A Phase I Study of Dinaciclib in Combination With MK-2206 in Patients With Advanced Pancreatic Cancer.

The combination of drugs targeting Ral and PI3K/AKT signaling has antitumor efficacy in preclinical models of pancreatic cancer. We combined dinaciclib (small molecule cyclin dependent kinase inhibitor with MK-2206 (Akt inhibitor) in patients with previously treated/metastatic pancreatic cancer. Patients were treated with dinaciclib (6-12 mg/m2 i.v.) and MK-2206 (60-135 mg p.o.) weekly. Tumor biopsies were performed to measure pAKT, pERK, and Ki67 at baseline and after one completed cycle (dose level 2 and beyond). Thirty-nine patients participated in the study. The maximum tolerated doses were dinaciclib 9 mg/m2 and MK-2206 135 mg. Treatment-related grade 3 and 4 toxicities included neutropenia, lymphopenia, anemia, hyperglycemia, hyponatremia, and leukopenia. No objectives responses were observed. Four patients (10%) had stable disease as their best response. At the recommended dose, median survival was 2.2 months. Survival rates at 6 and 12 months were 11% and 5%, respectively. There was a nonsignificant reduction in pAKT composite scores between pretreatment and post-treatment biopsies (mean 0.76 vs. 0.63; P = 0.635). The combination of dinaciclib and MK-2206 was a safe regimen in patients with metastatic pancreatic cancer, although without clinical benefit, possibly due to not attaining biologically effective doses. Given the strong preclinical evidence of Ral and AKT inhibition, further studies with better tolerated agents should be considered.

Paraherquamide J, a new prenylated indole alkaloid from the marine-derived fungus Penicillium janthinellum HK1-6.

A new prenylated indole alkaloid, named paraherquamide J (1), together with four known compounds (2-5), were isolated from the mangrove rhizosphere soil-derived fungus Penicillium janthinellum HK1-6. The planar structure and relative configuration of 1 were determined by detailed analysis of the spectroscopic data especially the NOESY spectrum. The absolute configuration of 1 was determined by ECD spectra. Compound 2 was first isolated as a natural product and named as paraherquamide K. All isolated metabolites were evaluated for their antibacterial, topoisomerase I (topo I) inhibitory activities and lethality towards brine shrimp Artemia salina.

Novel Series of Methyl 3-(Substituted Benzoyl)-7-Substituted-2-Phenylindolizine-1-Carboxylates as Promising Anti-Inflammatory Agents: Molecular Modeling Studies.

The cyclooxygenase-2 (COX-2) enzyme is considered to be an important target for developing novel anti-inflammatory agents. Selective COX-2 inhibitors offer the advantage of lower adverse effects that are commonly associated with non-selective COX inhibitors. In this work, a novel series of methyl 3-(substituted benzoyl)-7-substituted-2-phenylindolizine-1-carboxylates was synthesized and evaluated for COX-2 inhibitory activity. Compound 4e was identified as the most active compound of the series with an IC50 of 6.71 M, which is comparable to the IC50 of indomethacin, a marketed non-steroidal anti-inflammatory drug (NSAID). Molecular modeling and crystallographic studies were conducted to further characterize the compounds and gain better understanding of the binding interactions between the compounds and the residues at the active site of the COX-2 enzyme. The pharmacokinetic properties and potential toxic effects were predicted for all the synthesized compounds, which indicated good drug-like properties. Thus, these synthesized compounds can be considered as potential lead compounds for developing effective anti-inflammatory therapeutic agents.

Cytotoxicity of ungeremine towards multi-factorial drug resistant cancer cells and induction of apoptosis, ferroptosis, necroptosis and autophagy.

BACKGROUND: Successful cancer chemotherapy is hampered by resistance of cancer cells to established anticancer drugs. Numerous natural products reveal cytotoxicity towards tumor cells. PURPOSE: The present study was aimed to determine the cytotoxicity of a betaine-type alkaloid, ungeremine, towards 9 cancer cell lines including various sensitive and drug-resistant phenotypes. The mode of action of this compound was further investigated. METHODS: The cytotoxicity, ferroptotic and necroptotic cell death were determined by the resazurin reduction assay. Caspase activation was evaluated using the caspase-Glo assay. Flow cytometry was applied for the analysis of cell cycle analysis (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP) (JC-1) and reactive oxygen species (ROS) (H2DCFH-DA). Apoptotic, necroptotic and autophagic markers were determined by Western blotting. CCRF-CEM leukemia cells were used for all mechanistic studies. RESULTS: Ungeremine displayed cytotoxic activity towards the 9 cancer cell lines tested, including drug-sensitive and MDR phenotypes. The IC50values obtained varied from 3.67 µM (in MDA-MB-231-BCRP breast carcinoma cells) to 75.24 µM (against in CEM/ADR5000 leukemia cells) for ungeremine and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against CEM/ADR5000 cells) for doxorubicin (control drug). Ungeremine induced ferroptosis, necroptosis, autophagy as well as apoptosis mediated by caspase activation, MMP alteration and increase ROS production. CONCLUSION: The present investigation showed that ungeremine is a promising cytotoxic compoundthat could be further explored in the future to develop new anticancer drugs to fight sensitive and resistant phenotypes.

The discovery of fluazaindolizine: A new product for the control of plant parasitic nematodes.

Fluazaindolizine is a new highly effective and selective product for the control of plant parasitic nematodes. Specificity for nematodes coupled with absence of activity against the target sites of commercial nematicides suggests that fluazaindolizine has a novel mode of action. The discovery, structure-activity development and biological properties for this new class of nematicides are presented.

Anticancer activity of novel pyrido[2,3-b]indolizine derivatives: the relevance of phenolic substituents.

BACKGROUND/AIM: The potential of indolizine derivatives as anticancer agents has been shown through recent studies. Herein, we present our experimental results, showing that pyrido[2,3-b]indolizine derivatives are effective against colorectal cancer (CRC) cell lines. MATERIALS AND METHODS: Several pyrido[2,3-b]indolizine derivatives were synthesized and their anticancer potential was evaluated against three CRC cell lines and two normal fibroblast cultures. RESULTS: Our experiments identified 4-(3,4)-dihydroxyphenyl)-2-phenylpyrido[2,3-b]indolizine-10-carbonitrile (4f) as being active against all CRC cell lines at concentrations non-cytotoxic against fibroblast cultures. Additionally, cell-cycle analysis indicated that pyrido[2,3-b]indolizines can affect cell-cycle progression, with treated cells accumulating in the S- and G2/M-phase. CONCLUSION: The hydroxyl groups in both the 3- and 4- positions of the aromatic substituent on C4 of the indolizine nucleus are crucial for activity against CRC cell lines. Further manipulation of the number and position of hydroxyl substituents on the aromatic rings may lead to improved anticancer activity of this class of compounds.

A novel small molecule FL118 that selectively inhibits survivin, Mcl-1, XIAP and cIAP2 in a p53-independent manner, shows superior antitumor activity.

Drug/radiation resistance to treatment and tumor relapse are major obstacles in identifying a cure for cancer. Development of novel agents that address these challenges would therefore be of the upmost importance in the fight against cancer. In this regard, studies show that the antiapoptotic protein survivin is a central molecule involved in both hurdles. Using cancer cell-based survivin-reporter systems (US 7,569,221 B2) via high throughput screening (HTS) of compound libraries, followed by in vitro and in vivo analyses of HTS-derived hit-lead compounds, we identified a novel anticancer compound (designated FL118). FL118 shows structural similarity to irinotecan. However, while the inhibition of DNA topoisomerase 1 activity by FL118 was no better than the active form of irinotecan, SN-38 at 1 µM, FL118 effectively inhibited cancer cell growth at less than nM levels in a p53 status-independent manner. Moreover, FL118 selectively inhibited survivin promoter activity and gene expression also in a p53 status-independent manner. Although the survivin promoter-reporter system was used for the identification of FL118, our studies revealed that FL118 not only inhibits survivin expression but also selectively and independently inhibits three additional cancer-associated survival genes (Mcl-1, XIAP and cIAP2) in a p53 status-independent manner, while showing no inhibitory effects on control genes. Genetic silencing or overexpression of FL118 targets demonstrated a role for these targets in FL118's effects. Follow-up in vivo studies revealed that FL118 exhibits superior antitumor efficacy in human tumor xenograft models in comparison with irinotecan, topotecan, doxorubicin, 5-FU, gemcitabine, docetaxel, oxaliplatin, cytoxan and cisplatin, and a majority of mice treated with FL118 showed tumor regression with a weekly × 4 schedule. FL118 induced favorable body-weight-loss profiles (temporary and reversible) and was able to eliminate large tumors. Together, the molecular targeting features of FL118 plus its superior antitumor activity warrant its further development toward clinical trials.

Synthesis of phenanthroindolizidine alkaloids and evaluation of their antitumor activities and toxicities.

We previously reported that phenanthroindolizidine alkaloid 3 and its derivatives had markedly potent in vitro cytotoxicity. However, they had low in vivo antitumor activities and high in vivo toxicities, which was a serious problem. To address this problem, new phenanthroindolizidine derivatives were synthesized and their antitumor activities and toxicities were evaluated. This study describes the relationship between the chemical structures, antitumor activities, and toxicities of these phenanthroindolizidine derivatives. Based on its properties, compound 8 was found to be the most suitable potential antitumor agent.

Total synthesis of phenanthroindolizidine alkaloids (+/-)-antofine, (+/-)-deoxypergularinine, and their dehydro congeners and evaluation of their cytotoxic activity.

Due to their limited natural abundance and significant biochemical effects, we synthesized the alkaloids (+/-)-antofine (1a), (+/-)-deoxypergularinine (1b), and their dehydro congeners (2 and 3) starting from the corresponding phenanthrene-9-carboxaldehydes. We also evaluated their in vitro cytotoxic activity. Compounds 1a and 1b showed significant potency against various human tumor cell lines, including a drug-resistant variant, with EC(50) values ranging from 0.16 to 16ng/mL. Structure-activity correlations of these alkaloids and some of their synthetic intermediates were also ascertained. The non-planar structure between the two major moieties, phenanthrene and indolizidine, plays a crucial role in the cytotoxic activity of phenanthroindolizidines. Increasing the planarity and rigidity of the indolizidine moiety significantly reduced potency. A methoxy group at the 2-position (1a) was more favorable for cytotoxic activity than a hydrogen atom (1b).

Enantiospecific synthesis and cytotoxicity of 7-(4-methoxyphenyl)-6-phenyl-2,3,8,8a-tetrahydroindolizin-5(1H)-one enantiomers.

An enantiospecific synthesis was developed to generate both enantiomers of 7-(4-methoxyphenyl)-6-phenyl-2,3,8,8a-tetrahydroindolizin-5(1H)-one. A biological assay utilizing the HCT-116 colon cancer cell line to determine the cytotoxicity of these analogs revealed that only the (R)-enantiomer exhibited appreciable cytotoxicity with an IC(50) value of 0.2 microM.

Concise synthesis of 22-hydroxyacuminatine, cytotoxic camptothecinoid from Camptotheca acuminata, by pyridone benzannulation.

A short, efficient synthesis of 22-hydroxyacuminatine, starting from a readily accessible hydroxy pyridone, is presented; key steps include a Heck coupling with methyl pentadienoate, a flash vacuum pyrolytic cyclization, and a Friedländer condensation.

Scheloribatid mites as the source of pumiliotoxins in dendrobatid frogs.

The strawberry poison frog Dendrobates pumilio (Anura: Dendrobatidae) and related poison frogs contain a variety of dendrobatid alkaloids that are considered to be sequestered through the consumption of alkaloid-containing arthropods microsympatrically distributed in the habitat. In addition to ants, beetles, and millipedes, we found that adults of two species of oribatid mites belonging to the cohort Brachypylina, trophically a lower level of animal than ants and beetles, contain dendrobatid alkaloids. Gas chromatography/mass spectrometry (GC/MS) of hexane extracts of adult Scheloribates azumaensis (Oribatida: Acari) revealed the presence of not only pumiliotoxin 251D (8-hydroxy-8-methyl-6-(2'-methylhexylidene)-1-azabicyclo[4.3.0]nonane), but also precoccinelline 193C and another coccinelline-type alkaloid. From the corresponding extracts of an unidentified Scheloribates sp., pumiliotoxin 237A (8-hydroxy-8-methyl-6-(2'-methylpentylidene)-1-azabicyclo[4.3.0]nonane) was detected as a minor component, and identified by synthesis. The presence of related alkaloids, namely deoxypumiliotoxin 193H, a 6,8-diethyl-5-propenylindolizidine, and tentatively, a 1-ethyl-4-pentenynylquinolizidine, were indicated by the GC/MS fragmentation patterns, along with at least another six unidentified alkaloid components. Thus, one possible origin of pumiliotoxins, coccinellid alkaloids, and certain izidines found in poison frogs may be mites of the genus Scheloribates and perhaps related genera in the suborder Oribatida.

Novel mode of action of tylophorine analogs as antitumor compounds.

Tylophorine and its analogs are phenanthroindolizidine alkaloids, several of which have been isolated from the Tylophora genus of plants. Evaluation of (+)-S-tylophorine [DCB-3500 (NSC-717335)] and its analog DCB-3503 (NSC-716802) in the National Cancer Institute tumor screen showed a fairly uniform and potent inhibition of cell growth in all 60 cell lines (GI(50) approximately 10(-8) M). To further evaluate the antitumor potential of these compounds, we synthesized four tylophorine analogs, designated DCB-3500, DCB-3501, DCB-3502, and DCB-3503. All four tylophorine analogs exerted potent growth-inhibitory effects against HepG2, a human hepatocellular carcinoma cell line, and KB, a human nasopharyngeal carcinoma cell line. HepG2 cells were more sensitive than KB in terms of loss of clonogenicity. KB variants, which are resistant to etoposide, hydroxyurea, or camptothecin, have similar sensitivities to the tylophorine analogs, as do the parental KB cells. Treatment of nude mice bearing HepG2 tumor xenografts by i.p. injections of DCB-3503 at 6 mg/kg every 8 h on days 0 and 3 resulted in significant tumor growth suppression (P < 0.0001). Unlike conventional antitumor drugs, 3 micro M DCB-3503 did not cause DNA breaks or apoptosis in HepG2 cells. Tylophorine analogs induced albumin expression and decreased alpha-fetoprotein expression in HepG2 cells, which suggests that tylophorine analogs could induce HepG2 differentiation. Tylophorine analogs had an inhibitory effect on cyclic AMP response elements, activator protein-1 sites, or nuclear factor-kappaB binding site-mediated transcriptions. In summary, these tylophorine analogs are a unique class of antitumor compounds that have a mode of action different from known antitumor drugs.

Anthelmintic paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammals.

Oxindole alkaloids in the paraherquamide/marcfortine family exhibit broad-spectrum anthelmintic activity that includes drug-resistant strains of nematodes. Paraherquamide (PHQ), 2-deoxoparaherquamide (2DPHQ), and close structural analogs of these compounds rapidly induce flaccid paralysis in parasitic nematodes in vitro, without affecting adenosine triphosphate (ATP) levels. The mechanism of action of this anthelmintic class was investigated using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum. None of the compounds altered A. suum muscle tension or membrane potential. However, PHQ blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by acetylcholine (ACh) and the nicotinic agonists levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic activity of PHQ and marcfortines is due to blockade of cholinergic neuromuscular transmission. The effects of these compounds were also examined on subtypes of human nicotinic ACh receptors expressed in mammalian cells with a Ca2+ flux assay. 2DPHQ blocked nicotinic stimulation of cells expressing alpha3 ganglionic (IC50 approximately 9 microm) and muscle-type (IC50 approximately 3 microm) nicotinic cholinergic receptors, but was inactive at 100 microm vs. the alpha7 CNS subtype. PHQ anthelmintics are nicotinic cholinergic antagonists in both nematodes and mammals, and this mechanism appears to underlie both their efficacy and toxicity.

The effects of glycosylation inhibitors on the proliferation of a Wilms tumour cell line.

We have examined the effects of different glycosylation inhibitors on the proliferation of a human Wilms tumour derived cell line WCCS-1. It was found that two compounds that specifically inhibit distal steps in the glycosylation chain (swainsonone and castanospermine) only exerted marginal effects on cell multiplication and survival. In contrast, a proximal inhibitor (tunicamycin) efficiently increased necrosis in a dose dependent fashion. It is shown that this cell death was accompanied by a marked decrease in the incorporation of glucosamine, but rather unexpectedly, only caused a limited inhibition of de novo protein synthesis. Moreover, the entrance into S-phase was virtually unchanged in the cells surviving the exposure to tunicamycin. The effects of tunicamycin on cell multiplication and survival could not be reversed by concomitant addition of mevalonate as has been shown in other cell lines. Taken together this data suggests that tunicamycin does not operate in a cell cycle specific manner in Wilms tumour cells.

Castanospermine, an oligosaccharide processing inhibitor, reduces membrane expression of adhesion molecules and prolongs heart allograft survival in rats.

The inhibition of intracellular oligosaccharide processing is a new approach to immunosuppression in allotransplantation. The net effect of such inhibition is reduction in the membrane expression of certain glycoproteins. Hence cell-cell interaction in allorejection may be impaired in the presence of glycoprotein processing inhibitors because the expression of key ligand-receptor pairs of N-linked glycoproteins including adhesion molecules is inhibited. The aims of this study were to measure the immunosuppressive ability of castanospermine (CAST) in a rat heart allograft model, to measure its effect on membrane expression of adhesion molecules (LFA-1 alpha, LFA-1 beta, ICAM-1), class I and class II MHC antigens and on other T cell associated molecules (CD4, CD8, CD39, CD45, W3/13), to test its tolerogenic potential and its toxicity. Membrane expression of these molecules was measured by flow cytometry for single cells and by immunoperoxidase staining for the allograft. In grafted rats CAST significantly reduced the expression of LFA-1 alpha on lymphoid cells in the thymus, lymph node, spleen and heart allografts. ICAM-1 expression on endothelial cells of the allograft vasculature, class I and class II MHC expression on lymphoid cells in the thymus, class II MHC expression on lymphoid cells in the allograft; and CD4, CD8, CD45 and W3/13 expression on lymphoid cells in some organs. By contrast, in non-grafted rats CAST significantly upregulated expression of class I MHC and CD45 in the thymus, lymph node and spleen, ICAM-1 and CD4 on lymphoid cells in the spleen, but reduced expression of LFA-1 alpha on lymphoid cells in the thymus. It also prolonged rat heart allograft survival in a dose-dependent manner and with limited testing was relatively non-toxic. In conclusion, CAST is an immunosuppressive molecule which may work by downregulation of the ligand-receptor adhesion molecule pair, LFA-1 alpha-ICAM-1 although subtle downregulation of class I and II MHC, CD4 and CD8 molecules could also contribute to its immunosuppressive activity. Hence, both lymphocyte-endothelial cell binding and lymphocyte activation may be inhibited by CAST. This work suggests that CAST may hold significant potential as a transplant immunosuppressant probably as an adjuvant agent to inhibitors of interleukin 2 secretion.

The lesions of locoweed (Astragalus mollissimus), swainsonine, and castanospermine in rats.

To better characterize and compare the toxicity of and lesions produced by locoweed (Astragalus mollissimus) with those of swainsonine and a related glycoside inhibitor, castanospermine, 55 Sprague-Dawley rats were randomly divided into 11 groups of five animals each. The first eight groups were dosed via subcutaneous osmotic minipumps with swainsonine at 0, 0.1, 0.7, 3.0, 7.4, or 14.9 mg/kg/day or with castanospermine at 12.4 or 143.6 mg/kg/day for 28 days. The last three groups were fed alfalfa or locoweed pellets with swainsonine doses of 0, 0.9, or 7.2 mg/kg/day for 28 days. Swainsonine- and locoweed-treated rats gained less weight, ate less, and showed more signs of nervousness than did controls. Histologically, these animals developed vacuolar degeneration of the renal tubular epithelium, the thyroid follicular cells, and the macrophage-phagocytic cells of the lymph nodes, spleen, lung, liver, and thymus. Some rats also developed vacuolation of neurons, ependyma, adrenal cortex, exocrine pancreas, myocardial epicytes, interstitial cells, and gastric parietal cells. No differences in lesion severity or distribution were detected between animals dosed with swainsonine and those dosed with locoweed. Rats dosed with castanospermine were clinically normal; however, they developed mild vacuolation of the renal tubular epithelium, the thyroid follicular epithelium, hepatocytes, and skeletal myocytes. Special stains and lectin histochemical evaluation showed that swainsonine- and castanospermine-induced vacuoles contained mannose-rich oligosaccharides. Castanospermine-induced vacuoles also contained glycogen. These results suggest that 1) swainsonine causes lesions similar to those caused by locoweed and is probably the primary locoweed toxin; 2) castanospermine at high doses causes vacuolar changes in the kidney and thyroid gland; and 3) castanospermine intoxication results in degenerative vacuolation of hepatocytes and skeletal myocytes, similar to genetic glycogenosis.

Acute toxicity of paraherquamide and its potential as an anthelmintic.

Paraherquamide, an oxindole alkaloid metabolite of Penicillium paraherquei and P charlesii, is a new anthelmintic with potential broad-spectrum use. In initial trials, it had an excellent safety profile in cattle and sheep at doses efficacious against a dozen or more helminths, but recently it produced unexpected and severe toxicosis in dogs at doses far below those that were safe in the ruminants. To provide data on which to build rational safety tests in the future, we tested the acute toxicity of paraherquamide administered PO to male CD-1 mice and compared its profile with the most potent anthelmintic known, ivermectin. The estimated doses lethal to 50% of a group of mice were 14.9 and 29.5 mg/kg of body weight for paraherquamide and ivermectin, respectively. The no-effect doses were 5.6 and 18.0 mg/kg for paraherquamide and ivermectin, respectively. Signs of intoxication in paraherquamide-treated mice, if they developed, emanated within 30 minutes of administration, irrespective of dose, and consisted of either mild depression with complete recovery or a 5- to 10-minute period of breathing difficulty followed by respiratory failure and death by 1 hour after treatment. Gross necropsy findings in paraherquamide-treated mice that died in the high-dose group were normal. Ivermectin-related toxicity was slower and more predictable, taking place over a 3-day period, with dose-dependent signs of intoxication consisting of tremors, ataxia, recumbency, coma, and death. Necropsy of ivermectin-treated mice that died in the high-dose group revealed dehydration, a condition most likely resulting from the coma-induced state.(ABSTRACT TRUNCATED AT 250 WORDS)

6-0-butanoylcastanospermine (MDL 28,574) inhibits glycoprotein processing and the growth of HIVs.

The antiviral activity of 6-0-butanoylcastanospermine (MDL 28,574) [50% inhibitory concentration (IC50: 1.1 microM)] in JM cells infected with a recent isolate of HIV-1 (GB8), was compared with other inhibitors of glycoprotein-processing enzymes. N-butyldeoxynojirimycin (BuDNJ), deoxynojirimycin (DNJ), castanospermine (CAST) or the reverse transcriptase inhibitor 2'3'-dideoxycytidine (ddC) had activities of 56, 560, 29 and 0.1 microM, respectively. MDL 28,574 was at least 50 times more active than BuDNJ and less active but better tolerated in cell culture than ddC, two compounds currently undergoing clinical trials. The CAST derivative showed good protection in H9 cells infected with HIV-1 (RF; IIIB; U455), and HIV-2 (ROD), although the potency was less than that seen in the JM/GB8 system. HIV-1 glycoproteins, gp160 and gp120, synthesized in H9 cells chronically infected with HIV-1 (RF) and treated with MDL 28,574, were characterized by an increase in relative molecular weight of approximately 7-8000 kD. The ratio of gp120 to gp160 was markedly reduced in treated cells and provided further evidence that cleavage of the gp160 precursor molecule is a major consequence of the inhibition of glycoprotein processing. The intracellular target for MDL 28,574 was verified as alpha-glucosidase-I of the processing enzymes by the analysis of high-glucose glycopeptides recovered from treated mouse cells. This activity correlated with the antiviral effect observed against the growth of a mouse retrovirus, Moloney murine leukemia virus (MOLV), in mouse cells.