Miliutine C methyl ester, a new drimane sesquiterpene and bioactive alkaloids from the stems of Miliusa velutina.

Previous results from the our research group have isolated numerous compounds, including novel ones, but the anticancer activity of Miliusa velutina has not been demonstrated. In this study, from the most active ethyl acetate extract of the stems of Miliusa velutina, seven compounds were isolated and determined structures, including a new drimane sesquiterpenoid compound named miliutine C methyl ester (1) and three bioactive alkaloids (5-7). These three alkaloids (5-7) exhibited strong anticancer activities against various cancer cell lines such as MCF-7, HepG2, HeLa, NCI H460 and normal fibroblasts. Especially, on MCF-7 and normal fibroblasts with values of IC50 (µM) in order for compounds 5 (3.38, 31.15), 6 (21.96, 102.00), 7 (7.90, greater than 300), to compare with positive control camptothecin (0.020, 4.51); which is highly noteworthy. These results contribute to elucidating and confirming the value of Miliusa velutina, similar to other published and folkloric findings.

The Synergy between Nuclear Magnetic Resonance and Density Functional Theory Calculations.

This paper deals with the synergy between Nuclear Magnetic Resonance (NMR) spectroscopic investigations and DFT calculations, mainly of NMR parameters. Both the liquid and the solid states are discussed here. This text is a mix of published results supplemented with new findings. This paper deals with examples in which useful results could not have been obtained without combining NMR measurements and DFT calculations. Examples of such cases are tautomeric systems in which NMR data are calculated for the tautomers; hydrogen-bonded systems in which better XH bond lengths can be determined; cage compounds for which assignment cannot be made based on NMR data alone; revison of already published structures; ionic compounds for which reference data are not available; assignment of solid-state spectra and crystal forms; and the creation of libraries for biological molecules. In addition to these literature cases, a revision of a cage structure and substituent effects on pyrroles is also discussed.

Miliutine A acid, a new cyclofarnesane sesquiterpene from the stems of Miliusa velutina.

Six compounds were isolated from the ethyl acetate extract of the stems of Miliusa velutina, including miliutine A acid (1), a new cyclofarnesane sesquiterpenoid; miliutine B methyl ester (2), a cyclofarnesane sesquiterpenoid which was determined the absolute configuration for the first time and four known phenol derivatives (3-6). NMR spectroscopic and mass spectrometry were used for identifying relative configurations. The assignments of the absolute configurations were determined based on Electronic Circular Dichroism (ECD) and NOESY spectra analysis. All six compounds were screened for their in vitro cytotoxic activities against HepG2 cell line using the SRB assay and they showed weak or none activities.

Tautomerism of pyridinylbutane-1,3-diones: An NMR and DFT study.

The three possible 1-(n-pyridinyl)butane-1,3-diones (nPM) have been synthesized. Structures, tautomerism, and conformations are investigated by means of DFT calculations. 1 H and 13 C NMR spectra are assigned, and deuterium isotope effects on 13 C chemical shifts have been measured. Analysis of the isotope effects leads to the equilibrium constants of the keto-enol tautomers. Some interesting differences are seen between the three compounds and the phenyl analogs. The isotope effects can also rank the hydrogen bonds of the compounds, with the one with nitrogen in the three positions of the pyridine ring as the weakest. Structures, conformers, energies, and NMR nuclear shieldings are calculated using DFT calculations at the B3LYP/6-311++G(d,p) level.

A new sesquiterpene lactone from the leaves of Panax vietnamensis Ha et Grushv. (Vietnamese ginseng).

Panax vietnamensis (Vietnamese ginseng, Ngoc Linh ginseng) is an endemic Panax species of Vietnam. From the methanol extract of the leaves of Panax vietnamensis, five compounds (1-5) were isolated, including one new sesquiterpene lactone such as panaxolide (1) and four known compounds. The structures of the compounds (1-5) were elucidated by spectral techniques such as 1 D NMR (1H NMR, 13C NMR), 2 D NMR (COSY, HSQC, HMBC, NOESY) and mass spectrometry. The absolute configuration of 1 was determined based on the Cotton effects in the CD spectrum. All of the five compounds were screened for their in vitro growth inhibitory activities against cancerous cells (HepG2) and normal cells (fibroblast) using the SRB assay. Panaxolide (1) showed the highest potential for the growth inhibition of cancerous cells HepG2 with the IC50 values of 63.8 µM.

Steerable photonic jet for super-resolution microscopy.

A promising technique in optical super-resolution microscopy is the illumination of the sample by a highly localized beam, a photonic jet (also called photonic nanojet). We propose a method of computation of incident field amplitude and phase profiles that produce photonic jets at desired locations in the near field after interaction with a fixed micro-scale dielectric lens. We also describe a practical way of obtaining the incident field profiles using spatial light modulators. We expect our photonic jet design method to work for a wide range of lens shapes, and we demonstrate its application numerically using two-dimensional micro-lenses of circular and square cross-sections. We furthermore offer a theoretical analysis of the resolution of photonic jet design, predicting among other that a larger lens can produce a narrower photonic jet. Finally, we give both theoretical and numerical evidence that the waist width of the achieved designed jets is increasing linearly and slowly over a large interval of radial distances. With uniform plane wave illumination, the circular two-dimensional micro-lens produces a similar-sized jet at a fixed radial distance, while the square lens does not form a jet at all. We expect our steerable optical photonic jet probe to enable highly localized adaptive real-time measurements and drive advances in super-resolution optical microscopy and scatterometry, as well as fluorescence and Raman microscopy. Our relatively weak peak jet intensity allows application in biology and health sciences, which require high resolution imaging without damaging the sample bio-molecules.

Isotope Effects on Chemical Shifts in the Study of Hydrogen Bonds in Small Molecules.

This review is giving a short introduction to the techniques used to investigate isotope effects on NMR chemical shifts. The review is discussing how isotope effects on chemical shifts can be used to elucidate the importance of either intra- or intermolecular hydrogen bonding in ionic liquids, of ammonium ions in a confined space, how isotope effects can help define dimers, trimers, etc., how isotope effects can lead to structural parameters such as distances and give information about ion pairing. Tautomerism is by advantage investigated by isotope effects on chemical shifts both in symmetric and asymmetric systems. The relationship between hydrogen bond energies and two-bond deuterium isotope effects on chemical shifts is described. Finally, theoretical calculations to obtain isotope effects on chemical shifts are looked into.

Two new sesquiterpenes from the stems of Miliusa velutina.

From the ethyl acetate extract of the stems of Miliusa velutina, seven compounds (1-7) were isolated, including two new compounds such as mivelutina A acid (1), mivelutina B acid (2) and one known compound mivelutina B methyl ester (3). For this NMR data were not known previously. Their relative structures were elucidated based on NMR spectroscopic analysis. The absolute configuartions were determined based on DFT calculations of 13C chemical shifts. All of the seven compounds were screened for their in vitro cytotoxic activities against HepG2 cell line using the SRB assay. Epoxyconiferyl alcohol (7) showed the highest potential for the cytotoxicity of cancer cell lines HepG2 with the IC50 values of 95.94 µg/mL (527 µM).

NH Stretching Frequencies of Intramolecularly Hydrogen-Bonded Systems: An Experimental and Theoretical Study.

The vibrational NH stretching transitions in secondary amines with intramolecular NH···O hydrogen bonds were investigated by experimental and theoretical methods, considering a large number of compounds and covering a wide range of stretching wavenumbers. The assignment of the NH stretching transitions in the experimental IR spectra was, in several instances, supported by measurement of the corresponding ND wavenumbers and by correlation with the observed NH proton chemical shifts. The observed wavenumbers were correlated with theoretical wavenumbers predicted with B3LYP density functional theory, using the basis sets 6-311++G(d,p) and 6-31G(d) and considering the harmonic as well as the anharmonic VPT2 approximation. Excellent correlations were established between observed wavenumbers and calculated harmonic values. However, the correlations were non-linear, in contrast to the results of previous investigations of the corresponding OH···O systems. The anharmonic VPT2 wavenumbers were found to be linearly related to the corresponding harmonic values. The results provide correlation equations for the prediction of NH stretching bands on the basis of standard B3LYP/6-311++G(d,p) and B3LYP/6-31G(d) harmonic analyses, with standard deviations close to 38 cm-1. This is significant because the full anharmonic VPT2 analysis tends to be impractical for large molecules, requiring orders of magnitude more computing time than the harmonic analysis.

Structural Studies of ß-Diketones and Their Implications on Biological Effects.

The paper briefly summarizes methods to determine the structure of ß-diketones with emphasis on NMR methods. Density functional calculations are also briefly treated. Emphasis is on the tautomeric equilibria of ß-diketones in relation to biological effects. Relevant physical parameters such as acidity and solubility are treated. A series of biologically active molecules are treated with respect to structure (tautomerism). Characteristic molecules or groups of molecules are usnic acids, tetramic and tetronic acids, o-hydroxydibenzoylmethanes, curcumines, lupulones, and hyperforines.

Cellular Uptake and Intracellular Phosphorylation of GS-441524: Implications for Its Effectiveness against COVID-19.

GS-441524 is an adenosine analog and the parent nucleoside of the prodrug remdesivir, which has received emergency approval for treatment of COVID-19. Recently, GS-441524 has been proposed to be effective in the treatment of COVID-19, perhaps even being superior to remdesivir for treatment of this disease. Evaluation of the clinical effectiveness of GS-441524 requires understanding of its uptake and intracellular conversion to GS-441524 triphosphate, the active antiviral substance. We here discuss the potential impact of these pharmacokinetic steps of GS-441524 on the formation of its active antiviral substance and effectiveness for treatment of COVID-19. Available protein expression data suggest that several adenosine transporters are expressed at only low levels in the epithelial cells lining the alveoli in the lungs, i.e., the alveolar cells or pneumocytes from healthy lungs. This may limit uptake of GS-441524. Importantly, cellular uptake of GS-441524 may be reduced during hypoxia and inflammation due to decreased expression of adenosine transporters. Similarly, hypoxia and inflammation may lead to reduced expression of adenosine kinase, which is believed to convert GS-441524 to GS-441524 monophosphate, the perceived rate-limiting step in the intracellular formation of GS-441524 triphosphate. Moreover, increases in extracellular and intracellular levels of adenosine, which may occur during critical illnesses, has the potential to competitively decrease cellular uptake and phosphorylation of GS-441524. Taken together, tissue hypoxia and severe inflammation in COVID-19 may lead to reduced uptake and phosphorylation of GS-441524 with lowered therapeutic effectiveness as a potential outcome. Hypoxia may be particularly critical to the ability of GS-441524 to eliminate SARS-CoV-2 from tissues with low basal expression of adenosine transporters, such as alveolar cells. This knowledge may also be relevant to treatments with other antiviral adenosine analogs and anticancer adenosine analogs as well.

NMR of Natural Products as Potential Drugs.

This review outlines methods to investigate the structure of natural products with emphasis on intramolecular hydrogen bonding, tautomerism and ionic structures using NMR techniques. The focus is on 1H chemical shifts, isotope effects on chemical shifts and diffusion ordered spectroscopy. In addition, density functional theory calculations are performed to support NMR results. The review demonstrates how hydrogen bonding may lead to specific structures and how chemical equilibria, as well as tautomeric equilibria and ionic structures, can be detected. All these features are important for biological activity and a prerequisite for correct docking experiments and future use as drugs.

The best density functional theory functional for the prediction of 1 H and 13 C chemical shifts of protonated alkylpyrroles.

The prediction of 13 C chemical shifts can be challenging with density functional theory (DFT). In this study 39 different functionals and three different basis sets were tested on three neutral alkylpyrroles and their corresponding protonated species. The calculated shielding constants were compared to experimental data and results from previous calculations at the MP2. We find that the meta-hybrid functional TPSSh with either the Pople style basis set 6-311++G(2d,p) or the polarization consistent basis set pcSseg-1 gives the best results for the 13 C chemical shifts, whereas for the 1 H chemical shifts it is the TPSSh functional with either the 6-311++G(2d,p) or pcSseg-2 basis set. Including an explicit solvent molecule hydrogen bonded to NH in the alkylpyrroles improves the results slightly for the 13 C chemical shifts. On the other hand, for 1 H chemical shifts the opposite is true. Compared to calculations at the MP2 level none of the DFT functionals can compete with MP2 for the 13 C chemical shifts but for the 1 H chemical shifts the investigated DFT functionals are shown to give better agreement with experiment than MP2 calculations.

Azo-hydrazone molecular switches: Synthesis and NMR conformational investigation.

A series of five intramolecularly hydrogen-bonded arylhydrazone (aryl = phenol, p-nitrophenol, anisole, quinoline) derived molecular switches have been synthesized and characterized by NMR and HRMS techniques. It was found that the compounds exist as different isomers in solution. An investigation of both conformational and/or configurational changes of the azo-hydrazone compounds was carried out by 1D 1 H- and 13 C- spectra, 2D NOESY, COSY, HSQC, and HMBC techniques. It was found that these stimuli-responsive molecular switches exist mainly in the E form by intramolecularly hydrogen bonded between NH and the pyridine nitrogen at equilibrium. Deprotonation of the neutral E form yields the E' deprotonated isomer. Prediction of 13 C-NMR chemical shifts was achieved by DFT quantum mechanical calculations. Anions have traditionally been difficult to calculate correctly, so calculations of the anion using different functionals, basis sets, and solvent effects are also included. Deuterium isotope effects on the 13 C-NMR chemical shifts were employed in the assignments and furthermore utilized as indicators of intramolecular hydrogen bonding. Studies in various organic solvents including CDCl3 , CD3 CN, and DMSO-d6 were also performed aiming to monitor dynamic changes over several days. The effect of the hydrogen bonded solvents leads to Z forms.

A Spectroscopic Overview of Intramolecular Hydrogen Bonds of NH O,S,N Type.

Intramolecular NH…O,S,N interactions in non-tautomeric systems are reviewed in a broad range of compounds covering a variety of NH donors and hydrogen bond acceptors. 1H chemical shifts of NH donors are good tools to study intramolecular hydrogen bonding. However in some cases they have to be corrected for ring current effects. Deuterium isotope effects on 13C and 15N chemical shifts and primary isotope effects are usually used to judge the strength of hydrogen bonds. Primary isotope effects are investigated in a new range of magnitudes. Isotope ratios of NH stretching frequencies, νNH/ND, are revisited. Hydrogen bond energies are reviewed and two-bond deuterium isotope effects on 13C chemical shifts are investigated as a possible means of estimating hydrogen bond energies.

Derivatives of usnic acid cause cytostatic effect in Caco-2 cells.

Usnic acid has anti-cancer activity, however, low solubility and toxicity limit the potential. To investigate biological activity of usnic acid derivatives, enantiopure derivatives were synthesised by reacting usnic acid with ethylenediamine, which yielded one dimer product ((+)-1), and two tetra cyclic compounds ((+)-2 and (-)-2). The products were characterised with NMR, and evaluated in vitro in human colon cancer cell line Caco-2 by cell count, phase-contrast microscopy, MTT-assay, measurement of DNA content and cell cycle distribution. All compounds tested showed cytostatic effect in Caco-2 cells, but each compound had a distinct cellular effect. Compound (+)-1 showed anti-proliferative activity by increasing the percentage of cells in S-phase with 25% compared to the control. Compounds (+)-2 and (-)-2 induced paraptosis, but only compound (+)-2 modulated cell cycle distribution by accumulating cells in G2/M-phase by 47% and reduced DNA content by 60%. All compounds express interesting cellular and potential anti-proliferative activity.

Photoinduced and ground state conversions in a cyclic ß-thioxoketone.

The photochemistry of a cyclic ß-thioxoketone (2-methyl-1-(2-thioxycyclohexyl)propan-1-one (MTPO)) is investigated by NMR, UV, and IR experiments supported by DFT calculations. MTPO exists as a tautomeric mixture of an enol and a thiol form. Irradiation at low temperature led to a cis-trans isomerization of the thiol form resulting in a rather unusual enethiol (3). This is followed by a transfer of the isopropyl methine proton onto the carbonyl carbon resulting in yet another enethiol isomer (4). The photoconversion mechanisms without water present are discussed. Photochemical experiments at ambient temperature showed involvement of water in the excited state and resulted in another keto-form (5). The same species was also obtained when the products of the low temperature experiments were kept in the dark at ambient temperature.

Isotope effects on chemical shifts in the study of hydrogen bonded biological systems.

This review deals with biological systems and with deuterium isotope effects on chemical shifts caused by the replacement of OH, NH or SH protons by deuterons. Hydrogen bonding is clearly of central importance. Isotope effects on chemical shifts seems very suitable for use in studies of structures and reactions in the interior of proteins, as exchange of the label can be expected to be slow. One-bond deuterium isotope effects on 15N chemical shifts, and two-bond effects on 1H chemical shifts for N(D)Hx systems can be used to gauge hydrogen bond strength in proteins as well as in salt bridges. Solvent isotope effects on 19F chemical shifts show promise in monitoring solvent access. Equilibrium isotope effects need in some cases to be taken into account. Schemes for calculation of deuterium isotope effects on chemical shifts are discussed and it is demonstrated how calculations may be used in the study of complex biological systems.

One-bond 1 J(15 N,H) coupling constants at sp2 -hybridized nitrogen of Schiff bases, enaminones and similar compounds: A theoretical study.

1 J(15 N,H) coupling constants for enaminones and NH-forms of intramolecularly hydrogen-bonded Schiff bases as model compounds for sp2 -hybridized nitrogen atoms are evaluated using density functional theory (DFT) to find the optimal functionals and basis sets. Ammonia is used as a test molecule and its one-bond coupling constant is compared with experiment. A methylamine Schiff base of a truncated molecule of gossypol is used for checking the performance of selected B3LYP, O3LYP, PBE, BHandH, and APFD density functionals and standard, modified, and dedicated basis sets for coupling constants. Both in vacuum and in chloroform, modeled by the simple continuum model of solvent, the modified basis sets predict significantly better the 1 J(15 N,H) value in ammonia and in the methylamine Schiff base of a truncated molecule of gossypol than the standard basis sets. This procure is then used on a broad set of intramolecularly hydrogen-bonded molecules, and a good correlation between calculated and experimental one-bond NH coupling constants is obtained. The 1 J(15 N,H) couplings are slightly overestimated. The calculated data show for hydrogen-bonded NH interatomic distances that the calculated values depend on the NH bond lengths. The shorter the bond lengths, the larger the 1 J(15 N,H). A useful correlation between 1 J(15 N,H) and NH bond length is derived that enables realistic predictions of one-bond NH coupling constants. The calculations reproduce experimentally observed trends for the studied molecules.

Strong intramolecular hydrogen bonds and steric effects involving C═S groups: An NMR and computational study.

A number of 5-acyl rhodanines and thiorhodanines with bulky acyl groups (pivaloyl and adamantoyl), not previously available, have been synthesized. The compounds are shown to exist in the enol form. Structures have been calculated using both the MP2 approach and the B3LYP-GD3BJ functional and the 6-311++G(d,p) basis set. Hydrogen bond energies are estimated by subtracting energies of a structure with the OH group turned 180° from those of the intramolecularly hydrogen-bonded one. Properties such as OH chemical shifts, two-bond isotope effects on 13 C chemical shifts, electron densities at the bond critical point from atoms in molecules analysis, and the hydrogen bond energies show that the sterically hindered compounds have stronger hydrogen bonds than methyl or isopropyl derivatives. The combination of oxygen and sulfur derivatives enables a detailed analysis of hydrogen bond energies.