Structure and Additive-free Transamidation of Planar N-Cyano Amides.

Although transamidation of amides generally requires metals, additives, or harsh conditions, we present here a facile transamidation of N-cyano amides with various amines at ambient temperature without any additive. N-cyano amides preferred the trans conformation and have a reduced double bond character revealed by crystal analysis. The DFT study indicates that the transamidation reaction proceeds through the direct attack of amine on the amide carbonyl since the LUMO (or LUMO+1) is located at the carbonyl moiety.

Synthesis and Conformational Analysis of N-Aromatic Acetamides Bearing Thiophene: Effect of Intramolecular Chalcogen-Chalcogen Interaction on Amide Conformational Stability.

The conformations of aromatic amides bearing an N-(2-thienyl) or N-(3-thienyl) group were investigated in solution and in the crystal state. NMR spectral data indicate that the conformational preferences of these amides in solution are dependent not only on the relative π-electron densities of the N-aromatic moieties, but also on the three-dimensional relationship between carbonyl oxygen and the N-aromatic moieties. A comparison of the conformational preferences of N-(2-thienyl)amides and N-(3-thienyl)amides revealed that the Z-conformers of N-(2-thienyl)acetamides are stabilized by 1,5-type intramolecular S···O═C interactions between amide carbonyl and thiophene sulfur. The crystal structures of these compounds were similar to the solution structures. The stabilization energy due to 1,5-type intramolecular S···O═C interaction in N-aryl-N-(2-thienyl)acetamides and N-methyl-N-(2-thienyl)acetamide was estimated to be ca. 0.74 and 0.93 kcal/mol, respectively.

Structures and Reactivities of N-Alkenyl-Substituted Anilides: The "Magic" Methyl Effect on Alkene.

Methyl substitution at the double bond of N-alkenyl anilides influences both the preferred conformation and the susceptibility to acidic hydrolysis. The R1-substituted amide favors the trans conformation, whereas amides substituted at R2 or R3 favor the cis conformation. Substitution at the R1 and R3 positions increases the ratio of the trans conformer. DFT study indicated that these conformational preferences can be explained in terms of substituent-induced torsion twisting of the N-alkenyl moiety relative to the amide plane. R1 substitution enhances the susceptibility to acidic hydrolysis, whereas R2 or R3 substitution increases the stability. The effect of the double bond on the conformational effect was showcased by contrasting the preferred conformation of R1-substituted anilide (trans) and hydrogenated N-isopropyl amide (cis).

Conformational Switch of Benzanilide Derivative Induced by Acid; Effect of Pentafluorobenzoyl Group.

Amide-based molecular switches had its limitation on structural diversities. In this work, we designed and synthesized a series of pentafluorobenzoyl-based benzanilide compounds. The conformational ratio of these compounds in solution was correlated linearly with Hammett's σp value of the substituent on the anilide ring, reflecting the repulsive interaction between the carbonyl group and the electron-rich aryl group. The addition of acid into the solution of 6, bearing pentafluorobenzoyl group, switched the stable amide conformation. In addition, the sizeable rotational barrier of 6 induced by the pentafluorobenzoyl moiety enabled us to monitor the conformational transition by means of 1H NMR spectroscopy.

N-Ethynylation of Anilides Decreases the Double-Bond Character of Amide Bond while Retaining trans-Conformation and Planarity.

Activated amide bonds have been attracting intense attention; however, most of the studied moieties have twisted amide character. To add a new strategy to activate amide bonds while maintaining its planarity, we envisioned the introduction of an alkynyl group on the amide nitrogen to disrupt amide resonance by nN→Csp conjugation. In this context, the conformations and properties of N-ethynyl-substituted aromatic amides were investigated by DFT calculations, crystallography, and NMR spectroscopic analysis. In contrast to the cis conformational preference of N-ethyl- and vinyl-substituted acetanilides, N-ethynyl-substituted acetanilide favors the trans conformation in the crystal and in solution. It also has a decreased double bond character of the C(O)-N bond, without twisting of the amide. N-Ethynyl-substituted acetanilides undergo selective C(O)-N bond or N-C(sp) bond cleavage reactions and have potential applications as activated amides for coupling reactions or easily cleavable tethers.

Chemosynthetic homologues of Mycoplasma pneumoniae ß-glycolipid antigens for the diagnosis of mycoplasma infectious diseases.

Mycoplasma pneumoniae expresses ß-glycolipids (ß-GGLs) in cytoplasmic membranes, which possess a unique ß(1 → 6)-linked disaccharide epitope, which has high potential in biochemical and medicinal applications. In the present study, a series of ß-GGLs homologues with different acyl chains (C12, C14, C16, and C18) were prepared from a common precursor. An ELISA assay using an anti-(ß-GGLs) monoclonal antibody indicated that the synthetic homologues with long acyl chains had greater diagnostic potential in the order C18 > C16 > C14 > C12. Toward a simultaneous detection of natural glycolipids by mass spectrometry (MS), a deuterium-labeled C16 homologue (ß-GGL-C16-d3) was prepared and applied as an internal standard for a high-resolution electrospray ionization MS (ESI-MS) analysis. The ESI-MS analysis was used to identify and quantify acyl homologues (C16/C16, C16/C18, and C18/C18) of ß-GGL-C16 in cultured M. pneumoniae. A ß-GGLs homologue with a 1,2-diacetyl group (C2) was also prepared as a "water soluble" glycolipid homologue and characterized by 1H NMR spectroscopy. We envisage that each of these chemosynthetic homologues will provide promising approaches to solve medical and biological problems associated with mycoplasma infectious diseases (MIDs).

Spontaneous and Direct Transformation of N,O-Diaryl Carbamates into N,N'-Diarylureas.

We have discovered a spontaneous reaction of N,O-diaryl carbamates to afford symmetrical N,N'-diarylureas. Optimization of the conditions indicated that N,N-dimethylformamide (DMF) was the best solvent and triethylamine (Et3N) was the best additive for this transformation. The reaction requires the presence of aryl groups on the nitrogen and oxygen atoms of carbamates. Substrates bearing an electron-donating methoxy group on either of the aryl groups reacted slowly under these conditions.

Remarkable functions of sn-3 hydroxy and phosphocholine groups in 1,2-diacyl-sn-glycerolipids to induce clockwise (+)-helicity around the 1,2-diacyl moiety: Evidence from conformation analysis by 1H NMR spectroscopy.

Cell-membrane glycerolipids exhibit a common structural backbone of asymmetric 1,2-diacyl-sn-glycerol bearing polar head groups in the sn-3 position. In this study, the possible effects of sn-3 head groups on the helical conformational property around the 1,2-diacyl moiety in the solution state were examined. 1H NMR Karplus relation studies were carried out using a series of 1,2-dipalmitoyl-sn-glycerols bearing different sn-3 substituents (namely palmitoyl, benzyl, hydrogen, and phosphates). The 1H NMR analysis indicated that the helical property around the 1,2-diacyl moiety is considerably affected by these sn-3 substituents. The sn-3 hydroxy group induced a unique helical property, which was considerably dependent on the solvents used. In CDCl3 solution, three staggered conformers, namely gt(+), gg(-) and tg, were randomized, while in more polar solvents, the gt(+) conformer with (+)-helicity was amplified at the expense of gg(-) and tg conformers. The sn-3 phosphocholine in phosphatidylcholine exhibited a greater effect on the gt(+) conformer, which was independent of the solvents used. From the 1H NMR analysis, the helical conformational properties around the 1,2-diacyl moiety conformed to a simple empirical rule, which permitted the proposal of a conformational diagram for 1,2-dipalmitoyl-sn-glycerols in the solution states.

An easy α-glycosylation methodology for the synthesis and stereochemistry of mycoplasma α-glycolipid antigens.

Mycoplasma fermentans possesses unique α-glycolipid antigens (GGPL-I and GGPL-III) at the cytoplasm membrane, which carry a phosphocholine group at the sugar primary (6-OH) position. This paper describes a practical synthetic pathway to a GGPL-I homologue (C(16:0)) and its diastereomer, in which our one-pot α-glycosylation method was effectively applied. The synthetic GGPL-I isomers were characterized with (1)H NMR spectroscopy to determine the equilibrium among the three conformers (gg, gt, tg) at the acyclic glycerol moiety. The natural GGPL-I isomer was found to prefer gt (54%) and gg (39%) conformers around the lipid tail, while adopting all of the three conformers with equal probability around the sugar position. This property was very close to what we have observed with respect to the conformation of phosphatidylcholine (DPPC), suggesting that the Mycoplasma glycolipids GGPLs may constitute the cytoplasm fluid membrane together with ubiquitous phospholipids, without inducing stereochemical stress.

Hybrid molecule between platanic acid and LCL-161 as a yes-associated protein degrader.

Dysregulated yes-associated protein (YAP) is involved in several malignant cancers. However, discovering a druggable YAP inhibitor(s) is difficult because YAP itself does not have any enzymatic activity. In such cases, targeted protein degradation strategies based on hybrid molecules that bind to the target protein and an E3 ubiquitin ligase are useful for suppressing proteins that exhibit aberrant activation and/or excessive expression. Upon screening YAP-interacting small compounds, we identified HK13, a platanic acid, as a novel compound that interacts with YAP. Next, we synthesized hybrid compounds of platanic acid and LCL-161, which reportedly shows a high affinity for cIAP, one of E3 ubiquitin ligases. Among these compounds, HK24 possessed the ability to inhibit the growth of YAP overexpressing NCI-H290 cells. This inhibitory activity may be mediated by YAP degradation, although HK24 exhibited weak YAP degradation. Furthermore, we confirmed involvement of proteasome pathway in HK24-dependent YAP degradation by culturing NCI-H290 cells in the presence of a proteasome inhibitor. Therefore, it is possible that platanic acid is a potential candidate for molecular medicine targeting YAP.

Clostridium butyricum Modulates the Microbiome to Protect Intestinal Barrier Function in Mice with Antibiotic-Induced Dysbiosis.

Clostridium butyricum MIYAIRI 588 (CBM 588) is a probiotic bacterium that has previously been used to prevent antibiotic-associated diarrhea. However, the underlying mechanism by which CBM 588 protects the gut epithelial barrier remains unclear. Here, we show that CBM 588 increased the abundance of Bifidobacterium, Lactobacillus, and Lactococcus species in the gut microbiome and also enhanced the intestinal barrier function of mice with antibiotic-induced dysbiosis. Additionally, CBM 588 significantly promoted the expansion of IL-17A-producing γδT cells and IL-17A-producing CD4 cells in the colonic lamina propria (cLP), which was closely associated with changes in the intestinal microbial composition. Additionally, CBM 588 plays an important role in controlling antibiotic-induced gut inflammation through upregulation of anti-inflammatory lipid metabolites such as palmitoleic acid, 15d-prostaglandin J2, and protectin D1. This study reveals a previously unrecognized mechanism of CBM 588 and provides new insights into gut epithelial barrier protection with probiotics under conditions of antibiotic-induced dysbiosis.

HPLC-UV determination of metformin in human plasma for application in pharmacokinetics and bioequivalence studies.

In this study, a simple, rapid and sensitive HPLC method with UV detection is described for determination of metformin in plasma samples from bioequivalence assays. Sample preparation was accomplished through protein precipitation with acetonitrile and chromatographic separation was performed on a reversed-phase phenyl column at 40 degrees C. Mobile phase consisted of a mixture of phosphate buffer and acetonitrile at flow rate of 1.0 ml/min. Wavelength was set at 236 nm. The method was applied to a bioequivalence study of two drug products containing metformin, and allowed determination of metformin at low concentrations with a higher throughput than previously described methods.

Effect of Different Sampling Schedules on Results of Bioavailability and Bioequivalence Studies: Evaluation by Means of Monte Carlo Simulations.

Bioavailability and bioequivalence study is one of the most frequently performed investigations in clinical trials. Bioequivalence testing is based on the assumption that 2 drug products will be therapeutically equivalent when they are equivalent in the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed and becomes available at the site of drug action. In recent years there has been a significant growth in published papers that use in silico studies based on mathematical simulations to analyze pharmacokinetic and pharmacodynamic properties of drugs, including bioavailability and bioequivalence aspects. The goal of this study is to evaluate the usefulness of in silico studies as a tool in the planning of bioequivalence, bioavailability and other pharmacokinetic assays, e.g., to determine an appropriate sampling schedule. Monte Carlo simulations were used to define adequate blood sampling schedules for a bioequivalence assay comparing 2 different formulations of cefadroxil oral suspensions. In silico bioequivalence studies comparing different formulation of cefadroxil oral suspensions using various sampling schedules were performed using models. An in vivo study was conducted to confirm in silico results. The results of in silico and in vivo bioequivalence studies demonstrated that schedules with fewer sampling times are as efficient as schedules with larger numbers of sampling times in the assessment of bioequivalence, but only if Tmax is included as a sampling time. It was also concluded that in silico studies are useful tools in the planning of bioequivalence, bioavailability and other pharmacokinetic in vivo assays.

A recyclable heavy fluorous tag carrying an allyl alcohol pendant group: design and evaluation toward applications in synthetic carbohydrate chemistry.

Toward applications in synthetic carbohydrate chemistry, we converted our previous acid-resistant heavy fluorous tag [(Rf)3C-CH2-OH, 1] to allyl alcohol derivatives [(Rf)3C-CH2-O-(CH2)n-CH=CH-CH2-OH, 3 (n=1) or 4 (n=3)] by means of olefin cross metathesis. They were then subjected to ß-glycosylation reactions by using a series of glycosyl donors, including glycosyl bromide and trichloroacetimidates. The terminal OH group in 3 and 4 was found to be ß-glycosylated in moderate yield when 2,3,4,6-tetra-O-benzoyl-D-galactosyl trichloroacetimidate was used as the glycosyl donor. Upon a detachment reaction using Pd(PPh3)4, the initial heavy fluorous tag 1 was recovered in high yield (>90%) together with 1-hydroxy sugar, indicating that not only the allyl ether linkage in the glycosides but also the internal di-alkyl ether linkage in 4 be cleaved by the action of the Pd-catalyst enabling long-range olefin transmigration. Potential utility was demonstrated by using the tetra-O-benzoyl-ß-D-galactosylated derivative of 3 in a series of deprotection, protection and glycosylation reactions, which were conductible in high yields without using chromatographic purification process. These findings prompt us to propose a general scheme in which the acid-resistant heavy fluorous compound 1 is applied as a recyclable tag in synthetic carbohydrate chemistry.

Depression of the apparent chiral recognition ability obtained in the host-guest complexation systems by electrospray and nano-electrospray ionization mass spectrometry.

Chiral recognition in the host-guest complexation systems of chiral crown ether hosts and amino ester guests was thoroughly examined using the electrospray ionization (ESI) mass spectrometry/enantiomer labeled (EL)-guest method. In this method, the mass spectra of a mixture of three components in a solution, a chiral host (H), an equal amount of an (S)-enantiomer guest labeled with deuterium atoms (G(S-dn)(+)) and an unlabeled (R)-enantiomer guest (G(R)+), were measured and the relative peak intensity value [I(H + G(R))(+) / I(H + G(S-dn))(+) = IRIS] of the host-guest complex ions, observed with an excess guest concentration, was taken to provide the chiral recognition ability of the host. In our earlier report (1996), we demonstrated that the apparent chiral recognition abilities using a mass spectrometer with a homemade ESI interface were depressed by about one tenth compared with the corresponding abilities obtained by fast-atom bombardment (FAB) MS. In the present study, the enantioselective complexation behaviors of various combinations of chiral crown hosts with chiral guests were further investigated in detail mainly using a modern commercial ESI/ion trap (IT) mass spectrometer. Consequently, it was found that the apparent IRIS values from the ESI-MS/EL-guest method changed significantly, depending upon the instrument used, and in particular, upon the ESI interfaces. Moreover, under the specific measuring conditions in ESI-IT-MS, the degrees of depression of the apparent chiral recognition abilities are roughly grouped into three classes, depending upon the number (or probably the type) of the hydrophobic substituents of the hosts. Representing the degrees by the slopes when plotting the apparent IRIS values in ESI-MS versus those in FAB-MS, the slopes for the three classes are (1) 1.0, (2) 0.7 and (3) 0.3; the higher the hydrophobicity of the hosts (and then, the host-guest complex ions), the lower the slope (the apparent enantioselectivity). Strengthening the degree of depression may be caused by an increase in the local concentration of the host close to the surface of the droplets produced during the electrospary ionization process. The chiral recognition ability (K(R )/ K(S)) in an equilibrated solution agrees quite well with the IRIS value in FAB-MS rather than that in ESI-MS.