Design, synthesis, and evaluation of a novel prodrug, a S-trityl-l-cysteine derivative targeting kinesin spindle protein.

Kinesin spindle protein (KSP) is expressed only in cells undergoing cell division, and hence represents an attractive target for the treatment of cancer. Several KSP inhibitors have been developed and undergone clinical trial, but their clinical use is limited by their toxicity to rapidly proliferating non-cancerous cells. To create new KSP inhibitors that are highly selective for cancer cells, we optimized the amino acid moiety of S-trityl-l-cysteine (STLC) derivative 1 using in silico modeling. Molecular docking and molecular dynamics simulation were performed to investigate the binding mode of 1 with KSP. Consistent with the structure activity relationship studies, we found that a cysteine amino moiety plays an important role in stabilizing the interaction. Based on these findings and the structure of GSH, a substrate of γ-glutamyltransferase (GGT), we designed and synthesized the prodrug N-γ-glutamylated STLC derivative 9, which could be hydrolyzed by GGT to produce 1. The KSP ATPase inhibitory activity of 9 was lower than that of 1, and LC-MS analysis indicated that 9 was converted to 1 only in the presence of GGT in vitro. In addition, the cytotoxic activity of 9 was significantly attenuated in GGT-knockdown A549 cells. Since GGT is overexpressed on the cell membrane of various cancer cells, these results suggest that compound 9 could be a promising prodrug that selectively inhibits the proliferation of GGT-expressing cancer cells.

Structure activity study of S-trityl-cysteamine dimethylaminopyridine derivatives as SIRT2 inhibitors: Improvement of SIRT2 binding and inhibition.

Sirtuin proteins are a highly conserved class of nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacylases. The pleiotropic human isoform 2 of Sirtuins (SIRT2) has been engaged in the pathogenesis of cancer in a plethora of reports around the globe. Thus, SIRT2 modulation is deemed as a promising approach for pharmaceutical intervention. Previously, we reported S-Trityl-l-Cysteine (STLC)-ornamented dimethylaminopyridine chemical entity named STC4 with a significant SIRT2 inhibitory capacity; this was separate from the conventional application of STLC scaffold as a kinesin-5 inhibitor. An interactive molecular docking study of SIRT2 and STC4 showed interaction between Asn168 of SIRT2 and the methyl ester of STC4, that appears to hinder STC4 to reach the selective pocket of the protein unlike strong SIRT2 inhibitor SirReal2. To improve its activity, herein, we utilized S-trityl cysteamine pharmacophore lacking the methyl ester. Nine compounds were synthesized and assayed affording three biopertinent SIRT2 inhibitors, and two of them, STCY1 and STCY6 showed higher inhibitory activity than STC4. These compounds have pronounced anti-proliferative activities against different cancer cell lines. A molecular docking study was executed to shed light on the supposed binding mode of the lead compound, STCY1, into the selective pocket of SIRT2 by interaction of the nitrogen of pyridine ring of the compound and Ala135 of the protein. The outcome of the study exposes that the active compounds are effective intermediates to construct more potent biological agents.

Design and evaluation of self-nanoemulsifying drug delivery systems (SNEDDSs) for senicapoc.

Senicapoc (SEN), a potent antisickling agent, shows poor water solubility and poor oral bioavailability. To improve the solubility and cell permeation of SEN, self-nanoemulsifying drug delivery systems (SNEDDSs) were developed. Capryol PGMC®, which showed the highest solubilization capacity, was selected as the oil. The self-emulsification ability of two surfactants, viz., Cremophor-EL® and Tween® 80, was compared. Based on a solubility study and ternary phase diagrams, three optimized nanoemulsions with droplet sizes less than 200 nm were prepared. An in vitro dissolution study demonstrated the superior performance of the SNEDDS over the free drug. During in vitro lipolysis, 80% of SEN loaded in the SNEDDS remained solubilized. An in vitro cytotoxicity study using the Caco-2 cell line indicated the safety of the formulations at 1 mg/mL. The transport of SEN-SNEDDSs across Caco-2 monolayers was enhanced 115-fold (p < 0.01) compared to that of the free drug. According to these results, SNEDDS formulations could be promising tools for the oral delivery of SEN.

Synthetic, small-molecule photoantimicrobials - a realistic approach.

The search for suitable, low-molecular weight photoantimicrobials for use in infection control has strong foundations in conventional antiseptic research from the early-mid 20th Century. Many examples of dyes exist having conventional antimicrobial activity among the azine, acridine and triphenylmethane families which have since also been found to exhibit photosensitising capabilities. The prior employment of these examples in human antisepsis provides a practical basis in terms of low host toxicity, while extant structure-activity relationships for conventional antimicrobial activity can support the development of similar relationships for photoactivated cell killing. The range of chromophores covered allows progress to be made both in topical and deeper, fluid-involved infections.

Synthesis of Nanometer Sized Bis- and Tris-trityl Model Compounds with Different Extent of Spin-Spin Coupling.

Tris(2,3,5,6-tetrathiaaryl)methyl radicals, so-called trityl radicals, are emerging as spin labels for distance measurements in biological systems based on Electron Paramagnetic Resonance (EPR). Here, the synthesis and characterization of rigid model systems carrying either two or three trityl moieties is reported. The monofunctionalized trityl radicals are connected to the molecular bridging scaffold via an esterification reaction employing the Mukaiyama reagent 2-chloro-methylpyridinium iodide. The bis- and tris-trityl compounds exhibit different inter-spin distances, strength of electron-electron exchange and dipolar coupling and can give rise to multi-spin effects. They are to serve as benchmark systems in comparing EPR distance measurement methods.

EPR Oximetry Sensor-Developing a TAM Derivative for In Vivo Studies.

Oxygenation is one of the most important physiological parameters of biological systems. Low oxygen concentration (hypoxia) is associated with various pathophysiological processes in different organs. Hypoxia is of special importance in tumor therapy, causing poor response to treatment. Triaryl methyl (TAM) derivative radicals are commonly used in electron paramagnetic resonance (EPR) as sensors for quantitative spatial tissue oxygen mapping. They are also known as magnetic resonance imaging (MRI) contrast agents and fluorescence imaging compounds. We report the properties of the TAM radical tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl, (PTMTC), a potential multimodal (EPR/fluorescence) marker. PTMTC was spectrally analyzed using EPR and characterized by estimation of its sensitivity to the oxygen in liquid environment suitable for intravenous injection (1 mM PBS, pH = 7.4). Further, fluorescent emission of the radical was measured using the same solvent and its quantum yield was estimated. An in vitro cytotoxicity examination was conducted in two cancer cell lines, HT-29 (colorectal adenocarcinoma) and FaDu (squamous cell carcinoma) and followed by uptake studies. The stability of the radical in different solutions (PBS pH = 7.4, cell media used for HT-29 and FaDu cells culturing and cytotoxicity procedure, full rat blood and blood plasma) was determined. Finally, a primary toxicity test of PTMTC was carried out in mice. Results of spectral studies confirmed the multimodal properties of PTMTC. PTMTC was demonstrated to be not absorbed by cancer cells and did not interfere with luciferin-luciferase based assays. Also in vitro and in vivo tests showed that it was non-toxic and can be freely administrated till doses of 250 mg/kg BW via both i.v. and i.p. injections. This work illustrated that PTMTC is a perfect candidate for multimodal (EPR/fluorescence) contrast agent in preclinical studies.

Thiol-Dependent Reduction of the Triester and Triamide Derivatives of Finland Trityl Radical Triggers O2-Dependent Superoxide Production.

Tetrathiatriaylmethyl (trityl) radicals have found wide biomedical applications as magnetic resonance probes. Trityl radicals and their derivatives are generally stable toward biological reducing agents such as glutathione (GSH) and ascorbate. We demonstrate that the triester (ET-03) and triamide (AT-03) derivatives of the Finland trityl radical exhibit unique reduction by thiols such as GSH and cysteine (Cys) to generate the corresponding trityl carbanions as evidenced by the loss of EPR signal and appearance of characteristic UV-vis absorbance at 644 nm under anaerobic conditions. The trityl carbanions can be quickly converted back to the original trityl radicals by oxygen (O2) in air, thus rendering the reaction between the trityl derivative and biothiol undetectable under aerobic conditions. The reduction product of O2 by the trityl carbanions was shown to be superoxide radical (O2•-) by EPR spin-trapping. Kinetic studies showed that the reaction rate constants (k) depend on the types of both trityl radicals and thiols with the order of kET-03/Cys (0.336 M-1 s-1) > kET-03/GSH (0.070 M-1 s-1) > kAT-03/Cys (0.032 M-1 s-1) > kAT-03/GSH (0.027 M-1 s-1). The reactivity of trityl radicals with thiols is closely related to the para-substituents of trityl radicals as well as the pKa of the thiols and is further reflected by the rate of O2•- production and consumptions of O2 and thiols. This novel reaction represents a new metabolic process of trityl derivatives and should be considered in the design and application of new trityl radical probes.

Nitro-Triarylmethyl Radical as Dual Oxygen and Superoxide Probe.

Superoxide radical is involved in numerous physiological and pathophysiological processes. Tetrathiatriarylmethyl (TAM) radicals are known to react with superoxide allowing measurement of superoxide production in biological media. We report the synthesis of a Nitro conjugated TAM radical showing a rate constant of 7 × 105 M-1 s-1 which is two order of magnitude higher than other TAMs, allowing high sensitivity measurement of superoxide.

Versatile Trityl Spin Labels for Nanometer Distance Measurements on Biomolecules In†Vitro and within Cells.

Structure determination of biomacromolecules under in-cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site-directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well-suited for in-cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in-cell trityl-iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal-to-noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in-cell measurement.

Synthesis and Characterization of PEGylated Trityl Radicals: Effect of PEGylation on Physicochemical Properties.

Tetrathiatriarylmethyl (TAM, trityl) radicals have attracted considerable attention as spin probes for biological electron paramagnetic resonance (EPR) spectroscopy and imaging owing to their sharp EPR singlet signals and high biostability. However, their in vivo applications were limited by the short blood circulation lifetimes and strong binding with albumins. Our previous results showed that PEGylation is a feasible method to overcome the issues facing in vivo applications of TAM radicals. In the present study, we synthesized a series of new PEGylated TAM radicals (TTP1, TPP2, TNP1, TNP2, d-TNP1, and d-TNP3) containing various lengths and numbers of mPEG chains. Our results found that the pattern of PEGylation exerts an important effect on physicochemical properties of the resulting TAM radicals. Dendritic PEGylated TAM radicals, TNP1 and TNP2, have higher water solubility and lower susceptibility for self-aggregation than their linear analogues TPP1 and TPP2. Furthermore, dendritic PEGylated TAM radicals exhibit extremely high stability toward various biological oxidoreductants as well as in rat whole blood, liver homogenate, and following in vivo intravenous administration in mice. Importantly, the deuterated derivatives, especially d-TNP3, exhibit excellent properties including the sharp and O2-sensitive EPR singlet signal, good biocompatibility, and prolonged kinetics with half-life time of ≥10 h in mice. These PEGylated TAM radicals should be suitable for a wide range of applications in in vivo EPR spectroscopy and imaging.

Copper(I) halide complexes of 2,2,5,5-tetramethyl-imidazolidine-4-thione: Synthesis, structures, luminescence, thermal stability and interaction with DNA.

Five neutral mononuclear copper(I) halide complexes containing 2,2,5,5-tetramethylimidazolidine-4-thione (tmimdtH) and triphenylphosphane (PPh3) or tri-o-tolylphosphane (totp) have been prepared and structurally characterized by X-ray single-crystal analysis. The complexes containing PPh3 adopt the usual distorted tetrahedral geometry, while the presence of the bulkier totp forces the formation of three-coordinated trigonal planar species. The interaction of the compounds with calf-thymus DNA was monitored directly via UV-vis spectroscopy, DNA-viscosity measurements and indirectly via its competition with ethidium bromide for DNA studied by fluorescence emission spectroscopy. Intercalation was revealed as the most possible mode of binding. Furthermore, luminescent properties and thermal stabilities of the complexes were investigated.

Trityl Isocyanide as a Mechanistic Probe in Multicomponent Chemistry: Walking the Line between Ugi- and Strecker-type Reactions.

Herein, we describe the versatile application of triphenylmethyl (trityl) isocyanide in multicomponent chemistry. This reagent can be employed as a cyanide source in the Strecker reaction and as convertible isocyanide in the preparation of N-acyl amino acids by Ugi 4CR/detritylation and free imidazo[1,2-a]pyridin-3-amines by a Groebke-Blackburn-Bienaymé 3CR condensation/deprotection protocol. The mechanisms of these three classical MCRs intersect at the common N-trityl nitrilium ion intermediate, whose predictable reactivity can be exploited towards chemoselective transformations.

Synthesis, Characterization, and Nanoencapsulation of Tetrathiatriarylmethyl and Tetrachlorotriarylmethyl (Trityl) Radical Derivatives­A Study To Advance Their Applicability as in Vivo EPR Oxygen Sensors.

Tissue oxygenation plays an important role in the pathophysiology of various diseases and is often a marker of prognosis and therapeutic response. EPR (ESR) is a suitable noninvasive oximetry technique. However, to reliably deploy soluble EPR probes as oxygen sensors in complex biological systems, there is still a need to investigate and improve their specificity, sensitivity, and stability. We reproducibly synthesized various derivatives of tetrathiatriarylmethyl and tetrachlorotriarylmethyl (trityl) radicals. Hydrophilic radicals were investigated in aqueous solution mimicking physiological conditions by, e.g., variation of viscosity and ionic strength. Their specificity was satisfactory, but the oxygen sensitivity was low. To enhance the capability of trityl radicals as oxygen sensors, encapsulation into oily core nanocapsules was performed. Thus, different lipophilic triesters were prepared and characterized in oily solution employing oils typically used in drug formulations, i.e., middle-chain triglycerides and isopropyl myristate. Our screening identified the deuterated ethyl ester of D-TAM (radical 13) to be suitable. It had an extremely narrow single EPR line under anoxic conditions and excellent oxygen sensitivity. After encapsulation, it retained its oxygen responsiveness and was protected against reduction by ascorbic acid. These biocompatible and highly sensitive nanosensors offer great potential for future EPR oximetry applications in preclinical research.

EPR and electrochemical quantification of oxygen using newly synthesized para-silylated triarylmethyl radicals.

Novel silylated triarylmethyl (TAM) radicals based on TAM core CT-03 and their electron paramagnetic resonance (EPR) spectra are evaluated as a function of oxygen concentration. Combination of peak-to-peak linewidth of the EPR signal and electrochemical determination allows designing a method for oxygen quantification in phosphate buffer, dimethylsulfoxide, and dichloromethane, which can be extended to other solvents.

A high-throughput screening campaign for detection of ca(2+)-activated k(+) channel activators and inhibitors using a fluorometric imaging plate reader-based tl(+)-influx assay.

The intermediate-conductance Ca(2+)-activated K(+) channel (KCa3.1) has been proposed to play many physiological roles, and modulators of KCa3.1 activity are potentially interesting as new drugs. In order to identify new chemical scaffolds, high-throughput screening (HTS) assays are needed. In the current study, we present an HTS assay that has been optimized for the detection of inhibitors as well as activators of KCa3.1 in a combined assay. We used HEK293 cells heterologously expressing KCa3.1 in a fluorescence-based Tl(+) influx assay, where the permeability of potassium channels to Tl(+) is taken advantage of. We found the combined activator-and-inhibitor assay to be robust and insensitive to dimethyl sulfoxide (up to 1%), and conducted an HTS campaign of 217,119 small molecules. In total, 224 confirmed activators and 312 confirmed inhibitors were found, which corresponded to a hit rate of 0.10% and 0.14%, respectively. The confirmed hits were further characterized in a fluorometric imaging plate reader-based concentration-response assay, and selected compounds were subjected to secondary testing in an assay for endogenous KCa3.1 activity using human erythrocytes (red blood cell assay). Although the estimated potencies were slightly higher in the RBC assay, there was an overall good correlation across all clusters. The campaign led to the identification of several chemical series of KCa3.1 activators and inhibitors, comprising already known pharmacophores and new chemical series. One of these were the benzothiazinones that constitute a new class of highly potent KCa3.1 inhibitors, exemplified by 4-{[3-(trifluoromethyl)phenyl]methyl}-2H-1,4-benzothiazin-3(4H)-one (NS6180).

Expanding the scope of PNA-encoded synthesis (PES): Mtt-protected PNA fully orthogonal to fmoc chemistry and a broad array of robust diversity-generating reactions.

Nucleic acid-encoded libraries are emerging as an attractive and highly miniaturized format for the rapid identification of protein ligands. An important criterion in the synthesis of nucleic acid encoded libraries is the scope of reactions that can be used to introduce molecular diversity and devise divergent pathways for diversity-oriented synthesis (DOS). To date, the protecting group strategies that have been used in peptide nucleic acid (PNA) encoded synthesis (PES) have limited the choice of reactions used in the library synthesis to just a few prototypes. Herein, we describe the preparation of PNA monomers with a protecting group combination (Mtt/Boc) that is orthogonal to Fmoc-based synthesis and compatible with a large palette of reactions that have been productively used in DOS (palladium cross-couplings, metathesis, reductive amination, amidation, heterocycle formation, nucleophilic addition, conjugate additions, Pictet-Spengler cyclization). We incorporate γ-modifications in the PNA backbone that are known to enhance hybridization and solubility. We demonstrate the robustness of this strategy with a library synthesis that is characterized by MALDI MS analysis at every step.

Synthesis and characterization of tritylthioethanamine derivatives with potent KSP inhibitory activity.

Assembly of a bipolar mitotic spindle requires the action of class 5 kinesins, and inhibition or depletion of this motor results in mitotic arrest and apoptosis. S-Trityl-l-cysteine is an allosteric inhibitor of vertebrate Kinesin Spindle Protein (KSP) that has generated considerable interest due to its anti-cancer properties, however, poor pharmacological properties have limited the use of this compound. We have modified the triphenylmethyl and cysteine groups, guided by biochemical and cell-based assays, to yield new cysteinol and cysteamine derivatives with increased inhibitory activity, greater efficacy in model systems, and significantly enhanced potency against the NCI60 tumor panel. These results reveal a promising new class of conformationally-flexible small molecules as allosteric KSP inhibitors for use as research tools, with activities that provide impetus for further development as anti-tumor agents.

Synthesis of trityl radical-conjugated disulfide biradicals for measurement of thiol concentration.

Measurement of thiol concentrations is of great importance for characterizing their critical role in normal metabolism and disease. Low-frequency electron paramagnetic resonance (EPR) spectroscopy and imaging, coupled with the use of exogenous paramagnetic probes, have been indispensable techniques for the in vivo measurement of various physiological parameters owing to the specificity, noninvasiveness and good depth of magnetic field penetration in animal tissues. However, in vivo detection of thiol levels by EPR spectroscopy and imaging is limited due to the need for improved probes. We report the first synthesis of trityl radical-conjugated disulfide biradicals (TSSN and TSST) as paramagnetic thiol probes. The use of trityl radicals in the construction of these biradicals greatly facilitates thiol measurement by EPR spectroscopy since trityls have extraordinary stability in living tissues with a single narrow EPR line that enables high sensitivity and resolution for in vivo EPR spectroscopy and imaging. Both biradicals exhibit broad characteristic EPR spectra at room temperature because of their intramolecular spin-spin interaction. Reaction of these biradicals with thiol compounds such as glutathione (GSH) and cysteine results in the formation of trityl monoradicals which exhibit high spectral sensitivity to oxygen. The moderately slow reaction between the biradicals and GSH (k(2) ∼ 0.3 M(-1) s(-1) for TSSN and 0.2 M(-1) s(-1) for TSST) allows for in vivo measurement of GSH concentration without altering the redox environment in biological systems. The GSH concentration in rat liver was determined to be 3.49 ± 0.14 mM by TSSN and 3.67 ± 0.24 mM by TSST, consistent with the value (3.71 ± 0.09 mM) determined by the Ellman's reagent. Thus, these trityl-based thiol probes exhibit unique properties enabling measurement of thiols in biological systems and should be of great value for monitoring redox metabolism.

Unprecedented ipso aromatic nucleophilic substitution upon oxidative decarboxylation of tris(p-carboxyltetrathiaaryl)methyl (TAM) radicals: a new access to diversely substituted TAM radicals.

A new reaction of oxidative substitution of aromatic carboxyl groups on stable trityl radical derivatives (TAMs) by various nucleophiles is described; it leads to a wide variety of new persistent and diversely substituted TAM radicals.

Structure-activity relationship and multidrug resistance study of new S-trityl-L-cysteine derivatives as inhibitors of Eg5.

The mitotic spindle is a validated target for cancer chemotherapy. Drugs such as taxanes and vinca alkaloids specifically target microtubules and cause the mitotic spindle to collapse. However, toxicity and resistance are problems associated with these drugs. Thus, alternative approaches to inhibiting the mitotic spindle are being pursued. These include targeting Eg5, a human kinesin involved in the formation of the bipolar spindle. We previously identified S-trityl-L-cysteine (STLC) as a potent allosteric inhibitor of Eg5. Here, we report the synthesis of a new series of STLC-like compounds with in vitro inhibition in the low nanomolar range. We also performed a multidrug resistance study in cell lines overexpressing P-glycoprotein and showed that some of these inhibitors may have the potential to overcome susceptibility to this efflux pump. Finally, we performed molecular docking of the compounds and determined the structures of two Eg5-inhibitor complexes to explain the structure-activity relationship of these compounds.