PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Bioorg Med Chem. Author manuscript; available in PMC Jun 1, 2013.
Published in final edited form as:
PMCID: PMC3363956
NIHMSID: NIHMS375400
Syntheses and biological studies of novel spiropiperazinyl oxazolidinone antibacterial agents using a spirocyclic diene derived acylnitroso Diels Alder reaction
Cheng Ji,a Weimin Lin,a Garrett C. Moraski,a Jane A. Thanassi,b Michael J. Pucci,b Scott G. Franzblau,c Ute Möllmann,d and Marvin J. Millera*
aDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
bAchillion Pharmaceuticals, 300 George Street, New Haven, Connecticut, University of Illinois at Chicago, Chicago, Illinois 60612
cInstitute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612
dLeibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Beutenbergstrasse 11a, 07745 Jena, Germany
*Corresponding author. Marvin J. Miller, Tel.: +1 574 631 7571; Fax: +1 574 631 6652; mmiller1/at/nd.edu
Several novel oxazolidinone antibiotics with a spiropiperazinyl substituent at the 4’-position of the phenyl ring were synthesized through nitroso Diels–Alder chemistry and the in vitro antibacterial activities were evaluated against various Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and mycobacteria (Mycobacterium vaccae, Mycobacterium tuberculosis). Analogs (8a and 12) were active against selected drug resistant microbes, like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) and had no mammalian toxicity in a Hep-2 cellular assay (CC50 > 100 M).
Keywords: Oxazolidinone, Antibiotics, Nitroso Diels Alder reaction, Spirocyclic diene, MRSA
The rate of antimicrobial resistance has increased dramatically in the past few decades due to the misuse and overuse of antibiotics.12 Infections caused by resistant organisms such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae (PRSP) are often extremely difficult to treat. Therefore, efforts towards the discovery of new antibiotics are of great significance.
The oxazolidinones are a class of wholly synthetic, orally active antibiotics that has excellent activity against a variety of susceptible and resistant Gram-positive bacteria.36 Targeting bacterial protein synthesis, this class of compounds possesses a unique mode of action by preventing binding of the aminoacyl-tRNA to the A site of the ribosome.78 This unique activity indicates the potential for oxazolidinones to have low cross-resistance with existing protein synthesis inhibitors.911 To date, linezolid (Zyvox™, Figure 1) is the only oxazolidinone that has been approved for clinical use for the treatment of pneumonia and skin infections due to drug resistant Gram-positive bacteria such as MRSA, VRE and PRSP.3, 1213 Unfortunately, though in only a few cases, resistance against linezolid was observed not long after it was put into use.1417 The development of resistance encourages the design, syntheses, studies and hopefully development of novel oxazolidinone derivatives.
Figure 1
Figure 1
Structure-activity relationships of linezolid and several examples of different N-substituents at the 4'-position of the phenyl ring.
Extensive structure-activity relationships have been established for oxazolidinone derivatives (Figure 1).3 The S-configuration at the C-5 position of the oxazolidinone ring is necessary and the acetamide is good for antibacterial activity. Meanwhile, electron-donating nitrogen substituents at the 4’-position of the phenyl ring are well tolerated and often improve the overall safety profile.46, 18 For example, a number of linezolid derivatives with pyrroloaryl,19 azabicyclic2023 and cyanomethylpiperidinyl2425 N-substituents have been reported and some display improved properties relative to the parent drug.26
Nitroso Diels Alder (NDA) chemistry has been used as a remarkably efficient method for derivatization of complex diene-containing natural products by direct incorporation of a 1,4-amino-oxo group (Scheme 1).2730 This provides opportunities for Modular Enhancement of Nature’s Diversity (MEND).3132 NDA adducts derived from cyclopentadiene and other dienes are also synthetically valuable precursors for many biologically interesting molecules, such as carbocyclic nucleosides3336 and natural product analogs.3738 Herein we report the synthesis and biological activity evaluation of novel oxazolidinone derivatives with various spiropiperazinyl substituents using acylnitroso Diels Alder (NDA) reactions.
Scheme 1
Scheme 1
Acyl-nitroso Diels Alder (NDA) reaction.
2. 1 Chemistry
The spirocyclic diene 1 was prepared from cyclopentadienyl anion and N-Cbz-bis-(2-chloro-ethyl) amine, as reported previously.34 Diene 1 was reacted with the nitroso species generated from Boc-protected hydroxylamine by in situ oxidation with sodium periodate, providing spirocycloadduct 2 in 61% yield.39 To remove the Cbz protecting group, 2 was treated with a catalytic amount of 10% palladium on carbon and hydrogen gas (1 atm). However, a selective reduction of the alkene occurred to give compound 3 in 93% yield while leaving the Cbz group intact. The reason for the selectivity was proposed to be the steric effect of the bulky Boc group during the hydrogenolysis process. Increasing hydrogen gas to 50 psi and changing the catalyst to palladium hydroxide (20% palladium on carbon) simultaneously reduced the alkene and removed the Cbz protecting group, producing compound 4 in 91% yield for further reactions (Scheme 2).
Scheme 2
Scheme 2
Reagents: (a) NaIO4, BocNHOH, H2O/MeOH, rt, 8 h, 61%; (b) H2 (1 atm), 10% Pd/C, MeOH, rt, 8 h, 93%; (c) H2 (50 psi), Pd(OH)2, MeOH, rt, 8 h, 91%.
Compound 5 was prepared in 85% yield by an SNAr reaction of 4 with 3,4-difluoronitrobenzene in the presence of Hünig's base. The nitro group was reduced with 10% palladium on carbon and hydrogen gas (1 atm) to give amine 6 in 95% yield. The amine was then protected with a Cbz group to generate intermediate 7 in 90% yield. The oxazolidinone ring was constructed according to precedent by treatment of 7 with n-butyl lithium and R-glycidyl butyrate sequentially at −78 °C, producing alcohol derivative 8a in 85% yield with defined stereochemistry at the C-5 position.40 An ester derivative 8b was also formed in 5% yield in the same reaction. It was easily and quantitatively converted to 8a by treatment with sodium hydroxide. The hydroxyl group of 8a was converted to mesylate 9 in 90% yield. Sodium azide displacement of the mesylate provided compound 10 in 74% yield (Scheme 3).
Scheme 3
Scheme 3
Reagents: (a) 3,4-difluoronitrobenzene, DIPEA, CH3CN, rt, 8 h, 85%; (b) H2, 10% Pd/C, MeOH, rt, 1 h, 95%; (c) Cbz-Cl, NaHCO3, 0 °C to rt, 2 h, 90%; (d) (R)-(−)-glycidyl butyrate, n-BuLi, THF, −78 °C to rt, 2 h, 85%; (e) (more ...)
Exposure of 10 to hydrogen over palladium on carbon produced the corresponding amine 11 in 95% yield. Acetylation then afforded linezolid analog 12 in 70% yield. Compound 12 can serve as a common intermediate for various modifications on the spiropiperazine moiety without cleaving the N O bond, which retains the rigidity of the molecule. For example, after removal of the N-Boc protecting group of 12 with trifluoroacetic acid, reductive amination can be done as shown by incorporation of a nitrofuranyl substituent in moderated yield. Nitrofuranyl was chosen because of its structural similarity of 14 to the dual action antimicrobial agent RBx-7644 reported by the Ranbaxy Laboratories.41 Cleavage of the N O bond in the spiropiperazine moiety was anticipated to produce more flexible derivatives as the restriction of ring conformation would be eliminated. Indeed, molybdenum hexacarbonyl-mediated bond cleavage42 of compounds 11 and 12 provided the ring opened derivatives 15 and 16 in 64% and 80% yields, respectively (Scheme 4).
Scheme 4
Scheme 4
Reagents: (a) H2, 10% Pd/C, MeOH, rt, 8 h, 95%. (b) AcCl, pyridine, CH2Cl2, 0 °C to rt, 70%; (c) TFA, CH2Cl2, rt, 2 h, 95%; (d) 5-nitrofuraldehyde, NaBH(OAc)3, ClCH2CH2Cl, HOAc, rt, 2 h, 53%; (e) Mo(CO)6, NaBH4, CH3CN/H2O, 60 °C, 6 h, (more ...)
2. 2 Biological activity
Compounds 8a, 11, 12 and 16 were initially screened for antibiotic activity against representative Gram-positive and Gram-negative bacteria as well as Mycobacterium vaccae using agar diffusion assays (Table 1). Compounds 8a and 12 displayed in vitro antibacterial activities comparable to linezolid. Both showed good activity against all Gram-positive organisms tested including drug-resistant strains such as Staphylococcus aureus 134/93 (MRSA) and Enterococcus faecalis 1528 (VRE) as indicated by the large zones of inhibition they induced. Mycobacterium vaccae is a non-pathogenic model organism for Mycobacterium tuberculosis, the causative agent of tuberculosis. Although less active than linezolid, all compounds showed moderate to good activities against Mycobacterium vaccae, suggesting their potential in anti-tuberculosis agent development. Interestingly, compound 16 derived from reduction of the N–O bond showed decreased activity compared to its parent cycloadduct 12. Similar phenomena were observed in studies of C-5 side chain modification of linezolid through nitroso Diels–Alder chemistry.43 None of the compounds tested in this study displayed any noticeable significant activity against several Gram-negative indicator strains including E. coli and P. aeruginosa.
Table 1
Table 1
Antimicrobial activity in the agar diffusion assay (diameter of inhibition zone, measured in mm).
Cycloadducts 8a and 12, the two most active compounds in the agar diffusion assay, were subjected to further assays to determine their minimum inhibitory concentrations (MIC). Although both showed inferior antibacterial activity relative to linezolid,44 compound 12 has one dilution lower (better) MIC values against MSSA and MRSA than did compound 8a (Table 2), consistent with the expected advantage of having an acetamide on the C-5 side chain. To further assess their potential, the effect of protein binding on the activity was assessed by adding 50% mouse or human serum to the MIC assays, and calculating the fold shift in MIC values. Comparing to 8a which is relatively inactive in serum, compound 12 exhibited only 2 and 4-fold MIC shift in mouse and human serum, respectively, demonstrating that the spiropiperazinyl substituent at 4’-position of the phenyl ring of linezolid does not significantly change its stability or protein binding property in plasma. Potential mammalian toxicity was preliminarily assessed by screening the compounds against the Hep2 cancer cell line. Both compounds 8a and 12 did not show toxicity against the mammalian cell line.
Table 2
Table 2
MIC determination (g mL−1) of methicillin-susceptible (MS) and resistant (MR) Staphylococcus aureus, assessment of protein binding, and toxicity (M) of spiropiperazinyl compounds.
Additionally, since linezolid and its thiomorpholine analog (PNU-100480)45 are under consideration as potential antitubercular treatments, we decided to evaluate a set of compounds (5–9, 12 and 16) against replicating M. tuberculosis H37Rv in two different media, GAS46 and GAST47 (Table 3). The analogs lacking the oxazolidinone core (5, 6, and 7) all were inactive (>128 M). The incorporation of this moiety in compounds (8a, 8b, 9, 12, and 16) produced a range of antitubercular activity (MICs of 97, 19, 118, 24, >128 M, respectively in the GAS medium). As expected compound 12, the one most similar to linezolid, had one of the lowest MIC values at 24 M. Interestingly, compound 8b also had potency similar to that of compound 12 at 19 M despite missing the N-acyl moiety thought to be essential for antibacterial activity. Compound 16 derived from reduction of the N–O bond was inactive (>128 M), in sharp contrast to its parent cycloadduct 12 (24 M). This trend correlated to the observation in the agar diffusion assay against M. vaccae in which compound 16 gave only a small and unclear inhibition zone. Linezolid is reported to have excellent antitubercular potency45 (MIC range of 0.125 to 1 g mL−1) which was substantiated by our Mtb assay where it showed an MIC of 0.4 M (0.135 g mL−1) in the GAST medium and superior to our spiropiperazinyl analogs.
Table 3
Table 3
Activity of various compounds against replicating Mtb H37Rv in various media (M).
3. Conclusion
In conclusion, novel oxazolidinone antibiotics with a spiropiperazinyl substituent at the 4’-position of the phenyl ring were synthesized through nitroso Diels–Alder chemistry. The Gram-positive antibacterial activities of some of the compounds indicate that the diverse functionalities were well tolerated at phenyl 4’-position. The syntheses further exemplify the practicability of nitroso Diels–Alder chemistry as a powerful synthetic tool for the creation of unique structural and functional diversity.
4.1. General
All reactions were carried out under argon by using standard techniques. All solvents and reagents were obtained from commercial sources and used without further purification unless otherwise stated. Tetrahydrofuran (THF) was distilled from sodium and benzophenone. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel plates visualized under UV light, iodine or KMnO4 staining. Silica gel column chromatography was performed using Sorbent Technologies silica gel 60 (32–63 µm). NMR spectra were recorded on a Varian Unityplus 300 MHz spectrometer, Varian Inova 500 MHz spectrometer, or Varian 600 MHz spectrometer at ambient temperature. Benzyl 8-azaspiro[4.5]deca-1,3-diene-8-carboxylate (1) and 1'-benzyl 3-tert-butyl 2-oxa-3-azaspiro[bicyclo[2.2.1]hept[5]ene-7,4'-piperidine]-1',3-dicarboxylate (2) were prepared according to published procedures.39
4.2. Synthesis
4.2.1. 1'-Benzyl 3-tert-butyl 2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-1',3-dicarboxylate (3)
Compound 2 (1.5 g, 3.7 mmol) was dissolved in 10 mL of methanol in a 25-mL round-bottomed flask under argon. 10% Pd on carbon (0.2 g) was added and the flask was flushed with H2 gas and left to stir under a hydrogen atmosphere (balloon) for 8 h. After purging with argon, the mixture was filtered and concentrated in vacuo to give compound 3 (1.4 g, 90%) as a light yellow oil: IR (neat, cm−1): 2927, 1698, 1432; 1H NMR (600 MHz, CDCl3) δ 7.29–7.37 (m, 5H), 5.12 (s, 2H), 4.19–4.28 (m, 2H), 3.41–3.56 (m, 4H), 1.80–2.00 (m, 4H), 1.56–1.75 (m, 4H), 1.47 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 155.1, 136.6, 128.4, 127.9, 127.8, 82.7, 81.8, 67.0, 63.4, 62.2, 41.9, 28.1, 27.6, 26.6; HRMS(FAB) calcd. for C22H30N2O5 (M)+ : 402.2155; found 402.2165.
4.2.2. tert-Butyl 2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-3-carboxylate (4)
Compound 2 (1.0 g, 2.5 mmol) was dissolved in 15 mL of methanol in a 250-mL Parr bottle under argon. Pd(OH)2 (20% Pd on carbon, 0.2 g) was added and the bottle was flushed with H2 gas. The flask was shaken under 50 psi hydrogen using a Parr shaker at room temperature for 8 h. After purging with argon, the mixture was filtered and concentrated in vacuo to give compound 4 (0.61 g, 90%) as a light yellow oil: IR (neat, cm−1): 3485, 2978, 1697, 1368; 1H NMR (500 MHz, CDCl3) δ 4.12–4.45 (m, 2H), 3.20–3.35 (m, 4H), 1.86–2.10 (m, 8H), 1.48 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 157.0, 82.4, 81.4, 63.6, 61.7, 42.4, 42.3, 28.2, 27.4, 26.3, 25.2, 23.8; HRMS(FAB) calcd. for C14H25N2O3 (M+H)+: 269.1865; found, 269.1871.
4.2.3. tert-Butyl 1'-(2-fluoro-4-nitrophenyl)-2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-3-carboxylate (5)
A 10-mL oven-dried, round-bottomed flask was charged with compound 4 (0.35 g, 1.3 mmol), 3,4-difluoronitrobenzene (0.25 mL, 2.0 mmol) and DIPEA (0.45 mL, 2.6 mmol) in acetonitrile (5 mL). The reaction mixture was stirred at room temperature for 18 h and quenched by adding 1 N HCl (4 mL). The solution was extracted with ethyl acetate (5 mL×3) and the combined organic layers were washed sequentially with sat. NaHCO3 (5 mL), brine (5 mL), dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (hexanes:ethyl acetate = 3:1) to give compound 5 (0.45 g, 85%) as a yellow oil: IR (neat, cm−1): 2974, 1699, 1517; 1H NMR (500 MHz, CDCl3) δ 7.97 (dd, J = 8.9, 2.6 Hz, 1H), 7.90 (dd, J = 13.1, 2.6 Hz, 1H), 6.92 (t, J = 8.8 Hz, 1H), 4.10–4.34 (m, 2H), 3.22–3.38 (m, 4H), 1.82–2.01 (m, 8H), 1.58 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 153.9, 152.0, 145.6, 140.4, 121.0, 117.2, 112.6, 112.4, 83.2, 82.0, 64.0, 62.4, 48.5, 48.4, 28.3, 27.6, 26.6; HRMS(FAB) calcd. for C20H25FN3O5 (M-H)+: 406.1778; found 406.1787.
4.2.4. tert-Butyl 1'-(4-amino-2-fluorophenyl)-2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-3-carboxylate (6)
Compound 5 (0.30 g, 0.74 mmol) was dissolved in 4 mL of methanol in a 10-mL round-bottomed flask under argon. 10% Pd on carbon (30 mg) was added and the flask was flushed with H2 gas and left to stir under a hydrogen atmosphere (balloon) for 4 h. After purging with argon, the mixture was filtered and concentrated in vacuo to give compound 6 (0.27 g, 95%) as a viscous oil: IR (neat, cm−1): 2924, 1701, 1513; 1H NMR (300 MHz, CDCl3) δ 7.42–7.54 (m, 1H), 6.58–6.72 (m, 2H), 4.10–4.42 (m, 2H), 3.20–3.40 (m, 4H), 1.82–2.20 (m, 8H), 1.44 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 156.7, 155.1, 135.9, 132.3, 122.8, 113.8, 105.9, 82.3, 54.7, 51.4, 50.3, 46.1, 45.1, 41.1, 34.6, 28.2; HRMS(FAB) calcd. for C20H28FN3O3 (M)+ : 377.2115; found 377.2118.
4.2.5. tert-Butyl 1'-(4-(benzyloxycarbonylamino)-2-fluorophenyl)-2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-3-carboxylate (7)
A 10-mL oven-dried, round-bottomed flask was charged with compound 6 (0.38 g, 1.0 mmol), sodium bicarbonate (92 mg, 1.1 mmol) in a mixture of THF-H2O 1:1 (4 mL). After the solution was cooled to 0 °C, benzylchloroformate (0.18 mL, 1.1 mmol) was added and the mixture was stirred at the same temperature for 1 h. The reaction was quenched by adding 1 N HCl (4 mL) and extracted with ethyl acetate (5 mL×3). The combined organic layers were washed sequentially with sat. NaHCO3 (5 mL), brine (5 mL), dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (hexanes:ethyl acetate = 3:1) to give 7 (0.46 g, 90%) as a colorless oil: IR (neat, cm−1): 2936, 1704, 1516; 1H NMR (600 MHz, CDCl3) δ 7.28–7.40 (m, 5H), 6.94–7.00 (m, 1H), 6.86–6.89 (m, 1H), 6.72–6.76 (m, 1H), 5.19 (s, 2H), 4.08–4.33 (m, 2H), 2.88–3.08 (m, 4H), 1.76–1.98 (m, 8H), 1.49 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 166.7, 157.1, 153.9, 153.3, 135.9, 133.4, 128.6, 128.4, 128.3, 119.2, 114.3, 107.5, 83.2, 81.6, 66.8, 64.1, 62.5, 49.4, 28.6, 28.0, 27.5, 26.8; HRMS(FAB) calcd. for C28H34FN3O5 (M)+, 511.2482; found 511.2473.
4.2.6. 1'-(2-Fluoro-4-((R)-5-(hydroxymethyl)-2-oxooxazolidin-3-yl)phenyl)-2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-3-carboxylate (8a) and tert-butyl 1'-(4-((R)-5-(butyryloxymethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)-2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-3-carboxylate (8b)
Compound 7 (0.41 g, 0.80 mmol) was dissolved in 4 mL of anhydrous tetrahydrofuran in a 10-mL oven-dried, round-bottomed flask. The solution was cooled to −78 °C using dry ice-acetone bath and n-butyl lithium (1.6 M in hexane, 1.0 mL, 1.6 mmol) was added. After stirred at that temperature for 15 min, the solution was treated with (R)-glycidyl butyrate (0.15 mL, 1.0 mmol) and slowly warmed to 25 °C. After stirred for additional 2 h, the reaction mixture was quenched with sat. NH4Cl (3 mL) and extracted with ethyl acetate (5 mL×3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (hexanes:ethyl acetate = 1:1) to give 8a (0.32 g, 85%) and 8b (22 mg, 5%) as light yellow oils. 8a: IR (neat, cm−1): 3421 (br), 2968, 2360, 1732, 1516; 1H NMR (500 MHz, CDCl3) δ 7.43 (d, J = 14.2 Hz, 1H), 7.09 (m, 1H), 6.94 (m, 1H), 4.70–4.78 (m, 1H), 4.10–4.32 (m, 2H), 4.08 (m, 2H), 3.93–4.01 (m, 3H), 3.72–3.80 (m, 1H), 2.92–3.14 (m, 4H), 1.74–2.00 (m, 8H), 1.49 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 156.5, 154.6, 154.5, 136.6, 133.1, 119.3, 113.8, 107.4, 83.3, 81.9, 72.8, 64.3, 62.7, 49.4, 46.4, 28.8, 28.2, 28.0, 27.1; HRMS(FAB) calcd. for C24H32FN3O6 (M)+ : 477.2275; found 477.2293; 8b: IR (neat, cm−1): 2966, 1715, 1517; 1H NMR (600 MHz, CDCl3) δ 7.42 (dd, J = 14.0, 2.4 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 6.95 (t, J = 9.1 Hz, 1H), 4.85 (m, 1H), 4.38 (dd, J = 12.3, 3.8 Hz, 1H), 4.26–4.34 (m, 3H), 3.78 (dd, J = 8.8, 6.2 Hz, 1H), 3.00–3.12 (m, 4H), 2.32 (t, J = 7.6 Hz, 2H), 1.80–2.00 (m, 8H), 1.62–1.67 (m, 2H), 1.50 (s, 9H), 0.92 (t, J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 173.2, 156.4, 154.8, 154.0, 136.9, 136.8, 133.0, 119.4, 113.8, 107.5, 105.2, 81.7, 70.0. 63.8, 49.7, 47.2, 35.8, 28.3, 18.3, 13.6. Ester 8b can be converted to 8a by the following procedure: A 10-mL oven-dried, round-bottomed flask was charged with compound 8b (66 mg, 0.12 mmol) and sodium hydroxide (40 mg, 1.0 mmol) in a mixture of THF-H2O 1:1 (4 mL). After stirred at room temperature for 1 h, the solution was neutralized with 1 N HCl and extracted with ethyl acetate (5 mL×3). The combined organic layers were washed sequentially with sat. NaHCO3 (5 mL), brine (5 mL), dried over Na2SO4, filtered, and evaporated under reduced pressure to give 8a in quantitative yield.
4.2.7. tert-Butyl 1'-(2-fluoro-4-((R)-5-((methylsulfonyloxy)methyl)-2-oxooxazolidin-3-yl)phenyl)-2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-3-carboxylate (9)
Compound 8a (0.37 g, 0.78 mmol) was dissolved in 4 mL of dichloromethane in a 10-mL round-bottomed flask. Methyl sulfonyl chloride (0.10 mL, 1.2 mmol) and triethylamine (0.20 mL, 1.2 mmol) were added and the mixture was stirred at room temperature for 18 h. The solution was neutralized with 1 N HCl and extracted with ethyl acetate (5 mL×3). The combined organic layers were washed sequentially with sat. NaHCO3 (5 mL), brine (5 mL), dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (hexanes:ethyl acetate = 1:1) to give 9 (0.39 g, 90%) as a light yellow oil: IR (neat, cm−1): 2975, 2360, 1752, 1517, 1356; 1H NMR (300 MHz, CDCl3) δ 7.42 (dd, J = 14.0, 2.4 Hz, 1H), 7.07–7.13 (m, 1H), 6.95 (t, J = 9.0 Hz, 1H), 4.88–4.96 (m, 1H), 4.50 (dd, J =11.7, 3.7 Hz, 1H), 4.41 (dd, J =11.7, 4.2 Hz, 1H), 4.08–4.36 (m, 3H), 3.91 (dd, J = 9.1, 6.1 Hz, 1H), 3.00–3.12 (m, 7H), 1.76–2.00 (m, 8H), 1.49 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 156.5, 154.5, 153.6, 137.0, 132.4, 119.4, 114.0, 107.6, 83.0, 81.8, 69.4, 68.1, 63.9, 62.3, 49.4, 46.6, 37.8, 28.7, 28.5, 27.9, 26.8; HRMS(FAB) calcd. for C25H34FN3O8S (M)+ : 555.2051; found 555.2036.
4.2.8. tert-Butyl 1'-(4-((R)-5-(azidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)-2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-3-carboxylate (10)
A 10-mL oven-dried, round-bottomed flask was charged with compound 9 (0.28 g, 0.5 mmol) and sodium azide (65 mg, 1.0 mmol) in anhydrous dimethylsulfoxide (4 mL). The solution was stirred at 80 °C for 8 h. After the reaction mixture was cooled to room temperature, water (4 mL) was added. The mixture was extracted with ethyl acetate (5 mL×5). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (hexanes:ethyl acetate = 1:1) to give 10 (0.19 g, 74%) as a yellow oil: IR (neat, cm−1): 2921, 2105, 1717, 1514; 1H NMR (500 MHz, CDCl3) δ 7.42 (dd, J = 14.2, 2.4 Hz, 1H), 7.10–7.14 (m, 1H), 6.95 (t, J = 9.0 Hz, 1H), 4.76–4.81 (m, 1H), 4.10–4.34 (m, 2H), 4.04 (dd, J = 8.8, 8.8 Hz, 1H), 3.82 (dd, J = 8.8, 6.2 Hz, 1H), 3.70 (dd, J = 13.2, 4.6 Hz, 1H), 3.58 (dd, J = 13.2, 4.6 Hz, 1H), 2.94–3.12 (m, 4H), 1.82–2.00 (m, 8H), 1.50 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 156.7, 154.8, 154.0, 137.1, 133.1, 119.6, 114.0, 107.7, 83.3, 82.0, 70.7, 64.2, 53.2, 49.6, 47.9, 28.8, 28.5, 27.9, 27.1.
4.2.9. tert-Butyl 1'-(4-((S)-5-(aminomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)-2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-3-carboxylate (11)
Compound 10 (0.14 g, 0.28 mmol) was dissolved in 4 mL of methanol in a 10-mL round-bottomed flask under argon. 10% Pd on carbon (15 mg) was added and the flask was flushed with H2 gas and left to stir under a hydrogen atmosphere (balloon) for 8 h. After purging with argon, the mixture was filtered and concentrated in vacuo to give compound 11 (0.13 g, 95%) as a viscous oil: IR (neat, cm−1): 2980, 1515; 1H NMR (500 MHz, CDCl3) δ 7.45 (dd, J = 14.2, 2.4 Hz, 1H), 7.10–7.16 (m, 1H), 6.95 (dd, J = 9.1, 9.0 Hz, 1H), 4.65–4.83 (m, 1H), 4.10–4.34 (m, 2H), 4.00–4.04 (m, 1H), 3.81–3.84 (m, 1H), 2.89–3.14 (m, 6H), 1.80–2.00 (m, 8H), 1.50 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 156.6, 154.6, 136.6, 133.3, 119.3, 113.4, 107.4, 107.2, 83.3, 81.8, 73.7, 71.9, 64.2, 62.6, 54.5, 49.5, 47.8, 44.9, 36.4, 28.8, 28.5, 27.9, 27.1; HRMS(FAB) calcd. for C24H33FN4O5 (M)+ : 476.2435; found 476.2449.
4.2.10. tert-Butyl 1'-(4-((S)-5-(acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)-2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-3-carboxylate (12)
Compound 11 (0.11 g, 0.23 mmol) was dissolved in 4 mL of dichloromethane in a 10-mL round-bottomed flask. Acetic anhydride (0.08 mL, 0.80 mmol) and triethylamine (0.11 mL, 0.80 mmol) were added and the mixture was stirred at room temperature for 2 h. After neutralized with 1 N HCl, the mixture was extracted with ethyl acetate (5 mL×3). The combined organic layers were washed sequentially with sat. NaHCO3 (5 mL), brine (5 mL), dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (100% ethyl acetate) to give 12 (84 mg, 70%) as a colorless oil: IR (neat, cm−1): 2922, 1749, 1516; 1H NMR (500 MHz, CDCl3) δ 7.42 (dd, J = 14.2, 2.4 Hz, 1H), 7.04–7.09 (m, 1H), 6.93 (dd, J = 9.1, 9.0 Hz, 1H), 6.30 (t, J = 6.0 Hz 1H), 4.75–4.80 (m, 1H), 4.10–4.32 (m, 2H), 4.02 (d, J = 9.0, 9.0 Hz, 1H), 3.74–3.77 (m, 1H), 3.67–3.72 (m, 1H), 3.58–3.64 (m, 1H), 2.93–3.11 (m, 4H), 2.02 (s, 3H), 1.79–1.99 (m, 8H), 1.50 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 171.1, 156.5, 154.5, 154.3, 136.8, 133.0, 119.3, 113.8, 107.4, 83.3, 81.8, 71.8, 64.2, 62.6, 49.4, 47.6, 41.9, 28.7, 28.5, 27.9, 27.1, 23.3; HRMS(FAB) calcd. for C26H35FN4O6 (M)+ : 518.2541; found 518.2518.
4.2.11. N-(((5S)-3-(3-fluoro-4-(3-((5-nitrofuran-2-yl)methyl)-2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-1'-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13)
Compound 12 (20 mg, 0.04 mmol) was dissolved in 3 mL of dichloromethane in a 10-mL round-bottomed flask. Trifluoroacetic acid (0.5 mL) was added and the mixture was stirred at room temperature for 1 h. The solution was concentrated under reduced pressure to give the cyclic hydroxylamine which is used without further purification: IR (neat, cm−1): 2980, 1644, 1419; 1H NMR (300 MHz, CD3OD) δ 7.50 (dd, J = 14.6, 2.5 Hz, 1H), 7.08–7.21 (m, 2H), 4.74–4.83 (m, 2H), 4.24–4.27 (m, 1H), 4.12 (dd, J = 9.2, 9.1 Hz, 1H), 3.77–3.82 (m, 1H), 3.54–3.58 (m, 2H), 3.01–3.22 (m, 4H), 1.90–2.25 (m, 11H); HRMS calcd for C21H28FN4O4 (M+H)+, 419.2095; found, 419.2086.
4.2.12. N-(((5S)-3-(3-fluoro-4-(3-((5-nitrofuran-2-yl)methyl)-2-oxa-3-azaspiro[bicyclo[2.2.1]heptane-7,4'-piperidine]-1'-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (14)
Compound 13 (84 mg, 0.2 mmol) and 5-nitrofuraldehyde (84 mg, 0.6 mmol) were dissolved in 4 mL of 1,2-dichloroethane in a 10-mL round-bottomed flask. After stirred at room temperature for 15 min, the solution was treated with sodium triacetoxyborohydride (0.12 g, 0.6 mmol) and acetic acid (0.05 mL). The reaction was stirred at room temperature for 2 h and quenched with 5 mL of sat. NaHCO3 solution. The mixture was extracted with ethyl acetate (5 mL×3). The combined organic layers were sequentially washed with sat. NaHCO3 (5 mL), brine (5 mL), dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (100% ethyl acetate) to give 14 (58 mg, 53%) as a colorless oil: IR (neat, cm−1): 2929, 1674, 1517; 1H NMR (600 MHz, CDCl3) δ 7.43 (dd, J = 14.1, 2.6 Hz, 1H), 7.30 (d, J = 3.5 Hz, 1H), 7.07–7.10 (m, 1H), 6.95 (dd, J = 9.1, 9.1 Hz, 1H), 6.55 (d, 1H, J = 3.5 Hz), 5.9 (t, J = 6.2 Hz, 1H), 4.75–4.79 (m, 1H), 4.22 (d, 1H, J = 15.6 Hz), 4.07 (s, 1H), 4.03 (t, J = 9.1 Hz, 1H), 3.99 (d, J = 15.6 Hz, 1H), 3.70–3.76 (m, 2H), 3.58–3.63 (m, 1H), 3.37 (s, 1H), 2.96–3.17 (m, 4H), 2.16–2.27 (m, 4H), 2.03 (s, 3H), 1.85–1.99 (m, 4H); 13C NMR (150 MHz, CDCl3) δ 170.9, 156.5, 154.2, 112.9, 111.1, 88.9, 80.6, 71.8, 53.2, 49.6, 47.6, 42.0, 29.1, 28.9, 23.2; HRMS(FAB) calcd. for C26H30FN5O7 (M)+ : 543.2129; found 543.2115.
4.2.13. tert-Butyl 8-(4-((S)-5-(aminomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)-4-hydroxy-8-azaspiro[4.5]decan-1-ylcarbamate (15)
Compound 11 (0.12 g, 0.25 mmol) was dissolved in a mixture of 3 mL of acetonitrile and 1 mL of water in a 10-mL oven-dried, round-bottomed flask. Molybdenumhexacarbonyl (20 mg, 0.07 mmol) and sodium borohydride (15 mg, 0.50 mmol) were added to the solution in the indicated order. The mixture was stirred at 60 °C for 6 h. After the reaction mixture cooled to room temperature, all the solvent was removed under reduced pressure, and the residue was purified by chromatography on a silica gel column (ethyl acetate:MeOH = 10:1) to give compound 15 (76 mg, 64%) as a colorless oil: IR (neat, cm−1): 3487, 2927, 1747, 1616; 1H NMR (300 MHz, CDCl3) δ 7.40 (d, J = 14.1, 1.9 Hz, 1H), 6.98–7.06 (m, 1H), 6.86–6.96 (m, 1H), 5.46–5.54 (m, 1H), 4.58–4.76 (m, 1H), 3.92–4.06 (m, 3H), 3.73–3.79 (m, 1H), 2.84–3.12 (m, 6H), 2.14–2.32 (m, 4H), 1.66–2.00 (m, 6H), 1.40 (s, 9H). 13C NMR (75 MHz, CDCl3) δ 157.0, 155.5, 154.5, 153.7, 137.0, 132.7, 119.1, 113.6, 107.2, 106.9, 80.0, 78.8, 77.4, 73.7, 55.3, 48.2, 47.6, 44.8, 33.2, 31.1, 30.5, 28.3, 27.2; HRMS(FAB) calcd. for C24H35FN4O5 (M)+ : 478.2591; found 478.2592.
4.2.14. tert-Butyl 8-(4-((S)-5-(acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)-4-hydroxy-8-azaspiro[4.5]decan-1-ylcarbamate (16)
Compound 12 (21 mg, 0.04 mmol) was dissolved in a mixture of 3 mL of acetonitrile and 1 mL of water in a 10-mL round-bottomed flask. Molybdenumhexacarbonyl (5 mg, 0.02 mmol) and sodium borohydride (5 mg, 0.16 mmol) were added to the solution in the indicated order. The mixture was stirred at 60 °C for 6 h. After the reaction mixture cooled to room temperature, all the solvent was removed under reduced pressure, and the residue was purified by chromatography on a silica gel column (hexanes:ethyl acetate = 1:1) to give compound 16 (16 mg, 80%) as a colorless oil: IR (neat, cm−1): 3331 (br), 2962, 1686, 1518; 1H NMR (300 MHz, CDCl3) δ 7.39 (d, J = 14.2 Hz, 1H), 6.90–7.04 (m, 2H), 6.32–6.38 (m, 1H), 5.38–5.48 (m, 1H), 4.72–4.81 (m, 1H), 3.96–4.10 (m, 3H), 3.56–3.76 (m, 3H), 2.86–3.16 (m, 4H), 1.72–2.08 (m, 11H), 1.44 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 171.2, 157.0, 155.5, 154.4, 153.8, 149.9, 137.3, 132.4, 129.7, 128.7, 126.9, 126.2, 119.4, 113.8, 107.4, 80.2, 78.9, 77.4, 71.8, 55.6, 48.5, 47.9, 47.7, 42.2, 33.6, 31.4, 30.8, 30.1, 28.7, 27.4, 23.2; HRMS(FAB) calcd. for C26H37FN4O6 (M)+, 520.2697; found 520.2697.
Acknowledgments
This research was supported in part by grants GM 68012 and GM 075885 from the National Institutes of Health (NIH) as well as Eli Lilly and Company. We gratefully acknowledge the use of the NMR facilities provided by the Lizzadro Magnetic Resonance Research Center at the University of Notre Dame (UND) under the direction of Dr. Jaroslav Zajicek and the mass spectrometry services provided by The UND Mass Spectrometry & Proteomics Facility (Mrs. N. Sevova, Dr. W. Boggess, and Dr. M. V. Joyce; supported by the National Science Foundation under CHE-0741793). We thank Mrs. Patricia A. Miller (UND) for antibacterial susceptibility testing.
Footnotes
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1. Spellberg B, Guidos R, Gilbert D, Bradley J, Boucher HW, Scheld WM, Bartlett JG, Edwards J., Jr Clin. Infect. Dis. 2008;46:155. [PubMed]
2. Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, Scheld M, Spellberg B, Bartlett J. Clin. Infect. Dis. 2009;48:1. [PubMed]
3. Barbachyn MR, Ford CW. Angew. Chem,. Int. Ed. 2003;42:2010. [PubMed]
4. Gravestock MB. Curr. Opin. Drug Discovery Dev. 2005;8:469. [PubMed]
5. Hutchinson DK. Expert Opin. Ther. Pat. 2004;14:1309.
6. Renslo AR, Luehr GW, Gordeev MF. Bioorg. Med. Chem. 2006;14:4227. [PubMed]
7. Ippolito JA, Kanyo ZF, Wang DP, Franceschi FJ, Moore PB, Steitz TA, Duffy EM. J. Med. Chem. 2008;51:3353. [PubMed]
8. Leach KL, Swaney SM, Colca JR, McDonald WG, Blinn JR, Thomasco LM, Gadwood RC, Shinabarger D, Xiong L, Mankin AS. Mol. Cell. 2007;26:393. [PubMed]
9. Barry AL. Antimicrob. Agents Chemother. 1988;32:150. [PMC free article] [PubMed]
10. Neu HC, Novelli A, Saha G, Chin NX. Antimicrob. Agents Chemother. 1988;32:580. [PMC free article] [PubMed]
11. Slee AM, Wuonola MA, McRipley RJ, Zajac I, Zawada MJ, Bartholomew PT, Gregory WA, Forbes M. Antimicrob. Agents Chemother. 1987;31:1791. [PMC free article] [PubMed]
12. Brickner SJ, Hutchinson DK, Barbachyn MR, Manninen PR, Ulanowicz DA, Garmon SA, Grega KC, Hendges SK, Toops DS, Ford CW, Zurenko GE. J. Med. Chem. 1996;39:673. [PubMed]
13. Brickner SJ, Barbachyn MR, Hutchinson DK, Manninen PR. J. Med. Chem. 2008;51:1981. [PubMed]
14. Tsiodras S, Gold HS, Sakoulas G, Eliopoulos GM, Wennersten C, Venkataraman L, Moellering RC, Ferraro MJ. Lancet. 2001;358:207. [PubMed]
15. Gonzales RD, Schreckenberger PC, Graham MB, Kelkar S, DenBesten K, Quinn JP. Lancet. 2001;357:1179. [PubMed]
16. Auckland C, Teare L, Cooke F, Kaufmann ME, Warner M, Jones G, Bamford K, Ayles H, Johnson AP. J. Antimicrob. Chemother. 2002;50:743. [PubMed]
17. Seedat J, Zick G, Klare I, Konstabel C, Weiler N, Sahly H. Antimicrob. Agents Chemother. 2006;50:4217. [PMC free article] [PubMed]
18. Hutchinson DK. Curr. Top. Med. Chem. 2003;3:1021. [PubMed]
19. Paget SD, Foleno BD, Boggs CM, Goldschmidt RM, Hlasta DJ, Weidner-Wells MA, Werblood HM, Wira E, Bush K, Macielag MJ. Bioorg. Med. Chem. Lett. 2003;13:4173. [PubMed]
20. Fukuda YH, Hammond ML. 6,897,230 U.S. Patent. 2005
21. Renslo AR, Jaishankar P, Venkatachalam R, Hackbarth C, Lopez S, Patel DV, Gordeev MF. J. Med. Chem. 2005;48:5009. [PubMed]
22. Gordeev MF, Renslo A, Patel DV. 6,875,784 U.S. Patent. 2005
23. Barbachyn MR, Brickner SJ, Hutchinson DK. 6,090,820 U.S. Patent. 2000
24. Patel MV, Deshpande PK, Sindkhedkar MD, Gupte SV, Chugh Y, Shetty N, Shukla MC, Yeole RD, De Souza NJ. 2004/0063954 U.S. Patent Application. 2004
25. Chugh Y, Shetty N, Deshpande PK, Sindkhedkar MD, Jafri MD, Yeole RD, Shukla MC, Gupte SV, Patel MV, De Souza NJ. 2004/0235900 U.S. Patent Application. 2004
26. Prasad JV. Curr. Opin. Microbiol. 2007;10:454. [PubMed]
27. Tietze LF, Kettschau G. Top. Curr. Chem. 1997;189:1. (Stereoselective Heterocyclic Synthesis I)
28. Vogt PF, Miller MJ. Tetrahedron. 1998;54:1317.
29. Yamamoto H, Momiyama N. Chem. Commun. (Cambridge. U.K.) 2005:3514. [PubMed]
30. Bodnar BS, Miller MJ. Angew. Chem,. Int. Ed. 2011;50:5629.
31. Li FZ, Yang BY, Miller MJ, Zajicek J, Noll BC, Möllmann U, Dahse HM, Miller PA. Org. Lett. 2007;9:2923. [PubMed]
32. Krchnak V, Waring KR, Noll BC, Möllmann U, Dahse HM, Miller MJ. J. Org. Chem. 2008;73:4559. [PubMed]
33. Ji C, Miller MJ. Tetrahedron Lett. 2010;51:3789. [PMC free article] [PubMed]
34. Lin WM, Gupta A, Kirn KH, Mendel D, Miller MJ. Org. Lett. 2009;11:449. [PMC free article] [PubMed]
35. Li FZ, Brogan JB, Gage JL, Zhang DY, Miller MJ. J. Org. Chem. 2004;69:4538. [PubMed]
36. Kim KH, Miller MJ. Tetrahedron Lett. 2003;44:4571.
37. Li FZ, Miller MJ. J. Org. Chem. 2006;71:5221. [PubMed]
38. Li FZ, Warshakoon NC, Miller MJ. J. Org. Chem. 2004;69:8836. [PubMed]
39. Lin WM, Virga KG, Kim KH, Zajicek J, Mendel D, Miller MJ. J. Org. Chem. 2009;74:5941. [PMC free article] [PubMed]
40. Tucker JA, Allwine DA, Grega KC, Barbachyn MR, Klock JL, Adamski JL, Brickner SJ, Hutchinson DK, Ford CW, Zurenko GE, Conradi RA, Burton PS, Jensen RM. J. Med. Chem. 1998;41:3727. [PubMed]
41. Das B, Rudra S, Yadav A, Ray A, Rao A, Srinivas A, Soni A, Saini S, Shukla S, Pandya M, Bhateja P, Malhotra S, Mathur T, Arora SK, Rattan A, Mehta A. Bioorg. Med. Chem. Lett. 2005;15:4261. [PubMed]
42. Cicchi S, Goti A, Brandi A, Guarna A, Desarlo F. Tetrahedron Lett. 1990;31:3351.
43. Yan SS, Miller MJ, Wencewicz TA, Möllmann U. Bioorg. Med. Chem. Lett. 2010;20:1302. [PMC free article] [PubMed]
44. Typical MIC values of linezolid against MSSA and MRSA are 1–2 and 0.5–2 g mL−1, respectively. See Phillips OA, Udo EE, Ali AAM, Samuel SM Eur. J. Med. Chem. 2007;42:214. [PubMed]
45. Williams KN, Stover CK, Zhu T, Tasneen R, Tyagi S, Grosset JH, Nuermberger E. Antimicrob. Agents Chemother. 2009;53:1314. [PMC free article] [PubMed]
46. Collins LA, Franzblau SG. Antimicrob. Agents Chemother. 1997;41:1004. [PMC free article] [PubMed]
47. Cho SH, Warit S, Wan B, Hwang CH, Pauli GF, Franzblau SG. Antimicrob. Agents Chemother. 2007;51:1380. [PMC free article] [PubMed]