Norcantharidin toxicity profile: an in vivo murine study.

Norcantharidin (NCTD) is the demethylated analog of cantharidin, with allegedly reduced toxicity. However, there is still limited information regarding its posology and potential risk in its use in cancer treatment. Healthy BDF1 mice were intraperitoneally administered with norcantharidin (0, 3, 6, 12, and 25 mg/kg) every 24 h for 6 days. Survivor mice were euthanized, and the brain, lungs, kidneys, spleen, and liver were procured for enzymatic and histopathological analysis in the liver and kidney. DL50 were 8.86 mg/kg for females and 11.77 mg/kg for males. The treatments with 3.0 mg/kg and 6.0 mg/kg significantly modified the phosphorylase, alanine transaminase, and γ-glutamyl transferase activities; however, an organ-specific response was detected. A significant dose-dependent decrease was observed in the kidney for ROS, while the liver had the opposite effect. Histopathological analysis revealed a significant elevation in hepatocytes' nuclei average size and total area (3 mg/kg), as well as centrilobular vein and adjacent sinusoidal capillaries showed a significant difference. The portal triad presented a significant difference in veins and capillarity count in 6 mg/kg. Renal samples showed cortex convoluted tubules' average size significantly augmented in both doses' groups, and tubule count was found augmented in 6 mg/kg. These physiological effects of NCTD can be exploited as treatment strategies if able to operate in an established posology and proper testing.

The Combination of Venetoclax and Ixazomib Selectively and Efficiently Kills HIV-Infected Cell Lines but Has Unacceptable Toxicity in Primary Cell Models.

The antiapoptotic protein BCL2 inhibits death of HIV-infected cells. Previously, we showed that the BCL2 inhibitor venetoclax selectively kills acutely HIV-infected cells and reduces HIV DNA in latently infected CD4 T cells ex vivo after reactivation with anti-CD3/anti-CD28. However, there is a need to identify a combination therapy with venetoclax and a clinically relevant latency reversal agent. Ixazomib is an oral proteasome inhibitor which we have shown reactivates latent HIV and predisposes reactivated cells to cell death. Here, we determined that the combination of venetoclax and ixazomib kills more latently HIV-infected cells and leads to greater reduction in HIV replication than either treatment alone in vitro in a T cell model. However, combination treatment of ex vivo CD4 T cells from antiretroviral therapy (ART)-suppressed, HIV-positive participants resulted in unanticipated and unacceptable nonspecific toxicity in primary cells. Therefore, while we show proof of concept that multiple agents can enhance selective killing of HIV-infected cells, the combination of venetoclax and ixazomib has unacceptable toxicity in primary cells, and so further investigation is needed to identify a clinically relevant latency reversal agent to combine with venetoclax as a novel strategy to reduce the size of the HIV reservoir.IMPORTANCE A cure for HIV would require eliminating cells that contain the virus in a latent form from the body. Current antiretroviral medications are unable to rid the body of latently infected cells. Here, we show that a combination of investigational agents-ixazomib plus venetoclax-which reactivate latent virus and predispose infected cells to apoptosis may reduce latent virus in a T cell model, but at the expense of nonspecific toxicity in primary cells.

Paper spray ionization-high-resolution mass spectrometry (PSI-HRMS) of peroxide explosives in biological matrices.

Mitigation of the peroxide explosive threat, specifically triacetone triperoxide (TATP) and hexamethylene triperoxide diamine (HMTD), is a priority among the law enforcement community, as scientists and canine (K9) units are constantly working to improve detection. We propose the use of paper spray ionization-high-resolution mass spectrometry (PSI-HRMS) for detection of peroxide explosives in biological matrices. Occurrence of peroxide explosives and/or their metabolites in biological samples, obtained from urine or blood tests, give scientific evidence of peroxide explosives exposure. PSI-HRMS promote analysis of samples in situ by eliminating laborious sample preparation steps. However, it increases matrix background issues, which were overcome by the formation of multiple alkali metal adducts with the peroxide explosives. Multiple ion formation increases confidence when identifying these peroxide explosives in direct sample analysis. Our previous work examined aspects of TATP metabolism. Herein, we investigate the excretion of a TATP glucuronide conjugate in the urine of bomb-sniffing dogs and demonstrate its detection using PSI from the in vivo sample.

Acute toxicity of dihydroanatoxin-a from Microcoleus autumnalis in comparison to anatoxin-a.

The cyanobacterium Microcoleus autumnalis grows as thick benthic mats in rivers and is becoming increasingly prevalent around the world. M. autumnalis can produce high concentrations of anatoxins and ingestion of benthic mats has led to multiple dog deaths over the past two decades. M. autumnalis produces a suite of different anatoxin congeners including anatoxin-a (ATX), dihydroanatoxin-a, (dhATX), homoanatoxin-a and dihydrohomoanatoxin-a. Benthic mat samples often contain high levels of dhATX, but there is little toxicology information on this congener. In the present study, natural versions of dhATX and ATX were purified from cyanobacteria to determine the acute toxicity by different routes of administration using mice. Nuclear magnetic resonance spectroscopy was used to confirm the putative structure of dhATX. By intraperitoneal (ip) injection, the median lethal dose (LD50) for dhATX was 0.73 mg/kg, indicating a reduced toxicity compared to ATX (LD50 of 0.23 mg/kg). However, by oral administration (both gavage and feeding), dhATX was more toxic than ATX (gavage LD50 of 2.5 mg/kg for dhATX and 10.6 mg/kg for ATX; feeding LD50 of 8 mg/kg for dhATX and 25 mg/kg for ATX). The relative nicotinic acetylcholine receptor-binding affinities of ATX and dhATX were determined using the Torpedo electroplaque assay which showed consistency with the relative toxicity determined by ip injection. This work highlights that toxicity studies based solely on ip injection may not yield LD50 values that are relevant to those derived via oral administration, and hence, do not provide a good estimate of the risk posed to human and animal health in situations where oral ingestion is the likely route of exposure. The high acute oral toxicity of dhATX, and its abundance in M. autumnalis proliferations, demonstrates that it is an important environmental contaminant that warrants further investigation.

Toward Spontaneous Neuronal Differentiation of SH-SY5Y Cells Using Novel Three-Dimensional Electropolymerized Conductive Scaffolds.

Neuroblastoma-derived SH-SY5Y cells have become an excellent model for nervous system regeneration to treat neurodegenerative disorders. Many approaches achieved a mature population of derived neurons in in vitro plates. However, the importance of the third dimension in tissue regeneration has become indispensable to achieve a potential implant to replace the damaged tissue. Therefore, we have prepared porous 3D structures composed uniquely of carbon nanotubes (CNT) and poly(3,4-ethylenedioxythiophene) (PEDOT) that show great potential in the tridimensional differentiation of SH-SY5Y cells into mature neurons. The scaffolds have been manufactured through electropolymerization by applying 1.2 V in a three-electrode cell using a template of sucrose/CNT as a working electrode. By this method, PEDOT/CNT 3D scaffolds were obtained with homogeneous porosities and high conductivity. In vitro analyses showed that an excellent biocompatibility of the scaffold and the presence of high amount of ß-tubulin class III and MAP-II target proteins that mainly expresses in neurons, suggesting the differentiation into neuronal cells already after a week of incubation.

P14ARF: The Absence that Makes the Difference.

P14ARF is a tumor suppressor encoded by the CDKN2a locus that is frequently inactivated in human tumors. P14ARF protein quenches oncogene stimuli by inhibiting cell cycle progression and inducing apoptosis. P14ARF functions can be played through interactions with several proteins. However, the majority of its activities are notoriously mediated by the p53 protein. Interestingly, recent studies suggest a new role of p14ARF in the maintenance of chromosome stability. Here, we deepened this new facet of p14ARF which we believe is relevant to its tumor suppressive role in the cell. To this aim, we generated a monoclonal HCT116 cell line expressing the p14ARF cDNA cloned in the piggyback vector and then induced aneuploidy by treating HCT116 cells with the CENP-E inhibitor GSK923295. P14ARF ectopic re-expression restored the near-diploid phenotype of HCT116 cells, confirming that p14ARF counteracts aneuploid cell generation/proliferation.

A Role for Plant KASH Proteins in Regulating Stomatal Dynamics.

Stomatal movement, which regulates gas exchange in plants, is controlled by a variety of environmental factors, including biotic and abiotic stresses. The stress hormone abscisic acid (ABA) initiates a signaling cascade, which leads to increased H2O2 and Ca2+ levels and F-actin reorganization, but the mechanism of, and connection between, these events is unclear. SINE1, an outer nuclear envelope component of a plant Linker of Nucleoskeleton and Cytoskeleton complex, associates with F-actin and is, along with its putative paralog SINE2, expressed in guard cells. Here, we have determined that Arabidopsis (Arabidopsis thaliana) SINE1 and SINE2 play an important role in stomatal opening and closing. Loss of SINE1 or SINE2 results in ABA hyposensitivity and impaired stomatal dynamics but does not affect stomatal closure induced by the bacterial elicitor flg22. The ABA-induced stomatal closure phenotype is, in part, attributed to impairments in Ca2+ and F-actin regulation. Together, the data suggest that SINE1 and SINE2 act downstream of ABA but upstream of Ca2+ and F-actin. While there is a large degree of functional overlap between the two proteins, there are also critical differences. Our study makes an unanticipated connection between stomatal regulation and nuclear envelope-associated proteins, and adds two new players to the increasingly complex system of guard cell regulation.

Synthesis and anti-metastasis activities of norcantharidin-conjugated carboxymethyl chitosan as a novel drug delivery system.

Carboxymethyl chitosan (CMCS), a water-soluble derivative of chitosan possessing numerous enhanced physicochemical and biological properties, has emerged as a promising biopolymer carrier for new drug delivery. Norcantharidin (NCTD) is an effective anti-tumor compound with severe nephrotoxicity. In this study, norcantharidin-conjugated carboxymethyl chitosan (CMCS-NCTD) was synthesized to reduce systemic toxicity and improve anti-tumor efficiency of NCTD. Our results demonstrated that CMCS-NCTD could significantly inhibit migration of tumor cells both in vitro and in vivo in a dose-dependent manner (P < 0.05). The enhanced anti-tumor effects of CMCS-NCTD were confirmed by inhibiting the growth of solid tumors and extending survival time of tumor-bearing mice. Further investigation for the underlying mechanisms indicated that CMCS-NCTD could inhibit tumor angiogenesis and reduce degradation of extracellular matrix by regulating the expressions of VEGF, MMP-9 and TIMP-1. Overall, our findings suggested that CMCS-NCTD was an excellent polymer derivative for cancer treatment, and CMCS was a promising platform for efficient delivery of anti-cancer drugs.

Differential expression of cyclin-dependent kinases in the adult human retina in relation to CDK inhibitor retinotoxicity.

Cyclin-dependent kinases (CDKs) are a family of kinases associated predominantly with cell cycle control, making CDK inhibitors interesting candidates for anti-cancer therapeutics. However, retinal toxicity (loss of photoreceptors) has been associated with CDK inhibitors, including the pan-CDK inhibitor AG-012896. The purpose of this research was to use a novel planar sectioning technique to determine CDK expression profiles in the ex vivo human retina with the aim of identifying isoforms responsible for CDK retinotoxicity. Four CDK isoforms (CDK11, 16, 17 and 18) were selected as a result of IC50 data comparing neurotoxic (AG-012986 and NVP-1) and non-neurotoxic (dinaciclib and NVP-2) CDK inhibitors, with IC50s at CDK11 showing a clear difference between the neurotoxic and non-neurotoxic drugs. CDK11 was maximally expressed in the photoreceptor layer, whereas CDK16, 17 and 18 showed maximal expression in the inner nuclear layer. CDK5 (an isoform associated with retinal homeostasis) was maximally expressed in the retinal ganglion cell layer. Apart from CDK18, each isoform showed expression in the photoreceptor layer. The human Müller cell line MIO-M1 expressed CDK5, 11, 16 and 17 and AG-01298 (0.02-60 µM) caused a dose-dependent increase in MIO-M1 cell death. In conclusion, CDK11 appears the most likely candidate for mediation of photoreceptor toxicity. RNA profiling can be used to determine the distribution of genes of interest in relation to retinal toxicity in the human retina.

Wafer-Scale Fabrication of Conducting Polymer Hydrogels for Microelectrodes and Flexible Bioelectronics.

Future-oriented directions in neural interface technologies point towards the development of multimodal devices that combine different functionalities such as neural stimulation, neurotransmitter sensing, and drug release within one platform. Conducting polymer hydrogels (CPHs) are suggested as materials for the coating of standard metal electrodes to add functionalities such as local delivery of therapeutic drugs. However, to make such coatings truly useful for multimodal devices, it is necessary to develop process technologies that allow the micropatterning of CPHs onto selected electrode sites. In this study, a wafer-scale fabrication procedure is presented, which is used to coat the CPH, based on the hydrogel P(DMAA-co-5%MABP-co-2,5%SSNa) and the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT), onto flexible neural probes. The resulting material has favorable properties for the generation of recording electrodes and in addition offers a convenient platform for biofunctionalization. By controlling the PEDOT content within the hydrogel matrix, charge injection limits of up to 3.7 mC cm-2 are obtained. Long-term stability is tested by immersing coated samples in phosphate-buffered saline solution at 37 °C for 1 year. Non-cytotoxicity of the coatings is confirmed with a direct cell culture test using a fluorescent neuroblastoma cell line.

Microemulsion formulation of a new biopesticide to control the diamondback moth (Lepidoptera: Plutellidae).

This study was designed to develop a microemulsion formulation of norcantharidin for the control of the diamondback moth (DBM), Plutella xylostella (Linnaeus), a notorious pest of brassica crops worldwide. The oil phase was screened and selected based on norcantharidin solubility while the surfactants were selected on the basis of their efficiency to form microemulsion. Optimized batches were selected using pseudo ternary phase diagrams. The microemulsion system were stabilized using mixtures composed of norcantharidin, surfactants (Tx13 and Tw80), and cosurfactant (ethanol). Its physicochemical characteristics were also demonstrated to have a higher cloud point than 72 °C as well as good thermodynamic and dilution stability. In additon, a subsequent insecticidal bioassay indicated that the acute LC50 for norcantharidin microemulsion to P. xylostella was estimated to be 12.477 mg/L (11.58-13.41, 95% CL). Our results provide an environment-friendly promising alternative to control P. xylostella and possibly contribute to ameliorating any pesticide resistance in P. xylostella.

Benthic periphyton from Pennsylvania, USA is a source for both hepatotoxins (microcystins/nodularin) and neurotoxins (anatoxin-a/homoanatoxin-a).

In 2016, the Pennsylvania Department of Environmental Protection conducted a limited survey of streams in the Susquehanna River basin in Pennsylvania, USA, to screen for microcystins/nodularins, anatoxin-a (ATX) and homoanatoxin-a (HTX). Testing revealed the presence of HTX in samples collected from the Pine Creek basin, with ATX present at lower levels. Microcystins/nodularins (MCs/NODs) were also tested and found to be concomitant, with NOD-R confirmed present by LC-MS/MS.

The 7-phenyl benzoxaborole series is active against Mycobacterium tuberculosis.

We identified a series of novel 7-phenyl benzoxaborole compounds with activity against Mycobacterium tuberculosis. Compounds had a range of activity with inhibitory concentrations (IC90) as low as 5.1 µM and no cytotoxicity against eukaryotic cells (IC50 > 50 µM). Compounds were active against intracellular mycobacteria cultured in THP-1 macrophages. We isolated and characterized resistant mutants with mutations in NADH dehydrogenase (Ndh) or the regulatory protein Mce3R. Mutations suggest that Ndh may be the target of this series.

Do TRPV1 antagonists increase the risk for skin tumourigenesis? A collaborative in vitro and in vivo assessment.

A recent hypothesis suggesting that the pharmacological target TRPV1 (transient receptor potential vanilloid subfamily, member 1) may function as a tumour suppressor, which potentially impacts the development of TRPV1 antagonist therapeutics for a range of conditions. However, little is known about the long-term physiologic effects of TRPV1 blockade in the skin. In vitro and in vivo studies suggested that the potent TRPV1 competitive antagonist AMG-9810 promoted proliferation in N/TERT1 cells (telomerase-immortalised primary human keratinocytes 1) and tumour development in mouse skin that was mediated through EGFR/Akt/mTOR signalling. We attempted to reproduce the reported in vitro and in vivo findings to further explore this hypothesis to understand the underlying mechanism and the risk associated with TRPV1 antagonism in the skin. In vitro proliferation studies using multiple methods and topical application with AMG-9810 and structurally similar TRPV1 antagonists such as SB-705498 and PAC-14028 were performed. Although we confirmed expression of TRPV1 in primary human epidermal keratinocytes (HEKn) and spontaneously immortalised human keratinocytes (HaCaT), we were unable to demonstrate cell proliferation in either cell type or any clear evidence of increased expression of proteins in the EGFR/Akt/mTOR signalling pathway with these molecules. We were also unable to demonstrate skin tumour promotion or underlying molecular mechanisms involved in the EGFR/Akt/mTOR signalling pathway in a single-dose and two-stage carcinogenesis mouse study treated with TRPV1 antagonists. In conclusion, our data suggest that inhibiting the pharmacological function of TRPV1 in skin by specific antagonists has not been considered to be indicative of skin tumour development.

Palytoxin congeners.

Palytoxin, isolated from a zoanthid of the genus Palythoa, is the most potent marine toxin known. Intoxication by palytoxin leads to vasoconstriction, hemorrhage, ataxia, muscle weakness, ventricular fibrillation, pulmonary hypertension, ischemia and death. Palytoxin and its numerous derivatives (congeners) may enter the food chain and accumulate mainly in fishes and crabs, causing severe human intoxication and death following ingestion of contaminated products. Furthermore, toxic effects in individuals exposed via inhalation or skin contact to marine aerosol in coincidence with Ostreopsis blooms, have been reported. Blooms of the benthic dinoflagellate Ostreopsis cf. ovata are a concern in the Mediterranean Sea, since this species produces a wide range of palytoxin-like compounds listed among the most potent marine toxins. Thus, the formerly unsuspected broad distribution of the benthic dinoflagellate Ostreopsis spp. has recently posed a problem of risk assessment for human health. Palytoxin has a strong potential for toxicity in humans and animals, and currently this toxin is of great concern worldwide. This review summarized and discussed the pharmacology and toxicology data of palytoxin and its congeners, including their cytotoxicity, human and animal toxicities. Moreover, the risk assessment and their control strategies including prevention and treatment assays were evaluated.

ß-Catenin Cooperates with CREB Binding Protein to Promote the Growth of Tumor Cells.

BACKGROUND/AIMS: ß-catenin is an integral component of the canonical Wnt signaling pathway, and its mutations are an autosomal recessive cause of colorectal cancer (CRC), medulloblastoma (MDB), and ovarian cancer. Nevertheless, little is known about its function in lung cancers. METHODS: We first knocked down ß-catenin by siRNA to investigate its effects on lung cancer cell proliferation, migration and apoptosis. Then we verified the interaction between ß-catenin and CREB binding protein (CBP) by immunofluoresence and co-immunoprecipition assays. Finally, the expression of ß-catenin and CBP in human lung adenocarcinoma specimens were analyzed by immunohistochemistry assay. RESULTS: ß-catenin knockdown inhibited cell proliferation, promoted apoptosis and suppressed cell migration in A549 and H460 cells accompanied by the decreased expression of Myc, PCNA, VEGF, CD44, MMP-9, MMP-13 and activated bax/caspase-3 pathway. Furthermore, co-immunoprecipition and immunofluoresence analyses revealed that CBP interacted with ß-catenin and contributed to ß-catenin-mediated lung cancer cell growth. Abolishment of their interaction by the Wnt/ß-catenin inhibitor ICG-001 remarkably suppressed cell proliferation. Immunohistochemistry assay of tissue microarrays from patients with lung cancer indicated that both CBP and ß-catenin were highly expressed in tumor tissues and predicted poor prognosis in lung adenocarcinoma patients. CONCLUSIONS: Our study has provided new evidence for the role of ß-catenin in promoting the growth of lung cancer cells through cooperation with CBP, and suggested that dual targeting of ß-catenin and CBP could be a potential therapeutic strategy in lung cancer treatment.

Interacting amino acid replacements allow poison frogs to evolve epibatidine resistance.

Animals that wield toxins face self-intoxication. Poison frogs have a diverse arsenal of defensive alkaloids that target the nervous system. Among them is epibatidine, a nicotinic acetylcholine receptor (nAChR) agonist that is lethal at microgram doses. Epibatidine shares a highly conserved binding site with acetylcholine, making it difficult to evolve resistance yet maintain nAChR function. Electrophysiological assays of human and frog nAChR revealed that one amino acid replacement, which evolved three times in poison frogs, decreased epibatidine sensitivity but at a cost of acetylcholine sensitivity. However, receptor functionality was rescued by additional amino acid replacements that differed among poison frog lineages. Our results demonstrate how resistance to agonist toxins can evolve and that such genetic changes propel organisms toward an adaptive peak of chemical defense.

Mechanisms of chromosomal aberrations induced by sesamin metabolites in Chinese hamster lung cells.

Sesamin is a major lignan in sesame seeds and oil. We previously demonstrated that sesamin induces chromosomal aberrations (CA) in Chinese hamster lung (CHL/IU) cells in the presence of a metabolic activation system (S9 mix), although no genotoxicity was detected in vivo. To clarify the mechanism of CA induction by sesamin, we identified its principal active metabolite. A mono-catechol derivative, [2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabi-cyclo[3.3.0]octane (SC-1)], was previously identified in culture medium when sesamin was incubated with S9 mix. In the present study, we show that SC-1 induces CA in CHL/IU cells but not in human hepatoblastoma (HepG2) cells. SC-1 was unstable in culture medium. Addition of glutathione (GSH) to the incubation mixture decreased the rate of decomposition and also suppressed induction of CA in CHL/IU cells. These results indicate that SC-1 itself may not contribute to the induction of CA. Two GSH adducts of SC-1 were identified when SC-1 was incubated with GSH, suggesting that SC-1 was converted to the semiquinone/quinone form and then conjugated with GSH in the culture medium. Sodium sulfite (a quinone-responsive compound) also suppressed CA induction by SC-1. These findings strongly suggest that SC-1 is oxidized to semiquinone/quinone derivatives extracellularly in culture medium, that these derivatives are responsible for the induction of CA in CHL/IU cells, and therefore that the positive results obtained with sesamin in in vitro CA tests using CHL/IU cells may not be relevant to the assessment of in vivo activity.

Voltage gated ion channels blockade is the underlying mechanism of BIMU8 induced cardiotoxicity.

BIMU8 is a 5-HT4a receptor agonist and used as an experimental drug to counteract opioid induced respiratory depression. In preliminary experiments serious disturbances in ECG were observed in anesthetized rabbits which prompted us to explore the underlying cause of BIMU8 induced abnormal changes in ECG recordings. Electrophysiological experiments were performed on HEK-293 cells expressing hERG, CaV1.2 and NaV1.5 ion channels. In whole-cell recordings BIMU8 effectively blocked these three channels, with IC50 values of 0.06±0.05, 1.46±0.26 and 4.66±0.58µM for hERG, NaV1.5 and CaV1.2, respectively. Additionally it also produced a hyperpolarizing shift of 3.27mV in half maximal activation and 12.87mV in fast inactivation of NaV1.5 channel. These experimental findings indicate that BIMU8 is a potent blocker of hERG, NaV1.5 and CaV1.2 cardiac ion channels thus revealing its proarrhythmic potential.

Cytotoxicity of a Series of Norcantharidin-Inspired Tetrahydroepoxyisoindole Carboxamides.

A series of 28 norcantharidin (NorC)-inspired analogues were accessed via a robust two-step Ugi intramolecular Diels-Alder (IMDA) sequence. Four analogues displayed whole-cell cytotoxicity equipotent to that of NorC and cisplatin against a number of cancer cell lines and a normal breast cell line (MCF10A). Notably, (3S,3aS,6R)-2-benzyl-7-methyl-N-(naphthalen-2-yl)-1-oxo-1,2,3,6-tetrahydro-3a,6-epoxyisoindole-3-carboxamide (trans-27) displayed superior whole-cell activity against breast (MCF-7, GI50 =2.9 µm) and colon (HT29, GI50 =6.4 µm) cancer cell lines relative to the control (cisplatin), which elicited respective GI50 values of 6.5 and 11.3 µm against the aforementioned cell lines. This analogue also displayed improved activity relative to NorC across the breast (MCF-7, GI50 =2.9 µm; NorC GI50 =7.5 µm), ovarian (A2780, GI50 =2.2 µm; NorC GI50 =4.4 µm), and neuroblastoma (BE2-C, GI50 =2.2 µm; NorC GI50 =3.7 µm) cancer cell lines. Structure-activity relationship (SAR) investigations demonstrated that retention of sp2 hybridized connections within the tetrahydroepoxyisoindole carboxamide scaffold is crucial, as aromatization to a phenolic functionality decreased activity, whereas removal of a single olefin bond abolished cytotoxicity. Nonetheless, with respect to the latter, use of crotonic acid as opposed 2-butynoic acid in the Ugi-IMDA sequence imparted a significant improvement to diastereoselectivity, with the cis/trans isomer ratio shifting from ≈1:1.2 to ≈0.5:9.5.