Chemoenzymatic synthesis and immunological evaluation of sialyl-Thomsen-Friedenreich (sTF) antigen conjugate to CRM197.

sTF (sialyl-Thomsen-Friedenreich) is a type of tumor-associated carbohydrate antigens (TACAs) and is highly expressed in various human malignancies. To validate if sTF could be a valuable molecular target for future cancer vaccine development, in this work the sTF antigen was prepared by adopting a strategy combining chemical and enzymatic methods, and then was covalently conjugated to a carrier protein, CRM197. The preliminary immunological evaluation, performed on BALB/c mice, revealed that the sTF-CRM197 conjugate elicited high titers of specific IgG antibodies. FACS experiments showed that the antisera induced by sTF-CRM197 conjugate could specifically recognize and bind to sTF-positive cancer cells T-47D. Furthermore, the conjugate mediated effective and specific antibody-mediated complement-dependent cytotoxicity (CDC).

The novel selective TLR7 agonist GY101 suppresses colon cancer growth by stimulating immune cells.

Toll-like receptor (TLR) 7, a transmembrane signal transduction receptor expressed on the surface of endosomes, has become an attractive target for antiviral and cancer immunotherapies. TLR7 can induce signal transduction by recognizing single-stranded RNA or its analogs, leading to the release of cytokines such as IL-6, IL-12, TNF-α and type-I IFN. Activation of TLR7 helps to enhance immunogenicity and immune memory by stimulating immune cells. Herein, we identified a novel selective TLR7 agonist, GY101, and determined its ability to activate TLR7. In summary, in vitro, compound GY101 significantly induced the secretion of IL-6, IL-12, TNF-α and IFN-γ in mouse splenic lymphocytes; in vivo, peritumoral injection of GY101 significantly suppressed colon cancer CT26, as well as poorly immunogenic B16-F10 and 4T1 cancer cell-derived tumor growth by activating the infiltration of lymphocytes and polarization of M2-like macrophages into M1-like macrophages. These results demonstrate that GY101, as a potent TLR7 agonist, holds great potential for cancer immunotherapy.

An Investigation of Instability in Dried Blood Spot Samples for Pharmacokinetic Sampling in Phase 3 Trials of Verubecestat.

In-clinic dried blood spot (DBS) pharmacokinetic (PK) sampling was incorporated into two phase 3 studies of verubecestat for Alzheimer's disease (EPOCH [NCT01739348] and APECS [NCT01953601]), as a potential alternative to plasma PK sampling for improved logistical feasibility and decreased blood volume burden. However, an interim PK analysis revealed verubecestat concentrations in DBS samples declined with time to assay in both trials. An investigation revealed wide variation in implementation practices for DBS sample handling procedures resulting in insufficient desiccation which caused verubecestat instability. High-resolution mass spectrometry evaluations of stressed and aged verubecestat DBS samples revealed the presence of two hydrolysis degradants. To minimize instability, new DBS handling procedures were implemented that provided additional desiccant and minimized the time to analysis. Both verubecestat hydrolysis products were previously discovered and synthesized during active pharmaceutical ingredient stability characterization. A liquid chromatography-mass spectrometry assay to quantitate the dominant verubecestat degradant in DBS samples was developed and validated. The application of this method to stressed and aged verubecestat DBS samples confirmed that degradant concentrations accounted for the observed decreases in the verubecestat concentration. Furthermore, after increasing desiccant amounts, degradant concentrations accounted for approximately 7% of the verubecestat concentration in DBS clinical samples, indicating that issues with sample handling were minimized with new storage and shipping conditions. This case study illustrates the challenges with employing new sampling techniques in large, global trials, and the importance of anticipating and mitigating implementation risks.

Efficient Aliphatic Hydrogen-Isotope Exchange with Tritium Gas through the Merger of Photoredox and Hydrogenation Catalysts.

Employment of a combination of an organophotoredox catalyst with Wilkinson's catalyst (Rh(PPh3)3Cl) has given rise to an unprecedented method for hydrogen-isotope exchange (HIE) of aliphatic C(sp3)-H bonds of complex pharmaceuticals using T2 gas directly. Wilkinson's catalyst, commonly used for catalytic hydrogenations, was exploited as a precatalyst for activation of D2 or T2 and hydrogen atom transfer. In this combined methodology and mechanistic study, we demonstrate that by coupling photocatalysis with Rh catalysis, carbon-centered radicals generated via photoredox catalysis can be intercepted by Rh-hydride intermediates to deliver an effective hydrogen atom donor for hydrogen-isotope labeling of complex molecules in one step. By optimizing the ratio of the photocatalyst and Wilkinson's catalyst to balance the rate of the dual catalytic cycles, we can achieve efficient HIE and high recovery yield. This protocol was readily applied to direct HIE of C(sp3)-H bonds in 10 complex drug molecules, showing high isotope incorporation efficiency and exceptionally good functional group tolerance and demonstrating this approach as a practical and attractive labeling method for deuteration and tritiation.

The PET tracer [11C]MK-6884 quantifies M4 muscarinic receptor in rhesus monkeys and patients with Alzheimer's disease.

Positron emission tomography (PET) ligands play an important role in the development of therapeutics by serving as target engagement or pharmacodynamic biomarkers. Here, we describe the discovery and translation of the PET tracer [11C]MK-6884 from rhesus monkeys to patients with Alzheimer's disease (AD). [3H]MK-6884/[11C]MK-6884 binds with high binding affinity and good selectivity to an allosteric site on M4 muscarinic cholinergic receptors (M4Rs) in vitro and shows a regional distribution in the brain consistent with M4R localization in vivo. The tracer demonstrates target engagement of positive allosteric modulators of the M4R (M4 PAMs) through competitive binding interactions. [11C]MK-6884 binding is enhanced in vitro by the orthosteric M4R agonist carbachol and indirectly in vivo by the acetylcholinesterase inhibitor donepezil in rhesus monkeys and healthy volunteers, consistent with its pharmacology as a highly cooperative M4 PAM. PET imaging of [11C]MK-6884 in patients with AD identified substantial regional differences quantified as nondisplaceable binding potential (BPND) of [11C]MK-6884. These results suggest that [11C]MK-6884 is a useful target engagement biomarker for M4 PAMs but may also act as a sensitive probe of neuropathological changes in the brains of patients with AD.

Conjugate of ibrutinib with a TLR7 agonist suppresses melanoma progression and enhances antitumor immunity.

The use of large molecules for immunotherapy has led to exciting developments in cancer treatment, such as the development of PD-1/PD-L1 antibodies. However, small molecule targeted therapies still lack effective immune-functional classes. Ideal anticancer drugs should simultaneously generate immune memory when killing cancer cells to prevent tumor relapse and metastasis. To this end, we carried out a rationally designed strategy to develop novel classes of small molecule compounds with bifunctional targeting and immunostimulatory abilities by conjugating targeting compounds with TLR7 agonists, generating immune-targeting conjugates (ImmunTacs). GY161, as a representative ImmunTac, was synthesized via chemical conjugation of ibrutinib with a TLR7 agonist. In vitro, GY161 stimulated the production of cytokines by mouse spleen lymphocytes, promoted the maturation of dendritic cells (DCs), and inhibited the growth and induced the apoptosis of B16 melanoma cells by regulating the c-Met/ß-catenin pathway. In vivo, GY161 enhanced the frequency of CD8+ T cells in spleens and tumors, suppressed the growth of B16 melanoma cell-derived tumors and prolonged the survival time of mice. In summary, GY161 could prevent melanoma progression through direct tumor killing and by triggering specific immunity. These results strongly suggest that ImmunTacs are a reliable and promising strategy for developing small molecule immunogenic anticancer drugs.

Direct ß-Mannosylation of Primary Alcohol Acceptors: Trisaccharide Iteration Assembly of ß-1,6-Oligomannosides Corresponding to Kakelokelose.

Gold(I)-catalyzed stereoselective ß-glycosylation of primary alcohols is achieved using the orthogonally protected mannosyl α-ortho-hexynylbenzoate (OABz) donors devoid of 4,6-O-tethering groups used in conventionally constructing ß-mannosidic bonds. The potential of this methodology is showcased by the first assembly of ß-1,6-tri/hexa-/nonamannosides and related sulfated congeners through a convergent strategy. The synthesis features the stereocontrolled ß-glycosylation of α-trimannosyl OABz donors and the late-stage sulfonation. This work is expected to expedite the preparation of ß-1,6-mannans and functionalized derivatives.

Discovery of novel ibrutinib analogues to treat malignant melanoma.

A series of novel ibrutinib analogues was synthesized, and their proliferation inhibitory activities against various B lymphoma cell lines (DaudiB and Raji) and solid tumor cells (B16, CT26, HepG2 and 4T1) were evaluated. The most potent compound, YL7, exhibited strong antiproliferative activity in all cell lines, and its IC50 value in B16 cells was almost 9-fold better than that of ibrutinib. Mechanism of action studies showed that YL7 inhibited proliferation and migration and induced G1 cell cycle arrest, apoptosis and autophagy in B16 cells. Further assessment of in vivo antitumor efficacies demonstrated that YL7 significantly inhibited the growth of B16 melanoma. These preliminary studies suggest that it is reasonable to modify the structure of ibrutinib for antimelanoma treatment.

Synthesis and biological evaluation of novel cabazitaxel analogues.

Cabazitaxel is one of the most recently FDA-approved taxane anticancer agent. In view of the advantages in preclinical and clinical data of cabazitaxel over former toxoids, the synthesis and biological evaluation of novel cabazitaxel analogues were conducted. First, a novel semi-synthesis of cabazitaxel was described. This strategy is concise and efficient, which needs five steps from the 10-deacetylbaccatin III (10-DAB) moiety and a commercially available C13 side chain precursor with a 32% overall yield. Besides, this strategy avoids using many hazardous reagents that involved in the previously reported processes. Then, a panel of cabazitaxel analogues were prepared basing on this strategy. The cytotoxicity evaluations showed that the majority of these cabazitaxel analogues are potent against both A549 and KB cells and their corresponding drug-resistant cell lines KB/VCR, and A549/T, respectively. Further in vivo antitumor efficacies assessment of 7,10-di-O-methylthiomethyl (MTM) modified cabazitaxel (compounds 16 and 19) on SCID mice A549 xenograft model showed they both had similar antitumor activity to the cabazitaxel. Since compound 19 was observed causing more body wight loss on the mice than 16, these preliminary studies suggest 16 might be a potent drug candidate for further preclinical evaluation.

Late-Stage Carbon Isotope Exchange of Aryl Nitriles through Ni-Catalyzed C-CN Bond Activation.

A facile one-pot strategy for 13CN and 14CN exchange with aryl, heteroaryl, and alkenyl nitriles using a Ni phosphine catalyst and BPh3 is described. This late-stage carbon isotope exchange (CIE) strategy employs labeled Zn(CN)2 to facilitate enrichment using the nonlabeled parent compound as the starting material, eliminating de novo synthesis for precursor development. A broad substrate scope encompassing multiple pharmaceuticals is disclosed, including the preparation of [14C] belzutifan to illustrate the exceptional functional group tolerance and utility of this labeling approach. Preliminary experimental and computational studies suggest the Lewis acid BPh3 is not critical for the oxidative addition step and instead plays a role in facilitating CN exchange on Ni. This CIE method dramatically reduces the synthetic steps and radioactive waste involved in preparation of 14C labeled tracers for clinical development.

Metallaphotoredox aryl and alkyl radiomethylation for PET ligand discovery.

Positron emission tomography (PET) radioligands (radioactively labelled tracer compounds) are extremely useful for in vivo characterization of central nervous system drug candidates, neurodegenerative diseases and numerous oncology targets1. Both tritium and carbon-11 radioisotopologues are generally necessary for in vitro and in vivo characterization of radioligands2, yet there exist few radiolabelling protocols for the synthesis of either, inhibiting the development of PET radioligands. The synthesis of such radioligands also needs to be very rapid owing to the short half-life of carbon-11. Here we report a versatile and rapid metallaphotoredox-catalysed method for late-stage installation of both tritium and carbon-11 into the desired compounds via methylation of pharmaceutical precursors bearing aryl and alkyl bromides. Methyl groups are among the most prevalent structural elements found in bioactive molecules, and so this synthetic approach simplifies the discovery of radioligands. To demonstrate the breadth of applicability of this technique, we perform rapid synthesis of 20 tritiated and 10 carbon-11-labelled complex pharmaceuticals and PET radioligands, including a one-step radiosynthesis of the clinically used compounds [11C]UCB-J and [11C]PHNO. We further outline the direct utility of this protocol for preclinical PET imaging and its translation to automated radiosynthesis for routine radiotracer production in human clinical imaging. We also demonstrate this protocol for the installation of other diverse and pharmaceutically useful isotopes, including carbon-14, carbon-13 and deuterium.

Late-Stage 18 O Labeling of Primary Sulfonamides via a Degradation-Reconstruction Pathway.

A late-stage 18 O labeling approach of sulfonamides that employs the corresponding unlabeled molecule as the starting material was developed. Upon deamination of the sulfonamide, a sulfinate intermediate was isotopically enriched using eco-friendly reagents H2 18 O and 15 NH3 (aq) to afford a M+5 isotopologue of the parent compound. This degradation-reconstruction approach afforded isolated yields of up to 96 % for the stable isotope labeled (SIL) sulfonamides, and was compatible with multiple marketed therapeutics, including celecoxib, on a gram scale. The SIL products also exhibited no 18 O/16 O back exchange under extreme conditions, further validating the utility of this green strategy for drug labeling for both in vitro and in vivo use. This procedure was also adapted to include pharmaceutically relevant methyl sulfones by using 13 CH3 , affording M+5 isotopic enrichment, thereby illustrating the broad utility of this methodology.

Synthesis and biological evaluation of Ginsenoside Compound K analogues as a novel class of anti-asthmatic agents.

Ginsenoside Compound K (CK) showed potent activity against IgE for the treatment of asthma. A series of CK analogues were then synthesized by straightforward procedures. The in vivo anti-IgE activity evaluations using the OVA-induced asthmatic mouse model revealed preliminary SARs of the CK analogues, which showed that the sugar type, modifications on A-ring and the C20 side chain of CK all affected much on the activities. Primary SARs optimization led to the discovery of compounds T1, T2, T3, T8 and T12, which displayed superior or comparable anti-asthmatic effects (IgE value = 1237.11 ±â€¯106.28, 975.82 ±â€¯160.32, 1136.96 ±â€¯121.85, 1191.08 ±â€¯107.59 and 1258.27 ±â€¯148.70 ng/mL, respectively) in comparison with CK (1501.85 ±â€¯184.66 ng/mL). These potent compounds could serve as leads for further development.

Glycosylation of a Ketone with an O-Glycosyl Trichloroacetimidate Provides an Enol Glycoside.

An enol-type glycosylation reaction has been investigated. Enol glycosides can be obtained from the reaction between O-glycosyl trichloroacetimidates and the corresponding ketones promoted by an acid. The enol glycosides derived from cyclic ketones can be afforded efficiently and isolated in good yield, while those from acyclic ketones are prepared in low conversion or are too labile for isolation. Further studies on different glycosyl donor types indicate that only the O-glycosyl trichloroacetimidate works well as a donor for enol glycosylation.

Synthesis and biological evaluation of novel larotaxel analogues.

Taxoids are a class of successful drugs and have been successfully used in chemotherapy for a variety of cancer types. However, despite the hope and promises that these taxoids have engendered, their utility is hampered by some clinic limitations. Extensive structure-activity relationship (SAR) studies of toxoids have been performed in many different laboratories. Whereas, SAR studies that based on the new-generation toxoid, larotaxel, have not been reported yet. In view of the advantages in preclinical and clinical data of larotaxel over former toxoids, new taxoids that strategicly modified at the C3'/C3'-N and C2 positions of larotaxel were designed, semi-synthesized, and examined for their potency and efficacy in vitro. As a result, it has been shown that the majority of these larotaxel analogues are exceptionally potent against both drug-sensitive tumor cells and tumor cells with drug resistance arising from P-glycoprotein over expression. Further in vivo antitumor efficacies investigations revealed A2 might be a potent antitumor drug candidate for further preclinical evaluation.

Genistein Enhances or Reduces Glycosaminoglycan Quantity in a Cell Type-Specific Manner.

BACKGROUND/AIMS: Genistein is a natural isoflavone enriched in soybeans. It has beneficial effects for patients with mucopolysaccharidose type III through inhibiting glycosaminoglycan biosynthesis. However, other studies indicate that genistein does not always inhibit glycosaminoglycan biosynthesis. METHODS: To understand the underlying molecular mechanisms, CHOK1, CHO3.1, CHO3.3, and HCT116 cells were treated with genistein and the monosaccharide compositions and quantity of all glycans from the cell lysate were measured after thorough acid hydrolysis followed by HPLC analysis. In addition, the glycosaminoglycan disaccharide compositions were obtained by stable isotope labeling coupled with LC/MS analysis. RESULTS: Genistein treatment reduced the amount of glycans but increased the amount of glycosaminoglycans in HCT116 cells. In contrast, genistein treatment reduced both glycan and glycosaminoglycan quantities in CHOK1, CHO3.1, and CHO3.3 cells in addition to differential changes in glycosaminoglycan disaccharide compositions. CONCLUSION: Genistein treatment reduced overall glycan quantity but glycosaminoglycan quantities were either increased or decreased in a cell type-dependent manner.

34 S: A New Opportunity for the Efficient Synthesis of Stable Isotope Labeled Compounds.

The synthesis of stable isotope labeled (SIL) complex drug molecules with a ≥3 mass unit increase from the parent compound is essential for drug discovery and development. Typical approaches that rely on 2 H, 13 C, and 15 N isotopes can be very challenging or even intractable, and can delay the drug development process. This work introduces a new concept for the synthesis of labeled compounds that relies on the use of 34 S. The synthetic utility of 34 S was demonstrated with the efficient synthesis of [34 S]phosphorothioates [34 S2 ]-PS-ODNs-TTT and [13 C, 15 N, 34 S]-ceftolozane. In addition, a procedure for the direct oxidation of phosphites to [34 S]phosphorothioates using elemental 34 S without isotope dilution was developed.

Concise total synthesis of acylated phenolic glycosides vitexnegheteroin A and ovatoside D.

Starting from readily available vanillin and α-D-acetobromo glucose, two natural acylated phenolic glycosides vitexnegheteroin A and ovatoside D were synthesized for the first time in 4 steps with overall yields of 54% and 65%, respectively. The key steps involve the directly regioselective O-6 acylation of vanillin ß-D-glucopyranoside with acyl chlorides, and simultaneous removal of the allyl protecting groups on the phenolic acid moiety and reduction of the aldehyde in the aglycon moiety by using Pd(PPh)3-NaBH4 system in one pot.

Transition-Metal-Free Synthesis of C-Glycosylated Phenanthridines via K2S2O8-Mediated Oxidative Radical Decarboxylation of Uronic Acids.

We have developed an efficient protocol for the synthesis of C-glycosylated phenanthridines. Tetrafuranos-4-yl and pentapyranos-5-yl radicals, generated from K2S2O8-mediated oxidative decarboxylation of furan- and pyranuronic acids, undergo attack to 2-isocyanodiphenyls and ensuing homolytic aromatic substitution to provide diverse C-glycosylated phenanthridines in satisfactory yields without resort to transition metals. This reaction tolerates various functional groups, and enables ready synthesis of complex oligosaccharide-based phenanthridines. The C-glycosylated phenanthridine derived from ß-cyclodextrin has been prepared, which might be potential in medicinal and biological chemistry due to its flexible conformation.

Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycans Are Targeted by Bleomycin in Cancer Cells.

BACKGROUND/AIMS: Bleomycin is a clinically used anti-cancer drug that produces DNA breaks once inside of cells. However, bleomycin is a positively charged molecule and cannot get inside of cells by free diffusion. We previously reported that the cell surface negatively charged glycosaminoglycans (GAGs) may be involved in the cellular uptake of bleomycin. We also observed that a class of positively charged small molecules has Golgi localization once inside of the cells. We therefore hypothesized that bleomycin might perturb Golgi-operated GAG biosynthesis. METHODS: We used stable isotope labeling coupled with LC/MS analysis of GAG disaccharides simultaneously from bleomycin-treated and non-treated cancer cells. To further understand the cytotoxicity of bleomycin and its relationship to GAGs, we used sodium chlorate to inhibit GAG sulfation and commercially available GAGs to compete for cell surface GAG/bleomycin interactions in seven cell lines including CHO745 defective in both heparan sulfate and chondroitin sulfate biosynthesis. RESULTS: we discovered that heparan sulfate GAG was significantly undersulfated and the quantity and disaccharide compositions of GAGs were changed in bleomycin-treated cells in a concentration- and time-dependent manner. We revealed that bleomycin-induced cytotoxicity was directly related to cell surface GAGs. CONCLUSION: GAGs were targeted by bleomycin both at cell surface and at Golgi. Thus, GAGs might be the biological relevant molecules that might be related to the bleomycin-induced fibrosis in certain cancer patients, a severe side effect with largely unknown molecular mechanism.