Genotoxic Impurities in Pharmaceutical Manufacturing: Sources

Review pubs.acs.org/CR

Genotoxic Impurities in Pharmaceutical Manufacturing: Sources, Regulations, and Mitigation Gyorgy Szekely,*,† Miriam C. Amores de Sousa,‡ Marco Gil,§ Frederico Castelo Ferreira,*,‡ and William Heggie*,§ †

School of Chemical Engineering & Analytical Science, The University of Manchester, The Mill, Sackville Street, Manchester M13 9PL, United Kingdom ‡ Department of Bioengineering and Institute for Bioengineering and Biosciences (iBB), Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais, 1049-001, Lisbon, Portugal § Hovione FarmaCiencia SA, R&D, Sete Casas, 2674-506, Loures, Portugal S Supporting Information *

4.1.1. Altering the Synthesis 4.1.2. Adjusting Reaction Conditions To Mitigate GTI Formation 4.1.3. Quality by Design 4.2. API Purification 4.2.1. Purge Factors 4.2.2. Separation Technologies 5. Conclusions and Future Trends Associated Content Supporting Information Author Information Corresponding Authors Notes Biographies Acknowledgments References

CONTENTS 1. Introduction 2. Genotoxicity: Mechanisms, Risk and Regulation 3. Chemical Classes of Common Genotoxic Impurities 3.1. Genotoxic Compounds Used as Reactants 3.1.1. Alkyl Halides 3.1.2. Dialkyl Sulfates 3.1.3. Epoxides 3.1.4. Hydrazines 3.1.5. TEMPO 3.1.6. Aromatic Amines 3.1.7. Boronic Acids 3.2. Genotoxic Compounds Formed in Side Reactions 3.2.1. Sulfonate Esters and Their Precursors. Overview 3.2.2. Sulfonate Esters and Their Precursors Used in Stoichiometric Amounts 3.2.3. Sulfonate Esters and Their Precursors Used in Catalytic Amounts 3.2.4. Alkyl Halides 3.2.5. Acetamide 3.3. Genotoxicity and Carcinogenicity of Common Organic Solvents 4. Approaches for GTI Mitigation in the Pharmaceutical Industry 4.1. Chemical Synthetic Approaches © XXXX American Chemical Society

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AC AC AE AF AF AG AL AM AM AM AM AM AM AN AN

1. INTRODUCTION Most pharmaceutical products are manufactured either by applying a total synthesis approach or by modifying a naturally occurring product. In both cases, a wide range of reactive reagents are used. Therefore, it is natural that low levels of such reagents or side products are present in the final active pharmaceutical ingredient (API) or drug product as impurities. Such impurities may have unwanted toxicities, including genotoxicity and carcinogenicity. The risk for patient’s health caused by the presence of small molecules as impurities in APIs has become an increasing concern of pharmaceutical companies, regulatory authorities, patients, and doctors alike. Thus, pharmaceutical regulatory agencies such as the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have raised concerns regarding the presence of genotoxic impurities (GTIs) in APIs that could impact negatively on human health. There is an increasing scientific interest in this field, as is illustrated in Figure 1, from data obtained from the ISI Web of Science showing the number of publication hits on “genotoxicity” and on “genotoxic impurity”.1 The graph based

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Received: March 7, 2012

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DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX

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genotoxicity according to chemical structures. These systems follow either rule-based or quantitative structure−activity relationship models (QSAR). Rule-based systems are derived from identified mechanisms of action of chemicals in the cell genome or metabolic proteins. This approach was introduced by Miller and Miller in 19774 and followed by other authors. In spite of its mechanistic clarity, it has been criticized as being based only on single interactions and therefore failing to be comprehensive. QSAR models may use several inputs simultaneously, e.g., information on Ames test results, log P, molecule polarity and electrical distribution, and chemical substructures. The use of QSAR models is particularly useful for the prediction of the biological effect of a broad range of chemicals and new molecules with a high degree of accuracy. This subject is further explored in several studies, and examples include comparison of the use of three models for prediction of Ames genotoxicity5 and presentation of different case studies.6 QSAR models commonly used for determination of structural alerts to predict genotoxicity are the MULTICASE and the deductive estimation of risk from existing knowledge (DEREK) ones.7−9 However, for numerous chemical classes, structural alerts overpredict mutagenicity when they do not take into account factors such as high molecular weight, hydrophilicity, high reactivity, steric hindrance, molecular symmetry, and facile metabolism.10,11 On the other hand, their presence in the manufacture of APIs is not stochastic, since these genotoxic chemicals often have specific inherent roles in the chemical routes used in API synthesis. The presence of such chemical in the reaction is a result of their introduction into the reaction in stoichiometric or catalytic amounts or as solvents, as well as their formation as side products. The presence of genotoxins is usually inherently controlled during API manufacture, as several stages of intermediate API isolation and purification are included in the production process, during which most of the GTIs are, together with other impurities, removed. Additionally, many of the synthetic reaction sequences initially designed for production of new drugs are often further improved through optimization of reaction conditions or by substituting with different reaction steps. Such improvements aim at higher yields, reaction selectivity, and more efficient use of reactants, which results in lower amounts of unreacted compounds and side products formed. Nevertheless, production of APIs with low GTI content is a major concern for API-manufacturing companies. Ideal solutions consist of the simplest possible, robust process, using cost-effective reagents to obtain high product yields through selective reactions and purification steps. Development and validation of such processes in a timely manner are important for the industry, and as such, it is important to be aware of the chemical mechanisms in which genotoxic compounds are involved, whether as reagents or reaction side products, and of existing strategies to circumvent their use or remove them from postreaction streams. In addition to the Introduction and concluding remarks, this review includes the following sections: Section 2 provides a brief description of genotoxic mechanisms and a risk analysis, as well as the regulatory approaches taken concerning this issue. Further reviews on specific topics of risk assessment, 12 toxicology,13 and mechanism of action14 of such compounds can be found in the literature. Section 3 of this review focuses on GTIs related to starting materials, reagents, reactants, catalysts, and solvents with

Figure 1. Importance of genotoxicity demonstrated by the increasing number of publications on the topic, resulting from an ISI Web of Science search on “genotoxicity” and “genotoxic impurity”.

on the former search shows the overall importance of the field of genotoxicity, including chemistry, analytical methods, manufacturing, purification, diseases, medical aspects, genotoxicity tests, mechanism of action, assessment, and environment. The latter search illustrates the increasing attention of industry to GTIs, mainly related to drugs and food. Compounds categorized as GTIs actually include a broad range of unrelated chemicals with very different structures and from very different chemical families. From 4000 compounds tested, 44 molecular structures were correlated with mutagenicity and correlated highly with electrophilic reagents, such as epoxides (63%), aromatic amines (49%), and primary alkyl monohalides (46%).2 Aromatic amines are not electrophiles, but their decomposition leads to the formation of electrophilic reactive species such as aryl nitrenium ion. In section 3.1.6 examples of aromatic amine reactants are described. These compounds have a shared ability to react with DNA, resulting in an associated carcinogenic risk. However, from a chemical point of view, they do not have common chemical−physical properties or chemical structural elements that can contribute to easy identification. Experimental assessment of genotoxicity test models, such as the Ames test, allows direct study of genotoxicity, and the Committee for Medicinal Products for Human Use (CHMP) has defined GTIs as impurities that have been demonstrated to be genotoxic using such genotoxicity test models. The Ames test, developed in the early 1970s by Bruce N. Ames, is an experimental procedure to evaluate the potential carcinogenicity of chemicals, based on mutagenicity effects on Salmonella typhimurium histidine auxotrophic mutants strains. It became widely used due to its simplicity, low cost, and quick analysis without the need for animal testing. As discussed in ICH Q3A and Q3B, actual impurities in API are the ones that exceed the reported threshold when the lot is released or arise, for example, as degradation product, during storage and distribution over the shelf life of the API, whereas potential impurities may or not actually be present in the API but are identified as the ones that can theoretically arise during manufacture or storage. In the particular case of GTIs, “potential GTIs” are the ones that have structural alerts, i.e., functional chemical groups, for genotoxicity but have not been experimentally assessed; note that here potentially is not related with the presence or absence of the impurity.3 In silico systems are commonly used to identify structural alerts and predict B


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The authors believe the knowledge systematically gathered in the present review will help in the assessment of both new and alternative synthetic routes when taking into account the sources of GTIs and allow the pharmaceutical R&D scientists to make more confident decisions when embarking on the selection of alternative synthetic routes. In addition, reaction optimization or purification strategies should be greatly simplified. Early realization that a synthetic route could give rise to the presence of possible genotoxins in the API will improve timelines and safety by avoiding wasted effort on processes with no long-term future and, in addition, directing the focus on the relevant purification technology. Due to the interdisciplinary nature of drug manufacturing, the intended audience of this review covers organic chemists, process engineers, and project managers among other contributors in various phases of drug development. The fact that a wide audience is targeted by the present review calls for detailed descriptions at some points which might be common knowledge for experts in the particular field (this information is provided in eight charts throughout the review).

genotoxic effects and related genotoxic side products. Impurities structurally related to specific APIs (e.g., genotoxic sulfonate ester side products during the synthesis of the steroid mometasone15,16) are outside of the scope of this review (Figure 2). The book “Genotoxic ImpuritiesStrategies for

2. GENOTOXICITY: MECHANISMS, RISK AND REGULATION Although it has now been several years since the introduction of the first EMA guideline on limits of genotoxic impurities, the terms genotoxicity, carcinogenicity, and mutagenicity are often misused by chemists. The term genotoxicity covers a wider range of genetic damage, regardless if such damage is or is not corrected through a cell DNA-repairing mechanism. A mutation represents a permanent change in the genome, which can lead to phenotype change, and a mutagen is a substance able to increase the frequency of such changes. A carcinogen is a substance that induces unregulated growth processes in cells, through damage to the genome or cell metabolic effects, eventually leading to cancer. In other words, mutagenicity refers to processes leading to genetic change, and carcinogenicity refers to processes resulting in tumor development, which may result from mutagenic processes.18,19 The mechanism of action of genotoxins involves an electrophilic attack on the nucleophilic center(s) of the DNA, these being nitrogen and oxygen atoms of pyrimidine and purine bases and the phosphodiester backbone, which could, in some circumstances, lead to strand breaks (Figure 3). Bidentate genotoxic agents can react with two nucleophilic sites, resulting in (i) one single molecule giving a bicyclic or tricyclic system; (ii) involvment of two different molecules in the same or the opposite DNA strand, affording inter- or intrastrand crosslinkages, respectively; or (iii) linking a protein and a DNA strand, giving a DNA−protein adduct.14 Besides the chemical nature of the genotoxic agent, the stereospecificity of the reactions also depends on steric factors and nucleophilicity; for instance, the most nucleophilic sites of the DNA bases are endocyclic nitrogens, such as N3 and N7 of guanine and adenine, and on the contrary, exocyclic oxygens are less nucleophilic.18,20 Impurities, especially genotoxic impurities, have been at the center of increasing regulatory and industry attention in the past decade. The timeline of key actions toward those regulations is shown in Figure 4. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) was setup for the analysis of scientific and technical aspects of pharmaceutical product registration and includes the main players in the field,

Figure 2. Sources of GTIs in API streams.

Identif ication and Control” edited by Teasdale provides an exhaustive discussion on the identification and control of such impurities in API manufacturing.17 The major focus of this review is provided in section 3, which comprises a thorough description of chemical synthesis of several pharmaceuticals. The objective of this review is to contribute to an easy identification and mapping of GTI occurrence in chemical synthetic routes, highlighting the importance of GTIs in the manufacturing of APIs. Therefore, the focal point of this review is to improve awareness of different entry points for GTIs over the API synthesis. Case histories and synthetic examples selected include 93 different chemical schemes used in the synthetic routes of APIs or intermediates of 100 different drugs. Nine different chemical families are present because they are used as reagents or catalysts or are formed during synthesis. In recognition of the importance of risks posed by residual toxic solvents left in API formulations, an additional section highlights the potential genotoxicity and carcinogenicity of common solvents used in drug synthesis. Section 4 discusses approaches for mitigation of GTI content. A variety of different strategies are used to avoid/ reduce GTIs during industrial implementation, as disclosed in patents and academic publications. Section 4 includes two subsections focused on synthetic approaches and on API purifications. Examples include changing the reaction route involving a GTI reactant and the optimization of reaction conditions to mitigate the amount of unreacted genotoxic reactant left in the postreaction stream. The section on API purification strategies briefly describes (i) conventional purification techniques, such as recrystallization, chromatography, and distillation, and (ii) emerging technologies, such as organic solvent nanofiltration, supercritical extraction, and molecular imprinting−with the aim to expand the chemists’ and engineers’ toolbox to address API detoxification from GTIs. C


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Figure 3. Attack on the DNA by genotoxins, where the arrows indicate the targeted nucleophilic sites of the DNA bases (based on Madeleine Price Ball’s figure, GNU Free Documentation License).

namely, pharmaceutical regulatory authorities and experts from the pharmaceutical industry from Europe, the United States and Japan. In 1995, the ICH Q3 guidelines did not yet use the term genotoxic but “unusual toxicity”, which was a clear reference to many of the genotoxic impurities. Five years later, in 2000, PharmEuropa published the first article where a specific regulatory concern with genotoxic impurities, namely, the formation of sulfonate esters in API salt formation, was disclosed.21 Actually, the awareness of the formation of this class of genotoxic impurities has gained importance, and therefore, this review includes a specific subchapter dedicated to this particular class of GTIs. Two years later, the Committee for Proprietary Medicinal Products (CPMP) published the first draft position paper on GTIs showing sufficient evidence for the existence of a threshold mechanism in the toxicity of such compounds. This position paper challenged the scientific and industrial community to seek GTI-free routes to APIs or, when not possible, to provide a justification why the presence of GTIs is unavoidable. At that time, it was argued that in vivo studies would put test subjects at a nonjustifiable risk. Therefore, the model of a virtual safe dose concept, previously used in the food industry, was suggested as an alternative and the terminology “as low as technically feasible” was introduced. This model was the basis to the later introduction of the threshold of toxicological concern (TTC) concept. A draft guideline on the limits of GTIs was released in 2004 by the Committee on Human Medicinal Products (CHMP) from EMA and the TTC concept was introduced.22 The TTC is a concept that refers to the establishment of a level of exposure for all chemicals irrespective of the existence of chemicalspecific toxicity data, below which there is no appreciable risk to human health. It is assumed that a low level of exposure posing a negligible risk can be identified for any chemical based on its structure. The TTC concept was first introduced in the food

Figure 4. Timeline of key actions toward a regulation on GTI control in APIs.

industry and focused only on food additives; however, it evolved continuously as its broader applicability than simply to chemicals in food and its potential value in the assessment of risks in other exposure scenarios were realized.23 The brief history of TTC is summarized in Figure 5.24−26 The FDA and EMA have agreed on the implementation of the TTC concept that sets a limit of 1.5 μg day−1 for known and potential carcinogens, unless experimental evidence justifies higher limits. Higher levels can be applied in shorter-term studies during the clinical testing of the APIs (Table 1). The rational behind such low values is to ensure that even if a substance was later found to have negligible carcinogenic risk, no issues concerning safety would arise.27 An exhaustive effort has to be made by the industry to meet such requirements, and since many potentially genotoxic agents turned out to pose far less risk than originally supposed, the TTC approach is considered to be conservative. Nonetheless, such low GTI thresholds are here to stay, and the industry has been addressing this challenge with a variety of control and purification strategies. Moreover, the terminology of “as low as reasonably practical” (ALARP) was applied for GTIs; the requirement to introduce alternative routes or processes when available was dropped, and no guidance relating to permissible doses during short-term D


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Muller28 proposed acceptable limits for GTIs in APIs linked to duration of exposure, i.e., a staged TTC approach. The same document also defined five separate classes for the impurities based on a structure−activity relationship (SAR). A separate specific position paper addressing excipients was subsequently prepared in 2007 by the Committee For Human Medicinal Products (CHMP) of the European Medicines Agency.29 In 2007, EMEA was the first authority to issue and implement detailed guidelines30 on how such impurities should be controlled, shortly followed by the FDA, which issued a draft guideline in 2008.31 The main difference between the FDA draft and the EMEA guideline is in the requirements for the degree of lower GTI limits. FDA applied an additional safety factor of 3, while EMEA applied a factor of 10. These require specific genotoxicity tests for impurities above the ICH qualification thresholds and differences in staged TTC values. In 2010, the “Questions and Answers” of the Safety Working Party (SWP) introduced minor adjustments to the duration limits proposed by Muller, and stated that a “cause of concern” is a material with either a pre-existing or new genetic toxicology indications. Also, in 2008, the European Directorate for the Quality of Medicines & HealthCare (EDQM) in a PharmEuropa article commented that structurally alerting functionality alone does not constitute a “cause for concern” without actual toxicology data. More recently, the ICH Guideline M7 on Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals To Limit Potential Carcinogenic Risk was adopted by CHMP as of September 25, 2014,32 and information is provided on how to calculate TTC when several GTIs of similar structures are present with similar mechanisms of action, and recommendations are provided to harmonize the EMA guideline and FDA draft guideline. For further reading, the authors recommend the ICH M7 guideline and its critical evaluation.23,26,32,33

3. CHEMICAL CLASSES OF COMMON GENOTOXIC IMPURITIES Regardless of the strategy selected for GTI mitigation, before it is implemented it is crucial to identify and map the occurrence of the GTIs in each of the API manufacture steps. Table 244 provides a list of functional groups with structural alerts for genotoxic activity associated with the various reactions commonly employed today in pharmaceutical development and manufacturing. These include many of the “name reactions” of organic chemistry. Using this table as starting point, this review provides an overview of genotoxic impurities, including detailed individual chemical classes. The examples provided in this review include the use or appearance of GTIs from different chemical families, and within each family they are organized according to their role in the chemical reaction, namely, as a stoichiometric reagent, a catalytic reagent, or side product.

Figure 5. A brief histroy of the TTC principle.

clinical trials was provided. In 2006, the Pharmaceutical Research and Manufacturers of America (PhRMA) led by

Table 1. Proposed Allowable Daily Intake (ADI) for GTIs of Unknown Carcinogenic Potential during Clinical Development Duration of exposure (month) ADI (μg/day)

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120a or 0.5%,b whichever is lower




1.5b,c

Probability of not exceeding a 10−6 risk is 93%. bOther limits (higher or lower) may be appropriate. cProbability of not exceeding a 10−5 risk is 93%, which considers a 70-year exposure.

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Table 2. Common Synthetic Transformations Related to Genotoxic Impurities Bond formation Alerting group

C−O C−C C−N

AHCa

Associated reactions

Examples

(1) Genotoxic Reagents/Catalysts Applied Directly Phophonate esters Alkyl halides

X X

Dialkyl sulfates Epoxides Aldehydes Hydrazines TEMPO Aromatic amines compounds Aminoaryls Boronic acids Aromatic nitro compounds

X X X

X

X

X X X X X

X X

X X

X

X

Homer−Wadsworth−Emmons olefination Williamson ether synthesis; Heck, Sonogashira, Kumada Pd-catalyzed crosscouplings O- and N-alkylations Sharpless asymmetric epoxidation Aldol and Claisen condensation Fischer indol synthesis, common heterocyclic precursor Oxidation of alcohols Common feedstock for aromatic structures Common intermediate

X X

Common starting material and intermediate

refs 34−37 section 3.1.1 section 3.1.2 section 3.1.3 refs 38−40 section 3.1.4 section 3.1.5 section 3.1.6 section 3.1.7 refs 41−43

(2) Side Reactions Forming GTIs Sulfonates

Alkyl halides Acetamide

Stoichiometric amounts 3.2.2.1. API Salt Forming Agents 3.2.2.2. Good Leaving Groups 3.2.2.3. Cyclizations 3.2.2.4. Protecting Groups 3.2.2.5. Sulfonamide Formation 3.2.2.6. Chiral Auxiliary Group in Resolution of Enantiomers Catalytic amounts 3.2.3.1. Cyclizations 3.2.3.2 Protecting Group Manipulations 3.2.3.3. Mitsunobu Rearrangement 3.2.3.4. Double Bond Migration 3.2.3.5. Enamine−Amine Reduction 3.2.3.6. Esterification Reactions between alcohols and acids Cοmmon building blocks

section 3.2.2

section 3.2.3

section 3.2.4 section 3.2.5

(3) Solvents Solubilize reagents and reactants a

section 3.3

AHC means aromatic heterocycle.

3.1. Genotoxic Compounds Used as Reactants

have been shown to directly alkylate critical biologically active macromolecules, such as proteins and DNA.45 Geminal, vicinal, and ω-bifunctional alkyl halides are also directly used in API synthesis, of which ω-alkyl dihalides are common linking agents due to their ability to connect API intermediates via consecutive alkylation. It was hypothesized that bifunctional alkanes cause genotoxic damage by the glutathione-dependent pathway and consequent formation of toxic methanethiol.46 Nitrogen and sulfur mustards (e.g., 2,2-dichlorodiethyl sulfide) represent a special class of alkyl halides and have been used as chemical weapons. They are potential alkylating agents and their toxicity is attributed to cross-linking between DNA strands.47 The source of alkyl halide in APIs streams can be derived not only from direct use of alkyl halides but also from side reactions between alcoholic solvents and hydrogen halides or dequaternization of ammonium salts. It is worth mentioning that alkyl halides, such as methyl or ethyl chloride, deriving from low molecular weight alcohols, are volatile and readily purged from the API during the drying process. On the other

Reactants used in chemical synthesis are usually selected due to their appropriate reactivity; however, this very same reactivity could result in genotoxicity. Often such reactants are not fully consumed, persist in the reaction mixture, and can be carried forward in the reaction sequence. Seven classes of reactants used in API synthesis were selected as examples to be presented in this section, including two types of alkylating agents, alkyl halides, and dialkyl sulfate; epoxides used in several addition reactions; hydrazine, a strongly reducing nitrogen base; TEMPO, a cyclic amine oxide radical; aromatic amines, used as building blocks; and boronic acids, used in carbon−carbon coupling reactions. Other well-known classes of potentially genotoxic impurities, aldehydes and aromatic nitro compounds, which are mainly used as starting materials and not reactants, are not included in this section. 3.1.1. Alkyl Halides. Methyl, ethyl, and propyl halides are used widely as industrial alkylating agents. Although the mechanism of their toxicity is still not fully understood, they F


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the synthesis of ocaperidone53 and azimilide54 applying 1-chloro2-bromoethane, 1-chloro-4-bromobutane, respectively. During the synthesis of cerivastatin, the hydroxyl group of a carbinol is converted to the corresponding methyl ether with sodium hydride and methyl iodide (Scheme 3).55 S-Methylation with genotoxic methyl iodide is used during the synthesis of the amidine-based fibrinogen receptor antagonist lamif iban. In the final synthetic step, the tripeptide-like intermediate reacts with hydrogen sulfide, leading to the iminothiol addition intermediate, followed by alkylation with methyl iodide, which converts sulfur to the methylthio derivative. Treatment with ammonium acetate leads to displacement of the good leaving group, methyl mercaptide, by ammonia, affording lamifiban (Scheme 4).56 During the synthesis of eldacimibe, Meldrum’s acid reacts with carbon disulfide in the presence of a base, leading to condensation and formation of a bismercaptide. This transient dianion is reacts in situ with methyl iodide to give a highly reactive intermediate with two good leaving groups (Scheme 5).57 The synthesis of guanidine-containing fibrinogen antagonist efegatran involves the N-methylation of a N-carbobenzyloxy (Cbz) derivative with methyl iodide in the presence of a base, leading to the corresponding N-methyl derivative (Scheme 6).58 Alkyl halides are also used to obtain quaternary ammonium salts. During the first synthesis of the potential cholinergic agonist59 milameline, alkylation of the pyridine intermediate with methyl iodide leads to the quaternary salt as depicted in Scheme 7. Treatment with sodium borohydride leads to the dihydropyridine.60 Latanoprost is used for the treatment of high intraocular pressure in cases where the patient has open-angle glaucoma or ocular hypertension.61 During its synthesis, 2-iodopropane is directely used, which may result in the presence of this alkyl halide as an impurity in the final product (Scheme 8).62 Highly genotoxic nitrogen mustards are usually used for the formation of piperazine-type drug substances. The first synthesis of the potential antipsychotic agent mazapertine involves the use of genotoxic 2-bromopropane in an aromatic O-alkylation and N,N-bis(chloroethyl)amine in a cycloalkylation to give the piperazine precursor (Scheme 9).63 As in the previous example, the reaction of 2,3-dichloroaniline with nitrogen mustard gives an arylpiperazine derivative, a key intermediate during the synthesis of the antipsychotic agent aripiprazole. The side-chain connector is then incorporated by

hand, they can get trapped in the API crystal matrix and lead to trace impurities that are to be controlled.48 The sources for genotoxic alkyl halide impurities in APIs is summarized in Table 3 on the basis of the source of the impurity, reaction types, and examples of API synthesis. Table 3. Sources and Reaction Types That May Lead to Genotoxic Alkyl Halide Impurities in Drug Substances GTI source Dequaternization Direct use of alkyl halide reagents

Application

API synthesis example

DMTMMa coupling reactions C-alkylation

Antibiotics, peptides, alkaloids Fexofenadine, anastrozole Alisiren, cerivastation, mazapertin Lamifiban, eldacimibe Efegatran, aripiprazole Milameline Latanoprost Mazapertine, aripiprazole Capecitabine, sitagliptin Xemilofiban Sunitinib Bortezomib Pazopanib Conivaptan, pazopanib

O-alkylation S-alkylation N-alkylation Quaternization Esterification Cycloalkylation Use of hydrogen halides in alcoholic solvents

Cyclization Decyclization Decarboxylation Cleavage N-arylation Salt formation

a

DMTMM stands for 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride.

The synthesis of fexofenadine, an antihistaminic agent, involves the base-catalyzed C-methylation of 4-bromophenylacetonitrile with genotoxic methyl iodide, yielding the dimethyl derivative, as illustrated in Scheme 1.49 In a similar reaction, an intermediate in the synthesis of the bis-acetonitrile aromatase inhibitor anastrozole is submitted to exhaustive alkylation using sodium hydride and methyl iodide.50 Alkyl halides are often used directly for C-, N-, O- and Salkylation. Aliskiren was the first molecule of a new group of drugs, renin inhibitors, which treat primary hypertension.51 One of the first steps in its synthesis involves the O-alkylation of isovanillin with genotoxic 1,3-dibromopropane in a Williamson-type ether synthesis, as depicted in Scheme 2.52 Further examples for the use of 1,2- and 1,3-dihaloalkanes are

Scheme 1. C-Alkylation Step in the Synthesis of (a) Fexofenadine or (b) Anastrazole Can Result in Traces of Genotoxic Methyl Iodide

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Scheme 2. O-Alkylation of Isovanillin during the Synthesis of Aliskiren Using Genotoxic 1,3-Dibromopropane

Scheme 3. Use of Methyl Iodide in the Preparation of Cerivastatin

Scheme 4. S-Alkylation with Methyl Iodide during the Last Synthetic Step of Lamifiban

Scheme 7. Quaternization by Means of Methyl Iodide during Milameline Synthesis

Scheme 8. Direct Use of Genotoxic 2-Iodopropane in the Synthesis of Latanoprost

Scheme 9. Use of Genotoxic 2-Bromopropane and Nitrogen Mustard during the Synthesis of Mazapertine

Scheme 5. S-Alkylation with Methyl Iodide during the Synthesis of Eldacimibe

alkylation of the second nitrogen of the piperazine ring with the genotoxic reagent 4-chloro-1-bromobutane (Scheme 10).64 APIs streams may contain genotoxic alkyl halides impurities when a reaction takes place in alcoholic solvent at reflux temperature in the presence of hydrogen halides or alkali halides and strong acids. These reaction conditions may be applied in cyclization, decarboxylation, sulfonyl cleavage, Narylation and API salt formation. Note that this list is not

exhaustive. For instance, capecitabine is a chemotherapeutic agent used in the treatment of metastatic breast and colorectal cancers.65 During its synthesis, D-ribose is heated under reflux in methanol in the presence of concentrated HCI and acetone

Scheme 6. N-Methylation with Genotoxic Methyl Iodide during the Preparation of Efegatran

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Scheme 10. Use of Nitrogen Mustard and a Dihaloalkane in Piperazine Formation and N-Alkylation Reactions, Respectively, During the Synthesis of Aripiprazole

Scheme 13. Ethanolic Hydrogen Chloride Is Used to Open a β-Lactam during the Synthesis of Xemilofiban

Scheme 14. Ethanol−HCl-Assisted Decarboxylation May Result in Genotoxic Ethyl Chloride during Sunitinib Synthesis

to provide the cyclic methyl 2,3-O-isopropylidene-D-ribofuranoside; thus, the product may contain genotoxic methyl chloride (Scheme 11).66 Scheme 11. Methanol-HCl Assisted Cyclization May Result in Genotoxic Methyl Chloride during Capecitabine Synthesis

methanol to afford a primary amine with cleavage of the Nsulfinyl group (Scheme 15).72 Pazopanib is a tyrosine kinase inhibitor that blocks tumor growth and inhibits angiogenesis.73 During its synthesis, the 2chloro group of pyrimidine reacts with 5-amino-2-methylbenzenesulfonamide in 2-propanol and HCl at reflux to deliver pazopanib hydrochloride (Scheme 16).74 Since the 2propanol−HCl is introduced in the final step of the API synthetic route, there is a high potential for the presence of traces of genotoxic isopropyl chloride in the final drug substance. Conivaptan has been approved by the FDA for the treatment of hospitalized patients with euvolemic and hypervolemic hyponatremia.75 During the last synthetic step, ethanol−HCl is used as a salt-forming agent to form the imidazobenzazepine hydrochloride salt (Scheme 17).76 Therefore, once again the potential for the presence of traces of genotoxic ethyl chloride is high. 3.1.2. Dialkyl Sulfates. The most common dialkyl sulfates used in the pharmaceutical industry are the methyl and ethyl derivatives, the latter having been used as a chemical weapon.77 Being a strong methylating agent, dimethyl sulfate (DMS) is used to introduce a methyl group to atoms featuring unshared electron pairs, such as oxygen, nitrogen, carbon, sulfur, phosphorus, and some metals. Compared with the alkyl halide-type methylating agents, DMS is more favorable due the higher reaction rate and lower possibility of byproduct formation.78 Usually methylating with dimethyl sulfate requires the presence of a base, either (i) to intensify the reactivity of the reaction site (e.g., converting the phenolic hydroxyl group of vanillin to sodium phenolate during the first step of papaverine synthesis) or (ii) to neutralize the byproducts of the

A practical manufacturing route was developed for the synthesis of the triazole heterocycle of sitagliptin, which is a drug used to treat type 2 diabetes.67 Exposure of the amidine intermediate to methanol−HCl gives the desired triazole in a cyclization reaction, which can be directly isolated as its HCl salt by filtration (Scheme 12).68 Alcoholic hydrogen chloride can also be used for ring openings. Xemilof iban is used to treat cardiovascular disorders, and during its synthesis, the treatment of a β-lactam intermediate with ethanolic hydrogen chloride results in ring opening to afford the ethyl β-alaninate derivative (Scheme 13).69 During the synthesis of antiangiogenic sunitinib, selective hydrolysis of a tert-butyl ester is followed by decarboxylation, which is accomplished by stirring the tetrasubstituted pyrrole intermediate in HCl and ethanol, which may form genotoxic ethyl chloride (Scheme 14).70 Bortezomib is an intravenously administered, first-in-class, proteasome inhibitor.71 During its synthesis, the N-sulfinyl-αamino boronate ester intermediate is treated with HCl in

Scheme 12. Methanol−HCl-Assisted Cyclization May Result in Genotoxic Methyl Chloride during Sitagliptin Synthesis

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Scheme 15. Methanol−HCl-Assisted Cleavage of the N-Sulfinyl Group May Result in Genotoxic Methyl Chloride during Bortezomid Synthesis

Scheme 16. 2-Propanol−HCl-Assisted N-Arylation and Salt Formation May Result in Genotoxic Isopropyl Chloride during Pazopanib Synthesis

Table 4. Applications of Dialkyl Sulfates during API Manufacturing Application


O-alkylation S-alkylation N-alkylation Olefin alkylation Aromatic alkylation Amine transformation

Rotigotine Rofecoxib Ralitoline Alvimopan Telmisartan Clemastine

developed as an agent against vomiting and nausea, an antidote for nerve gas, a radiopaque medium for diagnostic aid, and a diuretic, respectively. A typical example of O-alkylation with dimethyl sulfate is the first synthetic step of the dopaminergic agent rotigotine, which was developed for the treatment of Parkinson’s disease83 and restless legs syndrome.84 Its preparation starts with the transformation of the dihydroxynaphthalene to its methyl ether by means of dimethyl sulfate (Scheme 18).85

Scheme 17. Salt Formation of Conivaptan with HCl in Ethanol May Result in Genotoxic Ethyl Chloride Formation

Scheme 18. Genotoxic Dimethyl Sulfate Is Typically Used To Form Ethers of Phenols

A typical example of N-alkylation is the dimethly sulfateassisted N-methylation of an N-heterocycle during the final synthetic step of antiepileptic ralitoline (Scheme 19).86

reaction, monomethyl sulfate (MMS) and sulfuric acid, for example, in the case of the methylation of aliphatic alcohols. Usually only one of the methyl groups of DMS reacts in the methylation reaction because the MMS formed is a much weaker alkylating agent than the original DMS. Although for research purposesin small-scale preparationsthere is little necessity to use both methyl groups, in manufacturinglarge scale productionin cases where the substrate is reactive enough to be methylated by MMS (e.g., the sodium salt of mercaptans), it is desirable to utilize both groups if possible. In order to do so one can adjust the reaction conditions as follows: (i) increase the reaction temperature and (ii) apply anhydrous conditions and avoid excess base to suppress the competing reactions with water and the hydroxide ion. To obtain the best results, the base can be introduced to the reaction mixture continuously in small portions as the reaction proceeds but limiting this to the extent of the acid formed to minimize the competing reaction with the hydroxide ion. Nonaqueous solvent−base systems such as DMF/K2CO3 can be used in certain cases. Most common applications of dialkyl sulfates in the pharmaceutical industry are summarized in Table 4. Dialkyl sulfates are used in the production of metoclopramide,79 pralidoxime,80 metrizoic acid,81 and merf ruside,82 APIs

Scheme 19. Methylation of a Pyrrolidine with Genotoxic Dimethyl Sulfate during Ralitoline Synthesis

The peripherally acting μ-opioid antagonist alvimopan87 can only cross the blood−brain barrier partially and does not have the usual side effects of the opioid agonists, such as constipation, while not losing the analgesic effect. During its synthesis (Scheme 20), the treatment of the styrene derivative with butyllithium and DMS leads to methylation at the 4position of the 1,2,3,4-tetrahydropyridine ring, since the negative charge on the quaternary carbon atom in that position is enhanced.88 J


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Scheme 22. S-Methylation of a Rofecoxib Intermediate

Scheme 20. Genotoxic Dimethyl Sulfate is Directly Used during the Synthesis of Alvimopana

manufacturing. They easily participate in epoxide-ring-opening reactions with alcohols, amines, halides, organometallics, cyanides, sulfides, aromatic compounds, and active methylene groups. On the other hand, the high reactivity of these compounds makes them genotoxic, as their two electrophilic carbon atoms can react with the DNA nucleophilic centers, giving alkylated products.14 Substituted epoxides, such as 2,3epoxypropanol (glycidol), 1-chloro-2,3-epoxypropane (epichlorohydrin), or 1,2-epoxy-3-butene, are often used as building blocks during the synthesis of APIs. They are often subjected to epoxide-ring-opening reactions, and since they are bifunctional, they usually act as linking agents or can form heterocycles. These substituted epoxides react primarily at the lesssubstituted and more-accessible carbon, due to steric hindrance. However, in the case of substituents that increase the positive charge at the adjacent carbon (such as aromatic or vinyl group), both carbons could potentially react.101 A recent review by Elder et al. provides guidance for analytical chemists faced by the need to control such impurities at trace levels due to their potential genotoxicity in drug products.102 An intermediate of the antiretroviral drug darunavir is prepared using phenylmagnesium bromide and commercially available 1,2-epoxy-3-butene in the presence of catalytic CuCN to furnish the corresponding allylic alcohol (Scheme 26).103 Rivaroxaban is an oral anticoagulant drug invented and manufactured by Bayer for the treatment of thromboembolic diseases.104 A key intermediate in the synthesis of rivaroxaban is (S)-2-(phthalimidomethyl)oxirane, of which alternatives are presented in the literature for its synthesis: (a) condensation of (S)-2,3-epoxy-1-propanol (glycidol) and phthalimide under Mitsunobu reaction conditions105 and (b) condensation of (S)1-chloro-2,3-epoxypropane (epichlorohydrin) and phthalimide in the presence of benzyltrimethylammonium chloride (BTMAC) as phase-transfer catalyst (Scheme 27).106 Azelnidipine is a calcium channel antagonist that selectively blocks voltage-dependent Ca2+ influx and is used for the treatment of hypertension.107 The patented synthesis of azelnidipine involves a heterocyclization reaction, in particular, an azetidine formation by means of reacting benzhydrylamine and epichlorohydrin without solvent to give 1-benzhydryl-3hydroxyazetidine in a slow process (Scheme 28).108 The structurally rather complex agent zosuquidar has shown promising activity against multidrug resistance in cancer chemotherapy.109 During its synthesis, 5-hydroxyquinoline

a

Note that the carbodiimide reagent forms a potentially genotoxic dialkylurea.

Dialkyl sulfates are also used to introduce an alkyl group into an aromatic ring; for instance, o-nitroaniline is methylated with dimethyl sulfate during the synthesis89 of telmisartan, which is an angiotensin II receptor antagonist (angiotensin receptor blocker, ARB) used in the management of hypertension (Scheme 21).90 S-Alkylation is demonstrated in the synthesis of a rofecoxib intermediate. Rofecoxib is a nonsteroidal anti-inflammatory drug (NSAID) marketed by Merck.91 Scheme 22 shows 1-(4mercaptophenyl)ethanone treatment with dimethyl sulfate in the presence of sodium hydroxide to form 4-(methylthio)acetophenone.92 Cimetidine was the first blockbuster drug, invented by Nobelprize winner James Black.93 It is a histamine H2-receptor antagonist that inhibits the production of acid in the stomach and it has been shown to have antitumor effects.94 After the reaction of carbon disulfide and cyanamide in the presence of a base, dimethyl sulfate is added to the reaction mixture in order to methylate both sulfur atoms in a consecutive reaction (Scheme 23).95 Quaternization of tertiary amines is often carried out with dimethyl sulfate,96 which is a preliminary step to an amine− nitrile transformation. Clemastine is used as an antihistamine and anticholinergic medicine with sedative effects.97 During its synthesis dimethyl sulfate is used to form a quaternary amine, which is a good leaving group and is displaced with a nitrile group in the following step (Scheme 24).98 Another example is the quarternization of an aromatic amine during the last synthetic step of neostigmine, a parasympathomimetic drug that acts as a reversible acetylcholinesterase inhibitor.99 Dimethyl sulfate is used in the last step of the API synthesis to form the quaternary ammonium salt, neostigmine, as shown in Scheme 25.100 3.1.3. Epoxides. Epoxides are the simplest cyclic ethers, featuring three ring atoms. Due to the large ring strain associated with the three-membered ring, epoxides are highly reactive molecules and thus are often used as reagents in API

Scheme 21. Use of Genotoxic Dimethyl Sulfate in the Synthesis of Telmisartan

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Scheme 23. Use of Dimethyl Sulfate for Consecutive S-Methylation during the Synthesis of a Cimetidine Intermediate

Scheme 24. Quaternization of a Tertiary Amine To Form a Good Leaving Group To Be Displaced with a Nitrile in Synthesis of Clemastine

Scheme 29. Glycidol Tosylate in an Aromatic O-Alkylation during the Synthesis of Zosuquidar

Scheme 25. Quaternary Ammonium Salt Formation by Means of Dimethyl Sulfate

Scheme 26. Use of 1,2-Epoxy-3-butene during the Synthesis of Darunavir

Scheme 30. Use of Chiral Glycidol Mesylate during the Synthesis of Lubazodone

Scheme 27. Condensation of (S)-2,3-Epoxypropanol (glycidol) or (S)-1-Chloro-2,3-epoxypropane (epichlorohydrin) and Phthalimide during the Synthesis of Rivaroxaban

carbon-centered radicals, and oxygen-centered radicals, which are considered to be highly reactive species. For these reactive intermediates, DNA alkylation and other DNA lesions have been reported.113 Recent reviews have been published on hydrazine and its derivatives related to (i) the mechanisms of chemical carcinogenicity by Benigni and Bossa14 and (ii) the control and trace analysis in drug substances by Elder et al.114 Since hydrazine is a highly reactive base that acts as a reducing agent, it has been used as a synthetic reagent in production of several different types of drugs. Table 5 summarizes the application of hydrazine and the synthetic examples discussed in detail in this section. Hydrazine is a green reducing agent, since only nitrogen gas and water are produced as by-products. A typical example where hydrazine is applied as a reducing agent is the Wolff− Kishner reaction, where a carbonyl groupboth ketone- and aldehyde-typeis transformed into a methylene or methyl

Scheme 28. Azetidine Formation with Epichlorohydrin during the Preparation of Azelnidipine

reacts with the tosyl derivative of glycidol in a convergent sequence, affording the epoxypropyl ether (Scheme 29).110 Lubazodone is an antidepressant compound belonging to the class of serotonin-selective reuptake inhibitors.111 The reaction of a fluoroindanol with the mesylate ester of (R)-glycidol in the presence of base leads to the epoxypropyl ether with retention of configuration (Scheme 30). Treatment of this intermediate with aminoethylsulfonic acid forms the morpholine ring and gives the enantiomerically pure final product lubazodone.112 3.1.4. Hydrazines. The toxicity of hydrazine and its derivatives is ascribed to the generation of carbocations,

Table 5. Applications of Hydrazine in API Synthesis

L

Application


Wolff−Kishner reduction Hydrazide formation Hydrazinolysis Electrophilic addition Heterocyclizations

Sunitinib, ziprasidone Sitagliptin, isoniazid Saquinavir, mofegiline Suritozole Sildenafil, sedoxantrone DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX

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Chart 1. Comparison of Different Wolff−Kishner Reduction Methods

group through a hydrazone intermediate. Chart 1 compares three different modifications of the Wolff−Kishner reduction. The first step is the formation of a hydrazone. Evolution of highly stable nitrogen after successive deprotonation−protonation reactions is the thermodynamic driving force of the transformation. Interestingly, this reaction used to be a method for distinguishing between aldehydes and ketones. The synthesis of tyrosine kinase receptor inhibitor sunitinib applies Wolff−Kishner reduction to form 5-fluorooxindole, as depicted in Scheme 31.115,116 A similar reaction is used for the synthesis of 5-chlorooxindole during the synthesis117 of ziprasidone, which is an antipsychotic agent for the treatment of schizophrenia.118 Reactive hydrazides are useful intermediates during API synthesis, being formed in the reaction of hydrazine with esters, amides, carboxylic acid, and acid halides. During the synthesis of the antidiabetic drug sitagliptin, trifluoroacetic acid ethyl ester is reacted with hydrazine to form the corresponding hydrazide, as shown in Scheme 32,119 which is then converted to the triazole. In another example of hydrazide formation is isoniazid, which is used for the treatment of tuberculosis and depression.120 Its synthesis involves the use of hydrazine in the final synthetic step, where an NH2 group is displaced by hydrazine.121 Hydrazinolysis is a chemical cleavage reaction, in which the hydrazine acts as a nucleophilic agent by attacking the carbon atom of a carbonyl group which has a partial positive charge.

Scheme 31. Hydrazine-Assisted Wolff−Kishner Reduction of Oxindoles during the Synthesis of Sunitinib and Ziprasidone

Such reaction is also used for a mild lysis of protection groups in peptide and sugar chemistry, but probably this scission reaction finds most common application in the Gabriel synthesis in which phthalylhydrazide is produced during the liberation of the desired amine from the phthalyl residue. The synthesis of saquinavir and mofegiline are examples of hydrazineassisted cleavage of N-alkylated phthalimide derivatives (Scheme 33). The peptide derivative saquinavir inhibits the HIV protease enzyme68 and mofegiline is a MAOB (monoamine oxidase B) inhibitor used in the treatment of Parkinson’s M


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Scheme 32. Hydrazide Formation during the Synthesis of (a) Sitagliptin and (b) Isoniazid

Scheme 34. Use of Methylhydrazine in an Electrophilic Addition during the Preparation of Suritozole

disease.122 Part of the synthesis of saquinavir entails the hydrazinolysis of the amido alcohol intermediate removing the phthalimide protecting group to produce the primary amine,123 while the final step of mofegiline synthesis is also characterized by the cleavage of the phthalimide protecting group with hydrazine leading to the free base.124 The side product formed from the hydrazine and the phthaloyl group is 2,3dihydrophthalazine-1,4-dione. Suritozole is a benzodiazepine reverse agonist, investigated as a potential treatment for Alzheimer’s disease.125 Its synthesis (the triazolothione portion) starts with the formation of a thiosemicarbazide by condensation of methylhydrazine with methyl isothiocyanate in an electrophilic addition, as depicted in Scheme 34.126 Hydrazine is a key bifunctional, with two NH2 groups, building block used in the preparation of various heterocyclic compounds via condensation reactions with a wide range of bifunctional electrophiles, such as 1,3-diones or 3-halo ketones or aldehydes, leading to pyrazoles, or imides, giving triazoles in the Einhorn−Brunner reaction. The resulting N-heterocycles are key intermediates in the synthetic routes to APIs. For instance, the preparation of sildenaf il, which is generally known as Viagra, a drug used for treatment of erectile disfunction,127 involves the hydrazine-assisted formation of a substituted pyrazole ring, as shown in Scheme 35.128 A similar reaction takes place during the synthesis of the topoisomerase inhibitor sedoxantrone.129 One of the steps of sedoxantrone synthesis is the condensation of a phenol intermediate with a substituted hydrazine, which leads to pyrazole formation. Though the order of the reaction steps has not been established, formation of the hydrazone, then displacement of the adjacent chlorine by the second nitrogen, and final closure of the pyrazole ring seems plausible.130,131 3.1.5. TEMPO. 2,2,6,6-Tetramethylpiperidin-1-oxyl free radical (trade name TEMPO) is a commonly employed

process reagent and potential process impurity. This compound was evaluated for genotoxic potential, and on the basis of the available, and somewhat conflicting, published data, it is considered to be genotoxic.132 TEMPO is widely used throughout chemical- and biochemistry-related industries as a stable nitroxyl radical. TEMPO is mainly used for oxidations of alcohols to yield aldehydes and ketones or carboxylic acids. A comprehensive review on the use of reactions mediated by TEMPO can be found elsewhere.133 An example of the use of TEMPO is the synthesis of a 5-HT2B receptor antagonist. Eli Lilly synthesizes 2-cyclohexylacetaldehyde by oxidizing 2cyclohexylethanol by the Anelli−Montanari protocol (Scheme 36), affording the aldehyde, which is then used as precursor of a tryptamine and further used as key synthon for the synthesis of the important 5-HT2B receptor antagonist.134 Antagonists of the coreceptor CCR5 have been an intense area of research within the HIV arena over the past decade. Maraviroc is the first-in-class CCR5 antagonist for the treatment of HIV.135 The initial synthesis of maraviroc that produced material for preclinical studies has been published.136,137 One of the reactions is catalyzed by TEMPO, where the alcohol is oxidized to give the required aldehyde, as shown in Scheme 37.138 Darunavir is an antiretroviral drug in the HIV-1 protease inhibitor class for the treatment of multidrug-resistant HIV.139 One step in the synthesis of darunavir is a cyclization that included a TEMPO oxidation, a NaBH4 reduction, and a lipase resolution to provide optically active bis-THF derivative, as illustrated in Scheme 38.140 Oseltamivir141 is a neuraminidase inhibitor and is the most commonly prescribed drug for treatment to combat influenza. The large number of synthetic approaches reported in the literature implicates the importance of this drug. In the example below, TEMPO is used with trichloroisocyanuric acid (TCCA) in the oxidation of a secondary alcohol to give the corresponding ketone intermediate (Scheme 39).142 An azabicyclooctanyl derivative was identified as another novel and potent DPP-4 (dipeptidylpeptidase-4) inhibitor at Novartis for treatment of type 2 diabetes. The preparation of its

Scheme 33. Hydrazinolysis after Gabriel Synthesis during the Preparation of (a) Saquinavir and (b) Mofegiline

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Scheme 35. Pyrazole Formation with Hydrazine during the Preparation of (a) Sildenafil and (b) Sedoxantrone

BYK405879 is a potassium-competitive acid blocker, a promising candidate for the treatment of gastroesophagealreflux-related diseases. The oxidation step of the alcoholic intermediate of BYK308944 was found to be crucial during the synthetic route, and a recently published article by Webel et al. describes in exhaustive detail the development and conditions of the TEMPO-mediated oxidation leading to the desired aldehyde (Scheme 43).150 3.1.6. Aromatic Amines. Although aromatic amines are generally not inherently genotoxic, during metabolic activation, electrophilic species are generated. The main transformation pathway of aromatic amine metabolism is oxidation, producing an N-hydroxy compound that is conjugated as an acetate, sulfate, or glucuronide. Further deconjugation results in a nitrenium ion (ArN+H), which is considered to be the active genotoxin that binds to DNA.151 Aromatic amines are often present as starting material, intermediate, or reagent in pharmaceutical synthesis. During the synthesis of steroids such as mometasone f uroate, in order to replace the 21-hydroxyl group with a chlorine, sulfonyl chlorides are used in a 4-dimethylaminopyridine (DMAP) base catalyzed sulfonylation reaction (Scheme 44).16 In order to control this reaction, a design of experiments to assist in trace analysis of DMAP in glucocorticoid matrices has recently been reported in the literature.152 Besides the sulfonylation reactions, DMAP is also used in acylations,153 esterifications,154,155 amino group protections with Boc,156,157 and silylations.158 Diclofenac is widely used as a nonsteroidal anti-inflammatory drug (NSAID). During its synthesis, the potentially genotoxic 2,6-dichloroaniline is used as a starting material.159 Cucatalyzed N-arylation with 2-chlorobenzoic acid takes place in the presence of KOH (Scheme 45A). The reaction may leave behind unreacted starting material, which has to be controlled.160 In a similar manner, the potentially genotoxic 2,6-dimethylaniline is used for the synthesis of the local anesthetic and antiarrhythmic drug lidocaine (Scheme 45B).161 2,6-Dimethylaniline is condensed with bromoacetic acid, as

Scheme 36. Synthesis of the 5-HT2B Receptor Antagonist Intermediate by Means of TEMPO

Scheme 37. TEMPO Oxidation during the Synthesis of the HIV Drug Maraviroc

azabicyclooctanyl intermediate involves the use of TEMPO in an alcohol−ketone oxidation (Scheme 40).143 SB-462795144 is an azepanone-based inhibitor of the protease cathepsin K, developed for the treatment of osteoarthritis and osteoporosis.145 Scheme 41 shows the oxidation of a carbinol in the final chemical stage of the synthesis by means of TEMPO.146 LY686017 is a potent NK1-II inhibitor for the treatment of depression, anxiety, and alcohol dependency.147,148 In its pilotplant synthesis, TEMPO is used as an oxidation agent with NaOCl, where a secondary alcohol is efficiently oxidized using the Anelli−Montanari protocol (Scheme 42).149

Scheme 38. TEMPO Oxidation Followed by Reduction and Cyclization in the Preparation of Darunavir

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Scheme 39. TEMPO-Assisted Oxidation of a Secondary Alcohol Intermediate to a Ketone during Oseltamivir Synthesis

Scheme 40. Oxidation Catalyzed by TEMPO in a DPP-4 Inhibitor Synthesis

Scheme 41. TEMPO-Mediated Oxidation of a Carbinol To Obtain the Final Drug Substance SB-462795

Scheme 42. Oxidation Step Catalyzed by TEMPO during the Pilot-Plant Synthesis of LY686017

Scheme 45. Synthesis of Diclofenac (a) and Lidocaine (b) with the Potentially Genotoxic 2,6-Dichloroaniline and 2,6Dimethylaniline Starting Materials, Respectively

Scheme 43. Alcohol−Aldehyde TEMPO-Mediated Oxidation during the Synthesis of BYK405879

mediated by 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) in N,N-dimethylformamide (DMF). Chlorhexidine was discovered more than 60 years ago and since then it has been used in more than 60 pharmaceuticals and medical devices.162 It is widely used as a disinfectant and topical antiseptic and has found applications in catheters and preoperative skin preparations. As shown in Scheme 46, the final step of its synthesis involves the use of potentially genotoxic 4-chloroaniline. 3.1.7. Boronic Acids. Boronic acids have been recently tested and identified as a novel family of bacterial mutagens. However, there is no direct evidence of direct covalent binding

Scheme 44. DMAP-Catalyzed Sulfonylation during the Synthesis of Mometasone Furoate

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advantage of all is that the coupling reaction proceeds with high regioselectivitynot affecting other functional groups in the substrateand high stereoselectivity, giving mainly one isomer of the desired product. An example of Suzuki coupling of interest for the pharmaceutical industry is the synthesis of garenoxacin, which is a quinolone antibiotic for the treatment of Gram-positive and -negative bacterial infections.167 As depicted in Scheme 47, in

Scheme 46. Use of Potentially Genotoxic 4-Chloroaniline in the Final Synthetic Step of Chlorhexidine

Scheme 47. Suzuki Coupling during the Synthesis of Garenoxacin between them and DNA. Twelve out of the 13 boronic acid derivatives recently tested by O’Donovan et al. were shown to be mutagenic.163 Boronic acids and their ester-type derivatives are important intermediates in synthetic organic chemistry because they are easy to handle and act as mild organic Lewis acids. They are also considered to be environmentally friendly, since they give boric acid as the side product during their application. These unique properties make them key intermediates in the manufacturing of active pharmaceutical ingredients.164 The boronic acids are easily converted into the cyclic trimeric boroxines by dehydration, although this reaction is readily reversible in aqueous media. To stabilize the monomeric species, it is convenient to convert these to cyclic boronate esters, the most frequently used being the pinacol ester.165 Usually, these esters can be used interchangeably in many reactions. Reactions that form carbon−carbon bonds are often key steps in the synthesis of candidate drugs. In recent years, some of the most important carbon−carbon bondforming methods involve the use of transition-metal-catalyzed reactions. Among these, the most frequently used is the Suzuki−Miyaura166 reaction, which uses boronic acids or esters as the key coupling partner. The mechanism is depicted in Chart 2. The main advantages of boronic acids and esters are that they are readily available and stable in both air and water. These compounds react under mild conditions, and the inorganic boron byproducts are easily removed after completion of the reaction. Probably the most important

the Suzuki strategy, a bromobenzene derivative is treated with butyllithium to afford an organolithium intermediate that is trapped with triisopropylborate to give the desired boronic acid upon workup of the reaction mixture. Since boronic ester derivatives are less sensitive to hydrolysis and air oxidation than the corresponding boronic acids, diethanolamine boronic ester was prepared in the next step by means of diethanolamine.168,169 Afterward, the boronate derivative is treated with AcOH, and the borate species formed reacts with the other key intermediate bromoquinolone in the presence of

Chart 2. Mechanism of Suzuki Coupling

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esterification. Notice that sulfonate esters are not the only potentially genotoxic side products; therefore, two additional small sections provide further examples on the formation of alkyl halides and acetamide. 3.2.1. Sulfonate Esters and Their Precursors. Overview. Sulfonate esters are alkylating agents, a class of potentially genotoxic compounds.178 They are called alkylating agents due to their ability per se, or after metabolic activation, of adding alkyl residues to the reactive nucleophile sites of the DNA bases. They cover a wide range of chemical structures from the simplest alkyl sulfonates to more complex structures featuring aromatic systems with various functional groups. Actually, this class of genotoxic impurities has drawn a high level of attention; the awareness for their presence is not straightforward, as sulfonate esters are most often side products, and formed many times with the solvent used in the reaction or even on cleanup procedures. A historically significant case of the presence of sulfonate esters in a final API formulation is the case of viracept, described below. The precursors of sulfonate esters are alkyl and aryl sulfonic acids and the corresponding halides and anhydrides (Scheme 49).

palladium bis(triphenylphosphine) dichloride and sodium carbonate. Angiotensin II receptor antagonist losartan is used for the treatment of hypertension.170 The synthetic step involving a Suzuki coupling in the synthesis of losartan developed by Merck research chemists is outlined in the following (Scheme 48).171,172 First, the trityl-protected phenyltetrazole was orthoScheme 48. Suzuki Coupling during the Merck Process for the Synthesis of Losartan

Scheme 49. Formation of Genotoxic Sulfonate Esters from the Corresponding Acids, Halides, and Anhydrides with Alcohols

lithiated by means of butyllithium and then quenched with triisopropyl borate, giving the boronic acid derivative after treatment with aqueous ammonium chloride. The resulting boronic acid participated in a Suzuki cross-coupling reaction with the other key intermediate, an imidazole alcohol. 3.2. Genotoxic Compounds Formed in Side Reactions

Examples of the previous section refer to genotoxic impurities introduced as reactants; however, genotoxic impurities can be formed as side products during API synthesis. A class that has drawn the most attention and that has been the most extensively studied when compared to other GTI classes is the sulfonate esters. This class of molecules is usually formed in side reactions with alcohols, and therefore, awareness for their presence in API synthesis could be, initially, not so straightforward. However, as a result of the work of several groups, in particular the efforts of the Working Group within the Product Quality Research Institute (PQRI), the industrial and academic community is today fully aware of this challenge.173 Therefore, taking into account the specific properties and widespread use of the alkyl sulfate acids as counterions in, but not exclusively, API salt formation in the presence of alcohols, special concern has been raised by the regulatory authorities. Several documents from regulatory authorities174−177 have specifically outlined controls to be taken. This has motivated an intensive effort to develop control and removal strategies and to understand their mechanism and kinetics of formation, and several industrial examples have been published. This section is organized into a subsection providing an exhaustive overview on the formation of sulfonate esters and their uses along with two additional subsections providing examples according to the use of sulfonate esters and their precursors in stoichiometric or catalytic amounts. The use of such compounds in stoichiometric amounts include their function as/in (a) API salt forming agents, (b) good leaving groups, (c) cyclization agents, (d) protecting agents, (e) Mitsunobu rearrangement; (f) sulfonamide formation, and (g) aids for the resolution of isomers. Sulfonate esters and their precursors can be used in catalytic amounts also in (a) cyclizations, (b) protecting group manipulations, (c) double bond migration, (d) enamine−amine reduction, and (e)

Since halides and anhydrides of sulfonic acids are alkylating agents, they are also considered genotoxic. Note that in many API syntheses it is, in some circumstances, challenging to substitute alcohols as solvents because of their ability to solubilize both the API and API salts. Examples of most common sulfonate esters and their precursors are summarized in Table 6. Due to their synthetic versatility, sulfonate derivatives are common and useful reagents in the pharmaceutical industry, especially in reactions where carbonium ion initiation is needed. Examples of such compounds are mesylates (methanesulfonate), triflates (trifluoromethanesulfonate), tosylates (p-toluenesulfonate), nosylate (4-nitrobenzenesulfonate), and besylates Table 6. Common Sulfonate Ester Impurities and Their Precursors

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(benzenesulfonate). Sulfonate ester impurities may be present in APIs or their intermediates (i) due to their production in side reactions between sulfonic acids or halides and alcohols or (ii) as reactants carried over from incomplete reactions. When any of the GTI precursors listed in Table 6 is used in an API synthesis, there is a possibility of the formation of genotoxic sulfonate esters. In particular, reactions containing sulfonic acids, sulfonic halides, or sulfonic anhydrides where an alcohol is also present, even if only in residual amounts, have the potential to yield sulfonate esters. Teasdale et al. pointed out that sulfonate esters decompose through alcoholysis to generate sulfonic acid and an ether, and this reaction, in conjunction with the reversibility of ester formation, limits the quantity of genotoxic esters produced.179 The European Pharmacopoeia makes it compulsory for APIs marketed as sulfonic acid salts to demonstrate that any sulfonate ester formed is removed during the purification process.2 Hence, it is crucial for scientists working in the field of API manufacturing to be aware of GTI precursors and their use in drug synthesis. How GTI precursors are used in the pharmaceutical industry is summarized in Table 7, and examples of each case, with reactions, are given later. 3.2.2. Sulfonate Esters and Their Precursors Used in Stoichiometric Amounts. 3.2.2.1. API Salt Forming Agents. Sulfonic acids are salt-forming agents used in the last step of the

API synthesis. Basic APIs are usually preferentially presented in the salt form due their higher aqueous solubility and subsequently higher bioavailability. The conversion of an API to a salt also can help to enhance stability and water solubility and helps isolation as final product (Chart 3). Elder et al.180 overviewed the utility, safety, and regulation of APIs formulated as sulfonic acid salts. For example, methanesulfonic acid is used in the production of viracept181 and delavirdine,182 while ptoluenesulfonic acid (TsOH) is used in the production of bretylium.183 The final manufacturing step of denagliptin, an API developed to treat diabetes mellitus, is forming a tosylate salt in ethanol, as illustrated in Scheme 50.184 There is potential for the formation of a potentially genotoxic p-toluenesulfonic acid ester with the alcoholic solvent. Teasdale et al. carried out a detailed study to understand the mechanism, kinetics, and processing parameters of sulfonate ester formation179,185,186 Note that these studies discuss the reactions between alcohols and sulfonic acids only and not sulfonyl halides. 18O-Labeled methanol was used to distinguish the different esterification pathways and the effect of water content, temperature, and API base to acid ratio, and solvolysis reaction rates were explored. The main findings and conclusions of the work are listed in Chart 3. These findings allow process chemists to control sulfonate ester formation during pharmaceutical manufacturing processes. Further discussion can be seen in section 4. The investigation concluded that sulfonate esters do not form if the acid is neutralized with even the slightest excess of API base. Therefore, the process controls elaborated by Teasdale et al. open the door for the pharmaceutical industry to demonstrate to the regulatory authority adequate control over the presence of sulfonic acid ester GTIs in APIs. A historically very important example, in which the formation of a GTI had a severe impact on API supply, is the case of viracept, the antiretroviral drug used to treat the human immunodeficiency virus (HIV).187 In June 2007, contamination with the genotoxic sulfonate ester ethyl mesylate (EtMs) led to the global recall of this drug.188 The case was investigated and it was established that the main reason for genotoxin accumulation in viracept took place in the final manufacturing step. In this step, the API salt nelfinavir mesylate is formed by addition of methanesulfonic acid (MsOH) to a suspension of nelfinavir in ethanol, and spray-drying is used to isolate the dissolved nelfinavir mesylate salt from the ethanolic solution (Scheme 51). After several patients reported a strange odor and nausea upon taking the medication,188,189 an investigation by the manufacturer revealed that the primary source of GTI contamination was due to an error in good manufacturing practices, more specifically, failure to dry the MsOH hold tank following ethanol cleaning. Additionally, although in negligible quantities, EtMs was also identified in some batches of MsOH. The long hold times, elevated temperatures, and cleaning of the spray dryer with ethanol, the vapors of which could potentially reach the MsOH hold tank through the ventilation system, all contributed to the formation of additional quantities of EtMs190 It is worth mentioning that a comprehensive preclinical toxicology program and safety follow-up registries of exposed patients were carried out by the manufacturer, which concluded that chromosomal damage and mutations only take place for EtMs doses higher than 60 and 25 mg/kg/day, respectively. A maximum intake of ∼0.055 mg/kg/day (for a daily dose of 2500 mg of viracept) was estimated for patients who took

Table 7. Applications of Sulfonate Ester GTI Precursors Application API salt forming agent Good leaving group

Cyclization reactions

Protecting group Protecting group removal Mitsunobu rearrangement Double bond migration Enamine−amine reduction Sulfonamide formation Esterification Resolution of enantiomers

Etherification

API synthesis example Viracept, delavirdine, denagliptin Betaxolol

Hydroxyl−halogene transformation Hydroxyl−sulfur transformation Hydroxyl−amine transformation Amine−nitrile transformation Amide−nitrile transformation Isocyanate−amine transformation Aziridine formation

Mometasone, clobetasone, halobetasole Tixocortol pivalate

Oxazoline formation Pyrrolidine formation Lactone formation Oxirane formation Cyclodehydratation

Ifetroban Napitane Orlistat Saquinavir Englitazone Dinoprost, tolterodine Oseltamivir, denagliptin, ABT-594 Fosinopril

Azaloxan, fluvoxamine, tolterodine Cromitril Denagliptin Temocillin Spiradoline, oseltamivir

Etonogestrel Titonavir Dofetilide Fosinopril Esomeprazole

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Chart 3. Utility of Sulfonate Salts in API Manufacturing and Process Control of Related GTIs

3.2.2.2. Good Leaving Groups. Genotoxic sulfonate esters can be produced by the reaction of sulfonyl chloride with alcohols. Sulfonyl chlorides are used to produce alkyl sulfonates with the aim to provide much better leaving groups than the corresponding alcohol, providing that the rate and yield of the reactions follow SN1 or SN2 mechanisms. Sulfonylation is carried out in the presence of a base, traditionally pyridine due to its high effectiveness: it forms a complex with the sulfonyl halide to favor the attack by the alcohol on the sulfur atom. Pyridine has an alerting structure and is considered a potential genotoxin, thus alternative base catalysis of the sulfonylation is being developed.191 Azaloxan is an antidepressant drug patented by CibaGeigy192 where tosyl chloride is used during its synthesis (Scheme 52) to form the good tosyloxy leaving group for use in

Scheme 50. Final Preparation Step of Denagliptin Is Salt Formation with TsOH in Ethanol

Scheme 51. Final Manufacturing Step of Viracept Drug Substance Nelfinavir Mesylate

Scheme 52. Synthesis of Azaloxan Where Tosyl Chloride Is Used as a Reagent

viracept with elevated levels; therefore, it was concluded that such patients are at no increased risk for carcinogenicity or teratogenicity over their background risk.189 Therefore, the conclusions from the viracept case study points out that GTI contamination of an API can result from a wide range of difficult to anticipate sources: cleaning procedures, pipelines and holding tanks (where reagents may be held for a lengthened period of time), problems with pH adjustment, charging speed of a chemical to the reaction mixture, raw material supply, drying procedures, prolonged reaction time, elevated temperatures, and the introduction of the genotoxic reagent during production of the API (more significant in the final steps).

a bimolecular nucleophilic substitution (SN2) displacement reaction193 transforming a hydroxyl group into an amine group. In a similar fashion, etherification of a hydroxyl group is carried out (Scheme 53) using mesyl chloride in the synthesis194 of Scheme 53. Using Mesyl Chloride during the Synthesis of Betaxolol

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Chart 4. Highlights of SN2 Reactions with the Example of a Sulfonate Leaving Group

betaxolol, a β(1)-selective adrenergic antagonist used in the treatment of hypertension and glaucoma.195 Chart 4 highlights the SN2 reaction mechanism. There is often a need for displacement of a hydroxyl group by an amine during API synthesis, for instance, in the synthesis of eperezolid196 and f luvoxamine.197 In the latter case, the terminal hydroxyl in the final step of the API synthesis is converted to a good leaving group by reaction with mesyl chloride, which is then converted to the terminal primary amine, fluvoxamine, by any of several methods, such as displacement with ammonia, as depicted in Scheme 54.

Scheme 55. Methanesulfonylation of Various Glucocorticoids

Scheme 54. Mesyl Chloride-Assisted Hydroxyl−Amine Transformation at the Last Manufacturing Step of Fluvoxamine

tory drug substance tixocortol pivalate has similar properties to hydrocortisone200 and is also used in patch testing in atopic dermatitis.201 During the final manufacturing steps, mesyl chloride is used to form a good leaving group. Displacement of the mesyloxy group with the anion from thiopivalic acid affords thioester-type API (Scheme 56), in the presence of a base, trimethylamine (Et3N).202 A similar hydroxyl−sulfur transformation takes place during the synthesis of microtubule inhibitor erbulozole.203 Sulfonyl halides are also useful tools to transform amines to nitriles. For instance, one synthesis of the antiasthmatic agent cromitril204 concludes in the classic manner by converting an ester to a carboxamide by ammonolysis, and dehydrating the

Mometasone, clobetasone, and halobetasol are glucocorticoids belonging to a class of steroid hormones that bind to the glucocorticoid receptor. They play a key role in regulating the feedback mechanism of the immune system during inflammation by turning the immune activity down.198 During the synthesis of these steroids, mesyl chloride is used in a methanesulfonylation catalyzed by a base, DMAP, and the mesylate group is consequently replaced by chlorine (Scheme 55).16,199 In other steroids, the 21-hydroxyl group is not converted to chlorine but to a sulfur-linked residue to achieve modified therapeutic activity. For example, the anti-inflammaU


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Scheme 56. Use of Mesyl Chloride in the Synthesis of a Thioester-Type Glucocorticoid

configuration at the former secondary alcohol is inverted as a consequence of the SN2 nature of the ring closure. Sulfonyl halides are also used in lactone formation. Orlistat is a drug designed to treat obesity,212 the synthesis of which involves treatment of the β-hydroxycarboxylic acid intermediate with benzenesulfonyl chloride, resulting in the formation of the butyrolactone ring present in the final API (Scheme 62).213 A rare but useful application of sulfonyl halides in API synthesis is in the formation of aziridines. In the synthesis of the opioid analgesic214 spiradoline, tosyl chloride initially converts the hydroxyl group to a chloride, followed by displacement of the halogen by the adjacent amine to form an aziridinium salt, as depicted in Scheme 63.215 The synthesis of oseltamivir, an antiviral drug,216 involves the formation of two aziridines in consecutive reaction steps by means of mesyl chloride. As depicted in Scheme 64, the secondary alcohol is first converted to the corresponding mesylate by means of mesyl chloride in the presence of triethylamine. The amine produced by reduction of the azide group in the second step with triphenylphosphine proceeds with a nucleophilic attack on the adjacent carbon, displacing the mesyloxy group in a nucleophilic substitution to provide the first aziridine. Due to the large angle strain of the threemembered heterocyclic system, ring opening of aziridine with sodium azide in the presence of ammonium chloride occurs easily. After deprotection of the MOM ether by acidic hydrolysis, the amino group is reacted with trityl chloride, the hydroxyl group is then transformed into a good leaving group by means of mesyl chloride in the presence of triethylamine, and in a one-pot process, a new aziridine-type intermediate is produced.217 The use of methanol as a solvent may lead to the formation of genotoxic sulfonate esters under certain conditions. Furthermore, the HCl/MeOH mixture used for the hydrolysis of the MOM ether may lead to the presence of genotoxic methyl chloride in the API. 3.2.2.4. Protecting Groups. Scheme 65 includes a synthetic step in the production tolterodine,218 an API used to manage urinary incontinence, showing the protection of a phenol group by the formation of a tosylate followed by the formation of the good leaving group nosylate, which is then easily displaced by diisopropylamine. Note that this synthesis provides an example involving three GTIs and one carcinogenic impurity of different chemical classes, sulfonate esters, alkyl halides, and acetamides. 3.2.2.5. Sulfonamide Formation. Sulfonamides provide part of the structural basis of several drugs (Chart 5). Originally, sulfonamides were used as synthetic antimicrobial agents, but

latter functionality to the nitrile with tosyl chloride in pyridine, followed by addition of sodium azide selectively across this functionality, produces the final API (Scheme 57)205 The βScheme 57. Amide Transformation to Nitrile with TsCl/Py during the Synthesis of Cromitril

lactamase-resistant carboxypenicillin drug temocillin is used for the treatment of multiresistant Gram-negative bacterial infections.206 During the synthesis, isocyanate−amine transformation takes place, where benzyl 6-β-isocyano-6-α-methylthiopenicillanate is treated with p-toluenesulfonic acid, giving one of the key intermediates to temocillin (Scheme 58).207 3.2.2.3. Cyclizations. Cyclization reactions where a sulfonate ester functions as a leaving group are common, as in the following examples. Oxazoline cyclization can be achieved by means of mesyl chloride, for instance, during the synthesis of ifetroban, which is a selective thromboxane receptor antagonist.208 First, the hydroxyl group is converted to a good living group by reaction with mesyl chloride. When this intermediate is treated with base, the mesylate is displaced by the enolate from the adjacent amide, giving the corresponding oxazoline (Scheme 59).209 Napitane is used as an antidepressant drug, and during the last synthetic step, 2 equiv of methanesulfonic acid is released.210 A diol is converted to a bidentate bis-mesylate with mesyl chloride followed by reaction of the required amine to give the pyrrolidine moiety of the final API (Scheme 60). During the large-scale synthesis of saquinavir, an anti-HIV protease inhibitor, a mesyloxy group is used to form an oxirane derivative in two steps. The secondary alcohol of a 1,2-diol is selectively converted to the methanesulfonate ester with mesyl chloride. Thereafter, strong base is used to produce an alkoxide at the primary alcohol, which then displaces the mesylate ion, giving an oxirane derivative.211 As depicted in Scheme 61, the

Scheme 58. Isocyanate Transformation to Amine with TsOH during the Synthesis of Temocillin

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Scheme 59. Oxazoline Heterocyclization with MsCl during Ifetroban Synthesis

Scheme 60. Formation of a Bidentate Mesylate, Which Eventually Gives Rise to the Liberation of 2 equiv of Methanesulfonic Acid in the Final Synthetic Step of Napitane

Scheme 64. Consecutive Aziridine Formation by Means of Mesyl Chloride in Oseltamivir Synthesis

Scheme 61. Formation of Mesyloxy Group as a Precursor of an Oxirane Derivative during the Synthesis of Saquinavir Scheme 65. Residues of Tosyl and Nosyl Chloride Can React with Alcohols, Forming Genotoxic Sulfonate Esters; Acetonitrile Can Be Contaminated with Carcinogenic Acetamide and also Forms Acetamide in the Presence of HCl and Water; and Due to the Elevated Temperature, Methanol Can Form Genotoxic Methyl Chloride in the Presence of HCl

Scheme 62. Sulfonyl Halide Assisted Lactone Formation during the Synthesis of Orlistat now there are novel drug families based on the original antibacterial sulfonamides, such as diuretics, anticonvulsants, and dermatologicals. The preparation of these APIs requires the use of sulfonyl halides. For example, the antiarrhythmic dofetilide is a bis-methanesulfonamide that is synthesized using mesyl chloride (Scheme 66).219 3.2.2.6. Chiral Auxiliary Group in Resolution of Enantiomers. Due to the identical scalar physical properties of enantiomers, one method for their separation can be carried out via “classical resolution” based on preferential crystallization of one of the diastereomeric derivatives. One of the most common natural, chiral resolving agents for the partition of racemic mixtures is camphorsulfonic acid. In a recent patent,220 its derivative, camphorsulfonyl chloride, is used to obtain enantiomerically pure esomeprazole, a proton pump inhibitor, used in the treatment of dyspepsia, peptic ulcer, and gastroesophageal reflux disease, via preferential resolution (Scheme 67).221 The enantiomers of omeprazole are converted into diastereomers by way of a chemical reaction with the resolving agent, temporarily introducing additional asymmetry into the molecule. The forming diastereomers are easily separated by recrystallization in alcohols; thereafter, cleavage of the resolving agent gives the enantiomerically pure esomeprazole. The side reaction of both camphorsulfonyl

Scheme 63. Tosyl Chloride-Assisted Formation of an Aziridin during the Synthesis of Spiradoline

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Chart 5. Highlights of Sulfonamide Drugs

Scheme 66. Use of Mesyl Chloride during the Synthesis of the Bis-methanesulfonamide Dofetilide

Scheme 68. TsOH-Assisted Cyclodehydration Leads to a Chroman Derivative in Englitazone Synthesis

useful way of protecting the hydroxyl group due to its stability toward a variety of harsh reaction conditions, such as strong bases, organometallic reagents, hydrides, and acylating and alkylation reagents. THP protecting groups are formed by the reaction of the hydroxyl compounds with dihydropyran under acidic conditions, for example, in the presence of ptoluenesulfonic acid.223 The naturally occurring prostaglandin dinoprost is used to induce labor and as an abortifacient. During its synthesis, the protection of the two hydroxyl groups as 2tetrahydropyranyl ethers by reaction with 3,4-dihydropyran (DHP) in the presence of p-toluenesulfonic acid is carried out (Scheme 69).224 Trifluoroacetic acid (TFA) was used originally for the removal of Boc protecting groups from amines. Since TFA is highly corrosive and difficult to recover and the HF generated during incineration causes problems, alternative conditions are being developed for large-scale Boc removal. Most of these apply TsOH to produce acidic conditions.225 During the synthesis of ABT-594, a potent, orally effective analgesic, TsOH is used both to remove a Boc-protecting group from an amine and as a salt-forming agent to obtain a stable API (Scheme 70). Neither HCl nor TFA can be used for deprotection in this particular case, as they gave a significant amount of dimer byproduct, which proved difficult to remove.226 Since the reaction and salt formation take place in ethanol and this is the

Scheme 67. Resolution of Racemic Omeprazole with Camphorsulfonyl Chloride To Obtain Enantiomerically Pure Esomeprazole

chloride and camphorsulfonic acid with the alcohol solvent can lead to the formation of potentially genotoxic sulfonate esters. 3.2.3. Sulfonate Esters and Their Precursors Used in Catalytic Amounts. 3.2.3.1. Cyclizations. A pharmaceutical example of a cyclization where a sulfonic acid is applied as catalyst is the TsOH-assisted cyclodehydration of a diol to afford the chroman framework during the synthesis process of the hypoglycemic agent englitazone (Scheme 68).222 3.2.3.2. Protecting Group Manipulations. The formation of tetrahydropyranyl (THP) ethers from alcohols and phenols is a X


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Scheme 69. Tetrahydropyranyl Ether Formation with DHP and TsOH during the Synthesis of Dinoprost

synthesis with TsOH in acetic acid, which causes the double bond at the 8,9-position to migrate to the adjacent 9,11position, effectively activating the otherwise unreactive C11 carbon.230 3.2.3.5. Enamine−Amine Reduction. The antiretroviral drug ritonavir belongs to the protease inhibitor family used for the treatment of HIV infection and AIDS.231 It is synthesized via an enamine intermediate, which is treated with sodium borohydride in the presence of methanesulfonic acid, resulting in the reduction of the enamine to a primary amine (Scheme 74).232 3.2.3.6. Esterification. Sulfonic acids are also used to catalyze esterifications. Fosinopril is an angiotensin converting enzyme (ACE) inhibitor. Its synthesis involves a manufacturing step where a carboxylic acid is esterified by methanol in the presence of TsOH (Scheme 75).233 Although the TsOH is used as a catalyst, the quantities used are generally quite high and may result in formation of considerable amounts of the corresponding ester. 3.2.4. Alkyl Halides. Examples of alkyl halides that have the potential to remain in solution as unreacted reagents were discussed previously. However, such species can also arise due to side reactions. This is illustrated by the recent use of 4-(4,6dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), which is an efficient coupling agent in a wide range of organic reactions,234,235 such as esterification,236,237 glycosidation238 and phosphonylation239 in the synthesis of antibiotics, 240 peptides, 241 and alkaloids. 242 However, DMTMM is unstable in organic solvents and reacts with itself, yielding 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-morpholine and genotoxic methyl chloride (Scheme 76). 3.2.5. Acetamide. Acetamide is a known carcinogen; thus, the awareness of its formation in API manufacturing is crucial.243 Although acetamide is not a genotoxin, it is sometimes referred to as such in the literature11 Acetamide is not commonly used directly in the synthesis of APIs, but its derivatives, such as 2- and N-bromoacetamide or trifluoroacetamide, are often used as building blocks in drug synthesis. These derivatives initially contain acetamide as an impurity, but also the 2- and N-derivatives have potential to form acetamide, depending on the reaction conditions. Another source for formation of carcinogenic acetamide is the hydrolysis of the

Scheme 70. Removing a Boc Group with TsOH in the Synthesis of ABT-594

final step of the synthetic route, the probability that the API contains genotoxic ethyl p-toluenesulfonate is high. An alternative synthetic route to oseltamivir uses TsOH in methanol to remove an acetonide protecting group of a diol intermediate. There is potential to form methyl p-toluenesulfonate in this reaction (Scheme 71).227 Scheme 71. Acetonide Protecting Group Removal by Means of TsOH in Methanol

3.2.3.3. Mitsunobu Rearrangement. Fosinopril, an inhibitor of angiotensin converting enzyme (ACE), is widely used for the treatment of hypertension, as well as in various types of chronic heart failure.228 Fosinopril manufacturing is another example of the use of sulfonic acids in the pharmaceutical industry, since MsOH is used as a reagent in a Mitsunobu rearrangement, as depicted in Scheme 72,229 wherein mesylation with inversion of configuration was accomplished by employing methanesulfonic acid, triphenylphosphine, diisopropyl azodicarboxylate, and triethylamine. A highly stereospecific Friedel−Crafts alkylation mediated by aluminum trichloride installed a 4-phenyl substituent with complete inversion. This shows an example how sulfonic acids are used in O−C transformation. Mitsunobu reactions are discussed in more details in Chart 6. 3.2.3.4. Double Bond Migration. Etonogestrel is a steroid used in hormonal contraceptives, and its synthesis involves a double bond migration assisted by p-toluenesulfonic acid. Scheme 73 illustrates the treatment of an intermediate in the

Scheme 72. Specific Mitsunobu Rearrangement with MsOH for an O−C Transformation during the Manufacturing of Fosinopril

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Chart 6. Mitsunobu Reaction and Its Use In API Synthesis

Scheme 73. TsOH-Assisted Double Bond Migration during the Synthesis of Etonogestrel

Scheme 74. Methanesulfonic Acid-Assisted Reduction of an Enamine during the Synthesis of Ritonavir

Scheme 77. Acetamide May Form from Acetonitrile during the Synthesis of Corontin

Scheme 75. Esterification of Fosinopril Intermediate Assisted by TsOH

ment phase (Scheme 78). A decision was made by the developers to mitigate the risk posed by the potential to form Scheme 78. Potential for Carcinogenic Acetamide Formation during the Final Synthetic Step of Zaurategrast

Scheme 76. Formation of Genotoxic Methyl Chloride from Coupling Agent DMTMM

acetamide by applying adequate chemical process design: the implementation of a workup sequence involving aqueous washes, followed by salt formation and crystallization, was proven to be successful.247 Sodelglitazar is an antidiabetic drug for treatment of type 2 diabetes. During one of the synthetic steps S-alkylation takes place under acidic conditions in acetonitrile, hence, the potential for formation of acetamide (Scheme 79).248 During the synthesis of the antiviral drug oseltamivir, a diene intermediate is converted to the bromodiamide derivative using a novel SnBr4-catalyzed bromoacetamidation with N-bromoa-

widely used solvent acetonitrile under acidic or basic conditions at elevated temperature. Acetonitrile is not only used as a solvent in the pharmaceutical industry but also as a reagent in API synthesis. For instance, it is directly used as a reagent in the synthesis244 of corontin, which is a drug used for treatment of angina pectoris (Scheme 77).245 Zaurategrast is a drug that reached phase II clinical development and was indicated for treatment of multiple sclerosis.246 Since the final synthetic step of the process takes place in acetonitrile under acidic conditions, the risk of acetamide formation was identified during the early developZ


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solvents, which include solvents such as ethanol and acetone, have permissible daily exposures of 50 mg, or up to 5000 ppm (0.5%) when it is assumed that 10 g is administered daily. There is no solvent recognized as being hazardous to human health at the average acceptable levels in pharmaceuticals in this group. They are less toxic in acute or short-term studies and have given negative results in genotoxicity studies. There is also an additional group, class 4 solvents. No toxicological data exists for this group, which would allow for the formulation of acceptable limits. When a manufacturer wishes to use class 4 solvents, a justification for the level of the solvent in the pharmaceutical product has to be submitted to the regulatory authorities. Note that most of the guidelines only address products on the market and not compounds under clinical trials. However, the Q7A guideline has a specific chapter dedicated to APIs used in clinical trials.254 Under the conditions of a 2-year gavage study, there was clear evidence of the carcinogenicity of benzene, which used to be a widely used solvent in the industry.256 Benzene was mainly replaced by toluene to carry out the same reactions that require nonreactive aromatic solvents. The genotoxicity of toluene is under investigation, although it is still widely used as a solvent.257 Although chlorobenzene showed positive results in some in vitro and in vivo genotoxicity studies, it showed negative results in the majority of the studies on “in vitro” gene mutation, chromosomal aberration, DNA damage, and UDS and in vivo SCE. From overall evaluation of these results, chlorobenzene is considered not to be genotoxic.258 Although the experimental group that was exposed to dimethylformamide (DMF) showed an increase in the incidences of chromosomal aberration,259 negative results were obtained in the majority of the in vitro and in vivo genotoxicity studies; thus, the overall evaluation of these data indicates that DMF is not genotoxic (categorized as group 3, i.e., not classifiable as to its carcinogenicity to humans by the IARC).260 Classification of dioxane (group 2B carcinogen by IARC) indicates that it is possibly carcinogenic to humans, since it is a known animal carcinogen.261 Dichloromethane (DCM) may be carcinogenic, as it has been linked to cancer of the lungs, liver, and pancreas in laboratory animals.262 Hydrolysis of the widely used solvent acetonitrile under acidic or basic conditions at elevated temperature can lead to the formation of acetamide, which is a nongenotoxic carcinogen.14

Scheme 79. S-Alkylation in Acetonitrile under Acidic Conditions May Lead to the Formation of Carcinogenic Acetamide during the Synthesis of Sodelglitazar

cetamide in acetonitrile (Scheme 80).249 In this reaction, both the reagent and the solvent can form carcinogenic acetamide. Scheme 80. Reagent N-Bromoacetamide and Solvent Acetonitrile May Lead to Acetamide Formation

The reagent 2-bromoacetamide is used during the synthesis of armodaf inil, which is used for the treatment of narcolepsy and sleeping disorders.250 2-Bromoacetamide, which may contain acetamide, is used in an S-alkylation in the presence of NaOH (Scheme 81).251 Scheme 81. Use of 2-Bromoacetamide May Result in the Presence of Acetamide in Armodafinil Synthesis

4. APPROACHES FOR GTI MITIGATION IN THE PHARMACEUTICAL INDUSTRY As illustrated in the previous section, the synthesis of pharmaceutical products often involves the use of highly reactive reagents for the production of APIs or their intermediates.263 Low levels of such reagents or corresponding side products may therefore be present in the final API or drug product as impurities. As briefly described in section 2, such chemically reactive impurities may have unwanted toxicities, including genotoxicity and carcinogenicity, and hence can have a severe impact on the product risk assessment.264 In some cases, these sources can be avoided. However, in many cases the presence of GTIs in postreaction streams during API synthesis is difficult to avoid. To overcome this, R&D scientists have to identify GTIs early on during process development, develop analytical methods, and implement synthetic processes to control and contain them. GTIs can be successfully reduced below the limits set by regulatory authorities either with carefully optimized synthetic approaches (preventive approach,

3.3. Genotoxicity and Carcinogenicity of Common Organic Solvents

Organic solvents are ubiquitously present in pharmaceutical production processes as reaction and purification media (e.g., extraction), separation phases (e.g., chromatographic mobile phases), and also for cleaning of the equipment. The pharmaceutical industry consumes the largest amount of organic solvents in relation to the final product gained.252 According to the Q3C guideline, solvents are divided into four groups.253 Classes 1 and 2 are considered “toxic” solvents, as summarized in Table 8. The first group (class 1) contains known human carcinogens, compounds strongly suspected of being human carcinogens, and those presenting environmental hazards. These solvents should be avoided, unless strongly justified. The limits for class 1 solvents are listed as absolute parts per million in a material under testing (drug or excipient). Class 2 solvents presented in Table 8 ought to be limited, because they are nongenotoxic animal carcinogens or associated with irreversible toxicity, such as teratogenicity. Class 3 AA


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Table 8. Classification of Solvents by the Q3C Guideline and Their Propertiesa Class 1

Class 2

a

Solvent

Concn limit (ppm)

Boiling point(°C)

Density (g/cm3)

Dielectric constant

Benzene Carbon tetrachloride 1,2-Dichloroethane 1,1-Dichloroethane 1,1,1-Trichloroethane 2-Methoxyethanol Methylbuthylketone Nitromethane Chloroform 1,1,2-Trichloroethylene 1,2-Dimethoxyethane Tetralin 2-Ethoxyethanol Sulfolane Pyridine Formamide Hexane Chlorobenzene 1,4-Dioxane Acetonitrile Dichloromethane Ethylene glycol N,N-dimethylformamide Toluene N,N-dimethylacetamide Methylcyclohexane 1,2-Dichloroethene Methanol Xylene Cyclohexane N-methylpyrrolidone

2 4 5 8 1500 50 50 50 60 80 100 100 160 160 200 220 290 360 380 410 600 620 880 890 1090 1180 1870 2000 2170 3880 4840

80.1 76.7 83.5 57.2 74 124 127 101.2 61.7 86.7 85 207 135 285 115.2 210 69 131.7 101.1 81.6 39.8 245 153 110.6 166.1 101 60.3 64.6 139.1 80.7 202

0.877 1.594 1.245 1.2 1.32 0.965 0.812 1.382 1.498 1.463 0.868 0.974 0.931 1.261 0.982 1.134 0.659 1.107 1.033 0.786 1.326 1.118 0.944 0.867 0.937 0.769 1.28 0.791 0.868 0.779 1.026

2.28 2.24 10.42 16.7 7.5 16.94 14.6 35.9 4.81 3.4 7.2 2.77 5.3 44 12.3 84 1.89 5.69 2.21 37.5 9.08 31.7 36.7 2.38 37.78 2.6 4.6 32.6 2.37 2.02 32.2

Data are from ref 255.

Scheme 82. Synthesis of Sodelglitazar: (a) Route with Genotoxic Mesylate Intermediate and (b) Alternative Route Avoiding the Use of Genotoxic Mesylate

However, in many cases, the use of reagents and intermediates that are reactive and synthetically useful, which in turn likely makes them interact with DNA, are often unavoidable. It may not be practical to change the synthetic steps during development to control or reduce GTIs, particularly when the process has reached the stage of being scaled up. Therefore, a second strategy to achieve GTI-free drug products is based on prevention, focusing on elimination or reduction of the concentration of GTI during the critical synthetic step. This can be achieved by altering appropriate reaction conditions, such as (i) proportions of reaction components, (ii)

hence preferred) or by implementing purification strategies as a last resort. 4.1. Chemical Synthetic Approaches

In this, the first strategy to mitigate GTIs in the production of APIs, R&D chemists avoid the use and generation of GTIs throughout the synthetic route, searching for different chemical sequences to reach the same API or intermediate or by optimizing the existing synthetic route.265 In very particular cases, this strategy can be achieved without significant reduction of yield. Examples of redesigning the synthetic process specifically to avoid GTIs can be found in section 4. AB


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β-D-ribofuranose (Scheme 84). This stereoselective coupling was initially mediated by N,O-bis(trimethylsilyl)acetamide and triflic acid. Since the workup leads to formation of stoichiometric amounts of genotoxic acetamide, N,O-bis(trimethylsilyl)acetamide was replaced by trimethylsilyl triflate as the coupling reagent. In the synthesis of Zeneca Pharmaceuticals’ ZD-2079268 for the treatment of noninsulin dependent diabetes, 1,2-dibromoethane is used to alkylate 4-hydroxylphenylacetamide (Scheme 85a). A side reaction leads to the formation of genotoxic vinyl bromide. In the scaled up reaction, the N-alkylethyl group was introduced via an oxathiazolidine S-oxide, obtained by reaction of N-benzylethanolamine with thionyl chloride, which on reaction with 4-hydroxylphenylacetamide gave the desired intermediate (Scheme 85b). Note that in this particular case, the main motivation to modify the synthetic step was safety rather than API purity. As a bonus, the new route gave a 64% yield while that for the dibromoethane-based route was 9%. 4.1.2. Adjusting Reaction Conditions To Mitigate GTI Formation. The feasibility of minimizing the formation of genotoxic impurities by simple adjustment of parameters such as reaction time, pH, temperature, and solvent matrix is demonstrated through the following examples. 4.1.2.1. Sulfonate Esters. The synthesis of the AstraZeneca drug for management of type 2 diabetes, tesaglitazar, includes a step where a potentially genotoxic bismesylate ester is added in excess to the phenolic key intermediate to form an ether269 at pH 10 and 100 °C for 4−5 h while using PEG-400 as phasetransfer catalyst in the presence of sodium carbonate (Scheme 86). Adjusting the pH to 7 and increasing the reflux time to 8− 9 h allow complete reaction and hydrolysis of the alkyl sulfonate ester without hydrolysis of carboxylate ester in the API. Such an approach, which takes advantage of different reactivities, can be applied in the elimination of other genotoxic sulfonate esters used in excess. Note, however, that the strategy employed in Scheme 86 is based on the use of a genotoxic sulfonate ester. Other studies have focused on routes where sulfonate esters are avoided and have been recently summarized by Elder et al.173 In the following paragraphs, the effect of pH, temperature and water content on the formation of sulfonate esters will be discussed. (i) pH: The elucidation of the mechanism of sulfonate ester formation using labeled 18O179 revealed that the formation of these species from the corresponding sulfonic acid and alcohol involves the protonation of the alcohol under acidic conditions. It was concluded that even a slight molar excess of a base prevents sulfonate esters formation. Therefore, avoidance of acidic conditions or even addition of a base is recommended to mitigate sulfonate ester formation. (ii) Temperature: It was observed that lower temperatures significantly reduce the rate of formation of sulfonate esters. Reduction of the reaction temperature from 40 to 10 °C showed a significant 4-fold reduction of sulfonate esters formation even without the addition of a base.179 Therefore, conducting both the reaction and workup at lower temperatures is recommended. (iii) Water: The presence of water, as it competes with alcohol for protonation and promotes ester hydrolysis, has a positive effect on reducing sulfonate esters formation, and even a small amount of water results in a 3-fold decrease of sulfonate esters without addition of base.185

interchanging the order of addition of the reactants, and (iii) changing the quality or method of preparation of key starting materials. Furthermore, a quality by design (QbD) approach can contribute to better control of GTIs.266 4.1.1. Altering the Synthesis. Three synthetic examples are given in which the chemical synthesis was changed to avoid the formation of a sulfonate ester. Two additional examples include side reactions with the potential to form genotoxic impurities vinyl bromide and acetamide. The syntheses of zaurategrast sulfate and sodelglitazar were previously mentioned in section 3.2.5 due to the potential formation of acetamide. However, there is a second source of GTI: sulfonate esters.247 In the case of zaurategrast sulfate, the use of methanesulfonic acid in the presence of ethanol posed the potential risk of generating genotoxic ethyl mesylate. This was mitigated by replacing the sulfonic acid with hydrochloride acid without affecting the yield.247 In the initial synthetic route of sodelglitazar (Scheme 82a), a genotoxic mesylate intermediate is used; therefore, the commercial application employs the corresponding alcohol instead of this mesylate ester (Scheme 82b) for the formation of the thioether linkage.248 Denagliptin was previously mentioned in section 3.2.2, illustrating API salt formation with sulfonic acids. However, in a previous step, a (S)-difluorophenyl amino acid is reacted with a fluoro amino amide mediated by n-propanephosphonic acid cyclic anhydride (T3P) and diisopropylethylamine (DIPEA).184 The next step involved dehydrating with p-toluenesulfonic anhydride with pyridine as base at 50 °C. Since ptoluenesulfonic anhydride was not available commercially, the scaled up reaction relied on the use of methanesulfonic anhydride (Scheme 83). In this case, the potential to form Scheme 83. Alternative Route for Large-Scale Synthesis of Denagliptin Tosylate, Avoiding the Formation of a Potentially Genotoxic Mesylate Ester

mesylate esters, which are potential genotoxins (as are the tosyl esters had the tosyl anhydride been used), was not desirable. The observation that partial dehydrating had occurred during the coupling reaction gave rise to the exploration of T3P as a dehydrating agent. A second equivalent of T3P, along with a higher temperature of 78 °C, gave satisfactory results (Scheme 83).184 The synthesis of the anti-inflammatory agent UK-371,104267 includes a glycosidation reaction of the adenosine key intermediate with a peracetylated sugar, 1,2,3,5-tetra-O-acetylAC


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Scheme 84. During the Synthesis of UK-371,104, the Workup Procedure Generates Genotoxic Acetamide from N,OBistrimethylsilylacetamide Reagent, Thus TMS Triflate Is Used Preferentially

Scheme 85. (a) Original Synthesis of ZD-2079 Led to the Formation of Genotoxic Vinyl Bromide as a Byproduct; (b) the Alternative Route Does Not Require the GTI precursor 1,2-Dibromoethane but Uses an Alternative Oxathiazolidinone SOxidea

a

MMP and NMP stand for N-methylmorpholine and N-methyl-2-pyrrolidone, respectively.

369,003-26, a candidate for treatment of benign prostatic hyperplasia, benzenesulfonic acid was used as salt forming agent.270 In this reaction potentially genotoxic ethyl besylate (EtBS) was formed because of the reaction between benzenesulfonic acid and API ethoxy side chain (Scheme 87). 4.1.2.2. Halides. As discussed in the previous paragraphs, sulfonic acids can form potentially genotoxic sulfonate esters, when used as API salt formation agents in alcoholic solutions. Similarly, halide acids (e.g., HCl), used as salt-forming agents, can form alkyl halides (e.g., MeCl and EtCl) by reaction with alcohol solvents (Scheme 88). Examples of the occurrence of

Scheme 86. Synthesis of Tesaglitazar, with a Change in pH for the Effective Hydrolysis of the Sulfonate Ester Precursor

Scheme 87. Formation of Potentially Genotoxic Ethyl Besylate (EtBS) through Salt Formationa (iv) Addition conditions: Minimizing residential time of sulfonic acids in alcoholic solutions, as well as minimizing the excess of sulfonic acid, is crucial to avoid the formation of sulfonate esters. Vigorous stirring and slow addition of the acid to the API solution allow for effective salt formation, avoiding a localized excess of acid, which can give rise to sulfate ester formation. Prolonged storage times of solutions containing both sulfonic acid and alcohol mixed should be avoided.173 Sulfonate ester impurities typically arise from the reaction of the respective acids with an alcohol which is usually present as a solvent. However, in particular cases other causes can be responsible. For example during the manufacture of UK-

a

Here it is not an alcoholic solvent but the ether substructure of the API that leads to GTI formation.

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in in situ formation of amine hydrochloride salts, also yields the genotoxic dimethylcarbamoyl chloride (DMCC). A hydrolysis study of DMCC concluded that elevated temperature (80 °C) and shorter reaction time decrease the amount of DMCC from 87 to 0.9 ppm in the reaction mixture while a yield of as high as 90% of product was maintained. 4.1.2.3. Nitro Aromatics. Scheme 9 describes a sequence that involves the catalytic reduction of a nitroaromatic group to an aniline derivative. A second example of such nitroaromatic reduction can be found in the synthesis of an adrenoreceptor antagonist, as represented in Scheme 90.272 The nitroaromatic reduction takes place through a hydrogenation catalyzed by Pd/ C, while workup involves removal of the catalyst by filtration, concentration, and crystallization. 4.1.3. Quality by Design. The use of the QbD approach has been suggested to develop synthetic routes or selection of conditions for API synthesis and can also be applied to control GTI formation below threshold values. In the pharmaceutic context, QbD aims to design and produce API formulations for which the final quality should be ensured a priori through the design of synthetic routes and the manufacture process. Generically, QbD includes four stages: (i) definition of the quality profile to be targeted; (ii) product and manufacture process design to achieve such quality; (iii) identification and selection of quality attributes, process parameters, and sources of variability; and (iv) control mechanisms to ensure quality over time. In the particular case of GTI risk control, the target for product quality requires one to maintain GTI below threshold numbers, while providing high API yields. The examples in section 4.1 provide cases of design of chemical synthesis that avoided the presence of GTI, and the following section 4.2 is focused on selection of parameters able to decrease the amounts of GTI present, in other words, give information that can be used in QbD stages 2 and 3 defined above (Figure 6). Quality by testing (QbT) is the main approach supported by regulatory agencies, which had resulted in an extremely robust effort to develop analytical tools and intensive screening for GTIs in raw material, intermediates, and APIs. The optimization of the process shown in Scheme 91 and described in section 4.1.2.3 employed QbD in order to minimize the presence of potential GTIs, namely, nitroso compounds and hydroxylamine.273 The potential genotoxicity of the compounds involved in the synthesis were first assessed using in silico approaches, such as DEREK, and toxicology data. Potential GTIs can be formed in the reduction of nitroaromatics to aniline derivatives, as illustrated in Scheme 91. The four compounds raised structural alerts according to DEREK,

Scheme 88. Competitive Formation of API Salt and Alkyl Halides in the Reaction of Halide Acids with an API Base in Alcoholic Solvents

alkyl halides in the synthesis of APIs are provided in Sections 3.1.1 and 3.2.4. The effect of reaction conditions on the mitigation and elimination of alkyl halides has been investigated using a quaternary amine as API model in the formation of the HCl salt.48 Drying of the product at 85 °C under vacuum failed to significantly decrease the alkyl halide content. Decreasing the rate of addition of HCl and increasing stirring times did not have a significant impact when applied alone. A reduction of the HCl load did decrease alkyl halide formation but resulted in lower yield of the salt. The reaction temperature proved to be crucial in managing the levels of alkyl halide. At 35 °C the formation of alkyl halides was favored, whereas a lower temperature of 10 °C proved to be an efficient strategy to mitigate formation of the genotoxin. When tested at larger scales a yield of 92% and GTI formation below 1 ppm, in compliance with TTC limits, was achieved. Another example, in which both temperature and reaction time were adjusted to mitigate GTI formation, was reported by AstraZeneca during the use of a Vilsmeyer chlorination reaction in a penultimate step of API synthesis (Scheme 89). This Scheme 89. Adjustment of Temperature and Reaction Time in a Chlorination Step Resulted in a Reduction in Formation of Genotoxic Dimethylcarbamoyl Chloride (DMCC)

particular reaction comprises simultaneous in situ formation of an amine hydrochloride salt.271 The reaction of the N,Ndimethylformamide and a chlorinating agent, POCl3, resulting

Scheme 90. A Nitroaromatic Catalytic Reduction Step in the Synthesis of an Adrenoreceptor Antagonist Candidate

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reaction time. The acceptable operating ranges were identified via the design of experiments (DoE) approach, which led to high product yield and GTI levels below TTC values. In a further example, the QbD approach was employed for the control of mesylate esters in the synthesis of fluoroarylamine mesylate266 Stage 8 of the synthetic route of fluoroaryl-amine mesylate (Scheme 92)266 is the salt formation and its crystallization using a combination of solvents, including ethyl acetate, acetone, and isooctane. During the risk assessment of this particular step, the manufacturer found that three possible genotoxic mesylate esters, namely, methyl mesylate, ethyl mesylate, and isopropyl mesylate could be present in the final drug. The sequential steps of stage 8 include crystallization, isolation, washings, and drying. Design of experiments were performed using GTIspiked drug samples to identify parameters having a crucial impact on the formation and purging of the GTIs. The main conclusions of the DoE-assisted investigation include that the amount of alcohol used in the various steps of stage 8 has no significant impact on the amount of GTIs formed; the drying operation does not generate any detectable GTIs, and the isolation effectively removes any GTI. Overall, the process understanding gained through QbD led to a robust control strategy with negligible levels of GTI, allowing testing of the final drug substance to be omitted.

Figure 6. Quality-by-design strategy for prevention of GTI formation.

Scheme 91. Nitroaromatic Catalytic Reduction (a) Showing Reaction Intermediates That Can Remain in the Product as Impurities (b)

4.2. API Purification

4.2.1. Purge Factors. As discussed in the previous section, the presence of GTIs can be, in many cases, avoided through novel designs of chemical routes or mitigated by control of reaction conditions. Additionally, it should be noted that during API synthesis, purification units are already in place at several steps. In spite of the fact that these steps are often not designed specifically to reduce GTIs, they have the ability to remove GTIs along with other impurities. Hence, there are several routes by which a given GTI can be eliminated during the synthesis. Previous works2 addressed the issue of purging, defining risk considering the number of synthetic steps between the appearance of GTI and the final production step. It was recommended that in cases where the presence of GTI is more than four steps away from the final synthetic step, chemical rational should be used to decide whether GTI specific impurity removal is required or not. However, such an empirical approach is not process specific. Therefore, Teasdale et al.39 developed a semiquantitative “assessment purge tool” focusing on the particular GTIs of concern and chemical properties of a given process in order to evaluate the risk of a GTI to be present in the final API. The proposed tool defined the following main purge factors: GTI’s reactivity, solubility (in the solvents used, e.g, for recrystallization, where the GTI is discharged with mother liquid), volatility (e.g., through GTI

but the nitroaromatic and aniline intermediates showed negative genotoxicity by the AMES test. The study by Looker et al.273 was focused on the optimization of reaction conditions to avoid the presence of these impurities and comply with the established specific thresholds. In particular, the process employed Pd/C catalyst for the hydrogenation of nitroaromatics. The purification steps consisted of filtration for the removal of Pd, followed by concentration of the resultant filtrate, addition of an antisolvent for recrystallization, and filtration/drying of the solid obtained. Solutions spiked with potential GTIs were used to monitor and assess the effectiveness of impurity purging at different stages of the purification units. The process parameters selected for optimization include temperature, amount of catalyst, and

Scheme 92. Key Stages of the Commercial Synthetic Route to a Fluoroaryl-Amine Mesylate Central Nervous System Agent

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added in isopropyl alcohol, which can form a second potentially genotoxic ingredient, namely, isopropyl chloride. Moreover, the HCl can react with the AZD9056 base, resulting in small amounts of AZD9056 chloride as a byproduct, a third potential GTI. The assessment purge tool was applied to these three potential GTIs and the predictive values were compared with experimental results, pointing out that the tool usually underestimates purging effects. The purging of the three potential GTIs were assessed by considering the following: (i) The main driver for purging AZD9056 aldehyde is its high reactivity, which leads to this compound’s full consumption, and thus it was scored as 100% in the first step. Since this compound is not volatile, a score of 1 was allocated to all the steps. A moderate solubility was considering in the last two steps; however, a larger removal of the AZD9056 aldehyde was experimentally measured in step 2, leading to an overall underprediction of the purging capacity of the process by 10 times. (ii) Isopropyl chloride is present in steps 2 and 3, and as defined by the established criteria for high solubility and volatility, a score of 10 for purge factors was allocated to these two parameters. The tool predicts the purging capacity of 10 000, again representing an underprediction of about 4 times, indicating that in spite of the relatively high formation of isopropyl chloride, its presence in the final product is highly improbable. (iii) AZD9056 chloride byproduct is actually not reactive, not volatile, and not particularly soluble in isopropyl alcohol. Therefore, an overall low purging factor of 3 was predicted against a measured value of 10, implying that action should be taken to either remove this compound or change the process to eliminate or mitigate the formation of this compound. 4.2.2. Separation Technologies. For the specific removal of GTIs, the selection of the purification method is intrinsically dependent on the physicochemical properties of the GTI, which will decide the relative “purge” factors. From a process chemistry point of view, it is also important to understand which separation operation units are involved in API purification. In this review, seven examples of conventional purification techniques and three emergent techniques are referred to (Chart 7). Usually the higher the selectivity of a purification process regarding a specific impurity, the lower the API loss and the higher the removal efficiency of the impurity in question. In many cases, delivering a safe API requires the application of a purification strategy where the GTI is reduced to acceptably low levels. To illustrate this, a specification of 70 ppb, calculated using the daily dose, was set by a pharmaceutical company for an especially potent genotoxin in a drug candidate (shown in eqs 1−4).275

removed with solvent during distillation for solvent exchange), and ionizability (e.g., for a partition of GTI and API between aqueous/organic, for example, for pH adjustments to change the ionized/un-ionized state of one of the compounds) and processes used for purification (e.g., chromatography). This tool used a score scale for each purge factor, as described in Table 9, where purge factor is defined as the ratio of GTI concentration before and after purging. Table 9. Example of Key Parameters in Purge Factors in the Tool by Teasdale et al.a Physicochemical parameters Reactivity

Solubility

Volatility

Ionisability Physical processes (e.g., chromatography)

Purge factor High reactivity = 100 Moderately reactivity = 10 Low/no reactivity = 1 Freely soluble = 10 Moderately soluble = 3 Sparingly soluble = 1 Boiling point >20 °C below that of the reaction/prcess solvent = 10 Boiling point ±10 °C that of the reaction/ prcess solvent = 3 Boiling point >20 °C above that of the reaction/prcess solvent = 1 Ionization potential of GTI significantly different Chromatographically, GTI elutes prior to the desired product = 10 Chromatographically, GTI elutes after the desired product = 10 Others processes are valuated on an individual basis

a

Adapted with permission from ref 39. Copyright 2013 American Chemical Society.

They also describe six case studies where each of the different existing purge factors was evaluated and their contribution assessed at different stages in the removal of GTIs. Such cases include the removal of thionyl chloride (two syntheses); nitropyridyl N-oxide (a starting material); and AZD9056 aldehyde and its respective byproduct AZD9056 chloride, together with the side products isopropyl chloride, methyl hydrazine, and hydrazine. When genotoxins are introduced as reactants, their reactivity is one of the main factors contributing to how they are purged as they are consumed in the chemical reaction. Consequently, the use of a genotoxic reactant in excess is, if possible, to be avoided. Note that some of these highly reactive compounds can also be eliminated by reaction with bases, acids, or even water in subsequent steps, as is the case for thionyl chloride in the examples provided. Volatility is an obvious route for the removal of low boiling point compounds, such as methyl hydrazine (88−90 °C), thionyl chloride (79 °C), and isopropyl chloride (36 °C) through distillation and drying. Solubility takes an important role in purging such compounds, in particular when crystallization or extraction operations are involved where GTIs can be dissolved either in mother liquors or the discarded phase. AZD9056 HCl/chloride case study clearly illustrates the use of this assessment purge tool (Scheme 93).39,274 In final steps of the API synthesis, the potentially genotoxic AZD9056 aldehyde reacted with 3-aminopropano-1-ol to produce an imide derivative that is subsequentiatly reduced to a free base (step 1), followed by HCl salt formation (step 2) and finally recrystallization (step 3). During the purification step, HCl is

GTI removal efficiency = GTIend /GTIstart

(1)

APIloss = APIend purification step /API fed purification step

(2)

GTIcontent = GTIend purification step/APIend purification step

(3)

GTIdaily intake = APIdaily dose per patient kg × GTIcontent × weight patient

(4)

Such ultralow levels in the specifications of APIs pose additional analytical and processing difficulties for efficient purification. The design of a synthetic process to produce an AG


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Scheme 93. Last Steps of Synthesis of AZD9056 (Potentially Genotoxic Impurities To Be Purged Are Shown in Red)

content through an increase in the number of cycles would lead to unacceptable API losses. The use of an additional “end-of-pipe” GTI purification could complement the already existing intercalated purification steps. Nevertheless, the removal efficiencies are usually concentration-dependent, decreasing with lower GTI concentrations. In such cases, it may be advantageous to follow a “point-of-source” GTI detoxification strategy. For implementation of this strategy, identification and mapping of the reactions where GTIs are present is crucial, and lessons taken from section 2 should be considered. Conventional purification steps during and after API synthesis include crystallization, precipitation, solvent extraction, silica gel or alumina column chromatography, and treatment with activated carbon and resins, as well as distillations. As in any separation, the efficiency of the separation depends of the differences in chemical and physical properties of the two entities to be separate and/or their relative affinities for a selective agent. In this review, we briefly report 10 different purification techniques, of which 7 can be perceived as conventional methodologies to remove impurities

API includes sequential reaction steps intercalated with purification steps. These conventional purification steps are already in place and already contribute to GTI removal, although not specifically designed to remove GTIs. The difference between point-of-source and end-of-pipe GTI removal is schematically illustrated in Figure 7. The removal of larger quantities of impurities can be usually achieved by increasing the number of cycles within a given purification step (e.g., the number of re-extractions, recrystallizations). However, increasing the number of cycles also leads to undesirably high API losses and may have diminishing efficiency with each new cycle. Consider, for example, an API stream with a GTI content of 1 g of GTI for each 100 g of API, and a theoretical purification operation in which for each step 80% of the GTI is constantly removed, along with the sacrifice of 3% of the API. To reduce the GTI from a concentration of 1g/L in solution (corresponding to an API concentration of 100 g/L) to 64 μg/L would require six cycles and a cumulative API loss of 17% in the purification alone. Therefore, the use of conventional purification procedures to reach ultralow GTI AH


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Chart 7. Conventional and Advanced API Purification Technologies

Figure 7. Point-of-source and end-of-pipe GTI removal.

along with the API or remain as part of the crystal lattice, depending on the efficiency of the washing procedure. Filtration is the normal technique used to isolate the crystalline solids. A particular example is illustrated in Chart 8, where acetamide is removed in a process that incorporates crystallization.247 4.2.2.2. Solvent liquid−liquid extraction. (2) Solvent liquid−liquid extraction is commonly used for API purification; API (or impurities) can be selectively transformed into salts and retained in an aqueous phase while the organic impurities (or API) are removed by a water immiscible organic solvent phase. The organic salt can then be converted to the neutral species by acidification or basification, according to the pKa of the API, and re-extracted into a second organic solvent, which is usually concentrated before isolation of the API. The efficiency of separation depends on the relative partition coefficients of API and GTI in the different solvents. Panel i of Figure 8 illustrates a purification process involving solvent phase exchanges and crystallization of the API, while panel ii maps the corresponding losses of API. 4.2.2.3. Precipitation. (3) Precipitation is commonly promoted by addition of a nonsolvent to a solution of the API (or vice versa). Similarly to crystallization, the impurities remain in the liquid and the API ends-up as a solid phase.

and 3 as advanced techniques proposed during past decade (Chart 7). 4.2.2.1. Crystallization. (1) Crystallization is one of the most important isolation and purification process for APIs. The API is isolated as a solid phase while the impurities remain dissolved in the liquid phase (the mother liquors). Crystallization is also broadly used in chiral separations, namely, through diastereomeric resolutions.276,277 In some cases, a two-solvent system, a solvent and a cosolvent, can be used to promote crystal formation in accordance with the respective phase diagrams. Robustness, kinetics, temperature, and pH of the crystallization system are also important parameters.278,279 Crystallization is a purification process that not only determines the purity and residual solvent content of the API but also establishes the crystalline properties in terms of polymorphic form, crystal habit, bulk density, and size distribution, all of which affect downstream processing, e.g., drying and formulation.280,281 More importantly, the crystalline properties and polymorphic forms can be responsible for drug bioavailability. Therefore, once a route is approved for API production, the crystallization step of the final API is usually retained. In some instances, depending on process optimization, a significant fraction, up to 30% of the API, can remain in the mother liquors282 or be lost through washes of the solids. Impurities may be washed out AI


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Chart 8. Example of a Conventional Process for API Purification from the Carcinogenic Acetamide

absorbents for metal impurities has been evaluated using microtubes.289,290 Other studies include removing formaldehyde using activated carbon containing amine groups291 and removal of an aldehyde impurity using polystyrene-based sulfonylhydrazine resin.292 GTIs such as p-toluenesulfonic acid methyl (MeTs), ethyl (EtTs), and isopropyl (i-PrTs) esters have also been evaluated using different commercially available nucleophilic resins.287 These studies used methyl, ethyl, and isopropyl esters of methanesulfonic, benzenesulfonic, and p-toluenesulfonic acids as model PGIs and screened the use of several amines, thiol, thiophenol, piperazine, and piperidine immobilized on silica and polystyrene. Removal was effective for methyl sulfate esters, whereas it proved to be more of a challenge to remove ethyl and isopropyl esters by this technique. This strategy was applied for the removal of MeTs from a 21-chlorodiflorasone solution. When trisamine was immobilized either on silica or macroporous polystyrene− divinylbenzene supports, 100% GTI removal was achieved.293 These adsorbents and resins can be used as stationary phases in chromatography. 4.2.2.6. Column chromatography. (7) Column chromatography is a typical postreaction technique applied in organic chemical synthesis to remove impurities. Sophisticated stationary phases are applied in the pharmaceutical industry, for example, in chiral separations.294,295 However, this review is focused on the removal of GTIs, and for this endeavor, preparative column chromatography using standard silica gel296 or alumina of pharmaceutical grade as stationary phase has been used. In this technique a solvent, such as ethyl acetate, ether, acetone, methylene chloride, and/or mixtures thereof, is used as eluent. Commercially available absorbents, such as polystyrenic or methacrylic matrices, with aqueous solutions at different pHs and ionic strengths have also been reported.297 Particle size,

However, the solid may be amorphous and not crystalline, but once more, the solvent system (final mixture of solvent and nonsolvent) selected should show higher solubility for the impurities than for the API. Solute solubility is, among other things, dependent on its polarity and the polarity of the solvent. Note that some of these polar solvents also have high boiling points and are potentially genotoxic themselves (Table 8); therefore, if they are not removed properly, they present an additional risk as a GTI in the API . Filtration is also used to separate liquid from solid, or distillation is used to evaporate low boiling point solvents. When the impurity is preferentially precipitated, it can be removed by filtration. 4.2.2.4. Fractional distillation. (4) Fractional distillation can be used to purify volatile APIs.283 However, distillation is also broadly used for removal of solvents and for solvent exchanges, particularly when switching from a low boiling point solvent to a higher boiling point solvent (see Table 8). Solvent exchanges from high boiling point solvent to lower boiling point solvents or when thermosensitive compounds are involved can be sustainably achieved using organic solvent nanofiltration (OSN).282 Volatile organic impurities, mainly resulting from residual solvents, 284,285 can also be removed through distillation. Many of the GTIs considered in this review have low volatility (e.g., hydrazine, MsCl, TsCl, DMS, 1,2-epoxy-3butene, acetamide, phenylboronic acid all have boiling points above 100 °C), and alkyl halides such as MeCl and EtCl have boiling points of −24.2 and 12.3 °C, respectively. 4.2.2.5. Adsorption processes. (5 and 6) Adsorbents such as granular activated carbon (GAC)286 and resins287 are broadly used to remove color and impurities.288 Adsorption-based separations rely on the different affinities of the disparate compounds for the adsorbent. Therefore, a high affinity of the GTI combined with lower binding of the API is desirable in this case. Screening of the different commercially available AJ


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4.2.2.8. Organic solvent nanofiltration. (9) Organic solvent nanof iltration (OSN) relies on separations based mainly on differences in molecule size, although other properties such as shape and polarity can also contribute.301 The use of OSN had been previously suggested for the purification of APIs302 and has recently been evaluated specifically for the removal of GTIs from API streams.303−305 The performance of this technique is highly dependent on the membrane selected and on the respective rejection curve. There are several commercially available polymeric and ceramic membranes that are stable in organic solvents. Examples include Koch SelRO membranes, the StarMem series developed by W. R. Grace & Co., the DuraMem series from Evonik MET, SolSep membranes, GMToNF-2 from Borsig Membrane Technology GmbH, and Novamem polymeric membranes, as well as Inopor or Pervap ceramic membranes. The role of OSN in API purification is illustrated in Figure 9, while the OSN-based API purification

Figure 8. (a) API purification by several cycles of phase exchanges and recrystallization and (b) the corresponding yields for each cycle. Figure 9. Role of OSN in API purification.

scheme is shown in Figure 10. The most crucial limitation of OSN in API purification is the low product yield due to insufficient rejection of the product.305 To overcome this limitation, Kim et al. recently proposed a two-stage membrane cascade.304 Through an API purification case study, the authors demonstrated that the proposed process significantly increases the API yield without compromising its purity. The second main drawback of OSN for API detoxification was the significant solvent consumption during diafiltration processes. However, OSN has markedly evolved in recent years, and the newest generations of solvent-resistant membranes can fully reject small molecules at the lower end of the nanofiltration range (50−2000 g·mol−1) and subsequently can be used for in situ solvent recovery.306 4.2.2.9. Molecular imprinting technology. (10) Molecular imprinted polymers (MIPs) are prepared by incorporating the

column dimensions, and eluent flow and pressure are critical parameters. 4.2.2.7. Supercritical extraction. (8) Supercritical extraction techniques utilize the properties of supercritical fluids, which have the high solvation power of a liquid and the enhanced diffusivity of a gas. Moreover, simply changing from the supercritical state to a gaseous state provides a straightforward method to isolate the solute. The relatively low critical point of CO2 (71 bar, 31 °C) had positioned it as an ideal supercritical solvent that can replace more hazardous solvents as reaction media and in extractions in chemical processes.298 Supercritical CO2 can be used to produce particles with controlled size and purity299 and also be used in packed column chromatography.300 Therefore, this purification technology has the potential to provide an effective and clean route for GTI removal from APIs. AK


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imprinting systems for given applications. Kecili et al. developed a protocol for the rapid identification of MIPs for genotoxic aminopyridine removal from piroxicam and tenoxicam via screening of MIP libraries.316

5. CONCLUSIONS AND FUTURE TRENDS Launching new pharmaceutical products involves the collaborative effort of R&D teams, physicians and hospitals, pharmaceutical companies, and investors, as well as regulatory authorities and reimbursing agents. The aim of launching a new pharmaceutical is always to treat or manage a specific disease and thus extend patient life or improve his/her quality of life. Therefore, efficacy and safety are the main end points for drug development. When highly reactive chemicals that can attack DNA or interfere in DNA replication are present as impurities in a pharmaceutical product, the administration of such drugs compromises safety, since it can become a vehicle for increasing genotoxic risk. The quantity of genotoxic impurities in drug products is strictly controlled by regulatory authorities that have set limits to ensure patient safety. To ensure compliance with the required low GTI concentrations, a significant effort during development is necessary. Three main strategies that contribute to producing APIs of acceptable quality can be identified: (1) At the most rigorous level, the strategy outlined in the regulatory authority’s guidelines is to avoid the use of any genotoxic chemical over the entire synthetic route, regardless of whether the genotoxic chemical is used as a reagent, starting compound, catalyst, or solvent. However, given the chemical nature and desirable reactive properties of chemicals, in many cases the direct use of a genotoxic chemical or a GTI precursor is unavoidable. (2) Even when genotoxic compounds are not applied directly, they can be formed during chemical reactions. Therefore, a second strategy for minimization of genotoxin formation can be achieved by thorough investigation of the particular reaction and adjusting the reaction parameters with assiduous care, in particular using QSAR strategies. (3) An alternative and complementary strategy is to design specific purification strategies targeting the removal of GTIs from the API once it has been established that they or their precursors are present. Either “point-of-source” or “end-ofpipe” strategies can be used. The full cycle of drug development, approval, and use has to work for the different stakeholders. This means safety and efficacy for patients, compensation for the efforts of the

Figure 10. OSN-based API purification, with potential use of organic reverse osmosis for solvent recycling.

target molecule into a polymeric matrix as template (Figure 11). The target molecule is therafter removed, leaving a potential binding site within the matrix. Thus, the final polymer structure usually provides enhanced affinity for removal of the molecules used as template. The use of MIPs for separations in the pharmaceutical industry has been suggested previously307 and used in bio- and pharmaceutical analysis.308 Exploring the high specificity achieved by MIPs, several studies have evaluated their use in chiral separations.309−311 Specific development and characterization of a MIP for potential GTI removal were recently reported.312,313 By exploring the ability of OSN to remove potential GTIs when at high concentrations and combining this with the better performance of MIPs to remove the target molecule at lower concentrations, a hybrid process using these two purification techniques also had been suggested.314 Possible limitations of the use of MIPs for GTI removal are as follows: specific MIPs need to be developed for individual (or similar) GTIs; removal is more effective at lower concentrations, and hence, high volumes are involved; and contamination is possible via leaching of impurities derived from the polymer. Hence, this technique has not become widespread at this time. Besides, hybrid processes imprinting and nanofiltration technologies have been combined in a molecularly imprinted organic solvent nanofiltration strategy.315 MIPs often show cross-reactivity, which can be exploited in the rapid screening of MIP libraries to identify suitable

Figure 11. Schematic of the molecular imprinting technique. Functional monomers, template, and cross-linker are allowed to self-assemble in solution, and subsequent polymerization yields the imprinted material. The template is extracted from the polymer, leaving a binding site with complementary topography and chemical functionality behind. The resulting MIP can selectively recognize the template molecule in complex mixtures. Reprinted with permission from ref 311. Copyright 2015 Americal Chemical Society. AL


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Biographies

pharmaceutical companies, and selling prices that are sustainable for reimbursing systems. The role of the regulatory agencies is not only to guarantee the safety of the patient, but also to ensure that the barriers to development of new products are not such that they result in a hindrance to the development of new medicines that target unmet medical needs or that improve the performance of current therapies. It is important that new drugs are not priced in such a way that both patients and reimbursing systems cannot support their cost. The risk of having GTIs present and the cost of the efforts to avoid or remove them from APIs are contributory factors to the cost of production of new drugs. Therefore, it is vital to develop costeffective strategies to remove or mitigate the presence of GTIs from APIs and to avoid inefficient strategies to attain levels lower than those where no adverse effects are evident. Therefore, a checklist for the pharmaceutical R&D community to manage the GTI risk can be outlined as follows: (1) Provide solid data for toxicological evaluation of potential GTIs with quantification of threshold values and highlight those that are higher than the general TTC value. (2) Develop analytical and monitoring techniques for ultalow levels of GTIs. (3) Develop new synthetic or process routes. Specifically, alternative reagents should be used to replace genotoxic or GTI precursor reagents. Identify new reaction media to replace genotoxic or carcinogenic reaction solvents. (4) Strive for a deeper understanding of existing reactions and process routes to be pursued, identifying and optimizing the crucial chemical and physical parameters in the process, to mitigate GTI presence. (5) Develop novel API purification techniques for removal of GTIs to the stringent limits required. During the past few years, research into genotoxic impurities has shown remarkable achievements, as shown by collaborative efforts between various R&D scientists. This has resulted in safe and profitable drug products. However, room for the expansion of our knowledge and the development and use of new technologies require the participation of innovative research from such diverse areas as chemistry, process engineering, material science, and biology.

Gyorgy Szekely received his M.Sc. degree in chemical engineering from the Technical University of Budapest (Budapest, Hungary), and he earned his Ph.D. degree in chemistry under Marie Curie Actions from the Technical University of Dortmund (Dortmund, Germany). He worked as an early stage researcher in the pharmaceutical research and development center of Hovione PharmaScience Ltd in Portugal and as an IAESTE fellow at the University of Tokyo (Tokyo, Japan. He was a visiting researcher at Biotage MIP Technologies AB in Sweden. He was a postdoctoral research associate at Imperial College London (London, UK). He is currently a lecturer at the School of Chemical Engineering & Analytical Science, The University of Manchester. His multidisciplinary professional background covers supramolecular chemistry, organic and analytical chemistry, molecular recognition, molecular imprinting, process development, membrane separations, and pharmaceutical impurity scavening. In addition, he is a board member of the Marie Curie Fellows Associationserving as Secretary Generaland is a member of the Royal Society of Chemistry, The Institution of Engineering and Technology, and the Institution of Chemical Engineers.

ASSOCIATED CONTENT S Supporting Information *

Complete author list for references with more than 10 authors. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/cr300095f.

Miriam C. Amores de Sousa graduated with a degree in applied chemistry (minor in biotechnology) in 2007 and concluded her Master’s degree in biotechnology in 2009, at Faculdade de Ciências e Tecnologia of Universidade Nova de Lisboa. She is currently a Ph.D. student at the Department of Bioengineering at Instituto Superior Técnico, Universidade de Lisboa, where she is a member of the BioEngineering Research Group at the Institute for Bioengineering and Biosciences. Previously, as a research assistant, she worked on the characterization of polysaccharides and explored biocompatible cellulose acetate membranes as potential drug delivery systems, focusing on the solid-state mobility properties of the materials. Currently, her research is focused on studying the interaction between electrospun functional nanofiber matrices and stem cells, to evaluate

AUTHOR INFORMATION Corresponding Authors

*G.S. e-mail: [email protected]. *F.C.F. e-mail: [email protected], frederico_castelo@ yahoo.com. *W.H. e-mail: [email protected]. Notes

The authors declare no competing financial interest. AM


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interests were broadened to the development of novel particle size reduction technologies aiming to produce engineered particles for inhalation. Currently, he heads the Process Chemistry Development Group, which is responsible for the development and scale-up of chemical processes for the production of active pharmaceutical ingredients.

the cell fateself-renewal and differentiationfor future applications in tissue engineering, as stem cells provide an interesting model to probe the cytotoxic effects of different compounds and materials.

Frederico Castelo Ferreira graduated with a degree in applied chemistry (minor in biotechnology) in 1999 from New University of Lisbon (UNL) and received his Ph.D. in chemical engineering from Imperial College London in 2004 and his MBA from UNL in 2008. He was a research associate (2004−2006) in a joint project of Imperial College London and GlaxoSmithKline (GSK). He was a visiting researcher (July 2007) at Institut Européen des Membranes (Montpellier, France) and a visiting scholar (Sept-Dec 2009) at the Massachusetts Institute of Technology, Deshpande Center for Technological Innovation. Since March 2009, he has been a member of the BioEngineering Research Group at the Institute for Bioengineering and Biosciences. He teaches at the Department of Bioengineering at Instituto Superior Técnico, Universidade de Lisboa, including the course “BioteamsTeams for Innovation” for Ph.D. students, and he launched two new elective M.Sc. courses: “Green Technologies and Strategic Management” and “Entrepreneurship in Bioengineering”. He also assists CoHiTec on translation of technology to the market. His current research interests balance between fundamental and applied research, for the development of new processes, reactors, and materials, with an emphasis on membranebased systems.

William Heggie received his Ph.D. in organic chemistry from The University of Manchester and held postdoctoral positions at Harvard, St. Andrews, and Oxford Universities. He held a teaching position at the New University of Lisbon from 1974 to 1976 and was a professor at Lisbon Superior Institute of Engineering from 1979 to 1998. He joined Hovione in 1980 and held various positions in Hovione’s R&D group before becoming Chief Scientific Officer in 2004, being responsible for innovation and introducing new technologies into the company. He is the author of more than 20 patents and scientific articles.

ACKNOWLEDGMENTS The authors acknowledge the support of NEMOPUR (New Molecular Purification Technology for Pharmaceutical Production), a Marie Curie Initial Training Network within the seventh Framework Programme of the European Commission’s Marie Curie Initiative, and the support of FCT (Fundaçaõ para a Ciência and Tecnologia) through the funding initiatives PTDC/QEQ-PRS/2757/2012, SFRH/BD/73560/2010, and IF/00442/2012. The authors would like to express their gratitude to the reviewers for their valuable comments that shaped the review. Many thanks go to Dr. Jozsef Kupai for useful discussions about organic chemistry. REFERENCES (1) Web of Science. http://thomsonreuters.com/products_services/ science/science_products/a-z/web_of_science/, accessed April 27, 2012. (2) Pierson, D. A.; Olsen, B. A.; Robbins, D. K.; DeVries, K. M.; Varie, D. L. Approaches to Assessment, Testing Decisions, and Analytical Determination of Genotoxic Impurities in Drug Substances. Org. Process Res. Dev. 2009, 13, 285−291. (3) Committee for Medicinal Products for Human Use (CHMP), Safety Working Party (SWP). Questions and Answers on the ‘Guideline on the Limits of Genotoxic Impurities’; EMA/CHMP/SWP/431994/ 2007 Rev. 3; European Medicines Agency: London, September 23, 2010. (4) Miller, J. A.; Miller, E. C. Ultimate Chemical Carcinogen as Reactive Mutagenic Electrophiles. In Origin of Human Cancers; Hiatt, H. H., Watson, J. D., Winsten, J. A., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1977; p 605. (5) Votano, J. R.; Parham, M.; Hall, L. H.; Kier, L. B.; Oloff, S.; Tropsha, A.; Xie, Q.; Tong, W. Three New Consensus QSAR Models

Marco Gil graduated with a degree in chemical engineering from Technical University of Lisbon, from which he obtained his Ph.D. in chemistry in 2006. The main focus of his thesis was the development of active ingredients for iron chelation therapy with improved in vivo behavior. In 2007, he joined the R&D department of Hovione as a scientist in the Particle Design Group. The focus of his work was the application of particle engineering technologies to improve bioavailability of poorly water soluble drugs and the scale-up of processes for the production of solid dispersions by spray-drying. Later on, his AN


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