Assessment of lisdexamfetamine dimesylate stability and identification of its degradation product by NMR spectroscopy.

Lisdexamfetamine dimesylate (LDX), a long-acting prodrug stimulant indicated for the treatment of the attention-deficit/hyperactivity disorder (ADHD), was subjected to forced degradation studies by acid and alkaline hydrolysis and the degradation profile was studied. To obtain between 10-30% of degraded product, acid and alkaline conditions were assessed with solutions of 0.01 M, 0.1 M, 0.5 M, and 1 M of DCl and NaOD. These solutions were analyzed through 1 H NMR spectra. Acid hydrolysis produced no degradation in 0.01 M and 0.1 M DCl and 4.38%, 9.69%, and 17.75% of degradation LDX, respectively, in 0.5 M, 1 M (4h) and 1 M (4 + 12 h) DCl. And alkaline hydrolysis produced no degradation in 0.01 M and 0.1 M DCl and a degradation LDX extension of 8.5%, 14.30%, and 22.91%, respectively, in 0.5 M, 1 M (4h) and 1 M (4 + 12 h) NaOD. LDX solutions subjected to 1 M (4 + 12 h) acid and alkaline hydrolysis were evaluated by NMR spectra (1 H NMR, 13 C NMR, HSQC and HMBC). LDX degradation product (DP) was identified and its structure elucidated as a diastereoisomer of LDX: (2R)-2,6-diamino-N-[(2S)-1-phenylpropan-2-yl] hexanamide without their physical separation.

Mass spectrometry study of N-alkylbenzenesulfonamides with potential antagonist activity to potassium channels.

Herein, we report the synthesis and mass spectrometry studies of several N-alkylbenzenesulfonamides structurally related to sulfanilic acid. The compounds were synthesized using a modified Schotten-Baumann reaction coupled with Meisenheimer arylation. Sequential mass spectrometry by negative mode electrospray ionization (ESI(-)-MS/MS) showed the formation of sulfoxylate anion (m/z 65) observed in the mass spectrum of p-chloro-N-alkylbenzenesulfonamides. Investigation of the unexpected loss of two water molecules, as observed by electron ionization mass spectrometry (EI-MS) analysis of p-(N-alkyl)lactam sulfonamides, led to the proposal of corresponding fragmentation pathways. These compounds showed loss of neutral iminosulfane dioxide molecule (M-79) with formation of ions observed at m/z 344 and 377. These ions were formed by rearrangement on ESI(+)-MS/MS analysis. Some of the molecules showed antagonistic activity against Kv3.1 voltage-gated potassium channels.

ESI(+)-MS and GC-MS study of the hydrolysis of N-azobenzyl derivatives of chitosan.

New N-p-chloro-, N-p-bromo-, and N-p-nitrophenylazobenzylchitosan derivatives, as well as the corresponding azophenyl and azophenyl-p-sulfonic acids, were synthesized by coupling N-benzylvchitosan with aryl diazonium salts. The synthesized molecules were analyzed by UV-Vis, FT-IR, 1H-NMR and 15N-NMR spectroscopy. The capacity of copper chelation by these materials was studied by AAS. Chitosan and the derivatives were subjected to hydrolysis and the products were analyzed by ESI(+)-MS and GC-MS, confirming the formation of N-benzyl chitosan. Furthermore, the MS results indicate that a nucleophilic aromatic substitution (SnAr) reaction occurs under hydrolysis conditions, yielding chloroaniline from N-p-bromo-, and N-p-nitrophenylazo-benzylchitosan as well as bromoaniline from N-p-chloro-, and N-p-nitrophenylazobenzyl-chitosan.

Exploring the Antimicrobial and Antitumoral Activities of Naphthoquinone-Grafted Chitosans.

Biopolymers obtained from natural macromolecules are noteworthy among materials presenting high biocompatibility and adequate biodegradability, as is the case of chitosan (CS), making this biopolymeric compound a suitable drug delivery system. Herein, chemically-modified CS were synthetized using 2,3-dichloro-1,4-naphthoquinone (1,4-NQ) and the sodium salt of 1,2-naphthoquinone-4-sulfonic acid (1,2-NQ), producing 1,4-NQ-CS and 1,2-NQ-CS by three different methods, employing an ethanol and water mixture (EtOH:H2O), EtOH:H2O plus triethylamine and dimethylformamide. The highest substitution degree (SD) of 0.12 was achieved using water/ethanol and triethylamine as the base for 1,4-NQ-CS and 0.54 for 1,2-NQ-CS. All synthesized products were characterized by FTIR, elemental analysis, SEM, TGA, DSC, Raman, and solid-state NMR, confirming the CS modification with 1,4-NQ and 1,2-NQ. Chitosan grafting to 1,4-NQ displayed superior antimicrobial activities against Staphylococcus aureus and Staphylococcus epidermidis associated with improved cytotoxicity and efficacy, indicated by high therapeutic indices, ensuring safe application to human tissue. Although 1,4-NQ-CS inhibited the growth of human mammary adenocarcinoma cells (MDA-MB-231), it is accompanied by cytotoxicity and should be considered with caution. The findings reported herein emphasize that 1,4-NQ-grafted CS may be useful in protecting injured tissue against bacteria, commonly found in skin infections, until complete tissue recovery.

Combining Conventional Organic Solvent Extraction, Ultrasound-Assisted Extraction, and Chromatographic Techniques to Obtain Pure Betanin from Beetroot for Clinical Purposes.

Red beetroot extract (E162) is a natural colorant that owes its color to betanin, its major red pigment. Betanin displays remarkable antioxidant, anti-inflammatory, and chemoprotective properties mediated by its structure and influence on gene expression. However, the betanin employed in most preclinical assays is a beetroot extract diluted in dextrin, not pure betanin, as no isolated compound is commercially available. This makes its use inaccurate concerning product content estimates and biological effect assessments. Herein, a combination of conventional extraction under orbital shaking and ultrasound-assisted extraction (UAE) to purify betanin by semi-preparative HPLC was performed. The employed methodology extracts betalains at over a 90% yield, achieving 1.74 ± 0.01 mg of pure betanin/g beetroot, a 41% yield from beetroot contents increasing to 50 %, considering the betalains pool. The purified betanin exhibited an 85% purity degree against 32 or 72% of a commercial standard evaluated by LC-MS or HPLC methods, respectively. The identity of purified betanin was confirmed by UV-Vis, LC-MS, and 1H NMR. The combination of a conventional extraction, UAE, and semi-preparative HPLC allowed for betanin purification with a high yield, superior purity, and almost three times more antioxidant power compared to commercial betanin, being, therefore, more suitable for clinical purposes.

Toward the reliable diagnosis of indeterminate thyroid lesions: a HRMAS NMR-based metabolomics case of study.

Cytological analysis of thyroid nodules detected using ultrasound-guided fine-needle aspiration technique is an efficient method for the diagnosis of well-differenciated tumors such as papillary thyroid carcinoma. However, for between 10 to 30% of all the nodules, the cytological analysis based on fine-needle aspiration biopsies leads to an "indeterminated" identification. Consequently, a surgical excision is then necessary for a definite histological diagnosis of the lesions, resulting in 85% of the patient with indeterminated nodules undergoing unnecessary surgery since their tumor is finally diagnosed as benign. In this work, we discuss how HRMAS (1)H NMR-based metabolomics could be a complementary tool for the diagnosis of these elusive cases. We first showed that our approach was able to discriminate clearly any types of thyroid lesions from healthy tissues. Then we proceeded to demonstrate that the information produced by (1)H HRMAS NMR spectra differentiate tumors according to their malignancy grade, even when they belong to the "indeterminate" category. Analysis of the discriminating spectral area in this last case points out toward a possible increase of phenylalanine, taurine, and lactate and a decrease of choline and choline derivatives, myo- and scyllo-inositol in the malignant tumors compared to the benign ones.

Novel Selective and Low-Toxic Inhibitor of LmCPB2.8ΔCTE (CPB) One Important Cysteine Protease for Leishmania Virulence.

Leishmaniasis is a highly prevalent, yet neglected disease caused by protozoan parasites of the genus Leishmania. In the search for newer, safer, and more effective antileishmanial compounds, we herein present a study of the mode of action in addition to a detailed structural and biological characterization of LQOF-G6 [N-benzoyl-N'-benzyl-N″-(4-tertbutylphenyl)guanidine]. X-ray crystallography and extensive NMR experiments revealed that LQOF-G6 nearly exclusively adopts the Z conformation stabilized by an intramolecular hydrogen bond. The investigated guanidine showed selective inhibitory activity on Leishmania major cysteine protease LmCPB2.8ΔCTE (CPB) with ~73% inhibition and an IC50-CPB of 6.0 µM. This compound did not show any activity against the mammalian homologues cathepsin L and B. LQOF-G6 has been found to be nontoxic toward both organs and several cell lines, and no signs of hepatotoxicity or nephrotoxicity were observed from the analysis of biochemical clinical plasma markers in the treated mice. Docking simulations and experimental NMR measurements showed a clear contribution of the conformational parameters to the strength of the binding in the active site of the enzyme, and thus fit the differences in the inhibition values of LQOF-G6 compared to the other guanidines. Furthermore, the resulting data render LQOF-G6 suitable for further development as an antileishmanial drug.

A solid-state NMR study of the structure and dynamics of the myristoylated N-terminus of the guanylate cyclase-activating protein-2.

Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca(2+) sensor protein. It plays a central role in shaping the photoreceptor light response and in light adaptation through the Ca(2+)-dependent regulation of the transmembrane retinal guanylate cyclase (GC). GCAP-2 is N-terminally myristoylated and the full activation of the GC requires this lipid modification. The structural and functional role of the N-terminus and particularly of the myristoyl moiety is currently not well understood. In particular, detailed structural information on the myristoylated N-terminus in the presence of membranes was not available. Therefore, we studied the structure and dynamics of a 19 amino acid peptide representing the myristoylated N-terminus of GCAP-2 bound to lipid membranes by solid-state NMR. (13)C isotropic chemical shifts revealed a random coiled secondary structure of the peptide. Peptide segments up to Ala(9) interact with the membrane surface. Order parameters for Calpha and side chain carbons obtained from DIPSHIFT experiments are relatively low, suggesting high mobility of the membrane-associated peptide. Static (2)H solid-state NMR measurements show that the myristoyl moiety is fully incorporated into the lipid membrane. The parameters of the myristoyl moiety and the DMPC host membrane are quite similar. Furthermore, dynamic parameters (obtained from (2)H NMR relaxation rates) of the peptide's myristic acid chain are also comparable to those of the lipid chains of the host matrix. Therefore, the myristoyl moiety of the N-terminal peptide of GCAP-2 fills a similar conformational space as the surrounding phospholipid chains.

Synthesis of potentially bioactive PABA-related N-(aminoalkyl)lactamic amino acids and esters via selective S(N)Ar reactions.

Potentially bioactive N-(aminoalkyl)lactamic amino acids and esters were synthesized in satisfactory to good yields by S(N)Ar reactions of aromatic acids with N-(3-aminopropyl)lactams followed by esterification with tertiary amino alcohols. The addition-elimination S(N)Ar mechanism was confirmed by NMR and MS measurements.

Integration of NMR studies, computational predictions, and in vitro assays in the search of marine diterpenes with antitumor activity.

Among the compounds of natural origin, diterpenes have proved useful as drugs for the treatment of cancer. Marine organisms, such as soft corals and algae, are a promising source of diterpenes, being a rich and unexplored source of cytotoxic agents. This study evaluated a library of 32 natural and semisynthetic marine diterpenes, including briarane, cembrane, and dolabellane nuclei, with the aim of determining their cytotoxicity against three human cancer cell lines (A549, MCF7, and PC3). The three most active compounds were submitted to a flow cytometry analysis in order to determine induction of apoptosis against the A549 cell line. An NMR analysis was conducted to determine and evaluate the interactions between active diterpenes and tubulin. These interactions were characterized by a computational study using molecular docking and MD simulations. With these results, two cembrane and one chlorinated briarane diterpenes were active against the three human cancer cell lines, induced apoptosis in the A549 cell line, and showed interactions with tubulin preferably at the taxane-binding site. This study is a starting point for the identification and optimization of the marine diterpenes selected for better antitumor activities. It also highlights the power of integrating NMR studies, computational predictions, and in vitro assays in the search for compounds with antitumor activity.

Bridging photochemistry and photomechanics with NMR crystallography: the molecular basis for the macroscopic expansion of an anthracene ester nanorod.

Crystals composed of photoreactive molecules represent a new class of photomechanical materials with the potential to generate large forces on fast timescales. An example is the photodimerization of 9-tert-butyl-anthracene ester (9TBAE) in molecular crystal nanorods that leads to an average elongation of 8%. Previous work showed that this expansion results from the formation of a metastable crystalline product. In this article, it is shown how a novel combination of ensemble oriented-crystal solid-state NMR, X-ray diffraction, and first principles computational modeling can be used to establish the absolute unit cell orientations relative to the shape change, revealing the atomic-resolution mechanism for the photomechanical response and enabling the construction of a model that predicts an elongation of 7.4%, in good agreement with the experimental value. According to this model, the nanorod expansion does not result from an overall change in the volume of the unit cell, but rather from an anisotropic rearrangement of the molecular contents. The ability to understand quantitatively how molecular-level photochemistry generates mechanical displacements allows us to predict that the expansion could be tuned from +9% to -9.5% by controlling the initial orientation of the unit cell with respect to the nanorod axis. This application of NMR-assisted crystallography provides a new tool capable of tying the atomic-level structural rearrangement of the reacting molecular species to the mechanical response of a nanostructured sample.

The effect of carbonyl group in the asymmetry of 3,4JCH coupling constants in norbornanones.

A rationalization of the known difference between the 3,4JC4H1 and 3,4JC1H4 couplings transmitted mainly through the 7-bridge in norbornanone is presented in terms of the effects of hyperconjugative interactions involving the carbonyl group. Theoretical and experimental studies of 3,4JCH couplings were carried out in 3-endo- and 3-exo-X-2-norbornanone derivatives (X = Cl, Br) and in exo- and endo-2-noborneol compounds. Hyperconjugative interactions were studied with the natural bond orbital (NBO) method. Hyperconjugative interactions involving the carbonyl pi*(C2=O) and sigma*(C2=O) antibonding orbitals produce a decrease of three-bond contribution to both 3,4JC4H1 and 3,4JC1H4 couplings. However, the latter antibonding orbital also undergoes a strong sigmaC3--C4 --> sigma*(C2=O) interaction, which defines an additional coupling pathway for 3,4JC4H1 but not for 3,4JC1H4. This pathway is similar to that known for homoallylic couplings, the only difference being the nature of the intermediate antibonding orbital; i.e. for 3,4JC4H1 it is of sigma*-type, while in homoallylic couplings it is of pi*-type.

Facile and innovative method for bioglass surface modification: Optimization studies.

In this work it is presented a facile and novel method for modification of bioglass surface based on (Camolten salt bath2+|Naglass+) ion exchange by immersion in molten salt bath. This method allows changing selectively the chemical composition of a surface layer of glass, creating a new and more reactive bioglass in a shell that surrounds the unchanged bulk of the original BG45S5 bioglass (core-shell type system). The modified bioglass conserves the non-crystalline structure of BG45S5 bioglass and presents a significant increase of surface reactivity in comparison with BG45S5. Melt-derived bioactive glasses BG45S5 with the nominal composition of 46.1mol% SiO2, 24.4mol% Na2O, 26.9mol% CaO, and 2.6mol% P2O5 have been subjected to ion exchange at 480°C in molten mixture of Ca(NO3)2 and NaNO3 with molar ratio of 70:30 for different time periods ranging from 0 to 60min. The optimization studies by using XRF and XRD showed that ion exchange time of 30min is enough to achieve higher changes on the glass surface without alters its non-crystalline structure. The chemical composition, morphology and structure of BG45S5 and bioglass with modified surface were studied by using several analytical techniques. FTIR and O1s XPS results showed that the modification of glass surface favors the formation of Si-ONBO groups at the expense of SiOBOSi bonds. 29Si MAS-NMR studies showed that the connectivity of SiQn species decreases from cross-linked SiQ3 units to chain-like SiQ2 units and finally to depolymerized SiQ1 and SiQ0 units after ion exchange. This result is consistent with the chemical model based on the enrichment with calcium ions of the bioglass surface such that the excess of positive charges is balanced by depolymerization of silicate network. The pH changes in the early steps of reaction of bioactive glasses BG45S5 and BG45Ca30, in deionized water or solutions buffered with HEPES were investigated. BG45Ca30 bioactive glass exhibited a significant increase in the pH during the early steps of the reaction compared to BG45S5.

5-Methoxypropacin, a novel coumarinolignoid from Protium unifoliolatum.

The novel coumarinolignoid 5-methoxypropacin was isolated from the ethereal extract of the wood of the Amazonian species Protium unifoliolatum. Its structure was elucidated by spectroscopic methods as IR, 1D and 2D (1)H, (13)C-NMR and mass spectrometry. The regioisomer and the position of coumarin and arylpropanoid coupling were determined by long-distance C-H correlation (HMBC) and NOE experiments. Scopoletin and beta-sitosterol were also isolated and identified by the same physical methods.

Mefenamic acid anti-inflammatory drug: probing its polymorphs by vibrational (IR and Raman) and solid-state NMR spectroscopies.

This work deals with the spectroscopic (supported by quantum chemistry calculations), structural, and morphological characterization of mefenamic acid (2-[(2,3-(dimethylphenyl)amino] benzoic acid) polymorphs, known as forms I and II. Polymorph I was obtained by recrystallization in ethanol, while form II was reached by heating form I up to 175 °C, to promote the solid phase transition. Experimental and theoretical vibrational band assignments were performed considering the presence of centrosymmetric dimers. Besides band shifts in the 3345-3310 cm(-1) range, important vibrational modes to distinguish the polymorphs are related to out-of-phase and in-phase N-H bending at 1582 (Raman)/1577 (IR) cm(-1) and 1575 (Raman)/1568 (IR) cm(-1) for forms I and II, respectively. In IR spectra, bands assigned to N-H bending out of plane are observed at 626 and 575 cm(-1) for polymorphs I and II, respectively. Solid-state (13)C NMR spectra pointed out distinct chemical shifts for the dimethylphenyl group: 135.8 to 127.6 ppm (carbon bonded to N) and 139.4 to 143.3 ppm (carbon bonded to methyl group) for forms I and II, respectively.

Prognostic relationship of metabolic profile obtained of melanoma B16F10.

Melanoma is a type of cancer that reaches more people in the world, characterized by genetic mutations that trigger the growth of disorganized cells. The diagnosis of skin tumors by invasive techniques has become a risk to the patients, so the search for new non-invasive techniques has been the subject of research in recent years. The objective of this work is to propose a non-invasive method prognosis based on the identification of specific biomarkers of the cancer, known as metabolomics analysis. For this study, we used B16F10 melanoma tumor cells and metabolic profiles were obtained at three time-periods by (1)HNMR and comparison with the cell cycle, apoptosis pathways and proliferation index. The metabolic profiles show the relationship between the metabolites found with energy metabolism, pathways of apoptosis and proliferation, which showed increases in proportion during growth and progression. Were found 29 metabolites, of which the differentially expressed are: lactate, aspartate, glycerol, lipids, alanine, myo-inositol, phosphocholine, choline, acetate, creatine and taurine. Choline and creatine are closely related with tumor progression, and are inversely expressed in later stages of tumor growth, which demonstrates the ability to be markers of tumor progression or monitoring the pharmacological efficacy when combined with other therapies. We conclude that the metabolome appeared as effective non-invasive technique predicts, besides providing possible biomarkers of melanoma.

Effect of hormonal contraceptives during breastfeeding on infant's milk ingestion and growth.

OBJECTIVE: To measure infants' breast milk intake and infant growth when their mothers initiated either combined oral contraceptive (COC), levonorgestrel-releasing intrauterine system, or etonogestrel-releasing implant, or copper intrauterine device (IUD) as a reference group. DESIGN: Prospective trial. SETTING: University-based hospital. PATIENT(S): On postpartum day 42, 40 women initiated a contraceptive method according to their choice. INTERVENTION(S): Deuterium (D2O; 0.5 g/kg mother's weight) was ingested by mothers on postpartum days 42, 52, and 63 as a marker of total body fluid. MAIN OUTCOME MEASURE(S): Infants' milk intake from 42 to 63 postpartum days was assessed by measurement of D2O levels in infants' saliva and infant growth by measuring their body weight, height, and tibia length. Women recorded all infant feed and changes of diapers wet with urine. Breastfeeding continuation was assessed at 6 months postpartum. RESULT(S): Infant mean milk intake, mean growth increase, mean number of breastfeeding episodes, daily wet diaper changes, and mean duration of exclusively breastfeeding (~5 months) were similar in the four groups. CONCLUSION(S): Use of a COC, the two progestin-only contraceptives, or copper IUD did not affect the amount of infant milk intake and growth up to 9 weeks of age. The incidence of full breastfeeding and breastfeeding continuation was similar with contraceptive hormonal use and no use. CLINICAL TRIALS REGISTRATION NUMBER: NCT01388582.

Flavonoids bearing an O-arabinofuranosyl-(1-->3)-rhamnoside moiety from Cladocolea micrantha: inhibitory effect on human melanoma cells.

Eleven known triterpenes (alpha-amyrin, beta-amyrin, lupeol, and their respective acetates, 3-O-acetyl derivatives of betulinic, oleanolic, and ursolic acids, cycloartenol, and tirucall-7,24-dienol), two new flavonols presenting an uncommon interglycosidic O-(1-->3) linkage (kaempferol 3-O-alpha-L-arabinofuranosyl(1-->3)-alpha-L-rhamnoside and quercetin 3-O-alpha-L-arabinofuranosyl-(1-->3)-alpha-L-rhamnoside), beta-sitosterol, stigmasterol, quercetin, and gallic acid were isolated from the Amazonian medicinal mistletoe, Cladocolea micrantha Kuijt (Loranthaceae). Their structures were established by spectral methods and eventual chromatographic comparisons. The quercetin derivative was not cytotoxic to MV3 human melanoma cells, but was able, when administered at 1 microg/mL, to promote a twofold inhibition of the migration of the cells through the transwell system when compared with paclitaxel at 5 microM.

Physical properties of pectin-high amylose starch mixtures cross-linked with sodium trimetaphosphate.

Pectin-high amylose starch mixtures (1:4; 1:1; 4:1) were cross-linked at different degrees and characterized by rheological, thermal, X-ray diffraction and NMR analyses. For comparison, samples without cross-linker addition were also prepared and characterized. Although all samples behaved as gels, the results evidenced that the phosphorylation reaction promotes the network strengthening, resulting in covalent gels (highest critical stress, G' and recovery %). Likewise, cross-linked samples presented the highest thermal stability. However, alkaline treatment without cross-linker allowed a structural reorganization of samples, as they also behaved as covalent gels, but weaker than those gels from cross-linked samples, and presented higher thermal stability than the physical mixtures. X-ray diffractograms also evidenced the occurrence of physical and chemical modifications due to the cross-linking process and indicated that samples without cross-linker underwent some structural reorganization, resulting in a decrease of crystallinity. The chemical shift of resonance signals corroborates the occurrence of structural modifications by both alkaline treatment and cross-linking reaction.

Metabolomics approach to thyroid nodules: a high-resolution magic-angle spinning nuclear magnetic resonance-based study.

BACKGROUND: Proton magnetic resonance spectroscopy of operative specimens has been reported to successfully differentiate normal tissue from malignant thyroid tissue. We used a new high-resolution magnetic resonance spectroscopy technique for the differentiation of benign and malignant thyroid neoplasms. METHODS: Histological specimens from 72 patients undergoing a total thyroidectomy were processed into a 4-mm ZrO(2) high-resolution magic angle spinning (HRMAS) rotor with 5 µL of D(2)O. A Bruker Avance spectrometer operating at 400 MHz for the (1)H frequency and equipped with a (1)H/(13)C/(31)P HRMAS probe was used. RESULTS: Normal and neoplastic thyroid tissues could be discriminated from each other by different relative concentrations of several amino acids and lipids, as well as benign and malignant neoplasms, that differed in terms of a greater lactate and taurine and a lesser lipid choline, phosphocholine, myo-inositol, and scyllo-inositol levels in malignant samples. A statistical analysis with a receiver operating characteristic curve revealed that 77% of the samples were accurately predicted. Similar results were obtained with specimens obtained from ex vivo aspirates. CONCLUSION: A further development of this project will be to use the metabolomics approach on specimens obtained from aspirates in vivo after the resolution of technical problems attributable to possible contamination.