In vitro studies evaluating the activity of imipenem in combination with relebactam against Pseudomonas aeruginosa.

BACKGROUND: The prevalence of antibiotic resistance is increasing, and multidrug-resistant Pseudomonas aeruginosa has been identified as a serious threat to human health. The production of ß-lactamase is a key mechanism contributing to imipenem resistance in P. aeruginosa. Relebactam is a novel ß-lactamase inhibitor, active against class A and C ß-lactamases, that has been shown to restore imipenem susceptibility. In a series of studies, we assessed the interaction of relebactam with key mechanisms involved in carbapenem resistance in P. aeruginosa and to what extent relebactam might overcome imipenem non-susceptibility. RESULTS: Relebactam demonstrated no intrinsic antibacterial activity against P. aeruginosa, had no inoculum effect, and was not subject to efflux. Enzymology studies showed relebactam is a potent (overall inhibition constant: 27 nM), practically irreversible inhibitor of P. aeruginosa AmpC. Among P. aeruginosa clinical isolates from the SMART global surveillance program (2009, n = 993; 2011, n = 1702; 2015, n = 5953; 2016, n = 6165), imipenem susceptibility rates were 68.4% in 2009, 67.4% in 2011, 70.4% in 2015, and 67.3% in 2016. With the addition of 4 µg/mL relebactam, imipenem susceptibility rates increased to 87.6, 86.0, 91.7, and 89.8%, respectively. When all imipenem-non-susceptible isolates were pooled, the addition of 4 µg/mL relebactam reduced the mode imipenem minimum inhibitory concentration (MIC) 8-fold (from 16 µg/mL to 2 µg/mL) among all imipenem-non-susceptible isolates. Of 3747 imipenem-non-susceptible isolates that underwent molecular profiling, 1200 (32%) remained non-susceptible to the combination imipenem/relebactam (IMI/REL); 42% of these encoded class B metallo-ß-lactamases, 11% encoded a class A GES enzyme, and no class D enzymes were detected. No relationship was observed between alleles of the chromosomally-encoded P. aeruginosa AmpC and IMI/REL MIC. CONCLUSIONS: IMI/REL exhibited potential in the treatment of carbapenem-resistant P. aeruginosa infections, with the exception of isolates encoding class B, some GES alleles, and class D carbapenemases.

Discovery of MK-7655, a ß-lactamase inhibitor for combination with Primaxin®.

ß-Lactamase inhibitors with a bicyclic urea core and a variety of heterocyclic side chains were prepared and evaluated as potential partners for combination with imipenem to overcome class A and C ß-lactamase mediated antibiotic resistance. The piperidine analog 3 (MK-7655) inhibited both class A and C ß-lactamases in vitro. It effectively restored imipenem's activity against imipenem-resistant Pseudomonas and Klebsiella strains at clinically achievable concentrations. A combination of MK-7655 and Primaxin® is currently in phase II clinical trials for the treatment of Gram-negative bacterial infections.

SAR studies on the central phenyl ring of substituted biphenyl oxazolidinone-potent CETP inhibitors.

SAR studies of the substitution effect on the central phenyl ring of the biphenyl scaffold were carried out using anacetrapib (9a) as the benchmark. The results revealed that the new analogs with substitutions to replace trifluoromethyl (9a) had a significant impact on CETP inhibition in vitro. In fact, analogs with some small groups were as potent or more potent than the CF(3) derivative for CETP inhibition. Five of these new analogs raised HDL-C significantly (>20mg/dL). None of them however was better than anacetrapib in vivo. The synthesis and biological evaluation of these CETP inhibitors are described.

Thiophenyl oxime-derived phosphonates as nano-molar class C beta-lactamase inhibitors reducing MIC of imipenem against Pseudomonas aeruginosa and Acinetobacter baumannii.

The preparation and characterization of a series of thiophenyl oxime phosphonate beta-lactamase inhibitors is described. A number of these analogs were potent and selective inhibitors of class C beta-lactamases from Pseudomonas aeruginosa and Enterobacter cloacae. Compounds 3b and 7 reduced the MIC of imipenem against an AmpC expressing strain of imipenem-resistant P. aeruginosa. A number of the title compounds retained micromolar potency against the class D OXA-40 beta-lactamase from Acinetobacter baumannii and at high concentrations compound 3b was shown to reduce the MIC of imipenem against a highly imipenem-resistant strain of A. baumanii expressing the OXA-40 beta-lactamase. In mice compound 3b exhibited phamacokinetics similar to imipenem.

Side chain SAR of bicyclic ß-lactamase inhibitors (BLIs). 2. N-Alkylated and open chain analogs of MK-8712.

The bridged monobactam ß-lactamase inhibitor MK-8712 (1) effectively inhibits class C ß-lactamases. Side chain N-alkylated and ring-opened analogs of 1 were prepared and evaluated for combination with imipenem to overcome class C ß-lactamase mediated resistance. Although some analogs were more potent inhibitors of AmpC, none exhibited better synergy with imipenem than 1.

2-(4-carbonylphenyl)benzoxazole inhibitors of CETP: scaffold design and advancement in HDLc-raising efficacy.

The development of 2-phenylbenzoxazoles as inhibitors of cholesteryl ester transfer protein (CETP) is described. Initial efforts aimed at engineering replacements for the aniline substructures in the benchmark molecule. Reversing the connectivity of the central aniline lead to a new class of 2-(4-carbonylphenyl)benzoxazoles. Structure-activity studies at the C-7 and terminal pyridine ring allowed for the optimization of potency and HDLc-raising efficacy in this new class of inhibitors. These efforts lead to the discovery of benzoxazole 11v, which raised HDLc by 24 mg/dl in our transgenic mouse PD model.

2-Arylbenzoxazoles as CETP inhibitors: raising HDL-C in cynoCETP transgenic mice.

We describe structure-activity studies leading to the discovery of 2-arylbenzoxazole 3, the first in a series to raise serum high-density lipoprotein cholesterol levels in transgenic mice. Replacement of the 4-piperidinyloxy moiety with piperazinyl provided a more synthetically tractable lead, which upon optimization resulted in compound 4, an excellent inhibitor of cholesteryl ester transfer protein function with good pharmacokinetic properties and in vivo efficacy.

2-Arylbenzoxazoles as CETP inhibitors: Substitution of the benzoxazole moiety.

A series of 2-arylbenzoxazole inhibitors of the cholesterol ester transfer protein (CETP) is described. Structure-activity studies focused on variation of the substitution of the benzoxazole moiety. Substitution at the 5- and 7-positions of the benzoxazole moiety was found to be beneficial for CETP inhibition. Compound 47 was found to be the most potent inhibitor in this series and inhibited CETP with an IC(50) of 28nM.

Discovery of pyrazolyl propionyl cyclohexenamide derivatives as full agonists for the high affinity niacin receptor GPR109A.

A series of pyrazolyl propionyl cyclohexenamides were discovered as full agonists for the high affinity niacin receptor GPR109A. The structure-activity relationship (SAR) studies were aimed to improve activity on GPR109A, reduce Cytochrome P450 2C8 (CYP2C8) and Cytochrome P450 2C9 (CYP2C9) inhibition, reduce serum shift and improve pharmacokinetic (PK) profiles.

2-Arylbenzoxazoles as CETP inhibitors: substitution and modification of the alpha-alkoxyamide moiety.

The development of a series of 2-arylbenzoxazole alpha-alkoxyamide and beta-alkoxyamine inhibitors of cholesteryl ester transfer protein (CETP) is described. Highly fluorinated alpha-alkoxyamides proved to be potent inhibitors of CETP in vitro, and the highly fluorinated 2-arylbenzoxazole beta-alkoxyamine 4 showed a desirable combination of in vitro potency (IC(50)=151 nM) and oral bioavailability in the mouse.

Design of a novel class of biphenyl CETP inhibitors.

A new class of CETP inhibitors was designed and prepared. These compounds are potent both in vitro and in vivo. The most active compound (12d) has shown an ability to raise HDL significantly in transgenic mouse PD model.

Side chain SAR of bicyclic beta-lactamase inhibitors (BLIs). 1. Discovery of a class C BLI for combination with imipinem.

Bridged monobactam beta-lactamase inhibitors were prepared and evaluated as potential partners for combination with imipenem to overcome class C beta-lactamase mediated resistance. The (S)-azepine analog 2 was found to be effective in both in vitro and in vivo assays and was selected for preclinical development.

Synthesis and biological evaluation of platensimycin analogs.

Platensimycin (1) displays antibacterial activity due to its inhibition of the elongation condensing enzyme (FabF), a novel mode of action that could potentially lead to a breakthrough in developing a new generation of antibiotics. The medicinal chemistry efforts were focused on the modification of the enone moiety of platensimycin and several analogs showed significant activity against FabF and possess antibacterial activity.

Agonist-like SERM effects on ERalpha-mediated repression of MMP1 promoter activity predict in vivo effects on bone and uterus.

Estradiol receptors (ER), ERalpha and ERbeta, are ligand-dependent transcription factors that regulate gene expression. Human and murine genetics suggest that ERalpha is the key target for estradiol action on bone, uterus and breast. To date, the molecular mode of action of estradiol and selective estradiol receptor modulators (SERMs) on bone is not fully understood. This is exemplified by a lack of in vitro assays that reliably predict SERM agonist activities in vivo. We hypothesized that ligand-dependent ERalpha transrepression, via protein-protein interactions at AP1, may predict estrogenic effects on bone. We modeled this using the MMP1 promoter, which encodes an AP1 binding site. We show that ICI-182780, raloxifene, 4-hydroxytamoxifen and estradiol all exhibit differential agonistic activities on the MMP1 promoter by suppressing activity by 20-80%. Transrepression efficacy and potency correlated with both uterotrophic (R(2)=0.98) and osteoprotective (R(2)=0.80) potential in the ovariectomized rat. This identifies MMP1 promoter transrepression as an agonist activity commonly shared by AF2 agonists and "antagonists" alike. Mutation analysis showed that the repression by estradiol and SERMs required correct amino acid sequences in the AF-2 domain. For instance, L540Q AF2 mutation did not alter responses to raloxifene, although it greatly increased responses to ICI-182780 (threefold) and reduced estradiol's effect by 20%. Furthermore, all tested ligands repressed the MMP1 promoter through the L540Q mutant with identical efficacy. Together, these data suggest that estradiol and SERMs share common agonist transcriptional activity via protein-protein interactions at AP1.

Discovery of pyrazolopyrimidines as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A.

Pyrazolopyrimidines were discovered as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A. In addition to its intrinsic activity, compound 9n significantly enhances nicotinic acid binding to the receptor, thereby potentiating the functional efficacy of nicotinic acid.

Tetrahydro anthranilic acid as a surrogate for anthranilic acid: application to the discovery of potent niacin receptor agonists.

The design, synthesis, and biological activity of a series of cycloalkene acid-based niacin receptor agonists are described. This led to the discovery that tetrahydro anthranilic acid is an excellent surrogate for anthranilic acid. Several compounds were identified that were potent against the niacin receptor, had enhanced cytochrome P450 selectivity against subtypes CYP2C8 and CYP2C9, and improved oral exposure in mice.

Discovery of betamethasone 17alpha-carbamates as dissociated glucocorticoid receptor modulators in the rat.

A series of betamethasone 17alpha-carbamates were designed, synthesized, and evaluated for their ability to dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepression, in rat cell lines. A number of alkyl substituted betamethasone 17alpha-carbamates were identified with excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC(50) 5.1 nM) and indicated dissociated profiles in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase, GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these compounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose, insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocorticoid receptor modulators can be identified in rodents.

Discovery of biaryl anthranilides as full agonists for the high affinity niacin receptor.

Biaryl anthranilides are reported as potent and selective full agonists for the high affinity niacin receptor GPR109A. The SAR presented outlines approaches to reduce serum shift and both CYPCYP2C8 and CYP2C9 liabilities, while improving PK and maintaining excellent receptor activity. Compound 2i exhibited good in vivo antilipolytic efficacy while providing a significantly improved therapeutic index over vasodilation (flushing) with respect to niacin in the mouse model.

Discovery of orally bioavailable and novel urea agonists of the high affinity niacin receptor GPR109A.

A urea class of high affinity niacin receptor agonists was discovered. Compound 1a displayed good PK, better in vivo efficacy in reducing FFA in mouse than niacin, and no vasodilation in a mouse model. Compound 1q demonstrated equal affinity to GPR109A as niacin.

Antagonist-induced, activation function-2-independent estrogen receptor alpha phosphorylation.

Estrogen receptor alpha (ERalpha) serine 118 (Ser118) phosphorylation modulates activation function-1 (AF1) function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cyclin-dependent kinase-7 to catalyze this event. In this study we show robust cyclin-dependent kinase-7-independent, AF2 antagonist-induced Ser118 phosphorylation. Estradiol (E2) and ICI-182,780 (ICI-780) induce Ser118 phosphorylation of wild-type ERalpha and either of two helix 12 mutants, suggesting AF2-independent action, probably via shedding of 90-kDa heat shock protein. With E2 treatment, the predominantly nuclear, phosphorylated ERalpha in COS-1 cells is detergent soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118-phosphorylated ERalpha is aggregated over the nucleus or in the cytoplasm, fractionating with the cell debris and making detection in cleared lysates improbable. Selective ER modulators (SERMs) elicit a mixed response with phosphorylated ERalpha in both detergent-soluble and -insoluble compartments. Apparent ligand-induced loss of ERalpha protein from cleared lysates is thus due to ligand-induced redistribution into the pellet, not degradation. The COS-1 response to ICI-780 can be mimicked in MCF-7 cells treated with a proteasome inhibitor to block authentic ligand-induced degradation. With SERMs and antagonists, the magnitude of Ser118-phosphorylated receptor redistribution into the insoluble fraction of COS-1 cells correlates with the magnitude of authentic ERalpha degradation in MCF-7 cells. A strong inverse correlation with ligand-induced uterotropism in vivo (P < 0.0001) and direct correlation with AF2-independent transrepression of the matrix metalloprotease-1 promoter in endometrial cells in vitro are seen. These data suggest that ligand-induced Ser118 phosphorylation of ERalpha can be AF2 independent. Furthermore, they identify translocation of Ser118-phosphorylated ERalpha out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.