Total Synthesis and Cytotoxicity Evaluation of Pareitropone and Analogues.

A concise synthesis of pareitropone by oxidative cyclization of a phenolic nitronate is delineated. The use of TMSOTf as an additive to promote the facile formation of a strained norcaradiene intermediate provides convenient access to highly condensed multicyclic tropones in high yields. This synthesis is modular, efficient, and scalable, highlighting the synthetic utility of radical anion coupling reactions in annulation reactions. This work is discussed in the context of total syntheses of the tropoloisoquinoline alkaloids. Also included are the preparation of several congeners and a brief description of their biological activities.

A Chlorine-Atom-Controlled Terminal-Epoxide-Initiated Bicyclization Cascade Enables a Synthesis of the Potent Cytotoxins Haterumaimides J and K.

Haterumaimide J (hatJ) is reportedly the most cytotoxic member of the lissoclimide family of labdane diterpenoids. The unusual functional group arrangement of hatJ-C18 oxygenation and C2 chlorination-resisted our efforts at synthesis until we adopted an approach based on rarely studied terminal epoxide-based cation-π bicyclizations that is described herein. Using the C2-chlorine atom as a key stereocontrol element and a furan as a nucleophilic terminator, the key structural features of hatJ were rapidly constructed. The 18-step stereoselective synthesis features applications of chiral pool starting materials, and catalyst-, substrate-, and auxiliary-based stereocontrol. Access to hatJ and its acetylated congener hatK permitted their biological evaluation against aggressive human cancer cell lines.

6BIO Enhances Oligonucleotide Activity in Cells: A Potential Combinatorial Anti-androgen Receptor Therapy in Prostate Cancer Cells.

Approximately 15%-25% of men diagnosed with prostate cancer do not survive their disease. The American Cancer Society estimated that for the year 2016 the number of prostate cancer deaths will be 26,120. Thus, there is a critical need for novel approaches to treat this deadly disease. Using high-throughput small-molecule screening, we found that the small molecule 6-bromo-indirubin-3'-oxime (6BIO) significantly improves the targeting of antisense oligonucleotides (ASOs) delivered by gymnosis (i.e., in the absence of any transfection reagents) in both the cell cytoplasm and the nucleus. Furthermore, as a single agent, 6BIO had the unexpected ability to simultaneously downregulate androgen receptor (AR) expression and AR signaling in prostate cancer cells. This includes downregulating levels of the AR-V7, a drug-resistance-related AR splice variant that is important in the progression of prostate cancer. Combining 6BIO and an anti-AR oligonucleotide (AR-ASO) can augment the downregulation of AR expression. We also demonstrated that 6BIO enhances ASO function and represses AR expression through the inhibition of the two main glycogen synthase kinase 3 (GSK-3) isoforms: GSK-3α and GSK-3ß activity. Our findings provide a rationale for the use of 6BIO as a single agent or as part of a combinatorial ASO-based therapy in the treatment of human prostate cancer.

Cinnamon extract reduces VEGF expression via suppressing HIF-1α gene expression and inhibits tumor growth in mice.

Although many anti-VEGF agents are available for cancer treatment, side effects of these agents limit their application for cancer treatment and prevention. Here we studied the potential use of a diet-based agent as an inhibitor for VEGF production. Using a VEGF reporter assay, our data showed that an extract from cinnamon (CE) was a potent inhibitor of VEGF production in human cancer cells and suggested inhibition might be mediated through the suppression of HIF-1α gene expression and protein synthesis. Furthermore, CE treatment was found to inhibit expression and phosphorylation of STAT3 and AKT, which are key factors in the regulation of HIF-1α expression, and significantly reduce angiogenesis potential of cancer cells by migration assay. Consistent with these results, we observed significant suppression of VEGF expression, blood vessel formation, and tumor growth in a human ovarian tumor model in mice treated with CE. Cinnamaldehyde, a major component in cinnamon, was identified as one active component in CE that inhibits VEGF expression. Taken together, our findings provide a novel mechanism underlying anti-angiogenic and anti-tumor actions of CE and support the potential use of CE in cancer prevention and treatment. © 2016 Wiley Periodicals, Inc.

Site-Selective Aliphatic C-H Chlorination Using N-Chloroamides Enables a Synthesis of Chlorolissoclimide.

Methods for the practical, intermolecular functionalization of aliphatic C-H bonds remain a paramount goal of organic synthesis. Free radical alkane chlorination is an important industrial process for the production of small molecule chloroalkanes from simple hydrocarbons, yet applications to fine chemical synthesis are rare. Herein, we report a site-selective chlorination of aliphatic C-H bonds using readily available N-chloroamides and apply this transformation to a synthesis of chlorolissoclimide, a potently cytotoxic labdane diterpenoid. These reactions deliver alkyl chlorides in useful chemical yields with substrate as the limiting reagent. Notably, this approach tolerates substrate unsaturation that normally poses major challenges in chemoselective, aliphatic C-H functionalization. The sterically and electronically dictated site selectivities of the C-H chlorination are among the most selective alkane functionalizations known, providing a unique tool for chemical synthesis. The short synthesis of chlorolissoclimide features a high yielding, gram-scale radical C-H chlorination of sclareolide and a three-step/two-pot process for the introduction of the ß-hydroxysuccinimide that is salient to all the lissoclimides and haterumaimides. Preliminary assays indicate that chlorolissoclimide and analogues are moderately active against aggressive melanoma and prostate cancer cell lines.

The discovery of new cytotoxic pyrazolopyridine derivatives.

A number of new 3,7-disubstituted pyrazolo[3,4-c]pyridines have been designed and synthesized from suitable 2-aminopyridines. The antiproliferative activity of the derivatives was determined against the pancreatic MIA PaCa-2 and ovarian SCOV3 cancer cell-lines. IC50 values of the most promising analogue 46 lie in the submicromolar or low micromolar range. Furthermore, compound 46 shows similar inhibitory activities against DU145, A2058 and PC-3 cancer cells, blocks the cell cycle at the G0/G1 phase and induce apoptosis, as determined by the appearance of apoptotic nuclei.

Natural-Based Indirubins Display Potent Cytotoxicity toward Wild-Type and T315I-Resistant Leukemia Cell Lines.

Drug resistance in chronic myelogenous leukemia (CML) requires the development of new CML chemotherapeutic drugs. Indirubin, a well-known mutikinase inhibitor, is the major active component of "Danggui Longhui Wan", a Chinese traditional medicine used for the treatment of CML symptoms. An in-house collection of indirubin derivatives was screened at 1 µM on wild-type and imatinib-resistant T315I mutant CML cells. Herein are reported that only 15 analogues of the natural 6-bromoindirubin displayed potent cytotoxicity in the submicromolar range. Kinase assays in vitro show that eight out of the 15 active molecules strongly inhibited both c-Src and Abl oncogenic kinases in the nanomolar range. Most importantly, these eight molecules blocked the activity of T315I mutant Abl kinase at the submicromolar level and with analogue 22 exhibiting inhibitory activity at the low nanomolar range. Docking calculations suggested that active indirubins might inhibit T315I Abl kinase through an unprecedented binding to both active and Src-like inactive conformations. Analogue 22 is the first derivative of a natural product identified as an inhibitor of wild-type and imatinib-resistant T315I mutant Abl kinases.

Stereocontrolled enantioselective total synthesis of the [2+2] quadrigemine alkaloids.

A unified strategy for enantioselective total synthesis of all stereoisomers of the 2+2 family of quadrigemine alkaloids is reported. In this approach, two enantioselective intramolecular Heck reactions are carried out at the same time on precursors fashioned in four steps from either meso- or (+)-chimonanthine to form the two critical quaternary carbons of the peripheral cyclotryptamine rings of these products. Useful levels of catalyst control are realized in either desymmetrizing a meso precursor or controlling diastereoselectivity in elaborating C2-symmetic intermediates. None of the synthetic quadrigemines are identical with alkaloids isolated previously and referred to as quadrigemines A and E. In addition, we report improvements in our previous total syntheses of (+)- or (-)-quadrigemine C that shortened the synthetic sequence to 10 steps and provided these products in 2.2% overall yield from tryptamine.

Enantioselective synthesis of α-secondary and α-tertiary piperazin-2-ones and piperazines by catalytic asymmetric allylic alkylation.

The asymmetric palladium-catalyzed decarboxylative allylic alkylation of differentially N-protected piperazin-2-ones allows the synthesis of a variety of highly enantioenriched tertiary piperazine-2-ones. Deprotection and reduction affords the corresponding tertiary piperazines, which can be employed for the synthesis of medicinally important analogues. The introduction of these chiral tertiary piperazines resulted in imatinib analogues which exhibited comparable antiproliferative activity to that of their corresponding imatinib counterparts.

Teriflunomide/leflunomide synergize with chemotherapeutics by decreasing mitochondrial fragmentation via DRP1 in SCLC.

Although up to 80% small cell lung cancer (SCLC) patients' response is good for first-line chemotherapy regimen, most patients develop recurrence of the disease within weeks to months. Here, we report cytostatic effect of leflunomide (Leflu) and teriflunomide (Teri) on SCLC cell proliferation through inhibition of DRP1 phosphorylation at Ser616 and decreased mitochondrial fragmentation. When administered together, Teri and carboplatin (Carbo) act synergistically to significantly inhibit cell proliferation and DRP1 phosphorylation, reduce abundance of intermediates in pyrimidine de novo pathway, and increase apoptosis and DNA damage. Combination of Leflu&Carbo has anti-tumorigenic effect in vivo. Additionally, lurbinectedin (Lur) and Teri potently and synergistically inhibited spheroid growth and depleted uridine and DRP1 phosphorylation in mouse tumors. Our results suggest combinations of Carbo and Lur with Teri or Leflu are efficacious and underscore how the relationship between DRP1/DHODH and mitochondrial plasticity serves as a potential therapeutic target to validate these treatment strategies in SCLC clinical trials.

General approach for preparing epidithiodioxopiperazines from trioxopiperazine precursors: enantioselective total syntheses of (+)- and (-)-gliocladine C, (+)-leptosin D, (+)-T988C, (+)-bionectin A, and (+)-gliocladin A.

A common strategy for preparing tryptophan-derived epidithiodioxopiperazine (ETP) natural products containing a hydroxyl substituent adjacent to a quaternary carbon stereocenter is reported. This strategy is exemplified by enantioselective total syntheses of four heptacyclic ETP natural products--gliocladine C (6), leptosin D (7), T988C (8), and bionectin A (9)--starting with the di-(tert-butoxycarbonyl) derivative 17 of the trioxopiperazine natural product gliocladin C, which is readily available by enantioselective chemical synthesis. In addition, total syntheses of the enantiomer of gliocladine C (ent-6) and gliocladin A (11), the di(methylthio) congener of bionectin A, are reported. These syntheses illustrate a synthetic strategy wherein diversity in the dioxopiperazine unit of ETP natural products is introduced at a late stage in a synthetic sequence. In vitro cytotoxicity of compounds in this series against invasive human prostrate (DU145) and melanoma (A2058) cancer cell lines is described and compared to that of chaetocin A (4).

Amphidinolide B: total synthesis, structural investigation, and biological evaluation.

The total syntheses of amphidinolide B1 and the proposed structure of amphidinolide B2 have been accomplished. Key aspects of this work include the development of a practical, non-transition-metal-mediated method for the construction of the C13-C15 diene, the identification of α-chelation and dipole minimization models for diastereoselective methyl ketone aldol reactions, the discovery of a spontaneous Horner-Wadsworth-Emmons macrocyclization strategy, and the development of a novel late stage method for construction of an allylic epoxide moiety. The originally proposed structure for amphidinolide B2 and diastereomers thereof display potent antitumor activities with IC50 values ranging from 3.3 to 94.5 nM against human solid and blood tumor cells. Of the different stereoisomers, the proposed structure of amphidinolide B2 is over 12-fold more potent than the C8,9-epimer and C18-epimer in human DU145 prostate cancer cells. These data suggest that the epoxide stereochemistry is a significant factor for anticancer activity.

Cytotoxic prenylated acetophenone dimers from Acronychia pedunculata.

Three new acetophenone dimers or Acronychia-type acetophenones, acropyrone (1), acropyranol A (2), and acropyranol B (3), were isolated from the trunk bark of Acronychia pedunculata and structurally characterized, together with four known acetophenone dimers, acrovestone (4), acrovestenol (5), acrofolione A (6), and acrofolione B (7), the acetophenone monomer acronyline (8), and four furoquinoline alkaloids. The chemical structures of the new isolated compounds were elucidated unambiguously by spectroscopic data analysis. The cytotoxic activities of the isolated acetophenone dimers were evaluated against the DU145 prostate and A2058 melanoma human cancer cell lines as well as the NHDF normal cell line. Acrovestone (4) and acrovestenol (5) exhibited substantial cytotoxicity, with IC(50) values of 0.38 and 2.8 µM against A2058 melanoma cells as well as 0.93 and 2.7 µM against DU145 prostate cancer cells, respectively.

Targeting the non-ATP-binding pocket of the MAP kinase p38γ mediates a novel mechanism of cytotoxicity in cutaneous T-cell lymphoma (CTCL).

We describe here for the first time a lipid-binding-domain (LBD) in p38γ mitogen-activated protein kinase (MAPK) involved in the response of T cells to a newly identified inhibitor, CSH71. We describe how CSH71, which binds to both the LBD and the ATP-binding pocket of p38γ, is selectively cytotoxic to CTCL Hut78 cells but spares normal healthy peripheral blood mononuclear (PBMC) cells, and propose possible molecular mechanisms for its action. p38γ is a key player in CTCL development, and we expect that the ability to regulate its expression by specifically targeting the lipid-binding domain will have important clinical relevance. Our findings characterize novel mechanisms of gene regulation in T lymphoma cells and validate the use of computational screening techniques to identify inhibitors for therapeutic development.

Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling.

As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for anti-angiogenesis treatment. However, the side effects associated with most anti-VEGF agents limit their chronic use. Identification of naturally occurring VEGF inhibitors derived from diet is a potential alternative approach, with the advantage of known safety. To isolate natural inhibitors of VEGF, we established an in vitro tyrosine kinase assay to screen for diet-based agents that suppress VEGFR2 kinase activity. We found that a water-based extract from cinnamon (cinnamon extract, CE), one of the oldest and most popular spices, was a potent inhibitor of VEGFR2 kinase activity, directly inhibiting kinase activity of purified VEGFR2 as well as mitogen-activated protein kinase- and Stat3-mediated signaling pathway in endothelial cells. As a result, CE inhibited VEGF-induced endothelial cell proliferation, migration and tube formation in vitro, sprout formation from aortic ring ex vivo and tumor-induced blood vessel formation in vivo. Depletion of polyphenol from CE with polyvinylpyrrolidone abolished its anti-angiogenesis activity. While cinnamaldehyde, a component responsible for CE aroma, had little effect on VEGFR2 kinase activity, high-performance liquid chromatography-purified components of CE, procyanidin type A trimer (molecular weight, 864) and a tetramer (molecular weight, 1152) were found to inhibit kinase activity of purified VEGFR2 and VEGFR2 signaling, implicating procyanidin oligomers as active components in CE that inhibit angiogenesis. Our data revealed a novel activity in cinnamon and identified a natural VEGF inhibitor that could potentially be useful in cancer prevention and/or treatment.

Synthesis and anticancer activities of ageladine A and structural analogs.

A series of ageladine A analogs that include 2-aminoimidazo[4,5-c]azepines (seven-membered rings) and 2-amino-3H-imidazo[4,5-c]pyridine (six-membered rings) derivatives were synthesized and evaluated for their anticancer effects against several human cancer cell lines and MMP-2 inhibition in vitro. Only compounds possessing the aromatic azepine (seven-membered ring) core showed anticancer activity with IC(50) values in the low micromolar range.

Bioactive Cycloperoxides Isolated from the Puerto Rican Sponge Plakortis halichondrioides.

Two new five-membered-ring polyketide endoperoxides, epiplakinic acid F methyl ester (1) and epiplakinidioic acid (3), and a peroxide-lactone, plakortolide J (2), were isolated from the Puerto Rican sponge Plakortis halichondrioides, along with two previously reported cyclic peroxides, 4 and 5. The structures of the new metabolites were determined by spectroscopic and chemical analyses. The absolute stereostructures of 1, 2, and 5 were determined by degradation reactions followed by application of Kishi's method for the assignment of absolute configuration of alcohols. Biological screening of cycloperoxides 1-5 and semisynthetic analogues 7-12 for cytotoxic activity against various human tumor cell lines revealed that compounds 3, 4, and 11 are very active. Upon assaying for antimalarial and antitubercular activity, some of the compounds tested showed strong activity against the pathogenic microbes Plasmodium falciparum and Mycobacterium tuberculosis.

Design and Synthesis of New Substituted Pyrazolopyridines with Potent Antiproliferative Activity.

BACKGROUND: Purine isosteres are often endowed with interesting pharmacological properties, due to their involvement in cellular processes replacing the natural purines. Among these compounds, pyrazolopyridines are under active investigation for potential anticancer properties. OBJECTIVES: Based on previously discovered substituted pyrazolopyridines with promising antiproliferative activity, we designed and synthesized new, suitably substituted analogues aiming to investigate their potential activity and contribute to SAR studies of this class of bioactive compounds. METHODS: The new compounds were synthesized using suitably substituted 2-amino-4-picolines, which upon ring-closure provided substituted pyrazolo[3,4-c] pyridine-5-carbonitriles that served as key intermediates for the preparation of the target 3,5,7 trisubstituted derivatives. The antiproliferative activity of 31 new target derivatives was evaluated against three cancer cell lines (MIA PaCa-2, PC-3 and SCOV3), whereas cell-cycle perturbations of exponentially growing PC-3 cells, using three selected derivatives were also performed. RESULTS: Eight compounds displayed IC50 values in the low µM range, allowing the extraction of interesting SAR's. Two of the most potent compounds against all cell lines share a common pattern, by accumulating cells at the G0/G1 phase. From this project, a new carboxamidine-substituted hit has emerged. CONCLUSION: Among the new compounds, those possessing the 3-phenylpyrazolo[3,4-c]pyridine scaffold, proved to be worth investigating and the majority of them showed strong cytotoxic activity against all cell lines, with IC50 values ranging from 0.87-4.3 µM. A carboxamidine analogue that resulted from the synthetic procedure, proved to be highly active against the cancer cells and could be considered as a useful lead for further optimization.

Sorafenib inhibits signal transducer and activator of transcription 3 signaling associated with growth arrest and apoptosis of medulloblastomas.

Medulloblastomas are the most frequent malignant brain tumors in children. Sorafenib (Nexavar, BAY43-9006), a multikinase inhibitor, blocks cell proliferation and induces apoptosis in a variety of tumor cells. Sorafenib inhibited proliferation and induced apoptosis in two established cell lines (Daoy and D283) and a primary culture (VC312) of human medulloblastomas. In addition, sorafenib inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) in both cell lines and primary tumor cells. The inhibition of phosphorylated STAT3 (Tyr(705)) occurs in a dose- and time-dependent manner. In contrast, AKT (protein kinase B) was only decreased in D283 and VC312 medulloblastoma cells and mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2) were not inhibited by sorafenib in these cells. Both D-type cyclins (D1, D2, and D3) and E-type cyclin were down-regulated by sorafenib. Also, expression of the antiapoptotic protein Mcl-1, a member of the Bcl-2 family, was decreased and correlated with apoptosis induced by sorafenib. Finally, sorafenib suppressed the growth of human medulloblastoma cells in a mouse xenograft model. Together, our data show that sorafenib blocks STAT3 signaling as well as expression of cell cycle and apoptosis regulatory proteins, associated with inhibition of cell proliferation and induction of apoptosis in medulloblastomas. These findings provide a rationale for treatment of pediatric medulloblastomas with sorafenib.

Preliminary pharmacokinetic study of the anticancer 6BIO in mice using an UHPLC-MS/MS approach.

Indirubins represent a group of natural and synthetic products with bio-activities against numerous human cancer cell lines acting by inhibiting protein kinases. The natural sources of indirubins are plants of Isatis sp., Indigofera sp., and Polygonum sp., recombinant bacteria, mammalian urine and some marine mollusks. Specifically, the halogenated derivative 6-bromo indirubin-3'-oxime (6BIO) possesses increased selectivity against GSK-3. However, to our knowledge, no analytical method to determine 6BIO in biological fluids has been developed till now. Therefore, a rapid, sensitive and high throughput UHPLC-MS/MS methods were developed and validated to evaluate the concentrations of 6BIO in mice plasma. Plasma samples were pre-treated by protein precipation using cold mixture of methanol: acetonitrile (9:1, v/v) and separations were carried out on a Hypersil Gold C18 column (50 × 2.1 mm i.d.; 1.9 µm p.s.) using 0.1% acetic acid and methanol as mobile phase at a flow rate of 500 mL/min in a gradient mode. For quantitation, a hybrid LTQ-Orbitrap MS equipped with an electro-spray ionization source was used applying a selected reaction monitoring (SRM) option. The monitored transitions were m/z 354.0 → 324.0 for 6BIO and 297.1 → 282.1 for afromorsin (used as the internal standard) in the negative mode. Following the EMA, ICH and FDA guidelines for validation of analytical procedures, the assay method was fully validated in terms of selectivity, linearity, recovery, matrix effect, accuracy, precision, stability, and robustness. The validated methods were successfully applied to the pharmacokinetic studies of 6BIO following an oral administration to mice at the dose of 50 mg/kg. The results indicated that 6BIO possesses a Tmax of 30 min, a half-life of 1 h, and low plasma bioavailability.