A diversity-oriented synthesis strategy enabling the combinatorial-type variation of macrocyclic peptidomimetic scaffolds

Article abstract of DOI:10.1039/c5ob00371g

Macrocyclic peptidomimetics are associated with a broad range of biological activities. However, despite such potentially valuable properties, the macrocyclic peptidomimetic structural class is generally considered as being poorly explored within drug discovery. This has been attributed to the lack of general methods for producing collections of macrocyclic peptidomimetics with high levels of structural, and thus shape, diversity. In particular, there is a lack of scaffold diversity in current macrocyclic peptidomimetic libraries; indeed, the efficient construction of diverse molecular scaffolds presents a formidable general challenge to the synthetic chemist. Herein we describe a new, advanced strategy for the diversity-oriented synthesis (DOS) of macrocyclic peptidomimetics that enables the combinatorial variation of molecular scaffolds (core macrocyclic ring architectures). The generality and robustness of this DOS strategy is demonstrated by the step-efficient synthesis of a structurally diverse library of over 200 macrocyclic peptidomimetic compounds, each based around a distinct molecular scaffold and isolated in milligram quantities, from readily available building-blocks. To the best of our knowledge this represents an unprecedented level of scaffold diversity in a synthetically derived library of macrocyclic peptidomimetics. Cheminformatic analysis indicated that the library compounds access regions of chemical space that are distinct from those addressed by top-selling brand-name drugs and macrocyclic natural products, illustrating the value of our DOS approach to sample regions of chemical space underexploited in current drug discovery efforts. An analysis of three-dimensional molecular shapes illustrated that the DOS library has a relatively high level of shape diversity.

Full text of DOI:10.1039/c5ob00371g

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Biomolecular Chemistry
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A diversity-oriented synthesis strategy enabling
the combinatorial-type variation of macrocyclic
peptidomimetic scaffolds†
Cite this: Org. Biomol. Chem., 2015,
13, 4570
Albert Isidro-Llobet, Kathy Hadje Georgiou, Warren R. J. D. Galloway,
Elisa Giacomini, Mette R. Hansen, Gabriela Méndez-Abt, Yaw Sing Tan,
Laura Carro, Hannah F. Sore and David R. Spring*
Macrocyclic peptidomimetics are associated with a broad range of biological activities. However, despite
such potentially valuable properties, the macrocyclic peptidomimetic structural class is generally con-
sidered as being poorly explored within drug discovery. This has been attributed to the lack of general
methods for producing collections of macrocyclic peptidomimetics with high levels of structural, and
thus shape, diversity. In particular, there is a lack of scaffold diversity in current macrocyclic peptidomi-
metic libraries; indeed, the efficient construction of diverse molecular scaffolds presents a formidable
general challenge to the synthetic chemist. Herein we describe a new, advanced strategy for the diversity-
oriented synthesis (DOS) of macrocyclic peptidomimetics that enables the combinatorial variation of
molecular scaffolds (core macrocyclic ring architectures). The generality and robustness of this DOS strat-
egy is demonstrated by the step-efficient synthesis of a structurally diverse library of over 200 macrocyclic
peptidomimetic compounds, each based around a distinct molecular scaffold and isolated in milligram
quantities, from readily available building-blocks. To the best of our knowledge this represents an unpre-
cedented level of scaffold diversity in a synthetically derived library of macrocyclic peptidomimetics.
Cheminformatic analysis indicated that the library compounds access regions of chemical space that are
distinct from those addressed by top-selling brand-name drugs and macrocyclic natural products, illus-
trating the value of our DOS approach to sample regions of chemical space underexploited in current
drug discovery efforts. An analysis of three-dimensional molecular shapes illustrated that the DOS library
has a relatively high level of shape diversity.
Received 24th February 2015,
Accepted 11th March 2015
DOI: 10.1039/c5ob00371g
www.rsc.org/obc
incorporation of an isosteric analogue of a peptide linkage
into the macrocyclic ring structure.4,5 Many macrocyclic pepti-
Introduction
Macrocycles are ring structures of 12 or more atoms. Where domimetic compounds are known to be capable of modulating
present in a small molecule, the macrocyclic ring architecture biological systems (Fig. 1).1,2,6–12 However, despite such poten-
typically serves as the molecular scaffold; that is, the core rigi- tially valuable properties, macrocyclic peptidomimetics are
difying structural feature of a molecule that has the largest considered to be a relatively poorly explored structural class
influence upon how it presents its chemical information (i.e. within drug discovery.1,2,9,13 This can be attributed to a lack of
functional groups and potential binding regions) in three general methods for producing macrocyclic peptidomimetics
dimensions (3D).1–4 Macrocyclic peptidomimetics are a sub- collections with broad structural (chemical) diversity and thus
class of macrocycles, designed to act as functional substitutes functional diversity (that is, displaying a broad range of bio-
for peptide motifs or proteins, whilst having more desirable logical activities).9 In the context of efficient exploration of the
biological properties.4,5 For example, this can involve the biological capabilities of this compound type, the key aspect of
structure is the molecular scaffold (that is, the macrocyclic
ring architecture).14 It is generally acknowledged that increas-
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge,
ing the scaffold diversity in a small molecule library is one of
CB2 1EW, UK. E-mail: spring@ch.cam.ac.uk; Fax: +44 (0)1223-336362;
the most effective ways of increasing its overall functional
Tel: +44 (0)1223-336498
†Electronic supplementary information (ESI) available: Experimental pro-
diversity.14,15 This has been attributed to a correlation between
the scaffold diversity of a compound set and its molecular
cedures, characterization data and details of the computational analyses. See
DOI: 10.1039/c5ob00371g
shape diversity.14,15 The overall shape of a molecule is the
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DOS-type strategies targeted at macrocyclic structures,3,26,27
including macrocyclic peptidomimetics in particular.28
Notable recent examples come from the research groups of
Wessjohann,28,29 Marcaurelle,30 Marsault9 and Harran.31
Overall however, there still remains a relative lack of DOS strat-
egies that are specifically directed towards macrocyclic pepti-
domimetics. Reports beyond the proof-of-principle stage (that
is, involving the application of such DOS strategies for the
generation of large numbers of structurally diverse compounds
based around a variety of macrocyclic peptidomimetic ring
architectures) are rare.9,18,28
A common synthetic algorithm underpinning many DOS
pathways is the so-called build/couple/pair (B/C/P) three-phase
strategy.32 The build phase involves the synthesis of starting
materials (or building blocks). These are then coupled together
in the couple phase to produce densely functionalized mole-
cules. In the pair phase intramolecular reactions that join pair-
wise combinations of functional groups incorporated in the
build phase are performed to generate diverse molecular
scaffolds. Recently, we reported the development of a DOS
pathway towards macrocyclic peptidomimetics that was based
around the B/C/P synthetic algorithm and two types of build-
ing blocks (Scheme 1).4 This approach was used to generate a
small proof-of-concept library of 14 structurally diverse pepti-
domimetic compounds based around four different general
structural types (2–5 in Scheme 1). Each compound contained
a triazole ring in place of an amide bond. The triazole moiety
acts as a peptide bond mimic (hence peptidomimetic; both
the trans- and the cis- amide bond configurations can be
mimicked by the 1,4- and 1,5-triazoles, respectively).4,33,34
Several compounds also featured a diketopiperazine (DKP)
motif in the macrocyclic framework; DKPs occur in numerous
natural products and are of significant importance in drug
discovery.35–37
Fig. 1 Chemical structures of some biologically active macrocyclic
peptidomimetics. MCP-1 is
a potent inhibitor of the menin-mixed
lineage leukemia 1 protein–protein interaction,17 Clicktophycin-52 dis-
plays in vitro activity against the multidrug resistant human cervix carci-
noma cell line KB-V18 and compound 1 is a motilin antagonist.18
most fundamental factor controlling its biological effects, and
this is intrinsically linked to the molecular scaffold that the
molecule possesses.14–16 Substantial ‘shape space coverage’
(that is, molecular shape diversity) has been correlated with
broad biological activity, and the scaffold diversity of any com-
pound set has a pivotal role in defining its overall molecular
shape diversity, with peripheral substituents being of consider-
ably less importance in this regard.14–16
There are synthetic methods that can provide collections of
macrocyclic peptidomimetics with varying appendages based
around similar core scaffolds (core macrocyclic ring architec-
tures), typically designed to modulate a specific biological
target or family of related targets.9–11,19–23 However, there is a
relative dearth of synthetic strategies to produce libraries of
macrocyclic peptidomimetics with high scaffold, and thus
functional, diversity. Indeed, the efficient construction of
diverse molecular scaffolds presents a formidable general chal-
lenge to the synthetic chemist.14,24
Over the course of the last decade, diversity-oriented syn-
thesis (DOS) has established itself as a field of organic chem-
istry directed towards the generation of structurally, and thus
functionally, diverse small molecule libraries.14 DOS involves
the conversion of simple and similar starting material(s) to
more complex and structurally diverse molecules through rela-
tively short synthetic sequences, with an emphasis typically
placed upon the efficient incorporation of multiple molecular
scaffolds in the library.14 Many ingenious DOS strategies have
been reported which have enabled the efficient synthesis of
libraries based on tens (up to 82)25 of different molecular
scaffolds.14 Recent years have seen the development of several
Recently, Nelson and co-workers have reported a DOS strat-
egy based around a modified B/C/P algorithm that incorpor-
ates triplets of building blocks and iterative couple steps.38 In
this approach, “initiating” building blocks were coupled with
“propagating” building blocks followed by “terminating”
building blocks, in two successive couple phases, to furnish a
diverse range of linear substrates for the subsequent pair
phase. This synthetic sequence can be thus described as B/C/
C/P, with the authors reporting the use of three “initiating”,
four “propagating” and three “terminating” building blocks in
the synthesis of approximately 14 different scaffolds (including
one macrocyclic framework). We envisaged that this elegant
concept could be leveraged in the design of an advanced DOS
strategy towards macrocyclic peptidomimetics that also incor-
porated iterative couple steps (B/C/C/P and B/C/C/C/P,
Scheme 1). This would allow for an increase in the diversity of
macrocyclic peptidomimetic scaffolds synthetically accessible
from a given set of building blocks relative to our previous
B/C/P sequence involving a single couple stage. Herein, we report
the successful development of this advanced DOS synthetic
algorithm. Application of this approach has enabled the step-
efficient synthesis of a library of over 200 compounds, each
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Scheme 1 Outline of our advanced DOS strategy towards the combinatorial variation of macrocyclic peptidomimetic scaffolds. The coloured
shapes represent major scaffold-defining elements (i.e. areas that can be varied to obtain different macrocyclic ring architectures).
containing a distinct macrocyclic peptidomimetic scaffold and synthetically-derived small molecule library. Computational
isolated in milligram quantities. To the best of our knowledge analyses indicated that the DOS library has a relatively high
this is the first time that over 100 distinct scaffolds have been level of molecular shape diversity and also samples attractive
generated in a DOS library. Thus this work represents a step- regions of chemical space underexploited in current drug-
change in the degree of scaffold diversity incorporated in a discovery efforts.
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information present and how it is displayed in 3D. This may
be important for any subsequent biological screening of the
DOS library compounds, as the nature of the appendages
Results and discussion
Outline of the synthetic strategy
Our advanced DOS strategy towards diverse macrocyclic pepti- around macrocyclic scaffolds can have a profound effect upon
domimetic scaffolds was based around the use of three biological activities (for example, it is well know that the sugar
general types of chiral building blocks (generated in the build appendages in erythromycin and other antibacterial macro-
phase) of the synthesis: “azido amines” (“initiating” building cycles are typically vital for the antibacterial activity of such
blocks), “Boc-amino-acids” (“propagating” building blocks) compounds39). In addition, the macrocycles resulting from
and “alkyne-acids” (“capping” building blocks, Scheme 1).38 iterative coupling sequences (B/C/C/P, B/C/C/C/P, etc.) would
We envisaged that these building blocks could be combined as be larger in size than those generated using a single couple
illustrated in Scheme 1 (in the couple phase) to yield linear phase. Consequently, they may be better suited to targeting
peptides containing both a terminal alkyne and an azide. extended binding interfaces, such as those associated with
These functionalities could then potentially be reacted protein–protein interactions (PPIs), which are traditionally
together in the pair phase to access diverse macrocyclic pepti- viewed as being notoriously difficult to modulate using small
domimetic scaffolds.
molecules.14,40–42
Specifically, the pair phase was to be comprised of two cycli-
zation steps. First, a regioselective metal-catalysed “click”-type
Library synthesis: build stage
1,3-dipolar cycloaddition, selectively generating 1,5-disubsti- In total six azido-amines (10a–f), 14 alkyne-acids (11a–n) and
tuted triazoles (ruthenium catalysis) or 1,4-disubstituted tri- four Boc-amino acids (12a–d) were readily prepared from
azoles (copper catalysis) with concomitant construction of the simple, commercially available amino acid starting materials
macrocyclic architecture (general structural types 2 and 3 (Fig. 2). These building blocks were then utilised for library
respectively). The second cyclization step involves an intra- formation, as outlined in Scheme 1. The full synthetic
molecular cyclization reaction between amine and carbonyl sequences leading to a representative selection of final macro-
groups to introduce a DKP motif into the macrocyclic frame- cyclic peptidomimetic compounds are discussed in more
work, leading to general structures 4 and 5.4 In our previous detail (Schemes 2–4). For a full list of the final compounds syn-
study, 14 B/C/P macrocyclic peptidomimetics based on general thesised and synthetic details consult the ESI.†
structures 2–5 were generated via the initial coupling of an
Library synthesis: couple stage
“initiating” building block with a “capping” building block in
the first couple stage of the DOS.4 It was envisaged that larger In the couple stage of the DOS, amide bond formation
B/C/C/P products of the general forms 6–9 could also be between various pairs of building-blocks (either “initiating”
accessed if “initiating” building blocks were instead first with “capping” or “initiating” with “propagating”), followed by
coupled with “propagating” building blocks before being com- optional Boc removal afforded a range of linear peptides,
bined with a “capping” building block. Indeed, it was antici- obtained as single stereoisomers after purification by column
pated that iterative coupling with “propagating” building chromatography on silica.43 Some examples are shown in
blocks to generate linear peptides of increasing length prior to Schemes 2–4 (compounds 13–15).
the installation of a “capping” building block should be poss-
ible. For example, two rounds of coupling with “propagating”
Library synthesis: pair stage
building blocks, followed by coupling with a “capping” build-
B/C/P sequence. In the first pair step of the DOS, “click”
ing block and then the pair phase reactions should lead to so- type 1,3-dipolar cycloadditions with Cu or Ru catalysts were
called B/C/C/C/P products. Overall, we anticipated that this carried out on the broad range of linear substrates generated
advanced DOS algorithm would allow efficient access to a large in the couple phase to furnish regioisomeric macrocycles con-
number of structurally diverse macrocyclic peptidomimetics taining 1,4- or 1,5-disubstituted triazoles respectively.44
A
1
from a small set of simple, readily available building blocks. single stereoisomer was detected in the crude H NMR of each
In principle, a combinatorial-type variation of molecular cyclized product, indicating that all the 1,3-dipolar cyclo-
scaffold should be possible, since the different building blocks addition reactions were highly regioselective and proceeded
could be assembled in varying combinations, with each without epimerization of the chiral centres present. Purifi-
different combination ultimately yielding a different macro- cation by column chromatography on silica gel was generally
cyclic scaffold. The DOS strategy should also allow for facile straightforward and led to the isolation of a range of pure Boc-
variation in the appendages (that is, the R and R′ groups and protected macrocycles, typically in good yields.45 Subsequent
other exocyclic groups present in the scaffold-defining Boc group removal furnished the final macrocyclic peptidomi-
elements in Scheme 1), and thus functionality, displayed metic library members.45 The yields calculated over these two
around the core scaffolds through the use of a range of “initiat- steps (macrocyclisation then deprotection) were generally
ing” and “propagating” building blocks (derived from a variety good, with both Cu- and Ru-catalysis giving similar results (the
of natural and non-natural amino acids). Thus, there is scope average yield over the two-step sequence was 79% for B/C/P
for achieving, in a compound efficient fashion, high levels of products whose synthesis involved a Cu-catalysed macrocycli-
diversity across the library in both the nature of chemical sation and 78% for B/C/P products whose synthesis involved a
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Fig. 2 Building blocks used in library synthesis. Complete experimental procedures for the synthesis of the building blocks are given in the ESI.†
Scheme 2 An illustrative example of the synthesis of final macrocyclic peptidomimetics using the B/C/P strategy. The lowest energy conformations
(molecular shapes) of two final library compounds 16 and 17 are shown46 (conformational search by Molecular Operating Environment (MOE) soft-
ware package47). Conditions: (a) EDC·HCl, HOBt, NEt3, CH2Cl2, rt; (b) (i) [Cp*RuCl]4, toluene, reflux; (ii) HCl–dioxane (4.0 M); (c) (i) CuI, DIPEA, THF,
reflux; (ii) HCl–dioxane (4.0 M); (d) AcOH–NMM* (1.25 : 1, molar ratio), 2-Butanol, microwave irradiation (T = 150 °C). NMM*: morpholinomethyl-
polystyrene (loading = 3.51 mmol g−1).
Ru-catalysed macrocyclisation). In terms of the average purity distinctive predicted molecular shapes (lowest energy confor-
of the final products, the Ru-catalysed process faired margin- mations).46 This illustrates the utility of the DOS strategy to
ally better (87% average purity compared to 79% as deter- generate scaffold, and thus shape diversity, in a step-efficient
mined by HPLC analysis).
fashion through variation in reaction conditions alone.
It is possible to identify some general underlying trends in
As a representative example of this sequence, macrocycles
16 and 17 were both obtained from 13 with purities of 95% the macrocyclisation reactions of the B/C/P sequence (if one
and 89% respectively (Scheme 2). These two macrocycles are assumes that the macrocyclisation reaction, rather then sub-
each derived from the same two building blocks, however, they sequent Boc group removal, is the main determinant of the
are each based on a different molecular scaffold and have very yield and purity of the final product). Linear precursors that
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Scheme 3 An illustrative example of the synthesis of some final macrocyclic peptidomimetics using a route incorporating iterative coupling steps
(i.e. B/C/C/P and B/C/C/C/P). (a) (i) EDC·HCl, HOBt, Boc-L-Ala-OH (12a), NEt3, CH2Cl2, rt; (ii) TMSCl, MeOH, 0 °C to rt; (b) (i) EDC·HCl, HOBt, Boc-L-
Phe-OH (12d), NEt3, CH2Cl2, rt; (ii) TMSCl, MeOH, 0 °C to rt; (c) EDC·HCl, HOBt, 11b, NEt3, CH2Cl2, rt; (d) (i) [Cp*RuCl]4, toluene, reflux; (ii) HCl–
dioxane (4.0 M); (e) (i) CuI, DIPEA, THF, reflux; (ii) HCl–dioxane (4.0 M).
incorporated capping building block 11a or 11b and/or initiat- more strained than those derived from 10d, which may dis-
ing building block 10a or 10b (which would be expected to favour their formation. However, in the case of the corres-
furnish macrocycles that incorporated an aromatic ring into ponding Ru-catalysed macrocyclisation reactions, there was no
the macrocyclic ring architecture) were found to especially noticeable difference in the yields obtained.
challenging substrates for the Cu-catalysed macrocyclisation
B/C/C/P and B/C/C/C/P sequences. The assembly and regio-
process, with little/no conversion typically observed. It is poss- selective cyclization of alternative linear peptides obtained
ible that the presence of the aromatic ring in these substrates from different pairs of building blocks allowed for a combina-
restricted their conformational flexibility such that any re- torial-type variation of macrocyclic scaffolds to be achieved.
organization needed to bring their reactive termini in close The cyclization of longer peptides generated by iterative coup-
spatial proximity would have been disfavoured (i.e. associated ling allowed step-economical access to a diverse range of larger
with a high degree of strain).48 Furthermore, there may be a peptidomimetic scaffolds. For example, the coupling of 10c
high degree of strain, in the resulting macrocycles themselves with 12a furnished compound 14 (Scheme 3). A second coup-
due to the incorporation of the aromatic sp2 carbons into the ling step with 11b (a “capping” building block) provided com-
macrocyclic ring architecture, which could have disfavoured pound 18, which could be converted to macrocycles 19 and 20
their formation. Interestingly, the Ru-catalysed macrocyclisa- by cycloaddition followed by Boc group removal; overall these
tions of linear precursors that incorporated one of these build- represent the products of a B/C/C/P sequence (Scheme 3).
ing blocks typically proceeded more smoothly. It is possible Although the yields over the two steps were only moderate in
that there is less strain associated with the incorporation of a both cases, the purities of the final compounds were good
1,5-, rather than a 1,4-, disubstituted triazole in the resultant (purities of 88% and 72% respectively). Alternatively, coupling
macrocycles. For B/C/P products whose synthesis involved a of 14 with 12d provided compound 21, which could then be
Cu-catalysed macrocyclisation, the yields obtained over the combined with 11b to furnish linear peptide 22. Subsequent
two-step macrocyclisation-deprotection sequence from sub- Ru-catalysed cycloaddition furnished 23, formally the product
strates derived from initiating building block 10c were often of a B/C/C/C/P sequence, in a good purity (80%). In another
below average, and also typically below the values obtained for example of the use of the B/C/C/P strategy, linear peptides 24,
analogous substrates incorporating building block 10d. It is 25 and 26 were converted to macrocycles 27, 28 and 29 respect-
possible that the differences in yields can be attributed to the ively (purities of 89%, 81% and 77% respectively) (Scheme 4).
shorter alkyl chain in 10c; the macrocyclic ring systems that
For the B/C/C/P and B/C/C/C/P sequences, the average
incorporate this unit would be smaller in size and perhaps yields over the final two steps (macrocyclisation then deprotec-
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Scheme 4 Illustrative example of the synthesis of some final macrocyclic peptidomimetics using a route incorporating two couple steps (i.e. B/C/
C/P). The lowest energy conformations (molecular shapes) of some final library compounds (27, 28, 32–34) are shown46 (conformational search by
MOE software package47). Conditions:(a) (i) EDC·HCl, HOBt, Boc-L-Glu-OMe (12b), NEt3, CH2Cl2, rt; (ii) TMSCl, MeOH, 0 °C to rt; (b) (i) EDC·HCl,
HOBt, 11i or 11m or 11j, NEt3, CH2Cl2, rt; (c) (i) [Cp*RuCl]4, toluene, reflux; (ii) HCl–dioxane (4.0 M); (d) AcOH–NMM* (1.25 : 1, molar ratio),
2-Butanol, Microwave irradiation (T = 150 °C). NMM*: morpholinomethyl-polystyrene (loading = 3.51 mmol g−1).
tion) were generally lower than those seen for the B/C/P case of the use of Ru-catalysis) were comparable to those for
sequence (68% for all the B/C/C/P and B/C/C/C/P products products obtained via the B/C/P sequence.
whose synthesis involved a Cu-catalysed macrocyclisation and
DKP formation. In our previous scoping study, we reported
55% for all the B/C/C/P and B/C/C/C/P products whose syn- the development of a unique method for introducing DKP
thesis involved a Ru-catalysed macrocyclisation). This can units into macrocyclic frameworks based around the use of
potentially be attributed to unfavourable entropic factors solid-supported N-methylmorpholine (NMM) (morpholino-
associated with the formation of the larger-sized macrocyclic methyl-polystyrene) in combination with microwave heating
ring systems or perhaps the increased distance between reac- under slightly acidic buffered conditions. In general, the final
tive termini in the acyclic precursors, which may favour com- products could be obtained with a good level of purity by
peting intermolecular reactions.47 Gratifyingly, the average simple filtration of the solid support and evaporation to
purities of the B/C/C/P and B/C/C/C/P products (83% for pro- dryness. Application of this protocol to the wider range of sub-
ducts whose synthesis involved Cu-catalysis and 82% in the strates employed in this work enabled efficient access to a
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diverse range of structurally complex DKP-containing macro- cycles in chemical space, as substantial overlap with the drug
cyclic peptidomimetics as final library members.49 Yields and library occurs in the plot of PC1 against PC3 (Fig. 3b). The
purities were typically moderate-good (the average yield of all most important contributors to the variance in PC2 are log P
successful DKP formations was 57%, with an average purity of (o/w), S log P (an alternative way of obtaining log P) and log S
77%). For example, 16 and 17 could be converted to 31 (94% (see ESI, Table S3†). It appears that an increase in hydrophili-
yield, 87% purity) and 30 (87% yield, 90% purity) respectively city (decrease in log P, increase in log S) shifts molecules in the
(Scheme 2). DKP units could also be introduced into macro- positive direction. This agrees with the fact that the DOS
cyclic scaffolds generated from cyclization precursors incorporat- library comprises of macrocycles with a large number of polar
ing more than two building blocks. For example, the B/C/C/P amide bonds and therefore high hydrophilicity, thus differen-
macrocycles 27–29 could be successfully converted to DKP-con- tiating them from the reference sets of drugs and macrocyclic
taining products 32–34 (Scheme 4). The fact that this single natural products.
synthetic transformation yielded products with unique mole-
We also evaluated the molecular shape diversity of the
cular scaffolds, and overall shapes,46 quite distinct from those same compound data sets by computing normalized ratios of
of their corresponding macrocyclic precursors, provides a clear principal moments of inertia (PMI) based on the lowest-energy
illustration of the utility of the overall DOS strategy in the conformations of the compounds.46 The PMI ratios were
context of step-efficient scaffold, and thus shape, diversity plotted on a triangular graph as previously described.15 This
generation.
PMI plot (Fig. 3d) intuitively visualizes the shape diversity of
For macrocycles that incorporated an aromatic ring in the each of the four collections in “molecular shape space”
cyclic architecture, DKP formation was found to be challen- spanned by the three basic extreme shape types “rod-like”,
ging; reactions were typically sluggish, with poor conversion or “disk-like” and “spherical”. The drug reference set predomi-
proceeding in a low yield, presumably because the high rigid- nantly contains compounds with rod-like shapes with some
ity of the macrocycle made additional ring closure difficult (as disc-like features, whereas the natural products display much
has previously been noted).4 The yields for DKP formations larger shape diversity. Notably, our DOS compounds exhibit
involving B/C/P macrocycles incorporating building block 10c almost the same range of shape diversity as the natural pro-
were generally found to be below average. This could possibly ducts (albeit lacking in some rod-like character), overlapping
be attributed to the fact that such macrocycles have core ring to a substantial extent with the drugs and macrocyclic natural
systems that are relatively small in size (due to the relatively products in the PMI plot; thus the library can be said to have a
short alkyl chain in 10c), which may have thus made further very high level of shape diversity. The structures of three
intramolecular cyclisation challenging due to high levels of macrocycle library compounds are shown in Fig. 3 to illustrate
associated strain.48,50
the diversity in molecular shapes that are obtained by our DOS
Overall, using the strategies outlined above, and a limited approach.46
number of building blocks, the DOS of a library of 219 macro-
cyclic peptidomimetics was achieved. Each final compound
contains a distinct molecular scaffold amongst other unique
Conclusions
structural features. The library was made using parallel syn-
thetic techniques, leading to at least 1 mg of each final product Herein, we have described the development of a new advanced
(typically 10 mg or above). All library members were assessed DOS strategy that enables the combinatorial-type variation of
for their identity and quality (HPLC and LCMS). Full character- macrocyclic peptidomimetic scaffolds. Our synthetic approach
ization of 46 (21%) of the final macrocyclic compounds was is based around a B/C/P algorithm that incorporates iterative
undertaken; HPLC and LCMS characterized the rest.
couple and pair stages. Application of this new DOS strategy
enabled concise, flexible and systematic access to a structurally
diverse library of over 200 unique macrocyclic peptidomi-
Diversity assessment
In order to assess the chemical space coverage of the DOS metics, each based around a distinct molecular scaffold, from
library, we compared it with three reference collections, com- simple, readily available amino acid starting materials. To the
prising of 40 top-selling brand-name drugs, 60 diverse natural best of our knowledge this represents an unprecedented level
products and 24 macrocyclic natural products51 in a principal of scaffold diversity in a synthetically-derived library of macro-
component analysis (PCA) based on various structural and cyclic peptidomimetic compounds. Each library compound is
physicochemical descriptors (see ESI, Table S1† for full structurally complex (an important feature for the specificity of
details). The distribution of these four sets of compounds in biological interactions52–54) and rich in functionality, contain-
chemical space is shown in Fig. 3a–c, which represent 87% of ing numerous potential bimolecular-interacting elements and
the information in the data set (see ESI, Table S2† for full structural motifs related to those found in numerous biologi-
details). The DOS macrocycle library clearly occupies a distinct cally active compounds. For example, 27% of macrocyclic
region of chemical space compared to the drug and macro- library members incorporate a DKP motif, a privileged struc-
cyclic natural product libraries, most evident in Fig. 3a and c. PC2 tural motif in terms of bio-relevance. The remaining library
is the principal component that mainly contributes to the sep- members incorporate a free amine and an ester group, which
aration of the DOS library from the reference drugs and macro- provide synthetic handles for further derivatisation. This may
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Fig. 3 Comparative PCA and PMI plots of 219 macrocyclic DOS library compounds (“DOS library”, solid red circles), 40 top-selling brand-name
drugs (“Drugs”, solid green squares), 60 diverse natural products (“Natural Products”, solid blue triangles) and 24 macrocyclic natural products
(“Macro Natural Products”, empty blue triangles). (a) PCA plot of PC1 versus PC2. (b) PCA plot of PC1 versus PC3. (c) PCA plot of PC3 versus PC2.
(d) PMI plot illustrating the molecular shape diversity of the DOS library. The lowest energy conformations (molecular shapes) of representative DOS
library compounds 35–37 based on each of the three extremes of molecular shape types are shown46 (conformational search by MOE software
package47). See ESI† for the structures of 35–37 and more details.
offer a means of tuning the biological profile of a compound screening in a large number of biological assays. The DOS
or enable the labelling of compounds for application as chemi- library compounds are now being screened against a wide
cal probes.4 PCA indicates that the compound library accesses range of biological targets, including challenging ones such as
regions of chemical space that overlap with some natural pro- PPIs and multi-drug resistant bacterial strains. The results
ducts but which are distinct from those addressed by top- from these studies, which will be reported in due course, will
selling brand-name drugs and macrocyclic natural products. provide much-needed further insight into the functional capa-
This illustrates the ability of our DOS approach to sample bilities of this class of molecules. The inherent modularity of
attractive regions of chemical space underexploited in current the DOS should facilitate the systematic modification and
drug-discovery efforts. In addition, PMI analysis shows that the optimisation of any library compounds that are found to
DOS library has a relatively high level of shape diversity, thus display interesting biological properties.
demonstrating the utility of the DOS strategy for the prepa-
In this report, we have demonstrated the generality and
ration of shape-diverse libraries.46 Another notable feature of robustness of our DOS strategy for the step-efficient synthesis
the DOS is that all library compounds were generated on milli- of hundreds of different macrocyclic peptidomimetic
gram (typically multi-milligram) scale. This enabled the iden- scaffolds. In principle, the DOS strategy could be applied on a
tity of all compounds to be unambiguously determined. In larger scale, using a greater number of each of the three types
addition, the quantities of each compound are sufficient for of building blocks, in order to access an even greater number
4578 | Org. Biomol. Chem., 2015, 13, 4570–4580
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Paper
of macrocyclic scaffolds. Currently, the most significant bottle-
neck in the DOS is the need for purification by column chrom-
atography.43 It may be possible to adapt the DOS methodology
for use on solid support, which would make the strategy more
high-throughput and thus more amenable to the preparation
of even larger numbers of compounds.
2 E. Marsault and M. L. Peterson, J. Med. Chem., 2001, 54, 1961.
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As previously highlighted, the nature of the appendages
around macrocyclic scaffolds can have a profound effect upon
their biological profiles. The main focus of the study described
herein was to develop a general and step-efficient DOS algor-
ithm towards varied macrocyclic peptidomimetic scaffolds;
appendage variation was not examined in detail. In principle,
there are two general approaches by which appendage diversity
could be introduced into macrocyclic libraries generated using
this DOS strategy. The first would involve post-pairing elabor-
ations of the macrocyclic scaffolds with different groups
through reactions of existing functional handles. For example,
with regards to the non-DKP macrocycles described in this
report, one could envisage using the free amine group as a site
for further elaboration via amide bond formation; where a
DKP unit is present, it may prove possible to introduce
additional chemical functionality around the macrocycle via,
for example, N-alkylation.55 The second approach to the vari-
ation of macrocyclic appendages would exploit the inherent
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17 H. Zhou, L. Liu, J. Huang, D. Bernard, H. Karatas,
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The research leading to these results has received funding
from the European Research Council under the European
Union’s Seventh Framework Programme (FP7/2007-2013)/ERC
grant agreement no [279337/DOS]. In addition, this work was
supported by grants from the Engineering and Physical
Sciences Research Council, Biotechnology and Biological
Sciences Research Council, Medical Research Council, Royal
Society, Frances and Augustus Newman Foundation, and
Welcome Trust. A.I.-L. thanks the European Union for a Marie-
Curie Intra-European Fellowship. Y.S.T. was supported by an
A*STAR Graduate Scholarship.
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A. Landry, M. Vézina, L. Ouellet, Z. Wang, M. Ramaseshan,
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