Professor Adam McCluskey

Many bacterial pathogens produce toxins that target intracellular molecules to alter or disrupt normal cellular function. To access the cell, these toxins often exploit endocytic pathways, recruit cellular machinery, or both to mediate their own internalization. Bacteria are becoming increasingly resistant to antibiotics, so alternatives to treat or prevent diseases caused by bacterial toxins are necessary. The advent of small molecule inhibitors, which target key proteins involved in endocytic trafficking pathways, provides an opportunity to develop these compounds as potential therapeutic inhibitors of bacterial toxicity. This chapter reviews the endocytic pathways commonly hijacked by these toxins and the drugs that are currently being developed to inhibit specific proteins involved in endocytosis. It further evaluates the feasibility of using these compounds to investigate the modalities of the endocytosis and trafficking of bacterial toxins, and the potential for the use of these drugs as therapeutics in the future.

Wiskostatin (1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol) (1) is a carbazole-based compound reported as a specific and relatively potent inhibitor of the N-WASP actin remodelling complex (S-isomer EC50 = 4.35 µM; R-isomer EC50 = 3.44 µM). An NMR solution structure showed that wiskostatin interacts with a cleft in the regulatory GTPase binding domain of N-WASP. However, numerous studies have reported wiskostatin's actions on membrane transport and cytokinesis that are independent of the N-WASP-Arp2/3 complex pathway, but offer limited alternative explanation. The large GTPase, dynamin has established functional roles in these pathways. This study reveals that wiskostatin and its analogues, as well as other carbazole-based compounds, are inhibitors of helical dynamin GTPase activity and endocytosis. We characterise the effects of wiskostatin on in vitro dynamin GTPase activity, in-cell endocytosis, and determine the importance of wiskostatin functional groups on these activities through design and synthesis of libraries of wiskostatin analogues. We also examine whether other carbazole-based scaffolds frequently used in research or the clinic also modulate dynamin and endocytosis. Understanding off-targets for compounds used as research tools is important to be able to confidently interpret their action on biological systems, particularly when the target and off-targets affect overlapping mechanisms (e.g. cytokinesis and endocytosis). Herein we demonstrate that wiskostatin is a dynamin inhibitor (IC50 20.7 ± 1.2 µM) and a potent inhibitor of clathrin mediated endocytosis (IC50 = 6.9 ± 0.3 µM). Synthesis of wiskostatin analogues gave rise to 1-(9H-carbazol-9-yl)-3-((4-methylbenzyl)amino)propan-2-ol (35) and 1-(9H-carbazol-9-yl)-3-((4-chlorobenzyl)amino)propan-2-ol (43) as potent dynamin inhibitors (IC50 = 1.0 ± 0.2 µM), and (S)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8a) and (R)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8b) that are amongst the most potent inhibitors of clathrin mediated endocytosis yet reported (IC50 = 2.3 ± 3.3 and 2.1 ± 1.7 µM, respectively).

We show that dansylcadaverine (1) a known in-cell inhibitor of clathrin mediated endocytosis (CME), moderately inhibits dynamin I (dynI) GTPase activity (IC50 45 µM) and transferrin (Tfn) endocytosis in U2OS cells (IC50 205 µM). Synthesis gave a new class of GTP-competitive dynamin inhibitors, the Sulfonadyns¿. The introduction of a terminal cinnamyl moiety greatly enhanced dynI inhibition. Rigid diamine or amide links between the dansyl and cinnamyl moieties were detrimental to dynI inhibition. Compounds with in vitro inhibition of dynI activity <10 µM were tested in-cell for inhibition of CME. These data unveiled a number of compounds, e.g. analogues 33 ((E)-N-(6-{[(3-(4-bromophenyl)-2-propen-1-yl]amino}hexyl)-5-isoquinolinesulfonamide)) and 47 ((E)-N-(3-{[3-(4-bromophenyl)-2-propen-1-yl]amino}propyl)-1-naphthalenesulfonamide)isomers that showed dyn IC50 <4 µM, IC50(CME) <30 µM and IC50(SVE) from 12-265 µM. Both analogues (33 and 47) are at least 10 times more potent that the initial lead, dansylcadaverine (1). Enzyme kinetics revealed these sulfonamide analogues as being GTP competitive inhibitors of dynI. Sulfonadyn-47, the most potent SVE inhibitor observed (IC50(SVE) = 12.3 µM), significantly increased seizure threshold in a 6 Hz mouse psychomotor seizure test at 30 (p = 0.003) and 100 mg kg-1 ip (p < 0.0001), with similar anti-seizure efficacy to the established anti-seizure medication, sodium valproate (400 mg kg-1). The Sulfonadyn¿ class of drugs target dynamin and show promise as novel leads for future anti-seizure medications.

In this study, we investigated the potential of an analogue of robenidine (NCL179) to expand its chemical diversity for the treatment of multidrug-resistant (MDR) bacterial infections. We show that NCL179 exhibits potent bactericidal activity, returning minimum inhibitory concentra-tion/minimum bactericidal concentrations (MICs/MBCs) of 1¿2 µg/mL against methicillin-resistant Staphylococcus aureus, MICs/MBCs of 1¿2 µg/mL against methicillin-resistant S. pseudintermedius and MICs/MBCs of 2¿4 µg/mL against vancomycin-resistant enterococci. NCL179 showed synergistic activity against clinical isolates and reference strains of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa in the presence of sub-inhibitory concentrations of colistin, whereas NCL179 alone had no activity. Mice given oral NCL179 at 10 mg/kg and 50 mg/kg (4 × doses, 4 h apart) showed no adverse clinical effects and no observable histological effects in any of the organs examined. In a bioluminescent S. aureus sepsis challenge model, mice that received four oral doses of NCL179 at 50 mg/kg at 4 h intervals exhibited significantly reduced bacterial loads, longer survival times and higher overall survival rates than the vehicle-only treated mice. These results support NCL179 as a valid candidate for further development to treat MDR bacterial infections as a stand-alone antibiotic or in combination with existing antibiotic classes.

Five focused compound libraries (forty-nine compounds), based on prior studies in our laboratory were synthesized and screened for antibiotic and anti-fungal activity against S. aureus, E. coli, K. pneumoniae, P. aeruginosa, A. baumannii, C. albicans and C. neoformans. Low levels of activity, at the initial screening concentration of 32 µg/mL, were noted with analogues of (Z)-2-(3,4-dichlorophenyl)-3-phenylacrylonitriles which made up the first two focused libraries produced. The most promising analogues possessing additional substituents on the terminal aromatic ring of the synthesised acrylonitriles. Modifications of the terminal aromatic moiety were explored through epoxide installation flowed by flow chemistry mediated ring opening aminolysis with discreet sets of amines to the corresponding amino alcohols. Three new focused libraries were developed from substituted anilines, cyclic amines, and phenyl linked heterocyclic amines. The aniline-based compounds were inactive against the bacterial and fungal lines screened. The introduction of a cyclic, such as piperidine, piperazine, or morpholine, showed >50% inhibition when evaluated at 32 µg/mL compound concentration against methicillin-resistant Staphylococcus aureus. Examination of the terminal aromatic substituent via oxirane aminolysis allowed for the synthesis of three new focused libraries of afforded amino alcohols. Aromatic substituted piperidine or piperazine switched library activity from antibacterial to anti-fungal activity with ((Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(4-methylpiperazin-1-yl)propoxy)phenyl)acrylonitrile), ((Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(4-(4-hydroxyphenyl)piperazin-1-yl)propoxy)-phenyl)acrylonitrile) and ((Z)-3-(4-(3-(4-cyclohexylpiperazin-1-yl)-2-hydroxypropoxy)-phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile) show-ing >95% inhibition of Cryptococcus neoformans var. grubii H99 growth at 32 µg/mL. While (Z)-3-(4-(3-(cyclohexylamino)-2-hydroxypropoxy)phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile, (S,Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(piperidin-1-yl)propoxy)phenyl)acrylonitrile, (R,Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(piperidin-1-yl)propoxy)phenyl)acrylonitrile, (Z)-2-(3,4-dichloro-phenyl)-3-(4-(2-hydroxy-3-(D-11-piperidin-1-yl)propoxy)phenyl)-acrylonitrile, and (Z)-3-(4-(3-(4-cyclo-hexylpiperazin-1-yl)-2-hydroxypropoxy)-phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile 32 µg/mL against Staphylococcus aureus.

In silico approaches identified 1, N-(6-((4-bromo- benzyl)amino)hexyl)-3,5-bis(trifluoromethyl)benzene sulfonamide, as a potential inhibitor of the S100A2-p53 protein-protein interaction, a validated pancreatic cancer drug target. Subsequent cytotoxicity screening revealed it to be a 2.97 µM cell growth inhibitor of the MiaPaCa-2 pancreatic cell line. This is in keeping with our hypothesis that inhibiting this interaction would have an anti-pancreatic cancer effect with S100A2, the validated PC drug target. A combination of focused library synthesis (three libraries, 24 compounds total) and cytotoxicity screening identified a propyl alkyl diamine spacer as optimal; the nature of the terminal phenyl substituent had limited impact on observed cytotoxicity, whereas N-methylation was detrimental to activity. In total 15 human cancer cell lines were examined, with most analogues showing broad-spectrum activity. Near uniform activity was observed against a panel of six pancreatic cancer cell lines: MiaPaCa-2, BxPC-3, AsPC-1, Capan-2, HPAC and PANC-1. In all cases there was good to excellent correlation between the predicted docking pose in the S100A2-p53 binding groove and the observed cytotoxicity, especially in the pancreatic cancer cell line with high endogenous S100A2 expression. This supports S100A2 as a pancreatic cancer drug target.

In silico screening predicted 1 (N-(4-((4-(3-(4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)piperazin-1-yl) sulfonyl)-phenyl)acetamide) as an inhibitor of the S100A2-p53 protein-protein interaction. S100A2 is a validated pancreatic cancer drug target. In the MiaPaCa-2 pancreatic cell line, 1 was a ~50 µM growth inhibitor. Synthesis of five focused compound libraries and cytotoxicity screening revealed increased activity from the presence of electron withdrawing moieties on the sulfonamide aromatic ring, with the 3,5-bis-CF3 Library 3 analogues the most active, with GI50 values of 0.91 (3-ClPh; 13 i; BxPC-3, Pancreas) to 9.0 µM (4-CH3; 13 d; PANC-1, Pancreas). Activity was retained against an expanded pancreatic cancer cell line panel (MiaPaCa-2, BxPC-3, AsPC-1, Capan-2, PANC-1 and HPAC) and the normal cell line MCF10A (breast). Bulky 4-disposed substituents on the terminal phenyl ring enhanced broad spectrum activity with growth inhibition values spanning 1.1 to 3.1 µM (4-C(CH3)3; 13 e; BxPC-3 and AsPC-1 (pancreas), respectively). Central alkyl spacer contraction from propyl to ethyl proved detrimental to activity with Library 4 and 5.5- to 10-fold less cytotoxic than the propyl linked Library 2 and Library 3. The data herein was consistent with the predicted binding poses of the compounds evaluated. The highest levels of cytotoxicity were observed with those analogues best capable of adopting a near identical pose to the p53-peptide in the S100A2-p53 binding groove.

The Arylhydrocarbon Receptor (AhR), a member of the Per-ARNT-SIM transcription factor family, has been as a potential new target to treat breast cancer sufferers. A series of 2-phenylacrylonitriles targeting AhR has been developed that have shown promising and selective activity against cancerous cell lines while sparing normal non-cancerous cells. A quantitative structure¿activity relationship (QSAR) modeling approach was pursued in order to generate a predictive model for cytotoxicity to support ongoing synthetic activities and provide important structure-activity information for new structure design. Recent work conducted by us has identified a number of compounds that exhibited false positive cytotoxicity values in the standard MTT assay. This work describes a good quality model that not only predicts the activity of compounds in the MCF-7 breast cancer cell line, but was also able to identify structures that subsequently gave false positive values in the MTT assay by identifying compounds with aberrant biological behavior. This work not only allows the design of future breast cancer cytotoxic activity in vitro, but allows the avoidance of the synthesis of those compounds anticipated to result in anomalous cytotoxic behavior, greatly enhancing the design of such compounds.

Endocytosis entails selective packaging of cell surface cargos in cytoplasmic vesicles, thereby controlling key intrinsic cellular processes as well as the response of normal and malignant cells to their microenvironment. The purpose of this review is to outline the latest advances in the development of endocytosis-targeting therapeutic strategies in hematological malignancies.

Bolinaquinone is a natural product that is a structurally complex, cytotoxic sesquiterpene quinone. A scaffold simplification and focused library approach using a microwave-assisted Suzuki coupling gave 32 bolinaquinone analogues with good-to-excellent cytotoxicity profiles. Mono-arylbenzoquinones, Library A, were preferentially toxic towards BE2-C (neuroblastoma) cells with growth inhibition (GI50) values of 4¿12 µM; only the 3,4-dimethoxyphenyl 23 and 3-biphenyl 28 variants were broad-spectrum active¿HT29 (colon carcinoma), U87 and SJ-G2 (glioblastoma), MCF-7 (breast carcinoma), A2780 (ovarian carcinoma), H460 (lung carcinoma), A431 (skin carcinoma), Du145 (prostate carcinoma), BE2-C (neuroblastoma), MIA (pancreatic carcinoma) and SMA (spontaneous murine astrocytoma). Library B with a second aryl moiety exhibited broad-spectrum cytotoxicity with MCF-7 cells' GI50 values of 5.6 ± 0.7 and 5.1 ± 0.5 µM for 2,5-dimethoxy-3-(naphthalene-1-yl)-6-(naphthalene-3-yl) 33 and 2,5-dimethoxy-3-(biaryl-2-yl)-6-(naphthalene-3-yl) 36, respectively. Similar potencies were also noted with 2,5-dimethoxy-3,6-diphenyl 30 against A2780 (GI50 = 5.9 ± 0.0 µM) and with 2,5-dimethoxy-3-(biaryl-3-yl)-6-(naphthalene-3-yl) 37 against HT29 (GI50 = 5.4 ± 0.4 µM), while the 3,4-dimethoxy mono-aryl analogue 23 exhibited good levels of activity against A2780 (GI50 = 3.8 ± 0.75 µM), the neuroblastoma cell line BE2-C (GI50 = 3 ± 0.35 µM) and SMA (GI50 = 3.9 ± 0.54 µM). Introduction of the amino-substituted Library C gave 2-(naphthalen-1-yl)-5-(naphthalen-3-yl)-3,6-bis(propylamino) 43, with excellent activity against HT29 (0.08 ± 0.0 µM), MCF-7 (0.17 ± 0.1 µM), A2780 (0.14 ± 0.1 µM), A431 (0.11 ± 0.0 µM), Du145 (0.16 ± 0.1 µM), BE2-C (0.08 ± 0.0 µM) and MIA (0.1 ± 0.0 µM).

Plants from the family Myoporaceae, which includes the genus Myoporum, are extremely prized by the Australian Aboriginal people for their medicinal properties. Leaves from a plant, which was subsequently identified as Myoporum montanum, were provided for chemical investigation by representatives of an Aboriginal community from the Northern Tablelands district of northern New South Wales, Australia. Acetone extraction of the leaves provided a complex mixture of compounds including sesquiterpene hydrocarbons and more polar furanosesquiterpenes, which were identified by gas-liquid chromatography and retention indices (sesquiterpene hydrocarbons) and spectrometric techniques (furanosesquiterpenes). The major compounds found in a water extract were studied for their antibacterial activity using a disc diffusion assay and for their cell growth inhibition activity. The acetone extract contained sesquiterpene hydrocarbons (~30% of the total extract) in which the major compounds were germacrene-D and bicyclogermacrene. In addition, the extract contained five known toxic furanosesquiterpenes: myoporum ketol, (-)-10,11-dehydroisomyodesmone, (+)-10,11-dehydromyodesmone, 10,11-dehydromyoporum ketol, (-)-10,11-dehydromyoporone, and (±)-myoporone. An aqueous extract of the leaves, emulating the medicinal tea used by the Australian Aboriginal community, was found not to contain significant quantities of the sesquiterpene hydrocarbons and the most toxic furanosesquiterpenes. (±)-Myoporone and (-)-10,11-dehydromyoporone remained in the extract as well as a new furanosesquiterpene, 11-hydroxymyoporone. These three compounds were found to have significant antibacterial activity against Staphylococcus epidermidis, Enterococcus faecalis, and Moraxella catarrhalis but low cytotoxicity against a range of cancer cell lines and normal breast cells at 25µM.

We describe a simple flow chemistry approach to libraries of ethyl 3-oxo-2-(substituted-phenylamino)-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxylates (12a-l) and N-ethyl-3-oxo-2-(substituted-phenylamino)-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxamides (13a-l) in 38-87% yields. This scaffold is poorly described in the chemical literature. Screening against a panel of 11 cancer and one normal cell line showed that the amide linked library 13a-l was devoid of toxicity. Whereas the ester linked analogues 12b, 12c, 12g, 12j and 12l were highly cytotoxic with growth inhibition (GI50) values from 0.34 to >50 µM across all cell lines, with the 2-OH-Ph substituted 12l analogue presenting with sub-micromolar potency against the A2780 (ovarian; 0.34 ± 0.04 µM), BEC-2 (glioblastoma; 0.35 ± 0.06 µM), MIA (pancreas; 0.91 ± 0.054 µM) and SMA (murine glioblastoma; 0.77 ± 0.029 µM) carcinoma cell lines. Interestingly, the U87 glioblastoma cell line showed inherent resistance to growth inhibition by all analogues (GI50 32 to >50 µM) while the A2780 cells were highly sensitive (GI50 3.8-0.34 µM), suggesting that the analogues developed herein may be valuable lead compounds for the development of ovarian carcinoma specific cytotoxic agents. The differences in amide versus ester cytotoxicity was consitent with esterase cleaveage to release the cytotoxic warhead.

The use of 3-dimensional (3D) collagen gels has yielded new insights into the migratory behaviour of cancer cells. While the large GTPase dynamin has emerged as an important regulator of cancer cell migration and invasion under 2D conditions, its role in 3D migration is unclear. We have used a potent dynamin modulator, a bis-tyrphostin derivative, Ryngo® 1-23, to investigate the role of dynamin in 3D migration in 3 different cell lines. The compound specifically inhibits persistent, elongated 3D migration in U87MG and SMA-560 cells. Treated U87MG cells adopt a rounded morphology that is not due to apoptosis, loss of matrix metalloprotease activity or inhibition of clathrin-mediated endocytosis. Given that Ryngo 1-23 is known to regulate dynamin oligomerisation and actin dynamics at the leading edge, we analysed actin filament distribution. Ryngo 1-23 induced a switch in actin filament organization in 3D cultures resulting in the generation of multiple short actin-rich microspikes. Correlated with the change in actin filament distribution, cells displayed reduced collagen gel contraction. Since acto-myosin force transmission to the extra-cellular matrix underpins persistent, elongated migration, our results suggest that Ryngo 1-23 modulates this process in 3D migration via dynamin-mediated regulation of acto-myosin force transmission to the extra-cellular matrix.

The acid dissociation constants (pKas) of a number of novel polymerisable vinyl biaryl compounds, 4-(4'-ethenylphenyl)-pyridine (M1), 4'-ethenyl-(1,1'-biphenyl)-4-ol (M2), 4'-ethenyl-N,N-dimethyl-(1,1'-biphenyl)-3-amine (M3), 4'-ethenyl-(1,1'-biphenyl)-4-methanol (M4), 4'-ethenyl-N,N-dimethyl-(1,1'-biphenyl)-4-amine (M5), 4'-ethenyl-(1,1'-biphenyl)-4-carboxylic acid (M6), 4'-ethenyl-4-hydroxy-5-methyl-(1,1'-biphenyl)-3-carboxaldehyde (M7) were determined in a mixed solvent (THF-water) potentiometric titration at 25 °C and subsequent extrapolation to pure water via the Yasuda-Shedlovsky method. The acidity and basicity of the compounds in THF-water mixtures was observed to decrease with increasing THF fraction and is attributed to the corresponding decrease in the dielectric constant of the solution. To the best of our knowledge, this is the first reported study of pKa values undertaken for this class of compounds. The biaryls, M1-M7, were prepared by microwave-assisted Suzuki cross coupling of 4-vinylphenyl boronic acid with the appropriate aryl bromide and were custom designed for use as functional monomers in the synthesis of molecularly imprinted polymers.

Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine-derived APDs inhibited dynamin I (dynI) in the 2-12µm range, the most potent being trifluoperazine (IC<inf>50</inf> 2.6±0.7µm). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8- to 10-fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine-derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine-derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin-active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure-activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP-2 recruitment in the formation of clathrin-coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP-2 interaction with amphiphysin or clathrin. Super-resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP-2, nor AP-2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine-derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in-cell target in endocytosis.

Abstract An efficient and divergent methodology for the synthesis of new anthracenone-pyranones and anthracenone-furans is described. Key reactions discussed in these syntheses include an aldehyde promoted annulation with a ß-keto-sulfoxide, a domino alkyne insertion/carbonylation/Nu-acylation and a DMEDA promoted Castro-Stephens reaction. We also report the in vitro growth inhibition of these compounds in a range of human cancer cells. The natural product BE-26554A displayed good cell growth activity on BE2-C neuroblastoma and SMA glioblastoma cell lines at 0.17 and 0.16 µM (GI50), respectively. Of note, were a CF3 functionalised anthracenone 4-pyranone (chromone) derivative 22, and an anthracenone-furan derivative 54 which displayed 0.20 µM and 0.38 µM growth inhibition, respectively, in the BE2-C neuroblastoma cell line.

Fragment-based in silico screening against dynamin I (dynI) GTPase activity identified the 1,8-naphthalimide framework as a potential scaffold for the design of new inhibitors targeting the GTP binding pocket of dynI. Structure-based design, synthesis and subsequent optimization resulted in the development of a library of 1,8-naphthalimide derivatives, called the Naphthaladyn¿ series, with compounds 23 and 29 being the most active (IC<inf>50</inf> of 19.1 ± 0.3 and 18.5 ± 1.7 µM respectively). Compound 29 showed effective inhibition of clathrin-mediated endocytosis (IC<inf>50(CME)</inf> 66 µM). The results introduce 29 as an optimised GTP-competitive lead Naphthaladyn¿ compound for the further development of naphthalimide-based dynI GTPase inhibitors.

The protein kinase inhibitor CTx-0152960 (6, 2-((5-chloro-2-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide), and the piperazinyl analogue, CTx-0294885 (7, 2-((5-chloro-2-((4-piperazin-1-ylphenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide), were prepared using a hybrid flow and microwave approach. The use of flow chemistry approaches avoided the need for Boc-protection of piperidine in the key SNAr coupling with 1-fluoro-4-nitrobenzene. Microwave coupling of 4-morphilinoaniline 8 and 4-(piperazine-1-yl)aniline 9 with 2-(2,5-dichloropyrimidine-4-ylamino)-N-methylbenzamide 10, proved to be the most efficacious route to the target analogues 6 and 7. This hybrid methodology reduced the number of synthetic steps, gave enhanced overall yields and increased atom economy through step reduction and minimal requirement for chromatographic purification, relative to the original batch synthesis approach.

Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by lowpH- dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-ß-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosislike pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, a-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFVrelated porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component.

Clathrin-mediated endocytosis (CME) is a process that regulates selective internalization of important cellular cargo using clathrin-coated vesicles. Perturbation of this process has been linked to many diseases including cancer and neurodegenerative conditions. Chemical proteomics identified the marine metabolite, 2-hydroxy-5-methoxy-3-(((1S,4aS,8aS)-1,4a,5-Trimethyl-1,2,3,4,4a,7, 8,8a-octahydronaphthalen-2-yl)methyl)cyclohexa-2,5-diene-1,4-dione (bolinaquinone) as a clathrin inhibitor. While being an attractive medicinal chemistry target, the lack of data about bolinaquinone's mode of binding to the clathrin enzyme represents a major limitation for its structural optimization. We have used a molecular modeling approach to rationalize the observed activity of bolinaquinone and to predict its mode of binding with the clathrin terminal domain (CTD). The applied protocol started by global rigid-protein docking followed by flexible docking, molecular dynamics and linear interaction energy calculations. The results revealed the potential of bolinaquinone to interact with various pockets within the CTD, including the clathrin-box binding site. The results also highlight the importance of electrostatic contacts over van der Waals interactions for proper binding between bolinaquinone and its possible binding sites. This study provides a novel model that has the potential to allow rapid elaboration of bolinaquinone analogues as a new class of clathrin inhibitors. © 2014 by the authors.

Two libraries of highly decorated norcantharidin analogues were accessed via a series of sequential chemoselective flow hydrogenations and solvent-free transformations. Utilising a 10% Pd/C catalyst, modifications to reaction parameters (H2 pressure, temperature and flow rate conditions) allowed facile access to effect selective direct reductive aminations and olefin reductions in the presence of furan, benzyl and nitrile moieties were established. The use of 20% Pd(OH)2/C; Pd tetrakis; 5% Pt/C (sulfided) gave mixtures of furan and olefin (both reduced) and olefin reduced products. RuO2; 0.5% Re/C and Re2O7 resulted in no reduction. Concurrent olefin and nitrile reduction was achieved in the presence of furan moieties by employing a RANEY® nickel catalyst. In total, 31 reaction conditions were examined using less than 200 mg of reagents allowing optimised conditions to be efficiently determined. These optimised hydrogenation conditions afforded desired analogues in near quantitative yields thus removing the requirements of reaction workup and chromatography. © 2014 The Royal Society of Chemistry.

The Knoevenagel condensation of a series of substituted phenyl acetonitriles with 1H-pyrrole-2-carbaldehyde was examined in seven 1-butyl-3-methylimidazolium based ionic liquids and three protic ionic liquids. Of these [BMIM][Br] and [BMIM][OH], with catalytic piperidine, proved most efficient affording 3-substituted-(1H-pyrrol-2-yl)acrylonitriles 3-17 in good to excellent yields (98%) whilst utilisation of the protic ionic liquid propyl ammonium nitrate resulted in reduced yields (0-66%). Screening of the 3-substituted-(1H-pyrrol-2-yl)acrylonitriles analogues 3-17 against a panel of 11 cancer cell lines and one normal cell line allowed the identification of a series of compounds with broad spectrum cytotoxicity, but more interestingly a significant degree of MCF-7 breast cancer cell line specificity was evident with 6 (7 to >25 fold) and 13 (5.7 to >80 fold). Other analogues show high level of efficacy against specific cell lines with 10 showing excellent activity against MCF-7 (GI50 = 1.7 µM) and A431 (GI50 = 2.8 µM) cell lines. The most promising of the compounds identified herein were the 4-CF3 substituted 10 and the 3,4-dichloro substituted 13 with excellent activities against MCF-7 and A431 cell lines. The 3,4-dichloro-13 was a 0.56 µM potent inhibitor of MCF-7 cell growth. © 2014 the Partner Organisations.

A commonly observed limitation of conducting hydrogenations under flow chemistry conditions is hydrodehalogenation. In a bid to circumvent this limitation a series of hydrogenation catalysts were screened, with 5% Pt/C (sulfided) catalyst identified as an effective catalyst to selectively effect reductive aminations, nitro reduction, and alkene reductions in the presence of halogen atoms. Additionally the optimised protocol cleanly reduced imines in the presence of a furan moiety indicating potential amenability to other labile functionalities.

Virtual screening of the ChemDiversity and ChemBridge compound databases against dynamin I (dynI) GTPase activity identified 2,5-bis-(benzylamino)-1,4- benzoquinone 1 as a 273 ± 106 µM inhibitor. In silico lead optimization and focused library-led synthesis resulted in the development of four discrete benzoquinone/naphthoquinone based compound libraries comprising 54 compounds in total. Sixteen analogues were more potent than lead 1, with 2,5-bis-(4-hydroxyanilino)-1,4-benzoquinone (45) and 2,5-bis(4-carboxyanilino)- 1,4-benzoquinone (49) the most active with IC50 values of 11.1 ± 3.6 and 10.6 ± 1.6 µM respectively. Molecular modelling suggested a number of hydrogen bonding and hydrophobic interactions were involved in stabilization of 49 within the dynI GTP binding site. Six of the most active inhibitors were evaluated for potential inhibition of clathrin-mediated endocytosis (CME). Quinone 45 was the most effective CME inhibitor with an IC50(CME) of 36 ± 16 µM. © 2014 Published by Elsevier Masson SAS. All rights reserved.

The selectivity and rebinding capacity of molecularly imprinted polymers selective for propranolol (1) using the room temperature ionic liquids [BMIM][BF4], [BMIM][PF6], [HMIM][PF6] and [OMIM][PF6] and CHCl3 were examined. The observed IF (imprinting factor) values for MIPBF4, MIPPF6 and MIP CHCl3 were 1.0, 1.98 and 4.64, respectively. The longer chain HMIM and OMIM systems returned lower IF values of 1.1 and 2.3, respectively. MIP PF6 also showed a ~25% binding capacity reduction vs. MIP CHCl3 (5 µmol g-1vs. 7 µmol g-1 respectively). MIPCHCl3 and MIPPF6 differed in terms of BET surface area (306 m2 g-1vs. 185 m2 g -1), pore size (1.10 and 2.19 nm vs. 0.97 and 7.06 nm), the relative number of pores (Type A: 10.4 vs. 7.5%; Type B: 8.5 vs. 3.0%), and surface zeta potential (-37.9 mV vs. -20.3 mV). The MIP specificity for 1 was examined by selective rebinding studies with caffeine (2) and ephedrine (3). MIP PF6 rebound higher quantities of 2 than MIPCHCl3, but this was largely due to non-specific binding. Both MIPCHCl3 and MIP PF6 showed a higher affinity for 3 than for 2. Reduction in the Room Temperature Ionic Liquid (RTIL) porogen volume had little impact on the polymer morphology, but did result in a modest decrease in IF from 2.6 to 2.3 and in the binding capacity (30% to 19%). MIPCHCl3 retained the highest template specificity on rebinding from CHCl3 (IF = 4.6) dropping to IF = 0.6 in MeOH/[BMIM][PF6]. The MIPCHCl3 binding capacity remained constant using CHCl3, CH2Cl2 and MeOH (46-52%), dropped to 6% on addition of [BMIM][PF6] and increased to 83% in H2O (but at the expense of specificity with IFH2O = 1.4). MIPPF6 rebinding from MeOH saw an increase in specific rebinding to IF = 4.9 and also an increase in binding capacity to 48% when rebinding 1 from MeOH and to 42% and 45% with H2O and CH2Cl2, respectively, although in the latter case the increased capacity was at the cost of specificity with IFCH2Cl2 = 1.2. Overall the MIPPF6 capacity and specificity were enhanced on addition of MeOH. This journal is © the Partner Organisations 2014.

The Hedgehog (Hh) signalling pathway plays a pivotal role in the spatial and temporal regulation of cell proliferation and differentiation. By controlling the correct maturation of developing tissues and ensuring attainment of the correct size, position and the presence of fully functioning cellular structures, the Hh plays a pivotal role in development. Conversely aberrant Hh signalling is involved in Gorlin syndrome, basal cell carcinoma (the most common cancer in the world), and more than one third of all human medulloblastoma cases. In all of these cases, it is believed that deregulated Hh signalling leads to increased cell proliferation and tumour formation. Inhibition of the Hedgehog signalling pathway, is a recently validated anti-cancer drug target, with vismodegib (Erivedge¿), approved by the U.S. Food and Drug Administration for the treatment of adult basal cell carcinoma. In this perspective we outline the current state of Hh pathway inhibitors with a particular focus on potential limitations of upstream Hh pathway inhibition in relation to resistance mutations and crosstalk pathways. Together, these limitations indicate that inhibition of downstream components, specifically the Gli family of transcription factors, may represent a next generation approach to suppress tumours associated with aberrant Hh pathway signalling. © 2014 The Royal Society of Chemistry.

Oroidin (1), (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-4,5-dibromo-1H- pyrrole-2-carboxamide, is a pyrrole alkaloid isolated from the marine sponge Agelas oroides. Routine screening in a panel of twelve cancer cell lines revealed 1 to be poorly cytotoxic with the 50% growth inhibition concentration (GI) of 42 µM in MCF-7 (breast) cells and 24 µM in A2780 (ovarian) cells and >50 µM in all other cell lines tested. The development of eight focused libraries comprising thirty compounds total identified N-(biphenyl-4-ylmethyl)-1H-pyrrole-2-carboxamide (4l), N-benzyl-4,5-dibromo-1H- pyrrole-2-carboxamide (5a) and N-(biphenyl-4-ylmethyl)-4,5-dibromo-1H-pyrrole-2- carboxamide (5l) as potent inhibitors of cell growth in our panel of cell lines. Of these compounds GI values of <5 µM were observed with 4l against HT29 (colon) and SW480 (colon); 5a against HT29; and 5l against HT29, SW480, MCF-7, A431 (skin), Du145 (prostate), BE2-C (neuroblastoma) and MIA (pancreas) cell lines. As a cancer class, colon cancer appears to be more sensitive to the oroidin series of compounds, with analogue 5l being the most active. © 2014 Elsevier Ltd. All rights reserved.

This protocol describes the synthesis of two classes of clathrin inhibitors, Pitstop 1 and Pitstop 2, along with two inactive analogs that can be used as negative controls (Pitstop inactive controls, Pitnot-2 and Pitnot-2-100). Pitstop-induced inhibition of clathrin TD function acutely interferes with clathrin-mediated endocytosis (CME), synaptic vesicle recycling and cellular entry of HIV, whereas clathrin-independent internalization pathways and secretory traffic proceed unperturbed; these reagents can, therefore, be used to investigate clathrin function, and they have potential pharmacological applications. Pitstop 1 is synthesized in two steps: sulfonation of 1,8-naphthalic anhydride and subsequent reaction with 4-amino(methyl)aniline. Pitnot-1 results from the reaction of 4-amino(methyl)aniline with commercially available 4-sulfo-1,8-naphthalic anhydride potassium salt. Reaction of 1-naphthalene sulfonyl chloride with pseudothiohydantoin followed by condensation with 4-bromobenzaldehyde yields Pitstop 2. The synthesis of the inactive control commences with the condensation of 4-bromobenzaldehyde with the rhodanine core. Thioketone methylation and displacement with 1-napthylamine affords the target compound. Although Pitstop 1-series compounds are not cell permeable, they can be used in biochemical assays or be introduced into cells via microinjection. The Pitstop 2-series compounds are cell permeable. The synthesis of these compounds does not require specialist equipment and can be completed in 3-4 d. Microwave irradiation can be used to reduce the synthesis time. The synthesis of the Pitstop 2 family is easily adaptable to enable the synthesis of related compounds such as Pitstop 2-100 and Pitnot-2-100. The procedures are also simple, efficient and amenable to scale-up, enabling cost-effective in-house synthesis for users of these inhibitor classes.

Dynamin is a large GTPase with roles in membrane fission during clathrin-mediated endocytosis, in actin dynamics and in cytokinesis. Defects in dynamin have been linked to human diseases. The synthesis of a dynamin modulator toolkit comprising two different inhibitor classes is described. The first series comprises Dynole 34-2, Dynole 2-24 and the inactive control Dynole 31-2. The Dynole compounds act on the dynamin G domain, are not GTP competitive and can be synthesized in 2-3 d. Knoevenagel condensation of 1-(3-(dimethylamino) propyl)-1H-indole-3-carbaldehyde (1) with cyanoamides (2 and 3) affords Dynole 31-2 and Dynole 34-2, respectively. Reductive amination of 1 with decylamine gives Dynole 2-24. The second series acts at an allosteric site in the G domain of dynamin and comprises Dyngo 4a and Dyngo Ø (inactive control). Both are synthesized in an overnight reaction via condensation of 3-hydroxy-2-naphthoic hydrazide with 2,4,5-trihydroxybenzaldehyde to afford Dyngo 4a, or with benzaldehyde to afford Dyngo Ø. © 2014 Nature America, Inc. All rights reserved.

Gold extraction from complex sulfidic ore occurs in l-butyl-3- methylimidazolium hydrogen sulfate (bmim+HS4-) ionic liquid in the presence of thiourea in a facile, high yielding (86%), recyclable process. Silver recovery from the ore is also enhanced. Gold recovery from ionic liquid / water environments directly by electrodeposition is described. Changing from aqueous acid to ionic liquid solvent causes a growth mode transition to dendritic growth patterns for deposited gold.

The role of the corticotropin releasing hormone in the onset of labour and the subsequent medicinal chemistry implications of CRH antagonists for the prevention of premature birth, and identification of the CRH type 1 receptor as the target for this drug design, are reviewed here.

Serine/threonine protein phosphatases have long been ignored as potential therapeutic targets for two reasons, one the biochemical significance of these proteins has not been appreciated and two, many natural protein phosphatase inhibitors are potent toxins and are considered unsuitable for clinical use. This review outlines the biochemical role of this protein family in cancer, cystic fibrosis, immunosuppression and, cardiac and neurological disorders. Particular emphasis is also given to the synthesis of selective small molecule inhibitors and their clinical exploitation.