Dipole-Transmissive 1,3-Dipolar Cycloadditions for the Rapid Construction of Polycyclic N-Heterocycles: Synthetic and Mechanistic Investigations.

The investigation of distinctive dipole-transmissive dipolar cycloaddition (DTDC) methodology and the formalisation of this concept is reported. A DTDC procedure was able to be developed by taking advantage of the structural complementarity of azide and diazoalkane 1,3-dipoles. Intramolecular azide-alkene 1,3-DCs followed by spontaneous dipole transmission upon work-up furnished intermediate α-diazoisoindole and α-diazoisoquinoline substrates bearing the key secondary diazoalkane 1,3-dipole. N-Derivatisation of the intermediate α-diazoisoindole and α-diazoisoquinolines with a tethered secondary dipolarophile followed by a subsequent 1,3-DC allowed for rapid construction of a range of functionalised polycyclic N-heterocycles. Integrated experimental and theoretical studies established requirements for product formation and revealed the likely mechanistic basis of divergent reactivity observed.

Strategies for the synthesis of Stemona alkaloids: an update.

Covering: 2009 to 2022The Stemona alkaloids, which are found in plant species from the family Stemonaceae, represent a tremendously large and structurally-diverse family of natural products. This review presents and discusses a selection of case studies, grouped by alkaloid class, that showcase the key strategies and overall progress that has been made in the synthesis of Stemona alkaloids and related compounds since 2009. Structural reassignments that have been reported over this period are also identified where necessary.

Identification of Active Site Residues of the Siderophore Synthesis Enzyme PvdF and Evidence for Interaction of PvdF with a Substrate-Providing Enzyme.

The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host-pathogen interactions.

Synthesis and Biological Evaluation of (-) and (+)-Spiroleucettadine and Analogues.

A second-generation enantiospecific synthesis of spiroleucettadine is described. The original reported antibacterial activity was not observed when the experiment was repeated on the synthetic samples; however, significant anti-proliferative activity was uncovered for both enantiomers of spiroleucettadine. Comparison of the optical rotational data and ORD-CD spectra of both enantiomers and the reported spectrum from the natural source have not provided a definitive answer regarding the absolute stereochemistry of naturally occurring spiroleucettadine. Efforts then focussed on alteration at the C-4 and C-5 positions of the slightly more active (-)-spiroleucettadine. Ten analogues were synthesised, with three analogues found to possess similar anti-proliferative profiles to spiroleucettadine against the H522 lung cancer cell line.

Does the Reaction of Cyclopropyl Acid Chlorides and Imines To Form 1,3-Oxazin-4-enone Heterocycles Proceed via a Ketene or an N-Acyl-iminium Mechanism?

The mechanism of the reaction between cyclopropyl acid chlorides and imines to form 1,3-oxazin-4-enones was probed through physical and computational experiments. The data gathered strongly support the reaction proceeding through an N-acyl iminium intermediate mechanism rather than a ketene intermediate mechanism.

Cytotoxicity of curcumin derivatives in ALK positive non-small cell lung cancer.

Non-small cell lung cancer with ALK rearrangements can be targeted effectively with ALK inhibitors such as crizotinib. However, cancer progression typically occurs within a year as drug resistance develops. One strategy to overcome this drug resistance is to determine if novel cytotoxic agents retain the ability to kill lung cancer cells that have developed ALK inhibitor resistance. We therefore examined curcumin, a drug with anticancer properties, and 2 s-generation curcumin derivatives (1-methyl-3,5-bis[(E)-4-pyridyl) methylidene]-4-piperidone (RL66) and 1-isopropyl-3,5-bis[(pyridine-3-yl) methylene]piperidin-4-one (RL118)) in lung cancer cell lines. The cytotoxicity of curcumin, RL66, and RL118 were tested in both ALK+ lung cancer cells (H3122), crizotinib resistant ALK+ cells (CR-H3122) and ALK- lung cancer cells (A549), both alone and in combination with crizotinib. ALK+ cells were 2-3x more sensitive to RL66 and RL118 than ALK- cells, with the drugs' eliciting IC50 values in the range of 0.7-1 µM in H3122 cells. Retained cytotoxic potency of the curcumin derivatives in crizotinib resistant cells indicated that mechanisms of resistance to the two drug types are independent, with resistance to ALK inhibitors not necessarily causing cross-resistance to curcumin derivatives. This was further corroborated by drug combination analysis where the effect of the drugs in combination was consistent with Bliss additivity, consistent with independent targets for crizotinib and curcumin derivatives. Results from Western blotting showed that RL118 (2 µM) inhibited p-ALK/ALK by ~50%, which was not as potent as the 90% inhibition elicited by crizotinib (0.25 µM). Since this is the primary mechanism of crizotinib cytotoxicity this provides further evidence of independent mechanisms of toxicity.

Chemical synthesis and characterization of a new quinazolinedione competitive antagonist for strigolactone receptors with an unexpected binding mode.

Strigolactones (SLs) are multifunctional plant hormones regulating essential physiological processes affecting growth and development. In vascular plants, SLs are recognized by α/ß hydrolase-fold proteins from the D14/DAD2 (Dwarf14/Decreased Apical Dominance 2) family in the initial step of the signaling pathway. We have previously discovered that N-phenylanthranilic acid derivatives (e.g. tolfenamic acid) are potent antagonists of SL receptors, prompting us to design quinazolinone and quinazolinedione derivatives (QADs and QADDs, respectively) as second-generation antagonists. Initial in silico docking studies suggested that these compounds would bind to DAD2, the petunia SL receptor, with higher affinity than the first-generation compounds. However, only one of the QADs/QADDs tested in in vitro assays acted as a competitive antagonist of SL receptors, with reduced affinity and potency compared with its N-phenylanthranilic acid 'parent'. X-ray crystal structure analysis revealed that the binding mode of the active QADD inside DAD2's cavity was not that predicted in silico, highlighting a novel inhibition mechanism for SL receptors. Despite a ∼10-fold difference in potency in vitro, the QADD and tolfenamic acid had comparable activity in planta, suggesting that the QADD compensates for lower potency with increased bioavailability. Altogether, our results establish this QADD as a novel lead compound towards the development of potent and bioavailable antagonists of SL receptors.

Benzannulated 6,5-Spiroketals from Donor-Acceptor Cyclopropanes.

A rapid and facile synthesis of benzannulated 6,5-spiroketals from vinyl 1,1-diacylcyclopropanes is reported. The method utilizes mild reaction conditions with good to excellent yields and high diastereoselectivity. This methodology was then used to construct the core of berkelic acid.

Selective Mono-allylation of 1,3-Diketones and Their Use in the Synthesis of 3-Allyl Chromones and Benzannulated 6,5-Bicyclic Ketals.

The selective mono-allylation of 1,3-diketone containing compounds is described. The reaction proceeds under mild reaction conditions and in moderate to high yield (66-99 %). Using this procedure to access the key mono-allylated intermediate, the hitherto difficult to access 3-allyl chromones were synthesized in excellent yield (87-98 %). Finally, the utility of this newly developed procedure was showcased through the rapid synthesis of the scaffold of the xyloketal family of natural products.

Strategies, Setbacks, and Successes in the Synthesis of (-)-Spiroleucettadine.

Various strategies toward the synthesis of the marine natural product (-)-spiroleucettadine are described. In the original strategy, a biomimetic inspired oxidation of a 2-imidazoline scaffold uncovered unexpected reactivity, where benzylic oxidation followed by a Baeyer-Villiger reaction was observed. The second generation approach examined oxidative dearomatization of the phenol ring system first, where a competing spirocyclization process was uncovered. Efforts to forge the scaffold via a carbocation mediated benzyl migration were unsuccessful. Highlights of the successful synthesis include two consecutive hypervalent iodine reactions: the first formed the spirocyclic center and the second facilitated installation of an acetate group at the C-5 position to allow for subsequent introduction of the methyl amine side chain.

Anticancer potential of novel curcumin analogs towards castrate-resistant prostate cancer.

Prostate cancer is initially sensitive to hormone therapy; however, over time the majority of patients progress to a hormone-insensitive form classified as castration-resistant prostate cancer (CRPC). CRPC is highly metastatic and patients have a poor prognosis. Thus, new drugs for the treatment of this disease are required. In this study, we therefore examined the cytotoxic effects and anticancer mechanism(s) of action of second generation curcumin analogs towards CRPC cells. For this purpose, PC3 and DU145 cells were treated with a series of curcumin analogs at 0-10 µM for 72 h and cytotoxicity was determined by the sulforhodamine B (SRB) assay. Two compounds, 1-isopropyl-3,5-bis(pyridin-3-ylmethylene)-4-piperidone (RL118) and 1-methyl-3,5-[(6-methoxynaphthalen-2-yl)methylene]-4-piperidone (RL121), were found to have the most potent cytotoxic effect with EC50 values of 0.50 and 0.58 µM in the PC3 cells and EC50 values of 0.76 and 0.69 µM in the DU145 cells, respectively. Thus, further experiments were performed focusing on these two compounds. Flow cytometry was performed to determine their effects on the cell cycle and apoptosis. Both analogs increased the number of cells in the G2/M phase of the cell cycle and induced apoptosis. Specifically, in the PC3 cells, RL121 increased the number of cells in the G2/M phase by 86% compared to the control, while RL118 increased the number of cells in the G2/M phase by 42% compared to the control after 24 h. Moreover, both RL118 and RL121 induced the apoptosis of both cell lines. In the DU145 cells, a 38-fold increase in the number of apoptotic cells was elicited by RL118 and a 78-fold increase by RL121 compared to the control. Furthermore, the effects of both analogs on the expression of key proteins involved in cell proliferation were also determined by western blot analysis. The results revealed that both analogs inhibited the expression of nuclear factor (NF)-κB (p65/RelA), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), p-4E-BP1, mammalian target of rapamycin (mTOR), p-mTOR, AKT and p-AKT. Thus, the findings of this study provide evidence that RL118 and RL121 have potent anticancer activity against CPRC cells, and both analogs warrant further investigation in vivo.

Total Synthesis of (-)-Spiroleucettadine.

One of a number of intriguing new alkaloids isolated from the Leucetta sp. sponge in 2004, spiroleucettadine displayed unique structural features on a restricted scaffold: a trans-fused 5,5-bicyclic ring system together with an amino hemiketal moiety. Attempts to synthesize the initially proposed structure failed, raising questions as to its veracity, and structure revision ensued in 2008; no successful synthetic approach has been reported to date. Herein, we describe the enantiospecific total synthesis of (-)-spiroleucettadine by a highly efficient biomimetic approach starting from l-tyrosine. One of two key hypervalent-iodine-mediated oxidation reactions forged the spirocyclic center, and the other enabled the installation of the methylamine side chain in the penultimate step. Our approach provides synthetic access to a new class of spiroannulated natural products and will enable future studies of the structure-biological-activity relationships of these antibacterial compounds.

Synthesis of Chromones from 1,1-Diacylcyclopropanes: Toward the Synthesis of Bromophycoic Acid E.

A tandem deprotection-cyclization reaction of 1,1-diacylcyclopropanes is described which allows rapid access to structurally diverse 2,3-disubstituted chromones in good yields, and with straightforward purification. The utility of this reaction is showcased by the construction of the potent antibacterial marine natural product bromophycoic acid E scaffold.

Curcumin and its cyclohexanone analogue inhibited human Equilibrative nucleoside transporter 1 (ENT1) in pancreatic cancer cells.

Our group investigated combining the phytochemical curcumin and gemcitabine in a liposome, to improve gemcitabine's activity against pancreatic tumours. While optimising the curcumin: gemcitabine ratio for co-encapsulation, we found that increasing curcumin concentrations relative to gemcitabine resulted in antagonistic interactions. As curcumin is a promiscuous transporter inhibitor; we suspected that increased resistance occurred via inhibition of Equilibrative nucleoside transporter 1 (ENT1)-mediated gemcitabine uptake. To test our hypothesis, we determined whether curcumin and a related analogue, 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)-cyclohexanone (or A13), inhibited ENT1-mediated accumulation of [3H]uridine and [3H]gemcitabine into pancreatic cancer cells. We then confirmed the inhibition of gemcitabine accumulation by investigating whether curcumin/A13 could increase gemcitabine resistance in growth inhibition assays. We found that curcumin and A13 concentration-dependently inhibited the ENT1-mediated accumulation of both uridine and gemcitabine in MIA PaCa-2 and PANC-1 cells. We also found that non-toxic concentrations of curcumin and A13 significantly increased the resistance of both cell lines to gemcitabine. Increased resistance only occurred when curcumin/A13 was co-incubated with gemcitabine, and not with sequential exposure (i.e., curcumin first, followed by gemcitabine, or vice versa). We also found that the curcumin analogue (3E,5E)-3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone (or EF24) did not inhibit gemcitabine accumulation, making it more suitable in combinations than curcumin/A13. From these results, we concluded that curcumin and A13 are inhibitors of the ENT1 transporter, but only at high concentrations (2-20µM). Curcumin is unlikely to inhibit gemcitabine uptake in tumours but may interfere with the oral absorption of ENT1 substrates due to high gut concentrations readily achievable from over-the-counter tablets/capsules.

Flavone C-glycosides from Trichuriella monsoniae (L.f.) Bennet.

In the first phytochemical investigation of Trichuriella monsoniae, three known flavonoidal C-glycosides, isoswertisin 1, 2″-O-ß-d-galactosyl isoswertisin 2 and 2″-O-ß-d-xylosyl isoswertisin 3 were isolated from the methanolic extract of the whole plant. Their structures were elucidated by extensive NMR spectroscopic studies including 2D NMR and HRMS, and the structure of 2 was supported by single crystal X-ray data studies. Further, NMR assignments for 3 are being reported for the first time.

The Synthesis of Multifunctionalized 1,3-Oxazin-4-ones from Donor-Acceptor Cyclopropanes.

A series of electronically diverse imines were found to readily react with various donor-acceptor cyclopropyl acid chlorides, with complete regioselectivity, to form 1,3-oxazin-4-ones in moderate yields (25-48% over two steps). Select oxazinones underwent a base induced rearrangement to afford the corresponding cycloheptene-fused oxazinones in good yields (up to 70%).

Cytotoxic Amides from Fruits of Kawakawa, Macropiper excelsum.

Cytotoxic amides have been isolated from the fruits of the endemic New Zealand medicinal plant kawakawa, Macropiper excelsum (Piperaceae). The main amide was piperchabamide A and this is the first report of this rare compound outside the genus Piper. Eleven other amides were purified including two new compounds with the unusual 3,4-dihydro-1(2H)-pyridinyl group. The new compounds were fully characterized by 2D NMR spectroscopy, which showed a slow exchange between two rotamers about the amide bond, and they were chemically synthesized. In view of the antitumor activity of the related piperlongumine, all of these amides plus four synthetic analogs were tested for cytotoxicity. The most active was the piperine homolog piperdardine, with an IC50 of 14 µM against HT 29 colon cancer cells.

Expedient metal-free synthesis of 1,3-oxazinen-4-ones.

1,3-Oxazinen-4-ones are medicinally important scaffolds which have traditionally been accessed using a hetero-Diels-Alder approach or more recently using a cobalt-catalyzed three-component cycloaddition. Herein we report a novel strategy to access this scaffold which allows for the rapid and high yielding synthesis of 1,3-oxazinen-4-ones under ambient temperature and pressures with improved substrate scope.

Heterocyclic cyclohexanone monocarbonyl analogs of curcumin can inhibit the activity of ATP-binding cassette transporters in cancer multidrug resistance.

Curcumin (CUR) is a phytochemical that inhibits the xenobiotic ABC efflux transporters implicated in cancer multidrug resistance (MDR), such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins 1 and 5 (MRP1 and MRP5). The use of CUR in the clinic however, is complicated by its instability and poor pharmacokinetic profile. Monocarbonyl analogs of CUR (MACs) are compounds without CUR's unstable ß-diketone moiety and were reported to have improved stability and in vivo disposition. Whether the MACs can be used as MDR reversal agents is less clear, as the absence of a ß-diketone may negatively impact transporter inhibition. In this study, we investigated 23 heterocyclic cyclohexanone MACs for inhibitory effects against P-gp, BCRP, MRP1 and MRP5. Using flow cytometry and resistance reversal assays, we found that many of these compounds inhibited the transport activity of the ABC transporters investigated, often with much greater potency than CUR. Overall the analogs were most effective at inhibiting BCRP and we identified three compounds, A12 (2,6-bis((E)-2,5-dimethoxy-benzylidene)cyclohexanone), A13 (2,6-bis((E)-4-hydroxyl-3-methoxybenzylidene)-cyclohexanone) and B11 (3,5-bis((E)-2-fluoro-4,5-dimethoxybenzylidene)-1-methylpiperidin-4-one), as the most promising BCRP inhibitors. These compounds inhibited BCRP activity in a non-cell line, non-substrate-specific manner. Their inhibition occurred by direct transporter interaction rather than modulating protein or cell surface expression. From these results, we concluded that MACs, such as the heterocyclic cyclohexanone analogs in this study, also have potential as MDR reversal agents and may be superior alternatives to the unstable parent compound, CUR.

Simplified silvestrol analogues with potent cytotoxic activity.

The complex natural products silvestrol (1) and episilvestrol (2) are inhibitors of translation initiation through binding to the DEAD-box helicase eukaryotic initiation factor 4A (eIF4A). Both compounds are potently cytotoxic to cancer cells in vitro, and 1 has demonstrated efficacy in vivo in several xenograft cancer models. Here we show that 2 has limited plasma membrane permeability and is metabolized in liver microsomes in a manner consistent with that reported for 1. In addition, we have prepared a series of analogues of these compounds where the complex pseudo-sugar at C6 has been replaced with chemically simpler moieties to improve drug-likeness. Selected compounds from this work possess excellent activity in biochemical and cellular translation assays with potent activity against leukemia cell lines.