Attaching Metal-Containing Moieties to ß-Lactam Antibiotics: The Case of Penicillin and Cephalosporin.

Procedures for the preparation of transition metal complexes having intact bicyclic cepham or penam systems as ligands have been developed. Starting from readily available 4-azido-2-azetidinones, a synthetic approach has been tuned using a copper-catalyzed azide-alkyne cycloaddition between 3-azido-2-azetinones and alkynes, followed by methylation and transmetalation to Au(I) and Ir(III) complexes from the mesoionic carbene Ag(I) complexes. This methodology was applied to 6-azido penam and 7-azido cepham derivatives to build 6-(1,2,3-triazolyl)penam and 7-(1,2,3-triazolyl)cepham proligands, which upon methylation and metalation with Au(I) and Ir(III) complexes yielded products derived from the coordination of the metal to the penam C6 and cepham C7 positions, preserving intact the bicyclic structure of the penicillin and cephalosporin scaffolds. The crystal structure of complex 28b, which has an Ir atom directly bonded to the intact penicillin bicycle, was determined by X-ray diffraction. This is the first structural report of a penicillin-transition-metal complex having the bicyclic system of these antibiotics intact. The selectivity of the coordination processes was interpreted using DFT calculations.

Synthesis and in vitro antibacterial activity of new aminothiazole-oximepiperidone cephalosporins.

Four new aminothiazole-oximepiperidone cephalosporins (10a-10d) were synthesized, with their in vitro antibacterial activities against hospital isolated Gram-negative bacteria assessed. The results showed that compounds 10a-10d effectively inhibit a variety of Gram-negative bacteria. Compound 10a was the most potent compound, with comparable activity as ceftazidime. The combination of compound 10a and Avibactam was very active against almost all bacteria tested, which including multidrug resistant K. pneumoniae and A. baumannii. Compared to Avycaz, this combination is more potent against ESBL producing K. pneumoniae. Thus, the combination of 10a and Avibactam is of interest for further studies.

New Methods for the Synthesis of Spirocyclic Cephalosporin Analogues.

Spiro compounds provide attractive targets in drug discovery due to their inherent three-dimensional structures, which enhance protein interactions, aid solubility and facilitate molecular modelling. However, synthetic methodology for the spiro-functionalisation of important classes of penicillin and cephalosporin ß-lactam antibiotics is comparatively limited. We report a novel method for the generation of spiro-cephalosporin compounds through a Michael-type addition to the dihydrothiazine ring. Coupling of a range of catechols is achieved under mildly basic conditions (K2CO3, DMF), giving the stereoselective formation of spiro-cephalosporins (d.r. 14:1 to 8:1) in moderate to good yields (28-65%).

Modified Penicillin Molecule with Carbapenem-Like Stereochemistry Specifically Inhibits Class C ß-Lactamases.

Bacterial ß-lactamases readily inactivate most penicillins and cephalosporins by hydrolyzing and "opening" their signature ß-lactam ring. In contrast, carbapenems resist hydrolysis by many serine-based class A, C, and D ß-lactamases due to their unique stereochemical features. To improve the resistance profile of penicillins, we synthesized a modified penicillin molecule, MPC-1, by "grafting" carbapenem-like stereochemistry onto the penicillin core. Chemical modifications include the trans conformation of hydrogen atoms at C-5 and C-6 instead of cis, and a 6-α hydroxyethyl moiety to replace the original 6-ß aminoacyl group. MPC-1 selectively inhibits class C ß-lactamases, such as P99, by forming a nonhydrolyzable acyl adduct, and its inhibitory potency is ∼2 to 5 times higher than that for clinically used ß-lactamase inhibitors clavulanate and sulbactam. The crystal structure of MPC-1 forming the acyl adduct with P99 reveals a novel binding mode for MPC-1 that resembles carbapenem bound in the active site of class A ß-lactamases. Furthermore, in this novel binding mode, the carboxyl group of MPC-1 blocks the deacylation reaction by occluding the critical catalytic water molecule and renders the acyl adduct nonhydrolyzable. Our results suggest that by incorporating carbapenem-like stereochemistry, the current collection of over 100 penicillins and cephalosporins can be modified into candidate compounds for development of novel ß-lactamase inhibitors.

Targeted synthesis of novel ß-lactam antibiotics by laccase-catalyzed reaction of aromatic substrates selected by pre-testing for their antimicrobial and cytotoxic activity.

The rapidly increasing problem of antimicrobial-drug resistance requires the development of new antimicrobial agents. The laccase-catalyzed amination of dihydroxy aromatics is a new and promising method to enlarge the range of currently available antibiotics. Thirty-eight potential 1,2- and 1,4-hydroquinoid laccase substrates were screened for their antibacterial and cytotoxic activity to select the best substrates for laccase-catalyzed coupling reaction resulting in potent antibacterial derivatives. As a result, methyl-1,4-hydroquinone and 2,3-dimethyl-1,4-hydroquinone were used as parent compounds and 14 novel cephalosporins, penicillins, and carbacephems were synthesized by amination with amino-ß-lactam structures. All purified products were stable in aqueous buffer and resistant to the action of ß-lactamases, and in agar diffusion and broth micro-dilution assays, they inhibited the growth of several Gram-positive bacterial strains including multidrug-resistant Staphylococcus aureus and Enterococci. Their in vivo activity and cytotoxicity in a Staphylococcus-infected, immune-suppressed mouse model are discussed.

Assay for drug discovery: Synthesis and testing of nitrocefin analogues for use as ß-lactamase substrates.

We report on the synthesis of three nitrocefin analogues and their evaluation as substrates for the detection of ß-lactamase activity. These compounds are hydrolyzed by all four Ambler classes of ß-lactamases. Kinetic parameters were determined with eight different ß-lactamases, including VIM-2, NDM-1, KPC-2, and SPM-1. The compounds do not inhibit the growth of clinically important antibiotic-resistant gram-negative bacteria in vitro. These chromogenic compounds have a distinct absorbance spectrum and turn purple when hydrolyzed by ß-lactamases. One of these compounds, UW154, is easier to synthesize from commercial starting materials than nitrocefin and should be significantly less expensive to produce.

[Enzymatic Synthesis of Acidic ß-Lactams (Review)].

The currently known methods of enzymatic ß-lactam synthesis, as well as the enzymes and heterogeneous biocatalysts used for this purpose, are presented, and the published reports on advances in the field of enzymatic synthesis of selected antibiotics belonging to the groups of acidic penicillins and acidic cephalosporins are summarized in the present review. The key conditions and parameters of biocatalytic processes, such as the biocatalyst form, concentration of the precursor compounds, solvent type, pH, temperature, etc. are analyzed and compared, and guidelines for further optimization of ß-lactam synthesis are given. The present review may be of use for a wide range of readers, as well as to enzymology and biotechnology experts.

Radiation induced decomposition of a refractory cefathiamidine intermediate.

Diisopropylthiourea (DPT), an intermediate of a widely used cephalosporin, has been found to be one of the most refractory components in cephalosporin synthesis wastewater. This compound cannot be completely removed by conventional biological processes due to its antimicrobial property. Ionizing radiation has been applied in the decomposition of refractory pollutants in recent years and has proved effective. Therefore, the decomposition of DPT by γ-irradiation was studied. The compound was irradiated at the dose of 150-2000 Gy before a change of concentration and UV absorption of the solutions was detected. Furthermore, the decomposition kinetics and radiation yield (G-value) of DPT was investigated. The results of radiation experiments on DPT-containing aqueous showed that the DPT can be effectively degraded by γ-radiation. DPT concentration decreased with increasing absorbed doses. G-values of radiolytic decomposition for DPT (20 mg/L) were 1.04 and 0.47 for absorbed doses of 150 and 2000 Gy, respectively. The initial concentration and pH of the solutions affected the degradation. As the concentration of substrate increased, the decomposition was reduced. The decrease of removal rate and radiation efficacy under alkaline condition suggested that lower pH values benefit the γ-induced degradation. UV absorption from 190 to 250 nm decreased after radiation while that from 250 to 300 nm increased, indicating the formation of by-products.

Fluorogenic probes with substitutions at the 2 and 7 positions of cephalosporin are highly BlaC-specific for rapid Mycobacterium tuberculosis detection.

Current methods for the detection of Mycobacterium tuberculosis (Mtb) are either time consuming or require expensive instruments and are thus are not suitable for point-of-care diagnosis. The design, synthesis, and evaluation of fluorogenic probes with high specificity for BlaC, a biomarker expressed by Mtb, are described. The fluorogenic probe CDG-3 is based on cephalosporin with substitutions at the 2 and 7 positions and it demonstrates over 120,000-fold selectivity for BlaC over TEM-1 Bla, the most common ß-lactamase. CDG-3 can detect 10 colony-forming units of the attenuated Mycobacterium bovis strain BCG in human sputum in the presence of high levels of contaminating ß-lactamases expressed by other clinically prevalent bacterial strains. In a trial with 50 clinical samples, CDG-3 detected tuberculosis with 90% sensitivity and 73% specificity relative to Mtb culture within one hour, thus demonstrating its potential as a low-cost point-of-care test for use in resource-limited areas.

Discovery of YFJ-36: Design, Synthesis, and Antibacterial Activities of Catechol-Conjugated ß-Lactams against Gram-Negative Bacteria.

Cefiderocol is the first approved catechol-conjugated cephalosporin against multidrug-resistant Gram-negative bacteria, while its application was limited by poor chemical stability associated with the pyrrolidinium linker, moderate potency against Klebsiella pneumoniae and Acinetobacter baumannii, intricate procedures for salt preparation, and potential hypersensitivity. To address these issues, a series of novel catechol-conjugated derivatives were designed, synthesized, and evaluated. Extensive structure-activity relationships and structure-metabolism relationships (SMR) were conducted, leading to the discovery of a promising compound 86b (Code no. YFJ-36) with a new thioether linker. 86b exhibited superior and broad-spectrum in vitro antibacterial activity, especially against A. baumannii and K. pneumoniae, compared with cefiderocol. Potent in vivo efficacy was observed in a murine systemic infection model. Furthermore, the physicochemical stability of 86b in fluid medium at pH 6-8 was enhanced. 86b also reduced potential the risk of allergy owing to the quaternary ammonium linker. The improved properties of 86b supported its further research and development.

Design, synthesis and antibacterial activities of a series of new 2-oxaisocephems.

A series of 2-oxaisocephems with a thio-substituted methyl group at the 3-position and a [2-(5-amino-1,2,4-thiadizol-3-yl)-2-(Z)-alkoxyimino]acetamido moiety at 7-position were synthesized and tested for their antibacterial activities. The analogs 17c and 17f have well-balanced potency and significantly enhanced activity as compared with the reference compound ceftazidime.

Synthesis and preliminary antimicrobial activities of new arylideneamino-1,3,4-thiadiazole-(thio/dithio)-acetamido cephalosporanic acids.

New derivatives of 7-aminocephalosporanic acid 1-8 were synthesized by acylation of the 7-amino group of the cephem nucleus with various arylidinimino-1,3,4-thiadiazole-thio(or dithio)-acetic acid intermediates 3a-d and 5a-d, respectively, so the acyl side chains of these new cephalosporins contained a sulfide or disulfide bond. This unique combination of a Schiff base with the sulfide or disulfide bonds in the acyl side chain afforded new cephalosporins of reasonable potencies, some of which were found to possess moderate activities against the tested microorganisms. Their chemical structures were characterized by ¹H-NMR, IR spectroscopy and elemental microanalysis. Preliminary in vitro antimicrobial activities of the prepared cephalosporins were investigated using a panel of selected microorganisms. Results indicated that the newly synthesized cephalosporins containing disulfide bonds (compounds 5-8) exhibited better activities against Staphylococcus aureus and Escherichia coli. The cephalosporins cross-linked by a sulfide bond (compounds 1-4) showed a slight change in antimicrobial activities when compared with that of the reference cephalosporin (cephalexin).

Novel Cephalosporin Conjugates Display Potent and Selective Inhibition of Imipenemase-Type Metallo-ß-Lactamases.

In an attempt to exploit the hydrolytic mechanism by which ß-lactamases degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based inhibitors of metallo-ß-lactamases (MBLs) in a spatiotemporally controlled fashion. While enzymatic hydrolysis of the ß-lactam ring was observed, it was not accompanied by inhibitor release. Nonetheless, the cephalosporin prodrugs, especially thiomandelic acid conjugate (8), demonstrated potent inhibition of IMP-type MBLs. In addition, conjugate 8 was also found to greatly reduce the minimum inhibitory concentration of meropenem against IMP-producing bacteria. The results of kinetic experiments indicate that these prodrugs inhibit IMP-type MBLs by acting as slowly turned-over substrates. Structure-activity relationship studies revealed that both phenyl and carboxyl moieties of 8 are crucial for its potency. Furthermore, modeling studies indicate that productive interactions of the thiomandelic acid moiety of 8 with Trp28 within the IMP active site may contribute to its potency and selectivity.

Characterization and study of the orientation of immobilized enzymes by tryptic digestion and HPLC-MS: design of an efficient catalyst for the synthesis of cephalosporins.

An innovative approach to determine the orientation of penicillin G acylase (PGA) from Escherichia coli covalently immobilized onto solid supports has been developed. This method is based on tryptic digestion of immobilized PGA followed by HPLC-MS analysis of the released peptides which are supposed to be only those exposed toward the reaction medium and not directly bound to the solid support. To this purpose, PGA was immobilized on Eupergit C (acrylic hydrophobic resin) and glyoxyl-agarose (hydrophilic resin) functionalized with epoxy and aldehyde groups, respectively, both involving the Lys residues of the protein. The peptide maps obtained were analyzed to derive the orientation of immobilized PGA, as the position of the detected Lys gave indication concerning the accessibility of the different areas of the protein. The results indicate that PGA immobilization on both supports involves mainly Lys located near the binding pocket (70%). Some differences in the enzyme orientation on the two supports can be deduced by the presence of different unbound Lys residues in the released peptides, specific to each support (Lys 117alpha for PGA-Eupergit C; Lys 163alpha and Lys 165alpha for PGA-glyoxyl-agarose). These results have been correlated with the data obtained in the kinetically controlled synthesis and indicate that the orientation of PGA on both supports is partially unfavorable, driving the active site near the support surface. This type of orientation of the enzyme enhances the effect of the nature of the support and of the binding chemistry on the catalytic properties. The information obtained indicated the most suitable support and activation strategy to design an immobilized acylase with good synthetic properties for preparative processes. The glyoxyl-Eupergit C support with enhanced porosity synergically combines the mechanical stability and synthetic performances of immobilized PGA and was successfully used in the synthesis of several cephalosporins.

Ferrier-Petasis rearrangement of 4-(vinyloxy)azetidin-2-ones: an entry to carbapenams and carbacephams.

Trimethylsilyl triflate promotes Ferrier-Petasis rearrangement of 4-(vinyloxy)-, 4-(propenyloxy)-, and 4-(isopropenyloxy)azetidin-2-ones to corresponding 4-(carbonylmethyl)azetidin-2-ones. The latter compounds may serve as attractive intermediates in the synthesis of carbapenem antibiotics. To illustrate the potential of this reaction, selected rearrangement products have been transformed into carbapenams.

Enzymatic deprotection of the cephalosporin 3'-acetoxy group using Candida antarctica lipase B.

Cephalosporins remain one of the most important classes of antibiotics. A useful site for derivatization involves generation of and chemistry at the 3'-hydroxymethyl position. While 3'-acetoxymethyl-substituted cephalosporins are readily available, deacetylation to access the free 3'-hydroxymethyl group is problematic when the carboxylic acid is protected as an ester. Herein we report that this important transformation has been efficiently accomplished using Candida antarctica lipase B. Although this transformation is difficult to carry out using chemical methods, the enzymatic deacetylation has been successful on gram scale, when the cephalosporin is protected as either the benzhydryl or tert-butyl esters and on the corresponding sulfoxide and sulfone of the tert-butyl ester.

Propenylamide and propenylsulfonamide cephalosporins as a novel class of anti-MRSA beta-lactams.

Novel C(3) propenylamide and propenylsulfonamide cephalosporins have been synthesized and tested for their ability to inhibit the penicillin-binding protein 2' (PBP2') from Staphylococcus epidermidis and the growth of a panel of clinically relevant bacterial species, including methicillin-resistant Staphylococcus aureus (MRSA). The most potent compounds inhibited the growth of MRSA strains with minimum inhibitory concentrations (MIC) as low as 1 microg/mL. The structure-activity relationship revealed the potential for further optimization of this new cephalosporin class.

An Overview of the Activities of Cefiderocol Against Sensitive and Multidrug- Resistant (MDR) Bacteria.

The need for new therapeutics and drug delivery systems has become necessary owing to the public health concern associated with the emergence of multidrug-resistant microorganisms. Among the newly discovered therapeutic agents is cefiderocol, which was discovered by Shionogi Company, Japan as an injectable siderophore cephalosporin. Just like the other ß-lactam antibiotics, cefiderocol exhibits antibacterial activity via cell wall synthesis inhibition, especially in Gram negative bacteria (GNB); it binds to the penicillin-binding proteins, but its unique attribute is that it crosses the periplasmic space of bacteria owing to its siderophore-like attribute; it also resists the activity of ß-lactamases. Among all the synthesized compounds with the modified C-7 side chain, cefiderocol (3) presented the best and well-balanced activity against multi-drug resistant (MDR) Gram negative bacteria, including those that are resistant to carbapenem. In this article, an overview of the recent studies on cefiderocol was presented.

Approaches to the simultaneous inactivation of metallo- and serine-beta-lactamases.

A series of cephalosporin-derived reverse hydroxamates and oximes were prepared and evaluated as inhibitors of representative metallo- and serine-beta-lactamases. The reverse hydroxamates showed submicromolar inhibition of the GIM-1 metallo-beta-lactamase. With respect to interactions with the classes A, C, and D serine beta-lactamases, as judged by their correspondingly low K(m) values, the reverse hydroxamates were recognized in a manner similar to the non-hydroxylated N-H amide side chains of the natural substrates of these enzymes. This indicates that, with respect to recognition in the active site of the serine beta-lactamases, the OC-NR-OH functionality can function as a structural isostere of the OC-NR-H group, with the N-O-H group presumably replacing the amide N-H group as a hydrogen bond donor to the appropriate backbone carbonyl oxygen of the protein. The reverse hydroxamates, however, displayed k(cat) values up to three orders of magnitude lower than the natural substrates, thus indicating substantial slowing of the hydrolytic action of these serine beta-lactamases. Although the degree of inactivation is not yet enough to be clinically useful, these initial results are promising. The substitution of the amide N-H bond by N-OH may represent a useful strategy for the inhibition of other serine hydrolases.

Design and synthesis of a beta-lactamase activated 5-fluorouracil prodrug.

An efficient synthesis of a 5-fluorouracil-cephalosporin prodrug is described for use against colorectal and other cancers in antibody and gene-directed therapies. The compound shows stability in aqueous media until specifically activated by beta-lactamase (betaL). The kinetic parameters of the 5-fluorouracil-cephalosporin conjugate were determined in the presence of Enterobacter cloacae P99 betaL (ECl betaL) revealing a K(m)=95.4 microM and V(max)=3.21 microMol min(-1) mg(-1). The data compare favorably to related systems that have been reported and enable testing of this prodrug against cancer cell lines in vitro and in vivo.