Diversity-oriented synthesis of macrocyclic peptidomimetics

Article abstract of DOI:10.1073/pnas.1015267108

Structurally diverse libraries of novel small molecules represent important sources of biologically active agents. In this paper we report the development of a diversity-oriented synthesis strategy for the generation of diverse small molecules based around a common macrocyclic peptidomimetic framework, containing structural motifs present in many naturally occurring bioactive compounds. Macrocyclic peptidomimetics are largely underrepresented in current small-molecule screening collections owing primarily to synthetic intractability; thus novel molecules based around these structures represent targets of significant interest, both from a biological and a synthetic perspective. In a proof-of-concept study, the synthesis of a library of 14 such compounds was achieved. Analysis of chemical space coverage confirmed that the compound structures indeed occupy underrepresented areas of chemistry in screening collections. Crucial to the success of this approach was the development of novel methodologies for the macrocyclic ring closure of chiral α-azido acids and for the synthesis of diketopiperazines using solid-supported N methylmorpholine. Owing to their robust and flexible natures, it is envisaged that both new methodologies will prove to be valuable in a wider synthetic context.

Full text of DOI:10.1073/pnas.1015267108

Diversity-oriented synthesis of macrocyclic
peptidomimetics
Albert Isidro-Llobeta, Tiffanie Murilloa, Paula Belloa, Agostino Cilibrizzia, James T. Hodgkinsona,
Warren R. J. D. Gallowaya, Andreas Benderb, Martin Welchc, and David R. Springa,1
aDepartment of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom; bUnilever Centre for Molecular Sciences
c
Informatics, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom; and Department of Biochemistry,
University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom
Edited by Stuart L. Schreiber, Broad Institute, Cambridge, MA, and approved January 13, 2011 (received for review November 15, 2010)
Structurally diverse libraries of novel small molecules represent
important sources of biologically active agents. In this paper we
report the development of a diversity-oriented synthesis strategy
for the generation of diverse small molecules based around a com-
mon macrocyclic peptidomimetic framework, containing structural
motifs present in many naturally occurring bioactive compounds.
Macrocyclic peptidomimetics are largely underrepresented in
current small-molecule screening collections owing primarily to
synthetic intractability; thus novel molecules based around these
structures represent targets of significant interest, both from a
biological and a synthetic perspective. In a proof-of-concept study,
the synthesis of a library of 14 such compounds was achieved.
Analysis of chemical space coverage confirmed that the compound
structures indeed occupy underrepresented areas of chemistry in
screening collections. Crucial to the success of this approach was
the development of novel methodologies for the macrocyclic ring
closure of chiral α-azido acids and for the synthesis of diketopiper-
azines using solid-supported N methylmorpholine. Owing to their
robust and flexible natures, it is envisaged that both new meth-
odologies will prove to be valuable in a wider synthetic context.
in this context, and this contribution is likely to be considerable
in the flexible macrocycles discussed later in this work. Because
the overall shape adopted by a given molecule is intrinsically
linked to its structure, the overall functional diversity of a small-
molecule library can be correlated (to some extent) with its
overall structural diversity (which in turn is proportional to the
amount of chemical space the library occupies) (6, 8, 9, 11). In
the context of biological screening, the “ideal” library is one of
such high structural (and thus functional) diversity that, for any
aspect of any biological process, there exists a library compound
that is able to modulate that aspect (8, 9, 15). Although the term
“diversity” is somewhat subjective, there are four principle
components of structural diversity that have been consistently
identified in the literature (8, 9):
1. Appendage diversity (or building-block diversity)—variation in
structural moieties around a common scaffold;
2. Functional group diversity—variation in the functional groups
present;
3. Stereochemical diversity—variation in the orientation of poten-
tial macromolecule-interacting elements;
4. Scaffold diversity—presence of a range of distinct molecular
scaffolds.
cheminformatics ∣ macrocycles ∣ natural products
It has been demonstrated that the overall molecular shape
diversity of a small-molecule library is mainly dependent upon
the nature of molecular scaffolds present, with the peripheral
substituents being of minor importance (12). Indeed, there is a
widespread consensus that increasing the scaffold diversity in a
small-molecule library is one of the most effective ways to in-
crease its overall structural diversity (6, 8, 14, 16, 17). Scaffold
diversity is thus intrinsically linked to shape, and thus functional,
diversity. Structural complexity is another desirable feature in
small-molecule libraries (6, 8, 18). It is generally considered
that the more structurally complex a molecule, the more likely
it is to interact with a biological macromolecule in a selective
and specific manner (6–8, 19–22).
he screening of libraries of small organic molecules (so-called
Tsmall molecules) to identify useful modulators of biological
systems is fundamental in the drug-discovery process and chemi-
cal biology studies in general (1–6). However, a crucial considera-
tion is what compounds to use (6–9). In “unbiased” screening
processes, where the precise nature of the biological target is
unknown (for example, in a random drug-discovery screen), the
selection criteria for small molecules is dramatically complicated
by the absence of any existing structural guidelines (1, 6, 7, 9, 10).
In such situations, it is not possible to define a priori the struc-
tural features that are required in the small molecules for them
to be able to affect the biological system. This problem is also
encountered when structurally unique modulators of a given bio-
logical system are desired (e.g., molecules that are structurally
distinct from existing ligands).
It has been argued that the greater the area of total bioactive
chemical space (i.e., the chemical space defined by all bioactive
molecules) covered by a given small-molecule library, the higher
the probability there is of identifying a compound with the de-
sired properties in any screening experiment involving that library
(6, 8, 9, 11, 12). Thus, in general, the identification of biologically
active small molecules may be aided by screening functionally
diverse compounds libraries (9).
Traditionally, nature has served as a rich source of biologically
active molecules (23–25). Natural products exhibit enormous
structural diversity (26); however, there are well-documented
problems associated with their use in screening experiments
(6, 9). These difficulties have spurred the development of
synthetic strategies for the de novo creation of small-molecule
collections (6). However, the choice of what molecules to make
is of paramount concern. Achieving a large coverage of chemical
space simply by a random synthesis of compounds is not practic-
Nature “sees” molecules as 3D surfaces of chemical informa-
tion. Therefore the biological activity of any given molecule is
intrinsically dependent upon its 3D shape (6, 13, 14). It has there-
fore been argued that the molecular shape diversity of a small-
molecule library is the most fundamental indicator of overall
functional diversity; indeed, substantial “shape space” coverage
has been correlated with broad biological activity (12). In addi-
tion, conformational flexibility does contribute to shape diversity
Author contributions: A.I.-L., M.W., and D.R.S. designed research; A.I.-L., T.M., P.B., A.C.,
J.T.H., W.R.J.D.G., and A.B. performed research; A.B. analyzed data; and A.I.-L., W.R.J.D.G.,
A.B., M.W., and D.R.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: spring@ch.cam.ac.uk.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1015267108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1015267108
PNAS ∣ April 26, 2011 ∣ vol. 108 ∣ no. 17 ∣ 6793–6798

able or desirable; it is widely accepted that it is not synthetically
feasible to produce all theoretically stable, small carbon-based
molecules (6, 10, 20), and making and screening molecules cost,
both in terms of time and money (6–8). The ideal synthesis of
a diverse small-molecule library is one in which diversity is
achieved in the most efficient manner possible; this presents a
formidable challenge to the synthetic chemist (6). Diversity-
oriented synthesis (DOS) is an approach toward small-molecule
library synthesis, which seeks to achieve this goal (9, 10, 13, 27).
The overall aim of a DOS is to efficiently generate a library of
small molecules with a high degree of structural, and thus func-
tional, diversity that interrogates large areas of chemical space
(9, 28). This includes known bioactive chemical space as, by de-
finition, this is a fruitful region for the discovery of biologically
active agents and regions of chemical space that are not accessed
by existing compound collections; these areas may contain struc-
turally unique molecules with exciting and unusual biological
properties (8). In principle, the screening of such libraries should
provide hits against a broad range of biological targets with in-
creased frequency and decreased cost (9). This includes so-called
“unduggable” targets and processes that have traditionally been
seen as difficult or even impossible to modulate with small mo-
lecules (15). Indeed, molecules capable of modulating protein–
protein interactions (29–31), transcription factor activity (32, 33),
and multidrug resistance in pathogens (34, 35) have all been
discovered using DOS libraries, whereas the typical compound
libraries employed by pharmaceutical companies or commercial
vendors have been largely unsuccessful in providing hits against
such challenging and underexploited targets (9, 36, 37). This has
been attributed to deficiencies in these compound libraries; the
candidate small molecules seem to be well-suited to modulating
“traditional” medicinal chemistry targets, but lack the necessary
structural elements required to affect other processes (9, 15,
36–40).
the low-hanging fruit have already been picked as a consequence
of decades of focused investigation, principally by pharmaceutical
companies (8, 9). In addition, molecules from such regions of
chemical space have proven to be largely inadequate when it
comes to modifying more challenging biological targets (vide
supra). As outlined previously, there is undoubted value in gen-
erating molecules with atypical molecular scaffolds that access
unexplored regions of chemical space as these areas may contain
molecules with exciting, unique biological properties, which inter-
act with previously undescribed target molecules or act via unique
modes of action (8). It can be argued that small-molecule libraries
that incorporate molecules based on both unique scaffolds and
known scaffolds of proven biological relevance represent a pru-
dent method of increasing “hit” frequency (by targeting areas of
chemical space “close” to known bioactive molecules) without
compromising too much on the exploration of uncharted regions
of chemical space.
Peptide motifs are present in many biologically active mole-
cules, and many peptides are drugs (45, 46). The biological prop-
erties of peptides can be tuned by introducing bioisosteric
modifications on them. The resulting molecules are known as
peptidomimetics, and they usually present improved stability,
bioavailability, and selectivity toward a particular target. An
important example of these bioisosteric modifications is the re-
placement of an amide bond by a triazole ring, which introduces
rigidity to the molecule and mimics either the cis- or the trans-like
configuration of the amide bond (47, 48).
There are many biologically active molecules that contain
cyclic peptide and peptidomimetic structural units including
diketopiperazines (DKPs, the smallest possible cyclic peptides,
Fig. 1) (49, 50). Many compounds incorporating macrocyclic pep-
tides and peptidomimetics (51–54) are also known to be capable
of modulating biological systems. Despite such valuable proper-
ties, macrocyclic peptides (indeed macrocyclic compounds in
general) are arguably a poorly explored structural class within
drug discovery (54), and such compounds are underrepresented
in current small-molecule libraries. This relative paucity of
compounds can be attributed primarily to synthetic intractability
(54). Thus there is a need for the development of efficient
methods of broad utility for the synthesis of a diverse range of
macrocyclic peptides and peptidomimetics so that the biological
usefulness of these structural moieties can be investigated and
exploited further.
It is important to emphasize that the ultimate goal of any
small-molecule library synthesis and screening endeavor is the
identification of biologically active compounds (6–8). If the
library does not yield hits in a chosen screening experiment, it
will be deemed a failure, no matter how structurally diverse it
is (6, 8, 9). Thus a DOS should aim to efficiently and specifically
access biologically relevant chemical space, rather than chemical
space that cannot provide biologically useful molecules (8, 9).
However, the boundaries of biologically relevant chemical space
have yet to be defined (if indeed it is ever possible to do so) and
thus the structural constraints that this consideration imposes
upon molecules are not precisely known (8, 9). In terms of
increasing the overall efficiency of the library synthesis and
screening sequence (i.e., maximizing the number of hits), it can
be argued that, because it is impossible to access all possible
chemical space (vide supra), the incorporation into a compound
set of some bias toward known biorelevant chemical space may
be desirable. There are several synthetic strategies, such as bio-
logically oriented synthesis (41), biology-inspired synthesis (1),
privileged structure synthesis (42), and diverted-total synthesis
(43), which seek to generate libraries of compounds which, while
structurally unique, are each based around the core structures of
known biologically active molecules, typically natural product
templates (8, 9). It has been argued that evolutionary pressure
has “prevalidated” natural products, and thus compounds that
are structurally similar, to be able to modulate biological systems
(1, 6, 8, 44). However, such methods inevitably generate com-
pound collections with a relatively low degree of overall scaffold
diversity and thus chemical space coverage (9), with an emphasis
toward known bioactive regions; that is, the compounds gener-
ated would be expected to explore areas of chemical space within
the boundaries of, or “close to,” those occupied by known bioac-
tive small molecules. Although the biological relevance of com-
pounds from such regions is expected to be high, it is likely that
Within our research group we are interested in the develop-
ment of previously undescribed synthetic methodologies that
O
O
H
O
N
OH
O
N
H
N
H
O
NH
OH
O
O
N
HN
HN
Kahalalide F
OH
O
O
O
Kendomycin
NH
O
O
N
H2N
O
N
N
O
O
O
O
NH
NH
MeO
Cl
O
O
O
Clicktophycin-52
O
NH
NH
O
O
H
N
NH
NH
OH
O
O
HN
OH
O
O
OH
Bicyclomycin
Fig. 1. Chemical structures of some biologically active macrocycles (49–53).
6794
∣
www.pnas.org/cgi/doi/10.1073/pnas.1015267108
Isidro-Llobet et al.

can be applied in a DOS context for the construction of libraries
of structurally unique compounds with increased biological rele-
vance. Toward this end, we wanted to explore the incorporation
of structural motifs from known bioactive molecules (or isosteres
thereof) into macrocyclic ring structures. In this paper we report
the development of a DOS strategy for the generation of struc-
turally diverse small molecules based around biologically relevant
macrocyclic peptidomimetic frameworks. In a proof-of-concept
study this approach was applied to the synthesis of a library of
14 structurally diverse and complex compounds containing these
motifs. The modular nature of the synthetic routes developed
should enable their application in future DOS studies for the
creation of larger libraries of these derivatives incorporating
greater levels of structural diversity. In addition, we envisage that
these chemistries will prove to be useful in a wide variety of other
synthetic applications.
Results and Discussion
Outline of the Synthetic Strategy. We targeted the synthesis of
structurally unique and diverse small molecules based upon two
general macrocyclic peptidomimetic structural types (A and B,
Fig. 2). Both structures have a trizaole ring that replaces an
amide bond, and structure B also incorporates a DKP in the
macrocycle. Additionally, both A and B contain seven points
where stereochemical and scaffold diversity can be easily intro-
duced. The presence of free amine groups in the library com-
pounds was desired as these would provide synthetic handles
for further derivatization; this should allow a means of tuning
the biological profile of any “hits” or enable labeling of the com-
pounds for use as chemical probes.
Initial proof-of-concept studies were directed toward the
synthesis of a library of 12 compounds based around four differ-
ent scaffolds: cis-DKPs, trans-DKPs, 1,4-, and 1,5-triazoles.
Stereochemical diversity in this collection is high as every mole-
cule contains three stereogenic centers that are changed through-
out the library.
Scheme 1. Outline of the B/C/P strategy for the preparation of the target
library.
merization of the chiral centers needs to be avoided at all stages
of the synthesis in order to obtain the products as single stereo-
isomers (and thus access the full matrix of stereochemical isomers
in the library, i.e., maximizing stereochemical diversity). In addi-
tion, generation of both 1,4- and 1,5-subsituted triazoles from
each tripetide derivative is desired so as to be able to access
the desired scaffold diversity in the library; thus the 1,3-dipolar
cycloaddition needs to operate in a highly controllable and regio-
selective fashion.
Our strategy for the synthesis was based around the elegant
build/couple/pair (B/C/P) approach described by Nielsen and
Schreiber (55). The outline of our B/C/P pathway is shown in
Scheme 1. The build step involves the preparation of two types
of chiral building blocks: The first contains a free amine and an
azide (“azido-amine” building blocks), whereas the second con-
tains a carboxylic acid and an alkyne (“alkyne-acid” building
blocks). Coupling of three of these building blocks via amide
bond formation would furnish a range of tripeptide derivatives;
this process provides the basis for stereochemical diversity. The
subsequent pair phase provides the basis for scaffold diversity and
will comprise of two cyclization steps. First, a 1,3-dipolar cycload-
dition will be used to selectively combine the azide and alkyne
functionalities of these tripeptides, thus generating the desired
macrocyclic peptidomimetic architecture. Intramolecular cycliza-
tion reactions between amine and carbonyl moieties would
then introduce the DKP motif into the macrocyclic framework.
There are several key synthetic challenges associated with the
proposed route. Two are particularly worthy of note. First, epi-
Synthesis of the Proof-of-Concept Library. All building blocks were
prepared starting from simple, commercially available and rela-
tively cheap amino acid derivatives (Scheme 2). For the synthesis
of the “azido-amine” building blocks, α-azido acids ðSÞ-2-azido-3-
phenylpropanoic acid 1a and the corresponding enantiomer 1b
were obtained from L phenylalanine (L-Phe) and D phenylala-
nine (D-Phe) by azido transfer using in situ generated trifluoro-
methanesulfonyl azide (56). Boc-L-Lys(Z)-OH was methylated
using EDC·HCl and 4-(N;N-dimethylamino)pyridine and cataly-
O
L-Phe or D-Phe
O
S
CF3
O
S
F3C
N3
CuSO4, K2CO3
O
O
NaN3
*
O
O
OH
O
S
H2O, MeOH,
CH2Cl2
(overnight)
H2O, CH2Cl2
2 h, 0 ˚C to rt
F3C
N3
Bn
1a: (S) 74%
1b: (R) 63%
EDC·HCl (1 eq)
DMAP (0.1 eq)
H2 Pd/C
Boc-L-Lys(Z)-OH
Boc-L-Lys(Z)-OMe
Boc-L-Lys-OMe
MeOH (4 eq)
MeOH
81 %
2
TFA·H-L-Lys-OMe
EDC·HCl
Boc-L-Lys-OMe
N3
R
N
O
*
TFA,
HOBt
Et3N
O
R
N
O
A
B
N3
*
CH2Cl2
N
*
O
Bn
N
*
1a,1b + 2
N
HN
N
HN
CH2Cl2
overnight, rt
(1:1)
azido-amine
4a: (S) 77% (2 steps)
4b: (R) 65% (2 steps)
3a: (S)
3b: (R)
Ph
O
O
*
*
O
*
O
HN
NH
O
O
NH OMe
NH2
Boc-Lys-OH
O
*
NH
O
OH
O
OSu
Boc-L-Lys-OH
or
HN
HOSu, DCC
Boc-D-Lys-OH
5
alkyne-acid
6a: (S) 86%
6b: (R) 84%
DIPEA, DMF
4 h, rt
EtOAc, dioxane
Fig. 2. General structures of the library compounds: (A) macrocyclic pepti-
domimetics; (B) macrocyclic DKPs. Diversity can be introduced in each struc-
ture by changing the building blocks and reaction conditions. Asterisks
indicate chiral centers.
Scheme 2. Build step: Building-block synthesis.
Isidro-Llobet et al.
PNAS
∣
April 26, 2011
∣
vol. 108
∣
no. 17
∣
6795

O
O
O
OMe
tically hydrogenated to yield Boc-L-Lys-OMe 2 that was coupled
to 1a and 1b. Subsequent Boc removal using TFA yielded the two
azido-amine building blocks 4a,b. The necessary alkyne-acid
building blocks Boc-L-Lys(N-4-pentynoic acid)-OH 6a and
Boc-D-Lys(N-4-pentynoic acid)-OH 6b were obtained by acyla-
tion of Boc-L-Lys-OH and Boc-D-Lys-OH with 4-pentynoic acid
succinimidyl ester (57).
In the couple step, stereochemical diversity was efficiently
generated by combination of building blocks 4a,b with 6a,b.
Three different tripeptide derivatives 7a–c were obtained as
single stereoisomers (Scheme 3).
1,3-dipolar
DKP
O
1
cycloaddition
formation
*
1
HN
*
H3N
O
N
H
2
(a) or (b)
(c) or (d)
*
2
*
NH
HN
O
7a-c
3
Bn
*
N
N
HN
N
N
N
HN
N
N
H
O
O
3
*
7-11a:
(1S,2S,3S)
7-11b:
(1S,2R,3S)
7-11c:
(1S,2S,3R)
1,4-triazole : 10a 10b 10c
(c) : 63% 41% 49%
Bn
1,4-triazole : 8a 8b 8c
(d) : 94% 93% 90%
(a) : 90% 81% 68%
1,5-triazole :
(c) :
11a 11b 11c
46% 35% 23%
91% 50% 91%
1,5-triazole :
9a 9b 9c
90% 90% 87%
(b) :
(d) :
With tripeptide derivatives 7a–c in hand, we were ready to
attempt the key 1,3-dipolar cycloaddition process to generate the
1,4- and 1,5-disubstituted triazoles with concomitant construction
of the macrocyclic architecture. Though there are numerous
reported examples of the use of regioselective 1,3-dipolar cyclo-
additions to furnish 1,4-triazole containing macrocycles (58), only
a handful of these employed chiral α-azido acids as the substrates
(48, 59, 60). The synthesis of 1,5-triazole containing macrocycles
via 1,3-dipolar cycloadditions (61) is much less common, and in
the very limited number of examples that use α-azido acid con-
taining substrates, the yields of the isolated products are typically
very low (47). A method where the formation of the macrocycle
proceeded with good regioselectivity (1,5-triazole over 1,4-tria-
zole) and moderate yields has been recently described, but there
were no stereogenic centers adjacent to the azide present in the
substrates (62, 63). We faced the challenge of performing the
same reaction with chiral α-azido acids, desiring both high yields
and no epimerization of the chiral center adjacent to the azide.
Such a reaction would be expected to be of significant interest for
the synthesis of natural product derivatives and for other applica-
tions in synthetic organic chemistry.
We envisaged that regioisomeric macrocycles containing
1,4- and 1,5-disubstituted triazoles could be obtained by reacting
peptides 7a–c with Cu or Ru catalysts, respectively, in a “click”
type 1,3-dipolar cycloaddition reaction (Scheme 4) (64, 65). In
the final optimized conditions tripeptide derivatives 7a–c were
refluxed overnight in the presence of CuI and N;N-diisopropy-
lethylamine (DIPEA) in THF (“copper route”) or ½CpꢀRuClꢁ4
in toluene (“ruthenium route”). The first six members of the
library (8a–c and 9a–c) were obtained after subsequent Boc re-
moval. Under these optimized conditions, the 1,3-dipolar cyclo-
addition reactions were completely regioselective and proceeded
without epimerization of the chiral centers (a single stereoisomer
was detected in the crude 1H NMR of each cyclization product).
The purification was typically straightforward (flash chromato-
graphy on silica gel), and the products were obtained in very good
yields (68–90%).
Scheme 4. Pair Steps. 1,3-Dipolar cycloaddition followed by DKP formation.
Conditions: (A) (i) CuI (2 eq), DIPEA (3 eq), THF, reflux, overnight; (ii) HCl-
MeOH (1.25 M); (B) (i) ½CpꢀRuClꢁ4, toluene, reflux, overnight; (ii) HCl-MeOH
(1.25 M); (C) AcOH-NMM (1∶1.5), 2-butanol, overnight reflux (D) AcOH-
NMM* (1∶1.5), 2-butanol, microwave 150 ºC, 3–6 h. NMM*: morpholino-
methyl-polystyrene (3.51 mmol∕g).
fore, during our optimization process, we decided to use slightly
acidic buffered conditions [Scheme 4, conditions (c)] (67). Using
these conditions we typically observed 100% conversion. How-
ever, separation of the N methylmorpholine (NMM) salts from
the desired products proved to be difficult due to the high water
solubility of the DKPs. Thus, the yields after separation either by
precipitation or flash column chromatography on silica gel were
relatively low. To solve this problem we decided to use solid-
supported NMM (morpholinomethyl-polystyrene). Also thermal
heating was replaced by microwave heating. When these condi-
tions were used [Scheme 4, conditions (d)], the only necessary
work up was filtration of the solid support and evaporation to
dryness. Thus, the yields were typically higher than 90%. The
only exception was compound 11b, which was obtained with
50% yield because an extra recrystallization was necessary to
obtain the pure product. To the best of our knowledge the use
of solid supported NMM in DKP formation is previously unde-
scribed. It is envisaged that this will prove to be a valuable general
method for the synthesis of DKPs that do not precipitate in the
reaction media. In addition, the combination of solid-supported
NMM with AcOH and microwave heating represents valuable
previously undescribed methodology for the formation of difficult
DKPs without epimerization of chiral centers.
Rigidifying the Macrocyclic Scaffold. It is a widely accepted concept
in medicinal chemistry that increasing the rigidity of a compound
can increase its affinity for a target (68); more rigid molecules
might also be expected to interact more selectively with a parti-
cular target. In addition, the introduction of extra rigidity into
a given molecular framework may drastically affect its overall
3D structure and consequently provide an additional means of
generating additional shape diversity. We were thus interested
in generating previously undescribed macrocyclic peptidomi-
metics with increased structural rigidity as a means of probing
areas of biologically relevant chemical space beyond those
accessed by the library compounds described above. Toward this
end we were inspired by the structure of (þ)-piperazinomycin,
a naturally occurring macrocyclic piperazine derivative with anti-
microbial and antifungal activity (69). This molecule contains a
biaryl ether ring linkage that is thought to confer rigidity upon the
structure. We envisaged that extra rigidity in our general macro-
cyclic peptidomimetic scaffolds could be achieved by replacing
a linear alkyl chain by an aromatic ring. Thus two previously
undescribed macrocyclic peptidomimetics 13 and 14 were tar-
geted (Fig. 3). Both compounds were prepared from linear pep-
tide 12 using the same B/C/P strategy as for the rest of the library
compounds. In this case, the best conditions for the 1,3-dipolar
cycloaddition to obtain 13 involved refluxing in toluene in the
presence of CuI and 1,8-diazabicyclo[5.4.0]undec-7-ene (45%
The next pair step involved introduction of the DKP unit into
the macrocyclic framework of the cycloaddition products. DKP
formation is a common side reaction in peptide synthesis, which
is especially favored in basic conditions or under acetic acid
catalysis (66). Preliminary experiments explored DKP formation
under basic conditions. However, these reactions were found to
be extremely sluggish, and attempts to accelerate the rate and
increase conversion through the use of higher reaction tempera-
tures led to epimerization of the chiral centers present. There-
O
OMe
TFA·H-L-Lys-OMe
Boc-Lys-OH
O
EDC·HCl, HOBt
2
1
N3
*
BocNH
+
*
*
O
O
alkyne-acid
6a, 6b
Et3N, CH2Cl2
overnight, rt
N
H
Bn
azido-amine
4a, 4b
NH
4a + 6a
4a + 6b
4b + 6a
7a: (1S,2S,3S) 37%
7b: (1S,2R,3S) 38%
7c: (1S,2S,3R) 43%
3
*
HN
Bn
2
O
O
N3
Scheme 3. Couple step: Coupling of building blocks 4a,b and 6a,b via amide
bond formation.
6796
∣
www.pnas.org/cgi/doi/10.1073/pnas.1015267108
Isidro-Llobet et al.

O
OMe
O
OH
BocNH
O
N
H
NH
NH
HN
HN
N3
O
O
O
(+)-piperazinomycin
O
12
Bn
OMe
O
HN
NH
HCl·H2N
N
H
NH
Bn
O
O
N
HN
N
N
HN
N
N
HN
O
O
N
Bn
13
14
O
Fig. 3. Rigidified library compounds 13 and 14 inspired by the antibiotic (+)
piperazinomycin and prepared from linear peptide 12. Compared to the
other library compounds, a linear chain has been replaced by an aromatic
ring (highlighted in blue).
Fig. 4. Location of novel macrocyclic peptidomimetics in chemical space de-
fined by shape- and charge-based molecular surface properties. Randomly
selected FDA-approved drugs and ChemBridge screening library compounds
are also represented in the graphic. For all structures TAE/RECON electrostatic
potential and electrostatic potential autocorrelation descriptors (75) were
calculated as implemented in the molecular operating environment and
the resulting variables were subject to principal component analysis in the
same program. The first two principle components are displayed in this figure
and the exploration of underrepresented chemical space by our DOS library
described in this work can clearly be seen.
yield). The DKP formation to obtain 14 was performed using
condition (d) in Scheme 4, but in this case, a reaction time of
9 h of reaction was required to reach 100% conversion. This
could possibly be attributed to the higher rigidity of the macro-
cycle that would be expected to make the additional ring closure
more difficult. The synthesis of these two compounds demon-
strates the versatility of the methodology employed; important
structural modifications can be introduced into the macrocyclic
derivatives in a simple way through variation in the building
blocks employed.
ated. Computational analyses demonstrated that the library
accesses both chemical space not explored by molecules from
“traditional” medicinal chemistry collections and also more po-
pulated areas, showing the versatility of the developed synthetic
methodology. In preliminary biological assays a number of
library compounds showed interesting biological properties; in
particular, compound 14 showed significant antibacterial activity
against Staphylococcus aureus. Further biological screening is
currently ongoing, and full results will be reported in due course.
Because of the modular nature of our synthetic strategy, we an-
ticipate that it holds significant potential for the DOS of libraries
of macrocyclic peptidomimetics with greater levels of diversity. A
wide variety of natural and unnatural α-, β-, and γ-amino acids
could be used in the build step of the synthesis, which would al-
low the generation of considerable stereochemical and scaffold
diversity. In addition, the final library compounds should prove
amenable to further modification and diversification. The gen-
eration of the proof-of-concept library required the development
of two previously undescribed pieces of synthetic methodology.
First, we have described a protocol for the macrocyclic ring
closure of chiral α-azido acids via regioselective and epimeriza-
tion-free 1,5-disubstituted triazole formation. The macrocycle
products were obtained in superior yields to those described
in the literature (47). Second, we have reported the development
of a unique method for the DKP synthesis using solid-supported
N methylmorpholine in combination with microwave heating.
The procedure allowed for the synthesis of structurally complex
DKPs in excellent yields. Crucially, this method may represent a
previously undescribed general strategy for the synthesis of
DKP-based structures that could be difficult to isolate by other
means. Owing to their robust and flexible natures, and given
the presence of macrocyclic peptidomimetics and DKPs in many
molecules of biological interest, it is envisaged that both pre-
viously undescribed methodologies will prove to be useful in a
wider synthetic context.
Assessing the Diversity of the Library Compounds. We have utilized a
computational process for diversity assessment based around the
calculation of various shape- and charge-based molecular surface
descriptors followed by principal component analysis (PCA) (6, 9,
70, 71). Essentially this provides a measure of overall chemical
space coverage (9). In brief, we analyzed various properties of
compounds 8–11a–c, 13 and 14 with respect to their similarity
to wider chemical space, as represented by US Food and Drug
Administration (FDA)-approved drugs and
a commercial
small-molecule library (see SI Appendix for more detail). A set
of 657 approved drugs was randomly selected from DrugBank
(72), and 746 compounds were selected randomly from the
ChemBridge screening library, as downloaded from ZINC (73).
The distribution of these three compound sets in chemical space
is illustrated in Fig. 4. It can be seen that FDA-approved drugs
and the ChemBridge screening library compounds occupy similar
regions of chemical space. On the other hand, 12 of the unique
compounds (8–11a–c) occupy an area of chemical space away
from this region defined by conventional medicinal chemistry
(74). This result illustrates the value of our synthetic strategy
for accessing previously undescribed areas of chemical space.
Overall, a reasonable level of diversity (chemical space coverage)
was achieved by the synthetic library. Interestingly, compounds 13
and 14 are very clearly separated from the other macrocyclic
peptidomimetic derivatives, occupying a more populated area
of chemical space due to a more “typical” aromatic ring replacing
a previously linear chain in the molecules. Hence, by selecting a
suitable synthesis scheme one can simultaneously decide which
region of chemical space to explore, while at the same time en-
suring sufficient diversity in the compounds being synthesized.
Conclusions
In conclusion, we have described a strategy for the DOS of a
library of structurally unique and diverse macrocyclic peptidomi-
metics from simple, readily available amino acid starting materi-
als. In a proof-of-concept study, a library of 14 such compounds
displaying both scaffold and stereochemical diversity was gener-
Materials and Methods
General experimental remarks, complete experimental procedures for the
synthesis of the building blocks, full characterization of all unique com-
pounds, and details of the analysis of chemical space method are contained
in SI Appendix.
Isidro-Llobet et al.
PNAS
∣
April 26, 2011
∣
vol. 108
∣
no. 17
∣
6797

Society, Frances and Augustus Newman Foundation, and Wellcome Trust
(to D.R.S. and M.W.). A.I.-L. thanks the EU for a Marie-Curie Intra-European
Fellowship.
ACKNOWLEDGMENTS. This work was supported by grants from the European
Union, Engineering and Physical Sciences Research Council, Biotechnology
and Biological Sciences Research Council, Medical Research Council, Royal
1. Kaiser M, Wetzel S, Kumar K, Waldmann
compound libraries. Cell Mol Life Sci 65:1186–1201.
H
(2008) Biology-inspired synthesis of
40. Taylor AM, Schreiber SL (2009) Aziridines as intermediates in diversity-oriented synth-
eses of alkaloids. Tetrahedron Lett 50:3230–3233.
2. Spring DR (2005) Chemical genetics to chemical genomics: Small molecules offer big
insights. Chem Soc Rev 34:472–482.
3. Kawasumi M, Nghiem P (2007) Chemical genetics: Elucidating biological systems with
small-molecule compounds. J Invest Dermatol 127:1577–1584.
41. Noren-Muller A, et al. (2006) Discovery of protein phosphatase inhibitor classes by
biology-oriented synthesis. Proc Natl Acad Sci USA 103:10606–10611.
42. DeSimone RW, Currie KS, Mitchell SA, Darrow JW, Pippin DA (2004) Privileged struc-
tures: Applications in drug discovery. Comb Chem High T Scr 7:473–493.
43. Danishefsky S (2010) On the potential of natural products in the discovery of pharma
leads: A case for reassessment. Nat Prod Rep 27:1114–1116.
44. Koch MA, et al. (2005) Charting biologically relevant chemical space: A structural
classification of natural products (SCONP). Proc Natl Acad Sci USA 102:17272–17277.
45. Wild CT, Shugars DC, Greenwell TK, McDanal CB, Matthews TJ (1994) Peptides corre-
sponding to a predictive alpha-helical domain of human immunodeficiency virus type
1 gp41 are potent inhibitors of virus infection. Proc Natl Acad Sci USA 91:9770–9774.
46. Kilby JM, et al. (1998) Potent suppression of HIV-1 replication in humans by T-20, a
peptide inhibitor of gp41-mediated virus entry. Nat Med 4:1302–1307.
47. Horne W, Olsen C, Beierle J, Montero A, Ghadiri M (2009) Probing the bioactive
conformation of an archetypal natural product HDAC inhibitor with conformationally
homogeneous triazole-modified cyclic tetrapeptides. Angew Chem Int Edit
48:4718–4724.
4. Spiegel DA, Schroeder FC, Duvall JR, Schreiber SL (2006) An oligomer-based approach
to skeletal diversity in small-molecule synthesis. J Am Chem Soc 128:14766–14767.
5. Walsh DP, Chang YT (2006) Chemical genetics. Chem Rev 106:2476–2530.
6. Galloway WRJD, Bender A, Welch M, Spring DR (2009) The discovery of antibacterial
agents using diversity-oriented synthesis. Chem Commun 2446–2462.
7. Galloway WRJD, Diaz-Gavilan M, Isidro-Llobet A, Spring DR (2009) Synthesis of unpre-
cedented scaffold diversity. Angew Chem Int Edit 48:1194–1196.
8. Galloway WRJD, Spring DR (2009) Is synthesis the main hurdle for the generation of
diversity in compound libraries for screening? Expert Opin Drug Dis 4:467–472.
9. Galloway WRJD, Isidro-Llobet A, Spring DR (2010) Diversity-oriented synthesis as a
tool for the discovery of novel biologically active small molecules. Nat Commun
1:80, 10.1038/ncomms1081.
10. Spring DR (2003) Diversity-oriented synthesis; a challenge for synthetic chemists. Org
Biomol Chem 1:3867–3870.
11. Haggarty SJ (2005) The principle of complementarity: Chemical versus biological space.
Curr Opin Chem Biol 9:296–303.
48. Bock VD, Speijer D, Hiemstra H, van Maarseveen JH (2007) 1,2,3-Triazoles as peptide
bond isosteres: Synthesis and biological evaluation of cyclotetrapeptide mimics. Org
Biomol Chem 5:971–975.
12. Schwarz MK, Sauer WHB (2003) Molecular shape diversity of combinatorial libraries:
A prerequisite for broad bioactivity. J Chem Inf Comput Sci 43:987–1003.
13. Burke MD, Schreiber SL (2004) A planning strategy for diversity-oriented synthesis.
Angew Chem Int Edit 43:46–58.
49. Williams RM, Durham CA (1988) Bicyclomycin: Synthetic, mechanistic, and biological
studies. Chem Rev 88:511–540.
50. Campbell J, Blackwell HE (2009) Efficient construction of diketopiperazine macroar-
rays through
a cyclative-cleavage strategy and their evaluation as luminescence
14. Burke MD, Berger EM, Schreiber SL (2003) Generating diverse skeletons of small
molecules combinatorially. Science 302:613–618.
15. Schreiber SL (2009) Molecular diversity by design. Nature 457:153–154.
16. Kennedy JP, et al. (2008) Application of combinatorial chemistry science on modern
drug discovery. J Comb Chem 10:345–354.
17. Shelat AA, Guy RK (2007) Scaffold composition and biological relevance of screening
libraries. Nat Chem Biol 3:442–446.
18. Lovering F, Bikker J, Humblet C (2009) Escape from flatland: Increasing saturation as an
approach to improving clinical success. J Med Chem 52:6752–6756.
19. Spandl RJ, Bender A, Spring DR (2008) Diversity-oriented synthesis; a spectrum of
approaches and results. Org Biomol Chem 6:1149–1158.
inhibitors in the bacterial symbiont Vibrio fischeri. J Comb Chem 11:1094–1099.
51. Hamann MT (2004) Technology evaluation: Kahalalide F (PharmaMar). Curr Opin Mol
Ther 6:657–665.
52. Magauer T, Martin H, Mulzer J (2009) Total synthesis of the antibiotic kendomycin
by macrocyclization using photo-fries rearrangement and ring-closing metathesis.
Angew Chem Int Edit 48:6032–6036.
53. Nahrwold M, Bogner T, Eissler S, Verma S, Sewald N “Clicktophycin-52”: A bioactive
cryptophycin-52 triazole analogue. Org Lett 12:1064–1067.
54. Driggers EM, Hale SP, Lee J, Terrett NK (2008) The exploration of macrocycles for drug
discovery—An underexploited structural class. Nat Rev Drug Discov 7:608–624.
55. Nielsen T, Schreiber S (2008) Towards the optimal screening collection: A synthesis
strategy. Angew Chem Int Edit 47:48–56.
20. Lipinski C, Hopkins A (2004) Navigating chemical space for biology and medicine.
Nature 432:855–861.
56. Lundquist JT, Pelletier JC (2001) Improved solid-phase peptide synthesis method utiliz-
21. Bender A, et al. (2006) Diversity oriented synthesis: A challenge for synthetic chemists.
Chemical Genomics: Small Molecule Probes to Study Cellular Function, eds S Jaroch
and H Weinmann (Springer, Berlin), pp 47–60.
22. Hopkins AL, Mason JS, Overington JP (2006) Can we rationally design promiscuous
drugs? Curr Opin Struct Biol 16:127–136.
23. Thomas GL, Wyatt EE, Spring DR (2006) Enriching chemical space with diversity-
oriented synthesis. Curr Opin Drug Disc Dev 700–712.
24. Clardy J, Walsh C (2004) Lessons from natural molecules. Nature 432:829–837.
25. Pucheault M (2008) Natural products: Chemical instruments to apprehend biological
symphony. Org Biomol Chem 6:424–432.
26. Schneider G, Grabowski K (2007) Properties and architecture of drugs and natural
products revisited. Curr Chem Biol 1:115–127.
27. Schreiber SL (2000) Target-oriented and diversity-oriented organic synthesis in drug
discovery. Science 287:1964–1969.
28. Comer E, Rohan E, Deng L, Porco JA, Jr (2007) An approach to skeletal diversity using
functional group pairing of multifunctional scaffolds. Org Lett 9:2123–2126.
29. Di Micco S, et al. (2009) Identification of lead compounds as antagonists of protein Bcl
xL with a diversity-oriented multidisciplinary approach. J Med Chem 52:7856–7867.
30. Kuruvilla FG, Shamji AF, Sternson SM, Hergenrother PJ, Schreiber SL (2002) Dissecting
glucose signalling with diversity-oriented synthesis and small-molecule microarrays.
Nature 416:653–657.
ing α-azide-protected amino acids. Org Lett 3:781–783.
57. Galibert M, Dumy P, Boturyn
D (2009) One-pot approach to well-defined bio-
molecular assemblies by orthogonal chemoselective ligations. Angew Chem Int Edit
48:2576–2579.
58. Looper RE, Pizzirani D, Schreiber SL (2006) Macrocycloadditions leading to conforma-
tionally restricted small molecules. Org Lett 8:2063–2066.
59. Bock VD, Perciaccante R, Jansen TP, Hiemstra H, van Maarseveen JH (2006) Click
chemistry as a route to cyclic tetrapeptide analogues: Synthesis of cyclo-[Pro-Val-Ψ
(triazole)-Pro-Tyr]. Org Lett 8:919–922.
60. Springer J, et al. (2008) Backbone amide linker strategy for the synthesis of 1,4-tria-
zole-containing cyclic tetra- and pentapeptides. Eur J Org Chem 2008:2592–2600.
61. Zhang L, et al. (2005) Ruthenium-catalyzed cycloaddition of alkynes and organic
azides. J Am Chem Soc 127:15998–15999.
62. Kelly AR, et al. (2009) Accessing skeletal diversity using catalyst control: Formation of n
and n þ 1 macrocyclic triazole rings. Org Lett 11:2257–2260.
63. Marcaurelle LA, et al. (2010) An aldol-based build/couple/pair strategy for the synth-
esis of medium- and large-sized rings: Discovery of macrocyclic histone deacetylase
inhibitor. J Am Chem Soc 132:16962–16976.
64. Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: Diverse chemical function from
a few good reactions. Angew Chem Int Edit 40:2004–2021.
65. Huisgen R (1984) 1,3-Dipolar cycloadditions—Introduction, survey, mechanism. 1,3-Di-
polar Cycloaddition Chemistry, ed A Padwa (Wiley, New York), Vol 1, pp 1–176.
66. Gisin BF, Merrifield RB (1972) Carboxyl-catalyzed intramolecular aminolysis. A side
reaction in solid-phase peptide synthesis. J Am Chem Soc 94:3102–3106.
67. Suzuki K, Sasaki Y, Endo N, Mihara Y (1981) Acetic acid-catalyzed diketopiperazine
synthesis. Chem Pharm Bull 29:233–237.
31. Stanton BZ, et al. (2009) A small molecule that binds Hedgehog and blocks its signaling
in human cells. Nat Chem Biol 5:154–156.
32. Ng PY, Tang YC, Knosp WM, Stadler HS, Shaw JT (2007) Synthesis of diverse lactam
carboxamides leading to the discovery of a new transcription-factor inhibitor. Angew
Chem Int Edit 46:5352–5355.
33. Koehler AN, Shamji AF, Schreiber SL (2003) Discovery of an inhibitor of a transcription
factor using small molecule microarrays and diversity-oriented synthesis. J Am Chem
Soc 125:8420–8421.
34. Wyatt EE, et al. (2008) Identification of an anti-MRSA dihydrofolate reductase inhibi-
tor from a diversity-oriented synthesis. Chem Commun 4962–4964.
35. Thomas GL, et al. (2008) Anti-MRSA agent discovery using diversity-oriented synthesis.
Angew Chem Int Edit 47:2808–2812.
68. Lumley JA (2005) Compound selection and filtering in library design. QSAR Comb Sci
24:1066–1075.
69. Boger DL, Zhou JC (1993) Total synthesis of (+)-piperazinomycin. J Am Chem Soc
115:11426–11433.
70. Ringner M What is principal component analysis? Nat Biotechnol 26:303–304.
71. Bauer RA, DiBlasi CM, Tan DS (2010) The tert-butylsulfinamide lynchpin in transition-
metal-mediated multiscaffold library synthesis. Org Lett 12:2084–2087.
72. Wishart DS, et al. (2008) DrugBank: A knowledgebase for drugs, drug actions and
drug targets. Nucleic Acids Res 36:D901–D906.
36. Dandapani S, Marcaurelle LA (2010) Current strategies for diversity-oriented synthesis.
Curr Opin Chem Biol 14:362–370.
37. Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL (2007) Drugs for bad bugs: Confront-
ing the challenges of antibacterial discovery. Nat Rev Drug Discov 6:29–40.
38. Pizzirani D, Kaya T, Clemons PA, Schreiber SL (2010) Stereochemical and skeletal
diversity arising from amino propargylic alcohols. Org Lett 12:2822–2825.
39. Schreiber SL (2009) The state of the art of chemical biology. The gap between scien-
tists’ aspirations and society’s expectations. ChemBioChem 10:16–29.
73. Irwin JJ, Shoichet BK (2005) ZINC—A free database of commercially available com-
pounds for virtual screening. J Chem Inf Model 45:177–182.
74. Fergus S, Bender A, Spring DR (2005) Assessment of structural diversity in combinator-
ial synthesis. Curr Opin Chem Biol 9:304–309.
75. Breneman CM, et al. (2003) New developments in PEST shape/property hybrid descrip-
tors. J Comput Aid Mol Des 17:231–240.
6798
∣
www.pnas.org/cgi/doi/10.1073/pnas.1015267108
Isidro-Llobet et al.