The Use of Anhydrous Barium Hydroxide for Selective Alkylation of Dialkyloxy-tert-butyl-calix[4]arenes.

A practical method of selective alkylation of the third hydroxyl group of disubstituted tert-butyl-calix[4]arenes using anhydrous barium hydroxide as a base was developed in this study. The use of this method in the synthesis of inherently chiral derivatives is shown herein.

Inhibition of glutathione S-transferases by photoactive calix[4]arene α-ketophosphonic acids.

Calix[4]arenes bearing photoactive α-ketophosphonic acid groups at the upper rim of the macrocycle were synthesized and evaluated as inhibitors of glutathione S-transferases. Irradiation at 365 nm increased the inhibition effects of some macrocyclic compounds on GSTP1-1 by more than two orders of magnitude. Calix[4]arene bis-α-ketophosphonic acids substituted at the lower rim by n-propyl or n-butyl groups showed IC50 values in the low micromolar range. Kinetics of the irreversible inhibition was described by pseudo-first-order rate constants dependent on inhibitor concentration. The values of second-order rate constants were higher for glutathione S-transferase from human placenta than for the enzyme from equine liver. Molecular docking suggested that photoactive macrocyclic compounds cover the active site of glutathione S-transferase, providing the possibility to modify the catalytically important amino acid residues during irradiation.

Synthesis and separation of diastereomeric N-(1-phenylethyl)amides of inherently chiral hydroxydibenzoyloxy-calix[4]arene acetic acids.

A preparative method for the synthesis of optically pure N-(1-phenylethyl)amides of inherently chiral (cR)- and (cS)-dibenzoyloxy-calix[4]arene acetic acids has been developed. Their absolute stereochemical configuration was determined by X-ray diffraction analysis.

Calixarene-based phosphinic acids as inhibitors of protein tyrosine phosphatases.

In the present work, the derivatives of calix[4]arene, thiacalix[4]arene, and sulfonylcalix[4]arene bearing four methylene(phenyl)phosphinic acid groups on the upper rim of the macrocycle were synthesized and studied as inhibitors of human protein tyrosine phosphatases. The inhibitory capacities of the three compounds towards PTP1B were higher than those for protein tyrosine phosphatases TC-PTP, MEG1, MEG2, and SHP2. The most potent sulfonylcalix[4]arene phosphinic acid displayed Ki value of 32 nM. The thiacalix[4]arene phosphinic acid was found to be a low micromolar inhibitor of PTP1B with selectivity over the other PTPs. The kinetic experiments showed that the inhibitors compete with the substrate for the active site of the enzyme. Molecular docking was performed to explain possible binding modes of the calixarene-based phosphinic inhibitors of PTP1B.

Synthesis of an Enantiomerically Pure Inherently Chiral Calix[4]Arene Phosphonic Acid and Its Evaluation as an Organocatalyst.

A facile method for the preparation of enantiomerically pure inherently chiral calix[4]arene phosphonic acid (cR,pR)-7 in four steps starting from the readily available and previously synthesized (cS)-enantiomer of calix[4]arene acetic acid 1 or its methyl ester 2 was developed. The first tests of this unique calixarene Brönsted acid with inherent chirality in organocatalysis of the aza-Diels-Alder reaction of imines with Danishefsky's diene and epoxide ring opening by benzoic acid were performed. The calixarene phosphonic acid (cR,pR)-7 shows good catalytic activities but with low enantioselectivities in these reactions.

Selective inhibition of smooth muscle plasma membrane transport Са2+,Mg2+-АТРase by calixarene C-90 and its activation by IPT-35 compound.

We investigated the influence of calixarene C-90 and IPT-35 on plasma membrane Ca2+- pumping АТРase (PMCA), intracellular calcium homeostasis and myometrium smooth muscle strain contractions. It has been shown that both effectors (100 µM) affect PMCA enzymatic activity: calixarene C-90 inhibits it by 75% and IPT-35 activates it by 40%. These compounds don't affect the Mg2+-АТРase, Mg2+-independent Са2+-АТРase and Na+,K+-АТРase enzymatic activities. C-90 inhibition coefficient I0.5 magnitude was approximately 20 µM and the Hill coefficient nH was 0.55. For IPT-35 activation, constant А0.5 was 6.4 and nH was 0.7. Mathematical modeling demonstrated the implication of calixarene C-90 on unexcited myocytes, which allows for a precise change in cytoplasm Ca2+ concentration and an influence on basal muscle tonus. By the same method, we determined that IPT-35 has a little influence on Ca2+ concentration in unexcited myocytes. It was also shown that calixarene C-90 in vitro can increase velocity of oxytocin-initiated contractions, whereas IPT-35 can suppress this aforementioned parameter. These results are promising for the design of new pharmacological compounds as better regulators of uterine contractions. Calixarene C-90 can be used in obstetric cases for the simultaneous use of oxytocin for enhancing uterine contractions, and IPT-35 for its antispasmodic effect on uterine tone.

Protein-Sized Bright Fluorogenic Nanoparticles Based on Cross-Linked Calixarene Micelles with Cyanine Corona.

The key challenge in the field of fluorescent nanoparticles (NPs) for biological applications is to achieve superior brightness for sizes equivalent to single proteins (3-7 nm). We propose a concept of shell-cross-linked fluorescent micelles, in which PEGylated cyanine 3 and 5 bis-azides form a covalently attached corona on micelles of amphiphilic calixarene bearing four alkyne groups. The fluorescence quantum yield of the obtained monodisperse NPs, with a size of 7 nm, is a function of viscosity and reached up to 15 % in glycerol. In the on-state they are circa 2-fold brighter than quantum dots (QD-585), which makes them the smallest PEGylated organic NPs of this high brightness. FRET between cyanine 3 and 5 cross-linkers at the surface of NPs suggests their integrity in physiological media, organic solvents, and living cells, in which the NPs rapidly internalize, showing excellent imaging contrast. Calixarene micelles with a cyanine corona constitute a new platform for the development of protein-sized ultrabright fluorescent NPs.

Phosphonate monoesters on a thiacalix[4]arene framework as potential inhibitors of protein tyrosine phosphatase 1B.

Monoester derivatives of thiacalix[4]arene tetrakis(methylphosphonic) acid were found to be capable of inhibiting protein tyrosine phosphatase 1B. In addition, these compounds can strongly bind to human serum albumin.

Anion carrier formation by calix[4]arene-bis-hydroxymethylphosphonic acid in bilayer membranes.

The action of calix[4]arenes C-91, C-97, C-99, C-107 and C-160 on solvent-containing planar bilayer membranes made of cholesterol and egg phosphatidylcholine (egg PC) or synthetic 18-carbon-tail phospholipid DOPC has been investigated in a voltage-clamp mode. Within the range of calix[4]arenes tested, a steady-state voltage-dependent transmembrane current was achieved only after addition of calix[4]-arene C-99 (calix[4]arene-bis-hydroxymethylphosphonic acid) from the side of the membrane the positive potential was applied to. This current exhibited anion selectivity passing more chloride at negative potentials applied from the side of the membrane to which calix[4]arene C-99 was introduced. The kinetics and temperature-dependence determined for calix[4]arene C-99-mediated ionic transport suggest a carrier mode of facilitated diffusion.

Use of thiacalix[4]arene C-1193 for a directed influence on the functional activity of mitochondria and simulation of this process using a Petri nets.

In molecular biological studies, considerable attention is paid to macrocyclic nanoscale compounds known as calix[4]arenes. An imperative concern in biochemical membranology and molecular biotechnology is the exploration of effectors capable of modifying the intensity of redox reactions within the inner mitochondrial membrane and influencing the activity of its Ca2+ transport systems. The simulation model development is relevant to formalize and generalize the experimental data and assess the conformity of experimental results with theoretical predictions. Experiments were carried out on a suspension of isolated rat myometrial mitochondria. The synthesized thiacalix[4]arene C-1193, containing four sulfur atoms, was employed. Demonstrations of time-dependent and concentration-dependent (0.01-10 µM) inhibition of Ca2+ accumulation and reactive oxygen species (ROS) formation by mitochondria in the presence of C-1193 were observed. While C-1193 inhibited the oxidation of NADH and FADH2, it did not induce mitochondrial swelling. The thiacalix[4]arene also inhibited the synthesis of nitric oxide, with a Ki of 5.5 ± 1.7 nM, positioning it as a high-affinity blocker of endogenous NO generation in mitochondria. These results are the basis for the possible application of the synthesized thiacalix[4]arene as a tool in researching biochemical processes in mitochondria. A simulation model employing functional hybrid Petri nets was developed, reproducing the functional activity of mitochondria, including simultaneous NADH oxidation, ROS formation, NO synthesis, and Ca2+ accumulation. The derived equations formalize and describe the time dependencies of the listed processes in the medium under the influence of thiacalix[4]arene C-1193.

Substitution of bridge carbons for sulphur in calix[4]arene-bis-α-hydroxymethylphosphonic acid transformed mobile carrier into ionic channel accompanied with evoked muscle contraction and impaired neurotransmission powered by membrane action of resulting thiocalix[4]arene-bis-α-hydroxymethylphosphonic acid.

The action of calix[4]arenes C-424, C-425 and C-1193 has been investigated on suspended cholesterol/egg phosphatidylcholine lipid bilayer in a voltage-clamp mode. Comparative analysis with the membrane action by calix[4]arene-bis-α-hydroxymethylphosphonic acid (C-99) has shown that the substitution of bridge carbons for sulphur and addition of another methyl group to two alkyl tales in the lower rim of former dipropoxycalix[4]arene C-99 transformed mobile carrier that C-99 created in lipid bilayer (Shatursky et al., 2014) into a transmembrane pore as exposure of the bilayer membrane to sulphur-containing derivative dibutoxythiocalix[4]arene C-1193 resulted in microscopic transmembrane current patterns indicative of a channel-like mode of facilitated diffusion. Within all calix[4]arenes tested a net steady-state voltage-dependent transmembrane current was readily achieved only after addition of calix[4]-arene C-1193. In comparison with the membrane action of C-99 the current induced by calix[4]-arene C-1193 exhibited a much weakened anion selectivity passing slightly more current at positive potentials applied from the side of bilayer membrane to which the calix[4]-arene was added. Testing C-1193 for the membrane action against smooth muscle cells of rat uterus or swine myometrium and synaptosomes of rat brain nerve terminals revealed an increase in intracellular concentration of Ca2+ with reduction of the effective hydrodynamic diameter of the smooth muscle cells and enhanced basal extracellular level of neurotransmitters (glutamate and γ-aminobutyric acid) after C-1193-induced depolarization of the nerve terminals.

Calix[4]arene methylenebisphosphonic acids as inhibitors of protein tyrosine phosphatase 1B.

Сalix[4]arenes bearing methylenebisphosphonic or hydroxymethylenebisphosphonic acid fragments at the wide rim of the macrocycle were studied as inhibitors of PTP1B. Some of the inhibitors showed IC50 values in the micromolar range and good selectivity in comparison with other protein tyrosine phosphatases such as TC-PTP, PTPß, LAR, and CD45. Kinetic studies indicated that the calix[4]arene derivatives influence PTP1B activity as slow-binding inhibitors. Based on molecular docking results, the binding modes of the macrocyclic bisphosphonates in the active centre of PTP1B are discussed.

Data for characterisation of nanoformulations formed by cationic 1,4-dihydopyridine and calix[4]arene compositions.

In this data file the characterisation of nanoformulations obtained from calix[4]arene/1,4-dihydropyridine (1,4-DHP) compositions in the various component ratio in an aqueous medium was performed by dynamic light scattering (DLS) technique. The hydrodynamic diameters of nanoparticle main population, polydispersity index and stability of nanoformulation were determined. In this article provided data are directly related to the previously published research articles - "Gene delivery agents possessing antiradical activity: Self-assembling cationic amphiphilic 1,4-dihydropyridine derivatives" [1], and "Studies of the physicochemical and structural properties of self-assembling cationic pyridine derivatives as gene delivery agents" [2] where was described synthesis, transfection activity of 1,1'-((3,5-bis((dodecyloxy)carbonyl)-4-phenyl-1,4-dihydropyridine-2,6-diyl)bis(methylene))bis(pyridin-1-ium) dibromide presented in this data file; and with articles "Cationic amphiphilic calixarenes to compact DNA into small nanoparticles for gene delivery" [3] and "Self-aggregation in aqueous solution of amphiphilic cationic calix[4]arenes. Potential use as vectors and nanocarriers" [4] where was described synthesis and ability to condense DNA for also mentioned calix[4]arenes - 5,11,17,23-tetra-(3-methylimidazolium)-methylene-25,26,27,28-etradodecyloxycalix[4]arene tetrachloride, 5,11,17,23-tetra(N,N-dimethyl-N-hydroxyethylammonium)-methylene-25,26,27,28-tetradodecyloxycalix[4]arene tetrachloride and 5,11,17,23-tetra(N,N-dimethyl-N-hydroxyethylammonium)-methylene-25,26,27,28-tetrahexadecyloxycalix[4]arene tetrachloride. Information provided in this data file can be used in medicinal chemistry for development of novel synthetic lipid nanoformulations.

Anionic amphiphilic calixarenes for peptide assembly and delivery.

Shape-persistent macrocycles enable superior control on molecular self-assembly, allowing the preparation of well-defined nanostructures with new functions. Here, we report on anionic amphiphilic calixarenes of conic shape and their self-assembly behavior in aqueous media for application in intracellular delivery of peptides. Newly synthesized calixarenes bearing four phosphonate groups and two or four long alkyl chains were found to form micelles of âˆ¼ 10 nm diameter, in contrast to an analogue with short alkyl chains. These amphiphilic calixarenes are able to complex model (oligo-lysine) and biologically relevant (HIV-1 nucleocapsid peptide) cationic peptides into small nanoparticles (20-40 nm). By contrast, a control anionic calixarene with short alkyl chains fails to form small nanoparticles with peptides, highlighting the importance of micellar assembly of amphiphilic calixarenes for peptide complexation. Cellular studies reveal that anionic amphiphilic calixarenes exhibit low cytotoxicity and enable internalization of fluorescently labelled peptides into live cells. These findings suggest anionic amphiphilic macrocycles as promising building blocks for the preparation of peptide delivery vehicles.

Virus-sized DNA nanoparticles for gene delivery based on micelles of cationic calixarenes.

Macrocyclic amphiphilic molecules based on calix[4]arenes are highly attractive for controlled supramolecular assembly of DNA into small nanoparticles, since they present a unique conical architecture and can bear multiple charged groups. In the present work, we synthesized new amphiphilic calixarenes bearing cationic groups at the upper rim and alkyl chains at the lower rim. Their self-assembly in aqueous solution was characterized by fluorescent probes, fluorescence correlation spectroscopy, dynamic light scattering, gel electrophoresis and atomic force microscopy. We found that calixarenes bearing long alkyl chains (octyl) self-assemble into micelles of 6 nm diameter at low critical micellar concentration and present the unique ability to condense DNA into small nanoparticles of about 50 nm diameter. In contrast, the short-chain (propyl) analogues that cannot form micelles at low concentrations failed to condense DNA, giving large polydisperse DNA complexes. Thus, formation of small DNA nanoparticles is hierarchical, requiring assembly of calixarenes into micellar building blocks that further co-assemble with DNA into small virus-sized particles. The latter showed much better gene transfection efficiency in cell cultures relative to the large DNA complexes with the short-chain analogues, which indicates that gene delivery of calixarene/DNA complexes depends strongly on their structure. Moreover, all cationic calixarenes studied showed low cytotoxicity. Thus, this work presents a two-step hierarchical assembly of small DNA nanoparticles for gene delivery based on amphiphilic cone-shaped cationic calixarenes.

Multivalent Calixarene-Based Liposomes as Platforms for Gene and Drug Delivery.

The formation of calixarene-based liposomes was investigated, and the characterization of these nanostructures was carried out using several techniques. Four amphiphilic calixarenes were used. The length of the hydrophobic chains attached to the lower rim as well as the nature of the polar group present in the upper rim of the calixarenes were varied. The lipid bilayer was formed with one calixarene and with the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE. The cytotoxicity of the liposomes for various cell lines was also studied. From the results obtained, the liposomes formed with the least cytotoxic calixarene, (TEAC12)4, were used as nanocarriers of both nucleic acids and the antineoplastic drug doxorubicin, DOX. Results showed that (TEAC12)4/DOPE/p-EGFP-C1 lipoplexes, of a given composition, can transfect the genetic material, although the transfection efficiency substantially increases in the presence of an additional amount of DOPE as coadjuvant. On the other hand, the (TEAC12)4/DOPE liposomes present a high doxorubicin encapsulation efficiency, and a slow controlled release, which could diminish the side effects of the drug.

Inhibition of Yersinia protein tyrosine phosphatase by phosphonate derivatives of calixarenes.

Inhibition of Yersinia protein tyrosine phosphatase by calix[4]arene mono-, bis-, and tetrakis(methylenebisphosphonic) acids as well as calix[4]arene and thiacalix[4]arene tetrakis(methylphosphonic) acids have been investigated. The kinetic studies revealed that some compounds in this class are potent competitive inhibitors of Yersinia PTP with inhibition constants in the low micromolar range. The binding modes of macrocyclic phosphonate derivatives in the enzyme active center have been explained using computational docking approach. The results obtained indicate that calix[4]arenes are promising scaffolds for the development of inhibitors of Yersinia PTP.

(Thia)calixarenephosphonic Acids as Potent Inhibitors of the Nucleic Acid Chaperone Activity of the HIV-1 Nucleocapsid Protein with a New Binding Mode and Multitarget Antiviral Activity.

The nucleocapsid protein (NC) is a highly conserved protein that plays key roles in HIV-1 replication through its nucleic acid chaperone properties mediated by its two zinc fingers and basic residues. NC is a promising target for antiviral therapy, particularly to control viral strains resistant to currently available drugs. Since calixarenes with antiviral properties have been described, we explored the ability of calixarene hydroxymethylphosphonic or sulfonic acids to inhibit NC chaperone properties and exhibit antiviral activity. By using fluorescence-based assays, we selected four calixarenes inhibiting NC chaperone activity with submicromolar IC50 values. These compounds were further shown by mass spectrometry, isothermal titration calorimetry, and fluorescence anisotropy to bind NC with no zinc ejection and to compete with nucleic acids for the binding to NC. Molecular dynamic simulations further indicated that these compounds interact via their phosphonate or sulfonate groups with the basic surface of NC but not with the hydrophobic plateau at the top of the folded fingers. Cellular studies showed that the most soluble compound CIP201 inhibited the infectivity of wild-type and drug-resistant HIV-1 strains at low micromolar concentrations, primarily targeting the early steps of HIV-1 replication. Moreover, CIP201 was also found to inhibit the flipping and polymerization activity of reverse transcriptase. Calixarenes thus form a class of noncovalent NC inhibitors, endowed with a new binding mode and multitarget antiviral activity.

Calixarenes in bio-medical researches.

Application of calixarene derivatives in bio-medical researches is reviewed in this article. Antiviral, bactericidal, antithrombothic, antituberculosis, anticancer activity as well as specific protein complexation, membranotropic properties and toxicity of modified calixarenes are discussed.

Thiacalix[4]arenes Remove the Inhibitory Effects of Zn Cations on the Myosin ATPase Activity.

Numerous female reproductive abnormalities are caused by uterine smooth muscle (myometrium) disorders. Heavy metals have an adverse effect on the contractility of the uterine smooth muscle. Although zinc is an essential biogenic element for most of the organisms, high doses of this element are toxic. The study of 0.5-5 mM Zn2+ effect on myosin S1 ATPase activity from the uterus found that 5 mM Zn2+ cations have the most pronounced inhibitory effect. The calculation of the kinetic parameters (Km and Vmax, ATP) revealed that the apparent maximum velocity of the hydrolysis ATP catalyzed by myosin in the presence of 5 mM Zn2+ decreased by 1.6 times. The value of Кm for ATP hydrolysis by myosin S1 in the presence of Zn2+ does not change statistically, although it tends to decrease. It was determined that uterine myosin S1 ATPase activity does not depend on the concentration of Mg2+ in the presence of 5 mM Zn2+. Also, it was demonstrated that tetrahydroxythiacalix[4]arene-tetrasulfosphonate (C-798) and tetrahydroxythiacalix[4]arene-tetraphosphonate (C-800) restored myosin S1 ATPase activity to the control level in the presence of 5 mM Zn2+. One of the most probable mechanisms of restoring the action of these thiacalix[4]arenes protective effect is based on its ability to chelate heavy metal cations from the incubation medium. The molecular docking of C-798 and C-800 into the myosin S1 region showed that these thiacalix[4]arenes could interact with Zn cation bond by myosin amino acid residues near the ATPase active site. Therefore, thiacalix[4]arenes may weaken the interaction between this cation and myosin S1. It was speculated that the obtained results could be used for further research with the aim of using this thiacalix[4]arenes as pharmacological compounds in the case of poisoning with high concentrations of zinc.