Elizabeth Farrant is Head of Chemical Technologies and Analytical Sciences at Pfizer. Previously Assistant Director, Technology development GSK, Harlow. Dr Farrant has more than10 years experience in pharmaceutical research working in combinatorial chemistry, medicinal chemistry and new technologies at the forefront of developing new approaches to synthetic chemistry including parallel chemistry and purification and flow chemistry.

New Synthetic Technologies in Medicinal Chemistry

By Elizabeth Farrant

The Royal Society of Chemistry

Copyright © 2012 Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-017-4

Contents

Chapter 1 Introduction Elizabeth Farrant, 1,
Chapter 2 High Throughput Chemistry in Drug Discovery Andy Merritt, 6,
Chapter 3 High Throughput Reaction Screening Andrew I. Morrell, 42,
Chapter 4 Microwave Assisted Chemistry Rachel Osborne, 63,
Chapter 5 Continuous Flow Chemistry in Medicinal Chemistry Martyn Deal, 90,
Chapter 6 Emerging Synthetic Technologies Brian H. Warrington, 126,
Subject Index, 154,

CHAPTER 1

Introduction

ELIZABETH FARRANT

Worldwide Medicinal Chemistry, Pfizer Ltd., Ramsgate Road, Sandwich, CT13 9NJ, UK

1.1 Introduction

When I was training as a synthetic chemist just under 15 years ago, the range of technologies we were expected to become familiar with as postdoctoral chemists was very limited: we were expected to pack a perfect flash column, be adept with an inert gas/vacuum line and to be able to shim a 250 MHz NMR instrument. In some special cases we might have been required to use a HPLC. Now, a standard industrial lab is likely to be equipped with automated HPLC, flash chromatography, microwave reactors, maybe a flow reactor and parallel synthesis is expected as routine to maintain productivity. Analytically, the chemist has routine access to LC-MS, automated NMR instruments running complex experiments and open-access accurate mass determination. The synthetic chemistry laboratory has become a highly technology-enabled environment.

1.2 The Legacy of Combinatorial Chemistry

Many of the technologies now routinely used in synthesis have their roots in the combinatorial chemistry paradigm of the late 1990’s. As the possibilities in drug discovery resulting from the sequencing of the human genome culminated in a rough draft announced by the Sanger Institute in 2001, the need to discover ligands for these estimate 3 000 to 10 000 potential disease genes led to the implementation of bead-based combinatorial mixture libraries. Using this technique, libraries of compounds of immense theoretical size could be manufactured but very soon it became clear that their utility was severely hampered by the deconvolution of any active products, the range of chemistry suitable for use with solid support and the close structural similarity of all the molecules generated.

The field evolved gradually into what is now practiced as high throughput medicinal chemistry, focusing on the synthesis of pure single compounds through solution-phase methods using diverse and imaginative chemistries with short cycle times from array design to biological test.

Many of the analytical and purification technologies developed during this time, including high throughput open-access LC-MS with UV and evaporative light scattering detection, mass-directed high throughput purification, automated medium-pressure liquid chromatography and high throughput flow NMR are now in routine use in standard synthetic chemistry labs.

In addition, the methods developed to carry out high throughput plate-based chemistry have evolved as an approach to generating rich data sets to guide the optimisation of chemical reactions where there is an array of reactant, solvent and condition combinations. This has also been extended to applications as diverse as biotransformation screening and de-racemisation via chiral salt formation. The power of this approach to find optimal reaction conditions for key reactions as well as to discover and enable new synthetic transformations has only begun to be exploited.

A recent addition to the synthetic chemist’s tool box has been the use of microwave energy to heat reactions. In many cases this more efficient heating method has been shown to dramatically shorten reaction times and also improve impurity profiles.

Another key innovation of the last 15 years has been the application of microfluidics, an approach that was initiated in the analytical community, to synthetic chemistry. In its true microfluidic format this technology is being explored as a methodology for combining the efficiency of combinatorial chemistry with the fast biological feedback needed to reduce the time to go from a hit molecule to a lead. In addition, conducting chemistry in larger (mesofluidic) tube reactors has also grown in popularity due to the ability to improve reproducibility of heating and mixing over the standard round-bottomed flask. One interesting and fruitful application has been to use this approach to help control reactions using unstable intermediates and as a scale-up route for reactions that progress well due to the efficient heating observed in a microwave reactor.

All of these technology solutions have contributed to the chemist’s toolbox, supplementing traditional approaches and equipment, and have revolutionised the way synthetic chemists design and carry out their syntheses. The impact they have had has not been the explosion in hits and lead molecules (and drug molecules) promised by the early vision of the combinatorial chemistry — that is still an underlying problem the industry is attempting to address on many fronts. However, it has been an enabling of the creativity of the synthetic chemist to build molecules and enter novel chemical space.

The case study of sorafenib illustrates beautifully the impact these technologies have been having in a drug discovery programme which used the true power of combinatorial chemistry to solve a problem that would have blocked progress to the discovery of an important cancer therapy.

1.3 Case Study: Sorafenib

The time scale for drug discovery programmes is frustratingly slow and attrition is high; however, the mid-2000’s have seen drugs entering the market whose discovery has relied heavily on the application of these novel technologies. One example is the Bayer molecule sorafenib (Nexavar®) (Figure 1.1).

Sorafenib was the first oral multikinase inhibitor on the market and was designed to target Raf which is important in tumor signaling and vasculature. It was first approved for the treatment of advanced renal cell carcinoma in 2005. Despite extensive traditional analoguing and structure–activity relationship (SAR) generation around the 17 µM high throughput screening hit 1 (Figure 1.2), the chemists were unable to improve the IC50 beyond 10-fold from this hit.

A high throughput chemistry programme was initiated in parallel with the later stages of this work and among the 1000 compounds efficiently generated in this manner, chemists identified compound 4 (Figure 1.3) which had an IC50 of 0.54 µM. Crucially, during traditional analoguing, compounds 2 and 3 had been synthesised and proved essentially inactive. These data would normally significantly deprioritise the synthesis of 4 when made by resource intensive single compound synthesis as they indicate that compound 4, a combination of the ringed groups, would lie outside the established SAR. In this case the chemists asserted that they would not have synthesised this compound in the normal course of the drug discovery programme.

Further traditional medicinal chemistry then led to the discovery of sorafenib. The researchers observed that this discovery programme shows the power of high throughput chemistry to explore efficiently the additive effects of medicinal chemistry modifications outside the normal SAR; in this case they postulate that compound 4 may adopt a binding conformation different from that of compounds 2 and 3, explaining the divergence from the initially proposed SAR.

1.4 Conclusion

In many ways the flowering of technology development in the 1990’s was largely about a wish to increase productivity in response to the increases in capacity in genomics and the promise of thousands of new drug discovery targets. In practice, however, as has often been observed, this resulted early on in an increase in the size of the drug discovery haystack rather than a rise in the number of needles found. As the following chapters of this book will demonstrate, the true result has been routine use in the synthesis lab of a range of new tools. These are used to their greatest effect when it is not merely to increase productivity by a numerical measure but to expand the access of the synthetic chemist to new chemical space which would not have been accessible by traditional approaches. The sorafenib story illustrates this in a programme that resulted in a marketed drug but the ensuing chapters will also show the many examples where wise use of technology has contributed to drug discovery, be it in target validation, medicinal chemistry design or the provision of a compound for drug discovery programmes.

CHAPTER 2

High Throughput Chemistry in Drug Discovery

ANDY MERRITT

MRCT Centre for Therapeutics Discovery, 1–3 Burtonhole Lane, Mill Hill, London, NW7 1AD, UK

2.1 Introduction

Combinatorial chemistry (Combichem), the technique of preparing (large) numbers of compounds by common reactions in parallel using building blocks drawn from groups of molecules with a specific reactive functionality, is now an established approach with widespread application across a number of fields. However, 20 years ago, when combinatorial chemistry was in its earliest days, pharmaceutical drug discovery research became one of the earliest adopters of the approach. Indeed, as will be discussed below, it was to do so with great commitment and expenditure. Up until the early 1990’s chemistry approaches to drug discovery had remained generally consistent over a large number of years. Although there were many developments in both synthetic methodology and analytical technology that gave medicinal chemists greater tools, allowing the synthesis of more and more complex drug structures, the general concept of single compound synthesis, biological testing and subsequent design of the next target molecule was standard throughout the industry. So when chemistries and technologies developed in peptide chemistry came to the attention of medicinal chemists at a time when productivity and efficiency were becoming more challenged, the opportunity for a revolutionary change in drug discovery clearly presented itself. The era of ‘Combichem’ was about to commence.

The fact that this chapter is titled ‘High Throughput Chemistry’ rather than ‘Combichem’ illustrates how, in its application to drug discovery and medicinal chemistry, combinatorial chemistry has changed since those early days of the start of the 1990’s, and how rocky a path that has been. Beginning with hype, when Combichem promised a solution to all lead discovery programmes through the concept of universal libraries, coupled to a predicted large scale reduction in medicinal chemistry requirements (and therefore resources), it was only after many endeavours that the realisation struck home that if you are looking for a needle, then maybe making the haystack bigger isn’t always the best approach. The terms combinatorial chemistry and Combichem fell from favour, associated with brute force ‘industrialisation’ that discarded the history and science of drug discovery with predictable lack of success (20/20 hindsight is, of course, always accurate), only to be reinvented quietly as the current practices of ‘high throughput chemistry’ and ‘parallel lead optimisation’. Beneath the headlines, be they the positive ‘great opportunities’ of the 1990’s or the negative ‘plethora of data but where are the drugs’ of the 2000’s, the application of combinatorial chemistry to drug discovery has had a wide impact on medicinal chemistry research. Techniques, strategies, design and synthetic methodologies have all been developed and are in constant use in most drug discovery labs of today. The intent of this chapter is to illustrate with several recent examples how such approaches are in common use; however, to understand these it is worth first reviewing some of the key developments of combinatorial chemistry and their application to drug discovery.

It is beyond the scope of this chapter to provide anything like a comprehensive coverage of the field of combinatorial chemistry, whether its historical development or its application to current lead discovery and optimisation. There are many comprehensive reviews, tabulated summaries, books and whole journals which the reader may wish to consult if greater depth of understanding or historical context is desired. Instead this chapter will provide a more personal view of the key moments and developments of combinatorial chemistry in drug discovery to hopefully illustrate the many aspects of combinatorial chemistry, drawing on experience in large diversity library production and technology development, technology transfer and change management in lead optimisation groups, targeted small array approaches to lead discovery and lead optimisation using array technologies.

The development of high throughput chemistry has also led to rapid changes in the IT infrastructure to support drug discovery, in areas as diverse as sample management, process automation and electronic registration; however, these areas are beyond the scope of this chapter and the references supplied should be followed if more information is required.

2.2 The Potential of High Throughput Chemistry in Drug Discovery

Before considering the historical development of combinatorial chemistry and the current best practices, it is worth reviewing how drug discovery typically progresses and identify those areas where combinatorial and high throughput chemistry may have a significant impact.

As described by Hughes, drug discovery can be roughly divided into 4 stages. The initial stage, that of therapeutic target definition, is a biology driven component which relies on chemical tools to help clarify mechanisms and pathways. As such, the opportunities for parallel chemistry methods to have an impact are limited, though they may be used in the identification and optimisation of tool compounds, especially if these are peptidic.

Once a target has been defined then lead identification begins, often using high throughput screening approaches. The goal of this stage is to identify compounds that have significant potency against the target, such that they warrant further exploration to optimise against a wider range of drug-like parameters. In this phase the demand for large screening collections is often met in part through compound collection enhancement using parallel chemistry approaches, either driven by chemical diversity or targeted towards particular structural motifs associated with certain protein classes.

The third stage, once a range of lead molecules has been identified, is lead optimisation, where the goal is to optimise against several parameters leading to a compound (or preferably several compounds) suitable for full pharmaceutical development. It is in this arena that the need to generate quality data on chemical series to support SARs (structure–activity relationships) can be rapidly facilitated through parallel chemistry approaches, at a scale of 10s to 100s of compounds at a time. Not only potency, but measured components for absorption, distribution, metabolism and elimination (ADME) properties, toxicity profiles, P450 enzyme profiles and selectivity profiles may also be explored using parallel approaches.

The final stage of the drug discovery process, that of development into a drug, is the most costly component of the discovery process. Attrition, especially at later stages, has a significant impact on the overall costs of drug discovery and as such the quantity and quality of data generated in the earlier stages can have a significant bearing on the final quality (and therefore potential for success) of any drug through development.

2.3 The Start of Combichem in Drug Discovery

The development of miniaturised screening leading to high throughput approaches was a significant advancement of drug discovery. The standardisation of the assay format into microtitre plates, initially a 96-well format, alongside the development of automated processing, radically changed the opportunity for screening to deliver new leads for drug discovery programmes. Automation of plate movement, liquid handling and plate reading processes meant that where a few 10s of compounds may have been tested in a day by manual techniques, suddenly 1000s were possible in enzyme, (membrane-bound) receptor and even whole cell assay format. Further enhanced by the miniaturisation of wells on the plates, from 96 to 384 (and subsequently 1536), high throughput screening of compound collections of 100 000s or more became clearly feasible and, when run alongside mechanism and knowledge/ structure-based targeted screening approaches, provided much greater opportunities to identify novel lead series and structural classes.

(Continues…)Excerpted from New Synthetic Technologies in Medicinal Chemistry by Elizabeth Farrant. Copyright © 2012 Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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