Selective but not pan-CDK inhibition abrogates 5-FU-driven tissue factor upregulation in colon cancer.

Thromboembolic events are complications in cancer patients and hypercoagulability has been linked to the tissue factor (TF) pathway, making this an attractive target. Here, we investigated the effects of chemotherapeutics and CDK inhibitors (CDKI) abemaciclib/palbociclib (CDK4/6), THZ-1 (CDK7/12/13), and dinaciclib (CDK1/2/5/9) alone and in combination regimens on TF abundance and coagulation. The human colorectal cancer (CRC) cell line HROC173 was treated with 5-FU or gemcitabine to stimulate TF expression. TF+ cells were sorted, recultured, and re-analyzed. The effect of treatment alone or in combination was assessed by functional assays. Low-dose chemotherapy induced a hypercoagulable state and significantly upregulated TF, even after reculture without treatment. Cells exhibited characteristics of epithelial-mesenchymal transition, including high expression of vimentin and mucin. Dinaciclib and THZ-1 also upregulated TF, while abemaciclib and palbociclib downregulated it. Similar results were observed in coagulation assays. The same anticoagulant activity of abemaciclib was seen after incubation with peripheral immune cells from healthy donors and CRC patients. Abemaciclib reversed 5-FU-induced TF upregulation and prolonged clotting times in second-line treatment. Effects were independent of cytotoxicity, senescence, and p27kip1 induction. TF-antibody blocking experiments confirmed the importance of TF in plasma coagulation, with Factor XII playing a minor role. Short-term abemaciclib counteracts 5-FU-induced hypercoagulation and eventually even prevents thromboembolic events.

Design, synthesis, and biological evaluation of dinaciclib and CAN508 hybrids as CDK inhibitors.

The scaffolds of two known CDK inhibitors (CAN508 and dinaciclib) were the starting point for synthesizing two series of pyarazolo[1,5-a]pyrimidines to obtain potent inhibitors with proper selectivity. The study presented four promising compounds; 10d, 10e, 16a, and 16c based on cytotoxic studies. Compound 16a revealed superior activity in the preliminary anticancer screening with GI % = 79.02-99.13 against 15 cancer cell lines at 10 µM from NCI full panel 60 cancer cell lines and was then selected for further investigation. Furthermore, the four compounds revealed good safety profile toward the normal cell lines WI-38. These four compounds were subjected to CDK inhibitory activity against four different isoforms. All of them showed potent inhibition against CDK5/P25 and CDK9/CYCLINT. Compound 10d revealed the best activity against CDK5/P25 (IC50 = 0.063 µM) with proper selectivity index against CDK1 and CDK2. Compound 16c exhibited the highest inhibitory activity against CDK9/CYCLINT (IC50 = 0.074 µM) with good selectivity index against other isoforms. Finally, docking simulations were performed for compounds 10e and 16c accompanied by molecular dynamic simulations to understand their behavior in the active site of the two CDKs with respect to both CAN508 and dinaciclib.

Design and Synthesis of 2-Phenylindolizine Acetamides: Molecular Docking, in Vitro Antimicrobial and Anticancer Activity Evaluation.

In the present work, we synthesized a small library of 2-phenylindolizine acetamide derivatives 7a-i and studied their biological activity. The synthesis was accomplished starting with easily available starting material phenacyl bromide 1 proceeding through the key intermediate 6-methyl-7-nitro-2-phenylindolizine 4. All the compounds 7a-i were characterized using spectroscopy viz., 1H-NMR, 13C NMR, FTIR, and mass spectrometry. Interestingly, 2-phenylindolizine scaffolds 7c, 7f and 7g revealed a remarkable antibacterial activity against relevant organisms S. aureus, E. coli, S. pneumoniae, P. aeruginosa. The target compounds 7e and 7h showed excellent anticancer activity against Colo-205 and MDA-MB-231 cell lines with IC50 values of 68.62, 62.91, 54.23 and 46.34 µM respectively. Additionally, all the 2-phenylindolizine acetamide derivatives 7a-i were subjected to molecular docking prediction by Autodock 4.2. Compounds 7a, 7f and 7c exhibited very good hydrogen bonding amino acid interactions Asp83 (2.23 Å), Asp83 (2.08 Å), His74 (2.05 Å), His76 (1.71 Å), Ser80 (1.05 Å) with active site of Topoisomerase-IV from S. pneumoniae (4KPE). Further, the compounds 7a-i have revealed acceptable ranges for drug-likeliness properties upon evaluation using SwissADME for ADMET and physiochemical properties.

Exploring novel HIV-1 reverse transcriptase inhibitors with drug-resistant mutants: A double mutant surprise.

HIV-1 reverse transcriptase (RT) remains a key target for HIV drug development. As successful management of the disease requires lifelong treatment, the emergence of resistance mutations is inevitable, making development of new RT inhibitors, which remain effective against resistant variants crucial. To this end, previous computationally guided drug design efforts have resulted in catechol diether compounds, which inhibit wildtype RT with picomolar affinities and appear to be promising preclinical candidates. To confirm that these compounds remain potent against Y181C, a widespread mutation conferring resistance to first generation inhibitors, they were screened against the HIV-1 N119 clinical isolate, reported as a Y181C single mutant. In comparison to a molecular clone with the same mutation, N119 appears less susceptible to inhibition by our preclinical candidate compounds. A more detailed sequencing effort determined that N119 was misidentified and carries V106A in combination with Y181C. While both indolizine and naphthalene substituted catechol diethers are potent against the classical Y181C single mutant, the addition of V106A confers more resistance against the indolizine derivatives than the naphthalene derivatives. Crystal structures presented in this study highlight key features of the naphthyl group, which allow these compounds to remain potent in the double mutant, including stronger interactions with F227 and less reliance on V106 for stabilization of the ethoxy-uracil ring, which makes critical hydrogen bonds with other residues in the binding pocket.

Pyrrole-Based Enaminones as Building Blocks for the Synthesis of Indolizines and Pyrrolo[1,2-a]pyrazines Showing Potent Antifungal Activity.

As a new approach, pyrrolo[1,2-a]pyrazines were synthesized through the cyclization of 2-formylpyrrole-based enaminones in the presence of ammonium acetate. The enaminones were prepared with a straightforward method, reacting the corresponding alkyl 2-(2-formyl-1H-pyrrol-1-yl)acetates, 2-(2-formyl-1H-pyrrol-1-yl)acetonitrile, and 2-(2-formyl-1H-pyrrol-1-yl)acetophenones with DMFDMA. Analogous enaminones elaborated from alkyl (E)-3-(1H-pyrrol-2-yl)acrylates were treated with a Lewis acid to afford indolizines. The antifungal activity of the series of substituted pyrroles, pyrrole-based enaminones, pyrrolo[1,2-a]pyrazines, and indolizines was evaluated on six Candida spp., including two multidrug-resistant ones. Compared to the reference drugs, most test compounds produced a more robust antifungal effect. Docking analysis suggests that the inhibition of yeast growth was probably mediated by the interaction of the compounds with the catalytic site of HMGR of the Candida species.

Reevaluation of bromodomain ligands targeting BAZ2A.

BAZ2A promotes migration and invasion in prostate cancer. Two chemical probes, the specific BAZ2-ICR, and the BAZ2/BRD9 cross-reactive GSK2801, interfere with the recognition of acetylated lysines in histones by the bromodomains of BAZ2A and of its BAZ2B paralog. The two chemical probes were tested in prostate cancer cell lines with opposite androgen susceptibility. BAZ2-ICR and GSK2801 showed different cellular efficacies in accordance with their unequal selectivity profiles. Concurrent inhibition of BAZ2 and BRD9 did not reproduce the effects observed with GSK2801, indicating possible off-targets for this chemical probe. On the other hand, the single BAZ2 inhibition by BAZ2-ICR did not phenocopy genetic ablation, demonstrating that bromodomain interference is not sufficient to strongly affect BAZ2A functionality and suggesting a PROTAC-based chemical ablation as an alternative optimization strategy and a possible therapeutic approach. In this context, we also present the crystallographic structures of BAZ2A in complex with the above chemical probes. Binding poses of TP-238 and GSK4027, chemical probes for the bromodomain subfamily I, and two ligands of the CBP/EP300 bromodomains identify additional headgroups for the development of BAZ2A ligands.

Structural basis of CDK3 activation by cyclin E1 and inhibition by dinaciclib.

Cell cycle transitions are controlled by multiple cell cycle regulators, especially CDKs. Several CDKs, including CDK1-4 and CDK6, promote cell cycle progression directly. Among them, CDK3 is critically important because it triggers the transitions of G0 to G1 and G1 to S phase through binding to cyclin C and cyclin E1, respectively. In contrast to its highly related homologs, the molecular basis of CDK3 activation remains elusive due to the lack of structural information of CDK3, particularly in cyclin bound form. Here we report the crystal structure of CDK3 in complex with cyclin E1 at 2.25 Å resolution. CDK3 resembles CDK2 in that both adopt a similar fold and bind cyclin E1 in a similar way. The structural discrepancy between CDK3 and CDK2 may reflect their substrate specificity. Profiling a panel of CDK inhibitors reveals that dinaciclib inhibits CDK3-cyclin E1 potently and specifically. The structure of CDK3-cyclin E1 bound to dinaciclib reveals the inhibitory mechanism. The structural and biochemical results uncover the mechanism of CDK3 activation by cyclin E1 and lays a foundation for structural-based drug design.

Involvement of the serotonergic system in the antidepressant-like effect of 1-(phenylselanyl)-2-(p-tolyl)indolizine in mice.

RATIONALE: Depression is a mental disorder that affects approximately 280 million people worldwide. In the search for new treatments for mood disorders, compounds containing selenium and indolizine derivatives show promising results. OBJECTIVES AND METHODS: To evaluate the antidepressant-like effect of 1-(phenylselanyl)-2-(p-tolyl)indolizine (MeSeI) (0.5-50 mg/kg, intragastric-i.g.) on the tail suspension test (TST) and the forced swim test (FST) in adult male Swiss mice and to elucidate the role of the serotonergic system in this effect through pharmacological and in silico approaches, as well to evaluate acute oral toxicity at a high dose (300 mg/kg). RESULTS: MeSeI administered 30 min before the FST and the TST reduced immobility time at doses from 1 mg/kg and at 50 mg/kg and increased the latency time for the first episode of immobility, demonstrating an antidepressant-like effect. In the open field test (OFT), MeSeI did not change the locomotor activity. The antidepressant-like effect of MeSeI (50 mg/kg, i.g.) was prevented by the pre-treatment with p-chlorophenylalanine (p-CPA), a selective tryptophan hydroxylase inhibitor (100 mg/kg, intraperitoneally-i.p. for 4 days), with ketanserin, a 5-HT2A/2C receptor antagonist (1 mg/kg, i.p.), and with GR113808, a 5-HT4 receptor antagonist (0.1 mg/kg, i.p.), but not with WAY100635, a selective 5-HT1A receptor antagonist (0.1 mg/kg, subcutaneous-s.c.) and ondansetron, a 5-HT3 receptor antagonist (1 mg/kg, i.p.). MeSeI showed a binding affinity with 5-HT2A, 5 -HT2C, and 5-HT4 receptors by molecular docking. MeSeI (300 mg/kg, i.g.) demonstrated low potential to cause acute toxicity in adult female Swiss mice. CONCLUSION: In summary, MeSeI exhibits an antidepressant-like effect mediated by the serotonergic system and could be considered for the development of new treatment strategies for depression.

An indolizine squaraine-based water-soluble NIR dye for fluorescence imaging of multidrug-resistant bacteria and antibacterial/antibiofilm activity using the photothermal effect.

The majority of nosocomial infections are caused by bacteria with antimicrobial resistance and the formation of biofilms, such as implant-related bacterial infections and sepsis. There is an urgent need to develop new strategies for early-stage screening, destruction of multidrug-resistant bacteria, and efficient inhibition of biofilms. Organic dyes that absorb and emit in the near-infrared (NIR) region are potentially non-invasive, high-resolution, and rapid biological imaging materials. In this study, a non-toxic and biocompatible indolizine squaraine dye with water-solubilizing sulfonate groups (SO3SQ) is studied for bacterial imaging and photothermal therapy (PTT). PTT is efficient in eliminating microorganisms through local hyperthermia without the risk of developing drug-resistant bacteria. The optical properties of SO3SQ are studied extensively in phosphate-buffered saline (PBS). UV-Vis-NIR absorption spectra analysis shows a strong absorption between 650 nm - 1000 nm. SO3SQ allows for the wash-free fluorescence imaging of drug-resistant bacteria via NIR fluorescence imaging due to a "turn-on" fluorescence property of the dye when interacting with bacteria. Although SO3SQ exhibits no toxicity against both Gram-positive bacteria and Gram-negative bacteria, the PTT property of SO3SQ is efficient in killing bacteria as well as inhibiting and eradicating biofilms. PTT experiments demonstrate that SO3SQ reduces 90% of cell viability in bacterial strains under NIR radiation with a minimum inhibition concentration (MIC90) of >450 µg/mL. The PTT property of SO3SQ can also inhibit biofilms (BIC90 = 1000-2000 µg/mL) and eradicate both preformed young and mature biofilms (MBEC90 = 1500-2000 µg/mL) as observed by crystal violet assays.

Design, synthesis and chemical stability of indolizine derivatives for antidiabetic activity.

Prodrugs of metformin were synthesized with the goal of enhancing biological activity of metformin. They were synthesized by combining metformin with 2-substituted indolizine (C7-C12). The synthesized prodrugs were characterized by IR, 1H NMR, 13C NMR, and mass spectroscopy. The chemical hydrolysis of C7-C12 was carried out at pH 1.2, 6.8, and 7.4. All compounds showed encouraging chemical stability at pH 1.2 and 6.8, whereas mild hydrolysis was shown at pH 7.4. Further prodrugs were screened for antidiabetic activity using a streptozotocin-induced model in rat. These derivatives showed substantial results. Among them C8 showed significant activity in the reduction of streptozotocin-induced blood glucose in rats when compared to that of metformin, indicating the effectiveness of prodrug.

Generation of a poly-functionalized indolizine scaffold and its anticancer activity in pancreatic cancer cells.

A highly efficient domino [4 + 2] annulation process was employed to construct a novel indolizine chemical scaffold. Biological investigation led us to identify 6w as a potent anticancer agent. 6w significantly inhibited cell viability in BxPC3 pancreatic cancer, MCF7 breast cancer, and PC3 prostate cancer cell lines with IC50 values of 0.47 ± 0.04, 1.82 ± 0.08 and 2.68 ± 0.08 µM, respectively. Remarkably, 6w showed a weak effect on cell viability of nontumorigenic human keratinocyte cell line HaCaT compared to the above three types of cancer cells. 6w most potently inhibited cell viability of BxPC3 cells, and 6w also potently reduced cell migration and induced apoptosis in BxPC3 cells through activation of caspase-3 and cleavage of PARP in a dose-dependent manner. These results suggest that 6w can be used for the development of potential anticancer drugs for the treatment of pancreatic cancer.

Insight on pyrimido[5,4-g]indolizine and pyrimido[4,5-c]pyrrolo[1,2-a]azepine systems as promising photosensitizers on malignant cells.

Searching for new small molecules as photosensitizing agents, we have developed a class of twenty-five pyrimido[5,4-g]indolizine and pyrimido[4,5-c]pyrrolo[1,2-a]azepines with a good substitution pattern defining a versatile synthetic pathway to approach the title ring system. All compounds were evaluated for their photocytotoxicity on a triple negative human breast cancer cell line (MDA-MB-231) in the dark and under UVA light (2.0 J/cm2). The most effective compounds exhibited a photoantiproliferative activity with IC50 values up to nanomolar ranges. Interestingly, these new developed compounds showed high selectivity towards cancerous cells with respect to non-cancerous ones. Moreover, four representative derivatives demonstrated to be phototoxic also against an additional human HER2 positive breast cancer cell line (HCC1954), and against the HER2 positive vesical cancer cell line (T24) harboring Hras mutation. Mechanistic studies performed in triple negative MDA-MB-231 cancer cells revealed the ability of the compounds to increase reactive oxygen species (ROS) production and to induce a thiol redox stress, thus triggering cancer cell death through apoptosis. Apoptotic cell death was also induced in highly aggressive and metastatic HER2 positive Hras mutated T24-treated bladder cancer cells. Overall, our data confirm that these new small photosensitizing agents may represent very promising candidates for phototherapy application against highly aggressive and resistant cancers.

Design, synthesis and chemical stability of indolizine derivatives for antidiabetic activity.

Prodrugs of metformin were synthesized with the goal of enhancing biological activity of metformin. They were synthesized by combining metformin with 2-substituted indolizine (C7-C12). The synthesized prodrugs were characterized by IR, 1H NMR, 13C NMR, and mass spectroscopy. The chemical hydrolysis of C7-C12 was carried out at pH 1.2, 6.8, and 7.4. All compounds showed encouraging chemical stability at pH 1.2 and 6.8, whereas mild hydrolysis was shown at pH 7.4. Further prodrugs were screened for antidiabetic activity using a streptozotocin-induced model in rat. These derivatives showed notable results. Among them C8 showed significant activity in the reduction of STZ-induced blood glucose in rats when compared to that of metformin, indicating the effectiveness of prodrug.

Dinaciclib inhibits the stemness of two subtypes of human breast cancer cells by targeting the FoxM1 and Hedgehog signaling pathway.

Cyclin­dependent kinase (CDK)4/6 inhibitors in combination with endocrine therapy are the current standard of care used in the first­line treatment of hormone receptor­positive/HER2­negative metastatic breast cancer (BC). Although CDK4/6 inhibitors mainly target the cell cycle, emerging evidence has indicated further potential roles of CDKs other than regulating cell cycle progression. The G1 and G2/M transition regulators, including cyclins D and E, as well as their catalytic partners, CDK2, CDK4 and CDK6, have been reported to play crucial roles in pluripotency maintenance and cell fate decisions of human pluripotent stem cells by controlling transcription factors, signaling pathways and epigenetic regulators. Dinaciclib, a CDK1/2/5/9 inhibitor, is currently being evaluated in clinical trials against various cancer types, including BC. However, the underlying molecular mechanisms of CDK1/2/5/9 inhibitors in regulating BC stemness remain poorly understood. The present study aimed to examine the stemness­inhibitory effects of dinaciclib in MCF­7 (luminal) and HCC­1806 (triple­negative) BC cells. We found that this drug not only effectively reduced the self­renewal abilities and other malignant properties, but also dose­dependently decreased the protein expression levels of three BC stem cell markers, CD44, aldehyde dehydrogenase 1 family member A1 (ALDH1A1) and BMI1 proto­oncogene, polycomb ring finger (Bmi1), as well as three embryonic stem cell markers, Oct4, Nanog and Sox2. Moreover, the dinaciclib­induced decrease of Oct4 and Nanog protein expression was able to be restored by co­treatment with MG­132, a proteasome inhibitor. Forkhead box M1 (FoxM1), both a stemness­stimulating transcription factor and a cell cycle regulator, along with the Hedgehog signaling pathway, were identified as the therapeutic targets of dinaciclib. Collectively, the present results demonstrated a novel role of dinaciclib in suppressing BC stemness and indicated its potential use for future cancer treatments.

Enhanced O-GlcNAc modification induced by the RAS/MAPK/CDK1 pathway is required for SOX2 protein expression and generation of cancer stem cells.

Cancer stem cells (CSCs) have tumour initiation, self-renewal, and long-term tumour repopulation properties, and it is postulated that differentiated somatic cells can be reprogrammed to CSCs by oncogenic signals. We previously showed that oncogenic HRASV12 conferred tumour initiation capacity in tumour suppressor p53-deficient (p53-/-) primary mouse embryonic fibroblasts (MEFs) through transcription factor NF-κB-mediated enhancement of glucose uptake; however, the underlying mechanisms of RAS oncogene-induced CSC reprogramming have not been elucidated. Here, we found that the expression of the reprogramming factor SOX2 was induced by HRASV12 in p53-/- MEFs. Moreover, gene knockout studies revealed that SOX2 is an essential factor for the generation of CSCs by HRASV12 in mouse and human fibroblasts. We demonstrated that HRASV12-induced cyclin-dependent kinase 1 (CDK1) activity and subsequent enhancement of protein O-GlcNAcylation were required for SOX2 induction and CSC generation in these fibroblasts and cancer cell lines containing RAS mutations. Moreover, the CDK inhibitor dinaciclib and O-GlcNAcylation inhibitor OSMI1 reduced the number of CSCs derived from these cells. Taken together, our results reveal a signalling pathway and mechanism for CSC generation by oncogenic RAS and suggest the possibility that this signalling pathway is a therapeutic target for CSCs.

Extracellular vesicle-mediated co-delivery of TRAIL and dinaciclib for targeted therapy of resistant tumors.

Extracellular vesicle (EV) delivery of TNF-related apoptosis-inducing ligand (TRAIL) (EV-T) has been shown to be highly efficient for cancer treatment when combined with the potent cyclin-dependent kinase (CDK) inhibitor dinaciclib (SCH727965, Dina). However, only topical administration was previously tested for cancer treatment, leaving unknown the efficacy of systemic therapy by EV-T and Dina. In this study we hypothesize that the systemic application of EV-T and Dina can be performed through EV-mediated co-delivery of TRAIL and Dina. Dina was first post-loaded into EV-Ts by sonication to prepare EV-mediated co-delivery of TRAIL and Dina, designated Dina@EV-T. Then Dina@EV-Ts were shown to be stable, readily endocytosed into cancer cells, and highly effective at inducing intensive apoptosis in resistant cancer lines but not in normal cells. Moreover, systemically infused Dina@EV-Ts showed evident tumor tropism suggesting their good potential for tumour-targeted delivery of therapeutics. Importantly, the systemic therapy with Dina@EV-Ts showed the best efficacy in vivo when compared with other treatments. The augmented therapeutic efficacy appeared to be associated with the concomitant suppression of prosurvival CDK1 and anti-apoptotic proteins including CDK9, cFLIP, MCL-1, BCL-2 and Survivin by Dina@EV-T treatment. Additionally, there were no adverse side effects observed for the systemic Dina@EV-T therapy. In conclusion, our data suggest that the co-delivery of TRAIL and Dina by EVs potentially constitutes a novel tumour-targeted therapy, which is highly effective and safe for the treatment of refractory tumors.

Phosphorylation-Induced Ubiquitination and Degradation of PXR through CDK2-TRIM21 Axis.

Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that is activated by a variety of endogenous metabolites or xenobiotics. Its downstream target genes are involved in metabolism, inflammation and processes closely related to cancer. However, the stability regulation of PXR protein resulting from post-translational modification is still largely undefined. In the present study, primary mouse hepatocytes, hepatoma HepG2 cells and HEK 293T cells were used to investigate gene expression and protein interactions. The role of kinases was evaluated by RNA interference and overexpression constructs with or without PXR phosphorylation site mutations. The activity of CYP3A4 and P-gp was determined by enzymatic and substrate accumulation assays. It was found that E3 ubiquitin ligase TRIM21 mediates the ubiquitination and degradation of PXR and plays an important role in regulating the activity of PXR. On this basis, PXR phosphorylation-associated kinases were evaluated regarding regulation of the stability of PXR. We found cyclin dependent kinase 2 (CDK2) exclusively phosphorylates PXR at Ser350, promotes its disassociation with Hsp90/DNAJC7, and leads to subsequent TRIM21-mediated PXR ubiquitination and degradation. As well-known CDK inhibitors, dinaciclib and kenpaullone stabilize PXR and result in elevated expression and activity of PXR-targeted DMETs, including carboxylesterases, CYP3A4 and P-gp. The suppressed degradation of PXR by CDK2 inhibitors denotes dinaciclib-induced promotion of PXR-targeted genes. The findings of CDK2-mediated PXR degradation indicate a wide range of potential drug-drug interactions during clinical cancer therapy using CDK inhibitors and imply an alternative direction for the development of novel PXR antagonists.

Pyrrolizine/Indolizine-NSAID Hybrids: Design, Synthesis, Biological Evaluation, and Molecular Docking Studies.

In the current study, eight new hybrids of the NSAIDs, ibuprofen and ketoprofen with five pyrrolizine/indolizine derivatives were designed and synthesized. The chemical structures of these hybrids were confirmed by spectral and elemental analyses. The antiproliferative activities of these hybrids (5 µM) was investigated against MCF-7, A549, and HT-29 cancer cell lines using the cell viability assay, MTT assay. The results revealed 4-71% inhibition of the growth of the three cancer cell lines, where 8a,e,f were the most active. In addition, an investigation of the antiproliferative activity of 8a,e,f against MCF-7 cells revealed IC50 values of 7.61, 1.07, and 3.16 µM, respectively. Cell cycle analysis of MCF-7 cells treated with the three hybrids at 5 µM revealed a pro-apoptotic increase in cells at preG1 and cell cycle arrest at the G1 and S phases. In addition, the three hybrids induced early apoptotic events in MCF-7 cells. The results of the molecular docking of the three hybrids into COX-1/2 revealed higher binding free energies than their parent compounds 5a,c and the co-crystallized ligands, ibuprofen and SC-558. The results also indicated higher binding free energies toward COX-2 over COX-1. Moreover, analysis of the binding modes of 8a,e,f into COX-2 revealed partial superposition with the co-crystallized ligand, SC-558 with the formation of essential hydrogen bonds, electrostatic, or hydrophobic interactions with the key amino acid His90 and Arg513. The new hybrids also showed drug-likeness scores in the range of 1.06-2.03 compared to ibuprofen (0.65) and ketoprofen (0.57). These results above indicated that compounds 8a,e,f deserve additional investigation as potential anticancer candidates.

Interest of novel N-alkylpyridinium-indolizine hybrids in the field of Alzheimer's disease: Synthesis, characterization and evaluation of antioxidant activity, cholinesterase inhibition, and amyloid fibrillation interference.

A small library of molecules combining indolizine and N-alkyl pyridinium was synthesized and evaluated in a multi-target-directed-ligand strategy for Alzheimer's disease (AD) treatment. The new compounds were classified in three series depending on the number of methylene residues linking the two heterocycles (Ind-PyCx with x = 0, 2 or 3). The molecules were synthesized from the corresponding bis-pyridines by two-step formation of the indolizine core including mono-alkylation of pyridine and 1,3-dipolar cycloaddition with an alkylpropiolate. Their activities against AD's key-targets were evaluated in vitro: acetyl- and butyrylcholinesterase (AChE and BChE) inhibition, antioxidant properties and inhibition of amyloid fibril formation. None of the three series showed significant activities against all the targets. The Ind-PyC2 and Ind-PyC3 series are active on eeAChE and hAChE (µM IC50 values). Most of the positively charged molecules from these two series also appeared active against eqBChE, however they lost their activity on hBChE. Comparative molecular modeling of 13 and 15 docked in hAChE and hBChE highlighted the importance of the substituent (p-methoxybenzoyl or methyloxycarbonyl, respectively) located on the indolizine C-3 for the binding. The larger molecule 13 fits more tightly at the active site of the two enzymes than 15 that shows a larger degree of freedom. The Ind-PyC2 and Ind-PyC3 hybrids displayed some antioxidant activity when tested at 750 µg/mL (up to 95% inhibition of DPPH radical scavenging for 10). In both series, most hybrids were also able to interact with amyloid fibers, even if the inhibitory effect was observed at a high 100 µM concentration. The Ind-PyC0 molecules stand out completely due to their spectroscopic properties which prevent their evaluation by Ellman's and ThT assays. However, these molecules showed interesting features in the presence of preformed fibers. In particular, the strong increase in fluorescence of 3 in the presence of amyloid fibers is very promising for its use as a fibrillation fluorescent reporter dye.

Anti-tubercular activity and molecular docking studies of indolizine derivatives targeting mycobacterial InhA enzyme.

A series of 1,2,3-trisubstituted indolizines (2a-2f, 3a-3d, and 4a-4c) were screened for in vitro whole-cell anti-tubercular activity against the susceptible H37Rv and multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) strains. Compounds 2b-2d, 3a-3d, and 4a-4c were active against the H37Rv-MTB strain with minimum inhibitory concentration (MIC) ranging from 4 to 32 µg/mL, whereas the indolizines 4a-4c, with ethyl ester group at the 4-position of the benzoyl ring also exhibited anti-MDR-MTB activity (MIC = 16-64 µg/mL). In silico docking study revealed the enoyl-acyl carrier protein reductase (InhA) and anthranilate phosphoribosyltransferase as potential molecular targets for the indolizines. The X-ray diffraction analysis of the compound 4b was also carried out. Further, a safety study (in silico and in vitro) demonstrated no toxicity for these compounds. Thus, the indolizines warrant further development and may represent a novel promising class of InhA inhibitors and multi-targeting agents to combat drug-sensitive and drug-resistant MTB strains.