A Practical Approach to Accelerated Process ... - ACS Publications

Organic Process Research & Development 1999, 3, 281−288

A Practical Approach to Accelerated Process Screening and Optimisation David F. Emiabata-Smith,* Derek L. Crookes, and Martin R. Owen Glaxo Wellcome Research and DeVelopment, Medicines Research Centre, Gunnels Wood Road, SteVenage, SG1 2NY, U.K.

Abstract: The development and operation of an automated workstation for performing solution-phase organic synthesis and on-line HPLC analysis is described. A wide scope of applications and chemistries, typically encountered in process development laboratories, are reported. The application of this workstation to process screening and optimisation studies coupled with statistical design of experiments (DOE) is illustrated using three simple case studies. The utility of this approach in accelerating core aspects of process research and development by performing multiple experiments in parallel and generating quality data more efficiently has been explored.

Introduction The discovery of new drugs is a cornerstone of the development strategy of any pharmaceutical company. Combinatorial chemistry and other techniques generate a stream of new compounds with pharmaceutical potential. Whilst the full impact of these new techniques1-4 on chemical development is yet to be felt, it is critical that new strategies are developed to respond to the potentially large increase in development candidates. The experimental conditions first used to synthesise these candidates may not be optimal in terms of yield, purity, or in production terms, economy. A large number of experiments may be necessary to adequately investigate the wide range of parameters potentially influencing process performance. To perform these manually (which normally implies sequentially), can be a very time-consuming and repetitive task, a potential bottleneck in the whole development programme. Attempts to overcome this by reducing the number of experiments could clearly result in lower data quality, leading to interpretation problems. A statistical approach to experiment design is useful,5-7 but unless experimental conditions can be controlled reproducibly, important results may be masked by test-to-test variability. Automation of solution-phase chemistry using highly parallel techniques is already well established.8-11 These (1) Kubinyi, H. Curr. Opin. Drug Disc. DeV. 1998, 1, 16-27. (2) Veber, D. F.; Drake, F. H.; Gowen, M. Curr. Opin. Chem. Biol. 1997, 1, 151-156. (3) Beeley, L. J.; Duckworth, D. M. Drug Disc. Today; 1996, 1, 474-480. (4) Kleinberg, M. L.; Wanke, L. A. Am. J. Health-Syst. Pharm. 1995, 52, 13231336. (5) Carlson, R. Design and Optimization in Organic Synthesis; Elsevier: Amsterdam, 1992. (6) Box, G. E. P. The Design and Analysis of Industrial Experiments, 4th ed.; Longmans: London, 1978. (7) Stansbury, W. F. Chemom. Intell. Lab. Syst. 1997, 36, 199-206. (8) Gayo, L. M. Biotechnol. Bioeng. 1998, 61, 95-106. (9) Pavia, M. R. Annu. Rep. Comb. Chem. Mol. DiVersity 1997, 1, 3-5.

techniques offer a partial solution to the increase in efficiency demanded by the goal to successfully speed up the drug development time cycle. Although a number of elegant systems have been reported12-19 they are often very complex, expensive, or not well suited to the standard laboratory environment. The requirement for a simple-to-operate, costeffective system capable of performing automated solutionphase organic chemistry with on-line analysis remained an unmet need. This paper describes the development and application of a flexible solution to this problem that is capable of handling a large proportion of the chemistries routinely encountered in the pharmaceutical industry, and with potential applications in any industry where organic synthesis is an important part of product development. Evolution of the System As part of a strategic approach to accelerating process optimisation in the process development laboratories at Glaxo Wellcome in 1995, the original concept of an automated workstation has evolved into a custom-built system allowing the chemist to automatically prepare and run up to 20 reactions simultaneously, and to carry out on-line HPLC analysis as the reaction proceeds. In its first manifestation a Gilson 231XL Autosampler was employed with HPLC vials as reaction vessels mounted in a Gilson Peltier-effect temperature-controlled rack. The sampling, quenching/dilution, and subsequent HPLC analysis of the reaction was programmed via the autosampler keypad. This simple setup proved particularly useful in the optimisation of a desilylation reaction of a key intermediate in the synthesis of a novel Trinem antibiotic.20-22 However, this system did (10) Garr, C. D.; Peterson, J. R.; Schultz, L. O.; Amy, R.; Underiner, T. L.; Cramer, R. D.; Ferguson, A. M.; Lawless, M. S.; Patterson, D. E. J. Biomol. Screening. 1996, 1(4), 179-186. (11) Lindsey, J. S. Chemom. Intell. Lab. Syst. 1992, 17(1), 15-45. (12) Owen, M. R.; DeWitt, S. H. Automation in Chemical Development Process Chemistry in the Pharmaceutical Industry; Marcel Dekker: New York, 1999. (13) Porco, J. A.; Deegan, T. L.; Devonport, W.; Gooding, O.; Labadie, J. W.; MacDonald, A. A.; Newcomb, W. S.; Van Eikeren, P. Drugs Future 1998, 23, 71-78. (14) Rudge, D. A. Lab. Autom. Inf. Manage. 1997, 33, 81-86. (15) Sugawara, T.; Cork, D. G. Lab. Rob. Autom. 1996, 8, 221-230. (16) Harness, J. R. Mol. DiVersity Comb. Chem.: Libr. Drug DiscoVery, Conf. 1996, 188-198. (17) Hilberink, P.; Aelst, S. V.; Vink, T. Q.; Gool, E. V.; Kasperson, F. ISLAR Proc. 1996. (18) Delacroix, A.; Desmoineaux, V.; Guette, J. P.; Petit, J.; Porte, C. Lab. Rob. Autom. 1993, 5, 3-9. (19) Josses, P.; Joux, B.; Barrier, R.; Desmurs, J. R.; Bulliot, H.; Ploquin, Y.; Metivier, P. AdV. Lab. Autom. Rob. 1990, 6, 463-475. (20) Owen, M. R.; Emiabata-Smith, D. F.; Crookes, D. L.; Luscombe, C.; Godbert, S.; Lai, L. W. Org. Proc. Res. DeV. 1999, manuscript submitted for publication. (21) Ghiron, C.; Rossi, T. Targets Heterocycl. Syst. 1997, 1, 161-186. (22) Lester, T.; Owen, M.; Martin, K. Chem. Br. 1996, 34, 45-47.

10.1021/op990016d CCC: $18.00 © 1999 American Chemical Society and The Royal Society of Chemistry Published on Web 06/25/1999

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Figure 1. STEM RS1000 (with forced air condenser).

Figure 3. The DART (development automated reaction toolkit).

Figure 2. STEM RS1050.

not adequately model typical reaction conditions: temperature control was limited (4-40 °C), the vessels were not stirred, and there was no reflux capability. The next progression used automation of the sampling, dilution, and HPLC analysis of aliquots taken from a conventional reaction vessel. However, neither of these approaches proved a suitable solution for a wide range of chemistries. Furthermore, the available memory of the autosampler keypad was insufficient to handle the increasingly complex programs required. It was quickly realised that this type of autosampler, with its ability to accurately position the probe in all three axes, had the potential to handle not only the sampling and dilution but also the preparation of the reactions themselves. The next step was to use a larger autosampler, with a larger workspace and the flexibility to dispense reagents and solvents as well as to handle sample processing. Two purpose-made reaction stations were also developed to replace the standard Gilson autosampler rack. The STEM model RS1000 (Figure 1) with temperature-control range from ambient to 150 °C and the STEM model RS1050 (Figure 2) from -30 to +70 °C. Both have 10 reaction positions, magnetic stirring, and provision for inert gas blanketing; on the RS1000 there is reflux capability. By using Gilson’s 719 Sampler Manager software as the underlying control tool and with assistance from Glaxo Wellcome’s IT department and Anachem Ltd (distributors of Gilson products in the U.K.), software was developed to adequately control the reaction preparation, sampling and dilution procedures, and the complicated timing sequences involved. More recently, a more sophisticated software package has been developed in a jointly funded collaboration 282

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with Anachem Ltd known as SK233 software. With these refinements the system has now developed to the point that it has become a routine tool in several chemical development laboratories in Glaxo Wellcome, where it is known as DART (development automated reaction toolkit) (Figure 3). The system is available commercially as the Anachem SK233 Workstation. Applications Scope The DART (or SK233 Workstation) is based on standard analytical hardware and has proved capable of handling a very wide range of different experiments. These have included: •Process screening: selecting the best solvent, catalyst, or reagent combination to bring about a synthetic transformation. •Process optimisation: determining the levels of continuous variables such as temperature, reagent concentration, time to optimise yield, purity, cost, etc. •Robustness testing: studying the effect of small changes in process variables in order to define PDRs (process deviation ranges) for use on pilot plant/manufacturing scale. •Reaction profiling: determination of reaction endpoints, presence of intermediates, impurities, etc. •Stability studies: determination of the solution stability of drug substance or intermediates. These have all been performed using the on-line HPLC to monitor the reactions. The system has also been used for other experiments such as crystallisation and polymorphism studies, but using the reaction preparation and control facilities only. System Description The system is based on a Gilson 233XL Autosampler, with up to five racks for reaction vessels, reagents, solvents,

Figure 4. Gas/vacuum manifold.

and analytical samples. An additional 15 liquids can be handled via “transfer ports” if required. This unit combined with either a single- or dual-channel Gilson 402 dilutor, carries out all liquid-handling operations. In the present version on-line HPLC with UV detection is the chosen analytical tool, with an automatic Rheodyne valve for sample injection. A standard Pentium PC controls the system via a Windows 95 or NT user interface. The programmability of the autosampler arm in all three axes allows flexibility in the choice of racks, including the possibility of fully customised racks. A 183-mm piercing probe is used to access the various vessels. The probe is coaxial in design, which allows efficient washing, and permits inert gas purging of reaction vessels and reagent vials as well as simultaneous gas delivery while samples are being processed. Furthermore, the wide bore probe (0.7-mm i.d.) is particularly suited to sampling and HPLC analysis of heterogeneous reaction mixtures. Reactions are conducted in round-bottomed Kimble culture tubes (25 × 150 mm) fitted with screw caps and sealed with a Teflon-lined silicone septum. If required, an inert gas atmosphere can be maintained using a custom-made manifold device (Figure 4). Control of the probe in the Z-axis (i.e., sampling depth) is sufficiently accurate to allow the reliable sampling of reaction mixtures. Sampling of biphasic mixtures, or the supernatant liquid after solid particulates have settled is also possible. Clearly, stirring must be interrupted during sampling for this to be successful, and provision is made for this in the control software. A number of types of magnetic stirrer bar have been investigated as the degree of agitation required for different chemistries varies. For most applications, including homogeneous and heterogeneous chemistry, a standard stirrer bar with centre rim can be used. However, for biphasic mixtures and very dense suspensions a Spinbar Spinvane24 magnetic stirrer provides more effective agitation. Precise control of the reaction environment is essential if quality data is to be obtained. The specially developed STEM (23) Available from STEM Corporation, Woorolfe Road, Tollesbury, Essex, CM9 8SJ U.K. (24) Available from RADLEYS, Shire Hill, Saffron Waldon, Essex, CB11 3AZ U.K.

Figure 5. Solvent loss data using RS1000 and forced air condenser, 21 h at reflux.

reaction stations (Figures 1 and 2) house up to 10 tubes, each of which is provided with a separate magnetic stirrer drive capable of speeds up to 1500 rpm. Typical working volumes are 4-15 mL, so that normally the tube is 55 °C in a standard tube with a pierced septum. A summary of the studies carried out on solvent loss using the forced air condenser is shown in Figure 5. Two reaction station racks can be used at once if required, increasing the number of reactions to 20 or allowing simultaneous experiments at different temperatures. The reaction station is positioned within the working envelope of the autosampler with reagent and analytical racks. Standard Gilson racks are used for these purposes, and the present system supports a number of different options 14 × 20 mL, 40 mL, or 50 mL vials 3 × 250 mL polypropylene or 150 mL glass bottles Analytical racks 60 × 1.8 mL vials 108 × 0.7 mL vials Reagent racks

The probe can also be programmed to visit up to 15 fixed “transfer ports” for additional reagents or solvents if required. The control software includes graphical representation of the working area, displaying the racks and allowing any vessel to be identified by a simple mouse click (Figure 6). All liquid volumes are dispensed by a Gilson model 402 dilutor. Whilst the 402 dilutor delivers good accuracy for addition and dilution tasks, it should be remembered that these experiments are essentially of a comparative nature, and overall reproducibility is therefore the prime consideration. In view of the widely different volumes used for different operations (from 1 to 20 µL for sampling from the reaction vessel to several mL for reagents) a two-channel dilutor is usually employed, fitted with syringes appropriate to the volumes to be measured. Reproducibility and accuracy checks on these processes were performed both by weight (i.e., absolute weights of material transferred) and by peak Vol. 3, No. 4, 1999 / Organic Process Research & Development

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Figure 6. Screenshot of the SK233 software rack setup.

area measurements on the HPLC system used for reaction monitoring. Typical results were: Additions using coaxial probe CV better than 0.4% (using water) Serial dilutions (1:100) CV 1.0% showing linear correlation HPLC injections CV typically 0.5%

One of the most important steps in the process is analysis of the reaction mixture. A small volume of reaction mixture is withdrawn from the reaction vessel at a user-defined time and transferred to one of the analytical vials where a quenching solvent or diluent is added. The sample is then mixed and an automatic injection made onto the HPLC system. Diluent and/or quench solvent are drawn in to the autosampler needle before the sample of reaction mixture, to ensure effective washout and minimise cross-contamination. HPLC and LC-MS are the analytical techniques we have employed on-line thus far, but other methods, like GC or UV spectrometry, could be adapted to the system or used off-line. Experimental Considerations DOE (statistical design of experiments) has been successfully used with this apparatus.25-27 When transferring an experiment onto the system, however, there are a number of practical considerations. The first step is to select and set up the HPLC method. This involves running a test mix of the reaction mixture to check that the components of interest are adequately resolved by the chromatography and to give an indication of the dilution parameters required for sample processing prior to HPLC injection. The diluent used to prepare the sample for analysis must be capable of quenching the reaction and of completely dissolving all the components of interest in the sample. Ideally it would be identical to the mobile phase used for the HPLC separation. This is not always possible; for example, if the reaction solvent is (25) Owen, M. R.; Crookes, D. L.; Emiabata-Smith, D. F.; Smith, L.; Lai, L. W. Presented at the Laboratory Automation in Chemical Process R&D Symposium, Leeds, 1997. (26) Emiabata-Smith, D. F.; Owen, M. R.; Crookes, D. L.; Smith, L.; Lai, L. W. Presented at the 1st International Conference on Organic Process R&D, San Francisco, 1997. (27) Emiabata-Smith, D. F.; Owen, M. R.; Crookes, D. L.; Lai, L. W. Presented at the 14th SCI Process DeVelopment Symposium, Manchester, 1996. 284

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nonpolar, a diluent intermediate between the reaction solvent and the (normally polar) mobile phase used for the HPLC may be needed. A “dilute and inject” protocol is available in the SK233 software to automatically prepare a range of dilutions for injection and analysis, allowing the optimum to be selected. Generic HPLC solvent gradients have been developed,28 and in most cases fast HPLC methods with a 10 min cycle time using them can be applied without further method development. The use of an internal standard has been found to have many benefits. It allows the solution yield of the reaction to be easily calculated, possibly avoiding the need for separate isolation of the reaction product. It reduces ambiguity when relative response factors of product and starting materials are not known or when components are present which cannot be detected by the HPLC system. Furthermore, it allows the overall reproducibility of the campaign to be monitored. Clearly, the internal standard must be inert under the reaction conditions in use. It can be added at the start of the chemistry, allowing the yield to be monitored throughout the reaction or at the end-point to enable a final quantitative assessment of reaction yield. One of the key factors in setting up the system for an experimental run is the time taken for the HPLC analytical cycle, which clearly determines the maximum rate at which samples can be injected. If the reaction is slow or the “chromatography time” is short, parallel operation may be possible, where all reactions are prepared before the first sample is taken for analysis. Alternatively, to prepare reactions in smaller batches or even singly gives the possibility of higher sampling frequency, but at the expense of throughput and efficiency. A feature of the control software is that reactions can be run in “synchronous” or “nonsynchronous” mode. In synchronous mode the sample from the reaction is diluted and then immediately injected onto the HPLC system, whereas the sample is diluted and only injected when the HPLC system is ready in nonsynchronous mode. The operator can decide which mode to use based on the sampling frequency demanded by the fixed reaction and chromatography conditions. Having established the analytical protocol, the next step is to develop the reaction protocol, which ideally should duplicate the manual procedure originally used to derive the compound of interest. To ensure that the chemistry is compatible, control reactions are normally conducted and the results compared with manually prepared samples from previous experiments. When manual additions of solids are necessary and the reactions are being run at high temperature, the thermal stability of the solid needs to be considered. Liquid reagent mixtures can be prepared as they are needed, but scheduling may require the solid reagent to sit in a reaction vessel for a period of time before anything else is added to it. Again, control reactions placed at the beginning and end of a run allow any potential problems to be identified. Once the reaction protocol is settled, reagent quantities are calculated and converted to volume (µL) so that a method can be set up in the software. (28) Mutton, I. M. Chromatographia 1998, 47, 291-298.

System Set-Up and Control As mentioned above, the control software is in the Windows environment, and has evolved to provide a very user-friendly, graphical user interface. To cope with the potentially large array of parameters and to avoid mistakes and oversights, a “Wizard” approach has been adopted, so that the operator is led step-by-step through the many choices available. Error-checking is also built in, so that any incompatible control settings and liquid transfers are detected and displayed. A full description is beyond the scope of this article, but to give a flavour of the control facilities and flexibility of the system, let us outline the steps involved in setting up an experiment. Step 1: The operator first defines the dilutor syringe defaults, including single or dual channel, syringe size, the aspiration rate, size of air gap, and so on. Step 2: Next, the volumes are set for reaction quenching and preanalysis dilution. The needle height can be defined at this stage as well as a time delay between quenching/ dilution and HPLC injection for the settling of particulates or biphasic mixtures. The main analysis parameters (synchronous or nonsynchronous, chromatography time, injection parameters, etc.) are also set. Step 3: The next step is to identify the autosampler racks according to their function (reaction, reagent, or analytical) and type (number and size of vessels, STEM RS1000, or STEM RS1050). If STEM racks are used, the temperature and stirring parameters are selected at this stage. Step 4: The locations and names of reagents and solvents in the racks or at the transfer ports are specified (Figure 6). Automatic rinsing of the probe between quench and dilution and after injection can be selected. Step 5: Finally, the detailed tasks to be applied to each reaction vessel are defined. These would include solvent additions, preparation and analytical sequences, and timing. At this stage, the automatic error checks are carried out. The software scheduler then interleaves the reactions according to their individual sequences to provide an overall schedule with maximum parallel operation. The user interface allows the experimental conditions applied to a particular vessel, or the complete experiment, to be conveniently viewed on a single screen (Figure 7). The set-up times for running chemistry vary depending on the complexity of the chemistry, the level of information required and whether an internal standard is being used. For a set of 10 reactions that require no analytical method development and do not use an internal standard, the set-up time will be short: typically 1 h for the preparation of reagents and solvents, 1 h to run HPLC test mixes and blank, and 15 min to set up the software. However, if an internal standard is being used, then response factors must also be calculated by running HPLC samples of both the internal standard and product, and this information can take up to a day to generate. The collection, integration, and manipulation of HPLC analytical data is carried out at Glaxo Wellcome using MultiChrom software.29 Sample tracking is managed by the (29) VG Data Systems, St. George’s Court, Altrincham, Cheshire, WA14 5UG U.K.

Figure 7. Screenshot of the SK233 software method setup.

SK233 software using tool tips on the graphical representation of each analytical rack during the run and on a textual description in the log file after a run is complete. To handle the large amount of data generated during a typical run, a custom program was written to automatically transfer all of the analytical data from MultiChrom directly to Microsoft Excel. The production of tables and graphs of reaction profiles and the transfer of data into Design Expert software therefore require little effort, and transcription errors are reduced to a minimum. Case Studies The DART system has been in use in Glaxo Wellcome chemical development laboratories for approximately 3 years. In that time, several thousands of experiments have been carried out across a wide range of projects worldwide. A full description of this chemistry is beyond the scope of this article, but to demonstrate the utility of the system a summary of example reactions along with three case studies are presented. A range of chemistries has been performed on the system including homogeneous, heterogeneous, air/moisture sensitive reactions from subambient to reflux. A selection of these reactions is listed in Table 1. They range from very simple to handle, homogeneous reactions such as the epoxide ringopening reaction (entry 3), to the rather more complex boronic acid formation (entry 9) which required 40 min addition times for both nBuLi and triisopropylborate. Process Screening Studies. It was alluded to earlier that process screening is one of the core activities in a drug development programme. The first two case studies illustrate successful applications of the system to this type of study, one, a solvent-screening study and the other, a reagentscreening exercise. A number of other more complex twoand three-dimensional screening studies (involving reagent, solvent, and catalyst) have been performed and will be reported at a later date. In studies of this type, other process variables such as temperature, concentration, stoichiometry, etc. may be held constant, and the reaction is performed with the desired range of solvents or reagents as discrete variables. Of course, the effects of the other variables can also be evaluated by extending the study. Vol. 3, No. 4, 1999 / Organic Process Research & Development

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Table 1. Examples of chemistry performed on DART system

Figure 8. Vorbruggen coupling solvent screen. Yield and purity plot. Scheme 2. Cyanomethylene deprotection

Scheme 1. Vorbruggen coupling

In the first example, the effect of solvent on the yield of product and impurity profile of the Vorbruggen coupling30 shown in Scheme 1 was investigated. Ten different solvents were selected, on the basis of the current knowledge of the chemistry, literature precedent, and diversity of properties. The reactions were carried out at 70 °C in two batches of five over 2 days. The system automatically monitored the (30) Vorbruggen, H.; Bennua, B. Chem. Ber. 1981, 114, 1279-1286. 286

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impurity profile and yield by performing ninety HPLC analyses over an 18 h reaction period at 2 h intervals. The results are presented graphically in Figure 8 using the best data point for each reaction. (note that “impurity profile” is an arbitrary scale where 0 represents gross levels of impurities and 1 represents a pure profile). Solvents in the upper right quadrant offer better yields and lower levels of impurities. Four of the 10 solvents fall in this area: acetonitrile, ethyl acetate, methyl acetate, and toluene, of which clearly ethyl acetate performs the best. This solvent was therefore chosen for subsequent scale-up of the chemistry, and its use yielded significant process advantages over acetonitrile, which was the current solvent in use, as two stages of chemistry could now be telescoped. Subsequent pilot plant campaigns using this solvent were performed successfully, affording consistent results with the small-scale experiments. The second example is a reagent-screening study to identify the optimum reagent for the cyanomethylene deprotection reaction shown in Scheme 2. In this case eight reagents were evaluated in a single day, with the generation of 64 data points (i.e., HPLC results). Figure 9 shows that only three reagents appeared in the preferred region. Although methoxylamine hydrochloride only performs marginally better than hydrazine hydrochloride (the reagent in current use for this reaction at the time) it was chosen for scale-up because of significant safety and handling advantages in the pilot plant. Both of these examples illustrate the advantage which automation confers of being able to select a wide range of variables, giving an improved chance of success in process screening studies. At the same time a large amount of experimental data was efficiently collected with minimum operator involvement.

Figure 9. Cyanomethylene deprotection reagent screen. Yield and purity plot. Scheme 3. Mitsunobu coupling

Process Optimisation Study. The third case study, a process optimisation exercise, shows the ability to combine both the efficiency of the system with the added effectiveness of DOE. In this case the objective was to investigate variability of the Mitsunobu31 coupling chemistry shown in Scheme 3, which had previously given isolated yields anywhere between 50%th and 70%th. (Note: DIAD ) Diisopropylazodicarboxylate.) An experimental plan was developed, part of which was a DOE optimisation of the Mitsunobu coupling reaction itself. The variables selected to be studied were concentration, temperature, addition rate (of DIAD), and stoichiometry (DIAD, alcohol 6). The reaction sequence involved addition of the DIAD reagent over 30-90min and HPLC analysis after 5 min and then 1 h. Therefore the reactions could only be run sequentially. A series of 20 experiments were performed over 5 days (2-level factorial design) to investigate the effect of these variables on yield and impurity profile. The early results clearly demonstrated that the coupling reaction itself was not responsible for the original variations, which were subsequently traced to the downstream process. Nevertheless, we continued with the investigation to gain a better understanding of the process and to identify the optimum reaction conditions. The results of the initial study are summarised in Figure 10 in the form of a half-normal plot. The factors having the greatest influence appear towards the right-hand side of the graph. It can be clearly seen that the equivalents of alcohol 6 and the two-factor interaction of the alcohol 6 and DIAD are the most important factors, with temperature also being significant. (31) Mitsunobu, O. Synthesis 1981, 1, 1-28.

Figure 10. Half-normal plot showing importance of factors in Mitsunobu coupling.

An optimisation design (small central composite) involving a further 16 experiments (foldover) over 4 days was then carried out to generate a predictive model of the process. This showed that the optimum conditions for high solution yield are 1.3 equiv of alcohol 6, 1.3 equiv of DIAD, and a temperature of 50 °C, giving conversions of g93%. However the purity of the isolated product was adversely affected by the presence of these reagents in such large excess. A more realistic optimum for the overall process, taking workup and isolation and purity of the product into account is nearer to an 89% conversion in solution, (achieved at 1.1 equiv of alcohol 6, 1.1 equiv of DIAD, and a temperature of 50 °C). The results also confirmed what had been suspected from previous findings, that there was a two-factor interaction between the equivalents of DIAD and alcohol 6. It had been postulated that optimum yield is obtained with a DIAD: alcohol 6 ratio of ∼1:1, which was clearly supported by the yield data observed in the 3-D surface plot of DIAD against alcohol 6 as shown in Figure 11. Conclusion An automated reaction preparation and on-line HPLC monitoring system has been developed which offers, at relatively low cost, the ability to investigate process variables on a wide range of the reactions employed by the pharmaceutical industry. It is estimated that about 80% of the chemistries encountered in this laboratory can be handled by the system (based on data gathered from an internal training course). Automation not only releases staff from the tedium of repetitive tasks but also yields improvements in reproducibility which, allied to a statistical DOE approach to experimentation, can give significant improvements in data quality and laboratory efficiency. Constraints imposed on experimental programmes by working hours are also removed. Being configured from standard, readily available modules, the system affords a high degree of module interchangeability and flexibility. Vol. 3, No. 4, 1999 / Organic Process Research & Development

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Figure 11. Three-dimensional surface plot for Mitsunobu optimisation showing highest yields at a ∼1:1 ratio of DIAD to alcohol 6.

BSA (1.09 mL) were then added automatically followed by addition of the Lewis acid (0.5 mL). The reactions were stirred at 70 °C over 18 h, and samples (10 µL) were taken at 2 h intervals for HPLC analysis. General Procedure for Cyanomethylene Deprotection. The protected piperidine 4 (0.5 g) and the appropriate amine‚ HCl were manually weighed into each reaction vessel. Ethanol (3.5 mL) followed by water (1.5 mL) was then added automatically, and the reactions were stirred at 75 °C for 16 h. Samples (20 µL) were taken every 2 h and analysed by HPLC. General Procedure for Mitsunobu Coupling. The alcohol 6 and phenol 7 were manually weighed into each reaction vessel. Toluene was then added automatically followed by addition of a solution of triphenyphosphine in toluene (350 mg/mL). DIAD was then added automatically over a period of 30-90 min. The reactions were stirred at the appropriate temperature for a period of 1 h. (The quantities of alcohol 6, phenol 7, toluene, triphenylphosphine, and DIAD and the temperature and addition period for DIAD were determined by the experimental design). Samples (20 µL) were taken for analysis directly after the addition of DIAD was complete and then again after 1 h.

Experimental Section The equipment used for automated chemistry was based on a Gilson 233XL autosampler, Gilson 402 dilutor (5 mL single or 5 and 0.5 mL dual channel), 183-mm piercing probe, custom mounting frame, STEM model RS1000 or STEM model RS1050 reacto-station and WinDART32 or SK233 control software. Analytical HPLC data was collected using either Gilson, Shimadzu, Perkin-Elmer or Waters instruments and MultiChrom software. Design Expert software was used for all DOE studies. General Procedure for Vorbruggen Coupling. Imidazole 2 (1 g) and ribofuranose 1 (1.6 g) were manually weighed into each reaction vessel. The solvent (10 mL) and

Acknowledgment The authors thank Stephen Skittrall, John MacKinnon, and Andrew Walsh for their scientific contribution. We also wish to thank Bruce Porteous for his assistance in developing the first rendition of the control software and Chris Hall and Andy Jeffrey for the software to transfer analytical data to Excel. Thanks are due to Graham Doherty, Steve Mount, Lai Wah Lai, and Mike Anson for their practical contributions to this work and Sonya Godbert for valuable statistical support. Special thanks are extended to Chris Orlopp of STEM corporation for the development of the reaction blocks and Mark Harding and Jim Stirling of Anachem Ltd and Aitken Scientific for their support in developing the SK233 software.

(32) WinDART software was developed at Glaxo Wellcome and used to control the DART system prior to the development and implementation of the SK233 software.

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Received for review March 8, 1999. OP990016D

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