Platinum(IV) Prodrugs Incorporating an Indole-Based Derivative, 5-Benzyloxyindole-3-Acetic Acid in the Axial Position Exhibit Prominent Anticancer Activity.

Kinetically inert platinum(IV) complexes are a chemical strategy to overcome the impediments of standard platinum(II) antineoplastic drugs like cisplatin, oxaliplatin and carboplatin. In this study, we reported the syntheses and structural characterisation of three platinum(IV) complexes that incorporate 5-benzyloxyindole-3-acetic acid, a bioactive ligand that integrates an indole pharmacophore. The purity and chemical structures of the resultant complexes, P-5B3A, 5-5B3A and 56-5B3A were confirmed via spectroscopic means. The complexes were evaluated for anticancer activity against multiple human cell lines. All complexes proved to be considerably more active than cisplatin, oxaliplatin and carboplatin in most cell lines tested. Remarkably, 56-5B3A demonstrated the greatest anticancer activity, displaying GI50 values between 1.2 and 150 nM. Enhanced production of reactive oxygen species paired with the decline in mitochondrial activity as well as inhibition of histone deacetylase were also demonstrated by the complexes in HT29 colon cells.

3,5-Bis(trifluoromethyl)phenylsulfonamides, a novel pancreatic cancer active lead. Investigation of the terminal aromatic moiety.

Virtual screening identified N-(6-((4-bromobenzyl)amino)hexyl)-3,5-bis(trifluoromethyl)benzenesulfonamide (1) a lead compound that bound to the S100A2-p53 binding groove. S100A2 is a Ca2+ binding protein with implications in cell signaling and is known to be upregulated in pancreatic cancer. It is a validated pancreatic cancer drug target. Lead 1, inhibited the growth of the MiaPaCa-2 pancreatic cancer cell line (GI50 = 2.97 µM). Focused compound libraries were developed to explore the SAR of this compound class with 4 libraries and 43 compounds total. Focused library (Library 1) development identified lipophillic sulfonamides as preferred for MiaPaCa-2 activity, with -CF3 and -C(CH3)3 substituents well tolerated (MiaPaCa-2 GI50 < 6 µM). Contraction of the hexylamino spacer to ethyl (Library 2) and propyl (Library 3) proved beneficial to activity against a broad spectrum panel of cancer cell lines: HT29 (lung), MCF-7 (breast), A2780 (ovarian), H460 (colon), A431 (skin), Du145 (prostate), BE2-C (neuroblastoma), U87 and SJ-G2 (glioblastoma) (cohort-1); and a pancreatic cancer cell line panel: MiaPaCa-2, BxPC-3, AsPC-1, Capan-2, HPAC and PANC-1 (cohort-2). With a marked preference for a propyl linker the observed GI50 values ranged from 1.4 to 30 µM against cohort-1 and 1.4-30 µM against cohort-2 cell lines. In Library 4 the terminal aromatic moiety was explored with 4-substituted analogues preferred (with activity of 48 (4-Cl) > 47 (3-Cl) > 46 (2-Cl)) against the cell lines examined. The introduction of bulky aromatic moieties was well tolerated, e.g. dihydrobenzo[b][1,4]dioxine (51) returned cohort-2 GI50 values of 1.2-3.4 µM. In all instances the observed docked binding poses and binding scores were consistent with the observed cytotoxicity. This in turn supports, but does not prove, that these analogues function via S100A2-p53 binding groove inhibition.

Potent Chlorambucil-Platinum(IV) Prodrugs.

The DNA-alkylating derivative chlorambucil was coordinated in the axial position to atypical cytotoxic, heterocyclic, and non-DNA coordinating platinum(IV) complexes of type, [PtIV(HL)(AL)(OH)2](NO3)2 (where HL is 1,10-phenanthroline, 5-methyl-1,10-phenanthroline or 5,6-dimethyl-1,10-phenanthroline, AL is 1S,2S-diaminocyclohexane). The resultant platinum(IV)-chlorambucil prodrugs, PCLB, 5CLB, and 56CLB, were characterized using high-performance liquid chromatography, nuclear magnetic resonance, ultraviolet-visible, circular dichroism spectroscopy, and electrospray ionization mass spectrometry. The prodrugs displayed remarkable antitumor potential across multiple human cancer cell lines compared to chlorambucil, cisplatin, oxaliplatin, and carboplatin, as well as their platinum(II) precursors, PHENSS, 5MESS, and 56MESS. Notably, 56CLB was exceptionally potent in HT29 colon, Du145 prostate, MCF10A breast, MIA pancreas, H460 lung, A2780, and ADDP ovarian cell lines, with GI50 values ranging between 2.7 and 21 nM. Moreover, significant production of reactive oxygen species was detected in HT29 cells after treatment with PCLB, 5CLB, and 56CLB up to 72 h compared to chlorambucil and the platinum(II) and (IV) precursors.

Borylated 2,3,4,5-Tetrachlorophthalimide and Their 2,3,4,5-Tetrachlorobenzamide Analogues: Synthesis, Their Glycosidase Inhibition and Anticancer Properties in View to Boron Neutron Capture Therapy.

Tetrachlorinated phthalimide analogues bearing a boron-pinacolate ester group were synthesised via two synthetic routes and evaluated in their glycosidase modulating and anticancer properties, with a view to use them in boron neutron capture therapy (BNCT), a promising radiation type for cancer, as this therapy does little damage to biological tissue. An unexpected decarbonylation/decarboxylation to five 2,3,4,5-tetrachlorobenzamides was observed and confirmed by X-ray crystallography studies, thus, giving access to a family of borylated 2,3,4,5-tetrachlorobenzamides. Biological evaluation showed the benzamide drugs to possess good to weak potencies (74.7-870 µM) in the inhibition of glycosidases, and to have good to moderate selectivity in the inhibition of a panel of 18 glycosidases. Furthermore, in the inhibition of selected glycosidases, there is a core subset of three animal glycosidases, which is always inhibited (rat intestinal maltase α-glucosidase, bovine liver ß-glucosidase and ß-galactosidase). This could indicate the involvement of the boron atom in the binding. These glycosidases are targeted for the management of diabetes, viral infections (via a broad-spectrum approach) and lysosomal storage disorders. Assays against cancer cell lines revealed potency in growth inhibition for three molecules, and selectivity for one of these molecules, with the growth of the normal cell line MCF10A not being affected by this compound. One of these molecules showed both potency and selectivity; thus, it is a candidate for further study in this area. This paper provides numerous novel aspects, including expedited access to borylated 2,3,4,5-tetrachlorophthalimides and to 2,3,4,5-tetrachlorobenzamides. The latter constitutes a novel family of glycosidase modulating drugs. Furthermore, a greener synthetic access to such structures is described.

Bioactive Platinum(IV) Complexes Incorporating Halogenated Phenylacetates.

A new series of cytotoxic platinum(IV) complexes (1-8) incorporating halogenated phenylacetic acid derivatives (4-chlorophenylacetic acid, 4-fluorophenylacetic acid, 4-bromophenylacetic acid and 4-iodophenylacetic acid) were synthesised and characterised using spectroscopic and spectrometric techniques. Complexes 1-8 were assessed on a panel of cell lines including HT29 colon, U87 glioblastoma, MCF-7 breast, A2780 ovarian, H460 lung, A431 skin, Du145 prostate, BE2-C neuroblastoma, SJ-G2 glioblastoma, MIA pancreas, the ADDP-resistant ovarian variant, and the non-tumour-derived MCF10A breast line. The in vitro cytotoxicity results confirmed the superior biological activity of the studied complexes, especially those containing 4-fluorophenylacetic acid and 4-bromophenylacetic acid ligands, namely 4 and 6, eliciting an average GI50 value of 20 nM over the range of cell lines tested. In the Du145 prostate cell line, 4 exhibited the highest degree of potency amongst the derivatives, displaying a GI50 value of 0.7 nM, which makes it 1700-fold more potent than cisplatin (1200 nM) and nearly 7-fold more potent than our lead complex, 56MESS (4.6 nM) in this cell line. Notably, in the ADDP-resistant ovarian variant cell line, 4 (6 nM) was found to be almost 4700-fold more potent than cisplatin. Reduction reaction experiments were also undertaken, along with studies aimed at determining the complexes' solubility, stability, lipophilicity, and reactive oxygen species production.

The aryl hydrocarbon receptor (AhR) as a breast cancer drug target.

Breast cancer is the most common cancer in women, with more than 1.7 million diagnoses worldwide per annum. Metastatic breast cancer remains incurable, and the presence of triple-negative phenotypes makes targeted treatment impossible. The aryl hydrocarbon receptor (AhR), most commonly associated with the metabolism of xenobiotic ligands, has emerged as a promising biological target for the treatment of this deadly disease. Ligands for the AhR can be classed as exogenous or endogenous and may have agonistic or antagonistic activity. It has been well reported that agonistic ligands may have potent and selective growth inhibition activity in a number of oncogenic cell lines, and one (aminoflavone) has progressed to phase I clinical trials for breast cancer sufferers. In this study, we examine the current state of the literature in this area and elucidate the promising advances that are being made in hijacking the cytosolic-to-nuclear pathway of the AhR for the possible future treatment of breast cancer.

Phytochemical, antioxidant, anti-proliferative and antimicrobial properties of Catharanthus roseus root extract, saponin-enriched and aqueous fractions.

Catharanthus roseus (L.) G. Don (C. roseus) is a well-known medicinal plant for its source of alkaloids solely found in the leaves. Other parts including the root are usually discarded after the alkaloid extraction. This study sought to investigate phytochemical profiles, antioxidant, antimicrobial and cytotoxic properties of the C. roseus root extract (RE) and its two sub-fractions including saponin-enriched (SE) and aqueous (AQ) fractions. The results showed that the RE was a rich source of saponins (1744.44 mg ESE/g) and phenolics (51.27 mg GAE/g), which comprised of gallic acid (25.74 mg/g), apigenin (1.45 mg/g) and kaempferol (1.58 mg/g). The SE fraction was enriched with 31% of saponins and 63% of phenolics higher than those of the RE; whereas the concentrations of saponins and phenolics of the AQ fraction were lower than those of the RE by 40% and 74%, respectively. The content of gallic acid in the SE fraction was 1.4-fold and 1.5-fold higher than those of the RE or AQ fraction, respectively. The SE fraction demonstrated potent antioxidant capacity, which was significantly higher than the RE or AQ fraction, and also exhibited strong anti-proliferative activity against 11 cancer cell lines including A2780 (ovarian), H460 (lung), A431 (skin), MIA PaCa-2 (pancreas), Du145 (prostate), HT29 (colon), MCF-7 (breast), BE2-C (neuroblastoma), SJ-G2, U87 and SMA (glioblastoma) with low GI50 values (≤ 2.00 µg/mL). The SE fraction was also shown to effectively inhibit the growth of both bacteria (Escherichia coli, Enterobacter aerogenes and Staphylococccus lugdunensis) and fungi (Candida albicans and Aspergillus niger). These findings warrant further investigation to isolate major compounds from the SE fraction and further test their antioxidant, anticancer and antimicrobial activities.

o-Vanillin Derived Schiff Bases and Their Organotin(IV) Compounds: Synthesis, Structural Characterisation, In-Silico Studies and Cytotoxicity.

Six new organotin(IV) compounds of Schiff bases derived from S-R-dithiocarbazate [R = benzyl (B), 2- or 4-methylbenzyl (2M and 4M, respectively)] condensed with 2-hydroxy-3-methoxybenzaldehyde (oVa) were synthesised and characterised by elemental analysis, various spectroscopic techniques including infrared, UV-vis, multinuclear (¹H, 13C, 119Sn) NMR and mass spectrometry, and single crystal X-ray diffraction. The organotin(IV) compounds were synthesised from the reaction of Ph2SnCl2 or Me2SnCl2 with the Schiff bases (S2MoVaH/S4MoVaH/SBoVaH) to form a total of six new organotin(IV) compounds that had a general formula of [R2Sn(L)] (where L = Schiff base; R = Ph or Me). The molecular geometries of Me2Sn(S2MoVa), Me2Sn(S4MoVa) and Me2Sn(SBoVa) were established by X-ray crystallography and verified using density functional theory calculations. Interestingly, each experimental structure contained two independent but chemically similar molecules in the crystallographic asymmetric unit. The coordination geometry for each molecule was defined by thiolate-sulphur, phenoxide-oxygen and imine-nitrogen atoms derived from a dinegative, tridentate dithiocarbazate ligand with the remaining positions occupied by the methyl-carbon atoms of the organo groups. In each case, the resulting five-coordinate C2NOS geometry was almost exactly intermediate between ideal trigonal-bipyramidal and square-pyramidal geometries. The cytotoxic activities of the Schiff bases and organotin(IV) compounds were investigated against EJ-28 and RT-112 (bladder), HT29 (colon), U87 and SJ-G2 (glioblastoma), MCF-7 (breast) A2780 (ovarian), H460 (lung), A431 (skin), DU145 (prostate), BE2-C (neuroblastoma) and MIA (pancreatic) cancer cell lines and one normal breast cell line (MCF-10A). Diphenyltin(IV) compounds exhibited greater potency than either the Schiff bases or the respective dimethyltin(IV) compounds. Mechanistic studies on the action of these compounds against bladder cancer cells revealed that they induced the production of reactive oxygen species (ROS). The bladder cancer cells were apoptotic after 24 h post-treatment with the diphenyltin(IV) compounds. The interactions of the organotin(IV) compounds with calf thymus DNA (CT-DNA) were experimentally explored using UV-vis absorption spectroscopy. This study revealed that the organotin(IV) compounds have strong DNA binding affinity, verified via molecular docking simulations, which suggests that these organotin(IV) compounds interact with DNA via groove-binding interactions.

(Z)-2-(3,4-Dichlorophenyl)-3-(1H-Pyrrol-2-yl)Acrylonitrile Exhibits Selective Antitumor Activity in Breast Cancer Cell Lines via the Aryl Hydrocarbon Receptor Pathway.

We have previously reported the synthesis and breast cancer selectivity of (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile (ANI-7) in cancer cell lines. To further evaluate the selectivity of ANI-7, we have expanded upon the initial cell line panel to now include the breast cancer cell lines (MCF7, MCF7/VP16, BT474, T47D, ZR-75-1, SKBR3, MDA-MB-468, BT20, MDA-MB-231); normal breast cells (MCF-10A); and cell lines derived from colon (HT29), ovarian (A2780), lung (H460), skin (A431), neuronal (BE2C), glial (U87, SJG2), and pancreatic (MIA) cancers. We now show that ANI-7 is up to 263-fold more potent at inhibiting the growth of breast cancer cell lines (MCF7, MCF7/VP16, BT474, T47D, ZR-75-1, SKBR3, MDA-MB-468) than normal breast cells (MCF-10A) or cell lines derived from other tumor types. Measures of growth inhibition, cell cycle analysis, morphologic assessment, Western blotting, receptor binding, gene expression, small interfering RNA technology, reporter activity, and enzyme inhibition assays were exploited to define the mechanism of action of ANI-7. In this work, we report that ANI-7 mediates its effects via the activation of the aryl hydrocarbon receptor (AhR) pathway and the subsequent induction of CYP1-metabolizing mono-oxygenases. The metabolic conversion of ANI-7 induces DNA damage, checkpoint activation, S-phase cell cycle arrest, and cell death in sensitive breast cancer cell lines. Basal expression of AhR, the AhR nuclear translocator, and the CYP1 family members do not predict for sensitivity; however, inherent expression of the phase II-metabolizing enzyme sulfur transferase 1A1 does. For the first time, we identify (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile as a new AhR ligand.

In vitro antibacterial and anticancer properties of Helicteres hirsuta Lour. leaf and stem extracts and their fractions.

Helicteres hirsuta Lour. (H. hirsuta) has been considered as a herbal medicine for the treatment of malaria and diabetes but limited studies have been conducted on its anticancer and antibacterial properties. In this study, the in vitro antibacterial and anticancer properties of the leaf and stem extracts and their two sub-fractions (aqueous and saponin-enriched butanol fractions) prepared from H. hirsuta were elucidated. MTT and CCK-8 assays were employed to assess their in vitro anticancer properties against various cancer cell lines. The antibacterial activity was assessed using the disc diffusion method and minimum inhibitory concentration (MIC) values were determined. The results revealed that the saponin-enriched fractions from H. hirsuta leaves and stems showed the highest antibacterial activity against E. coli (MIC values of 2.50 and 5.00 mg/mL, respectively) and S. lugdunensis (MIC values of 0.35 and 0.50 mg/mL, respectively). Importantly, these saponin-enriched fractions possessed strong anticancer activity in vitro towards a range of cancer cell lines including MIA PaCa-2 (pancreas); A2780 (ovarian); H460 (lung); A431 (skin); Du145 (prostate); HT29 (colon); MCF-7 (breast); SJ-G2, U87, SMA (glioblastoma) and BE2-C (neuroblastoma) at low doses (GI50 values of 0.36-11.17 µg/mL). They especially revealed potent anti-pancreatic cancer activity in vitro against MIA PaCa-2, BxPC-3 and CFPAC-1 cells with IC50 values of 1.80-6.43 µg/mL. This finding provides scientific evidence of the cytotoxic activity of the extracts prepared from H. hirsuta leaves and stems, and suggests further studies to isolate active compounds for development of new anticancer agents from these plant extracts.

Physicochemical, Antioxidant, and Cytotoxic Properties of Xao Tam Phan (Paramignya trimera) Root Extract and Its Fractions.

Xao tam phan (Paramignya trimera (Oliv.) Guillaum) has been used as a medicinal plant for cancer prevention and treatment in recent years. The objective of this study was to determine the physicochemical, antioxidant, and cytotoxic properties of crude P. trimera root (PTR) extract and its fractions using MeOH as a solvent and microwave-assisted extraction as an advanced technique for preparation of the PTR extract. The results showed that the PTR extract had high contents of saponins, phenolics, flavonoids, and proanthocyanidins (7731.05 mg escin equiv. (EE), 238.13 mg gallic acid equiv. (GAE), 81.49 mg rutin equiv., and 58.08 mg catechin equiv. (CE)/g dried extract, resp.). Antioxidant activity of PTR extract was significantly higher (P < 0.05) than those of four its fractions and ostruthin, a key bioactive compound in the P. trimera, while potent cytotoxic capacity of PTR extract on various cancer cell lines in terms of MiaPaCa-2 (pancreas), HT29 (colon), A2780 (ovarian), H460 (lung), A431 (skin), Du145 (prostate), BE2-C (neuroblastoma), MCF-7 (breast), MCF-10A (normal breast), and U87, SJ-G2, SMA (glioblastoma) was observed with GI50 values ranging from 15 to 32 µg/ml. Cytotoxic potential on pancreatic cancer cells of PTR extract (100 - 200 µg/ml) was significantly higher (P < 0.05) than those of its four fractions (50 µg/ml), ostruthin (20 µg/ml) and gemcitabine (50 nm), and being comparable to a saponin-enriched extract from quillajia bark, a commercial product. Based on the results achieved, we can conclude that the PTR extract is a potential source for application of in the nutraceutical, medical, and pharmaceutical industries.

Physicochemical Properties, Antioxidant and Anti-proliferative Capacities of Dried Leaf and Its Extract from Xao tam phan (Paramignya trimera).

Xao tam phan (Paramignya trimera) has been used for the treatment of cancer and cancer-like aliments. Among different parts of the P. trimera plant, leaf is considered as a residual part after harvesting of the root. This study aimed to determine the physiochemical properties and the antioxidant and anti-proliferative capacities of P. trimera leaf (PTL) using microwave drying for the preparation of dry sample; MeOH and microwave-assisted extraction for the preparation of crude extract; and freeze-drying for the preparation of powdered extract. The results showed that total phenolic, total flavonoid, proanthocyanidin, and saponin contents of PTL prepared by microwave drying at 450 W were 25.4 mg gallic acid equiv. (GAE), 86.3 mg rutin equiv. (RE), 5.6 mg catechin equiv. (CE), and 702.1 mg escin equiv. (EE) per gram dried sample, respectively. Gallic acid, protocatechuic acid, ellagic acid, rutin, and quercetin were identified in the PTL MeOH extract. Dried PTL displayed potent antioxidant activity, while the powdered PTL extract exhibited great anti-proliferative capacity on various cancer cell lines including MiaPaCa-2 (pancreas), HT29 (colon), A2780 (ovarian), H460 (lung), A431 (skin), Du145 (prostate), BE2-C (neuroblastoma), MCF-7 (breast), MCF-10A (normal breast), and U87, SJ-G2, and SMA (glioblastoma). Anti-proliferative capacity on pancreatic cancer cells (MiaCaPa2, BxPc3, and CFPAC1) of PTL extract (200 µg/ml) was significantly higher (P < 0.05) than those of ostruthin (20 µg/ml) and gemcitabine (50 nm), and to be comparable to the powdered P. trimera root extract and a saponin-enriched extract from quillajia bark (a commercial product). The findings from this study allow us to conclude that the PTL is a rich source of phytochemicals that possess promising antioxidant and anti-proliferative activities, therefore it shows potential as lead compounds for application in the nutraceutical, medicinal and pharmaceutical industries.

A facile hybrid 'flow and batch' access to substituted 3,4-dihydro-2H-benzo[b][1,4]oxazinones.

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.

Quinolone-1-(2H)-ones as hedgehog signalling pathway inhibitors.

A series of quinolone-2-(1H)-ones derived from the Ugi-Knoevenagel three- and four-component reaction were prepared exhibiting low micromolar cytotoxicity against a panel of eight human cancer cell lines known to possess the Hedgehog Signalling Pathway (HSP) components, as well as the seminoma TCAM-2 cell line. A focused SAR study was conducted and revealed core characteristics of the quinolone-2-(1H)-ones required for cytotoxicity. These requirements included a C3-tethered indole moiety, an indole C5-methyl moiety, an aliphatic tail or an ester, as well as an additional aromatic moiety. Further investigation in the SAG-activated Shh-LIGHT2 cell line with the most active analogues: 2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(1-methyl-1H-indol-3-yl)-N-(pentan-2-yl)acetamide (5), 2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(5-methyl-1H-indol-3-yl)-N-(pentan-2-yl)acetamide (23) and ethyl (2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(5-methyl-1H-indol-3-yl)acetyl)glycinate (24) demonstrated a down regulation of the HSP via a reduction in Gli expression, and in the mRNA levels of Ptch1 and Gli2. Analogues 5, 23 and 24 returned in cell inhibition values of 11.6, 2.9 and 3.1 µM, respectively, making this new HSP-inhibitor pharmacophore amongst the most potent non-Smo targeted inhibitors thus far reported.

Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis.

Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC50 ~ 15 µM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC50 = 479 µM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC50 of 5.7 and 5.8 µM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.

The synthesis and biological activity of novel anthracenone-pyranones and anthracenone-furans.

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 0.16µM (GI50), respectively. Of note, were a CF3 functionalised anthracenone 4-pyranone (chromone) derivative 22, and an anthracenone-furan derivative 54 which displayed 0.20µM and 0.38µM growth inhibition, respectively, in the BE2-C neuroblastoma cell line.

Synthesis and anticancer activity of focused compound libraries from the natural product lead, oroidin.

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 (GI50) 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 GI50 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.

2,3-Dihydroquinazolin-4(1H)-ones and quinazolin-4(3H)-ones as broad-spectrum cytotoxic agents and their impact on tubulin polymerisation.

Tubulin plays a central role in mitosis and has been the target of multiple anticancer drugs, including paclitaxel. Herein two separate families of 2,3-dihydroquinazoline-4(1H)-ones and quinazoline-4(3H) ones, comprising 57 compounds in total, were synthesised. Screening against a broad panel of human cancer cell lines (HT29 colon, U87 and SJ-G2 glioblastoma, MCF-7 breast, A2780 ovarian, H460 lung, A431 skin, Du145 prostate, BE2-C neuroblastoma, and MIA pancreas) reveals these analogues to be broad spectrum cytotoxic compounds. Of particular note, 2-styrylquinazolin-4(3H)-one 51, 2-(4-hydroxystyryl)quinazolin-4(3H)-one 63, 2-(2-methoxystyryl)quinazolin-4(3H)-one 64 and 2-(3-methoxystyryl)quinazolin-4(3H)-one 65 and 2-(naphthalen-1-yl)-2,3-dihydroquinazolin-4(1H)-one 39 exhibited sub-µM potency growth inhibition values. Of these 1-naphthyl 39 has activity <50 nM against the HT29, U87, A2780, H460 and BE2-C cell lines. Molecular modelling of these compounds, e.g. 2-(naphthalen-1-yl)-2,3-dihydroquinazolin-4(1H)-one 39, 2-(2-methoxystyryl)quinazolin-4(3H)-one 64, 2-(3-methoxystyryl)quinazolin-4(3H)-one 65, and 2-(4-methoxystyryl)quinazolin-4(3H)-one 50 docked to the known tubulin polymerisation inhibitor sites highlighted well conserved interactions within the colchicine binding pocket. These compounds were examined in a tubulin polymerisation assay alongside the known tubulin polymerisation promotor, paclitaxel (69), and tubulin inhibitor, nocodazole (68). Of the analogues examined, indoles 43 and 47 were modest promotors of tubulin polymerisation, but less effective than paclitaxel. Analogues 39, 64, and 65 showed reduced microtubule formation consistent with tubulin inhibition. The variation in ring methoxy substituent with 50, 64 and 65, from o- to m- to p-, results in a concomitant reduction in cytotoxicity and a reduction in tubulin polymerisation, with p-OCH350 being the least active in this series of analogues. This presents 64 as a tubulin polymerisation inhibitor possessing novel chemotype and sub micromolar cytotoxicity. Naphthyl 39, with complete inhibition of tubulin polymerisation, gave rise to a sub 0.2 µM cell line cytotoxicity. Compounds 39 and 64 induced G2 + M cell cycle arrest indicative of inhibition of tubulin polymerisation, with 39 inducing an equivalent effect on cell cycle arrest as nocodazole (68).

Next-generation of BBQ analogues that selectively target breast cancer.

We previously reported on the interaction of 10-chloro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (10-Cl-BBQ) with the Aryl hydrocarbon Receptor (AhR) and selective growth inhibition in breast cancer cell lines. We now report on a library of BBQ analogues with substituents on the phenyl and naphthyl rings for biological screening. Herein, we show that absence of the phenyl Cl of 10-Cl-BBQ to produce the simple BBQ molecule substantially enhanced the growth inhibitory effect with GI50 values of 0.001-2.1 µM in select breast cancer cell lines MCF-7, T47D, ZR-75-1, SKBR3, MDA-MB-468, BT20, BT474 cells, while having modest effects of 2.1-7 µM in other cell lines including HT29, U87, SJ-G2, A2780, DU145, BE2-C, MIA, MDA-MB-231 or normal breast cells, MCF10A (3.2 µM). The most potent growth inhibitory effect of BBQ was observed in the triple negative cell line, MDA-MB-468 with a GI50 value of 0.001 µM, presenting a 3,200-fold greater response than in the normal MCF10A breast cells. Additions of Cl, CH3, CN to the phenyl ring and ring expansion from benzoimidazole to dihydroquinazoline hindered the growth inhibitory potency of the BBQ analogues by blocking potential sites of CYP1 oxidative metabolism, while addition of Cl or NO2 to the naphthyl rings restored potency. In a cell-based reporter assay all analogues induced 1.2 to 10-fold AhR transcription activation. Gene expression analysis confirmed the induction of CYP1 oxygenases by BBQ. The CYP1 inhibitor α-naphthoflavone, and the SULT1A1 inhibitor quercetin significantly reduced the growth inhibitory effect of BBQ, confirming the importance of both phase I and II metabolic activation for growth inhibition. Conventional molecular modelling/docking revealed no significant differences between the binding poses of the most and least active analogues. More detailed DFT analysis at the DSD-PBEP86/Def-TZVPP level of theory could not identify significant geometric or electronic changes which would account for this varied AhR activation. Generation of Fukui functions at the same level of theory showed that CYP1 metabolism will primarily occur at the phenyl head group of the analogues, and substituents within this ring lead to lower cytotoxicity.

Inhibition Clathrin Mediated Endocytosis: Pitstop 1 and Pitstop 2 Chimeras.

Twenty-five chimera compounds of Pitstop 1 and 2 were synthesised and screened for their ability to block the clathrin terminal domain-amphiphysin protein-protein interaction (NTD-PPI using an ELISA) and clathrin mediated endocytosis (CME) in cells. Library 1 was based on Pitstop 2, but no notable clathrin PPI or in-cell activity was observed. With the Pitstop 1, 16 analogues were produced with 1,8-naphthalic imide core as a foundation. Analogues with methylene spaced linkers and simple amides showed a modest to good range of PPI inhibition (7.6-42.5 µM, naphthyl 39 and 4-nitrophenyl 40 respectively) activity. These data reveal the importance of the naphthalene sulfonate moiety, with no des-SO3 analogue displaying PPI inhibition. This was consistent with the observed analogue docked poses within the clathrin terminal domain Site 1 binding pocket. Further modifications targeted the naphthalene imide moiety, with the installation of 5-Br (45 a), 5-OH (45 c) and 5-propyl ether (45 d) moieties. Among them, the OH 45 c and propyl ether 45 d retained PPI inhibition, with propyl ether 45 d being the most active with a PPI inhibition IC50=7.3 µM. This is 2x more potent than Pitstop 2 and 3x more potent than Pitstop 1.