Segmented Tube Reactors (STR): a Simple Tool to Screen Multiple

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Segmented Tube Reactors (STR): a Simple Tool to Screen Multiple Reactions in Parallel in Batch Mode Within a Single Tube Franz J Weiberth, Matthew R Powers, Connor Gallin, and David McDonald Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00009 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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Organic Process Research & Development

Segmented Tube Reactors (STR): a Simple Tool to Screen Multiple Reactions in Parallel in Batch Mode Within a Single Tube Franz J. Weiberth,*,# Matthew R. Powers,# Connor Gallin+ and David McDonald+ # +

Sanofi US R&D, Synthesis Development, 153 Second Ave., Waltham, MA 02451, USA Sanofi US R&D, Synthesis Development, Northeastern University co-op student, 153 Second Ave., Waltham, MA 02451, USA

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Graphical Abstract:

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Abstract A simple tool to perform multiple reactions in parallel in batch mode in static segments within a single tube is described. The Segmented Tube Reactor (STR) involves using syringe pumps to load a section of teflon tubing with solutions of reaction components while forming a preprogramed gradient of one of the components (e.g. equivalents, concentration) along its length. Simultaneously, a chemically inert spacer is loaded to break the gradient into discrete static segments. Ten or more distinct sets of conditions can be quickly set up in a single experiment. Although the STR is set up in flow mode, reactions occur in batch mode within the tubes, usually by heating the STR for a desired duration. Segments are then individually sampled and analyzed to identify the conditions that provide the best performance. The technology is translatable: STR was employed to screen for preferred reaction stoichiometries that were then duplicated on larger scale in batch mode in traditional lab equipment. Key words: STR, Segmented Tube Reactor, screening, optimization

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Introduction A shift in technology from batch to continuous mode in the pharmaceutical industry for the manufacture of drug products and drug substances has become evident.1 For example, FDA has approved formulated drugs prepared using continuous manufacturing and an existing drug product where the production method changed from batch to continuous manufacturing.2 In addition, a team at Lilly has used continuous flow technology in a cGMP3 environment in a multi-step synthesis of a drug substance intended to be used in clinical trials in humans.4 This evolution is being driven by the benefits intrinsic to continuous processing, including improved mixing and heat transfer, shorter reaction times, enhanced real-time analysis and process control, processing of highly reactive intermediates, ease of developability and scalability, improved safety due to minimization of reaction space, and minimization of waste to name just a few.5 Despite these advantages, the shift in chemical processing is expected to be gradual due to a variety of challenges. For example, although some companies have built state-of-the-art facilities for continuous manufacturing, the vast majority of current global pharma manufacturing capacity and know-how remains batch mode. Sustained commitments and investments in lab, pilot plant and manufacturing facilities for continuous processing will be challenging to execute in the short term in a bottom-line driven and perhaps still contracting pharma industry despite the economic advantages offered in the longer term. Also, companies will need to invest in their work forces and train large numbers of chemists, engineers and regulatory folks to grow the expertise required to successfully navigate the progression from discovery to the market for new drugs intended to be manufactured in a continuous fashion. Further, not all chemical transformations are amenable to flow and some are preferred to be performed in batch mode, perhaps up to 50%6 including some homogeneous reactions and those that require long reaction times.7 Thus, in the context that organic syntheses performed in batch mode will continue to be pertinent in the pharmaceutical industry, we set out to develop a tool that would leverage aspects of flow technology but with intended direct application to batch chemistry. Specifically, we envisioned performing multiple reactions in segments in batch mode within a single tube as a tool to screen reactions that are intended to be performed batch-wise on larger scale. The Segmented Tube Reactor (STR; Fig. 1) tool involves generating a series of reaction slugs by pumping solutions of reaction components via multiple syringes into a length of a teflon tube while using a chemically inert spacer (liquid or gas phase) to keep slugs discreet. A step gradient of one component is generated across the segments via a pre-programmed pumping method so that each slug is comprised of a unique combination of components. Once the gradient is completed, the pumping is stopped. Each static segment is considered a distinct reactor separated by an inert spacer phase. The tube containing the set of static segments is subjected to desired batch-mode conditions (e.g., held 18 h at 75 oC), and then is cooled, sampled and analyzed to identify segments that provide the best reaction performance. These preferred micro-meso scale

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conditions are then scaled using traditional batch lab reactors to verify performance on macro scale before further scale up.

Figure 1. Diagram and picture of STR

The STR concept adapts existing technologies,8 but reactions are intended to occur in parallel in a tube in batch mode and thus differs from most other segmented-flow technologies that typically screen reactions9 or generate compound libraries10 in flow mode. In other words, the STR is set up in flow mode, but reactions are performed in batch mode and have long residence times.11 Further, the STR tool is being developed with the advantage of being simple in design and comprised of equipment routinely found in organic synthesis laboratories to enable it to be more broadly utilized by organic chemists who may not have access to more sophisticated and expensive screening instruments and techniques.12 The STR requires minimal time and materials to set up: preparation of two solutions using small amounts of reactants to perform reactions in 0.3 mL segments. Basic synthetic transformations were utilized in this study with first focus being on developing the technique rather than chemistry; namely, demonstrating reliable formation of a gradient of a reactant component using syringe pumps in segments along a length of tubing, performing parallel reactions in batch mode in the STR, then sampling post- reaction to determine performance in each segment. Results and Discussion Selection of spacer. Perfluorodecaline (PFD) was initially explored as a spacer for the STR system. PFD is commonly used as a spacer in segmented-flow applications because it is chemically inert, has a relatively high boiling point (142 oC) and it favors the formation of distinct liquid phases due to its apparent immiscibility in many organic solvents and its high density (1.9 g/mL). In initial experiments, the STR was placed on a flat surface and the coils were positioned horizontally. Upon simultaneously dispensing reagent solutions and PFD into teflon tubing, mini slugs were formed and were stable while in motion, but would merge into a single lower PFD phase and a single upper phase of reaction components across the STR after a few minutes while static. In contrast, when the coils were positioned vertically, by wrapping tubing around a small section of a rod, mini segments formed and persisted as they were in motion as the coils were being filled (Fig. 2a), and then, after the STR became static, merged and Page 5 of 18



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separated into more distinct and uniformly sized segments (Fig. 2b), one upper reaction phase and one lower PFD phase per loop segment.

Figure 2. Mini segments: a. In motion. b. Converged, one reaction upper phase per loop

Although we were able to form gradients of components in distinct segments and perform reactions, several limitations using PFD as the spacer were observed: • For reaction solutions with density greater than 1, several minutes or longer were required for complete separation of the spacer from the reaction phase into distinct segments. • The high propensity for PFD to dissolve gases13 negatively impacted performance. Gas bubbles evolved in the tubing during heating that caused fracturing and movement of segments. Attempts to eliminate this behavior by degassing PFD by sonication or by distillation just prior to use were unsuccessful. • Although PFD is mainly chemically inert and highly immiscible, it still affected the concentration of reagents in the reaction phases. When calibration experiments were performed to confirm the reliability of concentration gradients that were generated using the STR system, the absolute concentrations in segments tended to be lower compared to independently prepared solutions with known concentrations due to some solubilization of components into the PFD phase. Similarly, even minor solubilization of PFD into the reaction phase could impact the desired reaction and thus affect translation in performance to the intended larger scale in batch mode. As a result of these limitations, focus shifted to developing nitrogen as a more preferred spacer rather than PFD. Nitrogen as Spacer. The STR tool with N2 as spacer was developed as follows: •

• • •

A programmable, dual syringe pump equipped with 1 mL to 5 mL syringes loaded with solutions of reactants, together with a third syringe pump that dispensed nitrogen, provides accurate and reproducible performance in loading a concentration gradient across segments in the STR. Mini segments are formed along the length of the STR during loading that can be collapsed to one reaction segment per loop. A loop diameter of 1.5-in per segment is effective (3/16-in teflon tubing coiled around a 1.66-in diameter14 core rod or tube). In theory, the STR can be set up to have as many segments as desired. In practice, a 5-10 step gradient corresponding to 5-10 loops is normally sufficient for screening a range of conditions. Page 6 of 18


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•

•

A 148-cm length of 3/16-in tubing is needed for a 10-loop STR and provides a total internal volume of 11.7 mL15 (1.17 mL per loop). With a total fill volume of 3.0 mL (10 segments, each containing 0.3 mL reaction solutions, ca. 25% filling), the balance volume of 8.7 mL is filled with nitrogen dispensed simultaneously during the loading of the reaction solutions. The ratio of fill space can be adjusted; lower if supplies of reactants are limiting. A change in number of loops, loop diameter, tubing i.d. and fill volume will change the volume parameters and must to accounted for in the experimental design and dispensing program.

STR setup and operation. The complete STR assembly (Figure 1) is set up in several distinct steps, namely, wrapping tubing around a core pipe, connecting the STR to syringe pumps, loading the STR with a pre-programmed gradient of reactants, performing the intended batch reaction, and then sampling and analyzing the segments to determine performance, as follows: A 10-segment STR is prepared by snuggly wrapping a 148-cm length of 3/16-in diameter FEP tubing by hand around a section of pipe 1.25-in in diameter.14 The wrapping is facilitated by using plastic zip ties that are looped lengthwise through the center of the pipe prior to coiling, initially loosely so that the tubing can be wrapped under the ties, then snugged up firmly to hold the fully coiled STR in place, especially the end sections (Fig. 3a).16 The STR is then clamped in place vertically17 in preparation for loading.18 The charging end (bottom) is connected to three syringe pumps (two filled with reactants, one filled with nitrogen) manifolded through a cross coupling using shortest possible lengths of 0.04-in i.d. teflon tubing. The discharge end (top) is connected via a coupling to a short length of tubing that drains to a waste vessel that collects any pre-rinses or any overflow (Fig. 1). The STR is then loaded using a dual syringe pump that dispenses solutions of the two reactants simultaneously using a preprogrammed ramping sequence of steps19 performed without interruption that provides a desired gradient of concentrations across the segments. Simultaneously, nitrogen is dispensed at a predetermined rate. Upon completion of the loading operation, the pumps are stopped automatically as programmed for the dual pumps and manually in the case of the nitrogen syringe20 to afford a series of mini-segments broken up by nitrogen gas, mainly uniformly sized and distributed across the loops of the STR (Fig. 3b). Carefully, to minimize movement of the mini-segments, the STR is positioned horizontally with its fittings facing up.21 The ends of the STR tubing are disconnected from the assembly and then coupled together using a ca. 9-in section of 1/8-in diameter teflon tubing while ensuring air-tightness by hand-tightening the ferruled connectors. Thus, reactions performed in the STR are performed in a closed system to minimize the movement of segments during heating and cooling cycles. While still horizontal and with the two end connections still facing directly up, the STR is firmly tapped directly downwards on a benchtop several times until the mini-segments are completely collapsed into more discreet segments due to gravity and the tapping motion, one segment per loop (Fig. 3c).22,23 The STR is then clamped and immersed in an oil bath (Fig 3d). After heating for a desired duration, the SRT is removed from the bath and allowed to cool. The STR is vented by Page 7 of 18



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loosening an end connector slowly and with care to minimize excessive movement and avoid possible fragmentation of segments due to an abrupt equilibration.24 In preparation for sampling, one end of the STR is plugged. The STR is clamped in a vertical position with the plugged end at the bottom (Fig. 3e). Each segment is sampled starting from the top vented segment using a syringe and piercing the tubing with a 22-gauge needle. The aliquots are then analyzed to determine the performance for the set of conditions across the gradient.

Figure 3. a. FEP Tubing wrapped around a core pipe in ten coils. b. Loaded STR, mini-segments. c. One distinct segment per loop after tapping (done while horizontal, but shown vertically). d. STR ends coupled, clamped in an oil bath. e. After reaction, slight shifting of segments but still distinct, ready for sampling

Verifying performance in forming gradients reproducibly in a STR. The accuracy and reproducibility of generating gradients across segments in the STR tool were assessed using 4-tbutylphenol (1) and mesitylene (2) as standards. These standards each possess a unique set of nine equivalent methyl protons, sharp singlets at 1.23 ppm and 2.22 ppm, respectively, that facilitated using 1H NMR to accurately determine relative concentrations in segments generated using STR technology. For example, 5-mL syringes of the dual syringe pump were loaded with 1.0 M solutions of 1 and 2 in DMF accurately weighed and prepared in volumetric flasks. A predetermined gradient spanning 1.1 to 2.0 equivalents of 1 relative 2 was generated by simultaneously dispensing the solutions in 0.1 eq increments in 10 pre-programmed steps without interruption (Fig. 4). Each step had the same fill time and total volume but different ratio of components whereby an incremental increase in volume from one syringe was synchronized with a proportional decrease in volume from the second syringe B. In the meantime, the nitrogen spacer was dispensed from a third syringe pump at a rate of 3.27 mL/min.25 Step (loop) 1 2 3 4 5 6 7 8 9 10 Totals

time (min/step) 0.267 0.267 0.267 0.267 0.267 0.267 0.267 0.267 0.267 0.267 2.667 min

Equiv 1 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00

1 M t-butylphenol (1) Volume Rate (mL) (mL/min) 0.1571 0.589 0.1636 0.614 0.1696 0.636 0.1750 0.656 0.1800 0.675 0.1846 0.692 0.1889 0.708 0.1929 0.723 0.1966 0.737 0.2000 0.750 1.8083

1 M mesitylene (2) Volume Rate (mL) (mL/min 0.1429 0.536 0.1364 0.511 0.1304 0.489 0.1250 0.469 0.1200 0.450 0.1154 0.433 0.1111 0.417 0.1071 0.402 0.1034 0.388 0.1000 0.375 1.1917

Total V (mL) 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 3.000

Figure 4. Gradient program: 1.1 to 2.0 equivalents of 1 Page 8 of 18


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Upon completion of the gradient program and collapsing of mini-segments into discreet segments following the procedures described earlier, each of the ten segments was sampled and analyzed by 1H NMR spectroscopy. Plots of actual versus theory of equivalents of 1 demonstrated highly linear correlations in standardization experiments that evaluated two separate gradient programs of 1, namely, 1.1-2.0 equiv (r2 = 0.997) and 0.6-1.5 equiv (r2 = 0.998), as shown in Figs. 5 and 6. In addition, good agreement with the theoretical lines was achieved for both gradients with average deviation of 95% when >4.0 eq of morpholine were employed.

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Scheme 1. Screening the conversion of 3 to 5

Step (coil) 1 2 3 4 5 6 7 8 9 10 Totals

time (min/step) 0.424 0.424 0.424 0.424 0.424 0.424 0.424 0.424 0.424 0.424 4.235

Equiv 4 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

2.0 M morpholine 4 Volume Rate (mL) (mL/min) 0.075 0.177 0.100 0.236 0.120 0.283 0.136 0.322 0.150 0.354 0.162 0.381 0.171 0.405 0.180 0.425 0.188 0.443 0.194 0.458 1.4759

0.67 M pyridine 3 Volume Rate (mL) (mL/min) 0.225 0.531 0.200 0.472 0.180 0.425 0.164 0.386 0.150 0.354 0.138 0.327 0.129 0.304 0.120 0.283 0.113 0.266 0.106 0.250 1.5241

Total V (mL) 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 3.0000

Figure 7. Gradient program of 3 and 4

100

A% Product 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


90 80 70 60 50 1.0 1.5

2.0 2.5 3.0

3.5 4.0 4.5

5.0 5.5

Equivalents of 4 Figure 8. A% conversion to 5

As an indication of the utility of STR technology to screen a range of conditions and predict performance in batch mode, segment 430 of this STR screening was scaled in conventional lab equipment. Thus, using 2.5 equiv of 4 (13.7 mmol) at 100 oC for 14 h in a batch-mode reaction performed in a 25-mL flask, a 83.7 A% conversion to 5 by HPLC assay was observed which agreed well with the 86.4% conversion observed in segment 4 of the STR gradient screen.

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Screening an amidation. In another application, the amidation of ester 6 by piperidine (7) catalyzed by varying amounts of TBD (8) to afford amide 9 was screened (Scheme 2). A gradient of 0.075-0.300 eq of TBD across ten STR segments was generated (Fig. 9) employing a solution prepared from 7.5 mmol (0.75 M) of 6 and 9.3 mmol (0.93 M, 1.25 eq) of piperidine in THF in one syringe and 5 mmol (0.50 M) of TBD in THF in a second syringe. In the meantime, the nitrogen spacer was dispensed from a syringe at a rate of 2.87 mL/min over the 3.034 min filling time. The loaded STR was then immersed in an oil bath, heated at 60 oC for 21 h and then cooled to 25 oC. Each segment was sampled and analyzed by HPLC. The results (Fig. 10) show maximum conversion was achieved using about 0.25 eq of TBD, consistent with literature reports.31 Scheme 2. Screening conversion of 6 to 9

Step (coil) 1 2 3 4 5 6 7 8 9 10 Totals

time (min/step) 0.303 0.303 0.303 0.303 0.303 0.303 0.303 0.303 0.303 0.303 3.034

Equiv 8 0.075 0.100 0.125 0.150 0.175 0.200 0.225 0.250 0.275 0.300

0.50 M TBD (8) Volume Rate (mL) (mL/min) 0.030 0.100 0.039 0.129 0.047 0.156 0.055 0.182 0.062 0.206 0.069 0.228 0.076 0.250 0.082 0.270 0.088 0.289 0.093 0.307 0.6418

0.75 M ester 6 Volume Rate (mL) (mL/min) 0.270 0.889 0.261 0.860 0.253 0.833 0.245 0.807 0.238 0.783 0.231 0.761 0.224 0.739 0.218 0.719 0.212 0.700 0.207 0.682 2.3582

Total V (mL) 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 3.0000


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100 95 90

A% Product 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


85 80 75 70

Normalized conversion to 9

65 Conversion to 9 60 0.075 0.1 0.125 0.15 0.175 0.2 0.225 0.25 0.275 0.3


Segment 7 of the STR conditions was scaled in conventional lab equipment to compare performance. Thus, using 0.23 equiv of TBD with 6 (1.05 mmol) and 7 in THF and heating at 60 °C for 21 h in a batch-mode reaction performed in a stirred 5-mL vial afforded 87.2 A% of product 9, 3.5 A% of unreacted 6 and 9.3A% of the hydrolysis by-product 4-bromobenzoic acid. This result compared favorably with 83.9 A% of 9, 3.2 A% of 6 and 12.9 A% of 4-bromobenzoic acid obtained from segment 7 of the STR gradient screening. Screening a reaction and temperature. In a related pair of STR experiments, both the equivalents of a reactant and temperature of reaction were screened. 3,6-Dichloropyridazine (8) was reacted with 1.00 to 1.90 eq of 1-boc-piperazine 9 in 10 segments each (0.10 eq increments, Fig. 11) in two STRs, one reacted at 80 oC and the other at 100 oC, to afford 12 (Scheme 3).32 The reactions were held at temperature for 8 h. After cooling, each segment was analyzed (Fig. 12). At 80 oC, only a 91 A% conversion to 12 was achieved when using 1.9 equiv of 11. In contrast, >95% conversion to 12 was achieved when >1.3 eq of morpholine were employed at 100 oC. Segment 6 on the 80 oC curve was scaled in conventional lab equipment to compare performance of this transformation in STR mode versus batch mode. Thus, using 1.5 equiv of 11 (10 mmol) at 80 oC for 8 h in a 25-mL flask afforded 85.5 A% of product 12 and 14.5 A% of unreacted 10 which compared favorably with 84.6 A% of 12 and 15.4 A% unreacted 10 obtained from the STR gradient screening.

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Scheme 3. Optimizing conversion 10 to 12

Step (coil) 1 2 3 4 5 6 7 8 9 10 Totals

time (min/step) 0.468 0.468 0.468 0.468 0.468 0.468 0.468 0.468 0.468 0.468 4.683

2.0 M piperidine 11 Volume Rate (mL) (mL/min) 0.075 0.160 0.080 0.172 0.086 0.183 0.091 0.194 0.095 0.204 0.100 0.213 0.104 0.223 0.108 0.232 0.112 0.240 0.116 0.248 0.9683

Equiv 11 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90

0.67 M pyridazine 10 Volume Rate (mL) (mL/min 0.225 0.481 0.220 0.469 0.214 0.458 0.209 0.447 0.205 0.437 0.200 0.427 0.196 0.418 0.192 0.409 0.188 0.401 0.184 0.392 2.0317

Total V (mL) 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 0.300 3.0000


100 95 90

A% Product 12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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85 80 75 70

A% prod 100 °C

65 A% prod 80 °C

60 1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9


Fig. 12 illustrates the utility of the STR technology as a screening tool: one set of reactants was used to set up two separate STR systems to evaluate performance of 10-segment gradients at two Page 14 of 18


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temperatures. Twenty data points were obtained using minimal resources, such as, manpower (only 2 solutions were prepared to set up the STR reactions), materials (gram quantities in 5-mL or 10 ml volumetric flasks) and common and inexpensive lab equipment (syringe pumps, teflon tubing, and oil baths). Although sampling and analysis could be laborious and time-consuming, specific data points could be cherry-picked to expedite the data analysis from a screening. For example, by first analyzing a segment in the middle of a gradient, subsequent analyses can be cherry picked to zero in on the segment with the best performance. Other data points could then be assayed to provide a more complete picture of performance, such as tracking the formation of impurities over the gradient. The reaction can then be further optimized by performing a second STR reaction under a tighter range of conditions. Conclusions The feasibility of using Segmented Tube Reactors (STR) to perform multiple reactions in parallel in segments separated by nitrogen within a single tube while static has been described. Nitrogen is shown to be a preferred spacer compared to PFD because of undesired solubility of reaction components in PFD. The technique involves forming a pre-programmed gradient of one of the components (e.g. equivalents, concentration) along the length of the tubing. The reactions are performed in batch mode rather than flow mode. The STR technique has advantages including a simple design employing programmable syringe pumps to enable it to be more broadly utilized by chemists who may not have access to more sophisticated and expensive screening instruments. In addition, the STR requires minimal time and materials to set up. In one example, a total of twenty batch-reaction data points were generated from just two STRs using minimal manpower (2 solutions prepared from < 2 g substrates). The technology is translatable: STR was employed to screen for preferred reaction stoichiometries that were then duplicated in batch mode in traditional lab equipment. Basic synthetic transformations were utilized in this study with the first iteration focused on developing the technique rather than chemistry. Experimental 1

H and 13C NMR data were recorded using either a Bruker 400 MHz Avance III or Bruker 600 MHz Avance III HD. HRMS data were collected using an Agilent 1200 HPLC coupled to an Agilent 6520 Accurate Mass Q-TOF MS. J-Kem Scientific dual syringe pump Model SYR 2400 with software version 5.09 MP was employed to generate the STR gradients. Other components of the STR system and further operating details are described in the Supporting Information Section. Preparation of 4-(3,5-dichloropyridin-4-yl)morpholine (5).28 A 25-mL, 3-necked round-bottomed flask equipped with magnetic stirring, static nitrogen, a thermocouple probe, a condenser and a heating mantle was charged with 3,4,5-trichloropyridine (1.00 g, 5.48 mmol, 1.00 eq.), morpholine (1.19 mL, 13.70 mmol, 2.50 eq.) and NMP (10 mL). Page 15 of 18



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After stirring for a few moments until all solids had dissolved, DIPEA (1.43 mL, 8.22 mmol, 1.50 eq.) was added and the solution was heated. After 14 h at 100 °C, an HPLC analysis of an aliquot indicated 83.7 A% of product 5 and 16.3 A% of unreacted 3 which compared favorably with 86.4 A% of 5 and 13.6 A% unreacted 3 obtained from a STR gradient screening (Fig. 14, segment 4, 2.5 eq of 4). The reaction was cooled, diluted with water (25 mL) and extracted with TBME (3 x 15 mL). The combined organic phase was washed with water (3 x 20 mL), dried over magnesium sulfate and evaporated to an oil which was crystallized by adding heptane to afford 1.03 g (82% yield) of 5 as a light-orange solid. 1H NMR (CDCl3, 101 MHz): δ = 8.35 (2H, s), 3.84 (4H, m), 3.37 (4H, m); 13C NMR (CDCl3, 400 MHz): δ = 150.8, 149.3, 128.4, 67.4, 50.4; HRMS–ESI+: m/z [M + H]+ calcd for C9H11Cl2N2O+: 233.02429; found: 233.02446. Preparation of (4-bromophenyl)(piperidin-1-yl)methanone (9).33 A 5-mL, oven-dried vial equipped with a septum, magnetic stirrer, static nitrogen and a thermocouple probe was charged with methyl 4-bromobenzoate (225 mg, 1.05 mmol, 1.00 eq), THF (2 mL), piperidine (0.11 g, 1.31 mmol, 1.25 eq.) and TBD (32.8 mg, 0.24 mmol, 0.23 eq). The solution was stirred and heated. After 21 h at 60 °C, an HPLC analysis of an aliquot indicated 87.2 A% of product 9, 3.5 A% of unreacted 6 and 9.3A% of the hydrolysis byproduct 4-bromobenzoic acid which compared favorably with 83.9 A% of 9, 3.2 A% of 6 and 12.9 A% of 4-bromobenzoic acid obtained from a STR gradient screening (Fig. 16, segment 7, 0.225 eq of TBD). The reaction was cooled to 20 °C, diluted with water (10 mL) and extracted with dichloromethane (2 x 5 mL). The combined dichloromethane phases were washed with water (3 x 5 mL), dried over magnesium sulfate and evaporated to obtain a clear oil which was crystallized from a mixture of heptane and TBME to afford 230 mg (82% yield) of 9 as a white solid. 1H NMR (CDCl3, 400 MHz): δ = 7.54 (2H, d), 7.28 (2H, d), 3.69 (2H, bs), 3.33 (2H, bs), 1.69 (4H, m), 1.52 (2H, m); 13C NMR (CDCl3, 101 MHz): δ = 169.2, 135.3, 131.6, 128.6, 123.6, 48.7, 43.2, 26.5, 25.6, 24.5; HRMS–ESI+: m/z [M + H]+ calcd for C12H15BrNO+: 268.03315; found: 268.03326. Preparation of t-butyl 4-(6-chloropyridazin-3-yl)piperazine-1-carboxylate (12).32 A 25-mL, 3-necked round-bottomed flask equipped with magnetic stirring, static nitrogen, a thermocouple probe, a condenser and a heating mantle was charged with 3,6-dichloropyridazine (1.00 g, 6.71 mmol, 1.00 eq.), 1-boc-piperidine (1.88 g, 10.07 mmol, 1.50 eq.) and NMP (10 mL). After stirring for a few moments until all solids had dissolved, DIPEA (1.75 mL, 10.07 mmol, 1.50 eq.) was added and the solution was heated. After 8 h at 80 °C, an HPLC analysis of an aliquot indicated 85.5 A% of product 12 and 14.5 A% of unreacted 10 which compared favorably with 84.6 A% of 12 and 15.4 A% unreacted 10 obtained from a STR gradient screening (Fig. 18, segment 6 on 80 oC curve, 1.5 eq of 11). The reaction was cooled to 20 °C, and then diluted with water (15 mL). After filtering, washing with water (3 x 10 mL) and drying, 1.67 g (81% yield) of 12 was obtained as a tan solid. 1H NMR (CDCl3, 400 MHz): δ = 7.24 (1H, d), 6.91 (1H, d), 3.62 (4H, m), 3.57 (4H, m), 1.48 (9H, s); 13C NMR Page 16 of 18


Page 17 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(CDCl3, 101 MHz): δ = 159.0, 154.7, 147.2, 128.9, 115.4, 80.0, 45.0, 43.2, 28.4; HRMS–ESI+: m/z [M + H]+ calcd for C13H20ClN4O2+: 299.12693; found: 299.12671. Supporting Information Available: Description List of the components of a STR, sample gradient spreadsheets, 1H NMR spectra from a calibration experiment, 1H and 13C NMR spectra for compounds 5, 9 and 12. Acknowledgments The authors are thankful to Andre Bourque and Scott Clark for analytical support and the reviewers of the draft submission, several of whom provided excellent feedback and suggestions that improved this manuscript. Corresponding Author E-mail: [email protected]

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References 1

For recent reviews on using continuous flow chemistry in the synthesis of pharmaceuticals, see: a. Continuous Manufacturing of Pharmaceuticals; Kleinebudde, P., Khinast J., Rantanen, J., Eds; John Wiley & Sons, Ltd.: Hoboken, 2017. b. Porta, R.; Benaglia, M.; Puglisi, A. Org. Process Res. Dev. 2016, 20, 2. c. Baumann, M.; Baxendale, I. R. Beil. J. Org. Chem. 2015, 11, 1194; and chapters and references cited therein. 2 Nasr, M. M.; Krumme, M.; Matsuda, Y.; Trout, B. L.; Badman, C.; Mascia, S.; Cooney, C. L.; Jensen, K. D.; Florence, A.; Johnston, C.; Konstantinov, K.; Lee., S. l. J. Pharm. Sci. 2017, 106, 3199. 3 Current good manufacturing practices 4 Cole, K. P.; Groh, J. McClary; Johnson, M. D.; Burcham, C. L.; Campbell, B. M.; Diseroad, W. D.; Heller, M. R.; Howell, J. R.; Kallman, N. J.; Koenig, T. M.; May, S. A.; Miller, R. D.; Mitchell, D.; Myers, D. P.; Myers, S. S.; Phillips, J. L.; Polster, C. S.; White, T. D.; Cashman, J.; Hurley, D.; Moylan, R.; Sheehan, P.; Spencer, R. D.; Desmond, K.; Desmond, P.; Gowran, O. Science 2017, 356, 1144. 5 For a sampling of recent reviews, see: a. Plutschack, M. B.; Pieber, B.; Gilmore, K.; Seeberger, P. H. Chem. Rev. 2017, 11796. b. Movsisyan, M.; Delbeke, E. I. P.; Berton, J. K. E. T.; Battilocchio, C.; Ley, S. V.; Stevens, C. V. Chem. Soc. Rev. 2016, 45, 4892. c. Baxendale, I. R. J. Chem. Technol. Biotechnol. 2013, 88, 519. d. Malet-Sanz, L.; Susanne, F. J. Med. Chem. 2012, 55, 4062. e. Wegner, J.; Ceylan, S,; Kirschning, A. Adv. Synth. Catal. 2012, 354, 17. 6 Roberge, D. M.; Ducry, L.; Bieler, N.; Cretton, P.; Zimmermann, B. Chem. Eng. Technol. 2005, 28, 318. 7 For discussions on making informed decisions on whether to go with flow, see: a. Hartman, R. L.; McMullen, J. P.; Jensen, K. F. Angew. Chem. Int. Ed. 2011, 50, 7502. b. Valera, F. E.; Quaranta, M.; Moran, A.; Blacker, J.; Armstrong, A.; Cabral, J. T.; Blackmond, D. G. Angew. Chem. Int. Ed. 2010, 49, 2478. 8 For the use of a segmented flow tubular reactor for optimizing the physical quality of powders, see: Jongen, N. et al. Chem. Eng. Technol. 2003, 26, 3. 9 For example, see: a. Benali, O.; Deal, M.; Farrant, E.; Taicepolczay, D.; Wheeler, R. Org. Process Res. Dev. 2008, 12, 1007. b. Wheeler, R. C.; Benali, O.; Deal, M.; Farrant, E.; MacDonald, S. J. F.; Warrington, B. H. Org. Process Res. Dev. 2007, 11, 704. c. Hawbaker, N.; Wittgrove, E.; Christensen, B.; Sach, N.; Blackmond, D. G. Org. Process Res. Dev. 2016, 20, 465, and references cited therein. d. Reizman, B. J.; Jenson, K. F. Chem. Commun. 2015, 51, 13290. e. Wiles, C.; Watts, P. Beil. J. Org. Chem. 2011, 7, 1360. . 10 a. Thompson, C. M.; Poole, J. L.; Cross, J. L.; Akritopoulou-Zanze, I.; Djuric, S. W.; Molecules 2011, 16, 9161. b. Stanley, C. E.; Wootton, R. C. R.; deMello, A. J. Chimia 2012, 66, 88. c. Lange, P. P.; James, K. ACS Comb. Sci. 2012, 14, 570. 11 For a miniature multi-stage and continuous stirred tank reactor system allowing relatively long reaction residence times, see: Chapman, M. R.; Kwan, M. H. T.; King, G.; Jolley, K. E.; Hussain, M.; Hussain, S.; Salama, I. E.; Niño, C. G.; Thompson, L. A.; Bayana, M. E.; Clayton, A. D.; Nguyen, B. N.; Turner, N. T.; Kapur, N.; Blacker, A. J. Org. Process Res. Dev. 2017, 21, 1294. 12 For a sampling of screening and optimization tools, see: a. Rubin, E. A.; Tummala, S.; Both, D. A.; Wang, C.; Delaney, E. J. Chem. Rev. 2006, 106, 2794. b. Stencel, L. M.; Leadbeater, N. E. New J. Chem. 2014, 38, 242. c. Weller, H. N.; Rubin, A. E.; Moshiri, B.; Ruediger, W.; Li, W.-J.; Allen, J.; Nolfo, J.; Bertok, A.; Rosso, V. W J. Assoc. Lab. Autom. 2005, 10(1), 59. d. Kotlyar, V.; Shahar L.; Lellouche, J.-P. Molecular Diversity 2006, 10, 255. e. Perera, D.; Tucker, J. W.; Brahmbhatt, S.; Helal, C. J.; Chong, A.; Farrell, W.; Richardson, P.; Sach, N. W. Science 2018, 359, 429. F. See also: iChemExplorer, Reaction Analytics Inc.: Wilmington, DE, 2008; http:// www.ichemexplorer.com 13 Dias, A. M. A. ; Freire, M.; Coutinho, J. A. P.; Marrucho. I. M. Fluid Phase Equil. 2004, 222-223, 325. 14 1.25-in nominal pipe, 1.66-in o.d.; about 4 inches in length. A stainless steel pipe was used at T>100 oC. PVC or other thermoplasic core pipes are also suitable but with lower operating temperatures.

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15

11.72 mL obtained by mathematical calculation (volume of cylinder) agrees with 11.76 mL average volume obtained by weighing the contents of tubes with length of 148 cm that were completely filled with water or NMP and converting mass to volume using the density values of these solvents, 1.00 g/mL and 1.03 g/mL, respectively. 16 In practice, it is more convenient to set up the STR by using an excess length of tubing (about 155-160 cm for 10 loops), completing the coiling operation, securing the coils in place using zip ties, then clipping off excess tubing on both ends leaving just enough length to accommodate couplings (see Fig. 3a for the STR after coiling). 17 Segments that were generated when the STR was positioned vertically appeared visually to be more uniform compared to those prepared when positioned horizontally, although this observation was not verified experimentally. 18 Some of these operations are best done with the STR momentarily clamped in placed to minimize movement. 19 The number of coiled loops corresponds to the number of segments (reactors) that will be generated and in turn the number of sequenced steps that need to be programmed. Essentially, there is no limit on number of segments, but practically and logistically, 5-10 segments are normally sufficient for screening a range of conditions in a single STR experiment. 20 Preferably, a programmable 3-syringe dispensing system could be employed. The nitrogen filling needs to cease immediately upon completion of the reaction filling, otherwise the nitrogen will displace the segments from the STR. 21 It may take a user several trials before the technique to properly handle a loaded STR to minimize movement of segments is perfected. For example, the STR should not be rotated, otherwise segments will shift and an end segment could drain out of the STR. 22 The tapping technique ensures convergence of mini-segments into segments aligned at the bottom of each loop. Alternatively, larger, complete single segments can be dispensed in concept but would likely require more sophisticated dispensing equipment than those used in this study to achieve uniform alignment of the segments. 23 In practice, an extra “dummy” segment, a duplicate of segment 1, is included in the program. This extra segment ensures that segment 1 is completely charged and can be fully formed during tapping and collapsing of mini segments from both the front and the back of a coil that feasible for the other segments. Excess volume from the dummy segment during charging flows into the waste receiver (Fig. 1) upon completing the loading operation. 24 To minimize movement of segments, the venting was done in 2-3 short cycles by cracking open for an instant a coupling at the end of the STR. If necessary, the segments were realigned between the vent cycles by tapping the STR while it was horizontal. 25 8.7 mL nitrogen based on dead volume in 10 segments (11.7 mL volume of STR less 3.0 mL reagents) needed for the duration of the charging reagents in 2.667 min corresponds to 3.27 mL/min of N2 during loading. 26 Richmond, P.C. Surface Tension-Organic Compounds. In Thermophysical Properties of Chemicals and Hydrocarbons; Yaw, Carl L. Ed.; Norwich, NY, 2008; pp 686-781. 27 As a further check on reliably forming the desired gradients, mesitylene was frequently employed as an internal NMR standard during early development of the STR. 28 Pichowicz, M.; Crumpler, S.; McDonald, E.; Blagg, J. Tetrahedron 2010, 66, 2398. 29 8.7 mL nitrogen dead volume in 10 segments (11.7 mL volume of STR less 3.0 mL reagents) needed for the duration of the charging reagents in 4.235 min corresponds to 2.05 mL/min of N2 during loading. 30 An intermediate point on a curve was selected rather than using conditions that were at a plateau in the STR as the preferred comparison to a reaction performed in batch mode. 31 a. Sabot, C.; Kumar, K. A.; Meunier, S.; Mioskowski, C. Tetrahedron Lett. 2007, 48, 3863. b. Weiberth, F. J.; Yu, Y.; Subotkowski, W.; Pemberton, C. Org. Process Res. Dev. 2012, 16, 1967. 32 Seki, M.; Tsuruta, O.; Aoyama, Y.; Soejima, A.; Shimada, H.; Nonaka, H. Chem. Pharm. Bull. 2012, 60, 488. 33 Zhu, M.; Fujita, K.: Yamaguchi, R. J. Org. Chem. 2012, 77, 9102.

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Segmented Tube Reactors (STR): a Simple Tool to Screen Multiple

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