IR and NMR Reaction Monitoring Techniques for Nucleophilic

Article pubs.acs.org/OPRD

IR and NMR Reaction Monitoring Techniques for Nucleophilic Addition Reactions: In Situ Monitoring of the Addition of Benzimidazole to a Pyridinium Salt Michele T. Drexler,*,† David A. Foley,‡ Howard W. Ward, II,‡ and Hugh J. Clarke‡ †

Chemical Research & Development and ‡Analytical Research & Development, Pfizer Worldwide Research & Development, Eastern Point Rd, Groton, Connecticut 06340, United States S Supporting Information *

ABSTRACT: This study centers on the use of in situ FTIR spectroscopy and online NMR to study the nucleophilic addition of benzimidazole analogues to an N-methylpyridinium salt. The reaction consists of two stages. First, the protected benzimidazole was lithiated with LDA to form the C-2 lithiated benzimidazole. The lithiated benzimidazole was then added to the pyridinium salt to generate the coupled product. The lithiated benzimidazole was not thermally stable, so offline sampling was challenging. A good understanding of the reaction completion of the lithiation was needed for process understanding as well as to provide a basis to determine the fate of the lithiated benzimidazole through the entire addition sequence. In situ FTIR and online NMR provided online methods for monitoring the reaction sequence and studying the temperature-dependent stability of the lithiated benzimidazole.

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INTRODUCTION The use of PAT (process analytical technology) in pharmaceutical process development provides in situ analysis and real time data of a chemical process under investigation.1 This type of technology is now routinely used in the biopharmaceutical industry in both development and manufacturing activities to optimize, scale up, and control reaction processes.2 PAT encompasses a multitude of analytical techniques applied in an in situ or online mode, which includes pH probes, optical spectroscopies,3 chromatography,4 mass spectrometry,5 and NMR (nuclear magnetic resonance) spectroscopy.6 Infrared (IR) spectroscopy is firmly established in PAT use.7 The technique has been employed in the examination of organic reactions to determine mechanistic details,8 aid process understanding, and reliably produce chemical entities.9 IR spectroscopy has been utilized across a range of chemistry operational modes including traditional batch, flow,10 and microwave chemistry.11 NMR is one of the most powerful analytical tools available to the organic chemist. It is the key technique in elucidating and characterizing structures of synthetic products, unknown impurities, and natural products. To date, reports of the use of NMR as a PAT tool are few, but the use of NMR to monitor the progress of reactions is becoming increasingly popular.12 The combination of the inherent quantitative nature of NMR and the structural detail that can be mined from the 2D analysis of reaction species in the solution state provides unrivaled process understanding. A number of online NMR experimental set-ups have been described in the literature, which facilitate the real-time monitoring of reaction processes.13 The chemistry that is used herein to demonstrate the power of in situ monitoring with both online NMR and IR is the first step of the proposed manufacturing process of an active pharmaceutical ingredient (API),14 which involves the © XXXX American Chemical Society

nucleophilic addition of a protected benzimidazoles 3 to a pyridinium triflate salt 2 to afford dihydropyridones 4 as the basic skeleton of the target molecule 1 (Scheme 1).15 The end product API (1) is achieved through the transformation of 4 through isolated intermediate 8 and is a potent and orally bioavailable inhibitor of the smoothened receptor (SMO) recently reported by Pfizer.16 The program was on an accelerated development timeline, so the ability to fully understand these reactions via in situ analytical techniques was key in ensuring efficient manufacture of the final API. Scheme 1 shows the favored synthetic strategy for the project development team. In the development of the synthetic route, a number of benzimidazole analogues (3a−3c) were evaluated as starting materials to achieve 1. The initial step of the synthesis involved the formation dihydropyridones 4, which became the focus of the investigation using PAT tools. In this reaction, the protected benzimidazoles 3 were deprotonated with LDA to generate lithiated benzimidazoles 6, which then underwent addition to 4-methoxy-N-methylpyridinium salt 2 to give the corresponding dihydropyridines 7. The subsequent mild acidic hydrolysis of the enol ethers 7 during workup furnished dihydropyridones 4 (Scheme 2). However, the reactions were unable to achieve full conversion for the protected benzimidazoles explored. For benzyl benzimidazole 3a, the reaction gave 50−60% (assay) desired product with 30−40% starting material 3a remaining. While the sulfonyl protected benzimidazoles (3b or 3c) gave higher yield of the desired products (70−80% assay), there were still 10−20% of the starting materials (3b or 3c) remaining in the reaction mixtures. Special Issue: Engineering Contributions to Process Chemistry Received: January 23, 2015

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DOI: 10.1021/acs.oprd.5b00029 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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Scheme 1. Nucleophilic addition of protected benzimidazoles 3 to N-methylpyridinium salt 2 for the synthesis of a SMO inhibitor 1

Scheme 2. Preparation of dihydropyridones 4 with the nucleophilic addition of benzimidazoles 3 to pyridinium salt 2

The LithiationIR Studies of Protected Benzimidazoles 3. It was determined that understanding the lithiation reaction was key to ensure that complete conversion of the benzimidazoles 3 to the corresponding anions occurred. Protected benzimidazoles 3 required treatment with LDA to activate the species for the pyridinium salt addition at the C-2 position. Formation of the lithiated benzimidazoles 6 was conducted first, followed by the addition of the pyridinium salt 2. Since 6 are not stable intermediates, offline sampling of the lithiation reaction was not practical. There was a need to track the reaction species through the lithiation to acquire an understanding of reaction completion for this transformation and determine the fate of 6 through the reaction sequence, since full conversion to 4 was not achieved. This was then followed by a study of temperature-dependent stability of the lithiated species as well as a look at the overall addition of the triflate salt 2. In situ IR proved to be the best option to monitor this temperature and air-sensitive reaction. The initial focus, therefore, was to determining the IR bands to monitor during the reaction. To identify compound related IR bands, references of the protected benzimidazoles 3 (benzyl, tosyl, and benzenesulfonyl) and N-methylpyridinium triflate 2 were scanned as solids and as THF solutions. The determination of IR features related to the intermediates was performed during the reaction, as both lithiated species 6 and the methyl enol ethers 7 were not isolable compounds. Characteristic IR bands were identified for each of the reaction components, and Table 1 provides the IR bands used during the course of this study and spectral overlays are included in the Supporting Information. With appropriate bands identified, monitoring the stability of the lithiated benzimidazole species 6 was the first objective of the IR studies. The lithiation reaction was performed using commercially available 2 M LDA in THF/heptane/ethylbenzene and monitored using IR. All reactions performed demonstrated that upon the addition of the LDA to a solution

The initial objective of the work presented herein was to determine the reason that the addition of the N-methylpyridinium triflate salt 2 to the protected benzimidazoles 3 was unable to achieve to full conversion. Since lithiated benzimidazoles 6 are unstable and transient, the use of in situ IR and online NMR was considered to investigate the sequence of reactions for a more detailed understanding.

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RESULTS AND DISCUSSION

As a result of the issues highlighted from the low assay yield of 4, a study was undertaken to investigate this synthetic step using PAT techniques with the following objectives: • To determine the feasibility of using in situ FTIR (Fourier transform infrared) spectroscopy to study the lithiation and addition sequence for the protected benzimidazoles 3 to the pyridinium salt 2. • To monitor the stability of the anion species 6 formed upon addition of LDA to the protected benzimidazoles 3. • To understand the low assay yield obtained from the addition of lithiated benzimidazoles 6 to the Nmethylpyridinium triflate salt 2. • To study the kinetics of the system with the use of online NMR and in situ FTIR to determine the feasibility of increasing the yield of the addition sequence. This investigation was split into two main partsin situ IR analysis of the lithiation reaction and the tandem IR/online NMR examination of the sequence of reactions (lithiation and the addition to the pyridinium salt). This study began with the benzyl protected benzimidazole 3a. The benzyl substrate gave low yield in the reaction sequence, and the removal of the benzyl protecting group in downstream chemistry proved difficult. Therefore, tosyl and benzenesulfonyl protected benzimidazoles (3b and 3c, respectively) were also studied using in situ techniques to compare the different protecting groups and their performance in the reaction. B



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Table 1. FTIR bands for the components of the lithiationaddition sequence component(s)

FTIR bands (cm−1)

Bn-benzimidazole (3a) Ts-benzimidazole (3b) SO2Ph-benzimidazole (3c) Lithiated Bn-benzimidazole (6a) Lithiated Ts- and SO2Ph-benzimidazole (6b and 6c) N-methylpyridinium triflate salt (2) Bn-substituted addition product (7a) Ts- and SO2Ph-substituted addition product (7b and 7c)

1618 1385 1386 1238 1314,1484 1530 1308, 1661 1255, 1652

of the protected benzimidazole of interest, the benzimidazole was observed to be completely consumed. Figure 1 shows a typical IR trend for the disappearance of 1-benzylbenzimidazole 3a. Concurrent with disappearance of the benzimidazole was the growth of another feature. This feature was assumed to be the lithiated species 6, as no other materials were being added and the shift in wavenumber could be attributed to the activation of the carbon at the benzimidazole 2-position. Several lithiation reactions were performed−as the 1benzylbenzimidazole 3a was the first protected benzimidazole to be used, the stability of the corresponding lithiated species 6a was studied at several temperatures (−40, −20, and −10 °C). A stack plot of the lithiated product trends for these experiments is shown in Figure 2 over a 24 h time period. Degradation of the anion was observed in all cases, but the relative level of degradation with time was similar between the three temperatures. Within 5.5 h following the formation of the anion (Figure 2b), the degradation rate was considerably less; in fact, the anion would be considered stable enough in a processing environment since the intermediate showed enough stability to derisk any possible processing delays that come with normal scale manufacture within this 6 h period. The tosyl and benzenesulfonyl protected benzimidazole anions (6b and 6c) were also tested for stability at −10 °C. The tosyl compound 6b performed similarly to the benzyl compound 6a, with degradation in the first 6−8 h being minimal, but with similar degradation over long time periods. The 1-(benzenesulfonyl)benzimidazole anion 6c, however, appeared to degrade faster (i.e., relative slope of the disappearance of the lithiated species IR band was steeper). The benzyl, tosyl, and benzenesulfonyl protected benzimidazole trends are compared in Figure 3. While performing these lithiation studies, it was observed that the tosyl and benzenesulfonyl protected benzimidazoles were more soluble in THF at the lower temperatures. 1Benzylbenzimidazole 3a forms a thin slurry at temperatures below −10 °C, which initially was a concern based on availability of the compound for the lithiation. Upon the addition of LDA, it was observed that 3a becomes fully soluble. The tosyl and benzenesulfonyl substrates (3b and 3c) were easier to work with, however, as there was no concern of incomplete dissolution in THF even at temperatures around −10 °C. This was desirable from a processing standpoint to ensure all starting material is dissolved and available for reaction. Addition Reaction SequenceIn Situ Study of the Addition of 2 to 3a Using IR and NMR. Using the IR data gathered from the lithiation reaction studies, the overall addition reaction sequence was studied more closely. At this

Figure 1. IR trends with time of the protected benzimidazoles and the lithiated products. (a) 1-benzylbenzimidazole 3a (lithiation only) (b) 1-tosylbenzimidazole 3b and (c) 1-(benzenesulfonyl)benzimidazole 3c.

point, online NMR was employed in combination with in situ IR to discern the different reaction components and to aid in assigning the trends observed by the IR. This combined approach was focused on the addition of the N-methylpyridinium triflate salt 2 to the newly formed benzimidazole anion 6a. Initial studies investigated standard conditions for the reaction −1:1 stoichiometry of 3a to 2. Figure 4 shows the IR trends (using second derivative treatment of the data) of the 1benzylbenzimidazole 3a, the lithiated species 6a, and the final methyl enol ether addition product 7a. The benzylbenzimidaC



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Figure 4. IR trends for the entire lithiation-addition reaction sequence using benzyl benzimidazole 3a (IR data is processed using seond derivative). No timed dose was performed for the pyridinium salt; it was added to the reaction mixture in one portion.

the reaction was monitored simultaneously using an in situ IR probe. The reaction was run under standard batch stoichiometry conditions, but for this experiment, a stream of the reaction mixture was recirculated through an NMR flow tube.17 NMR data was captured over time to obtain a profile of the reaction. The reaction sequence was also run more dilute than was typical to ensure that there was enough volume to fill the NMR sample loop. The transfer lines of the recirculation loop were temperature regulated to ensure that entire system (reaction vessel and sample loop) was at the reaction temperature (−20 °C). The concern for this particularly chemistry was that the transfer time would be too long to capture the rate of the reaction and that snapshot gathered from the NMR data might not be representative of the reactor. The loop had a volume of 14 mL with a recirculation time of ∼60 s; so a slight delay of approximately 20 s from the reaction vessel to the site of detection had to be considered. As all the samples were slightly offset from what was actually occurring in the pot, this was not an issue; the relative timing of the reaction was correct. Figure 5a shows a stack plot of the NMR traces obtained for a standard lithiation-addition reaction sequence for 1:1 benzyl benzimidazole 3a with N-methylpyridinium triflate 2. The trends can be seen a little more clearly in the reaction profile plot shown in Figure 5b. As is seen by NMR spectroscopy, conversion of the benzimidazole to the lithiated species is complete. It can also be clearly seen in Figure 5b that the benzyl benzimidazole starting material 3a reappears upon addition of the pyridinium salt 2 at approximately 30 min. The levels of the enol ether product 7a and reformed starting material 3a plateau at the 40 min mark. At this point, the additional 2 that is being added accumulates since all of the anion 6a has been consumed and there is no other species available for reaction. These findings were consistent with pKa studies on the acidity of the α-C−H of pyridinium ions.14,18 Peng et al. discuss the deprotonation potential of the α-C−H of 2 and the use of a deuterium quench after the addition of 2 to 3, which confirms the loss of the proton and the subsequent quench of the lithiated benzimidazoles 6. The fact that 2 will easily deprotonate (pKa ∼33), providing a proton source to quench the lithiated intermediate prior to the addition reaction explains the reaction profiles seen by online NMR/in situ FTIR

Figure 2. (a) IR trends to study the inherent stability of the lithiated 1benzylbenzimidazole species 6a over 24 h. (b) IR trends for the first 5.5 h following the lithiated 1-benzylbenzimidazole 6a at given temperatures.

Figure 3. IR trends for the benzimidazole anions at −10 °C over 18 h: Ts = tosyl (6b); SO2Ph = benzenesulfonyl (6c), and Bn = benzyl (6a).

zole 3a is shown to reform following the dose of the pyridinium salt 2. To confirm these findings and try to further understand the implications, an online NMR setup was used. The progress of D



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higher yield of 7a. The experiments were performed as a sequence of reactions, starting with the activation of 3a with LDA to form the lithiated 1-benzylbenzimidazole 6a followed by the addition of N-methylpyridinium triflate 2 to obtain the methyl enol ether 7a. 3a was used at the limiting reagent, and the triflate salt 2 stoichiometry was varied to look at the concentration effect of this reagent. Understanding the IR response for each component as a function of concentration was necessary for studying the relative rates of the reactions. For the solid starting materials, 3a and 2, reference solutions at specific concentrations were made up in THF to understand the IR response. The in situ intermediates, such as 6a and 7a, were more challenging to work with as the inherent nature of the lithiation reaction created some problems for obtaining response vs concentration data. The lithiated species 6a is transient and thermally unstable, so sampling offline is impractical. An assumption of the maximum level of 6a had to be made for modeling.19 The in situ addition product, the methyl enol ether 7a, was not directly isolable. Therefore, the concentration of 7a was related to the corresponding IR signal using UPLC data of the quenched enone product 4a (Scheme 2), which should be the only species produced on quench of 7a. Figure 6 shows the

Figure 5. (a) NMR stack plot and (b) reaction profile generated from online NMR monitoring of lithiation-addition sequence using 1benzylbenzimidazole (3a).

and depressed yields obtained in that step. There are competing reactionsthe desired addition reaction of the lithiated benzimidazoles 6 to the pyridinium salt 2 and the undesired quench of the lithiated species. Competing ReactionsKinetic Comparison. In addition to determining the fate of the lithiated benzimidazole species (6) during the addition reaction, experiments were conducted to understand the basic kinetics of the overall reaction sequence. The objective was to determine if the quench of the lithiated benzimidazole species 6 by the pyridinium salt 2 was kinetically faster, slower, or of similar speed to the desired addition reaction of those two species. Findings from the online NMR spectroscopy experiments indicated that the benzimidazole species 3 was reformed during the reaction with the addition of the pyridinium triflate in a 2:1 ratio of desired addition product 7 to undesired reversion to starting material 3. With the potential quench of the active intermediate species to starting material 3, improvements to throughput and yield were paramount for commercial viability, and understanding the relative rates of the two competing reactions of 6 was key to making that improvement. In situ FTIR was used to describe the systems in terms of concentrations for the different species involved. Kinetic experiments were conducted using 1-benzylbenzimidazole 3a as the starting material to fit the concentration profiles to a basic kinetic model of the system. The basic model would then be used to determine if a more suitable mode of operation could be designed to run this chemistry robustly and achieve a

Figure 6. UPLC traces overlaid to show the growth of the addition product over time. The bottom trace (black) is the initial sample at the beginning of the dose, and moving up the figure shows the addition product 4a growing in at a retention time of about 2 min.

overlaid UPLC traces for a standard triflate addition and the enone produced. For LDA concentration, the reagent is purchased as a sealed solution that is also potentially unstable, so offline sample preparation was not attempted due to the likelihood of sample degradation. Therefore, LDA was assumed to be consumed at one equivalent for the model. The IR data from the four experiments was converted to concentration profiles for the 1-benzylbenzimidazole 3a, trifalte salt 2, lithiated species 6a, and product 7a. The IR shows the starting material 3a with characteristic band at 1618 cm−1 and the N-methylpyridinium triflate salt 2 with feature at 1530 cm−1. The lithiated species 6a is evident with the LDA addition at 1238 cm−1, and the addition product 7a is seen with the triflate salt addition at both 1308 and 1661 cm−1. Figure 7 shows the reaction concentration profiles at varying equivalents of 2. These profiles were used to perform a fit of a basic kinetic model. The model is shown in Table 2. The first equation in this model describes the lithiation as a second order reaction, first order in both the 1-benzylbenzimidazole 3a (BB) and LDA, forming the lithiated species 6a E



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the quench reactions being of the same magnitude (k2 = k3). The final case was looking at where the rate of the quench is greater than that of the addition, which would indicate a situation that was undesired. Figure 8 shows this final case,

Figure 7. Component profiles over time for the lithiation and pyridinium triflate addition sequence, varying the stoichiometry of the salt (2) added: (a) Excess (2 equiv), (b) standard conditions (1 equiv), and (c) substoichiometric (2 portions of 0.5 equiv added; total of 1 equiv).

Table 2

(lithiated). This is followed by the consumption of 6a to the addition reaction with the N-methylpyridinium triflate 2 (triflate) to give the methyl enol ether product 7a (product). There is a third reaction, which also occurs with the addition of 2. This reaction consumes 6a, but instead of forming 7a, there is a quench of the anion that occurs and converts 6a back to the 1-benzylbenzimidazole starting material 3a (BB). This model was designed to explain the data collected to this point in basic kinetic terms. The model was approached in a few different waysthe key was to look at how the data obtained at various pyridinium salt 2 stoichiometries fit relative rate scenarios. The initial fit of the data to the model was to assume that the rate of the first reaction, r1, was greater than the rates of the other two reactions. The initial fit also assumed that the rate of the desired addition, r2, was faster than the rate of the undesired quench of the anion, r3 (i.e., k2 > k3); this would present a case that the desired reaction should be favored. Using these assumptions as a starting point, the data were fit using DynoChem in simulator mode. A similar fit was performed simulating the addition and

Figure 8. Looking at the standard stoichiometry (1 equiv 3a and 1 equiv 2) data for the three different rate constant scenarios: k2 > k3, k2 = k3, and k2 < k3. The second case shows that the relative magnitude of the 3a (BB) and the addition product 7a are closer to the experimental data.

where k3 is double k2. If the absolute magnitude of the curves is ignored, the shape of the curves and the relative magnitudes of the addition product 7a and 1-benzylbenzimidazole 3a curves seem to indicate that the case in which k2 < k3 appears more accurate for this system than either of the other two options (k2 > k3 or k2 = k3). These data present the case that the reformation of 1-benzylbenzimidazole 3a is competitive kinetically with the desired addition product 7a formation in F



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Table 3. Scenarios used in DynoChem fits with the experimental data rate constants scenario

k1 (L/mol s)

k2 k3 (L/mol s) (L/mol s)

initial rate constants lithiation favored (r1); addition (r2) faster than quench (r3)

0.06

0.02

0.01

Lithiation favored (r1); addition (r2) equal to quench (r3)

0.06

0.01

0.01

Lithiation favored (r1); addition (r2) less than quench (r3)

0.06

0.01

0.02

results The lithiated species is under predicted The triflate salt is actually shown by the model to build up in the system initially (a spike); this is not the case in any of the experimental data. There is little reversion of the lithiated species to starting material The lithiated species is under predicted There is little reversion of the lithiated species to starting material The lithiated species is under predicted The shape of the curves does match nicely with the data in all cases, even if the actual magnitude is not correct.

development of a synthetic route. Online NMR was used to provide structural information on intermediate species in solution, which is very beneficial in the process development stage. Having simultaneous IR and NMR reaction profiles, allows the learnings to be translated to the IR technique which is more amenable to providing PAT support in a manufacturing environment.

the given method of synthesis. It would also indicate that it will be difficult to achieve higher yields of 7a given the affinity of the anion 6a to be quenched by the salt 2. Table 3 shows the rate constant values and the results from the fit.

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CONCLUSIONS In situ IR and online NMR were used to investigate the two stage reaction of benzimidazoles 3 lithiation and subsequent addition to the pyridinium triflate salt 2, a transformation which was low yielding in initial development experiments. The use of these PAT tools determined that the low product yield obtained was due to a competitive quench of the lithiated benzimidazole species 6. The online NMR and in situ IR data confirm that when the pyridinium salt 2 is added to the reaction there is a reversion of some of the lithiated species 6 back to starting material 3. It appears that the lithiated species 6 is being quenched by the pyridinium salt 2, specifically deprotonation of the α-C−H, as a competitive reaction to the formation of the enol ether 7. Therefore, 6 can either then add to 2 or be quenched by the proton that it is losing. Based on a kinetic screen of the various 3a:2 stoichiometries, the quench of the anion 6a appears to be competitive to the addition of 2 to 6a. The data gathered indicate that the addition product 7a is seen at about two times what the level of protected benzimidazole 3a is seen, but the reaction rate constants indicate that the experimental data fits a model where the quench is kinetically competitive to the addition in the current synthesis and will hinder the overall yield of the desired product 7a. The kinetic work was done only on the benzyl protected benzimidazole substrate; the tosyl and sulfonyl protected substrates showed evidence in the FTIR profiles of the reversion to starting material 3 during the addition of 2, but rate data was not collected for these substrates at the time of the study. In the course of this work, stability of the intermediate 6 in the reaction system was evaluated. It was determined that the lithiated benzyl benzimidazole 6a species would degrade over timethe IR data indicates that over a 24 h period the anion will degrade by approximately 60% given the temperature is less than −10 °C. The tosyl substituted benzimidazole 6b has similar stability to 6a, while the relative stability of the benzenesulfonyl protected benzimidazole 6c was lowerin the first 8 h, 6c appeared to degrade faster. In practical terms, the tosyl substituted and benzenesulfonyl protected benzimidazoles had a higher relative solubility at the low operating temperatures as compared to the benzyl substrate. This study represents a good example of the use these two PAT technologies and how they can be applied to the

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EXPERIMENTAL SECTION Online NMR Experiment. Online NMR setup overview. A flowing stream of the reaction mixture was transferred from the reaction vessel to the coil detection region of the NMR magnet, at a flow rate (∼3 mL/min) which was adequate to provide accurate quantitation of the NMR signal. All transfer lines were temperature regulated using Syltherm XLT and were maintained at the temperature of the reaction vessel. NMR spectra were acquired at 70 s intervals. Each acquisition consisted of 4 scans with a 30° pulse angle and a relaxation delay of 10 s. The equipment setup used for this type of experiment has been reported previously.4b,17a THF (20 mL) was charged to a 50 mL reaction vessel, and the temperature was cooled to −20 °C. 1-Benzylbenzimidazole 3a (2.00 g, 9.60 mmol) was added in one portion, and the solution was circulated around the sample loop, which was also regulated at −20 °C. LDA (2.0 M in THF/heptane/ ethylbenzene, 5.8 mL) was added at 0.5 mL/min. The progress of the reaction was monitored by 1H NMR. The reaction mixture was stirred at −20 °C for 15 min. The pyridinium salt 2 (2.49 g, 9.62 mmol) was dissolved in THF (10 mL), and the resulting solution was added to the reaction mixture at 1.0 mL/ min. The progress of the reaction was followed by NMR and IR. FTIR Set-up Overview. Mettler Toledo-iC10 and ReactIR 45m FTIR’s with MCT detectors were used to collect all IR reaction spectra. The instruments were outfitted with 6.3 mm DiComp (Diamond) probes connected via 1.5 m Silver Halide fibers. All data were collected at a 4 cm−1 resolution from 2000 to 650 cm−1. Scan intervals ranged from 1 to 5 min as appropriate for the given experiment. An air background was collected prior to all experiments, and Mettler Toledo iCIR software versions 4.2 and 4.3 were used for instrument control and data analysis. The probe was installed in the reactor using custom Teflon fittings so as to set the probe depth in an area away from the impeller vortex. A Thermo Nicolet Nexus 470 benchtop FTIR with a DTGS detector and KBr beam splitter coupled with a Golden Gate Diamond ATR sampling accessory was used to collect the IR spectra of solid references. These references were collected over G



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the region from 2800 to 650 cm−1 using 64 scans/spectrum and a resolution off 4 cm−1. The controlling software was Omnic v8.0. Kinetic Experiment Setup. THF (12 mL/g) was added to a reaction vessel equipped with overhead stirring, nitrogen purge, temperature probe, and FTIR probe. The vessel was cooled to −25 °C. 1-Benzylbenzimidazole 3a (limiting reagent, 2 g) was added, and the FTIR instrument scanning started (see FTIR setup). A thin slurry resulted. LDA (2 M in THF/ heptane/ethylbenzene; 1.2 equiv) was added to the vessel at a rate of 0.5 mL/min. The reaction mixture was allowed to stir at −25 °C for 20 min after dose completed and the thin slurry changed to a yellow solution. N-methylpyridinium triflate 2 (1 equiv) in THF (7.5 mL/g) is added to the reaction mixture at a rate of 1 mL/min. Samples were taken for offline LC analysis during the pyridinium salt addition; samples were quenched in a premeasured amount of 1 N HCl. Once the dose of pyridinium salt was complete, the reaction solution was stirred for 30 min. Approximately 46% conversion of the 1benzylbenzimidazole to enone product was observed. Assumptions for Modeling. LDA was assumed to be consumed at one equivalent for the model, due to the reagent instability once a sealed bottle was opened. The feature at 1238 cm−1 was followed as the lithiated species 6a. The reaction mixture was allowed to stir following the completion of the LDA addition to ensure that 3a had all been converted. Once there was no IR feature discernible for 3a, this was assumed to be full conversion to the lithiated species 6a (Figure 9). It was

AUTHOR INFORMATION

Corresponding Author

*E-mail: Michele.T.Drexler@pfizer.com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the following colleagues for valuable discussions during this work: Frank Busch, Zhihui Peng, Angela Puchlopek-Dermenci, and Nick Thompson.

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REFERENCES

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Figure 9. Plot of the lithiation of the 1-benzylbenzimidazole 3a indicating the consumption of all the benzimidazole that there is the maximum theoretical lithiated species available in solution.

at this IR intensity that the maximum number of moles for the lithiated species was set and allows for a calculation of the concentration of 6a over the course of the reaction as a portion of this maximum value. The methyl enol ether 7a is not directly isolable, and therefore, the concentration of 7a was related to the corresponding IR signal using UPLC data of the quenched enone product 4a.

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Article

ASSOCIATED CONTENT

S Supporting Information *

FTIR spectra showing key band used for each reaction component. This material is available free of charge via the Internet at http://pubs.acs.org. H



Article

(19) Please refer to Experimental section for details on data treatment and model assumptions.

I


IR and NMR Reaction Monitoring Techniques for Nucleophilic

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