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Online Inductive Electrospray Ionization Mass Spectrometry as a Process Analytical Technology Tool To Monitor the Synthetic Route to Anagliptin Xin Yan,† Ryan M. Bain,† Yafeng Li,† Ran Qiu,† Tawnya G. Flick,*,‡ and R. Graham Cooks*,† †

Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, United States Department of Analytical Research & Development, Amgen Inc., 1 Amgen Center Drive, Thousand Oaks, California 91320, United States

‡

S Supporting Information *

ABSTRACT: Inductive electrospray ionization (iESI) is an ambient ionization method that is particularly well-suited to online reaction monitoring. It allows the potential of electrospray mass spectrometry (MS) to be realized as a routine process analytical technology (PAT) tool to monitor practical synthetic reactions in real time. In this study, a synthetic route to Anagliptin (target API) was successfully monitored using online iESI-MS. Starting materials not seen by traditional reaction monitoring tools (HPLC-UV/Vis and GC-FID) were observed, as well as water-sensitive reagents and intermediates which cannot easily be followed by other methods. Online tandem mass spectrometry (MS/MS) was used to characterize chemical species in the reaction mixture. Impurities and byproducts were identified, and information on the progress of byproduct formation enabled implementation of strategies to eliminate these byproducts in the course of the reaction. This work demonstrates how iESI-MS can be employed to obtain comprehensive information and solutions to some practical problems that occur in small-molecule synthetic reaction monitoring.

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troscopy,21,22,31 and high-performance liquid chromatography (HPLC).31 However, little information is provided on chemical structures by many of these measurements, which only give signals for characteristic functional groups, and this remains one of the barriers to the application of spectroscopy in PAT applications. On the other hand, just as there is no single offline analytical tool that meets all needs for process development, understanding, or control strategy for any particular product, there is no single in situ analytical tool that will work for all applications. New analytical technologies need to be developed and added to the PAT toolbox. Because of its inherent sensitivity, speed, and molecular selectivity,32−37 mass spectrometry (MS) has seen some use in process analysis, including in-process monitoring of exhaust gases of fermentation processes,38 real-time deuterium abundance measurements in water vapor,39 analysis of trace gases in food products and environmental monitoring.40,41 However, MS has not been used as a routine PAT tool to monitor reactions in real time.42−44 Recent studies have shown that a particular version of electrospray mass spectrometry, namely, inductive electrospray ionization mass spectrometry (iESI-MS),45,46 can be used to continuously monitor reacting systems in a preparation-free online process. This analysis tool applies to organic chemical reactions and allows the study of reaction progress.47 This method has the notable advantage of being applicable to concentrated solutions of the type encountered in pharmaceutical manufacturing. An early version of such a system

INTRODUCTION Process analytical technology (PAT) methodology, as endorsed by the U.S. Food and Drug Administration (FDA),1,2 is a system of analytical techniques for real-time process characterization in the pharmaceutical industry, which is applied increasingly in pharmaceutical development, scale-up, and manufacture.3−7 The PAT concept is embraced in the FDA’s Quality-by-Design (QbD) framework,8−10 which aims at process understanding and control so that product quality is built into the manufacturing process.11 Such processes can produce consistent quality products, making product quality control less dependent on analytical testing at the end point, that is, the final active pharmaceutical ingredient (API).12−14 This initiative has encouraged the pharmaceutical industry to increase research and use of new analytical technologies to perform timely measurements on critical quality attributes of raw materials, intermediates, and products.15−17 Successful implementation of PAT requires the appropriate selection of process analytical methods, the particulars of which depend on the application and molecule. In the FDA’s PAT definition1,11 (“a system for designing, analyzing, and controlling manufacturing through timely measurements (i.e., during processing) of critical quality and performance attributes of raw and in-process materials and processes, with the goal of ensuring final product quality”), “analyzing” equates to the use of in situ analytical tools. A variety of analytical methods have been incorporated into the PAT toolbox for online process monitoring, including focused beam reflectance measurement (FBRM),18−20 particle video microscopy (PVM),18,19 infrared (IR) spectroscopy,11,21−28 UV−vis spectroscopy,20 Raman spectroscopy,22,29,30 nuclear magnetic resonance (NMR) spec© 2016 American Chemical Society

Received: February 10, 2016 Published: April 12, 2016 940

DOI: 10.1021/acs.oprd.6b00039 Org. Process Res. Dev. 2016, 20, 940−947

Organic Process Research & Development

Article

Scheme 1. Synthetic Route to API Anagliptin

Figure 1. Time-resolved mass spectra of amidation of (R)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile 2 with (2-amino-2-methyl-propyl)-carbamic acid tert-butyl ester 3 by iESI-MS.

(Figure S1) was used to automatically sample reaction mixtures in situ and deliver them to the MS inlet, thereby enabling the continuous reaction mixture monitoring and providing virtually real-time structural information on the intermediates and products in the mixture. Three important reactions in

pharmaceutical synthesis of different types including reductive amination, Negishi cross-coupling (an air and moisturesensitive reaction), and Pd/C-catalyzed hydrogenolysis (a heterogeneous reaction) were successfully monitored.47 This system enabled short-lived intermediates to be observed and 941



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Scheme 2. Structures and Molecular Masses of Impurities 10, Active Intermediate 11, and Byproduct 12, 13 in the Synthetic Route to Anagliptin 1

tracked through the synthetic reaction. The development of iESI-MS gave opportunities for online MS as a PAT tool in reaction monitoring. In the course of developing the system further to allow process analysis, the Anagliptin synthetic route (target API 1, Scheme 1)48 was monitored using online iESIMS. Solutions to practical problems which occurred in this realistic application were provided mainly by using additional capabilities of the mass spectrometer. Note that neither of the starting materials 2 or 3 can be detected by traditional reaction monitoring tools such as HPLC-UV−vis and GC-FID. Also, water-sensitive reagents and intermediates are involved, which cannot readily be followed by regular ESI-MS.

Figure 2. Reaction progress of the amidation of (R)-1-(2chloroacetyl)pyrrolidine-2-carbonitrile 2 with (2-amino-2-methylpropyl)-carbamic acid tert-butyl ester 3. Average total ion intensity is around 8E5.

RESULTS AND DISCUSSION Monitoring of Amidation of (S)-1-(2-Chloroacetyl)pyrrolidine-2-carbonitrile 2, with (2-Amino-2-methylpropyl)-carbamic Acid tert-Butyl Ester 3. The first step in the synthesis of Anagliptin, amidation of (S)-1-(2chloroacetyl)pyrrolidine-2-carbonitrile 2, with (2-amino-2methyl-propyl)-carbamic acid tert-butyl ester 3 (Scheme 1), leading to the intermediate product 4 was monitored by online iESI-MS. The reagent 3 was first mixed with sodium iodide and potassium carbonate in acetone. The reagent 2 was then added dropwise to an ice-cooled acetone suspension, and the reaction mixture was stirred at room temperature for 10 h. Timeresolved mass spectra (Figure 1) were recorded to show the progress of the reaction with designation of the detected ions, each time point requiring 1.8 s (average of 9 scans). Figure 1a was collected before the addition of reagent 2. Sodium adducts of the reagent 3 and its dimer were detected at m/z 211 and 399. Ions at m/z 311 and 499 are assigned as the sodium adduct of dicarbamate 10 (Scheme 2) and the cluster ion of [M3 + M10 + Na]+. Dicarbamate 10 arises from impurities in reagent 3 as shown by MS/MS (Figure S2). After the addition of reagent 2, the abundance of the ions at m/z 211 increased (Figure 1b), due to a contribution by the isobaric potassium adduct of reagent 2; that is, the ions of sodiated 3 overlap in nominal mass with potassiated 2 at m/z 211. This mass overlap was confirmed by the change of their 13C isotopic distributions (Figure S3). The proportion of each species was calculated from the experimentally observed isotopic pattern. With time, the signal due to product ion 4 observed at m/z 363 [M4 + K]+ increased, and it became dominant after 3 h (Figure 1d). The formation of dimers of product 4 at m/z 671 [2M4 + Na]+ and m/z 687 [2M4 + K]+ was also observed. The spectra then showed no significant changes out to 8 h (Figure 1f). Halogen exchange proceeded readily as was indicated by the formation of sodiated and potassiated ions of activated iodide intermediate 11 at m/z 287 and m/z 303 (Figure 1b,c,S6). The progression of the reaction is shown in Figure 2, which

displays the changing abundance (normalized) of the ions corresponding to the reagent 3, intermediate 11, and the product 4. The product 4 was isolated and purified by column chromatography. The mass spectrum of 4 after purification is shown in Figure S4a, and its identity was evident from its MS/ MS product ion spectrum (Figure S4b,c,d; tandem MS of 4 and its dimer in solution were shown in Figure S12). This reaction was also monitored by traditional off-line nanoelectrospray ionization (nESI) MS. The time-resolved mass spectra (Figure S5) show the formation of product 4 and impurity 12 (discussed later), but the ions of reagent 2 and 3 were suppressed by the strong signal from the protonated acetone dimer at m/z 117 and thus could not be followed. The fact that more species were ionized well in online-iESI makes this monitoring system an effective tool to follow the reactions. Ions at m/z 499 were observed before the addition of reagent 2, and the ions are assigned from the MS/MS data (Figure 3a,b) as corresponding to the sodium binding adduct of 10 and 3. The dicarbamate 10 also reacted with excess reagent 2 to form the product 4, resulting in the decreasing abundance of the ions at m/z 499 with full disappearance occurring within 2 h. However, the signal intensity at m/z 499 started to increase again at 5 h and reached 7% of the base peak at 8 h. This latter species was identified as the product 12 of a second amidation of 4 with reagent 2, and its identity was confirmed by the different tandem mass spectrum from that of the earlier species (Figure 3c,d). The MS spectrum of the latter species 12 after purification was in agreement with this assignment (Figure S7). These results demonstrate the advantage of using MS as a PAT tool to monitor low-level impurities that are formed and tracked through the synthetic reaction mixture. Another phenomenon worthy of note is the presence of both sodium adducts and potassium adducts of the same species. Both sodiated or potassiated ions are readily formed in the presence of sodium iodide and potassium carbonate in the reaction mixture. They include m/z 211 [M3 + Na]+, 227 [M3 + K]+; 287 [M11 + Na]+, 303 [M11 + K]+; 311 [M12 + Na]+, 327 [M12 + K]+ and 347 [M4 + Na]+, 363 [M4 + K]+ (Figure 1,

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Figure 3. (a,b) Tandem MS spectra of ions at m/z 499 formed before the addition of reagent 2; (c,d) tandem MS spectra of ions at m/z 499 formed after 5 h.

Figure 4. Time-resolved mass spectra of reaction of 4 with 4 N hydrochloric acid in 1,4-dioxane and dichloromethane by iESI-MS.

mass spectra (Figure 4) show the progress of the reaction. Figure 4a was recorded 1 min after the addition of 4 N hydrochloric acid in 1,4-dioxane. Protonated 4 and its dimer were detected at m/z 325 and 649, respectively. Meanwhile, a new ionic species at m/z 223 had started to be observed. In subsequent reaction monitoring MS spectra (representative spectra shown at 10, 20, and 50 min, Figure 4b−d), showed ions of protonated 4 and its dimers decreasing in abundance, whereas ions at m/z 223 continually increased and eventually dominated the spectra after 50 min. This latter signal represents the major product of this reaction. The observed cations at m/z 223 are designated as [M5 − H]+, loss of hydride from the neutral molecules with its MS/ MS shown in Figure S8b. The key step in ESI is normally the generation of the protonated form of the molecule: M + AH+ → [M + H]+ + A. However, hydride abstraction is a well-

S11), and their formation is competitive. The concentration of K+ is twice than that of Na+; however, all the ions formed are sodium adducts (Figure 1a,b) at the beginning of the reaction. This shows that potassium competes less well than sodium in adduct formation with this suite of compounds. As the reaction proceeded, the disappearance of sodiated ions and formation of potassiated ions for all these three species in the reaction mixture was observed. This is consistent with the preferential removal of sodium ions as an insoluble (in acetone) precipitate of sodium chloride.49 Monitoring the Deprotection of Compound 4. In step 2 (Scheme 1) of the synthetic route to Anagliptin, the reaction of 4 with 4 N hydrochloric acid in 1,4-dioxane and dichloromethane deprotects the Boc group. The reaction mixture was analyzed after dilution with the reaction solvent, 1,4-dioxane and dichloromethane (1:1, v:v). The time-resolved 943



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Figure 5. MS spectra of activation of 2-methylpyrazolo[1,5-a]pyrimidine-6-carboxylic acid 6 with N,N′-carbonyldiimidazole (CDI) 7 in THF analyzed by (a) online iESI-MS (b,c) traditional nESI. (b) Mass spectrum of pure CDI 7 in THF. (c) Mass spectrum of reaction of 6 with 7 in THF. (d) Tandem MS of acylimidazole (8).

Figure 6. Time-resolved mass spectra of amidation of (R)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile 6 with (2-amino-2-methyl-propyl)-carbamic acid tert-butyl ester 5 by iESI-MS.

moved to m/z 225, whereas the other major ions in the solution remained unaffected (Figure S8a, c). The ions at m/z 225 were assigned to [M5 + H]+ based on their fragmentation (Figure S8d). Monitoring of Formation of Anagliptin 1. The third step in the synthetic route to Anagliptin includes two reactions

known route to positive ion formation in chemical ionization (CI) MS. Factors such as the composition of the reaction mixture are known44 to affect the outcome of the competition between a proton donotion and hydride loss. In order to confirm the species at m/z 223, methanol was used as dilution solvent before analysis of the reaction mixture. Ions at m/z 223 944



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impurities generated in this reaction was provided from the above experiment.

which occur sequentially without separation of intermediates: activation of 2-methylpyrazolo[1,5-a]pyrimidine-6-carboxylic acid 6 with N,N′-carbonyldiimidazole (CDI) 7 to yield the corresponding acylimidazole 8, followed by amination with (S)1-((1-amino-2-methylpropan-2-yl)glycyl)pyrrolidine-2-carbonitrile 5, leading to the final product, Anagliptin 1 (Scheme 1). CDI 7 was first added to a solution of 6 in tetrahydrofuran (THF), and the mixture was stirred at room temperature. Figure S9a,b show spectra recorded before the addition of CDI 7. Reagent 6 is poorly protonated in the positive ion mode (Figure S9a), while it is easily deprotonated in the negative ion mode due to the carboxyl group (Figure S9b). Considering that other major ionic species involved in this reaction step will likely be observed as the protonated molecules, the positive ion mode was used as the ionization mode of choice when monitoring the reaction, but the negative ion mode offered complementary information. After adding CDI 7 to the reaction solution, protonated imidazole and its dimer were rapidly destroyed due to the sensitivity of CDI 7 to water. Protected by the use of a N2 sheath gas in online-iESI, the protonated CDI 7 at m/z 163 could still be seen with low intensity in the spectra (Figure 5a). The activated carboxylic acid product 8 is also water-sensitive, while protonated 8 was detected at m/z 288 and confirmed by MS/MS (Figure 5d). The comparison experiment was done using traditional nESIMS with CDI 7 in anhydrous THF (Figure 5b) and then reaction with 6 (Figure 5c). Neither protonated CDI 7 nor product 8 could be detected in the traditional nESI-MS. The current iESI reaction monitoring system is well-suited to follow moisture-sensitive reactions which are commonly applied in the pharmaceutical industry. The reaction mixture was added dropwise to 5 in an icecooled solution of triethylamine in THF in order to form the product 1. The mixture was warmed to room temperature and stirred. The spectrum shown in Figure 6a was recorded before adding the reaction mixture. Protonated 5, its dimer, water cluster, and triethylamine cluster were detected separately at m/ z 225, 449, 243, and 326, respectively. New ionic species at m/z 543 and 319 were seen right after the reaction mixture containing the acylimidazole was added dropwise to the solution (Figure 6b,c). Ions at m/z 319 are assigned to protonated 13 (Scheme 2) based on MS/MS data (Figure S10c,d). The major byproduct 13, originates from the side reaction of amine 5 with CDI 7, and the ions at m/z 543 are ionic clusters of 5 with protonated 13 (Figure S10a). The purity of commercial CDI 7 may be variable due to its water sensitivity, therefore it is common to employ excess in the activation step to ensure complete conversion of the carboxylic acid to the acylimidazole. The unreacted CDI 7 also reacted with the amine 5. From the reaction monitoring data, the amine 5 was shown to react faster with residual CDI 7 than with the acylimidazole 8 (Figure 6b,c). Once the byproduct formed, its signal remained relatively constant. This suggests the byproduct cannot be avoided by stopping the reaction early, instead reducing the amount of CDI 7 in the previous step can decrease the byproduct formation. After the reaction was allowed to occur for 30 min, the product ions, protonated Anagliptin 1 at m/z 384, started to form and reached its maximum at the end of the reaction period (Figure 6d,e,f). The structure of the product 1 was confirmed by MS/MS (Figure S10b). The other product, the imidazole 9, was observed at m/ z 69. The reported yield45 of this step is only 33% without corrections for impurities formed. The information on

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CONCLUSION The iESI-MS was investigated as a useful PAT tool on monitoring the synthetic route of Anagliptin in real time. The chemical species involved in the reactions were identified using MS/MS data. Active intermediates, impurities, and byproducts were identified in the reaction mixture. Two pairs of ions with the same m/z were differentiated on the basis of their isotopic distribution, and tandem MS and their structures were assigned. Moreover, the current iESI-MS reaction monitoring system shows advantages in better ionization of species in the reaction mixture compared with the analysis using traditional nESI-MS. It also shows the capability to monitor water-sensitive reagents and intermediates. The formation of byproducts due to the excess water-sensitive reagent were followed, and its reaction rate was found to be much faster than that of the target reaction. The work provides a comprehensive demonstration of process monitoring by mass spectrometry including solutions to some practical problems that occur in small molecule synthetic reaction monitoring. The probe used for this analysis could also be used to monitor reactions at different scales.

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EXPERIMENTAL SECTION Instrumentation. A linear ion trap mass spectrometer (LTQ, Thermo Fisher Scientific, San Jose, CA, U.S.A.) was used to record positive ion mode full scan mass and MSn spectra. Typical MS parameters included averaging of 3 microscans, 100 ms maximum injection time, 15 V capillary voltage, 150 °C capillary temperature, and 65 V tube lens voltage. Data were acquired and processed using Xcalibur 2.0 software (Thermo Fisher Scientific). The identification of analyte ions was confirmed by tandem mass spectrometry (MS/MS) using collision-induced dissociation (CID). An isolation window of 1.5 Th (mass/charge units) and normalized collision energy of 30−40% (manufacturer’s unit) were selected for the CID experiments. Chemicals and Reagents. All reagents and solvents were used directly without any further purification. (2-Amino-2methyl-propyl)-carbamic acid tert-butyl ester 3 was purchased from J&W Pharmlab, LLC; (S)-1-(2-chloroacetyl)pyrrolidine2-carbonitrile 2 was purchased from Alchem Pharmtech. 2Methylpyrazolo[1,5-a]pyrimidine-6-carboxylic acid 6 was purchased from Advanced ChemBlocks Inc.; N,N′-carbonyldiimidazole 7, sodium iodide, potassium carbonate, and acetone were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). OmniSolv HPLC grade methanol was purchased from EMD (Bedford, MA, U.S.A.). Water was purified and deionized using a Milli-Q system (Millipore, Bedford, MA, U.S.A.). Synthesis of (S)-t-Butyl (2-((2-(2-cyanopyrrolidin-1-yl)-2oxoethyl)amino)-2-methylpropyl)carbamate (4). A solution of (S)-1-(2-chloroacetyl) pyrrolidine-2-carbonitrile 2 (3 mmol, 673.4 mg) in acetone (6 mL) was added to an ice-cooled stirred suspension of (2-amino-2-methyl-propyl)-carbamic acid tertbutyl ester (3 mmol, 565 mg) 3, NaI (3.9 mmol, 584.8 mg), and K2CO3 (3.9 mmol, 539.2 mg) in acetone (14 mL). The reaction mixture was stirred at room temperature for 10 h. The resulting mixture was filtered to remove insoluble materials, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to give (S)-t-butyl (2945



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(( 2- (2 -c ya nopyrrolidi n -1- y l) -2 -ox o et hyl) ami n o)- 2methylpropyl)carbamate (4). Synthesis of (2-Amino-2-methyl-propyl)-carbamic Acid tert-Butyl Ester (5). (2-((2-(2-Cyanopyrrolidin-1-yl)-2oxoethyl)amino)-2-methylpropyl)carbamate 4 (210.3 mg) in dichloromethane (40 mL) was added to 4 N hydrochloric acid/ 1,4-dioxane (40 mL), and the mixture was stirred at room temperature until the reaction was complete in 1 h. The product was concentrated under reduced pressure to give 5. Synthesis of (S)-N-(2-((2-(2-Cyanopyrrolidin-1-yl)-2oxoethyl)amino)-2-methylpropyl)-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide (1). Carbonyl diimidazole 7 (17.8 mg, 0.11 mmol) was added to a solution of 6 (17.7 mg, 0.1 mmol) in tetrahydrofuran (5.7 mL), and the mixture was stirred at room temperature for 4 h. The reaction mixture was slowly added to 5 (29.7 mg, 0.1 mmol) in an ice-cooled solution of triethylamine (69 uL) in tetrahydrofuran (5.7 mL). The mixture was warmed to room temperature and stirred until reaction completion. The reaction mixture was concentrated under reduced pressure, and then dichloromethane was added to the residue. Insoluble materials were removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography (eluting solvent; dichloromethane/methanol, 50:1) to yield pure 1 (30%).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.oprd.6b00039.

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Additional information as noted in text (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail for T.G.F.: tfl[email protected]. *E-mail for R.G.C.: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors acknowledge funding support from Amgen Inc. and the National Science Foundation (CHE-1307264) as well as contributions of Dr. Laura Blue and Dr. Yuan-Qing Fang.

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REFERENCES

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