Syntheses and biological evaluation of new cephalosporin - oxazolidinone conjugates

Shanshan Yan; Marvin J. Miller; Timothy A. Wencewicz; Ute Möllmann

doi:10.1039/C0MD00015A

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DOI: 10.1039/C0MD00015A (Concise Article) Med. Chem. Commun., 2010, 1, 145-148

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Syntheses and biological evaluation of new cephalosporin-oxazolidinone conjugates†

Shanshan Yan ^a, Marvin J. Miller *^a, Timothy A. Wencewicz ^a and Ute Möllmann ^b
^aDepartment of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, Indiana 46556, USA. E-mail: mmiller1@nd.edu; Fax: +1 574 631 6652; Tel: +1 574 631 7571
^bLeibniz Institute for Natural Products Research and Infection Biology – Hans Knöell Institute, Beutenbergstrasse 11a, D-07745, Jena, Germany

Received 18th February 2010 , Accepted 31st March 2010

First published on 5th May 2010

Abstract

Two cephalosporin-oxazolidinone conjugates were synthesized by incorporation of a carbamate linker at the 3′-position of the cephalosporin. These compounds show stability in aqueous media until specifically activated by a β-lactamase, and retain antibacterial activities profiles reflecting both the individual cephalosporin and oxazolidinone components.

Introduction

Since the discovery of penicillin, β-lactam antibiotics have been the most important family of antibacterial agents. Cephalosporins, a class of β-lactam antibiotics, are known to exert their biological activity by reacting with bacterial enzymes to open the β-lactam ring. This process is accompanied by liberation of the 3′-substituent, when the substituent can function as a leaving group (Scheme 1).¹ Examples are known where cytotoxic components, including mitomycin C,² doxorubicin,³ as well as antimicrobials, such as quinolones,⁴ have been incorporated at the 3′ position. The cephalosporin-quinolone conjugates have been shown to exhibit a broad spectrum of antibacterial activity derived from both cephalosporin-like and quinolone-like components. These dual-action cephems can also be used to overcome the destructive action of β-lactamases (βL) (Enz = βL, Scheme 1), a main cause of bacterial resistance to β-lactam antibiotics.⁵ While, as indicated, many cephalosporin prodrug conjugates have been described, linezolid (Fig. 1), a relatively new FDA approved drug for treatment of MRSA and VRE, or any of the related oxazolidinone antibiotics, have not been evaluated in an analogous fashion. Linezolid is the first oxazolidinone drug approved for the treatment of Gram-positive bacterial infections.⁶ Cephalosporin-oxazolidinone conjugates are anticipated to possess activity against both Gram-negative and Gram-positive bacteria. Herein we report the syntheses and biological evaluation of two cephalosporin-oxazolidinone congeners and demonstrate the controlled release of the oxazolidinone in the presence of a β-lactamase GES-II.


	Scheme 1 Drug-release process from cephalosporin nucleus


	Fig. 1 Linezolid oxazolidinone antibiotic.

Results and discussion

Based on the ample precedent mentioned earlier for the use of the βL-induced release process as depicted in Scheme 1, we considered using a carbamate linkage between the 3′ position of a representative cephalosporin and piperazine-based linezolid analogs as depicted in Fig. 2. This strategy required the preparation and eventual conjugation of the appropriately functionalized cephalosporin and oxazolidinone components.


	Fig. 2 Cephalosporin-oxazolidinone conjugates.

The syntheses of the component linezolid analogs 3a and 3b are shown in Scheme 2. The syntheses utilized considerable literature precedent⁷ and began with a S_NAr reaction of 3,4-difluoronitrobenzene and mono Boc-protected piperazine 4 to afford para-substituted nitrobenzene derivative 5 in 49% yield. Palladium catalyzed hydrogenation of compound 5 followed by reaction with CbzCl gave protected aniline 6. Then, as reported,^7a compound 6 was sequentially treated with nBuLi and R-glycidyl butyrate at −78 °C to form the key oxazolidinone 7 in 71% yield with defined stereochemistry at the C-5 position of ring A. Reaction of 7 with acetic anhydride in the presence of DMAP and pyridine afforded the corresponding C-5 acetate analog, 8, in 95% yield. TFA-induced removal of the Boc group afforded the desired linezolid analog 3a in 92% isolated yield. Alternatively, stepwise reaction of 7 with methanesulfonyl chloride, ammonia and acetyl chloride produced the acetamidomethyl-containing analog, 9, in 61% overall yield for three steps. Analog 3b was then obtained in good yield by treatment of 9 with TFA to remove the Boc group.


	Scheme 2 Syntheses of linezolid analogs 3a and 3b

With oxazolidinones 3a and 3b in hand, the syntheses of carbamate-linked cephalosporin-oxazolidione compounds 1 and 2 were carried out as shown in Scheme 3. The syntheses began with a controlled hydrolysis to remove the acetyl group from the 3′-hydroxymethyl substituent of commercially available 7-amino cephalosporanic acid (7-ACA). Reaction with phenylacetyl chloride followed by treatment with freshly prepared diphenyldiazomethane⁸ provided cephem 10 in 29% overall yield for three steps.⁹ Subsequent reaction with tetrachloroethyl chloroformate gave intermediate carbonate 11 in 81% yield. Reaction of the activated carbonate 11 with 3a in the presence of pyridine and DMAP was attempted. Desired coupling product 12 was generated; however, as often during reactions of cephalosporins, partial isomerization of the cephem nucleus occurred,¹⁰ to give a nonseparable mixture of the Δ³ and Δ² double bond isomers (12 and 12b, Fig. 3). Cephalosporin Δ² isomers 12b are not effective antibiotics^10c,11 and also are not substrates for the planned β-lactamase induced prodrug process depicted earlier in Scheme 1. Further studies of the coupling reaction revealed that without any basic additives, reaction between 11 and 3a still took place and provided the desired protected conjugate 12 in 40% yield. No Δ²–Δ³ isomerization was observed. Subsequent removal of the benzhydryl protecting group from 12 gave the desired conjugate 1 in 28% yield after crystallization. With this approach validated, cephalosporin-oxazolidinone conjugate 2 was also synthesized in a similar manner, involving the formation of conjugate 13 with a C-5 acetamidomethyl substitute and subsequent removal of benzhydryl group of 13.


	Scheme 3 Syntheses of cephalosporin-oxazolidinone conjugates 1 and 2


	Fig. 3 General structures for Δ³–Δ² double bond cephem isomers.

To determine whether cephalosporin-oxazolidinone conjugates 1 and 2 could be specifically activated in the presence of β-lactamase as planned, a LC/MS assay was performed (Fig. 4).¹² Conjugates 1 and 2 were separately incubated with catalytic amounts of β-lactamase GES-II (0.4 mol%) in phosphate buffered saline (50 mM, pH 7.0) at room temperature. We were pleased to find that in the presence of β-lactamase, conjugates 1 and 2 were completely cleaved to the hydrolyzed cephalosporin and the oxazolidinone components 3a and 3b, respectively, after 4 h. Control experiments were also conducted by treatment of 1 and 2 under the same conditions in the absence of β-lactamase. As expected, conjugates 1 and 2 were stable even after 24 h. These data demonstrate that compounds 1 and 2 are good substrates for βL GES-II and are stable in a neutral aqueous environment until specifically activated by the β-lactamase.


	Fig. 4 HPLC trace of compounds 1, 2 and products 3a, 3b released by β-lactamase activation. Left to right: (*) 3a (with βL, 4 h), (◆) 1 (no βL, 24 h), (●) 3b (with βL, 4 h), (■) 2 (no βL, 24 h).

Cephalosporin-oxazolidinone conjugates 1 and 2, as well as linezolid analogs 3a and 3b, and ciprofloxacin as a control, were tested for their antibacterial activities against various strains of Gram-positive and Gram-negative bacteria as well as Mycobacterium vaccae, using an agar diffusion assay (Table 1). Cephalosporin derivative 14 was synthesized from 7-ACA (Scheme 4) and included in the assay as an additional control. In general, conjugates 1 and 2 were found to retain similar biological profiles compared to cephem 14 as well as individual oxazolidinones 3a and 3b. More interestingly, for most organisms tested, conjugates 1 and 2 displayed extended activity relative to linezolid itself. For example, they exhibited good activity against the Gram-negative strain Pseudomonas aeruginosa K799/61, while linezolid was relatively inactive. Conjugates 1 and 2, as well as components 3a and 3b, were also evaluated against MRSA and VRE, and good activity was observed. Again, conjugate and components matched in activity. All compounds, including conjugates 1 and 2, as well as oxazolidinone 3a and 3b, also exhibited antimycobacterial activity and could potentially be useful for treatment of M. tuberculosis as they induced large inhibition zones against M. vaccae, a common model for M. tuberculosis.¹³ Interestingly, linezolid components 3a and 3b were roughly equipotent in vitro with linezolid against several Gram-positive organisms, including Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis and Micrococcus luteus. These biological data suggested that the cephalosporin-oxazolidinone conjugates might possess additive biological activity corresponding to that of both the cephem and oxazolidinone components.

Table 1 Antibacterial activity of conjugates 1 and 2 in the agar diffusion assay

compds^a	Growth inhibition zones in mm (9 mm well diameter)
	Gram-positive bacteria							Gram-negative bacteria			M. vaccae IMET
	B. subtilis ATCC	S. aureus				E. faecalis	M. luteus ATCC	P. aeruginosa		E. coli	M. vaccae IMET
	6633^b	SG 511^b	Efs1^c	Efs4^c	134/93 MRSA^c	1528^cVRE	10240^b	K799/WT^c	K799/61^c	SG 458^b	10670^b
a Exactly 50 μL of a 2.0 or 1.0 mM solution in DMSO : MeOH (1:9) of each compound 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 a 5 μg/mL solution. b 1.0 mM solution of linezolid, 1–2, 3a–3b, and 14 was used. c 2.0 mM solution of linezolid, 1–2, 3a–3b, and 14 was used. d p, partially clear inhibition zone/colonies in the inhibition zone. e P, unclear inhibition zone/many colonies in the inhibition zone. f h, faint indication of inhibition zone.
linezolid	33/37p^d	35	34/39p	35/39p	42	34	38/45p-P^e	0	10P	23/28p	52
14	46/52p	39	43	35	13	23	38/44p-P	11h^f	23	35	0
3a	24/26p	21	31	19/23P	29	25	31p/40P	15h	16/26h	13P	42
3b	26	18	31/35p	14/18P	25	25	23/30p-P	11P/18h	20/30h	14P	35
1	43/47p	36	40	31	27	26	42	17h	22/31p	33	44
2	42/45p	34	40	30	17	23	38/42p	15P/20h	24/33h	33	30
cipro	31	18	0	0	0	15	0	29	35	34	23


	Scheme 4 Synthesis of cephem 14

Conclusions

We have described straightforward syntheses of cephalosporin-oxazolidinone conjugates. The conjugates retained antibacterial activity comparable to individual cephalosporin and oxazolidinone components, and showed stability in aqueous media until specifically activated by a β-lactamase. Future work will focus on in vitro enzyme assays to examine the release of oxazolidinone in the presence of β-lactamase producing bacteria. Additional structural analogs are also under consideration.

Acknowledgements

This work was supported by grant from the National Institutes of Health (GM 075855). We thank Professor Shahriar Mobashery for providing β-lactamase GES-II. We gratefully acknowledge Uta Wohlfeld for performing antibacterial assays, and Dr Viktor Krchnak for help with LC/MS. We also thank the Lizzadro Magnetic Resonance Research Center at Notre Dame for NMR facility and Nonka Sevova for mass spectroscopic analyses. TAW acknowledges the University of Notre Dame Chemistry-Biochemistry-Biology Interface (CBBI) Program and NIH Training Grant T32GM075762 for a fellowship.

Notes and references

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Footnote

† Electronic Supplementary Information (ESI) available: Experimental procedures, characterization data and copies of ¹H NMR and ¹³C NMR spectra, protocols of antibacterial assays. See DOI: 10.1039/C0MD00015A/

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