Agrochemical Discovery - American Chemical Society

Chapter 17 Parallel Synthesis at Novartis C r o p Protection:

Downloaded by UNIV MASSACHUSETTS AMHERST on September 17, 2012 | http://pubs.acs.org Publication Date: December 28, 2000 | doi: 10.1021/bk-2001-0774.ch017

Concept and Realization M . Diggelmann, J . Ehrler, and W. Lutz Novartis Crop Protection AG, Lead Discovery Department, P.O. Box, 4002 Basel, Switzerland

In order to increase synthesis capacity and to support lead finding and lead optimization activities, sizable investments into laboratory automation and state-of-the-art purification equipment were made at Novartis Crop Protection. Successful implementation of a high-speed-synthesis platform consisted of the modular assembly of different commercially available workstations following an off-line automation approach in combination with the design of cheap and primitive custom made reaction manifolds. Major emphasis was always given to the objective of optimizing overall throughput which led early to the simple conclusion that the bottleneck of isolating pure compounds is of utmost importance. The increased ability to derive reliable structure-activity relationships, fundamental for the tasks of chemists, should consequently lead to a fast return of capital investments spent to address this issue.

Combinatorial chemistry has emerged as a powerful tool for lead generation as well as lead optimization, and it's importance is demonstrated by the size of investments and the speed of implementation made by all major pharmaceutical and agrochemical companies. The clear driving force is the desire for faster drug discovery cycles (i). Since it's invention about a decade ago (2,5), a number of

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In Agrochemical Discovery; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.


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different techniques and ingenious concepts were described in the literature, ranging from truly combinatorial synthesis on solid support (4,5) towards more automated parallel synthesis approaches favoring liquid phase (6,7,8). While initially people were impressed by the huge number of compounds that can be prepared by these techniques, it also became quite clear over the years that simply preparing and screening more compounds does not automatically solve the drug optimization problem, among others probably one of the reasons why currently much focus is on improving the çheminformatics environment in our industry (9,10). Challenges and difficulties in finding marketable drugs or pesticides obviously still exist, and this will lead according to our view to a much healthier situation with respect to a realistic assessment of the potential of combinatorial chemistry. That such a potential certainly exists nobody seriously denies engaged in the art of drug design.

Impact on Drug Discovery The large number of existing combinatorial techniques provides today's researchers with a new set of tools, the selection of which largely depends on the characteristics of the problem to be solved. It's probably fair to say that the majority of applications in solid-phase synthesis are used in a true combinatorial fashion and applied more towards lead finding while solutionphase based approaches, supported by variable degrees of laboratory robotics, seem better geared for lead optimization or hit-follow up activities as indicated in Figure 1. Lead finding • • • •

Combinatorial Technologies

Lead Optimization

large numbers divers* design novelty diverse scaffolds

Molecular Diversity

• fast iterative cycles • smaller numbers • focused design • diverse building blocks - secured IP

Screen

OQI

[>

Figure 1: Impact on drug discovery by combinatorial technologies


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Automation Concepts At Novartis Crop Protection solid-phase as well as liquid-phase based approaches were and still are being pursued, this article only illustrating our activities in the latter one. Over the last years improvements in laboratory robotics and data management contributed significantly to the rapid growth of parallel synthesis methods. Conceptually two approaches can and should be distinguished One which we termed on-line automation, meaning that several différent tasks are performed by the same device, and if implemented successfully, allows long run times of the involved machine(s) without any manual intervention. The second one, off-line automation, refers to the modular integration of several different workstations into an assembly line, each performing a dedicated task. A clear distinction between these two automation approaches is more than just semantics since they differ significantly in overall performance and investment costs. Reasons for us to favor off-line automation are outlined in Table I.

Table I: Comparison of Automation Approaches Issue

On-Line

• # of reactors in parallel

small

high (scalable)

difficult

easy

• adaption to technical Improvements • removal of bottlenecks • user interface • throughput • manual steps

Off-Line

Impossible

possible

challenging

easy

low

high

no

yes (> racks)

As becomes clear from studying the table, the realization of a high throughput synthesis environment by an off-line automation concept basal on liquid-phase synthesis is easy, low tech and cheap. The reason for this statement is the fact that the synthesis and isolation processes involved (Figure 2) are the same as the ones performed traditionally by chemists for decades.


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j Labotiiig


QC Decision

Figure 2; Workflow in Parallel Synthesis Parallel liquid-phase synthesis can therefore be performed with rather simple devices (Table Π), and all that's left to do is basically to design reactors that allow increased parallel handling. For overall efficiency of course consideration about batch size and interfaces between the different devices are important and crucial for success, e.g. assigning a standard rack format is a clear must. In addition one has to ask oneself carefully what, how and how much to automate. Table Π: Devices for parallel liquid-phase Synthesis

Process

Devjce

1. A d d i n g B B ' s and Reagents

Pipette Robot

2. Heating

Hotplate

3. Quenching

Pipette Robot

4. Liquid Extraction

Pipette Robot

5. Evaporation

Evaporator

6. Dissolving

Pipette Robot

7. Aliquot Pipetting for Analysis

Pipette Robot

8. Evaporation

Evaporator

9. Weighing

Balance

10. Dissolve

Pipette Robot

11. Aliquot Pipetting for Screening

Pipette Robot

Reactor Design At the time of conception, no really cheap reactors were commercially available, and we decided to work with our own racks custom made from



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aluminum. Objectives guiding the design were overall low costs, flexible use and the ability to be used in connection with already existing commercially available liquid handling robots. We favored a 6x4 format similar to the one used by biologists for cell culturing and also relying on the same footprint as the traditional 96-well microtiter plates. This design gave us the flexibility to work in reasonable batch sizes, as well as the opportunity to work at different synthesis scales; and, additionaËy, e.g. the ability to perform liquid-liquid extraction steps directly out of the synthesis reactor. Depending on the reaction conditions to be applied, either disposable PP based tubes or reusable glass reactors can be used Heating and stirring is performed by traditional magnetic hot plate stirrers and magnetic stirrer bars while parallel sealing of the reactor device is achieved via mechanical clamping, analogous to the principle described many years ago by Meyers et al. (11). A schematic representation of the reactor manifold is shown in Figure 3.

Figure 3: Reactor Design

Feasible Chemistry In these admittedly rather simple reactor blocks, a wide range of reactions are possible allowing the chemist, in combination with simple liquid handlers and temperature controlled liquid cooling bathes, even to perform quite delicate


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organometalic chemistry (Figure 4). This may seem at first glance surprising, but more careful thinking about the physical prerequisites for such reactions leads to the conclusion that such experiments, reasonably scaled, are not a priori impossible. As it turns out the chemical transformations are in fact neither easier nor trickier to perform than in more traditionally set up experiments.

THF,-35°C

Figure 4: Chemistry Examples (performed)

Purification Concept Problems in parallel synthesis obviously arise not from the fact of the synthesis itself but rather from the wide range of physical and/or chemical behavior of all the involved reagents and reactions. Since this is an intrinsic characteristic of any combinatorial approach, one is confronted with a typical management problem; namely to find the line between targeting maximum diversity and increased speed resulting from parallel handling. In order to obtain a maximum number of clean products, the chemist has either the possibility to study all the reaction parameter carefully and subsequently choose a reduced set of no more maximally diverse reagents (!) or give up the parallel approach altogether and dedicate a maximum of commitment and attention to the synthesis of any individual compound. A partial way out of this dilemma,



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namely the application of chromatographic methods, is of course not new but has more recently become a very attractive option because significant improvements in the area of automation and software control led to higher throughput. Hyphenation of liquid chromatography with mass spectrometry (LC/MS) allows the simultaneous separation and identification of compounds and is undoubtedly the technical solution most often used in industry to fulfill the objective of increased throughput (i2,13). Such purification platforms are by definition not cheap, but an analysis of how chemists time is traditionally spent in most organic synthetic laboratories (Figure 5) shows the huge potential for a fast return on the capital investment without any sacrifice on behalf of their main task, which is the search for correlations between chemical structure and biological activity.

Figure 5: Saving Potential

We currently operate successfully several LC/MS systems in our research chemistry units across the whole research organization, i.e., at our research sites in Basel, Switzerland, as well as at RTP in the U.S. These purification platforms can be operated either in an analytical or preparative mode (Figure 6) and have overall significantly shortened the time to isolate and characterize compounds submitted for screening. Although it has to be clearly said that some limitations still exist, it is also true that these machines quickly become rather indispensable tools.



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Figure 6: Wiring Diagram LC/MS In summary, the high speed synthesis platform as just described together with a sizable and high quality chemicals stockroom, an efficient electronic building block management, automated data analysis tools as described earlier (14) and a good mix of experienced and dedicated chemists are the basis of a very efficient hit-follow up process at Novartis. And most importantly, once the platform is in place and the various workstations are up and running, the chemists can again focus on what they like most: designing and preparing compounds in order to improve their biological activity.

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