Structure–Uptake Relationship Studies of Oxazolidinones in Gram-Negative ESKAPE Pathogens

Abstract

The clinical success of linezolid for treating Grampositive infections paired with the high conservation of bacterial ribosomes predicts that if oxazolidinones were engineered to accumulate in Gram-negative bacteria, then this pharmacological class would find broad utility in eradicating infections. Here, we report an investigative study of a strategically designed library of oxazolidinones to determine the effects of molecular structure on accumulation and biological activity. Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa strains with varying degrees of compromise (in efflux and outer membrane) were used to identify motifs that hinder permeation across the outer membrane and/or enhance efflux susceptibility broadly and specifically between species. The results illustrate that small changes in molecular structure are enough to overcome the efflux and/or permeation issues of this scaffold. Three oxazolidinone analogues (3e, 8d, and 8o) were identified that exhibit activity against all three pathogens assessed, a biological profile not observed for linezolid.

Graphical Abstract

INTRODUCTION

The emergence and widespread prevalence of multidrug-resistant (MDR) bacteria pose a great threat to society.^1,2 A 2019 report from the Centers for Disease Control and Prevention estimates that >2.8 million MDR infections occur annually in the United States, giving rise to >35,000 deaths.³ MDR Gram-negative bacteria (GNB) have been pinpointed as the most urgent threats.^1,4,5 In fact, four (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) of the six leading contributors to hospital-acquired infections are GNB.⁶

It is well established that the encapsulation of the hydrophobic inner membrane by an asymmetric outer membrane (OM) presents a formidable barrier to small-molecule permeation in GNB. Molecules must either be amphipathic enough to permeate these antithetic membranes or capitalize on porin-mediated entry. Additionally, after a compound successfully crosses the outer or both membranes, it is subject to highly promiscuous efflux pumps.^7,8 As a result, while potent biochemical inhibitors can often be identified for new targets, developing them into compounds with whole-cell antibacterial activity has proven challenging.^9–11

Two approaches are typically taken to enhance the accumulation of molecules in GNB: (1) cotreatment with OM permeabilizing agents or (2) strategic structural modification. Sensitization of the OM upon cotreatment with permeabilizing agents (e.g., polymyxin B nonapeptide (PMBN)) provides a workaround for OM permeation issues.¹² Unfortunately, these permeabilization agents fail to address efflux issues and have demonstrated dose-limiting toxicities in the clinic. However, new PMBN analogues (e.g., NAB7061, SPR741/NAB741)¹² and permeabilizing chemotypes (e.g., tridecaptin A₁, teixobactin)^13,14 continue to provide inspiration for interrogating this strategy to broaden the spectrum of select agents. The second and more direct approach to improving compound accumulation is to rationally engineer the antibacterial molecule itself to render it capable of membrane penetration and efflux avoidance. Our poor understanding, however, of the structural features that correlate with small-molecule GNB permeation and accumulation persists as a major roadblock to this approach.

Retrospective efforts to summarize the physicochemical properties that coincide with Gram-negative activity^9,11,15 have inspired initial efforts to enhance Gram-negative compatibility of compounds in a more prospective manner. In fact, predictive models for chemical modification to impart activity against Escherichia coli through porin-mediated interactions have been achieved^16,17 and employed for diverse chemotypes.^18–26 For the deoxynybomycins and Debio-1452 series, lessons learned from E. coli have even proven useful in gaining antibacterial activity against A. baumannii and K. pneumoniae.^17,26 However, although the basic envelope composition is relatively conserved across GNB, significant variation exists between species and strains,²⁷ which often limits the generalization and extension of physicochemical predictors determined for E. coli permeation to other pathogens. As such, it is crucial to analyze compound behavior across not only species but also strains to gain insight into generally applicable “rules” versus those that are more organism-specific for each chemotype.

While novel antibacterial scaffolds are desperately needed, critical information remains to be learned from well-interrogated compound families, as the technology now exists to deconvolute permeation and efflux liabilities that lacked during the original period of chemotype development. Oxazolidinones, for example, were rationally developed in the 1990s as a new class of synthetic antibiotics that exert their activity by binding to the 23S rRNA of the 50S ribosomal subunit and inhibiting the initiation step of bacterial protein synthesis.²⁸ The clinical success of linezolid (LZD; Figure 1), yet susceptibility to resistance, has inspired pharmaceutical companies and academic groups to develop next-generation oxazolidinones.^29–31 Thus far, studies have focused mainly on overcoming target-driven LZD resistance,^32–34 exploring the utility in infections of the central nervous system,³⁵ and reducing toxic side effects.³⁶ However, the potential of this chemotype for broad-spectrum activity has been underexplored due to the Gram-positive specific activity and inability, thus far, to rationally design GNB-active derivatives.^37–40 High conservation of bacterial ribosomes, however, predicts that if oxazolidinones were engineered to accumulate in GNB, then this pharmacological class would find great utility in eradicating infections.⁴⁰ These reasons, paired with the knowledge that the biological activity of this chemotype can be maintained through broad C-ring diversification (Figure 1), establish the oxazolidinones as a seminal class to interrogate the effect of motif variation on GNB accumulation.

Figure 1.

Structure of linezolid and general SAR features of the oxazolidinone chemotype.

To expand the understanding of how specific motifs influence GNB accumulation and efflux susceptibility, we report the design, synthesis, and evaluation of oxazolidinones distinctly functionalized at the C-ring. Activity against wild-type E. coli, P. aeruginosa, and A. baumannii, and corresponding strains with varying degrees of the outer membrane and/or efflux pump efficiencies, was employed to allow for the deconvolution of structure–activity and structure–uptake relationships (SAR and SURs). Assessment in a ribosomal translation inhibition assay and against Staphylococcus aureus and LZD-resistant E. coli was used to verify the on-target activity of representative analogues. Motifs that prove problematic to OM permeation and/or efflux for each species were revealed. Three LZD analogues were identified that exhibit broad-spectrum activity against all three Gram-negative pathogens.

RESULTS AND DISCUSSION

Molecular Design.

An oxazolidinone A-ring containing an (S) C-5 methylene-linked acetamide and an N-aryl substituent (B-ring) comprises the bulk of the scaffold required for target engagement (Figure 1). A third ring system (C-ring) is appended to the para-position of the B-ring. Previous studies clearly demonstrate that the C-ring is the most amenable to modification and that the C-5 acetamide can be replaced with a more synthetically tractable hydroxyl group (Figure 1).⁴¹ As such, we aimed to design a library capable of leveraging known chemistry to provide common intermediates that could be exposed to reactants exhibiting diverse physicochemical attributes.

The design of the library was dictated by four factors: (1) the chemistry accessible from key synthetic intermediates, (2) reactant availability, (3) reagent compatibility with other libraries of interest, and (4) clustering-driven reagent selection. To begin the library design (Figure S1), we decided to use a single vendor, Enamine, which maintains a catalog of ~210 million building blocks. To narrow the focus, the catalog was filtered to provide building blocks with ≥250 mg of availability. These building blocks were then classified based on the molecular functionality present (e.g., amines, boronates, carboxylic acids, acid chlorides, alkyl halides, etc.), helping to identify available chemistries. Within these classifications, reagents with heavy atom counts ≥15 were removed to provide an inclusive list of reagents available for library generation. Because the targeted key intermediates (2a, 6, and 7; Scheme 1) used to synthesize the library congeners would be an aryl halide (or boronic acid/ester), we settled on Suzuki–Miyaura- and Sonogashira–Hagihara-compatible reactants and filtered the reagents to eliminate any noncompatible compositions. This narrowed the reagent classifications to alkynes, aryl boronic acids/esters, and aryl bromides. The remaining reagents were filtered to exclude structural alerts. At this point, protecting group compatibility was also considered for additional libraries we were targeting. Lastly, reagents were merged and clustered based on fingerprints comprised of the A log P atom type, with clusters being defined as a group of compounds exhibiting a ≥0.6 Tanimoto score. Samples within each cluster were sorted by increasing the size, and the smallest reagent was generally selected to represent each cluster. If, for any reason, the top priority compound was unavailable or otherwise triaged, the next smallest reagent within a cluster was chosen as the representative.

Scheme 1. General Synthetic Approach for Substituted Phenyl C-Ring Derivatives 3a–x^a.

aReagents and reaction conditions: (i) for 1a: benzyl chloroformate, NaHCO₃, tetrahydrofuran (THF), 25 °C, 16 h, 91%; for 1b: benzyl chloroformate, NaHCO₃, THF, 0 °C, 3 h, 99%; (ii) (R)-(–)-glycidyl butyrate, LHMDS (1 M in THF), THF, −78 °C to 25 °C, N₂, 16–20 h, 70–76%; (iii) aryl boronic acid/ester or aryl bromide, Pd(dppf)CI₂·dichloromethane (DCM), K₂CO₃, dioxane/H₂O (v/v = 9:1), 90 °C, N₂, 3 h, 11–98%; (iv) N,N′-di-Boc-1H-pyrazole-1-carboxamidine, N,N-diisopropylethylamine (DIPEA), THF, 60 °C, 3 h, 26%; (v) HCI/dioxane (4 M), 50 °C, 30 min, 56%; (vi) methyl chloromethyl ether, DIPEA, DCM, 25 °C, 5 h, 85%; (vii) HCI/dioxane (4 M), 25 °C, 1 h, 50–100%; (viii) N-iodosuccinimide, trifluoroacetic acid (TFA), 25 °C, 2 h, 77%; (ix) bis(pinacolato)diboron, Pd(dppf)CI₂·DCM, KOAc, DMSO, 80 °C, N₂,16 h, 49%; and (x) HCI/dioxane (4 M), 25 °C, 30 min, 91%.

Synthesis.

As mentioned, reagents resulting from the design workflow were amenable to Suzuki–Miyaura or Sonogashira–Hagihara coupling conditions to provide aryl–aryl- or alkynyl-linked compounds, respectively. For ease of discussion, we have classified the library into four series: phenyl, pyridyl, pyridinyl, and alkynyl (Figure 2).

Figure 2.

Subgroups represented within the library.

Phenyl Series.

Analogues 3a–x were synthesized according to Scheme 1. Treatment of 4-bromo-3-fluoroaniline or 3-fluoroaniline with benzyl chloroformate under mildly basic conditions afforded intermediate 1, which reacted with (R)-(–)-glycidyl butyrate to generate the corresponding oxazolidinone 2. With the exception of 3f, analogues 3a–o were obtained following a Suzuki cross-coupling between 2a and various aryl boronic acids/esters. Compound 3f was obtained by guanidinylation of compound 3e followed by deprotection of the resulting di-Bocguanidine to reveal the free guanidinium salt.

To synthesize analogues 3p–r, the hydroxy group of 2 was protected prior to the Suzuki coupling for purification purposes. Briefly, the primary hydroxy group of oxazolidinone 2a was protected as the methoxymethyl ether to give 4, which was coupled with select aryl boronic acids to provide the protected products 5a–c. Deprotection of 5a–c under typical acidic conditions yielded analogues 3p–r, respectively. To obtain analogues 3s–x, commercially available aryl bromides were utilized as the Suzuki coupling partner instead of aryl boronic acids/esters. Regioselective iodination of 2b by treatment with N-iodosuccinimide provided intermediate 6, which was subsequently transformed into boronic ester 7 through a palladium-catalyzed borylation reaction. Suzuki coupling between boronic ester 7 and select aryl bromides yielded compounds 3s–w. Removal of the Boc protecting group on 3w under acidic conditions yielded analogue 3x.

Pyridyl Series.

The general synthetic approach for pyridyl C-ring derivatives 8a–o is presented in Scheme 2. Analogues 8a,b were obtained following a Suzuki cross-coupling between 2a and the requisite boronic esters. Saponification of compound 8b generated carboxylic acid 8c. Suzuki coupling between boronic ester 7 and select aryl bromides yielded compounds 8d–m. Compound 8n was generated through a modified Suzuki reaction from the iodo-oxazolidinone 6. Additionally, the pendant hydroxyl group on 7 could be protected as the methoxymethyl ether to afford 9, which upon exposure to typical Suzuki conditions yielded the cyano pyridine 10. Attempts to isolate the free amine 8o after nitrile reduction proved problematic. Thus, we resorted to a strategy of trapping the free amine as the t-butylcarbamate for purification purposes. Once isolated, the resulting compound could be exposed to acidic conditions to simultaneously cleave the carbamate, methoxymethyl ether, and t-butyl group to reveal compound 8o.

Scheme 2. General Synthetic Approach for Substituted Pyridyl C-Ring Derivatives 8a–o^a.

aReagents and reaction conditions: (i) aryl boronic acid/ester or aryl bromide, Pd(dppf)CI₂·DCM, K₂CO₃, dioxane/H₂O (v/v = 9:1), 90 °C, N₂, 3 h, 38–90%; for compound 8g, 2-amino-5-iodopyrimidine, Pd(dppf)CI₂·DCM, Na₂CO₃, dioxane/H₂O (v/v = 9:1), 130 °C, N₂, 2.5 h, 20%; (ii) LiOH·H₂O, THF/MeOH/H₂O (v/v/v = 3:1:1), 25 °C, 1 h, 56%; (iii) isoxazole-4-ylboronic acid, Pd(dppf)CI₂·DCM, K₃PO₄·H₂O, THF/H₂O (v/v = 4:1), 80 °C, 16 h, 50%; (iv) methyl chloromethyl ether, DIPEA, DCM, 25 °C, 1 h, 58%; (v) (a) NiCI₂·6H₂O, NaBH₄, Boc₂O, MeOH, 25 °C, 16 h, 52%; and (b) TFA, DCM, 40 °C, 1 h, 66%.

Pyridinium Series.

The general synthetic route for substituted pyridinium C-ring derivatives 12a–1 is shown in Scheme 3. A Suzuki reaction between intermediate 9 and a corresponding heterocyclic bromide provided 11a–h, which were then reacted with a haloalkane of choice to form the N-alkylated pyridiniums. Removal of the methoxymethyl ether afforded final compounds 12a–1.

Scheme 3. General Synthetic Approach for Substituted Pyridinium C-Ring Derivatives 12a–l^a.

aReagents and reaction conditions: (i) aryl bromide, Pd(dppf)CI₂·DCM, K₂CO₃, dioxane/H₂O (v/v = 9:1), 90 °C, N₂, 3 h, 73–90%; (ii) (a) haloalkane, CH₃CN, 80 °C, 24 h; and (b) HCI/dioxane (4 M), 25 °C, 1 h, 14–65%.

Alkynyl Series.

For the last cohort of analogues, substituted alkynyl derivatives 13 were generated from 6 through a Sonogashira reaction with select coupling partners, as depicted in Scheme 4. In many cases, the resulting products were manipulated further to afford additional derivatives. For example, saponification of 13m provided alcohol 13n and hydrochloride salts 13t, 13v, 13x, 13z, and 13ab were obtained after Boc removal from 13s, 13u, 13w, 13y, and 13aa with trifluoroacetic acid followed by salt exchange, respectively.

Scheme 4. General Synthetic Approach for Substituted Akynyl Derivatives 13a–ab^a.

aReagents and reaction conditions: (i) alkyne, Pd(PPh₃)₄, Cul, DIPEA, N,N-dimethyl formamide (DMF), N₂, 25 °C, 16 h, 22–89%; (ii) LiOH·H₂O, THF/MeOH/H₂O (v/v/v = 3:1:1), 25 °C, 1 h, 47%; and (iii) TFA, DCM, 25 °C, 1 h, 40–91%.

Overview of Library Antibacterial Activity.

We next analyzed the activities of each compound series in three GNB, E. coli, P. aeruginosa, and A. baumannii. For all three species, wild-type and isogenic mutants lacking efflux pumps (Δ) and/or producing a large recombinant OM pore (Pore) were used to separate the contributions of active efflux and the OM permeability barriers on the activities of compounds.⁴² The MIC (Table S2) and IC₅₀ values (Tables 1–4) were determined first in the defined M9-MOPS medium, a better model (than nutrient-rich conditions) for bacterial growth observed in vivo during certain infections.^43–45 The dominant number of compounds had no activity in the wild-type strains of GNB (Tables 1–4 and S2). In contrast, at least half of the compounds inhibited the growth of the double-compromised (Δ+Pore) strains. Compounds 3e, 8d, and 8o exhibited activity against all three Gram-negative pathogens.

Table 1.

IC₅₀ Values of Substituted Phenyl C-Ring Derivatives in the M9-MOPS Medium^a

compound	ABWT	AB Pore	AB Δ	AB ΔPore	ECWT	EC Pore	EC Δ	EC ΔPore	PAWT	PA Pore	PA Δ	PA ΔPore
3e	54.5	14.3	17.7	8.2	18.1	2.6	1.7	2.6	23.0	26.3	3.3	8.1
3a	18.4	5.5	2.3	1.9	18.4	2.0	0.4	0.3	>100	13.1	2.3	1.4
3n	>100	>100	2.4	1.5	23.0	2.4	0.3	0.3	>100	>100	2.4	0.3
3r	>100	>100	0.7	0.6	23.3	3.4	0.2	0.3	>100	>100	0.7	1.2
3p	>100	>100	2.3	1.6	28.0	3.2	0.3	0.3	>100	>100	2.3	3.8
3b	63.5	23.5	9.1	8.1	35.1	9.4	6.9	4.9	>100	>100	78.2	6.9
3j	>100	59.1	33.2	24.8	42.6	46.3	9.3	21.9	>100	>100	75.6	29.9
3v	22.1	2.8	1.2	0.9	55.9	7.5	0.6	1.0	>100	63.6	5.1	1.8
3c	18.4	16.9	1.1	1.2	66.8	5.0	0.6	0.5	>100	5.9	1.1	0.02
3h	45.8	10.8	5.4	7.6	66.9	1.5	1.5	2.0	>100	>100	15.5	13.0
3g	45.3	5.7	4.4	4.6	67.9	17.6	3.9	3.7	>100	>100	5.3	5.5
3o	>100	>100	64.4	62.1	77.0	19.5	21.6	7.2	>100	>100	>100	>100
3s	>100	41.1	8.0	3.9	88.8	35.3	4.1	2.9	>100	>100	8.0	1.4
3q	>100	89.8	8.8	9.7	89.1	22.8	4.3	2.3	>100	89.8	8.8	2.2
3k	>100	>100	>100	>100	96.0	61.5	64.6	39.7	>100	>100	>100	>100
3l	>100	77.9	17.3	8.2	99.1	20.4	15.9	6.6	>100	>100	49.0	22.3
3d	>100	12.9	8.4	7.3	>100	22.1	2.0	2.3	>100	27.7	8.4	5.8
3u	>100	55.5	12.8	10.2	>100	>100	54.0	2.6	>100	>100	>100	8.9
3i	>100	>100	48.8	38.5	>100	21.2	19.3	12.5	>100	>100	>100	>100
3t	>100	25.6	16.3	6.4	>100	76.9	17.2	18.8	>100	>100	18.6	3.1
3f	>100	65.9	67.8	32.5	>100	56.1	63.8	55.7	>100	>100	12.0	25.7
3m	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
3w	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
3x	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
Linezolid	33.4	9.2	4.9	4.0	>100	18.0	8.7	9.3	>100	19.8	24.7	1.7

^aArranged in order of potency against ECWT. All values are reported in μM.

Table 4.

IC₅₀ Values of Substituted Alkynyl Derivatives in the M9-MOPS Medium^a

compound	ABWT	AB Pore	AB Δ	AB ΔPore	ECWT	EC Pore	EC Δ	EC ΔPore	PAWT	PA Pore	PA Δ	PA ΔPore
13f	>100	49.8	83.9	76.7	59.0	19.2	34.2	20.5	>100	>100	>100	>100
13l	>100	>100	61.9	34.7	62.4	24.7	30.2	13.5	>100	>100	>100	91.3
13a	>100	>100	>100	>100	74.8	12.8	26.1	3.6	>100	>100	64.9	16.2
13g	>100	>100	>100	>100	74.9	36.3	80.3	44.0	>100	>100	>100	>100
13h	>100	>100	>100	>100	94.9	35.1	65.9	47.8	>100	>100	>100	>100
13c	>100	>100	>100	>100	>100	1.2	86.3	3.4	>100	>100	>100	>100
13j	>100	>100	>100	>100	>100	>100	>100	11.2	>100	>100	>100	>100
13i	>100	60.3	44.0	30.3	>100	65.3	17.8	21.9	>100	>100	>100	30.1
13d	>100	>100	>100	62.0	>100	52.3	88.7	36.6	>100	>100	>100	>100
13k	>100	>100	>100	>100	>100	>100	64.3	39.0	>100	>100	>100	>100
13m	>100	>100	>100	>100	>100	44.4	72.4	39.7	>100	>100	>100	>100
13n	>100	>100	>100	>100	>100	>100	84.0	43.6	>100	>100	>100	>100
13u	>100	42.4	72.2	28.7	>100	>100	42.9	44.4	>100	>100	>100	46.9
13r	>100	>100	>100	>100	>100	51.4	13.3	56.1	>100	>100	>100	>100
13aa	>100	>100	>100	>100	>100	>100	50.9	62.6	>100	>100	>100	>100
13b	>100	>100	>100	>100	>100	>100	85.0	64.5	>100	>100	>100	>100
13s	>100	>100	>100	>100	>100	74.3	83.4	64.7	>100	>100	>100	>100
13w	>100	>100	>100	>100	>100	97.3	72.6	67.8	>100	>100	>100	>100
13e	>100	61.3	88.2	89.5	>100	>100	>100	>100	>100	>100	>100	>100
13p	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
13q	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
13o	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
13ab	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
13x	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
13y	> >100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
13z	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
13t	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
13v	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
Linezolid	33.4	9.2	4.9	4.0	>100	18.0	8.7	9.3	>100	19.8	24.7	1.7

^aArranged in order of potency against ECWT. All values are reported in μM.

With some exceptions, these compounds were active across species, including S. aureus (Table 5), suggesting that species-specific differences in binding to ribosomes are only minor factors in their activities. This was a bit surprising as interrogation of existing crystal structures reveals that while the cocrystal structure of LZD bound to the S. aureus 50S (SA50Slin) subunit indicates binding at the peptidyl transferase center (PTC), blocking the A-site in an orientation similar to that observed in other ribosome LZD complexes, a noteworthy difference is apparent. In the SA50Slin complex, the flexible nucleotide U2585 undergoes significant rotation and forms a hydrogen bond with the oxygen of the LZD morpholine ring, leading to a nonproductive conformation of the PTC.⁴⁶ This is different from other ribosome LZD complexes including Deinococcus radiodurans 50S, the whole ribosome of Thermus thermophilus, as well as the E. coli 70S structure. Hence, we anticipated that the alteration of the C-ring could affect target engagement and, by extension, antibacterial activity of LZD derivatives in a species-specific manner.

Table 5.

MICs of Select Oxazolidinone Analogues in LB Medium^a

compound	AB ΔPore	EC ΔPore	PA ΔPore	S. aureus ATCC 25923	SQ110	SQ110 DTC	SQ110 DTC G2032A	SQ110 DTC C2610A
8o	25	50	6.25	12.5	>100	25	100	50
3s	6.25	>100	100	3.12	>100	25	>100	>100
3t	25	>100	100	6.25	>100	100	>100	>100
3u	50	12.5	>100	6.25	>100	25	50	25
8d	6.25	6.25	3.125	1.56	>100	6.25	25	6.25
8e	12.5	25	6.25	1.56	>100	12.5	50	25
8g	50	100	12.5	6.25	>100	25	100	50
12a	100	100	100	>100	100	50	>100	100
12f	50	12.5	12.5	12.5	>100	3.12	25	12.5
12g	25	25	25	12.5	>100	12.5	50	25
3e	25	100	12.5	6.25	>100	50	100	50
3g	12.5	25	6.25	1.56	>100	12.5	50	25
Linezolid	25	25	12.5	6.25	>100	25	>100	50

^aAll values are reported in μM.

Structure–Activity Relationships.

E. coli is the best characterized, and we will first focus on SAR in this species and then identify species-specific differences for A. baumannii and P. aeruginosa.

Phenyl Series.

Among group 3 with substituted phenyl variants (Scheme 1), 16/24 analogues exhibited activity against ECWT (Table 1) at the concentrations tested (up to 100 μM). Within this series, 16/24 derivatives exhibited near-equal or better activity than LZD against the double-compromised ECΔPore strain. Compounds 3k, 3f, 3m, 3w, and 3x exhibited >4-fold less activity than LZD in the ECΔPore strain, suggesting that the motifs associated with these molecules are likely detrimental to target engagement. Inspection of the corresponding motifs reveals no obvious functionality or trend that may explain target engagement issues. Compounds 3d and 3u are interesting in that they exhibited improved activity against the ECΔPore strain but were inactive against ECWT. Efflux susceptibility seems to be the main issue for 3d, whereas 3u exhibits issues with both OM permeation and efflux susceptibility.

Activity of this series against ABWT decreased to include only 7/24 compounds, with 3j, 3k, 3l, and 3n–s dropping out from the series that was active against ECWT (Table 1). Seven compounds (3a–c, 3e, 3g, 3h, and 3v) exhibited activity against ECWT and ABWT. Contrary to activity against ECΔPore, only 7/24 analogues exhibited equal or better activity than LZD against the ABΔPore strain. In addition to the analogues identified as problematic for target engagement against E. coli, 3i, 3j and 3o also proved detrimental in A. baumannii. The list of compounds that exhibited activity against the ΔPore strain but not against the WT grew for A. baumannii to include 3l and 3n–s. This suggests that the OM and efflux proficiency of A. baumannii supersede that of E. coli for this chemotype.

Against P. aeruginosa, 3e was the only compound that exhibited activity against the WT strain (Table 1). Within this series, 5/24 compounds performed better than LZD against the PAΔPore strain, fewer than both E. coli and A. baumannii. In addition to the motifs identified that decreased the target engagement efficiency against E. coli and A. baumannii, 3h, 3j, and 3l, and 3u are included in the list of compounds exhibiting a >5-fold decreased activity than LZD against the ΔPore strain. Compound 3c is interesting to note, as it provided very good potency (20 nM) against PAΔPore, perhaps suggesting additional mechanisms of action. This is a reasonable hypothesis given the presence of a reactive aldehyde.

In summary, from this series, only 3e exhibited pan activity against E. coli, A. baumannii, and P. aeruginosa WT. No members of this subset that were inactive against ECWT exhibited activity against ABWT or PAWT. SAR trends observed in this series clearly indicate that barrier stringency exhibited between these species can be summarized as P. aeruginosa > A. baumannii > E. coli. Likewise, although the 23S rRNA target of the oxazolidinones is known to be highly conserved and the activity of analogues is mostly consistent across species, the observation that the list of motifs proving detrimental to target engagement increases from E. coli to A. baumannii to P. aeruginosa highlights that discernible differences in the target exist and can potentially be exploited for narrowing or broadening the activity.

Pyridyl Series.

Within this series, only 3/15 derivatives (8d, 8o, and 8m) were active against ECWT and 5/15 exhibited near-equal or better activity than LZD against the ECΔPore strain (Table 2). The list of compounds that exhibited >5-fold worse activity against ECΔPore than LZD includes (8f and 8h–m). It is worth noting that within this group that exhibits less efficient target engagement, motifs include extended pyridyls, comprising both rotatable (8k–m) and fused (8i and 8j) extensions. All analogues of this series that were nearly equal or more potent than LZD against ECΔPore exhibited weak/no activity against ECWT. Thus, while pyridyls may improve target engagement, they still pose OM permeation and efflux liabilities.

Table 2.

IC₅₀ Values of Substituted Pyridyl C-Ring Derivatives in the M9-MOPS Medium^a

compound	ABWT	AB Pore	AB Δ	AB ΔPore	ECWT	EC Pore	EC Δ	EC ΔPore	PAWT	PA Pore	PA Δ	PA ΔPore
8d	16.5	4.1	1.4	2.2	44.3	5.6	0.8	1.3	44.2	48.2	8.1	2.9
8o	37.6	5.8	15.4	6.3	66.1	23.3	10.0	10.3	55.9	40.8	2.2	0.2
8m	35.3	15.8	21.5	22.6	76.2	47.2	55.6	55.2	>100	>100	>100	>100
8g	>100	>100	53.0	13.6	>100	2.0	5.9	1.5	>100	>100	>100	33.0
8a	71.9	40.4	72.4	49.1	>100	4.0	15.4	1.5	>100	>100	>100	>100
8b	83.2	28.1	12.1	11.3	>100	4.7	2.7	2.2	>100	>100	32.9	38.7
8e	19.0	5.2	4.3	3.5	>100	14.5	2.8	3.2	>100	75.3	4.7	6.0
8n	>100	39.0	83.4	73.1	>100	56.1	23.6	30.6	>100	>100	22.7	32.9
8c	>100	>100	>100	>100	>100	>100	>100	42.2	>100	>100	>100	>100
8k	>100	>100	>100	6.2	>100	>100	>100	75.2	>100	>100	92.1	1.0
8f	>100	>100	>100	>100	>100	>100	76.8	79.6	>100	>100	64.2	47.4
8i	>100	35.3	5.6	3.4	>100	>100	>100	>100	>100	>100	67.7	37.9
8j	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	0.1
8l	>100	59.4	>100	20.4	>100	>100	>100	>100	>100	>100	>100	>100
8h	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
Linezolid	33.4	9.2	4.9	4.0	>100	18.0	8.7	9.3	>100	19.8	24.7	1.7

^aArranged in order of potency against ECWT. All values are reported in μM.

Against A. baumannii, 6/15 compounds were active against the WT strain, with four exhibiting equal or better activity than LZD. However, only 3/15 compounds exhibited near-equal or better activity than LZD against ABΔPore. Contrary to E. coli, compounds 8k and 8i exhibited good activity against ABΔPore. Compound 8l also exhibited activity against ABΔPore, whereas it was inactive against ECΔPore. These three analogues once again highlight the discernible differences in target engagement between the species. Against A. baumannii, in addition to 8f, 8h, 8j, 8l, and 8m identified in E. coli, derivatives 8a, 8c, and 8n proved to be detrimental to on-target activity.

This series produced two compounds active against PAWT (8d and 8o) and three that exhibited better activity than LZD in PAΔPore (8o, 8k, and 8j). Compound 8j is especially interesting, as it is inactive against ECΔPore and ABΔPore. This suggests either a secondary mechanism of action in P. aeruginosa or a special feature of the 23S rRNA binding pocket that can be selectively exploited. LZD exhibits activity against Pore and Δ6 strains, whereas most pyridyl analogues are inactive. This suggests that pyridyls succumb to additive/synergistic properties of OM permeation and efflux.

In summary, this series produced two analogues with pan WT activity (8d and 8o) and an additional compound (8m) that exhibited activity against ECWT and ABWT. Contrary to the substituted phenyl series, ECWT activity is not a preliminary qualifier for ABWT activity, as three compounds (8a, 8b, and 8e) exhibited activity against ABWT but were inactive against ECWT. For this series, the barrier stringency seems to be summarized as P. aeruginosa > E. coli > A. baumannii, thus highlighting that motif properties can influence species response.

Pyridinium Series.

Compound series 12 was comprised of various pyridinium motifs. From this series, 9/12 analogues were active against ECWT (Table 3). Only two derivatives (12c and 12b) were >5-fold less active than LZD against ECΔPore, indicating good complementarity of a pyridinium functionality with the 23S rRNA target. Comparison of the IC₅₀ values across all four strains reveals this series to be less of an issue for OM permeation and efflux susceptibility. This is consistent with previous observations that positively charged motifs may improve the accumulation of small molecules due to porin uptake mechanisms.

Table 3.

IC₅₀ Values of Substituted Pyridinium C-Ring Derivatives in the M9-MOPS Medium^a

compound	ABWT	AB Pore	AB Δ	AB Δ Pore	ECWT	EC Pore	EC Δ	EC ΔPore	PAWT	PA Pore	PA Δ	PA ΔPore
12a	59.4	13.8	23.3	13.8	13.5	12.3	10.0	11.0	>100	59.7	48.8	9.4
12f	>100	10.0	64.0	10.8	15.4	1.7	0.5	0.5	>100	>100	>100	>100
12i	>100	>100	>100	38.5	43.8	36.0	13.7	15.9	>100	>100	57.4	42.3
12j	>100	19.8	89.7	19.8	43.9	28.3	12.7	16.6	>100	>100	>100	>100
12l	>100	15.6	23.4	13.0	51.6	23.3	15.5	15.6	>100	41.7	4.6	1.1
12d	>100	20.1	29.7	27.9	63.6	30.0	4.0	11.4	>100	>100	>100	>100
12h	>100	13.3	20.4	10.2	72.6	13.2	3.1	4.4	>100	15.9	8.4	1.2
12k	89.5	22.0	34.5	22.3	81.6	42.8	16.9	16.6	>100	>100	35.1	36.6
12g	>100	13.9	29.7	12.6	98.4	7.8	1.2	2.1	>100	28.1	29.7	18.1
12e	>100	27.4	48.6	9.4	>100	21.0	5.6	9.6	>100	>100	>100	>100
12c	>100	>100	>100	33.3	>100	>100	72.3	60.4	>100	>100	>100	40.4
12b	>100	>100	>100	>100	>100	>100	70.6	88.8	>100	>100	>100	>100
Linezolid	33.4	9.2	4.9	4.0	>100	18.0	8.7	9.3	>100	19.8	24.7	1.7

^aArranged in order of potency against ECWT. All values are reported in μM.

Against A. baumannii, only two compounds were active against ABWT (12a and 12k), but both were less active than LZD. In addition, 12b–d, 12i, and 12k exhibited a >5-fold lower activity than LZD against ABΔPore. This suggests that the pyridinium motif may not be as complementary to target binding site as it is for E. coli. In fact, most analogues in this series are less active than LZD against any of the A. baumannii strains.

For P. aeruginosa, no analogues within this series were active against WT. In addition to motifs identified for being detrimental to target engagement in E. coli and A. baumannii, all compounds except 12l and 12h can be added to this list for P. aeruginosa. This further highlights the decrease in susceptibility of P. aeruginosa to modification of the oxazolidinone scaffold for 23S rRNA inhibition.

In summary, no pyridinium analogues were identified that exhibited a broad-spectrum activity against all three species. While this motif class seems rather beneficial for improving WT activity against E. coli, it is detrimental to whole-cell activity against A. baumannii and P. aeruginosa. The trend of motif SAR narrowing from E. coli to A. baumannii to P. aeruginosa, with P. aeruginosa being more stringent for allowable functionality, was consistent for this chemotype.

Alkynyl Series.

This series was the least active, with only 5/28 exhibiting activity against ECWT and no compounds active against ABWT of PAWT (Table 4). In fact, although 64% ofthe compounds in this series exhibited activity against ECΔPore, albeit with most having lower activity than LZD, only 21 and 14% were active against ABΔPore and PAΔPore, respectively. These results further suggest that the positioning and functionality at this location on the oxazolidinone chemotype are important for target engagement. The alkynyl attachment of this series likely extends beyond the binding pocket and is counterproductive to binding and by extension, activity. These results also confirm a trend of E. coli > A. baumannii > P. aeruginosa regarding compatibility with a broadened motif functionality.

Additional Trends from Cross-Series Comparisons.

While interrogation of each subset independently provided insight into each series, cross-series comparisons also provided interesting observations. For example, the comparison of IC₅₀ values of 3a, 8d–f, and 12a–c, elucidates the effect of pyridine inclusion, location, and conversion into a pyridinium on activity (Tables 1–3). This series demonstrates that activity against WT and ΔPore strains is relatively maintained against E. coli and A. baumannii after the incorporation of a pyridine with the nitrogen at the para-position (3a compared to 8d) and activity against PAWT is gained. Interestingly, if the position of the nitrogen is moved to the 3-position (8e) activity against ECWT and PAWT is lost, with both barriers now contributing to the loss of activity in E. coli and primarily efflux susceptibility becoming the main issue in P. aeruginosa. Conversion of the pyridine to the pyridinium, however, only improves WT activity against E. coli, further illustrating the potential limitation of the benefit that charge incorporation provides with general GNB activity. In fact, inherent charge incorporation was detrimental to ABWT and PAWT activities in every instance in this study.

When compounds 3a, 8d, 12a, and 12i–l are compared, IC₅₀ values highlight the effect of alkyl size appended to the nitrogen in the pyridinium groups. As mentioned previously, the incorporation of a charged pyridinium results in a slight loss in EC–Pore activity but the drop in activity is not magnified upon continued increases in the alkyl size. However, the activity against WT seems to correlate negatively with size, as groups larger than a methyl demonstrated decreased ECWT activity. Against A. baumannii and P. aeruginosa smaller or larger substituents exhibited more activity against the ΔPore strains, however, WT activity decreased in both of these species following the incorporation of a pyridinium.

Target Verification.

The above results show that the generated compounds vary broadly in their potencies against GNB. We next compared the MICs of select compounds against “barrier-less” strains of E. coli, A. baumannii, and P. aeruginosa to those against S. aureus, all grown in the nutrient-rich LB medium. Our results show consistent differences in MICs between the Gram-negative strains as well as in comparison to S. aureus (Table 5), albeit compounds seem to be more potent against cells grown in the M9-MOPS medium. In general, S. aureus was at least 2–4-fold more susceptible to growth inhibition induced by tested compounds, including the antibiotic LZD (Table 5). The most potent across the species were substituted pyridyls 8d and 8e and the substituted benzyl derivative 3g. Furthermore, these compounds were at least 4-fold more potent than LZD against S. aureus.

To confirm that the activities of compounds remain target-dependent, we analyzed the efficiency of the cell-free E. coli transcription–translation in the absence and presence of increasing concentrations of select compounds (Figure 3). We found that the apparent protein translation inhibition constant, K_i^app, of LZD is 2.8 μM (Figure 3A), which is in excellent agreement with K_i^app = 2.5 μM reported previously⁴⁷ and with the growth inhibition IC₅₀ = 9.3 μM for ECΔPore cells. The on-target activities of 3h, 8o, 8d, and 12f were all comparable to that of LZD and were consistent with their respective growth inhibition IC₅₀ values (Figure 3B).

Figure 3.

Effects of oxazolidinones on cell-free coupled transcription–translation in E. coli. A. Increasing concentrations of linezolid were added to transcription–translation reaction mixtures, and reactions were allowed to proceed for 5 h. To quantify the amounts of the synthesized protein, the reactions were treated with the Lumio reagent and were resolved on 16% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fluorescent bands corresponding to the target protein were quantified, normalized to the reaction that lacked the antibiotic, and plotted as a function of the antibiotic concentration. Error bars: standard deviation (SD) (n = 3). B. The same as in panel A but reactions were set with the indicated compounds at concentrations corresponding to their 1× and 0.1× MICs. The K_i^app values were calculated as in panel A and shown on top of the gel image along with IC₅₀ values, as determined in bacterial growth inhibition assays. M, protein size marker in kDa.

We also determined MICs against the E. coli SQ110 and SQDTC (ΔTolC) strains carrying only one copy of the rRNA operon and against SQDTC derivatives producing the LZD-resistant 23S rRNA variants with G2032A or C2610A substitutions.⁴⁸ We found that the SQ110 strain was resistant to all tested compounds (Table 5). With a few exceptions, SQ110 DTC was more susceptible to compounds than ECΔPore strain, suggesting that 23S rRNA copy number contributes to differences in MICs. However, for all compounds, MICs were higher in LZD-resistant strains. Thus, the antibacterial activities of compounds remain dependent on 23S rRNA inhibition.

Structure–Uptake Relationships.

We next identified problematic motifs associated with general or species-specific efflux susceptibility and poor OM permeation. All compounds exhibiting measurable IC₅₀ values against at least one double-compromised strain were included in the analysis. IC₅₀ ratios for WT(Pore)/ΔEfflux(Pore) (efflux impact ratio, P/PE) and ΔEfflux/ ΔEfflux(Pore) (OM impact ratio, E/PE) were calculated for each species and strain. Any IC₅₀ value >100 for a ΔEfflux or (Pore) strain was set to 100 to allow P/PE and E/PE ratios for each motif to be calculated. This approach ensures that, if anything, the ratios for compounds with >100 IC₅₀ values are underestimated. Ratios exceeding an arbitrary threshold of ≥5 were classified as a motif exhibiting problematic behavior for the corresponding barrier. For example, an E/PE ratio of 10 for a given analogue in E. coli would identify the corresponding motif as problematic to E. coli OM permeation. This ratio analysis was completed for each analogue over each of the three species. Motifs associated with poor OM permeation and/or increased efflux susceptibility are shown in Figures 4 and 5, respectively. Interestingly, no motifs were identified that exhibited poor OM permeation across all three species but six motifs (3c, 3n, 3p, 3r, 3s, and 3u) increased efflux susceptibility across all three species. In accordance with previous studies that indicate P. aeruginosa to exhibit higher levels of OM and efflux barrier synergy, 7 of the 10 motifs identified as being problematic for OM permeation were also exposed as efflux liabilities (3b, 3t, 8j, 8o, 12a, 12h, and LZD).

Figure 4.

Motifs identified as liabilities to OM permeation. E/PE = Δ/ΔPore IC₅₀ ratio for the indicated species.

Figure 5.

Motifs identified as liabilities to efflux susceptibility. P/PE = Pore/ΔPore IC₅₀ ratio for the indicated species.

Principal Component Analysis.

Important to the objective of advancing to a point of the rational design of GNB-accumulating chemotypes is the development of computational models. In an effort to confirm if observations identified from manual structural mapping (i.e., human interpretation of activity data) could be recapitulated via bioinformatics, we subjected the data set to principal component analysis (PCA). Three IC₅₀ ratios, WT/ΔEfflux(Pore) (total barrier ratio, W/PE), WT(Pore)/ΔEfflux(Pore) (efflux impact ratio, P/PE), and ΔEfflux/ ΔEfflux(Pore) (OM impact ratio, E/PE), were analyzed by PCA to provide insight into the influence of each barrier (i.e., efflux and porination) on activity for each species. For this PCA, only the 26 compounds that showed a detectable IC₅₀ in at least two barrier-variant strains across all three bacteria were used (Figure 6). The rest of the compounds had to be excluded from the comparative analysis because the effect of neither efflux nor porination could be quantified in at least one of the species.

Figure 6.

Principal component analysis of the three permeation ratios in A. baumannii (AB), E. coli (EC), and P. aeruginosa (PA).

In most cases for all three bacteria, the effect of efflux inactivation (P/PE ratio) dominated that of porination (E/PE ratio). Thus, any compound specificity observed for each species is primarily defined by the organism’s repertoire of efflux pumps. We further noted that A. baumannii was much closer to E. coli than P. aeruginosa, according to PCA. For both efflux and porination, the first principal component (PC1) was defined by the difference between P. aeruginosa and the other two bacteria, whereas the second principal component (PC2) was aligned with the distinction between E. coli and A. baumannii. Inspection of PCs (Table S1) revealed that the PC1 and PC2 in the efflux impact ratio (P/PE) were dominated, respectively, by 8o and 12l, and by 3p, 3r, and 3n. Thus, these compounds are the most reflective of the species-specific difference in the efficiency of their permeation barriers. In particular, both 8o and 12l were identified as efflux liabilities for P. aeruginosa but not for E. coli or A. baumannii (Figure 5). For the OM impact, most of the difference could be attributed to compounds 3c, 8o, and LZD (PC1) and 3m, 3c, 3e, and 12g (PC2). As seen in Figure 4, permeabilities of compounds 3c, 8o, and LZD were greatly affected by the presence of the pore for P. aeruginosa but not for E. coli or A. baumannii. Thus, the distinction of P. aeruginosa from two other bacteria is achieved through both the more efficient efflux and lower transmembrane diffusion. However, the two factors are differentially affected by compound structures. The PCA results are largely confirmatory of the observations ascertained through manual structural inspection, thus setting a foundation for more prospective designing of GNB-active compounds.

Growth Inhibition against Diverse Isolates.

To determine whether the trends established in the above experiments are applicable to other bacterial strains and species, we measured MICs for the select compounds with activities in the WT strains against multidrug-resistant A. baumannii (AYE and Ab5075), P. aeruginosa (BAA 2108 and BAA 2109, multidrug resistance ATCC panel), K. pneumoniae (ATCC13883 and ATCC43816), and K. aerogenes ATCC13048 (Table 6). The MICs were measured in the ion-adjusted Mueller-Hinton broth (per recommendations of CLSI) and in M9-MOPS. None of the compounds inhibited P. aeruginosa growth in concentrations up to 200 μM. However, all compounds except LZD inhibited the growth of K. pneumoniae ATCC13883 and K. aerogenes ATCC13048, especially in M9-MOPS medium with 8o having the lowest MIC = 12.5 μM against the ATCC13058 strain. In addition, compounds 3e and 8o inhibited the growth of K. pneumoniae ATCC43816 cells also grown in M9-MOPS. Compounds 3h, 8e, and 8d inhibited the growth of both A. baumannii strains when grown in M9-MOPS with MICs = 100–200 μM (Table 6).

Table 6.

MICs of Select Oxazolidinone Analogues in MHI and M9-MOPS Media^a

strain	medium	LZD	3h	3e	8o	8e	8d	12f
AB AYE	MHI	>200	>200	>200	>200	>200	>200	>200
	M9-MOPS	200	200	>200	>200	100	100	>200
AB Ab5075	MHI	>200	>200	>200	>200	>200	>200	>200
	M9-MOPS	≥200	200	>200	>200	100	100	200
PA BAA 2108	MHI	>200	>200	>200	>200	>200	>200	>200
	M9-MOPS	>200	>200	>200	>200	>200	≥200	>200
PA BAA 2109	MHI	>200	>200	>200	>200	>200	>200	>200
	M9-MOPS	>200	>200	>200	>200	>200	>200	>200
K. pneumoniae ATCC13883	MHI	>200	>200	>200	>200	>200	200	>200
	M9-MOPS	≥200	200	100–200	50–100	200	100–200	100
K. pneumoniae ATCC43816	MHI	>200	>200	>200	>200	>200	>200	>200
	M9-MOPS	>200	>200	200	100	>200	200	>200
K. aerogenes ATCC13048	MHI	>200	>200	>200	200	>200	200	>200
	M9-MOPS	>200	>200	100	12.5	200	200	50

^aAll values are reported in μM.

Thus, the uncovered trends can be applied to bacterial strains with diverse genetic backgrounds and could improve the activities of oxazolidinones and perhaps other antibiotics against Gram-negative pathogens. However, additional resistance mechanisms such as those present in MDR strains of P. aeruginosa require further studies.

CONCLUSIONS

In conclusion, we report the design, synthesis, and evaluation of a library of oxazolidinones against E. coli, P. aeruginosa, and A. baumannii and their isogenic strains with varying degrees of the outer membrane and/or efflux pump deficiencies. The data indicate that the evaluated permeation mechanisms do not account for all activity differences between organisms. This could suggest (1) secondary mechanisms of action or different off-target profiles for oxazolidinones in some species, (2) tangible differences in target engagement, and/or (3) involvement of additional permeation mechanisms such as alternative efflux pumps. While we do observe growth media-specific differences in activity, this was expected as growth and metabolic rates of bacteria differ in minimal versus nutrient-rich environments. It is important to note, however, that the overall trends of activity against certain species were not changed, suggesting that the growth medium does not contribute significantly to species-specific inhibitory differences.

The data clearly indicate that motif manipulation on a chemotype is enough to significantly modulate efflux and permeation. As such, the perception that the efflux and OM permeation issues are an inherent characteristic of the chemotype does not hold true for the oxazolidinone scaffold. Rather, the orientation, location, and composition of functional groups are the determinants of chemotype accumulation. In general, P. aeruginosa is more divergent from the other two bacteria and the least responsive to oxazolidinone treatment. It would be more challenging to engineer activity against P. aeruginosa for this chemotype, as efflux and OM barriers synergize strongly in this organism. A. baumannii and E. coli are more similar, with efflux being the major issue for this chemotype, with certain motifs also facing difficulties with OM permeation.

Determination of IC₅₀ values revealed three analogues (3e, 8d, and 8o) with a broadened spectrum of activity to include WT E. coli, A. baumannii, and P. aeruginosa. While these three analogues were previously reported to exhibit activity against another ECWT strain (MG1655),⁴⁰ this is the first report of their broadened activity to include two other ESKAPE pathogens A. baumannii and P. aeruginosa. Of the 80 compounds presented in this study, no single motif was identified that correlated with poor OM permeation across all three GNB species. On the contrary, six different motifs were identified that increased efflux susceptibility across all three species (3c, 3n, 3p, 3r, 3s, and 3u). These observations provide compelling evidence that molecular features that dictate compound accumulation are likely to vary from one species to the next, with OM permeation being the most species-specific and efflux susceptibility exhibiting more overlap.

While generating overarching Lipinski-like rules or guidelines for compound accumulation in Gram-negative bacteria is an enticing goal, we postulate that a more feasible first step may be to identify motifs for several chemotypes that significantly affect accumulation (positively or negatively). Once this has been completed for a variety of chemotypes and across several bacterial species, a clearer picture of general versus chemotype-and species-specific trends is expected to emerge. One can then imagine eventually advancing to a point wherein hierarchical ranking of motifs can be organized to provide Topliss-like trees⁴⁹ and/or bioisostere-like classifications⁵⁰ to guide accumulation improvement efforts and rational design for Gram-negative active molecules.

EXPERIMENTAL SECTION

Chemistry.

Starting materials, ACS-grade methylene chloride (DCM), methanol (MeOH), hexanes, ethyl acetate (EtOAc), acetone, acetonitrile (CH₃CN), dimethyl formamide (DMF), anhydrous dimethyl sulfoxide (DMSO), dioxane, ethanol, formic acid, anhydrous tetrahydrofuran (THF), tetrahydrofuran (THF), toluene, and trifluoroacetic acid (TFA) were purchased from TCI Chemicals, Oakwood, Alfa Aesar, Fisher Scientific, Enamine, or Sigma-Aldrich. Deionized water was used for all experimental procedures where “water” is indicated. All reactions requiring anhydrous conditions were run under a nitrogen atmosphere. Analytical and preparative thin-layer chromatography (TLC) was performed on silica gel 60 F₂₅₄ plates (Sigma-Aldrich 1.05715). Flash column chromatography was carried out on silica gel (70–230 mesh, SiliCycle). NMR data were collected on a 400, 500, and 600 MHz (specified below) Varian VNMRS Direct Drive spectrometer equipped with an indirect detection probe. NMR data were collected at 25 °C unless otherwise indicated. Pulse sequences were used as supplied by Varian VNMRJ 4.2 software. All NMR data were processed in MestReNova. High-resolution mass spectrometry was obtained from and analyzed by the Mass Spectrometry Facility at the University of Minnesota. All compounds evaluated in biological assays were >95% purity based on high-performance liquid chromatography (HPLC) and/or NMR.

Benzyl (4-Bromo-3-fluorophenyl)carbamate (1a).

To a solution of 4-bromo-3-fluoroaniline (3.8 g, 20 mmol) in THF (80 mL) was added sodium bicarbonate (3.36 g, 40 mmol) and the mixture was cooled to 0 °C. Benzyl chloroformate (4.23 mL, 30 mmol) was added dropwise with a syringe. The reaction mixture was stirred at 25 °C. After the reaction was judged to be completed by TLC (16 h), it was quenched with water and extracted with EtOAc three times. The combined organic layers were washed with water and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–20% EtOAc in hexanes) to give compound 1a as a yellow amorphous solid (5.90 g, 91%). ¹H NMR (300 MHz, chloroform-d) δ 7.71–7.31 (m, 7H), 6.93 (dd, J = 8.7, 2.5 Hz, 1H), 6.75 (s, 1H), 5.20 (s, 2H).

Benzyl (3-Fluorophenyl)carbamate (1b).

A mixture of 3-fluoroaniline (1.92 mL, 20 mmol) and sodium bicarbonate (3.36 g, 40 mmol) was suspended in THF (80 mL) and cooled to 0 °C. Benzyl chloroformate (4.23 mL, 30 mmol) was added dropwise with a syringe. The reaction mixture was stirred at 0 °C. After the reaction was judged to be completed by TLC (3 h), it was quenched with water and extracted with EtOAc three times. The combined organic layers were washed with water and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–15% EtOAc in hexanes) to give compound 1b as a white amorphous solid (4.9 g, 99%). ¹H NMR (300 MHz, chloroform-d) δ 7.47–7.29 (m, 5H), 7.29–7.16 (m, 1H), 7.01 (ddd, J = 8.4, 2.1, 0.9 Hz, 1H), 6.83–6.66 (m, 2H), 5.21 (s, 2H).

(R)-3-(4-Bromo-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one (2a).³⁸

Compound 1a (1.62 g, 5 mmol) was dissolved in anhydrous THF (25 mL) and cooled to −78 °C under a nitrogen atmosphere. Lithium bis(trimethylsilyl)amide solution (1 M in THF, 4.25 mL, 4.25 mmol) was added to the mixture slowly over a period of 40 min with a syringe. The mixture was stirred at −78 °C for 1 h under a nitrogen atmosphere followed by the addition of (R)-(–)-glycidyl butyrate (0.59 mL, 4.25 mmol) dropwise with a syringe at −78 °C. The mixture was stirred at this temperature for an additional 1 h and then gradually warmed to 25 °C. After the reaction was judged to be completed by TLC (20 h), it was quenched with water and extracted with EtOAc three times. The combined organic layers were washed with water and evaporated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) to afford compound 2a as a yellow amorphous solid (1.10 g, 76%). ¹H NMR (300 MHz, acetone-d₆) δ 7.74 (dd, J = 11.7, 2.7 Hz, 1H), 7.64 (dd, J = 8.9, 8.0 Hz, 1H), 7.36 (ddd, J = 8.9, 2.7, 1.0 Hz, 1H), 4.96–4.70 (m, 1H), 4.38 (dd, J = 6.2, 5.6 Hz, 1H), 4.20 (t, J = 8.8 Hz, 1H), 4.01 (dd, J = 8.8, 6.2 Hz, 1H), 3.90 (ddd, J = 12.3, 5.6, 3.4 Hz, 1H), 3.76 (ddd, J = 12.3, 6.2, 3.9 Hz, 1H).

(R)-3-(3-Fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one (2b).

Compound 1b (4.73 g, 19.27 mmol) was dissolved in anhydrous THF (100 mL) and cooled to −78 °C under a nitrogen atmosphere. Lithium bis(trimethylsilyl)amide solution (1 M in THF, 16.38 mL, 16.38 mmol) was added to the mixture slowly over a period of 1 h with a syringe. The mixture was stirred at −78 °C for 1 h under a nitrogen atmosphere followed by the addition of (R)-(–)-glycidyl butyrate (2.23 mL, 16.38 mmol) dropwise with a syringe at −78 °C. The mixture was stirred at this temperature for an additional 1 h and then gradually warmed to 25 °C. After the reaction was judged to be completed by TLC (16 h), it was quenched with water and evaporated under reduced pressure by rotary evaporation to remove most of THF. The mixture was diluted with water and extracted with EtOAc three times. The combined organic layers were washed with water and evaporated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–67% EtOAc in hexanes) to afford compound 2b as a white amorphous solid (2.86 g, 70%). ¹H NMR (400 MHz, acetone-d₆) δ 7.67–7.51 (m, 1H), 7.47–7.26 (m, 2H), 6.96–6.76 (m, 1H), 4.87–4.70 (m, 1H), 4.39 (t, J = 5.7 Hz, 1H), 4.25–4.09 (m, 1H), 4.04–3.95 (m, 1H), 3.94–3.83 (m, 1H), 3.81–3.71 (m, 1H).

General Procedure 1: Synthesis of Heterocyclic Oxazolidinone Analogues 3a–3o.⁴⁰

A mixture of compound 2a (29 mg, 0.1 mmol), aryl boronic acid or ester (0.12 mmol), potassium carbonate (55 mg, 0.4 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (8.2 mg, 0.01 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 90 °C under a nitrogen atmosphere for 3 h. The reaction mixture was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂) using an appropriate eluent as described below to afford compounds 3a–o and 8a,b. Various derivatives deviated slightly from this procedure, and full descriptions are included as needed for 3a–o and 8a,b.

(R)-3-(2-Fluoro-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)-oxazolidin-2-one (3a).

Using general procedure 1, employing phenylboronic acid (15 mg, 0.12 mmol), compound 3a was obtained after flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) as a white amorphous solid (20 mg, 70%). ¹H NMR (400 MHz, acetone-d₆) δ 7.70 (dd, J = 13.7, 2.3 Hz, 1H), 7.62–7.50 (m, 3H), 7.50–7.42 (m, 3H), 7.42–7.35 (m, 1H), 4.90–4.77 (m, 1H), 4.41 (t, J = 5.9 Hz, 1H), 4.24 (t, J = 8.8 Hz, 1H), 4.05 (dd, J = 8.8, 6.3 Hz, 1H), 3.91 (ddd, J = 12.3 5.9, 3.4 Hz, 1H), 3.78 (ddd, J = 12.3, 5.9, 3.9 Hz, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.5 (d, J = 244.5 Hz), 155.3, 140.9 (d, J = 11.3 Hz), 136.2 (d, J = 1.6 Hz), 131.7 (d, J = 5.0 Hz), 129.6 (d, J = 3.1 Hz, 2C), 129.4 (2C), 128.4, 124.1 (d, J = 14.0 Hz), 114.3 (d, J = 3.3 Hz), 106.2 (d, J = 29.3 Hz), 74.3, 63.2, 46.9.

(R)-3-(2-Fluoro-4′-((trifluoromethyl)thio)-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (3b).

Using general procedure 1, employing 4,4,5,5-tetramethyl-2-(4-((trifluoromethyl)thio)phenyl)-1,3,2-dioxaborolane (37 mg, 0.12 mmol), compound 3b was obtained after flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) as a yellow amorphous solid (26 mg, 67%). ¹H NMR (400 MHz, acetone-d₆) δ 7.82 (d, J = 8.1 Hz, 2H), 7.77–7.69 (m, 3H), 7.61 (t, J = 8.7 Hz, 1H), 7.50 (dd, J = 8.7, 2.3 Hz, 1H), 4.95–4.71 (m, 1H), 4.41 (s, 1H), 4.25 (t, J = 8.8 Hz, 1H), 4.06 (dd, J = 8.8, 6.2 Hz, 1H), 3.92 (dd, J = 12.6, 3.6 Hz, 1H), 3.83–3.70 (m, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.7 (d, J = 245.7 Hz), 155.4, 141.9 (d, J = 11.3 Hz), 139.5, 137.4 (2C), 131.8 (d, J = 4.6 Hz), 131.0 (d, J = 3.4 Hz, 2C), 130.9 (q, J = 306.9 Hz), 123.6, 122.5 (d, J = 13.3 Hz), 114.6 (d, J = 3.1 Hz), 106.4 (d, J = 29.1 Hz), 74.5, 63.3, 47.0.

(R)-2′-Fluoro-4′-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-[1,1′-biphenyl]-4-carbaldehyde (3c).

A mixture of compound 2a (58 mg, 0.2 mmol), 4-formylphenylboronic acid (36 mg, 0.24 mmol), potassium carbonate (110 mg, 0.8 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (16 mg, 0.02 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) to afford compound 3c as a yellow amorphous solid (40 mg, 56%). ¹H NMR (500 MHz, acetone-d₆) δ 10.09 (s, 1H), 8.01 (d, J = 8.3 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.74 (dd, J = 13.8, 2.3 Hz, 1H), 7.63 (t, J = 8.8 Hz, 1H), 7.51 (dd, J = 8.8, 2.3 Hz, 1H), 4.87–4.78 (m, 1H), 4.40 (t, J = 5.9 Hz, 1H), 4.25 (t, J = 8.9 Hz, 1H), 4.07 (dd, J = 8.9, 6.2 Hz, 1H), 3.96–3.87 (m, 1H), 3.82–3.74 (m, 1H). ¹³C NMR (126 MHz, acetone-d₆) δ 192.5, 160.6 (d, J = 246.0 Hz), 155.3, 142.1 (d, J = 1.7 Hz), 141.9 (d, J = 11.4 Hz), 136.6, 131.8 (d, J = 4.7 Hz), 130.5 (2C), 130.2 (d, J = 3.7 Hz, 2C), 122.8 (d, J = 13.3 Hz), 114.5 (d, J = 3.2 Hz), 106.3 (d, J = 29.3 Hz), 74.4, 63.2, 47.0.

(R)-3-(2-Fluoro-4′-hydroxy-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (3d).

A mixture of compound 2a (58 mg, 0.2 mmol), 4-hydroxyphenylboronic acid (33 mg, 0.24 mmol), potassium carbonate (110 mg, 0.8 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) complex with dichloromethane (16 mg, 0.02 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) to afford compound 3d as a yellow amorphous solid (7 mg, 11%). ¹H NMR (400 MHz, acetone-d₆) δ 8.48 (s, 1H), 7.66 (dd, J = 13.7, 2.3 Hz, 1H), 7.47 (t, J = 8.7 Hz, 1H), 7.44–7.39 (m, 3H), 6.96–6.91 (m, 2H), 4.86–4.76 (m, 1H), 4.36 (t, J = 5.9 Hz, 1H), 4.22 (t, J = 8.8 Hz, 1H), 4.03 (dd, J = 8.8, 6.2 Hz, 1H), 3.95–3.86 (m, 1H), 3.82–3.74 (m, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.5 (d, J = 245.4 Hz), 158.1, 155.4, 140.3, 131.4 (d, J = 5.1 Hz), 130.9 (d, J = 4.0 Hz, 2C), 127.5 (d, J = 2.0 Hz), 124.4 (d, J = 14.1 Hz), 116.4 (2C), 116.3, 114.5 (d, J = 4.0 Hz), 106.4 (d, J = 29.3 Hz), 74.4, 63.4, 47.1.

(R)-3-(4′-(Aminomethyl)-2-fluoro-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one Hydrochloride (3e).

A mixture of compound 2a (29 mg, 0.1 mmol), 4-aminomethylphenylboronic acid hydrochloride (23 mg, 0.12 mmol), potassium carbonate (55 mg, 0.4 mmol), and [1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) complex with dichloromethane (8 mg, 0.01 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature and concentrated under reduced pressure with a rotary evaporator. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–20% MeOH in DCM with 1% ammonia). The appropriate fractions were collected and evaporated under reduced pressure with a rotary evaporator. The resulting residue was dissolved in MeOH (1 mL), charged with the addition of HCl/dioxane (4 M, 50 μL), and concentrated under reduced pressure with a rotary evaporator. The resulting hydrochloride salt was washed with acetone (1 mL) and dried under high vacuum to afford compound 3e as a white amorphous solid (26 mg, 82%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.73 (s, 3H), 7.76–7.36 (m, 7H), 5.39 (t, J = 5.6 Hz, 1H), 4.80–4.65 (m, 1H), 4.12 (t, J = 8.9 Hz, 1H), 4.04 (s, 2H), 3.92 (dd, J = 8.9, 6.0 Hz, 1H), 3.75–3.64 (m, 1H), 3.61–3.50 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.1 (d, J = 244.7 Hz), 154.4, 139.6 (d, J = 11.1 Hz), 134.7, 133.6, 130.9 (d, J = 4.6 Hz), 129.4 (2C), 128.7 (d, J = 3.0 Hz, 2C), 122.1 (d, J = 13.4 Hz), 113.9 (d, J = 2.0 Hz), 105.4 (d, J = 28.8 Hz), 73.5, 61.5, 46.1, 41.8. MSESI m/z: 339.1125 (C₁₇H₁₇FN₂O₃ + Na⁺ requires 339.1115).

Bis-boc-(R)-1-((2′-fluoro-4′-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-[1,′-biphenyl]-4-yl)methyl)guanidine (3ea).

A mixture of compound 3e (182 mg, 0.51 mmol), N,N′-di-boc-1H-pyrazole-1-carboxamidine (240 mg, 0.77 mmol), and N,N-diisopropylethylamine (266 μL, 1.53 mmol) in THF (10 mL) was stirred at 60 °C. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure with rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–75% EtOAc in hexanes) to afford compound 3ea as a white amorphous solid (74 mg, 26%). ¹H NMR (400 MHz, acetone-d₆) δ 11.71 (s, 1H), 8.71 (s, 1H), 7.70 (dd, J = 13.7, 2.3 Hz, 1H), 7.59–7.42 (m, 6H), 4.86–4.76 (m, 1H), 4.70 (d, J = 5.8 Hz, 2H), 4.42 (s, 1H), 4.23 (t, J = 8.9 Hz, 1H), 4.05 (dd, J = 8.9, 6.2 Hz, 1H), 3.96–3.85 (m, 1H), 3.82–3.73 (m, 1H), 1.51 (s, 9H), 1.44 (s, 9H). ¹³C NMR (101 MHz, acetone-d₆) δ 164.6, 160.5 (d, J = 244.5 Hz), 157.0, 155.3, 153.8, 140.9 (d, J = 11.2 Hz), 138.8, 135.2 (d, J = 1.5 Hz), 131.6 (d, J = 5.0 Hz), 129.8 (d, J = 3.1 Hz, 2C), 128.7 (2C), 123.8 (d, J = 13.8 Hz), 114.3 (d, J = 3.2 Hz), 106.2 (d, J = 29.2 Hz), 83.8, 78.9, 74.3, 63.2, 46.9, 44.5, 28.4, 28.1.

(R)-1-((2′-Fluoro-4′-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-[1,1′-biphenyl]-4-yl)methyl)guanidine Hydrochloride (3f).

A mixture of 3ea (28 mg, 0.05 mmol) in HCl/dioxane (4 M, 1 mL) was stirred at 50 °C. After the reaction was judged to be completed by TLC (30 min), its solvent was removed under reduced pressure with rotary evaporation. The residue was washed with acetone (1 mL) and dried under high vacuum to give compound 3f as a white amorphous solid (11 mg, 56%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.31 (t, J = 6.2 Hz, 1H), 7.63 (dd, J = 13.5, 2.3 Hz, 1H), 7.60–7.49 (m, 3H), 7.48–7.35 (m, 3H), 5.31 (t, J = 5.6 Hz, 1H), 4.79–4.69 (m, 1H), 4.45 (d, J = 6.2 Hz, 2H), 4.13 (t, J = 9.0 Hz, 1H), 3.90 (dd, J = 9.0, 6.1 Hz, 1H), 3.69 (ddd, J = 12.4, 5.6, 3.3 Hz, 1H), 3.57 (ddd, J = 12.4, 5.6, 3.9 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.1 (d, J = 243.9 Hz), 157.2, 154.4, 139.5 (d, J = 11.1 Hz), 136.8, 133.9, 130.9 (d, J = 4.4 Hz), 128.8 (2C), 127.5 (2C), 122.3 (d, J = 12.9 Hz), 113.8, 105.4 (d, J = 28.9 Hz), 73.4, 61.6, 46.0, 43.6. MSESI m/z: 359.1532 (C₁₈H₁₉FN₄O₃ + H⁺ requires 359.1514).

(R)-3-(2-Fluoro-4′-(hydroxymethyl)-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (3g).

Using general procedure 1, employing 4-(hydroxymethyl)-benzeneboronic acid (18 mg, 0.12 mmol), compound 3g was obtained after flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) as a yellow amorphous solid (10 mg, 32%). ¹H NMR (500 MHz, DMSO-d₆) δ 7.62 (dd, J = 13.6, 2.2 Hz, 1H), 7.55 (t, J = 8.6 Hz, 1H), 7.52–7.48 (m, 2H), 7.44 (dd, J = 8.6, 2.2 Hz, 1H), 7.41 (d, J = 8.0 Hz, 2H), 5.29–5.09 (m, 2H), 4.81–4.70 (m, 1H), 4.54 (d, J = 5.3 Hz, 2H), 4.13 (t, J = 8.9 Hz, 1H), 3.88 (dd, J = 8.9, 6.1 Hz, 1H), 3.74–3.65 (m, 1H), 3.63–3.54 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.0 (d, J = 244.3 Hz), 154.3, 142.0, 139.2 (d, J = 11.1 Hz), 133.0, 130.8 (d, J = 4.8 Hz), 128.3 (d, J = 3.0 Hz, 2C), 126.7 (2C), 122.7 (d, J = 13.5 Hz), 113.8 (d, J = 3.1 Hz), 105.3 (d, J = 29.0 Hz), 73.4, 62.6, 61.6, 46.0. MSESI m/z: 340.0949 (C₁₇H₁₆FNO₄ + Na⁺ requires 340.0956).

(R)-3-(2-Fluoro-3′-(hydroxymethyl)-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (3h).

A mixture of compound 2a (58 mg, 0.2 mmol), 3-(hydroxymethyl)-phenylboronic acid (36 mg, 0.24 mmol), potassium carbonate (110 mg, 0.8 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) complex with dichloromethane (16 mg, 0.02 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) to afford compound 3h as a brown amorphous solid (55 mg, 87%). ¹HNMR (400 MHz, methanol-d₄) δ 7.63 (dd, J = 13.3, 2.3 Hz, 1H), 7.54–7.47 (m, 2H), 7.44–7.33 (m, 4H), 4.81–4.73 (m, 1H), 4.66 (s, 2H), 4.16 (t, J = 9.0 Hz, 1H), 3.97 (dd, J = 9.0, 6.4 Hz, 1H), 3.87 (dd, J = 12.6, 3.2 Hz, 1H), 3.71 (dd, J = 12.6, 4.0 Hz, 1H). ¹³C NMR (101 MHz, methanol-d₄) δ 161.0 (d, J = 246.4 Hz), 156.9, 143.1, 140.6 (d, J = 11.1 Hz), 136.7 (d, J = 1.0 Hz), 132.0 (d, J = 5.1 Hz), 129.6, 128.8 (d, J = 3.0 Hz), 128.4 (d, J = 3.0 Hz), 127.2, 125.5 (d, J = 13.1 Hz), 115.0 (d, J = 3.0 Hz), 107.2 (J = 29,3 Hz), 75.2, 65.1, 63.3, 47.6.

(R)-3-(2-Fluoro-3′-((trifluoromethyl)sulfonyl)-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (3i).

Using general procedure 1, employing 4,4,5,5-tetramethyl-2-(3-trifluoromethanesulfonylphenyl)-1,3,2-dioxaborolane (40 mg, 0.12 mmol), compound 3i was obtained after flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) as a foamy yellow amorphous solid (34 mg, 81%). ¹H NMR (400 MHz, acetone-d₆) δ 8.26 (s, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.14 (d, J = 7.9 Hz, 1H), 7.98 (t, J = 7.9 Hz, 1H), 7.78 (dd, J = 13.9, 2.3 Hz, 1H), 7.69 (t, J = 8.8 Hz, 1H), 7.55 (dd, J = 8.8, 2.3 Hz, 1H), 4.93–4.74 (m, 1H), 4.44 (s, 1H), 4.26 (t, J = 8.9 Hz, 1H), 4.08 (dd, J = 8.9, 6.2 Hz, 1H), 3.92 (dd, J = 12.4, 3.3 Hz, 1H), 3.79 (dd, J = 12.4, 3.8 Hz, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 159.7 (d, J = 245.7 Hz), 154.4, 141.4 (d, J = 11.4 Hz), 137.7 (d, J = 1.6 Hz), 137.3 (d, J = 3.0 Hz), 131.3 (d, J = 1.6 Hz), 130.9, 130.9, 130.3 (d, J = 4.2 Hz), 129.5, 120.2 (d, J = 13.3 Hz), 119.9 (q, J = 325.3 Hz), 113.8 (d, J = 3.1 Hz), 105.4 (d, J = 29.0 Hz), 73.5, 62.2, 46.1.

(R)-3-(2-Fluoro-2′-(trifluoromethoxy)-[1,1’-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (3j).

Using general procedure 1, employing 2-(trifluoromethoxy)-phenylboronic acid (25 mg, 0.12 mmol), compound 3j was obtained after flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) as a yellow oil (32 mg, 86%). ¹H NMR (400 MHz, acetone-d₆) δ 7.72 (dd, J = 12.8, 2.2 Hz, 1H), 7.59–7.44 (m, 5H), 7.41 (t, J = 8.4 Hz, 1H), 4.98–4.75 (m, 1H), 4.44 (t, J = 5.9 Hz, 1H), 4.24 (t, J = 8.9 Hz, 1H), 4.06 (dd, J = 8.9, 6.2 Hz, 1H), 3.99–3.86 (m, 1H), 3.84–3.73 (m, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.4 (d, J = 244.8 Hz), 155.3, 147.5, 141.8 (d, J = 11.2 Hz), 133.1, 132.6 (d, J = 4.6 Hz), 130.7, 129.9, 128.2, 121.7 (d, J = 1.6 Hz), 121.3 (q, J = 256.3 Hz), 119.4 (d, J = 16.4 Hz), 113.9 (d, J = 3.2 Hz), 105.6 (d, J = 28.7 Hz), 74.3, 63.2, 46.9.

Methyl (R)-2′-Fluoro-4′-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-[1,1′-biphenyl]-2-carboxylate (3k).

Using general procedure 1, employing 2-methoxycarbonylphenylboronic acid (22 mg, 0.12 mmol), compound 3k was obtained after flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) followed by preparative TLC (eluent 100% EtOAc) as a yellow oil (33 mg, 94%). ¹H NMR (400 MHz, acetone) δ 7.93 (d, J = 7.6 Hz, 1H), 7.74–7.59 (m, 2H), 7.52 (t, J = 7.6 Hz, 1H), 7.45–7.31 (m, 3H), 4.89–4.77 (m, 1H), 4.46 (s, 1H), 4.23 (t, J = 8.8 Hz, 1H), 4.04 (dd, J = 8.8, 6.2 Hz, 1H), 3.91 (dd, J = 12.4, 3.3 Hz, 1H), 3.79 (dd, J = 12.4, 3.8 Hz, 1H), 3.68 (s, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 168.2, 160.3 (d, J = 242.6 Hz), 155.3, 141.0 (d, J = 11.1 Hz), 136.7, 132.7, 132.3, 132.1, 131.5 (d, J = 5.0 Hz), 130.7, 128.7, 124.3 (d, J = 16.1 Hz), 113.7 (d, J = 3.1 Hz), 105.2 (d, J = 28.9 Hz), 74.3, 63.2, 52.2, 46.9.

(R)-3-(2-Fluoro-2′,4′-diisopropyl-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (3l).

Using general procedure 1, employing [2,4-bis(propan-2-yl)phenyl]-boronic acid (25 mg, 0.12 mmol), compound 3l was obtained after flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) as a white amorphous solid (33 mg, 89%). ¹H NMR (400 MHz, acetone-d₆) δ 7.67 (dd, J = 12.3, 2.3 Hz, 1H), 7.43 (dd, J = 8.5, 2.3 Hz, 1H), 7.32 (s, 1H), 7.27 (t, J = 8.5 Hz, 1H), 7.13 (d, J = 7.9, 1H), 7.6 (d, J = 7.9 Hz, 1H), 4.91–4.71 (m, 1H), 4.40 (t, J = 5.9 Hz, 1H), 4.24 (t, J = 8.8 Hz, 1H), 4.05 (dd, J = 8.8, 6.2 Hz, 1H), 3.92 (ddd, J = 12.4, 5.9, 3.5 Hz, 1H), 3.79 (ddd, J = 12.4, 5.9, 4.0 Hz, 1H), 2.96 (p, J = 7.0 Hz, 1H), 2.82–2.78 (m, 1H), 1.28 (d, J = 7.0 Hz, 6H), 1.25–1.01 (m, 6H). ¹H NMR (400 MHz, acetone) δ 7.67 (dd, J = 12.3, 2.3 Hz, 1H), 7.43 (dd, J = 8.5, 2.2 Hz, 1H), 7.32 (d, J = 1.9 Hz, 1H), 7.27 (t, J = 8.5 Hz, 1H), 7.13 (dd, J = 7.8, 1.8 Hz, 1H), 7.06 (d, J = 7.8 Hz, 1H), 4.89–4.75 (m, 1H), 4.40 (t, J = 5.9 Hz, 1H), 4.24 (t, J = 8.8 Hz, 1H), 4.5 (dd, J = 8.8, 6.2 Hz, 1H), 3.92 (ddd, J = 12.4, 5.9, 3.5 Hz, 1H), 3.79 (ddd, J = 12.4, 5.9, 4.0 Hz, 1H), 2.96 (p, J = 6.9 Hz, 1H), 2.84–2.77 (m, 1H), 1.28 (d, J = 6.9 Hz, 6H), 1.22–1.05 (m, 6H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.6 (d, J = 241.5 Hz), 155.4, 149.8, 148.0, 140.9 (d, J = 10.7 Hz), 132.9 (d, J = 5.0 Hz), 132.5, 131.2, 124.6 (d, J = 17.6 Hz), 124.4 (d, J = 5.2 Hz), 113.9 (d, J = 3.2 Hz), 105.7 (d, J = 29.1 Hz), 74.3, 63.3, 47.1, 35.0, 31.1, 24.5.

(R)-5-(Hydroxymethyl)-3-(2,3′,5′-trifluoro-[1,1′-biphenyl]-4-yl)-oxazolidin-2-one (3m).

A mixture of compound 2a (58 mg, 0.2 mmol), 3,5-difluorophenylboronic acid (38 mg, 0.24 mmol), potassium carbonate (110 mg, 0.8 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) complex with dichloromethane (16 mg, 0.02 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) to afford compound 3m as a yellow amorphous solid (48 mg, 66%). ¹H NMR (300 MHz, acetone) δ 7.73 (dd, J = 13.9, 2.3 Hz, 1H), 7.62 (t, J = 8.8 Hz, 1H), 7.53–7.44 (m, 1H), 7.34–7.18 (m, 2H), 7.05 (tt, J = 9.2, 2.3 Hz, 1H), 4.95–4.71 (m, 1H), 4.47–4.37 (m, 1H), 4.25 (t, J = 8.8 Hz, 1H), 4.06 (dd, J = 8.8, 6.2 Hz, 1H), 3.99–3.85 (m, 1H), 3.84–3.67 (m, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 163.9 (dd, J = 246.4, 13.1 Hz, 2C), 160.5 (d, J = 246.4 Hz), 155.3, 142.0 (d, J = 12.1 Hz), 139. 7 (td, J = 10.1, 1.0 Hz), 131.6 (d, J = 5.1 Hz), 114.5 (d, J = 3.0 Hz), 112.5 (dd, J = 26.3, 3.0 Hz, 2C), 112.5 (d, J = 12.1, 3.0 Hz), 106.3 (d, J = 29.3 Hz), 103.5 (t, J = 26.3 Hz), 74.4, 63.2, 47.0.

(R)-3-(4-(Benzo[d][1,3]dioxol-5-yl)-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one (3n).

A mixture of compound 2a (58 mg, 0.2 mmol), 3,4-(methylenedioxy)-phenylboronic acid (40 mg, 0.24 mmol), potassium carbonate (110 mg, 0.8 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) complex with dichloromethane (16 mg, 0.02 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–67% EtOAc in hexanes) to afford compound 3n as a brown amorphous solid (48 mg, 64%). ¹H NMR (300 MHz, acetone-d₆) δ 7.67 (dd, J = 13.7, 2.2 Hz, 1H), 7.55–7.35 (m, 2H), 7.12–7.01 (m, 2H), 6.99–6.83 (m, 1H), 6.05 (s, 2H), 4.93–4.68 (m, 1H), 4.39 (t, J = 5.9 Hz, 1H), 4.22 (t, J = 8.9 Hz, 1H), 4.3 (dd, J = 8.9, 6.2 Hz, 1H), 3.91 (ddd, J = 12.3, 5.9, 3.4 Hz, 1H), 3.78 (ddd, J = 12.3, 5.9, 3.9 Hz, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.5 (d, J = 245.4 Hz), 155.4, 148.9, 148.3, 140.7 (d, J = 11.1 Hz), 131.6 (d, J = 5.1 Hz), 130.1 (d, J = 1.0 Hz), 124.1 (d, J = 14.1 Hz), 123.4 (d, J = 3.0 Hz), 114.4 (d, J = 3.0 Hz), 110.0 (d, J = 4.0 Hz), 109.3, 106.4 (d, J = 29.3 Hz), 102.4, 74.4, 63.3, 47.1.

(R)-3-(3-Fluoro-4-(1H-indol-7-yl)phenyl)-5-(hydroxymethyl)-oxazolidin-2-one (3o).

Using general procedure 1, employing 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (29 mg, 0.12 mmol), compound 3o was obtained after flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) as a brown amorphous solid (25 mg, 76%). ¹H NMR (400 MHz, acetone-d₆) δ 10.16 (s, 1H), 7.74 (dd, J = 13.0, 2.2 Hz, 1H), 7.63 (t, J = 4.5 Hz, 1H), 7.55 (t, J = 8.4 Hz, 1H), 7.45 (dd, J = 8.4, 2.2 Hz, 1H), 7.34 (t, J = 2.6 Hz, 1H), 7.19–6.98 (m, 2H), 6.56 (t, J = 2.6 Hz, 1H), 5.04–4.75 (m, 1H), 4.44 (s, 1H), 4.25 (t, J = 8.8 Hz, 1H), 4.07 (dd, J = 8.8, 6.1 Hz, 1H), 3.93 (dd, J = 12.4, 3.3 Hz, 1H), 3.79 (dd, J = 12.4, 3.8 Hz, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.8 (d, J = 244.3 Hz), 155.4, 141.0 (d, J = 11.0 Hz), 135.3, 132.6 (d, J = 5.6 Hz), 129.6, 126.1, 123.4, 122.1 (d, J = 16.4 Hz), 121.1, 120.2, 120.2, 114.4 (d, J = 3.1 Hz), 106.3 (d, J = 28.8 Hz), 102.7, 74.3, 63.2, 47.1.

(R)-3-(3-Fluoro-4-(2-(pyrrolidin-1-yl)pyridin-4-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (8a).

Using general procedure 1, employing 2-(pyrrolidino)pyridine-4-boronic acid pinacol ester (33 mg, 0.12 mmol), compound 8a was obtained after flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) followed by washing with acetone (1 mL) as a gray amorphous solid (29 mg, 81%). 1H NMR (600 MHz, acetone-d6) δ 8.12 (d, J = 5.2 Hz, 1H), 7.70 (dd, J = 13.7, 2.2 Hz, 1H),7.60 (t, J = 8.7 Hz, 1H), 7.51–7.44 (m, 1H), 6.70 (d, J = 5.2 Hz, 1H), 6.55 (s, 1H), 4.91–4.75 (m, 1H), 4.39 (t, J = 5.9 Hz, 1H), 4.24 (t, J = 8.7 Hz, 1H), 4.3 (dd, J = 8.7, 6.2 Hz, 1H), 3.95–3.85 (m, 1H), 3.82–3.72 (m, 1H), 3.63–3.37 (m, 4H), 2.02–1.88 (m, 4H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.9 (d, J = 246.0 Hz), 158.9, 155.4, 149.3, 144.6 (d, J = 1.0 Hz), 141.8 (d, J = 11.4 Hz), 131.6 (d, J = 4.9 Hz), 122.9 (d, J = 13.2 Hz), 114.5 (d, J = 3.1 Hz), 112.1 (d, J = 3.4 Hz), 106.6, 106.4 (d, J = 26.9 Hz), 74.5, 63.3, 47.4, 47.1, 26.3. MSESI m/z: 358.1572 (C₁₉H₂₀FN₃O₃ + H⁺ requires 358.1561).

Methyl (R)-5-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)nicotinate (8b).

Using general procedure 1, employing methyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinate (32 mg, 0.12 mmol), compound 8b was obtained after flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) as a yellow amorphous solid (27 mg, 77%). ¹H NMR (600 MHz, acetone-d₆) δ 9.11 (d, J = 1.9 Hz, 1H), 8.99 (d, J = 1.9 Hz, 1H), 8.46 (s, 1H), 7.79 (dd, J = 13.7, 2.3 Hz, 1H), 7.69 (t, J = 8.7 Hz, 1H), 7.55 (dd, J = 8.7, 2.3 Hz, 1H), 4.90–4.75 (m, 1H), 4.40 (t, J = 5.9 Hz, 1H), 4.27 (t, J = 8.8 Hz, 1H), 4.08 (dd, J = 8.8, 6.1 Hz, 1H), 3.96 (s, 3H), 3.95–3.89 (m, 1H), 3.82–3.73 (m, 1H). ¹³C NMR (151 MHz, acetone-d₆) δ 166.3, 160.9 (d, J = 245.6 Hz), 155.4, 154.0 (d, J = 3.6 Hz), 150.1, 142.4 (d, J = 11.3 Hz), 137.3 (d, J = 3.5 Hz), 132.2 (d, J = 1.5 Hz), 131.9 (d, J = 4.2 Hz), 127.0, 119.8 (d, J = 13.9 Hz), 114.9 (d, J = 3.4 Hz), 106.5 (d, J = 28.8 Hz), 74.6, 63.3, 53.0, 47.2. MSESI m/z: 369.0856 (C₁₇H₁₅FN₂O₅ + Na⁺ requires 369.0857).

(R)-5-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)nicotinic Acid (8c).

Lithium hydroxide monohydrate (42 mg, 1 mmol) was added to a solution of 8b (35 mg, 0.1 mmol) in THF (1.5 mL), MeOH (0.5 mL), and H₂O (0.5 mL). The mixture was stirred at 25 °C. After the reaction was judged to be completed by TLC (1 h), it was acidified to pH = 1 with the addition of formic acid and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent 10% MeOH in DCM and then 10% MeOH in DCM with 1% formic acid) to give compound 8c as a yellow amorphous solid (19 mg, 56%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.05 (s, 1H), 8.92 (s, 1H), 8.37 (s, 1H), 8.21 (s, 1H), 7.79–7.63 (m, 2H), 7.55–7.41 (m, 1H), 4.81–4.70 (m, 1H), 4.15 (t, J = 8.9 Hz, 1H), 3.90 (dd, J = 8.9, 5.9 Hz, 1H), 3.70 (dd, J = 12.4, 3.2 Hz, 1H), 3.58 (dd, J = 12.4, 3.9 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 163.5, 159.3 (d, J = 245.2 Hz), 154.4, 151.9 (d, J = 3.6 Hz), 149.2, 140.5 (d, J = 11.4 Hz), 136.3, 131.0 (d, J = 4.1 Hz), 130.3, 128.1, 118.6 (d, J = 13.4 Hz), 114.0 (d, J = 3.0 Hz), 105.3 (d, J = 28.4 Hz), 73.5, 61.7, 46.0. MSESI m/z: 355.0733 (C₁₆H₁₃FN₂O₅ + Na⁺ requires 355.0701).

(R)-3-(4-Bromo-3-fluorophenyl)-5-((methoxymethoxy)methyl)-oxazolidin-2-one (4).

To a solution of compound 2a (300 mg, 1.04 mmol) in DCM (5 mL), N,N-diisopropylethylamine (0.54 mL, 3.11 mmol) and methyl chloromethyl ether (0.24 mL, 3.11 mmol) were added. The reaction mixture was stirred at 25 °C. After the reaction was judged to be completed by TLC (5 h), it was diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–67% EtOAc in hexanes) to afford compound 4 as a white amorphous solid (296 mg, 85%). ¹H NMR (300 MHz, acetone-d₆) δ 7.74 (dd, J = 11.6, 2.6 Hz, 1H), 7.64 (dd, J = 8.9, 8.0 Hz, 1H), 7.37 (ddd, J = 8.9, 2.6, 1.0 Hz, 1H), 4.95 (dddd, J = 9.0, 6.2, 4.4, 3.5 Hz, 1H), 4.66 (s, 2H), 4.26 (t, J = 9.0 Hz, 1H), 4.00 (dd, J = 9.0, 6.2 Hz, 1H), 3.86 (dd, J = 11.3, 3.5 Hz, 1H), 3.80 (dd, J = 11.3, 4.4 Hz, 1H), 3.32 (s, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 159.7 (d, J = 243.1 Hz), 155.0, 141.2 (d, J = 10.0 Hz), 134.3 (d, J = 1.8 Hz), 115.6 (d, J = 3.4 Hz), 106.9 (d, J = 28.2 Hz), 102.1 (d, J = 21.1 Hz), 97.3, 72.7, 68.6, 55.4, 47.4.

General Procedure 2: Synthesis of Heterocyclic Oxazolidinone Analogues 3p–r.

A mixture of compound 4 (67 mg, 0.2 mmol), aryl boronic acid (0.24 mmol), potassium carbonate (110 mg, 0.8 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (16 mg, 0.02 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–40% EtOAc in hexanes) to afford compound 5. Compound 5 (25 mg) was added to HCl/dioxane (4 M, 1 mL), and the mixture was stirred at 25 °C. After the reaction was judged to be completed by TLC (1 h), its solvent was evaporated (the residue was purified by flash column chromatography (SiO₂) if necessary) to afford compounds 3p–r.

(R)-3-(2-Fluoro-4′-methyl-[1,1′-biphenyl]-4-yl)-5-((methoxymethoxy)methyl)oxazolidin-2-one (5a).

Using general procedure 2, employing 4-methylphenylboronic acid (33 mg, 0.24 mmol), compound 5a was obtained as a yellow amorphous solid (68 mg, 98%). ¹H NMR (300 MHz, chloroform-d) δ 7.51 (dd, J = 12.8, 2.3 Hz, 1H), 7.47–7.38 (m, 3H), 7.34 (dd, J = 8.6, 2.3 Hz, 1H), 7.28–7.21 (m, 2H), 4.92–4.74 (m, 1H), 4.69 (s, 2H), 4.33–4.04 (m, 1H), 3.96 (dd, J = 8.7, 6.2 Hz, 1H), 3.85 (dd, J = 11.1, 4.2 Hz, 1H), 3.78 (dd, J = 11.1, 4.1 Hz, 1H), 3.39 (s, 3H), 2.40 (s, 3H).

(R)-3-(2-Fluoro-4′-methyl-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (3p).

Using general procedure 2, employing 5a (25 mg, 0.072 mmol), compound 3p was obtained (without flash column chromatography) as a yellow amorphous solid (22 mg, 100%). ¹H NMR (300 MHz, methanol-d₄) δ 7.61 (dd, J = 13.4, 2.2 Hz, 1H), 7.50–7.32 (m, 4H), 7.25 (d, J = 7.9 Hz, 2H), 4.83–4.71 (m, 1H), 4.16 (t, J = 8.9 Hz, 1H), 3.96 (dd, J = 8.9, 6.4 Hz, 1H), 3.87 (dd, J = 12.5, 3.2 Hz, 1H), 3.71 (dd, J = 12.5, 4.0 Hz, 1H), 2.38 (s, 3H). ¹³C NMR (101 MHz, methanol-d₄) δ 161.0 (d, J = 245.2 Hz), 156.9, 140.3 (d, J = 11.0 Hz), 138.5, 133.7, 131.8 (d, J = 5.0 Hz), 130.2 (2C), 129.7 (d, J = 3.2 Hz, 2C), 125.5 (d, J = 13.8 Hz), 115.0 (d, J = 3.4 Hz), 107.2 (d, J = 29.3 Hz), 75.2, 63.3, 47.6, 21.2.

(R)-3-(2,4′-Difluoro-[1,1′-biphenyl]-4-yl)-5-((methoxymethoxy)-methyl)oxazolidin-2-one (5b).

A mixture of compound 4 (50 mg, 0.15 mmol), 4-fluorophenylboronic acid (25 mg, 0.18 mmol), potassium carbonate (83 mg, 0.6 mmol), and [1,1’-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (12 mg, 0.015 mmol) in dioxane/H₂O (v/v = 9:1, 0.4 mL) was stirred at 90 °C under a nitrogen atmosphere for 2 h. The mixture was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–40% EtOAc in hexanes) to afford compound 5b as a yellow amorphous solid (45 mg, 85%). ¹H NMR (300 MHz, CDCl₃) δ 7.63–7.32 (m, 5H), 7.21–7.08 (m, 2H), 4.93–4.76 (m, 1H), 4.69 (s, 2H), 4.10 (t, J = 8.8 Hz, 1H), 3.97 (dd, J = 8.8, 6.2 Hz, 1H), 3.91–3.73 (m, 2H), 3.39 (s, 3H).

(R)-3-(2,4′-Difluoro-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl-oxazolidin-2-one (3q).

Using general procedure 2, employing 5b (25 mg, 0.072 mmol), compound 3q was obtained after flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) as a white amorphous solid (11 mg, 50%). ¹H NMR (400 MHz, acetone-d₆) δ 7.70 (dd, J = 13.7, 2.2 Hz, 1H), 7.65–7.57 (m, 2H), 7.57–7.50 (m, 1H), 7.46 (dd, J = 8.6, 2.2 Hz, 1H), 7.35–7.11 (m, 2H), 4.93–4.76 (m, 1H), 4.52–4.31 (m, 1H), 4.23 (t, J = 9.0 Hz, 1H), 4.11–3.98 (m, 1H), 3.96–3.85 (m, 1H), 3.82–3.71 (m, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 163.2 (d, J = 245.2 Hz), 160.4 (d, J = 244.2 Hz), 155.3, 141.1 (d, J = 11.1 Hz), 132. 5 (dd, J = 3.0, 1.0 Hz), 131.6 (d, J = 3.8 Hz, 2C), 131.5 (d, J = 3.1 Hz), 123. 1 (d, J = 13.9 Hz), 116.2 (d, J = 21.6 Hz, 2C), 114.4 (d, J = 3.2 Hz), 106.3 (d, J = 29.1 Hz), 74.3, 63.2, 47.0.

(R)-3-(2-Fluoro-4′-methoxy-[1,1′-biphenyl]-4-yl)-5-((methoxymethoxy)methyl)oxazolidin-2-one (5c).

Using general procedure 2, employing 4-methoxyphenylboronic acid (36 mg, 0.24 mmol), compound 5c was obtained as a yellow amorphous solid (66 mg, 90%). ¹H NMR (300 MHz, chloroform-d) δ 7.57–7.30 (m, 5H), 6.98 (d, J = 8.7 Hz, 2H), 4.83 (ddt, J = 8.6, 6.2, 4.2 Hz, 1H), 4.69 (s, 2H), 4.10 (t, J = 8.6 Hz, 1H), 3.96 (dd, J = 8.6, 6.2 Hz, 1H), 3.93–3.63 (m, 5H), 3.39 (s, 3H).

(R)-3-(2-Fluoro-4′-methoxy-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (3r).

Using general procedure 2, employing 5c (25 mg, 0.069 mmol), compound 3r was obtained (without flash column chromatography) as a yellow amorphous solid (22 mg, 100%). ¹H NMR (300 MHz, acetone-d₆) δ 7.67 (dd, J = 13.7, 2.3 Hz, 1H), 7.55–7.38 (m, 4H), 7.09–6.98 (m, 2H), 4.91–4.72 (m, 1H), 4.39 (dd, J = 6.2, 5.6 Hz, 1H), 4.22 (t, J = 8.9 Hz, 1H), 4.03 (dd, J = 8.9, 6.3 Hz, 1H), 3.95–3.87 (m, 1H), 3.84 (s, 3H), 3.83–3.73 (m, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.4 (d, J = 245.4 Hz), 160.3, 155.3, 140.4 (d, J = 11.1 Hz), 131.4 (d, J = 5.0 Hz), 130.7 (d, J = 3.1 Hz, 2C), 128.4 (d, J = 1.3 Hz), 124.0 (d, J = 13.7 Hz), 114.9 (2C), 114.4 (d, J = 3.3 Hz), 106.3 (d, J = 29.3 Hz), 74.3, 63.2, 55.6, 47.0. MSESI m/z: 340.0943 (C₁₇H₁₆FNO₄ + Na⁺ requires 340.0956).

(R)-3-(3-Fluoro-4-iodophenyl)-5-(hydroxymethyl)oxazolidin-2-one (6).³⁸

N-Iodosuccinimide (6.31 g, 28.0 mmol) was added to a solution of compound 2b (5.64 g, 26.7 mmol) in TFA (130 mL). The mixture was stirred at 25 °C. After the reaction was judged to be completed by TLC (2 h), it was concentrated under reduced pressure by rotary evaporation. The residue was dissolved in EtOAc and washed with saturated aqueous sodium carbonate until the aqueous phase was basic. The organic layer was evaporated under reduced pressure by rotary evaporation, and the residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) to afford compound 6 as a gray amorphous solid (6.9 g, 77%). ¹H NMR (400 MHz, acetone-d₆) δ 7.87 (dd, J = 8.8, 7.4 Hz, 1H), 7.67 (dd, J = 11.0, 2.5 Hz, 1H), 7.27 (ddd, J = 8.8, 2.5, 0.7 Hz, 1H), 4.80 (dddd, J = 9.3, 6.2, 4.1, 3.3 Hz, 1H), 4.13 (t, J = 9.3 Hz, 1H), 4.03–3.84 (m, 2H), 3.80–3.70 (m, 1H), 3.33 (t, J = 5.9 Hz, 1H).

(R)-3-(3-Fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-phenyl)-5-(hydroxymethyl)oxazolidin-2-one (7).³⁸

To a solution of compound 6 (3.37 g, 10 mmol) in anhydrous DMSO (40 mL), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium-(II) complex with dichloromethane (408 mg, 0.5 mmol), potassium acetate (4.91 g, 50 mmol), and bis(pinacolato)diboron (5.08 g, 20 mmol) were added. The reaction mixture was stirred at 80 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (16 h), it was cooled to room temperature, treated with the addition of MeOH (20 mL), and filtered through celite. The filtrate was concentrated under reduced pressure by rotary evaporation to remove MeOH, dissolved in EtOAc, washed with water, and evaporated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) to afford compound 7 as a yellow amorphous solid (1.64 g, 49%). ¹H NMR (400 MHz, acetone-d₆) δ 7.75–7.67 (m, 1H), 7.55 (dd, J = 12.2, 2.0 Hz, 1H), 7.37 (dd, J = 8.3, 2.0 Hz, 1H), 4.91–4.74 (m, 1H), 4.40 (t, J = 5.9 Hz, 1H), 4.20 (t, J = 8.9 Hz, 1H), 4.01 (dd, J = 8.9, 6.2 Hz, 1H), 3.90 (ddd, J = 12.3, 5.9, 3.3 Hz, 1H), 3.76 (ddd, J = 12.3, 5.9, 4.0 Hz, 1H), 1.33 (s, 12H).

General Procedure 3: Synthesis of Heterocyclic Oxazolidinone Analogues 3s–x, 8d–m.

A mixture of compound 7 (34 mg, 0.1 mmol), heterocyclic bromide, potassium carbonate (55 mg, 0.4 mmol), and [1,1′-bis-(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (8.2 mg, 0.01 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 90 °C under a nitrogen atmosphere for 3 h. The reaction mixture was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) to afford compound 3s–x and 8d–m. If not pure, compounds 3s–x and 8d–m were further purified by washing with EtOAc (1 mL).

(R)-3-(2-Fluoro-4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (3s).

Using general procedure 3, employing 1-bromo-4-(trifluoromethyl)-benzene (45 mg, 0.2 mmol), compound 3s was obtained by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) as a white amorphous solid (16 mg, 45%). ¹H NMR (400 MHz, acetone-d₆) δ 7.81 (s, 4H), 7.75 (dd, J = 13.8, 2.2 Hz, 1H), 7.62 (t, J = 8.6 Hz, 1H), 7.51 (dd, J = 8.6, 2.3 Hz, 1H), 4.91–4.75 (m, 1H), 4.41 (s, 1H), 4.25 (t, J = 8.9 Hz, 1H), 4.07 (dd, J = 8.9, 6.2 Hz, 1H), 3.95–3.88 (m, 1H), 3.84–3.74 (m, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.6 (d, J = 245.5 Hz), 155.3, 141.8 (d, J = 11.4 Hz), 140.3, 131.8 (d, J = 4.5 Hz), 130.3 (d, J = 3.4 Hz, 2C), 129.7 (d, J = 32.2 Hz), 126.3 (q, J = 3.9 Hz, 2C), 125.2 (q, J = 253.6 Hz), 122.5 (d, J = 13.4 Hz), 114.5 (d, J = 3.3 Hz), 106.3 (d, J = 29.1 Hz), 74.4, 63.2, 47.0. MSESI m/z: 356.0900 (C₁₇H₁₃F₄NO₃ + H⁺ requires 356.0904).

(R)-3-(2-Fluoro-4′-(trifluoromethoxy)-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (3t).

Using general procedure 3, employing 1-bromo-4-(trifluoromethoxy)-benzene (48 mg, 0.2 mmol), compound 3t was obtained by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) as a white amorphous solid (17 mg, 46%). ¹H NMR (400 MHz, acetone-d₆) δ 7.78–7.65 (m, 3H), 7.57 (t, J = 8.7 Hz, 1H), 7.48 (dd, J = 8.7, 2.3 Hz, 1H), 7.46–7.40 (m, 2H), 4.93–4.73 (m, 1H), 4.40 (s, 1H), 4.24 (t, J = 8.9 Hz, 1H), 4.06 (dd, J = 8.9, 6.2 Hz, 1H), 3.95–3.87 (m, 1H), 3.81–3.75 (m, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.6 (d, J = 245.0 Hz), 155.4, 149.4 (d, J = 1.9 Hz), 141.5 (d, J = 11.2 Hz), 135.6 (d, J = 1.6 Hz), 131.8 (d, J = 4.6 Hz), 131.5 (d, J = 3.3 Hz, 2C), 122.7 (d, J = 13.7 Hz), 122.1 (2C), 121.6 (q, J = 255.6 Hz), 114.6 (d, J = 3.2 Hz), 106.4 (d, J = 29.0 Hz), 74.5, 63.3, 47.1. MSEI m/z: 371.0775 (C₁₇H₁₃F₄NO₃ (M⁺) requires 371.0781).

(R)-2′-Fluoro-4′-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-[1,1′-biphenyl]-4-carboxylic Acid (3u).

A mixture of compound 7 (40 mg, 0.12 mmol), 4-bromobenzoic acid (20 mg, 0.1 mmol), potassium carbonate (55 mg, 0.4 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (8.2 mg, 0.01 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), formic acid (1 mL) was added to acidify the reaction. The mixture was concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–15% MeOH in DCM with 1% formic acid). The appropriate fractions were concentrated under reduced pressure by rotary evaporation, and the resulting residue was washed with MeOH (2 mL) to give compound 3u as a white amorphous solid (10 mg, 30%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.03 (d, J = 8.4 Hz, 2H), 7.74–7.59 (m, 4H), 7.49 (dd, J = 8.6, 2.3 Hz, 1H), 5.27 (s, 1H), 4.86–4.68 (m, 1H), 4.14 (t, J = 8.9 Hz, 1H), 3.89 (dd, J = 8.9, 6.1 Hz, 1H), 3.70 (dd, J = 12.4, 3.3 Hz, 1H), 3.57 (dd, J = 12.4, 3.9 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167.1, 159.1 (d, J = 245.4 Hz), 154.4, 140.1 (d, J = 11.2 Hz), 139.0, 131.0 (d, J = 4.5 Hz), 129.8, 129.7 (2C), 128.8 (d, J = 3.3 Hz, 2C), 121.6 (d, J = 13.0 Hz), 113.9 (d, J = 3.0 Hz), 105.4 (d, J = 28.6 Hz), 73.5, 61.6, 46.0. MSESI m/z: 330.0766 (C₁₇H₁₃FNO₅⁻ requires 330.0783).

(R)-2′-Fluoro-N′-hydroxy-4′-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-[1,1′-biphenyl]-4-carboximidamide (3v).

A mixture of compound 7 (40 mg, 0.12 mmol), compound 14 (22 mg, 0.1 mmol), potassium carbonate (55 mg, 0.4 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (8.2 mg, 0.01 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–20% MeOH in DCM) and preparative TLC (eluent, 15% MeOH in DCM) to give compound 3v as a yellow amorphous solid (17 mg, 49%). ¹H NMR (300 MHz, DMSO-d₆) δ 9.71 (s, 1H), 7.85–7.72 (m, 2H), 7.68–7.52 (m, 4H), 7.46 (dd, J = 8.6, 2.2 Hz, 1H), 5.87 (s, 2H), 5.25 (t, J = 5.6 Hz, 1H), 4.80–4.68 (m, 1H), 4.14 (t, J = 9.0 Hz, 1H), 3.88 (dd, J = 9.0, 6.1 Hz, 1H), 3.70 (ddd, J = 12.3, 5.6, 3.3 Hz, 1H), 3.57 (ddd, J = 12.3, 5.6, 4.0 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.1 (d, J = 244.8 Hz), 154.4, 150.5, 139.6 (d, J = 11.2 Hz), 135.1 (d, J = 1.7 Hz), 132.6, 130.8 (d, J = 5.0 Hz), 128.3 (d, J = 3.3 Hz, 2C), 125.6 (2C), 122.1 (d, J = 13.3 Hz), 113.8 (d, J = 3.1 Hz), 105.4 (d, J = 28.8 Hz), 73.4, 61.6, 46.0. MSESI m/z: 346.1187 (C₁₇H₁₆FN₃O₄ + H⁺ requires 346.1198).

(S)-2-((tert-Butoxycarbonyl)amino)-3-(2′-fluoro-4′-((R)-5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-[1,1′-biphenyl]-3-yl)propanoic Acid (3w).

A mixture of compound 7 (80 mg, 0.24 mmol), (S)-N-boc-3-bromophenylalanine (69 mg, 0.2 mmol), potassium carbonate (110 mg, 0.8 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II), complex with dichloromethane (16 mg, 0.02 mmol) in dioxane/H₂O (v/v = 9:1, 1.0 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM with 1% formic acid) to afford compound 3w as a yellow amorphous solid (34 mg, 36%). ¹H NMR (400 MHz, acetone-d₆) δ 7.70 (dd, J = 13.6, 2.3 Hz, 1H), 7.55 (t, J = 8.7 Hz, 1H), 7.50–7.42 (m, 3H), 7.39 (t, J = 7.5 Hz, 1H), 7.30 (d, J = 7.5 Hz, 1H), 6.11 (d, J = 8.4 Hz, 1H), 4.95–4.71 (m, 1H), 4.57–4.42 (m, 1H), 4.41–4.35 (m, 1H), 4.22 (t, J = 8.9 Hz, 1H), 4.04 (dd, J = 8.9, 6.2 Hz, 1H), 3.91 (dd, J = 12.3, 3.4 Hz, 1H), 3.78 (dd, J = 12.3, 3.9 Hz, 1H), 3.29 (dd, J = 13.8, 4.9 Hz, 1H), 3.08 (dd, J = 13.8, 8.8 Hz, 1H), 1.34 (s, 9H). ¹³C NMR (101 MHz, acetone-d₆) δ 173.8, 160.5 (d, J = 245.0 Hz), 156.2, 155.3, 140.8 (d, J = 11.2 Hz), 138.9, 136.1, 131.7 (d, J = 5.0 Hz), 130.6 (d, J = 2.7 Hz), 129.4, 129.3, 127.9 (d, J = 3.6 Hz), 124.1 (d, J = 13.5 Hz), 114.3 (d, J = 3.4 Hz), 106.2 (d, J = 29.1 Hz), 79.2, 74.3, 63.1, 55.7, 46.9, 38.1, 28.5.

(S)-2-Amino-3-(2′-fluoro-4′-((R)-5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-[1,1′-biphenyl]-3-yl)propanoic Acid Hydrochloride (3x).

A mixture of compound 3w (20 mg, 0.042 mmol) in HCl/dioxane (4 M, 1 mL) was stirred at 25 °C. After the reaction was judged to be completed by TLC (30 min), its solvent was removed under reduced pressure by rotary evaporation. The resulting residue was washed with acetone (1 mL) and dried under high vacuum to give 3x as a yellow amorphous solid (16 mg, 91%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.51 (s, 3H), 7.69–7.56 (m, 2H), 7.53–7.38 (m, 4H), 7.30 (d, J = 7.4 Hz, 1H), 5.33 (s, 1H), 4.81–4.67 (m, 1H), 4.22 (t, J = 6.1 Hz, 1H), 4.13 (t, J = 8.9 Hz, 1H), 3.91 (dd, J = 8.9, 6.0 Hz, 1H), 3.69 (dd, J = 12.3, 3.3 Hz, 1H), 3.62–3.52 (m, 1H), 3.21 (d, J = 6.1 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.4, 159.0 (d, J = 244.9 Hz), 154.4, 139.5 (d, J = 11.1 Hz), 135.5, 134.9, 131.0 (d, J = 4.8 Hz), 129.8, 128.9, 128.8, 127.6 (d, J = 3.2 Hz), 122.5 (d, J = 13.1 Hz), 113.8 (d, J = 2.5 Hz), 105.4 (d, J = 28.8 Hz), 73.4, 61.6, 53.1, 46.0, 35.6. MSESI m/z: 375.1360 (C₁₉H₁₉FN₂O₅ + H⁺ requires 375.1351).

(R)-3-(3-Fluoro-4-(pyridin-4-yl)phenyl)-5-(hydroxymethyl)-oxazolidin-2-one (8d).

Using general procedure 3, employing 4-bromopyridine hydrochloride (25 mg, 0.13 mmol), compound 8d was obtained as a brown amorphous solid (21 mg, 73%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (d, J = 5.0 Hz, 2H), 7.78–7.64 (m, 2H), 7.59 (d, J = 5.0 Hz, 2H), 7.54–7.47 (m, 1H), 5.27 (t, J = 5.5 Hz, 1H), 4.82–4.70 (m, 1H), 4.14 (t, J = 8.9 Hz, 1H), 3.89 (dd, J = 8.9, 6.1 Hz, 1H), 3.74–3.65 (m, 1H), 3.63–3.52 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.4 (d, J = 247.0 Hz), 154.4, 150.0 (2C), 142.1, 140.9 (d, J = 11.4 Hz), 130.8 (d, J = 4.5 Hz), 123.1 (d, J = 3.8 Hz, 2C), 119.7 (d, J = 12.4 Hz), 113.9 (d, J = 3.0 Hz), 105.4 (d, J = 28.6 Hz), 73.5, 61.6, 46.0. MSESI m/z: 289.0965 (C₁₅H₁₃FN₂O₃ + H⁺ requires 289.0983).

(R)-3-(3-Fluoro-4-(pyridin-3-yl)phenyl)-5-(hydroxymethyl)-oxazolidin-2-one (8e).

Using general procedure 3, employing 3-bromopyridine (12.5 μL, 0.13 mmol), compound 8e was obtained as a white amorphous solid (20 mg, 70%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.76 (s, 1H), 8.63–8.53 (m, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.75–7.60 (m, 2H), 7.55–7.43 (m, 2H), 5.28 (s, 1H), 4.84–4.69 (m, 1H), 4.14 (t, J = 8.9 Hz, 1H), 3.89 (dd, J = 8.9, 6.1 Hz, 1H), 3.79–3.64 (m, 1H), 3.63–3.52 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.2 (d, J = 245.0 Hz), 154.4, 149.0 (d, J = 3.7 Hz), 148.6, 140.2 (d, J = 11.3 Hz), 136.0 (d, J = 3.2 Hz), 131.0 (d, J = 4.5 Hz), 130.6, 123.7, 119.4 (d, J = 13.7 Hz), 114.0 (d, J = 3.1 Hz), 105.3 (d, J = 28.5 Hz), 73.5, 61.6, 46.0. MSESI m/z: 289.0965 (C₁₅H₁₃FN₂O₃ + H⁺ requires 289.0983).

(R)-3-(3-Fluoro-4-(pyridin-2-yl)phenyl)-5-(hydroxymethyl)-oxazolidin-2-one (8f).

Using general procedure 3, employing 2-bromopyridine (13.1 μL, 0.13 mmol), compound 8f was obtained as a yellow amorphous solid (20 mg, 70%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (dd, J = 5.1, 1.8 Hz, 1H), 8.01 (t, J = 8.9 Hz, 1H), 7.96–7.85 (m, 1H), 7.83–7.75 (m, 1H), 7.65 (dd, J = 14.4, 2.2 Hz, 1H), 7.49 (dd, J = 8.7, 2.2 Hz, 1H), 7.46–7.35 (m, 1H), 5.28 (s, 1H), 4.84–4.67 (m, 1H), 4.15 (t, J = 8.9 Hz, 1H), 3.89 (dd, J = 8.9, 6.1 Hz, 1H), 3.75–3.65 (m, 1H), 3.62–3.54 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.9 (d, J = 246.9 Hz), 154.4, 152.0 (d, J = 2.9 Hz), 149.8, 140.6 (d, J = 11.4 Hz), 137.0, 131.1 (d, J = 4.5 Hz), 123.8 (d, J = 9.5 Hz), 122.7, 121.3 (d, J = 11.8 Hz), 113.6 (d, J = 2.9 Hz), 105.2 (d, J = 29.2 Hz), 73.5, 61.6, 46.0. MSESI m/z: 289.0980 (C₁₅H₁₃FN₂O₃ + H⁺ requires 289.0983).

(R)-3-(4-(2-Aminopyrimidin-5-yl)-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one (8g).

A mixture of compound 7 (34 mg, 0.1 mmol), 2-amino-5-iodopyrimidine (33 mg, 0.15 mmol), sodium carbonate (23 mg, 0.2 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) complex with dichloromethane (8 mg, 0.01 mmol) in dioxane/H₂O (v/v = 9:1, 0.5 mL) was stirred at 130 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (2.5 h), it was cooled to room temperature and concentrated under reduced pressure by rotary evaporation. The resulting residue was purified by flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) to afford compound 8g as a gray amorphous solid (6 mg, 20%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.47–8.33 (m, 2H), 7.62 (dd, J = 13.6, 2.3 Hz, 1H), 7.57 (t, J = 8.6 Hz, 1H), 7.41 (dd, J = 8.6, 2.3 Hz, 1H), 6.87 (s, 2H), 5.26 (s, 1H), 4.81–4.64 (m, 1H), 4.12 (t, J = 8.9 Hz, 1H), 3.86 (dd, J = 8.9, 6.1 Hz, 1H), 3.73–3.65 (m, 1H), 3.61–3.51 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.7, 158.9 (d, J = 243.5 Hz), 157.3 (d, J = 3.9 Hz, 2C), 154.4, 139.1 (d, J = 11.1 Hz), 129.7 (d, J = 4.9 Hz), 117.7 (d, J = 14.2 Hz), 117.0 (d, J = 2.2 Hz), 113.9 (d, J = 3.1 Hz), 105.4 (d, J = 28.5 Hz), 73.4, 61.6, 46.0. MSESI m/z: 305.1038 (C₁₄H₁₃FN₄O₃ + H⁺ requires 305.1044).

(R)-3-(tert-Butoxy)-5-(2-fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)picolinonitrile (8h).

A mixture of compound 7 (121 mg, 0.36 mmol), compound 15 (77 mg, 0.3 mmol), potassium carbonate (166 mg, 1.2 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (24 mg, 0.03 mmol) in dioxane/H₂O (v/v = 9:1, 1.5 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) to afford compound 8h as a yellow amorphous solid (46 mg, 61%). ¹H NMR (400 MHz, acetone-d₆) δ 8.59 (d, J = 1.6 Hz, 1H), 7.94 (d, J = 1.6 Hz, 1H), 7.78 (dd, J = 13.9, 2.2 Hz, 1H), 7.71 (t, J = 8.8 Hz, 1H), 7.54 (dd, J = 8.8, 2.2 Hz, 1H), 4.95–4.76 (m, 1H), 4.42 (t, J = 5.8 Hz, 1H), 4.26 (t, J = 8.8 Hz, 1H), 4.8 (dd, J = 8.8, 6.1 Hz, 1H), 3.92 (ddd, J = 12.3, 5.8, 3.3 Hz, 1H), 3.78 (ddd, J = 12.3, 5.8, 3.7 Hz, 1H), 1.55 (s, 9H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.8 (d, J = 246.4 Hz), 156.4, 155.2, 145.3 (d, J = 3.9 Hz), 142.7 (d, J = 11.4 Hz), 136.2 (d, J = 2.2 Hz), 131.9 (d, J = 4.2 Hz), 130.4 (d, J = 4.0 Hz), 128.3, 118.8 (d, J = 13.4 Hz), 116.9, 114.6 (d, J = 3.1 Hz), 106.2 (d, J = 28.8 Hz), 84.4, 74.4, 63.1, 46.9, 29.0. MSESI m/z: 386.1517 (C₂₀H₂₀FN₃O₄ + H⁺ requires 386.1511).

(R)-3-(3-Fluoro-4-(isoquinolin-6-yl)phenyl)-5-(hydroxymethyl)-oxazolidin-2-one (8i).

Using general procedure 3, employing 6-bromoisoquinoline (42 mg, 0.2 mmol), compound 8i was obtained as a yellow amorphous solid (20 mg, 59%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.35 (s, 1H), 8.54 (d, J = 5.7 Hz, 1H), 8.21 (d, J = 8.6 Hz, 1H), 8.15 (s, 1H), 7.96–7.85 (m, 2H), 7.80–7.64 (m, 2H), 7.60–7.47 (m, 1H), 5.27 (s, 1H), 4.83–4.70 (m, 1H), 4.17 (t, J = 8.9 Hz, 1H), 3.91 (dd, J = 8.9, 6.1 Hz, 1H), 3.75–3.67 (m, 1H), 3.64–3.57 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.3 (d, J = 245.6 Hz), 154.4, 152.1, 143.3, 140.2 (d, J = 11.3 Hz), 136.7, 135.3, 131.3 (d, J = 4.5 Hz), 128.2 (d, J = 3.2 Hz), 127.9, 127.2, 126.0 (d, J = 3.4 Hz), 121.8 (d, J = 13.2 Hz), 120.5, 113.9 (d, J = 3.3 Hz), 105.4 (d, J = 28.7 Hz), 73.5, 61.6, 46.0. MSESI m/z: 339.1135 (C₁₉H₁₅FN₂O₃ + H⁺ requires 339.1139).

(R)-3-(3-Fluoro-4-(isoquinolin-7-yl)phenyl)-5-(hydroxymethyl)-oxazolidin-2-one (8j).

Using general procedure 3, employing 7-bromoisoquinoline (42 mg, 0.2 mmol), compound 8j was obtained as a yellow amorphous solid (16 mg, 47%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.39 (s, 1H), 8.54 (d, J = 5.7 Hz, 1H), 8.31 (s, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.87 (d, J = 5.7 Hz, 1H), 7.81–7.67 (m, 2H), 7.52 (dd, J = 8.5, 2.2 Hz, 1H), 5.27 (s, 1H), 4.96–4.71 (m, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.91 (dd, J = 9.0, 6.1 Hz, 1H), 3.77–3.68 (m, 1H), 3.64–3.55 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.2 (d, J = 245.2 Hz), 154.4, 152.7, 143.2, 139.9 (d, J = 11.2 Hz), 134.3, 133.6, 131.2 (d, J = 2.4 Hz), 131.2, 128.3, 127.1 (d, J = 3.3 Hz), 126.8, 121.8 (d, J = 13.2 Hz), 120.1, 113.9 (d, J = 3.1 Hz), 105.4 (d, J = 28.8 Hz), 73.4, 61.6, 46.0. MSESI m/z: 339.1130 (C₁₉H₁₅FN₂O₃ + H⁺ requires 339.1139).

(R)-3-(2-Fluoro-4′-(pyridin-4-yl)-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (8k).

Using general procedure 3, employing 4-(4-bromophenyl)pyridine (47 mg, 0.2 mmol), compound 8k was obtained as a gray amorphous solid (14 mg, 38%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.66 (d, J = 5.1 Hz, 2H), 7.92 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 5.1 Hz, 2H), 7.71 (d, J = 8.0 Hz, 2H), 7.68–7.58 (m, 2H), 7.54–7.43 (m, 1H), 5.25 (t, J = 5.7 Hz, 1H), 4.86–4.68 (m, 1H), 4.15 (t, J = 9.0 Hz, 1H), 3.90 (dd, J = 9.0, 6.1 Hz, 1H), 3.78–3.67 (m, 1H), 3.63–3.52 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.1 (d, J = 245.2 Hz), 154.4, 150.3 (2C), 146.4, 139.8 (d, J = 11.4 Hz), 136.2, 135.5, 130.8 (d, J = 4.7 Hz), 129.3 (d, J = 3.2 Hz, 2C), 127.1 (2C), 121.8 (d, J = 13.1 Hz), 121.1 (2C), 113.9 (d, J = 2.9 Hz), 105.4 (d, J = 28.7 Hz), 73.4, 61.6, 46.0. MSESI m/z: 365.1316 (C₂₁H₁₇FN₂O₃ + H⁺ requires 365.1296).

(R)-3-(2-Fluoro-4′-(pyridin-3-yl)-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (8l).

Using general procedure 3, employing 3-(4-bromophenyl)pyridine (47 mg, 0.2 mmol), compound 8l was obtained by flash column chromatography (SiO₂, eluent gradient 0–6% MeOH in DCM) as a yellow amorphous solid (17 mg, 47%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.96 (d, J = 1.9 Hz, 1H), 8.60 (dd, J = 4.7, 1.9 Hz, 1H), 8.14 (dt, J = 8.1, 1.9 Hz, 1H), 7.93–7.82 (m, 2H), 7.75–7.59 (m, 4H), 7.55–7.45 (m, 2H), 5.37–5.18 (m, 1H), 4.84–4.71 (m, 1H), 4.15 (t, J = 8.9 Hz, 1H), 3.90 (dd, J = 8.9, 6.1 Hz, 1H), 3.77–3.66 (m, 1H), 3.63–3.54 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.1 (d, J = 244.7 Hz), 154.4, 148.6, 147.6, 139.6 (d, J = 11.2 Hz), 136.2, 135.0, 134.4, 134.1, 130.8 (d, J = 4.7 Hz), 129.3 (d, J = 3.3 Hz, 2C), 127.1 (2C), 123.9, 122.0 (d, J = 13.1 Hz), 113.9 (d, J = 3.0 Hz), 105.4 (d, J = 28.8 Hz), 73.4, 61.6, 46.0. MSESI m/z: 365.1279 (C₂₁H₁₇FN₂O₃ + H⁺ requires 365.1296).

(R)-3-(2-Fluoro-4′-(pyridin-2-yl)-[1,1′-biphenyl]-4-yl)-5-(hydroxymethyl)oxazolidin-2-one (8m).

Using general procedure 3, employing 2-(4-bromophenyl)pyridine (47 mg, 0.2 mmol), compound 8m was obtained by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) as a yellow amorphous solid (24 mg, 66%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.75–8.63 (m, 1H), 8.20 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.0 Hz, 1H), 7.91 (td, J = 8.0, 1.8 Hz, 1H), 7.74–7.59 (m, 4H), 7.48 (dd, J = 8.6, 2.2 Hz, 1H), 7.38 (ddd, J = 8.6, 4.8, 1.0 Hz, 1H), 5.25 (t, J = 5.6 Hz, 1H), 4.83–4.71 (m, 1H), 4.15 (t, J = 8.9 Hz, 1H), 3.90 (dd, J = 8.9, 6.1 Hz, 1H), 3.71 (ddd, J = 12.4, 5.6, 3.3 Hz, 1H), 3.59 (ddd, J = 12.4, 5.6, 4.0 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.1 (d, J = 245.3 Hz), 155.4, 154.4, 149.6, 139.6 (d, J = 11.3 Hz), 137.8, 137.3, 135.2 (d, J = 1.7 Hz), 130.8 (d, J = 4.8 Hz), 128.9 (d, J = 3.2 Hz, 2C), 126.7 (2C), 122.7, 122.1 (d, J = 13.3 Hz), 120.3, 113.8 (d, J = 3.0 Hz), 105.4 (d, J = 28.9 Hz), 73.4, 61.6, 46.0. MSESI m/z: 365.1307 (C₂₁H₁₇FN₂O₃ + H⁺ requires 365.1296).

(R)-3-(3-Fluoro-4-(isoxazol-4-yl)phenyl)-5-(hydroxymethyl)-oxazolidin-2-one (8n).⁵¹

A mixture of compound 6 (34 mg, 0.1 mmol), isoxazole-4-boronic acid (17 mg, 0.15 mmol), potassium phosphate monohydrate (46 mg, 0.2 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II), complex with dichloromethane (8.2 mg, 0.01 mmol) in THF/H₂O (v/v = 4:1, 2 mL) was stirred at 80 °C. After the reaction was judged to be completed by TLC (16 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The resulting residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) to afford compound 8n as a yellow amorphous solid (14 mg, 50%). ¹H NMR (300 MHz, acetone-d₆) δ 9.13 (d, J = 2.2 Hz, 1H), 8.94 (d, J = 2.2 Hz, 1H), 7.85–7.66 (m, 2H), 7.51–7.41 (m, 1H), 4.82 (ddt, J = 9.0, 6.2, 3.7 Hz, 1H), 4.44 (s, 1H), 4.22 (t, J = 9.0 Hz, 1H), 4.04 (dd, J = 9.0, 6.2 Hz, 1H), 3.95–3.85 (m, 1H), 3.83–3.71 (m, 1H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.3 (d, J = 245.4 Hz), 156.3 (d, J = 8.7 Hz), 155.2, 148.9 (d, J = 2.7 Hz), 141.0 (d, J = 11.3 Hz), 129.5 (d, J = 5.3 Hz), 115.3 (d, J = 2.3 Hz), 114.4 (d, J = 3.0 Hz), 112.0 (d, J = 14.8 Hz), 106.1 (d, J = 28.2 Hz), 74.3, 63.1, 46.9. MSESI m/z: 279.0787 (C₁₃H₁₁FN₂O₄ + H⁺ requires 279.0776).

(R)-3-(3-Fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-phenyl)-5-((methoxymethoxy)methyl)oxazolidin-2-one (9).

To a solution of compound 7 (1.44 g, 4.27 mmol) in DCM (30 mL), N,N-diisopropylethylamine (2.23 mL, 12.81 mmol) and methyl chloromethyl ether (0.97 mL, 12.81 mmol) were added. The reaction mixture was stirred at 25 °C. After the reaction was judged to be completed by TLC (1 h), it was diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–66% EtOAc in hexanes) to afford compound 9 as a white amorphous solid (940 mg, 58%). ¹H NMR (400 MHz, chloroform-d) δ 7.72 (dd, J = 8.3, 6.9 Hz, 1H), 7.39 (dd, J = 11.7, 2.1 Hz, 1H), 7.28 (dd, J = 8.3, 2.1 Hz, 1H), 4.88–4.75 (m, 1H), 4.67 (s, 2H), 4.07 (t, J = 8.8 Hz, 1H), 3.93 (dd, J = 8.8, 6.3 Hz, 1H), 3.83 (dd, J = 11.2, 4.2 Hz, 1H), 3.76 (dd, J = 11.2, 4.1 Hz, 1H), 3.37 (s, 3H), 1.35 (s, 12H).

(R)-3-(tert-Butoxy)-5-(2-fluoro-4-(5-((methoxymethoxy)methyl)-2-oxooxazolidin-3-yl)phenyl)picolinonitrile (10).

A mixture of compound 9 (152 mg, 0.4 mmol), compound 15 (122 mg, 0.48 mmol), potassium carbonate (221 mg, 1.6 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (33 mg, 0.04 mmol) in dioxane/H₂O (v/v = 9:1, 2 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) to afford compound 10 as a yellow amorphous solid (154 mg, 90%). ¹H NMR (400 MHz, acetone) δ 8.59 (t, J = 1.6 Hz, 1H), 7.94 (t, J = 1.6 Hz, 1H), 7.78 (dd, J = 13.8, 2.3 Hz, 1H), 7.71 (t, J = 8.6 Hz, 1H), 7.56 (dd, J = 8.6, 2.3 Hz, 1H), 5.05–4.90 (m, 1H), 4.67 (s, 2H), 4.33 (t, J = 9.0 Hz, 1H), 4.07 (dd, J = 9.0, 6.1 Hz, 1H), 3.94–3.75 (m, 2H), 3.33 (s, 3H), 1.56 (s, 9H).

(R)-3-(4-(6-(Aminomethyl)-5-hydroxypyridin-3-yl)-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one Hydrochloride (8o).

To a solution of compound 10 (86 mg, 0.2 mmol) in methanol (3 mL) in an ice bath were added nickel(II) chloride hexahydrate (24 mg, 0.1 mmol) and di-tert-butyl dicarbonate (30% in dioxane, 0.73 mL, 1 mmol). The resulting solution was charged with the addition of sodium borohydride (76 mg, 2 mmol) portionwise and stirred at 25 °C. After the reaction was judged to be completed by TLC (16 h), it was evaporated under reduced pressure by rotary evaporation. The resulting residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc) to afford compound 10a as a colorless oil (56 mg, 52%). ¹H NMR (500 MHz, acetone) δ 8.37 (t, J = 1.6 Hz, 1H), 7.73 (dd, J = 13.6, 2.3 Hz, 1H), 7.66 (t, J = 1.6 Hz, 1H), 7.61 (t, J = 8.6 Hz, 1H), 7.50 (dd, J = 8.6, 2.3 Hz, 1H), 6.20 (s, 1H), 4.97 (dddd, J = 9.0, 6.1, 4.5, 3.5 Hz, 1H), 4.67 (s, 2H), 4.44 (d, J = 5.0 Hz, 2H), 4.31 (t, J = 9.0 Hz, 1H), 4.05 (dd, J = 9.0, 6.1 Hz, 1H), 3.87 (dd, J = 11.3, 3.5 Hz, 1H), 3.82 (dd, J = 11.3, 4.5 Hz, 1H), 1.51 (s, 9H), 1.45 (s, 9H). A solution of compound 10a (28 mg, 0.05 mmol) in DCM (1 mL) and TFA (0.5 mL) was stirred at 40 °C. After the reaction was judged to be completed by TLC (1 h), its solvent was evaporated under reduced pressure by rotary evaporation. The resulting residue was dissolved in MeOH (1 mL), charged with the addition of HCl/dioxane (4 M, 0.1 mL), and concentrated under reduced pressure by rotary evaporation. The resulting solid was washed with acetone (2 × 1 mL) and dried under high vacuum to give 8o as a yellow amorphous solid (12 mg, 66%). ¹H NMR (400 MHz, CD₃OD) δ 8.42 (t, J = 1.5 Hz, 1H), 7.75 (dd, J = 13.6, 2.2 Hz, 1H), 7.69 (t, J = 1.5 Hz, 1h), 7.61 (t, J = 8.6 Hz, 1H), 7.49 (dd, J = 8.6, 2.2 Hz, 1H), 4.84–4.76 (m, 1H), 4.37 (s, 2H), 4.19 (t, J = 8.9 Hz, 1H), 4.00 (dd, J = 8.9, 6.3 Hz, 1H), 3.89 (dd, J = 12.6, 3.1 Hz, 1H), 3.72 (dd, J = 12.6, 3.8 Hz, 1H). ¹³C NMR (101 MHz, CD₃OD) δ 161.2 (d, J = 246.6 Hz), 156.8, 153.9, 142.4 (d, J = 11.3 Hz), 138.5, 138.3 (d, J = 3.7 Hz), 135.2, 131.8 (d, J = 4.2 Hz), 125.9, 119.8 (d, J = 13.7 Hz), 115.3 (d, J = 3.1 Hz), 107.1 (d, J = 28.9 Hz), 75.3, 63.2, 47.5, 39.3. MSESI m/z: 334.1196 (C₁₆H₁₆FN₃O₄ + H⁺ requires 334.1198).

General Procedure 4: Synthesis of Heterocyclic MOM-Protected Oxazolidinone Analogues 11a–h.

A mixture of compound 9 (76 mg, 0.2 mmol), heterocyclic bromide (0.26 mmol), potassium carbonate (110 mg, 0.8 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (16 mg, 0.02 mmol) in dioxane/H₂O (v/v = 9:1, 1 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–8% MeOH in DCM) to afford compounds 11a–h.

(R)-3-(3-Fluoro-4-(pyridin-4-yl)phenyl)-5-((methoxymethoxy)-methyl)oxazolidin-2-one (11a).

A mixture of compound 9 (125 mg, 0.33 mmol), 4-bromopyridine hydrochloride (83 mg, 0.43 mmol), potassium carbonate (182 mg, 1.32 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) complex with dichloromethane (27 mg, 0.033 mmol) in dioxane/H₂O (v/v = 9:1, 2 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (3 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) followed by preparative TLC (SiO₂, eluent gradient 100% EtOAc) to afford compound 11a as a brown amorphous solid (80 mg, 73%). ¹H NMR (300 MHz, acetone-d₆) δ 8.65 (d, J = 6.2 Hz, 2H), 7.75 (dd, J = 13.9, 2.3 Hz, 1H), 7.67 (t, J = 8.7 Hz, 1H), 7.61–7.47 (m, 3H), 5.02–4.90 (m, 1H), 4.67 (s, 2H), 4.32 (t, J = 9.0 Hz, 1H), 4.06 (dd, J = 9.0, 6.1 Hz, 1H), 3.93–3.70 (m, 2H), 3.33 (s, 3H).

(R)-3-(3-Fluoro-4-(pyridin-3-yl)phenyl)-5-((methoxymethoxy)-methyl)oxazolidin-2-one (11b).

A mixture of compound 9 (67.5 mg, 0.177 mmol), 3-bromopyridine (25 μL, 0.26 mmol), potassium carbonate (110 mg, 0.8 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (16 mg, 0.02 mmol) in dioxane/H₂O (v/ v = 9:1, 1 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (2 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) to afford compound 11b as a colorless oil (42 mg, 71%). ¹H NMR (300 MHz, acetone-d₆) δ δ 8.78 (s, 1H), 8.58 (dd, J = 4.8, 1.7 Hz, 1H), 8.03–7.89 (m, 1H), 7.75 (dd, J = 13.6, 2.3 Hz, 1H), 7.61 (t, J = 8.6 Hz, 1H), 7.57–7.41 (m, 2H), 5.03–4.90 (m, 1H), 4.68 (s, 2H), 4.32 (t, J = 9.0 Hz, 1H), 4.06 (dd, J = 9.0, 6.1 Hz, 1H), 3.94–3.70 (m, 2H), 3.34 (s, 3H).

(R)-3-(3-Fluoro-4-(pyridin-2-yl)phenyl)-5-((methoxymethoxy)-methyl)oxazolidin-2-one (11c).

A mixture of compound 9 (67.5 mg, 0.177 mmol), 2-bromopyridine (26.1 μL, 0.26 mmol), potassium carbonate (110 mg, 0.8 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (16 mg, 0.02 mmol) in dioxane/H₂O (v/ v = 9:1, 1 mL) was stirred at 90 °C under a nitrogen atmosphere. After the reaction was judged to be completed by TLC (2 h), it was cooled to room temperature, diluted with EtOAc, washed with water, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) to afford compound 11c as a white amorphous solid (41 mg, 70%). ¹H NMR (300 MHz, acetone-d₆) δ 8.70 (dd, J = 4.8, 1.5 Hz, 1H), 8.13 (t, J = 8.9 Hz, 1H), 7.92–7.80 (m, 2H), 7.74 (dd, J = 14.4, 2.3 Hz, 1H), 7.49 (dd, J = 8.8, 2.3 Hz, 1H), 7.38–7.29 (m, 1H), 5.07–4.93 (m, 1H), 4.68 (s, 2H), 4.32 (t, J = 9.0 Hz, 1H), 4.06 (dd, J = 9.0, 6.1 Hz, 1H), 3.96–3.74 (m, 2H), 3.33 (s, 3H).

(R)-3-(3-Fluoro-4-(isoquinolin-6-yl)phenyl)-5-((methoxymethoxy)methyl)oxazolidin-2-one (11d).

Using general procedure 4, employing 6-bromoisoquinoline (54 mg), compound 11d was obtained as a yellow amorphous solid (69 mg, 90%). ¹H NMR (400 MHz, acetone-d₆) δ 9.33 (s, 1H), 8.54 (d, J = 5.7 Hz, 1H), 8.19 (d, J = 8.5 Hz, 1H), 8.13 (s, 1H), 7.89 (dt, J = 8.5, 1.8 Hz, 1H), 7.84 (d, J = 5.7 Hz, 1H), 7.77 (dd, J = 13.8, 2.3 Hz, 1H), 7.71 (t, J = 8.6 Hz, 1H), 7.55 (dd, J = 8.6, 2.3 Hz, 1H), 5.04–4.94 (m, 1H),4.68 (s, 2H), 4.33 (t, J = 8.9 Hz, 1H), 4.07 (dd, J = 8.9, 6.1 Hz, 1H), 3.89 (dd, J = 11.3, 3.4 Hz, 1H), 3.83 (dd, J = 11.3, 4.5 Hz, 1H), 3.34 (s, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.8 (d, J = 245.5 Hz), 155.1, 153.1, 144.5, 141.6 (d, J = 11.3 Hz), 138.1 (d, J = 1.6 Hz), 136.7, 132.1 (d, J = 4.6, Hz), 129.2 (d, J = 3.2 Hz), 128.6, 127.0 (d, J = 3.3 Hz), 123.3 (d, J = 13.5 Hz), 121.3, 114.6 (d, J = 3.1 Hz), 106.4 (d, J = 29.0 Hz), 97.3, 72.7, 68.7, 55.5, 47.4.

(R)-3-(3-Fluoro-4-(isoquinolin-7-yl)phenyl)-5-((methoxymethoxy)methyl)oxazolidin-2-one (11e).

Using general procedure 4, employing 7-bromoisoquinoline (54 mg), compound 11e was obtained as a yellow amorphous solid (68 mg, 89%). ¹H NMR (400 MHz, acetone-d₆) δ 9.37 (s, 1H), 8.54 (d, J = 5.7 Hz, 1H), 8.29 (s, 1H), 8.05 (d, J = 8.6 Hz, 1H), 8.01–7.96 (m, 1H), 7.82 (d, J = 5.7 Hz, 1H), 7.77 (dd, J = 13.9, 2.2 Hz, 1H), 7.72 (t, J = 8.6 Hz, 1H), 7.55 (dd, J = 8.6, 2.2 Hz, 1H), 5.04–4.93 (m, 1H), 4.68 (s, 2H), 4.33 (t, J = 8.9 Hz, 1H), 4.07 (dd, J = 8.9, 6.2 Hz, 1H), 3.89 (dd, J = 11.3, 3.4 Hz, 1H), 3.83 (dd, J = 11.3, 4.4 Hz, 1H), 3.34 (s, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.7 (d, J = 245.0 Hz), 155.1, 153.6, 144.3, 141.3 (d, J = 11.4 Hz), 135.6, 135.2 (d, J = 1.7 Hz), 132.1 (d, J = 3.3 Hz), 132.0 (d, J = 4.8 Hz), 129.7, 128.1 (d, J = 3.3 Hz), 127.6, 123.3 (d, J = 13.4 Hz), 120.9, 114.6 (d, J = 3.1 Hz), 106.3 (d, J = 29.1 Hz), 97.3, 72.7, 68.7, 55.4, 47.4.

(R)-3-(2-Fluoro-4′-(pyridin-4-yl)-[1,1′-biphenyl]-4-yl)-5-((methoxymethoxy)methyl)oxazolidin-2-one (11f).

Using general procedure 4, employing 4-(4-bromophenyl)pyridine (61 mg), compound 11f was obtained as a yellow amorphous solid (75 mg, 90%). ¹H NMR (400 MHz, acetone-d₆) δ 8.75–8.61 (m, 2H), 7.90 (d, J = 8.1 Hz, 2H), 7.81–7.65 (m, 5H), 7.63 (t, J = 8.7 Hz, 1H), 7.51 (dd, J = 8.7, 2.2 Hz, 1H), 5.04–4.93 (m, 1H), 4.68 (s, 2H), 4.32 (t, J = 8.9 Hz, 1H), 4.06 (dd, J = 8.9, 6.1 Hz, 1H), 3.88 (dd, J = 11.3, 3.4 Hz, 1H), 3.82 (dd, J = 11.3, 4.4 Hz, 1H), 3.34 (s, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.6 (d, J = 245.0 Hz), 155.1, 151.3 (2C), 148.0, 141.1 (d, J = 11.4 Hz), 137.9, 137.0, 131.6 (d, J = 4.8 Hz), 130.3 (d, J = 3.4 Hz, 2C), 127.9 (2C), 123.3 (d, J = 13.2 Hz), 122.1 (2C), 114.5 (d, J = 3.3 Hz), 106.3, (d, J = 29.1 Hz), 97.3, 72.6, 68.7, 55.4, 47.4.

(R)-3-(2-Fluoro-4′-(pyridin-3-yl)-[1,1′-biphenyl]-4-yl)-5-((methoxymethoxy)methyl)oxazolidin-2-one (11g).

Using general procedure 4, employing 3-(4-bromophenyl)pyridine (61 mg), compound 11g was obtained as a yellow amorphous solid (68 mg, 83%). ¹H NMR (400 MHz, acetone-d₆) δ 8.94 (d, J = 2.4 Hz, 1H), 8.73–8.55 (m, 1H), 8.18–8.03 (m, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.78–7.68 (m, 3H), 7.63 (t, J = 8.8 Hz, 1H), 7.57–7.44 (m, 2H), 5.03–4.93 (m, 1H), 4.68 (s, 2H), 4.32 (t, J = 8.9 Hz, 1H), 4.06 (dd, J = 8.9, 6.2 Hz, 1H), 3.88 (dd, J = 11.3, 3.4 Hz, 1H), 3.83 (dd, J = 11.3, 4.4 Hz, 1H), 3.35 (s, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 160.6 (d, J = 245.2 Hz), 155.1, 149.6, 148.8, 141.0 (d, J = 11.3 Hz), 137.8, 136.6, 136.0, 134.8, 131.6 (d, J = 4.9 Hz), 130.3 (d, J = 3.3 Hz, 2C), 128.0 (2C), 124.6, 123.5 (d, J = 13.0 Hz), 114.5 (d, J = 3.2 Hz), 106.3 (d, J = 29.1 Hz), 97.3, 72.6, 68.7, 55.4, 47.4.

(R)-3-(2-Fluoro-4′-(pyridin-2-yl)-[1,1′-biphenyl]-4-yl)-5-((methoxymethoxy)methyl)oxazolidin-2-one (11h).

Using general procedure 4, employing 2-(4-bromophenyl)pyridine (61 mg), compound 11h was obtained after flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) as a yellow amorphous solid (64 mg, 78%). ¹H NMR (400 MHz, acetone-d₆) δ 8.69 (ddd, J = 4.7, 1.8, 0.9 Hz, 1H), 8.23 (d, J = 8.5 Hz, 2H), 8.03–7.97 (m, 1H), 7.89 (td, J = 7.7, 1.8 Hz, 1H), 7.77–7.67 (m, 3H), 7.63 (t, J = 8.8 Hz, 1H), 7.50 (dd, J = 8.8, 2.3 Hz, 1H), 7.34 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 5.04–4.92 (m, 1H), 4.68 (s, 2H), 4.31 (t, J = 8.9 Hz, 1H), 4.05 (dd, J = 8.9, 6.2 Hz, 1H), 3.88 (dd, J = 11.3, 3.5 Hz, 1H), 3.82 (dd, J = 11.3, 4.5 Hz, 1H), 3.34 (s, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 159.7 (d, J = 245.2 Hz), 156.2, 154.21, 149.7, 140.1 (d, J = 11.2 Hz), 138.4, 136.9, 135.8 (d, J = 1.9 Hz), 130.7 (d, J = 4.8 Hz), 129.0 (d, J = 3.4 Hz, 2C), 126.8, 122.8 (d, J = 13.6 Hz), 122.4 (2C), 120.0, 113.6 (d, J = 3.2 Hz), 105.4 (d, J = 29.3 Hz), 96.4, 71.7, 67.8, 54.5, 46.5.

General Procedure 5: Synthesis of Positively Charged Oxazolidinone Analogues 12a–l.

A mixture of compound 11 (0.1 mmol) and haloalkane (0.5 mmol) in CH₃CN (1 mL) was stirred in a sealed tube at 80 °C for 24 h. The reaction mixture was cooled to room temperature, purged with a nitrogen flow for 5 min to remove most of the haloalkane, and concentrated under reduced pressure by rotary evaporation. The resulting residue was charged with the addition of HCl/dioxane (4 M, 1 mL) [if necessary, MeOH (1 mL) was employed as a cosolvent]. The reaction mixture was stirred at 25 °C for 1 h, purged with a nitrogen flow for 5 min to remove most of the hydrogen chloride, and concentrated under reduced pressure by rotary evaporation. The residue was further purified to give compounds 12 (their purification protocols are reported individually).

(R)-4-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)-1-methylpyridin-1-ium Chloride (12a).

A mixture of compound 11a (72 mg, 0.22 mmol) and methyl iodide (30 μL, 0.54 mmol) in CH₃CN (2 mL) was stirred in a sealed tube at 80 °C for 24 h. The reaction mixture was cooled to room temperature, purged with a nitrogen flow for 5 min to remove most of methyl iodide, and concentrated under reduced pressure by rotary evaporation. The resulting residue was charged with the addition of HCl/dioxane (4 M, 2 mL). The reaction mixture was stirred at 25 °C for 1 h, purged with a nitrogen flow for 5 min to remove most of the hydrogen chloride, and concentrated under reduced pressure by rotary evaporation. The crude residue was dissolved in methanol (2 mL). The solution was precipitated with the addition of acetone (4 mL) and kept still overnight. The supernatant was removed carefully, and the solid was resuspended in acetone (4 mL). The acetone supernatant was removed carefully, and the solid was dried under high vacuum to afford 12a as a yellow amorphous solid (30 mg, 40%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.05 (d, J = 6.6 Hz, 2H), 8.35 (d, J = 6.6 Hz, 2H), 7.97 (t, J = 8.8 Hz, 1H), 7.77 (dd, J = 14.4, 2.2 Hz, 1H), 7.62 (dd, J = 8.8, 2.2 Hz, 1H), 5.44–5.32 (m, 1H), 4.88–4.74 (m, 1H), 4.36 (s, 3H), 4.17 (t, J = 9.0 Hz, 1H), 3.97 (dd, J = 9.0, 5.9 Hz, 1H), 3.76–3.66 (m, 1H), 3.66–3.55 (m, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 160.2 (d, J = 250.4 Hz), 154.3, 149.7, 145.5 (2C), 143.2 (d, J = 11.9 Hz), 131.7 (d, J = 3.5 Hz), 125.9 (d, J = 5.3 Hz, 2C), 116.3 (d, J = 11.5 Hz), 114.1 (d, J = 2.9 Hz), 105.4 (d, J = 28.4 Hz), 73.7, 61.4, 47.2, 46.0. MSESI m/z: 303.1145 (C₁₆H₁₆FN₂O₃⁺ requires 303.1139).

(R)-3-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)-1-methylpyridin-1-ium Chloride (12b).

Using general procedure 5, employing 11b (33 mg, 0.1 mmol) and methyl iodide (31.1 μL, 0.5 mmol), compound 12b was obtained as a yellow amorphous solid (10 mg, 30%). Pyridinium 12b purification protocol: the crude residue was dissolved in methanol (1 mL). The solution was precipitated with the addition of acetone (5 mL) and kept still overnight. The supernatant was removed carefully, and the solid was resuspended in acetone (2 mL). The acetone supernatant was removed carefully, and the solid was dried under high vacuum to afford 12b. ¹H NMR (400 MHz, DMSO-d₆) δ 9.34 (s, 1H), 9.02 (d, J = 6.0 Hz, 1H), 8.78 (d, J = 8.2 Hz, 1H), 8.22 (dd, J = 8.2, 6.0 Hz, 1H), 7.89–7.71 (m, 2H), 7.59 (dd, J = 8.7, 2.3 Hz, 1H), 5.36 (t, J = 5.5 Hz, 1H), 4.85–4.72 (m, 1H), 4.43 (s, 3H), 4.17 (t, J = 9.0 Hz, 1H), 3.95 (dd, J = 9.0, 6.0 Hz, 1H), 3.70 (ddd, J = 12.3, 5.5, 3.1 Hz, 1H), 3.58 (ddd, J = 12.3, 5.5, 3.7 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.3 (d, J = 247.2 Hz), 154.4, 144.8 (d, J = 3.3 Hz), 144.1, 144.0, 141.9 (d, J = 11.6 Hz), 134.1 (d, J = 1.3 Hz), 131.3 (d, J = 3.6 Hz), 127.5, 115.5 (d, J = 12.8 Hz), 114.1 (d, J = 2.9 Hz), 105.3 (d, J = 27.9 Hz), 73.6, 61.5, 48.2, 46.0. MSESI m/z: 303.1122 (C₁₆H₁₆FN₂O₃⁺ requires 303.1139).

(R)-2-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)-1-methylpyridin-1-ium Chloride (12c).

Using general procedure 5, employing 11c (33 mg, 0.1 mmol) and methyl iodide (31.1 μL, 0.5 mmol), compound 12c was obtained as a yellow amorphous solid (5 mg, 15%). Pyridinium 12c purification protocol: the crude residue was dissolved in methanol (1 mL). The solution was precipitated with the addition of acetone (5 mL) and kept still overnight. The supernatant was removed carefully, and the solid was resuspended in acetone (2 mL). The acetone supernatant was removed carefully, and the solid was dried under high vacuum to afford 12c. ¹H NMR (400 MHz, methanol-d₄) δ 9.10 (d, J = 6.1 Hz, 1H), 8.70–8.59 (m, 1H), 8.20–8.05 (m, 2H), 7.90 (dd, J = 13.0, 2.1 Hz, 1H), 7.71–7.53 (m, 2H), 4.84–4.75 (m, 1H), 4.27–4.19 (m, 4H), 4.02 (dd, J = 9.0, 6.2 Hz, 1H), 3.88 (dd, J = 12.6, 3.0 Hz, 1H), 3.71 (dd, J = 12.6, 3.7 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 158.8 (d, J = 246.7 Hz), 154.3, 149.2, 147.5, 145.6, 143.2 (d, J = 11.4Hz), 131.9 (d, J = 2.9 Hz), 130.8, 127.3, 113.8 (d, J = 3.0 Hz), 113.3 (d, J = 15.0 Hz), 104.9 (d, J = 26.8 Hz), 73.6, 61.5, 46.6 (d, J = 2.6 Hz), 46.0. MSESI m/z: 303.1156 (C₁₆H₁₆FN₂O₃⁺ requires 303.1139).

(R)-6-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)-2-methylisoquinolin-2-ium Chloride (12d).

Using general procedure 5, employing 11d (38 mg, 0.1 mmol) and methyl iodide (31.1 μL, 0.5 mmol), employing MeOH (1 mL) as the cosolvent in the deprotection step, compound 12d was obtained as a yellow amorphous solid (25 mg, 64%). Pyridinium 12d purification protocol: the crude residue was dissolved in methanol (2 mL), and the solution was added to EtOAc (20 mL). The suspension was kept still overnight. The supernatant was removed carefully. The solid was washed with EtOAc (5 mL) and dried under high vacuum to afford 12d. ¹H NMR (400 MHz, methanol-d₄) δ 9.82 (s, 1H), 8.58 (d, J = 6.7 Hz, 1H), 8.53–8.42 (m, 3H), 8.25 (dd, J = 8.7, 2.0 Hz, 1H), 7.85–7.70 (m, 2H), 7.51 (dd, J = 8.7, 2.2 Hz, 1H), 4.82–4.77 (m, 1H), 4.55 (s, 3H), 4.20 (t, J = 8.9 Hz, 1H),4.01 (dd, J = 8.9, 6.3 Hz, 1H), 3.90 (dd, J = 12.6, 3.1 Hz, 1H), 3.73 (dd, J = 12.6, 3.8 Hz, 1H). ¹³C NMR (101 MHz, methanol-d₄) δ 161.5 (d, J = 248.2 Hz), 156.7, 151.3, 145.4 (d, J = 2.0 Hz), 142.9 (d, J = 11.5 Hz), 138.9, 137.0, 133.3 (d, J = 3.9 Hz), 132.6 (d, J = 4.0 Hz), 131.5, 128.0, 127.6 (d, J = 4.1 Hz), 127.1, 122.3 (d, J = 12.7 Hz), 115.4 (d, J = 3.1 Hz), 107.1 (d, J = 29.1 Hz), 75.3, 63.1, 48.7, 47.5. MSESI m/z: 353.1300 (C₂₀H₁₈FN₂O₃⁺ requires 353.1296).

(R)-7-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)-2-methylisoquinolin-2-ium Chloride (12e).

Using general procedure 5, employing 11e (38 mg, 0.1 mmol) and methyl iodide (31.1 μL, 0.5 mmol), employing MeOH (1 mL) as the cosolvent in the deprotection step, compound 12e was obtained as a yellow amorphous solid (25 mg, 64%). Pyridinium 12e purification protocol: the crude residue was dissolved in a mixture of methanol (2 mL) and acetone (4 mL). The solution was precipitated with the addition of EtOAc (10 mL) and kept still overnight. The suspension was centrifuged, and the supernatant was removed carefully. The solid was resuspended in EtOAc (5 mL), and the suspension was centrifuged. The EtOAc supernatant was removed carefully, and the solid was dried under high vacuum to afford 12e. ¹H NMR (400 MHz, DMSO-d₆) δ 10.05 (s, 1H), 8.73 (d, J = 6.8 Hz, 1H), 8.65 (s, 1H), 8.60 (d, J = 6.8 Hz, 1H), 8.44 (s, 2H), 7.91–7.71 (m, 2H), 7.59 (d, J = 8.8 Hz, 1H), 5.32 (t, J = 5.6 Hz, 1H), 4.87–4.69 (m, 1H), 4.49 (s, 3H), 4.17 (t, J = 9.0 Hz, 1H), 3.98–3.88 (m, 1H), 3.78–3.67 (m, 1H), 3.65–3.55 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.4 (d, J = 246.4 Hz), 154.4, 150.9, 140.9 (d, J = 11.6 Hz), 137.1 (d, J = 2.3 Hz), 137.0, 136.2, 135.7, 131.4 (d, J = 4.1 Hz), 129.1 (d, J = 4.2 Hz), 127.8, 127.4, 125.3, 120.2 (d, J = 12.8 Hz), 114.1 (d, J = 3.0 Hz), 105.4 (d, J = 28.4 Hz), 73.5, 61.6, 48.1, 46.0 MSESI m/z: 353.1306 (C₂₀H₁₈FN₂O₃⁺ requires 353.1296).

(R)-4-(2′-Fluoro-4′-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)- [1,1′-biphenyl]-4-yl)-1-methylpyridin-1-ium Chloride (12f).

Using general procedure 5, employing 11f (41 mg, 0.1 mmol) and methyl iodide (31.1 μL, 0.5 mmol), compound 12f was obtained as a brown amorphous solid (20 mg, 48%). Pyridinium 12f purification protocol: the crude residue was suspended in acetone (3 mL), and the suspension was kept still for 30 min. The supernatant was removed carefully, and the solid was resuspended in acetone (3 mL). The acetone supernatant was removed carefully, and the solid was dried under high vacuum to afford 12f. ¹H NMR (400 MHz, DMSO-d₆) δ 9.05 (d, J = 6.6 Hz, 2H), 8.57 (d, J = 6.6 Hz, 2H), 8.39–8.15 (m, 2H), 7.86–7.81 (m, 2H), 7.74–7.64 (m, 2H), 7.52 (dd, J = 8.6, 2.3 Hz, 1H), 5.31 (s, 1H), 4.86–4.69 (m, 1H), 4.34 (s, 3H), 4.15 (t, J = 9.0 Hz, 1H), 3.91 (dd, J = 9.0, 6.1 Hz, 1H), 3.76–3.66 (m, 1H), 3.64–3.55 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.3 (d, J = 245.4 Hz), 154.4, 153.6, 145.7 (2C), 140.3 (d, J = 11.1 Hz), 138.3 (d, J = 1.9 Hz), 132.5, 131.0 (d, J = 4.4 Hz), 129.7 (d, J = 3.5 Hz, 2C), 128.4 (2C), 124.0 (2C), 121.2 (d, J = 13.0 Hz), 114.0 (d, J = 3.0 Hz), 105.4 (d, J = 28.6 Hz), 73.5, 61.6, 47.1, 46.0. MSESI m/z: 379.1418 (C₂₂H₂₀FN₂O₃⁺ requires 379.1452).

(R)-3-(2′-Fluoro-4′-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-[1,1′-biphenyl]-4-yl)-1-methylpyridin-1-ium Chloride (12g).

Using general procedure 5, employing 11g (41 mg, 0.1 mmol) and methyl iodide (31.1 μL, 0.5 mmol), compound 12g was obtained as a gray amorphous solid (20 mg, 48%). Pyridinium 12g purification protocol: the crude residue was suspended in acetone (3 mL), and the suspension was kept still for 30 min. The supernatant was removed carefully, and the solid was resuspended in acetone (3 mL). The acetone supernatant was removed carefully. The solid was suspended in MeOH (2 mL), and the suspension was added to EtOAc (10 mL). The resulting suspension was kept still overnight. The supernatant was removed carefully, and the solid was further washed with EtOAc (5 mL) and then dried under high vacuum to afford 12g. ¹H NMR (400 MHz, DMSO-d₆) δ 9.53 (s, 1H), 9.00 (d, J = 6.0 Hz, 1H), 8.96 (d, J = 8.1 Hz, 1H), 8.23 (dd, J = 8.1, 6.0 Hz, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H), 7.73–7.60 (m, 2H), 7.50 (dd, J = 8.6, 2.2 Hz, 1H), 5.33 (t, J = 5.6 Hz, 1H), 4.85–4.71 (m, 1H), 4.44 (s, 3H), 4.15 (t, J = 9.0 Hz, 1H), 3.92 (dd, J = 9.0, 6.1 Hz, 1H), 3.78–3.66 (m, 1H), 3.63–3.52 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.2 (d, J = 245.1 Hz), 154.4, 143.9, 143.8, 142.0, 140.0 (d, J = 11.4 Hz), 138.6, 136.4, 132.3, 131.0 (d, J = 4.8 Hz), 129.6 (d, J = 3.4 Hz,2C), 127.73, 127.71 (2C), 121.5 (d, J = 13.0 Hz), 113.9 (d, J = 3.0 Hz), 105.4 (d, J = 28.6 Hz), 73.5, 61.6, 48.1, 46.0. MSESI m/z: 379.1468 (C₂₂H₂₀FN₂O₃⁺ requires 379.1452).

(R)-2-(2′-Fluoro-4′-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-[1,1′-biphenyl]-4-yl)-1-methylpyridin-1-ium Chloride (12h).

Using general procedure 5, employing 11h (41 mg, 0.1 mmol) and methyl iodide (31.1 μL, 0.5 mmol), compound 12h was obtained as a gray amorphous solid (10 mg, 24%). Pyridinium 12h purification protocol: the crude residue was suspended in MeOH (2 mL), and the suspension was added to acetone (10 mL). The resulting suspension was kept still overnight. The supernatant was removed carefully, and the solid was further washed with acetone (2 × 10 mL) and then dried under high vacuum to afford 12h. ¹H NMR (400 MHz, DMSO-d₆) δ 9.19 (d, J = 6.1 Hz, 1H), 8.66 (dd, J = 8.6, 7.2 Hz, 1H), 8.33–8.10 (m, 2H), 7.93–7.76 (m, 4H), 7.74–7.63 (m, 2H), 7.53 (dd, J = 8.7, 2.3 Hz, 1H), 5.30 (s, 1H), 4.82–4.70 (m, 1H), 4.39–4.08 (m, 4H), 3.91 (dd, J = 8.9, 6.0 Hz, 1H), 3.71 (dd, J = 12.4, 3.3 Hz, 1H), 3.58 (dd, J = 12.4, 3.8 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.2 (d, J = 245.5 Hz), 154.7, 154.4, 146.8, 145.5, 140.2 (d, J = 11.5 Hz), 137.3, 131.1 (d, J = 4.5 Hz), 130.9, 129.9, 129.7 (2C), 129.1 (d, J = 3.3 Hz, 2C), 126.8, 121.3 (d, J = 13.0 Hz), 114.0 (d, J = 3.1 Hz), 105.4 (d, J = 28.6 Hz), 73.5, 61.6, 47.2, 46.0. MSESI m/z: 379.1451 (C₂₂H₂₀FN₂O₃⁺ requires 379.1452).

(R)-1-Ethyl-4-(2-fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)pyridin-1-ium Chloride (12i).

Using general procedure 5, employing 11a (33 mg, 0.1 mmol) and ethyl iodide (40 μL, 0.5 mmol), compound 12i was obtained as a yellow amorphous solid (15 mg, 42%). Pyridinium 12i purification protocol: the crude residue was dissolved in MeOH (2 mL), and the solution was added to EtOAc (80 mL). The resulting suspension was kept still overnight. The supernatant was removed carefully, and the solid was further washed with EtOAc (10 mL) and then dried under high vacuum to afford 12i. ¹H NMR (500 MHz, DMSO-d₆) δ 9.14 (d, J = 6.3 Hz, 2H), 8.37 (d, J = 6.3 Hz, 2H), 7.97 (t, J = 8.8 Hz, 1H), 7.85–7.73 (m, 1H), 7.66–7.55 (m, 1H), 5.31 (t, J = 5.6 Hz, 1H), 4.88–4.74 (m, 1H), 4.64 (q, J = 7.2 Hz, 2H), 4.18 (t, J = 9.1 Hz, 1H), 3.95 (dd, J = 9.1, 5.8 Hz, 1H), 3.78–3.66 (m, 1H), 3.62–3.54 (m, 1H), 1.57 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 160.2 (d, J = 250.6 Hz), 154.3, 150.1 (d, J = 1.9 Hz), 144.4 (2C), 143.3 (d, J = 11.9 Hz), 131.7 (d, J = 3.3 Hz), 126.3 (d, J = 5.3 Hz, 2C), 116.3 (d, J = 11.3 Hz), 114.1 (d, J = 2.8 Hz), 105.4 (d, J = 28.5 Hz), 73.7, 61.5, 55.7, 46.0, 16.3. MSESI m/z: 317.1291 (C₁₇H₁₈FN₂O₃⁺ requires 317.1296).

(R)-4-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)-1-propylpyridin-1-ium Chloride (12j).

Using general procedure 5, employing 11a (33 mg, 0.1 mmol) and propyl iodide (48.7 μL, 0.5 mmol), compound 12j was obtained as a gray amorphous solid (15 mg, 41%). Pyridinium 12j purification protocol: the crude residue was dissolved in MeOH (2 mL), and the solution was added to EtOAc (60 mL). The resulting suspension was kept still overnight. The supernatant was removed carefully, and the solid was further washed with EtOAc (2 × 20 mL) and then dried under high vacuum to afford 12j. ¹H NMR (500 MHz, DMSO-d₆) δ 9.13 (d, J = 6.4 Hz, 2H), 8.38 (d, J = 6.4 Hz, 2H), 7.99 (t, J = 8.8 Hz, 1H), 7.78 (dd, J = 14.4, 2.2 Hz, 1H), 7.62 (dd, J = 8.8, 2.2 Hz, 1H), 5.31 (s, 1H), 4.89–4.75 (m, 1H), 4.58 (t, J = 7.3 Hz, 2H), 4.18 (t, J = 9.0 Hz, 1H), 3.95 (dd, J = 9.0, 5.9 Hz, 1H), 3.71 (dd, J = 12.5, 3.2 Hz, 1H), 3.59 (dd, J = 12.5, 3.8 Hz, 1H), 2.10–1.89 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 160.2 (d, J = 250.6 Hz), 154.3, 150.1 (d, J = 2.0 Hz), 144.6 (2C), 143.3 (d, J = 11.9 Hz), 131.7 (d, J = 3.4 Hz), 126.2 (d, J = 5.3 Hz, 2C), 116.2 (d, J = 11.4 Hz), 114.1 (d, J = 2.9 Hz), 105.4 (d, J = 28.4 Hz), 73.7, 61.5, 61.4, 46.0, 24.1, 10.3. MSESI m/z: 331.1463 (C₁₈H₂₀FN₂O₃⁺ requires 331.1452).

(R)-4-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)-1-isopropylpyridin-1-ium Chloride (12k).

Using general procedure 5, employing 11a (33 mg, 0.1 mmol) and isopropyl iodide (50 μL, 0.5 mmol), compound 12k was obtained as a yellow amorphous solid (5 mg, 14%). Pyridinium 12k purification protocol: the crude residue was dissolved in MeOH (1 mL), and the solution was added to EtOAc (80 mL). The resulting suspension was kept still overnight. The supernatant was removed carefully, and the solid was further washed with EtOAc (2 × 20 mL) and then dried under high vacuum to afford 12k. ¹H NMR (400 MHz, methanol-d₄) δ 9.05 (d, J = 6.4 Hz, 2H), 8.31 (d, J = 6.4 Hz, 2H), 7.99–7.86 (m, 1H), 7.83–7.75 (m, 1H), 7.55 (dd, J = 9.5, 2.1 Hz, 1H), 5.02 (p, J = 6.7 Hz, 1H), 4.84–4.72 (m, 1H), 4.30–4.06 (m, 1H), 4.04–3.92 (m, 1H), 3.92–3.83 (m, 1H), 3.74–3.66 (m, 1H), 1.72 (d, J = 6.7 Hz, 6H). ¹³C NMR (101 MHz, methanol-d₄) δ 162.3 (d, J = 251.4 Hz), 156.4, 153.1 (d, J = 2.1 Hz), 145.1 (d, J = 12.1 Hz), 143.8 (2C), 132.6 (d, J = 3.3 Hz), 127.9 (d, J = 5.9 Hz, 2C), 117.9 (d, J = 11.4 Hz), 115.5 (d, J = 3.0 Hz), 107.1 (d, J = 29.0 Hz), 75.4, 65.7, 63.1, 47.4, 23.1. MSESI m/z: 331.1446 (C₁₈H₂₀FN₂O₃⁺ requires 331.1452).

(R)-1-Benzyl-4-(2-fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)pyridin-1-ium Chloride (12l).

Using general procedure 5, employing 11a (33 mg, 0.1 mmol) and benzyl bromide (59.4 μL, 0.5 mmol), compound 12l was obtained as a gray amorphous solid (27 mg, 65%). Pyridinium 12l purification protocol: the crude residue was dissolved in MeOH (2 mL), and the solution was added to EtOAc (15 mL). The resulting suspension was kept still overnight. The supernatant was removed carefully, and the solid was further washed with EtOAc (5 mL) and then dried under high vacuum to afford 12l. ¹H NMR (500 MHz, DMSO-d₆) δ 9.23 (d, J = 6.5 Hz, 2H), 8.40 (d, J = 6.3 Hz, 2H), 7.97 (t, J = 8.8 Hz, 1H), 7.77 (dd, J = 14.4, 2.2 Hz, 1H), 7.62 (dd, J = 8.8, 2.2 Hz, 1H), 7.60–7.56 (m, 2H), 7.50–7.42 (m, 3H), 5.87 (s, 2H), 5.27 (t, J = 5.6 Hz, 1H), 4.84–4.69 (m, 1H), 4.17 (t, J = 9.1 Hz, 1H), 3.93 (dd, J = 9.1, 6.0 Hz, 1H), 3.71 (ddd, J = 12.4, 5.6, 3.2 Hz, 1H), 3.58 (ddd, J = 12.4, 5.6, 3.8 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 160.3 (d, J = 250.8 Hz), 154.2, 150.6 (d, J = 2.1 Hz), 144.6 (2C), 143.4 (d, J = 12.3 Hz), 134.4, 131.8 (d, J = 3.3 Hz), 129.4, 129.3 (2C), 128.8 (2C), 126.6 (d, J = 5.5 Hz, 2C), 116.2 (d, J = 11.1 Hz), 114.1 (d, J = 2.7 Hz), 105.4 (d, J = 28.3 Hz), 73.7, 62.6, 61.5, 46.0. MSESI m/z: 379.1469 (C₂₂H₂₀FN₂O₃⁺ requires 379.1452).

General Procedure 6: Synthesis of Alkynyl Oxazolidinone Analogues 13a–ab.

A solution of compound 6 (34 mg, 0.1 mmol), alkyne (0.15 mmol), tetrakis(triphenylphosphine)palladium(0) (12 mg, 0.01 mmol), and copper(I) iodide (2 mg, 0.01 mmol) in DMF (1 mL) was evacuated and purged with nitrogen three times. N,N-Diisopropylethylamine (0.5 mL) was added under the protection of a nitrogen atmosphere. The mixture was stirred at 25 °C. After the reaction was judged to be completed by TLC (16 h), it was diluted with EtOAc, washed with water three times, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂) to afford compound 13.

(R)-3-(4-((3,5-Dichlorophenyl)ethynyl)-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one (13a).

Using general procedure 6, employing 1,3-dichloro-5-ethynylbenzene (26 mg, 0.15 mmol), compound 13a was obtained after flash column chromatography (SiO₂, 0–100% EtOAc in hexanes) as a yellow amorphous solid (34 mg, 89%). ¹H NMR (400 MHz, acetone-d₆) δ 7.72 (dd, J = 12.5, 2.2 Hz, 1H), 7.62 (t, J = 8.4 Hz, 1H), 7.54 (s, 3H), 7.45 (dd, J = 8.4, 2.2 Hz, 1H), 4.93–4.75 (m, 1H), 4.43 (s, 1H), 4.23 (t, J = 8.9 Hz, 1H), 4.05 (dd, J = 8.9, 6.3 Hz, 1H), 3.95–3.87 (m, 1H), 3.78 (dd, J = 12.5, 3.6 Hz, 1H). ¹³C NMR (101 MHz, Acetone-d₆) δ 163.7 (d, J = 248.8 Hz), 155.2, 142.7 (d, J = 11.0 Hz), 135.9 (2C), 134.9 (d, J = 2.4 Hz), 130.5 (2C), 129.6, 127.0, 114.2 (d, J = 3.1 Hz), 105.7 (d, J = 27.1 Hz), 105.4 (d, J = 16.2 Hz), 91.4 (d, J = 3.0 Hz), 86.0, 74.5, 63.2, 47.0.

(R)-1-(3-((2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)ethynyl)phenyl)thiourea (13b).

Using general procedure 6, employing (3-ethynylphenyl)thiourea (26 mg, 0.15 mmol), compound 13b was obtained after flash column chromatography (SiO₂, 0–15% MeOH in DCM) followed by high-performance liquid chromatography purification as a yellow amorphous solid (17 mg, 44%). ¹H NMR (400 MHz, DMSO) δ 9.77 (s, 1H), 7.76–7.60 (m, 3H), 7.43 (d, J = 8.8 Hz, 2H), 7.37 (t, J = 7.8 Hz, 1H), 7.31–7.26 (m, 1H), 5.21 (t, J = 5.6 Hz, 1H), 4.79–4.65 (m, 1H), 4.13 (t, J = 9.0 Hz, 1H), 3.87 (dd, J = 9.0, 6.0 Hz, 2H), 3.75–3.63 (m, 1H), 3.64–3.51 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 181.1, 162.0 (d, J = 247.4 Hz), 154.3, 140.7 (d, J = 11.1 Hz), 139.7, 133.8 (d, J = 2.5 Hz), 129.3, 127.1, 125.2, 123.5, 122.1, 113.5 (d, J = 3.0 Hz), 104.7 (d, J = 26.8 Hz), 104.6 (d, J = 15.7 Hz), 93.5 (d, J = 2.02 Hz), 82.5, 73.5, 61.6, 46.0.

(R)-3-(3-Fluoro-4-((1,3,5-trimethyl-1H-pyrazol-4-yl)ethynyl)-phenyl)-5-(hydroxymethyl)oxazolidin-2-one (13c).

Using general procedure 6, employing 4-ethynyl-1,3,5-trimethyl-1H-pyrazole (20 mg, 0.15 mmol), compound 13c was obtained after flash column chromatography (SiO₂, 0–10% MeOH in DCM) as a white amorphous solid (20 mg, 59%). ¹H NMR (300 MHz, DMSO-d₆) δ 7.75–7.49 (m, 2H), 7.38 (dd, J = 8.6, 2.2 Hz, 1H), 5.25 (t, J = 5.6 Hz, 1H), 4.84–4.53 (m, 1H), 4.11 (t, J = 9.0 Hz, 1H), 3.85 (dd, J = 9.0, 6.1 Hz, 1H), 3.76–3.62 (m, 4H), 3.56 (ddd, J = 12.4, 5.6, 3.9 Hz, 1H), 2.30 (s, 3H), 2.18 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 161.4 (d, J = 246.4 Hz), 154.2, 147.7, 142.0, 139.6 (d, J = 10.6 Hz), 133.0, 113.4 (d, J = 2.8 Hz), 105.9 (d, J = 15.9 Hz), 104.7 (d, J = 26.9 Hz), 100.1, 86.7 (d, J = 1.5 Hz), 85.2, 73.4, 61.5, 45.9, 36.0, 12.1, 10.0.

Methyl (R)-2-(4-((2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)ethynyl)-1H-pyrazol-1-yl)acetate (13d).

Using general procedure 6, employing methyl (4-ethynyl-1H-pyrazol- 1-yl)acetate (25 mg, 0.15 mmol), compound 13d was obtained after flash column chromatography (SiO₂, 0–10% MeOH in DCM) as a white amorphous solid (17 mg, 46%). ¹H NMR (400 MHz, acetone-d₆) δ 8.03 (s, 1H), 7.73–7.63 (m, 2H), 7.52 (t, J = 8.6 Hz, 1H), 7.40 (dd, J = 8.6, 2.3 Hz, 1H), 5.09 (s, 2H), 4.89–4.72 (m, 1H), 4.44 (s, 1H), 4.22 (t, J = 8.9 Hz, 1H), 4.03 (dd, J = 8.9, 6.2 Hz, 1H), 3.90 (d, J = 12.7 Hz, 1H), 3.81–3.64 (m, 4H). ¹³C NMR (101 MHz, acetone-d₆) δ 168.9, 163.2 (d, J = 247.2 Hz), 155.1, 142.5, 141.5 (d, J = 10.9 Hz), 135.0, 134.2 (d, J = 2.9 Hz), 114.0 (d, J = 3.1 Hz), 106.8 (d, J = 16.3 Hz), 105.6 (d, J = 27.2 Hz), 104.0, 86.0 (d, J = 2.9 Hz), 83.5, 74.4, 63.1, 53.5, 52.7, 46.9.

(R)-4-((2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)ethynyl)-1-methyl-1H-pyrazole-5-carboxylic Acid (13e).

Using general procedure 6, employing 4-ethynyl-1-methyl-1H-pyrazole-5-carboxylic acid (23 mg, 0.15 mmol), compound 13e was obtained after flash column chromatography (SiO₂, 0–15% MeOH in DCM) followed by high-performance liquid chromatography purification as a gray amorphous solid (11 mg, 42%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.97 (s, 1H), 7.82 (t, J = 8.9 Hz, 1H), 7.70 (dd, J = 14.6, 2.3 Hz, 1H), 7.49 (dd, J = 8.9, 2.3 Hz, 1H), 7.25 (s, 1H), 5.27 (t, J = 5.6 Hz, 1H), 4.82–4.70 (m, 1H), 4.21 (s, 3H), 4.13 (t, J = 8.9 Hz, 1H), 3.88 (dd, J = 8.9, 6.0 Hz, 1H), 3.70 (ddd, J = 12.3, 5.6, 3.2 Hz, 1H), 3.57 (ddd, J = 12.3, 5.6, 3.9 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.0 (d, J = 248.9 Hz), 154.3, 153.5, 146.6 (d, J = 4.8 Hz), 140.7 (d, J = 11.8 Hz), 133.9, 128.7 (d, J = 3.4 Hz), 125.9, 124.9, 114.3 (d, J = 10.8 Hz), 113.6 (d, J = 2.8 Hz), 105.4 (d, J = 28.7 Hz), 100.7 (d, J = 13.0 Hz), 73.6, 61.6, 46.0, 38.4.

(R)-3-(4-((6-Aminopyridin-2-yl)ethynyl)-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one (13f).

Using general procedure 6, employing 6-ethynylpyridin-2-amine (18 mg, 0.15 mmol), compound 13f was obtained after flash column chromatography (SiO₂, 0–8% MeOH in DCM) followed by high-performance liquid chromatography purification as a yellow amorphous solid (14 mg, 42%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.73–7.56 (m, 2H), 7.48–7.33 (m, 2H), 6.74 (d, J = 7.2 Hz, 1H), 6.46 (d, J = 8.4 Hz, 1H), 6.19 (s, 2H), 5.33–5.16 (m, 1H), 4.82–4.67 (m, 1H), 4.12 (t, J = 9.0 Hz, 1H), 3.86 (dd, J = 9.0, 6.0 Hz, 1H), 3.72–3.64 (m, 1H), 3.61–3.52 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.2 (d, J = 247.7 Hz), 159.8, 154.2, 140.8 (d, J = 11.1 Hz), 139.8, 137.4, 133.9 (d, J = 2.5 Hz), 115.6, 113.5 (d, J = 3.0 Hz), 108.8, 104.8 (d, J = 26.8 Hz), 104.3 (d, J = 15.8 Hz), 94.1 (d, J = 2.9 Hz), 79.7, 73.5, 61.6, 46.0.

(R)-3-(3-Fluoro-4-(3-methoxyprop-1-yn-1-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (13g).

Using general procedure 6, employing methyl propargyl ether (12.7 μL, 0.15 mmol), compound 13g was obtained after flash column chromatography (SiO₂, 0–100% EtOAc in hexanes) as a white amorphous solid (22 mg, 79%). ¹H NMR (600 MHz, acetone-d₆) δ 7.67 (dd, J = 12.4, 2.1 Hz, 1H), 7.50 (t, J = 8.5 Hz, 1H), 7.38 (dd, J = 8.5, 2.1 Hz, 1H), 4.85–4.75 (m, 1H), 4.38 (t, J = 6.0 Hz, 1H), 4.33 (s, 2H), 4.21 (t, J = 8.9 Hz, 1H), 4.02 (dd, J = 8.9, 6.2 Hz, 1H), 3.92–3.86 (m, 1H), 3.79–3.72 (m, 1H), 3.38 (s, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 163.7 (d, J = 247.8 Hz), 155.1, 142.0 (d, J = 10.9 Hz), 134.6 (d, J = 2.8 Hz), 113.9 (d, J = 3.2 Hz), 105.9 (d, J = 16.3 Hz), 105.6 (d, J = 27.3 Hz), 90.9 (d, J = 3.1 Hz), 79.7, 74.4, 63.1, 60.5, 57.5, 46.9.

(R)-3-(3-Fluoro-4-(3-(methylthio)prop-1-yn-1-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (13h).

A solution of compound 6 (34 mg, 0.1 mmol), 3-(methylsulfanyl)-1-propyne (39 mg, 0.3 mmol), tetrakis(triphenylphosphine)- palladium(0) (23 mg, 0.02 mmol), and copper(I) iodide (3.8 mg, 0.02 mmol) in DMF (1 mL) was evacuated and purged with nitrogen three times. N,N-Diisopropylethylamine (0.5 mL) was added under the protection of a nitrogen atmosphere. The mixture was stirred at 25 °C. After the reaction was judged to be completed by TLC (16 h), it was diluted with EtOAc, washed with water three times, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, 0–100% EtOAc in hexanes) to afford compound 13h as a yellow amorphous solid (20 mg, 68%). ¹H NMR (400 MHz, acetone-d₆) δ 7.65 (dd, J = 12.4, 2.3 Hz, 1H), 7.47 (t, J = 8.5 Hz, 1H), 7.36 (dd, J = 8.5, 2.3 Hz, 1H), 4.89–4.73 (m, 1H), 4.41 (t, J = 5.9 Hz, 1H), 4.20 (t, J = 8.9 Hz, 1H), 4.01 (dd, J = 8.9, 6.2 Hz, 1H), 3.90 (ddd, J = 12.3, 5.9, 3.3 Hz, 1H), 3.76 (ddd, J = 12.3, 5.9, 3.9 Hz, 1H), 3.57 (s, 2H), 2.26 (s, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 163.6 (d, J = 247.3 Hz), 155.1, 141.6 (d, J = 10.9 Hz), 134.5 (d, J = 2.9 Hz), 113.9 (d, J = 3.1 Hz), 106.4 (d, J = 16.2 Hz), 105.5 (d, J = 27.3 Hz), 91.2 (d, J = 3.1 Hz), 76.3, 74.3, 63.1, 46.9, 22.3, 15.1.

Ethyl (R)-5-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)-2,2-dimethylpent-4-ynoate (13i).

Using general procedure 6, employing ethyl 2,2-dimethyl-4-pentynoate (23 mg, 0.15 mmol), compound 13i was obtained after flash column chromatography (SiO₂, 0–100% EtOAc in hexanes) as a yellow oil (21 mg, 58%). ¹H NMR (400 MHz, acetone-d₆) δ 7.63 (dd, J = 12.4, 2.3 Hz, 1H), 7.42 (t, J = 8.6 Hz, 1H), 7.34 (dd, J = 8.6, 2.3 Hz, 1H), 4.85–4.68 (m, 1H), 4.39 (t, J = 5.7 Hz, 1H), 4.24–4.07 (m, 3H), 4.00 (dd, J = 8.9, 6.2 Hz, 1H), 3.93–3.81 (m, 1H), 3.82–3.69 (m, 1H), 2.70 (s, 2H), 1.31 (s, 6H), 1.23 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 176.5, 163.6 (d, J = 247.0 Hz), 155.1, 141.3 (d, J = 10.8 Hz), 134.4 (d, J = 2.9 Hz), 113.8 (d, J = 3.1 Hz), 106.9 (d, J = 16.3 Hz), 105.6 (d, J = 27.3 Hz), 92.5 (d, J = 3.0 Hz), 76.2, 74.3, 63.1, 61.1, 46.9, 43.0, 31.1, 24.9, 14.5.

(R)-1-(3-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)prop-2-yn-1-yl)-3-methylurea (13j).

Using general procedure 6, employing N-methyl-N′-(2-propynyl)urea (17 mg, 0.15 mmol), compound 13j was obtained after flash column chromatography (SiO₂, 0–10% MeOH in DCM) as a yellow amorphous solid (7 mg, 22%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.58 (dd, J = 12.3, 2.3 Hz, 1H), 7.49 (t, J = 8.6 Hz, 1H), 7.37 (dd, J = 8.6, 2.3 Hz, 1H), 6.39 (t, J = 5.8 Hz, 1H), 5.92 (q, J = 4.6 Hz, 1H), 5.25 (t, J = 5.5 Hz, 1H), 4.78–4.67 (m, 1H), 4.15–3.98 (m, 3H), 3.83 (dd, J = 8.9, 6.0 Hz, 1H), 3.73–3.62 (m, 1H), 3.60–3.51 (m, 1H), 2.56 (d, J = 4.6 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.2 (d, J = 246.9 Hz), 158.1, 154.2, 140.2 (d, J = 10.9 Hz), 133.8 (d, J = 2.7 Hz), 113.4 (d, J = 3.0 Hz), 104.9 (d, J = 15.6 Hz), 104.7 (d, J = 26.8 Hz), 93.3 (d, J = 3.0 Hz), 74.2, 73.5, 61.6, 45.9, 29.7, 26.5.

(R)-3-(3-Fluoro-4-(3-(2-oxoimidazolidin-1-yl)prop-1-yn-1-yl)-phenyl)-5-(hydroxymethyl)oxazolidin-2-one (13k).

Using general procedure 6, employing 1-(2-propynyl)-2-imidazolidinone (19 mg, 0.15 mmol), compound 13k was obtained after flash column chromatography (SiO₂, 0–10% MeOH in DCM) as a yellow amorphous solid (10 mg, 30%). 1H NMR (400 MHz, DMSO-d₆) δ 7.59 (dd, J = 12.4, 2.2 Hz, 1H), 7.53 (t, J = 8.5 Hz, 1H), 7.37 (dd, J = 8.5, 2.2 Hz, 1H), 6.61 (s, 1H), 5.22 (t, J = 5.6 Hz, 1H), 4.83–4.57 (m, 1H), 4.16 (s, 2H), 4.09 (t, J = 9.0 Hz, 1H), 3.84 (dd, J = 9.0, 6.0 Hz, 1H), 3.67 (ddd, J = 12.5, 5.6, 3.2 Hz, 1H), 3.56 (ddd, J = 12.5, 7.9, 5.6 Hz, 1H), 3.50–3.37 (m, 2H), 3.30–3.15 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.2 (d, J = 247.2 Hz), 161.6, 154.2, 140.4 (d, J = 10.9 Hz), 133.8 (d, J = 2.6 Hz), 113.4 (d, J = 3.0 Hz), 104.6 (d, J = 42.8 Hz), 104.5, 89.7 (d, J = 2.9 Hz), 76.4, 73.4, 61.5, 45.9, 44.0, 37.2, 33.7.

(5R)-3-(3-Fluoro-4-(3-(4-(1-hydroxyethyl)phenoxy)prop-1-yn-1-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (13l).

Using general procedure 6, employing 1-[4-(2-propynyloxy)phenyl]-ethanol (26 mg, 0.15 mmol), compound 13l was obtained after flash column chromatography (SiO₂, 0–10% MeOH in DCM) as a yellow oil (30 mg, 79%). ¹H NMR (300 MHz, acetone-d₆) δ 7.66 (dd, J = 12.4, 2.2 Hz, 1H), 7.49 (t, J = 8.3 Hz, 1H), 7.40–7.28 (m, 3H), 7.01 (d, J = 8.2 Hz, 2H), 5.02 (s, 2H), 4.88–4.71 (m, 2H), 4.42 (t, J = 5.8 Hz, 1H), 4.19 (t, J = 8.9 Hz, 1H), 4.09 (d, J = 4.0 Hz, 1H),4.01 (dd, J = 8.9, 6.2 Hz, 1H), 3.93–3.84 (m, 1H), 3.81–3.68 (m, 1H), 1.37 (d, J = 6.4 Hz, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 163.7 (d, J = 248.3 Hz), 157.6, 155.1, 142.2 (d, J = 11.0 Hz), 141.1, 134.7 (d, J = 2.8 Hz), 127.3 (2C), 115.3 (2C), 113.9 (d, J = 3.1 Hz), 105.5 (d, J = 27.2 Hz), 105.5 (d, J = 16.2 Hz), 90.1 (d, J = 3.0 Hz), 80.4, 74.4, 69.5, 63.1, 57.0, 46.9, 26.2.

(R)-3-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)prop-2-yn-1-yl Acetate (13m).

Using general procedure 6, employing propargyl acetate (15 mg, 0.15 mmol), compound 13m was obtained after flash column chromatography (SiO₂, 0–100% EtOAc in hexanes) as a yellow amorphous solid (20 mg, 65%). ¹H NMR (400 MHz, acetone-d₆) δ 7.67 (dd, J = 12.5, 2.3 Hz, 1H), 7.50 (t, J = 8.3 Hz, 1H), 7.39 (dd, J = 8.3, 2.3 Hz, 1H), 4.94 (s, 2H), 4.86–4.72 (m, 1H), 4.39 (t, J = 5.8 Hz, 1H), 4.21 (t, J = 8.9 Hz, 1H), 4.02 (dd, J = 8.9, 6.1 Hz, 1H), 3.90 (ddd, J = 12.4, 5.8, 3.3 Hz, 1H), 3.77 (ddd, J = 12.4, 5.8, 3.8 Hz, 1H), 2.09 (s, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 170.4, 163.8 (d, J = 248.2 Hz), 155.1, 142.3 (d, J = 10.9 Hz), 134.8 (d, J = 2.7 Hz), 114.0 (d, J = 3.1 Hz), 105.6 (d, J = 27.2 Hz), 105.4 (d, J = 16.1 Hz), 89.1 (d, J = 3.2 Hz), 79.7, 74.4, 63.1, 52.9, 46.9, 20.6.

(R)-3-(3-Fluoro-4-(3-hydroxyprop-1-yn-1-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (13n).

To a solution of compound 13m (39 mg, 0.127 mmol) in THF (1.5 mL), MeOH (0.5 mL), and H₂O (0.5 mL) was added lithium hydroxide monohydrate (53 mg, 1.27 mmol). The reaction mixture was stirred at 25 °C for 1 h, quenched with the addition of water, and extracted with EtOAc four times. The combined organic layers were concentrated under reduced pressure by rotary evaporation. The residue was purified by flash column chromatography (SiO₂, 0–10% MeOH in DCM) followed by preparative TLC (SiO₂, 10% MeOH in DCM) to give compound 13n as a yellow amorphous solid (16 mg, 47%). ¹H NMR (400 MHz, methanol-d₄) δ 7.56 (d, J = 12.1 Hz, 1H), 7.42 (t, J = 8.6 Hz, 1H), 7.26 (d, J = 8.6 Hz, 1H), 4.77–4.66 (m, 1H), 4.39 (s, 2H), 4.20–4.03 (m, 1H), 3.95–3.86 (m, 1H), 3.85–3.78 (m, 1H), 3.70–3.63 (m, 1H). ¹³C NMR (101 MHz, methanol-d₄) δ 164.2 (d, J = 248.6 Hz), 156.6, 141.6 (d, J = 10.8 Hz), 134.9 (d, J = 2.8 Hz), 114.4 (d, J = 3.2 Hz), 107.4 (d, J = 16.1 Hz), 106.4 (d, J = 27.3 Hz), 93.7 (d, J = 3.0 Hz), 78.4, 75.2, 63.2, 51.2, 47.5.

(R)-6-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)hex-5-ynoic Acid (13o).

A solution of compound 6 (34 mg, 0.1 mmol), 5-hexynoic acid (17 mg, 0.15 mmol), tetrakis(triphenylphosphine)palladium(0) (12 mg, 0.01 mmol), and copper(I) iodide (2 mg, 0.01 mmol) in CH₃CN (1 mL) was evacuated and purged with nitrogen three times. N,N-Diisopropylethylamine (0.5 mL) was added under the protection of a nitrogen atmosphere. The mixture was stirred at 25 °C. After the reaction was judged to be complete by TLC (16 h), its solvent was concentrated under reduced pressure by rotary evaporation. The residue was diluted with MeOH (10 mL), acidified with formic acid (2 mL), and concentrated under reduced pressure by a rotary evaporator. The residue was purified by flash column chromatography (SiO₂, 10–20% MeOH in DCM with 0.1% formic acid) to give compound 13o as a yellow amorphous solid (10 mg, 31%). ¹H NMR (400 MHz, methanol-d₄) δ 7.51 (dd, J = 12.1, 2.3 Hz, 1H), 7.34 (t, J = 8.6 Hz, 1H), 7.20 (dd, J = 8.6, 2.3 Hz, 1H), 4.75–4.63 (m, 1H), 4.06 (t, J = 8.9 Hz, 1H), 3.87 (dd, J = 8.9, 6.3 Hz, 1H), 3.80 (dd, J = 12.5, 3.2 Hz, 1H), 3.64 (dd, J = 3.9 Hz, 1H), 2.53–2.36 (m, 4H), 1.91–1.76 (m, 2H). ¹³C NMR (101 MHz, methanol-d₄) δ 177.4, 164.1 (d, J = 247.8 Hz), 156.7, 140.9 (d, J = 10.6 Hz), 134.7 (d, J = 2.9 Hz), 114.4 (d, J = 3.4 Hz), 108.4 (d, J = 16.5 Hz), 106.4 (d, J = 27.4 Hz), 95.0 (d, J = 3.0 Hz), 75.2, 75.0, 63.2, 47.5, 34.2, 25.3, 19.6.

(R)-3-(4-(3-(4-Acetylpiperazin-1-yl)prop-1-yn-1-yl)-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one (13p).

Using general procedure 6, employing 1-(4-(prop-2-yn-1-yl)piperazin-1-yl)ethan-1-one (25 mg, 0.15 mmol), compound 13p was obtained after flash column chromatography (SiO₂, 0–15% MeOH in DCM) as a white amorphous solid (27 mg, 71%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.64 (dd, J = 12.3, 2.2 Hz, 1H), 7.56 (t, J = 8.4 Hz, 1H), 7.41 (dd, J = 8.4, 2.1 Hz, 1H), 5.29 (t, J = 5.5 Hz, 1H), 4.89–4.63 (m, 1H), 4.13 (t, J = 9.0 Hz, 1H), 3.88 (dd, J = 9.0, 6.1 Hz, 1H), 3.72 (ddd, J = 12.4, 5.5, 3.2 Hz, 1H), 3.69–3.55 (m, 3H), 3.55–3.45 (m, 4H), 2.73–2.52 (m, 4H), 2.03 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 168.1, 162.1 (d, J = 246.9 Hz), 154.2, 140.2 (d, J = 10.8 Hz), 133.7 (d, J = 2.7 Hz), 113.3 (d, J = 3.0 Hz), 104.7 (d, J = 16.1 Hz), 104.7 (d, J = 26.9 Hz), 89.8 (d, J = 3.0 Hz), 78.0, 73.4, 61.5, 51.5, 51.0, 46.7, 45.9, 45.5, 40.6, 21.2.

Ethyl (R)-1-(3-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)prop-2-yn-1-yl)piperidine-4-carboxylate (13q).

Using general procedure 6, employing ethyl 1-(prop-2-yn-1-yl)- piperidine-4-carboxylate (29 mg, 0.15 mmol), compound 13q was obtained after flash column chromatography (SiO₂, 0–15% MeOH in DCM) as a yellow amorphous solid (23 mg, 57%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.59 (d, J = 12.4 Hz, 1H), 7.51 (t, J = 8.6 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 5.24 (t, J = 5.6 Hz, 1H), 4.87–4.61 (m, 1H), 4.17–4.00 (m, 3H), 3.84 (t, J = 7.6 Hz, 1H), 3.67 (dt, J = 8.5, 4.1 Hz, 1H), 3.60–3.45 (m, 3H), 2.90–2.70 (m, 2H), 2.44–2.10 (m, 3H), 1.90–1.70 (m, 2H), 1.70–1.46 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 174.3, 162.1 (d, J = 246.9 Hz), 154.2, 140.1 (d, J = 10.9 Hz), 133.7 (d, J = 2.6 Hz), 113.3 (d, J = 3.0 Hz), 104.9 (d, J = 16.1 Hz), 104.7 (d, J = 27.0 Hz), 90.3 (d, J = 3.1 Hz), 77.8, 73.4, 61.6, 59.8, 51.0 (2C), 47.2, 45.9, 39.8, 27.9 (2C), 14.1.

tert-Butyl 2-(2-((tert-Butoxycarbonyl)amino)-5-(2-fluoro-4-((R)-5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)pent-4-ynoyl)- hydrazine-1-carboxylate (13r).

A solution of compound 6 (34 mg, 0.1 mmol), compound 17 (49 mg, 0.15 mmol), tetrakis(triphenylphosphine)palladium(0) (30 mg, 0.025 mmol), and copper(I) iodide (4 mg, 0.02 mmol) in DMF (1 mL) was evacuated and purged with nitrogen three times. N,N-Diisopropylethylamine (0.5 mL) was added under the protection of a nitrogen atmosphere. The mixture was stirred at 50 °C. After the reaction was judged to be completed by TLC (12 h), it was diluted with EtOAc, washed with water three times, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂ eluent gradient 0–100% EtOAc in hexanes) followed by preparative TLC purification (SiO₂, eluent 100% EtOAc) to afford compound 13r as a white amorphous solid (32 mg, 59%). ¹H NMR (400 MHz, acetone-d₆) δ 9.17 (s, 1H), 7.97 (s, 1H), 7.63 (dd, J = 12.3, 2.3 Hz, 1H), 7.47 (t, J = 8.5 Hz, 1H), 7.33 (dd, J = 8.5, 2.3 Hz, 1H), 6.29 (d, J = 8.8 Hz, 1H), 4.87–4.74 (m, 1H), 4.55–4.29 (m, 2H), 4.19 (t, J = 8.9 Hz, 1H), 4.00 (dd, J = 8.9, 6.2 Hz, 1H), 3.89 (dd, J = 12.3, 3.3 Hz, 1H), 3.76 (dd, J = 12.3, 3.8 Hz, 1H), 2.98 (dd, J = 5.4 Hz, 1H), 2.86 (dd, J = 17.0, 8.0 Hz, 1H), 1.51–1.34 (m, 18H). ¹³C NMR (101 MHz, acetone-d₆) δ 170.9, 163.5 (d, J = 247.4 Hz), 156.2, 156.1, 155.1, 141.3 (d, J = 10.9 Hz), 134.8, 113.7 (d, J = 3.2 Hz), 106.8 (d, J = 16.0 Hz), 105.4 (d, J = 27.4 Hz), 91.3, 80.5, 79.7, 74.3, 63.1, 55.4, 52.9, 46.9, 28.5, 28.4, 24.2.

tert-Butyl (R)-(3-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)prop-2-yn-1-yl)carbamate (13s).

A solution of compound 6 (67 mg, 0.2 mmol), 18 (37 mg, 0.24 mmol), tetrakis(triphenylphosphine)palladium(0) (23 mg, 0.02 mmol), and copper(I) iodide (4 mg, 0.02 mmol) in DMF (2 mL) was evacuated and purged with nitrogen three times. N,N-Diisopropylethylamine (1 mL) was added under the protection of a nitrogen atmosphere. The mixture was stirred at 50 °C for 3 h, diluted with EtOAc, washed with water three times, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) followed by preparative TLC purification (SiO₂, eluent 100% EtOAc) to give 13s as a yellow amorphous solid (64 mg, 88%). ¹H NMR (400 MHz, acetone-d₆) δ 7.64 (dd, J = 12.4, 2.3 Hz, 1H), 7.45 (t, J = 8.5 Hz, 1H), 7.36 (dd, J = 8.5, 2.3 Hz, 1H), 6.48 (s, 1H), 4.88–4.72 (m, 1H), 4.40 (t, J = 5.8 Hz, 1H), 4.20 (t, J = 8.9 Hz, 1H), 4.13 (d, J = 5.8 Hz, 2H), 4.01 (dd, J = 8.9, 6.2 Hz, 1H), 3.89 (ddd, J = 12.3, 5.8, 3.2 Hz, 1H), 3.76 (ddd, J = 12.3, 5.8, 3.8 Hz, 1H), 1.42 (s, 9H). ¹³C NMR (101 MHz, acetone-d₆) δ 163.6 (d, J = 247.7 Hz), 156.3, 155.1, 141.7 (d, J = 10.8 Hz), 134.6 (d, J = 2.9 Hz), 113.9 (d, J = 3.1 Hz), 106.3 (d, J = 16.2 Hz), 105.5 (d, J = 27.5 Hz), 92.3, 79.3, 75.6, 74.3, 63.1, 46.9, 31.3, 28.6.

(R)-3-(4-(3-Aminoprop-1-yn-1-yl)-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one Hydrochloride (13t).

A solution of 13s (8 mg, 0.022 mmol) in TFA (0.2 mL) and DCM (0.6 mL) was stirred at 25 °C. After the reaction was judged to be completed by TLC (30 min), its solvent was removed under reduced pressure by rotary evaporation. The residue was dissolved in MeOH (1 mL), charged with the addition of HCl/dioxane (4 M, 20 μL), and concentrated under reduced pressure by rotary evaporation. The resulting residue was washed with acetone (1 mL) and dried under high vacuum to give compound 13t as a yellow amorphous solid (6 mg, 91%). ¹H NMR (400 MHz, methanol-d₄) δ 7.65 (dd, J = 12.2, 2.3 Hz, 1H), 7.52 (t, J = 8.3 Hz, 1H),7.35 (dd, J = 8.3, 2.3 Hz, 1H), 4.84–4.68 (m, 1H), 4.14 (t, J = 9.0 Hz, 1H), 4.08 (s, 2H), 3.94 (dd, J = 9.0, 6.2 Hz, 1H), 3.87 (dd, J = 12.6, 3.1 Hz, 1H), 3.69 (dd, J = 12.6, 3.8 Hz, 1H). ¹³C NMR (101 MHz, methanol-d₄) δ 164.5 (d, J = 249.4 Hz), 156.6, 142.6 (d, J = 10.9 Hz), 135.1 (d, J = 2.6 Hz), 114.6 (d, J = 3.1 Hz), 106.4 (d, J = 27.1 Hz), 105.7 (d, J = 16.0 Hz), 86.1 (d, J = 2.9 Hz), 81.0, 75.2, 63.1, 47.4, 30.8.

tert-Butyl (R)-(3-((3-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)prop-2-yn-1-yl)oxy)-4-methoxybenzyl)carbamate (13u).

A solution of compound 6 (34 mg, 0.1 mmol), 19 (44 mg, 0.15 mmol), tetrakis(triphenylphosphine)palladium(0) (12 mg, 0.01 mmol), and copper(I) iodide (2 mg, 0.01 mmol) in DMF (1 mL) was evacuated and purged with nitrogen three times. N,N-Diisopropylethylamine (0.5 mL) was added under the protection of a nitrogen atmosphere. The mixture was stirred at 50 °C. After the reaction was judged to be completed by TLC (3 h), it was diluted with EtOAc, washed with water three times, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–100% EtOAc in hexanes) followed by preparative TLC purification (SiO₂, eluent 100% EtOAc) to give 13u as a yellow amorphous solid (35 mg, 70%). ¹H NMR (300 MHz, acetone) δ 7.65 (dd, J = 12.4, 2.2 Hz, 1H), 7.51 (t, J = 8.7 Hz, 1H), 7.37 (dd, J = 8.7, 2.2 Hz, 1H), 7.15–7.10 (m, 1H), 6.99–6.85 (m, 2H), 6.38 (s, 1H), 5.00 (s, 2H), 4.89–4.69 (m, 1H), 4.41 (t, J = 5.9 Hz, 1H), 4.27–4.12 (m, 3H), 4.01 (dd, J = 8.9, 6.2 Hz, 1H), 3.89 (ddd, J = 5.5, 3.3 Hz, 1H), 3.84–3.67 (m, 4H), 1.41 (s, 9H). ¹³C NMR (75 MHz, acetone-d₆) δ 163.7 (d, J = 248.4 Hz), 156.8, 155.1, 150.0, 148.0, 142.1 (d, J = 10.9 Hz), 134.9 (d, J = 2.2 Hz), 133.6, 122.0, 115.6 (d, J = 1.5 Hz), 113.9 (d, J = 2.2 Hz), 112.9, 105.6 (d, J = 15.9 Hz), 105. 3 (d, J = 2.2 Hz), 90.1 (d, J = 2.9 Hz), 80.6, 78.7, 74.4, 63.0, 58.1, 56.13, 56.08, 46.9, 44.4.

(R)-3-(4-(3-(5-(Aminomethyl)-2-methoxyphenoxy)prop-1-yn-1-yl)-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one Hydrochloride (13v).

A solution of 13u (30 mg, 0.06 mmol) in TFA (0.3 mL) and DCM (0.9 mL) was stirred at 25 °C. After the reaction was judged to be completed by TLC (30 min), its solvent was removed under reduced pressure by rotary evaporation. The residue was dissolved in MeOH (2 mL), charged with the addition of HCl/dioxane (4 M, 80 μL), and concentrated under reduced pressure by rotary evaporation. The resulting residue was washed with acetone (2 mL) and dried under high vacuum to give compound 13v as a yellow amorphous solid (10 mg, 40%). ¹H NMR (400 MHz, methanol-d₄) δ 7.62 (dd, J = 12.2, 2.2 Hz, 1H), 7.45 (t, J = 8.6 Hz, 1H), 7.30 (dd, J = 8.6, 2.2 Hz, 1H), 7.24 (d, J = 2.0 Hz, 1H), 7.13–7.01 (m, 2H), 5.04 (s, 2H), 4.80–4.71 (m, 1H), 4.12 (t, J = 9.1 Hz, 1H), 4.06 (s, 2H), 3.97–3.77 (m, 5H), 3.68 (dd, J = 12.5, 3.8 Hz, 1H). ¹³C NMR (101 MHz, methanol-d₄) δ 164.4 (d, J = 248.8 Hz), 156.6, 152.3, 148.6, 142.1 (d, J = 10.9 Hz), 135.0 (d, J = 2.8 Hz), 126.7, 124.4, 117.4, 114.5 (d, J = 3.2 Hz), 113.6, 106.6 (d, J = 16.1 Hz), 106.3 (d, J = 27.3 Hz), 89.8 (d, J = 2.9 Hz), 81.2, 75.2, 63.1, 58.8, 56.5, 47.4, 44.1.

tert-Butyl (5-(2-Fluoro-4-((R)-5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)-1-hydroxypent-4-yn-2-yl)carbamate (13w).

A solution of compound 6 (52 mg, 0.155 mmol), 20 (37 mg, 0.186 mmol), tetrakis(triphenylphosphine)palladium(0) (18 mg, 0.0155 mmol), and copper(I) iodide (3 mg, 0.0155 mmol) in DMF (2 mL) was evacuated and purged with nitrogen three times. N,N-Diisopropylethylamine (1 mL) was added under the protection of a nitrogen atmosphere. The mixture was stirred at 50 °C. After the reaction was judged to be completed by TLC (5 h), it was diluted with EtOAc, washed with water three times, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) followed by preparative TLC purification (SiO₂, eluent 100% EtOAc) to give 13w as a white amorphous solid (34 mg, 54%). ¹H NMR (400 MHz, acetone-d₆) δ 7.63 (dd, J = 12.4, 2.3 Hz, 1H), 7.44 (t, J = 8.6 Hz, 1H), 7.34 (dd, J = 8.6, 2.3 Hz, 1H), 5.90 (d, J = 8.3 Hz, 1H), 4.89–4.75 (m, 1H), 4.40 (t, J = 5.8 Hz, 1H), 4.19 (t, J = 9.0 Hz, 1H), 4.11–3.95 (m, 2H), 3.94–3.58 (m, 5H), 2.86–2.65 (m, 2H), 1.40 (s, 9H). ¹³C NMR (101 MHz, acetone-d₆) δ 163.6 (d, J = 246.9 Hz), 156.3, 155.1, 141.2 (d, J = 10.7 Hz), 134.6 (d, J = 2.9 Hz), 113.8 (d, J = 3.2 Hz), 107.1 (d, J = 16.3 Hz), 105.5 (d, J = 27.4 Hz), 92.6 (d, J = 3.0 Hz), 78.9, 75.5, 74.3, 63.5, 63.1, 52.7, 46.9, 28.6, 22.7.

(5R)-3-(4-(4-Amino-5-hydroxypent-1-yn-1-yl)-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one Hydrochloride (13x).

A solution of 13w (16 mg, 0.039 mmol) in TFA (0.2 mL) and DCM (0.6 mL) was stirred at 25 °C. After the reaction was judged to be completed by TLC (30 min), its solvent was removed under reduced pressure by rotary evaporation. The residue was dissolved in MeOH (1 mL), charged with the addition of HCl/dioxane (4 M, 40 μL), and concentrated under reduced pressure by rotary evaporation. The resulting residue was washed with acetone (1 mL) and dried under high vacuum to give compound 13x as a yellow amorphous solid (7 mg, 52%). ¹H NMR (400 MHz, methanol-d₄) δ 7.61 (dd, J = 12.1, 2.3 Hz, 1H), 7.48 (t, J = 8.3 Hz, 1H), 7.31 (dd, J = 8.3, 2.3 Hz, 1H), 4.83–4.71 (m, 1H), 4.13 (t, J = 9.0 Hz, 1H), 3.97–3.83 (m, 3H), 3.78 (dd, J = 11.6, 6.0 Hz, 1H), 3.69 (dd, J = 11.6, 3.8 Hz, 1H), 3.56–3.42 (m, 1H), 2.89 (dd, J = 6.7, 4.1 Hz, 2H). ¹³C NMR (101 MHz, methanol-d₄) δ 164.4 (d, J = 248.4 Hz), 156.6, 141.7 (d, J = 10.8 Hz), 134.9 (d, J = 2.6 Hz), 114.4 (d, J = 3.3 Hz), 107.1 (d, J = 16.2 Hz), 106.3 (d, J = 27.3 Hz), 88.9 (d, J = 3.0 Hz), 77.9, 75.2, 63.1, 61.6, 53.2, 47.5, 21.1.

(R)-N-(tert-Butoxycarbonyl)-N-(3-(2-fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)prop-2-yn-1-yl)glycine (13y).

A solution of compound 6 (82 mg, 0.24 mmol), 21 (62 mg, 0.29 mmol), tetrakis(triphenylphosphine)palladium(0) (28 mg, 0.024 mmol), and copper(I) iodide (5 mg, 0.024 mmol) in CH₃CN (2 mL) was evacuated and purged with nitrogen three times. N,N-Diisopropylethylamine (1 mL) was added under the protection of a nitrogen atmosphere. The mixture was stirred at 50 °C. After the reaction was judged to be completed by TLC (4 h), its solvent was concentrated under reduced pressure by rotary evaporation. The residue was dissolved in acetone, acidified with the addition of formic acid (1 mL), and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM with 1% formic acid) followed by preparative TLC purification (SiO₂, eluent 10% MeOH in DCM) to give 13y as a white amorphous solid (84 mg, 83%). ¹H NMR (600 MHz, acetonitrile-d_3>) δ 7.54 (dd, J = 12.4, 2.3 Hz, 1H), 7.44 (t, J = 8.3 Hz, 1H), 7.29 (dd, J = 8.3 Hz, 2.3 Hz, 1H), 4.76–4.64 (m, 1H), 4.41–4.30 (m, 2H), 4.09–3.97 (m, 3H), 3.83 (dd, J = 8.9, 6.1 Hz, 1H), 3.78 (dd, J = 12.5, 3.3 Hz, 1H), 3.64 (dd, J = 12.5, 4.2 Hz, 1H), 1.57–1.19 (m, 9H). ¹³C NMR (101 MHz, acetonitril-d₃) δ 172.5, 163.7 (d, J = 247.3 Hz), 155.8, 155.5, 141.6 (d, J = 11.0 Hz), 134.8 (d, J = 2.0 Hz), 114.2 (d, J = 3.2 Hz), 105.8 (d, J = 27.2 Hz), 90.4 (d, J = 3.3 Hz), 81.3, 81.2, 77.5, 77.3, 74.4, 63.0, 48.6, 48.5, 48.4, 47.1, 39.0, 38.1, 28.4, 28.3.

(R)-(3-(2-Fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)prop-2-yn-1-yl)glycine Hydrochloride (13z).

A solution of 13y (10 mg, 0.024 mmol) in TFA (0.2 mL) and DCM (0.6 mL) was stirred at 25 °C. After the reaction was judged to be completed by TLC (30 min), its solvent was removed under reduced pressure by rotary evaporation. The residue was dissolved in MeOH (1 mL), charged with the addition of HCl/dioxane (4 M, 20 μL), and concentrated under reduced pressure by rotary evaporation. The resulting residue was washed with acetone (1 mL) and dried under high vacuum to give compound 13z as a yellow amorphous solid (6 mg, 70%). ¹H NMR (400 MHz, methanol-d₄) δ 7.67 (dd, J = 12.2, 2.2 Hz, 1H), 7.55 (t, J = 8.3 Hz, 1H), 7.36 (dd, J = 8.3, 2.2 Hz, 1H), 4.84–4.69 (m, 1H), 4.28 (s, 2H), 4.14 (t, J = 9.0 Hz, 1H), 4.06 (s, 2H), 3.95 (dd, J = 9.0, 6.2 Hz, 1H), 3.87 (dd, J = 12.6, 3.1 Hz, 1H), 3.70 (dd, J = 12.6, 3.8 Hz, 1H). ¹³C NMR (101 MHz, methanol-d₄) δ 168.7, 164.6 (d, J = 249.6 Hz), 156.5, 142.8 (d, J = 11.0 Hz), 135.2 (d, J = 2.4 Hz), 114.6 (d, J = 3.1 Hz), 106.3 (d, J = 27.1 Hz), 105.3 (d, J = 16.1 Hz), 83.9 (d, J = 3.0 Hz), 83.0, 75.2, 63.1, 47.4, 47.3, 38.0.

tert-Butyl 4-(1-((3-(2-Fluoro-4-((R)-5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)prop-2-yn-1-yl)amino)-1-oxopropan-2-yl)-piperazine-1-carboxylate (13aa).

A solution of compound 6 (34 mg, 0.1 mmol), 22 (35 mg, 0.12 mmol), tetrakis(triphenylphosphine)palladium(0) (12 mg, 0.01 mmol), and copper(I) iodide (2 mg, 0.01 mmol) in DMF (2 mL) was evacuated and purged with nitrogen three times. N,N-Diisopropylethylamine (1 mL) was added under the protection of a nitrogen atmosphere. The mixture was stirred at 50 °C. After the reaction was judged to be completed by TLC (2 h), it was diluted with EtOAc, washed with water three times, and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) followed by preparative TLC purification (SiO₂, eluent 100% EtOAc) to give 13aa as a white amorphous solid (45 mg, 89%). ¹H NMR (400 MHz, acetone-d₆) δ 7.93 (t, J = 5.7 Hz, 1H), 7.64 (dd, J = 12.4, 2.2 Hz, 1H), 7.43 (t, J = 8.7 Hz, 1H), 7.35 (dd, J = 8.7, 2.2 Hz, 1H), 4.90–4.74 (m, 1H), 4.43 (s, 1H), 4.25 (d, J = 5.7 Hz, 2H), 4.19 (t, J = 8.9 Hz, 1H), 4.00 (dd, J = 8.9, 6.2 Hz, 1H), 3.94–3.85 (m, 1H), 3.79–3.71 (m, 1H), 3.47–3.34 (m, 4H), 3.17 (q, J = 6.9 Hz, 1H), 2.57–2.35 (m, 4H), 1.42 (s, 9H), 1.17 (d, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, acetone-d₆) δ 173.2, 163.6 (d, J = 247.4 Hz), 155.1, 154.9, 141.7 (d, J = 10.9 Hz), 134.5 (d, J = 2.7 Hz), 113.9 (d, J = 3.1 Hz), 106.2 (d, J = 16.3 Hz), 105.5 (d, J = 27.3 Hz), 92.1 (d, J = 3.1 Hz), 79.5, 75.4, 74.3, 64.4, 63.1, 50.2, 46.9, 29.6, 28.5, 11.7.

N-(3-(2-Fluoro-4-((R)-5-(hydroxymethyl)-2-oxooxazolidin-3-yl)-phenyl)prop-2-yn-l-yl-(piperazin-1-yl)propenamide Dihydrochloride (13ab).

A solution of 13aa (40 mg, 0.079 mmol) in TFA (0.8 mL) and DCM (2.4 mL) was stirred at 25 °C. After the reaction was judged to be completed by TLC (1 h), its solvent was removed under reduced pressure by rotary evaporation. The residue was dissolved in MeOH (4 mL), charged with the addition of HCl/dioxane (4 M, 80 μL), and concentrated under reduced pressure by rotary evaporation. The resulting residue was washed with acetone (4 mL) and dried under high vacuum to give compound 13ab as a yellow amorphous solid (22 mg, 58%). ¹H NMR (400 MHz, methanol-d₄) δ 7.61 (dd, J = 12.2, 2.2 Hz, 1H), 7.44 (t, J = 8.3 Hz, 1H), 7.29 (dd, J = 8.3, 2.2 Hz, 1H), 4.83–4.70 (m, 1H), 4.31 (d, J = 2.0 Hz, 2H), 4.12 (t, J = 8.9 Hz, 1H), 4.02 (q, J = Hz, 1H), 3.93 (dd, J = 8.9, 6.3 Hz, 1H), 3.86 (dd, J = 12.6, 3.0 Hz, 1H), 3.77–3.45 (m, 9H), 1.59 (d, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, methanol-d₄) δ 169.8, 164.3 (d, J = 247.9 Hz), 156.6, 141.8 (d, J = Hz), 134.9 (d, J = 2.0 Hz), 114.5 (d, J = 3.0 Hz), 106.9 (d, J = 16.7 Hz), 106.3 (d, J = 27.4 Hz), 90.2 (d, J = 3.0 Hz), 77.0, 75.2, 65.1, 63.1, 47.9 (2C), 47.4, 42.7 (2C), 30.5, 14.3.

4-Bromo-N’-hydroxybenzimidamide (14).

A mixture of 4-bromobenzonitrile (182 mg, 1 mmol), hydroxylammonium chloride (70 mg, 1 mmol), and sodium hydroxide (40 mg, 1 mmol) in ethanol (2 mL) and water (70 μL) was stirred under a nitrogen atmosphere at 80 °C. After the reaction was judged to be completed by TLC (4 h), it was concentrated under reduced pressure by rotary evaporation. The residue was purified by flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) to afford compound 14 as a yellow amorphous solid (181 mg, 84%). ¹H NMR (400 MHz, DMSO) δ 9.73 (s, 1H), 8.20–7.05 (m, 4H), 5.86 (s, 2H).

5-Bromo-3-(tert-butoxy)picolinonitrile (15).

To a solution of 5-bromo-3-fluoropicolinonitrile (1 g, 5 mmol) in anhydrous DMF (25 mL), sodium tert-butoxide (625 mg, 6.5 mmol) was added. The reaction mixture was stirred at 25 °C for 4 h, quenched with the addition of water, and extracted with a mixture of hexanes/EtOAc (v/v = 1:2). The organic layer was washed with water three times and evaporated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–20% EtOAc in hexanes) to afford compound 15 as a yellow oil (419 mg, 33%). ¹H NMR (400 MHz, chloroform-d) δ 8.41 (d, J = 1.9 Hz, 1H), 7.67 (d, J = 1.9 Hz, 1H), 1.52 (s, 9H).

tert-Butyl 2-(2-((tert-Butoxycarbonyl)amino)pent-4-ynoyl)-hydrazine-1-carboxylate (17).⁵²

Sodium bicarbonate (336 mg, 4 mmol) and di-tert-butyl dicarbonate (327 mg, 1.5 mmol) were added to a solution of 2-amino-4-pentynoic acid hydrochloride (150 mg, 1 mmol) in THF/H₂O (v/v = 1:1, 5 mL) at 0 °C. The reaction mixture was stirred at 25 °C for 16 h. The mixture was diluted with Et₂O and extracted with water three times. The combined aqueous layers were acidified to pH = 4–5 by carefully adding saturated aqueous citric acid in an ice bath and extracted with dichloromethane three times. The combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure by rotary evaporation. Residue 16 was used in the next step without further purification (94 mg, 44%). Toa solution of compound 16 (94 mg, 0.44 mmol) in DMF (1.0 mL) were added tert-butyl carbazate (87 mg, 0.66 mmol), HATU (335 mg, 0.88 mmol), and N,N-diisopropylethylamine (0.3 mL). The reaction mixture was stirred at 25 °C for 16 h, quenched with the addition of water, and extracted with EtOAc. The organic layer was washed with water two times as well as brine and evaporated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–50% EtOAc in hexanes) to afford compound 17 as a yellow amorphous solid (119 mg, 83%). ¹H NMR (400 MHz, CDCl₃) δ 8.48 (s, 1H), 6.70 (s, 1H), 5.44 (d, J = 8.5 Hz, 1H), 4.42 (s, 1H), 2.83–2.55 (m, 2H), 2.08 (d, J = Hz, 1H), 1.58–1.35 (m, 18H).

tert-Butyl Prop-2-yn-1-ylcarbamate (18).

To a solution of propargylamine (110 mg, 2 mmol) in DCM (2 mL) was added a solution of di-tert-butyl dicarbonate (458 mg, 2.1 mmol) in DCM (3 mL) dropwise with a syringe at 0 °C. The reaction mixture was stirred at 25 °C for 30 min and concentrated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–10% MeOH in DCM) to afford compound 18 as a yellow amorphous solid (260 mg, 84%). ¹H NMR (400 MHz, CDCl₃) δ 4.69 (s, 1H), 3.92 (s, 2H), 2.22 (s, 1H), 1.45 (s, 9H).

tert-Butyl (4-Methoxy-3-(prop-2-yn-1-yloxy)benzyl)carbamate (19).

Di-tert-butyl dicarbonate (98 mg, 0.45 mmol) was added to a solution of [4-methoxy-3-(2-propynyloxy)phenyl]methanamine hydrochloride (67 mg, 0.3 mmol) and triethylamine (126 μL, 0.9 mmol) in DCM (2 mL). The reaction mixture was stirred at 25 °C for 2 h, quenched with water, and extracted with EtOAc three times. The combined organic layers were evaporated under reduced pressure by rotary evaporation. The crude residue was purified by flash column chromatography (SiO₂, eluent gradient 0–50% EtOAc in hexanes) to afford compound 19 as a white amorphous solid (64 mg, 73%). ¹H NMR (400 MHz, CDCl₃) δ 6.96 (s, 1H), 6.90–6.74 (m, 2H), 4.94–4.78 (m, 1H), 4.74 (d, J = 2.5 Hz, 2H), 4.24 (d, J = 5.8 Hz, 2H), 3.84 (s, 3H), 2.50 (t, J = 2.5 Hz, 1H), (s, 9H).

tert-Butyl (1-Hydroxypent-4-yn-2-yl)carbamate (20).

A mixture of 2-aminopent-4-yn-1-ol (20 mg, 0.2 mmol), di-tert-butyl dicarbonate (87 mg, 0.4 mmol), and triethylamine (56μL, 0.4 mmol) in CH₃CN (1 mL) was stirred at 50 °C for 5 h. The mixture was evaporated under reduced pressure by rotary evaporation, and the resulting residue was purified by flash column chromatography (SiO₂, eluent gradient 0–3% MeOH in DCM) to afford compound 20 as a yellow amorphous solid (39 mg, 99%). ¹HNMR (400 MHz, acetone) δ 3.95 (s, 1H), 3.77–3.50 (m, 2H), 2.60–2.30 (m, 3H), 1.77 (s, 1H), 1.40 (s, 9H).

N-(tert-Butoxycarbonyl)-N-(prop-2-yn-1-yl)glycine (21).⁵³

To a solution of 2-[(prop-2-yn-1-yl)amino]acetic acid hydrochloride (45 mg, 0.3 mmol) in H₂O (1 mL) were added di-tert-butyl dicarbonate (262 mg, 1.2 mmol) and triethylamine (293 μL, 2.1 mmol). The mixture was stirred at 25 °C for 16 h, diluted with water, and washed withhexanes (20 mL) to remove di-tert-butyl dicarbonate. The aqueous layer was acidified with the addition of aqueous HCl (1 M) to pH = 3 and extracted with EtOAc three times. The combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure by rotary evaporation to give compound 21 (63 mg, 99%), which was used in the next step without further purification. ¹H NMR (400 MHz, CDCl₃) δ 4.43–3.89 (m, 4H), 2.27 (s, 1H), 1.66–0.87 (m, 9H).

tert-Butyl 4-(1-Oxo-1-(prop-2-yn-1-ylamino)propan-2-yl)-piperazine-1-carboxylate (22).

A mixture of 2-(1-piperazinyl)-N-(2-propynyl)propanamide dihydrochloride (53 mg, 0.2 mmol), di-tert-butyl dicarbonate (174 mg, 0.8 mmol), and triethylamine (111 μL, 0.8 mmol) in CH₃CN (2 mL) was stirred at 50 °C for 16 h. The mixture was evaporated under reduced pressure by rotary evaporation, and the resulting residue was purified by flash column chromatography (SiO₂, eluent gradient 0–15% MeOH in DCM) to afford compound 22 as a yellow amorphous solid (30 mg, 51%). Compound 22 was not pure but used in the next step without further purification.

Bacterial Strains and Growth Conditions.

The construction of strains and their properties were described previously. Bacterial strains (see the Supporting Information (SI))^{42,48,54–57} were grown in either Luria-Bertani broth (10 g of Bacto tryptone, 5 g of yeast extract, and 5 g of NaCl per liter; pH 7.0), LB agar (LB broth with 15 g of agar per liter), or a minimal M9 medium (1×) supplemented with 50 mM MOPS buffer (pH 7.2), which were used for bacterial growth. The M9-MOPS medium was optimized to support the growth and expression of the OM pore in strains of all three species. We previously found that in E. coli and A. baumannii cells, the expression of the pore was the most consistent under an arabinose-inducible promoter, but in P. aeruginosa the most consistent expression was achieved under an IPTG-inducible promoter. Since glucose acts as a repressor for an arabinose-inducible promoter, this sugar could not be used as a carbon source in these experiments. Therefore, M9-MOPS medium was supplemented with 0.2% xylose (E. coli, A. baumannii, and P. aeruginosa) plus 0.5% sodium citrate (P. aeruginosa and A. baumannii only) as carbon sources.

Minimum Inhibitory Concentrations.

Compounds were dissolved in DMSO (10 mM). The twofold serial dilution method in 96-well plates was used to determine MICs. E. coli, A. baumannii, and P. aeruginosa cultures grown in indicated media were induced with 0.1%, 1% arabinose, or 0.1 mM IPTG, respectively. The MIC was noted visually after 24 h of incubation at 37 °C, and OD₆₀₀ was measured for all 96-well plates followed by the determination of IC₅₀ values, as described previously.⁵⁸

Cell-Free Coupled Transcription–Translation Assay.

The Expressway Lumio Cell-Free Expression and Detection System (Invitrogen) was used for cell-free transcription–translation of the target protein CALML3 (GenBank accession number NM_005185; molecular mass of 19.5 kDa) from the pEXP5-NT/CALML3 plasmid. For this purpose, CALML3 was tagged with the N-terminal tetracysteine Lumio tag (Cys-Cys-Pro-Gly-Cys-Cys) using Q5^R Site-Directed Mutagenesis Kit (New England BioLabs). We followed the manufacturer’s instructions for the cell-free transcription–translation. LZD was added to the reactions to the final concentrations 0.02, 0.2, 2, 20 μM, and 3h, 8o, 8d, and 12f were added at 1× and 0.1× MICs (Table S1). The amounts of the synthesized protein were detected using In-Gel Lumio Detection method. For this purpose, after 5 hours of incubation at 37 °C, the biarsenical Lumio Green Detection Reagent was added to the reactions. This reagent becomes fluorescent upon binding covalently to proteins containing the Lumio tag.² Samples were loaded on 16% SDS-PA gel and visualized immediately after electrophoresis (Figure 3B). The ImageJ software (ImageJ (nih.gov)) was used to calculate the area of the picks from the intensity of the fluorescent protein bands on gels. The amounts of the synthesized CALML3 in the absence of oxazolidinones were set as 100%. The nonlinear fit to sigmoidal curves was performed to calculate the drug concentration that yields a 50% inhibition of protein synthesis (K_i^app).

ABBREVIATIONS USED

Δ

efflux knockout

AB

Acinetobacter baumannii

CLSI

Clinical & Laboratory Standards Institute

DIPEA

diisopropylethyl-amine

E/PE

OM impact ratio

EC

Escherichia coli

GNB

Gram-negative bacteria

LB

Luria-Bertani broth

LZD

line-zolid

M9-MOPS

M9 medium supplemented with MOPS buffer

MDR

multidrug-resistant

MHI

Mueller-Hinton broth

OM

outer membrane

P/PE

efflux impact ratio

PA

Pseudomonas aeruginosa

PC

principal component

PMBN

polymyxin B nonapeptide

Pore

hyperporinated strain

PTC

peptidyl transferase center

SAR

structure–activity relationship

SUR

structure–uptake relationship

WT

wild type