Development of a PDEδ‐Targeting PROTACs that Impair Lipid Metabolism

The prenyl‐protein chaperone PDEδ modulates the localization of lipidated proteins in the cell, but current knowledge about its biological function is limited. Small‐molecule inhibitors that target the PDEδ prenyl‐binding site have proven invaluable in the analysis of biological processes mediated by PDEδ, like KRas cellular trafficking. However, allosteric inhibitor release from PDEδ by the Arl2/3 GTPases limits their application. We describe the development of new proteolysis‐targeting chimeras (PROTACs) that efficiently and selectively reduce PDEδ levels in cells through induced proteasomal degradation. Application of the PDEδ PROTACs increased sterol regulatory element binding protein (SREBP)‐mediated gene expression of enzymes involved in lipid metabolism, which was accompanied by elevated levels of cholesterol precursors. This finding for the first time demonstrates that PDEδ function plays a role in the regulation of enzymes of the mevalonate pathway.     

Development of a PDEδ-Targeting PROTACs that Impair Lipid Metabolism

The prenyl-protein chaperone PDEδ modulates the localization of lipidated proteins in the cell, but current knowledge about its biological function is limited. Small-molecule inhibitors that target the PDEδ prenyl-binding site have proven invaluable in the analysis of biological processes mediated by PDEδ, like KRas cellular trafficking. However, allosteric inhibitor release from PDEδ by the Arl2/3 GTPases limits their application. We describe the development of new proteolysis-targeting chimeras (PROTACs) that efficiently and selectively reduce PDEδ levels in cells through induced proteasomal degradation. Application of the PDEδ PROTACs increased sterol regulatory element binding protein (SREBP)-mediated gene expression of enzymes involved in lipid metabolism, which was accompanied by elevated levels of cholesterol precursors. This finding for the first time demonstrates that PDEδ function plays a role in the regulation of enzymes of the mevalonate pathway.     

Inhibitors of the Bifunctional 2‐C‐Methyl‐d‐erythritol 4‐Phosphate Cytidylyl Transferase/2‐C‐Methyl‐d‐erythritol‐2,4‐cyclopyrophosphate Synthase (IspDF) of Helicobacter pylori

A library of over 103 thousand compounds was screened for inhibitors of the IspD domain (2‐C‐methyl‐d ‐erythritol 4‐phosphate cytidylyl transferase domain) of the bifunctional IspDF protein from Helicobacter pylori using a photometric assay. Around 300 compounds showed IC₅₀ values below 100 μm , and three compounds had IC₅₀ values below 1 μm . A few IspD inhibitors could also inhibit the IspF domain (2‐C‐Methyl‐d ‐erythritol‐2,4‐cyclopyrophosphate synthase) of the IspDF protein. The most potent IspD inhibitors were tested as growth inhibitors of H. pylori. Several compounds showed inhibition of bacterial growth with IC₅₀ in the single‐digit μm range. The most potent growth inhibitor had an IC₅₀ value of 3.4 μm . The most potent growth inhibitor without measurable effect on eukaryotic cell viability had an IC₅₀ value of 7.2 μm .     

Synthesis of (E)‐5‐Methoxy‐2‐styryl‐4‐pyrones as Potent Growth‐Inhibitory Agents Against Hepatocellular Carcinoma Cells

The present study a series of (E)‐5‐methoxy‐2‐styryl‐4H‐pyran‐4‐ones 6a , 6b , 6c , 6d , 6e , 6f , 6g , 6h , 6i , 6j was synthesized and evaluated for growth inhibitory inhibition against carcinoma cells. The growth inhibition study of eight carcinoma cell lines was examined and demonstrated that SKHep cells exhibit significant structure‐activity relationship in response to the tested compounds. Among them, 6f showed the most potent activity against SKHep, A549, AGS, and H460 cell lines with GI₅₀ values of 0.17, 8.3, 3.6, 8.0 μM, respectively.     

Synthesis of 4‐Heteroaryl–Quinazoline Derivatives as Potential Anti‐breast Cancer Agents

4‐Heteroaryl or heteroalkyl–quinazoline derivatives were prepared as dual epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor‐2 (VEGFR‐2) inhibitors. The new compounds were tested for their dual enzyme inhibition as well as their cytotoxic activity on MCF7 cell line. The results indicated that almost all the compounds showed moderate dual inhibition of both enzymes. Compound 3 (methyl piperidine‐4‐carboxylate derivative) showed the highest inhibitory activity against both enzymes with IC₅₀ 97.6 and 64.0 µM against EGFR and VEGFR‐2 kinases, respectively. Most of the test compounds showed potent to moderate antitumor activity on MCF7 cell line. Five compounds ( 3 , 9c , 11 , 13 , and 15b ) showed potent cytotoxic activity with IC₅₀ values between 10 and 17 µM .     

Synthesis of optically active af‐(1‐Ethyl‐3‐methylhexahydro‐1,3‐diazin‐5‐yl)‐ and N‐(1‐Ethyl‐5‐methyloctahydro‐1,5‐diazocin‐3‐yl)pyridine‐3‐carboxamides

As part of the structure‐activity relationship of the dopamine D₂ and serotonin 5‐HT₃ receptors antagonist 1, which is a clinical candidate with a broad antiemetic activity, the synthesis and dopamine D₂ and serotonin 5‐HT₃ receptors binding affinity of (R)‐5‐bromo‐N‐(1‐ethyl‐3‐methylhexahydro‐1,3‐diazin‐5‐yl)‐ and (R)‐5‐bromo‐N‐(1‐ethyl‐5‐methyloctahydro‐1,5‐diazocin‐3‐yl)‐2‐methoxy‐6‐methylaminopyridine‐3‐carboxam‐ides ( 2 and 3 ) are described. Treatment of 1‐ethyl‐2‐(p‐toluenesulfonyl)amino‐3‐methylaminopropane dihy‐drochloride ( 4a ) with paraformaldehyde and successive deprotection gave the 5‐aminohexahydro‐1,3‐diazine 6 in excellent yield. 3‐Amino‐1‐ethyl‐5‐methyloctahydro‐1,5‐diazocine ( 15 ) was prepared from 2‐(benzyloxycarbonyl)amino‐3‐[[N‐(tert‐butoxycarbonyl)‐N‐methyl]amino]‐1‐ethylaminopropane ( 9 ) through the intramolecular amidation of (R)‐3‐[N‐[(2‐benzyloxycarbonylamino‐3‐methylamino)propyl]‐N‐ethyl]aminopropionic acid trifluoroacetate ( 12 ), followed by lithium aluminum hydride reduction of the resulting 6‐oxo‐1‐ethyl‐5‐methyloctahydrodiazocine ( 13 ) in 41% yield. Reaction of the amines 6 and 15 with 5‐bromo‐2‐methoxy‐6‐methylaminopyridine‐3‐carboxylic acid furnished the desired 2 and 3 , which showed much less potent affinity for dopamine D₂ receptors than 1 .     

Kinetics and Protein‐Inhibitor Docking Studies of Enantiomers of exo‐2‐Norbornyl‐N‐n‐butylcarbamates as Pseudomonas Lipase Inhibitors to Probe the Enantioselectivity of the Enzyme

The Pseudomonas species lipase inhibition shows enantioselectivity for R‐enantiomer over S‐enantiomer of exo‐2‐norbornyl‐N‐n‐butylcarbamates. R‐, S‐, and racemic‐exo‐2‐norbornyl‐N‐n‐butylcarbamates are all characterized as pseudo substrate inhibitors of the enzyme. Thus, the mechanism for Pseudomonas species lipase‐catalyzed hydrolysis of the inhibitor is formation of the first enzyme‐inhibitor Michaelis complex via nucleophilic attack of the active site serine to the inhibitor (K_i step) then formation of the butylcarbamyl enzyme intermediate from this complex (k₂ step). Comparison of bimolecular rate constants (k_i = k₂ / K_i) of the inhibitors indicates that R‐enantiomer is 1.8 times more potent than S‐enantiomer. Thus, Pseudomonas species lipase shows enantioselectivity of 1.8 for R‐exo‐2‐norbornyl‐N‐n‐butyl‐carbamate over S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate. Protein‐ligand interaction studies on both enantiomers of exo‐2‐norbornyl‐N‐n‐butylcarbamate as inhibitors of Pseudomonas species lipase using AutoDock suggest that R‐enantiomer binds more tightly into the active site of the enzyme than S‐enantiomer. The norbornyl ring of S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate is repulsive to Ser 82 and His 251 of the catalytic triad as well as to Met 16 of the oxyanion hole. These repulsions may create few unfavorable interactions between S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate and the enzyme and make this inhibitor a less potent one.     

6β‐Methyl‐B‐norandrostenedione

The title compound, C₁₉H₂₆O₂, a B‐norandrogen with a 6β‐methyl group, is a recently identified and experimentally tested potent new aromatase inhibitor. It shares structural and physicochemical similarities both with the natural substrate of the enzyme, androstenedione, and with exemestane, another potent aromatase inhibitor having a 6‐methylidene group. X‐ray diffraction results indicate that the B‐nor molecule and exemestane have nearly the same oxygen–oxygen and methyl–methyl separations, though they have distinct configurations of the hydrophobic groups at the 6‐position. These structural comparisons allow correlations to be inferred between the active site geometry of the molecules and the aromatase inhibition power of the studied compound.     

Synthesis of 2‐[4‐(Imidazolin‐2‐Ylideneamino)Benzyl]‐Indan‐1‐Ones as Novel Potent Prostacyclin Antagonists

Prostacyclin is involved in many pathological conditions, such as sensitization of inflammation induced pain and isovolumetic distention. Therefore, antagonism of prostacyclin action may be useful in the alleviation of these conditions. In this study, novel potent prostacyclin antagonists, 2‐[4‐(imidazolin‐2‐ylideneamino)benzyl]‐indan‐1‐ones were synthesized from their respective substituted indanones in three steps. The construction of the amino‐imidazole moiety of these derivatives is achieved by using in situ generation of chloro‐imidazole and reaction with their respective anilines. Thus, these N‐substituted 2‐imidazolines can be prepared safely and efficiently. Moreover, these compounds show potent prostacyclin antagonistic activity by inhibition of prostacyclin agonist induced ERK_1/2 phosphorylation in human erythroleukemia cells. Moreover, we observed an increase in activity with the increase in electro‐donating property of the substitution on the indanone aromatic ring. Prostacyclin antagonists with increased potency may be designed based on these findings. These compounds may also be invaluable tools for the study of the physiological functions of prostacyclin.     

Design,synthesis and biological evaluation of 2,4‐diamino‐6‐methyl‐5‐substitutedpyrrolo[2,3‐d]pyrimidines as dihydrofolate reductase inhibitors

Nine novel nonclassical 2,4‐diamino‐6‐methyl‐5‐mioarylsubstituted‐ 7H ‐pyrrolo[2,3‐d]pyrimidines 2‐10 were synthesized as potential inhibitors of dihydrofolate reductase and as antitumor agents. The analogues contain various electron donating and electron withdrawing substituents on the phenylsulfanyl ring of the side chains and were synthesized from the key intermediate 2,6‐diamino‐6‐methyl‐7H‐pyrrolo[2,3‐d]‐pyrimidine, 14 . Compound 14 , was in turn obtained by chlorination of 4‐position of 2‐amino‐6‐methylpyrrolo[2,3‐d]pyrimidin‐4(3H)‐one, 16 followed by displacement with ammonia. Appropriately substituted phenyl thiols were appended to the 5‐position of 14 via an oxidative addition reaction using iodine, ethanol and water. The compounds were evaluated against rat liver, rat‐derived Pneumocystis, Mycobacterium avium and Toxoplasma gondii dihydrofolate reductase. The most potent and selective inhibitor, (2) has a 1‐naphthyl side chain. In this series of compounds electron‐withdrawing and bulky substituents in the side chain afford marginally active dihydrofolate reductase inhibitors. The single atom sulfur bridge in the side chain of these compounds is not conducive to potent dihydrofolate reductase inhibition.     

Synthesis and Cytotoxicity of 6‐Pyrrolidinyl‐2‐(2‐Substituted Phenyl)‐4‐Quinazolinones

In our continuing search for potential anticancer candidates, 2‐(3‐methoxyphenyl)‐6‐pyrrolidinyl‐4‐quinazolinone ( JJC‐1 ) was selected as the lead compound. Starting 5‐pyrrolidinyl‐2‐aminobenzamide was prepared using standard methodology from 5‐chloro‐2‐nitrobenzoic acid by reaction with SOCl₂, NH₃, pyrrolidine, and H₂. The starting benzamide then was reacted with 2‐substituted benzaldehyde or benzoyl chloride in N,N‐dimethylacetamide (DMAC) in the presence of NaHSO₃ at 150 °C. Thermal cyclodehydration/dehydrogenation gave the target 6‐pyrrolidinyl‐2‐(2‐substituted phenyl)‐4‐quinazolinones ( 15–22 ). These target compounds were assayed for their cytotoxicity in vitro against six cancer cell lines, including human monocytic leukemia cells (U937), mouse monocytic leukemia cells (WEHI‐3), human hepatoma cells (HepG2, Hep3B) and human lung carcinoma cells (A549, CH27). Most of them exhibited significant cytotoxic effect toward U937 and WEHI‐3 cells, with EC₅₀ values ranging from 0.30 to 10.10 μM. Compound 19 was investigated further for its action mechanisms. Preliminary findings indicated that compound 19 induced G2/M arrest and apoptosis on U937 cells.     

Synthesis and Microtubule‐Destabilizing Activity of N‐Cyclopropyl‐4‐((3,4‐dihydroquinolin‐1(2H)‐yl)sulfonyl)benzamide and its Analogs

While clinically useful, microtubule‐targeting agents are limited by factors that include their susceptibility to multidrug resistance. A series of aryl sulfonamides, terminally substituted with an amide or carboxylic acid, was synthesized and assayed for biological activity in two human cancer cell lines. The resulting antiproliferative activity data demonstrated that an amide was superior to a carboxylic acid in the para position. The most potent compound ( 3 ) had an IC₅₀ for growth inhibition in the low micromolar range, caused cells to accumulate in G₂M of the cell cycle, and led to depolymerization of microtubules. It was also not susceptible to the P‐glycoprotein drug efflux pump that underpins the resistance of cells to long‐term drug treatment schedules.     

Synthesis and Cytotoxic Activities of α‐Methylidene‐γ‐butyrolactones Bearing a Quinolin‐4(1H)‐one Moiety

The cytotoxicities of the α‐methylidene‐γ‐butyrolactones 4 , 5 , and 8 , which are linked to a quinolin‐4(1H)‐one moiety through a piperazine or O‐atom bridge were studied. These compounds were synthesized by alkylation of 1‐ethyl‐6‐fluoro‐1,4‐dihydro‐7‐hydroxy‐4‐oxoquinoline‐3‐carboxylic acid ( 6 ) followed by a Reformatsky‐type condensation. Compounds 4 , 5 , and 8 were evaluated in vitro against 60 human‐tumor cell lines derived from nine cancer‐cell types and demonstrated not only strong growth‐inhibitory activities against leukemia cancer cells, but also fairly good activities against the growth of certain solid tumors (see Table). The O‐bridged derivatives 8a and 8b exhibit both cytostatic (mean log GI₅₀=−5.20 and −5.82, resp.) and cytocidal (mean log LC₅₀=−4.30 and −4.93, resp.) effects, while the piperazine‐bridged analogues 4 and 5 possess only weak cytostatic (mean log GI₅₀=−5.19 and −4.74, resp.; mean log LC₅₀>−4.00) capability. Among them, 8b is the most potent, with log GI₅₀=−6.47, −6.72, −6.53, and −6.52 against leukemia, SW‐620 (colon), Lox IMV1, and SK‐MEL‐28 (melanoma) cancer cells, respectively.     

Cell‐ and Tissue‐Based Proteome Profiling and Bioimaging with Probes Derived from a Potent AXL Kinase Inhibitor

AXL has been defined as a novel target for cancer therapeutics. However, only a few potent and selective inhibitors targeting AXL are available to date. Recently, our group has developed a lead compound, 9im, capable of displaying potent and specific inhibition of AXL. To further identify the cellular on/off targets, in this study, competitive affinity‐based proteome profiling was carried out, leading to the discovery of several unknown cellular targets such as BCAP31, LPCAT3, POR, TM9SF3, SCCPDH and CANX. In addition, trans‐cyclooctene (TCO) and acedan‐containing probes were developed to image the binding between 9im and its target proteins inside live cells and tumor tissues. These probes would be useful tools in the detection of AXL in various biosystems.     

5，10，15，20-四 [4-(4''-乙基哌嗪基)苯基]卟啉的合成及其与DNA的相互作用

基于卟啉对癌细胞的特殊亲和作用和哌嗪化合物的抗肿瘤、抗病毒作用，设计并合成了具有哌嗪结构的新型卟啉化合物5，10，15，20-四[4-(4'-乙基哌嗪基)苯基]卟啉（TEPPPH₂），其结构经UV-Vis, 元素分析，¹H NMR等手段证明。采用UV-Vis光谱和荧光光谱研究了TEPPPH₂和小牛胸腺DNA 的相互作用模式和结合机理。实验发现，TEPPPH₂能嵌入到DNA的碱基对中，1个小牛胸腺DNA分子对TEPPPH₂分子的最大结合数n约为88，结合常数为8.4×10⁶mol•L^-1 。TEPPPH₂与DNA的结合数和结合常数大于已知的四（4-N-甲基吡啶基）卟啉和Ca/sal-his、Ni/sal–aln型席夫碱抗癌药物。     

Synthesis,Characterization, and Biological Evaluation of Novel 3‐(4‐Chlorophenyl)‐2‐(substituted)quinazolin‐4(3H)‐one Derivatives as Multi‐target Anti‐inflammatory Agents

A novel class of 3‐(4‐chlorophenyl)‐2‐(substituted)quinazolin‐4(3H)‐one derivatives were synthesized, and the structure of synthesized compounds was characterized by IR, ¹H NMR, and mass spectroscopy. The newly synthesized compounds ( 4a–g and 6a–g ) were tested for their in vitro cyclooxygenase (COX) inhibition activity. The compounds have inhibitory profile against both COX‐1 and COX‐2, and some of the compounds are found to be selective against COX‐2. The compound 6g showed distinct inhibitory activity on COXs. The synthesized compounds were evaluated for their potential anti‐inflammatory activity as inhibitors of the proinflammatory cytokines IL‐6. Compounds 4d – g showed the highest level of inhibition among all the tested compounds. Thus, our data suggested that these compounds may represent a new class of potent anti‐inflammatory agents.     

Cell‐based studies of the first‐in‐class half‐sandwich Ir(III) complex containing histone deacetylase inhibitor 4‐phenylbutyrate

We report on a cytotoxic half‐sandwich iridium(III) complex [Ir(η⁵‐Cp^ph)(phen)(PB)]PF₆ ( 1‐PB ), containing a monodentate coordinated O‐donor 4‐phenylbutyrato ligand (PB) belonging to the family of histone deacetylase inhibitors (HDACi); HCp^ph = (2,3,4,5‐tetramethylcyclopenta‐2,4‐dien‐1‐yl)benzene, phen = 1,?10‐phenanthroline. The solution behaviour studies indicated that complex 1‐PB partially hydrolysed in the mixture of methanol and water (1:4, v/v), resulting in the release of the PB ligand. The extent of the PB ligand release increased in the presence of 2 molar equiv. of the reduced glutathione (GSH). Complex 1‐PB exhibited comparable in vitro cytotoxicity against the cisplatin‐sensitive (IC₅₀ = 15.8 μM) and ‐resistant (IC₅₀ = 13.0 μM) variants of the A2780 human ovarian carcinoma cells, while its potency against the MRC‐5 human normal fibroblast cells was markedly lower (IC₅₀ = 124.1 μM). The cytotoxicity studies revealed an ability of complex 1‐PB to overcome the acquired resistance against cisplatin, with the resistance factor (RF = 0.8) being markedly lower than for complex 1‐Cl (RF = 1.8) and cisplatin (RF = 2.9). The A2780 cell‐based flow cytometry experiments showed different cell cycle modification induced by complex 1‐PB and cisplatin, induction of production of reactive oxygen species, and higher mitochondria membrane potential depleted cell populations after the treatment by complex 1‐PB as compared with cisplatin. In the cell‐free assay, complex 1‐PB inhibited the HDAC activity to ca 66% as compared to ca 74% valid for NaPB. The [Ir(η⁵‐Cp^ph)(phen)(H₂O)]²⁺ species ( 1‐OH ₂ ), representing the hydrolysis product of both complexes 1‐PB and 1‐Cl , induced hydroxyl radical from the hydrogen peroxide, as proved by the EPR spin trapping studies with the 5‐(diethoxyphosphoryl)‐5‐methyl‐1‐pyrroline‐N‐oxide (DEPMPO) spin trap.     

Synthesis and structures of photoactive rhenium carbonyl complexes derived from 2‐(pyridin‐2‐yl)‐1,3‐benzothiazole, 2‐(quinolin‐2‐yl)‐1,3‐benzothiazole and 1,10‐phenanthroline

Carbon monoxide (CO) has recently been identified as a gaseous signaling molecule that exerts various salutary effects in mammalian pathophysiology. Photoactive metal carbonyl complexes (photoCORMs) are ideal exogenous candidates for more controllable and site‐specific CO delivery compared to gaseous CO. Along this line, our group has been engaged for the past few years in developing group‐7‐based photoCORMs towards the efficient eradication of various malignant cells. Moreover, several such complexes can be tracked within cancerous cells by virtue of their luminescence. The inherent luminecscent nature of some photoCORMs and the change in emission wavelength upon CO release also provide a covenient means to track the entry of the prodrug and, in some cases, both the entry and CO release from the prodrug. In continuation of the research circumscribing the development of trackable photoCORMs and also to graft such molecules covalently to conventional delivery vehicles, we report herein the synthesis and structures of three rhenium carbonyl complexes, namely, fac‐tricarbonyl[2‐(pyridin‐2‐yl)‐1,3‐benzothiazole‐κ²N ,N ′](4‐vinylpyridine‐κN )rhenium(I) trifluoromethanesulfonate, [Re(C₇H₇N)(C₁₂H₈N₂S)(CO)₃](CF₃SO₃), ( 1 ), fac‐tricarbonyl[2‐(quinolin‐2‐yl)‐1,3‐benzothiazole‐κ²N ,N ′](4‐vinylpyridine‐κN )rhenium(I) trifluoromethanesulfonate, [Re(C₇H₇N)(C₁₆H₁₀N₂S)(CO)₃](CF₃SO₃), ( 2 ), and fac‐tricarbonyl[1,10‐phenanthroline‐κ²N ,N ′](4‐vinylpyridine‐κN )rhenium(I) trifluoromethanesulfonate, [Re(C₇H₇N)(C₁₂H₈N₂)(CO)₃](CF₃SO₃), ( 3 ). In all three complexes, the Re^I center resides in a distorted octahedral coordination environment. These complexes exhibit CO release upon exposure to low‐power UV light. The apparent CO release rates of the complexes have been measured to assess their comparative CO‐donating capacity. The three complexes are highly luminescent and this in turn provides a convenient way to track the entry of the prodrug molecules within biological targets.     

Synthesis and Fungicidal Activity of (E)‐5‐[1‐(2‐Oxo‐1‐oxaspiro[4,5]dec/non‐3‐en‐3‐yl)ethylidene]‐2‐aminoimidazolin‐4‐one Derivatives

The novel fungicidal agents, (E )‐5‐[1‐(2‐oxo‐1‐oxaspiro[4,5]dec/non‐3‐en‐3‐yl)ethylidene]‐2‐aminoimidazolin‐ 4‐one derivatives, were designed and synthesized in moderate to excellent yields in four steps using α ‐hydroxyketone and diketene as raw materials and characterized by HR‐ESI‐MS , ¹H NMR and X‐ray diffraction. The preliminary bioassay showed that some of these compounds, such as 5e , 6a , 6e , and 7 h exhibit 87.8%, 91.3%, 89.9% and 87.8% inhibition rates against Sclerotinia scleotiorum , 3b , 3c , 4c and 7 h exhibit 96.4%, 92.5%, 90.3% and 76.9% inhibition rates against Phytophthora capsici at the concentration of 50 µg/mL , respectively. These compounds exhibited significant fungicidal activities against S. scleotiorum and P. capsici with EC₅₀ values of 2.56–11.60 µg/mL , and compounds 6e and 7 h exhibited weak inhibition against the spore germination of S. scleotiorum , while the spore germination of P. capsici was strongly inhibited by compound 7 h solution. Scanning electron microscopy (SEM ) and transmission electron microscopy (TEM ) observation indicated that compound 7 h had a significant impact on the structure and function of the hyphal cell wall of P. capsici mycelium.     

Screening and identification of Caulis Sinomenii bioactive ingredients with dual‐target NF‐κB inhibition and β2‐AR agonizing activities

Caulis Sinomenii (CS) is a valuable traditional medicine in China. Its extract can act as an anti‐inflammatory agent and a vascular smooth muscle relaxant. However, the underlying mechanisms remain unknown. In this study, we developed a simple dual‐target method based on ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry combined with a dual‐target bioactive screening assay for anti‐inflammatory and antispasmodic activities to characterize the chemical structure of various bioactive compounds of CS rapidly. Seven potential NF‐κB inhibitors were identified, including laudanosoline‐1‐O‐xylopyranose, 6‐O‐methyl‐laudanosoline‐1‐O‐glucopyranoside, menisperine, sinomenine, laurifoline, magnoflorine and norsinoacutin. Furthermore, IL‐6 and IL‐8 assays confirmed the anti‐inflammatory effects of these potential NF‐κB inhibitors, in which laudanosoline‐1‐O‐d ‐xylopyranose and menisperine were revealed as novel NF‐κB inhibitors. Among the seven identified alkaloids, three potential β₂‐adrenergic receptor agonists, including sinomenine, magnoflorine and laurifoline, were characterized using a luciferase reporter system to measure for the activity of β₂‐adrenergic receptor agonists. Finally, sinomenine, magnoflorine and laurifoline were identified not only as potential NF‐κB inhibitors but also as potential β₂‐adrenegic receptor agonists, which is the first time this has been reported. Molecular dynamic simulation and docking results suggest that the three dual‐bioactive constituents could not only inhibit Pseudomonas aeruginosa PAK strain‐induced inflammatory responses via a negative regulation of the Braf protein that participates in MAPK signaling pathway but also activate the β₂‐adrenegic receptor. These results suggest that CS extract has dual signaling activities with potential clinical application as a novel drug for asthma.