Synthesis of portimines reveals the basis of their anti-cancer activity.

Marine-derived cyclic imine toxins, portimine A and portimine B, have attracted attention because of their chemical structure and notable anti-cancer therapeutic potential1-4. However, access to large quantities of these toxins is currently not feasible, and the molecular mechanism underlying their potent activity remains unknown until now. To address this, a scalable and concise synthesis of portimines is presented, which benefits from the logic used in the two-phase terpenoid synthesis5,6 along with other tactics such as exploiting ring-chain tautomerization and skeletal reorganization to minimize protecting group chemistry through self-protection. Notably, this total synthesis enabled a structural reassignment of portimine B and an in-depth functional evaluation of portimine A, revealing that it induces apoptosis selectively in human cancer cell lines with high potency and is efficacious in vivo in tumour-clearance models. Finally, practical access to the portimines and their analogues simplified the development of photoaffinity analogues, which were used in chemical proteomic experiments to identify a primary target of portimine A as the 60S ribosomal export protein NMD3.

Design, synthesis, and antitumor study of a series of novel 1-Oxa-4-azaspironenone derivatives.

A series of 1-oxa-4-azaspiro[4,5]deca-6,9-diene-3,8-dione derivatives containing structural fragments of conjugated dienone have been synthesized previously by our group, however the Michael addition reaction between conjugated dienone and nucleophilic groups in the body may generate harmful and adverse effects. To reduce harmful side effects, the authors started with p-aminophenol to make 1-oxo-4- azaspirodecanedione derivatives, then utilized the Michael addition and cyclopropanation to eliminate α, ß unsaturated olefinic bond and lower the Michael reactivity of the compounds in vivo for optimization. At the same time, heteroatoms are put into the molecules in order to improve the hydrophilicity of the molecules and the binding sites of the molecules and the target molecules, establishing the groundwork for improved antitumor activity. The majority of the compounds had moderate to potent activity against A549 human lung cancer cells, MDA-MB-231 breast cancer cells, and Hela human cervical cancer cells. Among them, the compound 6d showed the strongest effect on A549 cell line with IC50 of 0.26 µM; the compound 8d showed the strongest cytotoxicity on MDA-MB-231 cell line with IC50 of 0.10 µM; and the compound 6b showed the strongest activity on Hela cell line with IC50 of 0.18 µM.

Structure-activity and structure-property relationship studies of spirocyclic chromanes with antimalarial activity.

Malaria is a prevalent and lethal disease. The fast emergence and spread of resistance to current therapies is a major concern and the development of a novel line of therapy that could overcome, the problem of drug resistance, is imperative. Screening of a set of compounds with drug/natural product-based sub-structural motifs led to the identification of spirocyclic chroman-4-one 1 with promising antimalarial activity against the chloroquine-resistant Dd2 and chloroquine-sensitive 3D7 strains of the parasite. Extensive structure-activity and structure-property relationship studies were conducted to identify the essential features necessary for its activity and properties.

Positioning of an unprecedented 1,5-oxaza spiroquinone scaffold into SMYD2 inhibitors in epigenetic space.

Lysine methyltransferases are important regulators of epigenetic signaling and are emerging as a novel drug target for drug discovery. This work demonstrates the positioning of novel 1,5-oxaza spiroquinone scaffold into selective SET and MYND domain-containing proteins 2 methyltransferases inhibitors. Selectivity of the scaffold was identified by epigenetic target screening followed by SAR study for the scaffold. The optimization was performed iteratively by two-step optimization consisting of iterative synthesis and computational studies (docking, metadynamics simulations). Computational binding studies guided the important interactions of the spiro[5.5]undeca scaffold in pocket 1 and Lysine channel and suggested extension of tail length for the improvement of potency (IC50: up to 399 nM). The effective performance of cell proliferation assay for chosen compounds (IC50: up to 11.9 nM) led to further evaluation in xenograft assay. The potent compound 24 demonstrated desirable in vivo efficacy with growth inhibition rate of 77.7% (4 fold decrease of tumor weight and 3 fold decrease of tumor volume). Moreover, mirosomal assay and pharmacokinetic profile suggested further developability of this scaffold through the identification of major metabolites (dealkylation at silyl group, reversible hydration product, the absence of toxic quinone fragments) and enough exposure of the testing compound 24 in plasma. Such spiro[5.5]undeca framework or ring system was neither been reported nor suggested as a modulator of methyltransferases. The chemo-centric target positioning and structural novelty can lead to potential pharmacological benefit.

Design, synthesis and biological evaluation of a novel spiro oxazolidinedione as potent p300/CBP HAT inhibitor for the treatment of ovarian cancer.

Histone acetylation is one of the most essential parts of epigenetic modification, mediating a variety of complex biological functions. In these procedure, p300/CBP could catalyze the acetylation of lysine 27 on histone 3 (H3K27ac), and had been reported to mediate tumorigenesis and development in a variety of tumors by enhancing chromatin transcription activity. Ovarian cancer, as an extremely malignant tumor, has also been observed to undergo abnormal acetylation of histones. However, whether the treatment of ovarian cancer could be achieved by inhibiting the acetylation activity of p300/CBP on H3K27 has not been well investigated. In this article, we modified the structure of p300/CBP HAT domain inhibitor A-485 and obtained a highly active small molecule known as 13f, which has an IC50 value of 0.49 nM for inhibiting the in vitro enzyme activity of p300, as well as the anti-proliferation IC50 value on ovarian cancer cell line OVCAR-3 was 153 nM. In addition, 13f had strong acetylase family selectivity, good metabolic stability and promising in vivo anti-tumor activity in OVCAR-3 xenograft model. The discovery of 13f revealed a more active chemical entity of the HATs domain of p300/CBP and provided a novel idea for the application of epigenetic inhibitors in the treatment of ovarian cancer.

Molecular hybridization design and synthesis of novel spirooxindole-based MDM2 inhibitors endowed with BCL2 signaling attenuation; a step towards the next generation p53 activators.

Despite the achieved progress in developing efficient MDM2-p53 protein-protein interaction inhibitors (MDM2 inhibitors), the acquired resistance of tumor cells to such p53 activators posed an argument about the druggability of the pathway. Combination studies disclosed that concomitant inhibition of MDM2 and BCL2 functions can sensitize the tumor cells and synergistically induce apoptosis. Herein, we employed a rapid combinatorial approach to generate a novel series of hybrid spirooxindole-based MDM2 inhibitors (5a-s) endowed with BCL2 signaling attenuation. The adducts were designed to mimic the thematic features of the chemically stable potent spiro[3H-indole-3,2'-pyrrolidin]-2(1H)-ones MDM2 inhibitors while installing a pyrrole ring on the core via a carbonyl spacer inspired by the natural product marinopyrrole A that efficiently inhibits BCL2 family functions by various mechanisms. NCI 60 cell-line panel screening revealed their promising broad-spectrum antiproliferative activities. The NCI-selected derivatives were screened for cytotoxic activities against normal fibroblasts, MDA-MB 231, HepG-2, and Caco-2 cells via MTT assay, subjected to mechanistic apoptosis studies for assessment of p53, BCL2, p21, and caspase 3/7 status, then evaluated for potential MDM2 inhibition utilizing MST assay. The most balanced potent and safe derivatives; 5i and 5q were more active than 5-fluorouracil, exhibited low µmrange MDM2 binding (KD=1.32and 1.72 µm, respectively), induced apoptosis-dependent anticancer activities up to 50%, activated p53 by 47-63%, downregulated the BCL2 gene to 59.8%, and reduced its protein level (13.75%) in the treated cancer cells. Further downstream p53 signaling studies revealed > 2 folds p21 upregulation and > 3 folds caspase 3/7 activation. Docking simulations displayed that the active MDM2 inhibitors resided well into the p53 binding sites of MDM2, and shared key interactions with the co-crystalized inhibitor posed by the indolinone scaffold (5i, 5p, and 5q), the halogen substituents (5r), or the installed spiro ring (5s). Finally, in silico ADMET profiling predicted acceptable drug-like properties with full accordance to Lipinski's, Veber's, and Muegge's bioavailability parameters for 5i and a single violation for 5q.

Identification of Spiro-Fused Pyrrolo[3,4-a]pyrrolizines and Tryptanthrines as Potential Antitumor Agents: Synthesis and In Vitro Evaluation.

A series of heterocyclic compounds containing a spiro-fused pyrrolo[3,4-a]pyrrolizine and tryptanthrin framework have been synthesized and studied as potential antitumor agents. Cytotoxicity of products was screened against human erythroleukemia (K562) and human cervical carcinoma (HeLa) cell lines. Among the screened compounds. 4a, 4b and 5a were active against human erythroleukemia (K562) cell line, while 4a and 5a were active against cervical carcinoma (HeLa) cell line. In agreement with the DNA cytometry studies, the tested compounds have achieved significant cell-cycle perturbation with higher accumulation of cells in G2/M phase and induced apoptosis. Using confocal microscopy, we found that with 4a and 5a treatment of HeLa cells, actin filaments disappeared, and granular actin was distributed diffusely in the cytoplasm in 76-91% of cells. We discovered that HeLa cells after treatment with compounds 4a and 5a significantly reduced the number of cells with filopodium-like membrane protrusions (from 63 % in control cells to 29% after treatment) and a decrease in cell motility.

Microwave-assisted rapid synthesis of spirooxindole-pyrrolizidine analogues and their activity as anti-amyloidogenic agents.

A library of Sox-pyrrolizidines was rapidly prepared by microwave-assisted, one-pot, three-component, 1,3-dipolar cycloaddition of azomethine ylides from l-proline and isatin, with various ß-nitrostyrenes. Nitro-Sox compounds, 4b, 4d and 4e inhibit HEWL amyloid fibril formation by ThT studies with percentages of fluorescence intensity of 55.4, 42.9 and 40.3%, respectively. Further studies with MTT assay, Raman spectroscopy, TEM and molecular docking supported these promising candidates for activity against amyloid misfolding, a phenomenon leading to Alzheimer's disease pathology.

Design, synthesis and biological evaluation of exiguamine A analogues as IDO1 inhibitors.

A series of exiguamine A analogues were designed and synthesized via 15 steps. Their inhibitory activities against IDO1 were tested and the structure-activity relationships were studied. Most compounds exhibited potent IDO1 inhibitory activities with IC50 values at the level of 10-7-10-8 M. Compound 21f was the most potent IDO1 inhibitor with an IC50 value of 65.3 nM, which was comparable with the positive control drug epacadostat (IC50 = 46 nM). Moreover, compound 21f showed higher selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO) and no cytotoxicity at its effective concentration, rending it justifiable for further optimization and evaluation.

Seeking Illumination: The Path to Chemiluminescent 1,2-Dioxetanes for Quantitative Measurements and In Vivo Imaging.

Chemiluminescence is a fascinating phenomenon that evolved in nature and has been harnessed by chemists in diverse ways to improve life. This Account tells the story of our research group's efforts to formulate and manifest spiroadamantane 1,2-dioxetanes with triggerable chemiluminescence for imaging and monitoring important reactive analytes in living cells, animals, and human clinical samples. Analytes like reactive sulfur, oxygen and nitrogen species, as well as pH and hypoxia can be indicators of cellular function or dysfunction and are often implicated in the causes and effects of disease. We begin with a foundation in binding-based and activity-based fluorescence imaging that has provided transformative tools for understanding biological systems. The intense light sources required for fluorescence excitation, however, introduce autofluorescence and light scattering that reduces sensitivity and complicates in vivo imaging. Our work and the work of our collaborators were the first to demonstrate that spiroadamantane 1,2-dioxetanes had sufficient brightness and biological compatibility for in vivo imaging of enzyme activity and reactive analytes like hydrogen sulfide (H2S) inside of living mice. This launched an era of renewed interest in 1,2-dioxetanes that has resulted in a plethora of new chemiluminescence imaging agents developed by groups around the world. Our own research group focused its efforts on reactive sulfur, oxygen, and nitrogen species, pH, and hypoxia, resulting in a large family of bright chemiluminescent 1,2-dioxetanes validated for cell monitoring and in vivo imaging. These chemiluminescent probes feature low background and high sensitivity that have been proven quite useful for studying signaling, for example, the generation of peroxynitrite (ONOO-) in cellular models of immune function and phagocytosis. This high sensitivity has also enabled real-time quantitative reporting of oxygen-dependent enzyme activity and hypoxia in living cells and tumor xenograft models. We reported some of the first ratiometric chemiluminescent 1,2-dioxetane systems for imaging pH and have introduced a powerful kinetics-based approach for quantification of reactive species like azanone (nitroxyl, HNO) and enzyme activity in living cells. These tools have been applied to untangle complex signaling pathways of peroxynitrite production in radiation therapy and as substrates in a split esterase system to provide an enzyme/substrate pair to rival luciferase/luciferin. Furthermore, we have pushed chemiluminescence toward commercialization and clinical translation by demonstrating the ability to monitor airway hydrogen peroxide in the exhaled breath of asthma patients using transiently produced chemiluminescent 1,2-dioxetanedione intermediates. This body of work shows the powerful possibilities that can emerge when working at the interface of light and chemistry, and we hope that it will inspire future scientists to seek out ever brighter and more illuminating ideas.

Recent Strategies in the Synthesis of Spiroindole and Spirooxindole Scaffolds.

Spiroindole and spirooxindole scaffolds are very important spiro-heterocyclic compounds in drug design processes. Significant attention has been directed at obtaining molecules based on spiroindole and spirooxindole derivatives that have bioactivity against cancer cells, microbes, and different types of disease affecting the human body. Due to their inherent three-dimensional nature and ability to project functionalities in all three dimensions, they have become biological targets. Considering reports on spiroindole and spirooxindole-containing scaffolds in the past decades, introducing novel synthetic procedures has been an active research field of organic chemistry for well over a century and will be useful in creating new therapeutic agents. This review summarizes the pharmacological significance of spiroindole and spirooxindole scaffolds and highlights the latest strategies for their synthesis, focusing particularly on the past 2 years with typical examples. The spiroindole and spirooxindoles in this review are divided by the type and ring size of the spirocycle that is fused to indole or oxindole. Summarizing these procedures will be very beneficial for discovering novel therapeutic candidate molecules.

Design, synthesis and biological evaluation of 7-(5-((substituted - amino)-methyl)-thiophen-2-yl)-spiro-[chroman-2,4'-piperidin]-4-one hydrochloride analogues as anticancer agents.

A series of thirty-one novel 7-(5-((amino)-methyl)-thiophen-2-yl)-spiro-[chroman-2,4'-piperidin]-4-one hydrochloride analogues (Cst 1 - 31) have been designed, synthesized and characterized by 1H NMR, 13C NMR and MS spectral analysis. Here, we evaluated the anticancer potential and biological results of low-molecular-weight bridgehead oxygen and nitrogen-containing spirochromanones on proliferation and apoptosis of the human breast cancer cell line (MCF-7) and Murine melanoma (B16F10). The anticancer activity ranged from 2.9 to 35.0 µM. The most potent compounds Cst-22, Cst-24 and Cst-31 were found to be less toxic against human embryonic kidney (HEK-293) cell lines. Cst-24 and Cst-31 were found to be causing significant cytotoxicity through apoptotic cell death and also G2 phase arrest of cell cycle in B16F10 cells. In-silico ADME prediction stidies of the titled compounds were found within the rules outlined, and these compounds may not face any pharmacokinetic associated issues in the mere future upon developmental stage. These conjugates may serve as a lead for the discovery of potential anticancer drug candidate with better therapeutic profile.

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay.

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring systems is challenging. An efficient methodology to synthesize these compounds was recently published that described the solid phase synthesis in four steps rather than the previously reported five steps. The advantage of this shorter synthesis is the elimination of the use of toxic reagents. Low-loading Regenerating Michael (REM) linker-based resin was found to be crucial in the synthesis as high-loading versions prevented the addition of reagents containing bulky phenyl and aromatic side chains. The colorimetric 3-(4',5'-dimethylthiazol-2'-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to examine the cytotoxicity of micromolar concentrations of these novel spirocyclic molecules in vitro. MTT is readily available commercially and produces relatively fast, reliable results, making this assay ideal for these spirocyclic heterocycles. Orthogonal ring structures as well as furfurylamine (a precursor in the synthesis method containing a similar 5-member ring motif) were tested.

Total Synthesis of Ritterazine B.

The first total synthesis of the cytotoxic alkaloid ritterazine B is reported. The synthesis features a unified approach to both steroid subunits, employing a titanium-mediated propargylation reaction to achieve divergence from a common precursor. Other key steps include gold-catalyzed cycloisomerizations that install both spiroketals and late stage C-H oxidation to incorporate the C7' alcohol.

Novel 4,5-dihydrospiro[benzo[c]azepine-1,1'-cyclohexan]-3(2H)-one derivatives as PARP-1 inhibitors: Design, synthesis and biological evaluation.

To further explore the research of novel PARP-1 inhibitors, we designed and synthesized a series of novel amide PARP-1 inhibitors based on our previous research. Most compounds displayed certain antitumor activities against four tumor cell lines (A549, HepG2, HCT-116, and MCF-7). Specifically, the candidate compound R8e possessed strong anti-proliferative potency toward A549 cells with the IC50 value of 2.01 µM. Compound R8e had low toxicity to lung cancer cell line. And the in vitro enzyme inhibitory activity of compound R8e was better than rucaparib. Molecular docking studies provided a rational binding model of compound R8e in complex with rucaparib. The following cell cycle and apoptosis assays revealed that compound R8e could arrest cell cycle in the S phase and induce cell apoptosis. Western blot analysis further showed that compound R8e could effectively inhibit the PAR's biosynthesis and was more effective than rucaparib. Overall, based on the biological activity evaluation, compound R8e could be a potential lead compound for further developing novel amide PARP-1 inhibitors.

An insight into the structure of 5-spiro aromatic derivatives of imidazolidine-2,4-dione, a new group of very potent inhibitors of tumor multidrug resistance in T-lymphoma cells.

A series of 17 arylpiperazine derivatives of the 5-spiroimidazolidine-2,4-diones (6-22) has been explored, including variations in (i) the number of aromatic rings at position 5, (ii) the length of the linker, as well as (iii) the kind and position of the linked arylpiperazine terminal fragment. Synthesis (6-16) and X-ray crystallographic studies for representative compounds (8, 10, 14 and 18) have been performed. The ability to inhibit the tumor multidrug resistance (MDR) efflux pump P-glycoprotein (P-gp, ABCB1) overexpressed in mouse T-lymphoma cells was investigated. The cytotoxic and antiproliferative actions of the compounds on both the reference and the ABCB1-overproducing cells were also examined. The pharmacophore-based molecular modeling studies have been performed. ADMET properties in vitro of selected most active derivatives (6, 11 and 12) have been determined. All compounds, excluding 18, inhibited the cancer P-gp efflux pump with higher potency than that of reference verapamil. The spirofluorene derivatives with amine alkyl substituents at position 1, and the methyl group at position 3 (6-16), occurred the most potent P-gp inhibitors in the MDR T-lymphoma cell line. In particular, compounds 7 and 12 were 100-fold more potent than verapamil. Crystallography-supported pharmacophore-based SAR analysis has postulated specific structural properties that could explain this excellent cancer MDR-inhibitory action.

Evaluation of the cellular protection by novel spiropyrazole compounds in dopaminergic cell death.

The loss of neurons is strongly correlated with aging and aging-associated disorders. In this study, cell viability assays and mitochondrial function were performed to evaluate the effect of new spiro-pyrazole derivatives, prepared from aldehydes and 3-amino-1-phenyl-2-pyrazolin-5-one, on neuroprotection in an in vitro model of dopaminergic cell death induced by 1-methyl-4-phenylpyridinium (MPP+). The percentages of neuroprotection by derivatives were found between 21.26% and 52.67% at selected concentrations (10-50 µM) with compound 4d exerting the best neuroprotective effect. The results show that the studied spiropyrazolones perform important roles in dopaminergic neuroprotection and can be used for potential new therapies in the treatment of neurodegenerative disorders including Parkinson's disease.

New Spirocyclic Hydroxamic Acids as Effective Antiproliferative Agents.

AIMS: The main goal of this work is to synthesize new original spirocyclic hydroxamic acids, investigate their cytotoxicity against the panel of tumor cell lines and possible mechanism of action of these active compounds. BACKGROUND: Hydroxamic acids are one of the promising classes of chemical compounds with proven potential anticancer properties. This is manifested in the presence of metal chelating and antioxidant activities, the ability to inhibit histone deacetylase enzymes and a chemosensitizing effect against well known cytostatics. OBJECTIVE: Original spirocyclic hydroxamic acids were synthesized and spectra of their antiproliferative activities were investigated. METHODS: The cytotoxic activities on different tumor lines (SH-SY5Y, HeLa and healthy cells HEK-293) were investigated and determined possible underlying mechanisms of their activity. RESULTS: New original spirocyclic hydroxamic acids were synthesized. These compounds exhibit antiproliferative properties against various tumor cultures cells and also exhibit antioxidant activity, a depolarizing effect on the mitochondrial membrane, inhibit the activity of the histone deacetylase enzyme, and also decrease of basal glycolysis and glycolytic capacity reserve of HeLa and SH-SY5Y tumor cell lines. CONCLUSION: The most promising are compounds 5j-l containing two chlorine atoms as substituents in the quinazoline part of the molecule and hydroxamate function. Therefore, these compounds can be considered as hit compounds for the development on their basis multi-target anticancer agents.

Exploiting the antiproliferative potential of spiropyrazoline oxindoles in a human ovarian cancer cell line.

Cancer is still one of the deadliest diseases worldwide despite the efforts in its early detection and treatment strategies. However, most chemotherapeutic agents still present side effects in normal tissues and acquired resistance that limit their efficacy. Spiropyrazoline oxindoles might be good alternatives as they have shown antiproliferative activity in human breast and colon cancer cell lines, without eliciting cytotoxicity in healthy cells. However, their potential for ovarian cancer was never tested. In this work, the antiproliferative activity of five spiropyrazoline oxindoles was assessed in ovarian cancer cells A2780 and the biological targets and mechanism of action of the most promising compound evaluated. Compound 1a showed the highest antiproliferative effect, as well as the highest selectivity for A2780 cells compared to healthy fibroblasts. This antiproliferative effect results from the induction of cell death by mitochondria-mediated apoptosis and autophagy. In vitro DNA interaction studies demonstrated that 1a interacts with DNA by groove-binding, without triggering genotoxicity. In addition, 1a showed a strong affinity to bovine serum albumin that might be important for further inclusion in drug delivery platforms. Proteomic studies reinforced 1a role in promoting A2780 endoplasmatic reticulum (ER) stress by destabilizing the correct protein folding which triggers cell death via apoptosis and autophagy.

Synthetic Spirocyclohexadienones as New Anti-Migratory Compounds in Triple- Negative Breast Cancer Cell Migration.

BACKGROUND: Triple-negative BC is the most aggressive type of breast cancer and its lack of responsiveness to conventional therapies requires screening of new chemical entities. Anti-migratory compounds are promising to treat metastatic cancer since they inhibit one of the main steps of the metastatic cascade. Spirocyclic compounds are non-conventional structures used as building blocks for the synthesis of biologically active molecules and considered interesting structures in the search for new targets in cancer research. OBJECTIVE: Here, we evaluated the potential of eight synthetic spirocyclohexadienones as cell migration inhibitors. METHODS: The anti-migratory ability of compounds was tested by wound healing and Boyden chamber approaches. Experiments in tubulin were performed by fluorescence and tubulin polymerization techniques. Finally, compounds were submitted to cell proliferation inhibition and flow cytometry assays to explore the mechanism by which they inhibit cell migration. RESULTS: Four compounds inhibited cell migration significantly. Analogs containing the 3,4,5-trimethoxyphenil ring at R1 position were the most potent and, thus, selected for additional experiments. Tubulin polymerization and fluorescence assays highlighted a possible binding of spirocyclohexadienones in the colchicine binding site; however, these compounds did not affect the cell cycle to the same extent as colchicine. Cell proliferation was affected and, notably, the most potent analogs induced apoptosis of tumor cells, suggesting a different mechanism by which they inhibit cell migration. CONCLUSION: We presented, for the first time, a series of eight synthetic spirocyclohexadienones with the ability to inhibit TNBC cell migration. These compounds represent a new category to be explored as anticancer agents.