A Small Molecule Benzothiophene with In Vivo Efficacy in a Mouse Model of Drug-Resistant Enterococcus faecium Infection

Abstract

Hospital-acquired infections, caused by ESKAPE bacteria, are a challenging global public health concern in part due to the emergence of drug-resistant strains. While profiling a diverse set of compounds for in vitro activity versus this class of bacteria, we noted the benzothiophene JSF-2827 exhibited promising antibacterial activity against Enterococcus faecium. A hit evolution campaign ensued, involving the design, synthesis, and biological assay of analogs designed to address early issues such as a short mouse liver microsome half-life and a modest mouse pharmacokinetic profile. Among these derivatives, JSF-3269 was found to exhibit an enhanced profile and in vivo efficacy in an immunocompetent mouse model of acute, drug-resistant E. faecium infection. The findings suggest a rationale for the further evolution of this promising series to afford a novel therapeutic strategy to treat drug-resistant E. faecium infection.

Graphical Abstract

INTRODUCTION

Antibacterial drug resistance has become one of the most serious public health concerns worldwide.¹ With hospital-acquired infections, the ESKAPE bacteria² (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are amongst the leading causes.³ E. faecium, a Gram-positive facultative anaerobe, is a commensal of the gastrointestinal tract of humans. E. faecium infections can present clinically as urinary tract infections, bacteremia, meningitis, endocarditis, and wound infections.⁴

The common therapy for drug-susceptible E. faecium strains is based on combining an aminoglycoside with a β-lactam, such as ampicillin or penicillin, or vancomycin.⁴ However, the emergence of clinical drug-resistant strains towards β-lactams, aminoglycosides, macrolides, and glycopeptides has been reported.^{5, 6} Vancomycin-resistant enterococci (VRE) are especially challenging to treat and necessitate either daptomycin or linezolid therapy. However, the emerging resistance towards either drug is a significant concern. Therefore, it is of the utmost importance to develop innovative therapeutic agents to treat E. faecium infections, leveraging a novel mechanism of action as to avoid cross-resistance with approved antibacterials.

During the course of a program focused on the optimization of an antitubercular family of N-benzyl-5-nitrofuran-2-carboxamides,⁷ we noted the antibacterial efficacy of benzothiophene JSF-2827 (Figure 1). JSF-2827 exhibited significant activity against the Enterococcus faecium NCTC 7171 reference strain (ATCC 19434; all E. faecium assays utilize this strain unless noted otherwise) with a minimum inhibitory concentration (MIC; the minimum compound concentration required to achieve 90% growth inhibition of the bacterium in culture) of 0.75 μM and low cytotoxicity toward Vero cells (ATCC CCL-81) as a model mammalian cell line, quantified by a CC₅₀ (the minimum compound concentration required to attain 50% growth inhibition of this cell line in culture) of 75 μM. Given the presence of the nitrofuran in JSF-2827, we were drawn to consider parallels to nitrofurantoin, an approved drug used for urinary tract infections, but with limited in vitro efficacy versus E. faecium.⁸ Based on the antibacterial activity of our hit compound towards E. faecium, we decided to pursue optimization of JSF-2827 through structure-activity relationship (SAR) studies. We report herein the discovery of JSF-3269 (Figure 1), which evolved from JSF-2827 to demonstrate an improved pharmacokinetic (PK) profile in mice and in vivo efficacy in a mouse peritonitis-sepsis model of vancomycin-resistant E. faecium infection.

Figure 1. The chemical structures of hit compound JSF-2827 and optimized analog JSF-3269.

RESULTS AND DISCUSSION

JSF-2827, synthesized in 2 steps from commercial materials (Scheme 1), demonstrated selective activity versus E. faecium as it was inactive (MIC > 150 μM) against representative strains of Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa (Table S1). Given the clinical need for new agents to treat E. faecium infections, we decided to further profile this compound and began with assays for mouse liver microsome (MLM) stability and solubility. JSF-2827 showed an MLM half-life (t_1/2) of 3.30 min and a kinetic aqueous solubility (S) of 103 μM in pH 7.4 PBS. In addition, we obtained the mouse snapshot pharmacokinetic (PK) profile^{9, 10} of JSF-2827 which demonstrated modest plasma exposure as quantified by the 0 – 5 h area under the curve (AUC_0-5h = 1280 h*hg/mL) with oral (po) dosing (Figure S1). Based on this preliminary profiling of JSF-2827 which highlighted the insufficient MLM t_1/2 (goal value of ≥60 min¹¹), we focused our efforts to synthesize a family of JSF-2827 analogs to improve metabolic stability and antibacterial activity towards E. faecium.

Scheme 1. Synthesis of JSF-2827 and Hit Analogs with a Focus on the Core Heterocycle.

R¹ may be H, NO₂, or Cl, W may be O or S, and R² is delineated in Tables 1, 3, 4, and 5.

Our first goal was to synthesize a series of JSF-2827 analogs and evaluate their respective antibacterial activity towards E. faecium. We observed the requirement for the nitro moiety as des-nitro JSF-2827, prepared in two steps from the commercially available benzothiophene 2-carboxylic acid (Scheme 1), exhibited an MIC > 174 μM towards E. faecium. Thus, we proceeded to pursue, via the same synthetic chemistry route, the preparation of a set of analogs with replacements for the benzothiophene-2-yl moiety while maintaining the 5-nitrofuryl hydrazide portion of JSF-2827 (Table 1). The synthesis of these and structurally related analogs were achieved by coupling commercially available heterocyclic carboxylic acids with the appropriate furan-2-carbohydrazide, using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)¹² in the presence of DIPEA in CH₃CN (Scheme 1). Interestingly, movement of the benzothiophene acyl linkage from the 2- (cf., JSF-2827) to the 3-position (cf., 1) led to a twentyfold loss of whole-cell activity. Furthermore, replacement of the benzothiophene-2-yl with different heterocycles (i.e., benzofuran-2-yl, 1H-indole-2-yl, benzothiazole-2-yl, 4,5,6,7-tetrahydrobenzothiophene-2-yl, and thieno[2,3-b]pyridine-2-yl) in compounds 2 – 6 all afforded unacceptable increases in MIC.

Table 1.

Exploration of heterocyclic replacements in the hit series.

Compound	R	E. faecium MIC (μM)a,b	Vero Cell CC50 (μM)b
JSF-2827	benzothiophene-2-yl	0.75	75
1	benzothiophene-3-yl	15	75
2	benzofuran-2-yl	>100	39
3	1H-indole-2-yl	>100	>160
4	benzothiazole-2-yl	30	150
5	4,5,6,7-tetrahydrobenzo[b]thiophene-2-yl	37	37
6	thieno[2,3-b]pyridine-2-yl	>100	150
vancomycin		1.1	ND
linezolid		4.6	ND
daptomycin		10	ND
meropenem		19	ND
kanamycin		>24	ND

^aThe E. faecium strain was NCTC 7171.

^bEach measurement was determined as the average from at least two runs.

Subsequently, we explored replacement of the central hydrazide group with different linkers. For instance, gem-dimethyl 7, piperazine 8, ethylenediamine 9, hydrazone 10, and oxadiazole 11 were prepared (Scheme 2). 7 was obtained through the reduction of benzothiophene-2-carbonitrile to the α,α-dimethylamine,¹³ which was then coupled with 5-nitrofuran-2-carbonyl chloride. The synthesis of 8 and 9 was performed by formation of the acid chloride from benzothiophene-2-carboxylic acid and then coupling of it with the appropriate diamine and 5-nitrofuran-2-carbonyl chloride in a one-pot three component reaction. 10 was obtained via condensation of benzothiophene-2-carboxaldehyde with 5-nitrofuran-2-carbohydrazide. Subsequently, 11 was obtained by cyclization of 10 in the presence of (diacetoxyiodo)benzene¹⁴. Unfortunately, these linkers did not effectively substitute for the hydrazide linker of JSF-2827 as their MIC values were >130 μM.

Scheme 2. The Synthetic Route to Analogs with Replacements of the Hydrazide Linker.

We next focused our optimization on the benzothiophene ring and examined the differential placement of a single chlorine around the ring system. Among the mono-chloro substituted benzothiophenes, prepared via the synthetic route in Scheme 1, 16, the 7-Cl analog, exhibited the best antibacterial activity with an MIC of 4.2 μM (Table 3). 3-Cl 12 and 4-Cl 13 demonstrated reduced activity in comparison. 14 and 15, the 5-Cl and 6-Cl analogs respectively, both lacked significant antibacterial activity.

Table 3.

Exploration of Chloro Substitution of the Benzothiophene in the Hit Series.

Compound	R	E. faecium MIC (μM)a,b	Vero Cell CC50 (μM)b
12	3-Cl	17	68
13	4-Cl	34	136
14	5-Cl	>137	>137
15	6-Cl	>136	>136
16	7-Cl	4.2	68

^aThe E. faecium strain was NCTC 7171.

^bEach measurement was determined as the average from at least two runs.

Dichlorinated analogs were examined subsequently and were prepared similarly as the mono-chloro analogs (Scheme 1). 19 (heretofore referred to as JSF-3269), the 3,6-dichloro analog, and 5,7-dichloro 22 were the two most potent compounds in this series (Table 4) with improved Vero cell cytotoxicity and slightly diminished whole-cell efficacy as compared to JSF-2827. The remaining analogs were less cytotoxic to Vero cells than JSF-2827 but suffered unacceptable losses in MIC.

Table 4.

Exploration of Dichloro Substitution of the Benzothiophene in the Hit Series.

Compound	R	E. faecium MIC (μM)a,b	Vero Cell CC50 (μM)b
17	3,4-diCl	7.8	>124
18	3,5-diCl	>124	>124
19 (JSF-3269)	3,6-diCl	1.9 – 3.8	>120
20	3,7-diCl	62	>120
21	4,6-diCl	62	>124
22	5,7-diCl	0.75	124
23	3-Cl,6-CF3	3.6	110
24	3-Cl,6-F	>130	>130
25	5-CF3,7-Cl	1.8	>110

^aThe E. faecium strain was NCTC 7171.

^bEach measurement was determined as the average from at least two runs.

To further expand our SAR around JSF-3269, we sought to replace the chlorine atom in the 6-position with other electron-withdrawing groups such as CF₃ and F (Table 4). These analogs were synthesized similarly as the mono- and dichloro analogs (Scheme 1). 23 retained antibacterial activity comparable to JSF-3269. In contrast, 24 lacked significant antibacterial activity (MIC > 130 μM) towards E. faecium. In the case of 22, we prepared the 5-CF₃, 7-Cl analog 25. 25 was twofold less potent than 22 and approximately equipotent with JSF-3269.

Finally, we sought to replace the nitrofuran heterocycle, maintaining the 3,6-dichlorobenzothiophene scaffold of JSF-3269. 5-Nitrothiophene 26, 5-chlorofuran 27, and 5-chlorothiophene 28 were prepared in two steps as described in Scheme 1. In each case, it was clear that removal of the nitrofuran led to a loss of whole-cell efficacy (Table 5).

Table 5.

Exploration of Substitution for the Nitrofuran in the Hit Series.

Compound	R	W	E. faecium MIC (μM)a,b	Vero Cell CC50 (μM)b
26	NO2	S	>120	>120
27	Cl	O	>128	128
28	Cl	S	>123	123

^aThe E. faecium strain was NCTC 7171.

^bEach measurement was determined as the average from at least two runs.

At this juncture in the hit optimization, given their MIC values, JSF-3269, 22, and 25 were assayed for MLM stability, kinetic aqueous solubility, and mouse PK profile (Table 6). The MLM t_1/2 values for JSF-3269, 25, and 22 were 16.6 min, 57.8 min, and 13.3 min, respectively. 22 and JSF-3269 exhibited comparable solubilities of 0.55 and 0.35 μM, respectively; the solubility of 25 was quite low (S = 0.06 μM). From PK studies (Figures S2 – S4), their AUC_0-5h values were in the order of 25 > JSF-3269 > 22.

Table 6.

Mouse PK, Metabolic Stability, and Aqueous Solubility data for Select Compounds.

Compound	MLM t1/2a (min)	Solubility (S) in pH 7.4 PBSb (μM)	AUC0-5hc (h*ng/mL)
JSF-3269	16.6	0.35	75175
25	57.8	0.060	110845
22	13.3	0.55	43776

^aThe t_1/2 measurement was determined from a 5-point curve.

^bThe S measurement was determined as the average from three replicates.

^cThe AUC was determined as the average value from 2 mice.

Given consideration of compound MIC, Vero cell CC₅₀, MLM t_1/2, S, and mouse PK AUC_0-5h with the relatively poor solubility of 25 being particularly concerning, JSF-3269 was selected for further study. We began by determining its minimal bactericidal concentration (MBC) versus E. faecium NCTC 7171 to be 7.7 – 15 μM (Table S2). JSF-3269 was found to exhibit modest whole-cell activity versus the reference Staphylococcus aureus strain (MIC = 16 μM) while not demonstrating significant efficacy (MIC > 120 μM) versus the A. baumannii, K. pneumoniae, and P. aeruginosa reference strains (Table S1).

Furthermore, JSF-3269 exhibited acceptable stability in the presence of human liver microsomes (t_1/2 = 85.6 min), human plasma (76.6% remaining at t = 5 h), and mouse plasma (89.2% remaining at t = 5 h). The compound demonstrated high levels of binding to human plasma (99.3%) and mouse plasma (99.99%). Finally, JSF-3269 was not a significant inhibitor of human cytochrome P450 isoforms 1A2, 2C9, 2C19, 2D6, and 3A4 (Table S3).

With an eye towards an in vivo efficacy study in mice, we further characterized the PK profile of JSF-3269. JSF-3269 exhibited an oral bioavailability of 47% (Figure S5). Single doses of JSF-3269 were tolerated in mice up to the highest level tested of 300 mg/kg po, although exposure as quantified by AUC_0-24h appeared to plateau between 150 and 300 mg/kg. (Figure S6 and Table S4). Therefore, JSF-3269 was studied at 150 mg/kg in an immunocompetent mouse model of acute peritonitis-sepsis.¹⁵ A single 150 mg/kg po dose of JSF-3269 was compared to vancomycin (100 mg/kg subcutaneous (sc)) and linezolid (150 mg/kg po) (Figure 2). Mice were infected with the vancomycin-resistant E. faecium EN 2966 (MIC ≥ 30 μM (vancomycin), MIC = 4.7 μM (linezolid)) for which JSF-3269 exhibited the same MIC as versus the NCTC 7171 strain. While JSF-3269 afforded a similar level of protection as high-dose vancomycin at t = 3 d post-infection, both compounds were inferior to linezolid.

Figure 2. Efficacy study in a peritonitis-sepsis model of vancomycin-resistant E. faecium infection (EN 2966 strain), comparing JSF-3269, vancomycin, and linezolid.

Via a Mantel-Cox test, all three treatments were significantly different than vehicle (JSF-3269 (p < 0.05); vancomycin (p < 0.01); linezolid (p < 0.0001)).

The modest in vivo efficacy of JSF-3269 against a clinical drug-resistant E. faecium strain suggests that further efforts will be necessary to optimize this series (e.g., improving in vivo efficacy by enhancing the compound MIC and mouse PK plasma exposure). This will necessitate additional chemistry resources that ideally will be informed as to the mechanism of action of JSF-3269. An initial effort probed as to whether the essential nitro group is responsible for the release of NO•. Intracellular bioactivation of the nitro group may lead to formation of NO• and other reactive nitrogen species.^{16, 17} We quantified intrabacterial NO• release via the Griess reagent to detect the presence of nitrite in the supernatant,^{18, 19} which is the oxidation product of NO• that undergoes efflux from the bacterium. However, no evidence was found to support the intracellular release of NO• in E. faecium (Figure S7). Further studies will be required, including the attempts to raise spontaneous JSF-3269-resistant mutants and validate that one or more mutations confer/s resistance, to comment on this compound’s mechanism of action.

CONCLUSIONS

In summary, we commenced with benzothiophene JSF-2827 – a hit exhibiting in vitro potency versus E. faecium but with a modest PK profile most likely due to low mouse metabolic stability. SAR studies around JSF-2827 led to JSF-3269, featuring a 3,6-dichlorobenzothiophene scaffold, with improved MLM stability and mouse PK profile. Significantly, JSF-3269 demonstrated in vivo efficacy in a peritonitis-sepsis mouse model of vancomycin-resistant E. faecium infection. A search of the literature through SciFinder (https://scifinder.cas.org) and PubChem (https://pubchem.ncbi.nlm.nih.gov) did not evidence publications disclosing the core structure and its link to antibacterial efficacy. The sum total of results support future studies to further optimize the efficacy of this series which will hinge on additional medicinal chemistry efforts and mechanism of action studies.

EXPERIMENTAL SECTION

Chemistry

General Methods.

All reagents were purchased from commercial suppliers and used without further purification unless noted otherwise. All chemical reactions occurring solely in an anhydrous organic solvent were B under an inert atmosphere of argon or nitrogen unless noted otherwise. Reactions performed at rt were typically at 21 – 24 °C. Analytical TLC was performed with Merck silica gel 60 F₂₅₄ plates. Silica gel column chromatography was conducted with Teledyne Isco CombiFlash Companion or Rf+ systems. ¹H NMR spectra were acquired on Bruker 500 MHz instruments and are listed in parts per million downfield from TMS. LC-MS was performed on an Agilent 1260 HPLC coupled to an Agilent 6120 MS. All synthesized All compounds were >95% pure by HPLC analysis and were characterized by the expected parent ion/s in the MS.

General Procedure A – Synthesis of N'-(benzo[b]thiophene-2-carbonyl)-5-nitrofuran-2-carbohydrazide (JSF-2827):

Synthesis of 5-nitrofuran-2-carbohydrazide:

To a stirred solution of 5-nitrofuroyl chloride (1.5 g, 8.2 mmol) in THF (40 mL) was added EtOH (0.97 mL, 17 mmol, 2 eq) at 0 °C followed by slow addition of Et₃N (2.36 mL, 16.9 mmol, 2 eq). The reaction was allowed to stir at rt overnight. Then, the crude reaction mixture was extracted with EtOAc, and washed sequentially with saturated aqueous NaHCO₃ solution and saturated aqueous brine solution. The organic layer was dried over anhydrous Na₂SO₄ and concentrated in vacuo. The crude was dissolved in EtOH (30 mL) and cooled at 0 °C. Then, hydrazine monohydrate (0.67 mL, 14 mmol, 2.0 equiv) was added slowly and the reaction was stirred at 0 °C for 1 h and then at rt for 1h. The product was filtered off and obtained as a yellow solid (0.75 g, 4.4 mmol, 53% yield) which was used without further purification: ¹H NMR (500 MHz, d₆-DMSO) δ 10.2 (br s, 1H), 7.74 (d, J = 4.0 Hz, 1H), 7.36 (d, J = 4.0 Hz, 1H), 4.70 (br s, 2H). Calculated for C₅H₆N₃O₄ [M+H]⁺ 172.0, found 172.1.

To a stirred solution of benzothiophene-2-carboxylic acid (0.050 g, 0.29 mmol) and 5-nitrofuran-2-carbohydrazide (0.050 g, 0.29 mmol, 1 eq) in CH₃CN (4 mL) was added TBTU (0.16 g, 0.32 mmol, 1.1 eq), followed by DIPEA (0.070 mL, 0.38 mmol, 1.3 eq). The reaction mixture was allowed to stir at rt overnight. The crude reaction was quenched with saturated NaHCO_3(aq) solution and extracted with EtOAc. Afterwards, the organic layer was washed with 0.25 N HCl_(aq) (4 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. The crude product was purified via flash chromatography over silica gel eluting with MeOH/DCM (0 – 10%) to obtain the title product as a white solid (9.1 mg, 0.027 mmol, 9.7% yield): ¹H NMR (500 MHz, CD₃CN) δ 9.11 (br s, 2H), 8.01 (s, 1H), 7.99 (d, J = 7.0 Hz, 2H), 7.51 (m, 3H), 7.36 (s, 1H). Also noted 3.08 (s), 2.13 (s, H₂O), and 1.77 (s). Calculated for C₁₄H₁₀N₃O₅S [M+H]⁺ 332.0, found 332.3.

Compounds des-nitro JSF-2827, 1 – 6, and 12 – 28 were synthesized according to general procedure A and their spectroscopic characterization data may be found in the Supporting Information.

Synthesis of N-(2-(benzo[b]thiophen-2-yl)propan-2-yl)-5-nitrofuran-2-carboxamide (7):

Anhydrous CeCl₃ (1.53 g, 6.20 mmol, 3.0 eq) was stirred in THF (15 mL) at rt for 2 h. Then, the solution was cooled to −78 °C and followed by the dropwise addition of MeLi (3.9 mL, 1.6 M, 3.0 eq), and allowed to stir for 30 min at −78 °C. Then, a solution of benzo[b]thiophene-2-carbonitrile (0.32 g, 2.0 mmol) in THF (5 mL) was added dropwise to the above solution. The crude reaction mixture was stirred at rt overnight, quenched by adding 1.5 mL concentrated NH₄OH_(aq), and allowed to stir for 1 h. After 1 h, the crude reaction was extracted with EtOAc (20 mL) and washed with saturated aqueous brine solution. The organic layer was then separated, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The crude product (0.29 g, 1.6 mmol) was dissolved in DCM (5 mL) and a solution of 5-nitro-2-furoyl chloride (0.27 g, 1.6 mmol, 1.0 eq) in DCM (5 mL) was added dropwise, followed by the dropwise addition of Et₃N (0.48 mL, 3.4 mmol, 2.2 eq) at rt. The reaction was stirred for 5 h until completion as judged by LC-MS. The reaction mixture was diluted with DCM (10 mL) and washed with saturated aqueous brine solution. The organic layer was separated, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The reaction product was purified via flash chromatography over silica gel eluting with 95% DCM/5% MeOH to obtain the product as a light brown solid (100 mg, 0.348 mmol, 40.1%): ¹H NMR (500 MHz, d₆-DMSO) δ 9.03 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.75 (m, 2H), 7.52 (d, J = 4.0 Hz, 1H), 7.33 (t, J = 7.0 Hz, 1H), 7.28 (m, 2H), 1.82 (s, 6H). ¹³C NMR (125 MHz, d₆-DMSO) δ 155.7, 153.3, 148.2, 139.5, 138.2, 124.2, 123.9, 123.2, 122.2, 119.2, 115.8, 113.3, 54.7, 29.5. Calculated for C₁₆H₁₈N₃O₄S [M+NH₄]⁺ 348.1, found 347.9.

General Procedure B – Synthesis of benzo[b]thiophen-2-yl(4-(5-nitrofuran-2-carbonyl)piperazin-1-yl)methanone (8):

Synthesis of benzo[b]thiophene-2-carbonyl chloride: To a stirred solution of benzo[b]thiophene-2-carboxylic acid (0.10 g, 0.56 mmol, 1 eq) in DCM (5 mL) at 0 °C was added oxalyl chloride (0.050 mL, 0.60 mmol, 1.1 eq) dropwise, followed by DMF (1 drop). The reaction mixture was stirred at rt overnight. The crude reaction mixture was concentrated in vacuo and used for next reaction.

To a stirred solution of benzo[b]thiophene-2-carbonyl chloride (0.11 g, 0.56 mmol) and 5-nitro-2-furoyl chloride (0.11 g, 0.56 mmol, 1 eq) in THF (5 mL) at 0 °C was added a solution of piperazine (0.11 g, 1.1 mmol, 2.0 eq) in THF (3 mL) dropwise. The reaction mixture was allowed to warm up to rt overnight. The crude reaction was extracted with DCM (50 mL) and washed with saturated aqueous brine solution. The organic layer was dried over anhydrous Na₂SO₄ and concentrated in vacuo. The reaction product was purified via flash chromatography over silica gel eluting with 10% MeOH/DCM to obtain the title product as a white solid (65 mg, 0.17 mmol, 30%): ¹H NMR (500 MHz, d₆-DMSO) δ 8.04 (m, 1H), 7.94 (m, 1H), 7.81 (s, 1H), 7.80 (d, J = 3.5 Hz, 1H), 7.46 (m, 2H), 7.33 (d, J = 4.0 Hz, 1H), 3.86-3.75 (m, 8H). ¹³C NMR (125 MHz, d₆-DMSO) δ 162.8, 156.9, 147.3, 139.4, 138.6, 136.5, 126.0, 125.9, 125.0, 125.0, 122.5, 117.3, 112.9, 39.8, 39.6. Calculated for C₁₈H₁₆N₃O₅S [M+H]⁺ 386.1, found 386.4.

Compound 9 was synthesized according to general procedure B and its spectroscopic characterization data may be found in the Supporting Information.

Synthesis of (E)-N'-(benzo[b]thiophen-2-ylmethylene)-5-nitrofuran-2-carbohydrazide (10):

A stirred solution of benzo[b]thiophene-2-carbaldehyde (0.090 g, 0.57 mmol) and 5-nitrofuran carbohydrazide (0.10 g, 0.57 mmol, 1.0 eq) in MeOH (15 mL) was subjected to the addition glacial acetic acid (0.5 mL) and then was heated at reflux for 3 h. After cooling to rt, the crude reaction mixture was concentrated in vacuo, the resulting residue was washed with diethyl ether, and filtered to obtain pure product as an orange solid (0.17 g, 0.54 mmol, 95% yield): ¹H NMR (500 MHz, d₆-DMSO) δ 8.83 (s, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.89 (m, 2H), 7.82 (d, J = 3.5 Hz, 1H), 7.58 (d, J = 4.0 Hz, 1H), 7.42 (m, 3H). One hydrogen was unaccounted for. ¹³C NMR (125 MHz, d₆-DMSO) δ 152.6, 147.0, 144.9, 139.5, 139.1, 139.0, 129.0, 126.3, 124.9, 124.5, 122.7, 116.9, 113.4. One carbon was unaccounted for. Calculated for C₁₄H₁₀N₃O₄S [M+H]⁺ 316.0, found 316.0.

Synthesis of 2-(benzo[b]thiophen-2-yl)-5-(5-nitrofuran-2-yl)-1,3,4-oxadiazole (11):

To a stirred solution of (E)-N'-(benzo[b]thiophen-2-ylmethylene)-5-nitrofuran-2-carbohydrazide (10) (0.060 g, 0.20 mmol) in DCM (5 mL) was added (diacetoxyiodo)benzene (0.060 g, 0.20 mmol, 1.0 eq). The reaction mixture was stirred at rt overnight. The crude reaction mixture was concentrated in vacuo, washed with diethyl ether, and filtered to obtain pure product as a yellow solid (0.050 g, 0.16 mmol, 80% yield): ¹H NMR (500 MHz, d₆-DMSO) δ 8.38 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 8.09 (d, J = 7.5 Hz, 1H), 7.98 (d, J = 4.0 Hz, 1H), 7.80 (d, J = 4.0 Hz, 1H), 7.55 (m, 2H). ¹³C NMR (125 MHz, d₆-DMSO) δ 160.8, 155.6, 140.4, 139.8, 138.8, 128.5, 127.3, 125.6, 125.6, 123.0, 117.0, 114.2. Calculated for C₁₄H₈N₃O₄S [M+H]⁺ 314.0, found 314.0.

Biological Assays

Bacterial Strains.

E. faecium NCTC 7171 was purchased from the ATCC ATCC (19434) and the E. faecium clinical strain EN 2966 was supplied by the Kreiswirth lab (Public Health Research Institute, Rutgers University).

Antibacterial Growth Inhibition Assays.

Minimal inhibitory concentration (MIC) assays were performed in 96-well plates. Briefly, the test compounds were serially diluted in 50 μL of growth media (BBL^™ Muller Hilton II cation adjusted broth (MH) (Becton, Dickinson and Company)) with the addition of 1% IsoVitaleX^™ (Becton, Dickinson and Company). Supplemented 50 μL diluted cultures (1:1000) of E. faecium grown to mid-log phase (OD₆₀₀ = 0.2 – 0.3) were added to each well to reach a final volume of 100 μL per well. Typical test compound concentrations typically ranged from 200 to 0.1 μg/mL. After incubation overnight at 37 °C, MIC values were determined by visual inspection of cell pellet inside the 96-well plates. MIC values were read as the minimum test compound concentration where cell pellets were invisible in media. A similar approach was taken with the pertinent S. aureus, K. pneumoniae, A. baumannii, and P. aeruginosa strains. MH medium was used for these bacteria.

Minimum Bactericidal Concentration (MBC) Assay.

MBC assays were performed for E. faecium using the 96-well plate of the MIC assay. Briefly, after checking the wells for pellets, the wells were mixed by pipetting and 10-fold dilutions were made in Dulbecco’s Phosphate Buffered Saline Solution (ThermoFisher Scientific, Inc.). Dilutions were plated on MH, with the addition of 1% IsoVitaleX^™ agar plates. Plates were incubated for 2 d at 37 °C and then bacterial colonies were quantified.

Mammalian Cell Cytotoxicity Assay.

Vero cells (African green monkey kidney epithelial cells; ATCC CCL-81) were cultured in a 96-well plate, at a concentration of 10⁵ cells/well, and incubated for 2 to 3 h to allow cells to settle. Test compounds were diluted separately in Eagle’s minimal essential medium to generate test concentrations typically ranging from 200 to 0.1 μg/mL. The serial dilutions were then added to the plated cells and incubated for 48 h at 37 °C. The viability of Vero cells exposed to each compound was determined using the MTT [3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide] cell viability kit (Promega). The CC₅₀ was determined as the minimum test compound concentration to afford 50% growth inhibition of the Vero cells.

Mouse Liver Microsomal Stability Assay.

This assay was carried out by BioDuro, Incorporated. Solutions of the test compound were made in DMSO and diluted to a final concentration of 100 μM in 50 mM phosphate buffer (pH 7.4). Aliquots of mouse liver microsome working solution were added to Eppendorf tubes via a multichannel pipette. A positive control (midazolam) and test compound working solutions were added to the tubes. The mixtures were vortexed gently and then pre-incubated at 37 °C. Buffer with or without 5 mM NADPH was added to the tubes with a multichannel pipette and vortexed gently. At each time point of 0, 5, 15, 30, and 60 min with NADPH or 0, 30, and 60 min without NADPH, terfenadine/tolbutamide in acetonitrile/MeOH (1:1 v/v) was added to the reaction mixture to quench and precipitate the microsomal incubations. Samples were capped and vigorously vortexed and then centrifuged at 4 °C. An aliquot of each supernatant was attained for LC-MS/MS analysis. The MS detection was achieved with a SCIEX API 4000 QTRAP instrument. Each compound was analyzed by reverse-phase HPLC using a Kinetex 2.6μ C18 100Å column (3.0 mm X 30 mm, Phenomenex) with the mobile phase of solvent A: water with 0.1% formic acid, solvent B: acetonitrile with 0.1% formic acid. The amount of parent compound was quantified on the basis of the peak area ratio (compound area to internal standard area) at each time point, allowing the determination of the compound half-life, t_1/2.

Kinetic Aqueous Solubility Assay.

This assay was carried out by BioDuro, Incorporated. Dilutions of test compound solution were made in DMSO. 4 μL of each dilution of the test compound in DMSO was added to 396 μL of the universal aqueous buffer (pH = 7.4; 45 mM ethanolamine, 45 mM KH₂PO₄, 45 mM potassium acetate, 75 mM KCl) to provide a final DMSO concentration between 0.002 μM and 200 μM. Three replicates of each test compound were made per concentration. After 4 h shaking at rt, the mixture was further incubated without shaking for 30 min at rt and was then filtered. The filtrate was diluted 10x and 30x with DMSO before LC-MS/MS analysis. Standard solutions were made as follows: stock solutions were diluted to ten defined concentration points from 60 μM to 0.002 μM with DMSO. Aliquots of samples and standard solutions were filtered and mixed with acetonitrile/H₂O, then vortexed and used for LC-MS/MS analysis. The MS detection was achieved with a SCIEX API 4000 QTRAP instrument. Each compound was analyzed by reverse-phase HPLC using a Kinetex 2.6μ C18 100 Å column (3.0 mm X 30 mm, Phenomenex) with the mobile phase consisting of solvent A: water with 0.1% formic acid, solvent B: acetonitrile with 0.1% formic acid. The amount of parent compound was quantified on the basis of the peak area ratio (compound area to internal standard area) for each time point. The solubility of the test compound was found based on the largest calculated concentration amongst the samples.

Mouse Plasma Protein Binding and Plasma Stability Determination.

This assay was carried out by BioDuro, Incorporated. Working solutions (1 mM in DMSO) were made for each test and control compound. The dosing solutions were made by diluting the working solutions to 5 μM in mouse plasma. The dialysis plate was made by adding buffer to one chamber and dosing solution to the other chamber. The plate was sealed with an adhesive film and incubated at 37 °C while shaking for 5 h. Equal volumes of post dialysis samples were removed from both the plasma and the buffer chambers and put in separate microcentrifuge tubes and equal volumes (50 μL) of fresh phosphate buffer and plasma were added to the tubes, respectively. Plasma samples were diluted 5-fold and then all samples were subjected to the addition of quenching solution (terfenadine/tolbutamide in 1:1 v/v methanol/acetonitrile). Sample mixtures were then centrifuged, and the supernatant was subjected to LC-MS/MS analysis. To assess plasma stability, aliquots of dosing solution were stored at 4 °C (t = 0 h sample) and at 37 °C for 5 h (t = 5 h sample). Following incubation, aliquots were subjected to LC-MS/MS analysis. The MS detection was via a SCIEX API 4000 QTRAP instrument. Each compound was analyzed by reverse-phase HPLC using a Kinetex 2.6μ C18 100 Å column (3.0 mm X 30 mm, Phenomenex) with the mobile phase consisting of solvent A: water with 0.1% formic acid, solvent B: acetonitrile with 0.1% formic acid. The amount of parent compound was quantified via the peak area ratio (compound area to internal standard area) for each time point. The percent plasma protein binding was determined according to equation 1, where Cpe is the concentration of test compound in plasma at equilibrium and Cb is the concentration of test compound in buffer at equilibrium, and the percent plasma stability was determined via equation 2:

$$\%Binding=\frac{Cpe-Cb}{Cpe}\times 100$$ [Eqn. 1]

$$\%Stabilityoftestcompound=\frac{[stabilitysample]}{[timezerosample]}\times 100$$ [Eqn. 2]

Human Cytochrome P450 Inhibition Assay.

This assay was carried out by BioDuro, Incorporated. Pooled human liver microsomes were used as the enzyme source, and phenacetin (CYP1A2, 10 μM), diclofenac (CYP2C9, 10 μM), omeprazole (CYP2C19, 0.5 μM), dextromethorphan (CYP2D6, 5 μM), and midazolam (CYP3A4, 5 μM) as probe substrates. The assay mixture (200 μL total volume) contained test compound (each with final concentrations in the 0 – 50 μM range) and human liver microsomes (final concentration of 0.25 mg protein per mL) with or without NADPH (final concentration of 1.0 mM) in 100 mM phosphate buffer (pH 7.4). After a 20 min incubation at 37 °C, the mixture was quenched by addition of 300 μL of methanol/acetonitrile (1:1 v/v) containing terfenadine and tolbutamide. The sample was then centrifuged at 4,000 rpm for 15 min at 4 °C. 100 μL supernatant was analyzed via LC-MS/MS. The MS detection was with a SCIEX API 4000 QTRAP instrument. Each compound was analyzed by reverse-phase HPLC using a Kinetex 2.6μ C18 100 Å column (3.0 mm X 30 mm, Phenomenex) with the mobile phase consisting of solvent A: water with 0.1% formic acid, solvent B: acetonitrile with 0.1% formic acid. The amount of parent compound was quantified on the basis of the peak area ratio (compound area to internal standard area) for each time point. Residual enzyme activity was monitored by measuring area ratio with respect to the internal standard of the corresponding metabolite for each substrate. The IC₅₀ was fit using the GraphPad Prism software program (version 6.0) according to equation 3:

$$\%Residualactivity=\frac{1}{1+e^{\frac{lnIC50-\text{ln}[\mathrm{I}]}{P}}}\times 100$$ [Eqn. 3]

where [I] and P are inhibitor concentration and Hill slope, respectively.

Mouse Pharmacokinetics (PK) and Dose Tolerability Studies.

Animal studies were carried out in accordance with the guide for the care and use of Laboratory Animals of the National Institutes of Health, with approval from the Institutional Animal Care and Use Committee (IACUC) of the New Jersey Medical School, Rutgers University, Newark. All animals were maintained under specific pathogen-free conditions and fed water and chow ad libitum, and all efforts were made to minimize suffering or discomfort. In the 5 h PK studies, two female CD-1 mice received a single dose of experimental compound administered orally at 25 mg/kg in 5% DMA/60% PEG300/35% D5W (5% dextrose in water), and blood samples were collected in K₂EDTA coated tubes pre-dose, 0.5, 1, 3 and 5 h post-dose. In iv/po PK studies to determine oral bioavailability, groups of three female CD-1 mice received a single dose of experimental compound administered orally at 25 mg/kg in 0.5% CMC/0.5% Tween 80 suspension, or intravenously at 5 mg/kg in 5% DMA/95% (4 % Cremophor EL). Blood samples were collected in K₂EDTA coated tubes 0.25, 0.5, 1, 3, 5 and 8 h post-dose in the oral arm, and 0.033, 0.25, 0.5, 1 and 3 h post dose in the intravenous arm. Blood was kept on ice and centrifuged to recover plasma, which was stored at −80 °C until analyzed by HPLC coupled to tandem mass spectrometry (LC-MS/MS). Oral bioavailability was reported as the 100% multiplied by the dose-normalized plasma exposure of compound with oral dosing divided by the dose-normalized plasma exposure of compound with intravenous dosing. In the dose tolerability/proportionality study, five female CD-1 mice were dosed by oral gavage daily for 5 d with JSF-3269 (50, 150, and 300 mg/kg) formulated in 0.5% CMC/0.5% Tween 80 in water. Prior to dosing, JSF-3269 was mixed and vortexed. The mice were weighed and observed daily. Their behavior, drinking and feeding patterns, and feces were monitored and recorded. Plasma samples were drawn on day 1 after JSF-3269 administration at 0.5, 1, 3, 5, 8, and 24 h. Upon necropsy, liver, gallbladder, kidney and spleen pathology were observed for abnormalities.

LC/MS-MS quantitative analysis for all test compounds was performed on a Sciex Applied Biosystems Qtrap 4000 triple-quadrupole mass spectrometer coupled to an Agilent 1260 HPLC system, and chromatography was performed on an Agilent Zorbax SB-C8 column (2.1x30 mm; particle size, 3.5 μm) using a reverse phase gradient elution. Milli-Q deionized water with 0.1% formic acid (A) was utilized for the aqueous mobile phase and 0.1% formic acid in acetonitrile (B) for the organic mobile phase. The gradient was: 5-90% B over 2 min, 1 min at 90% B, followed by an immediate drop to 5% B and 1 min at 5% B. Multiple-reaction monitoring of parent/daughter transitions in electrospray positive-ionization mode was used to quantify all molecules. Sample analysis was accepted if the concentrations of the quality control samples and standards were within 20% of the nominal concentration. Data processing was performed using Analyst software (version 1.6.2; Applied Biosystems Sciex). Neat 1 mg/mL DMSO stocks for all compounds were first serial diluted in 50/50 acetonitrile/water and subsequently serial diluted in drug free CD-1 mouse plasma (K₂EDTA, Bioreclamation IVT, NY) to create standard curves (linear regression with 1/x^2 weighting) and quality control (QC) spiking solutions. 20 μL of standards, QCs, control plasma, and study samples were extracted by adding 200 μL of acetonitrile/methanol 50/50 protein precipitation solvent containing the internal standard (10 ng/mL verapamil). Extracts were vortexed for 5 min and centrifuged at 4000 rpm for 5 min. 100 μL of supernatant was transferred for HPLC-MS/MS analysis and diluted with 100 μL of Milli-Q deionized water. Plasma AUC_0-t was determined for each dosing group by trapezoidal integration.

Mouse Peritonitis-Sepsis Model.

VRE Strain EN 2966 was grown in Brain Heart Infusion (BHI) broth at 37 °C with shaking overnight. The culture was centrifuged, supernatant aspirated, and the bacteria were gently washed once in sterile saline. The optical density was determined at 600 nm. The bacterial suspension was diluted to provide challenge inoculate of approximately 4.0 x10⁹ CFU per mouse in a volume of 0.5 mL in 5% hog mucin and 0.9% NaCl. Inoculum count was verified by viable counts on BHI agar plates spread with proper dilutions of the inoculum and incubated at 37 °C for 24 – 48 h. On day 0, mice were infected with 3.7 x10⁹ CFU per mouse in a volume of 0.5 mL with 5% hog mucin and 0.9% NaCl via intraperitoneal injection. Groups consisting of 10 mice were given single doses of vehicle (sterile CMC 0.5% + Tween 80 0.5%), JSF-3269 150 mg/kg, linezolid 150 mg/kg or vancomycin 100 mg/kg, at 1 h post-infection. All drugs and vehicle were administered by oral gavage (po) or sc injection at a volume of 0.2 mL. Mice were observed twice daily for mortality and morbidity and possible signs of acute toxicity. Abnormal clinical signs were recorded if observed. Mice were maintained in accordance with American Association for Accreditation of Laboratory Care criteria. The Rutgers University Institutional Animal Care and Use Committee approved all animal procedures.

Griess Assay.

Sodium nitrite stock solutions ranging from 50 – 0.78 μM (2-fold dilutions) were prepared in Mueller-Hinton media. A single colony of E. faecium NCTC 7171 was inoculated in fresh Mueller-Hinton media and grown to an OD₆₀₀ of 0.4. The resulting culture was split into 3 equal volume batches. The first batch served as the negative control, the second volume was treated with JSF-3269 (100 μM), and the third volume was treated with DEA-NONOate (100 μM). Following incubations of 24 h and 48 h, 1 mL of culture was sampled and centrifuged. The supernatant was placed in the same plate as mentioned above along with the nitrite standards (180 μL per well). To each of these wells, 20 μL of freshly prepared Griess reagent mix was added, and the plate was allowed to sit at rt for 30 min. The plate was read with a Biotek Synergy Neo 2 plate reader at an absorbance of 548 nm. Reads from the sodium nitrite standards were used to correlate absorbance to concentration, which facilitated estimation of nitrite production.

Supplementary Material

JSF-2827 mouse PK profile, JSF-3269 mouse PK profile, Compound 25 mouse PK profile, Compound 22 mouse PK profile, JSF-3269 mouse oral bioavailability study, JSF-3269 mouse dose proportionality study, Detection of NO• generation from JSF-3296 treated E. faecium NCTC 7171 via the Griess assay (Figures S1 – 7) and In vitro activity of JSF-2827 and JSF-3269 versus select ESKAPE bacteria, JSF-3269 MBC assay data with the E. faecium NCTC 7171 strain, JSF-3269 profiling for inhibition IC₅₀ value in μM versus human cytochrome P450 enzymes, JSF-3269 plasma exposure data from a dose-ranging mouse PK study (Tables S1 – S4). (PDF) Molecular formula strings (CSV)

Table 2.

Exploration of Different Linker Strategies in the Hit Series.

Compound	R	E. faecium MIC (μM)a,b	Vero Cell CC50 (μM)b
7		>150	38
8		>130	16
9		>140	139
10		>158	39
11		>160	160

^aThe E. faecium strain was NCTC 7171.

^bEach measurement was determined as the average from at least two runs.

ABBREVIATIONS USED

AUC_0-5h

plasma exposure area under the curve for the 0 – 5 h window

CC₅₀

cellular cytotoxicity inhibitory concentration at the 50% level

DIPEA

N,N-diisopropylethylamine

MBC

minimum bactericidal concentration

MIC

minimum inhibitory concentration

MLM

mouse liver microsome

PK

pharmacokinetic

po

oral

S

solubility

sc

subcutaneous

SAR

structure-activity relationship/s

t_1/2

half-life

TBTU

2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

sc

subcutaneous

VRE

vancomycin-resistant enterococci