Synthesis and optimisation of P3 substituted vinyl sulfone-based inhibitors as anti-trypanosomal agents.

A series of lysine-based vinyl sulfone peptidomimetics were synthesised and evaluated for anti-trypanosomal activity against bloodstream forms of T. brucei. This focused set of compounds, varying in the P3 position, were accessed in a divergent manner from a common intermediate (ammonium salt 8). Several P3 analogues exhibited sub-micromolar EC50 values, with thiourea 14, urea 15 and amide 21 representing the most potent anti-trypanosomal derivatives of the series. In order to establish an in vitro selectivity index the most active anti-trypanosomal compounds were also assessed for their impact on cell viability and cytotoxity effects in mammalian cells. Encouragingly, all compounds only reduced cellular metabolic activity in mammalian cells to a modest level and little, or no cytotoxicity, was observed with the series.

Optimisation of estrogen receptor subtype-selectivity of a 4-Aryl-4H-chromene scaffold previously identified by virtual screening.

4-Aryl-4H-Chromene derivatives have been previously shown to exhibit anti-proliferative, apoptotic and anti-angiogenic activity in a variety of tumor models in vitro and in vivo generally via activation of caspases through inhibition of tubulin polymerisation. We have previously identified by Virtual Screening (VS) a 4-aryl-4H-chromene scaffold, of which two examples were shown to bind Estrogen Receptor α and ß with low nanomolar affinity and <20-fold selectivity for α over ß and low micromolar anti-proliferative activity in the MCF-7 cell line. Thus, using the 4-aryl-4H-chromene scaffold as a starting point, a series of compounds with a range of basic arylethers at C-4 and modifications at the C3-ester substituent of the benzopyran ring were synthesised, producing some potent ER antagonists in the MCF-7 cell line which were highly selective for ERα (compound 35; 350-fold selectivity) or ERß (compound 42; 170-fold selectivity).

Microfluidics in Haemostasis: A Review.

Haemostatic disorders are both complex and costly in relation to both their treatment and subsequent management. As leading causes of mortality worldwide, there is an ever-increasing drive to improve the diagnosis and prevention of haemostatic disorders. The field of microfluidic and Lab on a Chip (LOC) technologies is rapidly advancing and the important role of miniaturised diagnostics is becoming more evident in the healthcare system, with particular importance in near patient testing (NPT) and point of care (POC) settings. Microfluidic technologies present innovative solutions to diagnostic and clinical challenges which have the knock-on effect of improving health care and quality of life. In this review, both advanced microfluidic devices (R&D) and commercially available devices for the diagnosis and monitoring of haemostasis-related disorders and antithrombotic therapies, respectively, are discussed. Innovative design specifications, fabrication techniques, and modes of detection in addition to the materials used in developing micro-channels are reviewed in the context of application to the field of haemostasis.

Development of the ß-lactam type molecular scaffold for selective estrogen receptor α modulator action: synthesis and cytotoxic effects in MCF-7 breast cancer cells.

The estrogen receptors (ERα and ERß) which are ligand inducible nuclear receptors are recognized as pharmaceutical targets for diseases such as osteoporosis and breast cancer. There is an increasing interest in the discovery of subtype Selective Estrogen Receptor Modulators (SERMs). A series of novel ß-lactam compounds with estrogen receptor modulator properties have been synthesized. The antiproliferative effects of these compounds on human MCF-7 breast tumor cells are reported, together with binding affinity for the ERα and ERß receptors. The most potent compound 15g demonstrated antiproliferative effects on MCF-7 breast tumor cells (IC50 = 186 nM) and ERα binding (IC50 = 4.3 nM) with 75-fold ERα/ß receptor binding selectivity. The effect of positioning of the characteristic amine containing substituted aryl ring (on C-4 or N-1 of the ß-lactam scaffold) on the antiproliferative activity and ER-binding properties of the ß-lactam compounds is rationalized in a molecular modeling study.

Two novel homozygous SLC2A9 mutations cause renal hypouricemia type 2.

BACKGROUND: Elevated serum uric acid (UA) is associated with gout, hypertension, cardiovascular and renal disease. Hereditary renal hypouricemia type 1 (RHUC1) is caused by mutations in the renal tubular UA transporter URAT1 and can be complicated by nephrolithiasis and exercise-induced acute renal failure (EIARF). We have recently shown that loss-of-function homozygous mutations of another UA transporter, GLUT9, cause a severe type of hereditary renal hypouricemia with similar complications (RHUC2). METHODS: Two unrelated families with renal hypouricemia were clinically characterized. DNA was extracted and SLC22A12 and SLC2A9 coding for URAT1 and GLUT9, respectively, were sequenced. Transport studies into Xenopus laevis oocytes were utilized to evaluate the function of the GLUT9 mutations found. A molecular modeling study was undertaken to structurally characterize and probe the effects of these mutations. RESULTS: Two novel homozygous GLUT9 missense mutations were identified: R171C and T125M. Mean serum UA level of the four homozygous subjects was 0.15 ± 0.06 mg/dL and fractional excretion of UA was 89-150%. None of the affected subjects had nephrolithiasis, EIARF or any other complications. Transport assays revealed that both mutant proteins had a dramatically reduced ability to transport UA. Modeling showed that both R171C and T125M mutations are located within the inner channel that transports UA between the cytoplasmic and extracellular regions. CONCLUSIONS: This is the second report of renal hypouricemia caused by homozygous GLUT9 mutations. Our findings confirm the pivotal role of GLUT9 in UA transport and highlight the similarities and differences between RHUC1 and RHUC2.

Lead identification of ß-lactam and related imine inhibitors of the molecular chaperone heat shock protein 90.

Heat shock protein 90 is an emerging target for oncology therapeutics. Inhibitors of this molecular chaperone, which is responsible for the maintenance of a number of oncogenic proteins, have shown promise in clinical trials and represent a new and exciting area in the treatment of cancer. Heat shock protein 90 inhibitors have huge structural diversity, and here we present the lead identification of novel inhibitors based on ß-lactam and imine templates. ß-Lactam 5 and imines 12 and 18 exhibit binding to heat shock protein 90-α with IC(50) values of 5.6 µM, 14.5 µM, and 22.1 µM, respectively. The binding affinity displayed by these compounds positions them as lead compounds for the design of future inhibitors of heat shock protein 90 based on the ß-lactam and imine templates.

Synthesis and serotonin transporter activity of 1,3-bis(aryl)-2-nitro-1-propenes as a new class of anticancer agents.

Structural derivatives of 4-MTA, an illegal amphetamine analogue have been previously shown to have anticancer effects in vitro. In this study we report the synthesis of a series of novel 1,3-bis(aryl)-2-nitro-1-propene derivatives related in structure to 4-MTA. A number of these compounds containing a classic nitrostyrene structure are shown to have antiproliferative activities in vitro in a range of malignant cell lines, particularly against Burkitt's lymphoma derived cell lines, whilst having no effect on 'normal' peripheral blood mononuclear cells. Such effects appear to be independent of the serotonin transporter, a high affinity target for amphetamines and independent of protein tyrosine phosphatases and tubulin dynamics both of which have been previously associated with nitrostyrene-induced cell death. We demonstrate that a number of these compounds induce caspase activation, PARP cleavage, chromatin condensation and membrane blebbing in a Burkitt's lymphoma derived cell line, consistent with these compounds inducing apoptosis in vitro. Although no specific target has yet been identified for the action of these compounds, the cell death elicited is potent, selective and worthy of further investigation.

Modulating the Blood-Brain Barrier: A Comprehensive Review.

The highly secure blood-brain barrier (BBB) restricts drug access to the brain, limiting the molecular toolkit for treating central nervous system (CNS) diseases to small, lipophilic drugs. Development of a safe and effective BBB modulator would revolutionise the treatment of CNS diseases and future drug development in the area. Naturally, the field has garnered a great deal of attention, leading to a vast and diverse range of BBB modulators. In this review, we summarise and compare the various classes of BBB modulators developed over the last five decades-their recent advancements, advantages and disadvantages, while providing some insight into their future as BBB modulators.

Lead identification of conformationally restricted benzoxepin type combretastatin analogs: synthesis, antiproliferative activity, and tubulin effects.

We have synthesized a series of polymethoxylated rigid analogs of combretastatin A-4 which contain a benzoxepin ring in place of the usual ethylene bridge present in the natural combretastatin products. The compounds display antiproliferative activity when evaluated against the MCF-7 and MDA human breast carcinoma cell lines. 5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-benzoxepine (11g) was found to be the most potent product when evaluated against the MCF-7 breast cancer cell line. A brief computational study of the structure-activity relationship for the synthesized compounds is presented. These 4,5-diarylbenzoxepins are identified as potentially useful scaffolds for the further development of antitumor agents which target tubulin.

Synthesis, biological evaluation, structural-activity relationship, and docking study for a series of benzoxepin-derived estrogen receptor modulators.

The estrogen receptors ERalpha and ERbeta are recognized as important pharmaceutical targets for a variety of diseases including osteoporosis and breast cancer. A series of novel benzoxepin-derived compounds are described as potent selective modulators of the human estrogen receptor modulators (SERMs). We report the antiproliferative effects of these compounds on human MCF-7 breast tumor cells. These heterocyclic compounds contain the triarylethylene arrangement as exemplified by tamoxifen, conformationally restrained through the incorporation of the benzoxepin ring system. The compounds demonstrate potency at nanomolar concentrations in antiproliferative assays against an MCF-7 human breast cancer cell line with low cytotoxicity together with low nanomolar binding affinity for the estrogen receptor. The compounds also demonstrate potent antiestrogenic properties in the human uterine Ishikawa cell line. The effect of a number of functional group substitutions on the ER binding properties of the benzoxepin molecular scaffold is examined through a detailed docking and 2D-QSAR computational investigation. The best QSAR model developed for ERalphabeta selectivity yielded R(2) of 0.84 with an RMSE for the training set of 0.30. The predictive quality of the model was Q(2) of 0.72 and RMSE of 0.18 for the test set. One particular compound bearing a 4-fluoro substituent, exhibits 15-fold selectivity for ERbeta and both our docking and QSAR studies converge on the correlation between enhanced lipophilicity and enhanced ERbeta binding for this benzoxepin ring scaffold.

Beta-lactam type molecular scaffolds for antiproliferative activity: synthesis and cytotoxic effects in breast cancer cells.

A series of novel beta-lactam containing compounds are described as antiproliferative agents and potential selective modulators of the oestrogen receptor. The purpose of the study is to evaluate the antiproliferative effects of these compounds on human MCF-7 and MDA MB-231 breast cancer cells. The compounds are designed to contain three aryl ring substituents arranged on the heterocyclic azetidin-2-one (beta-lactam), thus providing conformationally restrained analogues of the triarylethylene arrangement exemplified in the tamoxifen type structure. The compounds demonstrated potency in antiproliferative assays against MCF-7 human breast cancer cell line at low micromolar to nanomolar concentrations with low cytotoxicity and moderate binding affinity to the oestrogen receptor. The effect of a number of aryl and amine functional group substitutions on the antiproliferative activity of the beta-lactam products was explored and a brief computational structure-activity relationship investigation with molecular simulation was investigated.

Lead Optimization of Benzoxepin-Type Selective Estrogen Receptor (ER) Modulators and Downregulators with Subtype-Specific ERα and ERß Activity.

Estrogen receptor α (ERα) is an important target for the design of drugs such as tamoxifen (2a) and fulvestrant (5). Three series of ER-ligands based on the benzoxepin scaffold structure were synthesized: series I containing an acrylic acid, series II with an acrylamide, and series III with a saturated carboxylic acid substituent. These compounds were shown to be high affinity ligands for the ER with nanomolar IC50 binding values. Series I acrylic acid ligands were generally ERα selective. In particular, compound 13e featuring a phenylpenta-2,4-dienoic acid substituent was shown to be antiproliferative and downregulated ERα and ERß expression in MCF-7 breast cancer cells. Interestingly, from series III, the phenoxybutyric acid derivative compound 22 was not antiproliferative and selectively downregulated ERß. A docking study of the benzoxepin ligands was undertaken. Compound 13e is a promising lead for development as a clinically relevant SERD, while compound 22 will be a useful experimental probe for helping to elucidate the role of ERß in cancer cells.

Target specific virtual screening: optimization of an estrogen receptor screening platform.

In this work, we introduce a four-step scoring and filtering procedure, furnishing target specific virtual screening (TS-VS), which serves to minimize false positives resulting from conformational artifacts of the docking process and is optimized to converge on novel chemotypes of estrogen receptor alpha (ERalpha). As a proof of concept, VS of a commercial compound database was undertaken (SPECs database release: Aug 2005, 202 054 compounds in total), resulting in the identification of both previously known and novel putative ER scaffolds. Application of distance constraints within TS-VS allowed facile identification of three novel active ligands with ERalpha binding affinities (IC50) of 1.4 microM, 57 nM, and 53 nM. Importantly, they all exhibited ERalpha over ERbeta selectivity, with the most selective being 17-fold. The ligands also displayed low micomolar antiproliferative activity (7-15 microM) in the human MCF-7 breast cancer cell line.

Flexible estrogen receptor modulators: synthesis, biochemistry and molecular modeling studies for 3-benzyl-4,6-diarylhex-3-ene and 3,4,6-triarylhex-3-ene derivatives.

Selective estrogen receptor modulators (SERMs) such as tamoxifen and toremifene are clinically useful drugs in the endocrine treatment of estrogen receptor positive breast cancer while raloxifene is an effective intervention for osteoporosis. In an ongoing SERM discovery programme we now report the synthesis of a series of 3-benzyl-4,6-diarylhex-3-enes and 3,4,6-triarylhex-3-enes containing an extended flexible core structure. In these novel structures, the ethylene group acts as a flexible spacing group linking the aryl Ring A or Ring B with the core alkene group. In the benzyl-4,6-diarylhex-3-ene series an additional methylene group is inserted as a spacing group between the aryl ring C and the ethylene core group. These products demonstrated antiproliferative activity against the MCF-7 human breast cancer cell line. The alkene compounds were also shown to have binding affinity for the estrogen receptor alpha (IC50 values for the most active compounds in the range 0.110-0.293 microM) together with selectivity for ER alpha/beta. The compounds demonstrated antiestrogenic activity in Ishikawa cells with low estrogenic stimulation. The structure-activity relationships for the active ligands were further explored in a computational study where docked structures of the active compounds were compared with the X-ray crystal structures for the complexes of ER alpha with 4-hydroxytamoxifen and ER beta with raloxifene. The alignment of the aromatic rings B and C of the compounds within the ligand binding domain could then be correlated with their observed ER alpha/beta selectivity.

Oncology exploration: charting cancer medicinal chemistry space.

Approaches for the experimental determination of protein-ligand molecular interactions are reliant on the quality of the compounds being tested. The application of large, randomly designed combinatorial libraries has given way to the creation of more-focused 'drug-like' libraries. Prior to synthesis, we wish to screen the potential compounds to remove undesired chemical moieties and to be within a required range of physiochemical properties. We have used a principal-component analysis (PCA) computational approach to analyze the 3D descriptor space of active and non-active (hit-like) cancer medicinal chemistry compounds. We define hit-like those molecules passing the unmodified OpenEye FILTER program. Our analysis indicates that these compounds occupy quite different regions in space. Cancer-active compounds exist in a much greater volume of space than generic hit-like space and most of them fail the commonly applied filters for orally bioavailable drugs. This is of great significance when designing orally bioavailable cancer target drugs.

Estrogen receptors: molecular interactions, virtual screening and future prospects.

Identification of the Estrogen Receptor (ER) as a key mediator of the proliferation of breast cancer, and its involvement in pathways leading to osteoporosis and coronary heart disease, has resulted in a surge to discover and design compounds with the ability to modulate its actions (SERMs). Concurrently, a dramatic increase in the number of crystal structures of the ER has led to a more in depth understanding of the governing mechanisms involved in ER modulation. Entwining computational techniques with the availability of 3D structural data, has allowed not only the rational design of potent inhibitors of the ER, but also its incorporation in Virtual Screening (VS) in the search for novel chemotypes that can modulate the ER. An important initial step in the VS process is to filter towards molecules that occupy similar chemical space to a set of known actives prior to docking. We illustrate through Principal Component Analysis (PCA) of 145 descriptors the region of chemical space antiestrogens occupy compared with 'drug-like' space. We also review all available studies involving validation of several docking algorithms utilizing the ER, ultimately focusing on analysis of Enrichment (E) rates and False Positive (FP) rates to illustrate the successes attributed to each docking algorithm. Finally, we relate the recent discovery of non-genomic mechanisms of the ER and subsequently present a model involving a recently identified alternative, second binding-pocket of the ER in our laboratory through cavity analysis that suggests how the same receptor can invoke these, 'classical' and rapid responses concurrently.

ß-Lactam estrogen receptor antagonists and a dual-targeting estrogen receptor/tubulin ligand.

Twelve novel ß-lactams were synthesized and their antiproliferative effects and binding affinity for the predominant isoforms of the estrogen receptor (ER), ERα and ERß, were determined. ß-Lactams 23 and 26 had the strongest binding affinities for ERα (IC50 values: 40 and 8 nM, respectively) and ERß (IC50 values: 19 and 15 nM). ß-Lactam 26 was the most potent in antiproliferative assays using MCF-7 breast cancer cells, and further biochemical analysis showed that it caused accumulation of cells in G2/M phase (mitotic blockade) and depolymerization of tubulin in MCF-7 cells. Compound 26 also induced apoptosis and downregulation of the expression of pro-survival proteins Bcl-2 and Mcl-1. Computational modeling predicted binding preferences for the dual ER/tubulin ligand 26. This series is an important addition to the known pool of ER antagonists and ß-lactam 26 is the first reported compound that has dual-targeting properties for both the ER and tubulin.

Vinyl sulfone-based peptidomimetics as anti-trypanosomal agents: design, synthesis, biological and computational evaluation.

A series of vinyl sulfone-containing peptidomimetics were rationally designed, synthesized, and evaluated against Trypanosoma brucei brucei . These electrophilic compounds are likely to exert their antitrypanosomal activity via inhibition of trypanosomal cysteine proteases, TbCatB and rhodesain, through alkylation of a key cysteine residue within the protease active site. The series was designed to present complementary groups to naturally recognized peptide substrates while probing tolerance to a range of substitutions at the P1, P1', and P2 positions. The most potent compound, 29 (EC50 = 70 nM, T. b. brucei whole cell assay), displayed minimal toxicity (>785 times selectivity) when assayed for cytotoxicity against the human promyelocytic leukemia (HL-60) cell line. Cells treated with compound 29, as with K777 (2), exhibited an increase in both the number of multinucleated cells and cells with swollen flagellar pockets. Computational analysis revealed a strong correlation between the hypothetical binding mode in TbCatB/rhodesain and trypanocidal activity in vitro.

Synthesis, biochemical and molecular modelling studies of antiproliferative azetidinones causing microtubule disruption and mitotic catastrophe.

The structure-activity relationships of antiproliferative ß-lactams, focusing on modifications at the 4-position of the ß-lactam ring, is described. Synthesis of this series of compounds was achieved utilizing the Staudinger and Reformatsky reactions. The antiproliferative activity was assessed in MCF-7 cells, where the 4-(4-ethoxy)phenyl substituted compound 26 displayed the most potent activity with an IC(50) value of 0.22 µM. The mechanism of action was demonstrated to be by inhibition of tubulin polymerisation. Cell exposure to combretastatin A-4 and 26 led to arrest of MCF-7 cells in the G2/M phase of the cell cycle and induction of apoptosis. Additionally, mitotic catastrophe for combretastatin A-4 and for 26 was demonstrated in breast cancer cells for the first time, as evidenced by the formation of giant, multinucleated cells.

Lead identification of conformationally restricted ß-lactam type combretastatin analogues: synthesis, antiproliferative activity and tubulin targeting effects.

The synthesis and study of the structure-activity relationships of a series of rigid analogues of combretastatin A-4 are described which contain the 1,4-diaryl-2-azetidinone (ß-lactam) ring system in place of the usual ethylene bridge present in the natural combretastatin stilbene products. The 1,4-diaryl-2-azetidinones are unsubstituted at C-3, or contain methyl substituent(s) at C-3. The most potent compounds 12d and 12e display antiproliferative activity at nanomolar concentrations when evaluated against the MCF-7 and MDA-MB-231 human breast carcinoma cell lines. 12d exerts antimitotic effects through an inhibition of tubulin polymerisation and subsequent G2/M arrest of the cell cycle in human MDA-MB-231 breast cancer cells, with similar activity to that of CA-4. These novel ß-lactam compounds are identified as potentially useful scaffolds for the further development of antitumour agents which target tubulin.