Syntheses and antibacterial activity studies of new oxazolidinones from nitroso Diels–Alder chemistry

Abstract

A series of novel oxazolidinone antibiotics having [2.2.1] and [2.2.2] bicyclic oxazine moieties at the C-5 side chain of the A ring was synthesized by nitroso Diels–Alder reactions, from three linezolid analogs containing morpholine, piperazine and thiomorpholine, respectively, as the C-ring components. Subsequent N–O bond cleavage generated oxazolidinones with 4-amino cyclo-2- en-1-ol substituents. The in vitro antibacterial activities of these oxazolidinone analogs were evaluated.

Oxazolidinones are a new class of synthetic antimicrobial agents which are now clinically useful. They inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit at the translation step.¹ The most promising feature of these compounds is their oral activity against multidrug-resistant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) as well as select anaerobic organisms.² Linezolid (Figure 1) is the first oxazolidinone drug approved for the treatment of Gram-positive bacterial infections in humans.^2b,3 Unfortunately, resistance to linezolid has already been observed in Gram-positive bacteria such as S. aureus and Enterococcus faecium.⁴ More recently, resistant strains of MRSA, E. coli and other bacteria have also been identified.⁵ Thus, there is significant need for the rapid development of novel antibacterial agents. Four Types of chemical modifications of linezolid and oxazolidinone-type antibiotics have been reported, including modifications on each of the A-, B- and C-rings as well as the C-5 side chain of the A ring substructure.^2b,6 The structure-activity- relationship studies of oxazolidinone antibiotics show that the stereochemistry (S) at the C-5 position of the A ring is critical in terms of biological activity. While an acetamide substituent on the 5-methyl group is found to produce optimal antibacterial activity, modification of the C-5 side chain would still benefit the development of new oxazolidinone antibiotics. In this regard, a frequently employed strategy involves the addition of heterocycles, such as triazoles,⁷ and pyridones.⁸ However, to the best of our knowledge, the incorporation of bicyclic oxazines into this class of antibiotics has not been disclosed.

Figure 1.

Linezolid antibiotic (1).

Syntheses of interesting biologically relevant molecules using nitroso Diels–Alder chemistry (NDA) is of growing interest. As a synthetic tool for the formation of 4-amino cyclopent-2-en-1-ol derivatives in one step, acylnitroso Diels–Alder reactions are useful for the creation of unique structural and functional diversity.⁹ We and others have demonstrated that the nitroso bicyclic oxazine 3 derived from NDA reaction between in situ generated acylnitroso 2 and 1,3-cyclopentadiene can undergo various chemical transformations (Scheme 1).^9b We envisioned that modification at the A-ring C-5 side chain of linezolid-type oxazolidinone antibiotics using nitroso Diels–Alder chemistry would generate novel analogs with incorporation of bicyclic oxazine functionalities. These new analogs were anticipated to not only enhance the available set of oxazolidinone antibiotics, but also provide new scaffolds for further transformations. Herein we report the syntheses and biological evaluation of a series of oxazolidinone antibiotics in which the C-5 side chain has been modified by nitroso Diels–Alder reactions and subsequent chemical transformations.

Scheme 1.

Acylnitroso Diels–Alder (NDA) reaction

To begin our investigation, three types of oxazolidinone precursors 14–16 were synthesized from commercial piperazine (4), morpholine (6) and thiomorpholine (7), respectively, as shown in Scheme 2. The syntheses began by separate S_NAr reactions of 3,4-difluoronitrobenzene with mono Boc-protected piperazine, 5, morpholine, 6, and thiomorpholine, 7, under basic conditions to afford para-substituted nitrobenzene derivatives 8–10 in moderate to good yields. Palladium catalyzed hydrogenolyses of 8 and 9, followed by reactions with CbzCl gave protected anilines 11–12. The synthesis of thiomorpholine analog 13 required modification to avoid poisoning of the catalyst by the sulfur atom during the hydrogenation step. Thus, use of indium powder and ammonium chloride¹⁰ successfully reduced the nitro group of 10 and gave compound 13 in 62% yield after Cbz protection. Then, as reported,¹¹ compounds 11–13 were treated with nBuLi and R-glycidyl butyrate sequentially at −78 °C to form oxazolidinone analogs 14–16 in 70–80% yields with defined stereochemistry at the A-ring C-5 position.

Scheme 2.

Reagents and conditions: (a) Boc₂O, DCM, 50%; (b) 3,4-difluoronitrobenzene, Hunig’s base, CH₃CN, 49% (for 8), 84% (for 9), 70% (for 10); (c) (i) Pd/C, H₂, MeOH, (ii) CbzCl, NaHCO₃, THF/H₂O, 89% (for 11), 92% (for 12); (d) (i) In, NH₄Cl, EtOH, reflux, (ii) CbzCl, NaHCO₃, THF/H₂O, 62% (for 13); (e) (i) nBuLi, THF, −78 °C, (ii) R-glycidyl butyrate, 71% (for 14), 70% (for 15), 80% (for 16).

With the desired aza-, oxa-, and thia-analogs, 14–16, in hand, introduction of the N-hydroxy carbamate moiety at the C-5 side chain and subsequent nitroso Diels–Alder reactions were explored (Scheme 3). Reactions of14 and 15 with O-benzylhydroxylamine hydrochloride (OBHA·HCl) and carbonyldiimidazole (CDI) generated protected N-hydroxy carbamates 17 and 18 in 96% and 94% yields, respectively. Hydrogenolyses afforded the desired free N-hydroxy carbamates, 20 and 21, which were directly used in nitroso Diels–Alder reactions without further purification. Toward this end, N-hydroxy carbamate 20 was chosen as the model substrate. The cycloaddition reaction was carried out by in situ oxidation of 20 in the presence of 1,3-cyclohexadiene. Among several oxidants attempted, including sodium periodate (NaIO₄), Dess-Martin and FeCl₃/H₂O₂,¹² we found that use of sodium periodate gave cycloadduct 23b in the best yield (56%) as one regioisomer after chromatography purification. For the thiomorpholine series, in order to again avoid catalyst poisoning during the subsequent deprotection step, O-p-methoxybenzyl hydroxylamine hydrochloride (PMBONH₂·HCl)¹³ instead of OBHA·HCl was used in the coupling reaction with 16. The PMB group was removed under acidic conditions to give N-hydroxy carbamate 22 in good yield. Using the optimized NDA reaction conditions, a series of new oxazolidinones 23a–25b with bicyclic oxazines at the C-5 side chain were synthesized. Among them, cycloadducts 25a and 25b were obtained with the sulfur atom in the thiomorpholinyl ring oxidized. N–O bond cleavage reactions of these cycloadducts were also carried out, with intent to generate the corresponding oxazolidinones with additional 1,4-amino alcohol substituents. Thus, Mo(CO)₆¹⁴ successfully reduced the N–O bond of compounds 23a–25b to generate the corresponding oxazolidinone analogs, 26a–28b, that contain aminocycloalkenol functional groups, in moderate yields. In addition, under Mo(CO)₆ conditions, the sulfoxide group of 25a and 25b was also reduced. For comparison during the biological testing, an oxazolidinone analog 29 with a C-5 acetate substituent was synthesized from compound 15 (Scheme 4).

Scheme 3.

Reagents and conditions: (a) (i) CDI, CH₃CN (ii) OBHA·HCl, imidazole, 96% (for 17), 94% (for 18); (b) CDI, CH₃CN, (ii) PMBONH₂·HCl, imidazole, 92% (for 19); (c) Pd/C, H₂, MeOH; (d) (i) 10% TFA, DCM, (ii) sat. Na₂CO₃; (e) NaIO₄, 1,3-cyclopentadiene, MeOH/H₂O, 0°C, 50% (for 23a, from 17), 49% (for 24a, from 18), 40% (for 25a, from 19); (e) NaIO₄, 1,3-cyclopentadiene, MeOH/H₂O, 0°C, 50% (for 23a, from 17), 49% (for 24a, from 18), 40% (25a, from 19); (f) NaIO₄, 1,3-cyclohexadiene, MeOH/H₂O, 0°C, 56% (for 23b, from 17), 47% (for 24b, from 18), 42% (25b, from 19); (g) Mo(CO)₆, CH₃CN/H₂O, 80°C, 50% (for 26a), 43% (for 26b), 42% (for 27a), 62% (for 27b), 54% (for 28a), 55% (for 28b).

Scheme 4.

Reagents and conditions: (a) Ac₂O, Pyridine, DMAP, DCM, 75%.

With the biological profile of parent linezolid, 1, known for comparison, all the oxazolidinone analogs 23a–28b, 29, as well as precursors 14–16, and ciprofloxacin, as a positive control, were subjected to broad antibacterial studies against various strains of Gram-positive and Gram-negative bacteria as well as Mycobacterium vaccae, using agar diffusion assays (Table 1).¹⁵ Compound 29 was found to be comparable to linezolid (1) itself for all tested organisms, as expected. Interestingly, all the oxazolidinone precursors 14–16 were roughly equipotent in vitro with linezolid (1) against several Gram-positive organisms, including Bacillus subtilis, S. aureus, E. faecalis and M. luteus. They also exhibited antimycobacterial activity and could potentially be useful for treatment of M. tuberculosis as they induced large inhibition zones against Mycobacterium vaccae, a common model for M. tuberculosis. Most oxazolidinone analogs with bicyclic oxazine or aminocycloalkenol moieties at the C-5 side chain, generated from NDA chemistry, had antimicrobial profiles similar to linezolid (1), but at a generally diminished level of activity. In general, oxazolidinone analogs substituted with [2.2.1] bicyclic oxazines (23a–25a) were more active than those with [2.2.2] bicyclic oxazines (23b–25b). Among them, compound 25a derived from thiomorpholine exhibited the best activity. Analogs 26a–28b, derived from reduction of the N–O bonds were relatively inactive compared to their parent cycloadducts.

Table 1.

Antimicrobial activity in the agar diffusion assay (Diameter of inhibition zone, measured in mm).

	Gram-positive bacteria						Gram-negative bacteria
Compds	Bacillus subtilis	Micrococcus luteus	Staphylococcus aureus		Enterococcus faecalis		Escherichia coli	Pseudomonas aeruginosa		Mycobacterium vaccae
	ATCC 6633	ATCC 10240	SG 511a	134/93 (MRSA)	ATCC 49532	1528 (VRE)	ATCC 25922	K799/WTa	K799/61b	IMET 10670
1	31/35P	38	27/36P	NT	25	NT	0	0	10P	42
29	26.5	38/46p	26/35P	NT	24	NT	0	0	17h	43
14	23/26P	34.5	24/33P	NT	21	NT	0	0	10P	NT
15	26/29P	38	23/32P	NT	21	NT	0	0	11P	43
16	26/29P	41	24/35P	NT	21.5	NT	0	0	11P	43
23a	12/18P	16	13/19p	19	19	16	0	14/20p	0	19p
23b	18P	NT	16P	14p	NT	12P	NT	NT	0	13
24a	17/30P	NT	15/21p	25	NT	19	NT	NT	0	30
24b	19P	NT	16P	18p	NT	16	NT	NT	0	15
25a	21/28P	22/32P	18/22p	NT	19	NT	0	0	11P	35
25b	17P	15p	15p	NT	17	NT	0	0	10P	18
26a	13h	12P	14p	NT	12.5	NT	0	0	10P	12h
26b	0	0	0	NT	12p	NT	0	0	12P	0
27a	14P	NT	15P	14p	NT	12P	NT	NT	0	18
27b	13P	NT	14P	14P	NT	12P	NT	NT	0	0
28a	14P	15h	12.5p	NT	12.5	NT	0	0	10P	18
28b	12h	14	0	NT	12	NT	0	0	10P	0
cipro	29.5	0	24	0	13	15	31/40P	35	39	19p

^awild type.

^bpermeability mutant.

p, partially clear inhibition zone/colonies in the inhibition zone.

P, unclear inhibition zone/many colonies in the inhibition zone.

h, faint indication of inhibition zone.

Exactly 50 µl of a 2.0 mM solution of each compound dissolved in 1:9 DMSO:MeOH was filled in 9 mm wells in agar media (Standard I Nutrient Agar, Serva or Mueller Hinton II Agar, Becton, Dickinson and Company). Inhibition zones read after incubation at 37 °C for 24 h. Cipro (ciprofloxacin) was dissolved in H₂O to give 5 µg/mL solution.

In summary, we have synthesized a series of novel oxazolidinone antibiotics with [2.2.1] and [2.2.2] bicyclic oxazine as well as aminocycloalkenol moieties at the C-5 side chain through nitroso Diels–Alder chemistry. These oxazolidinone analogs exhibited in vitro antibacterial profiles similar to that of linezolid.