N‐Benzyl Substitution of Polyhydroxypyrrolidines: The Way to Selective Inhibitors of Golgi α‐Mannosidase II

Inhibition of the biosynthesis of complex N‐glycans in the Golgi apparatus influences progress of tumor growth and metastasis. Golgi α‐mannosidase II (GMII) has become a therapeutic target for drugs with anticancer activities. One critical task for successful application of GMII drugs in medical treatments is to decrease their unwanted co‐inhibition of lysosomal α‐mannosidase (LMan), a weakness of all known potent GMII inhibitors. A series of novel N‐substituted polyhydroxypyrrolidines was synthesized and tested with modeled GH38 α‐mannosidases from Drosophila melanogaster (GMIIb and LManII). The most potent structures inhibited GMIIb (K_i=50–76 μm , as determined by enzyme assays) with a significant selectivity index of IC₅₀(LManII)/IC₅₀(GMIIb) >100. These compounds also showed inhibitory activities in in vitro assays with cancer cell lines (leukemia, IC₅₀=92–200 μm ) and low cytotoxic activities in normal fibroblast cell lines (IC₅₀>200 μm ). In addition, they did not show any significant inhibitory activity toward GH47 Aspergillus saitoiα1,2‐mannosidase. An appropriate stereo configuration of hydroxymethyl and benzyl functional groups on the pyrrolidine ring of the inhibitor may lead to an inhibitor with the required selectivity for the active site of a target α‐mannosidase.     

Antagonism of the Stat3-Stat3 protein dimer with salicylic acid based small molecules

More than 50 new inhibitors of the oncogenic Stat3 protein were identified through a structure–activity relationship (SAR) study based on the previously identified inhibitor S3I‐201 (IC₅₀=86 μM , K_i>300 μM ). A key structural feature of these inhibitors is a salicylic acid moiety, which, by acting as a phosphotyrosine mimetic, is believed to facilitate binding to the Stat3 SH2 domain. Several of the analogues exhibit higher potency than the lead compound in inhibiting Stat3 DNA binding activity, with an in vitro IC₅₀ range of 18.7–51.9 μM , and disruption of Stat3–pTyr peptide interactions with K_i values in the 15.5–41 μM range. One agent in particular exhibited potent inhibition of Stat3 phosphorylation in both breast and multiple myeloma tumor cells, suppressed the expression of Stat3 target genes, and induced antitumor effects in tumor cells harboring activated Stat3 protein.     

2-amino-3,4,5-trimethoxybenzophenones as potent tubulin polymerization inhibitors

A series of novel 2‐amino‐3,4,5‐trimethoxybenzophenone analogues exhibited excellent activity as tubulin polymerization inhibitors by targeting the colchicine binding site of microtubules. The lead compound 17 exhibited an IC₅₀ value of 1.6 μM , similar to that of combretastatin A‐4 (IC₅₀=1.9 μM ). It also displayed remarkable anti‐proliferative activity, with IC₅₀ values ranging from 7–16 nM against a variety of human cancer cell lines and one MDR(+) cancer cell line. SAR information indicated that the introduction of an amino group at the C2 position of benzophenone ring A and the C3’ position of benzophenone ring B play important roles in maximizing activity.     

Discovery of 3‐Hydroxy‐3‐phenacyloxindole Analogues of Isatin as Potential Monoamine Oxidase Inhibitors

A series of 3‐hydroxy‐3‐phenacyloxindole analogues of isatin were designed, synthesized, and evaluated in vitro for their inhibitory activity toward monoamine oxidase (MAO) A and B. Most of the synthesized compounds proved to be potent and selective inhibitors of MAO‐A rather than MAO‐B. 1‐Benzyl‐3‐hydroxy‐3‐(4′‐hydroxyphenacyl)oxindole (compound 18 ) showed the highest MAO‐A inhibitory activity (IC₅₀: 0.009±0.001 μm , K_i: 3.69±0.003 nm ) and good selectivity (selectivity index: 60.44). Kinetic studies revealed that compounds 18 and 16 (1‐benzyl‐3‐hydroxy‐3‐(4′‐bromophenacyl)oxindole) exhibit competitive inhibition against MAO‐A and MAO‐B, respectively. Structure–activity relationship studies suggested that the 3‐hydroxy group is an essential feature for these analogues to exhibit potent MAO‐A inhibitory activity. Computational studies revealed the possible molecular interactions between the inhibitors and MAO isozymes. The computational data obtained are congruent with experimental results. Further studies on the lead inhibitors, including co‐crystallization of inhibitor–MAO complexes and in vivo evaluations, are essential for their development as potential therapeutic agents for the treatment of MAO‐associated neurological disorders.     

Synthesis and Biological Evaluation of 1‐Methyl‐1H‐indole–Pyrazoline Hybrids as Potential Tubulin Polymerization Inhibitors

A series of 1‐methyl‐1H‐indole–pyrazoline hybrids were designed, synthesized, and biologically evaluated as potential tubulin polymerization inhibitors. Among them, compound e19 [5‐(5‐bromo‐1‐methyl‐1H‐indol‐3‐yl)‐3‐(3,4,5‐trimethoxyphenyl)‐4,5‐dihydro‐1H‐pyrazole‐1‐carboxamide] showed the most potent inhibitory effect on tubulin assembly (IC₅₀=2.12 μm ) and in vitro growth inhibitory activity against a panel of four human cancer cell lines (IC₅₀ values of 0.21–0.31 μm ). Further studies confirmed that compound e19 can induce HeLa cell apoptosis, cause cell‐cycle arrest in G₂/M phase, and disrupt the cellular microtubule network. These studies, along with molecular docking and 3D‐QSAR modeling, provide an important basis for further optimization of compound e19 as a potential anticancer agent.     

Norcantharidin Analogues: Synthesis,Anticancer Activity and Protein Phosphatase 1 and 2A Inhibition

Cantharidin ( 1 ) and its derivatives are of significant interest as serine/threonine protein phosphatase 1 and 2A inhibitors. Additionally, compounds of this type have displayed growth inhibition of various tumour cell lines. To further explore both of these inhibition pathways, a number of amide–acid norcantharidin analogues ( 15 – 26 ) were prepared. Compounds 23 and 24 , containing two carboxylic acid residues, showed good PP1 and PP2A activity, with IC₅₀ values of ～15 and ～3 μm, respectively. Substituted aromatic amide analogues 45 , 48 , 49 , 52 , 53 , and 54 also displayed good PP1 and PP2A inhibition, with IC₅₀ values in the range of 15–10 μM (PP1) and 11–5 μM (PP2A). However, bulky ortho substituents on the aromatic ring caused the aromatic ring to be skewed from the NCO planarity, leading to a decrease in PP1 and PP2A inhibition. A number of analogues, 20 , 22, 25 and 46 , showed excellent tumour growth inhibition, with 46 in particular being more potent than the lead, norcantharidin 2 .     

Nucleobase Modified Adefovir (PMEA) Analogues as Potent and Selective Inhibitors of Adenylate Cyclases from Bordetella pertussis and Bacillus anthracis

A series of 13 acyclic nucleoside phosphonates (ANPs) as bisamidate prodrugs was prepared. Five compounds were found to be non‐cytotoxic and selective inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT) in J774A.1 macrophage cell‐based assays. The 8‐aza‐7‐deazapurine derivative of adefovir (PMEA) was found to be the most potent ACT inhibitor in the series (IC₅₀=16 nm ) with substantial selectivity over mammalian adenylate cyclases (mACs). AC inhibitory properties of the most potent analogues were confirmed by direct evaluation of the corresponding phosphonodiphosphates in cell‐free assays and were found to be potent inhibitors of both ACT and edema factor (EF) from Bacillus anthracis (IC₅₀ values ranging from 0.5 to 21 nm ). Moreover, 7‐halo‐7‐deazapurine analogues of PMEA were discovered to be potent and selective mammalian AC1 inhibitors (no inhibition of AC2 and AC5) with IC₅₀ values ranging from 4.1 to 5.6 μm in HEK293 cell‐based assays.     

Synthesis and Evaluation of 1‐(1‐(Benzo[b]thiophen‐2‐yl)cyclohexyl)piperidine (BTCP) Analogues as Inhibitors of Trypanothione Reductase

Thirty two analogues of phencyclidine were synthesised and tested as inhibitors of trypanothione reductase (TryR), a potential drug target in trypanosome and leishmania parasites. The lead compound BTCP ( 1 , 1‐(1‐benzo[b]thiophen‐2‐yl‐cyclohexyl) piperidine) was found to be a competitive inhibitor of the enzyme (K_i=1 μM ) and biologically active against bloodstream T. brucei (EC₅₀=10 μM ), but with poor selectivity against mammalian MRC5 cells (EC₅₀=29 μM ). Analogues with improved enzymatic and biological activity were obtained. The structure–activity relationships of this novel series are discussed.     

Anti‐Dengue‐Virus Activity and Structure–Activity Relationship Studies of Lycorine Derivatives

Dengue is a systemic viral infection that is transmitted to humans by Aedes mosquitoes. No vaccines or specific therapeutics are currently available for dengue. Lycorine, which is a natural plant alkaloid, has been shown to possess antiviral activities against flaviviruses. In this study, a series of novel lycorine derivatives were synthesized and assayed for their inhibition of dengue virus (DENV) in cell cultures. Among the lycorine analogues, 1‐acetyllycorine exhibited the most potent anti‐DENV activity (EC₅₀=0.4 μM ) with a reduced cytotoxicity (CC₅₀>300 μM ), which resulted in a selectivity index (CC₅₀/EC₅₀) of more than 750. The ketones 1‐acetyl‐2‐oxolycorine (EC₅₀=1.8 μM ) and 2‐oxolycorine (EC₅₀=0.5 μM ) also exhibited excellent antiviral activities with low cytotoxicity. Structure–activity relationships for the lycorine derivatives against DENV are discussed. A three‐dimensional quantitative structure–activity relationship model was established by using a comparative molecular‐field analysis protocol in order to rationalize the experimental results. Further modifications of the hydroxy group at the C1 position with retention of a ketone at the C2 position could potentially lead to inhibitors with improved overall properties.     

A Structure‐Based Virtual Screening Approach toward the Discovery of Histone Deacetylase Inhibitors: Identification of Promising Zinc‐Chelating Groups

The inhibitors of histone deacetylases (HDACs) have drawn a great deal of attention due to their promising potential as small‐molecule therapeutics for the treatment of cancer. By means of virtual screening with docking simulations under consideration of the effects of ligand solvation, we were able to identify six novel HDAC inhibitors with IC₅₀ values ranging from 1 to 100 μM . These newly identified inhibitors are structurally diverse and have various chelating groups for the active site zinc ion, including N‐[1,3,4]thiadiazol‐2‐yl sulfonamide, N‐thiazol‐2‐yl sulfonamide, and hydroxamic acid moieties. The former two groups are included in many drugs in current clinical use and have not yet been reported as HDAC inhibitors. Therefore, they can be considered as new inhibitor scaffolds for the development of anticancer drugs by structure–activity relationship studies to improve the inhibitory activities against HDACs. Interactions with the HDAC1 active site residues responsible for stabilizing these new inhibitors are addressed in detail.     

Engineered s-Triazine-Based Dendrimer-Honokiol Conjugates as Targeted MMP-2/9 Inhibitors for Halting Hepatocellular Carcinoma

Despite the advances in developing MMP-2/9 inhibitors, off-target side effects and pharmacokinetics problems remain major challenges hindering their clinical success in cancer therapy. However, recent targeting strategies have clearly revitalized MMP research. Herein, we introduce new s-triazine-based dendrimers endowed with intrinsic MMP-2/9 inhibitory potential and tetherable to hepatocellular carcinoma-specific targeting ligands and anticancer agents via biodegradable linkages for targeted therapy. The designed dendrimeric platform was built with potential zinc-binding branching linkers (hydrazides) and termini (carboxylic acids and hydrazides) to confer potency against MMP-2/9. Preliminary cytotoxicity screening and MMP-2/9 inhibition assay of the free dendrimers revealed promising potency (MMP-9; IC₅₀=0.35–0.57 μM, MMP-2; IC₅₀=0.39–0.77 μM) within their safe doses (EC₁₀₀=94.15–42.75 μM). The hydrazide dendrimer was comparable to NNGH and superior to the carboxylic acid analogue. MTT assay showed that the free dendrimers were superior to the reference anticancer agent honokiol. Their anticancer potency was enhanced by HK conjugation, targeting ligands installation and PEGylation as exemplified by the hydrazide dendrimer conjugate (TPG₃−NH₂)-SuHK-FA-SuPEG (Huh-7; IC₅₀=5.54 μM, HepG-2; IC₅₀=10.07 μM) being 4 folds more active than HK, followed by the carboxylic acid conjugate (TPG₃−OH)-HK-LA-PEG (Huh-7; IC₅₀=14.97, HepG-2; IC₅₀=21.29 μM). This was consistent with apoptosis studies.     

Structure‐Guided Design of Thiazolidine Derivatives as Mycobacterium tuberculosis Pantothenate Synthetase Inhibitors

The pantothenate biosynthetic pathway is essential for the persistent growth and virulence of Mycobacterium tuberculosis (Mtb) and one of the enzymes in the pathway, pantothenate synthetase (PS, EC: 6.3.2.1), encoded by the panC gene, has become an appropriate target for new therapeutics to treat tuberculosis. Herein, we report nanomolar thiazolidine inhibitors of Mtb PS developed by a rational inhibitor design approach. The thiazolidine compounds were discovered by using energy‐based pharmacophore modelling and subsequent in vitro screening, which resulted in compounds with a half maximal inhibitory concentration (IC₅₀) value of (1.12±0.12) μM . These compounds were subsequently optimised by a combination of modelling and synthetic chemistry. Hit expansion of the lead by chemical synthesis led to an improved inhibitor with an IC₅₀ value of 350 nM and an Mtb minimum inhibitory concentration (MIC) of 1.55 μM . Some of these compounds also showed good activity against dormant Mtb cells.     

Development of Second‐Generation Small‐Molecule RhoA Inhibitors with Enhanced Water Solubility,Tissue Potency,and Significant in vivo Efficacy

RhoA, a member of the Rho GTPases, is involved in a variety of cellular functions and could be a suitable therapeutic target for the treatment of cardiovascular diseases. However, few small‐molecule RhoA inhibitors have been reported. Based on our previously reported lead compounds, 32 new 2‐substituted quinoline (or quinoxaline) derivatives were synthesized and tested in biological assays. Six compounds showed high RhoA inhibitory activities, with IC₅₀ values of 1.17–1.84 μM . Among these, (E)‐3‐(3‐(ethyl(quinolin‐2‐yl)amino)phenyl)acrylic acid ( 26 b ) and (E)‐3‐(3‐(butyl(quinolin‐2‐yl)amino)phenyl)acrylic acid ( 26 d ) demonstrated noticeable vasorelaxation effects against phenylephrine‐induced contraction in thoracic aorta artery rings, and compound 26 b had good water solubility and showed significant in vivo efficacy, which was similar to that of 5‐(1,4‐diazepane‐1‐sulfonyl)isoquinoline (fasudil) in a subarachnoid hemorrhage–cardiovascular model. To the best of our knowledge, compound 26 b is the first example of a small‐ molecule RhoA inhibitor with potent in vivo efficacy, which could serve as a good lead for designing cardiovascular agents.     

Fluorophore‐Labeled Cyclooxygenase‐2 Inhibitors for the Imaging of Cyclooxygenase‐2 Overexpression in Cancer: Synthesis and Biological Studies

A group of cyclooxygenase‐2 (COX‐2)‐specific fluorescent cancer biomarkers were synthesized by linking the anti‐inflammatory drugs ibuprofen, (S)‐naproxen, and celecoxib to the 7‐nitrobenzofurazan (NBD) fluorophore. In vitro COX‐1/COX‐2 inhibition studies indicated that all of these fluorescent conjugates are COX‐2 inhibitors (IC₅₀ range: 0.19–23.0 μM ) with an appreciable COX‐2 selectivity index (SI≥4.3–444). In this study the celecoxib–NBD conjugate N‐(2‐((7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)amino)ethyl)‐4‐(5‐(p‐tolyl)‐3‐(trifluoromethyl)‐1H‐pyrazol‐1‐yl)benzenesulfonamide ( 14 ), which displayed the highest COX‐2 inhibitory potency and selectivity (COX‐2 IC₅₀=0.19 μM ; SI=443.6), was identified as an impending COX‐2‐specific biomarker for the fluorescence imaging of cancer using a COX‐2‐expressing human colon cancer cell line (HCA‐7).     

Design,Synthesis, and Evaluation of an 18F‐Labeled Radiotracer Based on Celecoxib–NBD for Positron Emission Tomography (PET) Imaging of Cyclooxygenase‐2 (COX‐2)

A series of novel fluorine‐containing cyclooxygenase‐2 (COX‐2) inhibitors was designed and synthesized based on the previously reported fluorescent COX‐2 imaging agent celecoxib–NBD ( 3 ; NBD=7‐nitrobenzofurazan). In vitro COX‐1/COX‐2 inhibitory data show that N‐(4‐fluorobenzyl)‐4‐(5‐p‐tolyl‐3‐trifluoromethylpyrazol‐1‐yl)benzenesulfonamide ( 5 ; IC₅₀=0.36 μM , SI>277) and N‐fluoromethyl‐4‐(5‐p‐tolyl‐3‐trifluoromethylpyrazol‐1‐yl)benzenesulfonamide ( 6 ; IC₅₀=0.24 μM , SI>416) are potent and selective COX‐2 inhibitors. Compound 5 was selected for radiolabeling with the short‐lived positron emitter fluorine‐18 (¹⁸F) and evaluated as a positron emission tomography (PET) imaging agent. Radiotracer [¹⁸F] 5 was analyzed in vitro and in vivo using human colorectal cancer model HCA‐7. Although radiotracer uptake into COX‐2‐expressing HCA‐7 cells was high, no evidence for COX‐2‐specific binding was found. Radiotracer uptake into HCA‐7 tumors in vivo was low and similar to that of muscle, used as reference tissue.     

Aryl Bis‐Sulfonamide Inhibitors of IspF from Arabidopsis thaliana and Plasmodium falciparum

2‐Methylerythritol 2,4‐cyclodiphosphate synthase (IspF) is an essential enzyme for the biosynthesis of isoprenoid precursors in plants and many human pathogens. The protein is an attractive target for the development of anti‐infectives and herbicides. Using a photometric assay, a screen of 40 000 compounds on IspF from Arabidopsis thaliana afforded symmetrical aryl bis‐sulfonamides that inhibit IspF from A. thaliana (AtIspF) and Plasmodium falciparum (PfIspF) with IC₅₀ values in the micromolar range. The ortho‐bis‐sulfonamide structural motif is essential for inhibitory activity. The best derivatives obtained by parallel synthesis showed IC₅₀ values of 1.4 μm against PfIspF and 240 nm against AtIspF. Substantial herbicidal activity was observed at a dose of 2 kg ha^?1. Molecular modeling studies served as the basis for an in silico search targeted at the discovery of novel, non‐symmetrical sulfonamide IspF inhibitors. The designed compounds were found to exhibit inhibitory activities in the double‐digit micromolar IC₅₀ range.     

Aryloxyethyl Thiocyanates Are Potent Growth Inhibitors of Trypanosoma cruzi and Toxoplasma gondii

As a part of our project aimed at searching for new safe chemotherapeutic agents against parasitic diseases, several compounds structurally related to the antiparasitic agent WC‐9 (4‐phenoxyphenoxyethyl thiocyanate), which were modified at the terminal phenyl ring, were designed, synthesized, and evaluated as growth inhibitors against Trypanosoma cruzi, the etiological agent of Chagas disease, and Toxoplasma gondii, the parasite responsible of toxoplasmosis. Most of the synthetic analogues exhibited similar antiparasitic activity and were slightly more potent than our lead WC‐9. For example, two trifluoromethylated derivatives exhibited ED₅₀ values of 10.0 and 9.2 μM against intracellular T. cruzi, whereas they showed potent action against tachyzoites of T. gondii (ED₅₀ values of 1.6 and 1.9 μM against T. gondii). In addition, analogues of WC‐9 in which the terminal aryl group is in the meta position with respect to the alkyl chain bearing the thiocyanate group showed potent inhibitory action against both T. cruzi and T. gondii at the very low micromolar range, which suggests that a para‐phenyl substitution pattern is not necessary for biological activity.     

Multimerization of Peptide Mimotopes for Blocking of Factor VIII Neutralizing Antibodies

About 30 % of patients with severe hemophilia A develop neutralizing antibodies (inhibitors) to coagulation factor VIII (FVIII) upon treatment with exogenous factor preparations. Two peptides, C6 (NPVENMMDRDSQ) and H10 (QSPWQTWFTRAL), that mimic putative inhibitor epitopes (mimotopes), were previously selected by phage display screening of plasma samples from patients with inhibitors. Synthetic peptide mimotopes inhibited IgG binding to FVIII (IC₅₀: 30–50 μM ). This effect was increased by an equimolar combination of both mimotopes. Mimotopes were fused to the C‐terminal multimerization domain of the C4bp α‐chain and expressed as multimers in 293T cells. Multimerized mimotopes showed improved binding to anti‐FVIII IgG and prolonged in vitro half‐life relative to synthetic peptides. The two mimotopes were combined in heteromultimers by co‐transfection of 293T cells with respective vectors, resulting in bi‐specific molecules that almost completely blocked polyclonal antibody binding to FVIII (IC₅₀: 2–3 μM ). This strategy is capable of functionally improving synthetic peptides by multimerization and could provide a basis for novel therapeutic approaches for patients with hemophilia A and inhibitors.