Macrocyclic BACE1 inhibitors with hydrophobic cross-linked structures: Optimization of ring size and ring structure.

Based on the X-ray crystallography of recombinant BACE1 and a hydroxyethylamine-type peptidic inhibitor, we introduced a cross-linked structure between the P1 and P3 side chains of the inhibitor to enhance its inhibitory activity. The P1 and P3 fragments bearing terminal alkenes were synthesized, and a ring-closing metathesis of these alkenes was used to construct the cross-linked structure. Evaluation of ring size using P1 and P3 fragments with various side chain lengths revealed that 13-membered rings were optimal, although their activity was reduced compared to that of the parent compound. Furthermore, the optimal ring structure was found to be a macrocycle with a dimethyl branched substituent at the P3 ß-position, which was approximately 100-fold more active than the non-substituted macrocycle. In addition, the introduction of a 4-carboxymethylphenyl group at the P1' position further improved the activity.

Structure-activity relationship study of hydroxyethylamine isostere and P1' site structure of peptide mimetic BACE1 inhibitors.

An aromatic substituent has been introduced into a known hydroxyethylamine (HEA)-type BACE1 inhibitor containing the superior substrate sequence to enhance inhibitory activity. The HEA-type isosteres bearing different hydroxyl group and methyl group configurations were prepared through a branched synthesis approach using intra- and inter-molecular epoxide opening reactions. The effect of their configuration was evaluated, showing that an R-configuration improved the inhibitory activity, while introduction of a methyl group on the isostere decreased the activity. Based on the non-substituted isostere with an R-configuration, 21 derivatives containing various substituents at the P1' site were synthesized. Our evaluation of the derivatives showed that the structure of the P1' site had a clear effect on activity, and highly potent inhibitor 40g, which showed sub-micromolar activity against recombinant BACE1 (rBACE1), was identified. The docking simulation of 40g with rBACE1 suggested that a carboxymethyl group at the para-position of the P1' benzene ring interacted with Lys285 in the S1' pocket.

Synthesis of Novel Aryloxyethylamine Derivatives and Evaluation of Their in Vitro and in Vivo Neuroprotective Activities.

A series of aryloxyethylamine derivatives were designed, synthesized and evaluated for their biological activity. Their structures were confirmed by 1 H-NMR, 13 C-NMR, FT-IR and HR-ESI-MS. The preliminary screening of neuroprotection of compounds in vitro was detected by MTT, and the anti-ischemic activity in vivo was tested using bilateral common carotid artery occlusion in mice. Most of these compounds showed potential neuroprotective effects against the glutamate-induced cell death in differentiated rat pheochromocytoma cells (PC12 cells), especially for (4-fluorophenyl){1-[2-(4-methoxyphenoxy)ethyl]piperidin-4-yl}methanone, {1-[2-(4-methoxyphenoxy)ethyl]piperidin-4-yl}(4-methoxyphenyl)methanone, (4-bromophenyl){1-[2-(4-methoxyphenoxy)ethyl]piperidin-4-yl}methanone, {1-[2-(4-chlorophenoxy)ethyl]piperidin-4-yl}(4-chlorophenyl)methanone, (4-chlorophenyl)(1-{2-[(naphthalen-2-yl)oxy]ethyl}piperidin-4-yl)methanone, (4-chlorophenyl){1-[2-(4-methoxyphenoxy)ethyl]piperidin-4-yl}methanone and {1-[2-(4-bromophenoxy)ethyl]piperidin-4-yl}(4-chlorophenyl)methanone, which exhibited potent protection of PC12 cells at three doses (0.1, 1.0, 10 µM). Compounds (4-fluorophenyl){1-[2-(4-methoxyphenoxy)ethyl]piperidin-4-yl}methanone, (4-fluorophenyl){1-[2-(naphthalen-2-yloxy)ethyl]piperidin-4-yl}methanone, {1-[2-(4-methoxyphenoxy)ethyl]piperidin-4-yl}(4-methoxyphenyl)methanone and {1-[2-(4-chlorophenoxy)ethyl]piperidin-4-yl}(4-chlorophenyl)methanone possessed the significant prolongation of the survival time of mice subjected to acute cerebral ischemia and decreased the mortality rate at all five doses tested (200, 100, 50, 25, 12.5 mg/kg) and had significant neuroprotective activity. In addition, (4-fluorophenyl){1-[2-(4-methoxyphenoxy)ethyl]piperidin-4-yl}methanone, {1-[2-(4-methoxyphenoxy)ethyl]piperidin-4-yl}(4-methoxyphenyl)methanone and {1-[2-(4-chlorophenoxy)ethyl]piperidin-4-yl}(4-chlorophenyl)methanone possessed outstanding neuroprotection in vitro and in vivo. These compounds can be used as a promising neuroprotective agents for future development of new anti-ischemic stroke agents. Basic structure-activity relationships are also presented.

Effect of Four Novel Bio-Based DES (Deep Eutectic Solvents) on Hardwood Fractionation.

Using the basic principle of construction between a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD), four bio-based deep eutectic solvents (DESs) were prepared in a 1:2 molar ratio of HBA:HBD. 2,3-Dihydroxypropyl-1-triethylammonium chloride ([C9H22N+O2]Cl-) was synthesized from raw glycerol and used as an HBA. Lactic acid, urea, pure glycerol, and ethylene glycol were selected as HBD. Attempts to prepare DESs, using citric acid and benzoic acid as HBDs, were unsuccessful. All these DESs were characterized using FTIR and NMR techniques. Besides, physicochemical parameters such as pH, viscosity, density, and melting point were determined. The behavior of these DES to fractionate olive pomace was studied. Lignin recovery yields spanned between 27% and 39% (w/w) of the available lignin in olive pomace. The best DES, in terms of lignin yield ([C9H22N+O2]Cl- -lactic acid), was selected to perform a scale-up lignin extraction using 40 g of olive pomace. Lignin recovery on the multigram scale was similar to the mg scale (38% w/w). Similarly, for the holocellulose-rich fractions, recovery yields were 34% and 45% for mg and multi-gram scale, respectively. Finally, this DES was used to fractionate four fruit pruning samples. These results show that our novel DESs are alternative approaches to the ionic liquid:triethylammonium hydrogen sulfate and the widely used DES: choline chloride:lactic acid (1:10 molar ratio) for biomass processing.

Improving Covalent Organic Frameworks Fluorescence by Triethylamine Pinpoint Surgery as Selective Biomarker Sensor for Diabetes Mellitus Diagnosis.

The nitrogen-containing imine or hydrazone linked covalent organic frameworks (COFs) are poorly luminescent due to the fluorescence quenching by nitrogen atoms in the linkages, even if highly luminescent units and linkers are employed. The fluorescence quenching pathway to prevent linkage-originated to mitigate the inherent limitations of the linkage is a promising method for luminescent COFs. The generation of N- by deprotonation of the N-H unit eliminates the electron transfer from N lone pair to COF (TpPa-1) and enhances the luminescence. In this work, TpPa-1 achieved turn-on luminescence response with good sensitivity and reproducibility toward triethylamine (TEA) vapor in the process of deprotonation. The fabricated detector offers a viable approach for sensing ppm-level TEA, which can remind people to take timely measures to reduce the environmental hazards caused by TEA. The fluorescent sensor TpPa-1@LE constructed by the products of TpPa-1 and TEA can quantitatively trace biomarker methylglyoxal (MGO) for diabetes mellitus diagnosis in serum system. Furthermore, using TEA and MGO as input signals and the two fluorescence emissions G476 and Y525 as output signals, an advanced analytical device based on two Boolean logic gates with INH and AND function is constructed. This work provides a new strategy for improving the weak luminescence of COF in aqueous solution and realizes selective response to biomarker (MGO) for diabetes mellitus diagnosis.

Rationally designed organelle-specific thermally activated delayed fluorescence small molecule organic probes for time-resolved biological applications.

Two organelle-specific small molecule organic thermally activated delayed fluorescence (TADF) probes were developed, which exhibited the longest average fluorescence lifetime in time-resolved fluorescence imaging studies of cells among so far reported TADF probes.

Highly Selective and Potent Human ß-Secretase†2 (BACE2) Inhibitors against Type†2 Diabetes: Design, Synthesis, X-ray Structure and Structure-Activity Relationship Studies.

Herein we present the design, synthesis, and biological evaluation of potent and highly selective ß-secretase 2 (memapsin 1, beta-site amyloid precursor protein cleaving enzyme 2, or BACE 2) inhibitors. BACE2 has been recognized as an exciting new target for type 2 diabetes. The X-ray structure of BACE1 bound to inhibitor 2 a {N3 -[(1S,2R)-1-benzyl-2-hydroxy-3-[[(1S,2S)-2-hydroxy-1-(isobutylcarbamoyl)propyl]amino]propyl]-5-[methyl(methylsulfonyl)amino]-N1 -[(1R)-1-phenylpropyl]benzene-1,3-dicarboxamide} containing a hydroxyethylamine isostere was determined. Based on this structure, a computational docking study was performed which led to inhibitor 2 a-bound BACE2 models. These were used to optimize the potency and selectivity of inhibitors. A systematic structure-activity relationship study led to the identification of determinants of the inhibitors' potency and selectivity toward the BACE2 enzyme. Inhibitors 2 d [N3 -[(1S,2R)-1-benzyl-2-hydroxy-3-[[(1S,2S)-2-hydroxy-1-(isobutylcarbamoyl)pentyl]amino]propyl]-N1 -methyl-N1 -[(1R)-1-phenylpropyl]benzene-1,3-dicarboxamide; Ki =0.031 nm, selectivity over BACE1: ≈174 000-fold] and 3 l [N1 -((2S,3R)-3-hydroxy-1-phenyl-4-((3-(trifluoromethyl)benzyl)amino)butan-2-yl)-N3 ,5-dimethyl-N3 -((R)-1-phenylethyl)isophthalamide; Ki =1.6 nm, selectivity over BACE1: >500-fold] displayed outstanding potency and selectivity. Inhibitor 3 l is nonpeptide in nature and may pave the way to the development of a new class of potent and selective BACE2 inhibitors with clinical potential.

Varacin-1, a novel analog of varacin C, induces p53-independent apoptosis in cancer cells through ROS-mediated reduction of XIAP.

Varacin C is a promising anticancer agent and possesses acid-promoted and photo-induced DNA-damaging activities. In this study, we synthesized an analog varacin-1 (VCA-1) and examined its anticancer potentials. The results demonstrated that VCA-1 caused dose-dependent apoptotic cell death in cancer cells. Note that this action is independent of p53 status, because VCA-1 induced similar levels of apoptosis in two different panels of cell lines (HCT116 p53- wild-type vs. HCT116 p53-knockout colon cancer cells, and p53-expressing U2OS vs. p53-deficient saos2 osteosarcoma cancer cells). VCA-1-induced apoptosis was found to be mainly via the extrinsic apoptosis pathway involving caspase-8 activation and XIAP reduction. Forced over-expression of XIAP markedly prevented apoptosis, indicating its essential role in VCA-1 induced apoptosis. On the other hand, VCA-1 treatment enhanced intracellular ROS (reactive oxygen species) generation also in a p53-independent manner, and consequently promoted caspase activation. Pretreatment of N-acetyl cysteine (an antioxidant), rather than z-VAD (specific caspase inhibitor), markedly prevented XIAP reduction, suggesting that XIAP reduction may be resulted from oxidative stress. In conclusion, data from this study reveal the essential roles of ROS generation and XIAP reduction in VCA-1-induced apoptosis in cancer cells. VCA-1 may be a novel cancer therapeutic agent, especially in p53-mutant human cancers.

Fast-Acting Small Molecules Targeting Malarial Aspartyl Proteases, Plasmepsins, Inhibit Malaria Infection at Multiple Life Stages.

The eradication of malaria remains challenging due to the complex life cycle of Plasmodium and the rapid emergence of drug-resistant forms of Plasmodium falciparum and Plasmodium vivax. New, effective, and inexpensive antimalarials against multiple life stages of the parasite are urgently needed to combat the spread of malaria. Here, we synthesized a set of novel hydroxyethylamines and investigated their activities in vitro and in vivo. All of the compounds tested had an inhibitory effect on the blood stage of P. falciparum at submicromolar concentrations, with the best showing 50% inhibitory concentrations (IC50) of around 500 nM against drug-resistant P. falciparum parasites. These compounds showed inhibitory actions against plasmepsins, a family of malarial aspartyl proteases, and exhibited a marked killing effect on blood stage Plasmodium. In chloroquine-resistant Plasmodium berghei and P. berghei ANKA infected mouse models, treating mice with both compounds led to a significant decrease in blood parasite load. Importantly, two of the compounds displayed an inhibitory effect on the gametocyte stages (III-V) of P. falciparum in culture and the liver-stage infection of P. berghei both in in vitro and in vivo. Altogether, our findings suggest that fast-acting hydroxyethylamine-phthalimide analogs targeting multiple life stages of the parasite could be a valuable chemical lead for the development of novel antimalarial drugs.

Synthesis and Biological in vitro and in vivo Evaluation of 2-(5-Nitroindazol-1-yl)ethylamines and Related Compounds as Potential Therapeutic Alternatives for Chagas Disease.

Chagas disease, a neglected tropical disease caused by infection with the protozoan parasite Trypanosoma cruzi, is a potentially life-threatening illness that affects 5-8 million people in Latin America, and more than 10 million people worldwide. It is characterized by an acute phase, which is partly resolved by the immune system, but then develops as a chronic disease without an effective treatment. There is an urgent need for new antiprotozoal agents, as the current standard therapeutic options based on benznidazole and nifurtimox are characterized by limited efficacy, toxicity, and frequent failures in treatment. In vitro and in vivo assays were used to identify some new low-cost 5-nitroindazoles as a potential antichagasic therapeutic alternative. Compound 16 (3-benzyloxy-5-nitro-1-vinyl-1H-indazole) showed improved efficiency and lower toxicity than benznidazole in both in vitro and in vivo experiments, and its trypanocidal activity seems to be related to its effect at the mitochondrial level. Therefore, compound 16 is a promising candidate for the development of a new anti-Chagas agent, and further preclinical evaluation should be considered.

Synthesis of 1-Tetrasubstituted 2,2,2-Trifluoroethylamine Derivatives via Palladium-Catalyzed Allylation of sp3 C-H Bonds.

This note describes the construction of tetrasubstituted carbon stereocenters via palladium-catalyzed allylation of sp3 C-H bonds of 2,2,2-trifluoroethylamine derivatives. The presence of 2-pyridyl group of the imines derived from 1-substituted-2,2,2-trifluoroethylamine was key to promoting the reaction efficiently, allowing an access to a variety of 1-allylated 2,2,2-trifluoroethylamine derivatives with tetrasubstituted carbon stereocenters.

Synthesis and Evaluation of Antimicrobial Activity of [R4W4K]-Levofloxacin and [R4W4K]-Levofloxacin-Q Conjugates.

The development of a new class of antibiotics to fight bacterial resistance is a time-consuming effort associated with high-cost and commercial risks. Thus, modification, conjugation or combination of existing antibiotics to enhance their efficacy is a suitable strategy. We have previously reported that the amphiphilic cyclic peptide [R4W4] had antibacterial activity with a minimum inhibitory concentration (MIC) of 2.97 µg/mL against Methicillin-resistant Staphylococcus aureus (MRSA). Herein, we hypothesized that conjugation or combination of the amphiphilic cyclic peptide [R4W4] with levofloxacin or levofloxacin-Q could improve the antibacterial activity of levofloxacin and levofloxacin-Q. Fmoc/tBu solid-phase chemistry was employed to synthesize conjugates of [R4W4K]-levofloxacin-Q and [R4W4K]-levofloxacin. The carboxylic acid group of levofloxacin or levofloxacin-Q was conjugated with the amino group of ß-alanine attached to lysine in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) for 3 h to afford the products. Antibacterial assays were conducted to determine the potency of conjugates [R4W4K]-levofloxacin-Q and [R4W4K]-levofloxacin against MRSA and Klebsiella pneumoniae. Although levofloxacin-Q was inactive even at a concentration of 128 µg/mL, [R4W4K]-levofloxacin-Q conjugate and the corresponding physical mixture showed MIC values of 8 µg/mL and 32 µg/mL against MRSA and Klebsiella pneumonia, respectively, possibly due to the activity of the peptide. On the other hand, [R4W4K]-levofloxacin conjugate (MIC = 32 µg/mL and MIC = 128 µg/mL) and the physical mixture (MIC = 8 µg/mL and 32 µg/mL) was less active than levofloxacin (MIC = 2 µg/mL and 4 = µg/mL) against MRSA and Klebsiella pneumoniae, respectively. The data showed that the conjugation of levofloxacin with [R4W4K] significantly reduced the antibacterial activity compared to the parent analogs, while [R4W4K]-levofloxacin-Q conjugate was more significantly potent than levofloxacin-Q alone.

Design and validation of the first cell-impermeant melatonin receptor agonist.

BACKGROUND AND PURPOSE: The paradigm that GPCRs are able to prolong or initiate cellular signalling through intracellular receptors recently emerged. Melatonin binds to G protein-coupled MT1 and MT2 receptors. In contrast to most other hormones targeting GPCRs, melatonin and its synthetic analogues are amphiphilic molecules easily penetrating into cells, but the existence of intracellular receptors is still unclear mainly due to a lack of appropriate tools. EXPERIMENTAL APPROACH: We therefore designed and synthesized a series of hydrophilic melatonin receptor ligands coupled to the Cy3 cyanin fluorophore to reliably monitor its inability to penetrate cells. Two compounds, one lipophilic and one hydrophilic, were then functionally characterized in terms of their affinity for human and murine melatonin receptors expressed in HEK293 cells and their signalling efficacy. KEY RESULTS: Among the different ligands, ICOA-13 showed the desired properties as it was cell-impermeant and bound to human and mouse MT1 and MT2 receptors. ICOA-13 showed differential activities on melatonin receptors ranging from partial to full agonistic properties for the Gi /cAMP and ERK pathway and ß-arrestin 2 recruitment. Notably, ICOA-13 enabled us to discriminate between Gi /cAMP signalling of the MT1 receptor initiated at the cell surface and neuronal mitochondria. CONCLUSIONS AND IMPLICATIONS: We report here the first cell-impermeant melatonin receptor agonist, ICOA-13, which allows us to discriminate between signalling events initiated at the cell surface and intracellular compartments. Detection of mitochondrial MT1 receptors may have an important impact on the development of novel melatonin receptor ligands relevant for neurodegenerative diseases, such as Huntington disease.

Nickel-Catalyzed Enantioselective Reductive Cross-Coupling of Styrenyl Aziridines.

A Ni-catalyzed reductive cross-coupling of styrenyl aziridines with aryl iodides is reported. This reaction proceeds by a stereoconvergent mechanism and is thus amenable to asymmetric catalysis using a chiral bioxazoline ligand for Ni. The process allows facile access to highly enantioenriched 2-arylphenethylamines from racemic aziridines. Multivariate analysis revealed that ligand polarizability, among other features, influences the observed enantioselectivity, shedding light on the success of this emerging ligand class for enantioselective Ni catalysis.

In vivo evaluation of [18F]FECIMBI-36, an agonist 5-HT2A/2C receptor PET radioligand in nonhuman primate.

We recently reported the radiosynthesis and in vitro evaluation of [18F]-2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-(2-fluoroethoxy)benzyl)ethanamine, ([18F]FECIMBI-36) or ([18F]1), an agonist radioligand for 5HT2A/2C receptors in postmortem samples of human brain. Herein we describe the in vivo evaluation of [18F]FECIMBI-36 in vervet/African green monkeys by PET imaging. PET images show that [18F]FECIMBI-36 penetrates the blood-brain barrier and a low retention of radioactivity is observed in monkey brain. Although the time activity curves indicate a somehow heterogeneous distribution of the radioligand in the brain, the low level of [18F]FECIMBI-36 in brain may limit the use of this tracer for quantification of 5-HT2A/2C receptors by PET.

Convergent 18F-labeling and evaluation of N-benzyl-phenethylamines as 5-HT2A receptor PET ligands.

Positron emission tomography (PET) investigations of the 5-HT2A receptor (5-HT2AR) system can be used as a research tool in diseases such as depression, Alzheimer's disease and schizophrenia. We have previously developed a 11C-labeled agonist PET ligand ([11C]Cimbi-36), and the aim of this study was to identify a 18F-labeled analogue of this PET-ligand. Thus, we developed a convergent radiochemical approach giving easy access to 5 different 18F-labeled ligands structurally related to Cimbi-36 from a common 18F-labeled intermediate. After intravenous injection, all ligands entered the pig brain. However, since within-scan intervention with ketanserin, a known orthosteric 5-HT2A receptor antagonist, did not result in significant blocking, the radioligands seem unsuitable for neuroimaging of the 5-HT2AR in vivo.

Synthesis and Pharmacological Activity of a New Series of 1-(1H-Indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)propan-2-ol Analogs.

ß-Adrenergic receptor antagonists are important therapeutics for the treatment of cardiovascular disorders. In the group of ß-blockers, much attention is being paid to the third-generation drugs that possess important ancillary properties besides inhibiting ß-adrenoceptors. Vasodilating activity of these drugs is produced through different mechanisms, such as nitric oxide (NO) release, ß2 -agonistic action, α1 -blockade, antioxidant action, and Ca(2+) entry blockade. Here, a study on evaluation of the cardiovascular activity of five new compounds is presented. Compound 3a is a methyl and four of the tested compounds (3b-e) are dimethoxy derivatives of 1-(1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)propan-2-ol. The obtained results confirmed that the methyl and dimethoxy derivatives of 1-(1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)propan-2-ol and their enantiomers possess α1 - and ß1 -adrenolytic activities and that the antiarrhythmic and hypotensive effects of the tested compounds are related to their adrenolytic properties.

Synthesis of Chiral α-Trifluoromethylamines with 2,2,2-Trifluoroethylamine as a "Building Block".

The ß-isocupreidine, a cinchonine derived alkaloid, catalyzed asymmetric SN2'-SN2' reaction between N-2,2,2-trifluoroethylisatin ketimines and MBH type carbonates was realized in a simple and efficient way. A series of chiral α-trifluoromethylamines were prepared with excellent yields and stereoselectivities. A subsequent and easy process of deprotection gave γ-trifluoromethyl-α-methylenelactam in a stereoselective manner.

4-hydroxy-benzoic acid (4-diethylamino-2-hydroxy-benzylidene)hydrazide: DFT, antioxidant, spectroscopic and molecular docking studies with BSA.

The Schiff base 4-hydroxy-benzoic acid (4-diethylamino-2-hydroxy-benzylidene) hydrazide (SL) was synthesized and characterized. Its antioxidant activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging action. Being a potent antioxidant its binding ability to the transport protein bovine serum albumin (BSA) was studied using fluorescence quenching and circular dichroism (CD) studies. The binding distance has been calculated by fluorescence resonance energy transfer (FRET) to be 1.85 Å and the Stern-Volmer quenching constant has been calculated to be (3.23 ± 0.45) × 10(5) M(-1). Quantum chemical analysis was carried out for the Schiff base using DFT with B3LYP and 6-311G** and related to the experimentally obtained results. For a deeper understanding of the mechanism of the interaction, the experimental data were complemented by protein-Schiff base docking calculations using Argus Lab.

Synthesis and antiproliferative properties of a new ceramide analog of varacin.

A benzopentasulfane was synthesized in 8 steps with a ceramide attached through an amide bond to the 7-position of the heterocycle structure. The anticancer activity of this synthetic ceramide-benzopolysulfane drug conjugate was analyzed against five human cancer cell lines MDA-MB-231 (breast), DU145 (prostate), MIA PaCa-2 (pancreas), HeLa (cervix), and U251 (glioblastoma). The ceramide-benzopolysulfane conjugate had IC50 values ranging from 10 to >20 µM with complete cell killing at 12.5 µM for MDA-MB-231 and 20 µM for DU145 and HeLa cells. The ceramide-benzopolysulfane conjugate had IC50 values 1.8 and 4.0 times lower than a PEG benzopolysulfane, N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)benzo[f][1,2,3,4,5]-pentathiepine-7-carboxamide, for MDA-MB-231 and DU145 cells, respectively. The parent "unsubstituted" benzopolysulfane, o-C6H4S5, had IC50 values 4.2 times lower and 2.7 times higher than the ceramide benzopolysulfane for MDA-MB-231 and DU145 cells, respectively. The results indicate that the polysulfur linkage is needed for activity since benzenedithiol, o-C6H4(SH)2, had IC50 values greater than 30 µM with little effect on MDA-MB-231 and DU145 cells. Thus, to account for the bioactivity, a bimolecular reaction of cellular thiol with the ceramide benzopolysulfane is a proposed followed by thiozone (S3) extrusion.