Eravacycline (TP-434 or 7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline) is a novel fluorocycline that was evaluated for antimicrobial activity against panels of recently isolated aerobic and anaerobic Gram-negative and Gram-positive bacteria. Eravacycline showed potent broad-spectrum activity against 90% of the isolates (MIC 90 ) in each panel at concentrations ranging from <0.008 to 2 g/ml for all species panels except those of Pseudomonas aeruginosa and Burkholderia cenocepacia (MIC 90 values of 32 g/ml for both organisms). The antibacterial activity of eravacycline was minimally affected by expression of tetracycline-specific efflux and ribosomal protection mechanisms in clinical isolates. Furthermore, eravacycline was active against multidrug-resistant bacteria, including those expressing extended-spectrum ␤-lactamases and mechanisms conferring resistance to other classes of antibiotics, including carbapenem resistance. Eravacycline has the potential to be a promising new intravenous (i.v.)/oral antibiotic for the empirical treatment of complicated hospital/health care infections and moderate-to-severe community-acquired infections.

Macrolide resistance mechanisms can be target-based with a change in a 23S ribosomal RNA (rRNA) residue or a mutation in ribosomal protein L4 or L22 affecting the ribosome's interaction with the antibiotic. Alternatively, mono-or dimethylation of A2058 in domain V of the 23S rRNA by an acquired rRNA methyltransferase, the product of an erm (erythromycin ribosome methylation) gene, can interfere with antibiotic binding. Acquired genes encoding efflux pumps, most predominantly mef(A) þ msr(D) in pneumococci/streptococci and msr(A/B) in staphylococci, also mediate resistance. Drug-inactivating mechanisms include phosphorylation of the 2 0 -hydroxyl of the amino sugar found at position C5 by phosphotransferases and hydrolysis of the macrocyclic lactone by esterases. These acquired genes are regulated by either translation or transcription attenuation, largely because cells are less fit when these genes, especially the rRNA methyltransferases, are highly induced or constitutively expressed. The induction of gene expression is cleverly tied to the mechanism of action of macrolides, relying on antibiotic-bound ribosomes stalled at specific sequences of nascent polypeptides to promote transcription or translation of downstream sequences. Macrolide antibiotics are polyketides composed of a 14-, 15-, or 16-membered macrocyclic lactone ring (14-, 15-, and 16-membered) to which several sugars and/or side chains have been attached by the producing organism or as modifications during semisynthesis in the laboratory (Figs. 1 and 2). Newer semisynthetic derivations, like ketolides telithromycin, and solithromycin, have a C3-keto group in place of the C3 cladinose (akin to naturally occurring pikromycin) (Brockmann and Henkel 1950) and an 11,12-cyclic carbamate with an extended alkyl -aryl side chain that increases the affinity of the antibiotic for the ribosome by 10-to 100-fold (Hansen et al. 1999;Dunkle et al. 2010); in the case of solithromycin, a fluorine substituent at C2 provides an additional ribosomal interaction (Llano-Sotelo et al. 2010). Macrolides continue to be important in the therapeutic treatment of community-acquired pneumonia (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and atypicals Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae), sexually transmitted diseases (Neiserria gonorhoeae, Chlamydia trachomatis, Mycoplasma genitalium), shigellosis, and salmonellosis. With solithromycin heading for a new drug application (NDA) filing in 2016 and having the in vitro potency to treat erythromycin-resistant

c TP-434 is a novel, broad-spectrum fluorocycline antibiotic with activity against bacteria expressing major antibiotic resistance mechanisms, including tetracycline-specific efflux and ribosomal protection. The mechanism of action of TP-434 was assessed using both cell-based and in vitro assays. In Escherichia coli cells expressing recombinant tetracycline resistance genes, the MIC of TP-434 (0.063 g/ml) was unaffected by tet(M), tet(K), and tet(B) and increased to 0.25 and 4 g/ml in the presence of tet(A) and tet(X), respectively. Tetracycline, in contrast, was significantly less potent (MIC > 128 g/ml) against E. coli cells when any of these resistance mechanisms were present. TP-434 showed potent inhibition in E. coli in vitro transcription/translation (50% inhibitory concentration [IC 50 ] ‫؍‬ 0.29 ؎ 0.09 g/ml) and [ 3 H]tetracycline ribosome-binding competition (IC 50 ‫؍‬ 0.22 ؎ 0.07 M) assays. The antibacterial potencies of TP-434 and all other tetracycline class antibiotics tested were reduced by 4-to 16-fold, compared to that of the wild-type control strain, against Propionibacterium acnes strains carrying a 16S rRNA mutation, G1058C, a modification that changes the conformation of the primary binding site of tetracycline in the ribosome. Taken together, the findings support the idea that TP-434, like other tetracyclines, binds the ribosome and inhibits protein synthesis and that this activity is largely unaffected by the common tetracycline resistance mechanisms.

This and the accompanying report (DOI: 10.1021/jm201467r ) describe the design, synthesis, and evaluation of a new generation of tetracycline antibacterial agents, 7-fluoro-9-substituted-6-demethyl-6-deoxytetracyclines ("fluorocyclines"), accessible through a recently developed total synthesis approach. These fluorocyclines possess potent antibacterial activities against multidrug resistant (MDR) Gram-positive and Gram-negative pathogens. One of the fluorocyclines, 7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline (17j, also known as TP-434, 50th Interscience Conference on Antimicrobial Agents and Chemotherapy Conference , Boston, MA , September 12-15, 2010 , poster F1 - 2157 ), is currently undergoing phase 2 clinical trials in patients with complicated intra-abdominal infections (cIAI).

Eravacycline is a novel, fully synthetic fluorocycline antibiotic developed for the treatment of serious infections, including those caused by multidrug-resistant (MDR) pathogens. Here, we evaluated the in vitro activities of eravacycline and comparator antimicrobial agents against a global collection of frequently encountered clinical isolates of Gram-negative bacilli. The CLSI broth microdilution method was used to determine MIC data for isolates of Enterobacterales (n = 13,983), Acinetobacter baumannii (n = 2,097), Pseudomonas aeruginosa (n = 1,647), and Stenotrophomonas maltophilia (n = 1,210) isolated primarily from respiratory, intra-abdominal, and urinary specimens by clinical laboratories in 36 countries from 2013 to 2017. Susceptibilities were interpreted using both CLSI and EUCAST breakpoints. Multidrug-resistant (MDR) isolates were defined by resistance to agents from ≥3 different antimicrobial classes. The MIC90s ranged from 0.25 to 1 μg/ml for Enterobacteriaceae and were 1 μg/ml for A. baumannii and 2 μg/ml for S. maltophilia, Proteus mirabilis, and Serratia marcescens. Eravacycline’s potency was up to 4-fold greater than that of tigecycline against genera/species of Enterobacterales, A. baumannii, and S. maltophilia. The MIC90s for five of six individual genera/species of Enterobacterales and A. baumannii were within 2-fold of the MIC90s for their respective subsets of MDR isolates, while the MDR subpopulation of Klebsiella spp. demonstrated 4-fold higher MIC90s. Eravacycline demonstrated potent in vitro activity against the majority of clinical isolates of Gram-negative bacilli, including MDR isolates, collected over a 5-year period. This study further underscores the potential benefit of eravacycline in the treatment of infections caused by MDR Gram-negative pathogens.

Utilizing a fully synthetic route to tetracycline analogues, the C-9 side-chain of the fluorocyclines was optimized for both antibacterial activity and oral efficacy. Compounds were identified that overcome both efflux (tet(K), tet(A)) and ribosomal protection (tet(M)) tetracycline-resistance mechanisms and are active against Gram-positive and Gram-negative organisms. A murine systemic infection model was used as an oral efficacy screen to rapidly identify compounds with oral bioavailability. Two compounds were identified that exhibit both oral bioavailability in rat and clinically relevant bacterial susceptibility profiles against major respiratory pathogens. One compound demonstrated oral efficacy in rodent lung infection models that was comparable to marketed antibacterial agents.

TP-271 is a novel, fully synthetic fluorocycline antibiotic in clinical development for the treatment of respiratory infections caused by susceptible and multidrug-resistant pathogens. TP-271 was active in MIC assays against key community respiratory Gram-positive and Gram-negative pathogens, including Streptococcus pneumoniae (MIC 90 ϭ 0.03 g/ml), methicillin-sensitive Staphylococcus aureus (MSSA; MIC 90 ϭ 0.25 g/ml), methicillin-resistant S. aureus (MRSA; MIC 90 ϭ 0.12 g/ml), Streptococcus pyogenes (MIC 90 ϭ 0.03 g/ml), Haemophilus influenzae (MIC 90 ϭ 0.12 g/ml), and Moraxella catarrhalis (MIC 90 Յ0.016 g/ml). TP-271 showed activity (MIC 90 ϭ 0.12 g/ml) against community-acquired MRSA expressing PantonValentine leukocidin (PVL). MIC 90 values against Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydia pneumoniae were 0.004, 1, and 4 g/ml, respectively. TP-271 was efficacious in neutropenic and immunocompetent animal pneumonia models, generally showing, compared to the burden at the start of dosing,~2 to 5 log 10 CFU reductions against MRSA, S. pneumoniae, and H. influenzae infections when given intravenously (i.v.) and~1 to 4 log 10 CFU reductions when given orally (p.o.). TP-271 was potent against key community-acquired bacterial pneumonia (CABP) pathogens and was minimally affected, or unaffected, by tetracycline-specific resistance mechanisms and fluoroquinolone or macrolide drug resistance phenotypes. IMPORTANCE Rising resistance rates for macrolides, fluoroquinolones, and ␤-lactams in the most common pathogens associated with community-acquired bacterial pneumonia (CABP) are of concern, especially for cases of moderate to severe infections in vulnerable populations such as the very young and the elderly. New antibiotics that are active against multidrug-resistant Streptococcus pneumoniae and Staphylococcus aureus are needed for use in the empirical treatment of the most severe forms of this disease. TP-271 is a promising new fluorocycline antibiotic demonstrating in vitro potency and nonclinical efficacy by intravenous and oral administration against the major pathogens associated with moderate to severe CABP.KEYWORDS TP-271, community-acquired bacterial pneumonia, fluorocycline C ommunity-acquired bacterial pneumonia (CABP) is a serious condition associated with mortality rates estimated to be as high as 12 to 14% for hospitalized individuals and 25 to 40% for those admitted to intensive care units (1-3). Lower respiratory tract infections were the second greatest cause of deaths and years of life lost in 2013 as reported by The Global Burden of Disease Study, with the highest incidence occurring in children Ͻ5 years and adults Ͼ65 years (1, 4). The Centers for Disease

A novel series of fully synthetic 8-azatetracyclines was prepared and evaluated for antibacterial activity. Compounds were identified that overcome both efflux (tet(K)) and ribosomal protection (tet(M)) tetracycline resistance mechanisms and are active against Gram-positive and Gram-negative organisms. Two compounds were identified that exhibit comparable efficacy to marketed tetracyclines in in vivo models of bacterial infection.