In Vivo Bioimaging and Photodynamic Therapy Based on Two-Photon Fluorescent Conjugated Polymers Containing Dibenzothiophene-S,S-dioxide Derivatives.

As a critical component for photodynamic therapy toward cancer treatment, photosensitizers require high photoinduced reactive oxygen species generation efficiency, good biocompatibility, and high phototoxicity. Herein, a series of donor-acceptor conjugated polymers containing dibenzothiophene-S,S-dioxide derivatives are designed and synthesized, which can be used as effective photosensitizers. The resulting copolymer PTA5 shows strong green light emission with high photoluminescence quantum yields owing to the intercrossed excited state of local existed and charge transfer states. The PTA5 nanoparticles can be fabricated by encapsulation with a biocompatible polymer matrix. Upon excitation at 800 nm, these nanoparticles present a relatively large two-photon absorption cross section of 3.29 × 106 GM. These nanoparticles also exhibit good photostability in water and thus can be utilized for bioimaging. The tissue-penetrating depths of up to 170 µm for hepatic vessels and 380 µm for blood vessels of mouse ear were achieved using PTA5 nanoparticles. Furthermore, PTA5 nanoparticles show impressive reactive oxygen species generation capability under the irradiation of a white light source. This can be attributed to the effective intersystem crossing between high-level excited state. Upon irradiation with white light (400-700 nm) at 50 mW cm-2 for 5 min every other day, the tumor growth can be effectively suppressed in the presence of PTA5 nanoparticles. These findings demonstrate that PTA5 nanoparticles can be used as a photosensitizer for photodynamic therapy.

Formulation of Stattic as STAT3 inhibitor in nanostructured lipid carriers (NLCs) enhances efficacy of doxorubicin in melanoma cancer cells.

Nowadays, nanoparticle-based combination therapy has been emerging as huge innovation in cancer treatment. Here, we studied the effect of Stattic (STAT3 inhibitor) loaded in nanostructured lipid carriers (NLCs) on enhancing the efficacy, cytotoxicity, and induction of apoptosis of doxorubicin in B16F10 mouse melanoma cancer cell. The evaluation of Stattic-loaded NLCs has been done in terms of zeta potential, particle size, scanning electron microscope (SEM), and cellular uptake. MTT assay was applied to evaluate the cell proliferation. Apoptotic cell death and identification of early and late apoptosis were assessed by DAPI staining and Annexin V/PI staining, respectively. Real-time RT-PCR was applied to measure the effects of doxorubicin and/or Stattic on key apoptotic genes such as Bad, Survivin, HIF1, and STAT3. The Stattic formulated into NLCs shown mean particle size of 56 ± 7 nm which was confirmed by SEM. The IC50 values for Stattic and doxorubicin were 2.95 ± 0.52 µM and 1.21 ± 0.36 µM, respectively. Stattic-loaded NLCs diminished percent of cell proliferation from 68 ± 6.8 to 54 ± 3.7% (p < 0.05). Combinational treatment of the cells with Stattic-loaded nanoparticles and doxorubicin give rise to a significant increase in the percentage of apoptosis (p < 0.05). The study of gene expression profile has shown a remarkable decrease in anti-apoptotic gene, Survivin, along with smooth decline in HIF1 as angiogenesis intermediator and increase in Bad mRNA levels. Our results recommend that NLCs as novel technology have potent strategy to augment efficacy of current chemotherapeutic agent in melanoma cancer cells.

First Example of Catalytic Synthesis of Cyclic S-Containing Di- and Triperoxides.

An efficient method for the synthesis of tetraoxathiaspiroalkanes, tetraoxathiocanes, and hexaoxathiadispiroalkanes was developed by reactions of pentaoxacanes, pentaoxaspiroalkanes, and heptaoxadispiroalkanes with hydrogen sulfide in the presence of a catalyst, Sm(NO3)3·6H2O. We found that the synthesized S-containing di- and triperoxides exhibit high cytotoxic activity against Jurkat, K562, U937, and HL60 tumor cultures, and fibroblasts.

Enantioselective Radical Construction of 5-Membered Cyclic Sulfonamides by Metalloradical C-H Amination.

Both arylsulfonyl and alkylsulfonyl azides can be effectively activated by the cobalt(II) complexes of D2-symmetric chiral amidoporphyrins for enantioselective radical 1,5-C-H amination to stereoselectively construct 5-membered cyclic sulfonamides. In addition to C-H bonds with varied electronic properties, the Co(II)-based metalloradical system features chemoselective amination of allylic C-H bonds and is compatible with heteroaryl groups, producing functionalized 5-membered chiral cyclic sulfonamides in high yields with high enantioselectivities. The unique profile of reactivity and selectivity of the Co(II)-catalyzed C-H amination is attributed to its underlying stepwise radical mechanism, which is supported by several lines of experimental evidence.

Design, synthesis and molecular modelling of new bulky Fananserin derivatives with altered pharmacological profile as potential antidepressants.

It is now known that many neurotransmitter systems are responsible for diseases of the central nervous system (CNS). One of the most common CNS disease is depression. Considering that in the treatment and the genesis of depression, the most important are the serotonin receptors from 5-HT1A, 5-HT2A, 5-HT6 and 5-HT7 groups, and dopamine D2R this article describes searching for group of new ligands for mentioned receptors. In the searching for potentially useful compound, we decided to start from the structure of well-known Fananserin. We tried to developed new derivatives, with changed profile of activity compared to Fananserin. Literature analysis and virtual screening emerged group of halogenated long-chain arylpiperazines derivatives of 1,8 naphthosultam/lactam with hexyl carbon chain to synthesis. The compounds obtaining method was developed with a microwave assisted synthesis. Reactions were carried out in acetonitrile, water or in solvent-free conditions. The obtained compounds were tested for their affinity for the serotonin receptors mentioned above. The work managed to obtain compounds acting on selected serotonin receptors, including multifunctional 5-HT1A/5-HT7/D2 ligand 5k, dual 5-HT1A/D2 ligand 5j and selective 5-HT1A ligands 5r and 5c. The SAR analysis showed a visible dependence of affinity for the 5-HT6 receptors from structure of ligands. This relationship was discussed using molecular docking methods. A conformal analysis was also performed for selected ligands and the Fukui indexes were calculated using the DFT (B3LYP/6-311+G (d,p) level of theory) methods. The conducted research and analysis using molecular docking methods allows for selecting further pathways of structural modifications in the design of new ligands for serotonin receptors belonging to the group mentioned. What is more, conducted research show the potential using of Fukui indices to predict the biological activity of new molecules.

Varacin-1, a novel analog of varacin C, induces p53-independent apoptosis in cancer cells through ROS-mediated reduction of XIAP.

Varacin C is a promising anticancer agent and possesses acid-promoted and photo-induced DNA-damaging activities. In this study, we synthesized an analog varacin-1 (VCA-1) and examined its anticancer potentials. The results demonstrated that VCA-1 caused dose-dependent apoptotic cell death in cancer cells. Note that this action is independent of p53 status, because VCA-1 induced similar levels of apoptosis in two different panels of cell lines (HCT116 p53- wild-type vs. HCT116 p53-knockout colon cancer cells, and p53-expressing U2OS vs. p53-deficient saos2 osteosarcoma cancer cells). VCA-1-induced apoptosis was found to be mainly via the extrinsic apoptosis pathway involving caspase-8 activation and XIAP reduction. Forced over-expression of XIAP markedly prevented apoptosis, indicating its essential role in VCA-1 induced apoptosis. On the other hand, VCA-1 treatment enhanced intracellular ROS (reactive oxygen species) generation also in a p53-independent manner, and consequently promoted caspase activation. Pretreatment of N-acetyl cysteine (an antioxidant), rather than z-VAD (specific caspase inhibitor), markedly prevented XIAP reduction, suggesting that XIAP reduction may be resulted from oxidative stress. In conclusion, data from this study reveal the essential roles of ROS generation and XIAP reduction in VCA-1-induced apoptosis in cancer cells. VCA-1 may be a novel cancer therapeutic agent, especially in p53-mutant human cancers.

A Next Generation Synthesis of BACE1 Inhibitor Verubecestat (MK-8931).

The development of a commercial manufacturing route to verubecestat (MK-8931) is described, highlights of which include the application of a continuous processing step to outcompete fast proton transfer in a Mannich-type ketimine addition, a copper-catalyzed amidation reaction, and an optimized guanidinylation procedure to form the key iminothiadiazine dioxide core.

Macrocyclic glycopeptide chiral selectors bonded to core-shell particles enables enantiopurity analysis of the entire verubecestat synthetic route.

Verubecestat is an inhibitor of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) being evaluated in clinical trials for the treatment of Alzheimer's disease. Synthetic route development involves diastereoselective transformations with a need for enantiomeric excess (ee) determination of each intermediate and final active pharmaceutical ingredient (API). The analytical technical package of validated methods relies on enantioselective SFC and RPLC separations using multiple 3 and 5 µm coated polysaccharide-based chiral stationary phases (CSPs) and mobile phases combinations. Evaluation of recently developed chiral columns revealed a single chiral selector (Teicoplanin) bonded to 2.7 µm core-shell particles using H3PO4 in H2O/ACN and triethylammonium acetate: methanol based eluents at different isocratic compositions allowed good enatioseparation of all verubecestat intermediates. EE determination of verubecestat is easily performed on NicoShell, another macrocyclic glycopeptide chiral selector bonded to 2.7 µm superficially porous particles. This approach enables fast and reliable enantiopurity analysis of the entire verubecestat synthetic route using only two chiral columns and mobile phases on a conventional HPLC system, simplifying technical package preparation, method validation and transfer to manufacturing facilities.

Polymer-Supported Stereoselective Synthesis of Benzoxazino[4,3-b][1,2,5]thiadiazepinone 6,6-dioxides.

Herein, we report the stereoselective synthesis of trisubstituted benzoxazino[4,3-b][1,2,5]thiadiazepinone 6,6-dioxides from polymer-supported Fmoc-Ser(tBu)-OH and Fmoc-Thr(tBu)-OH. After the solid-phase synthesis of N-alkylated-N-sulfonylated intermediates using various 2-nitrobenzenesulfonyl chlorides and bromoketones, the target compounds were obtained via trifluoroacetic acid (TFA)-mediated cleavage from the resin, followed by cyclization of the diazepinone scaffold. Except for the threonine-based intermediates, the inclusion of triethylsilane (TES) in the cleavage cocktail yielded a specific configuration of the newly formed C3 chiral center. The final cyclization resulted in minor or no inversion of the C12a stereocenter configuration.

3H-1,2-benzoxathiepine 2,2-dioxides: a new class of isoform-selective carbonic anhydrase inhibitors.

A new chemotype with carbonic anhydrase (CA, EC 4.2.1.1) inhibitory action has been discovered, the homo-sulfocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) which have been designed considering the (sulfo)coumarins as lead molecules. An original synthetic strategy of a panel of such derivatives led to compounds with a unique inhibitory profile and very high selectivity for the inhibition of the tumour associated (CA IX/XII) over the cytosolic (CA I/II) isoforms. Although the CA inhibition mechanism with these new compounds is unknown for the moment, we hypothesize that it may be similar to that of the sulfocoumarins, i.e. hydrolysis to the corresponding sulfonic acids which thereafter anchor to the zinc-coordinated water molecule within the enzyme active site.

The anti-hepatocellular carcinoma cell activity by a novel mTOR kinase inhibitor CZ415.

Dysregulation of mammalian target of rapamycin (mTOR) in hepatocellular carcinoma (HCC) represents a valuable treatment target. Recent studies have developed a highly-selective and potent mTOR kinase inhibitor, CZ415. Here, we showed that nM concentrations of CZ415 efficiently inhibited survival and induced apoptosis in HCC cell lines (HepG2 and Huh-7) and primary-cultured human HCC cells. Meanwhile, CZ415 inhibited proliferation of HCC cells, more potently than mTORC1 inhibitors (rapamycin and RAD001). CZ415 was yet non-cytotoxic to the L02 human hepatocytes. Mechanistic studies showed that CZ415 disrupted assembly of mTOR complex 1 (mTORC1) and mTORC2 in HepG2 cells. Meanwhile, activation of mTORC1 (p-S6K1) and mTORC2 (p-AKT, Ser-473) was almost blocked by CZ415. In vivo studies revealed that oral administration of CZ415 significantly suppressed HepG2 xenograft tumor growth in severe combined immuno-deficient (SCID) mice. Activation of mTORC1/2 was also largely inhibited in CZ415-treated HepG2 tumor tissue. Together, these results show that CZ415 blocks mTORC1/2 activation and efficiently inhibits HCC cell growth in vitro and in vivo.

Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate.

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 102 M-1 min-1. Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

Synthesis of Verubecestat, a BACE1 Inhibitor for the Treatment of Alzheimer's Disease.

Verubecestat is an inhibitor of ß-secretase being evaluated for the treatment of Alzheimer's disease. The first-generation route relies on an amide coupling with a functionalized aniline, the preparation of which introduces synthetic inefficiencies. The second-generation route replaces this with a copper-catalyzed C-N coupling, allowing for more direct access to the target. Other features of the new route include a diastereoselective Mannich-type addition into an Ellman sulfinyl ketimine and a late-stage guanidinylation.

Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)-A ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer's Disease.

Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer's disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aß levels in rats and nonhuman primates and CSF Aß levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

Fused Ring Molecular Scaffold with 3D Architecture for Constrained Peptidomimetics: Polymer-Supported Stereoselective Synthesis of Tetrahydrobenzo[e]pyrazino[2,1-c][1,2,4]thiadiazinone 6,6-dioxide via N-Acyl Iminiums.

3,4,4a,5-Tetrahydrobenzo[e]pyrazino[2,1-c][1,2,4]thiadiazin-1(2H)-one 6,6-dioxides, molecular scaffolds with 3D architecture, were synthesized on solid supports via tandem N-acyl iminium ion cyclization followed by nucleophilic addition. The modular synthesis proceeded under mild conditions using commercially available building blocks and provided crude products with respectable purity. The synthesized compounds are applicable as fused nitrogenous heterocyclic compounds in drug discovery and as constrained peptidomimetics incorporated into a peptide backbone.

Design and synthesis of fused bicyclic inhibitors targeting the L5 loop site of centromere-associated protein E.

Centromere-associated protein-E (CENP-E) is a mitotic kinesin which plays roles in cell division, and is regarded as a promising therapeutic target for the next generation of anti-mitotic agents. We designed novel fused bicyclic CENP-E inhibitors starting from previous reported dihydrobenzofuran derivative (S)-(+)-1. Our design concept was to adjust the electron density distribution on the benzene ring of the dihydrobenzofuran moiety to increase the positive charge for targeting the negatively charged L5 loop of CENP-E, using predictions from electrostatic potential map (EPM) analysis. For the efficient synthesis of our 2,3-dihydro-1-benzothiophene 1,1-dioxide derivatives, a new synthetic method was developed. As a result, we discovered 6-cyano-7-trifluoromethyl-2,3-dihydro-1-benzothiophene 1,1-dioxide derivative (+)-5d (Compound A) as a potent CENP-E inhibitor with promising potential for in vivo activity. In this Letter, we discuss the design and synthetic strategy used in the discovery of (+)-5d and structure-activity relationships for its analogs possessing various fused bicyclic L5 binding moieties.

Enthalpy-Entropy Compensation in the Binding of Modulators at Ionotropic Glutamate Receptor GluA2.

The 1,2,4-benzothiadiazine 1,1-dioxide type of positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) are promising lead compounds for the treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind in a cleft formed by the interface of two neighboring ligand binding domains and act by stabilizing the agonist-bound open-channel conformation. The driving forces behind the binding of these modulators can be significantly altered with only minor substitutions to the parent molecules. In this study, we show that changing the 7-fluorine substituent of modulators BPAM97 (2) and BPAM344 (3) into a hydroxyl group (BPAM557 (4) and BPAM521 (5), respectively), leads to a more favorable binding enthalpy (ΔH, kcal/mol) from -4.9 (2) and -7.5 (3) to -6.2 (4) and -14.5 (5), but also a less favorable binding entropy (-TΔS, kcal/mol) from -2.3 (2) and -1.3 (3) to -0.5 (4) and 4.8 (5). Thus, the dissociation constants (Kd, µM) of 4 (11.2) and 5 (0.16) are similar to those of 2 (5.6) and 3 (0.35). Functionally, 4 and 5 potentiated responses of 10 µM L-glutamate at homomeric rat GluA2(Q)i receptors with EC50 values of 67.3 and 2.45 µM, respectively. The binding mode of 5 was examined with x-ray crystallography, showing that the only change compared to that of earlier compounds was the orientation of Ser-497 pointing toward the hydroxyl group of 5. The favorable enthalpy can be explained by the formation of a hydrogen bond from the side-chain hydroxyl group of Ser-497 to the hydroxyl group of 5, whereas the unfavorable entropy might be due to desolvation effects combined with a conformational restriction of Ser-497 and 5. In summary, this study shows a remarkable example of enthalpy-entropy compensation in drug development accompanied with a likely explanation of the underlying structural mechanism.

Synthesis of 2,6-dicarbethoxy-3,5-diaryltetrahydro-1,4-thiazine-1,1-dioxide derivatives as potent anticonvulsant agents.

An efficient synthesis of 2,6-dicarbethoxy-3,5-diaryltetrahydro-1,4-thiazine-1,1-dioxide derivatives has been achieved under aqueous medium for the first time in good to excellent yields. All the synthesized compounds were tested for anticonvulsant activity using the maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) screens, which are the most broadly employed seizure models for early identification of candidate anticonvulsants. Their neurotoxicity was determined applying the rotarod test. Seven compounds 4a, 4d, 4f, 4h, 4o, 4p and 4q showed promising anticonvulsant activities in both models employed for anticonvulsant evaluation. The most active compound 4d showed the MES-induced seizures with ED50 value of 10.2 mg/kg and TD50 value of 288.6 mg/kg after intraperitoneal injection to mice, which provided compound 4d with a protective index (TD50/ED50) of 28.3 in the MES test.

6-Substituted sulfocoumarins are selective carbonic anhdydrase IX and XII inhibitors with significant cytotoxicity against colorectal cancer cells.

6-Substituted sulfocoumarins bearing the carboxamido, trimethylammonium as well as the cyano and methoxy moieties with interesting inhibitory activity/selectivity against the tumor associated carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA IX and XII are reported. Moieties leading to the best inhibition were tert-butylcarboxamido, phenylcarboxamido, and 4-pyridylcarboxamido, with K(I) values of 2.1-8.1 nM. No inhibition of the off-target hCA II and I was observed. A number of these compounds were evaluated against HT-29 colon cancer cell lines ex vivo. Compounds 9c and 9e revealed effective cytotoxic effects after 72 h of incubation in both normoxic and hypoxic conditions, unlike sulfonamide CA inhibitors that show such effects only in hypoxia. These results may be of particular importance for the choice of future drug candidates targeting hypoxic tumors and metastases, considering the fact that a sulfonamide CA IX inhibitor (SLC-0111) is presently in phase I clinical trials.

Microwave-assisted radiosynthesis of [(18) F]ASEM, a radiolabeled α7-nicotinic acetylcholine receptor antagonist.

An improvement of the original radiochemical synthesis of [(18) F]ASEM, an α7-nicotinic acetylcholinergic receptor radioligand, is reported. The new procedure utilizes microwave-assisted radiofluorination. In addition, a new preparative HPLC method was developed to eliminate a chemical impurity in the final product. Quality control procedures were also enhanced to improve detection of product with enhanced resolution of potential impurities. [(18) F]ASEM was produced in 20.1 ± 8.9% non-decay corrected (NDC) yield with an average synthesis time of 57 min and an average specific radioactivity of 856 ± 332 GBq/µmol (23 ± 9 Ci/µmol).