Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE Advance Publication DOI:10.1248/cpb.c14-00510 October 9, 2014
Chem. Pharm. Bull.
Regular Article
Synthesis and in vitro evaluation of the antitubercular and antibacterial activity of novel oxazolidinones bearing octahydrocyclopenta[c]pyrrol-2-yl moieties
Deepak Bhattarai,a, b, † Ju-hyeon Lee,a, c, † Seon Hee Seo,a Ghilsoo Nam,a Hyunah Choo,a Soon Bang Kang,a Jin-Hwan Kwak,d Taegwon Oh,e Sang-Nae Cho,e Ae Nim Pae,a Eunice Eunkyeong Kim,a, b Nakcheol Jeong,c and Gyochang Keuma, b,* aCenter for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 5 Seongbuk-gu, Seoul 136-791, South Korea bDepartment of Biological Chemistry, University of Science and Technology, Gajungro 217, Youseong-gu, Daejeon 305-350, South Korea cDepartment of chemistry, Korea University, Anam-ro 145 Seongbuk-gu, Seoul 136-701, South Korea dSchool of Life Science, Handong Global University, Pohang 791-708, South Korea eDepartment of Microbiology and the Brain Korea 21 Project for the Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, South Korea
——— * To whom correspondence should be addressed. e-mail: [email protected] † These authors contributed equally to this work.
Ⓒ 2014 The Pharmaceutical Society of Japan Abstract
A novel series of oxazolidinone-class antimicrobial agents with 5-substituted octahydrocyclopenta [c]pyrrole moieties at the C-ring of linezolid and an acetamide or 1,2,3-triazole ring as the C-5 side chain of the oxazolidinone ring were prepared. The resulting series of compounds were evaluated for in vitro antimicrobial activity against Mycobacterium tuberculosis and a panel of clinically important resistant Gram-positive and -negative bacteria. Among them, endo-alcohol 2a and exo-alcohol 2b showed potent inhibitory activity against M. tuberculosis H37Rv, which was superior to that of linezolid. Several analogues in this series showed potent in vitro antibacterial activity against the clinically important vancomycin- resistant bacteria and showed similar or better potency against linezolid-resistant MRSA strains. The hydroxyl group in the azabicyclic C-ring interacted with the same hydrophobic pocket as linezolid based on a docking study. Selected compounds with high antimicrobial activity showed good human microsomal stability and low CYP isozyme and monoamine oxidase (MAO) inhibition.
Oxazolidinones are a novel class of orally active and synthetic antibacterial agents with a wide spectrum of antibacterial activity against various clinically significant resistant bacteria that pose a serious global health threat.1-3 Thus, oxazolidinones may be used to overcome the increasing incidence and prevalence of bacterial resistance to clinically effective antibiotics. Linezolid (Figure 1), the only available oxazolidinone antibacterial available at this time,4 was approved by the FDA in 2000 for the treatment of multidrug- resistant Gram-positive bacterial infections, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP) in humans.5 Linezolid and its thiomorpholine analogue sutezolid (PNU-100480) are active against Mycobacterium tuberculosis, including strains with multidrug and extensive drug resistance.6,7
Oxazolidinone antibacterials represent a well-characterized class of compounds that target RNA and modulate RNA function.8 Oxazolidinones selectively inhibit bacterial protein synthesis by binding to the
Chemical and Pharmaceutical Bulletin Advance Publication 23S rRNA of the 50S ribosomal subunit and prevent the formation of a functional 70S initiation complex at the translation process. The binding mode of linezolid was elucidated based on X-ray crystallography where linezolid was bound to the 50S A-site near the peptidyl transferase center.9 While linezolid has been successfully used in the clinic and is considered a drug of last resort against potentially intractable infections, clinical emergence of linezolid-resistant bacteria—such as staphylococci and enterococci—has been reported.10-12 Resistance to linezolid occurs mainly through point mutations in the 23S ribosomal RNA subunit and related proteins or through cfr-mediated methylation of 23S rRNA.13-15 Moreover, extended use of linezolid has some safety concerns such as myelosuppression, monoamine oxidase inhibition and mitochondrial toxicity.16,17 Thus, there is considerable unmet medical demand for newer oxazolidinone antibacterials with improved potency, spectrum, activity against resistant strains, and good safety profiles. The attempts to modify the structure of the oxazolidinone C-ring and C-5 side chain of the oxazolidinone A-ring were undertaken to improve the antibacterial activity of oxazolidinones.5,18-20 Among them, posizolid,21 torezolid phosphate,22 radezolid23 and ranbezolid,24 as shown in Figure 1, are under development for the clinical treatment of tuberculosis and serious Gram-positive bacterial skin infections. To improve the potency and broaden the spectrum of oxazolidinone antibacterials,25-27 we reported previously that modification of the morpholine C-ring of linezolid with an azabicylo[3.3.0]octanyl group as a conformationally constrained isostere enhanced in vitro antibacterial activity, which was comparable or superior to linezolid against Gram-positive bacteria and M. tuberculosis. Herein, we described the synthesis of novel oxazolidinones bearing an octahydrocyclopenta[c]pyrrol-2-yl ring with a stereoselective oxygen or nitrogen substituent at the 5-position, as shown in Figure 2, and evaluated their biological activity against clinically important bacterial strains. In addition, two series of target compounds with acetamide and 1,2,3- triazole at the C-5 side chain of oxazolidinone rings were prepared. Initial safety evaluations for selected compounds were also investigated against cytochrome P450 (CYP) isoforms and monoamine oxidase (MAO)-A and –B.
Results and Discussion
Chemistry
The synthesis of oxazolidinone derivatives bearing an acetamide (a) or 1,2,3-triazole (b) moiety at the C-5 position of the oxazolidinone A-ring was performed as described previously26 using azabicyclic ketone 1a and 1b, respectively, as shown in Chart 1. Reduction of ketone 1a and 1b with sodium borohydride
Chemical and Pharmaceutical Bulletin Advance Publication yielded the endo alcohols 2a and 2b at high yields, respectively. The stereochemistry of the endo alcohols was defined according to the stereochemistry of known N-Boc derivatives.28 The endo-selectivity was also confirmed by generating one meso-alcohol exclusively during the reduction of 2-(2-fluoro-4- nitrophenyl)hexahydrocyclopenta[c]pyrrol-5(1H)-one as an N-aryl azabicyclic ketone derivative (Chart 2).
< Chart 1>
< Chart 2>
Mesylation of the alcohols and substitution of the mesylates with sodium azide generated azide exo-3a and 3b by inversion. The 1,2,3-triazole derivatives exo-4a and 4b were prepared by cycloaddition of azide exo-3a and 3b, respectively, with vinyl acetate and spontaneous elimination.
Hydrogenolysis of azide exo-3a and 3b yielded amine exo-5a and 5b, and subsequent amidation afforded acetamide exo-6a and 6b and p-nitrobenzamide exo-7a and 7b, respectively, in good yields. The reaction of exo-amine 5a and 5b with thiocarbondiimidazole followed by ammonia treatment yielded exo-thiourea derivatives 8a and 8b, respectively. Phenylurea exo-9a and 9b were also prepared from the same amines, respectively, by reacting with phenylisocyanate. On the other hand, the exo alcohol compounds exo-2a and 2b were prepared from the corresponding endo alcohols 2a and 2b, respectively, through sequential Mitsunobu reactions and hydrolysis. Endo azides (endo-3a, 3b), endo triazole (endo-4a) and endo acetamides (endo-6a, 6b) were then obtained according to the preparation methods for their exo compounds, as shown in Chart 3.
< Chart 3>
Reductive amination of ketone 1a and 1b with benzylamine in the presence of sodium triacetoxyborohydride selectively yielded the corresponding endo-benzylamine derivatives 10a and 10b, respectively, with moderate to good yields combined with a reduced amount of alcohol as a side product. Removal of the benzyl group under hydrogenation conditions yielded the endo-amines 5a and 5b, respectively, at high yields (Chart 4).
< Chart 4>
Chemical and Pharmaceutical Bulletin Advance Publication Biological activity
The obtained oxazolidinones bearing the 5-substituted octahydrocyclopenta[c]pyrrol-2-ylphenyl moiety were subjected to evaluation of in vitro antimicrobial activities against a panel of clinically important resistant bacteria. Minimal inhibitory concentrations (MICs, μg/mL) of these compounds are summarized in Table 1.
MICs of the compounds against the virulent Mycobacterium tuberculosis H37Rv strain were determined using the microplate Alamar Blue Assay29 and compared to linezolid. The majority of prepared compounds, excluding benzylamine compounds endo-10a and endo-10b, showed good antitubercular activity. Among the C-5 acetamide oxazolidinones (series a), endo-alcohol 2a showed fourfold higher activity, and several exo compounds (such as ketone 1a,26 exo-alcohol 2a, exo-triazole 4a, exo-amide 7a and exo-thiourea 8a) showed twofold higher activity against M. tuberculosis compared to linezolid.
Among the C-5 1,2,3-triazole oxazolidinones (series b), exo-alcohol 2b showed fourfold higher activity, and ketone 1b,26 endo-alcohol 2b and exo-thiourea 8b showed twofold higher activities, than linezolid. Alcohol 2 and thiourea 8 showed potent mycobacterial activity, while amine 5 showed low mycobacterial activity. Azabicyclic compounds with 5-triazoles 4 and 5-amides 6 showed comparable or higher activity against M. tuberculosis compared to linezolid. Docking studies were performed using the crystal structure of the 50S ribosomal subunit of Deinococcus radiodurans (PDB ID 3DLL) to identify the binding mode of the new highly active antituberculosis compounds. The sequence of the 50S ribosomal subunit of D. radiodurans (D50S) showed more similarity
(79%) with that of M. tuberculosis H37Rv than those of other strains, such as Escherichia coli (77%) and Haloarcula marismortui (74%) based on sequence alignment using the BLASTN 2.2.29+ software.30 Docking studies revealed that the binding mode of these compounds to the ribosome was similar to those of linezolid and ranbezolid.24 The superposition of the docked configurations of the most active compounds, endo-2a and exo-2b, and D50S–linezolid structures is shown in Figure 3a. The oxazolidinone rings formed a partial stacking interaction with the face of the uracil U2483. The sugar moieties of G2484 are present on the sides of the oxazolidinones. The acetamide tails of endo- and exo-2a formed hydrogen bonds with a distance of 1.8 Å between their amide protons and the 5’-oxygen in the phosphate group of G2484, similar to the bonds formed with linezolid (Figure 3b). For the 1,2,3-triazole series endo- and exo-2b, the triazole ring tails form a face-to-face stacking interaction with uracil U2483, which resulted in hydrogen bonding between the carbonyl oxygen of the oxazolidinone ring and the O2’-hydrogen atom of G2484 (2.4 and 2.2 Å) (Figure 3c). The aromatic B-ring of the two series was located in the A-site hydrophobic cleft formed by
Chemical and Pharmaceutical Bulletin Advance Publication PTC site residues A2430 and U2485, and was stabilized by stacking interactions with the face of adenine A2430. In addition, the hydroxyl groups of the azabicyclic C-ring in endo/exo-2a and endo-2b formed two hydrogen bonding interactions with the 2-carbonyl oxygen in uracil U2563 (1.9–2.1 Å) and the amino hydrogen of guanine G2562 (1.8–1.9 Å). Otherwise, the hydroxyl group in exo-2b adopted different orientations through a hydrogen bonding interaction with the 2’-sugar oxygen of U2563 (1.9 Å). While linezolid was located between the P-site and A-site with weak hydrogen bonding between O4 of the morpholine C-ring and the N3 of U2564 in the D. radiodurans crystal structure,31 these compounds were closer to the A-site in the ribosomal PTC due to hydrogen bonding of the hydroxyl group extended by the azabicyclic C-ring. The results of docking experiments with the selected compounds are shown in Table 2. These results correlated well with their corresponding in vitro antitubercular activities. Note that, in general, endo-isomers showed stronger correlations than their corresponding exo-forms. Most of the active compounds endo-2a and exo-2b yielded total scores (10.9 and 8.63, respectively) that were higher than that of linezolid (7.80). Contrary to in vitro antitubercular activities, energy-minimized structures of exo-2b yielded total scores that were lower than those of endo-2b in docking studies where the position and binding mode of the triazole group in 2b differed from those of the acetamide group of 2a and linezolid. The lower antitubercular activity of endo-5a and endo-10a, in spite of their high total scores, can be explained by the low cell permeability of their ionized forms at pH 7.4. Amide compound endo-6a yielded a higher total score than that of exo-6a, but both isomers exhibited in vitro antitubercular activities similar to that of linezolid. High-scoring configurations of the amide and urea substituents in compounds 6–9a showed hydrogen bonding with the oxygen atom of U2485.
The MICs against six Gram-positive and nine Gram-negative vancomycin-resistant bacterial strains32 from the American Type Culture Collection (ATCC: Rockville, MD, USA) were determined using the twofold agar dilution method, as described by the Clinical and Laboratory Standard Institute,33 and linezolid was used as a reference compound. Vancomycin resistance of the strains was investigated; the results are shown in Table 1. Octahydrocyclopenta[c]pyrrol-2-yl oxazolidinones did not show activity against the tested Gram-negative vancomycin-resistant bacterial strains, but showed good antibacterial activity against vancomycin resistant Gram-positive bacteria, including Staphylococcus aureus ATCC65389, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC29212 and Bacillus cereus ATCC27348. Among the C-5 acetamide-substituted oxazolidinones (series a), ketone 1a, alcohols endo-2a and exo-2a, and endo-acetamide 6a showed comparable or higher activity than linezolid against the strains. The replacement of acetamide with triazole (series b) at the C-5 position of the oxazolidinone ring generated less potent compounds, while ketone 1b showed twofold higher activity than linezolid.
Chemical and Pharmaceutical Bulletin Advance Publication Ketone compound 1b showed higher activities against vancomycin-resistant S. aureus and E. faecalis than against the methicillin-resistant strains, as reported previously.26 The activities of the triazole-substituted bicyclic compound 4 decreased slightly and primary amine 5 and benzylamine 10 lost activity. The results also showed that oxazolidinone antibacterial agents, including linezolid, could be used to treat vancomycin- resistant Bacillus cereus. Furthermore, antibacterial activities of novel oxazolidinone compounds were evaluated against three clinically isolated linezolid-resistant MRSA strains containing the G2576U mutation in the peptidyl transferase center (NRS119,10 NRS12110 and NRS27134), and a linezolid-nonsusceptible MRSA with the ΔSer145 mutation in the ribosomal L3 protein (NRS127)35 obtained from the Network on Antimicrobial Resistance in S. aureus (NARSA). As shown in Table 3, ketone 1b and triazole-substituted compound 4a showed comparable activity to linezolid, and ketone 1a was >twofold more potent than linezolid against clinical linezolid-resistant staphylococcal strains containing the G2576U mutation. The similar potency of this series and linezolid against NRS127 (S. aureus with a Ser145 deletion mutation) demonstrated that the compounds and linezolid functioned through a similar coupling with the mutation in ribosomal protein L3. Because ribosome crystal structures have shown that G2576U mutations abrogate interactions with G2505 and U2506 to decrease allosteric binding of linezolid in the pocket,36 the additional hydrogen bonds with hydroxyl groups in 2a and 2b may not improve their binding affinities in the linezolid-binding pocket of the mutant strains compared to linezolid. However, the higher activity of ketone compound 1a against linezolid-resistant MRSA strains with the G2576U mutation can be explained by the increased hydrogen bonding acceptor properties of the ketone group compared to the morpholine oxygen of linezolid and the hydroxyl group in 2a and 2b.
With regard to drug metabolism and interactions, the CYP450 reversible inhibition of selected compounds with potent antimicrobial activity was determined (Table 3). All tested compounds showed low CYP inhibition towards CYP 2C19, 2D6 and 2C9 isoforms, but high CYP 1A2 and 3A4 inhibition were detected for compounds such as linezolid, excluding exo-alcohol 2a for CYP 3A4. The selected compounds were stable based on human microsomal incubation tests37 with a minimum of 95.2% activity after 30 min. Based on monoamine oxidase (MAO) inhibition assays using Amplex® Red Monoamine Oxidase Assay Kit (A12214, Invitrogen, Carlsbad, CA, USA), the compounds showed significantly reduced MAO-A and -B inhibition compared to linezolid, which is a weak reversible inhibitor of MAO that can interact with tyramine-containing food and should be used with caution in pediatrics.16
Chemical and Pharmaceutical Bulletin Advance Publication
Conclusions
Modification of the C-ring of linezolid with 5-substituted octahydropentacyclo[c]pyrrole moiety generated a series of oxazolidinone antimicrobial agents with potent activity against M. tuberculosis H37Rv. Among them, two alcohol compounds (endo-alcohol 2a and exo-alcohol 2b) showed fourfold higher antitubercular activity (MIC, 0.25 µg/mL), and six alcohol, triazole, and thiourea compounds showed twofold higher activity than linezolid. Docking studies suggested that the hydroxyl group in the azabicyclic C-ring formed additional interactions with the binding pocket. In addition, several alcohol and acetamide compounds showed potent antibacterial activity against clinical vancomycin-resistant S. aureus, E. faecalis and B. cereus that was comparable or superior to that of linezolid. Furthermore, ketones 1a and 1b showed higher in vitro antibacterial activities against clinical S. aureus strains resistant to methicillin and linezolid. Selected compounds with high antimicrobial activity showed good human microsomal stability, CYP- profiles, and low monoamine oxidase inhibition with improved pharmacological and safety profiles.
Experimental
General
All reactions were performed under an inert atmosphere unless otherwise mentioned. 1H and 13C-NMR spectra were recorded on a Bruker DPX 300- and 400-MHz spectrophotometer using CDCl3, CD3OD or DMSO-d6 as the NMR solvent. TMS was used as an internal standard and chemical shift data were reported in parts per million (ppm). s, d, t, q and m represent singlet, doublet, triplet, quartet and multiplet, respectively. Coupling constants (J) were reported in hertz (Hz). Mass spectra were recorded using a Waters Acquity UPLC/Synapt G2 QTOF MS mass spectrometer. The reaction progress was monitored using thin-layer chromatography (TLC) with 254-nm UV light, p-anisaldehyde, PMA, aq KMnO4, FeCl3 or ninhydrin for visualization. All reagents were purchased from Sigma-Aldrich, Alfa Aesar and TCI, and were used without purification unless otherwise indicated.
To the solution of starting material 1a26 (0.5 g, 1.33 mmol) in methanol (5 mL) was added sodium borohydride (50 mg, 1.33 mmol) at 0 ℃, and the reaction mixture was stirred at the same temperature for 1 h. Then the reaction mixture was concentrated under reduced pressure, diluted with water, and the mixture
Chemical and Pharmaceutical Bulletin Advance Publication was extracted with ethyl acetate (2 x 10 mL). The combined organic layer was washed with water, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel with a mixture of CH2Cl2 and MeOH (20:1) to give the title compound (0.49 1 g, 98%). H-NMR (CDCl3, 300 MHz) δ: 7.39 (dd, J = 14.1, 2.6 Hz, 1H), 7.05 (t, J = 4.3 Hz, 1H), 6.90 (dd, J = 9.0, 2.7, 1H), 6.71 (s, 1H), 5.19 (s, 1H), 4.76 (t, J = 2.6 Hz, 1H), 4.13 (s, 1H), 3.99 (d, J = 2.7 Hz, 1H), 3.76–3.60 (m, 3H), 3.39 (d, J = 7.2 Hz, 2H), 2.86 (m, 4H), 2.05 (m, 4H), 1.78 (d, J = 13.8 Hz, 2H); 13C-
To the mixture of endo-2a (0.6 g, 1.59 mmol), benzoic acid (0.21 g, 1.75 mmol), and triphenylphosphine (1.25 g, 4.78 mmol) in THF (10 mL) was added 40% solution of diethyl azodicarboxylate (DEAD, 0.83 g, 4.78 mmol) in toluene at 0 ℃ and the mixture was stirred at room temperature for 6 h. The mixture was concentrated under reduced pressure and the residue was dissolved in dichloromethane. Organic layer was washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel with a mixture of dichloromethane and methanol (50:1) to provide the corresponding benzoate as white solid.
The above white solid was dissolved in methanol and 1 N sodium hydroxide (84 mg, 2.1 mmol) solution was added. The mixture was stirred at room temperature for 4 h, concentrated under reduced pressure, and dissolved in dichloromethane. Organic layer was washed with water, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel with a 1 mixture of CH2Cl2 and MeOH (20:1) to provide the title compound (0.29 g, 49% in two steps). H-NMR
Chemical and Pharmaceutical Bulletin Advance Publication Triethylamine (0.45 mL, 3.20 mmol) was added slowly to the solution of exo-2a (0.5 g, 1.28 mmol) in dichloromethane (8 mL) at 0 ℃. Methanesulfonyl chloride (0.2 mL, 2.56 mmol) was added to the reaction mixture slowly. The mixture was stirred at the same temperature for 2 h, diluted with water, and extracted with dichloromethane (2 x 10 mL). The combined organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was dissolved in dimethylformamide and sodium azide (1.38 g, 21.26 mmol) was added. The mixture was stirred at 75 ℃ for 4 h, concentrated under reduced pressure and the residue was dissolved in ethyl acetate. Organic layer was washed with water, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel with a mixture of CH2Cl2 and MeOH (40:1) to provide endo-3a (0.48 g, 90%) as white solid.
Compound endo-3a (0.1 g, 0.24 mmol) was dissolved in vinyl acetate (3 mL) and then refluxed for 48 h. Reaction mixture was concentrated under reduced pressure and purified by flash chromatography on silica 1 gel with a mixture of CH2Cl2 and MeOH (30:1) to provide the title compound (45.6 mg, 43%). H-NMR
The mixture of compound endo-10a (0.25 g, 0.53 mmol) and 10% palladium on charcoal (57.1 mg, 10 mol%) in ethyl acetate/MeOH (5 mL, 1:1) was stirred under hydrogen gas (1 atm) at room temperature for
Chemical and Pharmaceutical Bulletin Advance Publication 12 h. The reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel with a mixture of CH2Cl2, MeOH 1 and NH4OH solution (40:2:1) to afford the title compound (0.16 g, 82%). H-NMR (CDCl3, 300 MHz) δ: 7.37 (dd, J = 11.2, 1.9 Hz, 1H), 7.02 (dd, J = 6.6, 1.7, 1H), 6.82 (t, J = 4.4 Hz, 1H), 6.76 (t, J = 6.9 Hz, 1H), 4.79–4.75 (m, 1H), 4.01 (t, J = 6.7 Hz, 1H), 3.75 (dd, J = 6.7, 5.1 Hz, 1H), 3.67–3.62 (m, 2H), 3.34–3.24 (m, 3H), 3.05 (dd, J = 6.5, 5.2 Hz, 2H), 2.69 (d, J = 2.1Hz, 2H), 2.28–2.22 (m, 2H), 2.03 (s, 3H), 1.84 (m, 13 2H), 1.36–1.28 (m, 2H); C-NMR (CDCl3, 100 MHz) δ: 171.27, 154.77, 154.57, 152.33, 134.68, 134.58, 130.46, 130.36, 117.59, 117.54, 114.28, 107.79, 107.52, 71.94, 57.10, 57.06, 54.58, 47.81, 43.72, 41.94, + 40.60, 23.04; HRMS (ESI-TOF) m/z: [M+H] Calcd for C19H26FN4O3: 377.1989; Found: 377.1987.
To the solution of exo-5a (80 mg, 0.21 mmol) in dichloromethane (2 mL) was added 1,1’- thiocarbonyldiimidazole (TCDI, 40.1 mg, 0.25 mmol) at room temperature and the mixture was stirred at same temperature for 3 h. Excess of 25% ammonium hydroxide solution was added to the reaction mixture and the mixture was stirred at room temperature for another 24 h. The precipitate was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on 1 silica gel with a mixture of CH2Cl2 and MeOH (25:1) to yield the title compound (43 mg, 47%). H-NMR
To a solution of ketone 1a (80 mg, 0.21 mmol) in dichloromethane (2 mL) was added benzylamine (35 µl, 0.32 mmol) and sodium triacetoxyborohydride (67.6 mg, 0.32 mmol) at room temperature. The reaction mixture was stirred at the same temperature for 24 h, diluted with water, and extracted with dichloromethane (2 x 5 mL). The combined organic layer was dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel with a mixture of 1 CH2Cl2, MeOH and NH4OH solution (40:2:1) to provide the title compound (44 mg, 45%). H-NMR
Chemical and Pharmaceutical Bulletin Advance Publication
Biological assay
Determination of minimum inhibitory concentrations (MIC) of antibacterial activity
The synthesized compounds were first assayed in vitro against fifteen bacterial strains with vancomycin resistance obtained from an American Type Culture Collection (ATCC: Rockville, MD, USA). The strains were six Gram-positive bacteria S. aureus ATCC65389, S. aureus ATCC25923, E. faecalis ATCC29212, M. luteus ATCC9341, B. cereus ATCC27348, B. subtilis ATCC6633, and nine different types of Gram- negative strains E. coli ATCC25922, E. coli ATCC10536, S. typhimurium ATCC13311, S. typhimurium ATCC14028, M. catarrhalis 25240, P. vulgaris 6059, E. cloacae ATCC27508, S. marcescens ATCC27117, A. calcoacetius ATCC15473. NRS119, NRS121, and NRS271, which are clinically isolated linezolid- resistant MRSA strains with G2576U mutation in the peptidyl transferase center, and NRS127, a linezolid- nonsusceptible MRSA with ΔSer145 mutation in ribosomal L3 protein, were obtained from the Network on Antimicrobial Resistance in S. aureus (NARSA).
Minimal inhibitory concentrations (MICs) were determined by two-fold agar dilution as described by the Clinical and Laboratory Standards Institute.33 Test strains were grown for 18 h at 37 °C in tryptic soy broth and diluted with the same fresh medium to a density of ca. 107 colony forming units (CFU)/mL. Suspensions were applied to Mueller–Hinton agar (MHA) plates containing serial dilutions of antimicrobial agents using a multipoint inoculator to yield 105 CFU/spot. Plates were incubated in air at 37 °C for 18 h and were examined for growth. The MIC was considered to be the lowest concentration that completely inhibited growth on agar plates, disregarding a single colony or a faint haze caused by the inoculum. Linezolid and vancomycin were used as reference compounds for comparison.
MIC determination against Mycobacterium tuberculosis
The MICs of compounds against Mycobacterium tuberculosis H37Rv was determined by the Microplate Alamar Blue Assay.29
Molecular docking studies
Docking studies of the ribosomal RNA were conducted using Surflex in the SYBYL-X 2.0 program (version 2.0; Tripos Associates Inc., St. Louis, MO)24 and Maestro program (version 9.2; Schrodinger LLC, New York)36. The docking model was created from the crystal structure of D. radiodurans 50S large subunit of ribosome (Protein Data Bank reference code 3DLL), using nucleotides within 9 Å of cocrystallized linezolid. And then, hydrogen atoms were added to the working model and neutralized at pH
Chemical and Pharmaceutical Bulletin Advance Publication 7.4 using the MCMM force field in MacroModel 9.9. All ligands including linezolid were minimized and ionized at pH 7.4 using LigPrep 2.5 mothod, which the stereoisomers were considered to retain the specified chiralities. The protomol of active site and the surflex-dock (sfxc) files were generated in ligand mode with threshold (0.50) and bloat (0) parameters. The fragment constraints were visualized in the active site, and the fluorophenyl (frg-018) and oxazolidinone ring (frg-010), which have 80 penalties for deviating, were selected to match with compounds. In the docking poses, protein movement was allowed with involving hydrogen heavy atoms. And then, covalent force field weighting was used as ligand 1.0 and protein 0.30 at that docking poses. Minimized cocrystal linezolid was docked into this docking protomol as a test with the ring flexible mode. The length of hydrogen bondings and Pi-orbital interactions were recognized in the Discovery Studio software 3.5 (Accelrys, Inc.).The conformational images of all compounds were drawn in the PyMoL program.
Determination of CYP inhibition, microsomal stability, and MAO inhibition
CYP450 reversible inhibition assay was conducted using Vivid® CYP450 Screening Kits Protocol (Invitrogen, Carlsbad, CA, USA) with recombinant human P450 enzymes, and human hepatic microsomal stability test was conducted by automated HPLC/Mass Spectrometry System.36 Monoamine oxidase MAO- A and MAO-B inhibition assays were performed in duplicate following the provided protocol using Amplex® Red Monoamine Oxidase Assay Kit (A12214, Invitrogen, Carlsbad, CA, USA).
Acknowledgments
This work was supported by the grant of Global Research Lab program from the National Research Foundation of Korea (20110021713), and by the KIST Institutional Program (2V02790).
Chemical and Pharmaceutical Bulletin Advance Publication References and notes
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Chemical and Pharmaceutical Bulletin Advance Publication [Figure s and Charts]
Figure 1. Linezolid and other oxazolidinone antibacterials under development
Figure 2. New oxazolidinone antibacterials
Figure 3. Overlapped structure of docked endo-2a (yellow) and exo-2b (cyan) with cocrystallized linezolid (green) in the active site of the D. radiodurans 50S ribosome (a). The docked conformations of compounds endo-2a (yellow) and exo-2a (blue) (b), and endo-2b (orange) and exo-2b (green) (c) and their interactions with the 50S ribosome residues; M. tuberculosis, D. radiodurans and E. coli are labeled in red, blue and green, respectively.
Chemical and Pharmaceutical Bulletin Advance Publication < Chart 1>
Chemical and Pharmaceutical Bulletin Advance Publication [Tables]
Table 1. In vitro antibacterial activities of oxazolidinone derivatives against Mycobacterium tuberculosis H37Rv and Gram-positive vancomycin-resistant strains (MICs in µg/mL).
Compd R R1, R2 M. t.a S. a.b S. a.c E. f.d B. c.e 1a a O 0.5f 1.56 3.12 1.56 1.56 endo-2a a H, OH 0.25 1.56 6.25 1.56 1.56 exo-2a a OH, H 0.5 1.56 1.56 1.56 0.78 endo-4a a H, triazole 1 3.12 6.25 3.12 3.12 exo-4a a triazole, H 0.5 1.56 3.12 1.56 1.56
endo-5a a H, NH2 2 3.12 >100 25 50 endo-6a a H, NHAc 1 0.78 1.56 1.56 0.78 exo-6a a NHAc, H 1 3.12 25 3.12 3.12
exo-7a a 4-NO2PhCONH, H 0.5 3.12 6.25 3.12 3.12
exo-8a a NH2CSNH, H 0.5 3.12 6.25 3.12 3.12 exo-9a a PhNHCONH, H 2 3.12 6.25 3.12 3.12 endo-10a a H, BnNH 32 25 >100 50 50 1b b O 0.5f 1.56 1.56 0.78 0.78 endo-2b b H, OH 0.5 3.12 6.25 6.25 6.25 exo-2b b OH, H 0.25 6.25 12.5 6.25 6.25 exo-4b b triazole, H 2 3.12 12.5 6.25 3.12
endo-5b b H, NH2 2 3.12 >100 25 100 endo-6b b H, NHAc 1 12.5 50 12.5 12.5 exo-6b b NHAc, H 1 3.12 12.5 6.25 6.25
exo-7b b 4-NO2PhCONH, H 1 1.56 6.25 6.25 3.12
exo-8b b NH2CSNH, H 0.5 6.25 >100 >100 3.12 exo-9b b PhNHCONH, H 4 6.25 12.5 6.25 6.25 endo-10b b H, BnNH 32 12.5 50 25 25 Linezolid 1 1.56 3.12 1.56 0.78 Vancomycin - 2 2 2 >64 aMycobacterium tuberculosis H37Rv; b Staphylococcus aureus ATCC65389; c Staphylococcus aureus ATCC 25923; d Enterococcus faecalis ATCC29212; e Bacillus cereus ATCC27348, f see reference 26.
Chemical and Pharmaceutical Bulletin Advance Publication Table 2. Docking scores for the linezolid and selected compounds.a Compd Total score FragRMSD G score PMF score D score Chemscore 1a 8.7288 0.54 -160.075 -89.671 -121.078 -14.146 endo-2a 10.8692 0.88 -181.175 -77.994 -123.848 -15.696 exo-2a 8.1564 0.78 -154.255 -81.299 -116.025 -13.033 endo-2b 9.5720 0.72 -210.699 -85.246 -162.723 -16.349 exo-2b 8.6289 0.84 -201.190 -84.746 -152.686 -14.811 endo-5a 10.2195 0.81 -172.098 -79.587 -55.581 -16.666 endo-6a 8.8220 0.45 -168.086 -90.617 -113.334 -12.475 exo-6a 7.7789 0.64 -201.221 -89.977 -130.845 -17.165 exo-7a 8.1991 0.53 -205.582 -115.972 -147.352 -17.043 exo-8a 10.0816 0.57 -188.558 -94.544 -126.041 -16.790 exo-9a 11.0435 0.83 -222.485 -106.868 -153.228 -19.049 endo-10a 9.4015 0.65 -277.318 -132.390 -94.713 -18.641 Linezolidb 7.7958 0.36 -129.714 -90.781 -100.845 -14.655 a Docking scores were calculated in SYBYL-X 2.0. b Cocrystallized linezolid was docked in the same condition using Surflex.
Table 3. In vitro antibacterial activities of oxazolidinone derivatives against both methicillin- and linezolid- resistant S. aureus strains (MICs in µg/mL). strains mutation linezolid 1a exo-2a 4ac 1b 2bc NRS119 MRSA G2576Ua >32 16 >32 32 32 >32 NRS121 MRSA G2576Ua >32 16 >32 32 32 >32 NRS127 MRSA ΔSer145b 8 8 8 16 8 16 NRS271 MRSA G2576Ua 32 16 32 32 16 32 aE. coli 23S rRNA numbering, bStaphylococcal numbering in the ribosomal L3 protein; c Both endo and exo isomers showed similar results.
Table 4. CYP450 inhibition, microsomal stability and hMAO-B inhibition assays.a CYP450 isoform, % remaining activity Microsomal hMAO inhibition (%) Stability 2C19 2D6 2C9 1A2 3A4 % remains after A B 30 min PCb 3.6 4.7 4.3 0.7 5.7 - 0 0 endo-2a 73.7 105.6 52.9 34.7 18.3 104.8 47.6 13.5 exo-2a 88.0 108.4 62.3 31.6 82.7 95.2 46.0 9.1 exo-4a 75.7 88.9 44.7 32.9 16.5 102.8 38.7 37.3 endo-6a 92.3 108.4 61.2 31.5 13.8 112.9 15.0 44.8 exo-2b 105.3 105.3 69.5 32.5 15.6 111.9 29.9 40.1 linezolid 111.8 116.3 82.1 35.0 15.6 109.0 60.8 68.6 a Ten micromolar solutions of compounds were used; b PC: Positive control of each isozyme; 2C19: miconazole; 2D6: quinidine; 2C9: sulfaphenazole; 1A2: α-naphthoflavone; 3A4: ketoconazole.
Chemical and Pharmaceutical Bulletin Advance Publication