A Kinetics-Based Approach for the Assignment of Reactivity Purge

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A Kinetics-Based Approach for the Assignment of Reactivity Purge Factors Rick C. Betori, Jeffrey M. Kallemeyn, and Dennie S Welch Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.5b00257 • Publication Date (Web): 05 Oct 2015 Downloaded from http://pubs.acs.org on October 5, 2015

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Organic Process Research & Development

A Kinetics-Based Approach for the Assignment of Reactivity Purge Factors Rick C. Betori, Jeffrey M. Kallemeyn*, and Dennie S. Welch* Process Chemistry, AbbVie, 1 N. Waukegan Rd. North Chicago, IL 60064 Corresponding Authors: [email protected]; [email protected]

Table of Contents Graphic

Abstract: The control of mutagenic impurities is of crucial interest to pharmaceutical companies and regulatory agencies alike. One risk-based methodology to assess the likelihood of impurity carry-over to drug substance entails evaluation of the physicochemical properties of the entity against the parameters of the chemical process to which it is exposed. This article details a simple experimental approach that utilizes kinetic analyses to facilitate the assignment of reactivity purge factors. These reactivity purge factors are important values in the semiquantitative risk assessment for impurity carry-over to drug substance.

Introduction: The manufacture of pharmaceutical drug substances often involves the use of reactive agents to facilitate the formation of new chemical bonds. The intrinsic electrophilicity of many

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of these agents and capacity to react with nucleophilic biological substrates, specifically DNA, invoke concern about their potential mutagenicity and carcinogenicity. Indeed, control of mutagenic impurities to ensure patient safety continues to be an imperative of pharmaceutical companies and expectation from regulatory authorities. In the recently adopted ICH M7 guideline,1 ‘Assessment and Control of DNA Reactive (Mutagenic) Impurities’, four options to demonstrate impurity control are described. One of the available options (Option 4) describes a control strategy that is based on process controls in lieu of analytical testing. Specifically, if an appropriately conducted risk assessment concludes that the process parameters impact the level of a mutagenic impurity such that the risk of the impurity being present in the drug substance above acceptable limits is negligible, no confirmatory analytical testing is necessary. Risk can be assessed through understanding the physicochemical properties of an impurity and the process parameters that may lead to consumption or removal of the impurity. Intrinsic properties such as reactivity, solubility, volatility, ionizability, or any additional physical removal processes (e.g. chromatography, resins) designed to eliminate impurities should factor into the risk assessment. A semiquantitative risk assessment method that incorporates these considerations was developed by Teasdale2,3,4 and subsequently cited in the ICH M7 guidance. In the ICH M7 guidance, a purge factor is defined as “the level of an impurity at an upstream point in a process divided by the level of an impurity at a downstream point in a process. Purge factors may be measured or predicted.”1 The output of the semiquantitative method is expressed as an overall purge factor, a numerical value that is a product of the standardized and individually assigned purge factors for each process unit operation to which the mutagen is subjected. Application of the overall purge factor to a given impurity provides insight into the risk that an impurity poses


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to product quality. The method described by Teasdale2,3,4 has been utilized to assess risk of impurity carry-over to drug substance by Elder5 and to support designation of Active Pharmaceutical Ingredient (API) Starting Materials by Faul.6 The assignment of a purge factor for a mutagenic impurity over a unit operation requires an understanding of the degree of consumption or removal of the impurity. Since a large number of mutagens are highly reactive species, degradation during processing subsequent to introduction or formation can be a path that leads to significant removal from the process stream. The degradation of a mutagen depends on numerous reaction parameters, such as its concentration versus the concentration of the consuming reactant, reaction temperature and duration. Quantification of impurity attrition or rejection through the development of analytical methods requires significant investment of resources due to the ppm-level sensitivity that is required and can be further complicated by the inherent reactivity of some mutagenic impurities. Confident assignment of a reactivity purge factor through expert inspection and literature review can still present a challenge in some cases for classes of compounds under reaction conditions to which these are not normally subjected. We sought to identify an experimentally-driven method to assign a purge factor based on reactivity for any given impurity or class of impurities in a process. To understand the interplay between the kinetic parameters of the desired reaction and the background consumption of an impurity, rate equations can be expressed to describe these simultaneously occurring processes. For the desired reaction, a starting material (SM) can react with a reagent (R) to provide product (P), as described by rate equation 1. In analogy to the starting material, the reactivity of the impurity with any reaction component (C) can also be expressed as rate equation 2. When applied to mutagenic impurities, which are often present in



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low molar levels relative to the reaction components, it may be possible to simplify the rate equation for the consumption of the impurity from second order to pseudo-first order (eq 3). In this scenario, the rate of consumption of an impurity is governed by the reaction rate constant (k) and concentration of the impurity. In principle, k' 7 can be determined by studying the reaction between the impurity and component C in the absence of any spectator molecules that are present in the reaction of interest.

Collection of time-course conversion data for a given impurity at multiple temperatures, either in a single experiment wherein the temperature is changed step-wise over the duration of the reaction or from multiple experiments that are conducted at different temperatures, permits determination of two fundamental kinetic parameters, the (1) rate constant (k) and (2) energy of activation (Ea).8 These parameters can be utilized to calculate the half-life of the impurity at any reaction temperature (eq 4). In this article, we describe an experimental protocol for determination of these values and the relationship between an impurity’s rate constant or half-life and the purge factor. This information can guide selection of one of the three standardized


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reactivity purge factors (1, 10, or 100) that are integral to the semiquantitative risk assessment developed by Teasdale.2,3,4

Results: To illustrate these concepts, the reaction of benzyl bromide (the model for a mutagenic impurity) with triethylamine was studied in both isolation under pseudo-first-order conditions and as a low-level impurity (5 mol %) in the TBS protection of benzyl alcohol using triethylamine as a base. The stand-alone reaction was performed in CH3CN (0.025 M) with triethylamine (20 equiv) at –13 °C. At 31 min, the reaction was warmed to 1 °C. Samples were collected over the duration of the reaction and the conversion was monitored by HPLC, aided by the inclusion of an internal standard. The collected data were analyzed in DynoChem 4.1.0.09 to calculate a k' at 1 °C of 6.86 × 10-4 s-1 and an Ea of 8.71 kcal/mol. From this data, the reaction of benzyl bromide with triethylamine in CH3CN was determined to have a half-life of 16.8 min at 1 °C. To evaluate the relevance of the kinetic information on this model mutagen as an impurity in a reaction of interest, 5 mol % benzyl bromide was spiked into the TBS protection of benzyl alcohol in CH3CN at 1 °C, using triethylamine as the base (Scheme 1). The consumption of benzyl bromide was monitored by HPLC analysis (Figure 1). The data was analyzed to determine the rate constant and half-life at 1 °C, which were 7.45 x10-4 s-1 and 15.5 min, respectively.



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Scheme 1. TBS Protection of Benzyl Alcohol in the Presence of Benzyl Bromide Impurity OH

+ TBS-Cl

+

NEt3

+

Br

CH 3CN, 1 °C (0.019 M in BnBr)

1.0 equiv

1.2 equiv

2.4 equiv

OTBS +

0.05 equiv

Figure 1. Measured versus predicted consumption of benzyl bromide in TBS protection reaction.

The alignment between the rate constants and half-lifes of the reaction of benzyl bromide with triethylamine in isolation and as a low-level impurity in the TBS protection of benzyl alcohol establishes the proof of concept that the kinetic information obtained from the standalone reaction can be used to predict impurity conversion in a more complex reaction matrix. The behavior of alkyl halides10 across a variety of reaction conditions is sufficiently well documented to, in many cases, guide selection of the standardized reactivity purge factors (1, 10, or 100) to input into the semiquantitative risk assessment and obviate this type of


NEt3 Br

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experimentation. We sought to further interrogate this approach for evaluating reactivity on a class of mutagens that is not as well understood as alkyl halides. The mutagenic potential of arylboronic acids has only recently been realized.11,12,13 While arylboronic acids are known to undergo oxidation with hydrogen peroxide to afford phenols, inspection of the literature reveals limited reaction rate information.14,15,16 The oxidation of phenylboronic acid with 30% aqueous H2O2 was chosen as a model system to investigate (Table 1). The reactions were conducted using a temperature step method wherein the reaction was initiated at, for example, –20 °C in DCM and the temperature was increased from –20 °C to 2 °C while quantifying the disappearance of phenylboronic acid over time. From the time-course data and temperature profiles, values of k' were determined at each temperature in the step profile and from these rate constants the Ea was calculated (Table 1, entry 1). To assess the impact of the reaction media on this process, similar experimentation was conducted in various solvents at multiple temperatures ranging from –20 to 65 °C, depending on the rate of the reaction in each solvent. The reaction constants at 21 °C were calculated using the previously determined Ea values, which allowed for facile comparison of the impact of different media on this oxidative process. The reaction proceeded the fastest in H2O (entry 4), the slowest in DMF (entry 7) and varied by one order of magnitude across the entire range of solvents.



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Table 1: Oxidation of Phenylboronic Acid in Various Solventsa

entry

solvent

k' (at 21 °C) (s-1)

Ea (kcal/mol)

1 2 3 4 5 6 7 8

DCM DCE acetone H2O MeOH EtOAc DMF CH3CN

1.2 × 10-3 2.0 × 10-3 5.1 × 10-4 3.7 × 10-3 3.62 × 10-4 2.37 × 10-4 1.34 × 10-4 2.29 × 10-4

8.59 11.57 8.62 11.72 7.55 10.66 8.45 7.12

half-life at 21 °C (min) 9.6 5.8 23 3.1 32 49 86 50

a

The reactions were carried out with 1 equiv of phenylboronic acid, and 20 equiv of H2O2 (30 w/w% in water) in 10 mL of the indicated solvent with naphthalene as an internal standard. See supporting information for additional details.

To test the applicability of the kinetic values from the variable temperature experiments, the oxidation of phenylboronic acid was conducted isothermally at –20 °C, 2 °C and 21 °C in DCM. The time-course data from the consumption of phenylboronic acid was used to determine the half-lifes at each temperature. The observed reaction and predicted half-lifes from the temperature step experiments show comparability (Table 2). This agreement supports the suitability of the temperature step method to determine the fundamental kinetic parameters for the purposes described in this paper.

Table 2: Comparison of Observed and Predicted Half-Lifes for Oxidation of Phenylboronic Acid temperature 22 °C 2 °C –20 °C

experimental half-life (min) 8.3 23.9 87.1

predicted halflife (min) 8.9 26.6 105


% difference 7% 10% 17%

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To assess the impact of electronic and steric effects on the reactivity of arylboronic acids, the oxidation of a variety of electron-rich, electron-deficient and sterically-hindered arylboronic acids were investigated by the same experimental protocol used for phenylboronic acid (Table 3) and the half-lifes were determined from the k' and Ea of the multiple-temperature reactions.17 Electron-rich substrates, such as 4-methoxyphenylboronic acid (entry 2), showed increased reactivity relative to electron-deficient substrates (entries 3-5). Sterically-hindered substrates, such as 2,6-dimethylphenylboronic acid (entry 6), displayed limited reactivity.



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Table 3: Survey of Arylboronic Acids and Calculated Half-Lifes

entry

boronic acid

calculated halflife at 21 °C (min)

1

32

2

20

3

53 B(OH)2

4

55 HO2C

5

78

6

2075

7

21

8

6

With this basic kinetic information determined, the next step in our evaluation of this kinetics-based approach was to identify a model reaction system in which to probe the reactivity of an arylboronic acid as an impurity. The oxidation of thioanisole with H2O2 was selected as the ‘main reaction’ due to the simplicity of the reaction matrix as well as a determined half-life (131 min), which was comparable to those observed with arylboronic acids under similar conditions.


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Monitoring the reaction of the arylboronic acid as an impurity (0.25 equiv) in the thioanisole oxidation allowed for comparison of the half-lifes under the two reaction modes—in the absence and presence of thioanisole.

In most cases the half-lifes were found to be

comparable (Table 4, entries 1–4, 6). A deviation was observed in the study with 4cyanophenylboronic acid (entry 5): a decrease in half-life of the arylboronic acid from 78 to 46 min was observed as well as a concomitant decrease in the half-life of thioanisole oxidation from 131to 53 min.

Table 4: Half-Life Comparison of Arylboronic Acid Oxidation

calculated half-life; in absence of PhSMe (min)

observed half-life; in presence of PhSMe (min)

1

32

41a

2

20

25

3

53

63b

55

43

5

78

46b

6

2075

3000

entry

boronic Acid

B(OH)2

4 HO2C

a

Average of 3 experiments. b Average of 2 experiments



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Oxidation of phenylboronic acid was also studied under another synthetically-relevant oxidation condition, namely in the presence of NaOH.18 This modification resulted in complete oxidation of phenylboronic acid in

A Kinetics-Based Approach for the Assignment of Reactivity Purge

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