A facile and efficient methanesulfonic acid-catalyzed, solvent-free, microwave-assisted method was developed for the synthesis of biologically active 3,3-bis(hydroxyaryl)oxindoles and spirooxindoles bearing a xanthene moiety. The scope of the procedure was investigated with a wide range of isatin and phenol derivatives; moreover, the reaction mechanism was studied by density functional theory calculations. Both 3,3-bis(hydroxyaryl)oxindoles and spirooxindoles bearing a xanthene moiety synthesized were evaluated for their anticancer and antimicrobial activity, and most of them showed promising or significant activity on six cancer cell lines and against Gram-positive bacteria.

A fast, mild, and efficient catalyst-free approach has been developed for the synthesis of chromonyl-substituted alpha-aminophosphine oxides by the three-component reaction of 3-formyl-6-methylchromone, primary amines, and secondary phosphine oxides at ambient temperature. Carrying out the reaction with aliphatic amines or aminoalcohols at a higher temperature (80 degrees C), phosphinoyl-functionalized 3-aminomethylene chromanones were formed instead of the corresponding chromonyl-substituted alpha-aminophosphine oxides. No reaction occurred when 3-formyl-6-methylchromone and secondary phosphine oxides were reacted with aromatic amines in the absence of any catalyst. Applying a basic catalyst, the formation of the phosphinoyl-functionalized 3-aminomethylene chromanones was observed; however, the reaction was not complete. Detailed experimental and quantum chemical studies were performed to study the transformation. Moreover, the in vitro cytotoxicity of phosphinoyl-functionalized 3-aminomethylene chromanones was also investigated in three different cell lines, such as human lung adenocarcinoma (A549), mouse fibroblast (NIH/3T3), and human promyelocytic leukemia (HL60) cells. Several derivatives showed modest activity against the human promyelocytic leukemia (HL60) cell line.

A fast, mild, and efficient catalyst-free approach has been developed for the synthesis of chromonyl-substituted α-aminophosphine oxides by the three-component reaction of 3-formyl-6-methylchromone, primary amines, and secondary phosphine oxides at ambient temperature. Carrying out the reaction with aliphatic amines or aminoalcohols at a higher temperature (80 °C), phosphinoyl-functionalized 3-aminomethylene chromanones were formed instead of the corresponding chromonyl-substituted α-aminophosphine oxides. No reaction occurred when 3-formyl-6-methylchromone and secondary phosphine oxides were reacted with aromatic amines in the absence of any catalyst. Applying a basic catalyst, the formation of the phosphinoyl-functionalized 3-aminomethylene chromanones was observed; however, the reaction was not complete. Detailed experimental and quantum chemical studies were performed to study the transformation. Moreover, the in vitro cytotoxicity of phosphinoyl-functionalized 3-aminomethylene chromanones was also investigated in three different cell lines, such as human lung adenocarcinoma (A549), mouse fibroblast (NIH/3T3), and human promyelocytic leukemia (HL60) cells. Several derivatives showed modest activity against the human promyelocytic leukemia (HL60) cell line.

Background:Vaccination has proven the potential to control the COVID-19 pandemic worldwide. Although recent evidence suggests a poor humoral response against SARS-CoV-2 in vaccinated hematological disease (HD) patients, data on vaccination in these patients is limited with the comparison of mRNA-based, vector-based or inactivated virus-based vaccines.Methods:Forty-nine HD patients and 46 healthy controls (HCs) were enrolled who received two-doses complete vaccination with BNT162b2, or AZD1222, or BBIBP-CorV, respectively. The antibodies reactive to the receptor binding domain of spike protein of SARS-CoV-2 were assayed by Siemens ADVIA Centaur assay. The reactive cellular immunity was assayed by flow cytometry. The PBMCs were reactivated with SARS-CoV-2 antigens and the production of activation-induced markers (TNF-α, IFN-γ, CD40L) was measured in CD4+ or CD8+ T-cells ex vivo.Results:The anti-RBD IgG level was the highest upon BNT162b2 vaccination in HDs (1264 BAU/mL) vs. HCs (1325 BAU/mL) among the studied groups. The BBIBP-CorV vaccination in HDs (339.8 BAU/mL ***p < 0.001) and AZD1222 in HDs (669.9 BAU/mL *p < 0.05) resulted in weaker antibody response vs. BNT162b2 in HCs. The response rate of IgG production of HC vs. HD patients above the diagnostic cut-off value was 100% vs. 72% for the mRNA-based BNT162b2 vaccine; 93% vs. 56% for the vector-based AZD1222, or 69% vs. 33% for the inactivated vaccine BBIBP-CorV, respectively. Cases that underwent the anti-CD20 therapy resulted in significantly weaker (**p < 0.01) anti-RBD IgG level (302 BAU/mL) than without CD20 blocking in the HD group (928 BAU/mL). The response rates of CD4+ TNF-α+, CD4+ IFN-γ+, or CD4+ CD40L+ cases were lower in HDs vs. HCs in all vaccine groups. However, the BBIBP-CorV vaccine resulted the highest CD4+ TNF-α and CD4+ IFN-γ+ T-cell mediated immunity in the HD group.Conclusion:We have demonstrated a significant weaker overall response to vaccines in the immunologically impaired HD population vs. HCs regardless of vaccine type. Although, the humoral immune activity against SARS-CoV-2 can be highly evoked by mRNA-based BNT162b2 vaccination compared to vector-based AZD1222 vaccine, or inactivated virus vaccine BBIBP-CorV, whereas the CD4+ T-cell mediated cellular activity was highest in HDs vaccinated with BBIBP-CorV.

Here, we describe the synthesis and biologic activity evaluation of 20 novel synthetic marine sponge alkaloid analogues with 2-amino-1H-imidazol (2-AI) core. Cytotoxicity was tested on murine 4T1 breast cancer, A549 human lung cancer, and HL-60 human myeloid leukemia cells by the resazurin assay. A total of 18 of 20 compounds showed cytotoxic effect on the cancer cell lines with different potential. Viability of healthy human fibroblasts and peripheral blood mononuclear cells upon treatment was less hampered compared to cancer cell lines supporting tumor cell specific cytotoxicity of our compounds. The most cytotoxic compounds resulted the following IC50 values 28: 2.91 mu M on HL-60 cells, and 29: 3.1 mu M on 4T1 cells. The A549 cells were less sensitive to the treatments with IC50 15 mu M for both 28 and 29. Flow cytometry demonstrated the apoptotic effect of the most active seven compounds inducing phosphatidylserine exposure and sub-G1 fragmentation of nuclear DNA. Cell cycle arrest was also observed. Four compounds caused depolarization of the mitochondrial membrane potential as an early event of apoptosis. Two lead compounds inhibited tumor growth in vivo in the 4T1 triple negative breast cancer and A549 human lung adenocarcinoma xenograft models. Novel marine sponge alkaloid analogues are demonstrated as potential anticancer agents for further development.

An efficient synthetic method for potentially biologically active new arylphosphinoyl-functionalized dihydroisoquinoline derivatives was developed by the Reissert-type reaction of isoquinoline, dialkyl acetylenedicarboxylates and secondary phosphine oxides or ethyl phenyl-H-phosphinate. The model reaction of isoquinoline, diethyl acetylenedicarboxylate and diphenylphosphine oxide was optimized in respect of the solvent, the temperature and the reaction time, as well as the dosing order of the starting materials. The protocol developed enabled the preparation of the target derivatives under mild conditions for a short reaction time. The in vitro cytotoxicity on different cell lines and the antibacterial effect of the phosphorylated dihydroisoquinolines synthesized were also investigated. Based on the IC50 values determined, the derivatives, which contain 3,5-dimethylphenyl or 2-naphtyl groups on the phosphorus atom, showed a promising activity against human promyelocytic leukemia (HL-60) cells and against Bacillus Subtilis Gram-positive bacteria. (C) 2022 The Authors. Published by Elsevier Ltd.

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging-related Alzheimer's disease (AD)-like pathology. However, concerns over adverse effects have slowed the transition of common CN-inhibiting drugs to the clinic for the treatment of AD and AD-related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN-mediated dephosphorylation of a non-NFAT target, either in vivo, or in vitro. Acute (≤1 week) oral delivery of Q134R to APP/PS1 (12 months old) or wild-type mice (3-4 months old) infused with oligomeric Aβ peptides led to improved Y maze performance. Chronic (≥3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild-type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal Aβ plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging-related disorders.

A new approach for the preparation of (2-amino-3-cyano-4H-chromen-4-yl)phosphonate derivatives is described. The multicomponent reaction of salicylaldehydes, malononitrile and dialkyl phosphites catalyzed by pentamethyldiethylenetriamine (PMDTA) provided the bicyclic derivatives in high yields. The method developed did not require chromatographic separation, since the products could be recovered from the reaction mixture by simple filtration. Our approach made also possible condensation with secondary phosphine oxides, and this reaction has not been previously reported in the literature. The crystal structures of five derivatives were studied by single-crystal XRD analysis. The in vitro cytotoxicity on different cell lines and the antibacterial activity of the (2-amino-4H-chromen-4-yl)phosphonates synthesized were also explored. According to the IC50 values determined, several derivatives showed moderate or promising activity against mouse fibroblast (NIH/3T3) and human promyelocytic leukemia (HL-60) cells. Furthermore, three (2-amino-3-cyano-4H-chromen-4-yl)phosphine oxides were active against selected Gram-positive bacteria.

A new method for the synthesis of 3-oxoisoindolin-1-ylphosphine oxides bearing same or different substituents on the phosphorus atom is described. The one-pot three-component reaction of 2-formylbenzoic acid, primary amines and achiral or P-stereogenic secondary phosphine oxides provided the target compounds under catalyst-free, mild conditions and for short reaction times. The deoxygenation of a 3-oxoisoindolin-1-ylphosphine oxide was also studied, and the phosphine obtained could be converted to a sulphide and to a platinum complex. The crystal structures of a selected phosphine oxide and the corresponding platinum species were investigated by X-ray diffraction analysis. The biological activity, such as in vitro cytotoxicity on different cell lines and antibacterial activity of the 3-oxoisoindolin-1-ylphosphine oxides was also investigated. Based on the IC50 values obtained, several derivatives showed moderate activity against the HL-60 cell line and two compounds containing 3,5-dimethylphenyl groups on the phosphorus atom showed promising activity against Bacillus subtilis bacteria.

The preparation and cytotoxic characterization of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-based beta-aminonitriles, beta-amino carboxamides, and their (thio)urea and annulated derivatives were accomplished. Following a synthetic route involving Gewald three-component reactions (G-3CR) and a Lewis acid-catalyzed iso (thio)cyanate coupling, 30 compounds were prepared for antitumor evaluation. For derivatizations, a catalytic amount of CuOAc2 (20 mol%) was essential for improving the reactivity of either the C-2 amino function of thiophene or isocyanates. The synthesized analogues demonstrated a weak to moderate antitumor activity in a low micromolar range against A549 and K562 cancer cell lines.

Novel 1,2,3-triazol-5-yl-phosphonates were prepared by the copper(I)-catalyzed domino reaction of phenylacetylene, organic azides and dialkyl phosphites. The process was optimized on the synthesis of the dibutyl (1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phosphonate in respect of the catalyst, the base and the solvent, as well as of the reaction parameters (molar ratio of the starting materials, atmosphere, temperature and reaction time). The method elaborated could be applied to a range of organic azides and dialkyl phosphites, which confirmed the large scope and the functional group tolerance. The in vitro cytotoxicity on different cell lines and the antibacterial activity of the synthesized 1,2,3-triazol-5-yl-phosphonates was explored. According to the IC50 values determined, only modest antibacterial effect was detected, while some derivatives showed moderate activity against human promyelocytic leukemia HL-60 cells.

The treatment of metastatic breast cancer remained a challenge despite the recent breakthrough in the immunotherapy regimens. Here, we addressed the multidimensional immunophenotyping of 4T1 metastatic breast cancer by the state-of-the-art single cell mass cytometry (CyTOF). We determined the dose and time dependent cytotoxicity of cisplatin on 4T1 cells by the xCelligence real-time electronic sensing assay. Cisplatin treatment reduced tumor growth, number of lung metastasis, and the splenomegaly of 4T1 tumor bearing mice. We showed that cisplatin inhibited the tumor stroma formation, the polarization of carcinoma-associated fibroblasts by the diminished proteolytic activity of fibroblast activating protein. The CyTOF analysis revealed the emergence of CD11b+/Gr-1+/CD44+ or CD11b+/Gr-1+/IL-17A+ myeloid-derived suppressor cells (MDSCs) and the absence of B220+ or CD62L+ B-cells, the CD62L+/CD4+ and CD62L+/CD8+ T-cells in the spleen of advanced cancer. We could show the immunomodulatory effect of cisplatin via the suppression of splenic MDSCs and via the promotion of peripheral IFN-γ+ myeloid cells. Our data could support the use of low dose chemotherapy with cisplatin as an immunomodulatory agent for metastatic triple negative breast cancer.

AbstractBackgroundHyperactivity of the protein phosphatase calcineurin (CN) in brain directly corresponds to key neuropathological and clinical features of Alzheimer’s disease (AD). While inhibition of CN ameliorates pathophysiologic and cognitive changes in mice with AD‐like pathology, concerns over adverse effects have slowed the transition of common CN‐inhibiting drugs to the clinic for treatment of AD and AD‐related disorders. Targeting substrates of CN, like the nuclear factor of activated T‐cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that modulates NFAT signaling, without inhibiting CN activity.Method Cell culture studies — Rat cortical astrocyte cultures were treated with a variety of pathogenic factors in the presence of Q134R or the CN inhibitor, cyclosporine. NFAT‐dependent luciferase activity and dephosphorylation of non‐NFAT substrates were assessed. Intact mouse studies — Q134R was delivered (via oral gavage) to APP/PS1 mice at different ages, or to wild type (WT) mice infused with oligomeric amyloid‐beta peptides. Cognition was assessed on a Y maze along with several postmortem measures including NFAT4 activity, glial activation (GFAP and Iba‐1 expression), amyloid deposition, long‐term survival, and synaptic function and plasticity.ResultIn primary astrocytes, Q134R significantly inhibited NFAT activation in a concentration‐dependent manner, but did not inhibit the CN‐dependent dephosphorylation of non‐NFAT substrates in vivo or in vitro. Short‐term treatment of APP/PS1 mice with Q134R inhibited the nuclear translocation of NFAT4 in hippocampal astrocytes while causing a significant drop in GFAP volume, a mild reduction in Iba1, and no change in amyloid pathology. Short‐term treatment with Q134R also improved cognitive function in APP/PS1 mice and amyloid‐infused WT mice. Long‐term oral delivery of Q134R over many months promoted survival in WT mice and improved synaptic function and plasticity in APP/PS1 mice.ConclusionQ134R has novel CN‐independent, NFAT modulatory properties and improves key neurologic function parameters in mouse models of AD‐like pathology. The results suggest that Q134R is a promising drug for treating AD and AD‐related disorders.

We developed and implemented a reconstituted system to screen for modulators of the ubiquitination of proliferating cell nuclear antigen, a process that activates pathways of DNA damage tolerance and drug resistance. We identified the primary putatively health-beneficial green tea polyphenol epigallocatechin gallate (EGCG) and certain related small molecules as potent inhibitors of ubiquitination. EGCG directly and reversibly targets the ubiquitin-activating enzyme Uba1, blocking formation of the Uba1~ubiquitin thioester conjugate and thus ubiquitination and in the cell. Structure–activity relationship profiles across multiple biochemical and cellular assays for a battery of EGCG analogues revealed distinct chemical and mechanism-of-action clusters of molecules, with catechin gallates, alkyl gallates, and myricetin potently inhibiting ubiquitination. This study defines a number of related though distinct first-in-class inhibitors of ubiquitination, each series with its own unique activity pattern and mechanistic signature.

The incidence of inflammatory bowel disease (IBD) increases gradually in Western countries with high need for novel therapeutic interventions. Mannich curcuminoids, C142 or C150 synthetized in our laboratory, have been tested for anti-inflammatory activity in a rat model of TNBS (2,4,6-trinitrobenzenesulphonic acid) induced colitis. Treatment with C142 or C150 reduced leukocyte infiltration to the submucosa and muscular propria of the inflamed gut. C142 or C150 rescued the loss of body weight and C150 decreased the weight of standard colon preparations proportional with 20% less tissue oedema. Both C142 and C150 curcumin analogues caused 25% decrease in the severity of colonic inflammation and haemorrhagic lesion size. Colonic MPO (myeloperoxidase) enzyme activity as an indicator of intense neutrophil infiltration was 50% decreased either by C142 or C150 Mannich curcuminoids. Lipopolysaccharide (LPS) co-treatment with Mannich curcuminoids inhibited NF-κB (nuclear factor kappa B) activity on a concentration-dependent manner in an NF-κB-driven luciferase expressing reporter cell line. Co-treatment with LPS and curcuminoids, C142 or C150, resulted in NF-κB inhibition with 3.57 μM or 1.6 μM half maximal effective concentration (EC50) values, respectively. C150 exerted a profound inhibition of the expression of inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-4 (IL-4) in human PBMCs (peripheral blood mononuclear cells) upon LPS stimulus. Mannich curcuminoids reported herein possess a powerful anti-inflammatory activity.

Hypoxia is a common feature of neurodegenerative diseases, including Alzheimer's disease that may be responsible for disease pathogenesis and progression. Therefore, the hypoxia-inducible factor (HIF)1 system, responsible for hypoxic adaptation, is a potential therapeutic target to combat these diseases by activators of cytoprotective protein induction. We have selected a candidate molecule from our cytoprotective hydroxyquinoline library and developed a novel enantioselective synthesis for the production of its enantiomers. The use of quinidine or quinine as a catalyst enabled the preparation of enantiomer-pure products. We have utilized in vitro assays to evaluate cytoprotective activity, a fluorescence-activated cell sorting (FACS) based assay measuring mitochondrial membrane potential changes, and gene and protein expression analysis. Our data showed that the enantiomers of Q134 showed potent and similar activity in all tested assays. We have concluded that the enantiomers exert their cytoprotective activity via the HIF1 system through HIF1A protein stabilization.

Leukemia, the malignancy of the hematopoietic system accounts for 10% of cancer cases with poor overall survival rate in adults; therefore, there is a high unmet medical need for the development of novel therapeutics. Eight imidazo[1,2-b]pyrazole-7-carboxamides have been tested for cytotoxic activity against five leukemia cell lines: Acute promyelocytic leukemia (HL-60), acute monocytic leukemia (THP-1), acute T-lymphoblastic leukemia (MOLT-4), biphenotypic B myelomonocytic leukemia (MV-4-11), and erythroleukemia (K-562) cells in vitro. Imidazo[1,2-b]pyrazole-7-carboxamides hampered the viability of all five leukemia cell lines with different potential. Optimization through structure activity relationship resulted in the following IC50 values for the most effective lead compound DU385: 16.54 nM, 27.24 nM, and 32.25 nM on HL-60, MOLT-4, MV-4-11 cells, respectively. Human primary fibroblasts were much less sensitive in the applied concentration range. Both monolayer or spheroid cultures of murine 4T1 and human MCF7 breast cancer cells were less sensitive to treatment with 1.5–10.8 μM IC50 values. Flow cytometry confirmed the absence of necrosis and revealed 60% late apoptotic population for MV-4-11, and 50% early apoptotic population for HL-60. MOLT-4 cells showed only about 30% of total apoptotic population. Toxicogenomic study of DU385 on the most sensitive MV-4-11 cells revealed altered expression of sixteen genes as early (6 h), midterm (12 h), and late response (24 h) genes upon treatment. Changes in ALOX5AP, TXN, and SOD1 expression suggested that DU385 causes oxidative stress, which was confirmed by depletion of cellular glutathione and mitochondrial membrane depolarization induction. Imidazo[1,2-b]pyrazole-7-carboxamides reported herein induced apoptosis in human leukemia cells at nanomolar concentrations.

The 8-hydroxyquinoline pharmacophore scaffold has been shown to possess a range of activities as metal chelation, enzyme inhibition, cytotoxicity, and cytoprotection. Based on our previous findings we set out to optimize the scaffold for cytoprotective activity for its potential application in central nervous system related diseases. A 48-membered Betti-library was constructed by the utilization of formic acid mediated industrial-compatible coupling with sets of aromatic primary amines such as anilines, oxazoles, pyridines, and pyrimidines, with (hetero)aromatic aldehydes and 8-hydroxiquinoline derivatives. After column chromatography and re-crystallization, the corresponding analogues were obtained in yields of 13–90%. The synthesized analogs were optimized with the utilization of a cytoprotection assay with chemically induced oxidative stress, and the most active compounds were further tested in orthogonal assays, a real time cell viability method, a fluorescence-activated cell sorting (FACS)-based assay measuring mitochondrial membrane potential changes, and gene expression analysis. The best candidates showed potent, nanomolar activity in all test systems and support the need for future studies in animal models of central nervous system (CNS) disorders.

The synthesis and in vitro cytotoxic characteristics of new imidazo[1,2‐b]pyrazole‐7‐carboxamides were investigated. Following a hit‐to‐lead optimization exploiting 2D and 3D cultures of MCF‐7 human breast, 4T1 mammary gland, and HL‐60 human promyelocytic leukemia cancer cell lines, a 67‐membered library was constructed and the structure–activity relationship (SAR) was determined. Seven synthesized analogues exhibited sub‐micromolar activities, from which compound 63 exerted the most significant potency with a remarkable HL‐60 sensitivity (IC50 = 0.183 μM).

Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2) and pancreatic cancer cell line (PANC-1). Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G(0)/G(1) cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER) and the up-regulation of ER stress-related unfolded protein response (UPR) genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.