Antigiardial activity of novel guanidine compounds

From four focused compound libraries based on the known anticoccidial agent robenidine, 44 compounds total were synthesised and screened for antigiardial activity. All active compounds were counter-screened for antibiotic and cytotoxic action. Of the analogues examined, 21 displayed IC50<5 μM, seven with IC50<1.0 μM. Most active were 2,2′-bis{[4-(trifluoromethoxy)phenyl]methylene}carbonimidic dihydrazide hydrochloride (30), 2,2′-bis{[4-(trifluoromethylsulfanyl)phenyl]methylene}carbonimidic dihydrazide hydrochloride (32), and 2,2′-bis[(2-bromo-4,5-dimethoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (41) with IC50=0.2 μM. The maximal observed activity was a 5 h IC50 value of 0.2 μM for 41. The clinically used metronidazole was inactive at this timepoint at a concentration of 25 μM. Robenidine off-target effects at bacteria and cell line toxicity were removed. Analogue 41 was well tolerated in mice treated orally (100 mg/kg). Following 5 h treatment with 41, no Giardia regrowth was noted after 48 h

Discovery of 4,6-bis(2-((E)-benzylidene)hydrazinyl)pyrimidin-2-Amine with antibiotic activity

Robenidine (E)-N'-((E)-1-(4-chlorophenyl)ethylidene)-2-(1-(4-chlorophenyl)ethylidene)hydrazine-1-carboximidhydrazide displays methicillin-resistant Staphyoccoccus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) MICs of 2 μg mL-1. Herein we describe the structure-activity relationship development of a novel series of guanidine to 2-aminopyrimidine isosteres that ameliorate the low levels of mammalian cytotoxicity in the lead compound while retaining good antibiotic activity. Removal of the 2-NH2 pyrimidine moiety renders these analogues inactive. Introduction of a central 2-NH2 triazine moiety saw a 10-fold activity reduction. Phenyl to cyclohexyl isosteres were inactive. The 4-BrPh and 4-CH3Ph with MIC values of 2 and 4 μg mL-1, against MRSA and VRE respectively, are promising candidates for future development.Cecilia C. Russe, Andrew Stevens, Kelly A. Young, Jennifer R. Baker ... Manouchehr Khazandi ... Abiodun Ogunniyi ... et al

In Vitro Activity of Robenidine Analogues NCL259 and NCL265 against Gram-Negative Pathogens

Published: 23 September 2022Multidrug-resistant (MDR) Gram-negative pathogens, especially Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Enterobacter spp., are recognized by the World Health Organization as the most critical priority pathogens in urgent need of drug development. In this study, the in vitro antimicrobial activity of robenidine analogues NCL259 and NCL265 was tested against key human and animal Gram-negative clinical isolates and reference strains. NCL259 and NCL265 demonstrated moderate antimicrobial activity against these Gram-negative priority pathogens with NCL265 consistently more active, achieving lower minimum inhibitory concentrations (MICs) in the range of 2–16 µg/mL. When used in combination with sub-inhibitory concentrations of polymyxin B to permeabilize the outer membrane, NCL259 and NCL265 elicited a synergistic or additive activity against the reference strains tested, reducing the MIC of NCL259 by 8- to 256- fold and the MIC of NCL265 by 4- to 256- fold. A small minority of Klebsiella spp. isolates (three) were resistant to both NCL259 and NCL265 with MICs &gt; 256 µg/mL. This resistance was completely reversed in the presence of the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PAβN) to yield MIC values of 8–16 µg/mL and 2–4 µg/mL for NCL259 and NCL256, respectively. When NCL259 and NCL265 were tested against wild-type E. coli isolate BW 25113 and its isogenic multidrug efflux pump subunit AcrB deletion mutant (∆AcrB), the MIC of both compounds against the mutant ∆AcrB isolate was reduced 16-fold compared to the wild-type parent, indicating a significant role for the AcrAB-TolC efflux pump from Enterobacterales in imparting resistance to these robenidine analogues. In vitro cytotoxicity testing revealed that NCL259 and NCL265 had much higher levels of toxicity to a range of human cell lines compared to the parent robenidine, thus precluding their further development as novel antibiotics against Gram-negative pathogens.Hongfei Pi, Henrietta Venter, Cecilia C. Russell, Kelly A. Young, Adam McCluskey, Stephen W. Page, Abiodun D. Ogunniyi and Darren J. Trot

Some Applications of the Extended Bendixson-Dulac Theorem

During the last years the authors have studied the number of limit cycles of several families of planar vector fields. The common tool has been the use of an extended version of the celebrated Bendixson-Dulac Theorem. The aim of this work is to present an unified approach of some of these results, together with their corresponding proofs. We also provide several applications.Comment: 19 pages, 3 figure

In vitro activity of robenidine Analog NCL195 in combination with outer membrane permeabilizers against gram-negative bacterial pathogens and impact on systemic gram-positive bacterial infection in mice

Multidrug-resistant (MDR) pathogens, particularly the ESKAPE group (Enterococcus faecalis/faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Enterobacter spp.), have become a public health threat worldwide. Development of new antimicrobial classes and the use of drugs in combination are potential strategies to treat MDR ESKAPE pathogen infections and promote optimal antimicrobial stewardship. Here, the in vitro antimicrobial activity of robenidine analog NCL195 alone or in combination with different concentrations of three outer membrane permeabilizers [ethylenediaminetetraacetic acid (EDTA), polymyxin B nonapeptide (PMBN), and polymyxin B (PMB)] was further evaluated against clinical isolates and reference strains of key Gram-negative bacteria. NCL195 alone was bactericidal against Neisseria meningitidis and Neisseria gonorrhoeae (MIC/MBC = 32 μg/mL) and demonstrated synergistic activity against P. aeruginosa, E. coli, K. pneumoniae, and Enterobacter spp. strains in the presence of subinhibitory concentrations of EDTA, PMBN, or PMB. The additive and/or synergistic effects of NCL195 in combination with EDTA, PMBN, or PMB are promising developments for a new chemical class scaffold to treat Gram-negative infections. Tokuyasu cryo ultramicrotomy was used to visualize the effect of NCL195 on bioluminescent S. aureus membrane morphology. Additionally, NCL195's favorable pharmacokinetic and pharmacodynamic profile was further explored in in vivo safety studies in mice and preliminary efficacy studies against Gram-positive bacteria. Mice administered two doses of NCL195 (50 mg/kg) by the intraperitoneal (IP) route 4 h apart showed no adverse clinical effects and no observable histological effects in major organs. In bioluminescent Streptococcus pneumoniae and S. aureus murine sepsis challenge models, mice that received two 50 mg/kg doses of NCL195 4 or 6 h apart exhibited significantly reduced bacterial loads and longer survival times than untreated mice. However, further medicinal chemistry and pharmaceutical development to improve potency, solubility, and selectivity is required before efficacy testing in Gram-negative infection models.Hongfei Pi, Hang Thi Nguyen, Henrietta Venter, Alexandra R. Boileau, Lucy Woolford, Sanjay Garg ... et al

Gram-positive and gram-negative antibiotic activity of asymmetric and monomeric robenidine analogues

Desymmetrisation of robenidine 1, N',2-bis((E)-4-chlorobenzylidene)hydrazine-1-carboximidhydrazide, and the introduction of imine alkyl substituents gave good antibiotic activity. Of note was the increased potency of 17 and 20 against VRE with 20 the most active, MIC of 0.5 μg mL-1. Analogues 2, 14, 17, 19 and 20 were equipotent or more potent than the lead 1. Introduction of an indole moiety, 30, resulted in the most MRSA active robenidine analogue, MIC of 1.0 μg mL-1. Imine C=NH isosteres (C=O / C=S) were inactive. Monomeric analogues, 33-35 were 16 - 64 μg mL-1 active against MRSA and VRE. Analogue 36, lacking the terminal hydrazide NH moiety showed modest Gram-negative activity at 64 μg mL-1. 4-t-Butyl 45 was Gram -positive and -negative active at of 16 - 64 μg mL-1. Typically additional aromatic moiety modification was poorly tolerated, except with concomitant introduction of an imine C-alkyl moiety. The activity of these analogues against MRSA and VRE ranged from 8 μg mL-1 with 64 and 68, to inactive (MIC > 128 μg mL-1) with the naphthyl 69 and 70 and the indole 73. Gram-negative activity was most promising with 62 and 68 at 16 μg mL-1 against E. coli. Against Ps. aeruginosa, the highest activity observed was with MIC values of 32 μg mL-1 with 62 and 64. Combined, these findings support the further development of the (E)-2-benzylidenehydrazine-1-carboximidamide scaffold as a promising Gram-positive and Gram-negative antibiotic development.Cecilia C. Russell, Andrew Stevens, Hongfei Pi, Manouchehr Khazandi, Abiodun D. Ogunniyi, Kelly A. Young, Jennifer R. Baker, Siobhann N. McCluskey, Stephen W. Page, Darren J. Trott and Adam McCluske

Antigiardial activity of novel guanidine compounds

From four focused compound libraries based on the known anticoccidial agent robenidine, 44 compounds total were synthesised and screened for antigiardial activity. All active compounds were counter-screened for antibiotic and cytotoxic action. Of the analogues examined, 21 displayed IC50<5 μM, seven with IC50<1.0 μM. Most active were 2,2′-bis{[4-(trifluoromethoxy)phenyl]methylene}carbonimidic dihydrazide hydrochloride (30), 2,2′-bis{[4-(trifluoromethylsulfanyl)phenyl]methylene}carbonimidic dihydrazide hydrochloride (32), and 2,2′-bis[(2-bromo-4,5-dimethoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (41) with IC50=0.2 μM. The maximal observed activity was a 5 h IC50 value of 0.2 μM for 41. The clinically used metronidazole was inactive at this timepoint at a concentration of 25 μM. Robenidine off-target effects at bacteria and cell line toxicity were removed. Analogue 41 was well tolerated in mice treated orally (100 mg/kg). Following 5 h treatment with 41, no Giardia regrowth was noted after 48 h.Andrew J. Stevens, Rebecca Abraham, Kelly A. Young, Cecilia C. Russell, Siobhann N. McCluskey, Jennifer R. Baker, Bertha Rusdi, Stephen W. Page, Ryan O'Handley, Mark O'Dea, Sam Abraham, Adam McCluske

Comparison of two transmission electron microscopy methods to visualize drug-induced alterations of gram-negative bacterial morphology

In this study, we optimized and compared different transmission electron microscopy (TEM) methods to visualize changes to Gram-negative bacterial morphology induced by treatment with a robenidine analogue (NCL195) and colistin combination. Aldehyde-fixed bacterial cells (untreated, treated with colistin or NCL195 + colistin) were prepared using conventional TEM methods and compared with ultrathin Tokuyasu cryo-sections. The results of this study indicate superiority of ultrathin cryo-sections in visualizing the membrane ultrastructure of Escherichia coli and Pseudomonas aeruginosa, with a clear delineation of the outer and inner membrane as well as the peptidoglycan layer. We suggest that the use of ultrathin cryo-sectioning can be used to better visualize and understand drug interaction mechanisms on the bacterial cell membrane.Hang Thi Nguyen, Lisa A. O’Donovan, Henrietta Venter, Cecilia C. Russell, Adam McCluskey, Stephen W. Page ... et al

A mathematical approach to the boolean minimization problem

Qualitative comparative analysis, Boolean minimization, Computer algorithms, Small-N research,