Quaternary Ammonium Derivatives Research Articles

Synthesis of quaternary ammonium saltsisonicotinic and nicotinic hydrazones acids and their anti-inflammatory activity

Currently, the number of infections caused by strains of bacteria that are not sensitive to antibiotics and antiseptics is growing worldwide. A similar increase in bacterial resistance is observed for both nosocomial infections and community-acquired human-to-human infections. An important task for chemists and pharmacologists is the synthesis of new compounds and the establishment of a structure-bioactivity relationship. The purpose of this work is to study the effect of the structure of quaternary ammonium derivatives of hydrazones of isonicotinic and nicotinic acids on the manifestation of their anti-inflammatory properties. Results and discussion. The alkylation of nicotinic acid hydrazones produced their new quaternary ammonium salts (43.7-70.7%). The methodology for the synthesis of quaternary ammonium compounds of hydrazones included obtaining a variety of structures by introducing one or two fluorine and bromine-containing fragments into their structure. The structure of compounds was confirmed by 1H, 13C NMR spectroscopy. Conclusion. New quaternary ammonium salts of nicotinic acid hydrazones have been obtained. The study of anti-inflammatory activity was carried out using the method of formalin paw edema in rats. The acute inflammatory reaction was reproduced by subplantar administration of 2% formalin solution. Compared with ibuprofen at a dosage of 100 mg/kg, these compounds were ineffective (p2<0.05). According to the results of the analysis of possible pharmacological effects, it was also revealed that new compounds may have an effect on the regulation and production of ketone bodies, the enzymatic activity of transaminases. New alkylated hydrazones of isonicotinic and nicotinic acids may be of interest for studying their antituberculous and antiviral activities.

New Quaternary Ammonium Derivatives Based on Citrus Pectin.

New citrus pectin derivatives carrying pendant N,N-dimethyl-N-alkyl-N-(2-hydroxy propyl) ammonium chloride groups were achieved via polysaccharide derivatization with a mixture of N,N-dimethyl-N-alkyl amine (alkyl = ethyl, butyl, benzyl, octyl, dodecyl) and epichlorohydrin in aqueous solution. The structural characteristics of the polymers were examined via elemental analysis, conductometric titration, Fourier Transform Infrared spectroscopy (FTIR) and 1D (1H and 13C) nuclear magnetic resonance (NMR). Capillary viscosity measurements allowed for the study of viscometric behavior as well as the determination of viscosity-average molar mass for pristine polysaccharide and intrinsic viscosity ([η]) values for pectin and its derivatives. Dynamic light scattering measurements (DLS) showed that pectin-based polymers formed aggregates in aqueous solution with a unimodal distribution. Critical aggregation concentration (cac) for the hydrophobic pectin derivatives were determined using fluorescence spectroscopy. Atom force microscopy (AFM) images allowed for the investigation of the morphology of polymeric populations obtained in aqueous solution, consisting of flocs and aggregates for crude pectin and its hydrophilic derivatives and well-organized aggregates for lipophilic pectin derivatives. Antimicrobial activity, examined using the disc diffusion method, proved that all polymers were active against Staphylococcus aureus bacterium and Candida albicans yeast.

Constructing a Photothermal and Quaternary Ammonium Cation Bactericidal Platform onto SEBS for Synergistic Therapy.

Poly(styrene-b-(ethylene-co-butylene)-b-styrene) (SEBS) with eminent elasticity, thermoplastic ability, and biological stability has aroused great interest in the medical area. However, bacteria can easily adhere to the hydrophobic SEBS surface to cause medical device-related infections. In this work, SEBS is modified to prepare the SEBS-polydopamine (PDA)-poly(lysine) quaternary ammonium derivative (PLQ) antibacterial surface by PDA deposition and surface grafting techniques to solve bacterial infections. PDA is used as an intermediate layer and presents an excellent photothermal effect. The grafted polymer PLQ has antimicrobial quaternary ammonium cation groups, which plays synergistic bactericidal therapy with PDA. The SEBS-PDA-PLQ surface almost totally suppresses the growth of bacteria with a surface bacterial survival rate of 0.05% under laser irradiation. The outstanding antibacterial activity of the SEBS-PDA-PLQ surface is attributed to the synergistic effects of the photothermal performance of PDA and quaternary ammonium cationic functional groups of PLQ. In addition, the membrane SEBS-PDA-PLQ shows good hydrophilicity, antiprotein adsorption ability, chemical stability, and biocompatibility. This antibiotic-free antimicrobial approach has great potential for practical application in solving infections associated with medical devices.

Preparation of Novel Chitosan Oligosaccharide Quaternary Ammonium Derivatives Bearing Quinoline with Antioxidative and Antibacterial Activities

AbstractIn this study, five new chitosan oligosaccharide quaternary ammonium derivatives bearing quinoline are synthesized by reaction between 6‐O‐chloroacetyl‐2‐N, N, N‐trimethyl quaternary ammonium salt chitosan oligosaccharide (CTCOS, compound 1) and quinoline derivatives. The derivatives are characterized by analytical techniques including Fourier Transform Infrared (FTIR), 1H Nuclear Magnetic Resonance (1H NMR) spectroscopy, and elemental analysis. The obtained results confirm that quinoline groups are successfully introduced into the CTCOS molecule. Meanwhile, the antioxidant activities of the prepared chitosan oligosaccharide derivatives are evaluated in vitro. The experimental results show that the derivatives have excellent free radical scavenging ability. The free radical scavenging abilities are all over 75% at the concentration of 1.6 mg mL−1. Moreover, antibacterial tests show that derivative 2e has the best antibacterial activities. Its minimum inhibitory concentration (MIC) value and minimum bactericidal concentration (MBC) value against Staphylococcus aureus and Escherichia coli are 0.0625 and 0.03125 mg mL−1, respectively. In addition, the cytotoxicity of chitosan oligosaccharide and its derivatives is examined by MTT colorimetric assay in RAW 264.7 macrophages, observing that all derivatives are non‐cytotoxic. Consequently, the findings indicate that the enhanced antioxidant activity, antibacterial activity, and biocompatibility of these chitosan oligosaccharide derivatives can enlarge the scope of the application of chitosan oligosaccharides, particularly as antioxidant and antibacterial agents in food packaging, pharmaceutical, cosmetics industries, and other fields.

Complexation between poly (styrene-co-methacrylic acid) and polyquaternium for use in shampoo formulations

The complexation between poly (styrene-co-methacrylic acid) (P(St-co-MAA)) and a series of cationically substituted quaternary ammonium derivatives of hydroxyethylcellulose, JR and LR (polyquaternium) was investigated through the viscosity change of the complexes at different mixing ratios of the two polymers. The performance of the shampoo containing the complexes was also evaluated. The results show that the viscosity of the mixed aqueous solution increased as the ratio of (P(St-co-MAA)) to JR or LR increased. The maximum viscosity was obtained when the molar ratio (r) of St in P(St-co-MAA) to cations in polyquaternium was near 1. As the mixing ratio increased further, syneresis occurred when the charge stoichiometry between MAA and the quaternary ammonium cation was 1:1. This result reveals that cation-π electron interactions rather than electrostatic attraction play a key role in forming complexes between polyquaternium and P(St-co-MAA). The higher the molecular weight of polyquaternium is, the higher the viscosity of the complex solution. The complexes maintained a large apparent viscosity at a temperature of 70 °C, a shear rate of 1 s−1 and a NaCl concentration of 1.2 mol/L. The complexes had good tolerance to temperature, salt and shearing. The addition of the complexes in shampoo was beneficial to thickening, which is difficult in amino acid anionic surfactant systems. Compared with the shampoo containing the polyquaternium normally, the addition of P(St-co-MAA) helped to generate more flocculation earlier during washing of the hair. Unexpectedly, the wet carding performance of the shampoo was significantly better than that of commercial silicon-containing shampoo (P < 0.01). The complex has potential as a thickener and a conditioner in shampoo formulations.

DOTAP: Structure, hydration, and the counterion effect

The cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) is one of the original synthetic cationic lipids used for the liposomal transfection of oligonucleotides in gene therapy. The key structural element of DOTAP is its quaternary ammonium headgroup that is responsible for interactions with both nucleic acids and target cell membranes. Because these interactions are fundamental to the design of a major class of transfection lipids, it is important to understand the structure of DOTAP and how it interacts with halide counterions. Here, we use x-ray and neutron diffraction techniques to examine the structure of DOTAP and how chloride (Cl−) and iodide (I−) counterions alter the hydration properties of the DOTAP headgroup. A problem of particular interest is the poor solubility of DOTAP/I− in water solutions. Our results show that the poor solubility results from very tight binding of the I− counterion to the headgroup and the consequent expulsion of water. The structural principles we report here are important for assessing the suitability of DOTAP and its quaternary ammonium derivatives for transfection.

Preparation of chitosan-rosmarinic acid derivatives with enhanced antioxidant and anti-inflammatory activities

In present study, several chitosan derivatives grafted with rosmarinic acid，including chitosan-rosmarinic acid conjugate (CS-RA), chitosan rosmarinic acid salt (CSRA), N,N,N-trimethylated chitosan rosmarinic acid salt (TMCRA), and N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan rosmarinic acid salt (HACRA), were prepared and the effects of different grafting methods on bioactivity of these derivatives were investigated. The structural characterization was identified by FTIR and 1H NMR spectroscopy. The chitosan derivatives were also evaluated biologically for antioxidant, cytotoxic, and anti-inflammatory activities. Derivatives CSRA, TMCRA, and HACRA prepared through ion exchange method exhibited significant antioxidant ability. All derivatives remarkably inhibited lipopolysaccharide-induced nitric oxide and TNF-α production in RAW 264.7 cells without cytotoxicity, especially derivative TMCRA, whose inhibition rate was >50 % at only 250 μg/mL. The results suggested that chitosan quaternary ammonium derivatives grafted with rosmarinic acid possessed better antioxidant and anti-inflammatory properties than chitosan, which could be used as drug materials against oxidative and inflammation-related pathological processes.

Topical Antibiofilm Agents With Potential Utility in the Treatment of Chronic Rhinosinusitis: A Narrative Review

The role of bacterial biofilms in chronic and recalcitrant diseases is widely appreciated, and the treatment of biofilm infection is an increasingly important area of research. Chronic rhinosinusitis (CRS) is a complex disease associated with sinonasal dysbiosis and the presence of bacterial biofilms. While most biofilm-related diseases are associated with highly persistent but relatively less severe inflammation, the presence of biofilms in CRS is associated with greater severity of inflammation and recalcitrance despite appropriate treatment. Oral antibiotics are commonly used to treat CRS but they are often ineffective, due to poor penetration of the sinonasal mucosa and the inherently antibiotic resistant nature of bacteria in biofilms. Topical non-antibiotic antibiofilm agents may prove more effective, but few such agents are available for sinonasal application. We review compounds with antibiofilm activity that may be useful for treating biofilm-associated CRS, including halogen-based compounds, quaternary ammonium compounds and derivatives, biguanides, antimicrobial peptides, chelating agents and natural products. These include preparations that are currently available and those still in development. For each compound, antibiofilm efficacy, mechanism of action, and toxicity as it relates to sinonasal application are summarised. We highlight the antibiofilm agents that we believe hold the greatest promise for the treatment of biofilm-associated CRS in order to inform future research on the management of this difficult condition.

Interleukin-12 Plasmid DNA Delivery by N-[(2-Hydroxy-3-trimethylammonium)propyl]chitosan-Based Nanoparticles.

Cationic polysaccharides are capable of forming polyplexes with nucleic acids and are considered promising polymeric gene carriers. The objective of this study was to evaluate the transfection efficiency and cytotoxicity of N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan salt (HTCS), a quaternary ammonium derivative of chitosan (CS), which benefits from non-ionizable positive charges. In this work, HTCS with a full quaternization of amino groups and a molar mass of 130,000 g·mol−1 was synthesized to use for delivery of a plasmid encoding the interleukin-12 (IL-12) gene. Thus, a polyplex based on HTCS and the IL-12 plasmid was prepared and then was characterized in terms of particle size, zeta potential, plasmid condensation ability, and protection of the plasmid against enzymatic degradation. We showed that HTCS was able to condense the IL-12 plasmid by the formation of polyplexes in the range of 74.5 ± 0.75 nm. The level of hIL-12 production following the transfection of the cells with HTCS polyplexes at a C/P ratio of 8:1 was around 4.8- and 2.2-fold higher than with CS and polyethylenimine polyplexes, respectively. These findings highlight the role of HTCS in the formation of polyplexes for the efficient delivery of plasmid DNA.

Synthesis and cholinesterase inhibitory activity study of Amaryllidaceae alkaloid analogues with N-methyl substitution.

Polycyclic compounds with N-methyl substitution, structurally related to Amaryllidaceae alkaloids, have been synthesised, together with their analogues bearing a quaternary nitrogen atom. To prevent the lone electron pair of the nitrogen from interfering with the reaction sequence, two approaches to the synthesis were investigated: N-oxidation and Boc protection of the nitrogen. The second method was more successful due to the limited stability of N-oxides in the halocyclisation step. An asymmetric version of the synthesis was also developed for this type of compounds. The prepared products were tested in vitro for their cholinesterase inhibitory activity and the results were rationalised by molecular docking studies with human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBuChE). In general, our products were more active against BuChE than against AChE, and it was noted that larger ligands should be prepared for future studies, since in some cases acetylcholine can still fit into the active site along with the bound ligand.

A Study of the Antibacterial Activities and the Mode of Action of L-Methionine and L-Cystine Based Surfactants and their Interaction with Bovine Serum Albumin Using Fluorescence Spectroscopy and in Silico Modelling

Surfactants are versatile excipients that are commonly employed in diverse pharmaceutical formulations given their broad range of antimicrobial activities. Sulfur-based amino acid surfactants are promising candidates as substitutes for conventional antibacterial agents in light of the current antibiotic resistance crisis. Dodecyl esters of the free amine (SURF1), cationic ester hydrochloride (SURF2), and quaternary ammonium (QUAT3) derivatives of methionine, and the Gemini diester of cystine (GEM4) were synthesized and evaluated for antibacterial activity against Gram-positive and Gram-negative pathogens. All surfactants displayed moderate to high antibacterial activities, particularly on Gram-positive bacteria, with QUAT3 showing the highest activity. Among Gram-negative bacteria, QUAT3, GEM4, and SURF2 were mostly active towards K. pneumoniae with minimum inhibitory concentrations (MIC) ranging from 0.004 to 0.441 mM. QUAT3 and GEM4 displayed a bacteriostatic behavior similar to that of tetracycline, as assessed by broth macrodilution assays. Fluorescence binding studies with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) revealed that the antibacterial activities could be attributed to a combination of electrostatic and hydrophobic interactions. Bovine serum albumin (BSA) fluorescence and molecular docking studies indicated that SURF1 and QUAT3 interact mainly via van der Waals' forces and hydrogen bonding while SURF2 binds through hydrophobic interactions. QUAT3, which displays broad-spectrum activity, has the potential to combat drug-resistant bacteria.

Porous membranes of quaternized chitosan composited with strontium-based nanobioceramic for periodontal tissue regeneration.

This report demonstrates the development of a degradable quaternary ammonium derivative of chitosan (QC) composited with strontium-containing nanoapatite (SA) for bioactivity. The material was made as porous membrane by solution casting and freeze drying, for guided tissue regeneration (GTR) applications. The micromorphology, tensile strength, suture pull-out strength, degradation (in vitro, in phosphate buffered saline), and cytocompatibility (using human periodontal ligament cells) were tested to investigate the effect of derivatization and SA addition. The porosity of the membranes increased with increasing SA content and so did the tensile strength and the degradation. The suture pull-out strength, however, showed a decrease. The cell culture evaluation endorsed biocompatibility. The composite with 1.5mg SA per 1mL QC was found to have optimal qualities for GTR applications.

New alkaloids from the noni juice with potential α-glucosidase inhibitory activity

Four new alkaloids, nonialkaloids A–D (1–4) and six known analogues (5–10) were isolated from the noni juice. Among the new compounds, 1 and 2 are indole alkaloids with a seven-membered fused N-heterocyclic ring, 3 and 4 are quaternary ammonium derivatives. The structures were elucidated by extensive NMR and MS analysis, while the absolute configurations of the stereogenic carbons were established based on quantum-chemical electronic circular dichroism calculations or the modified Mosher's method. All the isolates were tested for α-glucosidase inhibitory activities. Compounds 1 and 3 displayed potent inhibitory activity against α-glucosidase with the IC50 values of 413.7 and 364.4 μM, respectively.

An unusual acetylene–allene rearrangement in iodomethylates of cotarnine acetylene derivatives

N-Methylation of cotarnine–phenol propargyl ethers conjugates with MeI affords the corresponding quaternary ammonium derivatives with retained acetylenic moiety. 3-Hydroxyalk-1-yn-1-yl analogues in the course of such iodomethylation undergo isomerization into allenic derivatives which are transformed into acetylenic ones upon dissolution in DMSO.

Nanoparticles Based on Quaternary Ammonium Chitosan-methyl-β-cyclodextrin Conjugate for the Neuropeptide Dalargin Delivery to the Central Nervous System: An In Vitro Study.

Peptide oral administration is a hard goal to reach, especially if the brain is the target site. The purpose of the present study was to set up a vehicle apt to promote oral absorption of the neuropeptide dalargin (DAL), allowing it to cross the intestinal mucosal barrier, resist enzymatic degradation, and transport drugs to the brain after crossing the blood–brain barrier. Therefore, a chitosan quaternary ammonium derivative was synthesized and conjugated with methyl-β-cyclodextrin to prepare DAL-medicated nanoparticles (DAL-NP). DAL-NP particle size was 227.7 nm, zeta potential +8.60 mV, encapsulation efficiency 89%. DAL-NP protected DAL from degradation by chymotrypsin or pancreatin and tripled DAL degradation time compared to non-encapsulated DAL. Use of DAL-NP was safe for either Caco-2 or bEnd.3 cells, with the latter selected as a blood–brain barrier model. DAL-NP could also cross either the Caco-2 or bEnd.3 monolayer by the transepithelial route. The results suggest a potential DAL-NP ability to transport to the brain a DAL dose fraction administered orally, although in vivo experiments will be needed to confirm the present data obtained in vitro.

Synthesis of diosgenyl quaternary ammonium derivatives and their antitumor activity

Giosgenin is a naturally steroidal saponin exhibiting a variety of biological activities including antitumor ones. A series of novel diosgenyl quaternary ammonium derivatives were designed and synthesized to develop potential anti-tumor agents in our research. All novel derivatives were characterized by 1H NMR, 13C NMR and HR-MS, and evaluated for their in vitro anti-proliferative activities using MTT assay. The human cancer cell lines were A549 (Human lung cancer cell), H1975 (Human lung adenocarcinoma cell), A431 (Human skin squamous cell carcinoma), HCT-116 (Human colorectal adenocarcinoma cell), Aspc-1 (Human metastatic pancreatic cancer cell), Ramos (Human B lymphoma cell), HBE (Human bronchial epithelioid cell) and LO2 (Human normal hepatocyte).

Thiacalixarene based quaternary ammonium salts as promising antibacterial agents

The search for new antibacterial and antiseptic drugs is an urgent problem due to the resistance of microorganisms to existing drugs. In this work, for the first time, the design of antibacterial and bactericidal agents based on quaternary ammonium compounds on thiacalixarene macrocyclic platform was proposed and implemented. A series of tetrasubstituted quaternary ammonium salts with different nature and length of the substituent (-N+(CH3)2R, R = CH2Ph, CnH2n+1, n = 1, 4, 8, 10) based on p-tert-butylthiacalix[4]arene in cone and 1,3-alternate conformations was obtained with excellent yields. The obtained compounds have a high antibacterial effect against Gram-positive (S. aureus, S. epidermidis, B. subtilis) bacteria comparable with commercial antiseptics chlorhexidine, miramistin and benzalkonium chloride. It was found that quaternary ammonium derivatives of thiacalix[4]arene in 1,3-alternate conformation more effectively inhibit the growth of the tested bacterial strains in comparison with compounds in cone conformation. Cytotoxicity studies on human skin fibroblast (HSF) cells demonstrated that all compounds were less toxic compared to reference drugs. The different type of interaction of the studied compounds with model DPPC lipid membranes explains different antibacterial activity and cytotoxicity of compounds. The compounds in cone conformation are adsorbed on the DPPC vesicles membrane surface, while the incorporation of lipophilic alkyl fragments of macrocycles in 1,3-alternate conformation into the membrane leads to “clumping” of DPPC vesicles. It was shown the saving of antibacterial activity of thiacalixarene derivatives in 1,3-alternate conformation on Gram-positive clinical strains. The obtained results allow viewing the described thiacalixarene based quaternary ammonium compounds as promising molecules in the development of the new antibacterial agents.

Highly Effective Inactivation of SARS-CoV-2 by Conjugated Polymers and Oligomers.

In the present study, we examined the inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by synthetic conjugated polymers and oligomers developed in our laboratories as antimicrobials for bacteria, fungi, and nonenveloped viruses. The results show highly effective light-induced inactivation with several of these oligomers and polymers including irradiation with near-UV and visible light. In the best case, one oligomer induced a 5-log reduction in pfu/mL within 10 min. In general, the oligomers are more active than the polymers; however, the polymers are active with longer wavelength visible irradiation. Although not studied quantitatively, the results show that in the presence of the agents at concentrations similar to those used in the light studies, there is essentially no dark inactivation of the virus. Because three of the five materials/compounds examined are quaternary ammonium derivatives, this study indicates that conventional quaternary ammonium antimicrobials may not be active against SARS-CoV-2. Our results suggest several applications involving the incorporation of these materials in wipes, sprays, masks, and clothing and other personal protection equipment that can be useful in preventing infections and the spreading of this deadly virus and future outbreaks from similar viruses.

Phenolic-containing chitosan quaternary ammonium derivatives and their significantly enhanced antioxidant and antitumor properties

A class of phenolic-chitosan quaternary ammonium derivatives have been designed and synthesized. Three chitosan derivatives possess effective structure of hydroxycinnamic acid have been obtained through chemical modification to get chitosan derivatives owning high antioxidant activity and antitumor activity. In this study, the scavenging ability of DPPH, hydroxyl (•OH), and superoxide (O2•−) free radical and reducing power have been tested to evaluate the antioxidant activity of the synthesized chitosan derivatives. Base on the value of IC50, the chitosan derivatives have the best inhibitory property of 0.019 mg/mL (DPPH), 0.016 mg/mL (•OH), and 0.008 (O2•−), respectively; and the chitosan derivatives with conjugate structure of ferulic acid and sinapic acid (4b and 4c) show promising antitumor activity toward A549 cells with the IC50 of 0.046 and 0.052 mg/mL. These data indicate that the chitosan derivatives with phenolic group give much stronger antioxidant activity and antitumor activity. On the other hand, the synthesized chitosan derivatives show no cytotoxicity for L929 cells at the testing concentrations. These results demonstrate that the introduction of phenol group improves the antioxidant activity of chitosan obviously, and the antioxidant or free radical scavenger based on nature polymers and phenol shows potentials application.

Targeting pathogenic fungi, bacteria and fungal-bacterial biofilms by newly synthesized quaternary ammonium derivative of pyridoxine and terbinafine with dual action profile

Many pathogenic bacteria and microscopic fungi form rigid polymicrobial biofilms this way enhancing their resistant to treatment. A series of novel pyridoxine-based quaternary ammonium derivatives of terbinafine characterized by both antifungal and antibacterial activities was designed. The leading compound named KFU-127 exhibits promising antifungal and antibacterial activities against various bacteria and micromycetes in both planktonic and biofilm-embedded forms demonstrating MIC values comparable with those of conventional antifungals and antimicrobials. Similar to other antiseptics like benzalkonium chloride and miramistin, KFU-127 is considerably toxic for eukaryotic cells that limits is application to topical treatment options. On the other hand, KFU-127 reduces the number of viable biofilm-embedded bacteria and C. albicans by 3 orders of magnitude at concentrations 2–4 times lower than those of reference drugs and successfully eradicates S. aureus-C. albicans mixed biofilms. The mechanism of antimicrobial action of KFU-127 is bimodal including both membrane integrity damage and pyridoxal-dependent enzymes targeting. We expect that this bilateral mechanism would result in lower rates of resistance development in both fungal and bacterial pathogens. Taken together, our data suggest KFU-127 as a new promising broad spectrum topical antimicrobial capable of one-shot targeting of bacterial and fungal-bacterial biofilms.