Design, synthesis, and evaluation of formylpiperazine analogs of Ferrostatin-1 as novel improved ferroptosis inhibitors.

In this study, a series of new formylpiperazine-derived ferroptosis inhibitors were designed and synthesized based on the structure of a known ferroptosis inhibitor, ferrostatin-1 (Fer-1). The anti-ferroptosis activity of these synthetic compounds in human umbilical vein endothelial cells (HUVECs) induced by Erastin was evaluated. It was found that some of the new compounds, especially compound 26, showed potent anti-ferroptosis activity, as evidenced by its ability to restore cell viability, reduce iron accumulation, scavenge reactive oxygen species, maintain mitochondrial membrane potential, increase GSH levels, decrease LPO and MDA content, and upregulate GPX4 expression. Moreover, compound 26 exhibited superior microsomal stability than Fer-1. The present results suggest that compound 26 is a promising lead compound for the development of new ferroptosis inhibitors for the treatment of vascular diseases.

Synthesis and biological evaluation of NO-donor containing photosensitizers to induce ferroptosis of cancer cells.

Photodynamic therapy (PDT) is a non-invasive treatment method for tumors by exciting photosensitizers (PS) upon light irradiation to generate cytotoxic reactive oxygen species (ROS). However, the low oxygen concentration near the tumor tissue limits the therapeutic effect of PDT. Herein, we synthesized six chlorin e6 derivatives containing NO-donors to enhance their antitumor activity by synergistic effect of ROS and NO. The results revealed that the new NO-donor containing photosensitizers (PS-NO) exhibited more potent photodynamic activity than chlorin e6, and the introduction of NO donor moieties to chlorin e6 increased the level of NO and ROS in cells. The addition of Ferrostatin-1, a ferroptosis inhibitor, markedly reduced the photodynamic activity of PS-NO as well as the level of NO and ROS in cells. Mechanism studies further showed that PS-NO could reduce intracellular GSH level, inhibit GPX4 activity and promote malondialdehyde (MDA) accumulation upon light irradiation, which suggested the ferroptosis mechanism underlying the PDT effect of PS-NO.

Synthesis and biological study of class I selective HDAC inhibitors with NO releasing activity.

Based on the multi-mechanism antitumor strategy and the regulatory effect of nitric oxide (NO) on histone deacetylases (HDACs), a series of N-acyl-o-phenylenediamine-based HDAC inhibitors equipped with the phenylsulfonylfuroxan module as NO donor was designed, synthesized and biologically evaluated. The in vitro HDAC inhibitory assays revealed that compared with the clinical class I selective HDAC inhibitor MS275, compounds 7c, 7d and 7e possessed similar HDAC inhibitory potency and selective profile, which were confirmed by the results of western blot analysis. The western blot analysis also showed that NO scavenger N-acetyl cysteine (NAC) could weaken the intracellular HDAC inhibitory ability of compound 7c, supporting the HDAC inhibitory effect of NO generated by 7c. It is worth noting that compounds 7c, 7d and 7e exhibited more potent in vitro antiproliferative activities than MS275 against all four tested solid tumor cell lines. The promising in vivo antitumor potency of 7c was demonstrated in a HCT116 xenograft model.

Induced protein degradation of histone deacetylases 3 (HDAC3) by proteolysis targeting chimera (PROTAC).

Histone deacetylases (HDACs) play important roles in inflammatory diseases like asthma and chronic obstructive pulmonary disease (COPD). Unravelling of and interfering with the functions of specific isoenzymes contributing to inflammation provides opportunities for drug development. Here we synthesize proteolysis targeting chimeras (PROTACs) for degradation of class I HDACs in which o-aminoanilide-based class I HDAC inhibitors are tethered to the cereblon ligand pomalidomide. One of these PROTACs, denoted HD-TAC7, showed promising degradation effects for HDAC3 with a DC50 value of 0.32 µM. In contrast to biochemical evidence using siRNA, HD-TAC7 showed a minimal effect on gene expression in LPS/IFNγ-stimulated RAW 264.7 macrophages. The lack of effect can be attributed to downregulation of the NF-κB subunit p65, which is a known side effect of pomalidomide treatment. Altogether, we describe a novel PROTAC that enables selective downregulation of HDAC3 levels, however we note that concomitant downregulation of the NF-κB subunit p65 can confound the biological outcome.

Molecularly imprinted polymer-based sensor for electrochemical detection of erythromycin.

The increasing global reports on the occurrence of macrolide antibiotics resistance, especially erythromycin (Ery) resistant strains, suggests the possible presence of these antibiotics in the environment hence, their inclusion in the EU watchlist of water pollutants. Consequently, there is an urgent need for the development of portable and cost effective analytical sensing devices for their monitoring in water. The combination of molecularly imprinted polymer (MIP) as a sensing element with a portable electrochemical transducer such as screen printed electrode (SPE) may offer a valuable approach for the desired routine environmental monitoring. This work demonstrates the preparation of an electrochemical MIP-based sensor for Ery detection in aqueous media. Ery-selective MIP, Ery-MIP was generated directly on SPE, Ery-MIP/SPE via electrochemical polymerization of m-phenylenediamine (mPD). The optimization of sensor performance was achieved with special attention given to the selection of functional monomer, template removal, polymer thickness and incubation time. Ery-MIP/SPE sensor demonstrated the ability to discriminate target analyte against very close analogues i.e clarithromycin and azithromycin in both PBS and tap water. In addition, Ery-MIP/SPE could detect Ery down to low limits (LOD = 0.1 nM and LOQ = 0.4 nM) and exhibited good recovery in tap water. The presented analytical approach could be potentially suited and/or further developed for adequate monitoring of Ery as well as other macrolides in environmental water.

An activatable near-infrared fluorescent probe for methylglyoxal imaging in Alzheimer's disease mice.

Visual detection of the methylglyoxal (MGO) level in the brain is critical for understanding its role in the onset and progression of AD. Herein, we disclosed a NIR fluorescent probe, DBTPP, for detecting MGO by utilizing a thiadiazole-fused o-phenylenediamine moiety as a MGO-specific sensing unit. DBTPP exhibits a series of distinct advantages, such as NIR emission, high selectivity and sensitivity, excellent acid-stability, and a huge off-on ratio. The probe could accurately monitor both exogenous and endogenous MGO variations in SH-SY5Y cells. Besides, it was able to image the endogenous MGO in a transgenic AD mouse model successfully, suggesting the great potential of MGO as a biomarker for early AD diagnosis.

A New Approach in Cancer Treatment: Discovery of Chlorido[N,N'-disalicylidene-1,2-phenylenediamine]iron(III) Complexes as Ferroptosis Inducers.

Chlorido[N,N'-disalicylidene-1,2-phenylenediamine]iron(III) complexes generate lipid-based ROS and induce ferroptosis in leukemia and neuroblastoma cell lines. The extent of ferroptosis on the mode of action is regulated by simple modifications of the substituents at the 1,2-phenylenediamine moiety. In HL-60 cells, the unsubstituted lead exclusively caused ferroptosis. For instance, a 4-F substituent shifted the mode of action toward both ferroptosis and necroptosis, while the analogously chlorinated derivative exerted only necroptosis. Remarkably, cell-death in NB1 neuroblastoma cells was solely induced by ferroptosis, independent of the used substituents. The effects were higher than that of the therapeutically applied drug cisplatin. These data clearly demonstrate for the first time that not only iron ions but also iron salophene complexes are potent ferroptosis inducers, which can be optimized as antitumor agents.

Development of a Nitric Oxide-Responsive Labeling Reagent for Proteome Analysis of Live Cells.

Nitric oxide (NO) is a pleiotropic signaling molecule involved in the regulation of diverse physiological and pathophysiological mechanisms in cardiovascular, nervous, and immunological systems. To understand the biological functions of NO in detail, comprehensive characterization of proteins found in high-NO concentration environments is crucial. Herein, we describe the design of NO-responsive protein labeling reagents based on N-alkoxyacyl- o-phenylenediamine as an optimal reactive scaffold. The designed molecules can label proteins in murine macrophage cells in response to endogenously produced NO. The combination of NO-responsive protein labeling and liquid chromatography-tandem mass spectrometry technology allowed the characterization of the proteome under NO-generated conditions. Moreover, we demonstrated that our reagent was able to selectively mark and be used to fluorescently visualize NO-producing cells in a mixed cell culture system.

Three-dimensional Salphen-based Covalent-Organic Frameworks as Catalytic Antioxidants.

The development of three-dimensional (3D) functionalized covalent-organic frameworks (COFs) is of critical importance for expanding their potential applications. However, the introduction of functional groups in 3D COFs remains largely unexplored. Herein, we report the first example of 3D Salphen-based COFs (3D-Salphen-COFs) and their metal-containing counterparts (3D-M-Salphen-COFs). These Salphen-based COFs exhibit high crystallinity and specific surface area in addition to excellent chemical stability. Furthermore, the Cu(II)-Salphen COF displays high activity in the removal of superoxide radicals. This study not only presents a new pathway to construct 3D functionalized COFs but also promotes their applications in biology and medicine.

Effects of nitric oxide donor N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine on the expression of interferon-gamma in tumor infiltrating lymphocytes.

Nitric oxide (NO) has been proven to be a key regulator in the mammalian immune response, such as the innate and adaptive immune responses to tumors. The messenger NO involves T helper cell differentiation and lymphocyte biofunctions. In this study, we employed N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine as NO donor and released NO around tumor infiltrating lymphocytes in vitro by short-time blue light irradiation. The interferon-γ secretion of tumor infiltrating lymphocytes was investigated to study the functional changes caused by the accurate spatio-temporal delivery of NO. The downregulation of interferon-γ in tumor infiltrating lymphocytes after NO treatment indicates promising biological applications to potentially play a role in the treatment of autoimmune diseases. The study was approved by the Medical Ethics Committee of the Shenzhen Second People's Hospital, the First Affiliated Hospital of Shenzhen University, China (approved No. 065) on February 12, 2018.

A sensitive and fast responsive fluorescent probe for imaging hypoxic tumors.

Nitroreductase activities are positively associated with the hypoxic level of tumors, making it an attractive target for tumor detection. Herein, we have developed a 2,5-bis(methylsulfinyl)-1,4-diaminobenzene based probe (BBP), which is a nitroreductase (NTR) responsive fluorescent probe and can rapidly detect NTRs with high sensitivity and specificity. The BBP showed not only a selective response to NTRs over other biological reductants, but also high sensitivity to NTRs and could detect as low as 20 ng mL-1 NTRs. Furthermore, the BBP responded rapidly to NTRs in as fast as 10 minutes, enabling real-time monitoring of the production levels of NTRs. Most importantly, the BBP could identify NTR activities in 2D cell monolayers, 3D tumor spheroids, and even solid tumors in mice. Particularly, the BBP could monitor the early tumor formation and treatment response via measuring NTR activities. Overall, the BBP appears to be an ideal imaging probe for the detection of solid tumors, and possesses great potential in a broad range of diagnostic and therapeutic applications in the clinic.

Efficient Two-Photon Fluorescent Probe for Imaging of Nitric Oxide during Endoplasmic Reticulum Stress.

Nitric oxide (NO) is a vital gaseous signal molecule and plays an important role in diverse physiological and pathological processes including regulation of vascular functions. Endoplasmic reticulum (ER) stress is caused by the accumulation of misfolded or unfolded protein in the ER. Besides, ER stress induced by NO can be involved in the pathogenesis of various vascular diseases. Unfortunately, to the best of our knowledge, no ER-targeting probe for NO is reported to study the relationship between ER stress and the level of NO in a biological system. Herein, an ER-targeted fluorescent probe named ER-Nap-NO for imaging of NO is designed and synthesized. ER-Nap-NO consists of three main parts: naphthalimide (two-photon fluorophore), o-phenylenediamino (NO recognition group), and methyl sulfonamide (ER-targetable group). The probe itself is nonfluorescent because a photoinduced electron transfer (PET) process exists. After the addition of NO, the PET process is inhibited and thus strong fluorescence is released. Moreover, the response mechanism is confirmed by 1H NMR and mass spectra and DFT calculation in detail. In addition, from the experimental results, we can conclude that the probe displays several obvious advantages including high sensitivity, selectivity, and ER-targetable ability. Based on these excellent properties, the probe is used for the two-photon imaging of exogenous and endogenous NO in ER of living cells. Most importantly, the ER-targetable probe has potential capability as a tool for investigating the level of NO during tunicamycin-induced ER stress in cells and tissues, which is beneficial for revealing the role of NO in ER-associated vascular diseases.

Synthesis and characterization of a poly(p-phenylenediamine)-based electrospun nanofiber for the micro-solid-phase extraction of organophosphorus pesticides from drinking water and lemon and orange juice samples.

A new micro-solid-phase extraction sorbent was synthesized by electrospinning poly(p-phenylenediamine)/poly(vinyl alcohol) in the presence of cetyltrimethylammonium bromide. The modified nanofiber was prepared by removing the majority of the poly(vinyl alcohol) from the nanofiber blend by exposing it to the hot water. Scanning electron microscopy and surface analysis were performed to study the homogeneity and porosity of the electrospun nanofiber. In addition, Fourier transform infrared spectroscopy was applied for more characterization. The capability of the new nanofiber was explored by applying it in the extraction and preconcentration of organophosphorus pesticides from aqueous medium. After solvent desorption, the extracted analytes were analyzed by high-performance liquid chromatography with diode array detection. Under the optimum conditions, the relative standard deviation values at the concentration level of 50 ng/mL were in the range of 4.8-8.3%. The calibration curve showed linearity in the range of 0.5-500 ng/mL, and the limits of detection (S/N = 3) for the studied compounds were 0.15 ng/mL. By analyzing Tehran drinking water, lemon juice, sour lemon juice, orange juice and sour orange juice, the applicability of the presented method was investigated and the relative recoveries were in the range of 76-102%.

Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins are reversible allosteric inhibitors of PAR1-driven calcium mobilization in endothelial cells.

Several classes of ligands for Protease-Activated Receptors (PARs) have shown impressive anti-inflammatory and cytoprotective activities, including PAR2 antagonists and the PAR1-targeting parmodulins. In order to support medicinal chemistry studies with hundreds of compounds and to perform detailed mode-of-action studies, it became important to develop a reliable PAR assay that is operational with endothelial cells, which mediate the cytoprotective effects of interest. We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of known and new PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay is also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation.

Synthesis and Guest Recognition of Switchable Pt-Salphen Based Molecular Tweezers.

Molecular tweezers are artificial receptors that have an open cavity generated by two recognition units pre-organized by a spacer. Switchable molecular tweezers, using a stimuli-responsive spacer, are particularly appealing as prototypes of the molecular machines that combine mechanical motion and allosteric recognition properties. In this present study, the synthesis of switchable molecular tweezers composed of a central terpyridine unit substituted in 4,4″ positions by two Pt(II)-salphen complexes is reported. The terpyridine ligand can be reversibly converted upon Zn(II) coordination from a free 'U'-shaped closed form to a coordinated 'W' open form. This new substitution pattern enables a reverse control of the mechanical motion compared to the previously reported 6,6″ substituted terpyridine-based tweezers. Guest binding studies with aromatic guests showed an intercalation of coronene in the cavity created by the Pt-salphen moieties in the closed conformation. The formation of 1:1 host-guest complex was investigated by a combination of NMR studies and DFT calculations.

Glycoconjugate Oxime Formation Catalyzed at Neutral pH: Mechanistic Insights and Applications of 1,4-Diaminobenzene as a Superior Catalyst for Complex Carbohydrates.

The reaction of unprotected carbohydrates with aminooxy reagents to provide oximes is a key method for the construction of glycoconjugates. Aniline and derivatives serve as organocatalysts for the formation of oximes from simple aldehydes, and we have previously reported that aniline also catalyzes the formation of oximes from the more complex aldehydes, carbohydrates. Here, we present a comprehensive study of the effect of aniline analogues on the formation of carbohydrate oximes and related glycoconjugates depending on organocatalyst structure, pH, nucleophile, and carbohydrate, covering more than 150 different reaction conditions. The observed superiority of the 1,4-diaminobenzene (PDA) catalyst at neutral pH is rationalized by NMR analyses and DFT studies of reaction intermediates. Carbohydrate oxime formation at pH 7 is demonstrated by the formation of a bioactive glycoconjugate from a labile, decorated octasaccharide originating from exopolysaccharides of the soil bacterium Mesorhizobium loti. This study of glycoconjugate formation includes the first direct comparison of aniline-catalyzed reaction rates and equilibrium constants for different classes of nucleophiles, including primary oxyamines, secondary N-alkyl oxyamines, as well as aryl and arylsulfonyl hydrazides. We identified 1,4-diaminobenzene as a superior catalyst for the construction of oxime-linked glycoconjugates under mild conditions.

Development of a series of novel o-phenylenediamine-based indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

A novel series of o-phenylenediamine-based inhibitors of indoleamine 2,3-dioxygenase (IDO) has been identified. IDO is a heme-containing enzyme, overexpressed in the tumor microenvironment of many cancers, which can contribute to the suppression of the host immune system. Synthetic modifications to a previously described diarylether series resulted in an additional degree of molecular diversity which was exploited to afford compounds that demonstrated significant potency in the HeLa human cervical cancer IDO1 assay. .

Synthesis and anti-diabetic activity of new N,N-dimethylphenylenediamine-derivatized nitrilotriacetic acid vanadyl complexes.

Vanadium compounds are promising anti-diabetic agents. However, reducing the metal toxicity while keeping/improving the hypoglycemic effect is still a big challenge towards the success of anti-diabetic vanadium drugs. To improve the therapeutic potency using the anti-oxidative strategy, we synthesized new N,N-dimethylphenylenediamine (DMPD)-derivatized nitrilotriacetic acid vanadyl complexes ([VO(dmada)]). The in vitro biological evaluations revealed that the DMPD-derivatized complexes showed improved antioxidant capacity and lowered cytotoxicity on HK-2 cells than bis(maltolato)oxidovanadium (IV) (BMOV). In type II diabetic mice, [VO(p-dmada)] (0.15mmolkg-1/day) exhibited better hypoglycemic effects than BMOV especially on improving glucose tolerance and alleviating the hyperglycemia-induced liver damage. These insulin enhancement effects were associated with increased expression of peroxisome proliferator-activated receptor α and γ (PPARα/γ) in fat, activation of Akt (v-Akt murine thymoma viral oncogene)/PKB (protein kinase-B) in fat and liver, and inactivation of c-Jun NH2-terminal protein kinases (JNK) in liver. Moreover, [VO(p-dmada)] showed no tissue toxicity at the therapeutic dose in diabetic mice and the oral acute toxicity (LD50) was determined to be 1640mgkg-1. Overall, the experimental results indicated that [VO(p-dmada)] can be a potent insulin enhancement agent with improved efficacy-over- toxicity index for further drug development. In addition, the results on brain Tau phosphorylation suggested necessary investigation on the effects of vanadyl complexes on the pathology of the Alzheimer's disease in the future.

Synthesis, characterization and antimicrobial study of polymeric transition metal complexes of Mn(II), Co(II), Ni(II), Cu(II) and Zn(II).

Salen ligands comprising of o-phenylenediamine (salop) and p-phenylenediamine (salpp) have been synthesized. The salen ligand, salop undergo Schiff base reaction with Formaldehyde and Barbituric acid to generate novel polymeric Schiff base, SBOPA in one instance while the second salen ligand, salpp on Schiff base reaction with formaldehyde and piperazine gives another novel polymeric Schiff base, SBPBA. These polymeric Schiff base ligands, SBOPA and SBPBA generates polymeric metal complexes in high yields on reaction with transition metal acetates, M(CH3COO)2.xH2O where M = Mn(II), Co(II), Ni(II), Cu(II) and Zn(II). The polymeric Schiff bases, SBOPA and SBPBA and their transition metal complexes were systematically characterized, using various spectroscopic techniques. The structure, composition and geometry of SBOPA and SBPBA and their metal complexes were confirmed by spectral techniques (FT-IR, and 1H NMR), elemental analysis, and electronic spectra magnetic moment. On the basis of FT-IR, 1HNMR, electronic spectra and magnetic moment values Mn(II), Co(II) and Ni(II) ion were found to have octahedral geometry while Cu(II) and Zn(II) were found to be square-planar in nature. Thermogravimetric analysis (TGA) was used to evaluate their thermal behaviour and Cu(II)-SBOPA and Cu(II)-SBPBA were found to be thermally most stable. The polymeric Schiff base ligands, SBOPA and SBPBA and their metal complexes have also been screened for their plausible antimicrobial activity. Tetracyclin and Miconazole were used as standard drug to study the antibacterial and antifungal activity respectively. The Cu(II)-SBOPA and Cu(II)-SBPBA were found to be most potent antimicrobial agents.

Synthesis and evaluation of osimertinib derivatives as potent EGFR inhibitors.

Osimertinib has been identified as a promising therapeutic drug targeting for EGFR T790M mutant non-small cell lung cancer (NSCLC). A new series of N-oxidized and fluorinated osimertinib derivatives were designed and synthesized. The cellular anti-proliferative activity, kinase inhibitory activity and the activation of EGFR signaling pathways of 1-6 in vitro were determined against L858R/T790M and wild-type EGFR, the antitumor efficacy in NCI-H1975 xenografts in vivo were further studied. Compound 2, the newly synthesized N-oxide metabolite in N,N,N'-trimethylethylenediamine side chain of osimertinib, showed a comparable kinase selectivity in vitro and a slightly better antitumor efficacy in vivo to osimertinib, making it valuable and suitable for the potential lung cancer therapy.