Anti-MRSA cephems. Part 2: C-7 cinnamic acid derivatives
C-7 acidic cephems are described that have surprisingly good activity against methicillin-resistant Staphylococcus aureus (MRSA). The most active compound (41) displayed an MIC90 against MRSA of 1.0 μg/mL, and a PD50 of 0.8 mg/kg. A representative compound from this class was found to be very safe in a mouse model of acute toxicity.
Introduction
Post-surgical morbidity from bacterial infections of methicillin-resistant Staphylococcus aureus (MRSA) is increasing as most strains have become multi-drug resistant.1 The only effective treatment of MRSA currently in the clinic is vancomycin. The advent of vancomycin resistance in enterococci has raised concern that this resistance might eventually be transferred to MRSA, creating a lethal pathogen of epidemic proportions.2 Strains of MRSA with reduced susceptibility to vancomycin have begun to appear in the clinic, heralding the beginning of the end of vancomycin's utility in the management of these infections.3 Therefore, the search for new antibiotics with anti-MRSA activity is critical to the future maintenance of public health.4
We wished to develop an injectable anti-MRSA cephalosporin that would manage these life-threatening infections. Our biological and solubility criteria for a suitably active clinical candidate were as follows: MIC90s against MRSA ≤4 μg/mL; PD50 in a mouse systemic MRSA infection ≤5 mg/kg; safety in a mouse model of acute toxicity; and, aqueous solubility ≥20 mg/mL.
Our program began with the observation that C-7 2,5-dichlorothiophenyl acetamides imparted excellent anti-staphylococcal activity to cephems.5 The combination of this lipophilic C-7 group with C-3 thiopyridinium moieties provided compounds with exceptional anti-MRSA activity.6 An early lead, aminopropyl derivative 1, satisfied our goals for in vitro and in vivo activity. Unfortunately, compound 1 was found to be acutely toxic to mice upon iv bolus delivery to the tail vein. It was subsequently found that the C-3 amino acid derivative 2 had good activity against MRSA, and was less acutely toxic than 1 in mice.7 However, bis-zwitterion 2 was not very soluble in a variety of aqueous media (<3 mg/mL).8 As a result of this low solubility, a large number of derivatives were synthesized that had either a carboxylic or sulfonic acid group on the side-chain of the thiopyridinium moiety at C-3, or an acid substituted off the pyridinium ring itself.4i The goal was to move the acid group around the C-3 moiety in the hope of improving upon the solubility of our derivatives while maintaining sufficient anti-MRSA activity. As a result of these efforts, some general trends were observed regarding the inclusion of acid groups into our cephems. First, the extra acid functions improved the acute toxicity profiles of our compounds (vide supra regarding the toxicity of compound 1 vs 2). In general, it was observed that positively charged molecules (such as 1) were usually quite toxic in our acute toxicity assay. Zwitterions or bis-zwitterions (such as 2) usually fared better in the acute toxicity screen, while compounds carrying a net negative charge were usually non-toxic in our assay. While the inclusion of an extra acid improved the solubility of our compounds (except in the case of zwitterions), and helped improve their toxicity profile, the addition of each extra acid group at the C-3 position also resulted in a concomitant loss of potency in vitro (generally 4–8 fold less active by MIC). We therefore embarked upon a program to introduce an acid at C-7 of our cephems in the hopes of improving solubility, maintaining safety in the acute toxicity assay, and retaining excellent in vitro potency against MRSA. The results of some of our efforts in this area are hereby presented.
Section snippets
Chemistry
The C-7 amide-linked thiophenyl group is undoubtedly a major contributor to the lipophilicity of our derivatives. We had long maintained an interest in introducing functional groups onto the C-7 dichlorothiophenyl ring of our compounds that would improve their water solubility. One of our early routes to C-7 acid compounds is illustrated by Scheme 1. Iodination of 2,5-dichlorophenol in the para position, followed by conversion to the O-aryl dimethyl thiocarbamate, provided intermediate 3.
Results and discussion
The first aspect of SAR that was investigated was the effect of chlorine substitution on activity. As Table 1 demonstrates, the C-7 dichloro aryl derivative 25 is more active than the corresponding monochloro compounds 27 and 28. Similarly, the dichloro cephem 38 is more active than its related monochloro derivatives 39 and 40. Amongst the monochloro compounds, the ortho-substituted (relative to sulfur) 28 and 40 were less active than their meta-substituted counterparts 27 and 39. The bis meta
Conclusions
We have discovered a series C-7 cinnamic acid derivatives that often display excellent in vitro and in vivo activity against MRSA. The introduction of an extra acidic group at C-7 onto leads such as cephem 1 was found to be less deleterious to antibacterial potency than placing the acid group at C-3. Many of these compounds had good PK characteristics, and cephem 14 was shown to be a very safe compound in an acute toxicity assay in mice. Cephems such as 14, 25, 41, 50 and 60 are exciting leads
Biology
Antibacterial MICs were determined in broth according to the standard conditions recommended by the National Committee for Clinical Laboratory Standards (NCCLS). MIC assays against MRSA utilized Mueller-Hinton broth+2% NaCl, a bacterial inoculum size ∼5×105 CFU/mL, and were incubated at 35 °C for 24 h. MIC was defined as the lowest drug concentration inhibiting all visible growth. MIC90s were determined against a panel of 58 strains of MRSA.
The PD50 values reported represent the concentration of
Acknowledgements
We thank our colleagues in the Department of Microbiology in Wallingford, CT for the biological data reported in this work. We also thank the Department of Analytical Research in Wallingford, CT, for analytical data, and Mr. Robert Perrone of the Department of Pharmaceutics Research and Development in New Brunswick, NJ for solubility determinations in support of this work.
References (14)
Clinical Infectious Diseases
(1997)Curr. Pharm. Des.
(1999)- et al.
J. Infect. Dis. Pharm.
(1998) - et al.
J. Antimicrob. Chemother.
(1997)et al.New Eng. J. Med.
(1999) - et al.
J. Antibiot.
(2000)et al.J. Antibiot.
(2000)et al.J. Antibiot.
(2000)et al.J. Antibiot.
(2000)et al.Bioorg. Med. Chem.
(2000)et al.Tetrahedron
(2000)et al.J. Antibiot.
(2000)et al.J. Antibiot.
(2000)et al.Bioorg. Med. Chem. Lett.
(2001)et al.Tetrahedron
(2000)et al.Org. Process Res. Dev.
(2000)et al.Bioorg. Med. Chem. Lett.
(1997)et al.Bioorg. Med. Chem. Lett.
(1997)