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Macrocycles in new drug discovery
The use of drug-like macrocycles is emerging as an exciting area of medicinal chemistry, with several recent examples
highlighting the favorable changes in biological and physicochemical properties that macrocyclization can afford.
Natural product macrocycles and their synthetic derivatives have long been clinically useful and attention is now
being focused on the wider use of macrocyclic scaffolds in medicinal chemistry in the search for new drugs for
increasingly challenging targets. With the increasing awareness of concepts of drug-likeness and the dangers of
‘molecular obesity’, functionalized macrocyclic scaffolds could provide a way to generate ligand-efficient molecules
with enhanced properties. In this review we will separately discuss the effects of macrocyclization upon potency,
selectivity and physicochemical properties, concentrating on recent case histories in oncology drug discovery.
Additionally, we will highlight selected advances in the synthesis of macrocycles and provide an outlook on the
future use of macrocyclic scaffolds in medicinal chemistry.
Over the past several years, the prevalence
of biologically active macrocycles in medici­
nal chemistry literature has been increasing.
Numerous recent review articles have dis­
cussed the role that macrocycles can play in
medicinal chemistry, in particular looking
beyond the established importance of natural
product macrocycles in drug discovery [1–3] .
Driggers et al. have argued that macrocyclic
structures are underexploited in drug discov­
ery, and presented different classes of natural
product macrocycles and their applications
to highlight the suitability of the structural
class for further development [1] . Oyelere has
collated a selection of specialist articles with
a particular focus upon some of the challeng­
ing molecular targets that macrocycles might
be suited towards within the context of drug
development [2,4–9] . Marsault and Peterson pro­
vided a comprehensive overview of the use of
macrocycles in many therapeutic areas within
drug discovery, along with some of the strate­
gies used to synthesize these molecules [3] . In
the current review, we will focus on recent stud­
ies that aid the understanding of the effects of
macrocyclization upon target potency, selectiv­
ity and compound physicochemical properties.
The majority of examples presented here have
been taken from literature sources from the past
5 years and are largely focused on applications
within oncology drug discovery. Wherever pos­
sible, a comparison of the macrocycles to rel­
evant acyclic molecules has been highlighted, as
such pairwise comparisons are the most direct
means of determining the effects and potential
benefits of macrocyclization.
Macrocycles in drug discovery have been
defined as a ring system consisting of 12 or more
atoms [1] . Opinions differ on the definition of
macrocycles based on ring size, but this option
usefully captures the qualitative differences in
behavior between large macrocyclic rings (≥12
atoms) and medium rings (8–11 atoms). The
accessible conformations for these medium rings
are dominated by transannular interactions and
conformational strains that are not present in
the larger macrocycles [10] . Macrocycles have
a structure that affords a degree of conform­
ational pre-organization due to restricted rota­
tion. Many natural products have a macrocyclic
core, suggesting that an evolutionary advantage
may be associated with the production of second­
ary metabolites based upon these scaffolds [1,11] .
There are different classes that macrocycles could
fall into, including peptidic and nonpeptidic nat­
ural products, non-natural (synthetic) peptides
and non-natural (synthetic) macro­c ycles. This
review will primarily focus upon the last class,
since considerable scope exists for the greater
application of synthetic macrocycles to medicinal
chemistry.
The potential for macrocycles as drugs is already
evident. Exploitation of natural product macro­
cycles has yielded several oncology drugs that are
either approved for clinical use or have reached
late-stage clinical development (Table 1) , such
as the mTOR inhibitor Torisel® (temsirolimus)
[12,13] , the microtubulin stabilizer Ixempra®
(ixabepilone) [14–16] and the Hsp90 inhibitor
17-allylamino-geldanamycin [17] . A number
of synthetic macrocycles have entered clinical
development, such as the dual JAK2/FLT3
Jamie Mallinson &
Ian Collins*
10.4155/FMC.12.93 © 2012 Future Science Ltd
Future Med. Chem. (2012) 4(11), 1409–1438
ISSN 1756-8919
Cancer Research UK Cancer
Therapeutics Unit, Institute of Cancer
Research, 15 Cotswold Road, Sutton,
Surrey SM2 5NG, UK
*Author for correspondence:
Tel.: +44 2087224317
E-mail: [email protected]
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Review | Mallinson & Collins
Table 1. Examples of natural and non-natural macrocyclic oncology drugs and drug candidates.
Name and class
Structure
Target
Torisel (temsirolimus)
Natural product analogue
(rapamycin)
®
OH
Me
O
N
O
Me
O
HO
mTOR inhibitor
Microtubulin stabilizer
[14–16]
OH
O
H
Ref.
[12,13]
O
Me
OMe
O
O
O
Me
OH
Me
Me
MeO
O
MeO
Me
Me
Ixempra® (ixabepilone)
Natural product analogue
(epothilone B)
Me
O
S
Me
Me
Me
Me Me
N
HN
Me
OH
O
17-allylamino-geldanamycin
Natural product analogue
(benzoquinone ansamycin)
OH
O
[17,99]
Hsp90 inhibitor
O
H
N
O
Me
N
O
Me
OH MeO
Me
MeO
NH2
Me
O
O
Pacritinib (SB1518)
Synthetic macrocycle
O
JAK2/FLT3 kinase inhibitor
[18]
CDK2/JAK2/FLT3 kinase
inhibitor
[20]
O
O
N
N
N
N
H
SB1317/TG02
Synthetic macrocycle
O
NMe
N
N
Key Term
Macrocycle: Molecule with a
ring size of 12 or more atoms.
1410
N
H
inhib­itor pacritinib, now in advanced Phase II
trials [18,19] , the CDK2/JAK2/FLT3 inhibitor
SB1317 in Phase I trial [20] and cilengitide, a syn­
thetic cyclic peptide in Phase III investigations
Future Med. Chem. (2012) 4(11)
for the treatment of glioblastoma [21,22] . The
pan-CDK inhibitor Compound M is a further
example of a synthetic macrocycle proposed as a
development candidate [23] .
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Macrocycles in new drug discovery
| Review
Table 1. Examples of natural and non-natural macrocyclic oncology drugs and drug candidates (cont.).
Name and class
Structure
Cilengitide
Synthetic macrocycle
(cyclic peptide)
Target
Ref.
Anti-angiogenic
O
[21,22]
O
OH
N
NH H
O
HN
NH Me
N
N
H2N
O
NH2
Compound M
Synthetic macrocycle
O
O
N
O
Pan-CDK kinase inhibitor
Me
[23]
N
NH
O
N
O
It has been proposed that one possible barrier
to the wider adoption of macrocyclic scaffolds
in medicinal chemistry has been the percep­
tion that the structural class may be un-drug
like [1] . It is often assumed that natural prod­
uct macro­cycles will tend to violate the recom­
mended ranges in Lipinski’s rule-of-five and
other guidelines for balancing aqueous solubility
and lipid-membrane permeability to achieve oral
absorption [24–29] . However, this is not the case
for many natural products in clinical develop­
ment [30] . Furthermore, synthetic macrocycles in
the medicinal chemistry context that are struc­
turally unrelated to natural products tend to fol­
low the standard rule-of-five guidelines in terms
of their molecular descriptors [1] . The examples
presented in this review have been selected to
N
H
show how macrocyclic scaffolds, rather than pre­
senting an intrinsic liability, can serve as useful
platforms for the engineering of favorable new
properties into biologically active compounds.
The effect of restricting internal degrees of free­
dom upon target affinity and thermodynam­
ics, and how macrocyclization strategies have
improved target potency will be discussed in the
first section of this review, followed by analyses of
the effect of macrocyclization upon target select­
ivity, and changes in physicochemical properties
afforded through macrocycle functionalization.
We will briefly survey common linker motifs
and synthetic strategies used in the preparation
of biologically active macrocycles and conclude
with future perspectives on the further potential
for this approach in drug discovery.
Table 2. Isothermal titration calorimetry data for 1 and 2 , binding to the Grb2 SH2 domain.
HN
(HO)2OPO
NH
O
N
O
O
O
H
N
H
O
HN
HN
N
(HO)2OPO
O
O
HN
NH
NH HN
CONH2
O
1
Compound
Ka (M )
1
(6.5 ± 0.13) × 10
(1.0 ± 0. 20) × 107
2
CONH2
2
DG (kJ mol )
DH (kJ mol )
DS (J mol-1 K-1)
-TDS (kJ mol-1)
-38.9 ± 0.04
-40.2 ± 0.78
-26.4 ± 1.59
-18.0 ± 2.38
41.59 ± 0.71
74.1 ± 5.02
-12.6 ± 0.25
-22.2 ± 1.51
-1
6
O
O
O
-1
N
O
-1
T = 298 K.
Data from [36].
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Key Term
A
Entropy–enthalpy
compensation: Off-setting
of the effect on the Gibbs free
energy (DG) for binding of
changes in entropy terms by
opposing changes in enthalpic
contributions. For example,
reduction in the overall motion
of a receptor, although an
unfavorable entropic change,
may increase the strength of
intermolecular interactions to a
ligand, such as hydrogen bonds,
thus, increasing the favorable
enthalpic contribution.
Cl
C
H
N
O
N
NH
Cl
C6'
O
C2 O
H
N
O
N
NH
N
N
O
C2 O
C6'
N
4
3
D
B
Figure 1. Acyclic and macrocyclic CHK1 inhibitors. (A) Acyclic CHK1 inhibitor (3) and
macrocyclic CHK1 inhibitor formed by linking (C) C2 and C6’(4) . X-ray co-crystal structures of
inhibitors in CHK1: (B) acyclic inhibitor 3 (PDB: 2E9P); (D) macrocyclic inhibitor 4 (PDB: 2E9U).
Polar interactions within the pocket have been highlighted with dashed lines.
Improving potency through
macrocyclization
Conformational restriction, including cycliza­
tion of drug-like molecules, is commonly used
to improve potency towards a target [31] . To
successfully bind to a protein, a molecule has to
adopt a bioactive conformation, and by limit­
ing the number of conformations available to
the unbound molecule, there is a lower entro­
pic cost when the molecule binds. Macrocycles
have restricted internal bond rotations and are,
thus, conformationally constrained, although
not completely rigid. They are potentially
adaptive molecules with enough flexibility to
efficiently interact with flexible binding sites
in proteins, at the same time minimizing the
internal entropy penalty associated with the
change from the unbound to the bound state
of the ligand.
Table 3. Effect of macrocycle size on
potency for CHK1 inhibition.
H
N
O
Cl
NH
N
N
CN
O
O
n
Compound Ring size (n) CHK1 IC50 (nM)
5
6
7
1412
14 (1)
15 (2)
16 (3)
6
7
28
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Entropy is related to other thermodynamic
parameters for reversible binding by the Gibbs
free energy equations (Equation 1) :
DG = DH - TDS = -RTlnK
Equation 1
where DG is Gibbs free energy change on
binding; DH is enthalpy change; T is temp­
erature, DS is entropy change, R is gas constant
(8.13 Jmol-1K-1); and K is binding constant.
A more negative free energy for binding (DG)
results in improved binding affinity (lnK ), but
the individual enthalpic and entropic contribu­
tions to the binding event may compensate [32]
and assignment of the energetics of the binding
event into these two terms is often complex [33] .
While the decrease in ligand entropy on binding
may be unfavorable, the displacement of ordered
water molecules from within an otherwise unoc­
cupied binding site into bulk solvent is generally
assumed to provide a favorable entropic contri­
bution (classical hydrophobic effect) [34] , so
consideration of changes in ligand entropy must
be considered against this background. Prior
macro­c yclization of a molecule may minimize
the unfavorable reduction in internal degrees of
freedom experienced when a ligand binds, com­
pared with an uncyclized analogue. It is also
possible that ligand conformational restrictions,
such as macrocyclization, may result in reduced
overall motion in the bound complex when com­
pared with uncyclized analogues, due to this
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Macrocycles in new drug discovery
| Review
Table 4. SARs of 2,4-diaminopyrimidine CDK and VEGFR inhibitors
Br
N
Br
Br
H
N
N
N
HN
H
N
H
N
N
N
N
N
O
HN
HN
S
9
Compound
8
9
10
11
12
13
H
N
H
N
N
N
HN
S O
O
HN
O
8
Br
Br
NH2
Br
S
N
N
HN
O
S
O
O
10
11
Biochemical target potency
N
HN
HN
S O
O
NH2
O
12
13
Inhibition of cell growth
CDK1 IC50 (nM)
CDK2 IC50 (nM)
VEGF-R IC50 (nM)
MCF7 IC50 (nM)
100
NR
19
20
80
< 10
100
2400
40
140
130
<10
NR
NR
NR
40
140
<10
4000
4000
450
200
1300
500
NR: Not reported.
intrinsic rigidification. Thus, macro­c yclization
could be unfavorable in terms of the reduced
entropy of the bound state of the complex.
However, through entropy–enthalpy compensation, there may be increased enthalpic contri­
butions to binding in a less flexible complex, as
a result of reduced overall motion that increases
the strength of inter­molecular interactions, such
hydrogen bonds [35] . An example of the com­
plexity of analyzing the energetics of macro­
cyclization was reported by DeLorbe et al., who
carried out a study into the effect of macro­
cyclization of a Grb2 SH2 protein–protein
interaction (PPI) inhibitor on the enthalpic and
entropic components of binding using isother­
mal titration calorimetry [36] . Through pair­
wise comparison of acyclic (1) and macrocyclic
(2) ligands (Table 2) , they found that 2 had a
greater favorable entropic contribution to the
free energy of binding (74.1 ± 5.02 Jmol-1K-1)
over 1 (41.4 ± 0.71 Jmol-1K-1), but had a
smaller enthalpic contribution (-18.0 ± 2.38 vs
-26.4 ± 1.59 kJmol-1). From ana­lysis of x-ray cocrystal structures of 1 and 2 , the authors found
that the bound linear peptide 1 in fact formed
an intramolecular hydrogen bond between the
carbonyl oxygen of the phosphotyrosine residue
and the NH of the valine residue, while the
corresponding interaction was missing in the
macro­cycle 2 . The absence of the intra­molecular
interaction suggested that 2 might bind in a
higher energy conformation than the pseudocycle 1, which could explain the observed dif­
ferences in enthalpic contributions. In this case,
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macrocyclization did not necessarily result in the
most favorable constrained conformation, but
as a result of the high positive entropic contri­
bution to binding made by macrocycle 2 , this
ligand was nevertheless the most potent of all
those evaluated.
During the development of a urea-based
inhibitor (3) of CHK1, Tao et al. found from
an x-ray crystal structure that the C2 vector of
the phenyl ring and C6´ vector of the pyrazine
ring were both pointing into the ribose pocket
of the kinase (Figure 1) [37] . In particular, the
C6´ terminal dimethylamine group was close in
space to the C2 methoxy group on the phenyl
ring. The authors hypothesized that macrocy­
clization of the molecule between these posi­
tions would improve the potency of the inhibi­
tor. Compound 3 had an IC50 value of 22 nM,
Figure 2. An x-ray crystal structure of
macrocycle 11 bound to CDK2
(PDB: 2J9M). Hydrogen bonds indicated as
dashed lines.
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Table 5. Enzyme and cellular activity data for acyclic and macrocyclic
CK2 inhibitors.
Compound
Target affinity
Cancer cell growth inhibition
CK2 Ki (nM)
HCT116 IC50 (µM)
PC3 IC50 (µM)
14
0.26
2.3
5.1
15
24
0.29
0.88
while macrocycle 4 had an IC50 value of 10 nM,
representing retention of activity with respect to
the acyclic parent compound. However, com­
pound 3 was observed to form an ionic inter­
action between the tertiary amine and the acid
Glu91 within the ribose pocket, while 4 has an
undecorated alkyl chain and so cannot make this
interaction. This could suggest that an enthal­
pic contribution to affinity has been lost from 4
that must be compensated for, presumably by an
entropic gain attributed to macro­cyclization. An
alternative rationale could be that the protein is
less ordered (thus the entropy is increased) when
4 is bound, due to the loss of the ionic interac­
tion that is seen with 3, hence compensating for
the loss of the enthalpic contribution to bind­
ing. The cyclization reinforces the pyrazine-urea
intra­molecular hydrogen bond that holds the
cis–trans urea hinge-binding motif in the correct
conformation for binding.
It is important to note that macrocyclization
in itself does not guarantee to give the desired
bioactive conformation. It is often the case that
an investigation into macrocycle size is carried out
prior to the synthesis of macrocycles with more
complex functionalization. This is to ensure that
N
A
O
N
C
O
N
HN
N
HN
the initial macrocyclic scaffold is of a correct size
and conformation to be compatible with binding.
For example, a thorough investigation into the
effect of changing ring size was carried out in the
discovery of Hsp90 inhibitors based on the mac­
rocyclic natural product radicicol [38] . Analogues
of radicicol with different ring sizes were synthe­
sized, showing that Hsp90 could accommodate
macrocycles of differing size, which led to the
discovery of a potent inhibitor with a ring size
larger than that of the natural product.
The effect of macrocycle size upon CHK1
potency was investigated on a close analogue of
4, comparing 14- to 16-membered rings (Table 3)
[37] . Computational ana­lysis of the macrocycles
suggested that a ring size of 14–16 atoms would
be ideal – large enough not to be strained and
small enough to avoid clashes with the protein.
The macrocycles synthesized had broadly simi­
lar inhibitory activity, but the larger 16-mem­
bered ring (6) showed slightly less affinity, which
the authors suggested was due to the increased
flexibility of the molecule with more rotatable
bonds in the linker. The result of this flexibility
could be a greater entropic penalty upon bind­
ing that would not be expected of the more
conformationally defined smaller macrocycles.
The reduction in inhibitory activity could also
be due to increased steric clashes with the pro­
tein occurring as a larger alkyl linker has to be
accommodated in the binding cleft.
The activities of the 14- and 16-membered
macrocycles were compared with those of
the acyclic, ring-closing metathesis (RCM)
N
NH
N
N
HN
N
H
N
N
N
H
15
14
D
B
Key Term
Macrocyclic linker:
Structural motif within a
macrocycle that links proximal
regions of the binding
pharmacophore.
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Figure 3. Acyclic and macrocyclic CK2 inhibitors. Structures of (A) acyclic (14) and (C) macrocyclic
(15) CK2 inhibitors. X-ray crystal structures in corn CK2: (B) acyclic compound 14 (PDB: 2PVN);
(D) macrocyclic compound 15 (PDB: 3BE9). Hydrogen bonds are indicated as dashed lines.
Future Med. Chem. (2012) 4(11)
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Macrocycles in new drug discovery
O
O
O
Me
NH2
N
N
N
N
| Review
O
N
16
N
NH2
17
Me
O
N
O
O
N
N
19
O
N
NH2
N
NH2
N
18
Figure 4. Acyclic and macrocyclic b-secretase inhibitors. 3D structure of acyclic inhibitor 18
showing the hair-pin conformation on binding b-secretase (PDB: 2Q15).
pre­cursors, with the conclusion that the macro­
cycles showed significantly more CHK1 inhibi­
tion. This is perhaps unsurprising, as the bioac­
tive conformation for the uncyclized compounds
would require the restriction of the motion of the
flexible chains, with a larger entropic penalty. In
addition, the long chains could potentially intro­
duce steric clashes with the protein. Comparison
to a truncated acyclic analogue would be useful
to assess the contribution of the flexible chains
to binding.
Lücking et al. described the discovery of
macrocyclic inhibitors of CDKs and VEGF-R
tyrosine kinase, starting from the acyclic
2,4-diaminopyrimidine 8 found through a highthroughput screening (HTS) campaign [39] . A
crystal structure of compound 8 bound to CDK2
suggested that macrocyclization could stabilize
the bioactive conformation, thus increasing
binding affinity (Table 4) . Macrocyclization
was achieved by linking the terminal ethyl­
ene to the phenyl ring with a saturated chain
to form 9 ; however, the biological activity was
much lower than the initial hit. Incorporation
of a sulfonamide substituent to 8 gave improved
CDK2 potency in the acyclic molecule 10 due
to the formation of additional hydrogen bonds
to the protein, demonstrated by crystallogra­
phy. Incorporation of either endo- (11) and exosulfonamide (12) groups into the macrocycle
improved potency when compared with the
initial hit 8 and macrocycle 9. Both macrocycles
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11 and 12 were active against VEGF-R, while the
acyclic comparator 8 was not. Lead optimiza­
tion resulted in macrocycle 13 with high potency
against CDK1, CDK2 and VEGF-R. The only
change between macrocycles 11 and 13 is the
exchange of an amino group for a sulfur. The
x-ray crystal structure of 11 (Figure 2) confirmed
that the endo-sulfonamide formed a hydrogen
bond with the protein, contributing to an enthal­
pic gain in potency. The 4-amino substituent of
11 is located within a highly lipophilic region
of the pocket, which might better accommo­
date a more lipophilic sulfur and may account
for the increase in potency of macrocycle 13.
Interestingly, macrocycles 11 and 13, which both
contain an endo-sulfonamide, were more potent
in the cell-proliferation assay than 8, 9 & 12 . This
may be due to a change in solubility or increase
in membrane permeability, but data on physico­
chemical properties of these compounds were
not reported. We suggest that macrocycle 11 may
be an example where a macrocyclic linker, both
increases enthalpic contributions to affinity by
Table 6. BACE inhibition of
compounds 16–19.
Compound
BACE Ki (nM)
16
900
60
11
5
17
18
19
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Table 7. SARs for BACE macrocycles.
R1
n
N
O
O
N
NH2
N
Compound
R1
n
BACE Ki (nM)
19
Cyclohexyl
Cyclohexyl
1
2
1
5
31
17
1
20
1
>1000
20
21
O
22
O
23
O
OH
OH
Table 8. BACE-1 inhibition data for
acyclic (24) and macrocyclic (25)
compounds.
O O
S
NMe
Me
O O
S
NMe
Me
O
O
HN
NHMe
O
OH
O
NH
HN
O
MeO
24
25
Compound
BACE-1 IC50 (µM)
24
>100
2.9
25
O O
S
Me
NMe
O
O
HN
NH
OH
26
Figure 5. Structure and conformation of acyclic inhibitor 26 as bound to
BACE-1 (PDB: 2B8V).
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Future Med. Chem. (2012) 4(11)
OH
forming specific hydrogen bonding interactions
and also reduces the entropic penalty through
conformational restriction; although deter­
mination of thermodynamic parameters would
be required to test this. The compound showed
oral efficacy in vivo in a human HeLa-MaTu
breast cancer xenograft model.
An interesting observation was made during
the discovery of macrocyclic pyrazolo[1,5-a]
[1,3,5]triazine inhibitors of CK2, where the
macrocyclized form 15 was 100-fold less potent
in the enzyme assay when compared with the
acyclic compound 14 , but was tenfold more
potent in a cell-viability assay (Table 5) [40] .
The authors did not speculate as to whether
this was an on-target effect or due to off-target
cytotoxicity, but suggested that it was likely
due to increased cell permeability of the lessplanar conformation brought about by the
macro­c yclic structure, although the membrane
permeability was not experimentally measured.
An increase in the nonplanarity of a molecule
could improve aqueous solubility, as has been
discussed by Ishikawa and Hashimoto [41] and
Lovering et al., who suggested that an improve­
ment in solubility can be achieved by increasing
the number of sp3 over sp2 hybridized atoms
within a molecule [42] .
Crystal structures of compounds 14 & 15
were obtained bound to corn CK2 (Figure 3) .
The two points of connection to form the
macro­c ycle were proximal, as seen in the struc­
ture of the acyclic inhibitor 14. The structure
of macrocycle 15 showed key hydrogen bond­
ing interactions to Asp-175, Lys-68, Val-116
and Arg-43 in the ATP-binding site, while the
alkyl chain occupied a hydrophobic area deeper
within the pocket.
Two recent examples of macrocyclization
in a non-oncology context are provided by the
discovery of inhibitors of the aspartyl prote­
ase, b-secretase (BACE). From an ana­lysis of
the crystal structure of bound BACE inhibi­
tor 16, Huang et al. found that the molecule
adopted a hair-pin-like bioactive conformation
(F igure 4) [43] . They reasoned that formation
of a macrocycle would improve binding affin­
ity to BACE through conformational restric­
tion. Following in silico evaluation of several
macrocycles, compound 17 was synthesized
and found to be 150-fold more potent than
its acyclic progenitor (Table 6) . Previous work
had revealed that an (s)-cyclohexyl group
improved potency through occupancy of the
hydro­phobic S1´ pocket of the enzyme, as in
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Macrocycles in new drug discovery
compound 18 [44] , and macrocyclization of this
molecule to 19 slightly increased the potency of
BACE inhibition. There are as yet no published
crystal structures of the macrocycles in BACE
to confirm the binding mode predicted in silico.
An investigation into the size and functional­
ization of macrocycle 19 was carried out, with
selected examples shown in Table 7. The smaller
macro­c ycle size was optimal (n = 1), with the
lipophilic cyclohexyl group of 19 the best toler­
ated amide substituent (R1). Compound 19 was
a potent inhibitor of amyloid-b (Ab) protein
secretion from engineered CHO cells, an activ­
ity assigned, in part, to the presence of the two
lipophilic substituents conferring high cellular
permeability. Compounds from this macro­
cyclic series reduced Ab levels in plasma when
tested in vivo, but only trace levels were detected
in mouse brain, and no effect was seen on brain
Ab production. Preliminary data suggested the
compounds were P-glycoprotein transporter
substrates, accounting for the low brain pen­
etrance. This perhaps indicates a potential chal­
lenge with macrocyclic scaffolds, as with other
ligand classes when the compounds have a high
degree of lipophilic substitution.
Stachel et al. have also reported an example
of dramatically improved potency on macro­
cyclization of an acyclic BACE-1 inhibitor 24
(Table 8) [45] . A crystal structure of 26, an ana­
logue of the lead acyclic compound 24, clearly
showed that the phenyl rings were proximal and
in an orientation appropriate for macrocycliza­
tion (Figure 5) . The prototype macrocycle 25
was shown to have a 34-fold higher inhibitory
activity against BACE-1 than the acyclic ana­
logue, with a simple ethyl bridge forming the
linker. This is an example of a well-matched
acyclic and macrocyclic pair, where both
compounds have the same number of heavy
atoms, with minimal structural rearrange­
ment between them, and comparison shows
that macro­c yclization has a significant impact
upon the inhibition of BACE-1. The further
progression of this series is discussed in the sec­
tion devoted to the effects of macrocyclization
on physicochemical properties.
As shown above, the potency of inhibitors
can be retained or improved upon appropri­
ate macrocyclization. In the example of the
CHK1 macrocycle, inhibition was retained
at the same level as the acyclic analogue even
with the removal of a potentially important
ionic interaction to a protonated tertiary amine.
The CDK inhibitor macrocycles reported by
future science group
| Review
Table 9. Basic selectivity data for bisindolylmaleimide macrocycles.
Structure
Target
Pan-kinase
H
N
O
Staurosporine (27)
N
Me
N
O
H
MeO
NHMe
N
H
Cl
Pan-kinase
H
N
O
HO
O
Rebeccamycin (28)
N
H Cl
OH
O
OH
MeO
H
N
O
PKCb
IC50 = 5 nM
O
Ruboxistaurine (29)
N
N
O
NMe2
H
N
O
O
H
anti-conformer (30)
GSK3b
98% at 10 uM
Ki = 200 nM (estimated)
n = 4
H N
N
n = 2– 4
H
N
O
PKCb
Ki = 45 nM
n = 6
O
H
N
syn-conformer (31)
H N
n = 5–6
H
N
O
O
H
N
32
H N
N
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GSK3b
IC50 = 7 nM
PKCb
IC50 = 2400 nM
NMe2
1417
Review | Mallinson & Collins
Table 10. Structure–activity data for 2,4-diaminopyrimidine macrocyclic inhibitors
of CDK1, CDK2 and VEGFR.
Br
Br
N
H
N
H
N
N
N
HN
S O
O
HN
Br
N
N
HN
O
S
O
11
Compound
Br
H
N
N
N
N
HN
NH2
S
O
O
12
Me
H
N
N
O
S
33
Biochemical target potency
HN
HN
S O
O
34
Inhibition of cell
growth
CDK1 IC50 (nM)
CDK2 IC50 (nM)
VEGF-R IC50 (nM)
MCF7 IC50 (nM)
11
20
140
40
200
12
80
130
140
1300
33
NR
1000
970
6000
34
15
28
540
300
NR: Not reported.
Lücking et al. show that the macrocyclic linker
can both provide conformational restriction and
additional enthalpic contributions through the
integrated sulfonamide hydrogen bonding to
the kinase [39] . There are arguably still limited
examples where the manipulation of a macro­
cyclic linker is used to introduce new enthalpic
binding elements not present in the acyclic pre­
cursor, rather than acting solely to reduce entro­
pic penalties. Such scaffolds would likely have
higher ligand efficiencies, favorable for further
elaboration [46] . In all these examples, the avail­
ability of structural information on the bound
conformation of acyclic precursors was critical
in establishing the macrocyclization strategies.
Modulating selectivity through
macrocyclization
Staurosporine (27) and rebeccamycin (28) are
highly potent pan-kinase inhibitors, with the
planar indolocarbazole lactam ring system
mimicking the adenine ring found in ATP
(Table 9) . Breaking the link between the fused
indoles to form a macrocycle disrupted the pla­
narity, resulting in ruboxistaurin (29) , a selective
inhibitor for the b-isoform of PKCb [47] . The
kinase selectivity could be further modulated
by changing the length of the tether. Shorter
chains caused a conformational change of the
bisindoles to prefer the anti-conformer 30, pro­
viding some selectivity towards GSK3b inhibi­
tion. Longer chains preferred a syn-conformer
31, resulting in enhanced PKCb inhibition [48] .
1418
Future Med. Chem. (2012) 4(11)
The 2-dimethylaminopyridine analogue 32 also
showed a syn-conformation, but now with high
selectivity towards GSK3b over PKCb. These
examples show how macrocycle formation can
modulate the selectivity of a promiscuous kinase
inhibitor scaffold.
As described previously, Lücking et al. demon­
strated the use of macrocyclic aminopyrimidines
as multi-targeted CDK and VEGF-R inhibitors,
with cyclization resulting in potent inhibitors.
They also showed that subtle changes in selec­
tivity could be achieved upon modification of
the connectivity of the macrocyclic scaffold [39] ,
through positioning of the potency-enhancing
sulfonamide group (Table 10) . Moving from
an exo-sulfonamide 12 to the tertiary methyl­
sulfonamide 33 gave a large reduction in affinity.
Although the authors did not comment on this
result, the fall in activity could be due to losing a
hydrogen bond to a sulfonamide oxygen, which
was observed in the crystal structure of macro­
cycle 11 (Figure 5) [39] . Exchange of the disub­
stituted phenyl group within the macro­c yclic
framework for a thiophene generated macro­cycle
34, which brought modest selectivity towards
CDK1 and CDK2 over VEGF-R, while remain­
ing potent in the cellular assay ( 34 compared
with 11). It is worth noting that macrocycle 34
is one atom larger than all others within the
2,4-diaminopyrimidine series described. Small
differences in macrocycle size have been shown
to affect enzyme inhibition potency, as seen with
CHK1 macrocyclic ureas [37] .
future science group
Macrocycles in new drug discovery
| Review
Michael
acceptor
OH
Me
O
O
OH
Cl
OH
O
O
O
HO
Me
O
O
MeO
O
OH
OH
O
Radicicol 35
Selective HSP90 inhibitor
R3
X
RO
X
n R2
R1
General scaffold
Cysteine-containing kinase inhibitor
LL-Z1640-2 36
Selective kinase inhibitor
(TAK1)
Figure 6. Transformation of Hsp90 inhibitor radicicol to an irreversible kinase inhibitor through incorporation of a Michael
acceptor.
The kinase selectivity profiles of resorcylic
acid lactone analogues have been described,
showing that the ATPase-selective inhibi­
tor scaffold based upon radicicol (35) can be
modified to irreversibly inhibit protein kinases
instead (36) [49,50] . The ATP-conformation
adopted within the ATPase active site is distinct
from that of a protein kinase, thus high crossreactivity between synthetic small-molecule
reversible, ATP-competitive inhibitors of
ATPases and kinases is uncommon. Resorcylic
macrolides with changes in functionalization of
the macrocycle to incorporate a Michael accep­
tor showed that the scaffold could be modified
to inhibit kinases (Figure 6) . Irreversible inhibi­
tion of selected protein kinases arises by reaction
with an appropriately placed cysteine residue in
the ATP-binding site to form a covalent adduct.
Selectivity screening against a panel of kinases
revealed that most of the resorcinol analogues
inhibited kinases containing a cysteine residue.
Similarly, 84 analogues of the natural prod­
uct pochonin A (37) , also an Hsp90 inhibitor
[51] , were prepared and evaluated for kinase
selectivity [52] . The authors found that 12 of
these analogues showed >50% inhibition of
the kinases evaluated at 10 µM, while show­
ing no inhibition of Hsp90. For example, ana­
logue 38 inhibited 16 out of 24 kinases with an
IC50 <40 µM (Figure 7) . This exemplifies how
macro­cycle functionalization can not only direct
kinase selectivity, but also selectivity between
target classes.
During the discovery of antihypertensive
agents that target the renin–angiotensin–
aldosterone system, Sund et al. proposed that
a suitable agent should have desirable physico­
chemical properties and be a potent inhibitor
of the renin aspartyl protease, with good select­
ivity over cathepsin-D (Cat-D) and BACE-1
[53] . The group had already synthesized and
future science group
tested the linear peptidomimetic 39 [54] that
had good activity against renin, but also against
Cat-D and BACE-1, along with poor DMPK
properties, namely low permeability and high
human liver microsome metabolism, which are
typically associated with linear peptide inhibi­
tors [54,55] . The important areas contributing to
binding interactions were known for the renin
binding-site and linear peptide inhibitors, so
computational methods were used to determine
the ideal macrocycle size. Although details of
the modeling were not reported, a number of
15- or 16-membered macrocycles were synthe­
sized and tested; selected data are summarized
in Table 11.
Direct macrocyclization with an amide linker
to form macrocycle 40 reduced potency, with
no change in selectivity. Upon addition of the
N-benzylvaline group at R 2, with phenyl sub­
stitution restored at R1, 41 became a pico­molar
inhibitor of Cat-D and a potent inhibitor of
renin, while selectivity over BACE was intro­
duced. Truncation of the valine residue at R 2
to a methyl group (42) introduced select­ivity for
renin over both anti-targets by more significantly
reducing Cat-D and BACE-1 inhibition than
renin inhibition. When the amide macrocyclic
OH
Me
O
O
HO
OH
O
O
HO
Cl
O
pochonin A (37)
Hsp90 inhibitor
O
OMe
Cl
O
38
Kinase inhibitor
Figure 7. Natural product Hsp90 inhibitor pochonin A that was
transformed into a kinase inhibitor by changing macrocycle
functionalization.
www.future-science.com
1419
Review | Mallinson & Collins
Table 11. Structure–activity data for acyclic and macrocyclic renin inhibitors.
O
Me
N
S
Me
Me
H
N
Me
O
O
OH
H
N
O
O
H
N
S
O
Me
O
N
H
O
O
N
OH
H
N
X
O
Y
H
N
R2
O
O
R1
39
Compound
40-44
X
Y
R
R
C=O
C=O
C=O
CH2CH2
CH2
NH
NH
NH
CH2
CH2
Me
Ph
Ph
MeO(CH2)2O
MeO(CH2)2O
N-benzylvaline
N-benzylvaline
Me
Iso-butyl
Iso-butyl
1
Renin Ki (nM) Cat-D Ki (nM) BACE-1 Ki (nM)
2
39
40
41
42
43
44
21
3.3
30
94
3.3
340
940
>5000
13
0.75
1700
>5000
>5000
50
240
>10000
>10000
>10000
linker was replaced with an alkyl chain (43) ,
Cat-D and BACE-1 activity was removed com­
pletely; however, this was also at the cost of
potent renin inhibition. The smaller macro­
cycle 44 was inactive towards all three enzymes.
In this example, while macrocyclization of 39
modulated the selectivity profile of the scaffold
(compare 39 & 41), this was mainly sensitive
to modification of pendant groups outside the
macrocyclic linker, particularly at R1. Since
BACE and Cat-D appeared to be less tolerant
than renin of the lipophilic alkyl linker of 43,
Table 12. Initial acyclic ALK inhibitor (45) and general structures for subsequent
analogues (46–54) , with structure–activity data for ALK inhibition and selectivity
over insulin receptors.
Cl
N
HN
N
NH
MeO
Cl
N
HN
N
NH
HN
R
OMe
Cl
N
2
N
NH
2
1
R
R
X
N
MeO
N
N
N
O
N
Me
N
Me
45
Compound
X
46–47
48–54
R
R
H
H
OMe
H
OMe
H
OMe
H
OMe
H
OMe
H
N(Me)SO2Me
N(Me)SO­2Me
OMe
N(Me)SO2Me
1
2
45
46
47
48
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH=CH
CH=CH
49
50
51
52
53
54
ALK IC50 (nM) IR/ALK (-fold
selectivity)
7
67
22
92
3.1
5
3.4
0.51
259
7
1.3
3.6
2.8
67
58
75
173
>11
>140
IR: Insulin receptor.
1420
Future Med. Chem. (2012) 4(11)
future science group
Macrocycles in new drug discovery
| Review
Table 13. Kinase inhibition data for macrocyclic inhibitors of CDK2/JAK2/FLT3
O
O
HN
NH
O
NMe
NMe
N
N
H
N
N
N
H
55
N
H
N
56
N
H
N
57
O
58
O
N
N
N
CF3
O
N
N
NMe
NMe
N
N
N
O
N
H
O
N
N
N
59
N
N
H
60
Compound
CDK2 IC50 (nM)
JAK2 IC50 (nM)
FLT3 IC50 (nM)
55
4600
>10,000
13
660
>10,000
310
>10,000
>10,000
73
1700
220
150
2750
>10,000
56
210
110
29
56
57
58
59
60
it is possible that further modification of the
macrocyclic linker itself could also modulate the
selectivity within this group of targets.
Breslin et al. have discussed the discov­
ery of new anaplastic lymphoma kinase
(ALK) inhibitors based upon the acyclic
2,4-diaminopyrimidine inhibitor 45 (Table 12)
[56,57] . X-ray crystallography showed that the
conformation of 45 adopted a U-shape on bind­
ing to ALK, so it was suggested that macro­
cyclization would improve potency by locking
the molecule in this bioactive conformation.
In silico modeling was performed to find the
optimum macrocycle size for the simplified
scaffolds 46 and 47. By overlaying energy-mini­
mized structures of potential macrocycles with
the acyclic molecules, it was determined that an
ethylene linker between the two phenyl rings
provided the optimum macrocycle size.
Selectivity for ALK over the insulin receptor
tyrosine kinase was of importance for this class
of inhibitors. As shown in Table 12 , the direct
macrocyclic analogue 48 of the undecorated
scaffold 46 was of equal potency, with slightly
improved selectivity. The effect of macro­
cyclization on selectivity was more significantly
demonstrated by comparison of the more potent
future science group
methoxy-substituted acyclic analogue 47 to the
macrocycle 49, where a 20-fold improvement
in selectivity was attained. The authors sug­
gested that this confirmed that macro­cyclization
restrained the molecule preferentially in a con­
formation for ALK binding. Further gains in
potency and selectivity were achieved by optimi­
zation of peripheral substituents. Thus, addition
of a methyl sulfonamide group greatly improved
potency into the picomolar range (52) , with high
selectivity over insulin receptor. Macrocycles 53
and 54 contained a double-bond linker, which,
generally, decreased potency but increased
selectivity.
In this example, macrocyclization clearly
allowed an improvement in selectivity between
two specific targets, although to gain insight
into the effect of the macrocyclization on the
most potent inhibitors in the series, data for an
acyclic example corresponding to 51 would be
desirable. As the authors point out, the addition
of the methyl sulfonamide substituent could be
the main factor driving increased potency and
the macrocyclization provides additional control
over selectivity.
William et al. showed that subtle linker
variation in a series of macrocycles can lead to
www.future-science.com
1421
Review | Mallinson & Collins
Table 14. Selected SARs for acyclic (61) and macrocyclic (62–68) Hsp90 inhibitors, with varying ring size and linker
functionalization.
NH2
H
N
O
N
Me
Me
Me
O
O
Me
Me
NH
N
O
NH2
H
N
Me
Me
Me
Me
Me
Me
O
Me
O
NH
O
O
65
Me
O
O
67
66
N
Me
Me
Me
Me
NH2
H
N
N
N
Me
Me
N
Me
O
NH2
H
N
Me
N
NH
64
R
N
Me
O
O
NH2
H
N
Me
N
Me
Me
NH
63
62
O
N
Me
Me
O
Me
O
61
Me
Me
Me
Me
O
N
NH2
H
N
H
N
H
N
Me
O
NH2
NH2
68
Compound Ring size
Target activity Cell growth
Aqueous
Hsp90 IC50 (µM) inhibition
solubility
HCT116 EC50 (µM) (µg/ml)
Rat liver
cLog P
microsomal
stability t1/2 (min)
62
11
12
13
12
12
12
12
12.44
0.14
0.29
0.15
0.11
0.082
0.096
15
15
3
11
9
>30
6
63
64
65
66
67
68
>100
52
6
31
19
>100
2324
0.08
0.21
0.08
0.15
0.056
0.032
3.1
4.4
4.7
4.4
2.8
3.5
4.3
Table 15. Biological and physicochemical profile of two optimized macrocyclic Hsp90 inhibitors.
O
NH2
H
N
Me
O
Me
O
N
N
Me
H
N
Me
Me
O
NH2
N
N
Me
Me
NH2
H
N
Me
Me
Me
O
Me
O
69
70
Compound
Hsp90
IC50 (µM)
HCT116
EC50 (µM)
Aqueous
Microsomal
solubility (µg/ml) stability t1/2 (min)
Cyp inhibition
(% at 3 µM)
cLog P
69
0.068
0.037
>100
4.1
70
0.083
0.063
10
2 (2C9)
0 (2D6)
56 (3A4)
6 (2C9)
0 (2D6)
68 (3A4)
1422
Future Med. Chem. (2012) 4(11)
>30 (Human)
>30 (Mouse)
>30 (Rat)
>30 (Human)
>30 (Mouse)
>30 (Rat)
3.4
future science group
Macrocycles in new drug discovery
significant changes in kinase inhibitor selectiv­
ity profile [20] . While investigating macrocyclic
multi-targeted CDK2/JAK2/FLT3 inhibi­
tors (Table 13) such as 55, the authors found
that reversing the amide connectivity within
the linker removed activity against all three
kinases tested (56) . The phenolic linker 57
conferred much increased potency at all three
targets with some CDK2 selectivity. Selectivity
over JAK2 could be achieved by replacing the
phenolic ether 57 with a benzylic ether linker
58, although with a general decrease in potency.
Docking experiments were conducted to com­
pare 57 and 58 within CDK2, leading to the
suggestion that the drop in activity of 58 against
CDK2 was due to the ether oxygen clashing
with a backbone carbonyl.
Exploration of a wide range of linker amine
substitutions was carried out, described in more
detail in the section of this review devoted to
physicochemical properties, but no significant
changes in selectivity were observed. Larger
N-alkyl and N-aryl groups tend to be better
tolerated by CDK2, whereas these same groups
resulted in a slight loss of activity for JAK2 and
FLT3. When the aniline ring was substituted
with a pyrrolidinyl side chain, as in 59 and 60,
variation of the group appended to the nitro­
gen in the macrocyclic linker then provided
selectivity over CDK2, with the trifluoroethyl
group (59) removing all CDK2 inhibition,
while 60 had tenfold selectivity for JAK2 over
both CDK2 and FLT3. Optimization of this
series for in vivo properties led to the selection
of 57 (SB1317/TGO2, see Table 1) as a clinical
candidate for the treatment of leukemia.
The examples above show that macrocycle size
can modulate selectivity, as seen with the renin
inhibitors. Subtle differences in the macro­c yclic
linker will affect selectivity profiles; for example,
by introducing changes in the preferred ligand
| Review
Tyr-138
Lys-58
Asn-51
Asp-54
Thr-183
Figure 8. An x-ray crystal structure of macrocyclic Hsp90 inhibitor 70,
showing that the macrocyclic linker orientates the exocyclic amide to
interact with an extensive water-mediated hydrogen bonding network.
(PDB: 3RKZ).
conformation or introducing clashes with spe­
cific targets. There are as yet relatively few
examples detailing how macrocyclization may
directly affect the intrinsic selectivity profiles of
inhibitor scaffolds, as demonstrated for the ALK
inhibitors above. Tao et al. provided selectivity
data across a panel of kinases for three macro­
cyclic CHK1 inhibitors [37] , but no comparison
to acyclic analogues was included. More gen­
erally, comparison of matched pairs of acyclic
and macro­c yclic compounds screened against a
larger number of targets, such as a kinase panel
or a broader pharmacology screen, would be
Table 16. Solubility and activity data for macrocyclic CDK inhibitors.
O
N
X
N
Me
O
N
O
N
H
Compound
X
Solubility in saline Target activity
(µg/ml)
CDK4 IC50 (nM)
Cellular activity
E2F reporter
IC50 (nM)
cLogP
71
S
NH
20
990
13
77
2.30
0.96
72
future science group
11
13
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1423
Review | Mallinson & Collins
Table 17. Inhibitory and solubility data for macrocyclic JAK2/FLT3 inhibitor analogues.
O
O
R1
N
N
Compound
73
R1
N
N
Me
74
O
N
H
Solubility
(µg/ml)
cLogP
CDK2
IC50 (nM)
JAK2
IC50 (nM)
FLT3
IC50 (nM)
26
3.8
>10
41
30
150
3.2
3700
84
70
171
4.0
88
77
31
147
4.5
4700
24
29
>150
4.1
3900
23
22
OH
75
Me
N
76
O
77
O
NMe2
Me
N
Me
N
useful to assess if systematic changes in selec­
tivity can be brought about by macrocyclic
incorporation of pharmacophores.
Modulation of physicochemical &
ADMET properties
A recent series of publications from Zapf et al.
described the discovery of macrocyclic inhibi­
tors of the ATP-dependent molecular chap­
erone Hsp90, a well-established target in
oncology [58–61] . The authors described the
effect of varying a macrocyclic linker (Table 14)
in the previously disclosed [62] acyclic scaffold
61 upon several properties, including compre­
hensive data on Hsp90 inhibition, cell-growth
inhibition, aqueous solubility and microsomal
stability. Initial investigations to find the opti­
mum linker length were carried out by syn­
thesizing macrocycles of 11–13 atoms that
incorporated an amine within the linker, as
Table 18. Solubility and kinase inhibition data for N-substituted macrocycles.
O
N
R1
N
N
Compound R1
Solubility
(µg/ml) †
CDK2
IC50 (nM)
JAK2
IC50 (nM)
FLT3
IC50 (nM)
78
14.7
71.8
13.2
21
13
200
150
73
1500
110
56
190
105.1
530
120
140
79
H
Me
80
81
†
1424
N
H
O
H
N
Solubility in phosphate buffered saline +20% dimethylsulfoxide.
Future Med. Chem. (2012) 4(11)
future science group
Macrocycles in new drug discovery
shown in Table 14. Structural inform­ation on
the binding of the acyclic scaffold to Hsp90
informed the design the macrocycles, and crys­
tal structures of the compounds confirm­ed the
tether was oriented toward solvent, with the
basic nitrogen atom participating in watermediated hydrogen bonding to the protein
[58,59] . A wide range of potencies, aqueous solu­
bilities (from <10 to >100 µg/ml) and meta­
bolic stabilities were observed depending on
the length, constitution and substitution of the
macrocyclic tether. Although SARs appeared
complex, potency was sensitive to the degree and
chirality of alkyl substitution in the linker, sug­
gesting changing conformational preferences, as
well as steric clashes could drive affinity within
the series. Twelve-membered macrocycles were
determined as the most favorable platform for
multiparameter optimization. Interestingly, the
aqueous solubility and rat liver microsome sta­
bility of this series decreased as the macrocycle
size increased, exemplified by compounds 62–64
(Table 14) . Although, in part, reflecting increas­
ing lipophilicity with increasing ring size and
substitution, this may also indicate the smaller
macrocycle forcing the biaryl system to twist
and disrupt planarity, which has been shown to
improve solubility [41] . The larger macrocycles
should allow a more planar biaryl system.
The authors found that increasing the rigidity
of the macrocycle increased cell growth inhibi­
tion, so focused their attention upon incorpo­
rating rigidifying elements into the macrocyclic
linker. This may suggest that decreased rotational
degrees of freedom favored cell permeability, as
suggested in a previous ana­lysis of the factors
affecting oral absorption of drug molecules [28] .
Despite the observation of a water-mediated
contact to the protein, substitution of the linker
| Review
Table 19. Calculated ligand efficiencies for macrocyclic
CDK2/JAK2/FLT3 inhibitors.
Compound
Solubility
(µg/ml)
CDK2 LE†
JAK2 LE
FLT3 LE
78
14.7
71.8
105.1
171
147
<150
0.40
0.39
0.26
0.29
0.21
0.22
0.35
0.36
0.28
0.29
0.30
0.31
0.36
0.36
0.28
0.31
0.30
0.31
79
81
75
76
77
LE: Ligand efficiency = (-1.4*(logIC50 (M)/Heavy atoms) [46].
†
Me
Me
N
N
O
O
N
Me
Me
O
Me
H
N
N
O
O
H
N
Me
N
O
O
N
Me
O
Me
O
N
H
HO
Me
O
N
H
N
Me
O
82
Figure 9. Immunosuppressive natural product drug cyclosporin A.
Table 20. PAMPA data for linear and cyclic peptides.
Compound
Sequence
82 (Cyclosporin A)
-6.6
83 (Cyclic)
Cyclo[d-Leu- d-Leu-Leu- d-Leu-Pro-Tyr]
Ac- d-Leu- d-Leu-Leu- d-Leu-Pro-Tyr-OAllyl
84 (Linear)
†
Log P E
-6.2
<-8.1†
Outside assay range (-8.1 assay limit).
Table 21. Human liver microsome stability data for resorcylic macrolactone
analogues.
OH
O
O
HO
Cl
OH
Me
Radicicol 35
OH
O
O
O
N
HO
Me
HO
Cl
O
O
Cl
O
O
86
85
Compound
HLM% metabolized after 15 min
HLM% metabolized after 30 min
35 (Radicicol)
85
83.7
97.0
42.5
83.4
90.0
66.5
86
HLM: Human liver microsome.
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Review | Mallinson & Collins
Me
O
S
Me
N
OH
Me
Me Me
O
Me
Me
N
Me
Me
Me
HN
Me
OH
O
Me
O
S
Me
O
Me
O
87
Epothilone B
(Patupilone)
OH
O
OH
88
Aza-epothilone B
(Ixabepilone, lxempra®)
Figure 10. Structures of epothilone B (patupilone, 87) and aza-epothilone
B (ixabepilone, 88 ).
nitrogen allowed the modulation of hydrogen
bonding capability and basicity without reduc­
ing enzyme or cell-growth inhibition as seen for
compound 66. This was important, as unsub­
stituted basic amines tend to show unacceptable
inhibition of the human ether-a-go-go related
gene (hERG) potassium ion channel. Formation
of lactams such as 67 further improved enzyme
and cell-growth inhibition, while also increasing
microsome stability, aqueous solubility and
Table 22. Inhibition and parallel artificial membrane permeability
assay (apparent permeability, Papp) data for BACE-1 macrocycles.
O O
S
NMe
Me
O O
S
NMe
Me
NH
O
O
O
O
HN
OH
NH
HN
N
H
O
O
89
H
N
O
90
O O
S
NMe
Me
O O
S
N
Me
Me
Me
O
HN
O
N
H
H
N
HN
O
Compound
BACE-1 IC50 (nM)
89
2900
90
32
90
4
92
1426
H
N
O
92
91
91
N
H
Cell IC50 (µM)
Papp (10 -6 cm s-1)
5.4
1.1
0.076
0.5
19
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Future Med. Chem. (2012) 4(11)
reducing hERG activity ( 67, hERG IC50
>33 µM). The decreased hERG inhibition is
likely to be the result of removing the basic­
ity of the amine. However, it has been shown
in other studies that conformational restric­
tion can also alter ion channel activity profiles
[63,64] . Additional rigidification of the linker
was achieved by incorporating a pyrrolidine
or piperidine ring into the tether; however, in
general, Hsp90 inhibition remained the same,
along with a decrease in aqueous solubility and
microsomal stability. Pyrrolidine 68 showed high
cell-growth inhibition and demonstrated in vivo
efficacy in a xenograft model, suppressing tumor
growth at 100 mg/kg IV when dosed twice
per week.
The proline amide 69 (Table 15) displayed a
highly desirable biological and physico­chemical
profile, with high enzyme and cell growth
inhibition, high aqueous solubility and high
microsomal stability. Interestingly however,
the alanine analogue 70, which had a similar
profile but with substantially reduced aqueous
solubility, was progressed to in vivo studies and
showed significant tumor growth suppression
at 12.5 mg/kg intravenously, with once-weekly
dosing.
This thorough investigation showed that
the physicochemical and metabolic properties
of the inhibitors could be significantly altered
with subtle variation of the macrocyclic linker,
without detriment to enzyme or cell growth
inhibition. However, as acknowledged by the
authors, it is not straightforward to rational­
ize why the physicochemical properties change
with varying macrocycle functionalizations.
Additional comparison to acyclic analogues
(e.g., the initial core scaffold 61) would fur­
ther aid in highlighting the benefits that the
macrocyclic scaffolding provided across the
multiple parameters measured. In the original
amine-containing compounds, it is likely that
macrocyclization improved Hsp90 inhibitory
activity through conformational restriction and
by orienting the amine within the macrocyclic
tether to interact with the protein through a
water-mediated hydrogen bond. In the lactamand amide-substituted tethers, although this
interaction is removed, affinity is retained as the
amide instead participates in other inter­actions
to the complex water-mediated hydrogen
bonding networks in the protein, as seen in the
crystal structure of 70 (Figure 8) .
Kawanishi et al. showed how standard
strategies for improving solubility could be
future science group
Macrocycles in new drug discovery
incorporated into the elaboration of macrocy­
clic CDK1,2,4,6 inhibitors [65] . Exchanging
sulfur for nitrogen in 71 improved the solu­
bility of the macrocycle 72 in saline by nearly
50-fold (Table 16) . The authors suggested that
this improvement was due to the increased
hydrophilic nature of the compound, which is
supported by the calculated LogP of the com­
pounds. A consequence of the reduced lipophi­
licity can also be seen in the cellular potency,
with nearly a sixfold loss of activity, despite
maintained CDK4 inhibition in vitro, suggest­
ing that, in this case, the increased polarity also
compromised cell permeability.
William et al. reported the discovery and
development of macrocycles to inhibit JAK2
and FLT3, kinases that are implicated in myelo­
fibrosis and lymphoma [66] . The core macro­cyclic
scaffold was established by macrocyclization
of an HTS hit. During the lead optimization
of this macrocycle, selected compounds were
evaluated for their physicochemical properties,
which included solubility, passive membrane
permeability, microsomal stability and CYP450
inhibitory activity (Table 17) . All of these ana­
logues, except 73, have good solubility profiles.
In this case, the only change in the structure
was the pendant solubilizing group and not the
core macrocyclic scaffold, highlighting that wellestablished medicinal chemistry strategies can be
productively applied to macro­cyclic compounds.
Notably, the structure of the solubilizing group
also affected CYP450 interactions such that,
for example, 77 showed no CYP450 inhibition
(>5 µM) while 76 inhibited the 3A4 isoform
(IC50 = 2.8 µM). Orally bioavailable compound
77 was selected as a candidate for further develop­
ment, under the name pacritinib (SB1518), and
is currently in Phase II trials (Table 1) . A further
publication from the same authors presented the
optimization of the scaffold towards the inhibi­
tion of JAK2, FLT3 and CDKs [20] , as discussed
above in the section, ‘Modulating selectivity
through macro­cyclization’. A small modification
was made to the macrocyclic scaffold, exchang­
ing the ether for a basic amine. This change was
rationalized by in silico docking to be favorable
for CDK inhibition, through establishing an
important charge-assisted hydrogen bonding
interaction between the basic amine and Asp86
in the ATP site of CDK2. Following optimi­
zation of the linker, further substitution of the
amine was investigated, with a small selec­
tion of compounds undergoing solubility ana­
lysis (Table 18) . The macrocycles with highest
future science group
| Review
Table 23. Common linking motifs in the
examples highlighted in this review.
Linker motif
Y
X
Pharmacophore
Linker motif
Ref.
[37]
[37,57]
[60]
Me Me
[60]
Me
Me
[39]
HO
*
[38,83]
O
[60,65]
R
N
R = H, Me, Ac
[40,61]
O
N
H
H
N
H
N
O
H
N
O
[39]
S
O
[58,65]
X
H
N
*
[60]
N
X = CH2, NH, CH(CH3)
solubility were either a simple tertiary amine,
or an amide with a secondary amine substitu­
tion. Larger lipophilic groups reduced solubility
and, in most cases, also reduced target potency.
Compound 79 (SB1317/TG02) was selected for
development.
Comparing the two sets of compounds above,
in Table 19 the authors have calculated the ligand
efficiencies [46] for several of the most soluble
macrocycles in each class, showing that the
inhibitors with the nitrogen integral to the linker
have generally higher ligand efficiencies than the
macrocycles with appended solubilizing groups.
This suggests the potential benefit of a pharma­
ceutically and pharmacologically functional
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Review | Mallinson & Collins
macrocyclic linker when converting linear
precursors to their macrocyclic equivalents.
Rezai et al. investigated the prediction and
experimental determination of the membrane
permeability of cyclic peptides [67,68] , with the
most commonly used molecular descriptors in
predictive models of membrane permeability
being molecular weight and solvent-exposed
polar surface area. The authors suggested that
most cyclic peptides have poor membrane per­
meability, but there are notable exceptions,
including the orally active immunosuppressive
drug cyclosporin A (82; Figure 9) , which binds
to intracellular proteins. Cyclosporin A is a very
large molecule, violating the rule-of-five condi­
tions for high passive permeability [24–29] , yet it
is still an orally bioavailable drug. The backbone
of the macrocycle is highly N-methylated and
NMR studies have shown that in chloroform,
cyclosporine A adopts a conformation where
the remaining N–H bonds are involved in
intra­molecular hydrogen bonding. In aqueous
solution, the molecule changes its conformation
to allow the N–H bonds to interact with the
solvent [69–71] . It is this conformational switch­
ing to bury the N–H bonds when in a hydro­
phobic environment that permits such a large
compound to become membrane permeable,
transiently reducing the total polar surface area.
A small set of cyclic peptides was synthesized
and passive permeability was evaluated using
PAMPA (Table 20) [68] . As hypothesized, the lin­
ear peptide 84 was the least permeable, while its
cyclized form 83 was the most permeable of all
those tested, comparable to that of cyclosporin A
(82) . The NMR solution structure of 83 showed
extensive intramolecular hydrogen bonding
by the amide NH groups, and only one NH
exchanged rapidly in D2O, indicating that the
permeability of 83 was associated with masking
of the polar functionality.
Further investigation of the effect of macro­
cyclization upon membrane permeability was
carried out by first evaluating a virtual library
of cyclic peptides to determine a calculated DG
for transferring a low-energy conformation from
water to a hydrophobic environment, correlated
with the number of internal hydrogen bonds [67] .
The results were compared with experimental
PAMPA data, finding a strong linear correlation.
The authors identified caveats associated with this
study, in particular that the calculations do not
consider entropic losses of the peptides moving
into the hydrophobic environment. There is the
possibility of extending this hypothesis that mac­
rocyclization improves membrane permeability
further to include more drug-like macrocyclic
molecules, which could be used as a strategy for
improving cellular permeability. Terrett et al.
have demonstrated that this is a potentially viable
approach in drug discovery [72] . Their macro­
cyclic peptides, or ‘ensemblins’, contravene the
rule-of-five, yet are potent and cell membranepermeable compounds. Bogdan et al. have shown
that NMR-determined diffusion rates of druglike macrocycles were consistently higher than
for the corresponding matched acyclic molecules
in both low (CDCl 3 ) and high (d 6 -DMSO)
dielectric and viscosity solvents, across a range
of molecular weights (300–730 Da) [73] .
Day et al. investigated the in vitro microsomal
stability of selected Hsp90 macrocycles, com­
paring them to the natural product macrocyclic
inhibitor radicicol (35; Table 21) [74] . The data
showed that the macrolactones radicicol (35) and
85 were both highly metabolized after 15 min,
Table 24. Ring-closing metathesis macrocyclization yields and isomer ratio of CHK1 inhibitors, followed by
hydrogenation.
Cl
Cl
Cl
O
O
m
N
H
O
NH
Grubbs II
CH2Cl2, reflux
N
nO
93
N
O
m
O
NH
N
H
N
94a–c
N
nO
CN
NH
N
H
O
m
N
nO
CN
Pd/C (10%), H2
MeOH-THF (3:1), RT
95
N
CN
Compound
m
n
Yield (%)
cis:trans
94a
1
1
2
1
2
2
85
76
62
cis†
cis†
4:1
94b
94c
Ratio not given; however, the authors reported that the trans isomer was a trace component of the reaction [79].
†
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Future Med. Chem. (2012) 4(11)
future science group
Macrocycles in new drug discovery
O
O
O
TBDMSO
Grubbs II catalyst
S
S
O
O
S
S
OTBDPS
97
96
O
Cl
O
OH
OTBDPS
mCPBA
NaHCO3
CH2Cl2
O
HO
TBDMSO
CH2Cl2
37–99%
| Review
98
O
O
HO
Cl
36%
O
OH
99
Figure 11. Macrocyclization and subsequent alkene expoxidation to generate Hsp90 inhibitors. whereas macrolactam 86, the amide analogue of
85, was more stable to metabolism. This provides
an example of incorporating a standard strategy
for the replacement of suboptimal functional
groups into the context of macrocycles.
A similar strategy was used in the develop­
ment of the microtubule-stabilizing cancer drug
ixabepilone (88, Ixempra, Bristol-Myers Squibb),
which is the direct amide analogue of the natural
product macrocycle epothilone B (87, patupilone;
Figure 10 ) [15,75] . Ixapebilone displayed higher
free-drug concentrations and maintained cyto­
toxicity for a longer duration than the macro­
lactone natural product, suggesting that amide
cleavage was not occurring.
Stachel et al. showed that macro­c yclization
of an acyclic BACE-1 compound not only
significantly improved potency, but after fur­
ther optimization, also increased membrane
permeability (Table 22) [45] . Substituents were
added to 89 to enhance the interactions with
the enzyme, leading to the potent inhibitor
90. However, 90 showed poor cellular activity.
The amide bond in the macrocycle linker was
removed in an attempt to reduce the peptidic
character of the compounds, leading ultimately
to the ring-contracted, alkyl-linked macrocycle
91. The apparent permeability increased sig­
nificantly and corresponded with an increased
cellular potency. Further modification led to
Table 25. Ring-closing metathesis yields for ring-size variation.
Boc
Me Me
PMBO
N
Me
O
N
O2N
PMBO
O
Me
Ring-closing O N
2
metathesis
O
R
Me
N
O
Grubbs I
Toluene, 65 °C
n
Me
N
O
Boc
O
R
100
n
101a–f
Compound
Ring size (n)
R
Conversion (%) Product:dimer†
Yield (%)
101a
13 (0)
14 (1)
15 (2)
16 (3)
17 (4)
18 (5)
H
Me
Me
H
H
H
88
100
100
99
98
100
55
85‡
73
88
83
69
101b
101c
101d
101e
101f
81:19
100:0
98:2
98:2
98:2
98:2
UV ratio from LC-MS ana­lysis.
Hoveyda–Grubbs II catalyst.
†
‡
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1429
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(Z)
H
Cl
H
O
5 mol% W cat.
N
Toluene, 0.001 M
22°C, 1 torr, 8 h
N
O
N
N
N
63%, 94% Z
102
Cl
Nakadomarin A (103)
N
W
O
Ar
Ar
Ar = 2,4,6-(i-Pr)3C6H2
Figure 12. Synthesis of nakadomarin A using a Z isomer-selective ring-closing metathesis,
catalyzed by the tungsten catalyst shown.
compound 92 , which was brain penetrant and
active in vivo.
The examples in this section show that mod­
ulation of physicochemical properties can be
achieved by changing macrocycle functional­
ization. Zapf et al. have shown that multiple
physicochemical and ADMET properties can
be modulated through variation of the rigid­
ity, preferred conformation and basicity of a
macrocyclic scaffold. Kawanishi et al. showed
that a single atom substitution within a macro­
cycle can significantly improve aqueous solu­
bility [65] , which can also apply to improving
metabolic stability, as seen with the synthesis
of the microtubulin inhibitor ixabepilone [75] .
The development of SB1317/TG02 by William
et al. has exemplified how a macrocyclic linker
can simult­a neously restrict conformation,
improve solubility and provide enthalpic inter­
actions to the protein target to enhance ligand
performance [20] .
Common linking motifs & synthetic
strategies
The synthesis of macrocycles can be challenging
and, thus, a common design strategy is to join
the functionalized termini of otherwise linear
pharmacophores with simple linkers (Table 23) .
Br
H
N
Br
HN
N
N
Cl
O
S
O
4 M HCl
MeCN, H2O
0.008 M
85%
N
H
N
N
HN
HN
S O
O
H2N
104
11
Figure 13. SNAr macrocyclization of CDK inhibitor.
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Future Med. Chem. (2012) 4(11)
Often, the linker motif will be an unfunctional­
ized alkyl chain of varying length, linking two
proximal points of the uncyclized molecule. This
can give an idea of an optimal macrocycle size,
and a useful assessment of the effect of macro­
cyclic conformational constraint on the activity
of a compound series. The simple linkers provide
a platform from which further elaboration can
take place. This section gives examples of the
different methods of macro­c yclic ring-closure
used in a medicinal chemistry context, concen­
trating on the reports that are highlighted else­
where in this review. A comprehensive recent
review by Marsault and Peterson [3] contains
further examples of available macrocyclization
strategies. A more general overview of macro­
cyclization chemistry has been provided by
Wessjohann and Ruijter [76] .
Ring-closing metathesis (RCM) is a common
method of forming a macro­c ycle, providing
a double bond that can be further functional­
ized [77] . However, surprisingly there are only
a few examples in the literature to date where
this opportunity to introduce additional func­
tionality has been exploited in the context of
medicinal chemistry. Most often, the unsaturated
RCM products are subsequently reduced to give
the unfunctionalized alkyl chain. For example,
Tao et al. synthesized macrocycles of varying
size (94a–c) using a ruthenium-catalyzed RCM,
as shown in Table 24 [78] . Shorter olefin chains
resulted in an almost exclusive formation of the cis
isomer, as presumably a trans isomer would result
in a strained ring. As the chain length increased,
the formation of the trans isomer also increased.
The authors [78] suggested that the high yields
could be attributed to the molecule being held in
a favorable conform­ation for ring-closure, due to
the intramolecular hydrogen bond between the
urea NH and the pyrazine nitrogen. RCM was
future science group
Macrocycles in new drug discovery
OH
HO
O
BnO
OEt
l
I2, PPh3
Imidazole
O
HO
OEt
O
K2CO3
18-crown-6
Benzene
93%
OBn
| Review
OBn
0.01 M
DMF
64%
OBn
BnO
OBn
105
OEt
O
BnO
OBn
OBn
107
106
Figure 14. Synthesis of ether-linked macrocycle using an intramolecular SN2 reaction of a phenoxide and an iodide.
followed by hydrogenation of the double bond to
form macrocycles of varying size.
An example of derivatization of a double
bond following macrocyclization by RCM is
found in work from Proisy et al. to synthesize
Hsp90 inhibitors of varying ring size, where
the double bond was epoxidized ( F igure 11,
98 & 99 ) [38] . Similarly, Moulin et al. synthe­
sized functionalized pochonins, a class of
resorcylic acid lactone, initially forming the
macrocycle by RCM, followed by epoxidation
or dihydroxylation [52] .
Dandapani et al. have demonstrated the use
of RCM for the generation of a skeletally diverse
library by diversity-oriented synthesis (Table 25)
[79] . Several different catalyst and solvent systems
were evaluated to find conditions that reduced
the formation of dimers, and, for all but the
smallest of macrocycles synthesized, the choice
of catalyst had little effect. For the smallest
macrocycle (101a) , a lower conversion to prod­
uct and a higher proportion of dimerization was
observed. The cis:trans ratio of the RCM prod­
ucts was not discussed and final charac­­ter­ization
was carried out after hydrogenation of the alkene
and nitro groups.
The development of RCM reactions is still
ongoing, with much success. Yu et al. recently
OH
Me
showed that RCM can be used to synthesize
complex natural product macrocycles with pref­
erence for the Z isomer in high yields [80] . The
late-stage cyclization of pentacycle 102 using
a tungsten catalyst resulted in the synthesis of
the natural product nakadomarin A (103) with
high Z isomer selectivity (Figure 12) . When the
reaction was performed using Grubbs I catalyst,
the yield was equivalent (62%), but formed a
mixture of isomers (Z:E 63:37).
SNAr chemistry has been used to form macro­
cycles, as exemplified by Lücking et al. in the
synthesis of 2,4-diaminopyrimidines (Figure 13)
[39] . The synthesis was achieved by syringe pump
addition of the acyclic precursor 104 to a large
volume of acidified acetonitrile, which main­
tained high-dilution conditions to favor the
intramolecular SNAr reaction over competing
intermolecular reaction. Of note in this exam­
ple, the macrocyclization took place within the
pharmaco­phore of the inhibitors, allowing for
flexibility in the composition of the tethering
linker.
Kim et al. synthesized potential antidiabetic
agents, with the ring-closure carried out using an
SN2 reaction (Figure 14) [81] . From the diol 105, an
Appel reaction was performed to generate iodide
106, followed by an intra­molecular SN2 reaction
OMs
Me
N
O
N
i) MsCl, DIPEA
ii) TFA, H2O
N
NH
OMOM
N
MeO
Me
O
N
108
O
N
NH
N
MeO
N
NH
K2CO3, DMF
OH
85%
N
O
N
74%
MeO
N
N
109
110
Figure 15. Synthesis of ether-linked macrocycle using an SN2 reaction of a phenoxide and
a mesylate.
future science group
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1431
Review | Mallinson & Collins
A
EOMO
EOMO
Me
O
HO
OBz
OH
Me
O
O
PPh3, DIAD
Toluene, 0.05 M
23°C, 12 h
OMOE
S
OMOE
OBz
O
S
Me
O
Me
111
B
Me
OMe O
Me
OMe O
MeO
O
Cl
Me
O
RF-Ph3P, RF-DIAD
HO
OEOM
OH
Toluene, 0.01 M
23°C, 2 h
80%
O
Me
MeO
Cl
OEOM
O
Me
O
Me
112
Figure 16. Synthesis of resorcylic acid lactone analogues using the Mitsunobu reaction.
(A) solid-supported synthesis; (B) fluorous-tagged reagent synthesis.
to form macrocycle 107, using potassium carbon­
ate and a crown ether to form the phenoxide ion.
Similarly, Kawanishi et al. formed kinase inhibi­
tors using an intra­molecular SN2 reaction, first by
transforming secondary alcohol 108 into a mesyl­
ate leaving group 109, followed by ring closure
with inversion of stereo­chemistry, again using a
phenoxide as the nucleophile (Figure 15) [65] .
Lactonization has been used extensively in
natural product synthesis, with Yamaguchi,
Mukaiyama and Mitsunobu conditions regularly
employed in the early synthesis of resorcylic acid
lactones related to the natural product Hsp90
inhibitor, radicicol, as reviewed by Winssinger
and Barlugena [82] . Dakas et al. showed that
Mitsunobu conditions can generate resor­
cylic acid lactones 111 and 112 on both a solidsupported resin [83] and using fluorous-tagged
coupling reagents (Figure 16) [49] . The Mitsunobu
reaction was also used by Lücking et al. to form
O
OH
N
HN
O
NH2
N
1) HATU, i-Pr2NEt2
NMP, sonicate
N
N
H
N
N
HN
N
N
N
2) HATU, R R NH
NMP
1
NH
2
N
H
NR1R2
OH
O
O
113
114
Figure 17. One-pot, two-step macrolactam formation followed by amide
formation.
1432
Future Med. Chem. (2012) 4(11)
a macrocyclic ether, although with a low and
unoptimized yield [39] .
Macrolactam synthesis was performed by
Nie et al. during the synthesis of CK2 inhibi­
tors, using standard amide formation conditions
(Figure 17) [40] . This method was also used to
selectively form the macrocycle 114 in the pres­
ence of two carboxylic acids (113) , allowing a
one-pot, two-step diamide formation.
Zapf et al. [58–61] and Balraju et al. [84] have
used Buchwald–Hartwig couplings to synthesize
macrocycles (Figure 18) . The yields of the Hsp90
inhibitor macrocycle 115 showed large variation
(15–93%) depending upon substitution of the
linker [58–61] . The Buchwald–Hartwig coupling
in this case was versatile enough to generate
macrocycles of varying size and substitution.
Similarly, Balraju et al. also presented many
variations on the peptide scaffold 116 shown
here, varying amino acid substitution and chain
length, with the cyclization giving a moderate
yield of the cyclic peptide [84] .
Breslin et al. demonstrated the use of a Heck
coupling of an aryl bromide and a vinyl group to
form macrocyclic ALK inhibitors 117 (Figure 19) ,
which provided a double bond that was hydro­
genated [57] . It is interesting to note that the
palladium-mediated cyclization proceeded with
modest yields, given the conformationally rigid
nature of the triaryl substrates used.
Flow chemistry has been used to synthesize
libraries of drug-like macrocycles such as 118,
employing copper-catalyzed azide-alkyne cyclo­
addition (Figure 20) [85,86] . This technique has the
aim of avoiding the very high dilution conditions
future science group
Macrocycles in new drug discovery
typically associated with macrocyclization chem­
istry. This would facilitate the synthesis of macro­
cycles on a larger scale, which is one of the goals
of continuous flow chemistry [87] . Bogdan and
James also demonstrated that a new reaction
centre could be installed with this chemistry,
allowing rapid further functionalization through
Suzuki coupling, as with 119 [85] .
Conventional strategies for the synthesis of
cyclic peptides are improving, as reviewed by
White and Yudin [88] , but there is an increasing
trend for the generation of large libraries of cyclic
peptides inspired by biosynthetic methods. Terrett
has reviewed the various emerging combinations
of chemical and biological techniques to synth­
esize very large libraries of macrocycles, includ­
ing the generation of macrocyclic peptide librar­
ies using DNA-programmed chemistry [72] . For
example, Tse et al. described the design and synth­
esis of a library of 13,842 macrocycles [89] . The
reactions were carried out on a small scale using
36 amino acid building blocks on eight different
scaffolds, with each scaffold containing three of
the building block amino acids. Such libraries
are intended for screening as mixtures against a
target of interest, followed by deconvolution to
determine the active components.
Future perspective
Macrocyclic scaffolds have already shown them­
selves to be exciting scaffolds for drug discovery,
with several macrocyclic molecules based on natural
products approved as drugs and others, including
purely synthetic macrocyclic structures, in the
advanced stages of drug development [18,23,90,91] .
Macrocyclic inhibitors have shown improvements
in potency over uncyclized counterparts through
conformational restriction [37,40,43,45] . There is evi­
dence that macrocyclization intrinsically varies the
selectivity profiles of acyclic precursor pharmaco­
phores [20,39,47,48,52,53,57] . Modulation of permeabil­
ity through the formation of internal hydrogen
bonding networks was observed in the case of
peptidic macrocycles [67,68] . Generally, improved
solubility and decreased ADMET liabilities have
been achieved without detriment to target bio­
logical activity in multiparameter optimizations,
by strategic functionalization of the macrocyclic
scaffolds [20,58–61,66] .
A fruitful area for further research would be
to achieve more efficient macrocyclic ligands,
through addressing multiple properties by craft­
ing of functionality within the scaffold itself.
For example, a macrocycle could improve target
potency through conformational restriction,
future science group
| Review
N
A
N
Br
H2N
R
N
H
N
Pd2(dba)3
BINAP, NaOtBu
1,4-dioxane, toluene
110°C
N
H
N
R = H, 11%
R = Me, 50%
Me
O
R
N
H
115
O
B
Me
Ph
H
N
HN
O
O
Me
Me
Me
O
HN
O
NH
H
N
Ph HN
Pd(OAc)2, BINAP
t-BuOK, MeCN
100°C, 15 h
45%
Me
O
O
NH
O
N
H
Br
NH2
O
HN
Me
116
Figure 18. Buchwald–Hartwig macrocyclization. The reaction was used in the
synthesis of (A) substituted Hsp90 inhibitor macrocycles and (B) cyclic peptides.
while also incorporating polar functionality
within the core cycle, which could improve
physico­chemical properties such as solubility
and also directly interact with the protein target
through hydrogen bonding or charge–charge
interactions. With strategic positioning of this
additional functionality, which could be con­
trolled through spatial organization using a
fixed macrocyclic scaffold, changes in selectivity
against related targets could also be achieved.
This should lead to more efficient molecules, of
current interest given concerns over ‘molecular
obesity’ within drug discovery and the atten­
dant risks for development [92] . It is important
to note that in many of the cases highlighted in
this review, macro­cyclization was applied at a late
stage to established non-macrocyclic pharmaco­
phores. Driggers et al. have proposed the concept
of ‘domains’ within macrocycles, corresponding
to discrete structural segments of the molecules
making separate contributions to the overall
Cl
N
HN
N
NH
Br
Pd(OAc)2,
P(o-MePh)3
Cl
N
HN
N
NEt3, MeCN,
Microwave heating, 120°C
65%
NH
117
Figure 19. Macrocycle formation using the Heck reaction.
www.future-science.com
1433
Review | Mallinson & Collins
O
N3
Me
I
10 mol% TTTA
i-PrNEt2, MeCN
Me
Cu, Flow reactor, 10 min
100 °C
80%
HN
O
I
O
HN
O N
N
N
118
CF3
(HO)2B
10 mol% Pd(OAc)2
NBu4OAc,
PPh3, K2CO3
THF, 80 °C
68%
CF3
Me
O
HN
O N
N
N
119
Figure 20. Copper-catalyzed azide-alkyne cycloaddition cyclization
followed by Suzuki coupling to generate substituted macrocycles.
properties, for example, a binding domain or
a domain dominating the physicochemical or
ADME properties [1] . Interestingly, as noted
here, many current synthetic approaches to
macrocycles tend to enforce the dissection of the
molecules in this way, with large-ring formation
taking place at a site remote from the (preformed)
pharmacophore.
The wider adoption of macrocyclic scaffolds
could open up a new area of chemical space that
might be suited to finding new therapeutic agents
for targets that are currently challenging to modu­
late with small-molecule drugs. For example, it has
been noted that many natural macrocyclic prod­
ucts modulate PPIs [1] , such as rapamycin [93,94]
and the epothilones [95,96] . Access to new chemical
space also allows for the expansion of intellectual
property possibilities in otherwise crowded areas,
for example heteroaromatic inhibitor scaffolds for
ATP-binding sites.
To further advance the field of macrocycles in
medicinal chemistry, the progress in synthetic
methods available to generate macrocycles needs
to be continued. The application of RCM has
rapidly improved the accessibility of the struc­
tural class, but overall macrocyclization often
remains a challenge. Another current synthetic
limitation to the application of macrocycles in a
medicinal chemistry context is the need for rapid
generation of analogues with greater complexity
in the linker portions of the molecules. Several
effective ring-closing reactions are available, as
surveyed earlier, but these do not always lead
easily to highly functionalized or readily varied
linker regions. In addition, to incorporate highly
1434
Future Med. Chem. (2012) 4(11)
functionalized linkers there is a general require­
ment for chemical functionality with orthogonal
reactivity at either end of the macrocyclic pre­
cursors. Thus the preparation of the large rings
may often require more complex protecting group
strategies than are required for the synthesis of the
acyclic analogues.
Many of the initial chemical scaffolds used in
drug-discovery programs come from the HTS
of large libraries of compounds, with a propor­
tion of the library content origin­ating from
in-house drug-discovery programs. With the
popularity of sp2 C–C bond forming reactions,
hundreds of analogues can be synthesized
rapidly, which begins to reduce the diversity
of those screening libraries [97] . Several authors
have commented on the need for increasing
scaffold diversity and sp3 hybridized atom
content (i.e., increased 3D, non-aromatic
character in molecules) to improve long-term
drug-development outcomes [42,98] . To this end,
it would be beneficial if macro­c yclic scaffolds
had a greater representation in HTS libraries
to allow future drug-development programs to
exploit the unique properties that such mol­
ecules can provide. One example of a step
towards this is the creation of a commercially
available macrocycle HTS library, consisting
of 5000 compounds, designed to be screened
against PPIs, antibacterial and anti­viral targets
[101] . The identification of drug-like macrocyclic
scaffolds as primary hit matter from screening
would also circumvent the current reliance on
structural biology information on the binding
of acyclic molecules to guide the adoption of
macrocyclization strategies.
To make best use of this structural class in
the future, some changes in the mindset of mol­
ecule design within medicinal chemistry may
be needed; to consider generating macrocyclic
molecules earlier in the design process and to
consider the macrocycle as a functional scaffold,
rather than a nonfunctional conformational
restraint. It is clear that further systematic inves­
tigation of the behavior of macrocycles as poten­
tial drug molecules is needed, with perhaps the
most important factor to include comparisons
to acyclic analogues at regular stages. This will
aid in the understanding of the effect of macro­
cyclization upon the properties of the molecules,
and, thus, illuminate where macrocyclic scaffolds
can be most usefully applied in drug discovery.
Methods for the in silico prediction of the biologi­
cally relevant conform­ations of macro­cycles have
only recently been investigated. While a number
future science group
Macrocycles in new drug discovery
of methods are now available, more work in this
area is required. Often, significant prior x-ray
crystallographic data are still needed to guide
modeling of specific macrocyclic templates.
In the next 10 years, we would anticipate
that the use of macrocycles in drug discovery
will become routine and a significant number
of non-natural macrocyclic molecules would be
found in late-stage clinical trials. There are sev­
eral cases of this occurring, for example, pacri­
tinib (SB1518) is completing Phase II evalua­
tion. It is reasonable to expect that one or more
non-natural macrocycles will come into clinical
use in the next decade. We suggest that in the
future the in silico prediction of macrocyclic
conformations and their interactions with pro­
teins will become more robust, reducing the bar­
rier to incorporation of these scaffolds earlier in
the drug-discovery process. We also predict that
the distinctive space-filling properties of macro­
cyclic scaffolds will see their utility realized in
the modulation of more challenging drug tar­
gets, such as PPIs, for which effective medicinal
chemistry strategies are needed. An improved
| Review
understanding of how to control the physico­
chemical properties of macrocyclic scaffolds and
direct their biological activity profiles, will lead
to the incorporation of macrocycles into a new
generation of efficient drug molecules.
Financial & competing interests disclosure
I Collins is an employee of the Institute of Cancer Research,
which has a financial interest in CHK1 inhibitors. This
work was supported by Cancer Research UK [CUK] grants
C309/A8274 and C19524/A10795 (studentship to
J Mallinson), and by the Institute of Cancer Research.
I Collins has been involved in a research collaboration on
CHK1 inhibitors with Sareum Ltd. Please note that all
authors who are employed by The Institute of Cancer
Research are subject to a ‘Rewards to Inventors Scheme’,
which may reward contributors to a programme that is
subsequently licensed. The authors have no other relevant
affiliations or financial involvement with any organization
or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the
manuscript apart from those disclosed.
No writing assistance was utilized in the production of
this manuscript.
Executive summary
Macrocycles have clinical relevance
„„
Many macrocyclic natural products or their synthetic derivatives have become clinically useful drugs.
An increasing number of non-natural, synthetic macrocycles are being reported in the discovery of new therapeutic agents.
Macrocyclization changes biological & physicochemical properties
„„
„„
Macrocyclization can provide a method of improving target potency and modulating selectivity.
Physicochemical and ADME properties, such as aqueous solubility, membrane permeability and microsomal stability, can be modulated
by macrocyclization.
Synthetic routes to macrocycles remain challenging
„„
„„
The synthetic routes to generate macrocycles have improved with the advent of specialist reactions.
Further advances are needed in the efficient generation of complex, functionalized macrocycles, especially in a medicinal chemistry
context.
Macrocycles need to be incorporated earlier into the drug-discovery process
„„
„„
This could be achieved by increased inclusion of drug-like macrocyclic templates within high-throughput screening libraries and wider
adoption of macrocyclization as a hit expansion strategy in early stage medicinal chemistry design.
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| Review
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