Synthesis of Bioactive Molecules IV review

This item first appeared in 2008

Held at University College Cork, on 1 July 2008

The Clandestine Chemist
Getting to know Poison Frogs
Safer Pregnancies
Chemical Genomics Approaches
The Epothilone Story

This one-day conference held on 1 July 2008, and sponsored by SCI, the RSC, and ICI brought together scientists from the Republic of Ireland and from Northern Ireland who have varied interests in drug discovery and development and a common interest in synthesis. The 2008 meeting was the fourth in a series and establishes the event as an essential date in the Irish calendar. Professor Anita Maguire opened the conference with an introduction to the venue, The Analytical and Biological Chemistry Research Facility, University College Cork, which brings together researchers from the fields of analytical and biological chemistry and is currently hosting a new programme of undergraduate research. The Centre houses around 100 researchers who interact strongly with companies based in the region.

Professor Maguire underlined the central importance of organic synthesis to the field of chemical biology and the interfaces with drug development and process development. Expertise in synthesis allows researchers to attack interdisciplinary problems, to grapple with mechanistic problems and hence design superior processes. Synthesis remains a central, creative and exciting branch of chemistry.

Dr Nick Gathergood, Dublin City University, added that EuCheMS has belatedly recognised organic synthesis as a strategically important subject and it will henceforth have a much higher profile at European level. Professor Maguire also thanked the conference organisers, Simon Lawrence and Debbie Curran.

The Clandestine Chemist
Dr J J Keating (University College Cork) is one of a small group of chemists who synthesise drugs of abuse such as, for example, MDMA (more commonly known as ecstasy) and want the world to know about it! Of course, clandestine synthesis of ecstasy and other drugs of abuse is widespread. For example, around 120,000 ecstasy tablets were seized in Ireland in 2007. Why then, would an academic chemist want to engage in this activity?

Since the clandestine chemist is unlikely to engage in rigorous purification of their products, most drugs of abuse contain significant impurities. This may be of concern to the user, but Keating is most interested in the opportunities this gives for forensic linkage of recovered drugs to their production site. The clandestine chemist has to switch their method of production according to the availability of different starting materials, thus a number of synthetic routes are available to a given drug molecule. These will lead to different impurity profiles, which can often be characteristic of a particular method of production. Keating’s programme aims to elucidate full impurity profiles for drugs in relation to the full synthetic pathway employed.

For example, in the synthesis of amphetamines such as MDMA (ecstasy) and DOB (Dr Bob) an advanced intermediate ketone is converted to the desired amine. If the route known as the Leukart Route is used for this step, a range of pyridine and pyrimidine impurities is formed. This impurity profile depends on the impurities present in the intermediate ketone, which depend in turn on the synthetic route use to form the ketone. In particular, one pathway to the ketone intermediate results in dibenzylketone byproducts. These lead to very distinctive symmetrical pyridines. Hence, relatively straightforward analytical detection of this pyridine can instantly link, or not, a particular batch of drug to a particular clandestine production facility.


Getting to know Poison Frogs
A rich source of natural products has been the skin secretions of the 'poison frogs' of Central and South America, which sequester the alkaloids from their diet, and they have inspired the research programme of Dr Paul Stevenson from Queens University, Belfast. John Daly of the NIH in the USA dedicated his career to the isolation and characterisation of around 900 of these alkaloids and it was with sadness that Stevenson reported his recent death.

Stevenson’s talk reminded us that in many cases de novo synthesis of natural products is the only way to obtain sufficient quantities to permit their full structural characterisation and study in detail their biological activity. Indeed, one of the most famous poison frog alkaloids, epibatidine, which is a better painkiller than morphine, was originally isolated from a frog species that is now extinct!

Stevenson’s research has focused on the 5,8-disubstituted indolizidines, which are selective antagonists of the alpha-4, beta-2 and alpha-7 nicotinic acetylcholine receptors, and the pumiliotoxins, which cause contraction of striated muscle, in the case of allopumiliotoxin with very high potency. His strategy has been to use ring-closing metathesis as a powerful, generic method of installing the second heterocycle, while installing an alkene for further fictionalisation by for example epoxidation or dihydroxylation, which interestingly occur from the more hindered face. Another strategy has been the use of N-acyliminium ion chemistry.

Stevenson’s total syntheses of the structures believed to represent indolizidine 259B and pumiliotoxin 233C have shown these structural assignments to have been in error, which is unsurprising given the minute amounts that were isolated. In the latter case, Stevenson was able to work with Daly to identify the correct structure of the natural product, but in the indolizidine case, the identity of the natural product remains elusive. Importantly, the assumption that led to the erroneous assignment of this structure was also applied to a large number of related alkaloids, so Stevenson’s discovery calls into question the structure of a whole class of natural products.


Safer Pregnancies
6 to 15% of all births are 'pre-term', that is between 24 and 37 weeks. While in some cases pre-term births are unproblematic, the infant’s chances of survival are lower than with full-term births and pre-term infants are more susceptible to ongoing health problems. Therefore, there are significant social and economic drivers to reducing the number of pre-term births.

Dr Deirdre Hickey (GlaxoSmithKline, Harlow) leads a team that has been developing oxytonin antagonists that may eventually be used to alleviate the number of pre-term births. Such antagonists function by inhibiting uterine contractions. While there is already a peptide antagonist of oxytonin licensed for use in Europe, there is a pressing need to develop drugs that have superior selectivity for oxytonin with respect to the related vasopressin target. The lead molecules emerged from a screen of a combinatorially constructed library and are diketopiperazines.

Hickey drew the conference’s attention to the need to find a compromise between potency and pharmacokinetic properties. It is relatively straightforward to find analogues that are more potent by increasing lipophilicity. However, these lipophilic analogues are unlikely to possess the full range of properties needed to become candidates.

The synthesis of the lead molecules is a fine example of the use of multicomponent synthesis; in this case a four-component Ugi reaction. Nevertheless, it was necessary to investigate alternative routes to diketopiperazines, including asymmetric Strecker, symmetric hydrogenation, classical resolution, kinetic resolution and chiral chromatography methods. For example, successive kinetic resolutions with a hydantoinase and a carboxylase were employed to deliver products of high enantiomeric purity.

GW796679X emerged as an advanced lead, with a very promising pKi. However, the log D was too high and it was necessary 'to take a step back' on the potency and explore derivatives with better solubility characteristics. Finally, an azole derivative was designed that exhibited a very promising combination of potency and selectivity. Indeed, GSK221149A is now a clinical candidate.


Chemical Genomics Approaches
Only 500 drug targets have been identified from the 5000 believed to be available in the proteome. Molecules with known biological activity but no known target may well act on one of the other 4500! In the chemical genomics strategy, we start with the known activity of the small molecule and experimentally link it to the genome. Magic Tag® chemistry, developed by Paul Taylor and colleagues at the University of Warwick and commercialised by a2sp Limited, can be used to identify new protein targets for drugs and natural products.

Bacteriophage are viruses that infect bacteria. T7 bacteriophage, for example, are robust, stable and easy to handle safely in the lab. The genome of an organ or organism of interest, as represented in its mRNA, is 'cut up' and inserted into the DNA of different phage to create a phage cDNA 'library'. Adjacent to each inserted 'gene' is inserted a second gene that causes expression of the library protein on the surface of the phage. The idea of probing phage displayed libraries of proteins with small molecules is not new. However, examples of successful 'biopanning' with small molecules are rare, even when 'immobilised'. Non-specific binding adversely affects signal to noise and immobilising a small molecule can have a significant effect on its biological activity, reducing specific binding.

Taylor showed how photoimmobilisation offers ultrarapid surface synthesis of analogues of bioactive molecules. A range of photochemistries ensures bioavailable regioisomers are present on the surface enabling the desired specific binding. Incorporation of oligo(ethylene glycol) groups reduces non-specific binding. Biopanning amplifies the signal and reads out rapidly though DNA sequencing and competitive elution identifies the most 'solution like' binding events.

Taylor went on to relate how immobilization of biotin and digoxin with a fluorescent antibody assay proves the principle and that a Magic Tag® chemical genomics screen of abscisic acid against a lambda library from Arabidopsis revealed a putative target for ABA. Moving into the biomedical field, Taylor reported a Magic Tag® chemical genomics screen of salbutamol against a T7 library from human lungs that uncovered ATF4 as an 'off' target and a Magic Tag® chemical genomics screen of flecainide against a T7 library from Drosophila that revealed myosin regulatory light chain as a possible target for flecainide’s most striking adverse drug effect. Applications of this chemical genomics strategy in drug discovery, drug reprofiling and personalised medicine are clearly possible.


The Epothilone Story
If one needed proof that natural products remain of crucial importance to drug discovery, the epothilone story provides it. Dr Dieter Schinzer, now at the University of Magdeburg, has been at the heart of the research effort in this field. Epothilone, which is isolated from myxobacteria, has taxol-like anticancer activity, but its properties are more attractive with respect to formulation and delivery as a drug.

Schinzer and other groups around the World have carried out detailed studies on the structure of epothilones, their binding to tubulin and their structure activity relationships (SAR). These detailed studies, which required a great deal of high level synthetic work, provided the backdrop to the development of epothilone based drugs and drug candidates. For example, ixabepilone is an injectable anti-tumour drug. The drug can be prepared semi-synthetically in three steps from epothilone B, which is readily available from fermentation of the bacteria. Another analogue, ZK-Epo can be prepared by total synthesis in reasonable quantities and is in Phase II clinical trials. The key step is an aldol reaction, the whole synthesis showing again the central role that organic chemists play in the field.

The Schinzer group has also been engaged in the synthesis of another promising analogue, furanoepothilone. Among the elegant new methodology developed is an eye-catching, if more mundane, discovery that splinters of glass are an excellent mediator for hydrogen fluoride deprotection of silyl-protected alcohols! Finally, Schinzer’s conclusion is a fitting one for the whole meeting 'chemistry can be the hard way but is [ultimately] rewarding'.


Though a one-day meeting can only touch on the vast amount of synthetic effort underway internationally, the variety on show at the fourth Synthesis of Bioactive Molecules meeting was revealing. In the context of huge shifts within the drug discovery and development industry internationally it is clear that innovation in synthesis is becoming ever more important. Scientists equipped with expertise in synthesis will make significant contributions both to their own field and also through a basic chemical understanding that enables efficient new approaches to biological problems.

Paul Taylor, Department of Chemistry, University of Warwick, UK

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