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 Tecnico,́ 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 AC 4.1.2. Adjusting Reaction Conditions To Miti- gate GTI Formation AC 4.1.3. Quality by Design AE 4.2. API Purification AF 4.2.1. Purge Factors AF 4.2.2. Separation Technologies AG 5. Conclusions and Future Trends AL Associated Content AM Supporting Information AM Author Information AM Corresponding Authors AM Notes AM CONTENTS Biographies AM Acknowledgments AN 1. Introduction A References AN 2. Genotoxicity: Mechanisms, Risk and Regulation C 3. Chemical Classes of Common Genotoxic Impur- ities E 1. INTRODUCTION 3.1. Genotoxic Compounds Used as Reactants F Most pharmaceutical products are manufactured either by 3.1.1. Alkyl Halides F applying a total synthesis approach or by modifying a naturally 3.1.2. Dialkyl Sulfates I occurring product. In both cases, a wide range of reactive 3.1.3. Epoxides K reagents are used. Therefore, it is natural that low levels of such 3.1.4. Hydrazines L reagents or side products are present in the final active 3.1.5. TEMPO N pharmaceutical ingredient (API) or drug product as impurities. 3.1.6. Aromatic Amines O Such impurities may have unwanted toxicities, including 3.1.7. Boronic Acids P genotoxicity and carcinogenicity. The risk for patient’s health 3.2. Genotoxic Compounds Formed in Side caused by the presence of small molecules as impurities in APIs Reactions R has become an increasing concern of pharmaceutical 3.2.1. Sulfonate Esters and Their Precursors. companies, regulatory authorities, patients, and doctors alike. Overview R Thus, pharmaceutical regulatory agencies such as the Food and 3.2.2. Sulfonate Esters and Their Precursors Drug Administration (FDA) and the European Medicines Used in Stoichiometric Amounts S Agency (EMA) have raised concerns regarding the presence of 3.2.3. Sulfonate Esters and Their Precursors genotoxic impurities (GTIs) in APIs that could impact Used in Catalytic Amounts X negatively on human health. 3.2.4. Alkyl Halides Y There is an increasing scientific interest in this field, as is 3.2.5. Acetamide Y illustrated in Figure 1, from data obtained from the ISI Web of 3.3. Genotoxicity and Carcinogenicity of Com- Science showing the number of publication hits on mon Organic Solvents AA “genotoxicity” and on “genotoxic impurity”.1 The graph based 4. Approaches for GTI Mitigation in the Pharma- ceutical Industry AA 4.1. Chemical Synthetic Approaches AB Received: March 7, 2012

© XXXX American Chemical Society A DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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 5 ff 6 Figure 1. Importance of genotoxicity demonstrated by the increasing Ames genotoxicity and presentation of di erent case studies. number of publications on the topic, resulting from an ISI Web of QSAR models commonly used for determination of structural Science search on “genotoxicity” and “genotoxic impurity”. alerts to predict genotoxicity are the MULTICASE and the deductive estimation of risk from existing knowledge (DEREK) 7−9 on the former search shows the overall importance of the field ones. However, for numerous chemical classes, structural of genotoxicity, including chemistry, analytical methods, alerts overpredict mutagenicity when they do not take into manufacturing, purification, diseases, medical aspects, genotox- account factors such as high molecular weight, hydrophilicity, icity tests, mechanism of action, assessment, and environment. high reactivity, steric hindrance, molecular symmetry, and facile 10,11 The latter search illustrates the increasing attention of industry metabolism. to GTIs, mainly related to drugs and food. On the other hand, their presence in the manufacture of APIs Compounds categorized as GTIs actually include a broad is not stochastic, since these genotoxic chemicals often have range of unrelated chemicals with very different structures and specific inherent roles in the chemical routes used in API from very different chemical families. From 4000 compounds synthesis. The presence of such chemical in the reaction is a tested, 44 molecular structures were correlated with muta- result of their introduction into the reaction in stoichiometric genicity and correlated highly with electrophilic reagents, such or catalytic amounts or as solvents, as well as their formation as as epoxides (63%), aromatic amines (49%), and primary alkyl side products. The presence of genotoxins is usually inherently monohalides (46%).2 Aromatic amines are not electrophiles, controlled during API manufacture, as several stages of but their decomposition leads to the formation of electrophilic intermediate API isolation and purification are included in reactive species such as aryl nitrenium ion. In section 3.1.6 the production process, during which most of the GTIs are, examples of aromatic amine reactants are described. These together with other impurities, removed. Additionally, many of compounds have a shared ability to react with DNA, resulting the synthetic reaction sequences initially designed for in an associated carcinogenic risk. However, from a chemical production of new drugs are often further improved through point of view, they do not have common chemical−physical optimization of reaction conditions or by substituting with properties or chemical structural elements that can contribute different reaction steps. Such improvements aim at higher to easy identification. Experimental assessment of genotoxicity yields, reaction selectivity, and more efficient use of reactants, test models, such as the Ames test, allows direct study of which results in lower amounts of unreacted compounds and genotoxicity, and the Committee for Medicinal Products for side products formed. Nevertheless, production of APIs with Human Use (CHMP) has defined GTIs as impurities that have low GTI content is a major concern for API-manufacturing been demonstrated to be genotoxic using such genotoxicity test companies. Ideal solutions consist of the simplest possible, models. The Ames test, developed in the early 1970s by Bruce robust process, using cost-effective reagents to obtain high N. Ames, is an experimental procedure to evaluate the potential product yields through selective reactions and purification carcinogenicity of chemicals, based on mutagenicity effects on steps. Development and validation of such processes in a timely Salmonella typhimurium histidine auxotrophic mutants strains. manner are important for the industry, and as such, it is It became widely used due to its simplicity, low cost, and quick important to be aware of the chemical mechanisms in which analysis without the need for animal testing. genotoxic compounds are involved, whether as reagents or As discussed in ICH Q3A and Q3B, actual impurities in API reaction side products, and of existing strategies to circumvent are the ones that exceed the reported threshold when the lot is their use or remove them from postreaction streams. released or arise, for example, as degradation product, during In addition to the Introduction and concluding remarks, this storage and distribution over the shelf life of the API, whereas review includes the following sections: potential impurities may or not actually be present in the API Section 2 provides a brief description of genotoxic but are identified as the ones that can theoretically arise during mechanisms and a risk analysis, as well as the regulatory manufacture or storage. In the particular case of GTIs, approaches taken concerning this issue. Further reviews on “potential GTIs” are the ones that have structural alerts, i.e., specific topics of risk assessment,12 toxicology,13 and functional chemical groups, for genotoxicity but have not been mechanism of action14 of such compounds can be found in experimentally assessed; note that here potentially is not related the literature. with the presence or absence of the impurity.3 In silico systems Section 3 of this review focuses on GTIs related to starting are commonly used to identify structural alerts and predict materials, reagents, reactants, catalysts, and solvents with

B DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review genotoxic effects and related genotoxic side products. The authors believe the knowledge systematically gathered in Impurities structurally related to specific APIs (e.g., genotoxic the present review will help in the assessment of both new and sulfonate ester side products during the synthesis of the steroid alternative synthetic routes when taking into account the mometasone15,16) are outside of the scope of this review sources of GTIs and allow the pharmaceutical R&D scientists (Figure 2). The book “Genotoxic ImpuritiesStrategies for 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). 2. GENOTOXICITY: MECHANISMS, RISK AND REGULATION Although it has now been several years since the introduction Figure 2. Sources of GTIs in API streams. 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 Identification and Control” edited by Teasdale provides an range of genetic damage, regardless if such damage is or is not exhaustive discussion on the identification and control of such corrected through a cell DNA-repairing mechanism. A impurities in API manufacturing.17 mutation represents a permanent change in the genome, The major focus of this review is provided in section 3, which which can lead to phenotype change, and a mutagen is a comprises a thorough description of chemical synthesis of substance able to increase the frequency of such changes. A several pharmaceuticals. The objective of this review is to carcinogen is a substance that induces unregulated growth contribute to an easy identification and mapping of GTI processes in cells, through damage to the genome or cell occurrence in chemical synthetic routes, highlighting the metabolic effects, eventually leading to cancer. In other words, importance of GTIs in the manufacturing of APIs. Therefore, mutagenicity refers to processes leading to genetic change, and the focal point of this review is to improve awareness of carcinogenicity refers to processes resulting in tumor develop- different entry points for GTIs over the API synthesis. Case ment, which may result from mutagenic processes.18,19 histories and synthetic examples selected include 93 different The mechanism of action of genotoxins involves an chemical schemes used in the synthetic routes of APIs or electrophilic attack on the nucleophilic center(s) of the DNA, intermediates of 100 different drugs. Nine different chemical these being nitrogen and oxygen atoms of pyrimidine and families are present because they are used as reagents or purine bases and the phosphodiester backbone, which could, in catalysts or are formed during synthesis. In recognition of the some circumstances, lead to strand breaks (Figure 3). Bidentate importance of risks posed by residual toxic solvents left in API genotoxic agents can react with two nucleophilic sites, resulting formulations, an additional section highlights the potential in (i) one single molecule giving a bicyclic or tricyclic system; genotoxicity and carcinogenicity of common solvents used in (ii) involvment of two different molecules in the same or the drug synthesis. opposite DNA strand, affording inter- or intrastrand cross- Section 4 discusses approaches for mitigation of GTI linkages, respectively; or (iii) linking a protein and a DNA content. A variety of different strategies are used to avoid/ strand, giving a DNA−protein adduct.14 Besides the chemical reduce GTIs during industrial implementation, as disclosed in nature of the genotoxic agent, the stereospecificity of the patents and academic publications. Section 4 includes two reactions also depends on steric factors and nucleophilicity; for subsections focused on synthetic approaches and on API instance, the most nucleophilic sites of the DNA bases are purifications. Examples include changing the reaction route endocyclic nitrogens, such as N3 and N7 of guanine and involving a GTI reactant and the optimization of reaction adenine, and on the contrary, exocyclic oxygens are less conditions to mitigate the amount of unreacted genotoxic nucleophilic.18,20 reactant left in the postreaction stream. The section on API Impurities, especially genotoxic impurities, have been at the purification strategies briefly describes (i) conventional center of increasing regulatory and industry attention in the purification techniques, such as recrystallization, chromatog- past decade. The timeline of key actions toward those raphy, and distillation, and (ii) emerging technologies, such as regulations is shown in Figure 4. The International Conference organic solvent nanofiltration, supercritical extraction, and on Harmonisation of Technical Requirements for Registration molecular imprinting−with the aim to expand the chemists’ of Pharmaceuticals for Human Use (ICH) was setup for the and engineers’ toolbox to address API detoxification from analysis of scientific and technical aspects of pharmaceutical GTIs. product registration and includes the main players in the field,

C DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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 Figure 4. Timeline of key actions toward a regulation on GTI control therefore, this review includes a specific subchapter dedicated in APIs. to this particular class of GTIs. Two years later, the Committee for Proprietary Medicinal industry and focused only on food additives; however, it Products (CPMP) published the first draft position paper on evolved continuously as its broader applicability than simply to GTIs showing sufficient evidence for the existence of a chemicals in food and its potential value in the assessment of threshold mechanism in the toxicity of such compounds. This risks in other exposure scenarios were realized.23 The brief − position paper challenged the scientificandindustrial history of TTC is summarized in Figure 5.24 26 The FDA and community to seek GTI-free routes to APIs or, when not EMA have agreed on the implementation of the TTC concept possible, to provide a justification why the presence of GTIs is that sets a limit of 1.5 μg day−1 for known and potential unavoidable. At that time, it was argued that in vivo studies carcinogens, unless experimental evidence justifies higher limits. would put test subjects at a nonjustifiable risk. Therefore, the Higher levels can be applied in shorter-term studies during the model of a virtual safe dose concept, previously used in the clinical testing of the APIs (Table 1). The rational behind such food industry, was suggested as an alternative and the low values is to ensure that even if a substance was later found terminology “as low as technically feasible” was introduced. to have negligible carcinogenic risk, no issues concerning safety This model was the basis to the later introduction of the would arise.27 An exhaustive effort has to be made by the threshold of toxicological concern (TTC) concept. A draft industry to meet such requirements, and since many potentially guideline on the limits of GTIs was released in 2004 by the genotoxic agents turned out to pose far less risk than originally Committee on Human Medicinal Products (CHMP) from supposed, the TTC approach is considered to be conservative. EMA and the TTC concept was introduced.22 The TTC is a Nonetheless, such low GTI thresholds are here to stay, and the concept that refers to the establishment of a level of exposure industry has been addressing this challenge with a variety of for all chemicals irrespective of the existence of chemical- control and purification strategies. specific toxicity data, below which there is no appreciable risk to Moreover, the terminology of “as low as reasonably practical” human health. It is assumed that a low level of exposure posing (ALARP) was applied for GTIs; the requirement to introduce a negligible risk can be identified for any chemical based on its alternative routes or processes when available was dropped, and structure. The TTC concept was first introduced in the food no guidance relating to permissible doses during short-term

D DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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 PharmEur- opa 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 Figure 5. A brief histroy of the TTC principle. from different chemical families, and within each family they are organized according to their role in the chemical reaction, clinical trials was provided. In 2006, the Pharmaceutical namely, as a stoichiometric reagent, a catalytic reagent, or side Research and Manufacturers of America (PhRMA) led by product.

Table 1. Proposed Allowable Daily Intake (ADI) for GTIs of Unknown Carcinogenic Potential during Clinical Development

Duration of exposure <1 1−33−66−12 >12 (month) ADI (μg/day) 120a or 0.5%,b whichever is 40a or 0.5%,b whichever is 20a or 0.5%,b whichever is 10a or 0.5%,b whichever is 1.5b,c lower lower lower lower aProbability 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.

E DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Table 2. Common Synthetic Transformations Related to Genotoxic Impurities

Bond formation Alerting group C−OC−CC−N AHCa Associated reactions Examples

(1) Genotoxic Reagents/Catalysts Applied Directly

Phophonate esters X Homer−Wadsworth−Emmons olefination refs 34−37 Alkyl halides X X X Williamson ether synthesis; Heck, Sonogashira, Kumada Pd-catalyzed cross- section 3.1.1 couplings Dialkyl sulfates X X O- and N-alkylations section 3.1.2 Epoxides X X X Sharpless asymmetric epoxidation section 3.1.3 Aldehydes X X X Aldol and Claisen condensation refs 38−40 Hydrazines X X Fischer indol synthesis, common heterocyclic precursor section 3.1.4 TEMPO X Oxidation of alcohols section 3.1.5 Aromatic amines compounds X Common feedstock for aromatic structures section 3.1.6 Aminoaryls X X Common intermediate Boronic acids X section 3.1.7 Aromatic nitro compounds X Common starting material and intermediate refs 41−43

(2) Side Reactions Forming GTIs

Sulfonates Stoichiometric amounts 3.2.2.1. API Salt Forming Agents section 3.2.2 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 section 3.2.3 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 Alkyl halides Reactions between alcohols and acids section 3.2.4 Acetamide Cοmmon building blocks section 3.2.5

(3) Solvents

Solubilize reagents and reactants section 3.3 aAHC 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, Reactants used in chemical synthesis are usually selected due to ω their appropriate reactivity; however, this very same reactivity and -bifunctional alkyl halides are also directly used in API ω could result in genotoxicity. Often such reactants are not fully synthesis, of which -alkyl dihalides are common linking agents consumed, persist in the reaction mixture, and can be carried due to their ability to connect API intermediates via forward in the reaction sequence. Seven classes of reactants consecutive alkylation. It was hypothesized that bifunctional used in API synthesis were selected as examples to be presented alkanes cause genotoxic damage by the glutathione-dependent 46 in this section, including two types of alkylating agents, alkyl pathway and consequent formation of toxic methanethiol. halides, and dialkyl sulfate; epoxides used in several addition Nitrogen and sulfur mustards (e.g., 2,2-dichlorodiethyl sulfide) reactions; hydrazine, a strongly reducing nitrogen base; represent a special class of alkyl halides and have been used as TEMPO, a cyclic amine oxide radical; aromatic amines, used chemical weapons. They are potential alkylating agents and as building blocks; and boronic acids, used in carbon−carbon their toxicity is attributed to cross-linking between DNA 47 coupling reactions. Other well-known classes of potentially strands. The source of alkyl halide in APIs streams can be genotoxic impurities, aldehydes and aromatic nitro compounds, derived not only from direct use of alkyl halides but also from which are mainly used as starting materials and not reactants, side reactions between alcoholic solvents and hydrogen halides are not included in this section. or dequaternization of ammonium salts. It is worth mentioning 3.1.1. Alkyl Halides. Methyl, ethyl, and propyl halides are that alkyl halides, such as methyl or ethyl chloride, deriving used widely as industrial alkylating agents. Although the from low molecular weight alcohols, are volatile and readily mechanism of their toxicity is still not fully understood, they purged from the API during the drying process. On the other

F DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review hand, they can get trapped in the API crystal matrix and lead to the synthesis of ocaperidone53 and azimilide54 applying 1-chloro- trace impurities that are to be controlled.48 The sources for 2-bromoethane, 1-chloro-4-bromobutane, respectively. genotoxic alkyl halide impurities in APIs is summarized in During the synthesis of cerivastatin, the hydroxyl group of a Table 3 on the basis of the source of the impurity, reaction carbinol is converted to the corresponding methyl ether with types, and examples of API synthesis. sodium hydride and methyl iodide (Scheme 3).55 S-Methylation with genotoxic methyl iodide is used during Table 3. Sources and Reaction Types That May Lead to the synthesis of the amidine-based fibrinogen receptor Genotoxic Alkyl Halide Impurities in Drug Substances antagonist lamifiban.Inthefinal synthetic step, the tripeptide-like intermediate reacts with hydrogen sulfide, GTI source Application API synthesis example leading to the iminothiol addition intermediate, followed by a Dequaternization DMTMM Antibiotics, peptides, alkylation with methyl iodide, which converts sulfur to the coupling reactions alkaloids Direct use of alkyl halide C-alkylation Fexofenadine, methylthio derivative. Treatment with ammonium acetate leads reagents anastrozole to displacement of the good leaving group, methyl mercaptide, 56 O-alkylation Alisiren, cerivastation, by ammonia, affording lamifiban (Scheme 4). mazapertin During the synthesis of eldacimibe, Meldrum’s acid reacts S-alkylation Lamifiban, eldacimibe with carbon disulfide in the presence of a base, leading to N-alkylation Efegatran, aripiprazole condensation and formation of a bismercaptide. This transient Quaternization Milameline dianion is reacts in situ with methyl iodide to give a highly fi Esteri cation Latanoprost reactive intermediate with two good leaving groups (Scheme Cycloalkylation Mazapertine, 5).57 aripiprazole fi Use of hydrogen halides in Cyclization Capecitabine, The synthesis of guanidine-containing brinogen antagonist alcoholic solvents sitagliptin efegatran involves the N-methylation of a N-carbobenzyloxy Decyclization Xemilofiban (Cbz) derivative with methyl iodide in the presence of a base, Decarboxylation Sunitinib leading to the corresponding N-methyl derivative (Scheme Cleavage Bortezomib 6).58 N-arylation Pazopanib Alkyl halides are also used to obtain quaternary ammonium Salt formation Conivaptan, salts. During the first synthesis of the potential cholinergic pazopanib 59 a agonist milameline, alkylation of the pyridine intermediate DMTMM stands for 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl- with methyl iodide leads to the quaternary salt as depicted in morpholinium chloride. 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 The synthesis of fexofenadine, an antihistaminic agent, 61 involves the base-catalyzed C-methylation of 4-bromophenyla- ocular hypertension. During its synthesis, 2-iodopropane is cetonitrile with genotoxic methyl iodide, yielding the dimethyl directely used, which may result in the presence of this alkyl fi 62 derivative, as illustrated in Scheme 1.49 In a similar reaction, an halide as an impurity in the nal product (Scheme 8). intermediate in the synthesis of the bis-acetonitrile aromatase Highly genotoxic nitrogen mustards are usually used for the fi inhibitor anastrozole is submitted to exhaustive alkylation using formation of piperazine-type drug substances. The rst sodium hydride and methyl iodide.50 synthesis of the potential antipsychotic agent mazapertine Alkyl halides are often used directly for C-, N-, O- and S- involves the use of genotoxic 2-bromopropane in an aromatic alkylation. Aliskiren was the first molecule of a new group of O-alkylation and N,N-bis(chloroethyl)amine in a cycloalkyla- drugs, renin inhibitors, which treat primary hypertension.51 tion to give the piperazine precursor (Scheme 9).63 One of the first steps in its synthesis involves the O-alkylation As in the previous example, the reaction of 2,3-dichloroani- of isovanillin with genotoxic 1,3-dibromopropane in a line with nitrogen mustard gives an arylpiperazine derivative, a Williamson-type ether synthesis, as depicted in Scheme 2.52 key intermediate during the synthesis of the antipsychotic agent Further examples for the use of 1,2- and 1,3-dihaloalkanes are aripiprazole. The side-chain connector is then incorporated by

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

G DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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 Scheme 7. Quaternization by Means of Methyl Iodide during Synthetic Step of Lamifiban 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 exhaustive. For instance, capecitabine is a chemotherapeutic halides and strong acids. These reaction conditions may be agent used in the treatment of metastatic breast and colorectal 65 applied in cyclization, decarboxylation, sulfonyl cleavage, N- cancers. During its synthesis, D-ribose is heated under reflux arylation and API salt formation. Note that this list is not in methanol in the presence of concentrated HCI and acetone

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

H DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Scheme 10. Use of Nitrogen Mustard and a Dihaloalkane in Scheme 13. Ethanolic Hydrogen Chloride Is Used to Open a Piperazine Formation and N-Alkylation Reactions, β-Lactam during the Synthesis of Xemilofiban Respectively, During the Synthesis of Aripiprazole

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-ribofurano- side; 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 N- sulfinyl group (Scheme 15).72 Pazopanib is a tyrosine kinase inhibitor that blocks tumor growth and inhibits angiogenesis.73 During its synthesis, the 2- chloro group of pyrimidine reacts with 5-amino-2-methyl- benzenesulfonamide in 2-propanol and HCl at reflux to deliver pazopanib hydrochloride (Scheme 16).74 Since the 2- propanol−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 A practical manufacturing route was developed for the of hospitalized patients with euvolemic and hypervolemic synthesis of the triazole heterocycle of sitagliptin, which is a hyponatremia.75 During the last synthetic step, ethanol−HCl is drug used to treat type 2 diabetes.67 Exposure of the amidine used as a salt-forming agent to form the imidazobenzazepine intermediate to methanol−HCl gives the desired triazole in a hydrochloride salt (Scheme 17).76 Therefore, once again the cyclization reaction, which can be directly isolated as its HCl potential for the presence of traces of genotoxic ethyl chloride salt by filtration (Scheme 12).68 is high. Alcoholic hydrogen chloride can also be used for ring 3.1.2. Dialkyl Sulfates. The most common dialkyl sulfates openings. Xemilofiban is used to treat cardiovascular disorders, used in the pharmaceutical industry are the methyl and ethyl and during its synthesis, the treatment of a β-lactam derivatives, the latter having been used as a chemical weapon.77 intermediate with ethanolic hydrogen chloride results in ring Being a strong methylating agent, dimethyl sulfate (DMS) is opening to afford the ethyl β-alaninate derivative (Scheme used to introduce a methyl group to atoms featuring unshared 13).69 electron pairs, such as oxygen, nitrogen, carbon, sulfur, During the synthesis of antiangiogenic sunitinib, selective phosphorus, and some metals. Compared with the alkyl hydrolysis of a tert-butyl ester is followed by decarboxylation, halide-type methylating agents, DMS is more favorable due which is accomplished by stirring the tetrasubstituted pyrrole the higher reaction rate and lower possibility of byproduct intermediate in HCl and ethanol, which may form genotoxic formation.78 Usually methylating with dimethyl sulfate requires ethyl chloride (Scheme 14).70 the presence of a base, either (i) to intensify the reactivity of Bortezomib is an intravenously administered, first-in-class, the reaction site (e.g., converting the phenolic hydroxyl group proteasome inhibitor.71 During its synthesis, the N-sulfinyl-α- of vanillin to sodium phenolate during the first step of amino boronate ester intermediate is treated with HCl in papaverine synthesis) or (ii) to neutralize the byproducts of the

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

I DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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 Table 4. Applications of Dialkyl Sulfates during API Formation May Result in Genotoxic Isopropyl Chloride Manufacturing during Pazopanib Synthesis Application API synthesis example O-alkylation Rotigotine S-alkylation Rofecoxib N-alkylation Ralitoline Olefin alkylation Alvimopan Aromatic alkylation Telmisartan Amine transformation 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 Scheme 17. Salt Formation of Conivaptan with HCl in Parkinson’s disease83 and restless legs syndrome.84 Its Ethanol May Result in Genotoxic Ethyl Chloride Formation preparation starts with the transformation of the dihydrox- ynaphthalene to its methyl ether by means of dimethyl sulfate (Scheme 18).85

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

A typical example of N-alkylation is the dimethly sulfate- reaction, monomethyl sulfate (MMS) and sulfuric acid, for assisted N-methylation of an N-heterocycle during the final example, in the case of the methylation of aliphatic alcohols. synthetic step of antiepileptic ralitoline (Scheme 19).86 Usually only one of the methyl groups of DMS reacts in the methylation reaction because the MMS formed is a much Scheme 19. Methylation of a Pyrrolidine with Genotoxic weaker alkylating agent than the original DMS. Although for Dimethyl Sulfate during Ralitoline Synthesis 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 The peripherally acting μ-opioid antagonist alvimopan87 can proceeds but limiting this to the extent of the acid formed to only cross the blood−brain barrier partially and does not have minimize the competing reaction with the hydroxide ion. the usual side effects of the opioid agonists, such as − ff Nonaqueous solvent base systems such as DMF/K2CO3 can constipation, while not losing the analgesic e ect. During its be used in certain cases. Most common applications of dialkyl synthesis (Scheme 20), the treatment of the styrene derivative sulfates in the pharmaceutical industry are summarized in Table with butyllithium and DMS leads to methylation at the 4- 4. position of the 1,2,3,4-tetrahydropyridine ring, since the Dialkyl sulfates are used in the production of metoclopra- negative charge on the quaternary carbon atom in that position mide,79 pralidoxime,80 metrizoic acid,81 and merfruside,82 APIs is enhanced.88

J DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Scheme 20. Genotoxic Dimethyl Sulfate is Directly Used Scheme 22. S-Methylation of a Rofecoxib Intermediate a during the Synthesis of Alvimopan

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 a Note that the carbodiimide reagent forms a potentially genotoxic compounds makes them genotoxic, as their two electrophilic dialkylurea. carbon atoms can react with the DNA nucleophilic centers, giving alkylated products.14 Substituted epoxides, such as 2,3- Dialkyl sulfates are also used to introduce an alkyl group into epoxypropanol (glycidol), 1-chloro-2,3-epoxypropane (epi- an aromatic ring; for instance, o-nitroaniline is methylated with chlorohydrin), or 1,2-epoxy-3-butene, are often used as building dimethyl sulfate during the synthesis89 of telmisartan, which is blocks during the synthesis of APIs. They are often subjected to an angiotensin II receptor antagonist (angiotensin receptor epoxide-ring-opening reactions, and since they are bifunctional, blocker, ARB) used in the management of hypertension they usually act as linking agents or can form heterocycles. (Scheme 21).90 These substituted epoxides react primarily at the less- S-Alkylation is demonstrated in the synthesis of a rofecoxib substituted and more-accessible carbon, due to steric hindrance. intermediate. Rofecoxib is a nonsteroidal anti-inflammatory However, in the case of substituents that increase the positive drug (NSAID) marketed by Merck.91 Scheme 22 shows 1-(4- charge at the adjacent carbon (such as aromatic or vinyl group), mercaptophenyl)ethanone treatment with dimethyl sulfate in both carbons could potentially react.101 A recent review by the presence of sodium hydroxide to form 4-(methylthio)- Elder et al. provides guidance for analytical chemists faced by acetophenone.92 the need to control such impurities at trace levels due to their Cimetidine was the first blockbuster drug, invented by Nobel- potential genotoxicity in drug products.102 93 prize winner James Black. It is a histamine H2-receptor An intermediate of the antiretroviral drug darunavir is antagonist that inhibits the production of acid in the stomach prepared using phenylmagnesium bromide and commercially and it has been shown to have antitumor effects.94 After the available 1,2-epoxy-3-butene in the presence of catalytic CuCN reaction of carbon disulfide and cyanamide in the presence of a to furnish the corresponding allylic alcohol (Scheme 26).103 base, dimethyl sulfate is added to the reaction mixture in order Rivaroxaban is an oral anticoagulant drug invented and to methylate both sulfur atoms in a consecutive reaction manufactured by Bayer for the treatment of thromboembolic (Scheme 23).95 diseases.104 A key intermediate in the synthesis of rivaroxaban is Quaternization of tertiary amines is often carried out with (S)-2-(phthalimidomethyl)oxirane, of which alternatives are dimethyl sulfate,96 which is a preliminary step to an amine− presented in the literature for its synthesis: (a) condensation of nitrile transformation. Clemastine is used as an antihistamine (S)-2,3-epoxy-1-propanol (glycidol) and phthalimide under and anticholinergic medicine with sedative effects.97 During its Mitsunobu reaction conditions105 and (b) condensation of (S)- synthesis dimethyl sulfate is used to form a quaternary amine, 1-chloro-2,3-epoxypropane (epichlorohydrin) and phthalimide which is a good leaving group and is displaced with a nitrile inthepresenceofbenzyltrimethylammonium chloride group in the following step (Scheme 24).98 (BTMAC) as phase-transfer catalyst (Scheme 27).106 Another example is the quarternization of an aromatic amine Azelnidipine is a calcium channel antagonist that selectively during the last synthetic step of neostigmine, a parasympathomi- blocks voltage-dependent Ca2+ influx and is used for the metic drug that acts as a reversible acetylcholinesterase treatment of hypertension.107 The patented synthesis of inhibitor.99 Dimethyl sulfate is used in the last step of the azelnidipine involves a heterocyclization reaction, in particular, API synthesis to form the quaternary ammonium salt, an azetidine formation by means of reacting benzhydrylamine neostigmine, as shown in Scheme 25.100 and epichlorohydrin without solvent to give 1-benzhydryl-3- 3.1.3. Epoxides. Epoxides are the simplest cyclic ethers, hydroxyazetidine in a slow process (Scheme 28).108 featuring three ring atoms. Due to the large ring strain The structurally rather complex agent zosuquidar has shown associated with the three-membered ring, epoxides are highly promising activity against multidrug resistance in cancer reactive molecules and thus are often used as reagents in API chemotherapy.109 During its synthesis, 5-hydroxyquinoline

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

K DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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 25. Quaternary Ammonium Salt Formation by Scheme 29. Glycidol Tosylate in an Aromatic O-Alkylation Means of Dimethyl Sulfate during the Synthesis of Zosuquidar

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

Scheme 30. Use of Chiral Glycidol Mesylate during the Scheme 27. Condensation of (S)-2,3-Epoxypropanol Synthesis of Lubazodone (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 Scheme 28. Azetidine Formation with Epichlorohydrin control and trace analysis in drug substances by Elder et al.114 during the Preparation of Azelnidipine 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 reacts with the tosyl derivative of glycidol in a convergent aldehyde-typeis transformed into a methylene or methyl 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 Table 5. Applications of Hydrazine in API Synthesis of a fluoroindanol with the mesylate ester of (R)-glycidol in the presence of base leads to the epoxypropyl ether with retention Application API synthesis example of configuration (Scheme 30). Treatment of this intermediate Wolff−Kishner reduction Sunitinib, ziprasidone with aminoethylsulfonic acid forms the morpholine ring and Hydrazide formation Sitagliptin, isoniazid gives the enantiomerically pure final product lubazodone.112 Hydrazinolysis Saquinavir, mofegiline 3.1.4. Hydrazines. The toxicity of hydrazine and its Electrophilic addition Suritozole derivatives is ascribed to the generation of carbocations, Heterocyclizations Sildenafil, sedoxantrone

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Chart 1. Comparison of Different Wolff−Kishner Reduction Methods

group through a hydrazone intermediate. Chart 1 compares Scheme 31. Hydrazine-Assisted Wolff−Kishner Reduction of three different modifications of the Wolff−Kishner reduction. Oxindoles during the Synthesis of Sunitinib and Ziprasidone The first step is the formation of a hydrazone. Evolution of highly stable nitrogen after successive deprotonation−proto- nation 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 119 Such reaction is also used for a mild lysis of protection groups hydrazide, as shown in Scheme 32, which is then converted in peptide and sugar chemistry, but probably this scission to the triazole. In another example of hydrazide formation is reaction finds most common application in the Gabriel isoniazid, which is used for the treatment of tuberculosis and synthesis in which phthalylhydrazide is produced during the 120 depression. Its synthesis involves the use of hydrazine in the liberation of the desired amine from the phthalyl residue. The fi nal synthetic step, where an NH2 group is displaced by synthesis of saquinavir and mofegiline are examples of hydrazine- hydrazine.121 assisted cleavage of N-alkylated phthalimide derivatives Hydrazinolysis is a chemical cleavage reaction, in which the (Scheme 33). The peptide derivative saquinavir inhibits the hydrazine acts as a nucleophilic agent by attacking the carbon HIV protease enzyme68 and mofegiline is a MAOB (mono- atom of a carbonyl group which has a partial positive charge. amine oxidase B) inhibitor used in the treatment of Parkinson’s

M DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Scheme 32. Hydrazide Formation during the Synthesis of (a) Scheme 34. Use of Methylhydrazine in an Electrophilic Sitagliptin and (b) Isoniazid Addition during the Preparation of Suritozole

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 disease.122 Part of the synthesis of saquinavir entails the throughout chemical- and biochemistry-related industries as a hydrazinolysis of the amido alcohol intermediate removing the stable nitroxyl radical. TEMPO is mainly used for oxidations of phthalimide protecting group to produce the primary amine,123 alcohols to yield aldehydes and ketones or carboxylic acids. A while the final step of mofegiline synthesis is also characterized comprehensive review on the use of reactions mediated by by the cleavage of the phthalimide protecting group with TEMPO can be found elsewhere.133 An example of the use of hydrazine leading to the free base.124 The side product formed TEMPO is the synthesis of a 5-HT2B receptor antagonist. Eli from the hydrazine and the phthaloyl group is 2,3- Lilly synthesizes 2-cyclohexylacetaldehyde by oxidizing 2- dihydrophthalazine-1,4-dione. cyclohexylethanol by the Anelli−Montanari protocol (Scheme Suritozole is a benzodiazepine reverse agonist, investigated as 36), affording the aldehyde, which is then used as precursor of a a potential treatment for Alzheimer’s disease.125 Its synthesis tryptamine and further used as key synthon for the synthesis of (the triazolothione portion) starts with the formation of a the important 5-HT2B receptor antagonist.134 thiosemicarbazide by condensation of methylhydrazine with Antagonists of the coreceptor CCR5 have been an intense methyl isothiocyanate in an electrophilic addition, as depicted area of research within the HIV arena over the past decade. in Scheme 34.126 Maraviroc is the first-in-class CCR5 antagonist for the 135 Hydrazine is a key bifunctional, with two NH2 groups, treatment of HIV. The initial synthesis of maraviroc that building block used in the preparation of various heterocyclic produced material for preclinical studies has been pub- compounds via condensation reactions with a wide range of lished.136,137 One of the reactions is catalyzed by TEMPO, bifunctional electrophiles, such as 1,3-diones or 3-halo ketones where the alcohol is oxidized to give the required aldehyde, as or aldehydes, leading to pyrazoles, or imides, giving triazoles in shown in Scheme 37.138 the Einhorn−Brunner reaction. The resulting N-heterocycles Darunavir is an antiretroviral drug in the HIV-1 protease are key intermediates in the synthetic routes to APIs. For inhibitor class for the treatment of multidrug-resistant HIV.139 instance, the preparation of sildenafil, which is generally known One step in the synthesis of darunavir is a cyclization that 127 as Viagra, a drug used for treatment of erectile disfunction, included a TEMPO oxidation, a NaBH4 reduction, and a lipase involves the hydrazine-assisted formation of a substituted resolution to provide optically active bis-THF derivative, as pyrazole ring, as shown in Scheme 35.128 A similar reaction illustrated in Scheme 38.140 takes place during the synthesis of the topoisomerase inhibitor Oseltamivir141 is a neuraminidase inhibitor and is the most sedoxantrone.129 One of the steps of sedoxantrone synthesis is commonly prescribed drug for treatment to combat influenza. the condensation of a phenol intermediate with a substituted The large number of synthetic approaches reported in the hydrazine, which leads to pyrazole formation. Though the order literature implicates the importance of this drug. In the example of the reaction steps has not been established, formation of the below, TEMPO is used with trichloroisocyanuric acid (TCCA) hydrazone, then displacement of the adjacent chlorine by the in the oxidation of a secondary alcohol to give the second nitrogen, and final closure of the pyrazole ring seems corresponding ketone intermediate (Scheme 39).142 plausible.130,131 An azabicyclooctanyl derivative was identified as another novel 3.1.5. TEMPO. 2,2,6,6-Tetramethylpiperidin-1-oxyl free and potent DPP-4 (dipeptidylpeptidase-4) inhibitor at Novartis radical (trade name TEMPO) is a commonly employed 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

N DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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

Scheme 36. Synthesis of the 5-HT2B Receptor Antagonist BYK405879 is a potassium-competitive acid blocker, a Intermediate by Means of TEMPO promising candidate for the treatment of gastroesophageal- reflux-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 Scheme 37. TEMPO Oxidation during the Synthesis of the 3.1.6. Aromatic Amines. Although aromatic amines are HIV Drug Maraviroc 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 furoate, 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 esterifica- tions,154,155 amino group protections with Boc,156,157 and azabicyclooctanyl intermediate involves the use of TEMPO in 158 an alcohol−ketone oxidation (Scheme 40).143 silylations. SB-462795144 is an azepanone-based inhibitor of the protease Diclofenac is widely used as a nonsteroidal anti-inflammatory cathepsin K, developed for the treatment of osteoarthritis and drug (NSAID). During its synthesis, the potentially genotoxic 159 osteoporosis.145 Scheme 41 shows the oxidation of a carbinol in 2,6-dichloroaniline is used as a starting material. Cu- the final chemical stage of the synthesis by means of catalyzed N-arylation with 2-chlorobenzoic acid takes place in TEMPO.146 the presence of KOH (Scheme 45A). The reaction may leave LY686017 is a potent NK1-II inhibitor for the treatment of behind unreacted starting material, which has to be depression, anxiety, and alcohol dependency.147,148 In its pilot- controlled.160 In a similar manner, the potentially genotoxic plant synthesis, TEMPO is used as an oxidation agent with 2,6-dimethylaniline is used for the synthesis of the local NaOCl, where a secondary alcohol is efficiently oxidized using anesthetic and antiarrhythmic drug lidocaine (Scheme 45B).161 the Anelli−Montanari protocol (Scheme 42).149 2,6-Dimethylaniline is condensed with bromoacetic acid, as

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

O DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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 Scheme 45. Synthesis of Diclofenac (a) and Lidocaine (b) Pilot-Plant Synthesis of LY686017 with the Potentially Genotoxic 2,6-Dichloroaniline and 2,6- Dimethylaniline Starting Materials, Respectively

Scheme 43. Alcohol−Aldehyde TEMPO-Mediated mediated by 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide Oxidation during the Synthesis of BYK405879 (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

P DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Scheme 46. Use of Potentially Genotoxic 4-Chloroaniline in advantage of all is that the coupling reaction proceeds with high the Final Synthetic Step of Chlorhexidine 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 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 bond- the Suzuki strategy, a bromobenzene derivative is treated with forming methods involve the use of transition-metal-catalyzed butyllithium to afford an organolithium intermediate that is reactions. Among these, the most frequently used is the trapped with triisopropylborate to give the desired boronic acid Suzuki−Miyaura166 reaction, which uses boronic acids or esters upon workup of the reaction mixture. Since boronic ester as the key coupling partner. The mechanism is depicted in derivatives are less sensitive to hydrolysis and air oxidation than Chart 2. The main advantages of boronic acids and esters are the corresponding boronic acids, diethanolamine boronic ester that they are readily available and stable in both air and water. was prepared in the next step by means of diethanol- These compounds react under mild conditions, and the amine.168,169 Afterward, the boronate derivative is treated inorganic boron byproducts are easily removed after with AcOH, and the borate species formed reacts with the completion of the reaction. Probably the most important other key intermediate bromoquinolone in the presence of

Chart 2. Mechanism of Suzuki Coupling

Q DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review palladium bis(triphenylphosphine) dichloride and sodium esterification. Notice that sulfonate esters are not the only carbonate. potentially genotoxic side products; therefore, two additional Angiotensin II receptor antagonist losartan is used for the small sections provide further examples on the formation of treatment of hypertension.170 The synthetic step involving a alkyl halides and acetamide. Suzuki coupling in the synthesis of losartan developed by 3.2.1. Sulfonate Esters and Their Precursors. Over- Merck research chemists is outlined in the following (Scheme view. Sulfonate esters are alkylating agents, a class of 48).171,172 First, the trityl-protected phenyltetrazole was ortho- potentially genotoxic compounds.178 They are called alkylating agents due to their ability per se, or after metabolic activation, Scheme 48. Suzuki Coupling during the Merck Process for of adding alkyl residues to the reactive nucleophile sites of the the Synthesis of Losartan 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 sulfonateestersaremostoftenside 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).

Scheme 49. Formation of Genotoxic Sulfonate Esters from lithiated by means of butyllithium and then quenched with the Corresponding Acids, Halides, and Anhydrides with triisopropyl borate, giving the boronic acid derivative after Alcohols 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 Since halides and anhydrides of sulfonic acids are alkylating side reactions with alcohols, and therefore, awareness for their agents, they are also considered genotoxic. Note that in many presence in API synthesis could be, initially, not so API syntheses it is, in some circumstances, challenging to straightforward. However, as a result of the work of several substitute alcohols as solvents because of their ability to groups, in particular the efforts of the Working Group within solubilize both the API and API salts. Examples of most the Product Quality Research Institute (PQRI), the industrial common sulfonate esters and their precursors are summarized and academic community is today fully aware of this in Table 6. challenge.173 Therefore, taking into account the specific Due to their synthetic versatility, sulfonate derivatives are properties and widespread use of the alkyl sulfate acids as common and useful reagents in the pharmaceutical industry, counterions in, but not exclusively, API salt formation in the especially in reactions where carbonium ion initiation is needed. presence of alcohols, special concern has been raised by the Examples of such compounds are mesylates (methanesulfo- regulatory authorities. Several documents from regulatory nate), triflates (trifluoromethanesulfonate), tosylates (p-tolue- − authorities174 177 have specifically outlined controls to be nesulfonate), nosylate (4-nitrobenzenesulfonate), and besylates taken. This has motivated an intensive effort to develop control and removal strategies and to understand their mechanism and Table 6. Common Sulfonate Ester Impurities and Their kinetics of formation, and several industrial examples have been Precursors 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)

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(benzenesulfonate). Sulfonate ester impurities may be present API synthesis. Basic APIs are usually preferentially presented in in APIs or their intermediates (i) due to their production in the salt form due their higher aqueous solubility and side reactions between sulfonic acids or halides and alcohols or subsequently higher bioavailability. The conversion of an API (ii) as reactants carried over from incomplete reactions. When to a salt also can help to enhance stability and water solubility 180 any of the GTI precursors listed in Table 6 is used in an API and helps isolation as final product (Chart 3). Elder et al. synthesis, there is a possibility of the formation of genotoxic overviewed the utility, safety, and regulation of APIs formulated as sulfonic acid salts. For example, methanesulfonic acid is used sulfonate esters. In particular, reactions containing sulfonic 181 182 acids, sulfonic halides, or sulfonic anhydrides where an alcohol in the production of viracept and delavirdine, while p- is also present, even if only in residual amounts, have the toluenesulfonic acid (TsOH) is used in the production of 183 fi potential to yield sulfonate esters. Teasdale et al. pointed out bretylium. The nal manufacturing step of denagliptin,an API developed to treat diabetes mellitus, is forming a tosylate that sulfonate esters decompose through alcoholysis to generate 184 sulfonic acid and an ether, and this reaction, in conjunction salt in ethanol, as illustrated in Scheme 50. with the reversibility of ester formation, limits the quantity of There is potential for the formation of a potentially genotoxic p-toluenesulfonic acid ester with the alcoholic solvent. Teasdale genotoxic esters produced.179 The European Pharmacopoeia et al. carried out a detailed study to understand the mechanism, makes it compulsory for APIs marketed as sulfonic acid salts to kinetics, and processing parameters of sulfonate ester demonstrate that any sulfonate ester formed is removed during formation179,185,186 Note that these studies discuss the reactions the purification process.2 Hence, it is crucial for scientists fi between alcohols and sulfonic acids only and not sulfonyl working in the eld of API manufacturing to be aware of GTI halides. 18O-Labeled methanol was used to distinguish the precursors and their use in drug synthesis. different esterification pathways and the effect of water content, How GTI precursors are used in the pharmaceutical industry temperature, and API base to acid ratio, and solvolysis reaction is summarized in Table 7, and examples of each case, with rates were explored. The main findings and conclusions of the reactions, are given later. work are listed in Chart 3. These findings allow process 3.2.2. Sulfonate Esters and Their Precursors Used in chemists to control sulfonate ester formation during Stoichiometric Amounts. 3.2.2.1. API Salt Forming Agents. pharmaceutical manufacturing processes. Further discussion Sulfonic acids are salt-forming agents used in the last step of the can be seen in section 4. The investigation concluded that sulfonate esters do not form if the acid is neutralized with even Table 7. Applications of Sulfonate Ester GTI Precursors the slightest excess of API base. Therefore, the process controls elaborated by Teasdale et al. open the door for the Application API synthesis example pharmaceutical industry to demonstrate to the regulatory API salt forming Viracept, delavirdine, authority adequate control over the presence of sulfonic acid agent denagliptin ester GTIs in APIs. Good leaving Etherification Betaxolol A historically very important example, in which the formation group of a GTI had a severe impact on API supply, is the case of Hydroxyl−halogene Mometasone, clobetasone, transformation halobetasole viracept, the antiretroviral drug used to treat the human fi 187 Hydroxyl−sulfur Tixocortol pivalate immunode ciency virus (HIV). In June 2007, contamination transformation with the genotoxic sulfonate ester ethyl mesylate (EtMs) led to Hydroxyl−amine Azaloxan, fluvoxamine, the global recall of this drug.188 The case was investigated and it transformation tolterodine was established that the main reason for genotoxin accumu- − Amine nitrile Cromitril lation in viracept took place in the final manufacturing step. In transformation fi Amide−nitrile Denagliptin this step, the API salt nel navir mesylate is formed by addition transformation of methanesulfonic acid (MsOH) to a suspension of nelfinavir Isocyanate−amine Temocillin in ethanol, and spray-drying is used to isolate the dissolved transformation nelfinavir mesylate salt from the ethanolic solution (Scheme Cyclization Aziridine formation Spiradoline, oseltamivir 51). reactions After several patients reported a strange odor and nausea Oxazoline formation Ifetroban upon taking the medication,188,189 an investigation by the Pyrrolidine formation Napitane manufacturer revealed that the primary source of GTI Lactone formation Orlistat contamination was due to an error in good manufacturing Oxirane formation Saquinavir practices, more specifically, failure to dry the MsOH hold tank Cyclodehydratation Englitazone following ethanol cleaning. Additionally, although in negligible Protecting group Dinoprost, tolterodine quantities, EtMs was also identified in some batches of MsOH. Protecting group Oseltamivir, denagliptin, removal ABT-594 The long hold times, elevated temperatures, and cleaning of the Mitsunobu Fosinopril spray dryer with ethanol, the vapors of which could potentially rearrangement reach the MsOH hold tank through the ventilation system, all Double bond Etonogestrel contributed to the formation of additional quantities of EtMs190 migration It is worth mentioning that a comprehensive preclinical Enamine−amine Titonavir reduction toxicology program and safety follow-up registries of exposed Sulfonamide Dofetilide patients were carried out by the manufacturer, which concluded formation that chromosomal damage and mutations only take place for Esterification Fosinopril EtMs doses higher than 60 and 25 mg/kg/day, respectively. A Resolution of Esomeprazole maximum intake of ∼0.055 mg/kg/day (for a daily dose of enantiomers 2500 mg of viracept) was estimated for patients who took

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

Scheme 50. Final Preparation Step of Denagliptin Is Salt 3.2.2.2. Good Leaving Groups. Genotoxic sulfonate esters Formation with TsOH in Ethanol 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 SN1orSN2 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 Scheme 51. Final Manufacturing Step of Viracept Drug being developed.191 Substance Nelfinavir Mesylate Azaloxan is an antidepressant drug patented by Ciba- Geigy192 where tosyl chloride is used during its synthesis (Scheme 52) to form the good tosyloxy leaving group for use in

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 189 teratogenicity over their background risk. a bimolecular nucleophilic substitution (SN2) displacement 193 Therefore, the conclusions from the viracept case study reaction transforming a hydroxyl group into an amine group. points out that GTI contamination of an API can result from a In a similar fashion, etherification of a hydroxyl group is carried 194 wide range of difficult to anticipate sources: cleaning out (Scheme 53) using mesyl chloride in the synthesis of procedures, pipelines and holding tanks (where reagents may be held for a lengthened period of time), problems with pH Scheme 53. Using Mesyl Chloride during the Synthesis of adjustment, charging speed of a chemical to the reaction Betaxolol 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).

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

betaxolol,aβ(1)-selective adrenergic antagonist used in the Scheme 55. Methanesulfonylation of Various treatment of hypertension and glaucoma.195 Chart 4 highlights Glucocorticoids 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 fluvoxamine.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 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 Mometasone, clobetasone, and halobetasol are glucocorticoids dermatitis.201 During the final manufacturing steps, mesyl belonging to a class of steroid hormones that bind to the chloride is used to form a good leaving group. Displacement of glucocorticoid receptor. They play a key role in regulating the the mesyloxy group with the anion from thiopivalic acid affords feedback mechanism of the immune system during inflamma- thioester-type API (Scheme 56), in the presence of a base, 198 202 − tion by turning the immune activity down. During the trimethylamine (Et3N). A similar hydroxyl sulfur trans- synthesis of these steroids, mesyl chloride is used in a formation takes place during the synthesis of microtubule methanesulfonylation catalyzed by a base, DMAP, and the inhibitor erbulozole.203 mesylate group is consequently replaced by chlorine (Scheme Sulfonyl halides are also useful tools to transform amines to 55).16,199 In other steroids, the 21-hydroxyl group is not nitriles. For instance, one synthesis of the antiasthmatic agent converted to chlorine but to a sulfur-linked residue to achieve cromitril204 concludes in the classic manner by converting an modified therapeutic activity. For example, the anti-inflamma- ester to a carboxamide by ammonolysis, and dehydrating the

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

latter functionality to the nitrile with tosyl chloride in pyridine, configuration at the former secondary alcohol is inverted as a followed by addition of sodium azide selectively across this consequence of the SN2 nature of the ring closure. functionality, produces the final API (Scheme 57)205 The β- Sulfonyl halides are also used in lactone formation. Orlistat is a drug designed to treat obesity,212 the synthesis of which Scheme 57. Amide Transformation to Nitrile with TsCl/Py involves treatment of the β-hydroxycarboxylic acid intermediate during the Synthesis of Cromitril 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 lactamase-resistant carboxypenicillin drug temocillin is used for triethylamine. The amine produced by reduction of the azide the treatment of multiresistant Gram-negative bacterial group in the second step with triphenylphosphine proceeds infections.206 During the synthesis, isocyanate−amine trans- with a nucleophilic attack on the adjacent carbon, displacing the formation takes place, where benzyl 6-β-isocyano-6-α-methyl- mesyloxy group in a nucleophilic substitution to provide the thiopenicillanate is treated with p-toluenesulfonic acid, giving first aziridine. Due to the large angle strain of the three- one of the key intermediates to temocillin (Scheme 58).207 membered heterocyclic system, ring opening of aziridine with 3.2.2.3. Cyclizations. Cyclization reactions where a sulfonate sodium azide in the presence of ammonium chloride occurs ester functions as a leaving group are common, as in the easily. After deprotection of the MOM ether by acidic following examples. Oxazoline cyclization can be achieved by hydrolysis, the amino group is reacted with trityl chloride, the means of mesyl chloride, for instance, during the synthesis of hydroxyl group is then transformed into a good leaving group ifetroban, which is a selective thromboxane receptor antago- by means of mesyl chloride in the presence of triethylamine, nist.208 First, the hydroxyl group is converted to a good living and in a one-pot process, a new aziridine-type intermediate is group by reaction with mesyl chloride. When this intermediate produced.217 The use of methanol as a solvent may lead to the is treated with base, the mesylate is displaced by the enolate formation of genotoxic sulfonate esters under certain from the adjacent amide, giving the corresponding oxazoline conditions. Furthermore, the HCl/MeOH mixture used for (Scheme 59).209 the hydrolysis of the MOM ether may lead to the presence of Napitane is used as an antidepressant drug, and during the genotoxic methyl chloride in the API. last synthetic step, 2 equiv of methanesulfonic acid is 3.2.2.4. Protecting Groups. Scheme 65 includes a synthetic released.210 A diol is converted to a bidentate bis-mesylate step in the production tolterodine,218 an API used to manage with mesyl chloride followed by reaction of the required amine urinary incontinence, showing the protection of a phenol group to give the pyrrolidine moiety of the final API (Scheme 60). by the formation of a tosylate followed by the formation of the During the large-scale synthesis of saquinavir, an anti-HIV good leaving group nosylate, which is then easily displaced by protease inhibitor, a mesyloxy group is used to form an oxirane diisopropylamine. Note that this synthesis provides an example derivative in two steps. The secondary alcohol of a 1,2-diol is involving three GTIs and one carcinogenic impurity of different selectively converted to the methanesulfonate ester with mesyl chemical classes, sulfonate esters, alkyl halides, and acetamides. chloride. Thereafter, strong base is used to produce an alkoxide 3.2.2.5. Sulfonamide Formation. Sulfonamides provide part at the primary alcohol, which then displaces the mesylate ion, of the structural basis of several drugs (Chart 5). Originally, giving an oxirane derivative.211 As depicted in Scheme 61, the sulfonamides were used as synthetic antimicrobial agents, but

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 Scheme 64. Consecutive Aziridine Formation by Means of Eventually Gives Rise to the Liberation of 2 equiv of Mesyl Chloride in Oseltamivir Synthesis Methanesulfonic Acid in the Final Synthetic Step of Napitane

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 Scheme 63. Tosyl Chloride-Assisted Formation of an 3.2.2.6. Chiral Auxiliary Group in Resolution of Enan- Aziridin during the Synthesis of Spiradoline tiomers. 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

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Chart 5. Highlights of Sulfonamide Drugs

Scheme 66. Use of Mesyl Chloride during the Synthesis of Scheme 68. TsOH-Assisted Cyclodehydration Leads to a the Bis-methanesulfonamide Dofetilide Chroman Derivative in Englitazone Synthesis

useful way of protecting the hydroxyl group due to its stability Scheme 67. Resolution of Racemic Omeprazole with toward a variety of harsh reaction conditions, such as strong Camphorsulfonyl Chloride To Obtain Enantiomerically bases, organometallic reagents, hydrides, and acylating and Pure Esomeprazole 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 p- toluenesulfonic 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 2- tetrahydropyranyl 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 chloride and camphorsulfonic acid with the alcohol solvent can during incineration causes problems, alternative conditions are lead to the formation of potentially genotoxic sulfonate esters. being developed for large-scale Boc removal. Most of these 3.2.3. Sulfonate Esters and Their Precursors Used in apply TsOH to produce acidic conditions.225 During the Catalytic Amounts. 3.2.3.1. Cyclizations. A pharmaceutical synthesis of ABT-594, a potent, orally effective analgesic, TsOH example of a cyclization where a sulfonic acid is applied as is used both to remove a Boc-protecting group from an amine catalyst is the TsOH-assisted cyclodehydration of a diol to and as a salt-forming agent to obtain a stable API (Scheme 70). afford the chroman framework during the synthesis process of Neither HCl nor TFA can be used for deprotection in this the hypoglycemic agent englitazone (Scheme 68).222 particular case, as they gave a significant amount of dimer 3.2.3.2. Protecting Group Manipulations. The formation of byproduct, which proved difficult to remove.226 Since the tetrahydropyranyl (THP) ethers from alcohols and phenols is a reaction and salt formation take place in ethanol and this is the

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

Scheme 70. Removing a Boc Group with TsOH in the synthesis with TsOH in acetic acid, which causes the double Synthesis of ABT-594 bond at the 8,9-position to migrate to the adjacent 9,11- position, 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 final step of the synthetic route, the probability that the API acid, resulting in the reduction of the enamine to a primary contains genotoxic ethyl p-toluenesulfonate is high. amine (Scheme 74).232 An alternative synthetic route to oseltamivir uses TsOH in 3.2.3.6. Esterification. Sulfonic acids are also used to catalyze methanol to remove an acetonide protecting group of a diol esterifications. Fosinopril is an angiotensin converting enzyme intermediate. There is potential to form methyl p-toluenesul- (ACE) inhibitor. Its synthesis involves a manufacturing step fonate in this reaction (Scheme 71).227 where a carboxylic acid is esterified by methanol in the presence of TsOH (Scheme 75).233 Although the TsOH is used as a Scheme 71. Acetonide Protecting Group Removal by Means catalyst, the quantities used are generally quite high and may of TsOH in Methanol result in formation of considerable amounts of the correspond- ing 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-(4,6- dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride 3.2.3.3. Mitsunobu Rearrangement. Fosinopril, an inhibitor (DMTMM), which is an efficient coupling agent in a wide 234,235 236,237 of angiotensin converting enzyme (ACE), is widely used for the range of organic reactions, such as esterification, 238 239 treatment of hypertension, as well as in various types of chronic glycosidation and phosphonylation in the synthesis of 240 241 242 heart failure.228 Fosinopril manufacturing is another example of antibiotics, peptides, and alkaloids. However, the use of sulfonic acids in the pharmaceutical industry, since DMTMM is unstable in organic solvents and reacts with itself, MsOH is used as a reagent in a Mitsunobu rearrangement, as yielding 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-morpholine and depicted in Scheme 72,229 wherein mesylation with inversion of genotoxic methyl chloride (Scheme 76). configuration was accomplished by employing methanesulfonic 3.2.5. Acetamide. Acetamide is a known carcinogen; thus, acid, triphenylphosphine, diisopropyl azodicarboxylate, and the awareness of its formation in API manufacturing is triethylamine. A highly stereospecific Friedel−Crafts alkylation crucial.243 Although acetamide is not a genotoxin, it is mediated by aluminum trichloride installed a 4-phenyl sometimes referred to as such in the literature11 Acetamide is substituent with complete inversion. This shows an example not commonly used directly in the synthesis of APIs, but its how sulfonic acids are used in O−C transformation. Mitsunobu derivatives, such as 2- and N-bromoacetamide or trifluoroace- reactions are discussed in more details in Chart 6. tamide, are often used as building blocks in drug synthesis. 3.2.3.4. Double Bond Migration. Etonogestrel is a steroid These derivatives initially contain acetamide as an impurity, but used in hormonal contraceptives, and its synthesis involves a also the 2- and N-derivatives have potential to form acetamide, double bond migration assisted by p-toluenesulfonic acid. depending on the reaction conditions. Another source for Scheme 73 illustrates the treatment of an intermediate in the formation of carcinogenic acetamide is the hydrolysis of 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 Scheme 77. Acetamide May Form from Acetonitrile during Enamine during the Synthesis of Ritonavir 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

widely used solvent acetonitrile under acidic or basic conditions acetamide by applying adequate chemical process design: the at elevated temperature. Acetonitrile is not only used as a implementation of a workup sequence involving aqueous solvent in the pharmaceutical industry but also as a reagent in washes, followed by salt formation and crystallization, was API synthesis. For instance, it is directly used as a reagent in the proven to be successful.247 synthesis244 of corontin, which is a drug used for treatment of Sodelglitazar is an antidiabetic drug for treatment of type 2 angina pectoris (Scheme 77).245 diabetes. During one of the synthetic steps S-alkylation takes Zaurategrast is a drug that reached phase II clinical place under acidic conditions in acetonitrile, hence, the development and was indicated for treatment of multiple potential for formation of acetamide (Scheme 79).248 sclerosis.246 Since the final synthetic step of the process takes During the synthesis of the antiviral drug oseltamivir, a diene place in acetonitrile under acidic conditions, the risk of intermediate is converted to the bromodiamide derivative using fi acetamide formation was identi ed during the early develop- a novel SnBr4-catalyzed bromoacetamidation with N-bromoa-

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Scheme 79. S-Alkylation in Acetonitrile under Acidic solvents, which include solvents such as ethanol and acetone, Conditions May Lead to the Formation of Carcinogenic have permissible daily exposures of 50 mg, or up to 5000 ppm Acetamide during the Synthesis of Sodelglitazar (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 254 cetamide in acetonitrile (Scheme 80).249 In this reaction, both dedicated to APIs used in clinical trials. the reagent and the solvent can form carcinogenic acetamide. Under the conditions of a 2-year gavage study, there was clear evidence of the carcinogenicity of benzene, which used to 256 Scheme 80. Reagent N-Bromoacetamide and Solvent be a widely used solvent in the industry. Benzene was mainly Acetonitrile May Lead to Acetamide Formation 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 reagent 2-bromoacetamide is used during the synthesis the experimental group that was exposed to dimethylformamide of armodafinil, which is used for the treatment of narcolepsy 250 (DMF) showed an increase in the incidences of chromosomal and sleeping disorders. 2-Bromoacetamide, which may 259 aberration, negative results were obtained in the majority of contain acetamide, is used in an S-alkylation in the presence the in vitro and in vivo genotoxicity studies; thus, the overall of NaOH (Scheme 81).251 evaluation of these data indicates that DMF is not genotoxic (categorized as group 3, i.e., not classifiable as to its Scheme 81. Use of 2-Bromoacetamide May Result in the 260 fi fi carcinogenicity to humans by the IARC). Classi cation of Presence of Acetamide in Armoda nil Synthesis 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

3.3. Genotoxicity and Carcinogenicity of Common Organic 4. APPROACHES FOR GTI MITIGATION IN THE Solvents PHARMACEUTICAL INDUSTRY Organic solvents are ubiquitously present in pharmaceutical As illustrated in the previous section, the synthesis of production processes as reaction and purification media (e.g., pharmaceutical products often involves the use of highly extraction), separation phases (e.g., chromatographic mobile reactive reagents for the production of APIs or their phases), and also for cleaning of the equipment. The intermediates.263 Low levels of such reagents or corresponding pharmaceutical industry consumes the largest amount of side products may therefore be present in the final API or drug organic solvents in relation to the final product gained.252 product as impurities. As briefly described in section 2, such According to the Q3C guideline, solvents are divided into four chemically reactive impurities may have unwanted toxicities, groups.253 Classes 1 and 2 are considered “toxic” solvents, as including genotoxicity and carcinogenicity, and hence can have summarized in Table 8. The first group (class 1) contains a severe impact on the product risk assessment.264 In some known human carcinogens, compounds strongly suspected of cases, these sources can be avoided. However, in many cases being human carcinogens, and those presenting environmental the presence of GTIs in postreaction streams during API hazards. These solvents should be avoided, unless strongly synthesis is difficult to avoid. To overcome this, R&D scientists justified. The limits for class 1 solvents are listed as absolute have to identify GTIs early on during process development, parts per million in a material under testing (drug or excipient). develop analytical methods, and implement synthetic processes Class 2 solvents presented in Table 8 ought to be limited, to control and contain them. GTIs can be successfully reduced because they are nongenotoxic animal carcinogens or associated below the limits set by regulatory authorities either with with irreversible toxicity, such as teratogenicity. Class 3 carefully optimized synthetic approaches (preventive approach,

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a Table 8. Classification of Solvents by the Q3C Guideline and Their Properties

Solvent Concn limit (ppm) Boiling point(°C) Density (g/cm3) Dielectric constant Class 1 Benzene 2 80.1 0.877 2.28 Carbon tetrachloride 4 76.7 1.594 2.24 1,2-Dichloroethane 5 83.5 1.245 10.42 1,1-Dichloroethane 8 57.2 1.2 16.7 1,1,1-Trichloroethane 1500 74 1.32 7.5 Class 2 2-Methoxyethanol 50 124 0.965 16.94 Methylbuthylketone 50 127 0.812 14.6 Nitromethane 50 101.2 1.382 35.9 Chloroform 60 61.7 1.498 4.81 1,1,2-Trichloroethylene 80 86.7 1.463 3.4 1,2-Dimethoxyethane 100 85 0.868 7.2 Tetralin 100 207 0.974 2.77 2-Ethoxyethanol 160 135 0.931 5.3 Sulfolane 160 285 1.261 44 Pyridine 200 115.2 0.982 12.3 Formamide 220 210 1.134 84 Hexane 290 69 0.659 1.89 Chlorobenzene 360 131.7 1.107 5.69 1,4-Dioxane 380 101.1 1.033 2.21 Acetonitrile 410 81.6 0.786 37.5 Dichloromethane 600 39.8 1.326 9.08 Ethylene glycol 620 245 1.118 31.7 N,N-dimethylformamide 880 153 0.944 36.7 Toluene 890 110.6 0.867 2.38 N,N-dimethylacetamide 1090 166.1 0.937 37.78 Methylcyclohexane 1180 101 0.769 2.6 1,2-Dichloroethene 1870 60.3 1.28 4.6 Methanol 2000 64.6 0.791 32.6 Xylene 2170 139.1 0.868 2.37 Cyclohexane 3880 80.7 0.779 2.02 N-methylpyrrolidone 4840 202 1.026 32.2 aData 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

hence preferred) or by implementing purification strategies as a However, in many cases, the use of reagents and intermediates last resort. that are reactive and synthetically useful, which in turn likely 4.1. Chemical Synthetic Approaches makes them interact with DNA, are often unavoidable. It may In this, the first strategy to mitigate GTIs in the production of not be practical to change the synthetic steps during APIs, R&D chemists avoid the use and generation of GTIs development to control or reduce GTIs, particularly when throughout the synthetic route, searching for different chemical the process has reached the stage of being scaled up. Therefore, sequences to reach the same API or intermediate or by a second strategy to achieve GTI-free drug products is based on optimizing the existing synthetic route.265 In very particular prevention, focusing on elimination or reduction of the cases, this strategy can be achieved without significant concentration of GTI during the critical synthetic step. This reduction of yield. Examples of redesigning the synthetic can be achieved by altering appropriate reaction conditions, process specifically to avoid GTIs can be found in section 4. such as (i) proportions of reaction components, (ii)

AB DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review interchanging the order of addition of the reactants, and (iii) β-D-ribofuranose (Scheme 84). This stereoselective coupling changing the quality or method of preparation of key starting was initially mediated by N,O-bis(trimethylsilyl)acetamide and materials. Furthermore, a quality by design (QbD) approach triflic acid. Since the workup leads to formation of can contribute to better control of GTIs.266 stoichiometric amounts of genotoxic acetamide, N,O-bis- 4.1.1. Altering the Synthesis. Three synthetic examples (trimethylsilyl)acetamide was replaced by trimethylsilyl triflate are given in which the chemical synthesis was changed to avoid as the coupling reagent. the formation of a sulfonate ester. Two additional examples In the synthesis of Zeneca Pharmaceuticals’ ZD-2079268 for include side reactions with the potential to form genotoxic the treatment of noninsulin dependent diabetes, 1,2-dibromo- impurities vinyl bromide and acetamide. ethane is used to alkylate 4-hydroxylphenylacetamide (Scheme The syntheses of zaurategrast sulfate and sodelglitazar were 85a). A side reaction leads to the formation of genotoxic vinyl previously mentioned in section 3.2.5 due to the potential bromide. In the scaled up reaction, the N-alkylethyl group was formation of acetamide. However, there is a second source of 247 introduced via an oxathiazolidine S-oxide, obtained by reaction GTI: sulfonate esters. In the case of zaurategrast sulfate, the of N-benzylethanolamine with thionyl chloride, which on use of methanesulfonic acid in the presence of ethanol posed reaction with 4-hydroxylphenylacetamide gave the desired the potential risk of generating genotoxic ethyl mesylate. This intermediate (Scheme 85b). Note that in this particular case, was mitigated by replacing the sulfonic acid with hydrochloride the main motivation to modify the synthetic step was safety ff 247 acid without a ecting the yield. In the initial synthetic route rather than API purity. As a bonus, the new route gave a 64% of sodelglitazar (Scheme 82a), a genotoxic mesylate inter- yield while that for the dibromoethane-based route was 9%. mediate is used; therefore, the commercial application employs 4.1.2. Adjusting Reaction Conditions To Mitigate GTI the corresponding alcohol instead of this mesylate ester 248 Formation. The feasibility of minimizing the formation of (Scheme 82b) for the formation of the thioether linkage. genotoxic impurities by simple adjustment of parameters such Denagliptin was previously mentioned in section 3.2.2, as reaction time, pH, temperature, and solvent matrix is illustrating API salt formation with sulfonic acids. However, in a demonstrated through the following examples. previous step, a (S)-difluorophenyl amino acid is reacted with a 4.1.2.1. Sulfonate Esters. The synthesis of the AstraZeneca fluoro amino amide mediated by n-propanephosphonic acid drug for management of type 2 diabetes, tesaglitazar, includes a cyclic anhydride (T3P) and diisopropylethylamine (DIPEA).184 step where a potentially genotoxic bismesylate ester is added in The next step involved dehydrating with p-toluenesulfonic 269 ° excess to the phenolic key intermediate to form an ether at anhydride with pyridine as base at 50 C. Since p- ° − toluenesulfonic anhydride was not available commercially, the pH 10 and 100 C for 4 5 h while using PEG-400 as phase- transfer catalyst in the presence of sodium carbonate (Scheme scaled up reaction relied on the use of methanesulfonic fl − anhydride (Scheme 83). In this case, the potential to form 86). Adjusting the pH to 7 and increasing the re ux time to 8 9 h allow complete reaction and hydrolysis of the alkyl sulfonate ester without hydrolysis of carboxylate ester in the Scheme 83. Alternative Route for Large-Scale Synthesis of ff Denagliptin Tosylate, Avoiding the Formation of a API. Such an approach, which takes advantage of di erent Potentially Genotoxic Mesylate Ester 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. mesylate esters, which are potential genotoxins (as are the tosyl Reduction of the reaction temperature from 40 to 10 °C esters had the tosyl anhydride been used), was not desirable. showed a significant 4-fold reduction of sulfonate esters 179 The observation that partial dehydrating had occurred during formation even without the addition of a base. Therefore, the coupling reaction gave rise to the exploration of T3P as a conducting both the reaction and workup at lower temper- dehydrating agent. A second equivalent of T3P, along with a atures is recommended. higher temperature of 78 °C, gave satisfactory results (Scheme (iii) Water: The presence of water, as it competes with 83).184 alcohol for protonation and promotes ester hydrolysis, has a The synthesis of the anti-inflammatory agent UK-371,104267 positive effect on reducing sulfonate esters formation, and even includes a glycosidation reaction of the adenosine key a small amount of water results in a 3-fold decrease of sulfonate intermediate with a peracetylated sugar, 1,2,3,5-tetra-O-acetyl- esters without addition of base.185

AC DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Scheme 84. During the Synthesis of UK-371,104, the Workup Procedure Generates Genotoxic Acetamide from N,O- Bistrimethylsilylacetamide 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 S- a Oxide

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

Scheme 86. Synthesis of Tesaglitazar, with a Change in pH 369,003-26, a candidate for treatment of benign prostatic for the Effective Hydrolysis of the Sulfonate Ester Precursor 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 87. Formation of Potentially Genotoxic Ethyl a Besylate (EtBS) through Salt Formation

(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 aHere it is not an alcoholic solvent but the ether substructure of the responsible. For example during the manufacture of UK- API that leads to GTI formation.

AD DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Scheme 88. Competitive Formation of API Salt and Alkyl in in situ formation of amine hydrochloride salts, also yields the Halides in the Reaction of Halide Acids with an API Base in genotoxic dimethylcarbamoyl chloride (DMCC). A hydrolysis Alcoholic Solvents 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 alkyl halides in the synthesis of APIs are provided in Sections ff reduction can be found in the synthesis of an adrenoreceptor 3.1.1 and 3.2.4. The e ect of reaction conditions on the antagonist, as represented in Scheme 90.272 The nitroaromatic mitigation and elimination of alkyl halides has been investigated reduction takes place through a hydrogenation catalyzed by Pd/ using a quaternary amine as API model in the formation of the C, while workup involves removal of the catalyst by filtration, HCl salt.48 Drying of the product at 85 °C under vacuum failed concentration, and crystallization. to significantly decrease the alkyl halide content. Decreasing the 4.1.3. Quality by Design. The use of the QbD approach rate of addition of HCl and increasing stirring times did not has been suggested to develop synthetic routes or selection of have a significant impact when applied alone. A reduction of the conditions for API synthesis and can also be applied to control HCl load did decrease alkyl halide formation but resulted in GTI formation below threshold values. In the pharmaceutic lower yield of the salt. The reaction temperature proved to be context, QbD aims to design and produce API formulations for crucial in managing the levels of alkyl halide. At 35 °C the which the final quality should be ensured a priori through the formation of alkyl halides was favored, whereas a lower ° ffi design of synthetic routes and the manufacture process. temperature of 10 C proved to be an e cient strategy to fi mitigate formation of the genotoxin. When tested at larger Generically, QbD includes four stages: (i) de nition of the quality profile to be targeted; (ii) product and manufacture scales a yield of 92% and GTI formation below 1 ppm, in fi compliance with TTC limits, was achieved. process design to achieve such quality; (iii) identi cation and Another example, in which both temperature and reaction selection of quality attributes, process parameters, and sources time were adjusted to mitigate GTI formation, was reported by of variability; and (iv) control mechanisms to ensure quality AstraZeneca during the use of a Vilsmeyer chlorination reaction over time. In the particular case of GTI risk control, the target in a penultimate step of API synthesis (Scheme 89). This for product quality requires one to maintain GTI below threshold numbers, while providing high API yields. The Scheme 89. Adjustment of Temperature and Reaction Time examples in section 4.1 provide cases of design of chemical in a Chlorination Step Resulted in a Reduction in Formation synthesis that avoided the presence of GTI, and the following of Genotoxic Dimethylcarbamoyl Chloride (DMCC) 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. particular reaction comprises simultaneous in situ formation of Potential GTIs can be formed in the reduction of nitro- an amine hydrochloride salt.271 The reaction of the N,N- aromatics to aniline derivatives, as illustrated in Scheme 91. The dimethylformamide and a chlorinating agent, POCl3, resulting four compounds raised structural alerts according to DEREK,

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

AE DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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 fluoroaryl- amine 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 GTI- spiked 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 Figure 6. Quality-by-design strategy for prevention of GTI formation. amount of alcohol used in the various steps of stage 8 has no significant impact on the amount of GTIs formed; the drying Scheme 91. Nitroaromatic Catalytic Reduction (a) Showing operation does not generate any detectable GTIs, and the Reaction Intermediates That Can Remain in the Product as isolation effectively removes any GTI. Overall, the process Impurities (b) 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. 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, but the nitroaromatic and aniline intermediates showed defining risk considering the number of synthetic steps between negative genotoxicity by the AMES test. The study by Looker the appearance of GTI and the final production step. It was et al.273 was focused on the optimization of reaction conditions recommended that in cases where the presence of GTI is more to avoid the presence of these impurities and comply with the than four steps away from the final synthetic step, chemical established specific thresholds. In particular, the process rational should be used to decide whether GTI specific impurity employed Pd/C catalyst for the hydrogenation of nitro- removal is required or not. However, such an empirical aromatics. The purification steps consisted of filtration for the approach is not process specific. Therefore, Teasdale et al.39 removal of Pd, followed by concentration of the resultant developed a semiquantitative “assessment purge tool” focusing filtrate, addition of an antisolvent for recrystallization, and on the particular GTIs of concern and chemical properties of a filtration/drying of the solid obtained. Solutions spiked with given process in order to evaluate the risk of a GTI to be potential GTIs were used to monitor and assess the present in the final API. The proposed tool defined the effectiveness of impurity purging at different stages of the following main purge factors: GTI’s reactivity, solubility (in the purification units. The process parameters selected for solvents used, e.g, for recrystallization, where the GTI is optimization include temperature, amount of catalyst, and discharged with mother liquid), volatility (e.g., through GTI

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

AF DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review removed with solvent during distillation for solvent exchange), added in isopropyl alcohol, which can form a second potentially and ionizability (e.g., for a partition of GTI and API between genotoxic ingredient, namely, isopropyl chloride. Moreover, the aqueous/organic, for example, for pH adjustments to change HCl can react with the AZD9056 base, resulting in small the ionized/un-ionized state of one of the compounds) and amounts of AZD9056 chloride as a byproduct, a third potential processes used for purification (e.g., chromatography). This GTI. tool used a score scale for each purge factor, as described in The assessment purge tool was applied to these three Table 9, where purge factor is defined as the ratio of GTI potential GTIs and the predictive values were compared with concentration before and after purging. experimental results, pointing out that the tool usually underestimates purging effects. The purging of the three Table 9. Example of Key Parameters in Purge Factors in the potential GTIs were assessed by considering the following: a Tool by Teasdale et al. (i) The main driver for purging AZD9056 aldehyde is its high reactivity, which leads to this compound’s full con- Physicochemical parameters Purge factor sumption, and thus it was scored as 100% in the first step. Since Reactivity High reactivity = 100 this compound is not volatile, a score of 1 was allocated to all Moderately reactivity = 10 the steps. A moderate solubility was considering in the last two Low/no reactivity = 1 steps; however, a larger removal of the AZD9056 aldehyde was Solubility Freely soluble = 10 experimentally measured in step 2, leading to an overall Moderately soluble = 3 underprediction of the purging capacity of the process by 10 Sparingly soluble = 1 times. Volatility Boiling point >20 °C below that of the (ii) Isopropyl chloride is present in steps 2 and 3, and as reaction/prcess solvent = 10 defined by the established criteria for high solubility and Boiling point ±10 °C that of the reaction/ prcess solvent = 3 volatility, a score of 10 for purge factors was allocated to these Boiling point >20 °C above that of the two parameters. The tool predicts the purging capacity of reaction/prcess solvent = 1 10 000, again representing an underprediction of about 4 times, Ionisability Ionization potential of GTI significantly indicating that in spite of the relatively high formation of different isopropyl chloride, its presence in the final product is highly Physical processes (e.g., Chromatographically, GTI elutes prior to the improbable. chromatography) desired product = 10 (iii) AZD9056 chloride byproduct is actually not reactive, Chromatographically, GTI elutes after the desired product = 10 not volatile, and not particularly soluble in isopropyl alcohol. Others processes are valuated on an individual Therefore, an overall low purging factor of 3 was predicted basis against a measured value of 10, implying that action should be aAdapted with permission from ref 39. Copyright 2013 American taken to either remove this compound or change the process to Chemical Society. eliminate or mitigate the formation of this compound. 4.2.2. Separation Technologies. For the specific removal fi They also describe six case studies where each of the different of GTIs, the selection of the puri cation method is intrinsically existing purge factors was evaluated and their contribution dependent on the physicochemical properties of the GTI, “ ” assessed at different stages in the removal of GTIs. Such cases which will decide the relative purge factors. From a process include the removal of thionyl chloride (two syntheses); chemistry point of view, it is also important to understand nitropyridyl N-oxide (a starting material); and AZD9056 which separation operation units are involved in API fi aldehyde and its respective byproduct AZD9056 chloride, puri cation. In this review, seven examples of conventional fi together with the side products isopropyl chloride, methyl puri cation techniques and three emergent techniques are hydrazine, and hydrazine. When genotoxins are introduced as referred to (Chart 7). Usually the higher the selectivity of a fi fi reactants, their reactivity is one of the main factors contributing puri cation process regarding a speci c impurity, the lower the ffi to how they are purged as they are consumed in the chemical API loss and the higher the removal e ciency of the impurity reaction. Consequently, the use of a genotoxic reactant in in question. In many cases, delivering a safe API requires the fi excess is, if possible, to be avoided. Note that some of these application of a puri cation strategy where the GTI is reduced fi highly reactive compounds can also be eliminated by reaction to acceptably low levels. To illustrate this, a speci cation of 70 with bases, acids, or even water in subsequent steps, as is the ppb, calculated using the daily dose, was set by a case for thionyl chloride in the examples provided. Volatility is pharmaceutical company for an especially potent genotoxin in − 275 an obvious route for the removal of low boiling point a drug candidate (shown in eqs 1 4). compounds, such as methyl hydrazine (88−90 °C), thionyl GTI removal efficiency= GTIend /GTI start (1) chloride (79 °C), and isopropyl chloride (36 °C) through distillation and drying. Solubility takes an important role in APIloss= API end purification step /API fed purification step (2) purging such compounds, in particular when crystallization or extraction operations are involved where GTIs can be dissolved GTIcontent= GTI end purification step /API end purification step (3) either in mother liquors or the discarded phase. AZD9056 HCl/chloride case study clearly illustrates the use GTI=× API GTI of this assessment purge tool (Scheme 93).39,274 In final steps daily intake daily dose per patient kg content of the API synthesis, the potentially genotoxic AZD9056 × weight patient (4) aldehyde reacted with 3-aminopropano-1-ol to produce an imide derivative that is subsequentiatly reduced to a free base Such ultralow levels in the specifications of APIs pose (step 1), followed by HCl salt formation (step 2) and finally additional analytical and processing difficulties for efficient recrystallization (step 3). During the purification step, HCl is purification. The design of a synthetic process to produce an

AG DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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

API includes sequential reaction steps intercalated with content through an increase in the number of cycles would lead purification steps. These conventional purification steps are to unacceptable API losses. already in place and already contribute to GTI removal, The use of an additional “end-of-pipe” GTI purification although not specifically designed to remove GTIs. The could complement the already existing intercalated purification ffi difference between point-of-source and end-of-pipe GTI steps. Nevertheless, the removal e ciencies are usually removal is schematically illustrated in Figure 7. concentration-dependent, decreasing with lower GTI concen- The removal of larger quantities of impurities can be usually trations. In such cases, it may be advantageous to follow a “point-of-source” GTI detoxification strategy. For implementa- achieved by increasing the number of cycles within a given fi purification step (e.g., the number of re-extractions, recrystal- tion of this strategy, identi cation and mapping of the reactions where GTIs are present is crucial, and lessons taken from lizations). However, increasing the number of cycles also leads section 2 should be considered. to undesirably high API losses and may have diminishing fi ffi Conventional puri cation steps during and after API e ciency with each new cycle. Consider, for example, an API synthesis include crystallization, precipitation, solvent extrac- stream with a GTI content of 1 g of GTI for each 100 g of API, fi tion, silica gel or alumina column chromatography, and and a theoretical puri cation operation in which for each step treatment with activated carbon and resins, as well as fi 80% of the GTI is constantly removed, along with the sacri ce distillations. As in any separation, the efficiency of the of 3% of the API. To reduce the GTI from a concentration of separation depends of the differences in chemical and physical 1g/L in solution (corresponding to an API concentration of properties of the two entities to be separate and/or their 100 g/L) to 64 μg/L would require six cycles and a cumulative relative affinities for a selective agent. In this review, we briefly API loss of 17% in the purification alone. Therefore, the use of report 10 different purification techniques, of which 7 can be conventional purification procedures to reach ultralow GTI perceived as conventional methodologies to remove impurities

AH DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Chart 7. Conventional and Advanced API Purification Technologies

Figure 7. Point-of-source and end-of-pipe GTI removal. and 3 as advanced techniques proposed during past decade along with the API or remain as part of the crystal lattice, (Chart 7). depending on the efficiency of the washing procedure. 4.2.2.1. Crystallization. (1) Crystallization is one of the most Filtration is the normal technique used to isolate the crystalline important isolation and purification process for APIs. The API solids. A particular example is illustrated in Chart 8, where is isolated as a solid phase while the impurities remain dissolved acetamide is removed in a process that incorporates in the liquid phase (the mother liquors). Crystallization is also crystallization.247 broadly used in chiral separations, namely, through diastereo- 4.2.2.2. Solvent liquid−liquid extraction. (2) Solvent meric resolutions.276,277 In some cases, a two-solvent system, a liquid−liquid extraction is commonly used for API purification; solvent and a cosolvent, can be used to promote crystal API (or impurities) can be selectively transformed into salts formation in accordance with the respective phase diagrams. and retained in an aqueous phase while the organic impurities Robustness, kinetics, temperature, and pH of the crystallization (or API) are removed by a water immiscible organic solvent system are also important parameters.278,279 Crystallization is a phase. The organic salt can then be converted to the neutral fi fi fi puri cation process that not only determines the purity and species by acidi cation or basi cation, according to the pKa of residual solvent content of the API but also establishes the the API, and re-extracted into a second organic solvent, which crystalline properties in terms of polymorphic form, crystal is usually concentrated before isolation of the API. The habit, bulk density, and size distribution, all of which affect efficiency of separation depends on the relative partition downstream processing, e.g., drying and formulation.280,281 coefficients of API and GTI in the different solvents. Panel i of More importantly, the crystalline properties and polymorphic Figure 8 illustrates a purification process involving solvent forms can be responsible for drug bioavailability. Therefore, phase exchanges and crystallization of the API, while panel ii once a route is approved for API production, the crystallization maps the corresponding losses of API. step of the final API is usually retained. In some instances, 4.2.2.3. Precipitation. (3) Precipitation is commonly depending on process optimization, a significant fraction, up to promoted by addition of a nonsolvent to a solution of the 30% of the API, can remain in the mother liquors282 or be lost API (or vice versa). Similarly to crystallization, the impurities through washes of the solids. Impurities may be washed out remain in the liquid and the API ends-up as a solid phase.

AI DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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

However, the solid may be amorphous and not crystalline, but absorbents for metal impurities has been evaluated using once more, the solvent system (final mixture of solvent and microtubes.289,290 Other studies include removing formalde- nonsolvent) selected should show higher solubility for the hyde using activated carbon containing amine groups291 and impurities than for the API. Solute solubility is, among other removal of an aldehyde impurity using polystyrene-based things, dependent on its polarity and the polarity of the solvent. sulfonylhydrazine resin.292 GTIs such as p-toluenesulfonic Note that some of these polar solvents also have high boiling acid methyl (MeTs), ethyl (EtTs), and isopropyl (i-PrTs) points and are potentially genotoxic themselves (Table 8); esters have also been evaluated using different commercially therefore, if they are not removed properly, they present an available nucleophilic resins.287 These studies used methyl, additional risk as a GTI in the API . Filtration is also used to ethyl, and isopropyl esters of methanesulfonic, benzenesulfonic, separate liquid from solid, or distillation is used to evaporate and p-toluenesulfonic acids as model PGIs and screened the use low boiling point solvents. When the impurity is preferentially fi of several amines, thiol, thiophenol, piperazine, and piperidine precipitated, it can be removed by ltration. immobilized on silica and polystyrene. Removal was effective 4.2.2.4. Fractional distillation. (4) Fractional distillation can 283 for methyl sulfate esters, whereas it proved to be more of a be used to purify volatile APIs. However, distillation is also challenge to remove ethyl and isopropyl esters by this broadly used for removal of solvents and for solvent exchanges, technique. This strategy was applied for the removal of MeTs particularly when switching from a low boiling point solvent to from a 21-chlorodiflorasone solution. When trisamine was a higher boiling point solvent (see Table 8). Solvent exchanges immobilized either on silica or macroporous polystyrene− from high boiling point solvent to lower boiling point solvents divinylbenzene supports, 100% GTI removal was achieved.293 or when thermosensitive compounds are involved can be sustainably achieved using organic solvent nanofiltration These adsorbents and resins can be used as stationary phases in (OSN).282 Volatile organic impurities, mainly resulting from chromatography. residual solvents,284,285 can also be removed through 4.2.2.6. Column chromatography. (7) Column chromatog- distillation. Many of the GTIs considered in this review have raphy is a typical postreaction technique applied in organic low volatility (e.g., hydrazine, MsCl, TsCl, DMS, 1,2-epoxy-3- chemical synthesis to remove impurities. Sophisticated sta- tionary phases are applied in the pharmaceutical industry, for butene, acetamide, phenylboronic acid all have boiling points 294,295 above 100 °C), and alkyl halides such as MeCl and EtCl have example, in chiral separations. However, this review is boiling points of −24.2 and 12.3 °C, respectively. focused on the removal of GTIs, and for this endeavor, 296 4.2.2.5. Adsorption processes. (5 and 6) Adsorbents such as preparative column chromatography using standard silica gel granular activated carbon (GAC)286 and resins287 are broadly or alumina of pharmaceutical grade as stationary phase has been used to remove color and impurities.288 Adsorption-based used. In this technique a solvent, such as ethyl acetate, ether, separations rely on the different affinities of the disparate acetone, methylene chloride, and/or mixtures thereof, is used as compounds for the adsorbent. Therefore, a high affinity of the eluent. Commercially available absorbents, such as polystyrenic GTI combined with lower binding of the API is desirable in this or methacrylic matrices, with aqueous solutions at different pHs case. Screening of the different commercially available and ionic strengths have also been reported.297 Particle size,

AJ DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

4.2.2.8. Organic solvent nanofiltration. (9) Organic solvent nanofiltration (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, GMT- oNF-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 fl column dimensions, and eluent ow and pressure are critical OSN in API purification is the low product yield due to parameters. insufficient rejection of the product.305 To overcome this 4.2.2.7. Supercritical extraction. (8) Supercritical extraction fl limitation, Kim et al. recently proposed a two-stage membrane techniques utilize the properties of supercritical uids, which cascade.304 Through an API purification case study, the authors have the high solvation power of a liquid and the enhanced demonstrated that the proposed process significantly increases diffusivity of a gas. Moreover, simply changing from the the API yield without compromising its purity. The second supercritical state to a gaseous state provides a straightforward fi method to isolate the solute. The relatively low critical point of main drawback of OSN for API detoxi cation was the ° significant solvent consumption during diafiltration processes. CO2 (71 bar, 31 C) had positioned it as an ideal supercritical solvent that can replace more hazardous solvents as reaction However, OSN has markedly evolved in recent years, and the media and in extractions in chemical processes.298 Supercritical newest generations of solvent-resistant membranes can fully reject small molecules at the lower end of the nanofiltration CO2 can be used to produce particles with controlled size and − purity299 and also be used in packed column chromatog- range (50−2000 g·mol 1) and subsequently can be used for in raphy.300 Therefore, this purification technology has the situ solvent recovery.306 potential to provide an effective and clean route for GTI 4.2.2.9. Molecular imprinting technology. (10) Molecular removal from APIs. imprinted polymers (MIPs) are prepared by incorporating the

AK DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

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

5. CONCLUSIONS AND FUTURE TRENDS Launching new pharmaceutical products involves the collabo- rative 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 Figure 10. OSN-based API purification, with potential use of organic compromises safety, since it can become a vehicle for increasing reverse osmosis for solvent recycling. genotoxic risk. The quantity of genotoxic impurities in drug products is target molecule into a polymeric matrix as template (Figure strictly controlled by regulatory authorities that have set limits 11). The target molecule is therafter removed, leaving a to ensure patient safety. To ensure compliance with the potential binding site within the matrix. Thus, the final polymer required low GTI concentrations, a significant effort during structure usually provides enhanced affinity for removal of the development is necessary. Three main strategies that contribute molecules used as template. The use of MIPs for separations in to producing APIs of acceptable quality can be identified: the pharmaceutical industry has been suggested previously307 (1) At the most rigorous level, the strategy outlined in the and used in bio- and pharmaceutical analysis.308 Exploring the regulatory authority’s guidelines is to avoid the use of any high specificity achieved by MIPs, several studies have evaluated genotoxic chemical over the entire synthetic route, regardless of − their use in chiral separations.309 311 Specific development and whether the genotoxic chemical is used as a reagent, starting characterization of a MIP for potential GTI removal were compound, catalyst, or solvent. However, given the chemical recently reported.312,313 nature and desirable reactive properties of chemicals, in many By exploring the ability of OSN to remove potential GTIs cases the direct use of a genotoxic chemical or a GTI precursor when at high concentrations and combining this with the better is unavoidable. performance of MIPs to remove the target molecule at lower (2) Even when genotoxic compounds are not applied concentrations, a hybrid process using these two purification directly, they can be formed during chemical reactions. techniques also had been suggested.314 Possible limitations of Therefore, a second strategy for minimization of genotoxin the use of MIPs for GTI removal are as follows: specific MIPs formation can be achieved by thorough investigation of the need to be developed for individual (or similar) GTIs; removal particular reaction and adjusting the reaction parameters with is more effective at lower concentrations, and hence, high assiduous care, in particular using QSAR strategies. volumes are involved; and contamination is possible via (3) An alternative and complementary strategy is to design leaching of impurities derived from the polymer. Hence, this specific purification strategies targeting the removal of GTIs technique has not become widespread at this time. Besides, from the API once it has been established that they or their hybrid processes imprinting and nanofiltration technologies precursors are present. Either “point-of-source” or “end-of- have been combined in a molecularly imprinted organic solvent pipe” strategies can be used. nanofiltration strategy.315 The full cycle of drug development, approval, and use has to MIPs often show cross-reactivity, which can be exploited in work for the different stakeholders. This means safety and the rapid screening of MIP libraries to identify suitable efficacy for patients, compensation for the efforts of the

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 DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review pharmaceutical companies, and selling prices that are Biographies 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 cost- effective 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. Gyorgy Szekely received his M.Sc. degree in chemical engineering Therefore, a checklist for the pharmaceutical R&D from the Technical University of Budapest (Budapest, Hungary), and community to manage the GTI risk can be outlined as follows: he earned his Ph.D. degree in chemistry under Marie Curie Actions (1) Provide solid data for toxicological evaluation of potential from the Technical University of Dortmund (Dortmund, Germany). GTIs with quantification of threshold values and highlight He worked as an early stage researcher in the pharmaceutical research those that are higher than the general TTC value. and development center of Hovione PharmaScience Ltd in Portugal (2) Develop analytical and monitoring techniques for ultalow and as an IAESTE fellow at the University of Tokyo (Tokyo, Japan. He was a visiting researcher at Biotage MIP Technologies AB in levels of GTIs. fi Sweden. He was a postdoctoral research associate at Imperial College (3) Develop new synthetic or process routes. Speci cally, London (London, UK). He is currently a lecturer at the School of alternative reagents should be used to replace genotoxic or GTI Chemical Engineering & Analytical Science, The University of precursor reagents. Identify new reaction media to replace Manchester. His multidisciplinary professional background covers genotoxic or carcinogenic reaction solvents. supramolecular chemistry, organic and analytical chemistry, molecular (4) Strive for a deeper understanding of existing reactions recognition, molecular imprinting, process development, membrane and process routes to be pursued, identifying and optimizing separations, and pharmaceutical impurity scavening. In addition, he is a  the crucial chemical and physical parameters in the process, to board member of the Marie Curie Fellows Association serving as  mitigate GTI presence. Secretary General and is a member of the Royal Society of (5) Develop novel API purification techniques for removal of Chemistry, The Institution of Engineering and Technology, and the Institution of Chemical Engineers. 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.

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 Miriam C. Amores de Sousa graduated with a degree in applied ACS Publications website at DOI: 10.1021/cr300095f. chemistry (minor in biotechnology) in 2007 and concluded her Master’s degree in biotechnology in 2009, at Faculdade de Cienciaŝ e AUTHOR INFORMATION Tecnologia of Universidade Nova de Lisboa. She is currently a Ph.D. student at the Department of Bioengineering at Instituto Superior Corresponding Authors Tecnico,́ Universidade de Lisboa, where she is a member of the *G.S. e-mail: [email protected]. BioEngineering Research Group at the Institute for Bioengineering *F.C.F. e-mail: [email protected], frederico_castelo@ and Biosciences. Previously, as a research assistant, she worked on the yahoo.com. characterization of polysaccharides and explored biocompatible *W.H. e-mail: [email protected]. cellulose acetate membranes as potential drug delivery systems, focusing on the solid-state mobility properties of the materials. Notes Currently, her research is focused on studying the interaction between The authors declare no competing financial interest. electrospun functional nanofiber matrices and stem cells, to evaluate

AM DOI: 10.1021/cr300095f Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review the cell fateself-renewal and differentiationfor future applications interests were broadened to the development of novel particle size in tissue engineering, as stem cells provide an interesting model to reduction technologies aiming to produce engineered particles for probe the cytotoxic effects of different compounds and materials. 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.

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 William Heggie received his Ph.D. in organic chemistry from The was a research associate (2004−2006) in a joint project of Imperial University of Manchester and held postdoctoral positions at Harvard, College London and GlaxoSmithKline (GSK). He was a visiting St. Andrews, and Oxford Universities. He held a teaching position at researcher (July 2007) at Institut Europeeń des Membranes the New University of Lisbon from 1974 to 1976 and was a professor (Montpellier, France) and a visiting scholar (Sept-Dec 2009) at the at Lisbon Superior Institute of Engineering from 1979 to 1998. He Massachusetts Institute of Technology, Deshpande Center for joined Hovione in 1980 and held various positions in Hovione’s R&D Technological Innovation. Since March 2009, he has been a member group before becoming Chief ScientificOfficer in 2004, being of the BioEngineering Research Group at the Institute for responsible for innovation and introducing new technologies into fi Bioengineering and Biosciences. He teaches at the Department of the company. He is the author of more than 20 patents and scienti c Bioengineering at Instituto Superior Tecnico,́ Universidade de Lisboa, articles. including the course “BioteamsTeams for Innovation” for Ph.D. students, and he launched two new elective M.Sc. courses: “Green ACKNOWLEDGMENTS ” “ Technologies and Strategic Management and Entrepreneurship in The authors acknowledge the support of NEMOPUR (New ” Bioengineering . He also assists CoHiTec on translation of technology Molecular Purification Technology for Pharmaceutical Produc- to the market. His current research interests balance between tion), a Marie Curie Initial Training Network within the fundamental and applied research, for the development of new seventh Framework Programme of the European Commission’s processes, reactors, and materials, with an emphasis on membrane- Marie Curie Initiative, and the support of FCT (Fundaca̧õ para based systems. a Cienciâ 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.

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