Polycyclic N‐Heterocyclic Compounds. Part 82: Synthesis and Evaluation of Anti‐Platelet Aggregation Activity of 2,4‐Disubstituted 5,6‐Dihydro[1]benzothiepino[5,4‐d]pyrimidine and Related Compounds

We have synthesized a large number of tricyclic 2‐substituted 4‐alkylamino‐5,6‐dihydro[1]benzothiepino[5,4‐d]pyrimidines as part of our research to develop new effective anti‐platelet drugs. A variety of alkyl and aryl groups were used as substituents at the 2‐position. Evaluation of the effects of the newly synthesized compounds on collagen‐induced platelet aggregation revealed several promising anti‐platelet candidates with potencies superior to aspirin.     

Discovery of a Novel Inhibitor of the Hedgehog Signaling Pathway through Cell‐based Compound Discovery and Target Prediction

Cell‐based assays enable monitoring of small‐molecule bioactivity in a target‐agnostic manner and help uncover new biological mechanisms. Subsequent identification and validation of the small‐molecule targets, typically employing proteomics techniques, is very challenging and limited, in particular if the targets are membrane proteins. Herein, we demonstrate that the combination of cell‐based bioactive‐compound discovery with cheminformatic target prediction may provide an efficient approach to accelerate the process and render target identification and validation more efficient. Using a cell‐based assay, we identified the pyrazolo‐imidazole smoothib as a new inhibitor of hedgehog (Hh) signaling and an antagonist of the protein smoothened (SMO) with a novel chemotype. Smoothib targets the heptahelical bundle of SMO, prevents its ciliary localization, reduces the expression of Hh target genes, and suppresses the growth of Ptch^+/− medulloblastoma cells.     

Design and Synthesis of 3‐Aryl‐5‐Alicylic‐[1,2,4]‐oxadiazoles as Novel Platelet Aggregation Inhibitors

A series of 4‐[2‐(alicyclic‐[1,2,4]oxadiazol‐3‐yl)phenoxy]‐butyric acids were synthesized from N‐hydroxy‐2‐isopropoxy benzamidine in 4 steps with good yields. These [1,2,4]oxadiazoles are novel platelet aggregation inhibitors preventing human platelet aggregation induced by thromboxane derivative U44,619 and adenosine diphosphate. A structure‐activity‐relationship study revealed that the potency for these 5‐oxadiazoles increases with the increase in the ring size of the alicylic rings. Derivative 8f may be useful as a template for the design of more potent anti‐platelet agents.     

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays

Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC₅₀ value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP‐dependent kinase, inhibition of mTOR signaling, and induction of cell‐cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.     

Pharmacokinetics study of 16 active ingredients from Tabson‐2 decoction in normal and d‐galactose induced osteoporosis rats by liquid chromatography–tandem mass spectrometry

Tabson‐2 decoction is the traditional Mongolian formula for anti‐osteoporosis, and the ambiguous of active ingredient is an important factor in restricting its modernization and globalization. Although pharmacokinetic profiles research is a viable approach to find the components being responsible for formula efficacy, the pharmacokinetics study of Tabson‐2 decoction has not been elucidated yet. Owing to the existence of isomers, low bioavailability of some small molecule and interference of endogenous, the pharmacokinetics study of Tabson‐2 decoction are more difficult than that of chemical drugs. In our experiment, a specific and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for simultaneous determination of 16 active ingredients in Tabson‐2 decoction, which could fulfill the requirements of multi‐compounds pharmacokinetic study of Tabson‐2 decoction. Additionally, the ingredients with significant distributions in rats were gentianic acid, chlorogenic acid, and aucubin, which could be the main potential active components in Tabson‐2 decoction. The components with a significant bioavailability difference between normal and d ‐galactose induced osteoporosis rats were achieved as well. These data offer useful information for screening the active ingredients in Tabson‐2 decoction, and assessing the bioavailability of these active ingredients in different physiological status, which might provide a possible mechanism of anti‐osteoporosis efficacy of Tabson‐2 decoction.     

Evaluation of affinity interaction between small molecules and platelets by open tubular affinity capillary electrochromatography

In this paper, an open tubular affinity capillary electrochromatography (OT‐ACEC) was developed by physical adsorption of rabbit platelets on the inner surface of capillary. The interactions between small molecules include adenosine diphosphate (ADP) (positive control), protocatechuic acid (negative control) and seven natural products (salvianolic acid B, salvianic acid A sodium, hydroxysafflor yellow A, ferulic acid, chlorogenic acid, sinapic acid, caffeic acid) and platelets were evaluated by their retention factors and binding constants obtained based on peak‐shift assay. Then, the activities of anti‐platelet aggregation induced by thrombin (THR), ADP and arachidonic acid (AA) for those small molecules (except ADP) were evaluated by turbidimetric method. The results indicate that: (i) ADP, a platelet aggregation inducer, had strong interaction with platelet, while protocatechuic acid that had no inhibition on platelet aggregation behaved no specific interaction; (ii) there was a positive correlation between the anti‐platelet aggregation activities of small molecules and their interactions with platelet, generally those compounds with higher binding constants with platelet exhibited higher activities. Therefore, the OT‐ACEC method developed in the present study can be a potential method to evaluate affinity interactions between small molecules and platelets, so as to predict the biological activities such as anti‐platelet aggregation for the small molecules.     

Non‐Flat Bisbenzylisoquinoline Alkaloid Fangchinoline As a Class of Potent G‐Quadruplex Stabilizer with Anti‐cancer Activity

Compounds selectively binding and stabilizing G‐quadruplex structures could inhibit the telomerase or down‐ regulate the oncogenes and may act as anti‐cancer drugs. An alkaloid with non‐flat structure, fangchinoline, showed to strongly stabilize the intermolecular and intramolecular parallel stranded G‐quadruplex structure, increasing melting temperature by 20 and 23°C, respectively. The binding mode was investigated by using NMR and molecular modelling methods. Four human cell lines (HL‐60, BGC‐823, Bel‐7402 and KB) were taken to test the anti‐proliferation effects of fangchinoline and the IC₅₀ values were ranged from 16 to 32 µmol/L. These results showed that the fangchinoline or related moiety derivatives may represent a class of telomere‐targeted agents as potential anti‐cancer drugs.     

An Antibacterial β‐Lactone Kills Mycobacterium tuberculosis by Disrupting Mycolic Acid Biosynthesis

The spread of antibiotic resistance is a major challenge for the treatment of Mycobacterium tuberculosis infections. In addition, the efficacy of drugs is often limited by the restricted permeability of the mycomembrane. Frontline antibiotics inhibit mycomembrane biosynthesis, leading to rapid cell death. Inspired by this mechanism, we exploited β‐lactones as putative mycolic acid mimics to block serine hydrolases involved in their biosynthesis. Among a collection of β‐lactones, we found one hit with potent anti‐mycobacterial and bactericidal activity. Chemical proteomics using an alkynylated probe identified Pks13 and Ag85 serine hydrolases as major targets. Validation through enzyme assays and customized ¹³C metabolite profiling showed that both targets are functionally impaired by the β‐lactone. Co‐administration with front‐line antibiotics enhanced the potency against M. tuberculosis by more than 100‐fold, thus demonstrating the therapeutic potential of targeting mycomembrane biosynthesis serine hydrolases.     

One‐step hydrothermal synthesis of magnetic nitrogen‐doped graphene for magnetic solid‐phase extraction of nonsteroidal anti‐inflammatory drugs in environmental water samples

Graphene oxide has received extensive attention because of its unique properties and potential applications. In this study, magnetic nitrogen‐doped graphene was prepared by one‐step hydrothermal reaction using urea as the dopant and reductant, and ferroferric oxide nanoparticles were in situ deposited on the surface of the nanohybrids. The magnetic nitrogen‐doped graphene was characterized using various physical and chemical methods. It was used as a new adsorbent for the magnetic solid‐phase extraction of four nonsteroidal anti‐inflammatory drugs from the river water. The parameters influencing the extraction efficiency were optimized in detail. Under optimal conditions, this method provided a wide linear range (5–200 ng/mL). The limits of detection were in the range of 1.07–5.10 ng/mL. The recoveries varied from 81.2 to 121.5% with relative standard deviations of less than 10.8%. Overall, we can conclude that the proposed method offers an efficient pretreatment and enrichment and can be successfully applied for the extraction and determination of nonsteroidal anti‐inflammatory drugs in complex matrices.     

Drug Target Identification Using Affinity Core‐Shell Magnetic Nanoparticles and Mass Spectrometry

Affinity core‐shell magnetic nanoparticles (MNPs) were prepared for identifying the target proteins of drugs in the cell lysate when used in combination with nano‐high‐performance liquid chromatography tandem mass spectrometry (HPLC‐MS/MS)‐based shotgun proteomic analysis. A number of new potential targets of cyclosporine A (CsA) could be identified, owing to the high efficacy of the affinity MNPs in drug target identification. To the best of our knowledge, this is the first time to reveal such an abundant target spectrum of CsA.     

Anti‐cancer potential of selenium‐ and tellurium‐containing species: opportunities abound!

An overview highlighting the anti‐cancer potential of (bio‐essential) selenium‐ and tellurium‐containing species, with an emphasis on biological targets and mechanisms of action, is presented. Studies thus far have focused on selenium(II) compounds (along with – to a lesser extent – inorganic selenium and selenium nanoparticles) which often successfully exploit the inherent anti‐oxidizing ability of selenium. Significantly less work has been conducted in developing anti‐cancer tellurium compounds, yet two tellurium(IV) species are proven, clinically, as anti‐cancer agents. Given the prevalence of the disease and the accumulated insights into mechanisms of action, the continued development of selenium‐ and tellurium‐containing molecules is an area deserving greater attention. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis and Biological Evaluation of 2‐substituted‐6‐(morpholinyl/piperidinyl)pyridazin‐3(2H)‐ones as Potent and Safer Anti‐inflammatory and Analgesic Agents

A series of 2‐substituted‐6‐(morpholinyl/piperidinyl)pyridazin‐3(2H )‐ones was synthesized and the structures were established using various spectroscopic techniques. The target compounds were screened for anti‐inflammatory and analgesic activities at 20 and 40 mg/kg. The safety of the synthesized derivatives was evaluated by assessing anti‐platelet activity and ulcer index. The obtained pharmacological data revealed that 6‐morpholinyl derivatives 4a–12a were found to be somewhat more potent than 6‐piperidinyl derivatives 4b–6b. The 6‐morpholinyl substituted pyridazinone 12a exhibited maximum anti‐inflammatory and analgesic activities. Homoveratrylamine substituted compounds 6a and 6b emerged as promising leads in both the series with good anti‐inflammatory and analgesic activities without any ulcerogenicity. Anti‐platelet activity results of the compounds of both the series showed significantly low bleeding time in comparison with standard drug aspirin indicating the cardiovascular safety of new pyridazinones.     

Design,Synthesis and Biological Evaluation of Potent Azadipeptide Nitrile Inhibitors and Activity‐Based Probes as Promising Anti‐Trypanosoma brucei Agents

Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas disease and African sleeping sickness, respectively. There is an urgent need for the development of new drugs against both diseases due to the lack of adequate cures and emerging drug resistance. One promising strategy for the discovery of small‐molecule therapeutics against parasitic diseases has been to target the major cysteine proteases such as cruzain for T. cruzi, and rhodesain/TbCatB for T. brucei. Azadipeptide nitriles belong to a novel class of extremely potent cysteine protease inhibitors against papain‐like proteases. We herein report the design, synthesis, and evaluation of a series of azanitrile‐containing compounds, most of which were shown to potently inhibit both recombinant cruzain and rhodesain at low nanomolar/picomolar ranges. A strong correlation between the potency of rhodesain inhibition (i.e., target‐based screening) and trypanocidal activity (i.e., whole‐organism‐based screening) of the compounds was observed. To facilitate detailed studies of this important class of inhibitors, selected hit compounds from our screenings were chemically converted into activity‐based probes (ABPs), which were subsequently used for in situ proteome profiling and cellular localization studies to further elucidate potential cellular targets (on and off) in both the disease‐relevant bloodstream form (BSF) and the insect‐residing procyclic form (PCF) of Trypanosoma brucei. Overall, the inhibitors presented herein show great promise as a new class of anti‐trypanosome agents, which possess better activities than existing drugs. The activity‐based probes generated from this study could also serve as valuable tools for parasite‐based proteome profiling studies, as well as bioimaging agents for studies of cellular uptake and distribution of these drug candidates. Our studies therefore provide a good starting point for further development of these azanitrile‐containing compounds as potential anti‐parasitic agents.     

Natural Compounds against Alzheimer’s Disease: Molecular Recognition of Aβ1–42 Peptide by Salvia sclareoides Extract and its Major Component,Rosmarinic Acid,as Investigated by NMR

Amyloid peptides, Aβ1–40 and Aβ1–42, represent major molecular targets to develop potential drugs and diagnostic tools for Alzheimer’s Disease (AD). In fact, oligomeric and fibrillar aggregates generated by these peptides are amongst the principal components of amyloid plaques found post mortem in patients suffering from AD. Rosmarinic acid has been demonstrated to be effective in preventing the aggregation of amyloid peptides in vitro and to delay the progression of the disease in animal models. Nevertheless, no information is available about its molecular mechanism of action. Herein, we report the NMR characterization of the interaction of Salvia sclareoides extract and that of its major component, rosmarinic acid, with Aβ1–42 peptide, whose oligomers have been described as the most toxic Aβ species in vivo. Our data shed light on the structural determinants of rosmarinic acid–Aβ1–42 oligomers interaction, thus allowing the elucidation of its mechanism of action. They also provide important information for the rational design of new compounds with higher affinity for Aβ peptides to generate new anti‐amyloidogenic molecules and/or molecular tools for the specific targeting of amyloid aggregates in vivo. In addition, we identified methyl caffeate, another natural compound present in different plants and human diet, as a good ligand of Aβ1–42 oligomers, which also shows anti‐amyloidogenic activity. Finally, we demonstrated the possibility to exploit STD‐NMR and trNOESY experiments to screen extracts from natural sources for the presence of Aβ peptide ligands.     

Pharmacological Evaluation of Novel Organoiron Dendrimers as Antimicrobial and Anti‐Inflammatory Agents

The synthesis of a novel and attractive class of nonsteroidal anti‐inflammatory and antimicrobial organoiron dendrimers attached to the well‐known drug ibuprofen is achieved. The structures of these dendrimers are established by spectroscopic and analytical techniques. The antimicrobial activity of these dendrimers is investigated and tested against five human pathogenic Gram‐positive and Gram‐negative bacteria, and minimum inhibitory concentrations are reported. Some of these synthesized dendrimers exhibit higher inhibitory activity against methicillin‐resistant Staphylococcus aureus, vancomycin‐resistant Enterococcus faecium, and Staphylococcus warneri compare to the reference drugs. As well, the in vitro and in vivo anti‐inflammatory activities of these dendrimers are evaluated. The results of in vivo anti‐inflammatory activity and histopathology of inflamed paws show that all dendrimers display considerable anti‐inflammatory activity; however, second‐generation dendrimer ( G2‐D6 ) shows the best anti‐inflammatory activity, which is more potent than the commercial drug ibuprofen at the same tested dose. Results of the toxicity study reveal that G2‐D6  is the safest drug on biological tissues.     

Preparation and application of polyvinyl alcohol‐decorated cell membrane chromatography for screening anti‐osteoporosis components from Liuwei Dihuang decoction‐containing serum

Cell membrane chromatography is a powerful tool for screening active components from complex matrices. New cell membrane carriers need to be developed to increase the coverage of cell membranes on the surface of stationary phases, thereby improving cell membrane chromatographic retention. In this work, we prepared polyvinyl alcohol‐poly dimethyl diallyl ammonium chloride modified silica gel as a cell membrane carrier. Osteoblast cell was used as cell membrane source, which was widely used to evaluate the osteogenic activity of drugs. The new cell membrane chromatographic stationary phase was used to screen anti‐osteoporosis components in Liuwei Dihuang decoction‐containing serum. The chemical structures of the new modified materials were characterized by scanning electronic microscopy, Fourier transform infrared spectroscopy, and elemental analysis characterization. Compared with the common cell membrane column, the cell membrane coverage of this modified material was increased by 30%, and thus provided a stronger retention effect in positive drugs. Nineteen metabolites in rat serum samples were retained on the cell membrane chromatography and identified by ultra‐high‐performance liquid chromatography/time‐of‐flight mass spectrometry. Among those, four components (morroniside, catalpol, loganin, and acteoside) were selected for in vitro pharmacodynamics validation. They significantly increased the osteoblast proliferation. The new modified material was successfully applied to screen anti‐osteoporosis components from Liuwei Dihuang Decoction‐containing serum.     

Screening and isolation of cyclooxygenase‐2 inhibitors from the stem bark of Phellodendron amurense Ruprecht by ultrafiltration with liquid chromatography and tandem mass spectrometry,and complex chromatography

Nonsteroidal anti‐inflammatory drugs appear to reduce the risk of developing cancer. One mechanism through which nonsteroidal anti‐inflammatory drugs act to prevent carcinogenesis is inhibition of the activity of the enzyme cyclooxygenase‐2. The cyclooxygenase‐2 inhibitors are widely used to reduce the risk of developing cancer. Natural products are considered to be a promising source of several novel cyclooxygenase‐2 inhibitors. Ultrafiltration with liquid chromatography and mass spectrometry is an efficient method that can be applied to rapidly screen and identify the ligands from the barks of Phellodendron amurense Ruprecht. A continuous online method comprised of pressurized liquid extraction, countercurrent chromatography, and semi‐preparative liquid chromatography was developed for the efficient scaled‐up production of eight compounds with high purities. The bioactivities of the separated compounds were assessed by an in vitro enzyme inhibition assay. The use of bioactivity screening method combined with preparation method of bioactive compounds and an in vitro enzyme inhibition assay facilitated the efficient screening and isolation of the cyclooxygenase‐2 inhibitors from complex samples. This could be used as an efficient method for the large‐scale production of functional ingredients.     

An Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine‐56

The stress‐inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine‐targeted irreversible inhibitor. Using rational design, we adapted a validated 8‐N ‐benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine‐56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chemical probes and drugs.     

Comparison of reversed‐phase liquid chromatography and hydrophilic interaction chromatography for the fingerprint analysis of Radix isatidis

Radix isatidis is a famous anti‐influenza virus herbal medicine traditionally taken as a water decoction. However, the chemical fingerprint analysis of Radix isatidis is dominantly based on RPLC, from which it is difficult to obtain fingerprint information of hydrophilic compounds. Here, we developed the separation of Radix isatidis by RPLC and hydrophilic interaction chromatography, comparing the traditional RPLC fingerprint with the hydrophilic interaction chromatography fingerprint. Besides, an anti‐viral assay of Radix isatidis was conducted to evaluate its efficacy. The fingerprint–efficacy relationships between the fingerprints and the anti‐viral activity were further investigated with principal component regression analysis. The results showed that the anti‐viral activity correlated better with the hydrophilic interaction chromatography fingerprint than with the RPLC fingerprint. This study indicates that hydrophilic interaction chromatography could not only be a complementary method to increase the fingerprint coverage of conventional RPLC fingerprint, but also can better represent the efficacy and quality of Radix isatidis.     

Chemical Kinetic Strategies for High‐Throughput Screening of Protein Aggregation Modulators

Insoluble aggregates staining positive to amyloid dyes are known histological hallmarks of different neurodegenerative disorders and of type II diabetes. Soluble oligomers are smaller assemblies whose formation prior to or concomitant with amyloid deposition has been associated to the processes of disease propagation and cell death. While the pathogenic mechanisms are complex and differ from disease to disease, both types of aggregates are important biological targets subject to intense investigation in academia and industry. Here we review recent advances in the fundamental understanding of protein aggregation that can be used on the development of anti‐amyloid and anti‐oligomerization drugs. Specifically, we pinpoint the chemical kinetic aspects that should be attended during the development of high‐throughput screening assays and in the hit validation phase. The strategies here devised are expected to establish a connection between basic research and pharmaceutical innovation.