Synthetic Opportunities and Challenges for Macrocyclic Kinase
Inhibitors
Jennifer Alisa Amrhein, Stefan Knapp,* and Thomas Hanke*
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ABSTRACT: Macrocycles are typically cyclic variants of inhib￾itors derived from uncyclized canonical molecules or from natural
products. For medicinal chemistry, drug-like macrocycles have
received increasing interest over the past few years, since it has
been demonstrated that macrocyclization can favorably alter the
biological and physiochemical properties as well as selectivity in
comparison to the acyclic analogue. Recent drug approvals such as
Lorlatinib, glecaprevir, or voxilaprevir underline the clinical
relevance of drug-like macrocycles. However, the synthesis of
drug-like macrocycles can be challenging, since the ring-closing
reaction is generally challenging with yields depending on the size
and geometry of the bridging linker. Nevertheless, macrocycles are
one opportunity to expand the synthetic toolbox for medicinal
chemistry to provide bioactive molecules. Therefore, we reviewed the past literature of drug-like macrocycles highlighting reactions
that have been successfully used for the macrocyclization. We classified the cyclization reactions by their type, ring-size, yield, and
macrocyclization efficiency index.
■ INTRODUCTION
In recent years, macrocycles have received increasing attention
in drug discovery. The reason for this growing interest is based
on several approvals of drug candidates as well as data
suggesting that macrocyclization can change the biological and
physiochemical properties in comparison to the acyclic
counterparts.1,2 In general, one central focus of medicinal
chemistry is to improve the potency of a lead toward its target
of interest while also optimizing the selectivity of the
compound to closely related off-targets. It has been
demonstrated that both challenges can be addressed by
macrocylization, which makes this strategy attractive for the
drug discovery process. In addition, macrocycles offer an
opportunity for chemical novelty over the existing scaffolds.
For example, over 90% of the FDA-approved kinase inhibitors
are conventional type I/II inhibitors, which means they all
share often highly similar hinge-binding moieties. After more
than three decades of kinase drug discovery, the chemical
space for ATP-mimetic moieties is well explored, and
establishing new hinge-binding moieties can be a significant
challenge. Thus, macrocyclization has the potential to
contribute to a new chemical space for the design of novel
type I/II kinase inhibitors.3 The utility of macrocycles as
bioactive compounds or even as drugs was first limited to
natural products, such as rapamycin (1) and its closely related
rapalog derivatives, which are currently used as immunosup￾pressants or in the case of temsirolimus (2) as an oncology
drug. Besides these examples, macrolide antibiotics were
discovered almost 70 years ago with the first representative,
erythromycin, still in use for the treatment of a variety of
different bacterial infections. However, these first naturally
derived macrocycles discourage the medicinal chemistry
community from including macrocycles in a drug discovery
process, since they are usually very big molecules and often
violate the Lipinski rule of five.4 In addition, many macrocycles
based on natural products include stereocenters, which makes
the total synthesis a formidable challenge and slows down SAR
exploitation.5
Simpler macrocycles are synthetically more accessible and
favorable; the pharmacological properties of many of these
recently developed macrocycles has spawned research interest.
Conformational restriction, a consequence of macrocyclization,
is a common strategy in drug design to improve the potency of
a lead compound by minimizing the entropic cost.6 In
addition, minimizing the conformational freedom and trapping
small molecules in bioactive conformations would be expected
to lead to a gain of selectivity and potency. In large protein
families that harbor closely related active sites, selectivity is a
Received: February 4, 2021
Published: June 2, 2021
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Table 1. Overview of Macrocyclic Kinase Inhibitors in Clinical Trials
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major issue.7 One of these protein families is protein kinases, a
major drug target family which contains more than 500 closely
related proteins sharing a highly similar cofactor ATP binding
site and overall catalytic domain architecture. Thus, the
orthosteric binding pocket of protein kinases is highly
conserved over this protein family and the development of
macrocyclic kinase inhibitors is one approach that has been
successfully utilized to overcome selectivity issues.8,9 Many
synthetic strategies for the development of macrocyclic kinase
inhibitors have been elaborated, and some of these macro￾cycles have been approved as drugs or entered clinical trials
(Table 1). The first approved macrocyclic kinase inhibitors
were the mTOR inhibitors 1, 2, and 3, which were approved
between 1999 and 2009. Temsirolimus (2) and Everolimus
(3) are structurally related derivatives of Sirolimus (1), also
called rapamycin. Sirolimus (1) has been approved to prevent
transplant rejection after kidney transplantation,10 while 2 and
3 have been approved for the treatment of renal cell
Figure 1. A. R-Crizotinib (12) and the ALK/ROS1 selective inhibitor Lorlatinib (4) and the cellular IC50 values on ALK and ALK-L1196 M
gatekeeper mutation and the MDR efflux ratio.33,34 B. Selectivity panel of R-Crizotinib (12) against 456 recombinant human protein kinases with a
screening concentration of 1000 nM and Lorlatinib (4) against a panel of 206 kinases at a screening concentration of 1000 nM. The primary targets
ALK and ROS1 are highlighted in blue. Values were adopted from refs 35 and 33. C. IC50 values of Lorlatinib (4) against it closest off-targets.33 D.
Binding mode of acyclic compound R-Crizotinib (12) on human Anaplastic Lymphoma Kinase (ALK; PDB-ID: 2XP2). E. Binding mode of
macrocyclic compound Lorlatinib (4) on human Anaplastic Lymphoma Kinase (ALK; PDB-ID: 4CLI). F. Alignment of acyclic compound R￾Crizotinib (12) (orange) and macrocyclic compound Lorlatinib (4) (green) on human Anaplastic Lymphoma Kinase (PDB-ID: 2XP2) in surface
representation.
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cancer.11,12 Pacritinib (5), Zotiraciclib (7), and SB1578 (10)
are 2-aminopyrimidine-based macrocycles which target JAK2/
FLT3, CDK2/JAK2, and FLT3 and JAK2. All of them can be
synthesized via ring-closing metathesis.13,14 Pacritinib (5) is a
JAK2/FLT3 inhibitor for treatment of myelofibrosis and
lymphoma which is currently evaluated in phase 3 clinical
trials.15−17 Zotiraciclib (7) entered phase 1/2 clinical trials in
2010 as a potential new agent for treatment of leukemia and
multiple myeloma,18,19 whereas SB1578 (10) entered phase 1
clinical trials in 2010 for the treatment of autoimmune diseases
and inflammatory disorders such as psoriasis and rheumatoid
arthritis.14,20 E6201 (6) is a resorcyclic lactone, which was
synthesized via a Mukaiyama reaction.21 This MEK1 inhibitor
entered clinical trials in 2007 to study its efficacy treating
plaque psoriasis, and in 2017, a phase 1 study was initiated
evaluating E6201 (6) in malignant melanoma.22−24 Selitrecti￾nib (8) and Recrotrectinib (9) are pyrazolopyrimidine based
macrocycles, which have been synthesized via amide
coupling.25 Both inhibitors are potent TRKA/B/C inhibitors,
whereas Recrotrectinib (9) additionally targets ROS1/ALK.
Both compounds belong to the second-generation of TRK
inhibitors and are macrocyclic analogues of the recently
approved acyclic analogue Larotrectinib, a first-generation
TRK inhibitor. However, resistance to this first generation of
TRK inhibitors led to the development of both of these
macrocyclic compounds which were designed to overcome this
Figure 2. Overview over the optimization steps from the first to the third generation ALK/ROS inhibitors.38
Figure 3. Overview of PK/PD optimization steps from aminobenzimidazole 15 to macrocyclic aminobenzimidazole BI-4020 (19).50
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on-target resistance.26 Selitrectinib (8) and Recrotrectinib (9)
entered Phase1/2 clinical trials in 2017 for treatment of
advanced solid tumors. JNJ26483327 (11) is based on a
quinazoline scaffold, and it was synthesized via a Mitsunobu￾reaction.27 It was entered into a phase 1 clinical trial in 2008 as
a multitargeted tyrosine kinase inhibitor which was developed
for the treatment of advanced and/or refractory solid
malignancies.28 Lorlatinib (4) was approved in 2018 as a
highly potent and selective ROS1/ALK inhibitor, for the
treatment of NSCLC.29
The approval of the highly potent macrocyclic ALK/ROS
kinase inhibitor Lorlatinib (4) is an excellent example of the
implementation of macrocyclization in drug discovery.30,31
Lorlatinib (4) has outstanding central nervous system
(CNS) penetration, and it is highly selective for its designated
targeted ROS and ALK compared to its noncyclized template
R-Crizotinib (12), which has broad activity on receptor
tyrosine kinases as well as weaker potency on the main targets
ALK and ROS (Figure 1).32
Lorlatinib (4) was developed based on the bioactive
conformation of R-Crizotinib (12) to gain a potent and
more selective ALK/ROS inhibitor with improved CNS
availability (Figure 2). Lorlatinib (4) is effective against all
known resistant mutants of the first and second generation of
ALK inhibitors. Furthermore, it has considerably improved
blood-brain barrier and cell penetration due to low efflux in cell
lines overexpressing the efflux transporters P-glycoprotein and
breast cancer resistant protein. Thus, in addition to the
selectivity and potency gained, this macrocyclic third￾generation ALK inhibitor has also markedly improved
pharmacological properties.33,34
The optimization of R-Crizotinib (12) was carried out
stepwise via a second-generation ALK inhibitor, a molecular
design that was focused on avoiding the resistance of clinical
mutations after R-Crizotinib (12) treatment as well as the high
P-glycoprotein 1 efflux with a >2.5 MDR BA/AB ratio of its
parent compound.36 13 is about 6-fold more potent on ALK
wild-type, but it is also about 5-fold more potent on the ALK￾L1196 M gatekeeper mutation compared to R-Crizotinib.
Compound 13 showed a desirable low MDR efflux ratio, but it
still lacked potency and suitable in vivo clearance.33,37 For
further optimization, a macrocyclic inhibitor design was
performed. Though a first variation of the ring size of 12−14
membered rings, the cellular potencies of these compounds
reached 1- to 2-digit nanomolar EC50 values for ALK as well as
the L1196 M mutation. A picomolar binding affinity and a
LipE value of 4.4 were achieved for the macrocycle 14. The
lipophilicity was reduced by exchanging an ether with a lactam
linker in compound 4. This resulted in a higher LipE value in
comparison to the acyclic counterparts 12 and 13, but also
compared to the macrocycle 14. By these modifications, it was
possible to improve the potency of R-Crizotinib (12) by 62-
fold for wild-type ALK kinase and 40−825-fold for clinical
ALK mutants while also improving the selectivity.33,38
Lorlatinib (4) has a half-time of 19.0−28.8 h, and an in vivo
metabolite assay showed the main metabolism through
CYP3A4 and UGT1A4.39,40
Another excellent example of the improvement activity on
resistant mutants and of pharmacokinetic properties through
macrocyclization is BI-4020 (19). This inhibitor has been
explored for the inhibition of the tertiary EGFR mutation that
is present in 12−47% of non-small cell lung cancer (NSCLC)
tumors.41 The single mutations del19 and L858R can be
treated with the first-, second-, and third-generation EGFR
inhibitors such as Erlotinib, Gefitinib, Afatinib, or Osimerti￾nib.42−45 However, a second mutation, the gatekeeper
mutation T790M, occurs in 50−70% of patients who have
already been treated successfully.46 This mutation can only be
treated by covalent third-generation EGFR inhibitors, as it
weakens the inhibition activity of the first- and second￾generation inhibitors.47 However, even after successful treat￾ment of cancers bearing the gatekeeper mutation, 20−40% of
patients suffer a relapse due to the C797S mutation preventing
covalent binding of Osimertinib.48,49 Thus, the aim in
developing BI-4020 (19) was to have access to a new
generation of EGFR inhibitors, which are active against
gatekeeper and C797S mutations while sparing wild-type
EGFR and that maintain high selectivity across the human
kinome. Engelhardt et al. identified the aminobenzimidazole
15 as a starting point for further optimizations (Figure 3). This
inhibitor exhibited an IC50 value of 2100 nM and 250 nM for
EGFRL858R T790 M C797S and EGFRdel19 T790 M C797S while having
no activity on EGFRwt. Through interesting QM-torsion angle
scan optimizations and by additional interaction with the
protein, 16 was discovered. This inhibitor has good potency
for EGFRL858R T790 M C797S while sparing EGFRwt. However, the
improved activity on EGFR mutants resulted also in poorer
kinase selectivity. By removing the hydrogen-bond acceptor in
the phosphate binding region, the kinase selectivity of
compound 17 was improved while maintaining activity for
EGFR mutants. Critically, reduction of ligand entropy through
the macrocyclization led to a significant increase in potency.
For this optimization step, macrocycles with varying linker
length were synthesized. The first attempts showed a 5 times
better cellular potency compared to the linear precursor 17, in
which potency was affected by conformational restrictions,
which was addressed by introducing an N-methylpyrazole, and
better selectivity against EGFRwt was achieved through
installing an additional methyl group targeting a subpocket
and introducing a stereo center in the linker to address the
sugar binding pocket of EGFR. Compound 18 revealed an IC50
EGFRdel19 T790 M C797S of 1 nM, and the ratio of IC50 EGFRwt/
IC50 EGFRdel19 T790 M C797S > 1000. Further optimizations to
improve the solubility and reduce the plasma protein binding
were carried out by introducing a solubilizing group at position
6 at the phenyl core. BI-4020 (19) showed the most optimal
combination of properties, including solubility, permeability,
and fraction unbound in plasma. In addition, the cellular
activity was improved most likely by the improved cell
penetration of this macrocyclic compound to a subnanomolar
EC50 of 0.2 nM for EGFRdel19 T790 M C797S. The antiproliferative
effects against EGFRdel19 T790 M C797S-dependent BaF3 cells was
increased by a factor of 4000 in comparison to Osimertinib. In
addition, for the EGFRdel19 BaF3 cells BI-4020 (19) exhibited
the same activity as Osimertinib and on the NSCLS cell line
PC-9del19 T790 M C797S where it showed a potency of 1.3 nM.50
The examples of Lorlatinib (4) and BI-4020 (19)
convincingly demonstrated that locking the bioactive con￾formation of linear precursor molecules markedly improved
target potency, kinome wide selectivity, as well as physi￾ochemical and in vivo properties such as brain penetration.
Nevertheless, the synthetic intractability for the ring-closing
reaction is still a major concern for the medicinal chemist
limiting the number of published studies using a macro￾cyclization strategy. Therefore, in this review we focus on
synthetic strategies of small molecule macrocyclic kinase
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inhibitors and summarize data that have been published over
the past 15 years. In general, ring systems of both synthetic
macrocycles and analogues of natural products have been
considered for this review containing 12 or more atoms, as this
is a common definition for macrocycles.51 In order to obtain a
comprehensive overview of macrocyclic kinase inhibitors, we
extracted all macrocyclic kinase inhibitors from structures
deposited in the PDB. Additionally, we searched Pubmed and
Scifinder in order to gather a comprehensive data set on
synthetic efforts in this area.
1. MACROCYCLIZATION REACTIONS
The synthesis of macrocycles contains two major difficulties.
First, the ring-closing reaction involves typically two functional
groups, which should preferentially interact in an intra￾molecular reaction and not in an intermolecular reaction,
which would lead to dimerization, oligomerization, or
polymerization. Therefore, the cyclization step requires
typically highly diluted concentrations, which is an inefficient
strategy and is impractical to study these molecules in a
common SAR study. To identify at which concentration the
cyclization reaction, dimerization or polymerization, prevails,
Collins et al. established the macrocyclization efficiency index,
Emac, which is defined in formula 1.
52 This value describes the
Figure 4. Overview of the different synthetic reaction types that have been used for macrocyclization. Approximately 300 different macrocyclization
reactions have been reviewed, which have been performed in the past 15 years for the synthesis of macrocyclic kinase inhibitors with known yields.
The upper panel displays the percentage of the macrocyclization reactions used, whereas in the lower panel, the “macrolactonization and
macrolactamization”, the “palladium-catalyzed reactions”, the “C−O and C−N bond formations”, and “C−C bond formations” have been
subdivided into the individual macrocyclization reaction types.
Scheme 1. Example of Macrocyclization through a Ring-Closing Metathesis15
Figure 5. Mean values of yields and Emac values depending on the
ring size for the ring-closing metathesis reactions.
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feasibility of using a certain reaction in the drug discovery
process and indicates the probability of being able to carry out
the macrocyclization reaction on a sufficiently large scale.
Collins et al. identified 896 reactions and determined the Emac
values of this set. The determined values were between 2 and 9
with a mean of 5.8. Values between 8 and 9 are in an optimal
range and do not require further optimization, while an Emac
value of 6 represented an acceptable value, which, however,
still may be optimized further for future reactions. Values
between 2 and 5 were perceived as poor and needed to be
improved.
Emac log = [· Y C] 10
3
(1)
where Y = yield in % and C = concentration in mM.
Second, the ring-closing reaction itself can be challenging,
since several studies indicated that a conformational
preorganization is important for the macrocyclization reaction
to occur.53,54
Here, we searched the medicinal chemistry literature and
sorted the reported synthetic routes by different reaction types
used for the cyclization step. All in all, we identified 503
macrocyclization reactions, which have been published over
the past 15 years. However, out of the 503 macrocyclization
reactions just 302 have been considered here, since for the
remaining 201 reactions no yields have been reported, or the
yield has been reported only over several steps. Nevertheless,
for the approximately 300 reactions analyzed, we observed that
the most common reactions used to date are the ring-closing
metathesis55 and a nucleophilic substitution.53 However, some
rather unusual reaction types, which are rarely performed in a
classical medicinal chemistry SAR study, have been used such
as a Shiina esterification or Prins reactions (Figure 4).56−58
A set of 20 different macrocyclization reactions were
examined to determine the dependence of the ring size and
the reaction type on the yields. The following section is
organized according to the different reaction types that have
been identified.
1.1. Ring-Closing Metathesis. The ring-closing meta￾thesis (RCM) is the most commonly used reaction type for the
macrocyclization. 52% of the total summarized reactions were
metathesis reactions. This is an efficient and potent possibility
for the formation of a C−C bond, which goes back to the work
of Grubbs et al.59 An example of an RCM reaction is the
synthesis of the macrocyclic JAK2/FLT3 inhibitor Pacritinib
(5) (Scheme 1). For this reaction, a Grubbs catalyst of the
second generation was used with a moderate yield of 56%.15
The advantages of this ring-closing method are a high
tolerance toward many functional groups and the availability
of many different catalysts, which can be adapted to the
corresponding reaction requirements.51,60 The disadvantage of
this reaction type is the lack of control of the stereochemistry
of the olefin product that leads to E-/Z-isomers. Therefore, the
double bond was in most cases directly reduced to the
saturated linker, to avoid tedious purification of the isomers. In
addition, this double bond also provides an opportunity for the
introduction of more diverse functionalities, such as epox￾idation with further modifications.55 The importance of this
reaction type is also highlighted by 3 compounds which
entered clinical trials and have been produced by this
metathesis reaction.
The best yields were obtained with a ring size of 15 and 17,
whereas the synthesis of 13-membered macrocycles has been
Scheme 2. Example for a Macrocyclization Using an
Intermolecular Nucleophilic Substitution61
Figure 6. Mean values of yields and Emac values as a function of the
ring size for the nucleophilic substitution ring closure reactions.
Scheme 3. Example for a Macrocyclization Using Amide
Coupling62
Figure 7. Mean values of yields and Emac values depending on the
ring size for the amide coupling.
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associated with lower yields, which could indicate conforma￾tional constraints (Figure 5). For all analyzed reactions, the
Emac values were determined as a function of the ring size. All
of the Emac values were in a range between 5 and 6.
1.2. Nucleophilic Substitution. An example of a
nucleophilic aromatic substitution with a good yield (87%)
was reported for the macrocyclic PIM1/PIM2 inhibitor 22
(Scheme 2).61 Nucleophilic substitution represents 13% of the
total reactions, the second most common macrocyclization
type. This well-known reaction type often requires the
introduction of protecting groups, since depending on the
type of nucleophile, not every functional group is equally
tolerated. Nevertheless, the ring closure strategy offers a
versatile reaction type with multiple available conditions, is
easy to implement, and is also compatible for large ring
systems with over 20 atoms.51 The most common type of this
reaction for macrocyclization is the nucleophilic aromatic
substitution. A nitrogen or an oxygen has been typically used as
a nucleophile, while a halide or a mesityl group was used as a
favorable leaving group.
The nucleophilic reaction has been used for many large ring
systems (Figure 6). The yields vary greatly depending on the
ring size. While 15-, 16-, 46-, and 49-membered rings delivered
less than 40% yield, other ring-closing reactions achieved
moderate to good mean yields up to 82%. The Emac values for
Scheme 4. Examples of Macrolactonization and Macrolactamization Reactionsa
a
Example for a macrocyclization using a A, Yamaguchi esterfication;69 B, Mukaiyama reaction;72 C, Shiina esterfication;74 D, Steglich
esterfication.75
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all of the analyzed reactions were between 5 and 8, thus
moderate to good.
1.3. Amide Coupling. The third most common reaction is
the amide coupling. An example for this reaction type is the
synthesis of a macrocyclic ASK1 (MAP3K5) inhibitor 24
(Scheme 3).62 An amide bond can be formed by activating the
carboxylic acid, followed by the condensation with an amine.
The different strategies for this reaction are, for example, the
activation through uronium-derived reagents or the usage of
carbodiimides in combination with additives.63 Himmelbauer
et al. used propanephosphonic acid anhydride (T3P) as a
coupling reagent. However, other standard amide coupling
reagents have also been described for the macrocyclization
step, including HATU,33 TBTU,50 PyBOP,64 EDCl, and
HOBt.65 This step is usually preceded by deprotection so
that the functional groups can only react with each other in the
desired step. The FDA-approved kinase inhibitor Lorlatinib
(4) can also be obtained through an amide coupling which
highlights this type of reaction. After the optimization,
Lorlatinib (4) could be obtained with a yield of 56% for the
first scale-up synthesis.66
The yields for the closure of 12- to 16-membered rings were
generally rather low (<40%), while the larger macrocycles with
an 18- and 19-membered ring system had reported yields of
around 60%. The low yields might indicate a general problem
with macrocyclization under classical amide coupling con￾ditions. Usually, this reaction follows a two-step procedure
starting with the activation of the carboxylic acid by treating
the carboxylic acid with common coupling reagents. After
activating the carboxylic acid to the active ester, the amine is
added to start the amide formation. This two-step procedure is
typically conducted to avoid side reaction which occurs from
the reaction of the amine with the coupling reagents. However,
if both functionalities, the carboxylic acid and the amine, are in
the same molecule, a preactivation of the carboxylic acid to the
active ester is not possible and might be an explanation for the
detected low yields. Nevertheless, the Emac values for all of
them are reasonable and more or less in the same area. An
explanation for that could be that the dilution concentration of
the smaller rings was lower, and this leads to a low yield for the
smaller cycles; however, the Emac values are in the same range
as the bigger ring systems (Figure 7).
1.4. Macrolactonization and Macrolactamization. In
this section, we describe macrolactonization and macro￾lactamization reactions, which differ from the classical amide
coupling reagents. The macrolactonization is a very old and
well-studied reaction, since it is one of the most commonly
used reaction types for the total synthesis of many natural
products.67 Therefore, there are various synthetic options for
the macrolactonization and also the macrolactamization
reaction (Scheme 4). One of them is the Yamaguchi
esterification, where the carboxylic acid is activated by 2,4,6-
trichlorobenzoyl chloride (Yamaguchi reagent) to form a
mixed anhydride. Afterward, the ester bond can be formed by a
reaction with an alcohol in the presence of DMAP.68 Kraft et
al. used this strategy for the synthesis of 26, where the product
was isolated with 90% yield.69 Another option is the activation
through 2-chloro-1-methylpyridinium iodide (Mukaiyama
reagent). Mukaiyama et al. invented this reagent in order to
establish a method for the preparation of an ester bond by the
using an equimolar amount of a carboxylic acid, an alcohol,
and the Mukaiyama reagent.70 However, this reagent has
general utility as an efficient condensation agent, which can be
used for a variety of many different dehydrative coupling
reactions.71 With this reaction agent, 28, an example of a
macrolactam was achieved in modest yields.72 A third option is
the usage of an aromatic anhydride such as 2-methyl-6-
nitrobenzoic anhydride (MNBA). It is used as a dehydration
condensation agent for the Shiina esterfication under basic
conditions.73 Another option for the formation of an ester
bond with mixed anhydrides is in the presence of a Lewis acid
catalyst. This strategy was investigated by Shiina et al.56 30
resulted in 53% yields under basic conditions.74 Ley et al. used
the Steglich esterification for the macrocyclization step of 31 to
obtain 32 with 71% yield.75 It also offers the possibility of
coupling a wide variety of carboxylic acids and alcohols under
mild basic conditions.76
The Yamaguchi esterfication was used for smaller and larger
ring systems, but we observed a lack of published reactions for
creating 15- to 19-membered cycles. The yields were generally
Figure 8. Summary of esterification ring-closing reactions. Mean
values of yields and Emac values depending on the ring size for the
Yamaguchi esterfication. The values for the Mukaiyama reaction,
Shiina esterfication, and Steglich esterification are shown in Figure S1.
Scheme 5. Examples of Palladium-Catalyzed Cross￾Coupling Reactionsa
a
Example for a macrocyclization through A, Suzuki reaction;33 B,
Heck reaction;33 C, Buchwald-Hartwig amination.82
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between 50% and 80%, and the Emac values were in an
acceptable area between 4 and 6, indicating that these
procedures should still be improved. Due to the low number
of documented reactions, the chart for the Mukaiyama
reaction, Shiina and Steglich esterification showed only one
ring size per reaction type (Figure 8). For all of them, the
yields and Emac values were quite reasonable. Even though
macrolactonization is a well-studied reaction for the synthesis
of macrocycles, the usage of this reaction for the synthesis of
macrocyclic kinase inhibitors had less impact, probably because
ester linkages are considered prone to hydrolysis in the cellular
environment and are therefore usually not drug-like moieties.
1.5. Palladium-Catalyzed Reaction. Palladium-catalyzed
cross-coupling reactions are versatile reactions, which have
been met with great interest especially due to their utility in
drug discovery, since they enable the broad formation of
different C−C bonds (aryl−aryl, aryl−alkyl, aryl−alkenyl,
aryl−alkyne), but they can also be used for the formation of
various C−O or C−N bonds.77,78 Therefore, it is not a surprise
that attempts have been made to utilize palladium-catalyzed
cross-coupling reactions also for the macrocyclization reaction.
One of the most commonly used palladium-catalyzed C−C
bond formation reactions is the Suzuki cross-coupling. It is
typically a reaction between a boronic acid and an aryl halide
in the presence of a palladium (0) complex and a base.79
Johnson et al. used this reaction type for the formation of 34, a
macrocyclic ALK inhibitor; however, this reaction resulted
only in poor yields. For this reaction, the boronic acid was
generated in situ proceeding directly with the cross-coupling
step. Further studies of these coupling reactions suggested that
the yields could be potentially improved by carrying out the
borylation and the cross-coupling separately.33 Another way to
form a C−C bond is the frequently used Heck reaction. This
coupling reaction is a palladium-catalyzed cross-coupling
Scheme 6. Examples of C−O and C−N Bond Formationa
a
Example of a macrocyclization through A, Mitsunobu reaction;50 B, Prins-driven reaction;58 C, Click reaction;87 D, Aryl amidation.89
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between an unsaturated halide and an alkene in the presence of
a base,80 which has been used for the synthesis of Lorlatinib￾derivatives such as 36, a macrocyclic ALK/ROS1 inhibitor,
however in low yields.33 Therefore, in an exploratory process
development an alternatively synthetic route of Lorlatinib (4)
has been published that includes an amidation reaction as ring￾closing reaction.66 Another possibility for the palladium￾catalyzed formation of a C−N bond represents the
Buchwald−Hartwig amination. This is a cross-coupling
reaction between an amine and an aryl halide, and it offers
an alternative to a reductive amination or a nitration followed
by a reduction step with mild reaction conditions and with a
higher tolerance to the presence of functional groups.81 Wang
et al. used this kind of reaction for the synthesis of 38 by using
tris(dibenzylideneacetone)dipalladium(0) as a catalyst and
Xantphos as an ligand (Scheme 5). The desired product was
isolated with a yield of 28%.82
As already mentioned above, the mean yields for the Suzuki
coupling were quite low. The reason for this unsatisfying yield
was probably due to a strategy in which the boronic acid was
generated in situ and a potential improvement could be
separating the introduction of the boronic acid and the cross￾coupling step. The Emac values were in a moderate range. The
values for the Heck reaction and the Buchwald−Hartwig
amination were low to moderate. All these reactions were only
used for the synthesis of small macrocycles with a ring size
between 12 and 14 (Figure S2).
1.6. C−O and C−N Bond Formation. Besides the
palladium-catalyzed cross-coupling reactions and the classical
SN-reaction for the formation of C-heteroatom bonds (C−O/
C−N), many more reactions are known, which can also be
utilized for the macrocyclization reaction. The Mitsunobu
reaction is an opportunity to form C−O bonds and could be
used for a macrolactonization, as well as for the formation of
macrocyclic ethers. The reaction typically requires an acidic
functional group such as carboxylic acids, or phenols and an
alcohol group where the hydroxy group is replaced. For the
reaction, the alcohol group will be activated typically through
the usage of triphenylphosphine and diisopropyl azodicarbox￾ylate (DIAD) or diethyl azodicarboxylate (DEAD).83−85 40 is
an example for an macrocyclic EGFR inhibitor, which was
isolated with 65% yield using DIAD for the generation of the
ether bond.50 Another example for a C−O bond formation is a
Prins-driven reaction (Scheme 6B). The Prins reaction
requires typically the attendance of an aldehyde and an alkene
or alkyne, which reacts in an electrophilic addition and the
subsequent reaction with different nucleophiles.86 In 41, all
three different functionalities are present, which leads to the
formation of 42, which was achieved through this Prins-driven
reaction.58 This reaction type is quiet unusual for a cyclization
and introduces a 6-membered hydropyranyl ring systems,
which might serve as a driving force for the formation of the
20-membered macrocycle in excellent yields. 45 represents a
macrocyclic AXL kinase inhibitor. The macrocyclization step
was performed via an C−N bond formation by using double
Click chemistry.87 These reactions introduce a 1,2,3-triazole
moiety through a modified Huisgen 1,3-dipolar cycloaddition.
For that, an azide and an alkyne reacted in the presence of a
copper(I) catalyst.88 Another option for the formation of the
C−N bond of 47 is the aryl amidation.89 In addition, this
reaction represents a copper(I) mediated reaction, which
allows transformation of an aryl bromide or a aryl iodide into
an amide. It has a high tolerance to functional groups and takes
place in the presence of a base and a ligand like N,N′-
dimethylethylenediamine (DMEDA).90
The mean yield for the Mitsunobu coupling of a 13-
membered macrocycle is quite low at 17%, while the yields for
formation of larger ring systems containing 14 and 15 atoms
were around 65%. The effectiveness of the Click reactions was
surprisingly low (Figure 9). The yields for the formation of 15-
and 18-membered ring systems were lower than 25%. The
Prins-driven reaction and the aryl amidation were only used for
larger ring formation reactions and exhibited a good yield
around 80%. In addition, the Emac values for these reactions
were above 6.
1.7. C−C Bond Formation. Besides the aforementioned
olefin methathesis, a few more options for the C−C bond ring￾closing reaction have been described (Scheme 7; Figure 10).
Schaubach et al. described an alkyne metathesis for the
synthesis of 49, which was carried out with 78% yield by the
usage of a molybdenum catalyst 50.
91 The authors of this study
have shown that this reaction was equally applicable for the
synthesis of the sesquiterpene lactone manshurolide, which has
been described as a MAPK inhibitor.92,93 Another reaction we
found was the copper(I)-calatyzed [2 + 2 + 2] cycloaddition.
This reaction enables the selective creation of a C−C bond
that introduces a new ring system into the molecule. Alkynes as
well as nitriles or other unsaturated substances are possible
Figure 9. Summary of C−O and C−N bond formation. Mean values of yields and Emac values depending on the ring size for the A, Mitsunobu
reaction; B, Click reaction. The values for the Prins-driven reaction and aryl amidation are shown in Figure S3.
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educts for this reaction.94 Zhang et al. used this strategy to
synthesize 53, however, with a poor yield of 9%.95 The nickel￾mediated alkyne−aldehyde reductive coupling resulted in 55
with 57% yield. This reaction is an exo-seletive coupling with a
regioselectivity ≥95%.96 Interestingly, this method is tolerant
to a large number of functional groups and enables the
introduction of a stereogenic center.97 57 was produced by a
Wittig reaction, introducing a double bond. For that, the
Scheme 7. Examples of C−C Bond Formationa
a
Example of macrocyclization through A, alkyne metathesis;91 B, [2 + 2 + 2] cycloaddition;95 C, alkyne−aldehyde reductive coupling;96 D, Wittig
reaction;98 E, McMurry reaction;99 F, Nozaki−Hiyama−Kishi reaction.103
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triphenyl phosphonium ylide was first introduced, which then
reacted intramolecularly with the aldehyde to 57 with a yield of
68%.98 Jiang et al. used the McMurry reaction to produce 59.
For that, two aldehyde groups were coupled to a double bond,
using titanium(IV) chloride and a reducing agent such as zinc
or LiAlH4.
99,100 The Nozaki−Hiyama−Kishi reaction was used
for the synthesis of 61.
101 It is a reaction between an aldehyde
and an allyl or vinyl halide with nickel and chromium as a
catalyst. The reaction exhibits high selectively for aldehydes
even in the presence of ketones. In addition, it is tolerant to
many other functional groups.102 For 61, a yield of 35% was
achieved.103
The alkyne metathesis seems to be a versatile tool for the
macrocyclization. The yields for the smaller as well as the
larger ring systems were between 70% and 80%. The [2 + 2 +
2] cycloaddition seemed not that efficient with yields under
15% and less favorable Emac values below 4. The alkyne−
aldehyde reductive coupling was only used for smaller rings,
but it showed moderate to good yields around 40%. Due to the
low number of published ring closure reactions, the Wittig,
McMurry, and Nozaki−Hiyama−Kishi reactions covered only
one ring size per reaction type with moderate Emac values
around 6.
2. FUTURE PERSPECTIVES AND CONCLUSION
Over the past decades, macrocycles have received increasing
interest in the field of medicinal chemistry, due to their
remarkable pharmacological properties. Their suitability as
drugs is demonstrated by the rising numbers of FDA-approved
macrocycles and inhibitors currently tested clinically. In the
field of synthetic macrocyclic kinase inhibitors, just Lorlatinib
(4) has entered the market, but many more are currently being
evaluated in clinical trials such as Pacritinib (5) or its closely
related derivatives SB1578 (10),14,20,104 Zotiraciclib (TG02;
SB1317; 7),18,105−107 the quinazoline based derivative
JNJ26483327 (11),28 or the pyrazolopyrimidine-based TRK
inhibitors Selitrectinib (LOXO-195) (8) and Repotrectinib
(TPX-0005) (9). The macrocyclization reactions that have
been used for these compounds are the ring-closing metathesis,
Mitsunobu reactions, and amide coupling reactions. The
success of these inhibitors indicates that their macrocyclization
reactions are suitable also under scale-up conditions. Never￾theless, the synthesis of drug-like macrocycles is still
challenging, and macrocyclization is often difficult to integrate
into a classical SAR approach of a medicinal chemist. However,
the synthetic toolbox for medicinal chemistry is currently
poorly explored by focusing on a few reactions, which are
frequently used.78,108,109 The synthesis of drug-like macrocyclic
molecules represents an opportunity for new avenues
expanding the synthetic toolbox as well as the chemical
space for bioactive molecules. Therefore, we summarized in
this review published experience on macrocyclization reactions
that have been performed over the past 15 years to provide a
representative sample of drug-like macrocyclization reactions.
All in all, over 20 different reaction types were identified for the
ring-closing reaction (Figure 11). Brown and Boström et al.
made a comprehensive analysis of the synthetic methodologies
in medicinal chemistry over three decades.78,109 By a direct
comparison between their comprehensive analyses of these
reactions, which are frequently used in classical medicinal
chemistry SAR approaches and for the macrocyclization
reactions, it is obvious that a number of reactions appear in
both studies as most frequently used reactions such as amide
formation, aromatic nucleophilic substitution reaction (SNAr),
or Suzuki reaction. However, for the macrocyclizations
Figure 10. Summary of C−C bond formations. Mean values of yields
and Emac values depending on the ring size for the A, alkyne
metathesis; B, [2 + 2 + 2] cycloaddition; C, alkyne−aldehyde
reductive coupling. The values for the Wittig reaction, McMurry
reaction, and Nozaki−Hiyama−Kishi reaction are shown in Figure S4.
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reactions the most prominent reaction used by medicinal
chemists was the ring-closing metathesis that strongly differs
from the work of Brown and Boström.78 In addition, other
reactions such as Mitsunobu reaction, click-reaction, or alykne
metathesis may have the potential to be a versatile reaction for
the synthesis of drug-like macrocycles. One aspect to bring
potential candidates into clinical trials is the synthetic
accessibility of these compounds often requiring high dilution
in order to favor intermolecular reactions. Therefore, Collins et
al. introduced the Emac value that offers a convenient
comparative index for the assessment of macrocyclization
efficiency.52 In our analysis, we calculated the Emac values for
all the previously described reactions. In our comprehensive
analysis, we identified a mean value of 5.7 which is in good
agreement with the reported mean of 5.8 from Collins et al.52
The reaction types with the highest Emac values are the aryl
amidation and the Prins driven reaction with values of 6.8 and
6.7, respectively. The lowest Emac values were observed using
[2 + 2 + 2] cycloaddition and the Click reaction with values of
3.8 and 4.7, respectively. In general, the Emac value correlated
with the obtained yields. However, the RCM with a ring size of
13 had a low yield (34%), but the Emac was with a value of 5.9
slightly above the mean, suggesting additional synthetic
challenges. The same tendency was seen for the Click reaction.
For reactions resulting in a ring size of 18, yields of 23% were
achieved with an Emac value of 5.7. For these cases, it should
be tested whether the yield can be improved by changing other
reaction parameters, such as temperature or reaction time.
Albeit there are many different reaction types, the
implementation of macrocycles into the iterative process for
a classical SAR approach is still challenging. Usually, the
macrocyclization step is the last step in the synthesis, which
slows down the synthesis of a broad set of closely related
analogues. One option to overcome this obstacle would be to
introduce a mild ring-closing reaction during earlier steps to
create an opportunity for functionalization and diversification
of a macrocyclic scaffold. Cernak et al. have recently
summarized the possibilities for so-called late-stage function￾alization (LSF), which describes especially the process of C−H
activation to introduce new functional groups to highly
complex molecules that allows further derivatization.110
However, ring expansion, ring contraction, or rearrangement
reactions illustrate the diverse opportunities for late-stage
functionalization.111
The length of the linker has an enormous effect on
determining the lowest-energy conformation of the macrocycle
and the ability of the macrocyclic compound to adopt a
bioactive conformation. Therefore, the implementation of
different linkers should be included early in SAR approaches of
macrocycles. Cruz-Lopez et al. have developed an interesting
method for the ring-closing reaction by a double CuAAC
reaction, which attached a linker on the one side of the core
scaffold and closes the macrocycle by just one step. By using
various double-azide analogues, this reaction type offers an
option for the fast introduction of different linkers to form
macrocycles with distinct ring sizes.87 Future methodologies,
which could be implemented more often for the improvement
of cyclization reactions are, e.g., the continuous flow chemistry
or the multicomponent reaction. The flow chemistry is a
technique to reduce the high-dilution concentration and thus
reduce the waste and prevent the unwanted oligomers and
byproducts. Several examples have proven applicability for the
synthesis of macrocycles through continuous flow chemis￾try.112,113 In addition, due to the possibility to run the
macrocyclization reaction even under highly concentrated
conditions, it allows the continuous flow chemistry in general
as a more feasible scale-up of macrocyclic compounds. The
multicomponent reaction represents a possibility to reduce the
complexity of the synthesis of macrocycles. It is a method
yielding a product from the simultaneous reaction of three or
more compounds. An example and the most common MCR
macrocyclization is the Ugi four component reaction (U-4CR),
which involves a ketone or aldehyde, an isocyanide, an amine,
and a carboxylic acid to form a bis-amide.114 The MCR can be
used for the formation of linear precursors, which are then
closed in a cyclization reaction. However, it also can include
the cyclization step, and this strategy has the potential to be a
powerful tool for future synthesis of macrocyclic compounds
and the generation of chemical libraries.115
Figure 11. Comparison of the different macrocyclization methods and their dependence on the ring size.
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Many of the challenges synthesizing macrocyclic kinase
inhibitors have been addressed in the recent literature, and we
believe therefore that the reports on macrocyclic inhibitors will
increase in the near future. Lorlatinib (4) is the most
prominent example that has proven that macrocyclization
may have superior properties to its acyclic counterpart, which
is in that case crizotinib. By macrocyclization of R-Crizotinib
(12) to Lorlatinib (4), the in vitro and cellular potency was
increased on its main targets. In addition, the selectivity profile
of Lorlatinib (4) was superior to R-Crizotinib (12), and the
CNS availability was also improved, which finally led to the
approval of Lorlatinib (4) by the FDA as the first macrocyclic
kinase inhibitor. Therefore, their often-favorable selectivity
profiles, combined with the potential to increase the potency
and have an influence on the PK properties compared to
conventional inhibitors, makes macrocyclization strategies
particular interesting for the development of kinase inhibitors.
■ ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00217.

Figures: Summaries of esterification ring-closing reac￾tions, palladium-catalyzed cross-coupling reactions, C−
O, C−N, and C−C bond formation. All references used
for the statistical analysis. (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
Thomas Hanke − Institute for Pharmaceutical Chemistry,
Johann Wolfgang Goethe-University, D-60438 Frankfurt am
Main, Germany; Structure Genomics Consortium Buchmann
Institute for Molecular Life Sciences, Johann Wolfgang
Goethe-University, D-60438 Frankfurt am Main, Germany;
orcid.org/0000-0001-7202-9468; Phone: (+49)69 798-
29313; Email: [email protected]
Stefan Knapp − Institute for Pharmaceutical Chemistry,
Johann Wolfgang Goethe-University, D-60438 Frankfurt am
Main, Germany; Structure Genomics Consortium Buchmann
Institute for Molecular Life Sciences, Johann Wolfgang
Goethe-University, D-60438 Frankfurt am Main, Germany;
orcid.org/0000-0001-5995-6494; Phone: (+49)69 798-
29871; Email: [email protected]
Author
Jennifer Alisa Amrhein − Institute for Pharmaceutical
Chemistry, Johann Wolfgang Goethe-University, D-60438
Frankfurt am Main, Germany; Structure Genomics
Consortium Buchmann Institute for Molecular Life Sciences,
Johann Wolfgang Goethe-University, D-60438 Frankfurt am
Main, Germany
Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00217

Notes
The authors declare no competing financial interest.
Biographies
Jennifer Alisa Amrhein studied chemistry at the Goethe University in
Frankfurt. Currently, she is doing her PhD at the Institute of
Pharmaceutical Chemistry at the Goethe University under the
supervision of Prof. Dr. S. Knapp. Her main focus is on the
development and synthesis of macrocyclic kinase inhibitors.
Stefan Knapp studied Chemistry at the University of Marburg
(Germany) and at the University of Illinois (USA). He has a PhD in
protein crystallography (Karolinska Institute). In 1999, he joined
Pharmacia (Italy) and left the company in 2004 to set up a research
group at the Structural Genomics Consortium (Oxford University).
He was Professor of Structural Biology at Oxford University (UK)
and from 2012 to 2015 the director for Chemical Biology at the
Target Discovery Institute. He joined Frankfurt University in 2015 as
a Professor of Pharmaceutical Chemistry. He is also the CSO of the
SGC (Structure Genomics Consortium) node at the Goethe￾University Frankfurt. His research interest is focused on the design
of selective kinase inhibitors and inhibitors of protein interactions
domains.
Thomas Hanke studied Pharmacy at the Goethe University in
Frankfurt. He did his PhD in the group of Prof. Dr. Schubert￾Zsilavecz at Frankfurt University which included a sabbatical in the
group of Prof. Dr. Werz at the University of Tübingen to study the
anti-inflammatory responses of small molecules in vitro and in cellular
systems. In 2014, he completed his PhD in pharmaceutical chemistry,
investigating the synthesis and pharmacological characterization of
dual 5-LO and mPGES-1 inhibitors. In 2015, he joined the group of
Prof. Dr. Knapp where he is working as a senior scientist on the
development of selective chemical probes targeting kinases, with the
main focus on macrocyclic inhibitors.
■ ACKNOWLEDGMENTS
The SGC is a registered charity (no: 1097737) that receives
funds from AbbVie, Bayer AG, Boehringer Ingelheim, Canada
Foundation for Innovation, Eshelman Institute for Innovation,
Genentech, Genome Canada through Ontario Genomics
Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Dia￾mond, Innovative Medicines Initiative 2 Joint Undertaking
[EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD
in Canada and US), Merck & Co (aka MSD outside Canada
and US), Pfizer, Sao Paulo Research Foundation-FAPESP, ̃
Takeda and Wellcome [106169/ZZ14/Z].
■ ABBREVIATIONS
ALK, anaplastic lymphoma kinase; ASK1, apoptosis signal￾regulating kinase 1; ATP, adenosine triphosphate; AXL,
tyrosine-protein kinase receptor UFO; CNS, central nervous
system; CSF, cerebrospinal fluid; DEAD, diethyl azodicarbox￾ylate; DIAD, diisopropyl azodicarboxylate; DMAP, 4-dimethy￾laminopyridine; DMEDA, 1,2-dimethylethylenediamine;
EDCl, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide;
EGFR, epidermal growth factor receptor; EMA, European
Medicines Agency; FDA, Food and Drug Administration;
FLT3, fetal liver fms-related tyrosine kinase 3; HATU, 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate; HOBt, hydroxyben￾zotriazole; IC50, 50% inhibitory concentration; JAK2, janus
kinase 2; LipE, lipophilic efficiency; logD, octanol/buffer (pH
7.4) distribution coefficient; LSF, late-stage functionalization;
MAPK, mitogen-activated protein kinase; MCR, multicompo￾nent reaction; MNBA, 2-methyl-6-nitrobenzoic anhydride;
NSCLC, non-small-cell lung carcinoma; pALK, phospho￾ALK; PDB, protein data bank; PIM1, proto-oncogene
serine/threonine-protein kinase Pim-1; PIM2, serine/threo￾nine-protein kinase Pim-2; PK, pharmacokinetic; PD,
pharmacodynamics; PyBOP, benzotriazol-1-yl-oxytripyrrolidi￾nophosphonium hexafluorophosphate; QM, quantum mechan￾ics; RCM, ring-closing metathesis; ROS1, c-ros oncogene 1;
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SAR, structure−activity relationship; TBTU, 2-(1H-benzotria￾zole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate; wt,
wild-type
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