐b]pyridine (33d)

Compound 33d (1.8% over seven steps) was prepared in an analogous manner to the preparation of compound 33a starting from 1‐phenylethanone. ¹H NMR (600 MHz, CDCl₃) δ: 7.49 (br d, J = 7.2 Hz, 2 H), 7.41–7.30 (m, 4 H), 4.40–4.30 (m, 2 H), 2.89–2.78 (m, 2 H), 2.26 (s, 3 H), 2.09–1.97 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 159.0 (C), 154.3 (C), 142.1 (CH), 139.8 (C), 129.2 (CH), 128.0 (CH), 127.9 (CH), 123.9 (C), 116.0 (C), 67.4 (CH₂), 24.5 (CH₂), 22.1 (CH₂), 19.0 (CH₃). HRMS (ESI, m/z): calcd. for C₁₅H₁₆NO, 226.1236 [M + H]⁺; found 226.1232.

2.2.9. 7‐(2‐Chlorophenyl)‐3,4‐dihydro‐2H ‐pyrano[2,3‐b]pyridine (33e)

Compound 33e (4.2% over five steps) was prepared in an analogous manner to the preparation of compound 31 starting from 1‐(2‐chlorophenyl)ethanone. ¹H NMR (600 MHz, CDCl₃) δ: 7.64–7.60 (m, 1 H), 7.47–7.40 (m, 2 H), 7.34–7.26 (m, 2 H), 7.25–7.20 (m, 1 H), 4.45–4.33 (m, 2 H), 2.86 (t, J = 6.4 Hz, 2 H), 2.12–2.01 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 161.0 (C), 153.6 (C), 138.9 (C), 138.6 (CH), 132.2 (C), 131.9 (CH), 130.1 (CH), 129.4 (CH), 126.9 (CH), 118.4 (CH), 116.2 (C), 67.4 (CH₂), 24.9 (CH₂), 22.0 (CH₂). HRMS (ESI, m/z): calcd. for C₁₄H₁₃ClNO, 246.0680 [M + H]⁺; found 246.0686.

2.2.10. 7‐(o‐Tolyl)‐3,4‐dihydro‐2H ‐pyrano[2,3‐b]pyridine (33f)

Compound 33f (8.0% over five steps) was prepared in an analogous manner to the preparation of compound 31 starting from 1‐(o‐tolyl)ethenone. ¹H NMR (600 MHz, CDCl₃) δ: 7.44–7.38 (m, 2 H), 7.27–7.21 (m, 3 H), 6.95 (d, J = 7.5 Hz, 1 H), 4.40–4.37 (m, 2 H), 2.85 (t, J = 6.5 Hz, 2 H), 2.39 (s, 3 H), 2.08–2.04 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 160.7 (C), 157.0 (C), 140.2 (C), 138.9 (CH), 135.9 (C), 130.7 (CH), 129.8 (CH), 128.1 (CH), 125.8 (CH), 117.6 (CH), 115.2 (C), 67.3 (CH₂), 24.9 (CH₂), 22.1 (CH₂), 20.6 (CH₃). HRMS (ESI, m/z): calcd. for C₁₅H₁₆NO, 226.1226 [M + H]⁺; found 226.1242.

2.2.11. 6‐Bromo‐7‐(o‐tolyl)‐3,4‐dihydro‐2H ‐pyrano[2,3‐b]pyridine (33 g)

Compound 33 g (97 mg, 80%) was prepared from compound 33f (90 mg, 0.40 mmol) and N‐bromosuccinimide (110 mg, 0.60 mmol) in an analogous manner to the preparation of compound 32 starting from compound 31. ¹H NMR (600 MHz, CDCl₃) δ: 7.64 (s, 1 H), 7.31–7.26 (m, 1 H), 7.24–7.17 (m, 3 H), 4.42–4.30 (m, 2 H), 2.86 (t, J = 6.4 Hz, 2 H), 2.21–2.14 (m, 3 H), 2.11–2.00 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 159.7 (C), 155.9 (C), 142.3 (CH), 139.5 (C), 135.8 (C), 129.8 (CH), 128.8 (CH), 128.3 (CH), 125.4 (CH), 117.9 (C), 111.7 (C), 67.4 (CH₂), 24.5 (CH₂), 21.5 (CH₂), 19.5 (CH₃). HRMS (ESI, m/z): calcd. for C₁₅H₁₅BrNO, 304.0332 [M + H]⁺; found 304.0337.

2.2.12. 6‐Cyclopropyl‐7‐(2‐fluorophenyl)‐3,4‐dihydro‐2H ‐pyrano[2,3‐b]pyridine (33 h)

Compound 33 h (66 mg, 15%) was prepared from compound 32 (500 mg, 1.78 mmol) and cyclopropylboronic acid (152 mg, 1.78 mmol) in an analogous manner to the preparation of compound 33a starting from compound 32. ¹H NMR (600 MHz, CDCl₃) δ: 7.45 (br d, J = 1.7 Hz, 1 H), 7.38–7.31 (m, 1 H), 7.19 (br d, J = 0.7 Hz, 1 H), 7.09 (s, 1 H), 7.01 (s, 1 H), 4.38–4.28 (m, 2 H), 2.81 (t, J = 6.5 Hz, 2 H), 2.10–1.95 (m, 2 H), 1.79–1.68 (m, 1 H), 0.84–0.70 (m, 2 H), 0.57–0.42 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 160.0 (br d, J = 246.3 Hz, C), 159.1 (C), 150.7 (C), 135.9 (CH), 131.7 (d, J = 3.3 Hz, CH), 131.1 (C), 129.6 (d, J = 7.6 Hz, CH), 128.1 (br d, J = 16.3 Hz, C), 123.9 (d, J = 4.4 Hz, CH), 116.3 (C), 115.3 (d, J = 22.9 Hz, CH), 67.1 (C), 24.7 (CH₂), 22.0 (CH₂), 11.7 (d, J = 4.4 Hz, CH), 8.0 (CH₂). ¹⁹F NMR (565 MHz, CDCl₃) δ: −114.6 (m, F). HRMS (ESI, m/z): calcd. for C₁₇H₁₇FNO, 270.1289 [M + H]⁺; found 270.1294.

2.2.13. 6‐(2‐Fluorophenyl)‐2,3‐dihydrothieno[2,3‐b]pyridine (40)

To a stirred solution of 1‐(2‐fluorophenyl)ethanone (20, 11 g, 72 mmol) in 1,4‐dioxane (100 mL) was added selenium dioxide (8.0 g, 72 mmol) dissolved in a 20% water / 1,4‐dioxane (v/v) mixture After heating to reflux for 6 h, the reaction mixture was filtered, and the solvent was subsequently removed in vacuo. The oil remaining was purified by distillation to yield 2‐(2‐fluorophenyl)‐2‐oxo‐acetaldehyde (25), which was carried forward without further purification. Crude 2‐(2‐fluorophenyl)‐2‐oxo‐acetaldehyde (25) and thiosemicarbazide (34) were dissolved in a 25% water / ethanol (v/v) mixture (240 mL) and heated to reflux for 10 min before adding potassium carbonate (15 g, 110 mmol). After 8 h, the reaction mixture was cooled to rt and filtered. The filtrate was diluted with water and acidified with hydrochloric acid. The aqueous mixture was extracted with ethyl acetate (3 × 100 mL) and the combined organic phases were washed with brine (100 mL), dried over sodium sulfate, and concentrated to yield crude 5‐(2‐fluorophenyl)‐2H‐1,2,4‐triazine‐3‐thione (35, 7.9 g, 53% over two steps), which was carried forward without further purification. To a stirred solution of 5‐(2‐fluorophenyl)‐2H‐1,2,4‐triazine‐3‐thione (35, 12 g, 59 mmol) in tetrahydrofuran (120 mL) was added triethylamine (16.5 mL, 118 mmol) and 4‐iodobut‐1‐yne (36, 5.3 mL, 59 mmol) at rt. After 16 h, the reaction mixture was concentrated to dryness and water was added. The aqueous mixture was extracted with ethyl acetate and the combined organic phases were washed with brine, dried over sodium sulfate, and then concentrated. The crude material was then purified by column chromatography (10% ethyl acetate/hexane v/v) to give 2‐but‐3‐ynylsulfanyl‐6‐(2‐fluorophenyl)pyridine (38, 3.9 g, 27%). A stirred solution of 2‐but‐3‐ynylsulfanyl‐6‐(2‐fluorophenyl)pyridine (38, 2.02 g, 7.84 mmol) in 1,4‐dioxane (15 mL) was heated to reflux. After 72 h, the reaction mixture was concentrated and purified by column chromatography (20% ethyl acetate/hexane v/v) to give 6‐(2‐fluorophenyl)‐2,3‐dihydrothieno[2,3‐b]pyridine (40, 710 mg, 39%) as an off‐white solid. ¹H NMR (600 MHz, CDCl₃) δ: 7.99 (td, J = 7.9, 1.8 Hz, 1 H), 7.45–7.40 (m, 2 H), 7.38–7.28 (m, 1 H), 7.25–7.21 (m, 1 H), 7.12 (ddd, J = 11.4, 8.2, 0.9 Hz, 1 H), 3.45–3.42 (m, 2 H), 3.36–3.33 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 166.5 (C), 160.5 (br d, J = 249.6 Hz, C), 152.2 (br d, J = 2.2 Hz, C), 132.7 (C), 131.5 (CH), 131.1 (d, J = 3.3 Hz, CH), 130.3 (d, J = 7.6 Hz, CH), 127.2 (br d, J = 12.0 Hz, C), 124.6 (d, J = 3.3 Hz, CH), 119.8 (d, J = 9.8 Hz, CH), 116.2 (d, J = 22.9 Hz, CH), 33.6 (CH₂), 31.2 (CH₂). ¹⁹F NMR (565 MHz, CDCl₃) δ: −116.72 – −116.77 (m, F). HRMS (ESI, m/z): calcd. for C₁₃H₁₁FNS, 232.0591 [M + H]⁺; found 232.0592.

2.2.14. 6‐(2‐Fluorophenyl)‐2,3‐dihydrothieno[2,3‐b]pyridine 1‐oxide (42)

To a stirred solution of 6‐(2‐fluorophenyl)‐2,3‐dihydrothieno[2,3‐b]pyridine (40, 271 mg, 1.18 mmol) in dichloromethane (10 mL) was added m‐chloroperoxybenzoic acid (223 mg, 1.3 mmol) at rt. After 16 h, the reaction mixture was diluted with dichloromethane (10 mL) and washed sequentially with a saturated aqueous solution of sodium bicarbonate and brine. The organic phase was then dried over sodium sulfate and concentrated in vacuo. The crude residue was purified by preparative HPLC (Column 1) (mobile phase: 20 mM ammonium bicarbonate in water/acetonitrile) to yield 6‐(2‐fluorophenyl)‐2,3‐dihydrothieno[2,3‐b]pyridine 1‐oxide (42, 110 mg, 38%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ: 8.17 (br d, J = 15.0 Hz, 1 H), 8.00–7.83 (m, 2 H), 7.41 (q, J = 6.2 Hz, 1 H), 7.32–7.25 (m, 1 H), 7.17 (br dd, J = 11.1, 8.6 Hz, 1 H), 3.94–3.82 (m, 1 H), 3.48–3.27 (m, 3 H). ¹³C NMR (150 MHz, CDCl₃) δ: 165.4 (C), 160.7 (d, J = 249.6 Hz, C), 154.5 (d, J = 2.2 Hz, C), 135.3 (C), 135.2 (CH), 131.5 (d, J = 2.2 Hz, CH), 131.4 (d, J = 8.7 Hz, CH), 126.8 (d, J = 9.8 Hz, CH), 125.9 (d, J = 10.9 Hz, C), 124.9 (CH), 116.3 (d, J = 24.0 Hz, CH), 51.0 (CH₂), 29.3 (CH₂). ¹⁹F NMR (565 MHz, CDCl₃) δ: −116.82 – −116.86 (m, F). HRMS (ESI, m/z): calcd. for C₁₃H₁₁FNOS, 248.0540 [M + H]⁺; found 248.0545.

2.2.15. 6‐(2‐Fluorophenyl)‐2,3‐dihydrothieno[2,3‐b]pyridine 1,1‐dioxide (44)

To a stirred solution of compound 6‐(2‐fluorophenyl)‐2,3‐dihydrothieno[2,3‐b]pyridine (40, 180 mg, 0.78 mmol) in dichloromethane (10 mL) was added m‐chloroperoxybenzoic acid (400 mg, 2.4 mmol) at rt. After 16 h, the reaction mixture was diluted with dichloromethane and washed sequentially with a saturated aqueous solution of sodium bicarbonate and brine. The organic phase was then dried over sodium sulfate and concentrated in vacuo. The crude residue was purified by preparative HPLC (Column 1) (mobile phase: 20 mM ammonium bicarbonate in water/acetonitrile) to yield 6‐(2‐fluorophenyl)‐2,3‐dihydrothieno[2,3‐b]pyridine 1,1‐dioxide (44, 37 mg, 18%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ: 8.21 (td, J = 7.9, 1.3 Hz, 1 H), 8.09–7.77 (m, 2 H), 7.47–7.38 (m, 1 H), 7.29 (t, J = 7.5 Hz, 1 H), 7.17 (dd, J = 11.6, 8.3 Hz, 1 H), 3.63–3.50 (m, 2 H), 3.46–3.35 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 160.8 (d, J = 250.7 Hz, C), 156.5 (C), 155.4 (C), 136.6 (CH), 131.8 (d, J = 2.2 Hz, CH), 131.6 (d, J = 8.7 Hz, CH), 129.6 (C), 128.0 (d, J = 10.9 Hz, CH), 125.3 (d, J = 10.9 Hz, C), 125.0 (CH), 116.3 (d, J = 22.9 Hz, CH), 48.7 (CH₂), 22.4 (CH₂). ¹⁹F NMR (565 MHz, CDCl₃) δ: −116.58 – −116.61 (m, F). HRMS (ESI, m/z): calcd. for C₁₃H₁₁FNO₂S, 264.0489 [M + H]⁺; found 264.0505.

2.2.16. 7‐(2‐Fluorophenyl)‐3,4‐dihydro‐2H ‐thiopyrano[2,3‐b]pyridine (41)

To a stirred solution of 5‐(2‐fluorophenyl)‐2H‐1,2,4‐triazine‐3‐thione (35, 3.07 g, 14.8 mmol) in tetrahydrofuran (60 mL) was added triethylamine (4.1 mL, 30 mmol) and 5‐iodo‐pent‐1‐yne (37, 2.87 g, 14.8 mmol) at rt. After 16 h, the reaction mixture was concentrated to dryness and water was added. The aqueous mixture was extracted with ethyl acetate and the combined organic phases were washed with brine, dried over sodium sulfate, and then concentrated. The crude material was then purified by column chromatography (10% ethyl acetate/hexane v/v) to give 2‐(2‐fluorophenyl)‐6‐pent‐4‐ynylsulfanyl‐pyridine (39, 1.00 g, 25%). A stirred solution of 2‐(2‐fluorophenyl)‐6‐pent‐4‐ynylsulfanyl‐pyridine (39, 1.00 g, 3.72 mmol) in xylene (30 mL) was heated to reflux. After 120 h, the reaction mixture was concentrated and purified by column chromatography (15% ethyl acetate/hexane v/v) to give 7‐(2‐fluorophenyl)‐3,4‐dihydro‐2H‐thiopyrano[2,3‐b]pyridine (41, 320 mg, 35%) as a brown solid. ¹H NMR (600 MHz, CDCl₃) δ: 7.99 (td, J = 7.9, 1.8 Hz, 1 H), 7.40 (dd, J = 7.9, 2.3 Hz, 1 H), 7.35–7.28 (m, 2 H), 7.22 (td, J = 7.5, 1.0 Hz, 1 H), 7.11 (ddd, J = 11.4, 8.3, 0.8 Hz, 1 H), 3.20–3.15 (m, 2 H), 2.90–2.85 (m, 2 H), 2.19–2.12 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 160.7 (d, J = 249.6 Hz, C), 156.6 (C), 151.3 (C), 137.3 (CH), 131.2 (d, J = 2.2 Hz, CH), 130.3 (d, J = 8.7 Hz, CH), 129.0 (C), 127.0 (d, J = 12.0 Hz, C), 124.5 (d, J = 3.3 Hz, CH), 120.0 (d, J = 9.8 Hz, CH), 116.1 (d, J = 22.9 Hz, CH), 29.4 (CH₂), 28.8 (CH₂), 22.4 (CH₂). ¹⁹F NMR (565 MHz, CDCl₃) δ: −116.91 – −116.93 (m, F). HRMS (ESI, m/z): calcd. for C₁₄H₁₃FNS, 246.0747 [M + H]⁺; found 246.0761.

2.2.17. 7‐(2‐Fluorophenyl)‐3,4‐dihydro‐2H ‐thiopyrano[2,3‐b]pyridine 1‐oxide (43)

To a stirred solution of 7‐(2‐fluorophenyl)‐3,4‐dihydro‐2H‐thiopyrano[2,3‐b]pyridine (41, 204 mg, 0.83 mmol) in dichloromethane (10 mL) was added m‐chloroperoxybenzoic acid (157 mg, 0.83 mmol) at rt. After 16 h, the reaction mixture was diluted with dichloromethane and washed sequentially with a saturated aqueous solution of sodium bicarbonate and brine. The organic phase was then dried over sodium sulfate and concentrated in vacuo. The crude residue was purified by preparative HPLC (Column 1) (mobile phase: 20 mM ammonium bicarbonate in water/acetonitrile) to yield 7‐(2‐fluorophenyl)‐3,4‐dihydro‐2H‐thiopyrano[2,3‐b]pyridine 1‐oxide (43, 74 mg, 34%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ: 8.20 (td, J = 7.9, 1.9 Hz, 1 H), 7.90 (dd, J = 8.2, 2.0 Hz, 1 H), 7.65 (d, J = 8.1 Hz, 1 H), 7.41–7.37 (m, 1 H), 7.29–7.25 (m, 1 H), 7.14 (ddd, J = 11.6, 8.3, 1.0 Hz, 1 H), 3.41–3.36 (m, 1 H), 3.11 (dt, J = 17.6, 4.4 Hz, 1 H), 3.04 (ddd, J = 13.9, 12.4, 2.4 Hz, 1 H), 2.99–2.90 (m, 1 H), 2.72–2.63 (m, 1 H), 2.16–2.09 (m, 1 H). ¹³C NMR (150 MHz, CDCl₃) δ: 160.8 (br d, J = 249.6 Hz, C), 157.1 (C), 152.7 (br d, J = 2.2 Hz, C), 139.5 (CH), 131.6 (d, J = 2.2 Hz, CH), 131.4–130.9 (m, CH), 126.9 (d, J = 10.9 Hz, CH), 125.8 (br d, J = 10.9 Hz, C), 124.8 (d, J = 4.4 Hz, CH), 116.2 (d, J = 22.9 Hz, CH), 46.8 (CH₂), 28.2 (CH₂), 13.4 (CH₂). ¹⁹F NMR (565 MHz, CDCl₃) δ: −117.04 – −117.09 (m, F). HRMS (ESI, m/z): calcd. for C₁₄H₁₃FNOS, 262.0696 [M + H]⁺; found 262.0721.

2.2.18. 7‐(2‐Fluorophenyl)‐3,4‐dihydro‐2H ‐thiopyrano[2,3‐b]pyridine 1,1‐dioxide (45)

To a stirred solution of compound 7‐(2‐fluorophenyl)‐3,4‐dihydro‐2H‐thiopyrano[2,3‐b]pyridine (41, 204 mg, 832 μmol) in dichloromethane (10 mL) was added m‐chloroperoxybenzoic acid (428 mg, 2.49 mmol) at rt. After 16 h, the reaction mixture was diluted with dichloromethane and washed sequentially with a saturated aqueous solution of sodium bicarbonate and brine. The organic phase was then dried over sodium sulfate and concentrated in vacuo. The crude residue was purified by preparative HPLC (Column 1) (mobile phase: 20 mM ammonium bicarbonate in water/acetonitrile) to yield 7‐(2‐fluorophenyl)‐3,4‐dihydro‐2H‐thiopyrano[2,3‐b]pyridine 1,1‐dioxide (45, 59 mg, 27%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ: 8.23 (br t, J = 7.5 Hz, 1 H), 7.96 (br d, J = 8.0 Hz, 1 H), 7.66 (br d, J = 8.2 Hz, 1 H), 7.40 (q, J = 6.2 Hz, 1 H), 7.34–7.24 (m, 1 H), 7.14 (br dd, J = 11.3, 8.6 Hz, 1 H), 3.57–3.41 (m, 2 H), 3.07 (br t, J = 6.0 Hz, 2 H), 2.64–2.44 (m, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ: 160.9 (d, J = 249.6 Hz, C), 154.9 (C), 153.3 (d, J = 2.2 Hz, C), 138.9 (CH), 131.8 (d, J = 2.2 Hz, CH), 131.6 (d, J = 8.7 Hz, CH), 131.4 (C), 127.6 (d, J = 10.9 Hz, CH), 125.3 (d, J = 10.9 Hz, C), 124.9 (CH), 116.2 (d, J = 22.9 Hz, CH), 51.7 (CH₂), 28.4 (CH₂), 20.9 (CH₂). ¹⁹F NMR (565 MHz, CDCl₃) δ: −116.92 – −116.94 (m, F). HRMS (ESI, m/z): calcd. for C₁₄H₁₃FNO₂S, 278.0646 [M + H]⁺; found 278.0644.

2.2.19. 5‐(2‐Fluorophenyl)‐6‐methyl‐2H ‐[1,3]dioxolo[4,5‐b]pyridine (57)

To a stirred solution of 2‐bromo‐6‐iodopyridin‐3‐ol (46, 30.0 g, 100 mmol) and potassium carbonate (27.0 g, 195 mmol) in N,N‐dimethylformamide (300 mL) was added benzyl bromide (31.0 g, 181 mmol) portion wise at rt under a nitrogen atmosphere. After stirring overnight, the reaction was quenched with water and the aqueous mixture was extracted with ethyl acetate (3 × 500 mL). The combined organic phases were washed with brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel column chromatography (17% ethyl acetate/hexane v/v) to yield 3‐benzyloxy‐2‐bromo‐6‐iodo‐pyridine (47, 23.8 g, 61%) as a white solid. To a stirred solution of benzyl alcohol (16.6 g, 154 mmol) in N,N‐dimethylformamide (500 mL) was added sodium hydride (3.69 g, 154 mmol) portion wise at 0 °C under a nitrogen atmosphere. After stirring for 30 min, the reaction mixture was warmed to rt then 3‐benzyloxy‐2‐bromo‐6‐iodo‐pyridine (47, 30.0 g, 76.9 mmol) was added portion wise. After 1 h, the reaction was quenched with water (300 mL) at rt. The aqueous mixture was extracted with ethyl acetate (2 × 500 mL) and the combined organic phases were washed with brine (2 × 200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was then purified by silica gel column chromatography (9% ethyl acetate/petroleum ether v/v) to yield 2,3‐bis(benzyloxy)‐6‐iodopyridine (48, 30.0 g, 93%) as a white solid. To a stirred solution of 2,3‐bis(benzyloxy)‐6‐iodopyridine (48, 10.0 g, 24.0 mmol) and 2‐fluorophenylboronic acid (14, 6.67 g, 47.9 mmol) in 1,4‐dioxane (200 mL) was added Pd(dppf)Cl₂ (3.51 g, 4.79 mmol) and cesium carbonate (15.6 g, 47.9 mmol) at rt under a nitrogen atmosphere. The reaction mixture was then heated at 110 °C. After 3 h, the reaction was cooled to rt then extracted with ethyl acetate (3 × 200 mL). The combined organic layers were washed with brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was then purified by silica gel column chromatography (9% ethyl acetate/petroleum ether v/v) to yield 2,3‐bis(benzyloxy)‐6‐(2‐fluoro‐phenyl)pyridine (49, 8.30 g, 90%) as a yellow solid. To a stirred solution of 2,3‐bis(benzyloxy)‐6‐(2‐fluorophenyl)pyridine (49, 8.30 g, 28.1 mmol) in methanol (300 mL) was added palladium on carbon (10%, 120 mg) portion wise at rt under a nitrogen atmosphere. After changing to a hydrogen atmosphere (2 atm) and stirring overnight, the solid was filtered and the filter cake was washed with ethyl acetate (2 × 20 mL), then the filtrate was concentrated in vacuo. The crude material was purified by silica gel column chromatography (8% methanol/dichloromethane v/v) to yield 6‐(2‐fluorophenyl)‐pyridine‐2,3‐diol (50, 4.00 g, 91%). To a stirred mixture of 6‐(2‐fluorophenyl)pyridine‐2,3‐diol (50, 1.00 g, 4.87 mmol) and potassium carbonate (1.34 g, 9.75 mmol) in N,N‐dimethylformamide (50 mL) was added dibromomethane (51, 1.27 g, 7.31 mmol) dropwise at rt under a nitrogen atmosphere. The reaction mixture was then heated to 90 °C. After stirring overnight, the reaction was extracted with ethyl acetate (2 × 50 mL), and the combined organic layers were washed with brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel column chromatography (14% ethyl acetate/petroleum ether v/v) to yield 5‐(2‐fluorophenyl)‐2H‐[1,3]dioxolo[4,5‐b]pyridine (53, 400 mg, 38%). To a stirred solution of 5‐(2‐fluorophenyl)‐2H‐[1,3]dioxolo[4,5‐b]pyridine (53, 250 mg, 1.15 mmol) in glacial acetic acid (20 mL) was added N‐bromosuccinimide (307 mg, 1.72 mmol) portion wise at rt under a nitrogen atmosphere. The reaction mixture was then heated to 50 °C. After 5 h, the reaction was extracted with ethyl acetate (3 × 50 mL), and the combined organic layers were washed sequentially with aqueous sodium hydroxide and aqueous sodium thiosulfate. The organic layer was then dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel column chromatography (20% ethyl acetate/petroleum ether v/v) to yield 6‐bromo‐5‐(2‐fluorophenyl)‐[1,3]dioxolo[4,5‐b]pyridine (55, 120 mg, 35%). To a stirred solution of 6‐bromo‐5‐(2‐fluorophenyl)‐[1,3]dioxolo[4,5‐b]pyridine (55, 100 mg, 0.34 mmol) and methylboronic acid (41 mg, 0.68 mmol) in 1,4‐dioxane (5 mL) was added Pd(dppf)Cl₂ (50 mg, 0.067 mmol) and cesium carbonate (220 mg, 0.68 mmol) portion wise at rt under a nitrogen atmosphere. The reaction mixture was then heated to 110 °C. After 2 h, the reaction was extracted with ethyl acetate (3 × 30 mL), then the combined organic layers were washed with brine (2 × 10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel column chromatography (20% ethyl acetate/petroleum ether v/v) to yield 5‐(2‐fluorophenyl)‐6‐methyl‐2H‐[1,3]dioxolo[4,5‐b]pyridine (57, 58 mg, 74%) as a pale pink oil. ¹H NMR (600 MHz, CDCl₃) δ: 7.40–7.31 (m, 2 H), 7.20 (td, J = 7.5, 1.1 Hz, 1 H), 7.11 (ddd, J = 9.9, 8.4, 1.1 Hz, 1 H), 6.93 (s, 1 H), 6.07 (s, 2 H), 2.14 (d, J = 1.9 Hz, 3 H). ¹³C NMR (150 MHz, CDCl₃) δ: 159.9 (br d, J = 246.3 Hz, C), 156.3 (C), 142.4 (C), 139.7 (C), 131.8 (d, J = 3.3 Hz, CH), 129.9 (d, J = 8.7 Hz, CH), 127.8 (br d, J = 15.3 Hz, C), 126.5 (C), 124.3 (d, J = 3.3 Hz, CH), 116.8 (CH), 115.7 (d, J = 21.8 Hz, CH), 100.3 (CH₂), 18.9 (d, J = 4.4 Hz, CH₃). ¹⁹F NMR (565 MHz, CDCl₃) δ: −115.09 – −115.14 (m, F). HRMS (ESI, m/z): calcd. for C₁₃H₁₁FNO₂, 232.0768 [M + H]⁺; found 232.0779.

2.2.20. 6‐(2‐Fluorophenyl)‐7‐methyl‐2,3‐dihydro[1,4]dioxino[2,3‐b]pyridine (58)

To a stirred solution of 6‐(2‐fluorophenyl)pyridine‐2,3‐diol (50, 2.00 g, 9.74 mmol) and potassium carbonate (2.68 g, 19.5 mmol) in N,N‐dimethylformamide (50 mL) was added 1,2‐dibromoethane (52, 2.74 g, 14.6 mmol) dropwise at rt under a nitrogen atmosphere. The reaction mixture was then heated to 90 °C. After stirring overnight, the reaction was extracted with ethyl acetate (2 × 50 mL), and the combined organic layers were washed with brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel column chromatography (14% ethyl acetate/petroleum ether v/v) to yield 6‐(2‐fluorophenyl)‐2,3‐dihydro[1,4]dioxino[2,3‐b]pyridine (54, 1.00 g, 44%). To a stirred solution of 6‐(2‐fluorophenyl)‐2,3‐dihydro[1,4]dioxino[2,3‐b]pyridine (54, 500 mg, 2.16 mmol) in glacial acetic acid (20 mL) was added N‐bromosuccinimide (580 mg, 3.25 mmol) portion wise at rt under a nitrogen atmosphere. The reaction mixture was then heated to 50 °C. After 5 h, the reaction was extracted with ethyl acetate (3 × 50 mL), and the combined organic layers were washed sequentially with aqueous sodium hydroxide and aqueous sodium thiosulfate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel column chromatography (25% ethyl acetate/petroleum ether v/v) to yield 7‐bromo‐6‐(2‐fluorophenyl)‐2,3‐dihydro‐[1,4]dioxino[2,3‐b]pyridine (56, 120 mg, 18%). To a stirred solution of 7‐bromo‐6‐(2‐fluorophenyl)‐2,3‐dihydro‐[1,4]dioxino[2,3‐b]pyridine (56, 300 mg, 0.97 mmol) and methylboronic acid (116 mg, 1.94 mmol) in 1,4‐dioxane (5 mL) was added Pd(dppf)Cl₂ (151 mg, 0.193 mmol) and cesium carbonate (674 mg, 1.94 mmol) portion wise at rt under a nitrogen atmosphere. The reaction mixture was heated to 110 °C. After 2 h, the reaction was extracted with ethyl acetate (3 × 30 mL), then the combined organic layers were washed with brine (2 × 10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel column chromatography (20% ethyl acetate/petroleum ether v/v) to yield 6‐(2‐fluorophenyl)‐7‐methyl‐2,3‐dihydro[1,4]dioxino[2,3‐b]pyridine (58, 200 mg, 84%) as a colourless oil. ¹H NMR (600 MHz, CDCl₃) δ: 7.38 (td, J = 7.5, 1.8 Hz, 1 H), 7.35–7.31 (m, 1 H), 7.18 (td, J = 7.5, 1.1 Hz, 1 H), 7.11–7.07 (m, 2 H), 4.44–4.41 (m, 2 H), 4.28–4.24 (m, 2 H), 2.13 (d, J = 2.0 Hz, 3 H). ¹³C NMR (150 MHz, CDCl₃) δ: 159.9 (br d, J = 246.3 Hz, C), 148.6 (C), 143.6 (C), 138.7 (C), 131.8 (d, J = 4.4 Hz, CH), 129.8 (d, J = 8.7 Hz, CH), 127.8 (br d, J = 16.3 Hz, C), 127.6 (C), 126.6 (CH), 124.2 (d, J = 4.4 Hz, CH), 115.5 (d, J = 21.8 Hz, CH), 65.0 (CH₂), 64.2 (CH₂), 18.1 (d, J = 5.4 Hz, CH₃). ¹⁹F NMR (565 MHz, CDCl₃) δ: −115.29 – −115.35 (m, F). HRMS (ESI, m/z): calcd. for C₁₄H₁₃FNO₂, 246.0925 [M + H]⁺; found 246.0922.

2.2.21. 6‐(2‐fluorophenyl)‐7‐methyl‐3,4‐dihydro‐2H ‐pyrido[3,2‐b][1,4]oxazine (65)

To a stirred solution of 6‐bromo‐4H‐pyrido[3,2‐b][1,4]oxazin‐3‐one (59, 2.00 g, 8.73 mmol) and (2‐fluorophenyl)boronic acid (14, 1.34 g, 9.61 mmol) in 1,4‐dioxane (32 mL) and water (8 mL) was added Pd(dppf)Cl₂ (638 mg, 873 μmol) and potassium carbonate (2.41 g, 17.4 mmol). The resulting mixture was purged with nitrogen three times then heated to 90 °C under a nitrogen atmosphere. After 12 h, the reaction was filtered and poured into water (50 mL) and extracted with ethyl acetate (100 mL). The combined organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel chromatography (0–20% ethyl acetate/petroleum ether v/v) to yield 6‐(2‐fluorophenyl)‐4H‐pyrido[3,2‐b][1,4]oxazin‐3‐one (60, 1.80 g, 84%) as a white solid. To a stirred solution of 6‐(2‐fluorophenyl)‐4H‐pyrido[3,2‐b][1,4]oxazin‐3‐one (60, 3.23 g, 13.4 mmol) in tetrahydrofuran (35 mL) cooled to 0 °C was added BH_3•Me₂S (10.0 M, 4.03 mL, 40.2 mmol) dropwise. The reaction mixture was then heated to 60 °C. After 2 h, the reaction was cooled to 0 °C then quenched with methanol (10 mL) and warmed gradually to 25 °C and stirred for 1 h. The mixture was concentrated in vacuo and the crude material was purified by flash silica gel chromatography (0–21% ethyl acetate/petroleum ether v/v) to yield 6‐(2‐fluorophenyl)‐3,4‐dihydro‐2H‐pyrido[3,2‐b][1,4]oxazine (61, 1.32 g, 43%) as a white solid. To a stirred solution of 6‐(2‐fluorophenyl)‐3,4‐dihydro‐2H‐pyrido[3,2‐b][1,4]oxazine (61, 523 mg, 2.31 mmol) in dichloromethane (12 mL) was added acetic anhydride (216 μL, 2.31 mmol) and 4‐dimethylaminopyridine (56 mg, 0.46 mmol). The reaction mixture was then heated to 50 °C. After 8 h, the reaction was diluted with water (20 mL) and extracted with methyl tert‐butyl ether (45 mL). The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The crude material was purified by re‐crystallization from dichloromethane (5 mL) and petroleum ether (10 mL) at 25 °C to yield 1‐[6‐(2‐fluorophenyl)‐2,3‐dihydropyrido[3,2‐b][1,4]oxazin‐4‐yl]ethanone (62, 510 mg, 82%) as a white solid. To a stirred solution of 1‐[6‐(2‐fluorophenyl)‐2,3‐dihydropyrido[3,2‐b][1,4]oxazin‐4‐yl]ethanone (62, 2.24 g, 8.23 mmol) in acetonitrile (6 mL) was added N‐bromosuccinimide (2.20 g, 12.3 mmol) under a nitrogen atmosphere. The reaction mixture was then heated to 80 °C. After 16 h, the reaction was diluted with dichloromethane (3 × 20 mL) and water (20 mL). The combined organic layers were washed with brine, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel column chromatography (0–17% ethyl acetate/petroleum ether v/v) to yield 1‐[7‐bromo‐6‐(2‐fluorophenyl)‐2,3‐dihydropyrido[3,2‐b][1,4]oxazin‐4‐yl]ethanone (63, 2.66 g, 92%) as a yellow oil. To a stirred solution of 1‐[7‐bromo‐6‐(2‐fluorophenyl)‐2,3‐dihydropyrido[3,2‐b][1,4]oxazin‐4‐yl]ethanone (63, 500 mg, 1.42 mmol) in 17% water / 1,4‐dioxane (v/v) (12 mL) was added potassium carbonate (295 mg, 2.14 mmol), dichloro[1,1‐bis(di‐tert‐butylphosphino)ferrocene]palladium(II) (93 mg, 0.14 mmol) and 2,4,6‐trimethyl‐1,3,5,2,4,6‐trioxatriborinane (398 μL, 1.42 mmol). The reaction mixture was de‐gassed then heated to 110 °C under an argon atmosphere. After 16 h, the reaction was diluted with ethyl acetate (3 × 20 mL) and water (20 mL). The combined organic layers were washed with water (20 mL) and brine (20 mL), then dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The crude material was first purified by preparative HPLC (Column 2) (mobile phase: [water (0.05% hydrochloric acid)/acetonitrile]; B%: 40%–70%, 10 min) then isolated by SFC (Column 4) (mobile phase: [0.1% ammonium hydroxide/isopropyl alcohol]) to yield 1‐[6‐(2‐fluorophenyl)‐7‐methyl‐2,3‐dihydropyrido[3,2‐b][1,4]oxazin‐4‐yl]ethanone (64, 90 mg, 21%) as a white solid. A mixture of 1‐[6‐(2‐fluorophenyl)‐7‐methyl‐2,3‐dihydropyrido[3,2‐b][1,4]oxazin‐4‐yl]ethanone (64, 665 mg, 2.32 mmol) and hydrochloric acid (6.0 M, 1.6 mL) was heated to 80 °C. After 4 h, the reaction was diluted with water (20 mL) and adjusted to pH = 7 with a saturated aqueous solution of sodium bicarbonate (10 mL). The resulting white solid was filtered and the crude material was purified by preparative HPLC (Column 3) (mobile phase: [10 mM ammonium bicarbonate in water/acetonitrile]; B%: 46%–76%, 10 min). to yield 6‐(2‐fluorophenyl)‐7‐methyl‐3,4‐dihydro‐2H‐pyrido[3,2‐b][1,4]oxazine (65, 460 mg, 81%) as a yellow solid. ¹H NMR (600 MHz, CDCl₃) δ: 7.36–7.30 (m, 2 H), 7.21–7.17 (m, 1 H), 7.10 (t, J = 9.1 Hz, 1 H), 6.90 (s, 1 H), 4.96 (br s, 1 H), 4.23 (t, J = 4.3 Hz, 2 H), 3.47 (br s, 2 H), 2.05 (s, 3 H). ¹³C NMR (150 MHz, CDCl₃) δ: 159.9 (br d, J = 246.3 Hz, C), 145.0 (C), 143.3 (C), 139.3 (C), 131.6 (d, J = 3.3 Hz, CH), 129.5 (d, J = 8.7 Hz, CH), 128.6 (d, J = 16.3 Hz, C), 124.9–123.2 (m, CH), 122.9 (C), 115.7 (d, J = 22.9 Hz, CH), 65.0 (CH₂), 40.7 (CH₂), 18.1 (d, J = 3.3 Hz, CH₃). ¹⁹F NMR (565 MHz, CDCl₃) δ: −115.40 – −115.44 (m, F). HRMS (ESI, m/z): calcd. for C₁₄H₁₄FN₂O, 245.1085 [M + H]⁺; found 245.1107.

2.2.22. 6‐(2‐Fluorophenyl)‐4,7‐dimethyl‐2,3‐dihydropyrido[3,2‐b][1,4]oxazine (66)

To a stirred solution of 6‐(2‐fluorophenyl)‐7‐methyl‐3,4‐dihydro‐2H‐pyrido[3,2‐b][1,4]oxazine (65, 320 mg, 1.31 mmol) in tetrahydrofuran (7 mL) at 0 °C was added sodium hydride (105 mg, 2.62 mmol, 60% dispersion in oil) and methyl iodide (245 μL, 3.93 mmol). The reaction mixture was then heated to 35 °C. After 10 h, the reaction was diluted with water (20 mL) and extracted with ethyl acetate (3 × 30 mL), then dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The crude material was purified by preparative HPLC (Column 2) [mobile phase: [water (0.05% HCl)‐acetonitrile]; B%: 15%–40%, 10 min] to yield 6‐(2‐fluorophenyl)‐4,7‐dimethyl‐2,3‐dihydropyrido[3,2‐b][1,4]oxazine (66, 200 mg, 53%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ: 7.44–7.39 (m, 1 H), 7.39–7.34 (m, 1 H), 7.22 (br t, J = 7.2 Hz, 1 H), 7.16–7.09 (m, 1 H), 7.05 (s, 1 H), 4.27 (br s, 2 H), 3.60 (br s, 2 H), 3.56–3.48 (m, 3 H), 2.01 (s, 3 H). ¹³C NMR (150 MHz, CDCl₃) δ: 160.2 (br d, J = 248.5 Hz, C), 144.1 (C), 140.8 (C), 132.1 (d, J = 2.2 Hz, CH), 131.8 (br d, J = 8.7 Hz, CH), 126.3 (CH), 124.3 (d, J = 3.3 Hz, CH), 122.2 (C), 116.0 (d, J = 21.8 Hz, CH), 63.7 (CH₂), 49.2 (CH₂), 40.4 (CH₃), 17.5 (d, J = 2.2 Hz, CH₃). ¹⁹F NMR (565 MHz, CDCl₃) δ: −113.81 (br s, F). HRMS (ESI, m/z): calcd. for C₁₅H₁₆FN₂O, 259.1241 [M + H]⁺; found 259.1251.

2.2.23. 6‐(2‐Fluorophenyl)‐7‐methyl‐2H ,3H ,4H ‐pyrido[3,2‐b][1,4]thiazine (72)

A solution of 6‐(2‐fluorophenyl)pyridin‐2‐amine (67, 19.0 g, 111 mmol) and N‐bromosuccinimide (53.9 g, 303 mmol) in a 5% water / dimethylsulfoxide (v/v) mixture (210 mL) was stirred at 120 °C under a nitrogen atmosphere. After 2 days, the reaction mixture was cooled to rt and quenched by the addition of saturated aqueous ammonium chloride (200 mL). The resulting mixture was extracted with ethyl acetate (3 × 80 mL) and the combined organic phases were washed with brine (100 mL), dried over sodium sulfate, and then concentrated in vacuo. The crude residue was purified by reverse phase C18 flash chromatography [gradient changed over 10 min from 10% acetonitrile/water (v/v) to 50% acetonitrile/water (v/v), 254 nm UV detection] to yield 3,5‐dibromo‐6‐(2‐fluorophenyl)pyridin‐2‐amine (68, 5.90 g, 17%) was isolated as a white solid. To a stirred solution of 3,5‐dibromo‐6‐(2‐fluorophenyl)pyridin‐2‐amine (68, 13.0 g, 37.6 mmol) and ethyl thioglycolate (13.6 g, 113 mmol) in N,N‐dimethylformamide (130 mL) was added sodium hydride (2.60 g, 108 mmol) at 40 °C under a nitrogen atmosphere. The reaction mixture was stirred for 2 h at 70 °C and subsequently cooled to 0 °C and quenched by the addition of saturated aqueous ammonium chloride (200 mL). The resulting mixture was extracted with ethyl acetate (3 × 80 mL) and the combined organic phases were washed with brine (70 mL), dried over sodium sulfate, and then concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (17% ethyl acetate/petroleum ether v/v) to yield 7‐bromo‐6‐(2‐fluorophenyl)‐2H,4H‐pyrido[3,2‐b][1,4]thiazin‐3‐one (70, 4.00 g, 31%) as a white solid. To a stirred solution of 7‐bromo‐6‐(2‐fluorophenyl)‐2H,4H‐pyrido[3,2‐b][1,4]thiazin‐3‐one (70, 1.00 g, 2.95 mmol) and methylboronic acid (0.260 g, 4.42 mmol) in 1,4‐dioxane (10 mL) was added cesium carbonate (1.92 g, 5.90 mmol) and Pd(dppf)Cl₂•CH₂Cl₂ (240 mg, 0.295 mmol) at rt under a nitrogen atmosphere. The reaction mixture was then heated to reflux and left to stir overnight. After cooling to rt, the solvent was removed in vacuo and the crude residue was purified by silica gel column chromatography (17% ethyl acetate/petroleum ether v/v) to yield 6‐(2‐fluorophenyl)‐7‐methyl‐2H,4H‐pyrido[3,2‐b][1,4]thiazin‐3‐one (71, 400 mg, 49%) as a white solid. To a stirred solution of 6‐(2‐fluorophenyl)‐7‐methyl‐2H,4H‐pyrido[3,2‐b][1,4]thiazin‐3‐one (71, 1.00 g, 3.65 mmol) in tetrahydrofuran (5.0 mL) was added borane‐tetrahydrofuran complex (1.0 M, 36.5 mL) at rt under a nitrogen atmosphere. The reaction mixture was then heated to reflux. After 3 h, the reaction was cooled to rt and quenched by dropwise addition of methanol at rt. Following the removal of solvent in vacuo, the crude residue was purified by silica gel column chromatography (9% ethyl acetate/petroleum ether v/v) to yield 6‐(2‐fluorophenyl)‐7‐methyl‐2H,3H,4H‐pyrido[3,2‐b][1,4]thiazine (72, 500 mg, 53%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ: 7.37–7.32 (m, 2 H), 7.20 (td, J = 7.5, 1.0 Hz, 1 H), 7.16 (s, 1 H), 7.10 (t, J = 9.1 Hz, 1 H), 5.28 (br s, 1 H), 3.66–3.61 (m, 2 H), 3.02–2.98 (m, 2 H), 2.03–2.01 (m, 3 H). ¹³C NMR (150 MHz, CDCl₃) δ: 159.8 (br d, J = 247.4 Hz, C), 150.5 (C), 148.1 (C), 136.9 (CH), 131.4 (d, J = 3.3 Hz, CH), 129.7 (d, J = 8.7 Hz, CH), 128.4 (br d, J = 16.3 Hz, C), 124.3 (d, J = 3.3 Hz, CH), 122.1 (C), 115.7 (d, J = 22.9 Hz, CH), 112.0 (CH), 42.9 (CH₂), 25.6 (CH₂), 17.7 (d, J = 4.4 Hz, CH₃). ¹⁹F NMR (565 MHz, CDCl₃) δ: −115.17 – −115.22 (m, F). HRMS (ESI, m/z): calcd. for C₁₄H₁₄FN₂S, 261.0856 [M + H]⁺; found 261.0857.

2.2.24. 6‐(2‐Fluorophenyl)‐4,7‐dimethyl‐2H ,3H ‐pyrido[3,2‐b][1,4]thiazine (73)

To a stirred solution of 6‐(2‐fluorophenyl)‐7‐methyl‐2H,3H,4H‐pyrido[3,2‐b][1,4]thiazine (72, 150 mg, 587 μmol) and methyl iodide (164 mg, 1.15 mmol) in N,N‐dimethylformamide (2.0 mL) was added sodium hydride (28 mg, 1.2 mmol) at rt under a nitrogen atmosphere. The reaction mixture was then heated to 50 °C. After 4 h, the reaction was cooled to rt and quenched by dropwise addition of saturated aqueous ammonium chloride at rt. The resulting mixture was extracted with ethyl acetate (3 × 5.0 mL) and the combined organic phases were washed with brine (3 × 4.0 mL), dried over sodium sulfate, and then concentrated in vacuo. The crude residue was purified by reverse phase C18 flash chromatography [gradient changed over 10 min from 10% acetonitrile/water (v/v) to 50% acetonitrile/water (v/v), 254 nm UV detection] to yield 6‐(2‐fluorophenyl)‐4,7‐dimethyl‐2H,3H‐pyrido[3,2‐b][1,4]thiazine (73, 140 mg, 89%) as a yellow oil. ¹H NMR (600 MHz, CDCl₃) δ: 7.43 (td, J = 7.5, 1.8 Hz, 1 H), 7.39–7.28 (m, 1 H), 7.20 (td, J = 7.5, 1.1 Hz, 1 H), 7.14 (s, 1 H), 7.13–7.08 (m, 1 H), 3.75–3.72 (m, 2 H), 3.14 (s, 3 H), 3.08–3.05 (m, 2 H), 2.05 (d, J = 2.1 Hz, 3 H). ¹³C NMR (150 MHz, CDCl₃) δ: 159.9 (br d, J = 247.4 Hz, C), 151.6 (C), 147.7 (C), 136.3 (CH), 131.8 (d, J = 3.3 Hz, CH), 129.4 (d, J = 7.6 Hz, CH), 129.1 (br d, J = 16.3 Hz, C), 124.0 (d, J = 3.3 Hz, CH), 120.9 (C), 115.6 (d, J = 22.9 Hz, CH), 113.1 (C), 51.4 (CH₂), 37.9 (CH₃), 25.2 (CH₂), 17.6 (d, J = 5.4 Hz, CH₃). ¹⁹F NMR (565 MHz, CDCl₃) δ: −114.80 – −114.85 (m, F). HRMS (ESI, m/z): calcd. for C₁₅H₁₆FN₂S, 275.1013 [M + H]⁺; found 275.1018.

2.2.25. LpFAT A expression and purification

The fat a03 gene from Lemna paucicostata (Lp), in which the N‐terminal amino acids representing the chloroplast transit peptide were replaced by an N‐terminal 6x‐His‐tag, was cloned into a pET24 vector. ⁸ The LpFAT A protein was expressed in Escherichia coli BL21Star(DE3) cells. 5 mL of an overnight culture of E. coli cells grown in LB medium with 100 μg/mL carbenicillin were used to inoculate 0.5 L of autoinduction medium containing 100 μg mL⁻¹ carbenicillin. ²⁹ The bacteria were grown at 37 °C and 120 rpm for about 4.5 h to reach OD600 = 0.6 and then further cultivated at 21 °C overnight. The bacteria were harvested by centrifugation (20 min, 6000 g) and stored frozen at −80 °C. LpFAT A protein was purified using the Ni‐NTA Fast Start Kit (Qiagen GmbH, Germany) according to the instructions of the manufacturer. Active fractions were pooled and the buffer was exchanged into 25 mM K‐Phosphate buffer pH 7.3 containing 10% glycerol with PD10 columns (GE Healthcare). Aliquots of the protein solution were frozen in liquid nitrogen and stored at −80 °C.

2.2.26. LpFAT A fluorescence polarization assay

Fluorescence polarization (FP) competition assays were performed at rt in black 96‐well microtiter plates (Greiner, catalog No. 655900). The assay mixture contained 25 mM potassium phosphate buffer pH 7.3, 200 mM NaCl, 0.01% Triton X‐100, 2 nM fluorescent tracer, 0.4 μg of purified LpFAT A protein and different amounts of test compound in a total volume of 100 μL. FP was measured with a BMG CLARIOstar microtiter plate reader using the FP‐filter set for fluorescein (Ex 482‐16, Em 530‐40, LP504). FP is the difference between wells containing LpFAT A and wells containing only tracer. The pI₅₀ values were calculated from plots of inhibition values versus test compound concentration using Model 205 of the ID Business Solutions Ltd Xlfit software suite. The FAT A binding fluorescent tracer was synthesized from (2S,4S)‐4‐[(2,6‐difluorophenyl)methoxymethyl]‐4‐ethyl‐2‐methyl‐N‐(prop‐2‐ynylcarbamoyl)‐1,3‐dioxolane‐2‐carboxamide ⁸ and FAM azide 5‐isomer (Broadpharm BP‐22544, San Diego, CA, USA) by click‐chemistry, ³⁰ details of which have been previously reported. ¹⁵

2.2.27. Modeling

Protein modeling was carried out using the crystal structure of LpFAT A co‐crystallized with cinmethylin (1). ⁸ Compound editing, docking and the interaction analysis were accomplished using SeeSAR from BioSolveIT and the scoring function HyDe. ³¹

2.2.28. Herbicidal greenhouse screening

Pre‐emergence testing: Seeds of mono‐ and dicotyledonous weed plants and crop plants were sown in plastic pots (double sowings with one species of mono‐ and one species of dicotyledonous weed plants per pot) in sandy loam and covered with soil. The test compounds, formulated in the form of emulsifiable concentrates (EC), were sprayed onto the surface of the covering soil with the addition of 0.5% of an additive (e.g., rapeseed methyl ester), at an application rate of 600 L of water/ha. Following treatment, the pots are placed in a greenhouse and kept under optimum growth conditions for the test plants. The visual grading of the damage to the test plants was carried out 3 weeks after application in comparison to untreated controls. Post‐emergence testing: Seeds of mono‐ and dicotyledonous weed plants were sown in plastic pots (double sowings with one species of mono‐ and one species of dicotyledonous weed plants per pot) in sandy loam, covered with soil and grown in a greenhouse under controlled growth conditions. Two to three weeks after sowing, the test plants were sprayed in the single‐leaf stage with the test compounds, formulated in the form of EC, onto the green plant parts as an emulsion, with the addition of 0.5% of an additive (e.g. rapeseed methyl ester), at an application rate of 600 L of water/ha. The test plants were placed in the greenhouse for 3 weeks under optimum growth conditions and then the effect of the compounds was assessed visually in comparison with untreated controls.

3. RESULTS AND DISCUSSION

3.1. Compound design and chemistry

Encouraged by our work on the 2,3‐dihydro[1,3]thiazolo[4,5‐b]pyridine scaffold 11, we wanted to further investigate the effects of incorporating saturated ring systems into the bicyclic class of FAT inhibitors. In the first instance we sought to investigate the effect of integrating different heteroatoms into a saturated 5‐ or 6‐membered ring system, whilst maintaining the optimized right‐hand pyridine aryl motif which has been previously shown to give strong herbicidal efficacy (Fig. 2(i)). ¹⁵ This resulted in the proposal to prepare compounds of type I, where the heteroatoms are occupying the position that could allow them to bind the arginine residue found in the FAT A binding pocket. ⁸ After the synthesis of the first set of analogues we would then investigate compounds of type II, where an additional heteroatom has been introduced into the unsaturated ring system. During our literature searches to prepare for this research study, we found that the dihydrofuropyridine structure 12 had been previously reported as an herbicide in a patent from Imperial Chemical Industries in 1993 (Fig. 2(ii)). ²⁸ We therefore excluded this structure from our scaffold hopping activities and instead used it as a benchmark for our new compounds.

Figure 2.

(i) Scaffold hopping design strategy inspired by dihydrothiazolopyridine 11 to give the generic scaffolds I and II; (ii) Molecular structure of dihydrofuropyridine 12 found in Patent No. US 5260261.

To kick off our activities, we attempted to synthesize the 5‐ and 6‐membered ring compounds containing nitrogen, oxygen and sulfur in the arginine chelating position. Starting with the 5‐membered nitrogen compound (Scheme 1(i)), pyrrolopyridine 15 was prepared via a Suzuki coupling, followed by reduction of the pyrrole ring using Pd/C and a subsequent bromination with NBS to afford compound 17. Another Suzuki coupling then furnished the target dihydropyrrolopyridine 18 in 60% yield. For the synthesis of the 6‐membered ring system, we started from naphthyridine 21 which was prepared in good yield using a Friedländer synthesis (Scheme 1(ii)). ³² Reduction of the left‐hand pyridine ring with Pd/C then furnished tetrahydronaphthyridine 22 that smoothly underwent bromination with NBS. Lastly, a Suzuki coupling was used to install the methyl group and afford tetrahydronaphthyridine 24.

Scheme 1.

(i) Synthesis of dihydropyrrolopyridine 18: (a) Pd(dppf)Cl₂, Na₂CO₃, DMSO/H₂O, 90 °C, 96%; (b) Pd/C, H₂, aq. HCl, EtOH, 60 °C, 80%; (c) NBS, DMF, rt, 96%; (d) trimethylboroxine, Pd(OAc)₂, butyldi‐1‐adamantylphosphine, K₂CO₃, toluene/H₂O, 100 °C, 60%. (ii) Preparation of tetrahydronaphthyridine 24: (a) NaOH, DMF, 100 °C, 60%; (b) Pd/C, H₂, MeOH, rt, 84%; (c) NBS, MeCN, rt, 83%; (d) trimethylboroxine, Pd(PPh₃)₄, K₂CO₃, DMF, 80 °C, 64%.

Next, we focused on the preparation of the 6‐membered ring compounds featuring an oxygen atom (Scheme 2). In general, we utilized a previously reported inverse electron demand Diels−Alder strategy for the synthesis of this scaffold type. ³³ Firstly, an α‐oxidation in the presence of selenium dioxide enabled the preparation of dicarbonyl 25 which was then cyclized to triazine 27 using compound 26. Oxidation of the thioether functionality to a sulfone was facilitated by m‐CPBA and then an S_NAr reaction with hydroxyalkyne 29 afforded triazine 30. The stage was now set for the Diels−Alder reaction which was accomplished by heating to 130 °C in chlorobenzene, furnishing tetrahydropyranopyridine 31 in 85% yield. A subsequent bromination and Suzuki coupling then completed the synthesis of the desired target 33a.

Scheme 2.

Synthesis of dihydropyranopyridine 33a: (a) SeO₂, 1,4‐dioxane/H₂O, 90 °C, 67%; (b) NaHCO₃, EtOH/H₂O, rt → 0 °C, 50%; (c) m‐CPBA, CH₂Cl₂, rt, 89%; (d) NaH, THF, rt, 33%; (e) chlorobenzene, 130 °C, 85%; (f) NBS, DMF, 80 °C, 70%; g) methylboronic acid, Pd(PPh₃)₄, Cs₂CO₃, 1,4‐dioxane/H₂O, 80 °C, 33%.

Following on from the dihydropyranopyridine scaffold, we moved on to the sulfur containing compounds. Both the 5‐ and 6‐membered rings were prepared in an analogous fashion except for using a homologated iodoalkyne for the 6‐membered ring compared to the 5‐membered ring variant (Scheme 3). Cyclization of dicarbonyl 25 with substituted thiourea 34 gave triazine 35 in moderate yield. Alkylation of the sulfur atom with iodoalkynes 36 and 37 followed by Diels−Alder reactions conducted at reflux in 1,4‐dioxane and xylene afforded dihydrothienopyridine 40 and dihydrothiopyranopyridine 41 respectively. Unfortunately, our attempts to install a bromine atom on the pyridine ring were unsuccessful at this point. As a result, both compounds 40 and 41 were taken as final products for biological evaluation. We also converted the thioethers to the respective sulfoxides (42 and 43) and sulfones (44 and 45) using varying equivalents of m‐CPBA to further explore the structure–activity relationship (SAR) of these bicyclic FAT inhibitors.

Scheme 3.

Synthesis of the thioether (40 and 41), sulfoxide (42 and 43) and sulfone (44 and 45) containing scaffolds. (a) K₂CO₃, EtOH/H₂O, reflux, 53% over two steps starting from ketone 20; (b) Et₃N, THF, rt, 27% (for 38), 25% (for 39); (c) 1,4‐dioxane, reflux, 39% (for 40), xylene, reflux, 35% (for 41); (d) m‐CPBA, CH₂Cl₂, rt, 38% (for 42), 34% (for 43); (e) m‐CPBA, CH₂Cl₂, rt, 18% (for 44), 27% (for 45).

With preparation of the single heteroatom analogues complete, we moved on to the synthesis of scaffolds containing two heteroatoms in the saturated ring system. Initially we investigated the 5‐ and 6‐membered scaffolds containing two oxygen atoms (Scheme 4). Benzylation of hydroxypyridine 46 to pyridine 47 was followed by an S_NAr reaction to add the second oxygen atom protected with a benzyl group. A Suzuki coupling reaction then installed the substituted aryl ring with deprotection of both benzyl groups affording pyridine 50. Cyclization to the desired scaffolds was completed using 1,1‐dibromomethane (51) and 1,2‐dibromoethane (52) in the presence of potassium carbonate to afford compounds 53 and 54 respectively. Bromination with NBS followed by Suzuki coupling using Pd(dppf)Cl₂ gave the final compounds 57 and 58 for biological evaluation.

Scheme 4.

Preparation of dioxolopyridine 57 and dihydrodioxinopyridine 58. (a) benzyl bromide, K₂CO₃, DMF, rt, 61%; (b) benzyl alcohol, NaH, DMF, 0 °C → rt, 93%; (c) Pd(dppf)Cl₂, Cs₂CO₃, 1,4‐dioxane, 110 °C, 90%; (d) Pd/C, H₂, MeOH, rt, 91%; (e) K₂CO₃, DMF, 90 °C, 38% (for 53), 44% (for 54); (f) NBS, AcOH, 50 °C, 35% (for 55), 18% (for 56); (g) methylboronic acid, Pd(dppf)Cl₂, Cs₂CO₃, 1,4‐dioxane, 110 °C, 74% (for 57), 84% (for 58).

Next, we targeted the scaffold containing both a nitrogen and oxygen atom in a 6‐membered ring system (Scheme 5). Starting from heterocycle 59, a Suzuki coupling with boronic acid 14 afforded compound 60 and a subsequent reduction of the amide using borane‐dimethyl sulfide complex furnished compound 61 in 43% yield. At this stage, compound 61 was found to be unreactive with NBS. We therefore protected the free nitrogen atom as an amide which then allowed the bromination reaction to proceed, affording compound 63 which then had the desired methyl group installed using a Suzuki coupling reaction facilitated by the catalyst PdCl₂(dtbpf). Deprotection of the amide using aq. HCl successfully yielded the desired dihydropyrido‐oxazine 65, which was also methylated using methyl iodide with sodium hydride to afford the additional test compound 66.

Scheme 5.

Synthesis of dihydropyrido‐oxazines 65 and 66. (a) Pd(dppf)Cl₂, K₂CO₃, 1,4‐dioxane/H₂O, 90 °C, 84%; (b) BH₃•Me₂S, THF, 60 °C, 43%; (c) acetic anhydride, DMAP, CH₂Cl₂, 50 °C, 82%; (d) NBS, MeCN, 80 °C, 92%; (e) trimethylboroxine, PdCl₂(dtbpf), K₂CO₃, 1,4‐dioxane/H₂O, 110 °C, 21%; (f) aq. HCl, 80 °C, 81%; (g) NaH, MeI, THF, 35 °C, 53%.

With the completion of compounds 65 and 66, only the 6‐membered ring scaffold containing nitrogen and sulfur atoms remained. Pleasingly, we were able to access this scaffold starting from aminopyridine 67 (Scheme 6). A dibromination reaction gave rise to pyridine 68, which was then transformed into compound 70 via a cyclization reaction with thiolester 69. Reduction of the cyclic amide using borane THF complex then furnished dihydropyridothiazine 72 in 53% yield with dihydropyridothiazine 73 being accessed via methylation of the nitrogen atom.

Scheme 6.

Preparation of dihydropyridothiazines 72 and 73. (a) NBS, DMSO/H₂O, 120 °C, 17%; (b) NaH, DMF, 70 °C, 31%; (c) methylboronic acid, Pd(dppf)Cl₂•CH₂Cl₂, Cs₂CO₃, 1,4‐dioxane, reflux, 49%; (d) BH₃•THF, THF, reflux, 53%; (e) MeI, NaH, DMF, 50 °C, 89%.

3.2. In vitro activity of new scaffolds against FAT A

With the desired target molecules in hand, we wanted to evaluate their in vitro activity against FAT A to focus our subsequent activities on the most promising scaffolds that retain FAT A binding activity. Using a FP assay for FAT A, ¹⁵ we were able to obtain in vitro data for all of the prepared scaffolds (Table 1). The first nitrogen and oxygen containing scaffolds, dihydropyrrolopyridine 18, tetrahydronaphthyridine 24 and dihydropyranopyridine 33a all showed good in vitro efficacy, with dihydropyranopyridine 33a showing the highest value (pI₅₀ = 5.8). In contrast, the thioether containing 5‐ and 6‐membered ring scaffolds 40 and 41 did not exhibit in vitro activity against FAT A. Somewhat surprisingly, the sulfoxide containing compounds 42 and 43 and sulfone containing compounds 44 and 45 displayed improved in vitro inhibition compared to the thioethers, with sulfoxide 42 (pI₅₀ = 5.2) giving the best result. The dioxygen scaffolds 57 and 58 also showed a reduction in in vitro efficacy compared to the corresponding single oxygen scaffolds 12 and 33a respectively. Lastly, the dihydropyrido‐oxazines 65 and 66 were evaluated alongside the dihydropyrido‐thiazines 72 and 73. Interestingly, the compounds 65 and 72 bearing the secondary nitrogen atom were both shown to have almost no activity in the FAT A assay. This result was surprising given that tetrahydronaphthyridine 24 was found to have a pI₅₀ = 5.1, thus suggesting that the incorporation of an additional heteroatom into the bicyclic core at the secondary benzylic position significantly reduces the affinity of the scaffolds for the FAT A binding site. Conversely, the tertiary nitrogen compounds 66 and 73 both showed good levels of in vitro inhibition towards FAT A despite the presence an additional heteroatom in the left‐hand ring system. In comparison to the newly prepared compounds, the patent marker compound 12 displayed a high in vitro activity (pI₅₀ = 6.5) that was in the same range as those measured for the commercial FAT inhibitors cinmethylin (1) (pI₅₀ = 6.8) and methiozolin (2) (pI₅₀ = 6.8).

Table 1.

Prepared compounds and their corresponding FAT A pI₅₀ values^*

Compound structure	Compound no.	FAT A pI50	Compound structure	Compound no.	FAT A pI50
	18	4.9 ± 0.03		45	4.6 ± 0.09
	24	5.1 ± 0.03		57	4.9 ± 0.02
	33a	5.8 ± 0.09		58	5.2 ± 0.02
	40	< 4.0		65	< 4.0
	41	< 4.0		66	4.8 ± 0.04
	42	5.2 ± 0.08		72	< 4.0
	43	4.8 ± 0.03		73	5.3 ± 0.11
	44	4.7 ± 0.05		12	6.5 ± 0.06

^*FAT A pI₅₀ values were obtained using the methods described in the Materials and Methods section of the manuscript. Using the described methods the following FAT A pI₅₀ values were obtained for cinmethylin (1) (pI₅₀ = 6.8 ± 0.06) and methiozolin (2) (pI₅₀ = 6.8 ± 0.02).

3.3. Molecular modelling of new scaffolds using an X‐ray co‐crystal structure of FAT A

In an effort to rationalize some of the in vitro FAT A assay results that we had obtained, we docked some of our structures into a previously obtained X‐ray co‐crystal structure of FAT A bound to cinmethylin (1) (Fig. 3) ⁸ using Flex‐X placement and the scoring function HyDe. ³¹ The compounds 33a, 12 and 58, displayed in (a), (b) and (c) respectively, all have the same binding mode sharing the two H‐bonds to arginine 136 and the hydrophobic effect due to the burial of an aromatic ring moiety in the hydrophobic pocket formed by Val81, Cys126, Tyr177, Phe180, and Cys181. This is the typical binding mode to FAT A that has been observed with our previous thiazolopyridine compounds. ¹⁵ The docking of compound 58, which has an additional oxygen atom, suggests that the presence of an extra polar heteroatom that is unable to form an H‐bonding interaction destabilizes the binding and weakens the affinity. This would nicely explain the reduced in vitro affinity observed for compounds 57 and 58. The manual placement displayed for compound 73 (d) shows that this compound should only bind very weakly due to the steric clashes between the protein and the compound that are indicated by the arrows. This placement is in contrast with the measured weak activity of compound 73 (pI₅₀ = 5.3). However, the relative location of the H‐bonds can vary to a certain extent due to flexibility in the arginine residue. Perhaps this would explain the obtained pI₅₀ for this compound.

Figure 3.

Molecular docking showing the interactions of different compounds binding to FAT A. The interactions and most likely docking modes exhibited by dihydropyranopyridine 33a (a), dihydrofuropyridine 12 (b), dihydrodioxinopyridine 58 (c) and a manual fit for dihydropyridothiazine 73 (d). The contributions of individual atoms are marked by spheres with green spheres indicating a stabilizing contribution to affinity and red spheres a destabilizing contribution. Their diameter correlates with the size of the contribution.

3.4. Herbicidal activity in greenhouse screening assays

To further investigate our newly prepared compounds, we evaluated their herbicidal efficacy in greenhouse assays using a range of warm and cold season weeds (Table 2). To further focus on the compounds bearing the most promising scaffolds, we tested all of the compounds from Table 1 that exhibited a FAT A pI₅₀ value over 5.0 in an attempt to ensure that any herbicidal affect observed was caused by the inhibition of FAT. We also chose to test the compounds using pre‐emergence application as this has previously been demonstrated to give stronger herbicidal efficacy for FAT inhibitors than post‐emergence application (post‐emergence results can be found in the Supporting Information, Table S1). Firstly, tetrahdyronaphthyridine 24 was shown to have good efficacy against the warm season grasses Echinochloa crus‐galli and Setaria viridis, but only with moderate efficacy on the cold season grasses Alopecurus myosuroides and Lolium rigidum. Dihydropyranopyridine 33a displayed a very similar profile as that of compound 24 on the warm season weeds, but its efficacy on cold season weeds was far superior.

Table 2.

Herbicidal evaluation of compounds exhibiting FAT A pI₅₀ values over 5.0 in pre‐emergence application at 1280 g ha^{−1 *}

Compound structure	No.	ECHCG	SETVI	ABUTH	AMARE	ALOMY	LOLRI	MATIN	VERPE
	24	4	5	1	5	2	3	2	3
	33a	5	5	1	5	5	5	4	4
	42	1	1	0	2	0	0	4	0
	58	5	4	0	4 †	4	4	4	4
	73	1	0	0	0	0	1	0	0
	12	5	5	0	4	3	5	4	4

^*Rating scale: ‘5’ = 100% inhibition, ‘4’ = 80–99% inhibition, ‘3’ = 60–79% inhibition, ‘2’ = 40–59% inhibition, ‘1’ = 20–39% inhibition, ‘0’ = <20% inhibition and ‘−’ = no data captured.

^† AMAPA was tested instead of AMARE Abbreviations: Echinochloa crus‐galli (ECHCG), Setaria viridis (SETVI), Abutilon theophrasti (ABUTH), Amaranthus palmeri (AMAPA), Amaranthus retroflexus (AMARE), Alopecurus myosuroides (ALOMY), Lolium rigidum (LOLRI), Matricaria inodora (MATIN) and Veronica persica (VERPE).

Sulfoxide 42 showed a large reduction in efficacy compared to dihydropyranopyridine 33a, with only notable effects being observed on Matricaria inodora. The diheteroatom containing compound dihydrodioxinopyridine 58 exhibited a good level of weed control at the tested dose of 1280 g ha⁻¹, but it was deemed to be weaker than dihydropyranopyridine 33a because of reduced control of the cold season grasses coupled with having no effects on Abutilon theophrasti. The compound dihydropyridothiazine 73 showed almost no efficacy, suggesting that other barriers to achieving good in vivo activity could exist for this compound, such as fast metabolism of the active ingredient. As a comparison, we tested compound 12 and found that dihydropyranopyridine 33a showed improved performance against Amaranthus retroflexus and A. myosuroides weeds in our greenhouse screening assays.

Having found that the dihydropyranopyridine 33a was the best performing of our prepared compounds, we wanted to investigate the SAR of this class. Accordingly, we synthesized an additional nine compounds that explored new substituents in the 2‐ and 3‐positions of the pyridine ring of the dihydropyranopyridine scaffold (Table 3). The synthesis was accomplished in an analogous manner to the synthetic route shown in Scheme 2 (see the Materials and Methods section for details). Still using pre‐emergence application (post‐emergence results can be found in the Supporting Information, Table S2) but now using a dose rate of 320 g ha⁻¹, we found that compound 33a performed to the same degree as the results obtained at the higher does rate (1280 g ha⁻¹). Shifting the fluorine substituent to the meta‐position of the aromatic ring gave a significant reduction in herbicidal efficacy. The 2,6‐difluoro substituted compound 33c performed well on the warm season grasses but it did not retain the broad efficacy observed for compound 33a. Interestingly, the unsubstituted compound 33d had some efficacy against A. theophrasti, which is known to be a challenging species for FAT inhibitors to control, but there was reduced efficacy on A. myosuroides and Veronica persica. The compounds 31, 33e and 33f, with no substituent in the meta‐position of the pyridine, could effectively control the warm season grasses but had weak performance against the other weeds tested. A similar trend was observed with the bromine containing compounds 32 and 33g. These compounds only really exhibited efficacy on the warm grasses E. crus‐galli and S. viridis, even though the measured FAT A pI₅₀ values for both compounds were very favorable, especially for that of compound 32 (pI₅₀ = 7.7). The high pI₅₀ of compound 32 may be explained by an increased hydrophobic effect due to an improved burial of the bromine atom into the hydrophobic pocket formed by the side chain atoms of Thr91, Trp101 and Tyr177. From our studies there is no evidence to suggest that this high pI₅₀ is caused by a halogen bonding effect. ³⁴ The overall weaker in vivo efficacy of the bromine containing compounds, particularly that of compound 32, could be due to a multitude of reasons such as the uptake, bioavailability and metabolism of the compounds, as these factors have been previously shown to play a role in the overall performance of herbicidal compounds. ³⁵ , ³⁶ We also observed that the bromine substituted compound 32 exhibited a higher logP value (3.09) than that given by the methyl substituted compound 33a (2.37). This could also give insights as to why the in vivo efficacy of compound 32 is weaker than the in vitro result would suggest, as perhaps compound 32 is too lipophilic for optimal in vivo weed control when targeting FAT with the dihydropyranopyridine scaffold. Lastly, we prepared the cyclopropyl compound 33h via a palladium cross coupling reaction and found that it displayed a reduced in vitro activity (pI₅₀ = 5.5), as well as having herbicidal activity only on E. crus‐galli and M. inodora. For comparison purposes we tested dihydrofuropyridine 12 and the commercial FAT inhibitor methiozolin (2) in our greenhouse assays and found that a comparable level of herbicidal performance was achieved as that displayed by dihydropyranopyridine 33a at 320 g ha⁻¹. Thus, demonstrating that dihydropyranopyridine 33a could be a candidate for further investigations in the future. ³⁷

Table 3.

Herbicidal evaluation of dihydropyranopyridines in pre‐emergence application at 320 g ha^{−1 *}

Compound structure	No.	FAT A pI50	ECHCG	SETVI	ABUTH	AMARE	ALOMY	LOLRI	MATIN	VERPE
	33a	5.8 ± 0.09	5	5	1	5	5	5	4	4
	33b	5.2 ± 0.01	4	2	0	1	1	3	2	0
	33c	5.6 ± 0.01	5	5	0	1	2	4	4	2
	33d	6.2 ± 0.03	5	5	2	4	2	5	4	1
	31	6.6 ± 0.03	5	5	0	1	0	1	0	0
	33e	6.6 ± 0.03	5	4	0	0	−	1	0	−
	33f	6.2 ± 0.04	4	5	0	1	0	0	1	0
	32	7.7 ± 0.01	3	4	0	0	2	1	1	3
	33 g	6.6 ± 0.05	5	5	0	2	0	0	0	0
	33 h	5.5 ± 0.04	5	0	0	1	0	0	4	0
	12	6.5 ± 0.06	5	5	0	4	3	5	4	4
	2	6.8 ± 0.02	5	5	0	4	4	4	1	2

^*Rating scale: ‘5’ = 100% inhibition, ‘4’ = 80–99% inhibition, ‘3’ = 60–79% inhibition, ‘2’ = 40–59% inhibition, ‘1’ = 20–39% inhibition, ‘0’ = <20% inhibition and ‘−’ = no data captured. Abbreviations: Echinochloa crus‐galli (ECHCG), Setaria viridis (SETVI), Abutilon theophrasti (ABUTH), Amaranthus retroflexus (AMARE), Alopecurus myosuroides (ALOMY), Lolium rigidum (LOLRI), Matricaria inodora (MATIN) and Veronica persica (VERPE).

4. CONCLUSION

The scaffold hopping study outlined herein covers the design, synthesis and investigation into a series of novel structural motifs that were planned to target the herbicidal MoA FAT. Utilizing scaffold hopping and bioisosteric approaches, supported by molecular docking and in vitro FAT A assays, we were able to identify several active bicyclic scaffolds that featured a combination of saturated and unsaturated heterocycles. Evaluation of the herbicidal activity in greenhouse screening assays enabled us to determine that the dihydropyranopyridine scaffold exhibited a superior level of performance over the others that were prepared. Further exploration of the SAR around the class and additional greenhouse testing showed that dihydropyranopyridine 33a displayed good herbicidal efficacy, particularly against warm and cold season grasses, at 320 g ha⁻¹ using pre‐emergence application on a comparable level to that obtained using the commercially available FAT inhibitor methiozolin (2). Additional studies would be needed to evaluate in detail the full potential of dihydropyranopyridine 33a as a future herbicidal candidate. However, we believe that our study has shown that chemistry driven scaffold hopping approaches effectively enable the broadening of the SAR associated with modern herbicidal compound classes, as well as being a useful tool to address weed resistance and sustainability goals.

Supporting information

Data S1. Supporting Information.

DATA AVAILABILITY STATEMENT

The data that supports the findings of this study are available in the supporting information of this article.