On-line monitoring of continuous flow chemical synthesis using a portable, small footprint mass spectrometer

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1 n-line monitoring of continuous flow chemical synthesis using a portable, small footprint mass spectrometer Tony Bristow, 1 Andy Ray, 1 Louise Lim 2 and Anne Kearney-McMullan. 1 1 AstraZeneca, Pharmaceutical Development, Silk Road Business Park, Charter Way, Macclesfield, Cheshire, SK10 2NA. 2 University of Strathclyde, Institute of Pharmacy and Biomedical Sciences, 16 Richmond Street, Glasgow G1 1XQ. Introduction For on-line monitoring of chemical reactions (batch or continuous flow) mass spectrometry (MS) can provide data to (i) determine the fate of starting materials and reagents, (ii) confirm the presence of the desired product, (iii) identify intermediates and impurities, (iv) determine steady state conditions and (v) speed up process optimisation. Recent developments in portable mass spectrometers further enable this coupling as the MS system can be positioned with any reaction system to be studied. A major issue for this combination is the transfer of a sample that is representative of the reaction and also compatible with the mass spectrometer. This is particularly challenging as high concentrations of reagents and products can be encountered in organic synthesis. The application of a portable mass spectrometer for on-line characterisation of flow chemical synthesis has been evaluated by coupling a Microsaic 4000 MiD to the Future Chemistry Flow Start EV chemistry system. The consumption of the amide staring material as a function of reaction temperature is shown in Figure 4, which illustrates that steady state reaction conditions were established. Figure 4: Plots of the abundance of m/z 325 (amide starting material ion) as a function of reaction temperature and using different make-up flow compositions (1000:1 sample dilution). Figure 1: Photograph showing the combination of the Microsaic 4000 MiD coupled to the Flow Start EV and a schematic diagram of the experimental set-up. (ii) n-line monitoring of the formation of the amine product. The formation of the amine product following an in-line HCl reaction quench was also monitored on-line by MS. Figure 5 shows two representative mass spectra which contain both product and impurity ions. Experimental The potential of this approach was investigated using the Hofmann rearrangement shown in Figure 2. 40ºC H2 HCl -C2 60ºC Figure 2: A generic representation of the Hofmann Rearrangement. A number of flow chemistry and MS experimental parameters were varied and their effect monitored on-line using the 4000 MiD MS. Flow Reaction conditions: 20ºC, 30ºC, 40ºC, 50ºC, 60ºC, 70ºC and 80ºC (data acquisition time for each temperature was on average 6 minutes). The reaction was studied with and without the HCl quench. Reaction flow rates: Reagent 1 = 49.9 µl/min and Reagent 2 =50.1 µl/min. Figure 5: Mass spectra from on-line monitoring of the reaction at temperatures of 40ºC and 60 ºC, recorded using a make-up flow of H 2 :MeCN (80:20) 0.1% HCH Figure 6 shows the formation of the amine product (m/z 297) and consumption of the amide starting material (m/z 325) as a function of reaction temperature. MS Conditions: Composition of the make-up flow (1 ml/min): (i) MeCN: H 2 (50: 50) 0.1% formic acid. (ii) H 2 : MeCN (80: 20 ) 0.1% formic acid. (iii) MeCN: H 2 (50: 50) 0.05% TFA. Sample dilution factors: 1000:1, 500:1, 250:1, 100:1. Results and discussion (i) n-line monitoring of the formation of the isocyanate intermediate. Isocyanate ion Amide starting material ion Figure 6: Plots of the ion abundance of m/z 325, m/z 323 and m/z 297 as a function of reaction temperature using a make-up low of H 2 :MeCN (80:20) 0.1% HCH. Figure 3: Mass spectra from on-line monitoring of the formation of the isocyanate intermediate (m/z 323) at a reaction temperature of 20ºC and recorded using a make-up flow of MeCN: H 2 (50:50) 0.1% HCH. Conclusion The Microsaic 4000 MiD MS has been successfully coupled to the Flow Start EV chemistry system for on-line monitoring of the Hofmann Rearrangement reaction. The data was used to identify the optimum reaction temperature and the impurities formed in real time. Acknowledgements ur sincere thanks go to Bryan McCullough and Alessio Zammataro at Microsaic for their excellent support and advice throughout this project.

2 Multivariate analysis of a nucleophilic aromatic substitution by real-time on-line flow reaction monitoring using a miniaturized mass spectrometer INTRDUCTIN The Microsaic 4000 MiD is a miniaturised single quadrupole mass spectrometer (MS) designed with the chemist in mind. By utilising micro-electro-mechanical systems (MEMS) technology we have been able to miniaturise the key components of a MS which has allowed us to produce the smallest mass spectrometer on the market. The unit has been designed with the vacuum system, electronics and computer all inside the one box. This means the instrument can be installed in places where no other mass spectrometer can be normally deployed. For instance, the product can be put into a fume hood next to a flow reactor where it is needed. The system is portable with modular plug and play components for maximum application flexibility. For on-line monitoring of reactions in flow, MS can be used to analyse in real-time the chemical composition of the flow stream. This specific work-flow provides data to monitor starting materials, identify the presence of reactive transients and impurities, determine steady state conditions and optimize reaction yield. The use of automated flow systems combined with on-line MS analysis enables rapid screening and efficient optimisation for process development is fully scalable from laboratory, pilot plant to manufacturing plant. This produces a massive saving in the time and materials required if compared to common approaches. Specifically, we investigated the optimization of the nucleophilic aromatic substitution of 2,4-difluoro-nitrobenzene with morpholine over a wide variety of stochiometric ratios, residence times and temperatures to demonstrate all the benefits of using on-line flow reaction monitoring. Flow reactor Figure 1. Schematic representation of Microsaic 4000 MiD coupled via MiDas TM (interface module) to CDR Polar Bear Plus flow chemistry system. Scan mode Full scan Mass Range m/z Scan Rate 1 scan/sec Step size m/z 0.2 Ion polarity Tip voltage Gas flow Vacuum interface Tube lens Plate lens Ion guide Positive (ESI) 850 V 2500 ml/min 50 V 10 V MiDas TM 4000 MiD Table 1. Microsaic 4000 MiD settings for on-line flow reaction monitoring. 5 V 1 V The reaction of 2,4-difluoro-nitrobenzene and morpholine was carried out in ethanol and triethylamine (Scheme 1) over a wide range of stochiometric ratios, residence times and temperatures using the instrument set up reported in Table 2. EXPERIMENTAL Et3N The continuous flow optimization of the nucleophilic aromatic substitution has been carried out by coupling the Microsaic 4000 MiD mass spectrometer to the CDR Polar Bear Plus flow chemistry system through an interface module (Figure 1). EtH MiDas TM The time taken to install the Microsaic 4000 MiD and connect to the flow reactor to acquire meaningful data was less than 1 hour. Using a similar set up with a conventional MS system would take anything from 2 to 5 days. EtH 3 ml 4000 MiD The continuous flow optimization of the nucleophilic aromatic substitution was monitored by the MS using the settings reported in Table 1. Scheme 1. Flow route for the nucleophilic aromatic substitution.

3 Reactor PFA coiled tube Reactor size 3 ml Pressurization module 1 bar 2,4-difluoro-nitrobenzene (3.04 M) in ethanol Reagent A (11.44 M) Reagent B Morpholine (4.17 M) in triethylamine (4.58 M) Reagent C Ethanol Total flow rate (pump ABC) Ramped from 1 to 6 ml/min over 20 min Dynamically adjusted to achieve desired reagent Pump A,B and C flow rates ratios and residence times Residence time Ramped from 0.5 to 3 min over 20 min Stochiometric ratios A:B 1:1, 1:1.5, 1:2 and 1:3 Reactor temperatures 60, 80, 100 and 120 C Make-up pump solvent Methanol, 0.1% formic acid Make-up pump flow rate 1 ml/min Split ratio 3000:1 Table 2. Flow reactor-ms set up for the chemical transformation of 2,4-difluoronitrobenzene with morpholine. RESULTS AND DISCUSSIN The reaction expected is shown in the Figure 2, where 2,4- difluoro-nitrobenzene (1) reacts with the nucleophile morpholine (2) to afford substitution products (3,4 and 5) by an additionelimination mechanism. The reaction is conducted in triethylamine to neutralize the hydrofluoric acid generated as side product by forming triethylamide hydrofluoride (6). Figure 4 shows the mass spectrum of both reagents and products ions at a reaction temperature of 80 C. [(2)H] [Et3NH] [(3)&(4)H] [(5)H] [(3)&(4)Na] [(5)Na] Figure 4. Mass spectrum of protonated morpholine (2) at m/z 88.2, triethylamine at m/z 102.2, 4-fluoro-2-morpholino-nitrobenzene (3) and 2-fluoro-4-morpholino -nitrobenzene (4) at m/z 227.4, 2,4 dimorpholino-nitrobenzene (5) at m/z monitoring the reaction at 80 C. Typical sodium adducts of reaction products are also shown. To optimize the studied chemical transformation, the effect of the various reaction parameters was evaluated plotting the EICs of 2,4 dimorpholino-nitrobenzene (m/z 294.4) against residence times, stochiometric ratios and temperatures in a waterfall 3D graph. Figure 5 shows the effect on the product intensity and reaction yield of increasing the residence time, stochiometric ratio and reactor temperature. The completion of the whole set of tests took 16 hrs, a fraction of the time and chemicals needed if conventional off-line analysis was used. Figure 2. Reaction scheme for nucleophilic aromatic substitution of 2,4 difluoronitrobenzene with morpholine. The desired flow experimental conditions were inputted into a custom control program. The system used was solvent-free which means that the reagents stochiometric ratios and the residence times of the different components vary as the algorithm changes the flow rates of the reactants. The multivariate analysis of the reaction was carried out adopting a 4 x 4 grid of different 2,4-difluoronitrobenzene:morpholine stochiometric ratios (1:1, 1:1.5, 1:2, 1:3) and reactor temperatures (60, 80, 100, 120 C) ramping the total reagents residence time from 0.5 to 3 min over 20 min. This approach resulted in approximately 15,000 conditions monitored in real-time by analysing the reactor mixture on-line using the Microsaic 4000 MiD mass spectrometer. Figure 3 shows the extracted ion chromatogram (EIC) of a reaction product monitored in real-time under different stochiometric ratios and residence times at a specific reactor Figure 3. EIC of 2,4 dimorpholino-nitrobenzene (m/z 294.4) monitored ramping the residence time from 0.5 to 3 min in 20 min for stochiometric ratios 2,4- difluoro-nitrobenzene:morpholine 1:1, 1:1.5, 1:2 and 1:3 at 80 C. Figure 5. Waterfall 3D graph for the extracted ion chromatograms of 2,4 dimorpholino-nitrobenzene at m/z as a function of the residence time, stochiometric ratio and reaction temperature. CNCLUSINS A nucleophilic aromatic substitution was studied using a multivariate analysis approach by means of an automated continuous flow reactor with real-time on-line MS analysis. Using the Microsaic 4000 MiD mass spectrometer for on-line reaction monitoring enabled yield optimization evaluating a wide range of reaction conditions, resulting in a reduction of materials and time required compared to routine off-line approaches. With the results presented in this application note, Microsaic Systems has successfully deployed its miniaturised mass spectrometer in a multivariate analysis for rapid and low cost reaction yield optimization by using on-line flow reaction monitoring.

4 Automated reaction monitoring by direct analysis mass spectrometry using the 4000 MiD and MiDas TM Microsaic Systems, Woking, UK bjective Monitor and compare two Boc-deprotection reactions using Microsaic Systems unique mass detection and sampling platform, consisting of the MiDas TM, 4000 MiD and stand-alone on-board Masscape software. H 3C H Acid Solvent Background The Boc group can be added to amines to give N- tert-butoxycarbonyl or so-called t-bc derivatives. These derivatives do not behave as amines, which allows certain subsequent transformations to occur that would have otherwise affected the amine functional group. Removal of the t-bc amino acid can be accomplished with strong acids such as trifluoroacetic acid neat or in dichloromethane, or with HCl in methanol. MiDas - MiD fully integrated sampling, dilution, analysis and data manipulation The MiDas - MiD offers automated monitoring of a chemical process with real-time analysis of data, all possible with the use of the on-board Masscape software. With a small footprint and quick pump down time, the MiD is able to be deployed to the reactor, employing whichever modular arrangement is required for the chemistry and workspace. H CH2 H 3 C Figure 1. General scheme for the deprotection of N α -Boc-Lysine Sampling is achieved using a fully customisable dual syringe pump with multi-port valves, and controlled by Masscape. Integrated into operational methods are user defined values to allow for optimisation where a non-standard tube kit is used. Sampling volume, frequency and dilution parameters are customisable by the user to minimise sample consumption or sample transfer time. Reaction monitoring by Direct Analysis MS The 4000 MiD TM operates with generic, soft ionisation conditions that result in simple to interpret mass spectra, where pseudomolecular ions of known and unexpected species are easily determinable. Rapid direct analysis also allows for the profiling of reactions involving transient species that are not observed using conventional off-line techniques. In addition, from point of sampling to ionisation source the system is closed to outside atmosphere, and make-up solvents can be selected to preserve compounds of interest. Figure 2. Schematic representation of 4000 MiD coupled to a reaction vessel via the MiDas TM and dual syringe pump

5 Figure 3. Mass spectra of HCl reaction using the waterfall function to display as 3D stack Experimental A 25mg/mL solution of N α -Boc-Lysine was prepared in 1:1 Methanol : Acid (neat Trifluoroacetic acid or 25% HCl in water) Additional m/z values can be added to the profile dynamically as they appear. The reaction proceeds cleanly using TFA, however several additional species were detected where HCl was used. [MH] (2) [MH] (1) [MH] (3) [MH] (4) Table MiD settings Scan mode Full scan Mass range m/z Scan rate 1 scan/sec Step size m/z 0.2 Ion polarity Tip voltage Gas flow Vacuum interface Table 2. MiDas TM settings Purge & transfer solvent Positive (ESI) 850 V 2500 ml/min 30V Methanol Make-up solvent 50:50 methanol:water (0.1% formic acid) Make-up flow rate Sample volume 20µl 1mL/min Split ratio 1000:1 Sample interval 15 mins (TFA), 8.5 mins (HCl) Results and Discussion Masscape was used to analyse reaction profiles and changes in mass spectra over time: Figure 4. Additional species identified from mass spectra, available at any time during analysis The reaction profile indicates quantitative conversion of N α -Boc-Lysine to lysine in under 100mins where TFA is used. Where HCl is used the profile shows rapid consumption of m/z 246 (1) and generation of m/z 260 (3) and m/z 146 (2). This is followed by consumption of (3) and (2) and steady generation of (4), trends which are clearly visible in the 3D graph generated in Masscape (figure 3). The proposed scheme is shown below. H (1) MeH HCl (aq.) Figure 7. Reaction scheme for de-protection with HCl H H 2 N (2) (3) MeH H H (4) Summary Stand-alone automated sampling, dilution and direct analysis using the MiDas TM and 4000 MiD reliably monitored starting material and several products. Figure 3. Selected EIC reaction profile of TFA reaction (TP) and HCl reaction (BTTM) End of reaction determined for TFA deprotection. Profile of products fits with proposed chemistry.