In-depth characterization of the GOTCAB saponins in seven cultivated Gypsophila L. species (Caryophyllaceae) by liquid chromatography coupled with quadrupole-Orbitrap mass spectrometer

Roots of Gypsophila L. (Caryophyllaceae) have been shown to accumulate bidesmosides of triterpenoid carboxylic acids, also called GOTCAB saponins (Glucuronide Oleanane-type Triterpenoid Carboxylic Acid 3, 28-Bidesmosides). The study aimed at in-depth characterization of GOTCABs from root extracts of cultivated Gypsophila scorzonerifolia Ser., G. acutifolia Stev. ex Spreng., G. altissima L., G. pacifica Kom., G. paniculata L., G. oldhamiana Miq. and G. zhegualensis Krasnova using ultra high-performance liquid chromatography coupled with hybrid quadrupol-Orbitrap high resolution mass spectrometry (UHPLC-HRMS). Based on the accurate mass measurements, elemental composition, isotopic peak profiles, fragmentation pattern in tandem mass spectrometry (MS/MS) and literature data, a total of 53 GOTCABs were tentatively identified. In addition, 29 core structures, forming between 2 and 12 isobaric isomers were described. They possess gypsogenin, quillaic and gypsogenic acid as sapogenin, substituted at C-3 with O-β-d-galactopyranosyl-(1 → 2)-[pentosyl-(1 → 3)]-β-d-glucuronopyranoside (β-chain). According to the C-28 ester-bonded oligosaccharide (α-chain) saponins were classified into four groups: GOTCABs with C-28 tetra- and pentasaccharide (type I), GOTCABs with C-28 oligosaccharide substituted with methoxycinnamoyl group (type II), GOTCABs with mono- and diacetylated C-28 oligosaccharide (type III) and GOTCABs with C-28 oligosaccharide substituted with both acetyl and methoxycinamoyl groups (type IV). The possible fragmentation pathways of saponins were proposed. Eleven core structures forming between 2 and 7 isobars are undescribed in the literature. To examine the differences between the assayed Gypsophila species at the same environmental conditions, the variation of saponins was estimated by hierarchical clustering on isobaric fingerprints of GOTCABs. The clustering of the studied species revealed three well-defined clusters. The first cluster comprises G. scorzonerifolia (G1) and G. altissima (G3), characterized by GOTCABs from type III. G. acutifolia (G2) and G. pacifica (G4) formed the second cluster accumulating saponins from types II and III. The third cluster grouped G. paniculata (G5), G. oldhamiana (G6) and G. zhegualensis (G7) sharing GOTCABs from types IV in addition to II and III. This is the first report on the saponins from G. scorzonerifolia and G. zhegualensis. An in-depth depiction of the GOTCAB saponin composition of seven cultivated Gypsophila species was achieved. Therefore, saponins are worth investigating for better understanding of the potential use of Gypsophila roots for pharmaceutical purposes.     

Direct anabolic metabolism of three-carbon propionate to a six-carbon metabolite occurs in vivo across tissues and species

Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of ¹³C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.     

Antiviral properties and interaction of novel chalcone derivatives containing a purine and benzenesulfonamide moiety

A new concise and facile method was explored to synthesize a series of novel chalcone derivatives containing a purine and benzenesulfonamide moiety and their antiviral properties were evaluated against TMV and CMV. Biological assays indicated that several of the derivatives exhibited significant anti-TMV and anti-CMV activities in vivo. In particular, compound d2 displayed excellent inactivating activity against TMV, with the EC₅₀ value of 51.65?μg/mL, which was better than that of ribavirin (150.45?μg/mL). Molecular docking showed that there are four hydrogen bonds between compound d2 and TMV coat protein (TMV-CP). Compound d2 demonstrated strong binding capacity to TMV-CP with K_a?=?1.58?×?10⁵?L/mol and K_d?=?12.16?μM. These findings indicated that chalcone derivatives are worthy of further research and development as templates for new antiviral agents.     

UHPLC‐HRMS/MS Based Profiling of Algerian Lichens and Their Antimicrobial Activities

Lichens are complex symbiotic organisms able to produce a vast array of compounds. The Algerian lichen diversity has only prompted little interest even given the 1085 species listed. Herein, the chemodiversity of four Algerian lichens including Cladonia rangiformis, Ramalina farinaceae, R. fastigiata, and Roccella phycopsis was investigated. A dereplication strategy, using ultra high performance liquid chromatography‐high resolution‐electrospray ionization‐mass spectrometry (UHPLC‐HRMS/MS), was carried out for a comprehensive characterization of their substances including phenolics, depsides, depsidones, depsones, dibenzofurans, and aliphatic acids. Some known compounds were identified for the first time in some species. Additionally, the lichenic extracts were evaluated for their antifungal and antimicrobial activities on human pathogenic strains (Candida albicans, C. glabrata, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli). Cyclohexane extracts were found particularly active against human pathogenic fungi with MIC₈₀ values ranging from 8 to 62.5 μg/mL, without cytotoxicity. This study highlights the therapeutic and prophylactic potential of lichenic extracts as antibacterial and antifungal agents.     

Design,synthesis, in vitro and in vivo evaluation,and structure-activity relationship (SAR) discussion of novel dipeptidyl boronic acid proteasome inhibitors as orally available anti-cancer agents for the treatment of multiple myeloma and mechanism studies

A series of novel dipeptidyl boronic acid inhibitors of 20S proteasome were designed and synthesized. Aliphatic groups at R¹ position were designed for the first time to fully understand the SAR (structure–activity relationship). Among the screened compounds, novel inhibitor 5c inhibited the CT-L (chymotrypsin-like) activity with IC₅₀ of 8.21?nM and the MM (multiple myeloma) cells RPMI8226, U266B and ARH77 proliferations with the IC₅₀ of 8.99, 6.75 and 9.10?nM, respectively, which showed similar in vitro activities compared with the compound MLN2238 (biologically active form of marketed MLN9708). To investigate the oral availability, compound 5c was esterified to its prodrug 6a with the enzymatic IC₅₀ of 6.74?nM and RPMI8226, U266B and ARH77 cell proliferations IC₅₀ of 2.59, 4.32 and 3.68?nM, respectively. Furthermore, prodrug 6a exhibited good pharmacokinetic properties with oral bioavailability of 24.9%, similar with MLN9708 (27.8%). Moreover, compound 6a showed good microsomal stabilities and displayed stronger in vivo anticancer efficacy than MLN9708 in the human ARH77 xenograft mouse model. Finally, cell cycle results showed that compound 6a had a significant inhibitory effect on CT-L and inhibited cell cycle progression at the G2M stage.     

Discovery of a new class of valosine containing protein (VCP/P97) inhibitors for the treatment of non-small cell lung cancer

Valosine containing protein (VCP/p97) is a member of the AAA ATPase family involved in several essential cellular functions and plays an important role in the ubiquitin-mediated degradation of misfolded proteins. P97 has a significant role in maintaining the cellular protein homeostasis for tumor cell growth and survival and has been found overexpressed in many tumor types. No new molecule entities based on p97 target were approved in clinic. Herein, a series of novel pyrimidine structures as p97 inhibitors were designed and synthesized. After enzymatic evaluations, structure-activity relationships (SAR) were discussed in detailed. Among the screened compounds, derivative 35 showed excellent enzymatic inhibitory activity (IC₅₀, 36?nM). The cellular inhibition results showed that compound 35 had good antiproliferative activity against the non-small cell lung cancer A549 cells (IC₅₀, 1.61?μM). Liver microsome stability showed that the half-life of compound 35 in human liver microsome was 42.3?min, which was more stable than the control CB-5083 (25.8?min). The in vivo pharmacokinetic results showed that the elimination phase half-lives of compound 35 were 4.57?h for ig and 3.64?h for iv, respectively and the oral bioavailability was only 4.5%. These results indicated that compound 35 could be effective for intravenous treatment of non-small cell lung cancer.     

Phosphonodiamidate prodrugs of N-alkoxy analogs of a fosmidomycin surrogate as antimalarial and antitubercular agents

A series of N-alkoxy analogs of a l-leucine ethyl ester phosphonodiamidate prodrug of a fosmidomycin surrogate were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. These compounds originate by merging a previously reported successful phosphonate derivatisation with favorable modifications of the hydroxamate moiety. None of the synthesized compounds showed enhanced activity against either P. falciparum or M. tuberculosis in comparison with the parent free hydroxamate analog.     

Design, synthesis and activity as acid ceramidase inhibitors of 2-oxooctanoyl and N-oleoylethanolamine analogues

The synthesis of novel N-acylethanolamines and their use as inhibitors of the aCDase is reported here. The compounds are either 2-oxooctanamides or oleamides of sphingosine analogs featuring a 3-hydroxy-4,5-hexadecenyl tail replaced by ether or thioether moieties. It appears that, within the 2-oxooctanamide family, the C3-OH group of the sphingosine molecule is required for inhibition both in vitro and in cultured cells. Furthermore, although the (E)-4 double bond is not essential for inhibitory activity, the (E) configuration is required, since the analogue with a (Z)-4 unsaturation was not inhibitory. None of the oleamides inhibited the aCDase in vitro. Conversely, with the exception of N-oleoylethanolamine and its analogs with S-decyl and S-hexadecyl substituents, all the synthesized oleamides inhibited the aCDase in cultured cells, although with a relatively low potency. We conclude that novel aCDase inhibitors can evolve from N-acylation of sphingoid bases with electron deficient-acyl groups. In contrast, chemical modification of the N-oleoylsphingosine backbone does not seem to offer an appropriate strategy to obtain aCDase inhibitors.     

Determination of doxazosin and verapamil in human serum by fast LC-MS/MS: application to document non-compliance of patients

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of doxazosin and verapamil in human serum has been developed. Trimipramine-d₃ as an isotopic labelled internal standard was used for quantification. Serum samples were prepared by simple liquid–liquid extraction with mixture of tert butyl methyl ether and ethyl acetate (1:1, v:v). The analytes and internal standard were separated on C18 column using an isocratic elution with 5 mM ammonium formate with 0.02% formic acid and 0.02% formic acid in acetonitrile (55:45, v:v) at a flow rate of 1.1 mL/min. Positive TurboIonSpray mass spectrometry was used with multiple reaction monitoring of the transitions at: m/z 455.3 → 165.2 and 150.2 for verapamil, m/z 452.2 → 344.4 and 247.4 for doxazosin, m/z 298.2 → 103.1 for trimipramine-d₃. Linearity was achieved between 1 and 500 ng/mL (R² ≥ 0.997) for both analytes. An extensive pre-study method validation was carried out in accordance with FDA guidelines. This assay was successfully applied to determine the serum concentrations of doxazosin and verapamil in suspect non-compliance patients.