Discovery of pyrido[3,2-d]pyrimidin-6(5H)-one derivatives as checkpoint kinase 1 (CHK1) inhibitors with potent antitumor efficacy.

Checkpoint kinase 1 (CHK1) plays a crucial role in the DNA damage response pathway, making it an attractive target for cancer therapy. Herein, we present the synthesis, optimization, and evaluation of selective CHK1 inhibitors with a pyrido[3,2-d]pyrimidin-6(5H)-one scaffold. Among them, compound 11 showed single-digit nanomolar potency against CHK1 (IC50: 0.55 nM) with good kinase selectivity. Notably, 11 showed anti-proliferative effect in MV-4-11 cells singly (IC50 = 202 nM) and a synergistic effect in combination with gemcitabine in HT-29 cells (IC50 = 63.53 nM). Furthermore, the combination of 11 and gemcitabine exhibited synergistic effect in the HT-29 xenograft mouse model. Overall, this work provides a strong foundation for the development of selective CHK1 inhibitors and the therapeutic strategy for cancer.

Effect of Task-Oriented Biomechanical Perception-Balance Training on Motor Gait in Stroke Patients With Hemiplegia.

Objective: To investigate the effects of task-oriented biomechanical sensors-balance training on lower limb motor function and gait balance function in stroke patients with hemiplegia. Methods: Researchers divided 106 stroke patients with hemiplegia into observation and control groups. All received essential rehabilitation training treatment. The observation group's rehabilitation consisted of task-oriented biomechanical sensors-balance training. The modified Ashworth Scale score, FuGL-Meyer Motor Function Scale score, and other indicators measured the results of the two groups. Results: The Berg balance scale score and FuGL-Meyer Motor Function Scale score in the observation group were higher than in the control group (P < .05). The modified Ashworth Scale score of the triceps calf muscle in the observation group was significantly lower than that in the control group (P < .05). The observation group's step length, step frequency, maximum angle of hip extension, and knee flexion exceeded those of the control group. In contrast, the maximum angle of knee extension was smaller than those in the control group (P < .05). Conclusion: Basic rehabilitation training combined with task-oriented biomechanical perception-balance training can improve the lower limb motor function of stroke patients with hemiplegia.

Paeoniflorin inhibits hepatocellular carcinoma growth by reducing PD-L1 expression.

Abnormal expression of programmed death-ligand 1 (PD-L1) on cancer cells contributes to immune escape in hepatocellular carcinoma (HCC). Paeoniflorin has been shown to inhibit the growth of HCC; however, whether its inhibitory effect involves reducing PD-L1 expression on HCC cells remains unknown. We investigated the antitumor effects of paeoniflorin and its potential regulatory mechanisms in HCC. The effects of paeoniflorin on tumor growth and tumor immunity were determined in H22-xenografted mice and DEN-induced HCC rats. Small interfering RNA against suppressor of cytokine signaling 3 (SOCS3) was transfected into HepG2 cells to verify the effect of paeoniflorin on the SOCS3/signal transducer and activator of transcription 3 (STAT3)/PD-L1signaling pathway. The levels of SOCS3/STAT3/PD-L1 signaling pathway-related mRNAs and proteins were determined by real time-polymerase chain reaction and western blotting, respectively. Interleukin-2 (IL-2), interferon-γ (IFN-γ), granzyme B (GrB), and perforin 1 (PRF1) levels were detected in an H22 and mouse T cell co-culture system. Paeoniflorin can trigger T cell-mediated anti-tumor immune responses by increasing CD8+ T cell counts in tumor tissues, thereby inhibiting tumor growth. Moreover, paeoniflorin increased IL-2, IFN-γ, GrB, and PRF1 levels in the co-culture system. PD-L1 expression was suppressed by paeoniflorin, and this effect was mediated by the SOCS3/STAT3 signaling pathway. Paeoniflorin might thus act via enhancing SOCS3 to inhibit STAT3/PD-L1 signaling and subsequently restore T cell sensitivity to kill tumor cells. Our findings provide novel insights into the anticancer effects of paeoniflorin.

Discovery and Structural Optimization of Novel Quinolone Derivatives as Potent Irreversible Pan-Fibroblast Growth Factor Receptor Inhibitors for Treating Solid Tumors.

Aberrant activation of fibroblast growth factor receptors (FGFRs) has been identified as an oncogenic driver force for multiple cancer types, making FGFRs a compelling target for anticancer therapy. Because of the renewed interest in irreversible inhibitors, considerable efforts have been made to find irreversible FGFR inhibitors. Herein, we discovered a series of novel quinolone-based covalent pan-FGFR inhibitors by further optimizing the lead compound (lenvatinib) under the guidance of molecular docking. The representative pan-FGFR inhibitor I-5 exhibited significant inhibitory potency against FGFR1-4 with nanomolar activity and effectively suppressed the proliferation of Huh-7 and Hep3B HCC cells. I-5 displayed high selectivity against a panel of 369 kinases at 1 µM. The irreversible binding to target proteins was characterized by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Moreover, I-5 exhibited favorable PK properties in vivo and induced significant TGI in the Huh-7 and NCI-H1581 xenograft mouse models.

Rational design of 4-((6-phenoxypyrimidin-4-yl)amino)-N-(4-(piperazin-1-yl)phenyl)-1H-pyrazole-3-carboxamide (LT-540-717) as orally bioavailable FLT3 inhibitor.

In recent years, fms-like tyrosine kinase 3 (FLT3) was confirmed as an exciting target for treatment of AML. However, resistance to FLT3 inhibitors caused by acquired point mutations in tyrosine kinase domain (TKD) have limited their sustained efficacious. Thus, there remains an unmet need to develop high-efficacy FLT3 inhibitors against both FLT3 internal tandem duplication (ITD) and FLT3 (TKD) mutations. Herein, we describe the discovery of compound LT-540-717 (32), a potent FLT3 inhibitor (IC50: 0.62 nM), starting from FN-1501. Compound 32 exhibited highly inhibitory activity against several acquired FLT3 mutations including FLT3 (ITD, D835V), FLT3 (ITD, F691L), FLT3 (D835Y) and FLT3 (D835V). Additionally, 32 displayed potent antiproliferative activity against FLT3-mutation driven BaF3 and AML cells. Oral administration of 32 (25 mg/kg, QD) significantly prohibited tumor growth (tumor-inhibition rate is 94.18%), and no obvious side effect was observed even when increasing dose to 50 mg/kg (tumor-inhibition rate is 93.98%). Furthermore, 32 showed an acceptable bioavailability (F = 33.3% in rat and 72.7% in beagles), a suitable half-life time (T1/2 = 3.5 h in rat and T1/2 = 11.1 h in beagles), and a satisfactory metabolic stability. In summary, these results show the therapeutic potential of 32 to become a new anti-AML drug, especially for AML harboring dual FLT3 (ITD, TKD) mutations.

Discovery of pyrazolo[3,4-b]pyridine derivatives as novel and potent Mps1 inhibitors for the treatment of cancer.

Monopolar spindle kinase 1 (Mps1) is a key element of the mitotic checkpoint and clinically evaluated as a target in the treatment of aggressive tumors. With this aim, a set of pyrazolo[3,4-b]pyridine-based compounds as new Mps1 inhibitors was investigated through a multidisciplinary approach, based on virtual screening, chemical synthesis and biological evaluation. One of the representative compounds, 31, exhibited strong kinase inhibitory potency against Mps1 with an IC50 value of 2.596 nM and significantly inhibited proliferation of cancer cells, especially MDA-MB-468 and MV4-11 cells. Compound 31 also displayed reasonable kinome selectivity against a panel of 606 wild-type kinases at 1 µM. Moreover, compound 31 exhibited suitable preclinical pharmacokinetic parameters and a promising pharmacodynamic profile. Further, compound 31 showed good antitumor efficacy in MDA-MB-468 xenograft model with no obvious toxicity. Overall, compound 31 was identified as a potential Mps1 inhibitor for cancer therapy and deserve further research.

Rhizoma Paridis saponins suppresses vasculogenic mimicry formation and metastasis in osteosarcoma through regulating miR-520d-3p/MIG-7 axis.

Osteosarcoma (OS) is a highly metastatic bone cancer that usually affects children. Rhizoma Paridis saponins (RPS) have been identified to show a broad-spectrum anti-tumor activity. Our previous study has identified vasculogenic mimicry (VM) as an indicator of poor prognosis for OS. Rhizoma Paridis ethanol extract exhibits potent anti-OS property. However, the anti-metastatic effect of RPS on OS and the detailed mechanisms remain unknown. RPS was characterized by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS) analysis. The anti-OS, anti-metastasis and anti-VM activities of RPS were investigated using in vitro biological assays and a xenograft mouse model. Western blot, qRT-PCR, ELISA, Phalloidin staining and immunohistochemistry assays were conducted to investigate the molecular mechanism of RPS. A total of 34 phytochemicals from RPS were identified by LC/Q-TOF/MS. RPS dose-dependently suppressed the OS cell proliferation, metastasis and VM formation in vitro and in vivo. Mechanically, we found that RPS downregulated migration-inducing gene 7 (MIG-7) expression, resulting in inhibition of the PI3K/MMPs/Ln-5γ2 pathway and cell protrusion formation. Additionally, we confirmed that RPS downregulated MIG-7 by upregulating miR-520d-3p expression. Our results suggests that RPS inhibits the VM formation and metastasis of OS by modulating the miR-520d-3p/MIG-7 signaling axis.

Comparative effects of different enzymatic hydrolysates of konjac glucomannan on gut flora and constipation in rats.

This study aimed to compare the effects of different hydrolysates (named GKOS and MKOS) on constipated rats, which were obtained by degradation from konjac glucomannan by ß-glucanase and ß-mannanase, respectively. GKOS and MKOS were characterized and administered by gavage at 100 mg kg-1 to constipated rats. The variation of the gut flora, content of short-chain fatty acids (SCFAs), defecation function, gastrointestinal motility, and intestinal mucus secretion were determined to evaluate their regulatory effects on constipation. The results revealed the more prominent augmentation of species richness in MKOS than with GKOS. They also possessed diverse modulatory effects on different genera, such as Prevotella and Parabacteroides. Unexpectedly, there was no statistical divergence between GKOS and MKOS in defecation parameters, gastrointestinal transit, serum parameters, and mucous secretion. Overall, MKOS and GKOS exhibited differential regulatory function on gut microbiota in vivo, but with nearly consistent therapeutic effects on constipation.

Target identification and occupancy measurement of necrosis avid agent rhein using bioorthogonal chemistry-enabling probes.

Necrosis is an important biomarker, which only occurs in pathological situations. Tracking of necrosis avid agents is of crucial importance toward understanding their mechanisms. Herein, we developed a modular probe strategy based on bioorthogonal copper-free click chemistry. Structural modification of rhein with transcyclooctene (TCO) led to the identification of rhein-TCO2 as the most active probe with specific necrosis affinity. In a systematic evaluation, the colocalization of rhein-TCO2 in the nucleus (exposed DNA and rRNA) of necrotic cells was observed. This work provides a foundation for the development of target-identified of rhein compounds, and binding to exposed DNA and rRNA may be an important target of rhein compounds in necrotic cells.

Development of Duramycin-Based Molecular Probes for Cell Death Imaging.

Cell death is involved in numerous pathological conditions such as cardiovascular disorders, ischemic stroke and organ transplant rejection, and plays a critical role in the treatment of cancer. Cell death imaging can serve as a noninvasive means to detect the severity of tissue damage, monitor the progression of diseases, and evaluate the effectiveness of treatments, which help to provide prognostic information and guide the formulation of individualized treatment plans. The high abundance of phosphatidylethanolamine (PE), which is predominantly confined to the inner leaflet of the lipid bilayer membrane in healthy mammalian cells, becomes exposed on the cell surface in the early stages of apoptosis or accessible to the extracellular milieu when the cell suffers from necrosis, thus representing an attractive target for cell death imaging. Duramycin is a tetracyclic polypeptide that contains 19 amino acids and can bind to PE with excellent affinity and specificity. Additionally, this peptide has several favorable structural traits including relatively low molecular weight, stability to enzymatic hydrolysis, and ease of conjugation and labeling. All these highlight the potential of duramycin as a candidate ligand for developing PE-specific molecular probes. By far, a couple of duramycin-based molecular probes such as Tc-99 m-, F-18-, or Ga-68-labeled duramycin have been developed to target exposed PE for in vivo noninvasive imaging of cell death in different animal models. In this review article, we describe the state of the art with respect to in vivo imaging of cell death using duramycin-based molecular probes, as validated by immunohistopathology.

Direct Cyclopropanation of Quinolinium Zwitterionic Thiolates via Dearomative Reactions.

A one-pot, two-step protocol for the synthesis of libraries of remarkable functionalized sulfone analogues of 9b,10,10a,10b-tetrahydro-1H-cyclopropa[c][1,4]thiazino[4,3-a]quinolines is described. A class of various functionalized molecular skeletons was obtained by cyclopropanation of quinolinium zwitterionic thiolates. The reaction pathway involves the formation of a [2 + 1] cycloaddition intermediate followed by a [5 + 1] cycloaddition.

Pyrrolo[2,3-b]pyridine-3-one derivatives as novel fibroblast growth factor receptor 4 inhibitors for the treatment of hepatocellular carcinoma.

Aberrant signaling of the FGF/FGFR pathway occurs frequently in cancers and is an oncogenic driver in many solid tumors, especially liver cancer. With the resurgence of interest in irreversible inhibitors, efforts have been directed to the discovery of irreversible FGFR4 inhibitors. Currently, several selective irreversible inhibitors containing pyrrolo[2,3-b]pyridine-3-one and pyrrolo[2,3-d]pyrimidin-2-amine skeletons were designed and synthesized as FGFR4 inhibitors. Among the screened compounds, derivative 25 showed excellent enzymatic inhibitory activity (IC50, 51.6 nM) and antiproliferative potency of 0.1397 µM against Hep3B cell lines. Compound 25 exhibited good in vitro human liver microsomal stability with the half-life of 62.0 min, which was more stable than BLU9931 (46.7 min). But the in vivo pharmacokinetic results showed that the oral bioavailability was only 6.65%, which needs to be improved in the next work. These results showed that compound 25 might be an effective lead compound for further investigation to treat the hepatocellular carcinoma.

Untiring Pursuit for Glucarate-Based Molecular Imaging Probes.

Glucarate, a physiologic end-product of the D-glucuronic acid pathway in mammals, is a six-carbon dicarboxylic acid with a wide range of uses. Glucarate-based molecular imaging probes including [99mTc]glucarate and [18F]glucarate have been developed and demonstrated to have infarct/necrosis-avid and/or tumor-seeking properties, showing potential applications in early detection of myocardial infarction, evaluation of tissue viability, monitoring of therapeutic effectiveness, and noninvasive imaging of certain tumors including drug-resistant ones. The mechanism by which [99mTc]glucarate localizes in acute necrotic tissues has been demonstrated to be largely attributable to its binding to the positively charged histones, which become accessible after the disruption of the cell and nuclear membranes as a result of irreversible damage, while the tumor-seeking mechanism of [99mTc]glucarate has been found to be closely related to glucose transporter 5 expression. Moreover, the recently developed [18F]glucarate provides a new alternative probe for positron emission tomography imaging and may have potential advantages over [99mTc]glucarate. In this review, we present the untiring pursuit for glucarate-based molecular imaging probes as infarct/necrosis-avid agent and/or tumor-seeking agent. Moreover, the limitations and the prospects for future research of glucarate-based molecular probes are also discussed.

Design and Evaluation of Rhein-Based MRI Contrast Agents for Visualization of Tumor Necrosis Induced by Combretastatin A-4 Disodium Phosphate.

PURPOSE: Visualization of tumor necrosis can determine tumor response to therapy. Our previous study showed that the rhein-based magnetic resonance imaging (MRI) contrast agent with alkane linker (GdL2) could clearly image tumor necrosis. However, its water solubility and cell safety needed to be improved. Herein, three rhein-based MRI agents with ether or lysine linkers were designed. PROCEDURES: Three rhein-based MRI agents were synthesized with a tetracarbon ether (GdP1), a hexacarbon ether (GdP2), and a lysine (GdP3) linker, respectively. Their octanol-water partition coefficients (log P) and cytotoxicity were determined. Necrosis avidity of the leading agent was explored on HepG2 cells and ischemia reperfusion-induced liver necrosis (IRLN) rats by MRI. The effect of visualization of tumor necrosis was tested on nude mice with W256 tumor treated by combretastatin-A4 phosphate (CA4P). DNA binding assays were applied to evaluate the possible necrosis-avidity mechanism of the leading agent. RESULTS: The log P of three agents (- 1.66 ± 0.09, - 1.74 ± 0.01, - 1.95 ± 0.01) decreased when compared with GdL2, indicating higher water solubility. GdP1 not only presented lower cytotoxicity and good necrotic affinity in vitro and in vivo, but also can be fast excreted by renal. According to MRI results of tumor, distinct visualization of tumor necrosis can be discernible from 3 to 4.5 h post-injection of GdP1. In DNA-binding assays, the fluorescence quenching constant KSV (1.00 × 104 M-1) and the ultraviolet binding constant Kb (1.11 × 104 M-1) suggested that GdP1 may bind to DNA through intercalation. CONCLUSION: GdP1 may serve as a potential candidate for early evaluation of tumor response to CA4P treatment.

Discovery of necrosis avidity of rhein and its applications in necrosis imaging.

Necrosis-avid agents possess exploitable theragnostic utilities including evaluation of tissue viability, monitoring of therapeutic efficacy as well as diagnosis and treatment of necrosis-related disorders. Rhein (4,5-dihydroxyl-2-carboxylic-9,10-dihydrodiketoanthracene), a naturally occurring monomeric anthraquinone compound extensively found in medicinal herbs, was recently demonstrated to have a newly discovered necrosis-avid trait and to show promising application in necrosis imaging. In this overview, we present the discovering process of rhein as a new necrosis-avid agent as well as its potential imaging applications in visualisation of myocardial necrosis and early evaluation of tumour response to therapy. Moreover, the molecular mechanism exploration of necrosis avidity behind rhein are also presented. The discovery of necrosis avidity with rhein and the development of rhein-based molecular probes may further expand the scope of necrosis-avid compounds and highlight the potential utility of necrosis-avid molecular probes in necrosis imaging.

Imaging Cell Death: Focus on Early Evaluation of Tumor Response to Therapy.

Cell death plays a prominent role in the treatment of cancer, because most anticancer therapies act by the induction of cell death including apoptosis, necrosis, and other pathways of cell death. Imaging cell death helps to identify treatment responders from nonresponders and thus enables patient-tailored therapy, which will increase the likelihood of treatment response and ultimately lead to improved patient survival. By taking advantage of molecular probes that specifically target the biomarkers/biochemical processes of cell death, cell death imaging can be successfully achieved. In recent years, with the increased understanding of the molecular mechanism of cell death, a variety of well-defined biomarkers/biochemical processes of cell death have been identified. By targeting these established cell death biomarkers/biochemical processes, a set of molecular imaging probes have been developed and evaluated for early monitoring treatment response in tumors. In this review, we mainly present the recent advances in identifying useful biomarkers/biochemical processes for both apoptosis and necrosis imaging and in developing molecular imaging probes targeting these biomarkers/biochemical processes, with a focus on their application in early evaluation of tumor response to therapy.

A [3 + 2] cycloaddition/C-arylation of isatin N,N'-cyclic azomethine imine 1,3-dipole with arynes.

A [3 + 2] annulation/C-arylation of isatin N,N'-cyclic azomethine imine 1,3-dipole 1 with in situ generated arynes has been established for the synthesis of 3,3-disubstituted oxindole scaffolds. These highly functionalized scaffolds were assembled in moderate yields (up to 85% yield). The novel spirooxindole scaffolds displayed moderate antitumor activities, which represented promising lead compounds for antitumor drug discovery.

Synthesis and Evaluation of Ga-68-Labeled Rhein for Early Assessment of Treatment-Induced Tumor Necrosis.

PURPOSE: This study aimed to synthesize a necrosis-avid agent using rhein as a precursor and labeled with gallium-68 (Ga-68) for positron emission tomography/computed tomography (PET/CT) imaging, to evaluate response to anticancer treatment in a mouse model. PROCEDURES: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated rhein was radiolabeled with Ga-68 to formulate [68Ga]DOTA-rhein. The in vitro stability of [68Ga]DOTA-rhein was assessed by radio-HPLC. Necrosis avidity was evaluated in a mouse model of muscle necrosis by microPET/CT imaging, biodistribution study, histochemical staining, and autoradiography studies. Murine tumor models with the subcutaneous implantation of S180 cell lines were generated for the evaluation of therapeutic effect. Tumor necrosis was induced by the treatment of combretastatin A4 disodium phosphate (CA4P), and microPET/CT imaging was performed at 1 h post tracer injection. DNA binding studies were conducted to explore the necrosis avidity mechanism of the tracer. RESULTS: [68Ga]DOTA-rhein exhibited a satisfactory yield, a radiochemical purity over 97 %, and a good serum stability. The uptakes of [68Ga]DOTA-rhein in necrotic muscles and tumors were significantly higher than those in normal muscles and tumors (P < 0.05). The results of autoradiography and histochemical staining were consistent with the selective uptake of the radiotracer in necrotic regions. MicroPET/CT images showed a high uptake of the tracer in necrotic muscles and necrotic tumors. DNA binding studies suggested that necrosis avidity correlated with DNA binding to a certain extent. CONCLUSIONS: Our results demonstrated that [68Ga]DOTA-rhein showed a prominent necrosis avidity and could be a useful probe for early assessment of response to anticancer therapy by PET/CT imaging.

Synthesis and Evaluation of Diindole-Based MRI Contrast Agent for In Vivo Visualization of Necrosis.

PURPOSE: Noninvasive imaging of cell necrosis can provide an early evaluation of tumor response to treatments. Here, we aimed to design and synthesize a novel diindole-based magnetic resonance imaging (MRI) contrast agent (Gd-bis-DOTA-diindolylmethane, Gd-DIM) for assessment of tumor response to therapy at an early stage. PROCEDURES: The oil-water partition coefficient (Log P) and relaxivity of Gd-DIM were determined in vitro. Then, its necrosis avidity was examined in necrotic cells in vitro and in rat models with microwave ablation-induced muscle necrosis (MAMN) and ischemia reperfusion-induced liver necrosis (IRLN) by MRI. Visualization of tumor necrosis induced by combretastatin A-4 disodium phosphate (CA4P) was evaluated in rats bearing W256 orthotopic liver tumor by MRI. Finally, DNA binding assay was performed to explore the possible necrosis-avidity mechanism of Gd-DIM. RESULTS: The Log P value and T1 relaxivity of Gd-DIM is - 2.15 ± 0.01 and 6.61 mM-1 s-1, respectively. Gd-DIM showed predominant necrosis avidity in vitro and in vivo. Clear visualization of the tumor necrosis induced by CA4P was achieved at 60 min after administration of Gd-DIM. DNA binding study indicated that the necrosis-avidity mechanism of Gd-DIM may be due to its binding to exposed DNA in necrotic cells. CONCLUSION: Gd-DIM may serve as a promising necrosis-avid MRI contrast agent for early assessment of tumor response to therapy.