Review

pubs.acs.org/CR

Genotoxic Impurities in Pharmaceutical Manufacturing: Sources,

Regulations, and Mitigation
Gyorgy Szekely,*,† Miriam C. Amores de Sousa,‡ Marco Gil,§ Frederico Castelo Ferreira,*,‡
and William Heggie*,§
†
School of Chemical Engineering & Analytical Science, The University of Manchester, The Mill, Sackville Street, Manchester M13
9PL, United Kingdom
‡
Department of Bioengineering and Institute for Bioengineering and Biosciences (iBB), Instituto Superior Técnico, Universidade de
Lisboa, Avenida Rovisco Pais, 1049-001, Lisbon, Portugal
§
Hovione FarmaCiencia SA, R&D, Sete Casas, 2674-506, Loures, Portugal

*
S Supporting Information

4.1.1. Altering the Synthesis 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

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Figure 1. Importance of genotoxicity demonstrated by the increasing
number of publications on the topic, resulting from an ISI Web of
Science search on “genotoxicity” and “genotoxic impurity”.
on the former search shows the overall importance of the field
of genotoxicity, including chemistry, analytical methods,
manufacturing, purification, diseases, medical aspects, genotox-
icity tests, mechanism of action, assessment, and environment.
The latter search illustrates the increasing attention of industry
to GTIs, mainly related to drugs and food.
Compounds categorized as GTIs actually include a broad
range of unrelated chemicals with very different structures and
from very different chemical families. From 4000 compounds
tested, 44 molecular structures were correlated with muta-
genicity and correlated highly with electrophilic reagents, such
as epoxides (63%), aromatic amines (49%), and primary alkyl
monohalides (46%).2 Aromatic amines are not electrophiles,
but their decomposition leads to the formation of electrophilic
reactive species such as aryl nitrenium ion. In section 3.1.6
examples of aromatic amine reactants are described. These
compounds have a shared ability to react with DNA, resulting
in an associated carcinogenic risk. However, from a chemical
point of view, they do not have common chemical−physical
properties or chemical structural elements that can contribute
to easy identification. Experimental assessment of genotoxicity
test models, such as the Ames test, allows direct study of
genotoxicity, and the Committee for Medicinal Products for
Human Use (CHMP) has defined GTIs as impurities that have
been demonstrated to be genotoxic using such genotoxicity test
models. The Ames test, developed in the early 1970s by Bruce
N. Ames, is an experimental procedure to evaluate the potential
carcinogenicity of chemicals, based on mutagenicity effects on
Salmonella typhimurium histidine auxotrophic mutants strains.
It became widely used due to its simplicity, low cost, and quick
analysis without the need for animal testing.
As discussed in ICH Q3A and Q3B, actual impurities in API
are the ones that exceed the reported threshold when the lot is
released or arise, for example, as degradation product, during
storage and distribution over the shelf life of the API, whereas
potential impurities may or not actually be present in the API
but are identified as the ones that can theoretically arise during
manufacture or storage. In the particular case of GTIs,
“potential GTIs” are the ones that have structural alerts, i.e.,
functional chemical groups, for genotoxicity but have not been
experimentally assessed; note that here potentially is not related
with the presence or absence of the impurity.3 In silico systems
are commonly used to identify structural alerts and predict
B DOI: 10.1021/cr300095f
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genotoxicity according to chemical structures. These systems
follow either rule-based or quantitative structure−activity
relationship models (QSAR). Rule-based systems are derived
from identified mechanisms of action of chemicals in the cell
genome or metabolic proteins. This approach was introduced
by Miller and Miller in 19774 and followed by other authors. In
spite of its mechanistic clarity, it has been criticized as being
based only on single interactions and therefore failing to be
comprehensive. QSAR models may use several inputs
simultaneously, e.g., information on Ames test results, log P,
molecule polarity and electrical distribution, and chemical
substructures. The use of QSAR models is particularly useful
for the prediction of the biological effect of a broad range of
chemicals and new molecules with a high degree of accuracy.
This subject is further explored in several studies, and examples
include comparison of the use of three models for prediction of
Ames genotoxicity5 and presentation of different case studies.6
QSAR models commonly used for determination of structural
alerts to predict genotoxicity are the MULTICASE and the
deductive estimation of risk from existing knowledge (DEREK)
ones.7−9 However, for numerous chemical classes, structural
alerts overpredict mutagenicity when they do not take into
account factors such as high molecular weight, hydrophilicity,
high reactivity, steric hindrance, molecular symmetry, and facile
metabolism.10,11
On the other hand, their presence in the manufacture of APIs
is not stochastic, since these genotoxic chemicals often have
specific inherent roles in the chemical routes used in API
synthesis. The presence of such chemical in the reaction is a
result of their introduction into the reaction in stoichiometric
or catalytic amounts or as solvents, as well as their formation as
side products. The presence of genotoxins is usually inherently
controlled during API manufacture, as several stages of
intermediate API isolation and purification are included in
the production process, during which most of the GTIs are,
together with other impurities, removed. Additionally, many of
the synthetic reaction sequences initially designed for
production of new drugs are often further improved through
optimization of reaction conditions or by substituting with
different reaction steps. Such improvements aim at higher
yields, reaction selectivity, and more efficient use of reactants,
which results in lower amounts of unreacted compounds and
side products formed. Nevertheless, production of APIs with
low GTI content is a major concern for API-manufacturing
companies. Ideal solutions consist of the simplest possible,
robust process, using cost-effective reagents to obtain high
product yields through selective reactions and purification
steps. Development and validation of such processes in a timely
manner are important for the industry, and as such, it is
important to be aware of the chemical mechanisms in which
genotoxic compounds are involved, whether as reagents or
reaction side products, and of existing strategies to circumvent
their use or remove them from postreaction streams.
In addition to the Introduction and concluding remarks, this
review includes the following sections:
Section 2 provides a brief description of genotoxic
mechanisms and a risk analysis, as well as the regulatory
approaches taken concerning this issue. Further reviews on
specific topics of risk assessment, 12 toxicology,13 and
mechanism of action14 of such compounds can be found in
the literature.
Section 3 of this review focuses on GTIs related to starting
materials, reagents, reactants, catalysts, and solvents with
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Chemical Reviews
genotoxic effects and related genotoxic side products.
Impurities structurally related to specific APIs (e.g., genotoxic
sulfonate ester side products during the synthesis of the steroid
mometasone15,16) are outside of the scope of this review
(Figure 2). The book “Genotoxic ImpuritiesStrategies for
Figure 2. Sources of GTIs in API streams.
Identif ication and Control” edited by Teasdale provides an
exhaustive discussion on the identification and control of such
impurities in API manufacturing.17
The major focus of this review is provided in section 3, which
comprises a thorough description of chemical synthesis of
several pharmaceuticals. The objective of this review is to
contribute to an easy identification and mapping of GTI
occurrence in chemical synthetic routes, highlighting the
importance of GTIs in the manufacturing of APIs. Therefore,
the focal point of this review is to improve awareness of
different entry points for GTIs over the API synthesis. Case
histories and synthetic examples selected include 93 different
chemical schemes used in the synthetic routes of APIs or
intermediates of 100 different drugs. Nine different chemical
families are present because they are used as reagents or
catalysts or are formed during synthesis. In recognition of the
importance of risks posed by residual toxic solvents left in API
formulations, an additional section highlights the potential
genotoxicity and carcinogenicity of common solvents used in
drug synthesis.
Section 4 discusses approaches for mitigation of GTI
content. A variety of different strategies are used to avoid/
reduce GTIs during industrial implementation, as disclosed in
patents and academic publications. Section 4 includes two
subsections focused on synthetic approaches and on API
purifications. Examples include changing the reaction route
involving a GTI reactant and the optimization of reaction
conditions to mitigate the amount of unreacted genotoxic
reactant left in the postreaction stream. The section on API
purification strategies briefly describes (i) conventional
purification techniques, such as recrystallization, chromatog-
raphy, and distillation, and (ii) emerging technologies, such as
organic solvent nanofiltration, supercritical extraction, and
molecular imprinting−with the aim to expand the chemists’
and engineers’ toolbox to address API detoxification from
GTIs.
C DOI: 10.1021/cr300095f
Review
The authors believe the knowledge systematically gathered in
the present review will help in the assessment of both new and
alternative synthetic routes when taking into account the
sources of GTIs and allow the pharmaceutical R&D scientists
to make more confident decisions when embarking on the
selection of alternative synthetic routes. In addition, reaction
optimization or purification strategies should be greatly
simplified. Early realization that a synthetic route could give
rise to the presence of possible genotoxins in the API will
improve timelines and safety by avoiding wasted effort on
processes with no long-term future and, in addition, directing
the focus on the relevant purification technology.
Due to the interdisciplinary nature of drug manufacturing,
the intended audience of this review covers organic chemists,
process engineers, and project managers among other
contributors in various phases of drug development. The fact
that a wide audience is targeted by the present review calls for
detailed descriptions at some points which might be common
knowledge for experts in the particular field (this information is
provided in eight charts throughout the review).
2. GENOTOXICITY: MECHANISMS, RISK AND
REGULATION
Although it has now been several years since the introduction
of the first EMA guideline on limits of genotoxic impurities, the
terms genotoxicity, carcinogenicity, and mutagenicity are often
misused by chemists. The term genotoxicity covers a wider
range of genetic damage, regardless if such damage is or is not
corrected through a cell DNA-repairing mechanism. A
mutation represents a permanent change in the genome,
which can lead to phenotype change, and a mutagen is a
substance able to increase the frequency of such changes. A
carcinogen is a substance that induces unregulated growth
processes in cells, through damage to the genome or cell
metabolic effects, eventually leading to cancer. In other words,
mutagenicity refers to processes leading to genetic change, and
carcinogenicity refers to processes resulting in tumor develop-
ment, which may result from mutagenic processes.18,19
The mechanism of action of genotoxins involves an
electrophilic attack on the nucleophilic center(s) of the DNA,
these being nitrogen and oxygen atoms of pyrimidine and
purine bases and the phosphodiester backbone, which could, in
some circumstances, lead to strand breaks (Figure 3). Bidentate
genotoxic agents can react with two nucleophilic sites, resulting
in (i) one single molecule giving a bicyclic or tricyclic system;
(ii) involvment of two different molecules in the same or the
opposite DNA strand, affording inter- or intrastrand cross-
linkages, respectively; or (iii) linking a protein and a DNA
strand, giving a DNA−protein adduct.14 Besides the chemical
nature of the genotoxic agent, the stereospecificity of the
reactions also depends on steric factors and nucleophilicity; for
instance, the most nucleophilic sites of the DNA bases are
endocyclic nitrogens, such as N3 and N7 of guanine and
adenine, and on the contrary, exocyclic oxygens are less
nucleophilic.18,20
Impurities, especially genotoxic impurities, have been at the
center of increasing regulatory and industry attention in the
past decade. The timeline of key actions toward those
regulations is shown in Figure 4. The International Conference
on Harmonisation of Technical Requirements for Registration
of Pharmaceuticals for Human Use (ICH) was setup for the
analysis of scientific and technical aspects of pharmaceutical
product registration and includes the main players in the field,
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Chemical Reviews
Figure 3. Attack on the DNA by genotoxins, where the arrows indicate
the targeted nucleophilic sites of the DNA bases (based on Madeleine
Price Ball’s figure, GNU Free Documentation License).
namely, pharmaceutical regulatory authorities and experts from
the pharmaceutical industry from Europe, the United States
and Japan. In 1995, the ICH Q3 guidelines did not yet use the
term genotoxic but “unusual toxicity”, which was a clear
reference to many of the genotoxic impurities. Five years later,
in 2000, PharmEuropa published the first article where a specific
regulatory concern with genotoxic impurities, namely, the
formation of sulfonate esters in API salt formation, was
disclosed.21 Actually, the awareness of the formation of this
class of genotoxic impurities has gained importance, and
therefore, this review includes a specific subchapter dedicated
to this particular class of GTIs.
Two years later, the Committee for Proprietary Medicinal
Products (CPMP) published the first draft position paper on
GTIs showing sufficient evidence for the existence of a
threshold mechanism in the toxicity of such compounds. This
position paper challenged the scientific and industrial
community to seek GTI-free routes to APIs or, when not
possible, to provide a justification why the presence of GTIs is
unavoidable. At that time, it was argued that in vivo studies
would put test subjects at a nonjustifiable risk. Therefore, the
model of a virtual safe dose concept, previously used in the
food industry, was suggested as an alternative and the
terminology “as low as technically feasible” was introduced.
This model was the basis to the later introduction of the
threshold of toxicological concern (TTC) concept. A draft
guideline on the limits of GTIs was released in 2004 by the
Committee on Human Medicinal Products (CHMP) from
EMA and the TTC concept was introduced.22 The TTC is a
concept that refers to the establishment of a level of exposure
for all chemicals irrespective of the existence of chemical-
specific toxicity data, below which there is no appreciable risk to
human health. It is assumed that a low level of exposure posing
a negligible risk can be identified for any chemical based on its
structure. The TTC concept was first introduced in the food
D DOI: 10.1021/cr300095f
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Figure 4. Timeline of key actions toward a regulation on GTI control
in APIs.
industry and focused only on food additives; however, it
evolved continuously as its broader applicability than simply to
chemicals in food and its potential value in the assessment of
risks in other exposure scenarios were realized.23 The brief
history of TTC is summarized in Figure 5.24−26 The FDA and
EMA have agreed on the implementation of the TTC concept
that sets a limit of 1.5 μg day−1 for known and potential
carcinogens, unless experimental evidence justifies higher limits.
Higher levels can be applied in shorter-term studies during the
clinical testing of the APIs (Table 1). The rational behind such
low values is to ensure that even if a substance was later found
to have negligible carcinogenic risk, no issues concerning safety
would arise.27 An exhaustive effort has to be made by the
industry to meet such requirements, and since many potentially
genotoxic agents turned out to pose far less risk than originally
supposed, the TTC approach is considered to be conservative.
Nonetheless, such low GTI thresholds are here to stay, and the
industry has been addressing this challenge with a variety of
control and purification strategies.
Moreover, the terminology of “as low as reasonably practical”
(ALARP) was applied for GTIs; the requirement to introduce
alternative routes or processes when available was dropped, and
no guidance relating to permissible doses during short-term
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Muller28 proposed acceptable limits for GTIs in APIs linked to

duration of exposure, i.e., a staged TTC approach. The same
document also defined five separate classes for the impurities
based on a structure−activity relationship (SAR). A separate
specific position paper addressing excipients was subsequently
prepared in 2007 by the Committee For Human Medicinal
Products (CHMP) of the European Medicines Agency.29
In 2007, EMEA was the first authority to issue and
implement detailed guidelines30 on how such impurities should
be controlled, shortly followed by the FDA, which issued a draft
guideline in 2008.31 The main difference between the FDA
draft and the EMEA guideline is in the requirements for the
degree of lower GTI limits. FDA applied an additional safety
factor of 3, while EMEA applied a factor of 10. These require
specific genotoxicity tests for impurities above the ICH
qualification thresholds and differences in staged TTC values.
In 2010, the “Questions and Answers” of the Safety Working
Party (SWP) introduced minor adjustments to the duration
limits proposed by Muller, and stated that a “cause of concern”
is a material with either a pre-existing or new genetic toxicology
indications. Also, in 2008, the European Directorate for the
Quality of Medicines & HealthCare (EDQM) in a 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

Figure 5. A brief histroy of the TTC principle.
clinical trials was provided. In 2006, the Pharmaceutical
Research and Manufacturers of America (PhRMA) led by
IMPURITIES
Regardless of the strategy selected for GTI mitigation, before it
is implemented it is crucial to identify and map the occurrence
of the GTIs in each of the API manufacture steps. Table 244
provides a list of functional groups with structural alerts for
genotoxic activity associated with the various reactions
commonly employed today in pharmaceutical development
and manufacturing. These include many of the “name
reactions” of organic chemistry. Using this table as starting
point, this review provides an overview of genotoxic impurities,
including detailed individual chemical classes. The examples
provided in this review include the use or appearance of GTIs
from different chemical families, and within each family they are
organized according to their role in the chemical reaction,
namely, as a stoichiometric reagent, a catalytic reagent, or side
product.

Table 1. Proposed Allowable Daily Intake (ADI) for GTIs of Unknown Carcinogenic Potential during Clinical Development
Duration of exposure <1 1−3 3−6 6−12 >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

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

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Table 2. Common Synthetic Transformations Related to Genotoxic Impurities

Bond formation
Alerting group C−O C−C C−N AHCa Associated reactions Examples

(1) Genotoxic Reagents/Catalysts Applied Directly

Phophonate esters 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

a
AHC means aromatic heterocycle.

3.1. Genotoxic Compounds Used as Reactants
Reactants used in chemical synthesis are usually selected due to
their appropriate reactivity; however, this very same reactivity
could result in genotoxicity. Often such reactants are not fully
consumed, persist in the reaction mixture, and can be carried
forward in the reaction sequence. Seven classes of reactants
used in API synthesis were selected as examples to be presented
in this section, including two types of alkylating agents, alkyl
halides, and dialkyl sulfate; epoxides used in several addition
reactions; hydrazine, a strongly reducing nitrogen base;
TEMPO, a cyclic amine oxide radical; aromatic amines, used
as building blocks; and boronic acids, used in carbon−carbon
coupling reactions. Other well-known classes of potentially
genotoxic impurities, aldehydes and aromatic nitro compounds,
which are mainly used as starting materials and not reactants,
are not included in this section.
3.1.1. Alkyl Halides. Methyl, ethyl, and propyl halides are
used widely as industrial alkylating agents. Although the
mechanism of their toxicity is still not fully understood, they
F DOI: 10.1021/cr300095f
have been shown to directly alkylate critical biologically active
macromolecules, such as proteins and DNA.45 Geminal, vicinal,
and ω-bifunctional alkyl halides are also directly used in API
synthesis, of which ω-alkyl dihalides are common linking agents
due to their ability to connect API intermediates via
consecutive alkylation. It was hypothesized that bifunctional
alkanes cause genotoxic damage by the glutathione-dependent
pathway and consequent formation of toxic methanethiol.46
Nitrogen and sulfur mustards (e.g., 2,2-dichlorodiethyl sulfide)
represent a special class of alkyl halides and have been used as
chemical weapons. They are potential alkylating agents and
their toxicity is attributed to cross-linking between DNA
strands.47 The source of alkyl halide in APIs streams can be
derived not only from direct use of alkyl halides but also from
side reactions between alcoholic solvents and hydrogen halides
or dequaternization of ammonium salts. It is worth mentioning
that alkyl halides, such as methyl or ethyl chloride, deriving
from low molecular weight alcohols, are volatile and readily
purged from the API during the drying process. On the other
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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 lamif iban. In the final synthetic step, the
tripeptide-like intermediate reacts with hydrogen sulfide,
GTI source Application API synthesis example
leading to the iminothiol addition intermediate, followed by
Dequaternization DMTMMa 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,
O-alkylation Alisiren, cerivastation, by ammonia, affording lamifiban (Scheme 4).56
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
Esterification Latanoprost
reactive intermediate with two good leaving groups (Scheme
Cycloalkylation Mazapertine,
aripiprazole 5).57
Use of hydrogen halides in Cyclization Capecitabine, The synthesis of guanidine-containing fibrinogen 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
a
agonist59 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
The synthesis of fexofenadine, an antihistaminic agent, pressure in cases where the patient has open-angle glaucoma or
involves the base-catalyzed C-methylation of 4-bromophenyla- ocular hypertension.61 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
derivative, as illustrated in Scheme 1.49 In a similar reaction, an halide as an impurity in the final product (Scheme 8).62
intermediate in the synthesis of the bis-acetonitrile aromatase Highly genotoxic nitrogen mustards are usually used for the
inhibitor anastrozole is submitted to exhaustive alkylation using formation of piperazine-type drug substances. The first
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
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Scheme 2. O-Alkylation of Isovanillin during the Synthesis of Aliskiren Using Genotoxic 1,3-Dibromopropane

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

Scheme 4. S-Alkylation with Methyl Iodide during the Last 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
applied in cyclization, decarboxylation, sulfonyl cleavage, N- cancers.65 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
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Scheme 10. Use of Nitrogen Mustard and a Dihaloalkane in
Piperazine Formation and N-Alkylation Reactions,
Respectively, During the Synthesis of Aripiprazole
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
A practical manufacturing route was developed for the
synthesis of the triazole heterocycle of sitagliptin, which is a
drug used to treat type 2 diabetes.67 Exposure of the amidine
intermediate to methanol−HCl gives the desired triazole in a
cyclization reaction, which can be directly isolated as its HCl
salt by filtration (Scheme 12).68
Alcoholic hydrogen chloride can also be used for ring
openings. Xemilof iban is used to treat cardiovascular disorders,
and during its synthesis, the treatment of a β-lactam
intermediate with ethanolic hydrogen chloride results in ring
opening to afford the ethyl β-alaninate derivative (Scheme
13).69
During the synthesis of antiangiogenic sunitinib, selective
hydrolysis of a tert-butyl ester is followed by decarboxylation,
which is accomplished by stirring the tetrasubstituted pyrrole
intermediate in HCl and ethanol, which may form genotoxic
ethyl chloride (Scheme 14).70
Bortezomib is an intravenously administered, first-in-class,
proteasome inhibitor.71 During its synthesis, the N-sulfinyl-α-
amino boronate ester intermediate is treated with HCl in
Scheme 12. Methanol−HCl-Assisted Cyclization May Result in Genotoxic Methyl Chloride during Sitagliptin Synthesis
I DOI: 10.1021/cr300095f
Review
Scheme 13. Ethanolic Hydrogen Chloride Is Used to Open a
β-Lactam during the Synthesis of Xemilofiban
Scheme 14. Ethanol−HCl-Assisted Decarboxylation May
Result in Genotoxic Ethyl Chloride during Sunitinib
Synthesis
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
of hospitalized patients with euvolemic and hypervolemic
hyponatremia.75 During the last synthetic step, ethanol−HCl is
used as a salt-forming agent to form the imidazobenzazepine
hydrochloride salt (Scheme 17).76 Therefore, once again the
potential for the presence of traces of genotoxic ethyl chloride
is high.
3.1.2. Dialkyl Sulfates. The most common dialkyl sulfates
used in the pharmaceutical industry are the methyl and ethyl
derivatives, the latter having been used as a chemical weapon.77
Being a strong methylating agent, dimethyl sulfate (DMS) is
used to introduce a methyl group to atoms featuring unshared
electron pairs, such as oxygen, nitrogen, carbon, sulfur,
phosphorus, and some metals. Compared with the alkyl
halide-type methylating agents, DMS is more favorable due
the higher reaction rate and lower possibility of byproduct
formation.78 Usually methylating with dimethyl sulfate requires
the presence of a base, either (i) to intensify the reactivity of
the reaction site (e.g., converting the phenolic hydroxyl group
of vanillin to sodium phenolate during the first step of
papaverine synthesis) or (ii) to neutralize the byproducts of the
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Scheme 15. Methanol−HCl-Assisted Cleavage of the N-Sulfinyl Group May Result in Genotoxic Methyl Chloride during
Bortezomid Synthesis
Scheme 16. 2-Propanol−HCl-Assisted N-Arylation and Salt
Formation May Result in Genotoxic Isopropyl Chloride
during Pazopanib Synthesis
Scheme 17. Salt Formation of Conivaptan with HCl in
Ethanol May Result in Genotoxic Ethyl Chloride Formation
reaction, monomethyl sulfate (MMS) and sulfuric acid, for
example, in the case of the methylation of aliphatic alcohols.
Usually only one of the methyl groups of DMS reacts in the
methylation reaction because the MMS formed is a much
weaker alkylating agent than the original DMS. Although for
research purposesin small-scale preparationsthere is little
necessity to use both methyl groups, in manufacturinglarge
scale productionin cases where the substrate is reactive
enough to be methylated by MMS (e.g., the sodium salt of
mercaptans), it is desirable to utilize both groups if possible. In
order to do so one can adjust the reaction conditions as
follows: (i) increase the reaction temperature and (ii) apply
anhydrous conditions and avoid excess base to suppress the
competing reactions with water and the hydroxide ion. To
obtain the best results, the base can be introduced to the
reaction mixture continuously in small portions as the reaction
proceeds but limiting this to the extent of the acid formed to
minimize the competing reaction with the hydroxide ion.
Nonaqueous solvent−base systems such as DMF/K2CO3 can
be used in certain cases. Most common applications of dialkyl
sulfates in the pharmaceutical industry are summarized in Table
4.
Dialkyl sulfates are used in the production of metoclopra-
mide,79 pralidoxime,80 metrizoic acid,81 and merf ruside,82 APIs
J DOI: 10.1021/cr300095f
Review
Table 4. Applications of Dialkyl Sulfates during API
Manufacturing
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
Parkinson’s disease83 and restless legs syndrome.84 Its
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-
assisted N-methylation of an N-heterocycle during the final
synthetic step of antiepileptic ralitoline (Scheme 19).86
Scheme 19. Methylation of a Pyrrolidine with Genotoxic
Dimethyl Sulfate during Ralitoline Synthesis
The peripherally acting μ-opioid antagonist alvimopan87 can
only cross the blood−brain barrier partially and does not have
the usual side effects of the opioid agonists, such as
constipation, while not losing the analgesic effect. During its
synthesis (Scheme 20), the treatment of the styrene derivative
with butyllithium and DMS leads to methylation at the 4-
position of the 1,2,3,4-tetrahydropyridine ring, since the
negative charge on the quaternary carbon atom in that position
is enhanced.88
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Scheme 20. Genotoxic Dimethyl Sulfate is Directly Used
during the Synthesis of Alvimopana
a
Note that the carbodiimide reagent forms a potentially genotoxic
dialkylurea.
Dialkyl sulfates are also used to introduce an alkyl group into
an aromatic ring; for instance, o-nitroaniline is methylated with
dimethyl sulfate during the synthesis89 of telmisartan, which is
an angiotensin II receptor antagonist (angiotensin receptor
blocker, ARB) used in the management of hypertension
(Scheme 21).90
S-Alkylation is demonstrated in the synthesis of a rofecoxib
intermediate. Rofecoxib is a nonsteroidal anti-inflammatory
drug (NSAID) marketed by Merck.91 Scheme 22 shows 1-(4-
mercaptophenyl)ethanone treatment with dimethyl sulfate in
the presence of sodium hydroxide to form 4-(methylthio)-
acetophenone.92
Cimetidine was the first blockbuster drug, invented by Nobel-
prize winner James Black.93 It is a histamine H2-receptor
antagonist that inhibits the production of acid in the stomach
and it has been shown to have antitumor effects.94 After the
reaction of carbon disulfide and cyanamide in the presence of a
base, dimethyl sulfate is added to the reaction mixture in order
to methylate both sulfur atoms in a consecutive reaction
(Scheme 23).95
Quaternization of tertiary amines is often carried out with
dimethyl sulfate,96 which is a preliminary step to an amine−
nitrile transformation. Clemastine is used as an antihistamine
and anticholinergic medicine with sedative effects.97 During its
synthesis dimethyl sulfate is used to form a quaternary amine,
which is a good leaving group and is displaced with a nitrile
group in the following step (Scheme 24).98
Another example is the quarternization of an aromatic amine
during the last synthetic step of neostigmine, a parasympathomi-
metic drug that acts as a reversible acetylcholinesterase
inhibitor.99 Dimethyl sulfate is used in the last step of the
API synthesis to form the quaternary ammonium salt,
neostigmine, as shown in Scheme 25.100
3.1.3. Epoxides. Epoxides are the simplest cyclic ethers,
featuring three ring atoms. Due to the large ring strain
associated with the three-membered ring, epoxides are highly
reactive molecules and thus are often used as reagents in API
Scheme 21. Use of Genotoxic Dimethyl Sulfate in the Synthesis of Telmisartan
K DOI: 10.1021/cr300095f
Review
Scheme 22. S-Methylation of a Rofecoxib Intermediate
manufacturing. They easily participate in epoxide-ring-opening
reactions with alcohols, amines, halides, organometallics,
cyanides, sulfides, aromatic compounds, and active methylene
groups. On the other hand, the high reactivity of these
compounds makes them genotoxic, as their two electrophilic
carbon atoms can react with the DNA nucleophilic centers,
giving alkylated products.14 Substituted epoxides, such as 2,3-
epoxypropanol (glycidol), 1-chloro-2,3-epoxypropane (epi-
chlorohydrin), or 1,2-epoxy-3-butene, are often used as building
blocks during the synthesis of APIs. They are often subjected to
epoxide-ring-opening reactions, and since they are bifunctional,
they usually act as linking agents or can form heterocycles.
These substituted epoxides react primarily at the less-
substituted and more-accessible carbon, due to steric hindrance.
However, in the case of substituents that increase the positive
charge at the adjacent carbon (such as aromatic or vinyl group),
both carbons could potentially react.101 A recent review by
Elder et al. provides guidance for analytical chemists faced by
the need to control such impurities at trace levels due to their
potential genotoxicity in drug products.102
An intermediate of the antiretroviral drug darunavir is
prepared using phenylmagnesium bromide and commercially
available 1,2-epoxy-3-butene in the presence of catalytic CuCN
to furnish the corresponding allylic alcohol (Scheme 26).103
Rivaroxaban is an oral anticoagulant drug invented and
manufactured by Bayer for the treatment of thromboembolic
diseases.104 A key intermediate in the synthesis of rivaroxaban is
(S)-2-(phthalimidomethyl)oxirane, of which alternatives are
presented in the literature for its synthesis: (a) condensation of
(S)-2,3-epoxy-1-propanol (glycidol) and phthalimide under
Mitsunobu reaction conditions105 and (b) condensation of (S)-
1-chloro-2,3-epoxypropane (epichlorohydrin) and phthalimide
in the presence of benzyltrimethylammonium chloride
(BTMAC) as phase-transfer catalyst (Scheme 27).106
Azelnidipine is a calcium channel antagonist that selectively
blocks voltage-dependent Ca2+ influx and is used for the
treatment of hypertension.107 The patented synthesis of
azelnidipine involves a heterocyclization reaction, in particular,
an azetidine formation by means of reacting benzhydrylamine
and epichlorohydrin without solvent to give 1-benzhydryl-3-
hydroxyazetidine in a slow process (Scheme 28).108
The structurally rather complex agent zosuquidar has shown
promising activity against multidrug resistance in cancer
chemotherapy.109 During its synthesis, 5-hydroxyquinoline
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Chemical Reviews
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
Means of Dimethyl Sulfate
Scheme 26. Use of 1,2-Epoxy-3-butene during the Synthesis
of Darunavir
Scheme 27. Condensation of (S)-2,3-Epoxypropanol
(glycidol) or (S)-1-Chloro-2,3-epoxypropane
(epichlorohydrin) and Phthalimide during the Synthesis of
Rivaroxaban
Scheme 28. Azetidine Formation with Epichlorohydrin
during the Preparation of Azelnidipine
reacts with the tosyl derivative of glycidol in a convergent
sequence, affording the epoxypropyl ether (Scheme 29).110
Lubazodone is an antidepressant compound belonging to the
class of serotonin-selective reuptake inhibitors.111 The reaction
of a fluoroindanol with the mesylate ester of (R)-glycidol in the
presence of base leads to the epoxypropyl ether with retention
of configuration (Scheme 30). Treatment of this intermediate
with aminoethylsulfonic acid forms the morpholine ring and
gives the enantiomerically pure final product lubazodone.112
3.1.4. Hydrazines. The toxicity of hydrazine and its
derivatives is ascribed to the generation of carbocations,
L DOI: 10.1021/cr300095f
Review
Scheme 29. Glycidol Tosylate in an Aromatic O-Alkylation
during the Synthesis of Zosuquidar
Scheme 30. Use of Chiral Glycidol Mesylate during the
Synthesis of Lubazodone
carbon-centered radicals, and oxygen-centered radicals, which
are considered to be highly reactive species. For these reactive
intermediates, DNA alkylation and other DNA lesions have
been reported.113 Recent reviews have been published on
hydrazine and its derivatives related to (i) the mechanisms of
chemical carcinogenicity by Benigni and Bossa14 and (ii) the
control and trace analysis in drug substances by Elder et al.114
Since hydrazine is a highly reactive base that acts as a reducing
agent, it has been used as a synthetic reagent in production of
several different types of drugs. Table 5 summarizes the
application of hydrazine and the synthetic examples discussed
in detail in this section.
Hydrazine is a green reducing agent, since only nitrogen gas
and water are produced as by-products. A typical example
where hydrazine is applied as a reducing agent is the Wolff−
Kishner reaction, where a carbonyl groupboth ketone- and
aldehyde-typeis transformed into a methylene or methyl
Table 5. Applications of Hydrazine in API Synthesis
Application API synthesis example
Wolff−Kishner reduction Sunitinib, ziprasidone
Hydrazide formation Sitagliptin, isoniazid
Hydrazinolysis Saquinavir, mofegiline
Electrophilic addition Suritozole
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 Such reaction is also used for a mild lysis of protection groups
hydrazide, as shown in Scheme 32,119 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
depression.120 Its synthesis involves the use of hydrazine in the liberation of the desired amine from the phthalyl residue. The
final 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
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Scheme 32. Hydrazide Formation during the Synthesis of (a)
Sitagliptin and (b) Isoniazid
disease.122 Part of the synthesis of saquinavir entails the
hydrazinolysis of the amido alcohol intermediate removing the
phthalimide protecting group to produce the primary amine,123
while the final step of mofegiline synthesis is also characterized
by the cleavage of the phthalimide protecting group with
hydrazine leading to the free base.124 The side product formed
from the hydrazine and the phthaloyl group is 2,3-
dihydrophthalazine-1,4-dione.
Suritozole is a benzodiazepine reverse agonist, investigated as
a potential treatment for Alzheimer’s disease.125 Its synthesis
(the triazolothione portion) starts with the formation of a
thiosemicarbazide by condensation of methylhydrazine with
methyl isothiocyanate in an electrophilic addition, as depicted
in Scheme 34.126
Hydrazine is a key bifunctional, with two NH2 groups,
building block used in the preparation of various heterocyclic
compounds via condensation reactions with a wide range of
bifunctional electrophiles, such as 1,3-diones or 3-halo ketones
or aldehydes, leading to pyrazoles, or imides, giving triazoles in
the Einhorn−Brunner reaction. The resulting N-heterocycles
are key intermediates in the synthetic routes to APIs. For
instance, the preparation of sildenaf il, which is generally known
as Viagra, a drug used for treatment of erectile disfunction,127
involves the hydrazine-assisted formation of a substituted
pyrazole ring, as shown in Scheme 35.128 A similar reaction
takes place during the synthesis of the topoisomerase inhibitor
sedoxantrone.129 One of the steps of sedoxantrone synthesis is
the condensation of a phenol intermediate with a substituted
hydrazine, which leads to pyrazole formation. Though the order
of the reaction steps has not been established, formation of the
hydrazone, then displacement of the adjacent chlorine by the
second nitrogen, and final closure of the pyrazole ring seems
plausible.130,131
3.1.5. TEMPO. 2,2,6,6-Tetramethylpiperidin-1-oxyl free
radical (trade name TEMPO) is a commonly employed
Scheme 33. Hydrazinolysis after Gabriel Synthesis during the Preparation of (a) Saquinavir and (b) Mofegiline
N DOI: 10.1021/cr300095f
Review
Scheme 34. Use of Methylhydrazine in an Electrophilic
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
throughout chemical- and biochemistry-related industries as a
stable nitroxyl radical. TEMPO is mainly used for oxidations of
alcohols to yield aldehydes and ketones or carboxylic acids. A
comprehensive review on the use of reactions mediated by
TEMPO can be found elsewhere.133 An example of the use of
TEMPO is the synthesis of a 5-HT2B receptor antagonist. Eli
Lilly synthesizes 2-cyclohexylacetaldehyde by oxidizing 2-
cyclohexylethanol by the Anelli−Montanari protocol (Scheme
36), affording the aldehyde, which is then used as precursor of a
tryptamine and further used as key synthon for the synthesis of
the important 5-HT2B receptor antagonist.134
Antagonists of the coreceptor CCR5 have been an intense
area of research within the HIV arena over the past decade.
Maraviroc is the first-in-class CCR5 antagonist for the
treatment of HIV.135 The initial synthesis of maraviroc that
produced material for preclinical studies has been pub-
lished.136,137 One of the reactions is catalyzed by TEMPO,
where the alcohol is oxidized to give the required aldehyde, as
shown in Scheme 37.138
Darunavir is an antiretroviral drug in the HIV-1 protease
inhibitor class for the treatment of multidrug-resistant HIV.139
One step in the synthesis of darunavir is a cyclization that
included a TEMPO oxidation, a NaBH4 reduction, and a lipase
resolution to provide optically active bis-THF derivative, as
illustrated in Scheme 38.140
Oseltamivir141 is a neuraminidase inhibitor and is the most
commonly prescribed drug for treatment to combat influenza.
The large number of synthetic approaches reported in the
literature implicates the importance of this drug. In the example
below, TEMPO is used with trichloroisocyanuric acid (TCCA)
in the oxidation of a secondary alcohol to give the
corresponding ketone intermediate (Scheme 39).142
An azabicyclooctanyl derivative was identified as another novel
and potent DPP-4 (dipeptidylpeptidase-4) inhibitor at Novartis
for treatment of type 2 diabetes. The preparation of its
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Chemical Reviews
Scheme 35. Pyrazole Formation with Hydrazine during the Preparation of (a) Sildenafil and (b) Sedoxantrone
Scheme 36. Synthesis of the 5-HT2B Receptor Antagonist
Intermediate by Means of TEMPO
Scheme 37. TEMPO Oxidation during the Synthesis of the
HIV Drug Maraviroc
azabicyclooctanyl intermediate involves the use of TEMPO in
an alcohol−ketone oxidation (Scheme 40).143
SB-462795144 is an azepanone-based inhibitor of the protease
cathepsin K, developed for the treatment of osteoarthritis and
osteoporosis.145 Scheme 41 shows the oxidation of a carbinol in
the final chemical stage of the synthesis by means of
TEMPO.146
LY686017 is a potent NK1-II inhibitor for the treatment of
depression, anxiety, and alcohol dependency.147,148 In its pilot-
plant synthesis, TEMPO is used as an oxidation agent with
NaOCl, where a secondary alcohol is efficiently oxidized using
the Anelli−Montanari protocol (Scheme 42).149
Scheme 38. TEMPO Oxidation Followed by Reduction and Cyclization in the Preparation of Darunavir
O DOI: 10.1021/cr300095f
Review
BYK405879 is a potassium-competitive acid blocker, a
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
3.1.6. Aromatic Amines. Although aromatic amines are
generally not inherently genotoxic, during metabolic activation,
electrophilic species are generated. The main transformation
pathway of aromatic amine metabolism is oxidation, producing
an N-hydroxy compound that is conjugated as an acetate,
sulfate, or glucuronide. Further deconjugation results in a
nitrenium ion (ArN+H), which is considered to be the active
genotoxin that binds to DNA.151
Aromatic amines are often present as starting material,
intermediate, or reagent in pharmaceutical synthesis. During
the synthesis of steroids such as mometasone f uroate, in order to
replace the 21-hydroxyl group with a chlorine, sulfonyl
chlorides are used in a 4-dimethylaminopyridine (DMAP)
base catalyzed sulfonylation reaction (Scheme 44).16 In order
to control this reaction, a design of experiments to assist in
trace analysis of DMAP in glucocorticoid matrices has recently
been reported in the literature.152 Besides the sulfonylation
reactions, DMAP is also used in acylations,153 esterifica-
tions,154,155 amino group protections with Boc,156,157 and
silylations.158
Diclofenac is widely used as a nonsteroidal anti-inflammatory
drug (NSAID). During its synthesis, the potentially genotoxic
2,6-dichloroaniline is used as a starting material.159 Cu-
catalyzed N-arylation with 2-chlorobenzoic acid takes place in
the presence of KOH (Scheme 45A). The reaction may leave
behind unreacted starting material, which has to be
controlled.160 In a similar manner, the potentially genotoxic
2,6-dimethylaniline is used for the synthesis of the local
anesthetic and antiarrhythmic drug lidocaine (Scheme 45B).161
2,6-Dimethylaniline is condensed with bromoacetic acid, as
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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
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Chemical Reviews Review

Scheme 46. Use of Potentially Genotoxic 4-Chloroaniline in
the Final Synthetic Step of Chlorhexidine
advantage of all is that the coupling reaction proceeds with high
regioselectivitynot affecting other functional groups in the
substrateand high stereoselectivity, giving mainly one isomer
of the desired product.
An example of Suzuki coupling of interest for the
pharmaceutical industry is the synthesis of garenoxacin, which
is a quinolone antibiotic for the treatment of Gram-positive and
-negative bacterial infections.167 As depicted in Scheme 47, in

Scheme 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

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palladium bis(triphenylphosphine) dichloride and sodium
carbonate.
Angiotensin II receptor antagonist losartan is used for the
treatment of hypertension.170 The synthetic step involving a
Suzuki coupling in the synthesis of losartan developed by
Merck research chemists is outlined in the following (Scheme
48).171,172 First, the trityl-protected phenyltetrazole was ortho-
Scheme 48. Suzuki Coupling during the Merck Process for
the Synthesis of Losartan
lithiated by means of butyllithium and then quenched with
triisopropyl borate, giving the boronic acid derivative after
treatment with aqueous ammonium chloride. The resulting
boronic acid participated in a Suzuki cross-coupling reaction
with the other key intermediate, an imidazole alcohol.
3.2. Genotoxic Compounds Formed in Side Reactions
Examples of the previous section refer to genotoxic impurities
introduced as reactants; however, genotoxic impurities can be
formed as side products during API synthesis. A class that has
drawn the most attention and that has been the most
extensively studied when compared to other GTI classes is
the sulfonate esters. This class of molecules is usually formed in
side reactions with alcohols, and therefore, awareness for their
presence in API synthesis could be, initially, not so
straightforward. However, as a result of the work of several
groups, in particular the efforts of the Working Group within
the Product Quality Research Institute (PQRI), the industrial
and academic community is today fully aware of this
challenge.173 Therefore, taking into account the specific
properties and widespread use of the alkyl sulfate acids as
counterions in, but not exclusively, API salt formation in the
presence of alcohols, special concern has been raised by the
regulatory authorities. Several documents from regulatory
authorities174−177 have specifically outlined controls to be
taken. This has motivated an intensive effort to develop control
and removal strategies and to understand their mechanism and
kinetics of formation, and several industrial examples have been
published. This section is organized into a subsection providing
an exhaustive overview on the formation of sulfonate esters and
their uses along with two additional subsections providing
examples according to the use of sulfonate esters and their
precursors in stoichiometric or catalytic amounts. The use of
such compounds in stoichiometric amounts include their
function as/in (a) API salt forming agents, (b) good leaving
groups, (c) cyclization agents, (d) protecting agents, (e)
Mitsunobu rearrangement; (f) sulfonamide formation, and (g)
aids for the resolution of isomers. Sulfonate esters and their
precursors can be used in catalytic amounts also in (a)
cyclizations, (b) protecting group manipulations, (c) double
bond migration, (d) enamine−amine reduction, and (e)
R DOI: 10.1021/cr300095f
Review
esterification. Notice that sulfonate esters are not the only
potentially genotoxic side products; therefore, two additional
small sections provide further examples on the formation of
alkyl halides and acetamide.
3.2.1. Sulfonate Esters and Their Precursors. Over-
view. Sulfonate esters are alkylating agents, a class of
potentially genotoxic compounds.178 They are called alkylating
agents due to their ability per se, or after metabolic activation,
of adding alkyl residues to the reactive nucleophile sites of the
DNA bases. They cover a wide range of chemical structures
from the simplest alkyl sulfonates to more complex structures
featuring aromatic systems with various functional groups.
Actually, this class of genotoxic impurities has drawn a high
level of attention; the awareness for their presence is not
straightforward, as sulfonate esters are most often side
products, and formed many times with the solvent used in
the reaction or even on cleanup procedures. A historically
significant case of the presence of sulfonate esters in a final API
formulation is the case of viracept, described below. The
precursors of sulfonate esters are alkyl and aryl sulfonic acids
and the corresponding halides and anhydrides (Scheme 49).
Scheme 49. Formation of Genotoxic Sulfonate Esters from
the Corresponding Acids, Halides, and Anhydrides with
Alcohols
Since halides and anhydrides of sulfonic acids are alkylating
agents, they are also considered genotoxic. Note that in many
API syntheses it is, in some circumstances, challenging to
substitute alcohols as solvents because of their ability to
solubilize both the API and API salts. Examples of most
common sulfonate esters and their precursors are summarized
in Table 6.
Due to their synthetic versatility, sulfonate derivatives are
common and useful reagents in the pharmaceutical industry,
especially in reactions where carbonium ion initiation is needed.
Examples of such compounds are mesylates (methanesulfo-
nate), triflates (trifluoromethanesulfonate), tosylates (p-tolue-
nesulfonate), nosylate (4-nitrobenzenesulfonate), and besylates
Table 6. Common Sulfonate Ester Impurities and Their
Precursors
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(benzenesulfonate). Sulfonate ester impurities may be present 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
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.180
synthesis, there is a possibility of the formation of genotoxic overviewed the utility, safety, and regulation of APIs formulated
sulfonate esters. In particular, reactions containing sulfonic as sulfonic acid salts. For example, methanesulfonic acid is used
acids, sulfonic halides, or sulfonic anhydrides where an alcohol in the production of viracept181 and delavirdine,182 while p-
is also present, even if only in residual amounts, have the toluenesulfonic acid (TsOH) is used in the production of
potential to yield sulfonate esters. Teasdale et al. pointed out bretylium.183 The final manufacturing step of denagliptin, an
that sulfonate esters decompose through alcoholysis to generate API developed to treat diabetes mellitus, is forming a tosylate
sulfonic acid and an ether, and this reaction, in conjunction salt in ethanol, as illustrated in Scheme 50.184
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 between alcohols and sulfonic acids only and not sulfonyl
working in the field 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
Hydroxyl−sulfur Tixocortol pivalate immunodeficiency virus (HIV).187 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
transformation
Cromitril lation in viracept took place in the final manufacturing step. In
Amide−nitrile Denagliptin this step, the API salt nelfinavir 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
Protecting group Dinoprost, tolterodine
following ethanol cleaning. Additionally, although in negligible
Protecting group Oseltamivir, denagliptin,
quantities, EtMs was also identified in some batches of MsOH.
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
Formation with TsOH in Ethanol
Scheme 51. Final Manufacturing Step of Viracept Drug
Substance Nelfinavir Mesylate
viracept with elevated levels; therefore, it was concluded that
such patients are at no increased risk for carcinogenicity or
teratogenicity over their background risk.189
Therefore, the conclusions from the viracept case study
points out that GTI contamination of an API can result from a
wide range of difficult to anticipate sources: cleaning
procedures, pipelines and holding tanks (where reagents may
be held for a lengthened period of time), problems with pH
adjustment, charging speed of a chemical to the reaction
mixture, raw material supply, drying procedures, prolonged
reaction time, elevated temperatures, and the introduction of
the genotoxic reagent during production of the API (more
significant in the final steps).
T DOI: 10.1021/cr300095f
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3.2.2.2. Good Leaving Groups. Genotoxic sulfonate esters
can be produced by the reaction of sulfonyl chloride with
alcohols. Sulfonyl chlorides are used to produce alkyl sulfonates
with the aim to provide much better leaving groups than the
corresponding alcohol, providing that the rate and yield of the
reactions follow SN1 or SN2 mechanisms. Sulfonylation is
carried out in the presence of a base, traditionally pyridine due
to its high effectiveness: it forms a complex with the sulfonyl
halide to favor the attack by the alcohol on the sulfur atom.
Pyridine has an alerting structure and is considered a potential
genotoxin, thus alternative base catalysis of the sulfonylation is
being developed.191
Azaloxan is an antidepressant drug patented by 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
a bimolecular nucleophilic substitution (SN2) displacement
reaction193 transforming a hydroxyl group into an amine group.
In a similar fashion, etherification of a hydroxyl group is carried
out (Scheme 53) using mesyl chloride in the synthesis194 of
Scheme 53. Using Mesyl Chloride during the Synthesis of
Betaxolol
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Chart 4. Highlights of SN2 Reactions with the Example of a Sulfonate Leaving Group

betaxolol, a β(1)-selective adrenergic antagonist used in the 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 f luvoxamine.197 In the latter case, the
terminal hydroxyl in the final step of the API synthesis is
converted to a good leaving group by reaction with mesyl
chloride, which is then converted to the terminal primary
amine, fluvoxamine, by any of several methods, such as
displacement with ammonia, as depicted in Scheme 54.

Scheme 54. Mesyl Chloride-Assisted Hydroxyl−Amine

Transformation at the Last Manufacturing Step of
Fluvoxamine

tory drug substance tixocortol pivalate has similar properties to

Mometasone, clobetasone, and halobetasol are glucocorticoids
belonging to a class of steroid hormones that bind to the
glucocorticoid receptor. They play a key role in regulating the
feedback mechanism of the immune system during inflamma-
tion by turning the immune activity down.198 During the
synthesis of these steroids, mesyl chloride is used in a
methanesulfonylation catalyzed by a base, DMAP, and the
mesylate group is consequently replaced by chlorine (Scheme
55).16,199 In other steroids, the 21-hydroxyl group is not
converted to chlorine but to a sulfur-linked residue to achieve
modified therapeutic activity. For example, the anti-inflamma-
U DOI: 10.1021/cr300095f
hydrocortisone200 and is also used in patch testing in atopic
dermatitis.201 During the final manufacturing steps, mesyl
chloride is used to form a good leaving group. Displacement of
the mesyloxy group with the anion from thiopivalic acid affords
thioester-type API (Scheme 56), in the presence of a base,
trimethylamine (Et3N).202 A similar hydroxyl−sulfur trans-
formation takes place during the synthesis of microtubule
inhibitor erbulozole.203
Sulfonyl halides are also useful tools to transform amines to
nitriles. For instance, one synthesis of the antiasthmatic agent
cromitril204 concludes in the classic manner by converting an
ester to a carboxamide by ammonolysis, and dehydrating the
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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,
followed by addition of sodium azide selectively across this
functionality, produces the final API (Scheme 57)205 The β-
Scheme 57. Amide Transformation to Nitrile with TsCl/Py
during the Synthesis of Cromitril
lactamase-resistant carboxypenicillin drug temocillin is used for
the treatment of multiresistant Gram-negative bacterial
infections.206 During the synthesis, isocyanate−amine trans-
formation takes place, where benzyl 6-β-isocyano-6-α-methyl-
thiopenicillanate is treated with p-toluenesulfonic acid, giving
one of the key intermediates to temocillin (Scheme 58).207
3.2.2.3. Cyclizations. Cyclization reactions where a sulfonate
ester functions as a leaving group are common, as in the
following examples. Oxazoline cyclization can be achieved by
means of mesyl chloride, for instance, during the synthesis of
ifetroban, which is a selective thromboxane receptor antago-
nist.208 First, the hydroxyl group is converted to a good living
group by reaction with mesyl chloride. When this intermediate
is treated with base, the mesylate is displaced by the enolate
from the adjacent amide, giving the corresponding oxazoline
(Scheme 59).209
Napitane is used as an antidepressant drug, and during the
last synthetic step, 2 equiv of methanesulfonic acid is
released.210 A diol is converted to a bidentate bis-mesylate
with mesyl chloride followed by reaction of the required amine
to give the pyrrolidine moiety of the final API (Scheme 60).
During the large-scale synthesis of saquinavir, an anti-HIV
protease inhibitor, a mesyloxy group is used to form an oxirane
derivative in two steps. The secondary alcohol of a 1,2-diol is
selectively converted to the methanesulfonate ester with mesyl
chloride. Thereafter, strong base is used to produce an alkoxide
at the primary alcohol, which then displaces the mesylate ion,
giving an oxirane derivative.211 As depicted in Scheme 61, the
Scheme 58. Isocyanate Transformation to Amine with TsOH during the Synthesis of Temocillin
V DOI: 10.1021/cr300095f
Review
configuration at the former secondary alcohol is inverted as a
consequence of the SN2 nature of the ring closure.
Sulfonyl halides are also used in lactone formation. Orlistat is
a drug designed to treat obesity,212 the synthesis of which
involves treatment of the β-hydroxycarboxylic acid intermediate
with benzenesulfonyl chloride, resulting in the formation of the
butyrolactone ring present in the final API (Scheme 62).213
A rare but useful application of sulfonyl halides in API
synthesis is in the formation of aziridines. In the synthesis of
the opioid analgesic214 spiradoline, tosyl chloride initially
converts the hydroxyl group to a chloride, followed by
displacement of the halogen by the adjacent amine to form
an aziridinium salt, as depicted in Scheme 63.215
The synthesis of oseltamivir, an antiviral drug,216 involves the
formation of two aziridines in consecutive reaction steps by
means of mesyl chloride. As depicted in Scheme 64, the
secondary alcohol is first converted to the corresponding
mesylate by means of mesyl chloride in the presence of
triethylamine. The amine produced by reduction of the azide
group in the second step with triphenylphosphine proceeds
with a nucleophilic attack on the adjacent carbon, displacing the
mesyloxy group in a nucleophilic substitution to provide the
first aziridine. Due to the large angle strain of the three-
membered heterocyclic system, ring opening of aziridine with
sodium azide in the presence of ammonium chloride occurs
easily. After deprotection of the MOM ether by acidic
hydrolysis, the amino group is reacted with trityl chloride, the
hydroxyl group is then transformed into a good leaving group
by means of mesyl chloride in the presence of triethylamine,
and in a one-pot process, a new aziridine-type intermediate is
produced.217 The use of methanol as a solvent may lead to the
formation of genotoxic sulfonate esters under certain
conditions. Furthermore, the HCl/MeOH mixture used for
the hydrolysis of the MOM ether may lead to the presence of
genotoxic methyl chloride in the API.
3.2.2.4. Protecting Groups. Scheme 65 includes a synthetic
step in the production tolterodine,218 an API used to manage
urinary incontinence, showing the protection of a phenol group
by the formation of a tosylate followed by the formation of the
good leaving group nosylate, which is then easily displaced by
diisopropylamine. Note that this synthesis provides an example
involving three GTIs and one carcinogenic impurity of different
chemical classes, sulfonate esters, alkyl halides, and acetamides.
3.2.2.5. Sulfonamide Formation. Sulfonamides provide part
of the structural basis of several drugs (Chart 5). Originally,
sulfonamides were used as synthetic antimicrobial agents, but
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Scheme 59. Oxazoline Heterocyclization with MsCl during Ifetroban Synthesis
Scheme 60. Formation of a Bidentate Mesylate, Which
Eventually Gives Rise to the Liberation of 2 equiv of
Methanesulfonic Acid in the Final Synthetic Step of
Napitane
Scheme 61. Formation of Mesyloxy Group as a Precursor of
an Oxirane Derivative during the Synthesis of Saquinavir
Scheme 62. Sulfonyl Halide Assisted Lactone Formation
during the Synthesis of Orlistat
Scheme 63. Tosyl Chloride-Assisted Formation of an
Aziridin during the Synthesis of Spiradoline
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Review
Scheme 64. Consecutive Aziridine Formation by Means of
Mesyl Chloride in Oseltamivir Synthesis
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
now there are novel drug families based on the original
antibacterial sulfonamides, such as diuretics, anticonvulsants,
and dermatologicals. The preparation of these APIs requires the
use of sulfonyl halides. For example, the antiarrhythmic
dofetilide is a bis-methanesulfonamide that is synthesized
using mesyl chloride (Scheme 66).219
3.2.2.6. Chiral Auxiliary Group in Resolution of Enan-
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
the Bis-methanesulfonamide Dofetilide
Scheme 67. Resolution of Racemic Omeprazole with
Camphorsulfonyl Chloride To Obtain Enantiomerically
Pure Esomeprazole
chloride and camphorsulfonic acid with the alcohol solvent can
lead to the formation of potentially genotoxic sulfonate esters.
3.2.3. Sulfonate Esters and Their Precursors Used in
Catalytic Amounts. 3.2.3.1. Cyclizations. A pharmaceutical
example of a cyclization where a sulfonic acid is applied as
catalyst is the TsOH-assisted cyclodehydration of a diol to
afford the chroman framework during the synthesis process of
the hypoglycemic agent englitazone (Scheme 68).222
3.2.3.2. Protecting Group Manipulations. The formation of
tetrahydropyranyl (THP) ethers from alcohols and phenols is a
X DOI: 10.1021/cr300095f
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Scheme 68. TsOH-Assisted Cyclodehydration Leads to a
Chroman Derivative in Englitazone Synthesis
useful way of protecting the hydroxyl group due to its stability
toward a variety of harsh reaction conditions, such as strong
bases, organometallic reagents, hydrides, and acylating and
alkylation reagents. THP protecting groups are formed by the
reaction of the hydroxyl compounds with dihydropyran under
acidic conditions, for example, in the presence of 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
during incineration causes problems, alternative conditions are
being developed for large-scale Boc removal. Most of these
apply TsOH to produce acidic conditions.225 During the
synthesis of ABT-594, a potent, orally effective analgesic, TsOH
is used both to remove a Boc-protecting group from an amine
and as a salt-forming agent to obtain a stable API (Scheme 70).
Neither HCl nor TFA can be used for deprotection in this
particular case, as they gave a significant amount of dimer
byproduct, which proved difficult to remove.226 Since the
reaction and salt formation take place in ethanol and this is the
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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 of ABT-594
final step of the synthetic route, the probability that the API
contains genotoxic ethyl p-toluenesulfonate is high.
An alternative synthetic route to oseltamivir uses TsOH in
methanol to remove an acetonide protecting group of a diol
intermediate. There is potential to form methyl p-toluenesul-
fonate in this reaction (Scheme 71).227
Scheme 71. Acetonide Protecting Group Removal by Means
of TsOH in Methanol
3.2.3.3. Mitsunobu Rearrangement. Fosinopril, an inhibitor
of angiotensin converting enzyme (ACE), is widely used for the
treatment of hypertension, as well as in various types of chronic
heart failure.228 Fosinopril manufacturing is another example of
the use of sulfonic acids in the pharmaceutical industry, since
MsOH is used as a reagent in a Mitsunobu rearrangement, as
depicted in Scheme 72,229 wherein mesylation with inversion of
configuration was accomplished by employing methanesulfonic
acid, triphenylphosphine, diisopropyl azodicarboxylate, and
triethylamine. A highly stereospecific Friedel−Crafts alkylation
mediated by aluminum trichloride installed a 4-phenyl
substituent with complete inversion. This shows an example
how sulfonic acids are used in O−C transformation. Mitsunobu
reactions are discussed in more details in Chart 6.
3.2.3.4. Double Bond Migration. Etonogestrel is a steroid
used in hormonal contraceptives, and its synthesis involves a
double bond migration assisted by p-toluenesulfonic acid.
Scheme 73 illustrates the treatment of an intermediate in the
Scheme 72. Specific Mitsunobu Rearrangement with MsOH for an O−C Transformation during the Manufacturing of
Fosinopril
Y DOI: 10.1021/cr300095f
Review
synthesis with TsOH in acetic acid, which causes the double
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
acid, resulting in the reduction of the enamine to a primary
amine (Scheme 74).232
3.2.3.6. Esterification. Sulfonic acids are also used to catalyze
esterifications. Fosinopril is an angiotensin converting enzyme
(ACE) inhibitor. Its synthesis involves a manufacturing step
where a carboxylic acid is esterified by methanol in the presence
of TsOH (Scheme 75).233 Although the TsOH is used as a
catalyst, the quantities used are generally quite high and may
result in formation of considerable amounts of the 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
(DMTMM), which is an efficient coupling agent in a wide
range of organic reactions,234,235 such as esterification,236,237
glycosidation238 and phosphonylation239 in the synthesis of
antibiotics, 240 peptides, 241 and alkaloids. 242 However,
DMTMM is unstable in organic solvents and reacts with itself,
yielding 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-morpholine and
genotoxic methyl chloride (Scheme 76).
3.2.5. Acetamide. Acetamide is a known carcinogen; thus,
the awareness of its formation in API manufacturing is
crucial.243 Although acetamide is not a genotoxin, it is
sometimes referred to as such in the literature11 Acetamide is
not commonly used directly in the synthesis of APIs, but its
derivatives, such as 2- and N-bromoacetamide or trifluoroace-
tamide, are often used as building blocks in drug synthesis.
These derivatives initially contain acetamide as an impurity, but
also the 2- and N-derivatives have potential to form acetamide,
depending on the reaction conditions. Another source for
formation of carcinogenic acetamide is the hydrolysis of the
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Chart 6. Mitsunobu Reaction and Its Use In API Synthesis
Scheme 73. TsOH-Assisted Double Bond Migration during the Synthesis of Etonogestrel
Scheme 74. Methanesulfonic Acid-Assisted Reduction of an
Enamine during the Synthesis of Ritonavir
Scheme 75. Esterification of Fosinopril Intermediate
Assisted by TsOH
Scheme 76. Formation of Genotoxic Methyl Chloride from
Coupling Agent DMTMM
widely used solvent acetonitrile under acidic or basic conditions
at elevated temperature. Acetonitrile is not only used as a
solvent in the pharmaceutical industry but also as a reagent in
API synthesis. For instance, it is directly used as a reagent in the
synthesis244 of corontin, which is a drug used for treatment of
angina pectoris (Scheme 77).245
Zaurategrast is a drug that reached phase II clinical
development and was indicated for treatment of multiple
sclerosis.246 Since the final synthetic step of the process takes
place in acetonitrile under acidic conditions, the risk of
acetamide formation was identified during the early develop-
Z DOI: 10.1021/cr300095f
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Scheme 77. Acetamide May Form from Acetonitrile during
the Synthesis of Corontin
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
acetamide by applying adequate chemical process design: the
implementation of a workup sequence involving aqueous
washes, followed by salt formation and crystallization, was
proven to be successful.247
Sodelglitazar is an antidiabetic drug for treatment of type 2
diabetes. During one of the synthetic steps S-alkylation takes
place under acidic conditions in acetonitrile, hence, the
potential for formation of acetamide (Scheme 79).248
During the synthesis of the antiviral drug oseltamivir, a diene
intermediate is converted to the bromodiamide derivative using
a novel SnBr4-catalyzed bromoacetamidation with N-bromoa-
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Scheme 79. S-Alkylation in Acetonitrile under Acidic
Conditions May Lead to the Formation of Carcinogenic
Acetamide during the Synthesis of Sodelglitazar
cetamide in acetonitrile (Scheme 80).249 In this reaction, both
the reagent and the solvent can form carcinogenic acetamide.
Scheme 80. Reagent N-Bromoacetamide and Solvent
Acetonitrile May Lead to Acetamide Formation
The reagent 2-bromoacetamide is used during the synthesis
of armodaf inil, which is used for the treatment of narcolepsy
and sleeping disorders.250 2-Bromoacetamide, which may
contain acetamide, is used in an S-alkylation in the presence
of NaOH (Scheme 81).251
Scheme 81. Use of 2-Bromoacetamide May Result in the
Presence of Acetamide in Armodafinil Synthesis
3.3. Genotoxicity and Carcinogenicity of Common Organic
Solvents
Organic solvents are ubiquitously present in pharmaceutical
production processes as reaction and purification media (e.g.,
extraction), separation phases (e.g., chromatographic mobile
phases), and also for cleaning of the equipment. The
pharmaceutical industry consumes the largest amount of
organic solvents in relation to the final product gained.252
According to the Q3C guideline, solvents are divided into four
groups.253 Classes 1 and 2 are considered “toxic” solvents, as
summarized in Table 8. The first group (class 1) contains
known human carcinogens, compounds strongly suspected of
being human carcinogens, and those presenting environmental
hazards. These solvents should be avoided, unless strongly
justified. The limits for class 1 solvents are listed as absolute
parts per million in a material under testing (drug or excipient).
Class 2 solvents presented in Table 8 ought to be limited,
because they are nongenotoxic animal carcinogens or associated
with irreversible toxicity, such as teratogenicity. Class 3
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solvents, which include solvents such as ethanol and acetone,
have permissible daily exposures of 50 mg, or up to 5000 ppm
(0.5%) when it is assumed that 10 g is administered daily.
There is no solvent recognized as being hazardous to human
health at the average acceptable levels in pharmaceuticals in this
group. They are less toxic in acute or short-term studies and
have given negative results in genotoxicity studies. There is also
an additional group, class 4 solvents. No toxicological data
exists for this group, which would allow for the formulation of
acceptable limits. When a manufacturer wishes to use class 4
solvents, a justification for the level of the solvent in the
pharmaceutical product has to be submitted to the regulatory
authorities. Note that most of the guidelines only address
products on the market and not compounds under clinical
trials. However, the Q7A guideline has a specific chapter
dedicated to APIs used in clinical trials.254
Under the conditions of a 2-year gavage study, there was
clear evidence of the carcinogenicity of benzene, which used to
be a widely used solvent in the industry.256 Benzene was mainly
replaced by toluene to carry out the same reactions that require
nonreactive aromatic solvents. The genotoxicity of toluene is
under investigation, although it is still widely used as a
solvent.257 Although chlorobenzene showed positive results in
some in vitro and in vivo genotoxicity studies, it showed
negative results in the majority of the studies on “in vitro” gene
mutation, chromosomal aberration, DNA damage, and UDS
and in vivo SCE. From overall evaluation of these results,
chlorobenzene is considered not to be genotoxic.258 Although
the experimental group that was exposed to dimethylformamide
(DMF) showed an increase in the incidences of chromosomal
aberration,259 negative results were obtained in the majority of
the in vitro and in vivo genotoxicity studies; thus, the overall
evaluation of these data indicates that DMF is not genotoxic
(categorized as group 3, i.e., not classifiable as to its
carcinogenicity to humans by the IARC).260 Classification of
dioxane (group 2B carcinogen by IARC) indicates that it is
possibly carcinogenic to humans, since it is a known animal
carcinogen.261 Dichloromethane (DCM) may be carcinogenic, as
it has been linked to cancer of the lungs, liver, and pancreas in
laboratory animals.262 Hydrolysis of the widely used solvent
acetonitrile under acidic or basic conditions at elevated
temperature can lead to the formation of acetamide, which is
a nongenotoxic carcinogen.14
4. APPROACHES FOR GTI MITIGATION IN THE
PHARMACEUTICAL INDUSTRY
As illustrated in the previous section, the synthesis of
pharmaceutical products often involves the use of highly
reactive reagents for the production of APIs or their
intermediates.263 Low levels of such reagents or corresponding
side products may therefore be present in the final API or drug
product as impurities. As briefly described in section 2, such
chemically reactive impurities may have unwanted toxicities,
including genotoxicity and carcinogenicity, and hence can have
a severe impact on the product risk assessment.264 In some
cases, these sources can be avoided. However, in many cases
the presence of GTIs in postreaction streams during API
synthesis is difficult to avoid. To overcome this, R&D scientists
have to identify GTIs early on during process development,
develop analytical methods, and implement synthetic processes
to control and contain them. GTIs can be successfully reduced
below the limits set by regulatory authorities either with
carefully optimized synthetic approaches (preventive approach,
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Table 8. Classification of Solvents by the Q3C Guideline and Their Propertiesa

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
a
Data are from ref 255.

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

hence preferred) or by implementing purification strategies as a
last resort.
4.1. Chemical Synthetic Approaches
In this, the first strategy to mitigate GTIs in the production of
APIs, R&D chemists avoid the use and generation of GTIs
throughout the synthetic route, searching for different chemical
sequences to reach the same API or intermediate or by
optimizing the existing synthetic route.265 In very particular
cases, this strategy can be achieved without significant
reduction of yield. Examples of redesigning the synthetic
process specifically to avoid GTIs can be found in section 4.
AB
However, in many cases, the use of reagents and intermediates
that are reactive and synthetically useful, which in turn likely
makes them interact with DNA, are often unavoidable. It may
not be practical to change the synthetic steps during
development to control or reduce GTIs, particularly when
the process has reached the stage of being scaled up. Therefore,
a second strategy to achieve GTI-free drug products is based on
prevention, focusing on elimination or reduction of the
concentration of GTI during the critical synthetic step. This
can be achieved by altering appropriate reaction conditions,
such as (i) proportions of reaction components, (ii)
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interchanging the order of addition of the reactants, and (iii)
changing the quality or method of preparation of key starting
materials. Furthermore, a quality by design (QbD) approach
can contribute to better control of GTIs.266
4.1.1. Altering the Synthesis. Three synthetic examples
are given in which the chemical synthesis was changed to avoid
the formation of a sulfonate ester. Two additional examples
include side reactions with the potential to form genotoxic
impurities vinyl bromide and acetamide.
The syntheses of zaurategrast sulfate and sodelglitazar were
previously mentioned in section 3.2.5 due to the potential
formation of acetamide. However, there is a second source of
GTI: sulfonate esters.247 In the case of zaurategrast sulfate, the
use of methanesulfonic acid in the presence of ethanol posed
the potential risk of generating genotoxic ethyl mesylate. This
was mitigated by replacing the sulfonic acid with hydrochloride
acid without affecting the yield.247 In the initial synthetic route
of sodelglitazar (Scheme 82a), a genotoxic mesylate inter-
mediate is used; therefore, the commercial application employs
the corresponding alcohol instead of this mesylate ester
(Scheme 82b) for the formation of the thioether linkage.248
Denagliptin was previously mentioned in section 3.2.2,
illustrating API salt formation with sulfonic acids. However, in a
previous step, a (S)-difluorophenyl amino acid is reacted with a
fluoro amino amide mediated by n-propanephosphonic acid
cyclic anhydride (T3P) and diisopropylethylamine (DIPEA).184
The next step involved dehydrating with p-toluenesulfonic
anhydride with pyridine as base at 50 °C. Since p-
toluenesulfonic anhydride was not available commercially, the
scaled up reaction relied on the use of methanesulfonic
anhydride (Scheme 83). In this case, the potential to form
Scheme 83. Alternative Route for Large-Scale Synthesis of
Denagliptin Tosylate, Avoiding the Formation of a
Potentially Genotoxic Mesylate Ester
mesylate esters, which are potential genotoxins (as are the tosyl
esters had the tosyl anhydride been used), was not desirable.
The observation that partial dehydrating had occurred during
the coupling reaction gave rise to the exploration of T3P as a
dehydrating agent. A second equivalent of T3P, along with a
higher temperature of 78 °C, gave satisfactory results (Scheme
83).184
The synthesis of the anti-inflammatory agent UK-371,104267
includes a glycosidation reaction of the adenosine key
intermediate with a peracetylated sugar, 1,2,3,5-tetra-O-acetyl-
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β-D-ribofuranose (Scheme 84). This stereoselective coupling
was initially mediated by N,O-bis(trimethylsilyl)acetamide and
triflic acid. Since the workup leads to formation of
stoichiometric amounts of genotoxic acetamide, N,O-bis-
(trimethylsilyl)acetamide was replaced by trimethylsilyl triflate
as the coupling reagent.
In the synthesis of Zeneca Pharmaceuticals’ ZD-2079268 for
the treatment of noninsulin dependent diabetes, 1,2-dibromo-
ethane is used to alkylate 4-hydroxylphenylacetamide (Scheme
85a). A side reaction leads to the formation of genotoxic vinyl
bromide. In the scaled up reaction, the N-alkylethyl group was
introduced via an oxathiazolidine S-oxide, obtained by reaction
of N-benzylethanolamine with thionyl chloride, which on
reaction with 4-hydroxylphenylacetamide gave the desired
intermediate (Scheme 85b). Note that in this particular case,
the main motivation to modify the synthetic step was safety
rather than API purity. As a bonus, the new route gave a 64%
yield while that for the dibromoethane-based route was 9%.
4.1.2. Adjusting Reaction Conditions To Mitigate GTI
Formation. The feasibility of minimizing the formation of
genotoxic impurities by simple adjustment of parameters such
as reaction time, pH, temperature, and solvent matrix is
demonstrated through the following examples.
4.1.2.1. Sulfonate Esters. The synthesis of the AstraZeneca
drug for management of type 2 diabetes, tesaglitazar, includes a
step where a potentially genotoxic bismesylate ester is added in
excess to the phenolic key intermediate to form an ether269 at
pH 10 and 100 °C for 4−5 h while using PEG-400 as phase-
transfer catalyst in the presence of sodium carbonate (Scheme
86). Adjusting the pH to 7 and increasing the reflux time to 8−
9 h allow complete reaction and hydrolysis of the alkyl
sulfonate ester without hydrolysis of carboxylate ester in the
API. Such an approach, which takes advantage of different
reactivities, can be applied in the elimination of other genotoxic
sulfonate esters used in excess.
Note, however, that the strategy employed in Scheme 86 is
based on the use of a genotoxic sulfonate ester. Other studies
have focused on routes where sulfonate esters are avoided and
have been recently summarized by Elder et al.173
In the following paragraphs, the effect of pH, temperature
and water content on the formation of sulfonate esters will be
discussed.
(i) pH: The elucidation of the mechanism of sulfonate ester
formation using labeled 18O179 revealed that the formation of
these species from the corresponding sulfonic acid and alcohol
involves the protonation of the alcohol under acidic conditions.
It was concluded that even a slight molar excess of a base
prevents sulfonate esters formation. Therefore, avoidance of
acidic conditions or even addition of a base is recommended to
mitigate sulfonate ester formation.
(ii) Temperature: It was observed that lower temperatures
significantly reduce the rate of formation of sulfonate esters.
Reduction of the reaction temperature from 40 to 10 °C
showed a significant 4-fold reduction of sulfonate esters
formation even without the addition of a base.179 Therefore,
conducting both the reaction and workup at lower temper-
atures is recommended.
(iii) Water: The presence of water, as it competes with
alcohol for protonation and promotes ester hydrolysis, has a
positive effect on reducing sulfonate esters formation, and even
a small amount of water results in a 3-fold decrease of sulfonate
esters without addition of base.185
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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-
Oxidea

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

Scheme 86. Synthesis of Tesaglitazar, with a Change in pH
for the Effective Hydrolysis of the Sulfonate Ester Precursor
369,003-26, a candidate for treatment of benign prostatic
hyperplasia, benzenesulfonic acid was used as salt forming
agent.270 In this reaction potentially genotoxic ethyl besylate
(EtBS) was formed because of the reaction between
benzenesulfonic acid and API ethoxy side chain (Scheme 87).
4.1.2.2. Halides. As discussed in the previous paragraphs,
sulfonic acids can form potentially genotoxic sulfonate esters,
when used as API salt formation agents in alcoholic solutions.
Similarly, halide acids (e.g., HCl), used as salt-forming agents,
can form alkyl halides (e.g., MeCl and EtCl) by reaction with
alcohol solvents (Scheme 88). Examples of the occurrence of

Scheme 87. Formation of Potentially Genotoxic Ethyl

Besylate (EtBS) through Salt Formationa

(iv) Addition conditions: Minimizing residential time of

sulfonic acids in alcoholic solutions, as well as minimizing the
excess of sulfonic acid, is crucial to avoid the formation of
sulfonate esters. Vigorous stirring and slow addition of the acid
to the API solution allow for effective salt formation, avoiding a
localized excess of acid, which can give rise to sulfate ester
formation. Prolonged storage times of solutions containing
both sulfonic acid and alcohol mixed should be avoided.173
Sulfonate ester impurities typically arise from the reaction of
the respective acids with an alcohol which is usually present as a
a
solvent. However, in particular cases other causes can be Here 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.

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Scheme 88. Competitive Formation of API Salt and Alkyl
Halides in the Reaction of Halide Acids with an API Base in
Alcoholic Solvents
alkyl halides in the synthesis of APIs are provided in Sections
3.1.1 and 3.2.4. The effect of reaction conditions on the
mitigation and elimination of alkyl halides has been investigated
using a quaternary amine as API model in the formation of the
HCl salt.48 Drying of the product at 85 °C under vacuum failed
to significantly decrease the alkyl halide content. Decreasing the
rate of addition of HCl and increasing stirring times did not
have a significant impact when applied alone. A reduction of the
HCl load did decrease alkyl halide formation but resulted in
lower yield of the salt. The reaction temperature proved to be
crucial in managing the levels of alkyl halide. At 35 °C the
formation of alkyl halides was favored, whereas a lower
temperature of 10 °C proved to be an efficient strategy to
mitigate formation of the genotoxin. When tested at larger
scales a yield of 92% and GTI formation below 1 ppm, in
compliance with TTC limits, was achieved.
Another example, in which both temperature and reaction
time were adjusted to mitigate GTI formation, was reported by
AstraZeneca during the use of a Vilsmeyer chlorination reaction
in a penultimate step of API synthesis (Scheme 89). This
Scheme 89. Adjustment of Temperature and Reaction Time
in a Chlorination Step Resulted in a Reduction in Formation
of Genotoxic Dimethylcarbamoyl Chloride (DMCC)
particular reaction comprises simultaneous in situ formation of
an amine hydrochloride salt.271 The reaction of the N,N-
dimethylformamide and a chlorinating agent, POCl3, resulting
Scheme 90. A Nitroaromatic Catalytic Reduction Step in the Synthesis of an Adrenoreceptor Antagonist Candidate
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in in situ formation of amine hydrochloride salts, also yields the
genotoxic dimethylcarbamoyl chloride (DMCC). A hydrolysis
study of DMCC concluded that elevated temperature (80 °C)
and shorter reaction time decrease the amount of DMCC from
87 to 0.9 ppm in the reaction mixture while a yield of as high as
90% of product was maintained.
4.1.2.3. Nitro Aromatics. Scheme 9 describes a sequence
that involves the catalytic reduction of a nitroaromatic group to
an aniline derivative. A second example of such nitroaromatic
reduction can be found in the synthesis of an adrenoreceptor
antagonist, as represented in Scheme 90.272 The nitroaromatic
reduction takes place through a hydrogenation catalyzed by Pd/
C, while workup involves removal of the catalyst by filtration,
concentration, and crystallization.
4.1.3. Quality by Design. The use of the QbD approach
has been suggested to develop synthetic routes or selection of
conditions for API synthesis and can also be applied to control
GTI formation below threshold values. In the pharmaceutic
context, QbD aims to design and produce API formulations for
which the final quality should be ensured a priori through the
design of synthetic routes and the manufacture process.
Generically, QbD includes four stages: (i) definition of the
quality profile to be targeted; (ii) product and manufacture
process design to achieve such quality; (iii) identification and
selection of quality attributes, process parameters, and sources
of variability; and (iv) control mechanisms to ensure quality
over time. In the particular case of GTI risk control, the target
for product quality requires one to maintain GTI below
threshold numbers, while providing high API yields. The
examples in section 4.1 provide cases of design of chemical
synthesis that avoided the presence of GTI, and the following
section 4.2 is focused on selection of parameters able to
decrease the amounts of GTI present, in other words, give
information that can be used in QbD stages 2 and 3 defined
above (Figure 6). Quality by testing (QbT) is the main
approach supported by regulatory agencies, which had resulted
in an extremely robust effort to develop analytical tools and
intensive screening for GTIs in raw material, intermediates, and
APIs.
The optimization of the process shown in Scheme 91 and
described in section 4.1.2.3 employed QbD in order to
minimize the presence of potential GTIs, namely, nitroso
compounds and hydroxylamine.273 The potential genotoxicity
of the compounds involved in the synthesis were first assessed
using in silico approaches, such as DEREK, and toxicology data.
Potential GTIs can be formed in the reduction of nitro-
aromatics to aniline derivatives, as illustrated in Scheme 91. The
four compounds raised structural alerts according to DEREK,
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Figure 6. Quality-by-design strategy for prevention of GTI formation.
Scheme 91. Nitroaromatic Catalytic Reduction (a) Showing
Reaction Intermediates That Can Remain in the Product as
Impurities (b)
but the nitroaromatic and aniline intermediates showed
negative genotoxicity by the AMES test. The study by Looker
et al.273 was focused on the optimization of reaction conditions
to avoid the presence of these impurities and comply with the
established specific thresholds. In particular, the process
employed Pd/C catalyst for the hydrogenation of nitro-
aromatics. The purification steps consisted of filtration for the
removal of Pd, followed by concentration of the resultant
filtrate, addition of an antisolvent for recrystallization, and
filtration/drying of the solid obtained. Solutions spiked with
potential GTIs were used to monitor and assess the
effectiveness of impurity purging at different stages of the
purification units. The process parameters selected for
optimization include temperature, amount of catalyst, and
Scheme 92. Key Stages of the Commercial Synthetic Route to a Fluoroaryl-Amine Mesylate Central Nervous System Agent
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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
amount of alcohol used in the various steps of stage 8 has no
significant impact on the amount of GTIs formed; the drying
operation does not generate any detectable GTIs, and the
isolation effectively removes any GTI. Overall, the process
understanding gained through QbD led to a robust control
strategy with negligible levels of GTI, allowing testing of the
final drug substance to be omitted.
4.2. API Purification
4.2.1. Purge Factors. As discussed in the previous section,
the presence of GTIs can be, in many cases, avoided through
novel designs of chemical routes or mitigated by control of
reaction conditions. Additionally, it should be noted that during
API synthesis, purification units are already in place at several
steps. In spite of the fact that these steps are often not designed
specifically to reduce GTIs, they have the ability to remove
GTIs along with other impurities. Hence, there are several
routes by which a given GTI can be eliminated during the
synthesis. Previous works2 addressed the issue of purging,
defining risk considering the number of synthetic steps between
the appearance of GTI and the final production step. It was
recommended that in cases where the presence of GTI is more
than four steps away from the final synthetic step, chemical
rational should be used to decide whether GTI specific impurity
removal is required or not. However, such an empirical
approach is not process specific. Therefore, Teasdale et al.39
developed a semiquantitative “assessment purge tool” focusing
on the particular GTIs of concern and chemical properties of a
given process in order to evaluate the risk of a GTI to be
present in the final API. The proposed tool defined the
following main purge factors: GTI’s reactivity, solubility (in the
solvents used, e.g, for recrystallization, where the GTI is
discharged with mother liquid), volatility (e.g., through GTI
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removed with solvent during distillation for solvent exchange),
and ionizability (e.g., for a partition of GTI and API between
aqueous/organic, for example, for pH adjustments to change
the ionized/un-ionized state of one of the compounds) and
processes used for purification (e.g., chromatography). This
tool used a score scale for each purge factor, as described in
Table 9, where purge factor is defined as the ratio of GTI
concentration before and after purging.
Table 9. Example of Key Parameters in Purge Factors in the
Tool by Teasdale et al.a
Physicochemical parameters Purge factor
Reactivity High reactivity = 100
Moderately reactivity = 10
Low/no reactivity = 1
Solubility Freely soluble = 10
Moderately soluble = 3
Sparingly soluble = 1
Volatility Boiling point >20 °C below that of the
reaction/prcess solvent = 10
Boiling point ±10 °C that of the reaction/
prcess solvent = 3
Boiling point >20 °C above that of the
reaction/prcess solvent = 1
Ionisability Ionization potential of GTI significantly
different
Physical processes (e.g., Chromatographically, GTI elutes prior to the
chromatography) desired product = 10
Chromatographically, GTI elutes after the
desired product = 10
Others processes are valuated on an individual
basis
a
Adapted with permission from ref 39. Copyright 2013 American
Chemical Society.
They also describe six case studies where each of the different
existing purge factors was evaluated and their contribution
assessed at different stages in the removal of GTIs. Such cases
include the removal of thionyl chloride (two syntheses);
nitropyridyl N-oxide (a starting material); and AZD9056
aldehyde and its respective byproduct AZD9056 chloride,
together with the side products isopropyl chloride, methyl
hydrazine, and hydrazine. When genotoxins are introduced as
reactants, their reactivity is one of the main factors contributing
to how they are purged as they are consumed in the chemical
reaction. Consequently, the use of a genotoxic reactant in
excess is, if possible, to be avoided. Note that some of these
highly reactive compounds can also be eliminated by reaction
with bases, acids, or even water in subsequent steps, as is the
case for thionyl chloride in the examples provided. Volatility is
an obvious route for the removal of low boiling point
compounds, such as methyl hydrazine (88−90 °C), thionyl
chloride (79 °C), and isopropyl chloride (36 °C) through
distillation and drying. Solubility takes an important role in
purging such compounds, in particular when crystallization or
extraction operations are involved where GTIs can be dissolved
either in mother liquors or the discarded phase.
AZD9056 HCl/chloride case study clearly illustrates the use
of this assessment purge tool (Scheme 93).39,274 In final steps
of the API synthesis, the potentially genotoxic AZD9056
aldehyde reacted with 3-aminopropano-1-ol to produce an
imide derivative that is subsequentiatly reduced to a free base
(step 1), followed by HCl salt formation (step 2) and finally
recrystallization (step 3). During the purification step, HCl is
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added in isopropyl alcohol, which can form a second potentially
genotoxic ingredient, namely, isopropyl chloride. Moreover, the
HCl can react with the AZD9056 base, resulting in small
amounts of AZD9056 chloride as a byproduct, a third potential
GTI.
The assessment purge tool was applied to these three
potential GTIs and the predictive values were compared with
experimental results, pointing out that the tool usually
underestimates purging effects. The purging of the three
potential GTIs were assessed by considering the following:
(i) The main driver for purging AZD9056 aldehyde is its
high reactivity, which leads to this compound’s full con-
sumption, and thus it was scored as 100% in the first step. Since
this compound is not volatile, a score of 1 was allocated to all
the steps. A moderate solubility was considering in the last two
steps; however, a larger removal of the AZD9056 aldehyde was
experimentally measured in step 2, leading to an overall
underprediction of the purging capacity of the process by 10
times.
(ii) Isopropyl chloride is present in steps 2 and 3, and as
defined by the established criteria for high solubility and
volatility, a score of 10 for purge factors was allocated to these
two parameters. The tool predicts the purging capacity of
10 000, again representing an underprediction of about 4 times,
indicating that in spite of the relatively high formation of
isopropyl chloride, its presence in the final product is highly
improbable.
(iii) AZD9056 chloride byproduct is actually not reactive,
not volatile, and not particularly soluble in isopropyl alcohol.
Therefore, an overall low purging factor of 3 was predicted
against a measured value of 10, implying that action should be
taken to either remove this compound or change the process to
eliminate or mitigate the formation of this compound.
4.2.2. Separation Technologies. For the specific removal
of GTIs, the selection of the purification method is intrinsically
dependent on the physicochemical properties of the GTI,
which will decide the relative “purge” factors. From a process
chemistry point of view, it is also important to understand
which separation operation units are involved in API
purification. In this review, seven examples of conventional
purification techniques and three emergent techniques are
referred to (Chart 7). Usually the higher the selectivity of a
purification process regarding a specific impurity, the lower the
API loss and the higher the removal efficiency of the impurity
in question. In many cases, delivering a safe API requires the
application of a purification strategy where the GTI is reduced
to acceptably low levels. To illustrate this, a specification of 70
ppb, calculated using the daily dose, was set by a
pharmaceutical company for an especially potent genotoxin in
a drug candidate (shown in eqs 1−4).275
GTI removal efficiency = GTIend /GTIstart (1)
APIloss = APIend purification step /API fed purification step (2)
GTIcontent = GTIend purification step/APIend purification step (3)
GTIdaily intake = APIdaily dose per patient kg × GTIcontent
× weight patient (4)
Such ultralow levels in the specifications of APIs pose
additional analytical and processing difficulties for efficient
purification. The design of a synthetic process to produce an
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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
purification steps. These conventional purification steps are
already in place and already contribute to GTI removal,
although not specifically designed to remove GTIs. The
difference between point-of-source and end-of-pipe GTI
removal is schematically illustrated in Figure 7.
The removal of larger quantities of impurities can be usually
achieved by increasing the number of cycles within a given
purification step (e.g., the number of re-extractions, recrystal-
lizations). However, increasing the number of cycles also leads
to undesirably high API losses and may have diminishing
efficiency with each new cycle. Consider, for example, an API
stream with a GTI content of 1 g of GTI for each 100 g of API,
and a theoretical purification operation in which for each step
80% of the GTI is constantly removed, along with the sacrifice
of 3% of the API. To reduce the GTI from a concentration of
1g/L in solution (corresponding to an API concentration of
100 g/L) to 64 μg/L would require six cycles and a cumulative
API loss of 17% in the purification alone. Therefore, the use of
conventional purification procedures to reach ultralow GTI
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content through an increase in the number of cycles would lead
to unacceptable API losses.
The use of an additional “end-of-pipe” GTI purification
could complement the already existing intercalated purification
steps. Nevertheless, the removal efficiencies are usually
concentration-dependent, decreasing with lower GTI concen-
trations. In such cases, it may be advantageous to follow a
“point-of-source” GTI detoxification strategy. For implementa-
tion of this strategy, identification and mapping of the reactions
where GTIs are present is crucial, and lessons taken from
section 2 should be considered.
Conventional purification steps during and after API
synthesis include crystallization, precipitation, solvent extrac-
tion, silica gel or alumina column chromatography, and
treatment with activated carbon and resins, as well as
distillations. As in any separation, the efficiency of the
separation depends of the differences in chemical and physical
properties of the two entities to be separate and/or their
relative affinities for a selective agent. In this review, we briefly
report 10 different purification techniques, of which 7 can be
perceived as conventional methodologies to remove impurities
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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
(Chart 7).
4.2.2.1. Crystallization. (1) Crystallization is one of the most
important isolation and purification process for APIs. The API
is isolated as a solid phase while the impurities remain dissolved
in the liquid phase (the mother liquors). Crystallization is also
broadly used in chiral separations, namely, through diastereo-
meric resolutions.276,277 In some cases, a two-solvent system, a
solvent and a cosolvent, can be used to promote crystal
formation in accordance with the respective phase diagrams.
Robustness, kinetics, temperature, and pH of the crystallization
system are also important parameters.278,279 Crystallization is a
purification process that not only determines the purity and
residual solvent content of the API but also establishes the
crystalline properties in terms of polymorphic form, crystal
habit, bulk density, and size distribution, all of which affect
downstream processing, e.g., drying and formulation.280,281
More importantly, the crystalline properties and polymorphic
forms can be responsible for drug bioavailability. Therefore,
once a route is approved for API production, the crystallization
step of the final API is usually retained. In some instances,
depending on process optimization, a significant fraction, up to
30% of the API, can remain in the mother liquors282 or be lost
through washes of the solids. Impurities may be washed out
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along with the API or remain as part of the crystal lattice,
depending on the efficiency of the washing procedure.
Filtration is the normal technique used to isolate the crystalline
solids. A particular example is illustrated in Chart 8, where
acetamide is removed in a process that incorporates
crystallization.247
4.2.2.2. Solvent liquid−liquid extraction. (2) Solvent
liquid−liquid extraction is commonly used for API purification;
API (or impurities) can be selectively transformed into salts
and retained in an aqueous phase while the organic impurities
(or API) are removed by a water immiscible organic solvent
phase. The organic salt can then be converted to the neutral
species by acidification or basification, according to the pKa of
the API, and re-extracted into a second organic solvent, which
is usually concentrated before isolation of the API. The
efficiency of separation depends on the relative partition
coefficients of API and GTI in the different solvents. Panel i of
Figure 8 illustrates a purification process involving solvent
phase exchanges and crystallization of the API, while panel ii
maps the corresponding losses of API.
4.2.2.3. Precipitation. (3) Precipitation is commonly
promoted by addition of a nonsolvent to a solution of the
API (or vice versa). Similarly to crystallization, the impurities
remain in the liquid and the API ends-up as a solid phase.
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Chart 8. Example of a Conventional Process for API Purification from the Carcinogenic Acetamide
However, the solid may be amorphous and not crystalline, but
once more, the solvent system (final mixture of solvent and
nonsolvent) selected should show higher solubility for the
impurities than for the API. Solute solubility is, among other
things, dependent on its polarity and the polarity of the solvent.
Note that some of these polar solvents also have high boiling
points and are potentially genotoxic themselves (Table 8);
therefore, if they are not removed properly, they present an
additional risk as a GTI in the API . Filtration is also used to
separate liquid from solid, or distillation is used to evaporate
low boiling point solvents. When the impurity is preferentially
precipitated, it can be removed by filtration.
4.2.2.4. Fractional distillation. (4) Fractional distillation can
be used to purify volatile APIs.283 However, distillation is also
broadly used for removal of solvents and for solvent exchanges,
particularly when switching from a low boiling point solvent to
a higher boiling point solvent (see Table 8). Solvent exchanges
from high boiling point solvent to lower boiling point solvents
or when thermosensitive compounds are involved can be
sustainably achieved using organic solvent nanofiltration
(OSN).282 Volatile organic impurities, mainly resulting from
residual solvents, 284,285 can also be removed through
distillation. Many of the GTIs considered in this review have
low volatility (e.g., hydrazine, MsCl, TsCl, DMS, 1,2-epoxy-3-
butene, acetamide, phenylboronic acid all have boiling points
above 100 °C), and alkyl halides such as MeCl and EtCl have
boiling points of −24.2 and 12.3 °C, respectively.
4.2.2.5. Adsorption processes. (5 and 6) Adsorbents such as
granular activated carbon (GAC)286 and resins287 are broadly
used to remove color and impurities.288 Adsorption-based
separations rely on the different affinities of the disparate
compounds for the adsorbent. Therefore, a high affinity of the
GTI combined with lower binding of the API is desirable in this
case. Screening of the different commercially available
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absorbents for metal impurities has been evaluated using
microtubes.289,290 Other studies include removing formalde-
hyde using activated carbon containing amine groups291 and
removal of an aldehyde impurity using polystyrene-based
sulfonylhydrazine resin.292 GTIs such as p-toluenesulfonic
acid methyl (MeTs), ethyl (EtTs), and isopropyl (i-PrTs)
esters have also been evaluated using different commercially
available nucleophilic resins.287 These studies used methyl,
ethyl, and isopropyl esters of methanesulfonic, benzenesulfonic,
and p-toluenesulfonic acids as model PGIs and screened the use
of several amines, thiol, thiophenol, piperazine, and piperidine
immobilized on silica and polystyrene. Removal was effective
for methyl sulfate esters, whereas it proved to be more of a
challenge to remove ethyl and isopropyl esters by this
technique. This strategy was applied for the removal of MeTs
from a 21-chlorodiflorasone solution. When trisamine was
immobilized either on silica or macroporous polystyrene−
divinylbenzene supports, 100% GTI removal was achieved.293
These adsorbents and resins can be used as stationary phases in
chromatography.
4.2.2.6. Column chromatography. (7) Column chromatog-
raphy is a typical postreaction technique applied in organic
chemical synthesis to remove impurities. Sophisticated sta-
tionary phases are applied in the pharmaceutical industry, for
example, in chiral separations.294,295 However, this review is
focused on the removal of GTIs, and for this endeavor,
preparative column chromatography using standard silica gel296
or alumina of pharmaceutical grade as stationary phase has been
used. In this technique a solvent, such as ethyl acetate, ether,
acetone, methylene chloride, and/or mixtures thereof, is used as
eluent. Commercially available absorbents, such as polystyrenic
or methacrylic matrices, with aqueous solutions at different pHs
and ionic strengths have also been reported.297 Particle size,
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Figure 8. (a) API purification by several cycles of phase exchanges and
recrystallization and (b) the corresponding yields for each cycle.
column dimensions, and eluent flow and pressure are critical
parameters.
4.2.2.7. Supercritical extraction. (8) Supercritical extraction
techniques utilize the properties of supercritical fluids, which
have the high solvation power of a liquid and the enhanced
diffusivity of a gas. Moreover, simply changing from the
supercritical state to a gaseous state provides a straightforward
method to isolate the solute. The relatively low critical point of
CO2 (71 bar, 31 °C) had positioned it as an ideal supercritical
solvent that can replace more hazardous solvents as reaction
media and in extractions in chemical processes.298 Supercritical
CO2 can be used to produce particles with controlled size and
purity299 and also be used in packed column chromatog-
raphy.300 Therefore, this purification technology has the
potential to provide an effective and clean route for GTI
removal from APIs.
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4.2.2.8. Organic solvent nanofiltration. (9) Organic solvent
nanof iltration (OSN) relies on separations based mainly on
differences in molecule size, although other properties such as
shape and polarity can also contribute.301 The use of OSN had
been previously suggested for the purification of APIs302 and
has recently been evaluated specifically for the removal of GTIs
from API streams.303−305 The performance of this technique is
highly dependent on the membrane selected and on the
respective rejection curve. There are several commercially
available polymeric and ceramic membranes that are stable in
organic solvents. Examples include Koch SelRO membranes,
the StarMem series developed by W. R. Grace & Co., the
DuraMem series from Evonik MET, SolSep membranes, 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 9. Role of OSN in API purification.
scheme is shown in Figure 10. The most crucial limitation of
OSN in API purification is the low product yield due to
insufficient rejection of the product.305 To overcome this
limitation, Kim et al. recently proposed a two-stage membrane
cascade.304 Through an API purification case study, the authors
demonstrated that the proposed process significantly increases
the API yield without compromising its purity. The second
main drawback of OSN for API detoxification was the
significant solvent consumption during diafiltration processes.
However, OSN has markedly evolved in recent years, and the
newest generations of solvent-resistant membranes can fully
reject small molecules at the lower end of the nanofiltration
range (50−2000 g·mol−1) and subsequently can be used for in
situ solvent recovery.306
4.2.2.9. Molecular imprinting technology. (10) Molecular
imprinted polymers (MIPs) are prepared by incorporating the
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Figure 10. OSN-based API purification, with potential use of organic
reverse osmosis for solvent recycling.
target molecule into a polymeric matrix as template (Figure
11). The target molecule is therafter removed, leaving a
potential binding site within the matrix. Thus, the final polymer
structure usually provides enhanced affinity for removal of the
molecules used as template. The use of MIPs for separations in
the pharmaceutical industry has been suggested previously307
and used in bio- and pharmaceutical analysis.308 Exploring the
high specificity achieved by MIPs, several studies have evaluated
their use in chiral separations.309−311 Specific development and
characterization of a MIP for potential GTI removal were
recently reported.312,313
By exploring the ability of OSN to remove potential GTIs
when at high concentrations and combining this with the better
performance of MIPs to remove the target molecule at lower
concentrations, a hybrid process using these two purification
techniques also had been suggested.314 Possible limitations of
the use of MIPs for GTI removal are as follows: specific MIPs
need to be developed for individual (or similar) GTIs; removal
is more effective at lower concentrations, and hence, high
volumes are involved; and contamination is possible via
leaching of impurities derived from the polymer. Hence, this
technique has not become widespread at this time. Besides,
hybrid processes imprinting and nanofiltration technologies
have been combined in a molecularly imprinted organic solvent
nanofiltration strategy.315
MIPs often show cross-reactivity, which can be exploited in
the rapid screening of MIP libraries to identify suitable
Figure 11. Schematic of the molecular imprinting technique. Functional monomers, template, and cross-linker are allowed to self-assemble in
solution, and subsequent polymerization yields the imprinted material. The template is extracted from the polymer, leaving a binding site with
complementary topography and chemical functionality behind. The resulting MIP can selectively recognize the template molecule in complex
mixtures. Reprinted with permission from ref 311. Copyright 2015 Americal Chemical Society.
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imprinting systems for given applications. Kecili et al.
developed a protocol for the rapid identification of MIPs for
genotoxic aminopyridine removal from piroxicam and 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
compromises safety, since it can become a vehicle for increasing
genotoxic risk.
The quantity of genotoxic impurities in drug products is
strictly controlled by regulatory authorities that have set limits
to ensure patient safety. To ensure compliance with the
required low GTI concentrations, a significant effort during
development is necessary. Three main strategies that contribute
to producing APIs of acceptable quality can be identified:
(1) At the most rigorous level, the strategy outlined in the
regulatory authority’s guidelines is to avoid the use of any
genotoxic chemical over the entire synthetic route, regardless of
whether the genotoxic chemical is used as a reagent, starting
compound, catalyst, or solvent. However, given the chemical
nature and desirable reactive properties of chemicals, in many
cases the direct use of a genotoxic chemical or a GTI precursor
is unavoidable.
(2) Even when genotoxic compounds are not applied
directly, they can be formed during chemical reactions.
Therefore, a second strategy for minimization of genotoxin
formation can be achieved by thorough investigation of the
particular reaction and adjusting the reaction parameters with
assiduous care, in particular using QSAR strategies.
(3) An alternative and complementary strategy is to design
specific purification strategies targeting the removal of GTIs
from the API once it has been established that they or their
precursors are present. Either “point-of-source” or “end-of-
pipe” strategies can be used.
The full cycle of drug development, approval, and use has to
work for the different stakeholders. This means safety and
efficacy for patients, compensation for the efforts of the
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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
and as an IAESTE fellow at the University of Tokyo (Tokyo, Japan.
(2) Develop analytical and monitoring techniques for ultalow
He was a visiting researcher at Biotage MIP Technologies AB in
levels of GTIs.
Sweden. He was a postdoctoral research associate at Imperial College
(3) Develop new synthetic or process routes. Specifically, 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
Chemistry, The Institution of Engineering and Technology, and the
(5) Develop novel API purification techniques for removal of
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
ACS Publications website at DOI: 10.1021/cr300095f.
AUTHOR INFORMATION
Corresponding Authors
*G.S. e-mail: gyorgy.szekely@manchester.ac.uk.
*F.C.F. e-mail: frederico.ferreira@ist.utl.pt, frederico_castelo@
yahoo.com.
*W.H. e-mail: bheggie@hovione.com.
Notes
The authors declare no competing financial interest.
Miriam C. Amores de Sousa graduated with a degree in applied
chemistry (minor in biotechnology) in 2007 and concluded her
Master’s degree in biotechnology in 2009, at Faculdade de Ciências e
Tecnologia of Universidade Nova de Lisboa. She is currently a Ph.D.
student at the Department of Bioengineering at Instituto Superior
Técnico, Universidade de Lisboa, where she is a member of the
BioEngineering Research Group at the Institute for Bioengineering
and Biosciences. Previously, as a research assistant, she worked on the
characterization of polysaccharides and explored biocompatible
cellulose acetate membranes as potential drug delivery systems,
focusing on the solid-state mobility properties of the materials.
Currently, her research is focused on studying the interaction between
electrospun functional nanofiber matrices and stem cells, to evaluate

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the cell fateself-renewal and differentiationfor future applications
in tissue engineering, as stem cells provide an interesting model to
probe the cytotoxic effects of different compounds and materials.
Frederico Castelo Ferreira graduated with a degree in applied
chemistry (minor in biotechnology) in 1999 from New University
of Lisbon (UNL) and received his Ph.D. in chemical engineering from
Imperial College London in 2004 and his MBA from UNL in 2008. He
was a research associate (2004−2006) in a joint project of Imperial
College London and GlaxoSmithKline (GSK). He was a visiting
researcher (July 2007) at Institut Européen des Membranes
(Montpellier, France) and a visiting scholar (Sept-Dec 2009) at the
Massachusetts Institute of Technology, Deshpande Center for
Technological Innovation. Since March 2009, he has been a member
of the BioEngineering Research Group at the Institute for
Bioengineering and Biosciences. He teaches at the Department of
Bioengineering at Instituto Superior Técnico, Universidade de Lisboa,
including the course “BioteamsTeams for Innovation” for Ph.D.
students, and he launched two new elective M.Sc. courses: “Green
Technologies and Strategic Management” and “Entrepreneurship in
Bioengineering”. He also assists CoHiTec on translation of technology
to the market. His current research interests balance between
fundamental and applied research, for the development of new
processes, reactors, and materials, with an emphasis on membrane-
based systems.
Marco Gil graduated with a degree in chemical engineering from
Technical University of Lisbon, from which he obtained his Ph.D. in
chemistry in 2006. The main focus of his thesis was the development
of active ingredients for iron chelation therapy with improved in vivo
behavior. In 2007, he joined the R&D department of Hovione as a
scientist in the Particle Design Group. The focus of his work was the
application of particle engineering technologies to improve bioavail-
ability of poorly water soluble drugs and the scale-up of processes for
the production of solid dispersions by spray-drying. Later on, his
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interests were broadened to the development of novel particle size
reduction technologies aiming to produce engineered particles for
inhalation. Currently, he heads the Process Chemistry Development
Group, which is responsible for the development and scale-up of
chemical processes for the production of active pharmaceutical
ingredients.
William Heggie received his Ph.D. in organic chemistry from The
University of Manchester and held postdoctoral positions at Harvard,
St. Andrews, and Oxford Universities. He held a teaching position at
the New University of Lisbon from 1974 to 1976 and was a professor
at Lisbon Superior Institute of Engineering from 1979 to 1998. He
joined Hovione in 1980 and held various positions in Hovione’s R&D
group before becoming Chief Scientific Officer in 2004, being
responsible for innovation and introducing new technologies into
the company. He is the author of more than 20 patents and scientific
articles.
ACKNOWLEDGMENTS
The authors acknowledge the support of NEMOPUR (New
Molecular Purification Technology for Pharmaceutical Produc-
tion), a Marie Curie Initial Training Network within the
seventh Framework Programme of the European Commission’s
Marie Curie Initiative, and the support of FCT (Fundaçaõ para
a Ciência and Tecnologia) through the funding initiatives
PTDC/QEQ-PRS/2757/2012, SFRH/BD/73560/2010, and
IF/00442/2012. The authors would like to express their
gratitude to the reviewers for their valuable comments that
shaped the review. Many thanks go to Dr. Jozsef Kupai for
useful discussions about organic chemistry.
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