Phenylimino-2H-chromen-3-carboxamide derivatives as novel small molecule inhibitors of β-secretase (BACE1)

The inhibition of β secretase (BACE1) is potentially important approach to treatment of Alzheimer disease (AD). A novel series of 4-bromophenyl piperazine derivatives coupled to the phenylimino-2H-chromen-3-carboxamide scaffold were investigated as BACE1 inhibitors in this study. Docking study suggested that the phenyl-imino group of the scaffold establishes favorable π–π stacking interaction with side chain of Phe108 of flap pocket. Some of the docking proposed derivatives were synthesized and evaluated for BACE1 inhibitory activity using a FRET-based assay. High BACE1 inhibitory activities were observed from derivatives containing fused heteroaromtic groups attached through the aliphatic linkage to the N4-piperazine moiety, which may be attributed to the engagement of effective interactions with S1–S′1 sub-pocket residues. Of the most potent compounds, 9e displayed an IC₅₀ value for BACE1 of 98 nM. Some of these derivatives demonstrated good inhibitory activity on Aβ production in N2a-APPswe cells at 5 and 10 μM. These compounds might be considered as promising BACE1 inhibitory agents that could lower Aβ production in AD.     

Prolyl endopeptidase inhibitory activity of chitosan sulfates with different degree of deacetylation

Three kinds of partially deacetylated chitosan, 90% deacetylated chitosan, 75% deacetylated chitosan and 50% deacetylated chitosan, were prepared from crab chitin by N-deacetylation with 40% (w/w) sodium hydroxide solution for different durations. In order to improve biological activity and solubility, their sulfated derivatives were prepared, and prolyl endopeptidase (PEP) inhibitory activities were investigated. Fifty percent-deacetylated chitosan sulfate (50-CS) exhibited the highest inhibitory activity, and inhibition rate was a dose-dependant. In addition, Dixon plots suggested that 50-CS was act as competitive inhibitor, and the inhibition constant (K_i) was 2.6 mg/ml.     

Searching for the Multi-Target-Directed Ligands against Alzheimer's disease: discovery of quinoxaline-based hybrid compounds with AChE, H₃R and BACE 1 inhibitory activities

A novel series of quinoxaline derivatives, as Multi-Target-Directed Ligands (MTDLs) for AD treatment, were designed by lending the core structural elements required for H(3)R antagonists and hybridizing BACE 1 inhibitor 1 with AChE inhibitor BYYT-25. A virtual database consisting of quinoxaline derivatives was first screened on a pharmacophore model of BACE 1 inhibitors, and then filtered by a molecular docking model of AChE. Seventeen quinoxaline derivatives with high score values were picked out, synthesized and evaluated for their biological activities. Compound 11a, the most effective MTDL, showed the potent activity to H(3)R/AChE/BACE 1 (H(3)R antagonism, IC(50)=280.0 ± 98.0 nM; H(3)R inverse agonism, IC(50)=189.3 ± 95.7 nM; AChE, IC(50)=483 ± 5 nM; BACE 1, 46.64±2.55% inhibitory rate at 20 μM) and high selectivity over H(1)R/H(2)R/H(4)R. Furthermore, the protein binding patterns between 11a and AChE/BACE 1 showed that it makes several essential interactions with the enzymes.     

Triazolyl tryptoline derivatives as β-secretase inhibitors

Tryptoline, a core structure of ochrolifuanine E, which is a hit compound from virtual screening of the Thai herbal database against BACE1 was used as a scaffold for the design of BACE1 inhibitors. The tryptoline was linked with different side chains by 1,2,3-triazole ring readily synthesized by catalytic azide-alkyne cycloaddition reactions. Twenty two triazolyl tryptoline derivatives were synthesized and screened for the inhibitory action against BACE1. JJCA-140 was the most potent inhibitor (IC(50)=1.49 μM) and was 100 times more selective for BACE1 than for Cat-D.     

Dual-target-directed 1,3-diphenylurea derivatives: BACE 1 inhibitor and metal chelator against Alzheimer’s disease

Dual-target-directed 1,3-diphenylurea derivatives were designed by hybridizing BACE 1 inhibitor 1 with metal chelator LR-90. A database consisted of 1,3-diphenylurea derivatives was built and screened by the pharmacophore model (Hypo 1) of BACE 1 inhibitor. Based on the predicted results, 11 compounds (6a–d, 9a–g) with favorable Fitvalues were selected, synthesized and evaluated for their BACE 1 inhibitory activities, which showed that the predicted results were in good agreement with the experimental values. Besides, the synthesized compounds also displayed the ability to chelate metal ions. The most effective BACE 1 inhibitor 9f (27.85 ± 2.46 μmol/L) was selected for further receptor-binding studies, the result of which indicated that an essential hydrogen bonds was formed between the urea group of 9f and the catalytic aspartate Asp228.     

beta-Secretase inhibitors: modification at the P4 position and improvement of inhibitory activity in cultured cells

Recently, we reported potent and small-sized beta-secretase (BACE1) inhibitors KMI-570 and KMI-684 in which we replaced carboxylic acid groups at the P(1)(') position of KMI-420 and KMI-429, respectively, with tetrazole derivatives as carboxylic acid bioisosteres. These modifications improved significantly BACE1 inhibitory activity and chemical stability. In this study, the acidic tetrazole ring of the P(4) position of KMI-420 and KMI-570, respectively, was replaced with various hydrogen bond acceptor groups. We found BACE1 inhibitor KMI-574 that exhibited potent inhibitory activity in cultured cells as well as in vitro enzymatic assay.     

Design and synthesis of highly active Alzheimer's beta-secretase (BACE1) inhibitors, KMI-420 and KMI-429, with enhanced chemical stability

Recently, we reported potent and small-sized BACE1 inhibitors KMI-358 and KMI-370 in which the Glu residue is replaced by a beta-N-oxalyl-DAP (l-alpha,beta-diaminopropionyl) residue at the P(4) position. The beta-N-oxalyl-DAP group is important for enhancing BACE1 inhibitory activity, but these inhibitors isomerized to alpha-N-oxalyl-DAP derivatives in solvents. Hence, we used a tetrazole moiety as a bioisostere of the free carboxylic acid of the oxalyl group. KMI-420 and KMI-429, containing a tetrazole ring, showed improved stability and potent enzyme inhibitory activity.     

β-secretase inhibitory activity of phenolic acid conjugated chitooligosaccharides

Eight kinds of phenolic acid conjugated chitooligosaccharides (COSs) were synthesized using hydroxyl benzoic acid and hydroxyl cinnamic acid. These phenolic acid conjugated-COSs with different substitution groups, including p-hydroxyl, 3,4-dihydroxyl, 3-methoxyl-4-hydroxyl and 3,5-dimethoxyl-4-hydroxy groups, were evaluated for their inhibitory activities against β-site amyloid precursor protein (APP)-cleaving enzyme (BACE) and inhibited BACE with a ratio of 50.8%, 74.8%, 62.1%, 64.8% and 42.6%, respectively at the concentration of 1,000 μg/mL. BACE is a critical component to reduce the levels of Aβ amyloid peptide in Alzheimer’s disease (AD) which is based on the amyloid cascade theory in the brain, as this protease initiates the first step in Aβ production. Among them, Caffeic acid conjugated-COS (CFA-COS) was further analysed to determine mode of inhibition of BACE and it showed non-competitive inhibition. Hence in this study, we suggest that CFA-COS derivatives have potential to be used as novel BACE inhibitors to reduce the risk of AD.     

Carbazole-containing arylcarboxamides as BACE1 inhibitors

β-Secretase (BACE1) is widely recognized as a prime drug target for the treatment of Alzheimer's disease (AD). In this Letter, we report the synthesis and the BACE1 inhibitory activity of novel, variously substituted N-[3-(9H-carbazol-9-yl)-2-hydroxypropyl]-arylcarboxamides. The best results have been obtained with the introduction of a 4-OMe substituent (IC(50)=3.8 μM) or a 3,4-dichloro substituent (IC(50)=2.5 μM) in the amidic aromatic ring. The blood-brain barrier penetration predictions resulted to be promising for this type of compounds. To better understand the structure-activity relationships (SAR) of the new derivatives, a docking study procedure has been applied exploiting different conformational and ionic states of BACE1.     

Molecular modeling,synthesis, and activity studies of novel biaryl and fused-ring BACE1 inhibitors

A series of transition state analogues of beta-secretases 1 and 2 (BACE1, 2) inhibitors containing fused-ring or biaryl moieties were designed computationally to probe the S2 pocket, synthesized, and tested for BACE1 and BACE2 inhibitory activity. It has been shown that unlike the biaryl analogs, the fused-ring moiety is successfully accommodated in the BACE1 binding site resulting in the ligands with excellent inhibitory activity. Ligand 5b reduced 65% of Aβ40 production in N2a cells stably transfected with Swedish human APP.     

Synthesis and biological evaluation of quinazolinone-based hydrazones with potential use in Alzheimer’s disease

Discovering multifunctional agents for the treatment of Alzheimer’s disease (AD) is an attractive therapeutic approach. BACE1 (β-site amyloid precursor protein cleaving enzyme 1) inhibitors may play a pivotal role in treating AD. Therefore, the discovery of novel non-peptide BACE1 inhibitors with desirable blood brain barrier permeability is a favorable approach for treatment. Moreover, the antioxidant potential of a drug could serve as an added value for designing dual-acting therapeutic agents. Here, we report the design, synthesis and biological evaluation of quinazolinone-hydrazone derivatives as new multi-target candidates for the treatment of AD. The compounds were investigated for their in vitro BACE1 inhibitory potential using a FRET-based enzymatic assay and also screened for antioxidant activity using DPPH. Among them, compound 4h bearing a 2,3-dichlorophenyl moiety showed the highest activity with an IC₅₀ value of 3.7 μM against BACE1. In addition, compound 4i with a 2,4-dihydroxyphenyl scaffold demonstrated moderate BACE1 inhibitory activity (IC₅₀ = 27.6 μM) with a significant antioxidant effect (IC₅₀ = 8.4 μM). Furthermore, docking studies revealed strong interaction between compound 4h and the key residues of BACE1 active site. These results demonstrate that quinazolinone-hydrazone derivatives represent a valuable scaffold for the discovery of novel non-peptidic BACE1 inhibitors.     

Design,synthesis, and bioactivities of tasiamide B derivatives as cathepsin D inhibitors

Cathepsin D (Cath D) is overexpressed and hypersecreted by malignant tumors and involved in the progress of tumor invasion, proliferation, metastasis, and apoptosis. Cath D has been considered as a potential target to treat cancer. Our previous studies revealed that tasiamide B derivatives TB‐9 and TB‐11 exhibited high potent inhibition against Cath D and other aspartic proteases, but their molecular weights are still high, and the role of each residue is unknown yet. Based on this, two series of tasiamide B derivatives have been designed, synthesized, and evaluated for their inhibitory activity against Cath D/Cath E/BACE1. Enzymatic assays revealed that the target compound 1 with lower molecule weight showed good inhibitory activity against Cath D with IC₅₀ of 3.29 nM and satisfactory selectivity over Cath E (72‐fold) and BACE1 (295‐fold), which could be a valuable template for the design of highly potent and selective Cath D inhibitors.     

Antibacterial activity of quaternary ammonium chitosan containing mono or disaccharide moieties: Preparation and characterization

The 9 quaternary ammonium chitosans containing monosaccharides or disaccharides moieties were successfully synthesized by reductive N-alkylation then quaternized by N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (Quat-188). The chemical structures of quaternary ammonium chitosan derivatives were characterized by ATR-FTIR and ¹H NMR spectroscopy. The degree of N-substitution (DS) and the degree of quaternization (DQ) were determined by ¹H NMR spectroscopic method. It was found that the DS was in the range of 12–40% while the DQ was in the range of 90–97%. The results indicated that the O-alkylation was occured in this condition. Moreover, all quaternary ammonium chitosan derivatives were highly water-soluble at acidic, basic, and neutral pH. Minimum inhibitory concentration (MIC) antibacterial studies of these materials were carried out on Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria compared to quaternary ammonium N-octyl and N-benzyl chitosan derivatives. The quaternary ammonium mono and disaccharide chitosan derivatives showed very high MIC values which were in the range of 32 to >256 μg/mL against both bacteria. Also it was found that the antibacterial activity decreased with increasing the DS. This was due to the increased hydrophilicity of mono and disaccharide moieties. On the other hand, the low MIC values (8–32 μg/mL) were obviously observed when the DS of quaternary ammonium N-octyl and N-benzyl chitosan derivatives was lower than 18%. The results showed that the presence of hydrophobic moiety such as the N-benzyl group enhanced the antibacterial activity compared to the hydrophilic moiety against both bacteria.     

BACE1 inhibitory activities of enantiomerically pure, variously substituted N-(3-(4-benzhydrylpiperazin-1-yl)-2-hydroxypropyl) arylsulfonamides

β-Secretase (BACE1) has been widely recognized as one of the possible therapeutic targets for the treatment of Alzheimer's disease. In this paper, we report the synthesis and the BACE1 inhibitory activity of new, variously substituted N-(3-(4-benzhydrylpiperazin-1-yl)-2-hydroxypropyl) arylsulfonamides. Each enantiomeric form was separately evaluated in BACE1 inhibition assays and IC(50) values were obtained in the low micromolar range. According to our biological results and docking studies, it can be asserted that the stereochemistry around the OH group in the central hydroxyethylamino linker does not significantly influence the BACE1 inhibitory activity of this type of molecules.     

Inhibition of beta-secretase in vivo via antibody binding to unique loops (D and F) of BACE1

β-Secretase (BACE1) is an attractive drug target for Alzheimer disease. However, the design of clinical useful inhibitors targeting its active site has been extremely challenging. To identify alternative drug targeting sites we have generated a panel of BACE1 monoclonal antibodies (mAbs) that interfere with BACE1 activity in various assays and determined their binding epitopes. mAb 1A11 inhibited BACE1 in vitro using a large APP sequence based substrate (IC(50) ～0.76 nm), in primary neurons (EC(50) ～1.8 nm), and in mouse brain after stereotactic injection. Paradoxically, mAb 1A11 increased BACE1 activity in vitro when a short synthetic peptide was used as substrate, indicating that mAb 1A11 does not occupy the active-site. Epitope mapping revealed that mAb 1A11 binds to adjacent loops D and F, which together with nearby helix A, distinguishes BACE1 from other aspartyl proteases. Interestingly, mutagenesis of loop F and helix A decreased or increased BACE1 activity, identifying them as enzymatic regulatory elements and as potential alternative sites for inhibitor design. In contrast, mAb 5G7 was a potent BACE1 inhibitor in cell-free enzymatic assays (IC(50) ～0.47 nm) but displayed no inhibitory effect in primary neurons. Its epitope, a surface helix 299-312, is inaccessible in membrane-anchored BACE1. Remarkably, mutagenesis of helix 299-312 strongly reduced BACE1 ectodomain shedding, suggesting that this helix plays a role in BACE1 cellular biology. In conclusion, this study generated highly selective and potent BACE1 inhibitory mAbs, which recognize unique structural and functional elements in BACE1, and uncovered interesting alternative sites on BACE1 that could become targets for drug development.     

Structure and antimicrobial activities of benzoyl phenyl-thiosemicarbazone-chitosans

Previously, we had prepared acetyl phenyl-thiosemicarbazone derivatives of chitosan, and their antimicrobial activities were analyzed. The purpose of the present study was to further assess the relationship between the structure and antimicrobial activities of benzoyl phenyl-thiosemicarbazone-chitosan. Ten new benzoyl phenyl-thiosemicarbazone-chitosans were prepared and their structures were characterized by FT-IR and elemental analysis. The antimicrobial experiment against four species of bacteria and four crop-threatening pathogenic fungi were conducted based on the derivatives of chitosan with different molecular weight at different concentrations. The results indicated that the antimicrobial activities of benzoyl phenyl-thiosemicarbazone derivatives are much better than that of pure CS. The value of the minimum inhibition concentration (MIC) and the minimum bactericidal concentration (MBC) of the derivatives against Escherichia coli was 7.03 and 225 μg mL(-1) respectively. All of the derivatives had significant inhibiting effect on the investigated fungi in the concentration of 50-500 μg mL(-1), and the maximum inhibitory index was 94.74%. These results indicate that the derivatives have potential ability used as antibacterial reagent in agricultural field.     

Novel BACE1 inhibitors with a non-acidic heterocycle at the P1′ position

We have reported potent peptidic and non-peptidic BACE1 inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. However, our potent inhibitors possess a tetrazole ring at the P₁′ position. It is desirable that central nervous system (CNS) drugs do not possess an acidic moiety. In this study, we synthesized non-acidic BACE1 inhibitors with heterocyclic derivatives at the P₁′ position. KMI-1764 (27) exhibited potent inhibitory activity (IC₅₀ = 27 nM). Interestingly, these non-acidic inhibitors tended to follow the quantitative structure–activity relationship (QSAR) equation and interacted with BACE1-Arg235 in the binding model.     

Cytotoxic activities of water-soluble chitosan derivatives with different degree of deacetylation

Chitosans with different degree of deacetylation (DD) (90% and 50% deacetylated chitosan) were prepared by N-deacetylation followed by grafted onto chitosan to form water-soluble aminoethyl-chitosan (AE-chitosan), and dimetylaminoethyl-chitosan (DMAE-chitosan), diethylaminoethyl-chitosan (DEAE-chitosan). In the present study, cytotoxic activities of the chitosan derivatives were evaluated using three tumor cell lines and two normal cell lines, and structure-activity relationship was suggested. The cytotoxic activity was dependent on their DD and substituted group.     

Preparation and antimicrobial activity of hydroxypropyl chitosan

Water-soluble hydroxypropyl chitosan (HPCS) derivatives with different degrees of substitution (DS) and weight-average molecular weight (Mw) were synthesized from chitosan and propylene epoxide under basic conditions. Their structure was characterized by IR spectroscopy, NMR spectroscopy, and elemental analysis, which showed that both the OH groups at C-6 and C-3 and the NH2 group of chitosan were alkylated. The DS value of HPCS ranged from 1.5 to 3.1 and the Mw was between 2.1x10(4) and 9.2x10(4). In vitro antimicrobial activities of the HPCS derivatives were evaluated by the Kirby-Bauer disc diffusion method and the macrotube dilution broth method. The HPCS derivatives exhibited no inhibitory effect on two bacterial strains (Escherichia coli and Staphylococcus aureus); however, some inhibitory effect was found against four of the six pathogenic fruit fungi investigated. Some derivatives (HPCS1, HPCS2, HPCS3, HPCS3-1, and HPCS4) were effective against C. diplodiella and F. oxysporum. HPCS3-1 is the most effective one with MIC values of 5.0, 0.31, 0.31, and 0.16mg/mL against A. mali, C. diplodiella, F. oxysporum, and P. piricola, respectively. Antifungal effects were also observed for HPCS2 and HPCS3-1 against A. mali, as well as HPCS3 and HPCS3-1 against P. piricola. The results suggest that relatively lower DS and higher Mw value enhances the antifungal activity of HPCS derivatives.