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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...

The Bis-T series of compounds comprise some of the most potent inhibitors of dynamin GTPase activity yet reported, e. g., (2E,2 ' E)-N,N '-(propane-1,3-diyl)bis(2-cyano-3-(3,4-dihydroxyphenyl)acrylamide) (2), Bis-T-22. The catechol moieties are believed to limit cell permeability, rendering these compounds largely inactive in cells. To solve this problem, a prodrug strategy was envisaged and eight ester analogues were synthesised. The shortest and bulkiest esters (acetate and butyl/tert-butyl) were found to be insoluble under physiological conditions, whilst the remaining five were soluble and stable under these conditions. These five were analysed for plasma stability and half-lives ranged from similar to 2.3 min (propionic ester 4), increasing with size and bulk, to greater than 24 hr (dimethyl carbamate 10). Similar profiles where observed with the rate of formation of Bis-T-22 with half-lives ranging from similar to 25 mins (propionic ester 4). Propionic ester 4 was chosen to undergo further testing and was found to inhibit endocytosis in a dose-dependent manner with IC50 similar to 8 mu M, suggesting this compound is able to effectively cross the cell membrane where it is rapidly hydrolysed to the desired Bis-T-22 parent compound.

During folliculogenesis, oocytes are dependent on metabolic and molecular support from surrounding somatic cells. Here, we examined the role of the dynamin (DNM) family of mechanoenzymes in mediating endocytotic uptake into growing follicular oocytes. We found DNM1 and DNM2 to be highly expressed in growing follicular oocytes as well as in mature germinal vesicle (GV) and metaphase II (MII) stage oocytes. Moreover, oocyte-specific conditional knockout (cKO) of DNM2 (DNM2Δ) led to complete sterility, with follicles arresting at the preantral stage of development. In addition, DNM2Δ ovaries were characterized by disrupted follicular growth as well as oocyte and follicle apoptosis. Further, the loss of DNM activity, either through DNM2 cKO or through pharmacological inhibition (Dyngo 6a) led to the impairment of endocytotic pathways in preantral oocytes as well as in mature GV and MII oocytes, respectively. Loss of DNM activity resulted in the redistribution of endosomes and the misslocalization of clathrin and actin, suggesting dysfunctional endocytosis. Notably, there was no observable effect on the fertility of DNM1Δ females. Our study has provided new insight into the complex and dynamic nature of oocyte growth during folliculogenesis, suggesting a role for DNM2 in mediating the endocytotic events that are essential for oocyte development.

Photooxygenation reactions involving singlet oxygen (1O2) are utilized industrially as a mild and sustainable access to oxygenated products. Due to the usage of organic dyes as photosensitizers, these transformations can be successfully conducted using natural sunlight. Modern solar chemical reactors enable outdoor operations on the demonstration (multigram) to technical (multikilogram) scales and have subsequently been employed for the manufacturing of fine chemicals such as fragrances or biologically active compounds. This review will highlight examples of solar photooxygenations for the manufacturing of industrially relevant target compounds and will discuss current challenges and opportunities of this sustainable methodology.

An effective multi-step continuous flow approach towards N-diaminoalkylated 3-arylmethylene-2,3-dihydro-1H-isoindolin-1-ones, including the local anesthetic compound AL-12, has been realized. Compared to the traditional decoupled batch processes, the combined photochemical–thermal–thermal flow setup rapidly provides the desired target compounds in superior yields and significantly shorter reaction times.

The development of a (Z)-5-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (2), rhodanine-based lead that led to the Pitstop® 2 family of clathrin inhibitors is described herein. Head group substitution and bioisosteric replacement of the rhodanine core with a 2-aminothiazol-4(5H)-one scaffold eliminated off target dynamin activity. A series of N-substituents gave first phenylglycine (20, IC50 ∼ 20 μM) then phenyl (25, IC50 ∼ 7.1 μM) and 1-napthyl sulfonamide (26, Pitstop® 2 compound, IC50 ∼ 1.9 μM) analogues with good activity, validating this approach. A final library exploring the head group resulted in three analogues displaying either slight improvements or comparable activity (33, 38, and 29 with IC50 ∼ 1.4, 1.6 and 1.8 μM respectively) and nine others with IC50 < 10 μM. These results were rationalized using in silico docking studies. Docking studies predicted enhanced Pitstop® 2 family binding, not a loss of binding, within the Pistop® groove of the reported clathrin mutant invalidating recent assumptions of poor selectivity for this family of clathrin inhibitors.

A series of photochemical transformations has been successfully conducted under continuous-flow conditions in a concentrating solar trough reactor. Photoacylations and [2+2]-photocycloadditions involving 1,4-naphthoquinones gave the corresponding photoproducts in moderate to high yields with residence times of 70min. Likewise, acetone-sensitized photodecarboxylations involving phthalimides furnished the corresponding benzylated hydroxy phthalimidines in good to excellent yields and purity with residence times of 40min. Compared with corresponding exposures to direct sunlight conducted in a solar float, flow operation generally gave superior conversions and subsequent yields.

A series of photochemical transformations has been successfully conducted under continuous-flow conditions in a concentrating solar trough reactor. Photoacylations and [2+2]-photocycloadditions involving 1,4-naphthoquinones gave the corresponding photoproducts in moderate to high yields with residence times of 70min. Likewise, acetone-sensitized photodecarboxylations involving phthalimides furnished the corresponding benzylated hydroxy phthalimidines in good to excellent yields and purity with residence times of 40min. Compared with corresponding exposures to direct sunlight conducted in a solar float, flow operation generally gave superior conversions and subsequent yields.

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.

An effective multi-step continuous flow approach towards N-diaminoalkylated 3-arylmethylene-2,3-dihydro-1H-isoindolin-1-ones, including the local anesthetic compound AL-12, has been realized. Compared to the traditional decoupled batch processes, the combined photochemical&ndash, thermal&ndash, thermal flow setup rapidly provides the desired target compounds in superior yields and significantly shorter reaction times.

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.

We reported the first small molecule inhibitors of the interaction between the clathrin N-terminal domain (TD) and endocyctic accessory proteins (i.e., clathrin inhibition1). Initial screening of a ∼17 000 small molecule ChemBioNet library identified 1. Screening of an existing in-house propriety library identified four substituted 1,8-napthalimides as ∼80-120 μM clathrin inhibitors. Focused library development gave 3-sulfo-N-(4-aminobenzyl)-1,8-naphthalimide, potassium salt (18, IC50 ≈ 18 μM). A second library targeting the 4-aminobenzyl moiety was developed, and four analogues displayed comparable activity (26, 27, 28, 34 with IC50 values of 22, 16, 15, and 15 μM respectively) with a further four (24, 25, 32, 33) more active than 18 with IC50 values of 10, 6.9, 12, and 10 μM, respectively. Docking studies rationalized the structure-activity relationship (SAR) with the biological data. 3-Sulfo-N-benzyl-1,8-naphthalimide, potassium salt (25) with an IC50 ≈ 6.9 μM, is the most potent clathrin terminal domain-amphiphysin inhibitor reported to date.

Six focused rhodanine-based libraries, 60 compounds in total, were synthesized and evaluated as potential dynamin I GTPase inhibitors. Twenty-six were more potent than the lead compound with 13 returning IC50 values ≤10 μM, making the Rhodadyn series among the most active dynamin inhibitors reported. Two analogues were highly effective at blocking receptor-mediated endocytosis: C10 and D10 with IC50(RME) = 7.0 ± 2.2 and 5.9 ± 1.0 μM, respectively. These compounds are equipotent with the best reported in-cell dynamin inhibitors.

Acid-ester and acid-amide norcantharidin derivatives are prepared using a 'one-pot' synthetic procedure utilizing the ThalesNano H-cube™ flow hydrogenator. Traditionally, rapid library generation and reaction scale up of these analogues was limited by the batch wise hydrogenation of 5,6-dehydronorcantharidin. This was resolved with the use of flow chemistry. With no associated scale up issues, a method was devised to produce norcantharidin, along with acid-ester and acid-amide analogues on any scale necessary for biological screening.

The four component Ugi reaction of 2-furaldehyde, an alkenoic acid (three examples), an isonitrile (eight examples) and an amine (eight examples) affords rapid access to a family of acetylenic furan analogues, which on heating undergo an intramolecular Diels–Alder (IMDA) reaction yielding highly substituted tricyclic lactams (17 examples) in good to excellent yields (38–72% two-steps). This Ugi-IMDA reaction proved to be highly substituent tolerant across both the isonitriles and amines examined. In one instance, with N,N-dimethylaminopropylamine, a second equivalent of alkynoic acid was required to afford a good yield of the desired tricyclic lactam.

We report the development of a homology model for the GTP binding domain of human dynamin I based on the corresponding crystal structure of Dictyostelium discoidum dynamin A. Virtual screening identified 2-[(2-biphenyl-2-yl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl)amino]-4-chlorobenzoic acid (1) as a approximately 170 microM potent inhibitor. Homology modeling- and focused library-led synthesis resulted in development of a series of active compounds (the "pthaladyns") with 4-chloro-2-(2-(4-(hydroxymethyl)phenyl)-1,3-dioxoisoindoline-5-carboxamido)benzoic acid (29), a 4.58 +/- 0.06 microM dynamin I GTPase inhibitor. Pthaladyn-29 displays borderline selectivity for dynamin I relative to dynamin II ( approximately 5-10 fold). Only pthaladyn-23 (dynamin I IC(50) 17.4 +/- 5.8 microM) was an effective inhibitor of dynamin I mediated synaptic vesicle endocytosis in brain synaptosomes with an IC(50) of 12.9 +/- 5.9 microM. This compound was also competitive with respect to Mg(2+).GTP. Thus the pthaladyns are the first GTP competitive inhibitors of dynamin I and II GTPase and may be effective new tools for the study of neuronal endocytosis.

[Extract] While the cationic glycopeptide vancomycin has long been regarded as the gold standard for the treatment of recalcitrant Gram-positive bacterial infection, this position has been compromised by the emergence of resistant strains. The first report of such resistance emerged in 1988, and has subsequently widened amongst the enterococci and staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA); 1, 2 cross-resistance to linezolid is also a concern. Some recent chemical strategies for overcoming this resistance have centered on other high molecular weight cyclic peptides, elegantly crafted vancomycin11 or vancomycin aglycone analogues, potent dual-action vancomycin/β-lactam hybrid antibiotics,or large vancomycin dimers. An alternative strategy is to design smaller, simpler cationic peptoids with some related design features to vancomycin which could still interact with the altered peptide-glycan cell-wall moiety in both vancomycin-resistant and -sensitive strains and thus broaden the antibacterial spectrum. Svendsen et al. designed minimal cationic peptidomimetics, and a pharmacophore has been developed for dipeptides which includes the presence of two cationic charges and two hydrophobic units of steric bulk. Subsequently, cationic tripeptide analogues were developed that demonstrated good activity against both Gram-positive (including MRSA) and Gram-negative bacteria, but were not evaluated with respect to vancomycin-resistant strains.

The facility of aminoalcohol ligand synthesis via ring opening of cyclohexene oxide with polyamines including a piperazine ring is illustrated here with the syntheses and characterization of (2′-hydroxycyclohexyl)piperazine (1), bis(2′-hydroxycyclohexyl)-piperazine (2), 4-{(2″-hydroxycyclohexyl)-2′-aminoethyl)}piperazine (3), 1-(2″-hydroxycyclohexyl)-4-{(2″-hydroxycyclohexyl)-2′-aminoethyl)}piperazine (4), and 1,4-bis[(2″-hydroxycyclohexyl)-3′-aminopropyl]piperazine (5) described, along with an analogue of 4 in which a single –CH2–CH2– alkyl chain replaces the piperazine ring, 1,5-bis[(2′-hydroxycyclohexyl)amine]-3-azapentane (6). The viability of 5 as a hexadentate ligand was established by preparation of its copper(II) complex and subsequent X-ray crystal structure analysis. The complex [Cu(5)](ClO4)2 cation lies in a distorted octahedral environment with the four nitrogen donors in an approximate plane also incorporating the copper (Cu–Ntert 2.058(4) A; Cu–Nsec 2.072(4) A) and the two alcohol groups occupying axial sites with elongated bonds (Cu–O 2.415(3) A). The piperazine ring adopts a ‘butterfly wing’ geometry, whereas the two cyclohexane rings are in chair conformations. Significant bond angle distortions occur around the copper, exacerbated by the axial Jahn–Teller bond length distortion. The ability of the copper(II) complexes of the aminoalcohols to promote DNA cleavage was examined. Complexes of 2, 3 and 5 are effectively inactive whereas 4 is an efficient single strand cleavage promoter; however, the more flexible close analogue of 4, 6, also proved ineffective. Such observations for a closely related series indicate the subtle influences of spectator ligand rigidity and steric congestion on DNA cleavage promotion.

The four-strand and potentially N2O2-donor ligand 2,3-endo,endo-bis(aminomethyl)-5,6-endo,endo-bis(hydroxymethyl)bicyclo[2.2.1]heptane (L1), a close analogue of the known tetraalcohol 2,3,5,6-endo,endo,endo,endo-tetrakis(hydroxymethyl)bicyclo[2.2.1]heptane (L2), has been prepared via a multi-step synthesis and isolated as the copper(II) complex [Cu(L1)2](ClO4)2. An ESI-MS study of the complex and metal ion exchange with other transition metal ions (Fe2+, Co2+, Ni2+, Mn2+ or Zn2+) indicates that 1:1 complexes form readily. Apparently special stability for the Ni2+ species observed in the ESI-MS study suggests strong encapsulation of this ion.

l -3-Phenylpropane-1,2-diamine (dapp) was prepared by a three-step synthesis based on l -phenylalanine and characterized, including determination of stability constants with M 2+ ions (Ni, Cu, Zn, Cd). The reaction of L -3-phenylpropane-1,2-diamine as the [Cu(dapp) 2 ] 2+ complex ion with formaldehyde and nitroethane in basic solution yields the acyclic (5-methyl-5-nitro-1,9-diphenyl-3,7-diazanonane-1,9-diamine)copper(II) complex ion, [Cu( 1 )] 2+ , as the major product. In addition, small amounts of the macrocyclic complex ion (2,10-diphenyl-6,13-dimethyl-6,13-dinitro-1,4,8,11-tetraazacyclotetradecane)copper(II), [Cu( 2 )] 2+ , form. Reduction of the [Cu( 1 )] 2+ ion with zinc in aqueous acid yields the acyclic polyamine 5-methyl-1,9-diphenyl-3,7-diazanonane-1,5,9-triamine ( 3 ), an analogue of the previously reported pentaamine 5-methyl-3,7-diazanonane-1,5,9-triamine. Using the bis( l -3-phenylpropane-1,2-diamine)palladium(II) as precursor and an excess of other reagents, the macrocyclization reaction to produce [Pd( 2 )] 2+ proved more successful. Reduction and recomplexation to copper(II) allowed isolation of the 2,9-dibenzyl-6,13-diammonio-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane)copper(II) ion, [Cu( 4 H 2 2+ )] 4+ . The acyclic complex [Cu( 1 )] 2+ promotes the hydrolytic cleavage of plasmid DNA modestly; a mechanism to support this observation is presented.

Owing to their favourable photophysical and electrochemical properties, phthalimides undergo a variety of highly efficient photodecarboxylation reactions. These transformations have been applied to the synthesis of macrocyclic compounds as well as bioactive addition adducts. N-Acetoxyphthalimides are versatile precursors to imidyl and alkyl radicals through photodecarboxylation and have subsequently been used for a variety of coupling reactions. The generally mild reaction conditions make these reactions attractive for green chemical applications. The process protocols were successfully transferred to novel photoreactor devices, among these falling film or continuous flow reactors.

The saturated, stereodefined tetraalcohol 2,3,5,6- endo,endo,endo,endo- tetrakis(hydroxymethyl)bicyclo[2.2.1]heptane (tetol, L 1 ) and the simple alcohol butane-1,3-diol (L 2 ) form complexes with alkali metal ions (lithium, sodium, potassium, rubidium and caesium), alkali earth cations (magnesium, calcium, strontium and barium) and Ga(III) and Ce(IV) in aqueous solution, characterised by electrospray ionisation mass spectrometry (ESMS). Metal ion exchange between the Li + complex of L 1 and the other metal ions is rapid, with a range of M(L 1 ) n m + species detected, in addition to solvated species. With the alkal metal ions, M(L 1 ) + and M(L 1 ) 2 + are dominant, although speciation varies with metal ion size. For the alkaline earth ions, a range of complex ions up to n =8 are observed, although n =1–3 dominate. A preference for M(L 1 ) 2 2+ with Mg 2+ versus M(L 1 ) 3 2+ with Ca 2+ may again relate to a larger ion size. For the higher-charged Ga(III) and Ce(IV), hydroxo species M(OH)(L 1 ) n ( m −1)+ are dominant reflecting bulk solution behaviour, which the ESMS studies appear to map generally.

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 O (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 O.

The tetraalcohol 2,3,5,6-endo,endo,endo,endo-tetrakis(hydroxymethyl]bicyclo[2.2.1]heptane (tetol, 1) has been prepared and crystallises readily as the lithium(I) complex [Li(1)(2)]Cl, forming an oligomeric multi-chain structure in which pairs of alcohols from two crystallographically independent tetol molecules bind lithium ions tetrahedrally. However, formation of monomeric structures in solution is inferred from electrospray mass spectroscopy, which has also shown evidence of exchange of lithium ion in the complexed species by added alkaline earth ions. (C) 2000 Elsevier Science S.A. All rights reserved.

Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop® inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane.

Objective A mixed-methods study exploring the UK general public9s views towards consent for the use of biosamples for biomedical research. Setting Cross-sectional population-based focus groups followed by an online survey. Participants 12 focus groups (81 participants) selectively sampled to reflect a range of demographic groups; 1110 survey responders recruited through a stratified sampling method with quotas set on sex, age, geographical location, socioeconomic group and ethnicity. Main outcome measures (1) Views on the importance of consent when donating residual biosamples for medical research; (2) preferences for opt-in or opt-out consent approaches and (3) preferences for different consent models. Results Participants believed obtaining consent for use of residual biosamples was important as it was ‘morally correct’ to ask, and enabled people to make an active choice and retain control over their biosamples. Survey responders preferred opt-in consent (55%); the strongest predictor was being from a low socioeconomic group (OR 2.22, 95% CI 1.41 to 3.57, p=0.001) and having a religious affiliation (OR 1.36, 95% CI 1.01 to 1.81, p=0.04). Focus group participants had a slight preference for opt-out consent because by using this approach more biosamples would be available and facilitate research. Concerning preferred models of consent for research use of biosamples, survey responders preferred specific consent with recontact for each study for which their biosamples are eligible. Focus group participants preferred generic consent as it provided ‘flexibility for researchers’ and reduced the likelihood that biosamples would be wasted. The strongest predictor for preferring specific consent was preferring opt-in consent (OR 4.58, 95% CI 3.30 to 6.35, p=0.015) followed by non-‘White’ ethnicity (OR 2.94, 95% CI 1.23 to 7.14, p Conclusions There is a preference among the UK public for ongoing choice and control over donated biosamples; however, increased knowledge and opportunity for discussion is associated with acceptance of less restrictive consent models for some people.

Objective A mixed methods study exploring the UK general public's willingness to donate human biosamples (HBSs) for biomedical research.\ud \ud Setting Cross-sectional focus groups followed by an online survey.\ud \ud Participants Twelve focus groups (81 participants) selectively sampled to reflect a range of demographic groups; 1110 survey responders recruited through a stratified sampling method with quotas set on sex, age, geographical location, socioeconomic group and ethnicity.\ud \ud Main outcome measures (1) Identify participants’ willingness to donate HBSs for biomedical research, (2) explore acceptability towards donating different types of HBSs in various settings and (3) explore preferences regarding use and access to HBSs.\ud \ud Results 87% of survey participants thought donation of HBSs was important and 75% wanted to be asked to donate in general. Responders who self-reported having some or good knowledge of the medical research process were significantly more likely to want to donate (p\ud \ud Conclusions There is a high level of public support and willingness to donate HBSs for biomedical research. Underlying concerns exist regarding the use of certain types of HBSs and conditions under which they are used. Improved education and more controlled forms of consent for sensitive samples may mitigate such concerns.

Focused library development of our lead 2-cyano-3-(1-(3-(dimethylamino)propyl)-2-methyl-1H-indol-3-yl)-N-octylacrylamide (2) confirmed the tertiary dimethylamino-propyl moiety as critical for inhibition of dynamin GTPase. The cyanoamide moiety could be replaced with a thiazole-4(5H)-one isostere (19, IC(₅₀(dyn I)) = 7.7 μM), reduced under flow chemistry conditions (20, IC(₅₀(dyn I)) = 5.2 μM) or replaced by a simple amine. The latter provided a basis for a high yield library of compounds via a reductive amination by flow hydrogenation. Two compounds, 24 (IC(₅₀ (dyn I)) = 0.56 μM) and 25 (IC(₅₀(dyn I)) = 0.76 μM), stood out. Indole 24 is nontoxic and showed increased potency against dynamin I and II in vitro and in cells (IC(₅₀(CME)) = 1.9 μM). It also showed 4.4-fold selectivity for dynamin I. The indole 24 compound has improved isoform selectivity and is the most active in-cell inhibitor of clathrin-mediated endocytosis reported to date.

The four component Ugi reaction of 2-furaldehyde, an alkenoic acid (three examples), an isonitrile (eight examples) and an amine (eight examples) affords rapid access to a family of acetylenic furan analogues, which on heating undergo an intramolecular Diels–Alder (IMDA) reaction yielding highly substituted tricyclic lactams (17 examples) in good to excellent yields (38–72% two-steps). This Ugi-IMDA reaction proved to be highly substituent tolerant across both the isonitriles and amines examined. In one instance, with N,N-dimethylaminopropylamine, a second equivalent of alkynoic acid was required to afford a good yield of the desired tricyclic lactam.

In an investigation into their potential ecological role(s), a group of mainly diterpene isonitriles, nine in total, isolated from the tropical marine sponge Cymbastela hooperi, and the sesquiterpene axisonitrile-3, isolated from the tropical marine sponge Acanthella kletra, were evaluated in a series of bioassays including anti-fouling, anti-algal, anti-photosynthetic, anti-bacterial (Gram +ve and −ve), anti-fungal, and anti-tubercular. The results of these assays showed that all of the tested compounds, with the exception of diterpene 9, were active in at least two of the applied test systems, with axisonitrile-3 (10) and diterpene isonitrile 1 being the two most active compounds overall, closely followed by diterpene isonitrile 3. Based on the results of the photosynthetic study a molecular modelling investigation was undertaken with all of the compounds used in that study. The results showed a positive correlation between reduction in photosynthetic activity and the interaction of the modelled compounds with a potential enzyme active site.

The synthesis of eleven novel antibacterial agents is reported. The structures are based on a C 2 -symmetric binaphthyl scaffold which holds two identical chains consisting of a short linker, a basic amino acid and a small hydrophobic side chain. Antibacterial activity is revealed for a number of derivatives down to an MIC of 2 μg/mL (2 μM) against Staphylococcus aureus – comparable to vancomycin, and an MIC of 31 μg/mL (31 μM) against some vancomycin-resistant enterococcal strains.

Herein we report the synthesis of discrete iminochromene ("iminodyn") libraries (14-38) as potential inhibitors of dynamin GTPase. Thirteen iminodyns were active (IC(50) values of 260 nM to 100 microM), with N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (17), N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (22), and N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (23) (IC(50) values of 330 +/- 70, 450 +/- 50, and 260 +/- 80 nM, respectively) being the most potent. Five of the most potent iminodyns all inhibited dynamins I and II approximately equally. Iminodyn-22 displayed uncompetitive inhibition with respect to GTP. Selected iminodyns were evaluated for their ability to block receptor mediated endocytosis (RME, mediated by dynamin II) and synaptic vesicle endocytosis (SVE, mediated by dynamin I), with 17 showing no activity while 22 returned RME and SVE IC(50) values of 10.7 +/- 4.5 and 99.5 +/- 1.7 microM, respectively. The iminodyns reported herein represent a new chemical class of the first nanomolar potent dynamin inhibitors that are also effective endocytosis inhibitors.

An efficient synthesis of 29 new binaphthyl-based neutral, and mono- and di-cationic, peptoids is described. Some of these compounds had antibacterial activities with MIC values of 1.9-3.9microg/mL against Staphylococcus aureus. One peptoid had a MIC value of 6microg/mL against a methicillin-resistant strain of S. aureus (MRSA) and a MIC value of 2microg/mL against vancomycin-resistant strains of enterococci (VRE).

Estrogens play an important role in breast cancer development, with approximately 60% of premenopausal and 75% of postmenopausal breast cancer patients having estrogen-dependent carcinomas. In recent decades endocrine therapy has emerged for the treatment of estrogen receptor (ER) positive breast cancer, including aromatase inhibitors (anastrozole) and estrogen receptor antagonists (tamoxifen). However, drug resistance is often induced by these therapies, tumor selectivity is sometimes poor with non-tumor populations being affected, and target selectivity is also a problem with “off-target-effects” culminating in adverse toxicities and/or limited efficacy. For the past ten years collaboration between the Calvary Mater Newcastle Hospital and the University of Newcastle (Australia) has seen the development of many small molecule drugs for the treatment of malignancy. Our recent findings have discovered a family of 2-phenylacrylonitriles showing growth inhibition against a panel of human cancer derived cell lines. Focused library approaches facilitated the identification of a simple pharmacophore, comprising two terminal aromatic moieties linked via a conjugated cyano moiety. Multiple libraries led to the discovery of two key compounds, MT-CN-07 and MT-CN-18. Both exhibit broad spectrum growth inhibition with GI50 values of 3.2–46μM and 3.1–69μM, respectively, in a panel of nine human cancer cell lines derived from colon, ovarian, lung, skin, prostate and pancreatic carcinomas, neuroblastoma and glioblastoma1. An initial screen in one breast cancer ER+ve cell line (MCF-7) suggested that these compounds may selectively target estrogen dependent breast cancer cells. Indeed recent studies have shown that MT-CN-07 and MT-CN-18 are up to 645 fold more potent at inhibiting the growth of ER+ve breast cancer cells (ZR-75–1, T47D, MCF-7) with GI50 values of 0.08–0.6μM, and 0.107–2.25μM, respectively, when compared with the nine cell lines derived from other tumor types. Moreover, these compounds are up to 450 fold more potent in ER+ve breast cancer cell lines when compared with the estrogen independent cell line MDA-MB-231 (ER-ve, GI50 = 36μM and 26μM, respectively) and the non-tumorigenic breast epithelial cell line MCF10A (GI50 = 36μM and 37μM, respectively). Screening of these compounds in the drug resistant, etoposide-selected breast cancer cell line, MCF7/VP, which has amplification and over-expression of the ABCC1 gene, showed they were not substrates for this drug resistant mechanism. The ability to specifically target estrogen dependent tumors, while having little or no effect on normal breast cells, or other tumor types is a unique finding. Thus we have identified a novel group of molecules that could be exploited for the treatment of estrogen sensitive breast cancer. Reference: 1. Tarleton M, Gilbert J, Robertson MJ, McCluskey A, Sakoff JA. (2011) Library synthesis and cytotoxicity of a family of 2-phenylacrylonitriles and discovery of an estrogen dependent breast cancer lead compound. Med Chem Comm. 2, 31–37 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A127.

The unsymmetric mixed-donor Nam,Npy,O-ligand N-trans-(2'-hydroxycyclohexyl)-2-aminomethylpyridine (pyca, 1) was readily prepared by reaction of an epoxide (cyclohexene oxide) with (2-aminomethyl)pyridine. The ligand forms octahedral bis-tridentate complexes, exemplified with isolation of copper(II), nickel(II), zinc(II), and cobalt(III) compounds; in addition, a square-planar palladium(II) complex with the ligand behaving as bidentate was prepared. The bis-tridentate octahedral complexes may exist in six geometric isomeric forms, and molecular mechanics analysis was employed to predict the most stable isomer for copper(II), nickel(II), and cobalt(III) complexes. For the copper(II) and cobalt(III) complexes, the predictions match the X-ray crystal structures of the single isomer isolated in each case. Both the copper(II) complex [Cu . 12](ClO4)2 and the cobalt(III) complex [Co . 1 . (1–H)](ClO4)2 crystallized in the monoclinic space group P21/n (no. 14), with a 9.8400(13), b 15.434(2), c 10.0180(13) Å, and β 109.004(2)°, and a 14.162, b 18.583, c 21.596 Å, and β 100.04°, respectively. Both complexes adopt distorted octahedral structures. The copper(II) complex exists as the all-trans-fac isomer with Jahn–Teller elongation of the Cu–O bond [2.3795(19) Å] compared to Cu–Npy [2.040(2) Å] and Cu–Nam [2.024(2) Å] distances. The cobalt(III) complex appears as the cis,cis,trans(Npy) fac isomer, but deprotonation of one of the two alcohol groups leads to all six M–L distances differing: for each of two similar independent complex cations, Co–O [e.g. 1.9123(16), 1.9459(16) Å], Co–Nam [e.g. 1.949(2), 1.968(2) Å], and Co–Npy [e.g. 1.938(2), 1.940(2) Å] vary, with particularly the deprotonated alcohol bond significantly shorter than the protonated bond, despite a strong hydrogen bonding interaction with the protonated alcohol in the partner complex cation in the unit cell.

The chiral 2,5,8-triazabicyclo[7.4.01,9]tridecane (L1), an analogue of 1,4,7-triazacyclononane, (L2) that has a trans-cyclohexane ring fused to the tacn framework, forms bis complexes with a wide range of metal ions where two geometric forms may exist depending on the relative locations of the substituent cyclohexane units. For the crystal structure of bis(RR-2,5,8-triazabicyclo[7.4.01,9]tridecane)nickel(II) nitrate, two macrocyclic ligands each occupy a face on opposite sides of the metal-centred octahedron, with the cyclohexane rings, when viewed down an axis passing through the centre of these faces and the metal, arranged in an anti disposition. However, the crystal structure of bis(RR-2,5,8-triazabicyclo[7.4.01,9]tridecane)chromium(III) perchlorate shows the cyclohexane rings disposed in the alternate syn arrangement. Isomerism is defined simply by the spatial disposition of macrocycle substituents. Isomeric preference has been probed by a molecular mechanics analysis, including an energy profile analysis as one macrocycle is rotated relative to the other. The force field calculations predict very small differences between isomers, but a large barrier to interconversion by a twist mechanism. Complexation of L1 with a range of labile metal(II) ions has been probed by determination of apparent stability constants via spectrophotometric titrations. In general, formation constants of L1 complexes are similar to or even slightly higher than the unsubstituted 1,4,7-triazacyclononane (L2) analogues, consistent with no strong steric influence of the fused cyclohexane rings in L1 and with differences in properties reflecting minor electronic effects and ligand flexibility differences resulting from substitution.

SummaryClathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.

We examined the effect of chronic opiate treatment on the alpha 2-adrenergic receptor binding of both the agonist [3H]clonidine and the antagonist [3H]yohimbine in the cerebral cortex and kidney of rats. In addition, we demonstrated the presence of an adrenergic receptor of the alpha 2 type on the neurotumor cell line NCB20 and used this cell line as a model for studying the interaction between the opiate and alpha 2-adrenergic systems. In all the three above systems, chronic opiate treatment did not modify the number or the affinity of alpha 2-adrenergic receptors.