Facile extraction of berberine from different plants, separation, and identification by thin-layer chromatography, high-performance liquid chromatography, and biological evaluation against Leishmaniosis.

The extraction of berberine was carried out from Berberis vulgaris, Berberis aquifolium, and Hydrastis canadensis plants using ethanol and water (70:30, v/v). The extracted berberine was characterized by ultraviolet-visible and Fourier-transform infrared spectroscopy. The purity of berberine was ascertained by thin-layer chromatography using n-propanol-formic acid-water (95:1:4) and (90:1:9) solvents. hRf values were in the range of 44-49 with compact spots (diameter 0.2-0.4 cm). HPLC was carried out using ammonium acetate buffer and acetonitrile in gradient mode with Zodiac (4.6 × 150 mm, 3 µm) column. The flow rate was 1.0 mL/min and detection was at 220 nm. The values of separation and resolution factors of the standard and the extracted berberine were in the range of 1.13-1.16 and 1.40-1.71, respectively. A comparison has shown that both thin-layer chromatography and high-performance liquid chromatography (HPLC) methods found applications in different situations and requirements. The extracted berberine samples were used to treat Leishmaniosis and the results showed better activity of berberine in comparison to the standard drug Amphotericin B. Briefly, the reported research is a novel and may be used to extract berberine from plants, separation and identification of berberine by thin layer chromatography and HPLC and to treat Leishmaniosis.

A strategy for evaluation of isotopic enrichment and structural integrity of deuterium labelled compounds by using HR-MS and NMR.

Determining the purity of deuterium labelled compounds is important due to the increasing use of these compounds in mass spectrometry (MS) based quantitative analyses for targeting metabolic flux, reducing toxicity, confirming reaction mechanisms during synthesis, predicting enzyme mechanisms, and enhancing the efficacy of drugs, in quantitative proteomics, and also as internal standards. In the present study, a strategy using liquid chromatography electrospray ionization high resolution mass spectrometry (LC-ESI-HR-MS) and nuclear magnetic resonance (NMR) spectroscopy was proposed to determine the isotopic enrichment and structural integrity of deuterium labelled compounds. The proposed strategy involves recording full scan MS, extracting and integrating isotopic ions, and calculating the isotopic enrichment of the desired labelled compounds. NMR analysis confirms structural integrity or positions of labelled atoms and can provide insights into the relative percent isotopic purity. This strategy was used to evaluate the isotopic enrichment and structural integrity of in-house synthesized compounds as well as a series of commercially available deuterium labelled compounds. The % isotopic purity for labelled compounds of a benzofuranone derivative (BEN-d2), tamsulosin-d4 (TAM-d4), oxybutynin-d5 (OXY-d5), eplerenone-d3 (EPL-d3), and propafenone-d7 (PRO-d7) was calculated and found to be 94.7, 99.5, 98.8, 99.9, and 96.5, respectively. All the samples were run in triplicate and the results were observed to be reproducible.

Isolation and characterization of pseudo degradation products of acalabrutinib using ESI-HRMS/MS and NMR: Formation of possible geometrical isomers.

A forced degradation study of acalabrutinib (ACB), used to treat relapsed mantle cell lymphoma, was performed to identify and characterize all possible major degradation products formed under different stress conditions. The degradation products (DP) were separated using reverse phase UHPLC system on Kinetex EVO C18 column. Major DPs formed were isolated using semi-preparative HPLC and characterized by LC-ESI-HRMS/MS and NMR. ACB degraded to form seven major degradants (DP-I to DP-VII). DP-I and DP-V were formed under alkaline stress condition, whereas DP-II, DP-III, DP-VI and DP-VII were formed under both acidic and basic conditions. Further, DP-IV was formed when ACB drug was exposed to hydrogen peroxide stress condition. ACB was found to be stable when subjected to aqueous (neutral pH), thermal and UV radiation of 254 nm, as it has not shown any significant degradation under these conditions. Interestingly, two pairs of pseudo geometrical isomeric DPs (DP-II and DP-III, DP-VI and DP-VII) were observed. The plausible degradation pathway of ACB and fragmentation patterns of both ACB and major DPs were discussed.

RP-HPLC separation of interconvertible rotamers of a 5-tetrahydroisoquinolin-6-yl-pyridin-3-yl acetic acid derivative and confirmation by VT NMR and DFT study.

Due to emergence of drug resistance and drug tolerability, there is urgent need for discovery of new chemical entity for the treatment of HIV infection. As a part of in-house small molecule drug discovery program for HIV infection, sodium-2-(tert-butoxy)- 2-(5-(2-(2-chloro-6-methylbenzyl)- 1,2,3,4-tetrahydroisoquinolin-6-yl)- 4-(4,4-dimethylpiperidin-1-yl)- 2,6-dimethylpyridin-3-yl) acetate (SCMTDDA) was prepared as an intermediate for the synthesis of an API, designed as a HIV-1 integrase inhibitor. Initially, the final compound was isolated as a mixture of rotamers. In the current study, we have developed a simple and efficient achiral, reversed phase (RP) HPLC method to separate the interconvertible rotamers of SCMTDDA. The effect of several parameters, including stationary phase, buffer, modifiers and column temperature, were optimized for the chromatographic separation and it was observed that best separation was achieved on a SunFire C18 column using TFA/acetonitrile (ACN) - methanol (MeOH) (1:1 v/v) as the mobile phase at 10 0C. The chromatographic observations were complemented with variable-temperature NMR and energy barrier calculations using density functional theory (DFT).

Mass spectrometric-based investigation of differentially protected azatryptophan derivatives using Orbitrap mass spectrometry: Differentiation of positional isomers under protonation and alkali-cationization conditions.

RATIONALE: Differentiation and structural characterization of positional isomers of differentially protected azatryptophan derivatives using electrospray ionization high-resolution tandem mass spectrometry (ESI-HRMS/MS) is important from the perspective of drug discovery research. Also, these derivatives can be used as building blocks for the synthesis of various biologically active compounds and have attracted significant attention in the field of modern drug discovery, especially peptide-based drugs, protein folding and protein-protein interactions because of their interesting spectral properties. METHODS: ESI-HRMS/MS in positive ionization mode was used to differentiate and characterize positional isomers of protected azatryptophan derivatives. RESULTS: ESI-HRMS/MS of [M + H]+ and [M + Na]+ ions of positional isomers of differentially protected azatryptophan derivatives display distinct fragmentation patterns. The MS/MS of [M + H]+ ion of isomer 1 showed an additional ion at m/z 358.0846 ([M + H-Boc-C14 H10 -HF]+ ) which was not present for 4. The fragment ion at m/z 332.0857 was observed for 1 and not for 4 which would be formed by the expulsion of butyloxycarbonyl (Boc) and fluorenylmethyloxycarbonyl (Fmoc) groups. Moreover, the ions 422.0812 and 378.0912 are found to be relatively more abundant for isomer 4 which could be probably attributed to the formation of stable ions. Similarly, other positional isomers exhibited distinct fragmentation from one another. CONCLUSIONS: The present study demonstrates that ESI-HRMS/MS can be used for differentiation and structural characterization of positional isomers of protected azatryptophan derivatives. The MS/MS of [M + H]+ and [M + Na]+ ions of these positional isomers displayed differences in their fragmentation behaviour. The impact of different substitutions at different positions (1 and 6) of protected azatryptophan derivatives (1-6) on their fragmentation behaviour was also investigated in detail. Also, the nitrogen atom at different positions in the pyrrolopyridine ring led to different fragmentation patterns.

Lipidomic Profiling of Clinical Prostate Cancer Reveals Targetable Alterations in Membrane Lipid Composition.

Dysregulated lipid metabolism is a prominent feature of prostate cancer that is driven by androgen receptor (AR) signaling. Here we used quantitative mass spectrometry to define the "lipidome" in prostate tumors with matched benign tissues (n = 21), independent unmatched tissues (n = 47), and primary prostate explants cultured with the clinical AR antagonist enzalutamide (n = 43). Significant differences in lipid composition were detected and spatially visualized in tumors compared with matched benign samples. Notably, tumors featured higher proportions of monounsaturated lipids overall and elongated fatty acid chains in phosphatidylinositol and phosphatidylserine lipids. Significant associations between lipid profile and malignancy were validated in unmatched samples, and phospholipid composition was characteristically altered in patient tissues that responded to AR inhibition. Importantly, targeting tumor-related lipid features via inhibition of acetyl-CoA carboxylase 1 significantly reduced cellular proliferation and induced apoptosis in tissue explants. This characterization of the prostate cancer lipidome in clinical tissues reveals enhanced fatty acid synthesis, elongation, and desaturation as tumor-defining features, with potential for therapeutic targeting. SIGNIFICANCE: This study identifies malignancy and treatment-associated changes in lipid composition of clinical prostate cancer tissues, suggesting that mediators of these lipidomic changes could be targeted using existing metabolic agents.

Synthesis and characterization of a series of N,N'-substituted urea derivatives by using electrospray ionization tandem mass spectrometry: Differentiation of positional isomers.

RATIONALE: Characterization of N,N'-substituted ureas was found to be challenging by nuclear magnetic resonance (NMR) spectroscopy, particularly N-di- and tri-alkylated ureas because of the absence of adjacent protons. In the present study, electrospray ionization tandem mass spectrometry has been used to differentiate positional isomeric pairs and to characterize a series of N,N'-substituted ureas, as these compounds have significant importance for drug discovery. Additionally, urea is an essential functionality in several bioactive compounds as well as a variety of clinically approved therapies. METHODS: High-resolution electrospray ionization tandem mass spectrometry (ESI-HR-MS/MS) has been used to characterize a series of N,N'-substituted urea derivatives and differentiate two pairs of positional isomers. The data was acquired by Xcaliber application in positive ionization mode. RESULTS: ESI-HR-MS/MS spectra of [M + H]+ ions of the positional isomeric urea derivatives 8a and 8b show distinct fragmentation patterns. For example, the MS/MS spectrum of the [M + H]+ ion of isomer 8a displays the abundant fragment ion at m/z 285.1595, which was totally absent in isomer 8b. This would be plausibly formed by the cleavage of the C-N bond of the urea group with the elimination of the isocyanate moiety. In contrast, the MS/MS spectrum of the [M + H]+ ion of isomer 8b shows an intense ion at m/z 311.1389 which is completely absent in isomer 8a which would be formed by the cleavage of the C-N bond attached to the ring nitrogen. Similarly, another pair of positional isomers, 8c and 8d, have been clearly distinguished by their fragmentation behaviour. In addition, a series of N,N'-substituted urea derivatives were studied to investigate the impact of different substitution on the fragmentation behaviour. CONCLUSIONS: The present study demonstrates that ESI-HR-MS/MS can be used to differentiate pairs of N,N'-substituted urea positional isomers and characterize a series of derivatives. It was observed that a characteristic fragment ion was formed by the C-N bond cleavage with the elimination of an isocyanate moiety. The proposed mechanism of fragmentation was supported by the change in the fragmentation pathway upon alkylation of the NH. In order to generalize this fragmentation pattern, a series of N-alkylated ureas was synthesized and studied by MS/MS.

Lipidomics investigations into the tissue phospholipidomic landscape of invasive ductal carcinoma of the breast.

The need of identifying alternative therapeutic targets for invasive ductal carcinoma (IDC) of the breast with high specificity and sensitivity for effective therapeutic intervention is crucial for lowering the risk of fatality. Lipidomics has emerged as a key area for the discovery of potential candidates owing to its several shared pathways between cancer cell proliferation and survival. In the current study, we performed comparative phospholipidomic analysis of IDC, benign and control tissue samples of the breast to identify the significant lipid alterations associated with malignant transformation. A total of 33 each age-matched tissue samples from malignant, benign and control were analyzed to identify the altered phospholipids by using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). A combination of univariate and multivariate statistical approaches was used to select the phospholipid species with the highest contribution in group segregation. Furthermore, these altered phospholipids were structurally confirmed by tandem mass spectrometry. A total of 244 phospholipids were detected consistently at quantifiable levels, out of which 32 were significantly altered in IDC of the breast. Moreover, in pairwise comparison of IDC against benign and control samples, 11 phospholipids were found to be significantly differentially expressed. Particularly, LPI 20:3, PE (22:1/22:2), LPE 20:0 and PC (20:4/22:4) were observed to be most significantly associated with IDC tissue samples. Apart from that, we also identified that long-chain unsaturated fatty acids were enriched in the IDC tissue samples as compared to benign and control samples, indicating its possible association with the invasive phenotype.

Identification of drugs that restore primary cilium expression in cancer cells.

The development of cancer is often accompanied by a loss of the primary cilium, a microtubule-based cellular protrusion that functions as a cellular antenna and that puts a break on cell proliferation. Hence, restoration of the primary cilium in cancer cells may represent a novel promising approach to attenuate tumor growth. Using a high content analysis-based approach we screened a library of clinically evaluated compounds and marketed drugs for their ability to restore primary cilium expression in pancreatic ductal cancer cells. A diverse set of 118 compounds stimulating cilium expression was identified. These included glucocorticoids, fibrates and other nuclear receptor modulators, neurotransmitter regulators, ion channel modulators, tyrosine kinase inhibitors, DNA gyrase/topoisomerase inhibitors, antibacterial compounds, protein inhibitors, microtubule modulators, and COX inhibitors. Certain compounds also dramatically affected the length of the cilium. For a selection of compounds (Clofibrate, Gefitinib, Sirolimus, Imexon and Dexamethasone) their ability to restore ciliogenesis was confirmed in a panel of human cancer cell line models representing different cancer types (pancreas, lung, kidney, breast). Most compounds attenuated cell proliferation, at least in part through induction of the primary cilium, as demonstrated by cilium removal using chloral hydrate. These findings reveal that several commonly used drugs restore ciliogenesis in cancer cells, and warrant further investigation of their antineoplastic properties.

p53 attenuates AKT signaling by modulating membrane phospholipid composition.

The p53 tumor suppressor is the central component of a complex network of signaling pathways that protect organisms against the propagation of cells carrying oncogenic mutations. Here we report a previously unrecognized role of p53 in membrane phospholipids composition. By repressing the expression of stearoyl-CoA desaturase 1, SCD, the enzyme that converts saturated to mono-unsaturated fatty acids, p53 causes a shift in the content of phospholipids with mono-unsaturated acyl chains towards more saturated phospholipid species, particularly of the phosphatidylinositol headgroup class. This shift affects levels of phosphatidylinositol phosphates, attenuates the oncogenic AKT pathway, and contributes to the p53-mediated control of cell survival. These findings expand the p53 network to phospholipid metabolism and uncover a new molecular pathway connecting p53 to AKT signaling.

Non-small cell lung cancer is characterized by dramatic changes in phospholipid profiles.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death globally. To develop better diagnostics and more effective treatments, research in the past decades has focused on identification of molecular changes in the genome, transcriptome, proteome, and more recently also the metabolome. Phospholipids, which nevertheless play a central role in cell functioning, remain poorly explored. Here, using a mass spectrometry (MS)-based phospholipidomics approach, we profiled 179 phospholipid species in malignant and matched non-malignant lung tissue of 162 NSCLC patients (73 in a discovery cohort and 89 in a validation cohort). We identified 91 phospholipid species that were differentially expressed in cancer versus non-malignant tissues. Most prominent changes included a decrease in sphingomyelins (SMs) and an increase in specific phosphatidylinositols (PIs). Also a decrease in multiple phosphatidylserines (PSs) was observed, along with an increase in several phosphatidylethanolamine (PE) and phosphatidylcholine (PC) species, particularly those with 40 or 42 carbon atoms in both fatty acyl chains together. 2D-imaging MS of the most differentially expressed phospholipids confirmed their differential abundance in cancer cells. We identified lipid markers that can discriminate tumor versus normal tissue and different NSCLC subtypes with an AUC (area under the ROC curve) of 0.999 and 0.885, respectively. In conclusion, using both shotgun and 2D-imaging lipidomics analysis, we uncovered a hitherto unrecognized alteration in phospholipid profiles in NSCLC. These changes may have important biological implications and may have significant potential for biomarker development.