Preparation, biological & cheminformatics-based assessment of N2,N4-diphenylpyrimidine-2,4-diamine as potential Kinase-targeted antimalarials.

Twenty eight new N2,N4-diphenylpyrimidine-2,4-diamines have been prepared in order to expand our understanding of the anti-malarial SAR of the scaffold. The aim of the study was to make structural modifications to improve the overall potency, selectivity and solubility of the series by varying the anilino groups attached to the 2- and 4-position. We evaluated the activity of the compounds against Plasmodium falciparum (Pf) 3D7, cytotoxicity against HepG2, % inhibition at a panel of 10 human kinases, solubility, permeability and lipophilicity, and human and rat in vitro clearance. 11 was identified as a potent anti-malarial with an IC50 of 0.66 µM at the 3D7 strain and a selectivity (SI) of ~ 40 in terms of cytotoxicity against the HepG2 cell line. It also displayed low experimental logD7.4 (2.27), reasonable solubility (124 µg/ml), good metabolic stability, but low permeability. A proteo-chemometric workflow was employed to identify putative Pf targets of the most promising compounds. Ligand-based similarity searching of the ChEMBL database led to the identification of most probable human targets. These were then used as input for sequence-based searching of the Pf proteome. Homology modelling and molecular docking were used to evaluate whether compounds could indeed bind to these targets with valid binding modes. In vitro biological testing against close human analogs of these targets was subsequently undertaken. This allowed us to identify potential Pf targets and human anti-targets that could be exploited in future development.

Quantitative Assessment of Drug Delivery to Tissues and Association with Phospholipidosis: A Case Study with Two Structurally Related Diamines in Development.

Drug induced phospholipidosis (PLD) may be observed in the preclinical phase of drug development and pose strategic questions. As lysosomes have a central role in pathogenesis of PLD, assessment of lysosomal concentrations is important for understanding the pharmacokinetic basis of PLD manifestation and forecast of potential clinical appearance. Herein we present a systematic approach to provide insight into tissue-specific PLD by evaluation of unbound intracellular and lysosomal (reflecting acidic organelles) concentrations of two structurally related diprotic amines, GRT1 and GRT2. Their intratissue distribution was assessed using brain and lung slice assays. GRT1 induced PLD both in vitro and in vivo. GRT1 showed a high intracellular accumulation that was more pronounced in the lung, but did not cause cerebral PLD due to its effective efflux at the blood-brain barrier. Compared to GRT1, GRT2 revealed higher interstitial fluid concentrations in lung and brain, but more than 30-fold lower lysosomal trapping capacity. No signs of PLD were seen with GRT2. The different profile of GRT2 relative to GRT1 is due to a structural change resulting in a reduced basicity of one amino group. Hence, by distinct chemical modifications, undesired lysosomal trapping can be separated from desired drug delivery into different organs. In summary, assessment of intracellular unbound concentrations was instrumental in delineating the intercompound and intertissue differences in PLD induction in vivo and could be applied for identification of potential lysosomotropic compounds in drug development.

Glucoprotamin antimicrobial activity against selected standard antibiotic-resistant bacteria and reference strains used in the assessment of disinfection efficacy.

BACKGROUND: The ability of bacteria to develop common mechanisms of resistance to antibiotics and disinfectants raises doubts about the effectiveness of disinfection processes. Glucoprotamin (GP) is an antimicrobial active substance which is widely used to the disinfection in medical area. OBJECTIVE: The aim of study was to compare GP's effectiveness with susceptibility of reference strains used for the evaluation of bactericidal efficacy of disinfectants Staphylococcus aureus (S. aureus); Pseudomonas aeruginosa (P. aeruginosa) and standard antibiotic-resistant strains: meticillin-resistant S. aureus (MRSA) and tetracycline-resistant P. aeruginosa (PAO-LAC). MATERIAL AND METHODS: Minimum inhibitory concentrations (MICs) of GP and minimum bactericidal concentrations (MBCs) against tested strains were evaluated by serial broth dilution technique. GP's efficiency was examined according to qualitative (phenol coefficient GP-PC) and quantitative (EN 1040: 2006) test methods. RESULTS: Gram-negative strains were more tolerant to GP than Gram-positive strains among tested strains. MRSA and S. aureus exhibited similar susceptibility to GP. PAO-LAC had significantly lower susceptibility to GP than P. aeruginosa (P≤0,05). There were no differences in GP efficiency against these strains based on GP-PC. According to PN-EN 1040: 2006 standard average obligatory reduction≥5 log10 was demonstrated in the active concentration of GP (84 mg/l) at obligatory 5 min contact time for PAO-LAC and P. aeruginosa. The differences in basis bactericidal activity between PAO-LAC and P. aeruginosa were obtained in the active concentration at 10 and 15 min contact time (P≤0,05). CONCLUSIONS: Variation in a susceptibility of reference strains and antibiotic-resistant standard strains has no meaning at used clinically GP concentrations, which are higher than concentration causing basis bactericidal activity of GP.

Synthesis and biological evaluation of N,N'-squaramides with high in vivo efficacy and low toxicity: toward a low-cost drug against Chagas disease.

Access to basic drugs is a major issue in developing countries. Chagas disease caused by Trypanosoma cruzi is a paradigmatic example of a chronic disease without an effective treatment. Current treatments based on benznidazole and nifurtimox are expensive, ineffective, and toxic. N,N'-Squaramides are amide-type compounds that feature both hydrogen bond donor and acceptor groups and are capable of multiple interactions with complementary sites. When combined with amine and carboxylic groups, squaramide compounds have increased solubility and therefore make suitable therapeutic agents. In this work, we introduce a group of Lipinski's rule of five compliant squaramides as candidates for treating Chagas disease. The in vivo studies confirmed the positive expectations arising from the preliminary in vitro studies, revealing compound 17 to be the most effective for both acute and chronic phases. The activity, stability, low cost of starting materials, and straightforward synthesis make amino squaramides appropriate molecules for the development of an affordable anti-Chagasic agent.

Assessment of leishmanicidal and trypanocidal activities of aliphatic diamine derivatives.

Leishmanicidal and trypanocidal activity of seventeen lipophilic diamines was evaluated in vitro against Leishmania braziliensis, L. chagasi, and Trypanosoma cruzi. Twelve compounds presented anti-Leishmania and six showed anti-T. cruzi amastigote activity. Compound 14 (N-tetradecyl-1,4-butanediamine) was the most active against both L. braziliensis (IC50  = 2.6 µm) and L. chagasi (IC50  = 3.0 µm) which showed a selectivity index (SI) >100. N-decyl-1,6-hexanediamine (compound 9) presented an IC50  = 1.6 µm and SI >187 and was over six times more potent than the reference drug benznidazole against T. cruzi. Treatment of infected or uninfected macrophages with compounds 9 and 14 did not induce significant TNFα and NO production. Four compounds (15, 16, 22, and 23) inhibited 78.9%, 77.7%, 83.7%, and 70.1% of rTRLb activity, respectively, and compound 23 inhibited 73.3% of rTRTc activity at 100 µm. A concentration-dependent effect on mitochondrial membrane depolarization was observed in T. cruzi epimastigotes treated with compound 9, suggesting this mechanism may be involved in the trypanocidal effect. On the contrary, in L. braziliensis promastigotes treated with compound 14, no mitochondrial depolarization was observed. Our results demonstrate that N-decyl-1,6-hexanediamine and N-tetradecyl-1,4-butanediamine are promising molecules for the development of novel leading compounds against T. cruzi and Leishmania spp., particularly given a possible alternative mechanism of action.

Identification and characterization of bi-thiazole-2,2'-diamines as kinase inhibitory scaffolds.

Based on bioinformatics interrogation of the genome, >500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4'-methyl-N(2)-3-pyridinyl-4,5'-bi-1,3-thiazole-2,2'-diamine (JNK Docking (JD) compound 123, but not the related compound (4'-methyl-N~2~-(6-methyl-2-pyridinyl)-4,5'-bi-1,3-thiazole-2,2'-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-ß or p38-δ. Further screening of a broad panel of kinases using 10µM JD123, identified inhibition of kinases including protein kinase Bß (PKBß/Aktß). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBß/Aktß.

Epididymal and testicular enzymes as monitors for assessment of male antifertility drugs.

PIP: Since the synthesis and maturational processes of sperm are associated with characteristic alterations in different marker enzyme activities in testes and epididymis, it is possible to monitor these enzymes to investigate whether 3 antispermatogenic agents, WIN 18 446, alpha-chlorohydrin (AC), and cyproterone acetate (CA), have any characteristic effects on biochemical events associated with spermatogenesis and maturation of sperm; acid, neutral, and alkaline proteinases, particulate and soluble arylamidases, and sialidase were studied after treatment with 1 of the 3 agents in male albino rats (CIBA strain) by measuring these enzyme levels in rat testicular and epididymal tissues. After WIN treatment, sialidase activity as well as sialic acid content increased in the testis, whereas no such effect occurred in the epididymis at any dose. At low dose, AC (10 mg/kg/day for 7 days) and CA (50 mg/kg/day for 10 days) decreased the sialic acid content and the sialidase activity in the epididymis, whereas the sialic acid and sialidase activity in the testis remained unchanged. At higher dose levels, AC (25 mg) and CA (50 mg) both affected the tissues significantly, i.e., enhancing sialidase activity and lowering sialic acid content. Therefore, the effect of CA and AC is more prominent on the maturational phenomena than the testicular spermatogenesis. AC and CA deserve further investigation for use as a male contraceptive. The relationship between proteinase, sialidase, and arylamidase activities and different phases of spermatogenesis and maturation was established by these test results.^ieng