Structural Development of Salicylanilide-Based SPAK Inhibitors as Candidate Antihypertensive Agents.

Hypertension is an important target for drug discovery. We have focused on the with-no-lysine kinase (WNK)-oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)-NaCl cotransporter (NCC) signal cascade as a potential target, and we previously developed a screening system for inhibitors of WNK-OSR1/SPAK-NCC signaling. Herein we used this system to examine the structure-activity relationship (SAR) of salicylanilide derivatives as SPAK kinase inhibitors. Structural design and development based on our previous hit compound, aryloxybenzanilide derivative 2, and the veterinary anthelmintic closantel (3) led to the discovery of compound 10 a as a potent SPAK inhibitor with reduced toxicity. Compound 10 a decreased the phosphorylation level of NCC in mouse kidney in vivo, and appears to be a promising lead compound for a new class of antihypertensive drugs.

Exploiting the HSP60/10 chaperonin system as a chemotherapeutic target for colorectal cancer.

Over the past few decades, an increasing variety of molecular chaperones have been investigated for their role in tumorigenesis and as potential chemotherapeutic targets; however, the 60 kDa Heat Shock Protein (HSP60), along with its HSP10 co-chaperone, have received little attention in this regard. In the present study, we investigated two series of our previously developed inhibitors of the bacterial homolog of HSP60/10, called GroEL/ES, for their selective cytotoxicity to cancerous over non-cancerous colorectal cells. We further developed a third "hybrid" series of analogs to identify new candidates with superior properties than the two parent scaffolds. Using a series of well-established HSP60/10 biochemical screens and cell-viability assays, we identified 24 inhibitors (14%) that exhibited > 3-fold selectivity for targeting colorectal cancer over non-cancerous cells. Notably, cell viability EC50 results correlated with the relative expression of HSP60 in the mitochondria, suggesting a potential for this HSP60-targeting chemotherapeutic strategy as emerging evidence indicates that HSP60 is up-regulated in colorectal cancer tumors. Further examination of five lead candidates indicated their ability to inhibit the clonogenicity and migration of colorectal cancer cells. These promising results are the most thorough analysis and first reported instance of HSP60/10 inhibitors being able to selectively target colorectal cancer cells and highlight the potential of the HSP60/10 chaperonin system as a viable chemotherapeutic target.

Phenolic N-monosubstituted carbamates: Antitubercular and toxicity evaluation of multi-targeting compounds.

The research of novel antimycobacterial drugs represents a cutting-edge topic. Thirty phenolic N-monosubstituted carbamates, derivatives of salicylanilides and 4-chlorophenol, were investigated against Mycobacterium tuberculosis H37Ra, H37Rv including multidrug- and extensively drug-resistant strains, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium aurum and Mycobacterium smegmatis as representatives of nontuberculous mycobacteria (NTM) and for their cytotoxic and cytostatic properties in HepG2 cells. Since salicylanilides are multi-targeting compounds, we determined also inhibition of mycobacterial isocitrate lyase, an enzyme involved in the maintenance of persistent tuberculous infection. The minimum inhibitory concentrations were from ≤0.5 µM for both drug-susceptible and resistant M. tuberculosis and from ≤0.79 µM for NTM with no cross-resistance to established drugs. The presence of halogenated salicylanilide scaffold results into an improved activity. We have verified that isocitrate lyase is not a key target, presented carbamates showed only moderate inhibitory activity (up to 18% at a concentration of 10 µM). Most of the compounds showed no cytotoxicity for HepG2 cells and some of them were without cytostatic activity. Cytotoxicity-based selectivity indexes of several carbamates for M. tuberculosis, including resistant strains, were higher than 125, thus favouring some derivatives as promising features for future development.

Novel salicylanilides from 4,5-dihalogenated salicylic acids: Synthesis, antimicrobial activity and cytotoxicity.

Salicylanilides have proved their activity against tuberculosis (TB). One weak electron-withdrawing substituent is favored at the salicylic part, specially Cl or Br atoms at positions 4 or 5. On the other hand, the antimycobacterial activity of salicylanilides is negatively affected when a strong electron-withdrawing substituent (NO2) is present at the same positions. Herein we describe the synthesis and characterization of novel salicylanilides possessing two weak electron-withdrawing groups (halogen atoms) at their salicylic part and compare their antitubercular activity with their monohalogenated analogues. All dihalogenated derivatives proved to possess antitubercular activity at a very narrow micromolar range (MIC=1-4µM), similar with their most active monohalogenated analogues. More importantly, the most active final molecules were further screened against multidrug resistant strains and found to inhibit their growth at the range of 0.5-4µM.

Design, synthesis and SARs of novel salicylanilides as potent inhibitors of RANKL-induced osteoclastogenesis and bone resorption.

Inhibiting osteoclastogenesis is a promising therapeutic target for treating osteoclast-related diseases. Herein, we synthesized a series of modified salicylanilides and their corresponding 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione and 10-phenyldibenzo[b,f][1,4]oxazepin-11(10H)-one derivatives, and investigated the effects of such compounds on RANKL-induced osteoclast formation. Among them, a salicylanilide derivative (A04) and its 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivative (B04) markedly suppressed RANKL-induced osteoclast differentiation and showed no significant cytotoxic effects at doses higher than that required to inhibit osteoclast formation. Both compounds reduced osteoclast formation and bone resorptive activity of osteoclasts in a dose-dependent manner. Further, the anti-osteoclastogenic effects of A04 and B04 may operate through reducing the RANKL-induced nuclear translocation of NFATc1. Accordingly, we present the potent anti-osteoclastogenic compounds A04 and B04 as promising candidates for further optimization as anti-resorptive agents.

Salicylanilide N-monosubstituted carbamates: Synthesis and in vitro antimicrobial activity.

The research of innovative antimicrobial agents represents a cutting edge topic. Hence, we synthesized and characterised novel salicylanilide N-monosubstituted carbamates. Twenty compounds were evaluated in vitro against eight bacterial strains and eight fungal species. The lowest minimum inhibitory concentrations (MICs) were found to be ⩽0.49 µM. Genus Staphylococcus, including methicillin-resistant Staphylococcus aureus, and fungus Trichophyton mentagrophytes showed uniformly the highest rate of susceptibility, whilst Gram-negative bacteria and most of the fungi were less susceptible. A wide range of carbamates provided comparable or superior in vitro antimicrobial activity in comparison to established drugs. Interestingly, extended-spectrum ß-lactamase producing strain of Klebsiella pneumoniae was inhibited with MICs starting from 31.25 µM. With respect to Staphylococci, 2-[(4-bromophenyl)carbamoyl]-4-chlorophenyl phenylcarbamate exhibited the lowest MIC values (⩽0.98 µM). 2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl benzylcarbamate showed the widest spectrum of antifungal action. The results indicate that some salicylanilide carbamates can be considered to be promising candidates for future investigation.

Synthesis of p-O-Alkyl Salicylanilide Derivatives as Novel EGFR Inhibitors.

Preclinical Research Epidermal growth factor receptor (EGFR), a validated target for anticancer drugs, plays a critical role in tumorigenesis and tumor development. A series of p-O-alkyl salicylanilide derivatives were designed and synthesized as novel EGFR inhibitors using a salicylic acid scaffold. A simulated six-membered ring strategy formed through intramolecular hydrogen bonds was employed to mimic the planar quinazoline of the EGFR antagonist, gefitinib. The derived compounds with hydroxyl at the ortho position were more potent than ones with methoxyl group. In particular, compounds 5d and 5b displayed significant EGFR inhibitory (IC50 values = 0.30 and 0.45 µM, respectively) activity as well as potent antiproliferative activity in A431 and HCT-116 tumor cells. These salicylanilides could be considered as promising lead compounds for developing novel EGFR inhibitors.

Synthesis, Bioactivity Evaluation, and Toxicity Assessment of Novel Salicylanilide Ester Derivatives as Cercaricides against Schistosoma japonicum and Molluscicides against Oncomelania hupensis.

A series of novel salicylanilide ester derivatives were synthesized, characterized, and evaluated for cercaricidal potential against Schistosoma japonicum and molluscicidal potential against Oncomelania hupensis. Four derivatives exhibited remarkable cercaricidal activity superior to that of niclosamide. Among them, the most active compound, 4-chloro-2-((2-methoxy-4-nitrophenyl)carbamoyl)phenyl 4-methoxybenzoate (compound 4c), showed a marked minimum effective cercaricidal concentration as low as 0.43 µM and significant molluscicidal activity, with a 50% lethal concentration (LC50) of 0.206 g/m(2). Particularly, compound 4c displayed 88-fold decreased fish toxicity on Danio rerio and 44-fold reduced cytotoxicity on human kidney HEK293 cells in comparison with the toxicity of niclosamide. The results indicated that 4c could serve as a promising drug candidate, with environmental safety properties, against Schistosoma japonicum at transmission stages. The preliminary molecular mechanism of target compounds in Schistosoma japonicum cercariae was also investigated. Salicylanilide ester derivatives exhibited an inhibitory effect on nitric oxide synthase (NOS) but no effect on lactate dehydrogenase (LDH) and acetylcholinesterase (AChE), and a strong and significant correlation between NOS inhibitory efficacy and cercaricidal activity was observed. In addition, 4c could downregulate the expression of NOS in a dose-dependent manner. These results suggested that NOS was probably one of the drug targets of salicylanilide esters.

Combating highly resistant emerging pathogen Mycobacterium abscessus and Mycobacterium tuberculosis with novel salicylanilide esters and carbamates.

In the Mycobacterium genus over one hundred species are already described and new ones are periodically reported. Species that form colonies in a week are classified as rapid growers, those requiring longer periods (up to three months) are the mostly pathogenic slow growers. More recently, new emerging species have been identified to lengthen the list, all rapid growers. Of these, Mycobacterium abscessus is also an intracellular pathogen and it is the most chemotherapy-resistant rapid-growing mycobacterium. In addition, the cases of multidrug-resistant Mycobacterium tuberculosis infection are also increasing. Therefore there is an urgent need to find new active molecules against these threatening strains. Based on previous results, a series of salicylanilides, salicylanilide 5-chloropyrazinoates and carbamates was designed, synthesized and characterised. The compounds were evaluated for their in vitro activity on M. abscessus, susceptible M. tuberculosis H37Rv, multidrug-resistant (MDR) M. tuberculosis MDR A8, M. tuberculosis MDR 9449/2006 and on the extremely-resistant Praha 131 (XDR) strains. All derivatives exhibited a significant activity with minimum inhibitory concentrations (MICs) in the low micromolar range. Eight salicylanilide carbamates and two salicylanilide esters exhibited an excellent in vitro activity on M. abscessus with MICs from 0.2 to 2.1 µM, thus being more effective than ciprofloxacin and gentamicin. This finding is potentially promising, particularly, as M. abscessus is a threateningly chemotherapy-resistant species. M. tuberculosis H37Rv was inhibited with MICs from 0.2 µM, and eleven compounds have lower MICs than isoniazid. Salicylanilide esters and carbamates were found that they were effective also on MDR and XDR M. tuberculosis strains with MICs ≥1.0 µM. The in vitro cytotoxicity (IC50) was also determined on human MonoMac-6 cells, and selectivity index (SI) of the compounds was established. In general, salicylanilide derivatives substituted by halogens on both salicyl and aniline rings showed better activity, than 4-benzoylaniline derivatives. The ester or carbamate bond formation of parent salicylanilides mostly retained or improved antimycobacterial potency with moderate selectivity.

Design, synthesis, and cercaricidal activity of novel high-efficient, low-toxic self-spreading PEG-N-salicylanilide derivatives against cercariae larvae of Schistosome Japonicum floating on the water surface.

Novel cercaricides of PEG-N-salicylanilide derivatives that could self-spread and float on the water surface were designed and synthesized according to the particular habit of cercariae larvae of Schistosome japonicum. The structures of the cercaricides were characterized by the infrared spectra (IR), magnetic resonance ((1) H NMR), and mass spectrum (MS). The images of the floating cercaricides on the water surface were investigated by the Brewster angle microscopy (BAM). When the cercaricides were dropped on the water surface, they could spread along the air-water interface automatically and form thin membranes floating on the water surface immediately. The lethality rate of cercariae for 5a and 6a was more than 90% in 120 min at a surface concentration of 0.008 mg/cm(2) . The non-ionic surfactant-cercaricides not only showed strong cercaricidal activities against the cercariae larvae but also exhibited low toxicities, which offered an effective and environment-friendly approach for the reduction of population infection rate and the realization of schistosome control.

Modified salicylanilide and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives as novel inhibitors of osteoclast differentiation and bone resorption.

Inhibition of osteoclast formation is a potential strategy to prevent inflammatory bone resorption and to treat bone diseases. In the present work, the purpose was to discover modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives as potential antiosteoclastogenic agents. Their inhibitory effects on RANKL-induced osteoclastogenesis from RAW264.7 cells were evaluated by TRAP stain assay. The most potent compounds, 1d and 5d, suppressed RANKL-induced osteoclast formation and TRAP activity dose-dependently. The cytotoxicity assay on RAW264.7 cells suggested that the inhibition of osteoclastic bone resorption by these compounds did not result from their cytotoxicity. Moreover, both compounds downregulated RANKL-induced NF-κB and NFATc1 in the nucleus, suppressed the expression of osteoclastogenesis-related marker genes during osteoclastogenesis, and prevented osteoclastic bone resorption but did not impair osteoblast differentiation in MC3T3-E1. Therefore, these modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-diones could be potential lead compounds for the development of a new class of antiresorptive agents.