Design, synthesis, and mechanistic insight of novel imidazolones as potential EGFR inhibitors and apoptosis inducers.

As regards to the structural analysis and optimization of diverse potential EGFR inhibitors, two series of imidazolyl-2-cyanoprop-2-enimidothioates and ethyl imidazolylthiomethylacrylates were designed and constructed as potential EGFR suppressors. The cytotoxic effect of the prepared derivatives was assessed toward hepatic, breast, and prostate cancerous cells (Hep-G2, MCF-7, and PC-3). Three derivatives 3d, 3e, and 3f presented potent antiproliferative activity and selectivity against the examined tumor cells showing IC50 values at low micromolar levels. Hence, successive biological assays were applied to determine the probable mechanism of action of the new compounds. They exhibited significant EGFR suppression with an IC50 range of 0.137-0.507 µM. The most effective EGFR inhibitor 3f arrested the MCF-7 cell cycle at the S phase by inducing the apoptotic pathway that was confirmed via increasing the expression of Caspases 8, 9, and Bax, which are associated with Bcl-2 decline. Additionally, molecular docking displayed a distinctive interaction between 3f and EGFR binding pocket. Overall, this work introduces some novel imidazolyl-2-cyanoprop-2-enimidothioates and ethyl imidazolylthiomethylacrylates as potential cytotoxic and EGFR inhibitors that deserve further research in tumor therapy.

Design and synthesis of novel dithiazole carboxylic acid Derivatives: In vivo and in silico investigation of their Anti-Inflammatory and analgesic effects.

Inflammation is a complex set of interactions that can occur in tissues as the body's defensive response to infections, trauma, allergens, or toxic compounds. Therefore, in almost all diseases, it can be observed because of primary or secondary reasons. Since it is important to control and even eliminate the symptoms of inflammation in the treatment of many diseases, anti-inflammatory and analgesic drugs are always needed in the clinic. Therefore, the discovery of new anti-inflammatory/analgesic drugs with increased effectiveness and safer side effect profiles is among the popular topics of medicinal chemistry. Therefore, in this study, in order to synthesize and diversify new molecules, we focused on the N,N-dithiazole carboxylic acid core and linked it with the chalcone functional group. The final eleven molecules were analyzed via HRMS, 1H NMR, and 13C NMR. The antinociceptive effects of the test compounds were examined by tail-clip, hot-plate, and formalin methods in mice, while their anti-inflammatory activities were investigated by carrageenan-induced inflammation tests in rats. The motor activities of the experimental animals were evaluated using an activity-meter device. Obtained findings revealed that none of the test compounds (10 mg/kg) were effective in the tail-clip and hot-plate tests. However, compounds 4b, 4c, 4f, 4 h, and 4 k in the serial shortened the paw-licking times of mice in the late phase of the formalin test indicating that these compounds had peripherally-mediated antinociceptive effects. The same compounds, moreover, showed potent anti-inflammatory effects by significantly reducing paw edema of rats in the inflammation tests. To provide an approach to pharmacological findings regarding possible mechanisms of action, the binding modes of the most active compounds were investigated by in silico approaches. The results of molecular docking studies indicated that the anti-inflammatory and analgesic activities of the compounds might be related to the inhibition of both COX-1 and COX-2 isoenzymes. Findings obtained from in silico studies showed that 4 k, which was chosen as a model for its analogs in the series, forms strong bindings to the basic residues (Arg120, Tyr355), side pocket loop area and deep hydrophobic regions of the enzyme. Moreover, results of the molecular dynamics simulation studies revealed that ligand-COX enzyme complexes are quite stable. Obtained results of in vivo and in silico studies are in harmony, and all together point out that compounds 4b, 4c, 4f, 4 h, and 4 k have significant anti-inflammatory and analgesic activities with good ADME profiles. The potential of the derivatives, whose pharmacological activities were revealed for the first time in this study, as anti-inflammatory and analgesic drug candidates, needs to be evaluated through comprehensive clinical studies.

Determination and pharmacokinetic study of isothiouronium-modified pyrimidine-substituted curcumin analog (1G), a novel antitumor agent, in rat plasma by liquid chromatography-tandem mass spectrometry.

1G, a novel derivative of curcumin, exhibits promising anticancer activities in various cancer cell lines. To support its further pharmacological studies, a liquid chromatography-tandem mass spectrometry method was developed and validated in accordance with FDA's Guidance. After extraction by protein precipitation, analytes were separated by a 4.5 min gradient elution (water/0.1% formic acid and methanol) on a reverse-phase C18 column at 40 °C. The multiple reaction monitoring mode was used for quantification on a triple quadrupole mass spectrometer with positive ionization. The assay was linear over the concentration range of 5-1000 ng/mL with a correlation coefficient (r) greater than 0.99. Values of intra- and inter-day precision and accuracy were satisfactory, i.e. <10.1% for precision and within ± 14.5% for accuracy. No obvious matrix effect was observed. Recovery of the analyte was higher than 95.3%. 1G was stable during the whole analytic process. The validated method was successfully applied to the pharmacokinetic study of 1G after intravenous and intraperitoneal administration in rats. Favorable pharmacokinetic profiles were demonstrated, including good abdominal absorption (F = 62.58%), moderate clearance and high extravascular distribution. Results indicated that as a novel antitumor agent, 1G exhibited acceptable pharmacokinetic properties for further in vivo pharmacologic evaluation.

Isothiouronium modification empowers pyrimidine-substituted curcumin analogs potent cytotoxicity and Golgi localization.

Most of protein post-translational modifications occur in the Golgi and many human diseases are associated with abnormal Golgi function or improper post translational modifications of proteins in the Golgi. In this study, we designed and synthesized 4 × 6 series of novel isothiouronium-modified (E,E)-4,6-bis(styryl)-pyrimidine analogs and found that they localized at the Golgi as visualized by the intrinsic fluorescence of the analogs. The isothiouronium-modified analogs had potent cytotoxicity in both normal (Chinese Hamster Ovary or CHO) and cancer cells. Furthermore, permethylated isothiouronium-modified analogs showed cancer cell-selective cytotoxicity. The molecular mechanisms underlying Golgi localization of isothiouronium-modified compounds were investigated using 7 CHO and 4 human cancer cell lines and the results indicated that the compounds had binding partners in the Golgi. Thus, isothiouronium-modified analogs might be promising anticancer agents, novel Golgi staining reagents, and useful research tools for studying Golgi functions in normal or cancer cells and in Golgi-related human diseases.

Isothiouronium salts reduce NRAS expression, induce apoptosis and decrease invasion of melanoma cells.

Melanoma is a very aggressive type of skin cancer. Mutation in BRAF and NRAS are often found in patients with this disease. Therefore, in recent years the search for new molecules that inhibit these proteins has been intensified. After many years with no new treatments for melanoma, the U.S. Food and Drug Administration (FDA) recently approved vemurafenib. However, many patients have already acquired resistance and have experienced severe side effects. Therefore, this work aims to evaluate a new set of compounds including allylic isothiouronium salts (1, 2 and 3), N-phenyl-substituted analog (4) and isothiosemicarbazide salts (5 and 6) for their potential antimelanoma activity. To this end, viability assay, cell cycle analysis, expression of NRAS and BRAF, as well as migration and invasion assay were performed with different melanoma cell lines. Isothiouronium salts 1-3 presented CC50 (concentration required to reduce the cell number by 50%) in a range of 7-28 µM. Furthermore, salt 1 significantly decreased the expression of NRAS. However, cells incubated with these salts did not disturb the cell cycle phases; instead, an increase in the number of apoptotic cells was observed. Regarding potential antiinvasion effects, both 1 and 2 prevented cell migration as well as cell invasion. Finally, when salts 1 and 2 were associated with vemurafenib, a marked decrease in cell viability was observed when compared to the compounds incubated alone. Briefly, the salts exhibited interesting results, especially 1, which decreased the expression of NRAS, increased apoptotic cells and, when combined with vemurafenib, resulted in a synergistic effect. Therefore, we intend to test compound 1 in pre-clinical studies.

Nanospherical aggregation of isothiouronium-terminated amphiphilic polythiophene: preparation and vapor-phase detection of volatile organic compounds.

Cationic isothiouronium-terminated amphiphilic poly(3-octylthiophene) IPOT has been synthesized by the reaction of bromoethylene-terminated poly(3-octylthiophene) with 1-(2-(2-methoxyethoxy)ethyl)-3-methylthiourea. By performing gel-permeation chromatography analysis, we estimated the number-average molecular weight (M(n)) and the polydispersity index (PDI) to be 1.07 x 10(4) and 1.48, respectively. The incorporation of an isothiouronium unit at the terminal position renders the polymer amphiphilic. Well-dispersed spherical aggregates were formed with increasing H2O content in the THF solution and were characterized by FE-SEM and powder XRD measurements. We thus proposed a core/shell-type micelle with a poly(3-octylthiophene) segment as the core and an isothiouronium segment as the shell, exposing the hydrophilic methoxyethoxy pendant. The change in morphology to a spherical shape led to a bathochromic shift of the absorption band with decreasing fluorescence intensity, enabling us to use it as a fluorimetric "off-on" sensor for volatile organic compounds (VOCs) where a quick response to THF was observed on the IPOT aggregate-coated TLC plate.

Inverse neighboring group participation: explanation of an unusual S→N alkyl migration of isothiuronium salts containing a lactone group.

A detailed experimental and DFT study of the S to N alkyl migration of substituted S-(1(3H)-isobenzofuranon-3-yl)isothiuronium bromide to N,N'-dimethyl-N-(3-oxo-1,3-dihydro-2-benzofuran-1-yl)thiourea provided evidence for the existence of an unusual double displacement mechanism involving two consecutive back-side S(N)2 reactions where a carboxylate anion has a crucial role both as a leaving group as well as an internal nucleophile. The thiazetidine zwitterionic species that is involved in this mechanism as an intermediate was characterized by infrared multiphoton dissociation spectroscopy and was trapped with methyl iodide. It was found that the intermediate has a structure of a free ion pair. The double-displacement mechanism can be considered as a new type of inverse lactone neighboring group participation.

Synthetic studies on novel 1,4-dihydro-2-methylthio-4,4,6-trisubstituted pyrimidine-5-carboxylic acid esters and their tautomers.

A mixture of alkyl 1,4-dihydro-2-methylthio-4,4,6-trisubstituted pyrimidine-5-carboxylate 1 and its tautomeric isomer, alkyl 1,6-dihydro-2-methylthio-4,6,6-trisubstituted pyrimidine-5-carboxylate 2 is synthesized by the Atwal-Biginelli cyclocondensation reaction of S-methylisothiourea hemisulfate salt 3 with 2-(gem-disubstituted)methylene-3-oxoesters 4 that can be accessed by the Lehnert procedure for the Knoevenagel-type condensation. The structures of the tautomeric products of the Atwal-Biginelli cyclocondensation reaction, 1 and 2, which are inseparable from each other, are determined unambiguously by (1)H-NMR spectroscopy at various temperatures and nuclear Overhauser enhancement spectroscopy (NOESY) experiment. Because these dihydropyrimidine products are otherwise inaccessible and thus hitherto unavailable, the synthetic methods established in this study will help to expand the molecular diversity of their related derivatives.

Synthesis of novel deoxynucleoside S-methylphosphonic acids using S-(diisopropylphosphonomethyl)isothiouronium tosylate, a new equivalent of mercaptomethylphosphonate.

The synthesis of the novel nucleotide analogues 5'-deoxynucleoside-5'-S-methylphosphonates, starting from 5'-deoxy-5'-haloribonucleosides, 5'-O-tosylribonucleosides, and 2'-O-triflylnucleosides, is described. The phosphonothiolation of these compounds was achieved using S-(diisopropylphosphonomethyl)isothiouronium tosylate, a new, odourless, and efficient equivalent of mercaptomethylphosphonate. The thiolate anion of mercaptomethylphosphonate was generated in situ from the isothiouronium salt in both protic and aprotic solvents using two equivalents of sodium iso-propoxide. The prepared nucleoside 5'-S-methylphosphonates were deprotected, and the free phosphonic acids were transformed into diphosphoryl derivatives (the NTP analogues). Both mononucleotides and NTP analogues were studied as substrates/inhibitors of several enzymes that are involved in the nucleoside/nucleotide metabolism.

Kinetic evidence for the coexistence of zwitterionic (T+/-), neutral (T0) and anionic (T-) intermediates during rearrangement of S-(2-oxotetrahydrofuran-3-yl)-N-(4-methoxyphenyl)isothiuronium bromide to 5-(2-hydroxyethyl)-2-(4-methoxyphenylimino)-1,3-thiazolidin-4-one.

The kinetics and mechanism of rearrangement of S-(2-oxotetrahydrofuran-3-yl)-N-(4-methoxyphenyl)isothiuronium bromide (1) into 5-(2-hydroxyethyl)-2-[(4-methoxyphenyl)imino]-1,3-thiazolidin-4-one have been studied under pseudo-first-order reaction conditions in aqueous buffer solutions and in diluted HCl at 25 degrees C. Multiple breaks in the pH profile establish the formation of three different kinetically detectable intermediates T(+/-), T(0), and T(-). Treatment of 1 (pK(a) = 6.7) with base produces reactive isothiourea, which undergoes cyclization to give T(+/-) (rate limiting step at pH < 0.5). Intermediate T(+/-) then undergoes either general acid-catalyzed, concerted (alpha = -0.47) breakdown to 2 (rls at pH 2-3) or a water-mediated proton switch to T(0) which is followed by its general acid-catalyzed breakdown (pH 3-6). The last reaction pathway involves the formation of T(-) either from T(+/-) or from T(0) (pH > 6). The first possibility seems to be more likely because it is in accordance with kinetics observed in basic amine buffers, where the nonlinear increase of the k(obs) with the c(Buffer) changes to a linear increase as a general base-catalyzed pathway is introduced. Coexistence of all three kinetically detectable intermediates is very rare and is possibly due to relatively enhanced stability of these intermediates necessitating participation of an acid for progression to products.

Fluorescence sensing of phytate in water using an isothiouronium-attached polythiophene.

A water-soluble isothiouronium-attached polythiophene (1(poly)) is prepared for the first time by the oxidative polymerization of 3-(3-(N-(2-methoxyethoxy)ethyl-N-methyl-S-isothiouronio)propoxy-4-methylthiophene (1) with FeCl(3). The gel-permeation chromatography analysis allows us to estimate the number average molecular weight (M(n)) and the polydispersity index (PDI) to be 5.68x10(5) and 1.4, respectively. The polymers (excited at 438 nm) show an absorption band at 438 nm and fluorescence at 546 nm, with 1.2 % fluorescence quantum yield when fluorescein was used as a reference. The pH-dependent absorption spectra at varying pH, indicates that aggregation starts at neutral conditions. At pH 5.5, 1(poly) exhibits a significant quenching in the fluorescence upon the addition of phytate (IP(6)), as a plausible analyte. This response is higher than that in the case of other anions such as adenosine 5-triphosphate (ATP), pyrophosphate (PPi), cis,cis-1,3,5-cyclohexanetriol triphosphate (CTP(3)), and H(2)PO(4)(-). As inferred from the SEM measurement, the phytate-induced spectral change has been explained on the basis of the pi-stacked aggregation of the polymer.

Effects of high-affinity inhibitors on partial reactions, charge movements, and conformational States of the Ca2+ transport ATPase (sarco-endoplasmic reticulum Ca2+ ATPase).

The inhibitory effects of thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone, and 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene on the Ca(2+) ATPase were characterized by comparative measurements of sequential reactions of the catalytic and transport cycle, including biochemical measurements and detection of charge movements within a single cycle. In addition, patterns of ATPase proteolytic digestion with proteinase K were derived to follow conformational changes through the cycle or after inhibitor binding. We find that thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone inhibit Ca(2+) binding and catalytic activation as demonstrated with isotopic tracers and lack of charge movement upon addition of Ca(2+) in the absence of ATP. It has been shown previously that binding of these inhibitors requires the E2 conformational state of the ATPase, obtained in the absence of Ca(2+). We demonstrate here that E2 state conformational features are in fact induced by these inhibitors on the ATPase even in the presence of Ca(2+). The resulting dead-end complex interferes with progress of the catalytic and transport cycle. Inhibition by 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene, on the other hand, is related to interference with a conformational transition of the phosphorylated intermediate (E1 approximately P . 2Ca(2+) to E2-P . 2Ca(2+) transition), as demonstrated by increased phosphoenzyme levels and absence of bound Ca(2+) translocation upon addition of ATP. This transition includes large movements of ATPase headpiece domains and transmembrane segments, produced through utilization of ATP-free energy as the "conformational work" of the pump. We conclude that the mechanism of high-affinity Ca(2+) ATPase inhibitors is based on global effects on protein conformation that interfere with ATPase cycling.

Probing the structure and activity of trypsin with amidination.

Trypsin reacts with S-methylisothiourea for 1 to 2 h and the number of primary amine sites at which covalent labeling occurs is determined by mass spectrometry. By digesting the amidinated trypsin and mass analyzing the proteolytic peptides the sites of reaction are determined. The addition of cytochrome c to a solution of amidinated trypsin enables the proteolytic activity and autolytic properties of the enzyme to be studied.

Formation of water-dispersible nanotubular graphitic assembly decorated with isothiouronium ion groups and its supramolecular functionalization.

A newly designed Gemini-shaped hexabenzocoronene amphiphile (1), carrying an isothiouronium ion-appended side chain, self-assembles in CH2Cl2 to form a nanotubular object, whose graphitic wall is densely covered by a positively charged molecular layer of isothiouronium ion pendants. The graphitic nanotube can be dispersed uniformly in aqueous media owing to effective hydration as well as electrostatic repulsion. Post-supramolecular functionalization of the nanotube surface is possible, without disruption of the tubular morphology, by taking advantage of a specific interaction of the isothiouronium ion pendants with oxoanion guests. Mixing with sodium poly(4-styrenesulfonate) results in wrapping of the nanotube, while complexation with an electron-accepting oxoanion such as anthraquinone carboxylate allows photoinduced electron transfer from the graphitic wall to the bound guest molecules.

Surface and biocidal activity of some synthesized metallo azobenzene isothiouronium salts.

A novel series of azobenzene isothiouronium salts of different alkyl chains (propyl, hexyl and dodecyl) were synthesized by reaction of 4-((4-methylphenyl)azo)phenol with 1,3-dibromopropane, 1,6-dibromohexane and 1,12-dibromododecane, respectively. These salts were reacted with copper (II) halide to give their corresponding metallo complexes. The surface tension measurements for the synthesized compounds show that the metallo complexes have adsorption and micellization better than that of the parent azobenzene isothiouronium salts. The pathogenic Gram-negative bacteria, Gram-positive bacteria, fungi and yeast were used to determine the biocidal activity of these compounds using gradient plate technique. The results indicate that the copper complexes of the synthesized azobenzene isothiouronium salts have a relatively better biocidal activity than the parent salts.

Interference with phosphoenzyme isomerization and inhibition of the sarco-endoplasmic reticulum Ca2+ ATPase by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene.

ATP hydrolysis and Ca(2+) transport by the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) are inhibited by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene (Br(2)-TITU) in the micromolar range (Berman, M. C., and Karlish, S. J. (2003) Biochemistry 42, 3556-3566). In a study of the mechanism of inhibition, we found that Br(2)-TITU allows the enzyme to bind Ca(2+) and undergo phosphorylation by ATP. The level of ADP-sensitive phosphoenzyme (i.e. E1P-2Ca(2+)) observed in the transient state following addition of ATP is much higher in the presence than in the absence of the inhibitor. Br(2)-TITU does not interfere with enzyme phosphorylation by P(i) in the reverse direction of the cycle (i.e. E2P) and produces only a slight inhibition of its hydrolytic cleavage. The inhibitory effect of Br(2)-TITU on steady state ATPase velocity is attributed to interference with the E1P-2Ca(2+) to E2P-2Ca(2+) transition. In fact, experiments on conformation-dependent protection from proteolytic digestion suggest that, in the presence of Br(2)-TITU, the loops connecting the "A" domain to the ATPase transmembrane region undergo greater fluctuation than expected in the E2 and E2P states. Optimal stability of the gathered headpiece domains is thereby prevented. These effects are opposite to those of thapsigargin, in which the mechanism of inhibition is related to stabilization of a highly compact ATPase conformation and interference with Ca(2+) binding and phosphoenzyme formation. Our experiments with Br(2)-TITU provide the first demonstration of a kinetic limit posed by an inhibitor on the E1P-2Ca(2+) to E2P-2Ca(2+) transition in the wild-type enzyme.

A fast and parallel route to cyclic isothioureas and guanidines with use of microwave-assisted chemistry.

A fast and simple approach to novel cyclic isothioureas and related guanidine derivatives is presented in this study. The construction of the central basic scaffolds is achieved solely by the application of microwave-assisted chemistry, without any need of activating agents or protecting group manipulations. The product formation of various substituted guanidines from the corresponding isothiouronium salts was controlled by the nucleophilicity of the counterion and influenced by the reaction temperature. Further, a new fast-track access to tetrahydropyrimidin-2-ylamines was developed.

Synthesis of vesicles on polymer template.

Reactive single-tail cationic surfactants self-assemble on the anionic block copolymer templates. These systems spontaneously arrange in small vesicles of nanoscale size. The vesicles are further stabilized by dimerization of the assembled surfactant monomers forming double-tail surfactants bound to the block copolymer. The resulting systems are resistant to changes in environmental characteristics such as pH, ionic strength, and temperature variations. Hydrophilic macromolecules can be encapsulated in the internal aqueous volume of these vesicles. The simplicity of the preparation makes these systems promising as drug and gene delivery carriers.

Mapping the active site polarity in structures of endothelial nitric oxide synthase heme domain complexed with isothioureas.

Analyzing the active site topology and plasticity of nitric oxide synthase (NOS) and understanding enzyme-drug interactions are crucial for the development of potent, isoform-selective NOS inhibitors. A small hydrophobic pocket in the active site is identified in the bovine eNOS heme domain structures complexed with potent isothiourea inhibitors: seleno analogue of S-ethyl-isothiourea, S-isopropyl-isothiourea, and 2-aminothiazoline, respectively. These structures reveal the importance of nonpolar van der Waals contacts in addition to the well-known hydrogen bonding interactions between inhibitor and enzyme. The scaffold of a potent NOS inhibitor should be capable of donating hydrogen bonds to as well as making nonpolar contacts with amino acids in the NOS active site.

Controlled template-assisted assembly of plasmid DNA into nanometric particles with high DNA concentration.

A series of novel cationic detergents that contain cleavable hydrophilic isothiuronium headgroups was synthesized, and their utility in controlled assembly of plasmid DNA into small stable particles with high DNA concentration investigated. The detergents have alkyl chains of C(8)-C(12) and contain hydrophilic isothiuronium headgroups that give relatively high critical micelle concentration (CMC) to the detergents (>10 mM). The isothiuronium group masks a sulfhydryl group on the detergent and can be cleaved in a controlled manner under basic conditions to generate a reactive thiol group. The thiol group can undergo a further reaction after the detergents have accumulated on a DNA template to form a disulfide-linked lipid containing two alkyl chains. The pH-dependent kinetics of cleavage of the isothiuronium group, the CMC of the surfactants, the formation of the complexes, and the transfection efficiency of the DNA complexes have been investigated. Using the C(12) detergent, a approximately 6 kilo-basepair plasmid DNA was compacted into a small particle with an average diameter of around 40 nm with a approximately -13 mV zeta-potential at high DNA concentration (up to 0.3 mg/mL). The compounds were well tolerated in cell culture and showed no cytotoxicity under their CMCs. Under appropriate conditions, the small particle retained transfection activity.