Synthesis and in vitro antibacterial activity of novel fluoroalkyl-substituted pyrazolyl oxazolidinones

Abstract

A series of novel oxazolidinone derivatives bearing fluoroalkyl-substituted pyrazole as the C-ring structure were designed, synthesized and evaluated for their antibacterial activity against six Gram-positive bacterial pathogens. Most of the target compounds have good antibacterial activity. Especially, compounds 13f, 13i and 13l show excellent activity comparable to linezolid.

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1 Introduction

Common multi-drug-resistant Gram-positive bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), vancomycin-resistant Enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae (PRSP) have become serious health threats.^1–3 Oxazolidinones, as exemplified by linezolid, are a new class of synthetic antibacterial agents that have high activity against a broad range of Gram-positive bacteria.⁴ Linezolid was developed by Pharmacia and Upjohn Company and approved in 2000 for the treatment of serious infections caused by the above-mentioned strains.⁵ However, bacterial resistance to linezolid has been found recently in Gram-positive bacteria Staphylococcus aureus and Enterococcus faecium.⁶ Hence, numerous researches on the synthesis and bioassay of various oxazolidinones have been developed.^7–11 These studies have focused mainly on the modification of A-ring (oxazolidinone), B-ring (phenyl), C-ring (morpholine), and the C-5 side chain on the A-ring substructure of linezolid. Among them, series of oxazolidinones containing nitrogen heterocyclic structures as C-ring unit were reported and some analogues have good activity comparable or superior to linezolid against Gram-positive bacterial pathogens (Fig. 1).^12,13 Oxazolidinones containing a pyrroloaryl substituent have been synthesized by J&J Pharmaceutical, and one compound was approved to begin phase I clinical trial.¹⁴ Gyochang Keum has disclosed some oxazolidinones with 3-azabicyclo[3.3.0]octanyl rings as C-ring exhibit potent in vitro antibacterial activity.¹⁵ Potent oxazolidinone antibacterials with pyrazolo[1,5-a]pyridines as the C-ring substructure were described by Hideyuki Suzuki et al.¹⁶ Youfu Luo's group have synthesized an oxazolidinone compound with 4-(pyridin-2-yl)-1H-pyrrole, which has formed nanoassemblies without any carrier and display antimicrobial activity on MRSA.¹⁷

Fig. 1 Structure of linezolid and its analogs.

For decades, there is considerable interest in introducing fluorine-containing groups into organic molecules since the growing application of organic fluorine compounds in pharmaceuticals.^18,19 Many commercial drugs containing trifluoromethyl and difluoromethyl groups have come to market.¹⁹ However, literature survey showed that the incorporation of these fluoroalkyl groups into oxazolidinones has not been studied systematically. Consequently, we are interested in developing fluoroalkyl-containing oxazolidinones and expect these compounds might result in potent antibacterial activity. According to the literature, pyrazole cycle has been broadly used as a biologically active scaffold in medicine science.²⁰ Herein, we reported the synthesis of novel oxazolidinone derivatives bearing fluoroalkyl-substituted pyrazole as the C-ring structure and their antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-sensitive Staphylococcus epidermidis (MSSE), penicillin-resistant Streptococcus pneumoniae (PRSP) and Enterococcus faecium (EF). Three target compounds show good activity comparable to linezolid.

2 Result and discussion

2.1 Chemistry

The synthetic routes to fluoroalkyl-substituted pyrazolyl oxazolidinone analogues are outlined in Schemes 1–3. The key intermediate 3 was prepared following literature procedures and described in Scheme 1.²¹ (S)-Epichlorohydrin 1 reacted with benzaldehyde and aqueous ammonia in ethanol to form compound 2 after the addition of a solution of hydrochloric acid. The reaction of compound 2 and di-tert-butyldicarbonate provided the key intermediate 3 in high yield.

Scheme 1 Reagents and conditions: (a) PhCHO, NH₃, CH₂Cl₂, 40 °C for 6 h, then 25 °C for 14 h; (b) HCl, H₂O, toluene, 40–45 °C, 3.5 h; (c) (Boc)₂O, Et₃N, CH₂Cl₂, 25 °C, 12 h.

The synthesis of key intermediates 9 is depicted in Scheme 2. β-Ethyloxyvinyl fluoroalkyl ketones 5 were prepared from ethyl vinyl ether and its derivatives with fluoroalkylacetic anhydride under base condition.²² Treatment of compounds 5 with p-nitrophenylhydrazines 6 in ethanol gave exclusively the 5-hydroxy-4,5-dihydropyrazoles 7. Dehydration and reduction of the nitro group afforded the corresponding pyrazoles 9.²³

Scheme 2 Reagents and conditions: (d) (R_fCO)₂O, pyridine, CH₂Cl₂, 0 °C for 1 h, then 20 °C for 12 h; (e) EtOH, reflux with stirring, 24 h; (f) TFA, CH₂Cl₂, 25 °C, 12 h; (g) H₂, Pd/C, MeOH, 25 °C, 3 h.

Scheme 3 indicated the synthetic route of the target compounds 13.²⁴ The preparation involved a coupling reaction between the compound 3 and intermediates 9 to give compounds 10, which then underwent cyclization in the presence of carbonyl diimidazole (CDI) to form the 2-oxazolidinones 11. Access to the fluoroalkyl-substituted pyrazolyl oxazolidinones 13 were achieved via the N-Boc deprotection and subsequent acetylation reaction. The structures of these target compounds are shown in Table 1. All the ¹H-NMR, ¹³C-NMR, ¹⁹F-NMR and mass data of compounds 13a–l were in accordance with the proposed molecular structures.

Scheme 3 Reagents and conditions: (h) neat, 150 °C, 12 h; (i) CDI, Et₃N, THF, 25 °C, 12 h; (j) TFA, CH₂Cl₂, 25 °C, 12 h; (k) Ac₂O, Et₃N, CH₂Cl₂, 25 °C, 12 h.

Table 1 Structure of the target compounds 13a–l

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2.2 Antibacterial activity

The antibacterial activity of the target compounds 13a–l and linezolid, known for comparison were evaluated against Gram-positive bacterial strains: methicillin-resistant Staphylococcus aureus (MRSA) (5 strains), methicillin-sensitive Staphylococcus aureus (MSSA) (5 strains), methicillin-resistant Staphylococcus epidermidis (MRSE) (5 strains), methicillin-sensitive Staphylococcus epidermidis (MSSE) (5 strains), penicillin-resistant Streptococcus pneumoniae (PRSP) (5 strains), Enterococcus faecium (EF) (5 strains) and Staphylococcus aureus ATCC43300 and ATCC29213, as quality-control strains, using two-fold agar dilution method. The minimum inhibitory concentrations (MIC) of these compounds against the above bacteria are presented in Table 2.

Table 2 Minimal-Inhibitory Concentrations (MICs) of oxadiazoles 13a–l

Compound	Minimum inhibitory concentrations (MIC) (μg mL^−1)
	MRSA	MSSA	MRSE	MSSE	PRSP	EF	ATCC43300	ATCC29213
13a	2–4	2–4	4	2–4	2–4	4	4	2
13b	>16	>16	>16	>16	>16	>16	>16	16
13c	2–4	2–4	4	2–4	2–4	4	4	2
13d	8–16	8–16	8–16	8–16	8	8	8	8
13e	2–4	2–4	4	2–4	2–4	4	4	2
13f	0.5–2	0.5–1	1–2	1–2	1–2	1–2	2	0.5
13g	8–16	8–16	8–16	16	8–16	8–16	8	8
13h	2–4	2–4	4	2–4	2–4	4	4	2
13i	1–2	1–2	1–2	1–2	1–2	1–2	2	1
13j	4–8	4–8	4–8	4–8	4	4	8	4
13k	>16	>16	>16	>16	>16	>16	>16	>16
13l	1–2	1–2	1–2	1–2	1–2	1–2	2	1
Linezolid	1–2	1–2	1–2	1–2	1–2	1–2	2	1

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The results revealed that most of the compounds exhibited good to moderate antibacterial activity. Compounds 13f, 13i and 13l appear to exhibit similar antibacterial activity in comparison with linezolid, while compounds have 5-fluoroalkyl pyrazole moiety such as 13b, 13d, 13g and 13k showed poor activity against all the tested bacterial strains with MIC values greater than 8 μg mL⁻¹. In addition, compounds 13a, 13c, 13e and 13h were 2-fold less active than linezolid. According to the above result, it is found that compounds have 5-fluoroalkyl-4-methyl pyrazole moiety showed higher MIC value against Gram-positive bacteria than those have 5-fluoroalkyl-3-methyl pyrazole and 5-fluoroalkyl pyrazole moiety. When 4-methyl-5-trifluromethyl pyrazole was used as C-ring, compound with a fluorine atom at the 3-position of phenyl ring 13f showed 2–4 fold potency advantages compared to compound with no fluorine atom on phenyl ring 13c. Compounds with 5-difluoromethyl-4-methyl pyrazole as C-ring have equal activity no matter the fluorine atom substituted at the phenyl ring or not (13i vs. 13l).

2.3 Molecular docking

To elucidate the antibacterial activity of the target compounds at the molecular level, we explored the interaction mode between the representative compounds 13f, 13l, linezolid and the Deinococcus radiodurans linezolid-bound 50S ribosomal subunit crystal structure (Protein Data Bank accession codes 3DLL) using Surflex-dock.²⁵ The predicted binding affinity (total scores) of docking studies was shown in Table 3. The scores of linezolid, 13f and 13l are with values of 6.47, 7.92 and 8.29, respectively. The docking result was shown in Fig. 2. The Fig. 2A depicted the docking result of 13f and linezolid. From the Fig. 2A, we found the –CF₃ group occupy more space than the –H atom, which make the –CF₃ group combine with the residues of binding site very easy and result in high antibacterial activity of 13f. The Fig. 2B showed the docking result of 13l and linezolid, according to the figure, the –CHF₂ group has a huger space compared to the –H atom. Therefore compound 13l also showed high antibacterial activity comparable to linezolid. The steric space of –CF₃ is bigger than –CHF₂, and it is not conductive to the antibacterial activity. Hence, the docking result showed that compound 13l exhibit higher antibacterial activity than that of compound 13f. The molecular docking result, along with the biological assay data, suggests that compounds 13f and 13l prove to be potential antibacterials.

Table 3 Total-score of compounds 13f, 13l and linezolid

Compound	Total-score
13f	7.92
13l	8.29
Linezolid	6.47
13b	4.66
13k	6.10

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Fig. 2 The docking results of linezolid, 13f and 13l.

Next, we applied the molecular docking protocol to predict the affinity of compounds 13b and 13k, which both show low antibacterial activity in the bioassay, to the Deinococcus radiodurans linezolid-bound 50S ribosomal subunit crystal structure. The scores of 13b and 13k are both lower than linezolid (Table 3). It might due to that in both two compounds, the trifluoromethyl group and methyl group in the pyrazole ring occupy in meta position, which lead to a large steric space and make the target compounds hard to fit into the binding pocket of the subunit.

3 Conclusion

In summary, we have synthesized a series of oxazolidinone-type antibacterials bearing fluoroalkyl-substituted pyrazole as the C-ring structure and evaluated their activity against a panel of Gram-positive bacterial pathogens. Most of the target compounds exhibited potent in vitro antibacterial activity. Compounds have 5-fluoroalkyl-4-methyl pyrazole moiety 13f, 13i and 13l showed high antibacterial activity comparable to linezolid with minimum inhibitory concentration (MIC) values of 1–2, 1–2, 1–2 μg mL⁻¹. Molecular docking studies of linezolid and the representative compounds with most high activity 13f, 13l and low activity 13b, 13k, was performed and gave 6.47, 7.92, 8.20, 4.66, 6.10 total-score respectively. The synthesis of more fluoroalkyl-containing oxazolidinone analogues is ongoing for structure–activity relationship studies.

4 Experimental

4.1 Materials and measurements

All reagents were obtained from commercial suppliers and used without further purification. All NMR spectra were recorded on a Bruker Avance 500 (resonance frequencies 500 MHz for ¹H and 126 MHz for ¹³C) equipped with a 5 mm inverse broadband probe head with z-gradients at 295.8 K with standard Bruker pulse programs. The samples were dissolved in 0.6 mL CDCl₃ (99.8% D TMS). Chemical shifts were given in values of δ_H and δ_C referenced to residual solvent signals (δ_H 7.26 for ¹H, δ_C 77.0 for ¹³C in CDCl₃). The ¹⁹F NMR spectra were obtained using a 500 spectrometer (471 MHz) using trifluorotoluene as external standard. Conventional pulse sequences for NOESY, HMQC and HMBC with standard Bruker pulse programs; 200 ms mixing time for NOESY; J in Hz. High resolution mass spectra (HRMS) were recorded on a Bruker solan X 70 FT-MS (samples was dissolved in CH₃OH and the ion source was ESI), and the energy was 22.5 eV at MS/MS. Melting points are uncorrected.

4.2 Synthesis of key intermediate (S)-tert-butyl(3-chloro-2-hydroxypropyl)carbamate 3

The synthesis of (S)-tert-butyl(3-chloro-2-hydroxypropyl)carbamate 3 had been reported by William R. Perrault. We prepared this compound completely according to the literature procedure and obtained compound 3 in high yield.²³

4.3 General procedure for the preparation of 4-(5-(fluoroalkyl)-1H-pyrazol-1-yl)anilines 9a–l

To a stirred solution of pyridine (1.58 g, 20 mmol) and ethyl vinyl ether derivatives (22 mmol) in CH₂Cl₂ (50 mL) was dropwise added 2,2,2-trifluoroacetic anhydride or 2,2-difluoroacetic anhydride (20 mmol) at −5 °C. The reaction mixture was stirred for 1 h at 0 °C, and then for another 12 h at 20 °C. After the completion of reaction, the mixture was concentrated in vacuo, diluted with ethylene chloride (50 mL) and poured into saturated sodium bicarbonate solution (50 mL). The two phases were separated and the organic layer was washed with water, dried by anhydrous Na₂SO₄. The solvent was removed in reduced pressure to provide oil products 4-ethoxy-1-fluoroalkyl-but-3-en-2-ones 5a–f in high yields. Compounds 5a–f were used for next step without further purification.

Compound 5 (15 mmol) and (4-nitrophenyl)hydrazine 6 (15 mmol) in 50 mL ethanol was heated at reflux for 24 h, followed by cooling and concentration in vacuo. The residue was dissolved in EtOAc (100 mL), washed with water (3 × 100 mL) and brine (100 mL). The organic layer was dried by anhydrous Na₂SO₄ and concentrated in vacuo to afford the brown solid 7a–l. Then, the compounds 7a–l were dissolved in CH₂Cl₂ and added trifluoroacetic acid (3.42 g, 30 mmol) dropwise. The mixture was stirred for 12 h at room temperature. The reaction was quenched with water and extracted with CH₂Cl₂ (3 × 60 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography using petroleum ether/ethyl acetate (8 : 1) as eluent to give the pure products 8a–l in good yields.

A mixture of compound 8 (10 mmol) and 5% Pd/C (0.25 g) in methanol (30 mL) was stirred under H₂ for 3 h at room temperature. The reaction mixture was filtered through diatomaceous earth, and the residue was washed with methanol. The filter was concentrated under reduced pressure. The crude liquid was purified by flash column chromatography using petroleum ether/ethyl acetate (3 : 1) as eluent to yield the pure products 9a–l.

4.3.1 4-(5-(Trifluoromethyl)-1H-pyrazol-1-yl)aniline (9a). Light yellow liquid, yield: 90%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.64–7.62 (m, 1H, pyrazole–H), 7.23–7.21 (m, 2H, ArHs), 6.75–6.73 (m, 2H, ArHs), 6.67–6.65 (m, 1H, pyrazole–H), 3.81 (br, 2H, NH₂); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 147.7, 139.0, 132.7 (q, ²J_C–F = 39.0 Hz), 129.8, 127.0, 120.0 (q, ¹J_C–F = 268.7 Hz), 114.7, 108.2; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.3 (s, 3F).

4.3.2 4-(3-Methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)aniline (9b). Light yellow liquid, yield: 91%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.21–6.69 (m, 4H, ArHs), 6.54 (s, 1H, pyrazole–H), 3.53 (br, 2H, NH₂), 2.34 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 148.3, 147.3, 133.1 (q, ²J_C–F = 39.0 Hz), 130.0, 127.4, 120.5 (q, ¹J_C–F = 268.9 Hz), 114.7, 107.8, 13.3; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.7 (s, 3F).

4.3.3 4-(4-Methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)aniline (9c). Light yellow liquid, yield: 90%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.45–7.42 (m, 1H, pyrazole–H), 7.18–7.16 (m, 2H, ArHs), 6.65–6.62 (m, 2H, ArHs), 3.89 (br, 2H, NH₂), 2.22 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 147.5, 140.6, 130.6, 129.2 (q, ²J_C–F = 37.1 Hz), 127.3, 120.9 (q, ¹J_C–F = 269.6 Hz), 119.1, 114.6, 9.0; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.7 (s, 3F).

4.3.4 3-Fluoro-4-(5-(trifluoromethyl)-1H-pyrazol-1-yl)aniline (9d). Light yellow liquid, yield: 89%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.73–7.70 (m, 1H, pyrazole–H), 7.14–7.11 (m, 1H, ArHs), 6.79–6.76 (m, 1H, pyrazole–H), 6.43–6.40 (m, 2H, ArHs), 4.01 (br, 2H, NH₂); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 158.7 (d, ¹J_C–F = 250.8 Hz), 150.1 (d, ³J_C–F = 10.6 Hz), 139.8, 134.2 (q, ^2′J_C–F = 39.0 Hz), 129.8, 119.8 (q, ^1′J_C–F = 264.6 Hz), 116.4, 110.0, 108.0, 101.8 (d, ²J_C–F = 27.7 Hz); ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.8 (s, 3F), −121.9 (s, 1F).

4.3.5 3-Fluoro-4-(3-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)aniline (9e). Light yellow liquid, yield: 90%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.14–7.11 (m, 1H, ArH), 6.57–6.55 (m, 1H, pyrazole–H), 6.44–6.39 (m, 2H, ArHs), 3.93 (br, 2H, NH₂), 2.35 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 158.6 (d, ¹J_C–F = 250.9 Hz), 149.8 (d, ³J_C–F = 10.7 Hz), 149.1, 134.6 (q, ^2′J_C–F = 38.8 Hz), 130.0, 119.7 (q, ^1′J_C–F = 269.6 Hz), 116.6, 110.0, 107.6, 101.8 (d, ²J_C–F = 23.0 Hz), 13.4; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.7 (s, 3F), −121.9 (s, 1F).

4.3.6 3-Fluoro-4-(4-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)aniline (9f). Light yellow liquid, yield: 92%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.53–7.50 (m, 1H, pyrazole–H), 7.16–7.12 (m, 1H, ArH), 6.57–6.53 (m, 1H, ArH), 6.47–6.44 (m, 1H, ArH), 3.82 (br, 2H, NH₂), 2.36 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 158.8 (d, ¹J_C–F = 250.8 Hz), 149.6 (d, ³J_C–F = 10.8 Hz), 149.1, 141.4, 135.1 (q, ^2′J_C–F = 36.6 Hz), 130.0, 120.2 (q, ^1′J_C–F = 264.5 Hz), 110.0, 107.6, 101.9 (d, ²J_C–F = 22.9 Hz), 13.4; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.8 (s, 3F), −121.6 (s, 1F).

4.3.7 4-(5-(Difluoromethyl)-1H-pyrazol-1-yl)aniline (9g). Light yellow liquid, yield: 92%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.67–7.64 (m, 1H, pyrazole–H), 7.23–7.21 (m, 2H, ArHs), 6.75–6.70 (m, 2H, ArHs), 6.58 (t, ²J_H–F = 49.5 Hz, 1H, CF₂H), 6.48–6.44 (m, 1H, pyrazole–H), 3.88 (br, 2H, NH₂); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 147.4, 139.5, 136.3 (t, ²J_C–F = 30.1 Hz), 129.6, 126.5, 115.0, 108.7 (t, ¹J_C–F = 235.0 Hz), 106.4; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −110.1 (s, 2F).

4.3.8 4-(5-(Difluoromethyl)-3-methyl-1H-pyrazol-1-yl)aniline (9h). Light yellow liquid, yield: 89%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.21–7.19 (m, 2H, ArHs), 6.69–6.67 (m, 2H, ArHs), 6.52 (t, ²J_H–F = 53.9 Hz, 1H, CF₂H), 6.49–6.48 (m, 1H, pyrazole–H), 3.77 (br, 2H, NH₂), 2.35 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 148.8, 147.1, 136.9 (t, ²J_C–F = 29.9 Hz), 129.6, 126.5, 115.0, 108.7 (t, ¹J_C–F = 234.7 Hz), 105.9, 13.4; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −110.1 (s, 2F).

4.3.9 4-(5-(Difluoromethyl)-4-methyl-1H-pyrazol-1-yl)aniline (9i). Light yellow liquid, yield: 89%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.47–7.46 (m, 1H, pyrazole–H), 7.18–7.16 (m, 2H, ArHs), 6.67–6.66 (m, 2H, ArHs), 6.57 (t, ²J_H–F = 53.1 Hz, 1H, CF₂H), 3.92 (br, 2H, NH₂), 2.26 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 147.3, 140.7, 132.4 (t, ²J_C–F = 29.1 Hz), 129.8, 126.6, 117.8, 114.9, 109.7 (t, ¹J_C–F = 233.5 Hz), 8.5; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −111.4 (s, 2F).

4.3.10 4-(5-(Difluoromethyl)-1H-pyrazol-1-yl)-3-fluoroaniline (9j). Light yellow liquid, yield: 90%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.72–7.71 (m, 1H, pyrazole–H), 7.17–7.16 (m, 1H, ArH), 6.57 (t, ²J_H–F = 53.6 Hz, 1H, CF₂H), 6.46–6.44 (m, 2H, ArHs), 6.43–6.40 (m, 1H, pyrazole–H), 4.05 (br, 2H, NH₂); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 157.8 (d, ¹J_C–F = 249.4 Hz), 149.9 (d, ³J_C–F = 10.7 Hz), 140.2, 137.9 (t, ^2′J_C–F = 29.6 Hz), 129.6, 116.3 (d, ^3′J_C–F = 13.0 Hz), 110.5, 108.5 (t, ^1′J_C–F = 237.0 Hz), 106.4, 101.8 (d, ²J_C–F = 22.9 Hz); ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −112.4 (s, 2F), −122.7 (s, 1F).

4.3.11 4-(5-(Difluoromethyl)-3-methyl-1H-pyrazol-1-yl)-3-fluoroaniline (9k). Light yellow liquid, yield: 88%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.12–7.11 (m, 1H, ArH), 6.51 (t, ²J_H–F = 54.3 Hz, 1H, CF₂H), 6.40–6.39 (m, 2H, ArHs), 6.38–6.37 (m, 1H, pyrazole–H), 4.17 (br, 2H, NH₂), 2.33 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 157.8 (d, ¹J_C–F = 249.4 Hz), 158.9, 156.9, 149.7, 138.4 (t, ^2′J_C–F = 29.4 Hz), 116.3 (d, ¹J_C–F = 13.6 Hz), 110.4, 107.7 (t, ^1′J_C–F = 236.6 Hz), 107.0, 101.8 (d, ²J_C–F = 22.9 Hz), 13.4; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −112.4 (s, 2F), −122.7 (s, 1F).

4.3.12 4-(5-(Difluoromethyl)-4-methyl-1H-pyrazol-1-yl)-3-fluoroaniline (9l). Light yellow liquid, yield: 92%. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.58–7.55 (m, 1H, pyrazole–H), 7.53–7.48 (m, 1H, ArH), 6.77 (t, ²J_H–F = 54.3 Hz, 1H, CF₂H), 6.48–6.45 (m, 2H, ArHs), 3.97 (br, 2H, NH₂), 2.25 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 155.2 (d, ¹J_C–F = 247.6 Hz), 147.6 (d, ³J_C–F = 10.6 Hz), 145.1 (t, ^2′J_C–F = 28.6 Hz), 131.4 (d, ⁴J_C–F = 6.1 Hz), 126.0, 119.0 (d, ^3′J_C–F = 10.4 Hz), 116.8, 112.6 (t, ^1′J_C–F = 232.5k Hz), 110.9, 102.3 (d, ²J_C–F = 23.2 Hz), 8.0; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −112.1 (s, 2F), −124.7 (s, 1F).

4.4 General procedure for the preparation of (S)-tert-butyl(2-hydroxy-3-((4-(5-(fluoroalkyl)-1H-pyrazol-1-yl)phenyl)amino)propyl)carbamates 10a–l

The mixture of compound 9 (10 mmol) and compound 3 (3.14 g, 15 mmol) was heated to 150 °C and stirred overnight. After the completion of reaction, the residue was purified by flash column chromatography using petroleum ether/ethyl acetate (8 : 1) as eluent to give the pure products 10a–l.

4.4.1 (S)-tert-Butyl (2-hydroxy-3-((4-(5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)amino)propyl)carbamate (10a). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.67 (br, 1H, NHCO), 7.20–7.19 (m, 1H, pyrazole–H), 7.20–7.17 (m, 2H, ArHs), 6.80–6.70 (m, 2H, ArHs), 6.70–6.60 (m, 1H, pyrazole–H), 5.10–5.09 (m, 1H, Ar–NH) 4.01–4.00 (m, 1H, CH), 3.40–3.21 (m, 4H, 2CH₂), 1.48 (s, 9H, 3CH₃).

4.4.2 (S)-tert-Butyl (2-hydroxy-3-((4-(3-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)amino)propyl)carbamate (10b). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.67 (br, 1H, NHCO), 7.21–7.15 (m, 2H, ArHs), 6.90–6.85 (m, 2H, ArHs), 6.84–6.80 (m, 1H, pyrazole–H), 5.61–5.60 (m, 1H, Ar–NH), 4.00–3.98 (m, 1H, CH), 3.40–3.21 (m, 4H, 2CH₂), 2.37 (s, 3H, pyrazole–CH₃), 1.50 (s, 9H, 3CH₃).

4.4.3 (S)-tert-Butyl(2-hydroxy-3-((4-(4-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)amino)propyl)carbamate (10c). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.67 (br, 1H, NHCO), 7.43–7.40 (m, 1H, pyrazole–H), 7.21–7.15 (m, 2H, ArHs), 6.92–6.83 (m, 2H, ArHs), 6.00–5.98 (m, 1H, Ar–NH), 4.00–3.99 (m, 1H, CH), 3.39–3.18 (m, 4H), 2.26 (s, 3H, pyrazole–CH₃), 1.49 (s, 9H, 3CH₃).

4.4.4 (S)-tert-Butyl(3-((3-fluoro-4-(5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)amino)-2-hydroxypropyl)carbamate (10d). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.69 (br, 1H, NHCO), 7.44–7.41 (m, 1H, pyrazole–H), 7.40–7.37 (m, 1H, ArH), 6.88–6.82 (m, 2H, ArHs), 6.77–6.75 (m, 1H, pyrazole–H), 5.12–5.10 (m, 1H, Ar–NH), 4.04–4.02 (m, 1H, CH), 3.37–3.18 (m, 4H, 2CH₂), 1.49 (s, 9H, 3CH₃).

4.4.5 (S)-tert-Butyl(3-((3-fluoro-4-(3-methyl-5-(trifluoro-methyl)-1H-pyrazol-1-yl)phenyl)amino)-2-hydroxy propyl)carbamate (10e). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.72 (br, 1H, NHCO), 7.33–7.30 (m, 1H, ArH), 6.64–6.60 (m, 2H, ArHs), 6.23–6.20 (m, 1H, pyrazole–H), 4.03–4.01 (m, 1H, CH), 3.39–3.18 (m, 4H, 2CH₂), 2.38 (s, 3H, pyrazole–CH₃), 1.51 (s, 9H, 3CH₃).

4.4.6 (S)-tert-Butyl(3-((3-fluoro-4-(4-methyl-5-(trifluoro-methyl)-1H-pyrazol-1-yl)phenyl)amino)-2-hydroxypropyl)carbamate (10f). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.70 (br, 1H, NHCO), 7.44–7.41 (m, 1H, pyrazole–H), 6.64–6.60 (m, 3H, ArHs), 4.01–3.99 (m, 1H, CH), 3.37–3.14 (m, 4H), 2.27 (s, 3H, pyrazole–CH₃), 1.50 (s, 9H, 3CH₃).

4.4.7 (S)-tert-Butyl(3-((4-(5-(difluoromethyl)-1H-pyrazol-1-yl)phenyl)amino)-2-hydroxypropyl)carbamate (10g). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.70 (br, 1H, NHCO), 7.54–7.52 (m, 1H, pyrazole–H), 7.50–7.48 (m, 2H, ArHs), 6.77–6.68 (m, 2H, ArHs), 6.64–6.62 (m, 1H, pyrazole–H), 6.43 (t, ²J_H–F = 53.4 Hz, 1H, CF₂H), 4.00–3.99 (m, 1H, CH), 3.40–3.25 (m, 4H, 2CH₂), 1.51 (s, 9H, 3CH₃).

4.4.8 (S)-tert-Butyl(3-((4-(5-(difluoromethyl)-3-methyl-1H-pyrazol-1-yl)phenyl)amino)-2-hydroxypropyl)carbamate (10h). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.70 (br, 1H), 7.40–7.32 (m, 2H, ArHs), 6.65–6.63 (m, 2H, ArHs), 6.62–6.60 (m, 1H, pyrazole–H), 6.31 (t, ²J_H–F = 53.8 Hz, 1H, CF₂H), 4.00–3.99 (m, 1H, CH), 3.40–3.25 (m, 4H, 2CH₂), 2.33 (s, 3H, pyrazole–CH₃), 1.48 (s, 9H, 3CH₃).

4.4.9 (S)-tert-Butyl(3-((4-(5-(difluoromethyl)-4-methyl-1H-pyrazol-1-yl)phenyl)amino)-2-hydroxypropyl)carbamate (10i). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.71 (br, 1H, NHCO), 7.15–7.13 (m, 1H, pyrazole–H), 7.10–7.02 (m, 2H, ArHs), 6.67–6.60 (m, 2H, ArHs), 6.44 (t, ²J_H–F = 52.8 Hz, 1H, CF₂H), 4.02–4.01 (m, 1H, CH), 3.45–3.30 (m, 4H, 2CH₂), 2.23 (s, 3H, pyrazole–CH₃), 1.49 (s, 9H, 3CH₃).

4.4.10 (S)-tert-Butyl(3-((4-(5-(difluoromethyl)-1H-pyrazol-1-yl)-3-fluorophenyl)amino)-2-hydroxypropyl)carbamate (10j). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.73 (br, 1H, NHCO), 7.50–7.48 (m, 1H, pyrazole–H), 7.40–7.28 (m, 1H, ArH), 6.72–6.65 (m, 2H, ArHs), 6.62–6.60 (m, 1H, pyrazole–H), 6.42 (t, ²J_H–F = 54.4 Hz, 1H, CF₂H), 4.02–4.00 (m, 1H, CH), 3.44–3.32 (m, 4H, 2CH₂), 1.50 (s, 9H, 3CH₃).

4.4.11 (S)-tert-Butyl(3-((4-(5-(difluoromethyl)-3-methyl-1H-pyrazol-1-yl)-3-fluorophenyl)amino)-2-hydroxypropyl)carbamate (10k). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.69 (br, 1H, NHCO), 7.24–7.16 (m, 2H, ArHs), 6.72–6.64 (m, 1H, ArH), 6.62–6.60 (m, 1H, pyrazole–H), 6.44 (t, ²J_H–F = 54.4 Hz, 1H, CF₂H), 4.00–3.99 (m, 1H, CH), 3.39–3.30 (m, 4H, 2CH₂), 2.30 (s, 3H, pyrazole–CH₃), 1.53 (s, 9H, 3CH₃).

4.4.12 (S)-tert-Butyl(3-((4-(5-(difluoromethyl)-4-methyl-1H-pyrazol-1-yl)-3-fluorophenyl)amino)-2-hydroxypropyl)carbamate (10l). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.67 (br, 1H, NHCO), 7.19–7.16 (m, 1H, pyrazole–H), 7.12–7.08 (m, 1H, ArH), 6.80–6.55 (m, 2H, ArHs), 6.64 (t, ²J_H–F = 54.2 Hz, 1H, CF₂H), 4.00–3.99 (m, 1H, CH), 3.39–3.30 (m, 4H, 2CH₂), 2.20 (s, 3H, pyrazole–CH₃), 1.47 (s, 9H, 3CH₃).

4.5 General procedure for the preparation of (S)-tert-butyl ((2-oxo-3-(4-(5-(fluoroalkyl)-1H-pyrazol-1-yl)phenyl)oxazolidin-5-yl) methyl)carbamates 11a–l

Triethylamine (1.62 g, 16 mmol) and carbonyl diimidazole (CDI) (2.59 g, 16 mmol) was added dropwise to a solution of compound 10 (8 mmol) in THF (50 mL) at room temperature. The mixture was stirred for 14 h. After the completion of reaction, the reaction was quenched with water and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography using petroleum ether/ethyl acetate (4 : 1) as eluent to give the pure products 11a–l.

4.5.1 (S)-tert-Butyl((2-oxo-3-(4-(5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)oxazolidin-5-yl)methyl)carbamate (11a). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.75–7.74 (m, 1H, pyrazole–H), 7.73–7.71 (m, 2H, ArHs), 7.56–7.53 (m, 2H, ArHs), 6.79–6.77 (m, 1H, pyrazole–H), 6.19–6.18 (m, 1H, NH), 4.85–4.84 (m, 1H, CH), 4.10–4.09 (m, 1H, NCH₂–H_a), 3.89–3.86 (m, 1H, NCH₂–H_b), 3.73–3.70 (m, 2H, CH₂), 1.43 (s, 9H, 3CH₃).

4.5.2 (S)-tert-Butyl((3-(4-(3-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11b). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.71–7.59 (m, 4H, ArHs), 6.70 (s, 1H, pyrazole–H), 6.52–6.43 (m, 1H, NH), 4.93–4.90 (m, 1H, CH), 4.09–4.07 (m, 1H, NCH₂–H_a), 3.76–3.69 (m, 1H, NCH₂–H_b), 3.62–3.59 (m, 2H, CH₂), 1.97 (s, 3H, pyrazole–CH₃), 1.47 (s, 9H, 3CH₃).

4.5.3 (S)-tert-Butyl((3-(4-(4-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11c). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.69–7.65 (m, 1H, pyrazole–H), 7.64–7.60 (m, 1H, ArH), 7.32–7.30 (m, 3H, ArHs), 6.86–6.83 (m, 1H, NH), 4.72–4.70 (m, 1H, CH), 4.09–4.01 (m, 1H, NCH₂–H_a), 3.77–3.75 (m, 1H, NCH₂–H_b), 3.60–3.57 (m, 2H, CH₂), 1.99 (s, 3H, pyrazole–CH₃), 1.47 (s, 9H, 3CH₃).

4.5.4 (S)-tert-Butyl((3-(3-fluoro-4-(5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11d). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.69–7.67 (m, 1H, pyrazole–H), 7.65–7.63 (m, 1H, ArH), 7.34–7.30 (m, 2H, ArHs), 6.87–6.86 (m, 1H, pyrazole–H), 6.85–6.84 (m, 1H, NH), 4.74–4.72 (m, 1H, CH), 4.09–4.05 (m, 1H, NCH₂–H_a), 3.84–3.81 (m, 1H, NCH₂–H_b), 3.70–3.65 (m, 2H, CH₂), 1.45 (s, 9H, 3CH₃).

4.5.5 (S)-tert-Butyl((3-(3-fluoro-4-(3-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11e). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.74–7.71 (m, 1H, ArH), 7.54–7.50 (m, 1H, ArH), 7.40–7.37 (m, 1H, ArH), 6.76–6.74 (m, 1H, pyrazole–H), 6.34–6.31 (m, 1H, NH), 4.78–4.75 (m, 1H, CH), 4.09–4.06 (m, 1H, NCH₂–H_a), 3.86–3.84 (m, 1H, NCH₂–H_b), 3.77–3.68 (m, 2H, CH₂), 1.98 (s, 3H, pyrazole–CH₃), 1.49 (s, 9H, 3CH₃).

4.5.6 (S)-tert-Butyl((3-(3-fluoro-4-(4-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11f). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.71–7.68 (m, 1H, pyrazole–H), 7.50–7.48 (m, 1H, ArH), 7.42–7.39 (m, 1H, ArH), 6.78–6.76 (m, 1H, ArH), 5.94–5.92 (m, 1H, NH), 4.24–4.22 (m, 1H, CH), 4.02–4.00 (m, 1H, NCH₂–H_a), 3.79–3.76 (m, 1H, NCH₂–H_b), 3.68–3.65 (m, 2H, CH₂), 1.97 (s, 3H, pyrazole–CH₃), 1.47 (s, 9H, 3CH₃).

4.5.7 (S)-tert-Butyl((3-(4-(5-(difluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11g). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.69–7.67 (m, 1H, pyrazole–H), 7.66–7.60 (m, 2H, ArHs), 7.59–7.56 (m, 2H, ArHs), 6.68–6.66 (m, 1H, pyrazole–H), 6.58 (t, ²J_H–F = 55.7 Hz, 1H, CF₂H), 6.49–6.45 (m, 1H, NH), 4.83–4.80 (m, 1H, CH), 4.04–4.01 (m, 1H, NCH₂–H_a), 3.86–3.84 (m, 1H, NCH₂–H_b), 3.67–3.64 (m, 2H, CH₂), 1.43 (s, 9H, 3CH₃).

4.5.8 (S)-tert-Butyl((3-(4-(5-(difluoromethyl)-3-methyl-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11h). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.70–7.68 (m, 2H, ArHs), 7.51–7.48 (m, 2H, ArHs), 6.86–6.83 (m, 1H, pyrazole–H), 6.50 (t, ²J_H–F = 54.9 Hz, 1H, CF₂H), 6.41–6.39 (m, 1H, NH), 4.77–4.74 (m, 1H, CH), 4.09–4.06 (m, 1H, NCH₂–H_a), 3.78–3.75 (m, 1H, NCH₂–H_b), 3.63–3.61 (m, 2H, CH₂), 1.97 (s, 3H, pyrazole–CH₃), 1.50 (s, 9H, 3CH₃).

4.5.9 (S)-tert-Butyl((3-(4-(5-(difluoromethyl)-3-methyl-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11i). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.69–7.67 (m, 1H, pyrazole–H), 7.58–7.54 (m, 3H, ArHs), 7.10–7.07 (m, 1H, ArH), 6.70 (t, ²J_H–F = 53.7 Hz, 1H, CF₂H), 6.43–6.41 (m, 1H, NH), 4.71–4.68 (m, 1H, CH), 4.11–4.07 (m, 1H, NCH₂–H_a), 3.76–3.73 (m, 1H, NCH₂–H_b), 3.54–3.50 (m, 2H, CH₂), 1.93 (s, 3H, pyrazole–CH₃), 1.46 (s, 9H, 3CH₃).

4.5.10 (S)-tert-Butyl((3-(4-(5-(difluoromethyl)-1H-pyrazol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11j). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.71–7.70 (m, 1H, pyrazole–H), 7.69–7.68 (m, 1H, ArH), 7.53–7.35 (m, 2H, ArHs), 6.77–6.75 (m, 1H, pyrazole–H), 6.68 (t, ²J_H–F = 54.6 Hz, 1H, CF₂H), 6.55–6.53 (m, 1H, NH), 4.76–4.72 (m, 1H, CH), 4.09–4.07 (m, 1H, NCH₂–H_a), 3.78–3.74 (m, 1H, NCH₂–H_b), 3.61–3.59 (m, 2H, CH₂), 1.48 (s, 9H, 3CH₃).

4.5.11 (S)-tert-Butyl((3-(4-(5-(difluoromethyl)-3-methyl-1H-pyrazol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11k). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.69–7.37 (m, 3H, ArHs), 6.75–6.73 (m, 1H, pyrazole–H), 6.65 (t, ²J_H–F = 54.8 Hz, 1H, CF₂H), 6.57–6.53 (m, 1H, NH), 4.89–4.85 (m, 1H, CH), 4.11–4.09 (m, 1H, NCH₂–H_a), 3.78–3.76 (m, 1H, NCH₂–H_b), 3.59–3.55 (m, 2H, CH₂), 1.97 (s, 3H, pyrazole–CH₃), 1.51 (s, 9H, 3CH₃).

4.5.12 (S)-tert-Butyl((3-(4-(5-(difluoromethyl)-4-methyl-1H-pyrazol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11l). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.77–7.75 (m, 1H, pyrazole–H), 7.74–7.70 (m, 2H, ArHs), 7.30–7.29 (m, 1H, ArH), 6.97–6.95 (m, 1H, NH), 6.83 (t, ²J_H–F = 53.6 Hz, 1H, CF₂H), 4.90–4.88 (m, 1H, CH), 4.06–4.03 (m, 1H, NCH₂–H_a), 3.76–3.73 (m, 1H, NCH₂–H_b), 3.65–3.62 (m, 2H, CH₂), 1.96 (s, 3H, pyrazole–CH₃), 1.49 (s, 9H, 3CH₃).

4.6 General procedure for the preparation of (S)-N-((2-oxo-3-(4-(5-(fluoroalkyl)-1H-pyrazol-1-yl)phenyl)oxazolidin-5-yl)methyl)acetamides 13a–l

To a solution of compound 11 (6 mmol) in CH₂Cl₂ (50 mL) was added dropwise trifluoroacetic acid (1.37 g, 12 mmol). The mixture was stirred for 12 h at room temperature. The solvent was removed in reduced pressure to provide the crude products 12a–l, which were used for next step without further purification.

Acetic anhydride (1.23 g, 12 mmol) was added dropwise to a mixture of compound 12 (6 mmol) in CH₂Cl₂ (50 mL) and triethylamine (1.82 g, 18 mmol). The resulting solution was stirred for 12 h at room temperature. After the completion of reaction, the reaction was quenched with water and extracted with CH₂Cl₂ (3 × 50 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography using ethyl acetate as eluent to give the pure target products 13a–l.

4.6.1 (S)-N-((2-Oxo-3-(4-(5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)oxazolidin-5-yl)methyl)acetamide (13a). Light yellow solid, yield: 85%, mp: 145.2–147.8 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.72–7.71 (m, 1H, pyrazole–H), 7.70–7.68 (m, 2H, ArHs), 7.53–7.51 (m, 2H, ArHs), 6.83–6.82 (m, 1H, pyrazole–H), 6.21–6.20 (m, 1H, NH), 4.84–4.83 (m, 1H, CH), 4.16–4.12 (m, 1H, NCH₂–H_a), 3.90–3.87 (m, 1H, NCH₂–H_b), 3.76–3.61 (m, 2H, CH₂), 2.05 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 171.2, 154.3, 139.7, 138.8, 135.0, 132.6, 126.5, 119.9 (q, ¹J_C–F = 268.3 Hz), 118.3, 109.0, 72.1, 47.5, 41.9, 23.1; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.8 (s, 3F). HRMS calcd for C₁₆H₁₅F₃N₄O₃Na [M + Na]⁺: 391.0994, found: 391.0999.

4.6.2 (S)-N-((3-(4-(3-Methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13b). Light yellow solid, yield: 83%, mp: 148.4–150.2 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.66–7.48 (m, 4H, ArHs), 6.61 (s, 1H, pyrazole–H), 6.41–6.32 (m, 1H, NH), 4.83–4.82 (m, 1H, CH), 4.14–4.10 (m, 1H, NCH₂–H_a), 3.88–3.85 (m, 1H, NCH₂–H_b), 3.71–3.63 (m, 2H, CH₂), 2.37 (s, 3H, pyrazole–CH₃), 2.04 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 171.2, 154.3, 149.1, 138.5, 135.1, 133.1 (q, ²J_C–F = 37.8 Hz), 126.1, 119.8 (q, ¹J_C–F = 268.9 Hz), 118.2, 108.7, 72.1, 47.2, 41.9, 23.0, 13.3; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.8 (s, 3F). HRMS calcd for C₃₄H₃₄F₆N₈O₆Na [2M + Na]⁺: 787.2403, found: 787.2412.

4.6.3 (S)-N-((3-(4-(4-Methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13c). Off-white solid, yield: 82%, mp: 179.4–180.9 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.66–7.64 (m, 1H, pyrazole–H), 7.63–7.62 (m, 1H, ArH), 7.44–7.42 (m, 3H, ArHs), 6.75–6.73 (m, 1H, NH), 4.81–4.80 (m, 1H, CH), 4.12–4.08 (m, 1H, NCH₂–H_a), 3.88–3.85 (m, 1H, NCH₂–H_b), 3.69–3.60 (m, 2H, CH₂), 2.26 (s, 3H, pyrazole–CH₃), 2.02 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 171.3, 154.4, 141.4, 138.6, 135.6, 129.2 (q, ²J_C–F = 37.6 Hz), 126.7, 120.7 (q, ¹J_C–F = 269.4 Hz), 119.9, 118.2, 72.1, 47.5, 41.9, 23.0, 9.0; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.8 (s, 3F). HRMS calcd for C₁₇H₁₇F₃N₄O₃Na [M + Na]⁺: 405.1150, found: 405.1149.

4.6.4 (S)-N-((3-(3-Fluoro-4-(5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13d). Off-white solid, yield: 80%, mp: 165.1–167.3 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.74–7.71 (m, 1H, pyrazole–H), 7.70–7.67 (m, 1H, ArH), 7.44–7.30 (m, 2H, ArHs), 6.88–6.86 (m, 1H, pyrazole–H), 6.84–6.82 (m, 1H, NH), 4.84–4.82 (m, 1H, CH), 4.13–4.09 (m, 1H, NCH₂–H_a), 3.88–3.85 (m, 1H, NCH₂–H_b), 3.68–3.63 (m, 2H, CH₂), 2.01 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 171.5, 158.9 (d, ¹J_C–F = 253.3 Hz), 154.2, 141.0 (d, ³J_C–F = 10.6 Hz), 140.3, 134.1 (q, ^2′J_C–F = 38.8 Hz), 129.7, 122.1 (d, ^3′′J_C–F = 13.2 Hz), 119.6 (q, ^1′J_C–F = 268.8 Hz), 113.0, 108.5, 106.3 (d, ²J_C–F = 25.4 Hz), 72.3, 47.4, 41.8, 22.9; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.8 (s, 3F), −118.8 (s, 1F). HRMS calcd for C₁₆H₁₄F₄N₄O₃Na [M + Na]⁺: 409.0900, found: 409.0913.

4.6.5 (S)-N-((3-(3-Fluoro-4-(3-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13e). Off-white solid, yield: 87%, mp: 153.5–155.2 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.71–7.68 (m, 1H, ArH), 7.45–7.42 (m, 1H, ArH), 7.33–7.30 (m, 1H, ArH), 6.64–6.60 (m, 1H, pyrazole–H), 6.23–6.20 (m, 1H, NH), 4.85–4.84 (m, 1H, CH), 4.13–4.10 (m, 1H, NCH₂–H_a), 3.88–3.84 (m, 1H, NCH₂–H_b), 3.76–3.65 (m, 2H, CH₂), 2.38 (s, 3H, pyrazole–CH₃), 2.05 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 171.2, 158.0 (d, ¹J_C–F = 253.4 Hz), 153.9, 149.8, 140.7 (d, ³J_C–F = 10.5 Hz), 134.6 (q, ^2′J_C–F = 39.1 Hz), 129.8, 122.4 (d, ^3′J_C–F = 12.5 Hz), 119.5 (q, ^1′J_C–F = 269.6 Hz), 112.8, 108.2, 106.3 (d, ²J_C–F = 25.6 Hz), 72.1, 47.4, 41.9, 23.1, 13.4; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.8 (s, 3F), −118.6 (s, 1F). HRMS calcd for C₁₇H₁₆F₄N₄O₃Na [M + Na]⁺: 423.1056, found: 423.1055.

4.6.6 (S)-N-((3-(3-Fluoro-4-(4-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13f). Off-white solid, yield: 83%, mp: 160.3–162.2 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.69–7.66 (m, 1H, pyrazole–H), 7.65–7.28 (m, 3H, ArHs), 6.65–6.61 (m, 1H, NH), 4.85–4.83 (m, 1H, CH), 4.13–4.09 (m, 1H, NCH₂–H_a), 3.88–3.84 (m, 1H, NCH₂–H_b), 3.68–3.65 (m, 2H, CH₂), 2.36 (s, 3H, pyrazole–CH₃), 2.03 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 171.4, 158.0 (d, ¹J_C–F = 253.1 Hz), 154.1, 149.8, 142.0, 140.7 (d, ³J_C–F = 11.6 Hz), 134.6 (q, ^2′J_C–F = 38.8 Hz), 129.7 (d, ^3′J_C–F = 11.4 Hz), 120.5 (q, ^1′J_C–F = 269.2 Hz), 112.9, 108.2, 106.3 (d, ²J_C–F = 25.6 Hz), 72.2, 47.4, 41.9, 23.0, 13.4; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −62.8 (s, 3F), −118.6 (s, 1F). HRMS calcd for C₁₇H₁₆F₄N₄O₃Na [M + Na]⁺: 423.1056, found: 423.1055.

4.6.7 (S)-N-((3-(4-(5-(Difluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13g). Light yellow solid, yield: 82%, mp: 154.4–154.9 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.73–7.71 (m, 1H, pyrazole–H), 7.70–7.68 (m, 2H, ArHs), 7.55–7.51 (m, 2H, ArHs), 6.77–6.72 (m, 1H, pyrazole–H), 6.64 (t, ²J_H–F = 53.4 Hz, 1H, CF₂H), 6.59–6.55 (m, 1H, NH), 4.86–4.80 (m, 1H, CH), 4.14–4.10 (m, 1H, NCH₂–H_a), 3.90–3.86 (m, 1H, NCH₂–H_b), 3.70–3.67 (m, 2H, CH₂), 2.04 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 171.3, 154.4, 140.2, 138.4, 136.3 (t, ²J_C–F = 29.7 Hz), 134.9, 125.8, 118.7, 108.5 (t, ¹J_C–F = 236.1 Hz), 107.6, 72.2, 47.6, 42.0, 23.1; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −109.9 to −109.8 (m, 2F). HRMS calcd for C₁₆H₁₆F₂N₄O₃Na [M + Na]⁺: 373.1088, found: 373.1098.

4.6.8 (S)-N-((3-(4-(5-(Difluoromethyl)-3-methyl-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13h). Off-white solid, yield: 78%, mp: 148.0–149.3 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.64–7.63 (m, 2H, ArHs), 7.49–7.47 (m, 2H, ArHs), 6.75–6.72 (m, 1H, pyrazole–H), 6.57 (t, ²J_H–F = 53.8 Hz, 1H, CF₂H), 6.53–6.51 (m, 1H, NH), 4.83–4.80 (m, 1H, CH), 4.11–4.08 (m, 1H, NCH₂–H_a), 3.88–3.85 (m, 1H, NCH₂–H_b), 3.68–3.64 (m, 2H, CH₂), 2.35 (s, 3H, pyrazole–CH₃), 2.02 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 171.3, 154.4, 149.6, 138.1, 136.7 (t, ²J_C–F = 29.2 Hz), 135.0, 125.6, 118.7, 108.4 (t, ¹J_C–F = 235.5 Hz), 107.3, 72.1, 47.5, 41.9, 23.0, 13.4; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −109.9 to −109.8 (m, 2F). HRMS calcd for C₁₇H₁₈F₂N₄O₃Na [M + Na]⁺: 387.1245, found: 387.1249.

4.6.9 (S)-N-((3-(4-(5-(Difluoromethyl)-4-methyl-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13i). Off-white solid, yield: 83%, mp: 156.3–158.6 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.63–7.62 (m, 1H, pyrazole–H), 7.62–7.61 (m, 1H, ArH), 7.48–7.43 (m, 3H, ArHs), 7.06–7.02 (m, 1H, NH), 6.59 (t, ²J_H–F = 52.8 Hz, 1H, CF₂H), 4.80–4.76 (m, 1H, CH), 4.09–4.05 (m, 1H, NCH₂–H_a), 3.85–3.83 (m, 1H, NCH₂–H_b), 3.64–3.60 (m, 2H, CH₂), 2.23 (s, 3H, pyrazole–CH₃), 1.98 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 171.5, 154.5, 141.4, 138.3, 135.1, 132.2 (t, ²J_C–F = 28.2 Hz), 125.9, 118.9, 118.7, 109.2 (t, ¹J_C–F = 235.5 Hz), 72.2, 47.5, 41.9, 22.9, 8.4; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −111.0 (s, 2F). HRMS calcd for C₃₄H₃₆F₄N₈O₆Na [2M + Na]⁺: 751.2592, found: 751.2590.

4.6.10 (S)-N-((3-(4-(5-(Difluoromethyl)-1H-pyrazol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13j). Light yellow solid, yield: 79%, mp: 149.5–152.2 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.74–7.73 (m, 1H, pyrazole–H), 7.72–7.70 (m, 1H, ArH), 7.50–7.28 (m, 2H, ArHs), 6.72–6.71 (m, 1H, pyrazole–H), 6.70–6.68 (m, 1H, NH), 6.59 (t, ²J_H–F = 54.4 Hz, 1H, CF₂H), 4.86–4.82 (m, 1H, CH), 4.13–4.11 (m, 1H, NCH₂–H_a), 3.89–3.85 (m, 1H, NCH₂–H_b), 3.70–3.66 (m, 2H, CH₂), 2.03 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 171.4, 156.8 (d, ¹J_C–F = 251.5 Hz), 154.1, 140.8, 140.4 (d, ³J_C–F = 10.5 Hz), 137.8 (t, ^2′J_C–F = 27.8 Hz), 129.3, 122.4 (d, ^3′J_C–F = 12.9 Hz), 113.4, 108.5 (t, ^1′J_C–F = 237.0 Hz), 107.3, 106.4 (d, ²J_C–F = 25.4 Hz), 72.2, 47.4, 41.8, 23.0; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −112.6 (s, 2F), −119.9 (s, 1F). HRMS calcd for C₁₆H₁₅F₃N₄O₃Na [M + Na]⁺: 391.0994, found: 391.1010.

4.6.11 (S)-N-((3-(4-(5-(Difluoromethyl)-3-methyl-1H-pyrazol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13k). Light yellow solid, yield: 81%, mp: 151.3–152.7 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.64–7.27 (m, 3H, ArHs), 7.26–7.24 (m, 1H, pyrazole–H), 6.54 (t, ²J_H–F = 54.4 Hz, 1H, CF₂H), 6.47–6.42 (m, 1H, NH), 4.80–4.76 (m, 1H, CH), 4.08–4.05 (m, 1H, NCH₂–H_a), 3.83–3.78 (m, 1H, NCH₂–H_b), 3.62–3.57 (m, 2H, CH₂), 2.29 (s, 3H, pyrazole–CH₃), 1.96 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 172.1, 156.8 (d, ¹J_C–F = 251.0 Hz), 154.3, 150.2, 140.2 (d, ³J_C–F = 9.9 Hz), 138.3 (t, ^2′J_C–F = 28.9 Hz), 129.3, 122.4 (d, ^3′J_C–F = 12.6 Hz), 113.4, 108.5 (t, ^1′J_C–F = 236.6 Hz), 107.0, 106.3 (d, ²J_C–F = 26.0 Hz), 72.2, 47.4, 41.8, 22.7, 13.3; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −112.7 (s, 2F), −120.1 (s, 1F). HRMS calcd for C₁₇H₁₇F₃N₄O₃Na [M + Na]⁺: 405.1150, found: 405.1166.

4.6.12 (S)-N-((3-(4-(5-(Difluoromethyl)-4-methyl-1H-pyrazol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13l). Light yellow solid, yield: 80%, mp: 163.2–165.9 °C. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.79–7.77 (m, 1H, pyrazole–H), 7.76–7.64 (m, 2H, ArHs), 7.21–7.19 (m, 1H, ArH), 6.84–6.83 (m, 1H, NH), 6.74 (t, ²J_H–F = 54.2 Hz, 1H, CF₂H), 4.86–4.82 (m, 1H, CH), 4.10–4.06 (m, 1H, NCH₂–H_a), 3.86–3.83 (m, 1H, NCH₂–H_b), 3.70–3.68 (m, 2H, CH₂), 2.24 (s, 3H, pyrazole–CH₃), 2.03 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) 171.5, 154.3, 153.4 (d, ¹J_C–F = 248.4 Hz), 145.9 (t, ^2′J_C–F = 29.2 Hz), 137.7 (d, ³J_C–F = 10.1 Hz), 131.2 (d, ⁴J_C–F = 9.7 Hz), 124.4, 123.8 (d, ^3′J_C–F = 9.8 Hz), 118.6, 113.7, 112.3 (t, ^1′J_C–F = 237.0 Hz), 106.8 (d, ²J_C–F = 26.5 Hz), 72.2, 47.4, 41.8, 23.0, 8.0; ¹⁹F NMR (471 MHz, CDCl₃) δ (ppm) −112.6 (s, 2F), −122.4 (s, 1F). HRMS calcd for C₁₇H₁₇F₃N₄O₃Na [M + Na]⁺: 405.1150, found: 405.1167.

4.7 Antibacterial activity assay

The antibacterial activities of the target compounds were tested against six Gram-positive bacterial strains using linezolid as a positive control. The strains were isolated and identified by Jiangsu Province People's Hospital and kept in this laboratory at −80 °C. In vitro activities of the compounds were tested in nutrient broth (NB) for bacteria by two-fold serial agar dilution method using the method recommended by National Committee for Clinical Laboratory Standards (NCCLS). Compounds were dissolved in 50% water in DMSO to prepare a stock solution that had a concentration of 320 μg mL⁻¹. Serial 2-fold dilutions were prepared from the stock solution with sterile water and then 10-fold diluted with Mueller-Hinton (MH) agar medium to provide concentration ranges of 16–0.0312 μg mL⁻¹. The tested organisms were grown in MH broth medium (containing 5% sheep serum) at 35 °C for 8 h and were adjusted to the turbidity of the 0.5 McFarland standard to a concentration of 10⁸ CFU mL⁻¹. The bacterial suspensions were inoculated onto the drug-supplemented MH agar plates with a multipoint inoculator and incubated at 35 °C for 16–20 h. The minimum inhibitory concentrations (MIC) values were determined at the end of the incubation period. The lowest concentration of the compound that prevented visible growth was considered as MIC.

4.8 Molecule docking

4.8.1 Energy minimization. The conformations of compounds 13b, 13f, 13l, 13k and linezolid were optimized using the minimize module of SYBYL-X 2.1.²⁶ The force field was calculated with tripos field and the atom charges were also calculated using Gasteiger–Huckel method.²⁷

4.8.2 Docking calculations. The docking studies of compounds 13b, 13f, 13l, 13k and linezolid were performed by the Surflex-dock method.²⁵ The three compounds were docked into the Deinococcus radiodurans 50S ribosomal subunit crystal structure (PDB ID: 3DLL) by a posteriori scoring function in Surflex-dock. Before the docking program was performed, the ligand of the crystal structure was used to build the protomol, which is the representation of a ligand making every potential interaction with the binding pocket. In this paper, the protomol of SYBYL was built by automatic docking and other setups were default setting up. The binding affinity was characterized by the total-scores and expressed in −lg K_d unit.

Acknowledgements

We are grateful for financial supports from the National Natural Science Foundation of China (Grant No. 21472126, 21172148, 21302128).

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Footnote

† Electronic supplementary information (ESI) available: Copies of ¹H NMR, ¹³C NMR, ¹⁹F NMR and mass spectra of the target compounds can be found as supplementary material. See DOI: 10.1039/c5ra11782h

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