2-(3-Indolyl)acetamides and their oxazoline analogues: Anticancer SAR study.

We previously studied 2-aryl-2-(3-indolyl)acetohydroxamates as potential agents against melanoma. These compounds were ineffective in a mouse melanoma xenograft model, most likely due to unfavorable metabolic properties, specifically due to glucuronidation of the N-hydroxyl of the hydoxamic moiety. In the present work, we prepared a series of analogues, 2-aryl-2-(3-indolyl)acetamides and their oxazoline derivatives, which do not contain the N-hydroxyl group. We investigated the structure-activity relationship in both series of compounds and found that the 2-naphthyl is a preferred group at C-2 of the indole in the amide series, whereas the tetralin moiety is favorable in the same location in the oxazoline series. Overall, three compounds in the amide series have GI50 values as low as 0.2-0.3 µM and the results clearly indicate that the N-hydroxyl group is not necessary for high potency in vitro.

Red-Light Activation of a Microtubule Polymerization Inhibitor via Amide Functionalization of the Ruthenium Photocage.

Photoactivated chemotherapy (PACT) is a promising cancer treatment modality that kills cancer cells via photochemical uncaging of a cytotoxic drug. Most ruthenium-based photocages used for PACT are activated with blue or green light, which penetrates sub-optimally into tumor tissues. Here, we report amide functionalization as a tool to fine-tune the toxicity and excited states of a terpyridine-based ruthenium photocage. Due to conjugation of the amide group with the terpyridine π system in the excited state, the absorption of red light (630 nm) increased 8-fold, and the photosubstitution rate rose 5-fold. In vitro, red light activation triggered inhibition of tubulin polymerization, which led to apoptotic cell death both in normoxic (21 % O2 ) and hypoxic (1 % O2 ) cancer cells. In vivo, red light irradiation of tumor-bearing mice demonstrated significant tumor volume reduction (45 %) with improved biosafety, thereby demonstrating the clinical potential of this compound.

Synthesis of ß-Carbolines with Electrocyclic Cyclization of 3-Nitrovinylindoles.

The ß-carboline motif is common in drug discovery and among numerous biologically active natural products. However, its synthetic preparation relies on multistep sequences and heavily depends on the type of substitution required in the core of the desired ß-carboline target. Herein, we demonstrate that this structural motif can be accessed with the microwave-assisted electrocyclic cyclization of heterotrienic aci (alkylideneazinic acid) forms of 3-nitrovinylindoles. The reaction can start with 3-nitrovinylindoles themselves under two sets of conditions. The first one involves microwave irradiation of butanolic solutions of 3-nitrovinylindoles, whereas the second one consists of prior Boc protection of indolic nitrogen, where the protecting group cleanly comes off during the course of the reaction. Alternatively, the reaction can start with 3-nitrovinylindoles prepared in situ using various processes. Finally, the reaction may utilize indoles with ß-nitrostyrenes, likely involving the intermediacy of spirocyclic oxazolines, which rearrange to similar heterotrienic systems undergoing cyclization to ß-carbolines. As part of this study, several natural products, namely, alkaloids norharmane, harmane, and eudistomin N, were synthesized.

Discovery of 5-sulfonyltetrazoles as neuroblastoma differentiation agents.

Previously, we developed an innovative high-content screening (HCS) approach to quantify neuroblastoma cell differentiation based on neurite outgrowth, a morphological differentiation marker of neuroblastoma cells. Here, we report the utilization of this platform to identify 1-methyl-5-(ethylsulfonyl)-1H-tetrazole (3a) as a new neuroblastoma differentiation agent using the ChemBridge DiversetTM commercial synthetic small molecule compound library. We show that this activity can be extended to a group of analogues, which can be accessed via a short two-step synthetic sequence. A new analogue, 5-(allylsulfonyl)-1-methyl-1H-tetrazole (3c) was identified in this synthetic effort as a compound that has even more pronounced differentiation and cytotoxic activities than the original hit compound 3a.

Polygodial, a Sesquiterpene Dialdehyde, Activates Apoptotic Signaling in Castration-Resistant Prostate Cancer Cell Lines by Inducing Oxidative Stress.

Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. Surgery, radiation therapy, chemotherapy, and androgen deprivation therapy are currently the standard treatment options for PCa. These have poor outcomes and result in the development of castration-resistant prostate cancer (CRPC), which is the foremost underlying cause of mortality associated with PCa. Taxanes, diterpene compounds approved to treat hormonal refractory PCa, show poor outcomes in CRPC. Polygodial (PG) is a natural sesquiterpene isolated from water pepper (Persicaria hydropiper), Dorrigo pepper (Tasmannia stipitata), and mountain pepper (Tasmannia lanceolata). Previous reports show that PG has an anticancer effect. Our results show that PG robustly inhibits the cell viability, colony formation, and migration of taxane-resistant CRPC cell lines and induces cell cycle arrest at the G0 phase. A toxicity investigation shows that PG is not toxic to primary human hepatocytes, 3T3-J2 fibroblast co-cultures, and non-cancerous BPH-1 cells, implicating that PG is innocuous to healthy cells. In addition, PG induces oxidative stress and activates apoptosis in drug-resistant PCa cell lines. Our mechanistic evaluation by a proteome profiler-human apoptotic array in PC3-TXR cells shows that PG induces upregulation of cytochrome c and caspase-3 and downregulation of antiapoptotic markers. Western blot analysis reveals that PG activates apoptotic and DNA damage markers in PCa cells. Our results suggest that PG exhibits its anticancer effect by promoting reactive oxygen species generation and induction of apoptosis in CRPC cells.

Reductive Cleavage of 4'H-Spiro[indole-3,5'-isoxazoles] En Route to 2-(1H-Indol-3-yl)acetamides with Anticancer Activities.

Unusual cascade transformation involving ring opening and 1,2-alkyl shift was observed upon the reduction of 4'H-spiro[indole-3,5'-isoxazoles] or 2-(3-oxoindolin-2-yl)acetonitriles with sodium borohydride. This reaction allowed for expeditious and highly efficient preparation of 2-(1H-Indol-3-yl)acetamides with antiproliferative properties.

Amaryllidaceae Alkaloids Decrease the Proliferation, Invasion, and Secretion of Clinically Relevant Cytokines by Cultured Human Colon Cancer Cells.

Alkaloids isolated from members of the Amaryllidaceae plant family are promising anticancer agents. The purpose of the current study was to determine if the isocarbostyrils narciclasine, pancratistatin, lycorane, lycorine, crinane, and haemanthamine inhibit phenomena related to cancer progression in vitro. To achieve this, we examined the proliferation, adhesion, and invasion of cultured human colon cancer cells via MTT assay and Matrigel-coated Boyden chambers. In addition, Luminex assays were used to quantify the secretion of matrix metalloproteinases (MMP) and cytokines associated with poor clinical outcomes. We found that all alkaloids decreased cell proliferation regardless of TP53 status, with narciclasine exhibiting the greatest potency. The effects on cell proliferation also appear to be specific to cancer cells. Narciclasine, lycorine, and haemanthamine decrease both adhesion and invasion but with various potencies depending on the cell line. In addition, narciclasine, lycorine, and haemanthamine decreased the secretion of MMP-1, -2, and -7, as well as the secretion of the cytokines pentraxin 3 and vascular endothelial growth factor. In conclusion, the present study shows that Amaryllidaceae alkaloids decrease phenomena and cytokines associated with colorectal cancer progression, supporting future investigations regarding their potential as multifaceted drug candidates.

In Vitro Effects of Fungal Phytotoxins on Cancer Cell Viability: First Insight into Structure Activity Relationship of a Potent Metabolite of Cochliobolus australiensis Radicinin.

Natural compounds have always represented an important source for new drugs. Although fungi represent one such viable source, to date, no fungal metabolite has been marketed as an anticancer drug. Based on our work with phytotoxins as potential chemical scaffolds and our recent findings involving three phytopathogenic fungi, i.e., Cochliobolus australiensis, Kalmusia variispora and Hymenoscyphus fraxineus, herein, we evaluate the in vitro anti-cancer activity of the metabolites of these fungi by MTT assays on three cancer cell models harboring various resistance levels to chemotherapeutic drugs. Radicinin, a phytotoxic dihydropyranopyran-4,5-dione produced by Cochliobolus australiensis, with great potential for the biocontrol of the invasive weed buffelgrass (Cenchrus ciliaris), showed significant anticancer activity in the micromolar range. Furthermore, a SAR study was carried out using radicinin, some natural analogues and hemisynthetic derivatives prepared by synthetic methods developed as part of work aimed at the potential application of these molecules as bioherbicides. This investigation opens new avenues for the design and synthesis of novel radicinin analogues as potential anticancer agents.

Conversion of Natural Narciclasine to Its C-1 and C-6 Derivatives and Their Antitumor Activity Evaluation: Some Unusual Chemistry of Narciclasine.

During the search for a general, efficient route toward the synthesis of C-1 analogues of narciclasine, natural narciclasine was protected and converted to its C-1 enol derivative using a novel semi-synthetic route. Attempted conversion of this material to its triflate in order to conduct cross-coupling at C-1 resulted in a triflate at C-6 that was successfully coupled with several functionalities. Four novel compounds were fully deprotected after seven steps and subjected to evaluation for cytotoxic activity against three cancer cell lines. Only one derivative showed moderate activity compared to that of narciclasine. Spectral and physical data are provided for all new compounds.

Design and Synthesis of C-1 Methoxycarbonyl Derivative of Narciclasine and Its Biological Activity.

A 15-step chemoenzymatic total synthesis of C-1 methoxycarbonyl narciclasine (10) was accomplished. The synthesis began with the toluene dioxygenase-mediated dihydroxylation of ortho-dibromobenzene to provide the corresponding cis-dihydrodiol (12) as a single enantiomer. Further key steps included a nitroso Diels-Alder reaction and an intramolecular Heck cyclization. The C-1 homolog 10 was tested and evaluated for antiproliferative activity against natural narciclasine (1) as the positive control. Experimental and spectral data are reported for all novel compounds.

Three-component assembly of stabilized fluorescent isoindoles.

The tandem addition of an amine and a thiol to an aromatic dialdehyde engages a selective three-component assembly of a fluorescent isoindole. While an attractive approach for diversity-based fluorophore discovery, isoindoles are typically unstable and present considerable challenges for their practical utility. We found that introduction of electron-withdrawing substituents into the dialdehyde component affords stable isoindole products in one step with acceptable yields and high purity.

Medicinal chemical optimization of fluorescent pyrrole-based COX-2 probes.

Recent studies have demonstrated the ability of human prostaglandin-endoperoxide synthase 2 (COX-2) to guide the formation of fluorescent pyrroles through the Paal-Knorr reaction resulting in the discovery of a central motif. This initial discovery prompted further exploration of this motif for the design of COX-2 inhibitors through the modifications of the substituents on the pyrrole core. This effort led to the discovery of a set of pyrroles whose activity was comparable to Celecoxib, an orally prescribed nonsteroidal anti-inflammatory COX-2 inhibitor. Furthermore, structure-activity relationship (SAR) data, important for the discovery of COX-2 inhibitors, has been obtained.

Polygodial and Ophiobolin A Analogues for Covalent Crosslinking of Anticancer Targets.

In a search of small molecules active against apoptosis-resistant cancer cells, including glioma, melanoma, and non-small cell lung cancer, we previously prepared α,ß- and γ,δ-unsaturated ester analogues of polygodial and ophiobolin A, compounds capable of pyrrolylation of primary amines and demonstrating double-digit micromolar antiproliferative potencies in cancer cells. In the current work, we synthesized dimeric and trimeric variants of such compounds in an effort to discover compounds that could crosslink biological primary amine containing targets. We showed that such compounds retain the pyrrolylation ability and possess enhanced single-digit micromolar potencies toward apoptosis-resistant cancer cells. Target identification studies of these interesting compounds are underway.

[3 + 2]-Annulation of pyridinium ylides with 1-chloro-2-nitrostyrenes unveils a tubulin polymerization inhibitor.

Indolizines and pyrazolo[1,5-a]pyridines were prepared via [3 + 2]-cycloaddition of pyridinium ylides to 1-chloro-2-nitrostyrenes. The synthesized molecules were evaluated for antiproliferative activities against a BE(2)-C neuroblastoma cell line with several compounds decreasing the viability of cancer cells. Indolizine 9db showed higher potency than that of all-trans-retinoic acid, an approved cancer drug. Mechanistically, it was found to inhibit tubulin polymerization and it is thus proposed that the discovered chemistry can be exploited for the development of novel microtubule-targeting anticancer agents.

Lessons in Organic Fluorescent Probe Discovery.

Fluorescent probes have gained profound use in biotechnology, drug discovery, medical diagnostics, molecular and cell biology. The development of methods for the translation of fluorophores into fluorescent probes continues to be a robust field for medicinal chemists and chemical biologists, alike. Access to new experimental designs has enabled molecular diversification and led to the identification of new approaches to probe discovery. This review provides a synopsis of the recent lessons in modern fluorescent probe discovery.

Epithelial-mesenchymal transition sensitizes breast cancer cells to cell death via the fungus-derived sesterterpenoid ophiobolin A.

The epithelial-mesenchymal transition (EMT) imparts properties of cancer stem-like cells, including resistance to frequently used chemotherapies, necessitating the identification of molecules that induce cell death specifically in stem-like cells with EMT properties. Herein, we demonstrate that breast cancer cells enriched for EMT features are more sensitive to cytotoxicity induced by ophiobolin A (OpA), a sesterterpenoid natural product. Using a model of experimentally induced EMT in human mammary epithelial (HMLE) cells, we show that EMT is both necessary and sufficient for OpA sensitivity. Moreover prolonged, sub-cytotoxic exposure to OpA is sufficient to suppress EMT-imparted CSC features including sphere formation and resistance to doxorubicin. In vivo growth of CSC-rich mammary cell tumors, is suppressed by OpA treatment. These data identify a driver of EMT-driven cytotoxicity with significant potential for use either in combination with standard chemotherapy or for tumors enriched for EMT features.

Synthesis and biological evaluation of 10-benzyloxy-Narciclasine.

A chemoenzymatic convergent synthesis of 10-benzyloxy narciclasine from bromobenzene was accomplished in 16 steps. The key transformations included toluene dioxygenase-mediated hydroxylation, nitroso Diels-Alder reaction and intramolecular Heck cyclization. The unnatural derivative of narciclasine was subjected to biological evaluation and its activity was compared to other C-10 and C-7 compounds prepared previously.

Activity of natural and synthetic polygodial derivatives against Trypanosoma cruzi amastigotes, trypomastigotes and epimastigotes.

Our laboratories have been investigating biological effects of a sesquiterpenoid polygodial and its natural and synthetic analogues. Herein, we report the evaluation of these compounds against the three forms of Trypanosoma cruzi, amastigotes, trypomastigotes and epimastigotes. Although polygodial was found to be poorly active, its natural congener epipolygodial and synthetic Wittig-derived analogues showed low micromolar potency against all three forms of the parasite. Synthetic α,ß-unsaturated phosphonate 9 compared favorably with clinically approved drugs benznidazole and nifurtimox, and was effective against trypomastigotes, toward which benznidazole showed no activity.[Formula: see text].

Deciphering the chemical instability of sphaeropsidin A under physiological conditions - degradation studies and structural elucidation of the major metabolite.

The fungal metabolite sphaeropsidin A (SphA) has been recognised for its promising cytotoxicity, particularly towards apoptosis- and multidrug-resistant cancers. Owing to its intriguing activity, the development of SphA as a potential anticancer agent has been pursued. However, this endeavour is compromised since SphA exhibits poor physicochemical stability under physiological conditions. Herein, SphA's instability in biological media was explored utilizing LC-MS. Notably, the degradation tendency was found to be markedly enhanced in the presence of amino acids in the cell medium utilized. Furthermore, the study investigated the presence of degradation adducts, including the identification, isolation and structural elucidation of a major degradation metabolite, (4R)-4,4',4'-trimethyl-3'-oxo-4-vinyl-4',5',6',7'-tetrahydro-3'H-spiro[cyclohexane-1,1'-isobenzofuran]-2-ene-2-carboxylic acid. Considering the reduced cytotoxic potency of aged SphA solutions, as well as that of the isolated degradation metabolite, the reported antiproliferative activity has been attributed primarily to the parent compound (SphA) and not its degradation species. The fact that SphA continues to exhibit remarkable bioactivity, despite being susceptible to degradation, motivates future research efforts to address the challenges associated with this instability impediment.

Nitroalkanes as electrophiles: synthesis of triazole-fused heterocycles with neuroblastoma differentiation activity.

We discovered a reaction of nitroalkanes with 2-hydrazinylquinolines, 2-hydrazinylpyridines and bis-2,4-dihydrazinylpyrimidines in polyphosphoric acid (PPA) affording 1,2,4-triazolo[4,3-a]quinolines, 1,2,4-triazolo[4,3-a]pyridines and bis[1,2,4]triazolo[4,3-a:4',3'-c]pyrimidines, respectively. The reaction expands the scope of heterocyclic annulations involving phosphorylated nitronates, believed to be the electrophilic intermediates formed from nitroalkanes in PPA. Several of the synthesized triazoles showed promising anticancer activity by inducing differentiation in neuroblastoma cancer cells. Due to the urgent need for novel differentiation agents for neuroblastoma therapy, this finding warrants further evaluation of this class of compounds against neuroblastoma.