Synthesis of Novel CYP1 Activated Heterocyclic Anticancer Prodrugs

The cytochrome P450 superfamily of enzymes are critical in the metabolism of endogenous and exogenous substrates. CYP1A1 and CYP1B1 have been found to be over-expressed in tumour cells whilst undetected or present in very low levels in corresponding normal tissue. This presented a novel target for the development of anti-cancer prodrugs, which would remain non-toxic until undergoing metabolism to toxic species by CYP1 enzymes over-expressed at tumour sites. The chalcones have been shown to exhibit effective anti-cancer prodrug activity, but are labile to photoisomerisation reactions converting the potent trans isomer to the less toxic cis isomer. Several heterocyclic ring systems were incorporated across the α,β-unsaturated moiety of the chalcones to produce rigid structures, eliminating the possibility of photoisomerisation occurring whilst maintaining the substituted phenyl groups in a trans like geometry. Lead compounds were identified using an in vitro MTT screening assay against a panel of tumour cell lines characterised for their constitutive or inducible CYP1 expression. These were the MDA 468, MCF7 and MDA 231 cell lines. The non-tumour MCF10A cell line which has no basal CYP1 expression was used as the control. A library of eighteen 3,5-diarylpyrazoles were synthesised. The lead pyrazole DMU 10107 (3-(2,3,4-trimethoxyphenyl)-5-(3,4-methylenedioxyphenyl)pyrazole) gave an IC50 value of 8μM towards the MDA 468 cell line. The MCF7 cells, TCDD induced and non-induced gave IC50 values of 10μM each. Although the pyrazoles showed plausible tumour toxicity, an investigation into six membered pyrimidine heterocycles was undertaken in an attempt to obtain enhanced cytotoxicities than those observed from the five membered pyrazoles. Therefore, a library of fifteen 2-amino-4,6-diarylpyrimidines was synthesised. The lead amino-pyrimidine DMU 10212 (2-amino-4-(2,4-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)pyrimidine) showed significant cytotoxicity towards the MDA 468 cell line with an IC50 value of 0.01μM. Notable IC50 values of 0.3μM and 0.07μM were also observed towards the MCF7 and MCF7 cells induced with TCDD. The important toxicity seen from the 2-amino-4,6-diarylpyrimidines prompted the investigation of the 2-position of the pyrimidine ring, and to assess the tumour toxicities of the synthesised compounds. The 2-amino-4,6-diarylpyrimidines were converted to produce 4,6-diarylpyrimidones by a one-step conversion reaction using sodium nitrate. The pyrimidone DMU 10313 (4-(2-methoxyphenyl)-6-(3,4-methylenedioxyphenyl)pyrimidin-2-one) showed high toxicity with an IC50 value of 0.07μM towards the MDA 468 cells and IC50 values of 1.8μM and 0.5μM 3 towards the MCF7 and MCF7 cells induced with TCDD. A library of nine 2-morpholino-4,6-diarylpyrimidines was synthesised. The lead compound DMU 10405 (4-(2,4-dimethoxyphenyl)-6-(4-methoxyphenyl)-2-morpholinopyrimidine) gave an IC50 value of 10μM towards the MDA 468 cells. DMU 10600 (4-(2,4-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)-2-dimethylethylenediaminopyrimidine), showed an IC50 value of 7μM towards the MDA 468 cells and an identical IC50 value of 10μM towards the MCF7 and MCF7 cells treated with TCDD. DMU 10700 (2-methyl-4-(2,4-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)pyrimidine), a substituted pyrimidine based on the phenyl substitutions of DMU 10212 gave an IC50 value of 2.5μM towards the MDA 468 cells. DMU 10800 (4-(2,4-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)pyrimidine), also based on the phenyl substitutions of DMU 10212 showed an IC50 value of 0.08μM towards the MDA 468 cells and equal IC50 values of 0.2μM against the MCF7 and MCF7 cells induced with TCDD. All lead compounds did not show toxicity towards the non-tumour MCF10A cell line. DMU 10212 was selected as the overall lead compound due to the significant tumour toxicities recorded, and for the non-toxicity observed towards the MCF10A cells. Inhibition studies using the known CYP1 inhibitor α-naphthoflavone (α-NF) were conducted to show that DMU 10212 was a substrate of the CYP1 enzymes. The resulting data showed that the cytotoxicity of DMU 10212 was completely eliminated suggesting CYP1 enzymes play an activating role in the cytotoxic effect of DMU 10212. LCMS metabolism studies using isolated CYP1 isoforms were performed showing that DMU 10212 is metabolised to produce four metabolites (M1, M2, M3 and M4), determined from their individual retention times and molecular masses. The metabolites of DMU 10212 were also found to be generated at a greater rate with CYP1A1 than CYP1B1. Metabolite structures were proposed as CYP1 enzyme reactions are known. The metabolite M2 was synthesised and was identified to be an authentic metabolite of DMU 10212 via LCMS and co-elution studies. Screening of M2 against the tumour cells gave an IC50 value of 0.6μM towards the MDA 468 cells, and IC50 values of 0.6μM and 1μM against the MCF7 and MCF7 cells induced with TCDD. In conclusion, DMU 10212, a novel CYP1 activated anticancer prodrug with selective high toxicity towards tumour cells has been identified

Analysis of plant secondary metabolism using stable isotope‐labelled precursors

Special issue of Phytochemical Analysis on NMR-based analytical techniques. open access articleIntroduction Analysis of biochemical pathways typically involves feeding a labelled precursor to an organism, and then monitoring the metabolic fate of the label. Initial studies used radioisotopes as a label and then monitored radioactivity in the metabolic products. As analytical equipment improved and became more widely available, preference shifted the use stable ‘heavy’ isotopes like deuterium (2H)‐, carbon‐13 (13C)‐ and nitrogen‐15 (15N)‐atoms as labels. Incorporation of the labels could be monitored by mass spectrometry (MS), as part of a hyphenated tool kits, e.g. Liquid chromatography (LC)–MS, gas chromatography (GC)–MS, LC–MS/MS. MS offers great sensitivity but the exact location of an isotope label in a given metabolite cannot always be unambiguously established. Nuclear magnetic resonance (NMR) can also be used to pick up signals of stable isotopes, and can give information on the precise location of incorporated label in the metabolites. However, the detection limit for NMR is quite a bit higher than that for MS. Objectives A number of experiments involving feeding stable isotope‐labelled precursors followed by NMR analysis of the metabolites is presented. The aim is to highlight the use of NMR analysis in identifying the precise fate of isotope labels after precursor feeding experiments. As more powerful NMR equipment becomes available, applications as described in this review may become more commonplace in pathway analysis. Conclusion and Prospects NMR is a widely accepted tool for chemical structure elucidation and is now increasingly used in metabolomic studies. In addition, NMR, combined with stable isotope feeding, should be considered as a tool for metabolic flux analyses

Electrochemical reactivity of TiO2 nanoparticles adsorbed onto boron-doped diamond

TiO2 (anatase) nanoparticles of ca. 6–10 nm diameter are adsorbed from acidic aqueous solution onto polycrystalline industrially polished boron-doped diamond electrode surfaces. After immobilisation at the electrode surface, TiO2 nanoparticles are imaged in vacuum by electron microscopy (FEGSEM) and when immersed in a liquid film of aqueous 12 M LiCl by in situ scanning tunnelling microscopy (STM). Mono-layer films of TiO2 particles are studied voltammetrically in different electrolyte media. Boron-doped diamond as an inert substrate material allows the reduction of TiO2 particles in phosphate buffer solution to be studied and two distinct steps in the reduction–protonation process are identified: (i) a broad reduction signal associated with the binding of an outer layer of protons and (ii) a sharper second reduction signal associated with the binding of an inner (or deeper) layer of protons. Voltammetric experiments in aqueous 0.1 M NaClO4 with variable amounts of HClO4 suggest that the reduction of TiO2 particles is consistent with the formation of Ti(III) surface sites and accompanied by the adsorption of protons. Saturation occurs and the total amount of surface sites can be determined. Preliminary data for electron transfer processes at the reduced TiO2 surface such as the dihydrogen evolution process and the two-electron–two-proton reduction of maleic acid to succinic acid are discussed

Synthesis and antitrypanosomal activities of novel pyridylchalcones

Collaboration with the London School of Hygiene and Tropical Medicine. The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.A library of novel pyridylchalcones were synthesised and screened against Trypanosoma brucei rhodesiense. Eight were shown to have good activity with the most potent 8 having an IC50 value of 0.29 M. Cytotoxicity testing with human KB cells showed a good selectivity profile for this compound with a selectivity index of 47. Little activity was seen when the library was tested against Leishmania donovani. In conclusion, pyridylchalcones are promising leads in the development of novel compounds for the treatment of human African trypanosomiasis (HAT)

Specialized Plant Metabolism Characteristics and Impact on Target Molecule Biotechnological Production.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI linkPlant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation

Pyrogallol is the main antibacterial compound in the aqueous extract of Boswellia dalzielii bark

Plants belonging to the genus Boswellia (Burseraceae) have long been appreciated for their pharmacological properties such as anti-inflammatory, antioxidative and anticancer activities1. In the Northern part of Nigeria, the bark of B. dalzielii Hutch. is an important ingredient for the treatment of infections2. To identify water-soluble antibacterial compounds, powdered bark was macerated in water for 6 to 24h at a range of temperatures. The aqueous extracts were subsequently fractionated by column chromatography, and the fractions were initially screened against wild type and methicillin resistant strains of Staphylococcus aureus. Then, minimum inhibitory concentrations of purified fractions were determined using series of concentrations from 2 mg/mL to 8 μg/mL. Results showed that longer maceration resulted in stronger antibacterial activity. With the aid of NMR and accurate mass analysis, pyrogallol was identified as the main antibacterial agent, with MIC values ranging from 24-28 μg/mL for MRSA to 34-36 μg/mL for wild-type S. aureus. Gallic acid was found to play a lesser role (MIC >200 μg/mL). Pyrogallol was found not to be a plant secondary metabolite, but a metabolic product from microbial degradation of gallic acid from the bark. Only two bacterial species could be isolated from the plant material, which were identified as Raoultella planticola and Enterobacter cloacae. Of these two micro-organisms, R. planticola was shown to be responsible for the production of pyrogallol. This is an example of bacterial allelopathy, which results in an increased efficacy of the aqueous extract

Design, synthesis and antitrypanosomal activities of 2,6-disubstituted-4,5,7-Trifluorobenzothiophenes

Current treatments for Human African Trypanosomiasis (HAT) are limited in their application, have undesirable dosing regimens and unsatisfactory toxicities highlighting the need for the development of a safer drug pipeline. Our medicinal chemistry programme in developing rapidly accessible and modifiable heterocyclic scaffolds led to the design and synthesis of novel substituted benzothiophenes, with 6-benzimidazol-1-ylbenzothiophene derivatives demonstrating significant antitrypanosomal activities (IC50 <1 μM) against Trypanosoma brucei rhodesiense and no toxicity towards mammalian cells

Building a DMU e-Biology resource for health sciences’ students.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI linkThe BSc Biomedical Science (BMS) programme at De Montfort University (DMU, Leicester, UK) is accredited by the Institute of Biomedical Science (IBMS). Students enrolled within this programme acquire highly sought after skills related with human health sciences to work in: pathology departments in hospitals; research institutions; biotechnology and pharmaceutical industries; and the education sector to name a few. The degree recruits a large number of students with currently around 600 students enrolled on this programme at DMU. Despite pre-entry requirements of knowledge of subjects related to human biology, biology or chemistry, we have noted that first year students require basic support in STEM subjects (biology, chemistry and mathematics) in modules such as “Basic Microbiology”, “Basic Anatomy and Physiology” and “Chemistry for the Biosciences”. This support is especially necessary for students that come from non-traditional routes such as Business and Technology Education Council (BTEC) routes. Moreover, usually topics related with microbiology and human diseases are challenging for students, often causing stress impacting their overall performance and experience. A group of BMS academics at DMU in conjunction with universities in the European Union (EU; e.g. University of San Pablo CEU, Spain) have started to design, create and develop a series of e-learning resources or units in human biology and BMS for undergraduate students that study health sciences degrees in the EU. These units are being uploaded onto the DMU web server (http://parasitology.dmu.ac.uk/) and will be only accessible for students from participating universities during the first phase of this project (2017/18 course) in which comprehensive feedback will be collected. This web server space has three sections or modules (theoretical section, virtual laboratory and microscope) in which the new e-learning resources will be preliminary accommodated. These units will be interactive and easy to follow, and will cover basic human biology (e.g. cells, cell structure), human anatomy and physiology, histology and basic microbiology, which will be embedded in a theoretical module named DMU e-Biology within the above URL link. They will include formative assessments and case studies throughout each unit. In addition, a series of practical units are being developed which describe routine practical elements in any biomedical laboratory such as laboratory materials, pipetting, molecular techniques (e.g. PCR), cell culture (e.g. use of biological safety cabinet) and histological techniques (e.g. use of microtome, staining techniques). The development of this teaching and learning resource will cover a gap in the traditional teaching and learning methods that are currently used and provided in the participating universities. The DMU e-Biology will aid to our undergraduate students to gain knowledge in human biology and microbiology by promoting self-learning. We consider that the DMU e-Biology will help overcome spatiotemporal, equipment and resource barriers. Additionally, it may help student retention as currently about a 10% of our first year students fail to continue BMS at DMU. Finally, the creation of the DMU e-Biology will also provide support to the DMU Student Retention and Attainment Strategy 2016-2020 through the DMU Student Learning Hub, which is currently under development

Building on-line materials for teaching parasitology to health sciences’ students: initial impressions.

Background: It is widely recognised that the use of web-based teaching resources is an increasingly important method for delivering education, and it will be particularly important in the near future due to the progressively increasing number of health science students and the current number of academics in the “European Higher Education Area”. The study of parasitology and infectious diseases is essential to build professionals in the health sector with the key knowledge and skills to face global public health threats such as food-, water- or vector-borne infectious diseases outbreaks. However, the current time dedicated to the teaching of this discipline in all health sciences degrees at De Montfort University (DMU, Leicester, UK) is very little or non-existent depending on the degree/master. Methods: An innovative teaching group at DMU is trying to fill this gap in the currently available teaching offer in line with new trends in global health education, the large number of students enrolled in any health degree and the increasing number of students that would like to study this discipline (but due to different commitments do not have enough time or resources to study on a full time basis). Thus, an innovative teaching group from different EU Universities (DMU and the Spanish universities: University of San Pablo CEU, University of Alcalá, and University Miguel Hernández de Elche) and clinicians (University Hospitals of Leicester, UK) have started to design, create and develop a complete on-line package in Parasitology for undergraduate and postgraduate students that study health sciences. Results: The e-Parasitology package will be accessible through the DMU website (http://parasitology.dmu.ac.uk) in 2017 and will be focused on infection, prevention and treatment of major and emerging parasitological diseases. Conclusions: This teaching resource will aid our undergraduate and postgraduate students to gain a significant knowledge in parasitology by promoting self-learning and internationalization. This poster will explore one of the first mini-modules developed so far related with Toxocara, a helminthiasis with prevalence rates that can reach as high as 40% or more in parts of the world, and the challenges for its development

Novel fluorinated benzimidazole-based scaffolds and their anticancer activity in vitro

A small library of twelve, structurally diverse, fluoroaryl benzimidazoles was prepared using a simple synthetic strategy employing SNAr reactions. This allowed rapid assembly of heterocyclic structures containing linked and tethered fluoroaryl benzimidazoles. X-ray crystal structures of seven compounds were obtained including those of two macrocyclic compounds containing 21- and 24-membered rings. Three tethered fluoroaryl benzimidazole derivatives demonstrated micromolar inhibition against K-562 and MCF-7 cell lines. These compounds, in addition to 1-tetrafluoropyrid-4-yl-2-tetrafluoropyrid-4-ylsulfanyl-1H-benzimidazole, also demonstrated micromolar inhibition against G361 and HOS cell lines. Two of the compounds were found to activate caspases leading to apoptosis