In recent years, a confluence of spectacular advances in chemistry, molecular biology, genomic and chemical technology, synthesis and the cognate fields of spectroscopy, chromatography and crystallography has led to the discovery and development of numerous novel therapeutic agents for the treatment of a wide spectrum of diseases.
In order to facilitate this process, there has been a significant and noticeable effort aimed at improving the integration of novel discovery technologies, external collaborations for route selection and the delivery of APIs, drug product formulations, clinical trials and the use of information technologies.
The pharmaceuticals industry has undergone unprecedented changes in recent years. The rapidly increasing pace of regulatory reform, allied with the necessity for drastic reductions in the price of bulk drugs, has resulted in marked shifts in its strategic paradigms.
Multi-disciplinary and multi-functional teams focusing on lead generation and optimisation, drug discovery and development have replaced the traditional, specialised research groups. Pharma strategists have long envisioned departing from fragmented endeavours and knowledge directed to incremental improvements in a particular therapeutic area to a search for blockbuster products through centralised corporate wisdom, based on creating and accumulating knowledge of targets by bioinformatics and chemoinformatics approaches.
This approach promises to deliver the novelty demanded by regulatory authorities. However, the resources for creating chemical diversity, conducting high throughput screening and the curation and mining of informatic databases have been underutilised. The diversity generated has been deep rather than broad.
Our high standards of living and health owe much to the chemical synthesis of pharmaceuticals and consumer products. Chemists today have an enviable armoury of techniques and methodologies in organic synthesis but are facing heavy demands on their efficiency and creativity. The need of the hour is to improve productivity and efficiency and explore new approaches, strategies and tactics for compound synthesis.
Synthesis chemists are expected to make discoveries and prepare compounds at a phenomenal rate and with increasing levels of structural diversity. Hence, the new methods adopted need to provide alternatives to the labour-intensive practices of the past, such as manual optimisation, aqueous work-ups and extractions, difficult crystallisations or distillations and chromatographic purifications.
This has led to a noticeable increase in the use of automation, informatics and technology-based approaches in the laboratory. Furthermore, by combining the use of supported reagents with new equipment such as microfluidic flow reactors and focused microwaves and the practice of catch-and-release strategies, many new opportunities for synthesis are being recognised.
This paper highlights some of the advances being made in this field by scientists at Cambridge University in the UK, in two broad areas: flow chemistry in organic synthesis and the multi-step synthesis of biologically active compounds. It then goes on to discuss wider issues in flow chemistry.
Flow chemistry in organic synthesis
Concepts of increased mixing efficiency, controlled scaling factors, enhanced safety ratings and continuous processing capabilities have been well recognised but have not been generically leveraged into conventional synthetic chemistry. Traditionally, almost all chemical manipulations have been conducted as sequences of well-defined and laboriously optimised single-step transformations in batch mode.
The pioneering work of Professor Steve Ley's group has resulted in the application of immobilised systems to such complex synthetic challenges as multi-step integrated synthetic operations. Such continuous sequential or parallel processing of reactants, often involving several stages of reactive intermediates, has naturally led to the development of dynamic, coupled, multi-step, continuous flow procedures.
Solid supported reagents are reactive species associated with a support material and are used to transform substrates into new chemical products. An excess of the reagent drives a reaction to completion and helps to isolate the product very cleanly. Any highly toxic or obnoxious materials used can be rendered easier to manipulate and recover and therefore safer.
The immobilisation of expensive catalysts and ligands greatly facilitates their recycling potential. Furthermore, immobilisation intrinsically results in reagents becoming site-isolated and able to react with substrates in a solution but not readily with one another. Multi-reagent processes, such as oxidation and reduction, or other mutually incompatible transformations can be conducted simultaneously in single reaction vessels.
The immobilisation of a reagent is designed to facilitate reaction work-up and product purification, thereby increasing overall productivity and output. Immobilised scavengers are supported compounds that selectively sequester the by-products of a reaction, then render them insoluble and readily removable by filtration.
Their use can be highly effective at improving the purity profile of complex reaction streams without resorting to liquid-liquid extractions or column chromatography. Scavengers can exploit both ionic and covalent interactions and bind either organic or inorganic impurities. Electrophilic or nucleophilic by-products can be removed by reciprocally functionalised supports.
A related concept involves a process referred to as a 'catch-and-release'. A uitably functionalised support can be designed to react with the desired product in a reaction mixture. After filtration and washing with a suitable solvent to remove by-products or other unwanted materials, the captured compound can then be released from the support in a pure form by an appropriate chemical process.
The release mechanism can be driven by a variety of reactions, such as hydrolysis or acid-base exchange. In more sophisticated examples, the concept of 'catch-activate-and-release' can be invoked to provide a different product from the cleavage process. Similarly, the release step may involve a chemical functional group interchange, such as a reductive or oxidative conversion.
With the use of supported reagents, by-products and unwanted components can also be scavenged; this obviates the need for conventional, expensive or wasteful procedures such as chromatography, distillation or crystallisation. Purification can also be achieved via the addition of solid-bound species that are 'programmed' to recognise only the target product. This is not a serendipitous process; it requires specific chemical design.
Immobilisation can be accomplished on beads, active surfaces, colloids, dendrimers, plugs and laminates. Each of these formats has a variety of support materials available e.g. polymers, cellulose and silica gels, enabling a large selection of reagents to be built.
The immobilised reagents can be delivered in a variety of formats, most commonly as loose beads, or as beads contained within pouches or formed into composite plugs. The same reagents can be readily packed into tubes, cartridges and various flow assemblies, resulting in tremendous versatility - particularly so for complex molecule assemblies.
The effectiveness of supported reagents was initially validated by the efficient preparation of commercially available drugs. The compound Viagra (sildenafil) which is used for the treatment of male erectile dysfunction, has become one of the largest-selling globally marketed prescription drugs in recent history.
A key feature of this synthesis of sildenafil was the convergent strategy that was adopted. The reaction between the resin-bound activated ester and the heterocyclic amine formed the important amide bond, with any excess amine being scavenged by polymer-supported isocyanate.
The supported hydroxybenzotriazole used in the coupling step performed a dual function: it facilitated the simultaneous activation of the acid and also its purification from various impurities and by-products accumulated during its formation. Cyclodehydration was then effected using catalytic sodium ethoxide under focused microwave irradiation conditions to produce gram quantities of Sildenafil in excellent overall yield.
Synthesis of natural products
Natural products and their derivatives, with their exquisite molecular architectures, have adorned pharmaceutical research and have provided the impetus for drug discovery. The typically complex structures found in nature are suitable for synthesis using immobilised reagents, scavengers, quenching agents and catch-and-release techniques.
The Amaryllidaceae alkaloids have been studied extensively owing to their structural variation and biological activities. Using an orchestrated array of supported reagents, the first natural products ever to be synthesised by means of these methods were the alkaloid natural products oxomaritidine and epimaritidine, in just five and six steps respectively.
This landmark synthetic achievement demonstrated how these effective synthesis methods are robust and applicable to the production of gram quantities of the materials without recourse to chromatography, water washes, crystallisation or distillation work-up procedures. In fact, only filtrations had to be performed to remove the spent reagents, followed by solvent evaporation to yield the intermediates, giving the products in remarkably high yield and purity.
Integration of supported reagents
The flow-based integration of supported reagents replaces traditional glassware with pre-loaded columns and cartridges containing immobilised reagents and catalysts. It also incorporates precision-manufactured reaction chips that permit controlled mixing and precise temperature control of reaction sequences.
This transforms conventional batch reaction sequencing into a flowing, dynamic processing procedure, either by passing the starting materials through various immobilised reagents or by combining intermediates and reagents in specialised reactor blocks in pre-defined combinations.
The intermediate material generated in situ can be subjected to a series of reaction cascades and scavenging protocols before it eventually exits from the reactor into a chemically inactive environment, allowing for its immediate collection as a pure product. Throughout this process, the packed cartridges can be interacted upon by various physical means, such as heating/cooling, oscillation, ultrasound, microwaves or irradiation.
The incorporation of automated and real-time analysis facilitates rapid optimisation and excellent quality control, while also ensuring reproducibility. The extensive range of reagent formats from traditional silica or polymer micro-beads through to woven fibres, pressed disks and monolithic structures can all be customised rapidly for use in such flow reactor designs.
The development of a diverse toolkit of such functionalised materials can then be combined to facilitate multi-step synthetic sequences. Alternatively, when used in parallel, the reactor arrays could be sequenced for the production and purification of very large libraries of compounds.
Focused microwave irradiation has always offered an effective mechanism for rapidly heating flow reactions, especially when employing heterogeneous components. Flow chemistry comes into its own in the arena of catalysed reactions and continuous processing.
An excellent example of this is the Suzuki reaction for the generation of unsymmetrical biaryl compounds. The standard catalyst employed in this reaction is a palladium species, but the reactions suffer from high levels of residual palladium contamination of the final products. Microwave heating can be used to expedite the production of Suzuki-furnished compound arrays including the generation of multi-gram quantities of products in a single, continuous operation.
Stock solutions of the boronic acid, aryl halide and nBu4NOAc activator were prepared and fed constantly at a flow rate of 0.1 ml/minute through the flow reactor, which was resident in the microwave cavity. As the reaction mixture exited the reactor chamber, it was progressed through a column of Amberlyst 15 sulphonic acid resin to remove any residual base and boronic acid salts. The solution could then be collected and following evaporation the final product isolated without the need for further purification.
Use of microreactors
Microreactors can be used to create a tool for rapid discovery and optimisation of new drug leads via an integrated, micro-scale chemical synthesis and bioassay system, able to conduct fast cycling iterative searches of diverse chemical space as an enhanced method for identifying and optimising novel lead chemotypes.
This significant down-scaling and acceleration of the synthesis of potent but short-lived biomarkers of disease could provide a means of shortening the time and resources required to achieve drug approval.
Chemicals and pharmaceuticals producers can deploy flow microreactors to provide a more flexible production regime than is achievable with large-scale batch reactors. With efficient monitoring of output flow, long run-times or parallel reactors, microreactors can deliver exquisitely enhanced and inherently safer protocols for bulk production.
The flow microreactor affords several advantages in process enhancement and safety. Due to the small cross-sectional dimensions of the reaction zone, mixing occurs within a relatively small number of molecular collisions, thereby ensuring faster, more efficient and more reproducible mass and heat transfer than is achievable in a batch reactor. The effects of erratic mixing and thermal gradients within the reaction bulk are thus largely avoided.
A large increase in the surface area/volume ratio and the catalytic effect of interfaces, coupled with the non-equilibrium conditions persisting in the micro-flow environment can alter the outcome of the reaction, often beneficially. Stringent control of product quality and safety is achieved by monitoring output identity and quality and applying this feedback in real time to optimise operating conditions.
This leads to an inherently safer process and avoids the dangers that exist in the batch method, where the accumulation of added reagents can rapidly release substantial amounts of energy on the initiation of the reaction. Large quantities of material can be synthesised by running a single microreactor for a sufficiently long time, or several identical reactors in parallel, although each must be monitored.
Fast serial iterative chemistry coupled with similarly fast parallel assays can be enabled by deploying flow microreactors in both chemistry generation and the assays, as can be accomplished using the directly coupled flow assay system, which can operate on minute amounts of small molecule sample and biological reagents. The parallel assays would be used to assess potency at the target protein and effects in assays which would define cross-reactivity, toxicity and efficacy.
(Courtesy: spacechemonline.com)