De novo Design of SARS-CoV-2 Main Protease Inhibitors

Abstract

The COVID-19 pandemic prompted many scientists to investigate remedies against SARS-CoV-2 and related viruses that are likely to appear in the future. As the main protease of the virus, M^Pro, is highly conserved among coronaviruses, it has emerged as a prime target for developing inhibitors. Using a combination of virtual screening and molecular modeling, we identified small molecules that were easily accessible and could be quickly diversified. Biochemical assays confirmed a class of pyridones as low micromolar noncovalent inhibitors of the viral main protease.

Small molecules continue to play a crucial role in the fight against viral diseases. Examples for their success include medications for HIV and HCV, which have been made manageable or can be cured by inhibitors of proteases and RNA polymerases.[1] This will likely be the case for the current COVID-19 pandemic as well. Although vaccines based on chemically modified mRNA packaged in lipid nanoparticles have proven to be highly effective, their future usefulness may be impeded by rapid mutations in the viral envelope.[2] In addition, vaccines are less effective in the immunocompromised and are not embraced by a substantial portion of the population for a variety of reasons. Therefore, the development of easily applicable and stable small molecules that fight SARS-CoV-2 and related coronaviruses remains a priority.[3]

Amongst the limited set of viral target proteins, the SARS-CoV-2 main protease (M^Pro, also called 3CL^Pro) stands out. This enzyme, a cysteine protease, cleaves its substrate after a glutamine residue, which appears to be unknown for human cysteine proteases that could be responsible for off-target effects.[4] Over the course of the COVID-19 pandemic, the amino acid sequence of M^Pro has remained remarkably conserved,[5] although it has not been challenged yet by drugs on a large scale. Numerous X-ray structures of M^Pro with covalent and noncovalent inhibitors and small-molecule fragments bound are available to aid computational designs.[6] In addition, SARS-CoV-2 M^Pro is highly homologous to corresponding proteases of other coronaviruses and enteroviruses and likely to be related to proteases of harmful viruses that will emerge in the future.[7]

As such, efforts to develop M^Pro inhibitors have been launched on a large scale. To date, they have mostly been centered on covalent inhibitors derived from peptides that correspond to the natural cleavage site of the M^Pro substrate.[8] Given the rapid advance of this approach, which had been pursued for coronaviruses well before the emergence of SARS-CoV-2, we decided to take an alternative one: the de novo design of molecules through virtual screening and further refinement of the best candidates with molecular docking. This was always done with an eye on synthetic accessibility and the ability to quickly diversify successful candidates. As an additional distinguishing criterium, we decided to work on molecules that bind noncovalently, at least in the first phase of our program. We now disclose a series of small molecules that fulfill criteria for ‘druggability’ and can be assembled in a few synthetic steps. Our most successful ones inhibit M^Pro at single digit micromolar concentrations in biochemical assays.

The ASINEX PPI nonmacrocyclic screening library consists of 11870 fragments and compounds that were computationally docked into the catalytic site of the crystal structure of the SARS-CoV-2 main protease (PDB: 6LU7 and 6Y84; details can be found in the Supporting Information).[9] This provided a virtual spectrum of docking score vs ligand efficiency (Figure [1a], blue) in which some of the initial virtual hits were manually evaluated and optimized in terms of ligand efficiency, synthetic accessibility, and docking score (Figure [1a], red). Based on these parameters, we selected three distinct types of molecules, represented by compounds 1–3 for synthesis and biological testing (Figure [1b]).

N-Alkyl-3-arylpyridone 5-carboxamides of type 1 were recognized as an easily accessible scaffold as well as m-teraryl-linked cyclic ureas of type 2. In addition to these, short d-Phg-d-Lys peptides of type 3 were identified as potential inhibitors that should remain proteolytically stable and capable to disrupt the natural function of the protease. Methyl 6-hydroxynicotinate (4) is selectively N-alkylated with chloroacetamide 5a using K₂CO₃ (Scheme [1a]). Bromination and Suzuki cross-coupling allow access to the arylated pyridone methyl ester 6a, which is subsequently hydrolyzed. The resulting carboxylic acid is coupled using HATU to the benzimidazole amine 7 to form N-alkyl-3-arylpyridone 5-carboxamide 1. The cyclic urea 2 was synthesized in similar efficiency (Scheme [1b]). The initial isocyanate addition to 3,5-dibromoaniline (8) was followed by the cyclization of the urea moiety using NaH. Two sequential Suzuki cross-coupling reactions on the dibromide 9 with aryl boronic acid pinacol esters 10 and 11 rendered the desired m-teraryl-linked urea 2. The short d-Phg-d-Lys peptide 3 was manually assembled starting from Fmoc-d-Lys(Boc)-OH (12) by a tailored sequence of EDCI/HOBt-based couplings and protecting group removals (Scheme [1c]).

With our first target molecules in hand, we proceeded to test them in enzymatic activity assays that monitor the formation of fluorescent cleavage products (see the Supporting Information).[10] Despite their excellent docking scores, pyridone 1, the cyclic urea 2, and the d-peptide 3 failed to inhibit the viral main protease even at high concentrations. Gratifyingly, however, some synthetic intermediates that were also tested showed activities that were worth following up upon. For instance, the Boc-protected intermediate 16 inhibited M^Pro with an IC₅₀ of 121 μM. The truncated pyridone methyl ester intermediate 6a showed more substantial inhibition (IC₅₀ = 19 μM). Replacing the benzimidazole amide of 1 with a methyl ester not only reduces the molecular weight but also the lipophilicity (clogP lowered from ca. 4.4 to ca. 3.5). Therefore, this pyridone scaffold was identified as a promising lead for further optimization and our subsequent investigations focused on this class of compounds.

The succinct synthesis of pyridone methyl ester 6a, outlined in Scheme [1], was generally applicable to various other derivatives but also allowed a broad range of cross-coupling reactions (see the Supporting Information for details). Initial structure–activity relationship (SAR) investigations showed that a substituent next to the pyridine carbonyl was critical. For instance, unsubstituted pyridone 4′ did not show any activity (Figure [2]). Also, the activities of the unsubstituted phenyl 6b and the more lipophilic fluoro aryls 6c–e were negligibly low. While the CF₃ derivative 6f and the acetamide 6g showed improved IC₅₀ values, the sulfonamide 6h remained inactive. In the absence of an X-ray structure, systematic synthesis and biological evaluation were required to improve our understanding of how the pyridone series interacts with the binding site.

We anticipate that a favorable interaction (e.g., hydrogen bonding) very close to the 4-position of the aryl substituent is required to further lower the IC₅₀. Therefore, the 4-pyridyl 6i and 6j were synthesized, showing increased activity at 14 and 12 μM, respectively. Also, 4-anisole 6k as well as methoxy-pyrimidine 6l supported this hypothesis with an increase in activity, whereas the corresponding aryloxy ether 6m is likely missing such a key interaction. Possibly due to the orientation of the sulfur lone pairs the thiomorpholine 6n is less optimal. The more extended morpholine 6o, the sulfone analogue 6p, and the piperidine-4-carbonitrile 6q seem to be missing this favorable interaction as well. A stark increase in inhibition of the main protease was found with dihydropyrrolopyridine 6r having an IC₅₀ of 3.2 μM. To probe the length of the substituent further, we examined ethynylpyridine 6s in comparison to ethynylbenzene 6t. Both had diminishing effects on the potency. Hence a more favorable interaction could be imagined with a smaller functional group. Whereas the alkyne 6u was ineffective, the nitrile 6v was inhibiting the main protease with an IC₅₀ of 3.3 μM. In addition to the improved potency, the nitrile 6v had also a favorable calculated logP of 1.1 and a polar surface area of 101 Å, which promise good cell permeation.

During our SAR investigations, we realized that some of the pyridones, such as the acetylated derivative 6g, showed strong fluorescence (λ_abs 318 nm; λ_em 420 nm in aq. PBS with 1% DMSO). This reaffirmed our selection of a bathochromically shifted rhodamine-based fluorescent probe for the enzymatic assays that did not interfere with our inhibitors (see the Supporting Information for details).

To further improve the activity of the pyridone scaffold we decided to explore variations in the acetamide side chain. Unfortunately, Suzuki cross-coupling reactions on the free 4-bromo-6-hydroxynicotinate core were met with limited success. Based on reports by Gademann and co-workers the hydroxypyridine 18 was protected first using SEM-Cl (Scheme [2]).[11] This enabled derivatization like the nitrile substitution and Suzuki cross-coupling reaction. As anisole is a neutral, fairly stable moiety and showed an IC₅₀ of 23 μM in derivative 6k, it was chosen together with the more active nitrile for further exploration. After deprotection of the SEM group, the acetamides were installed under the previously introduced conditions. For the nitrile, both the morpholine amide 20a and the adamantyl amide 20b did not show any inhibition in the enzyme assay. Interestingly, for the anisole, a N,N-diethylamide 21a lost the activity completely. However, the adamantyl amide 21b and the morpholino amide 21c showed a nearly tenfold lower IC₅₀ of 3.5 μM and 3.4 μM, respectively.

Our concern of the pyridone methyl ester hydrolysis as a serious liability grew when we observed a significantly increased IC₅₀ of the corresponding carboxylic acid of the anisole derivative 22a. The installation of dimethyl amides, morpholino amides, and phenyl amides were examined for both 4-anisole and nitrile derivatives 22a–c and 23a,b (Scheme [3a]). Disappointingly, all derivatives showed a complete loss of activity. In search of another functional group, we decided to explore the installation of an aryl moiety instead. N-Alkylation of 5-bromopyridin-2-ol (25) using chloroacetamide 5a allowed the installation of the fluorophenyl via Suzuki cross-coupling (Scheme [3b]). Installation of a nitrile at the 3-position leads to 28, a significantly more active SARS-CoV-2 main protease inhibitor at an IC₅₀ of 2.8 μM.[12] This is our strongest inhibitor to date and is in stark contrast to the activity of compound 27, which lacks the nitrile (IC₅₀ = ca. 77 μM).

In sum, we have introduced small molecules that could potentially be developed into antivirals against SARS-CoV-2. Using a combination of virtual screening, molecular docking, and luck, we quickly were able to identify low micromolar inhibitors of the viral main protease M^Pro. Their further development will require X-ray crystallographic studies, which will provide insights into the binding site and pose of our inhibitors and can also serve to calibrate our docking results. Attempts in this direction are well underway and results will be reported in due course.

Conflict of Interest

The authors declare no conflict of interest.

• References and Notes

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• 12 Procedures a-d for the synthesis of compound 28: (a) To a mixture of 5‑bromo-2-hydroxypyridine (25) (522 mg, 3.00 mmol), K₂CO₃ (1.24 g, 9.00 mmol) in acetone (30 mL) was added 2-chloro-N-cyclohexylacetamide (5a) (738 mg, 4.20 mmol) at room temperature and the mixture was stirred at 50 °C for 18 hours. The suspension was treated with hexanes (50 mL), filtered and washed portionwisely with water (100 mL). The remaining solid was dried and recrystallized from acetone/hexanes to give intermediate 26 as an off-white solid (716 mg, 76%): R _f 0.31 (hexanes:EtOAc, 1:2); ¹H-NMR (400 MHz, CDCl₃): δ = 7.56 (1H, d, ⁴ J 2.6 Hz), 7.41 (1H, dd, ³ J 9.7 Hz, ⁴ J 2.7 Hz), 6.62 (1H, d, ³ J 5.8 Hz), 6.52 (1H, d, ³ J 9.7 Hz), 4.45 (2H, s), 3.65–3.74 (1H, m), 1.83–1.87 (2H, m), 1.65–1.71 (2H, m), 1.54–1.61 (1H, m), 1.28–1.39 (2H, m), 1.11–1.21 (3H, m); ¹³C‑NMR (101 MHz, CDCl₃): δ = 165.6, 161.4, 143.7, 138.1, 121.9, 99.0, 54.0, 48.8, 32.8, 25.6, 24.7; APCI-HRMS: m/z calcd. for [C₁₃H₁₈BrN₂O₂]⁺ 315.0526 found 315.0529 [M+H]⁺. (b) A mixture of intermediate 26 (94.0 mg, 0.300 mmol), 4-fluorophenylboronic acid (105 mg, 0.750 mmol), Na₂CO₃ (127 mg, 1.20 mmol), Pd(PPh₃)₄ (17.3 mg, 15.0 µmol, 5.00 mol%) was evacuated for 10 minutes under high vacuum and backfilled with N₂at room temperature. The mixture was treated with PhMe:EtOH:H₂O (1.9 mL, 0.74 mL, 0.26 mL) and stirred at 75 °C for 3 hours in a pre-heated sand bath. The mixture was treated with water and extracted with CH₂Cl₂. The combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. Precipitation from hexanes gives intermediate 27 as a white solid (85.3 mg, 87%): R _f 0.26 (hexanes:EtOAc, 1:2, UV); ¹H-NMR (400 MHz, CDCl₃): δ = 7.64 (1H, dd, ³ J 9.4 Hz, ⁴ J 2.6 Hz), 7.59 (1H, d, ⁴ J 2.4 Hz), 7.36–7.40 (2H, m), 7.09–7.13 (2H, m), 6.82 (1H, d, ³ J 6.7 Hz), 6.71 (1H, d, ³ J 9.4 Hz), 4.57 (2H, s), 3.67–3.76 (1H, m), 1.85–1.89 (2H, m), 1.66–1.71 (2H, m), 1.55–1.60 (1H, m), 1.28–1.36 (2H, m), 1.14–1.24 (3H, m); ¹³C‑NMR (101 MHz, CDCl₃): δ = 166.1, 162.6 (¹ J _CF 247 Hz), 162.2, 140.4, 135.4, 132.3 (⁴ J _CF 3.7 Hz), 127.8 (³ J _CF 8.1 Hz), 120.9, 120.5, 116.2 (² J _CF 22 Hz), 54.6, 48.7, 32.8, 25.6, 24.7; ¹⁹F-NMR (377 MHz, CDCl₃): δ = –114.66; APCI-HRMS: m/z calcd. for [C₁₉H₂₂FN₂O₂]⁺ 329.1660 found 329.1660 [M+H]⁺. (c) To a solution of intermediate 27 (32.8 mg, 0.100 mmol) in glacial acetic acid (1.0 mL) was added dropwise bromine (8.00 µL, 0.150 mmol) at room temperature. The orange mixture was stirred at 60 °C for 18 hours. When cooled to room temperature the mixture was treated with aqueous saturated Na₂S₂O₃ and adjusted to pH 7 with aqueous saturated NaHCO₃. The suspension was filtered and washed extensively with water. The filter cake was dried in vacuo and purified by column chromatography over silica gel with eluent 100% hexanes to hexanes:EtOAc 90:10 to 75:25 to 60:40 to 50:50 to give 2-(3-bromo-5-(4-fluorophenyl)-2-oxopyridin-1(2H)-yl)-N-cyclohexyl­acetamide (27.0 mg, 66%): R _f 0.30 (hexanes:EtOAc, 1:1, UV); ¹H‑NMR (400 MHz, CDCl₃): δ = 8.03 (1H, d, ⁴ J 2.4 Hz), 7.64 (1H, d, ⁴ J 2.3 Hz), 7.36–7.39 (2H, m), 7.09–7.13 (2H, m), 6.73 (1H, d, ³ J 8.1 Hz), 4.63 (2H, s), 3.67–3.76 (1H, m), 1.85–1.89 (2H, m), 1.67–1.72 (2H, m), 1.56–1.61 (1H, m), 1.28–1.38 (2H, m), 1.15–1.24 (3H, m); ¹³C‑NMR (101 MHz, CDCl₃): δ = 165.5, 162.8 (¹ J _CF 248 Hz), 158.6, 142.3, 135.1, 131.3 (⁴ J _CF 3.3 Hz), 128.0 (³ J _CF 8.8 Hz), 120.6, 116.3 (² J _CF 21 Hz), 55.0, 49.0, 32.8, 25.5, 24.8; ¹⁹F-NMR (377 MHz, CDCl₃): δ = – 13.99; APCI-HRMS: m/z calcd. for [C₁₉H₂₁BrFN₂O₂]⁺ 407.0765 found 407.0767 [M+H]⁺. (d) A solution of 2-(3-bromo-5-(4-fluorophenyl)-2-oxopyridin-1(2H)-yl)-N-cyclohexylacetamide (12.7 mg, 30.0 µmol) in dry DMF (0.30 mL) was treated with CuCN (8.40 mg, 90.0 µmol) at room temperature. The mixture was deareated by N₂ sparging for five minutes at room temperature and heated at 120 °C for 20 hours. The mixture was treated with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. Purification by column chromatography over silica gel with eluent 100% CH₂Cl₂ to 95:5 CH₂Cl₂:MeOH gives 2-(3-cyano-5-(4-fluorophenyl)-2-oxopyridin-1(2H)-yl)-N-cyclo­hexylacetamide (28) as a white solid (3.70 mg, 34%): R _f 0.21 (hexanes:EtOAc, 1:1, UV); ¹H-NMR (400 MHz, CDCl₃): δ = 8.07 (1H, d, ⁴ J 2.6 Hz), 7.87 (1H, d, ⁴ J 2.7 Hz), 7.34–7.41 (2H, m), 7.11–7.19 (2H, m), 6.40 (1H, d, ³ J 7.9 Hz), 4.62 (2H, s), 3.67–3.77 (1H, m), 1.85–1.94 (2H, m), 1.67–1.72 (2H, m), 1.58–1.64 (1H, m), 1.28–1.40 (2H, m), 1.13–1.26 (3H, m); ¹³C-NMR (101 MHz, CDCl₃): δ = 164.5, 163.0 (¹ J _CF 248 Hz), 159.2, 147.2, 141.0, 130.5 (⁴ J _CF 3.4 Hz), 128.1 (³ J _CF 8.3 Hz), 119.9, 116.6 (² J _CF 22 Hzs), 105.6, 53.7, 49.3, 32.9, 25.5, 24.8; ¹⁹F-NMR (377 MHz, CDCl₃): δ = –113.03; APCI-HRMS: m/z calcd. for [C₂₀H₂₁FN₃O₂]⁺ 354.1612 found 354.1609 [M+H]⁺. Further details can be found in the Supporting Information.

Corresponding Authors

Publication History

Received: 13 July 2021

Accepted after revision: 10 August 2021

Accepted Manuscript online:
10 August 2021

Article published online:
05 October 2021

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• References and Notes

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• 12 Procedures a-d for the synthesis of compound 28: (a) To a mixture of 5‑bromo-2-hydroxypyridine (25) (522 mg, 3.00 mmol), K₂CO₃ (1.24 g, 9.00 mmol) in acetone (30 mL) was added 2-chloro-N-cyclohexylacetamide (5a) (738 mg, 4.20 mmol) at room temperature and the mixture was stirred at 50 °C for 18 hours. The suspension was treated with hexanes (50 mL), filtered and washed portionwisely with water (100 mL). The remaining solid was dried and recrystallized from acetone/hexanes to give intermediate 26 as an off-white solid (716 mg, 76%): R _f 0.31 (hexanes:EtOAc, 1:2); ¹H-NMR (400 MHz, CDCl₃): δ = 7.56 (1H, d, ⁴ J 2.6 Hz), 7.41 (1H, dd, ³ J 9.7 Hz, ⁴ J 2.7 Hz), 6.62 (1H, d, ³ J 5.8 Hz), 6.52 (1H, d, ³ J 9.7 Hz), 4.45 (2H, s), 3.65–3.74 (1H, m), 1.83–1.87 (2H, m), 1.65–1.71 (2H, m), 1.54–1.61 (1H, m), 1.28–1.39 (2H, m), 1.11–1.21 (3H, m); ¹³C‑NMR (101 MHz, CDCl₃): δ = 165.6, 161.4, 143.7, 138.1, 121.9, 99.0, 54.0, 48.8, 32.8, 25.6, 24.7; APCI-HRMS: m/z calcd. for [C₁₃H₁₈BrN₂O₂]⁺ 315.0526 found 315.0529 [M+H]⁺. (b) A mixture of intermediate 26 (94.0 mg, 0.300 mmol), 4-fluorophenylboronic acid (105 mg, 0.750 mmol), Na₂CO₃ (127 mg, 1.20 mmol), Pd(PPh₃)₄ (17.3 mg, 15.0 µmol, 5.00 mol%) was evacuated for 10 minutes under high vacuum and backfilled with N₂at room temperature. The mixture was treated with PhMe:EtOH:H₂O (1.9 mL, 0.74 mL, 0.26 mL) and stirred at 75 °C for 3 hours in a pre-heated sand bath. The mixture was treated with water and extracted with CH₂Cl₂. The combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. Precipitation from hexanes gives intermediate 27 as a white solid (85.3 mg, 87%): R _f 0.26 (hexanes:EtOAc, 1:2, UV); ¹H-NMR (400 MHz, CDCl₃): δ = 7.64 (1H, dd, ³ J 9.4 Hz, ⁴ J 2.6 Hz), 7.59 (1H, d, ⁴ J 2.4 Hz), 7.36–7.40 (2H, m), 7.09–7.13 (2H, m), 6.82 (1H, d, ³ J 6.7 Hz), 6.71 (1H, d, ³ J 9.4 Hz), 4.57 (2H, s), 3.67–3.76 (1H, m), 1.85–1.89 (2H, m), 1.66–1.71 (2H, m), 1.55–1.60 (1H, m), 1.28–1.36 (2H, m), 1.14–1.24 (3H, m); ¹³C‑NMR (101 MHz, CDCl₃): δ = 166.1, 162.6 (¹ J _CF 247 Hz), 162.2, 140.4, 135.4, 132.3 (⁴ J _CF 3.7 Hz), 127.8 (³ J _CF 8.1 Hz), 120.9, 120.5, 116.2 (² J _CF 22 Hz), 54.6, 48.7, 32.8, 25.6, 24.7; ¹⁹F-NMR (377 MHz, CDCl₃): δ = –114.66; APCI-HRMS: m/z calcd. for [C₁₉H₂₂FN₂O₂]⁺ 329.1660 found 329.1660 [M+H]⁺. (c) To a solution of intermediate 27 (32.8 mg, 0.100 mmol) in glacial acetic acid (1.0 mL) was added dropwise bromine (8.00 µL, 0.150 mmol) at room temperature. The orange mixture was stirred at 60 °C for 18 hours. When cooled to room temperature the mixture was treated with aqueous saturated Na₂S₂O₃ and adjusted to pH 7 with aqueous saturated NaHCO₃. The suspension was filtered and washed extensively with water. The filter cake was dried in vacuo and purified by column chromatography over silica gel with eluent 100% hexanes to hexanes:EtOAc 90:10 to 75:25 to 60:40 to 50:50 to give 2-(3-bromo-5-(4-fluorophenyl)-2-oxopyridin-1(2H)-yl)-N-cyclohexyl­acetamide (27.0 mg, 66%): R _f 0.30 (hexanes:EtOAc, 1:1, UV); ¹H‑NMR (400 MHz, CDCl₃): δ = 8.03 (1H, d, ⁴ J 2.4 Hz), 7.64 (1H, d, ⁴ J 2.3 Hz), 7.36–7.39 (2H, m), 7.09–7.13 (2H, m), 6.73 (1H, d, ³ J 8.1 Hz), 4.63 (2H, s), 3.67–3.76 (1H, m), 1.85–1.89 (2H, m), 1.67–1.72 (2H, m), 1.56–1.61 (1H, m), 1.28–1.38 (2H, m), 1.15–1.24 (3H, m); ¹³C‑NMR (101 MHz, CDCl₃): δ = 165.5, 162.8 (¹ J _CF 248 Hz), 158.6, 142.3, 135.1, 131.3 (⁴ J _CF 3.3 Hz), 128.0 (³ J _CF 8.8 Hz), 120.6, 116.3 (² J _CF 21 Hz), 55.0, 49.0, 32.8, 25.5, 24.8; ¹⁹F-NMR (377 MHz, CDCl₃): δ = – 13.99; APCI-HRMS: m/z calcd. for [C₁₉H₂₁BrFN₂O₂]⁺ 407.0765 found 407.0767 [M+H]⁺. (d) A solution of 2-(3-bromo-5-(4-fluorophenyl)-2-oxopyridin-1(2H)-yl)-N-cyclohexylacetamide (12.7 mg, 30.0 µmol) in dry DMF (0.30 mL) was treated with CuCN (8.40 mg, 90.0 µmol) at room temperature. The mixture was deareated by N₂ sparging for five minutes at room temperature and heated at 120 °C for 20 hours. The mixture was treated with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. Purification by column chromatography over silica gel with eluent 100% CH₂Cl₂ to 95:5 CH₂Cl₂:MeOH gives 2-(3-cyano-5-(4-fluorophenyl)-2-oxopyridin-1(2H)-yl)-N-cyclo­hexylacetamide (28) as a white solid (3.70 mg, 34%): R _f 0.21 (hexanes:EtOAc, 1:1, UV); ¹H-NMR (400 MHz, CDCl₃): δ = 8.07 (1H, d, ⁴ J 2.6 Hz), 7.87 (1H, d, ⁴ J 2.7 Hz), 7.34–7.41 (2H, m), 7.11–7.19 (2H, m), 6.40 (1H, d, ³ J 7.9 Hz), 4.62 (2H, s), 3.67–3.77 (1H, m), 1.85–1.94 (2H, m), 1.67–1.72 (2H, m), 1.58–1.64 (1H, m), 1.28–1.40 (2H, m), 1.13–1.26 (3H, m); ¹³C-NMR (101 MHz, CDCl₃): δ = 164.5, 163.0 (¹ J _CF 248 Hz), 159.2, 147.2, 141.0, 130.5 (⁴ J _CF 3.4 Hz), 128.1 (³ J _CF 8.3 Hz), 119.9, 116.6 (² J _CF 22 Hzs), 105.6, 53.7, 49.3, 32.9, 25.5, 24.8; ¹⁹F-NMR (377 MHz, CDCl₃): δ = –113.03; APCI-HRMS: m/z calcd. for [C₂₀H₂₁FN₃O₂]⁺ 354.1612 found 354.1609 [M+H]⁺. Further details can be found in the Supporting Information.

• Supplementary Material