β-Lactam Antibiotics Renaissance

Since the 1940s β-lactam antibiotics have been used to treat bacterial infections. However, emergence and dissemination of β-lactam resistance has reached the point where many marketed β-lactams no longer are clinically effective. The increasing prevalence of multidrug-resistant bacteria and the progressive withdrawal of pharmaceutical companies from antibiotic research have evoked a strong reaction from health authorities, who have implemented initiatives to encourage the discovery of new antibacterials. Despite this gloomy scenario, several novel β-lactam antibiotics and β-lactamase inhibitors have recently progressed into clinical trials, and many more such compounds are being investigated. Here we seek to provide highlights of recent developments relating to the discovery of novel β-lactam antibiotics and β-lactamase inhibitors.


Introduction
The emergence and spread of resistance to antibiotics always has accompanied their clinical use.During the past 20 years, however, physicians have experienced a worrisome situation, described by Shlaes [1] and by Pendleton [2], involving a significant increase in morbidity and mortality due to bacterial infections in both community and hospital settings.In particular, two types of strains have compromised the clinical utility of currently available antibiotics and underscored the need for new compounds: OPEN ACCESS  multidrug-resistant strains (MDR), which are non-susceptible to one or more drugs belonging to ≥3 antimicrobial classes;  and extremely drug-resistant strains (XDR), which are non-susceptible (or nearly so) to all classes of antimicrobials [3].

Discussion
Since 2000 about 20 new antibiotics have been launched, covering five new drug classes for combating bacterial diseases.These five new classes are represented by linezolid (intravenous and oral oxazolidinone, active against Gram-positive cocci, approved 2000), daptomycin (intravenous lipopeptide, active against Gram-positive cocci, approved 2003), retapamulin (topical pleuromutilin, active against Gram-positive cocci, approved 2007), fidaxomicin (oral macrocycle, active against Clostridium difficile, approved 2010), and bedaquiline (intravenous diarylquinoline, active against Mycobacterium tuberculosis, approved 2012).Table 1 lists new antibiotics approved since the start of the second millennium.Widespread use of β-lactam antibiotics derives from their efficacy and safety profile, but emergence of new, more aggressive β-lactamases is obliging clinicians to resort to drugs with lower therapeutic windows, such as polymyxins and tigecycline.According to the Pharmaceutical Research and Manufacturers of America, there are 124 products in development for bacterial infections, the vast majority of which are in Phases II and III, with only a few of them being new chemical entities with novel mechanism of action, underlying the difficulties in fuelling the pipeline.To improve the situation and counter bacterial resistance, several government-supported initiatives have been undertaken, including the US Generating Antibiotic Incentives Act (-GAIN‖: automatic priority review and an additional 5-7 years of market exclusivity for qualified infectious disease products), and the Innovative Medicines Initiative New Drugs for Bad Bugs (IMI ND4BB), a fund to support clinical development of new antibiotics, particularly those effective against Gram-negative pathogens [9].Amongst other incentives to have been proposed [10][11][12][13], the Infectious Diseases Society of America launched (2010) its -10 × '20 Initiative‖ to develop 10 new safe and efficacious systemically administered antibiotics by 2020 [14].In all of this antibiotic development activity, novel β-lactam antibiotics or β-lactamase inhibitors play a significant role.One avenue of research has aimed at identifying novel β-lactams belonging to well established classes, but with an expanded spectrum, e.g., methicillin-resistant staphylococci (MRSA), P. aeruginosa, and/or A. baumannii.Another avenue has focused on identifying novel β-lactamase inhibitors that can protect existing β-lactams from emerging β-lactamases, like KPC, OXAs, and metallo-β-lactamases.This approach ought to enable the rejuvenation of old antibiotics that lately have lost their efficacy due to the appearance of new resistance.

β-Lactam and β-Lactamase Inhibitors in Development
Nearly all recently-developed β-lactam antibiotics have been studied in combination with β-lactamase inhibitors, because bacteria have evolved β-lactamases that hydrolyze (inactivate) all β-lactam classes, including carbapenems.The antibacterial pipeline contains several combinations of novel β-lactamase inhibitors with -old‖ β-lactam antibiotics and -old‖ β-lactamase inhibitors with new β-lactam products (Table 2).Presently the most important medical need lies with countering MDR and XDR Gram-negative pathogens [15].The decline in efficacy of available β-lactams has stimulated the study of creative antibiotic combinations (e.g., carbapenems + polymyxins, mecillinam + amoxicillin/clavulanate) in an effort to identify therapeutic alternatives to monotherapies [16][17][18]; publications on BAL30072, an experimental ESBL-susceptible β-lactam, suggest that for most indications this compound is being positioned as a partner with meropenem (vide infra).Due to the limitations of such approaches, that often are unable to overcome the emerging resistance mechanisms, research has focused on other directions, including new β-lactam/β-lactamase inhibitor combinations.CXA-101 (ceftolozane) is a cephalosporin [19] discovered in 2004 by the Fujisawa Pharmaceutical Co., Ltd.(now Astellas, Chertsey, UK) and originally named FR264205 [20,21].The compound is particularly active against MDR P. aeruginosa, including isolates from chronically-infected cystic fibrosis patients [22], due to enhanced affinity of the β-lactam for the PBPs of this species, reported stability to many β-lactamases including AmpCs (but not ESBLs, KPC carbapenemases, or metallo-β-lactamases), and indifference to efflux pumps [23][24][25][26].However, it also has (relatively) poor activity towards other Gram-negative pathogens, as well as towards multidrug-resistant Gram-positive cocci and anaerobes.Ceftolozane MICs against clinical isolates of P. aeruginosa are 8-to 16-fold lower than those of ceftazidime [23,24], and are little affected by MexAB-OprM overexpression and/or OprD deletion.Susceptibility of ceftolozane to ESBLs as evidenced by the 4-to 128-fold increase in MIC by producers of this β-lactamase [27].
CXA-101 (Figure 1) is a 2:1 (w/w) combination of ceftolozane and tazobactam, an old (ca.1990) sulfone penam β-lactamase inhibitor, that is being developed by Cubist.CXA-101 is in Phase 3 trials for treatment of complicated urinary tract infections (levofloxacin as comparator), and for treatment of complicated intra-abdominal infections (meropenem as comparator).Addition of tazobactam at a fixed concentration of 8 µg/mL restored the in vitro susceptibility of 93% of ESBL producers and 95% of the AmpC overproducers examined.However, tazobactam was unable to lower MICs, for Enterobacteriaceae producing KPCs, [28] and CXA-101 does not show better activity than ceftolozane alone against P. aeruginosa.Cubist recently filed a patent covering use of ceftolozane/tazobactam (2:1) for treating pulmonary infections [29].

CPT (Ceftaroline)
Ceftaroline fosamil (Figure 4), is a cephalosporin prodrug whose active principle, ceftatoline (CPT), is active against MRS and drug-resistant S. pneumoniae [44].This compound has the distinction of being the first anti-MRS β-lactam to be marketed in the USA (2010), where it received FDA approval for treatment of acute bacterial skin and skin structure infections (SSSI) and for community-acquired pneumonia (CAP) [45].Ceftaroline shows good clinical efficacy [46] against methicillin-resistant Staphylococcus aureus (MRSA) due to its ability to bind to PBP2A.Avibactam demonstrated synergy with ceftaroline against some β-lactamase-producing anaerobes (Bacteroides fragilis, Prevotella spp., and Finegoldia magna cultured from diabetic foot infections [47]).The antistaphylococcal activity of ceftaroline makes it a good partner antibiotic for certain polymicrobial wound infections where MRSA may be a dominant pathogen [48].The combination ceftaroline fosamil/avibactam currently is in Phase III trials [49,50].

Imipenem/Cilastatin/MK-7655
Imipenem, the first carbapenem to reach the market (invented and developed by Merck), is a potent, broad-spectrum β-lactam with antipseudomonas activity.It lacks a β-methyl substitutent at position 1 and, accordingly, is not stable to human renal dehydropeptidase I; therefore, imipenem is marketed in combination with cilastatin, a dehydropeptidase inhibitor specifically developed by Merck as a companion to imipenem.MK-7655 is a DBO β-lactamase inhibitor [51] that, combined with imipenem [52], showed good activity against imipenem-resistant Gram-negative isolates in vitro [53].The triple combination of imipenem/cilastin/MK-7655 (Figure 5) poses challenges in terms of a balanced pharmacokinetics, but provides improved activity against some carbapenemase-resistant P. aeruginosa and Enterobacteriaceae [54].A short, scalable, cost-effective route for production of MK-7655 recently has been reported (Figure 6) [55,56].Synthesis was made possible through the availability of optically pure cis-5-hydroxypipecolic acid, obtained by enzymatic oxidation of pipecolic acid [57].This route provides pure, crystalline MK7655 in 8 steps, with a 42% overall yield, allowing for a more cost-effective manufacture of the DBO on a multi-kilogram scale.2.1.5.BAL30072 BAL30072 (Figure 7) is a monobactam developed by Basilea Pharmaceutica AG and currently in Phase I trials [58]; it is a slight structural modification of tigemonam, where the acetate group has been replaced by dihydroxypyridone, the siderophoric moiety earlier used in the experimental monobactam PTX2416 [59].BAL30072 is taken up by bacteria via one of their iron transport systems [60]; like other monobactams (e.g., aztreonam), BAL30072 is stable towards metallo-β-lactamases and has some inhibitory activity towards class C β-lactamases, though the latter is unlikely to translate into clinical efficacy against most AmpC-producing pathogens.BAL30072 was active against multidrug-resistant (P.aeruginosa, Acinetobacter spp., Burkholderia spp.and Stenotrophomonas maltophilia).It was active against 70% of the carbapenem-resistant Enterobacteriaceae strains tested [61].In combination with different carbapenems, BAL30072 showed synergism against several Enterobacteriaceae and P. aeruginosa [62].In 2013 Basilea received a contract potentially totaling US$17 million from the United States Biomedical Advanced Research and Development Authority (BARDA) to continue Phase 1 studies of the safety and tolerability of BAL30072, alone and in combination with carbapenems.It is likely that its further development will involve its combination with carbapenems to extend their activity towards metallo β-lactamases producers [63].

ATM-AVI
ATM-AVI is a combination [65] of aztreonam (Figure 8), a monobactam launched in the US in 1984, and the DBO β-lactamase inhibitor avibactam, under development by AstraZeneca.ATM-AVI was evaluated in a Phase I trial (NCT01689207) but the study was recently suspended because of poor participant recruitment [9].

TD-1792
The antibacterial activity of TD-1792 (Figure 10), a hybrid glycopeptide-cephalosporin antibiotic invented by Theravance, is restricted to Gram-positive pathogens, and has completed a Phase II cSSSI trial [4].TD-1792 is highly potent towards MRSA and vancomycin-intermediate Staphylococcus aureus (VISA).TD-1792 was designed to allow the molecule to interact and inhibit simultaneously two molecular targets involved in murein biosynthesis that are in close proximity to one another, with the expectation that such a strategy would raise significantly the statistical barrier to resistance development [70].

Arylboronic Acids
The John Hopkins University is offering to out-licence a series of arylboronic acids (Figure 14), originally patented by Fulcrum Pharmaceuticals in 2004, showing activity against TEM-1 and AmpC β-lactamases (US7,183,267, also filed as EP1635812).
The synthesis of representative sulfonamido β-lactamase inhibitors is shown in Figure 19.The initial step is synthesis of (E)-2-(3-chlorophenyl)ethene-1-sulfonyl chloride from 3-chlorostyrene and sulfuryl chloride.In parallel, 3-aminophenol was protected at the nitrogen using Boc anhydride and O-alkylated using 3-chlorobenzylbromide.Deprotection of the nitrogen, using trifluoroacetic acid, afforded the O-alkylated aniline in 83% yield.The aniline and sulfonyl chloride were then coupled to produce (E)-N-(3-((3-chlorobenzyl)oxy)phenyl)-2-(3-chlorophenyl)ethene-1-sulfonamide, which was N-alkylated using sodium hydride and 3-chlorobenzylbromide to afford the final product in 11% yield.2.1.17.CB-027 CB-027 (Figure 21) is reported by Cubist to be an ultra-broad spectrum cephalosporin with in vitro activity against both MRSA and P. aeruginosa.The compound is claimed to be have in vivo activity against clinical isolates of MRSA comparable to that of vancomycin and of ceftaroline, and against ceftazidime-resistant P. aeruginosa and K. pneumonia [81].

Conclusions
The interest for novel β-lactam antibiotics or β-lactamase inhibitors has recently boosted with several companies actively involved in the development of new molecules.The antibacterial pipeline contains several combinations of novel β-lactamase inhibitors with -old‖ β-lactam antibiotics and -old‖ β-lactamase inhibitors with new β-lactam products.The main goal is to achieve antibacterial efficacy against multidrug resistant pathogens.In particular, two new families of β-lactamase inhibitors devoid of β-lactam structure are emerging, namely Diazabicycooctanes (DBOs) and boronic acids (see Table 2).These novel β-lactamase inhibitors have been combined with cephalosporins and carbapenems and have shown activity against β-lactamase-producing Gram-negative bacteria, including KPC producers.Despite the progress made so far, activity against certain class A (ESBL, KPC), class D (OXA) and class B (NDM) β-lactamases remains a challenge.Overall β-lactam antibiotics will continue to play an important role in the treatment of difficult infections, particularly addressing multidrug resistant pathogens.It is important to note that after more than 70 years of clinical use, β-lactam antibiotics are still widely prescribed because of their efficacy and safety profile.
Medicinal chemistry research in this field is intense, particularly in the β-lactamase inhibitors and more molecules are expected to enter in the pipeline.

Figure 6 .
Figure 6.Reaction scheme for the improved synthesis of MK-7655.