The discovery of antibacterial agents using diversity-oriented synthesis

17 Pages·2009·12.59 MB·English

FEATURE ARTICLE www.rsc.org/chemcomm | ChemComm
The discovery of antibacterial agents using diversity-oriented synthesis
Warren R. J. D. Galloway,a Andreas Bender,b Martin Welchc
and David R. Spring*a
Received(inCambridge,UK)25thSeptember2008,Accepted24thFebruary2009
FirstpublishedasanAdvanceArticleontheweb1stApril2009
DOI: 10.1039/b816852k
Theemergenceandincreasingprevalenceofmulti-drugresistancebacterialstrainsrepresentsa
clearandpresentdangertothestandardofhumanhealthcareasweknowit.Thesystematic
studyofmodulatingbiologicalsystemsusingsmallmolecules(so-calledchemicalgenetics)offersa
potentiallyfruitfulmeansofdiscoveringcriticallyneedednewantibacterialagents.Crucialtothe
successofthisapproachisthereadyavailabilityoffunctionallydiversesmallmoleculecollections.
Inthisfeaturearticlewefocusupontheuseofadiversity-orientedsynthesis(DOS)approachfor
theefficientgenerationofsuchcompoundcollections,anddiscusstheutilityofDOSforthe
discoveryofnewantibacterialagents.
Introduction in antibacterial research that the existing drugs we have in
handforthetreatmentofinfectiousdiseasesareinsufficientto
Thedevelopmentofantibacterialagentsisoneofthegreatest protect us in the long term.3 Thus, bacterial infection, parti-
successes of 20th century medicine.1 Since the serendipitous
cularly from multi-drug resistant strains, remains a serious
discovery of penicillin by Alexander Fleming in 1928, an threat to human lives3,4,7,8 and there is a clear and critical
arsenalofantibacterialagentshavebeendevelopedandfound
medicalneedforthediscoveryofnovelantibacterialagents.In
widespread clinical application. However, bacteria have
thiscontext,smallmolecularmasscompounds(so-calledsmall
quicklybecomeresistanttocommonlyprescribedantibiotics.2,3 moleculesw)arecertaintoplayaprominentrole.
Combined with the lack of fundamental antibiotic research
Small organic molecules have always been of interest in
carried out by pharmaceutical companies over recent
chemistry and biochemistry due to their ability to exert
decades we are left with a legacy of relatively few efficacious
powerful effects on the functions of macromolecules that
drugs.4–6Indeed,thereislittledoubtamongstmanyinvolved comprise living systems.9,10 Indeed, the underlying approach
of using small molecules to treat disease represents the basis
aDepartmentofChemistry,UniversityofCambridge,LensfieldRoad, for medicinal chemistry as we know it.11 There are a several
Cambridge,UK.E-mail:[email protected];
Fax:[phone];Tel:[phone]
bLeiden/AmsterdamCenterforDrugResearch,LeidenUniversity,
GorlaeusLaboratories,Leiden,2300,TheNetherlands wTheterm‘smallmolecules’hasnostrictdefinition,butitisusually
cDepartmentofBiochemistry,UniversityofCambridge,Cambridge, used to describe carbon-based compounds whose molecular weight
UK.E-mail:[email protected]; is generally under 2000 g mol(cid:2)1 and always less than that of
Tel:[phone] macromoleculessuchasDNA,RNAandproteins.128,129
WarrenGallowaywasbornin Andreas Bender is an Assis-
Dundee,Scotlandin1981and tantProfessor forCheminfor-
attended the University of matics with the Leiden/
Cambridge for his under- Amsterdam Center for Drug
graduate chemistry degree, Research (LACDR). Before
graduatingin2004.Hestayed joining the LACDR, hewasa
at Cambridge for his PhD Presidential Postdoctoral
studies which were funded by FellowwiththeNovartisInsti-
theEPSRCandPfizer.Under tutesforBiomedicalResearch
the supervision of Dr David in Cambridge/MA, working
Spring, his doctoral research on in silico chemogenomics
focused upon the development methods to understand small
of strategies for diversity- molecule bioactivity space.
oriented synthesis. Warren He earned his PhD from the
WarrenGalloway received his PhD in 2008 and AndreasBender University of Cambridge
is currently a postdoctoral re- workingwithProfRobertGlen
searchassistantforProfessorRobertGlenandDrDavidSpring on small-molecule data analysis. Andreas’ work is centered on
at Cambridge, working on the design and synthesis of novel cheminformatics topics, dealing with small molecule activity
modulatorsof5-HTreceptors. models,theanalysisofhigh-contentandcellularscreeningdata
andthepredictionofphysicochemicalpropertiesofmolecules.
2446 | Chem.Commun., 2009, 2446–2462 Thisjournalis(cid:3)c TheRoyalSocietyofChemistry2009
benefits associated with the use of small molecules as thera- is a demand for novel biologically active molecules that exert
peuticagentsincludingimprovedstabilityoverpeptidesinoral their biological effect through a unique mechanism. This is
administration (where peptide bonds are easily cleaved by perhaps mostpertinent in the fieldofantibacterial discovery,
proteases), synthetic accessibility and, perhaps most notably, whereitiswidelyrecognisedthatdealingwiththeproblemof
optimisation of compound bioactivity is considerably easier bacterial resistance is not simply a case of synthesising new
forasmallmoleculethanforcomplexmacromolecules. variants of existing drugs (see Fig. 1 for some examples of
Methodswhichutilisesmallmoleculesas‘chemicalprobes’to existingantibiotics).z
modulate biological systems can be described by the umbrella Established antibiotics are based around a limited number
term12 chemical genetics.13–15 Whereas traditional genetics uses of structural classes which interfere with a small number
geneknockouts(orknock-ins)ontheleveloftheDNA,chemical of biological targets and operate by a limited array of
genetics uses biologically active small molecules to directly mechanisms.17–20 Resistance to one antibiotic of a class
attenuatethecorrespondingbiologicalmacromolecularproduct frequentlyleadstoresistancetoits’wholeclass;21,22although
andthusaffectabiologicalresponse,forexample,theinhibition new derivatives of existing agents can improve efficacy
ofbacterialgrowth.Thefirststepofachemicalgeneticsexperi- temporarily, the underlying resistance mechanisms are still
ment involves the identification of a small molecule which presentintheenvironment.5Consequently,thereisanurgent
induces a desired phenotype (forward chemical genetics) or need for new antibacterial agents that have new modes of
modulates the function of a specific protein of interest (reverse action or interfere with novel targets23 and, as such, are not
chemical genetics). Thus, in the former case, investigations subject to the resistance mechanisms that have evolved to
proceed from phenotype to protein, whereas in the latter case combatpreviousgenerationsofantibacterials.24
investigationsprogressfromproteintophenotype.Smallmole- Inthequesttodiscovernovelantibacterialagentstheuseof
cules that exhibit biological effects can be discovered by both forwardchemicalgeneticsisparticularlypowerful;duetothe
forward and reverse chemical genetics approaches through the inherent lack of biological target-bias associated with the
screening of collections (or ‘libraries’) of small molecules to approach, a single forward chemical genetics experiment has
identifythosewiththedesiredcharacteristics(so-called‘hits’). the potential to identify numerous bioactive small molecules
Thisfeaturearticlediscussesvariousaspectsassociatedwith which could operate via several different modes of action.
the use of a forward chemical genetics approach for the Aforwardchemicalgeneticsprogrammegenerallyconsistsof
discovery of new antibacterial agents. In particular, we focus threedefinedstages:thesynthesisofa‘suitablydesigned’small
on the crucial importance of ‘high-quality’ rather than molecule library, a phenotypic screen to identify bioactive
‘high-quantity’ small molecule libraries, and the use of compounds and a biological target identification method.12
diversity-orientedsynthesis(DOS)togeneratesuchcompound Work has have been carried out within the author’s research
collections in a highly efficient manner. We conclude with
some representative examples from our own group which
highlight the utility of a DOS approach for the discovery of zAninherentproblemassociatedwiththe‘antibioticapproach’isthat
newantibacterialagents. byinhibitingbacterialcellularprocessesthatareessentialformicro-
bialsurvivalanintenseselectionpressurefordrug-resistantmutations
iscreated.Consequentlythedevelopmentofresistanceisinevitablythe
Chemical genetics and antibacterial discovery
resultofantibioticuseandultimatelylimitstheefficacyandlifespan
of every antibiotic.17 That is, bacterial resistance to antibiotics can
The ultimate aim of any biological screening project is to
never be overcome, it is simply a case of managing it as well as
identifybiologicallyactivesmallmolecules.Inparticular,there possible.
Martin Welch grew up near David Spring is currently an
Newmarket and went on to EPSRC Advanced Fellow at
study Biochemistry at Oxford the University of Cambridge
University. HisPhDwaswith Chemistry Department. Pre-
Prof Michael Eisenbach vious to this appointment he
(Weizmann Institute), where spent two and a half years as
he worked on the mechanism aWellcomeTrustPostdoctoral
of switching in the bacterial Fellow and Fulbright Scholar
flagellar motor. Next, he at Harvard University with
joined the laboratory of ProfessorStuartL.Schreiber.
Prof Jean-Pierre Samama He gained his DPhil for work
(Toulouse), where he solved on the proposed biosynthesis
the X-ray crystal structure of of the manzamine alkaloids
one of the protein complexes at Oxford University under
MartinWelch involved in bacterial chemo- DavidSpring the supervision of Professor
taxis. Later he joined the lab Sir Jack E. Baldwin FRS.
of Prof George Salmond, establishing an interest in quorum David’s research programme is focused on diversity-oriented
sensing. Presently, he is a lecturer in Biochemistry in synthesis,syntheticmethodologyandchemicalgenetics.
Cambridge. His research interests lie in understanding how
antibioticsaffectcellfunction.
Thisjournalis(cid:3)c TheRoyalSocietyofChemistry2009 Chem.Commun., 2009, 2446–2462 | 2447
Fig.1 Someselectedexamplesofantibiotics.Mostofthemajorclassesofantibioticsintherapeuticusearenaturalproductsorsemi-synthetic
derivatives thereof.16 There are three main classes of synthetic antibiotics in clinical use: sulfa drugs (e.g. sulfamethoxazole), the quinolones
(e.g.ciprofloxacin)andoxazolidinones(e.g.linezoild).16
groups to address all of these elements within the context of screening process are usually carefully designed based on
thediscoveryofnovelantibacterialagents. knowledge of the targets structure or the structure of known
natural ligands.27,31 However, if the screening process is
unbiased, such as in a forward chemical genetics experiment
Library design in the context of antibacterial
where the precise nature of the biological target is unknown,
discovery: biological and structural diversity the selection criteria for small molecules is dramatically
complicatedandcannotbedefinedapriori.26,32
Thereadyavailabilityofsmallmoleculesisofvitalimportance
Theidentificationofnovelbiologicallyactivesmallmolecules
inbiologicalscreeningexperiments.25However,afundamental
may be aided by screening functionally (biologically) diverse
consideration is what compounds should be synthesised and
compound libraries, since it has been argued that a greater
employed in these screening processes.26 Ultimately this is
sample of the bioactive chemical universe (i.e. of all bioactive
determined by the desired outcome of the experiment and
molecules)increasesthechanceofidentifyingacompoundwith
the requirements this places upon the compounds.27 For
the desired properties (for example, a small molecule with
example, if the goal of a screening process is to identify a
antibacterial properties that operates via a novel mode of
smallmoleculeforapplicationasanorallybioavailabledrug,
action).33,34 The overall functional (biological) diversity of a
then several observations have been made as to molecular
smallmoleculelibraryhasbeenshowntobedirectlycorrelated
characteristics that are desirable (most notably Lipinski’s
with its overall structural diversity, which in turnis related to
‘rule-of-five’ criteria).27–29 However, since antibacterial
theamountofchemicalspacethelibraryoccupies.33,35
compoundsaregenerallymorehydrophilicandhaveagreater
molecular weight than other drugs in general (Fig. 2), the
Structural diversity and chemical space
guidelines that govern the discovery of orally bioavailable
drugs are unlikely to be useful for the identification of new Chemicalcompoundscanbecharacterizedbyawidevarietyof
antibioticcompounds.5 physiochemical and topological ‘descriptors’ which contain
An important consideration is the nature of the biological informationabouteitherthebulkpropertiesofthecompound,
target of interest. When a specific biological molecule or such as molecular mass and lipophilicity, or its topological
family of molecules is targeted the compounds used in the featuressuchasdegreeofbranching.36–39‘Chemicalspace’isa
2448 | Chem.Commun., 2009, 2446–2462 Thisjournalis(cid:3)c TheRoyalSocietyofChemistry2009
Fig. 2 A plot of calculated logP(o/w) (partition coefficient between octanol and water)30 vs. molecular mass of antibacterial compounds
(redsquares)comparedwithcompoundsfromtheMDLDrugDataRepository(MDDR)database(bluecircles).Antibacterialmoleculesare,on
average,morehydrophilicandtypicallyhaveagreatermolecularweight,whichisinagreementwithpreviousreports.5
termoftenusedinplaceofmultidimensionaldescriptorspace; spacearedefinedbythespecificbindinginteractionsthatmust
itisaregiondefinedbyaparticularchoiceofdescriptorsand occurbetweensmallmoleculesandthethree-dimensional(3D)
thelimitsplaceduponthem.40Inthecontextofsmallmolecule molecularrecognitionpatternsonbiologicalmoleculessuchas
libraries,chemicalspacecanbedefinedasthetotaldescriptor proteins.29 What is uncertain is the size of this region in
space that encompasses all the small carbon-based molecules comparisontototalchemicalspace,i.e.whetherthebiologically
thatcould,inprinciple,becreated.40 relevant region is ‘small’ and most of the chemical universe
Each specific molecule will have a unique combination of is ‘empty’ (containing no therapeutically interesting
molecular properties and thus descriptor values, and will compounds).29 That is, are the regions of chemical space
therefore reside at a discrete point in chemical space. definedbynatural products andknown drugs the most fertile
Consequently, the structural features present within a regionsfor discoveringbiologicallyactivesmallmolecules,9or
collection of molecules will influence the overall distribution istherescopefordiscoveringusefulbioactivemolecules,parti-
of the collection in chemical space. Thus it follows that the cularly those with novel modes of action, from ‘un-tapped’
morestructurallydiversethelibrary,themorechemicalspace areasofchemicalspace?27,29Whatisclearisthatifwedonot
it interrogates. As molecular shape is intrinsically linked to tryandaccesssuchregions,wewillneverknow!Inparticular,
biologicalactivity,thegreaterthestructuraldiversityandthus since antibacterials are rather ‘un-drug-like’ structures in the
chemical space coverage of a library, the greater its overall sense that they are, on average, larger and more hydrophilic
functional (biological) diversity and consequently the better than other drug compounds (Fig. 2), it is likely that chemical
theoddsofidentifyingsmallmoleculemodulatorsforabroad space with these properties has not yet been sampled suffi-
range of biological targets.35 This viewpoint is supported by ciently. In spite of this controversy, structural diversity
the similar property principle41 which states that structurally (and thus total chemical space coverage), though rarely the
similar molecules will exhibit similar physiochemical and ‘end-game’ in a chemical genetics experiment, is generally
biologicalproperties.42 perceived to be an important consideration in small molecule
library synthesis, particularly when the precise nature of the
biologicaltargetmoleculeisunknownortheidentificationofa
Biologically relevant chemical space novelbiologicallyactivemoleculeisdesired.25,44,45
Biologically relevantchemical spacehas beendefined as those
partsofchemicalspaceinwhichbiologicallyactivecompounds Maximising structural diversity and the importance
reside.40 The degree of overlap between biologically relevant
of structural complexity
chemical space and total chemical space is somewhat of a
contentious issue and subject to much debate in the It is widely accepted that it is not synthetically feasible to
literature.9,26,27,43 The limits of biologically relevant chemical produce all theoretically stable, small carbon-based
Thisjournalis(cid:3)c TheRoyalSocietyofChemistry2009 Chem.Commun., 2009, 2446–2462 | 2449
molecules.27,29,32 Therefore the generation of a collection of andstructurallycomplex.Acollectionofsuchcompoundshas
structurally diverse small molecules offers a unique challenge the potential to provide hits against a panel of biological
tothesyntheticchemist.26,32Makingmoleculescosts,bothin targets, allowing the discovery of small molecules with
termsoftimeandmoney;thereforeselectivityinsynthesisisan previouslyunknown(andpotentiallynovel)biologicaleffects.51
important consideration. This issue has spurred the develop- Thereareanumberofpotentialsourcesofsmallmolecules
ment of several different approaches to efficiently interrogate foruseinbiologicalscreens.Naturehastraditionallyservedas
wide regions of chemical space simultaneously or to identify a rich source of biologically active molecules46,52,53 which
and access areas of chemical space which have an enhanced exhibit enormous structural diversity54 and usually a high
probabilityofcontainingbioactivecompounds.26 degree of specificity for their biological target. A multitude
Though the term ‘diversity’ is to some degree a subjective of natural products have proven to be useful as drugs or
one, there are four main components of structural diversity leads55,56andarestillamajorsourceofinnovativetherapeutic
thathavebeenconsistentlyidentifiedintheliterature:32,46 agents for infectious diseases.17,52 Indeed, most of the major
1. Appendage diversity (or building-block diversity)— classesofantibioticsintherapeuticusearenaturalproductsor
variation in different structural moieties around a common semi-synthetic derivatives thereof (Fig. 1).16 In addition, due
skeleton; the evolution of natural products to interact with target
2. Functional group diversity—variation in the functional proteins, hit rates in high-throughput screens are usually
groupspresent; several times higher for natural products compared to small
3. Stereochemical diversity—variation in the orientation of molecule libraries from conventional synthetic sources
potentialmacromolecule-interactingelements;9 (vide infra). Unfortunately there are several problems asso-
4. Skeletal diversity—presence of many distinct molecular ciated with using natural product compounds in screening
skeletons(orframeworks/scaffolds).y experiments including difficulties with purification, bioactive
Increasingtheskeletaldiversityinasmallmoleculelibraryis componentidentification,structuralassignmentandchemical
widelyregardedasoneofthemosteffectivewaysofincreasing modification and analogue synthesis (processes which are
the overall structural diversity of the library.25,35,47–49 pertinent in the drug development process).25 Of particular
Furthermore, computational analyses have been carried out importance for the discovery of novel antibacterial agents is
tosupportthenotionthatsmallmultiplescaffoldlibrariesare thefactthatnaturalproductsonlyoccupyasmallproportion
superior to large single-scaffold libraries in terms of of total chemical space29,40 which runs the risk of omitting a
bio-relevant diversity.34 Libraries which are based around a vastnumberofpossiblybiologicallyvaluablesmallmolecules
single scaffold, regardless of their size, are restricted to a fromanyscreeningprocess.9
limited number of molecular shapes, as opposed to The problems associated with the use of natural product
smaller libraries designed around multiple scaffolds.34,35 compounds in biological screening experiments has spurred
Consequently, the compounds in libraries which are based the development of several different synthetic approaches for
aroundacommonmolecularskeletontendtodisplaychemical thedenovocreationofsmallmoleculecollections.Itispossible
information similarly in 3D space, thus limiting the range of to divide these approaches into two broad categories based
potential binding partners to a small set of macromolecules uponthenatureofthemolecularskeletonspresentinthesmall
withacomplementary3Dbindingsurface.9,47 moleculesthataregenerated:
In addition to structural diversity, structural complexity is 1.Biasedapproaches:thesyntheticrouteisdesignedwitha
another characteristic that is important in small molecule pre-encoded skeletal bias such that all compounds are based
libraries. Although there is some debate in the literature, it aroundasimilarmolecularskeleton;
has been argued that molecules that are structurally complex 2. Non-biased approaches: the synthetic route is designed
aremorelikelytointeractwithbiologicalmacromoleculesina withnopre-encodedskeletalbiassuchthatarangeofdifferent
selectiveandspecificmanner.25,27,29,50 molecular skeletons are present in the final small molecule
collection.
It should be noted that this distinction is somewhat arbi-
Sources of small molecules trary and most approaches towards small molecule library
synthesisliesomewherealongacontinuumbetweenthesetwo
In the context of the discovery of novel biologically active
extremes. Nevertheless, this division does provide a useful
small molecules, including antibacterial agents, what is
frameworkforthefollowingdiscussion.
requiredisasmallmoleculelibrarythatefficientlyinterrogates
large areas of chemical space simultaneously and whose
Biased approaches: combinatorial synthesis and
members have the potential to act as selective modulators of
biological molecules. Thus what is needed are molecules that synthesis around privileged structures
arestructurallydiverse(andconsequentlyfunctionallydiverse)
Pharmaceutical proprietary compound collections are
yThe term ‘molecular skeleton’ has no strict definition. Within the traditionally very important sources of small molecules.35,54
contextofthisreport,thedescriptionrecentlyoutlinedbySchreiberis At the beginning of the 1990s a typical pharmaceutical
appropriate;thetermskeletonlooselydenotesrigidifyingelementsin company would have possessed numerous collections of
small molecules; these can be atom connectivities that yield either
structurally related compounds which had been synthesised
linked, fused, bridged or spiro rings, or acyclic conformational
during traditional medicinal chemistry-lead optimisation
elements that provide substantial rigidification by avoiding non-
bondinginteractions.96 campaigns.57 Such compound collections would have been
2450 | Chem.Commun., 2009, 2446–2462 Thisjournalis(cid:3)c TheRoyalSocietyofChemistry2009
pannedforpossibleantibacterials(andmanyotherdrugtypes) physicalchemicalpropertiesareimportantforantibacterials.5
overthecourseofmanyyearsanditwasacknowledgedatthis However,itisimportanttonotethatnewantibacterialagents
time that the ‘low hanging fruit’ from the antibiotic tree had with novel modes of action may exist outside of known
probably already been picked.5 Companies therefore sought ‘antibiotic physiochemical space’; consequently, small
new sources of compounds to augment their screening molecules libraries that simultaneously cover both known
collections.5,57 Combinatorial chemistry emerged as a key ‘antibioticchemicalspace’andunexploredregionsofchemical
strategyinthisregard. spacemayproveparticularlyvaluableinthiscontext.
In a very broad sense combinatorial chemistry may be Although molecules based around a specific core skeleton
defined as the rapid synthesis and screening of libraries of are usually limited to a smaller range of biological partners
varied compounds to identify agents with desired functional thanmoleculesbasedaroundawidevarietyofskeletons,ithas
properties.58 Combined with established high-throughput emerged that there is a sub-set of molecular skeletons whose
screening(HTS)techniques,thedevelopmentofcombinatorial presenceinmoleculesconfersuponthemmoreflexiblebinding
chemistry strategies in the early 1990s enabled the synthesis properties. These skeletons are known as ‘privileged’
and testing of libraries of hundreds of thousands of different frameworks (or substructures62) which have been defined as
compounds at comparably low cost.54 The approach was molecularscaffoldswithversatilebindingproperties,suchthat
quickly embraced by the pharmaceutical industry with the asinglescaffoldisabletoprovidepotentandselectiveligands
hope that new drug leads, including novel antibiotic classes, for a range of different biological targets (including multiple,
wouldbeproducedbysheerweightofnumbers.However,the unrelatedclassesofproteinreceptors63)throughmodification
expected surge in productivity, both in terms of antibiotic of functional groups.64,65 Privileged scaffolds are common to
discoveryanddrugdiscoveryingeneral,hasnotmaterialised. knownbiologicallyactivemolecules(usuallynaturalproducts)
Indeed,asoftheendofthecalendaryear2007therewasonly andthushaveprovenbiologicalrelevance.26,43,64
one reported de novo new chemical entity resulting from this Recentyearshavewitnessedanincreaseinthesynthesisof
method of chemical discovery which had been approved for small molecule libraries which are based around such
druguse(asanantitumouragent).56 privileged structures.66–71 Therationale behindusingsuchan
This‘innovationdeficit’isgenerallyattributedtodefectsin approach is based on two main hypotheses. Firstly, that
the nature of the libraries produced.59 Early combinatorial millions of years of evolutionary pressure has ‘pre-validated’
libraries have been described as being intrinsically useless for natural products, and thus compounds that are structurally
drug discovery60 because the compounds were too similar to similar, to be able to modulate protein function.26,43,72–74
each other, having limited structural diversity54 and conse- Secondly, that the chemical space explored by natural
quentlyofferingonlyanarrowsliceofchemicalspace.60This products and protein structure during evolution is strongly
limited degree of overall structural diversity can be primarily limited in size and highly conserved.26 That is, there is a
attributed to a lack of skeletal diversity. Traditionally, concept of ‘evolutionary convergence of structures’ in that
combinatoriallibrariescontainedlargenumbersofmolecules natural products have evolved to interact with multiple
which were each based around a specific molecular skeleton proteins.73,74 Synthesis around a privileged scaffold has been
(a ‘one-synthesis/one skeleton approach’) which resulted in a described as being distinct from the process of (focused)
high degree of appendage and functional group diversity combinatorial library synthesis because the ultimate goal is
(and possibly stereochemical diversity) but little variety in the identification of compounds with novel biological
the nature of the core molecular scaffold.9,25,35 Recent years properties distinct from those of the original privileged
havewitnessedthedevelopmentofmoreconsideredsynthetic compound.72 The screening of compound collections
strategies in an attempt to increase the structural diversity containing various privileged scaffold motifs has successfully
exhibitedbycombinatoriallibraries.35Neverthelesseventhese identified numerous novel compounds with antibacterial
approachesaregenerallylimitedtoknownbiologicallyactive activity(Fig.3,thoughatthetimeofwritingnonehavebeen
frameworks and, as such, have met with limited success in successfullydevelopedintomarketeddrugs).
identifyingnovelbiologicallyactivesmallmolecules. Criticsoftheprivilegedscaffoldapproachhavearguedthat
Inthecontextofantibacterialdiscoveryithasbeenargued many privileged structures have limited utility due to their
that pharmaceutical companies should place a greater promiscuous nature (that is, a lack of target specificity).62
emphasis on generating libraries which access the unique Typical privileged structure analyses have attempted to
physiochemical property space spanned by known anti- identify minimal substructures that are frequently found in
bacterial agents;61 existing antibacterials do not generally ligands associated with a particular biological target family
followLipinski’s‘rule-of-five’criteriawhereascorporatecom- (e.g. G-coupled protein receptors, serine proteases, protein
pound collections have always been heavily biased towards kinases and ligand-gated ion channels).78 However, this
compounds that do (vide supra).5 Therefore, to some process can easily detect structures that are merely drug-like
researchers it is unsurprising that such collections have been and/or promiscuous protein binders and therefore confer
found to be inadequate for the identification of new anti- bioactivity across a wide range of target families.78 In recent
bacterial agents.5,61 Indeed, in a recent review on antibiotic years the definition of privileged structure has been modified
discovery,researchersfromGlaxoSmithKlinecommentedthat by some researchers to describe commonly occurring
a new paradigm for library design in the context of anti- fragments within ligands associated with a particular target
bacterial discovery is needed, with an emphasis upon greater family; that is, the term has become identified with those
molecular diversity and a better understanding of which substructures found to be promiscuous within a given target
Thisjournalis(cid:3)c TheRoyalSocietyofChemistry2009 Chem.Commun., 2009, 2446–2462 | 2451
tomeetthechallengeofsynthesisingstructurallydiversesmall
moleculecollections.9,32,47,84
DOS has been defined as the deliberate, simultaneous and
efficient synthesis of more than one target compound in a
diversity-drivenapproach.32TheaimofaDOSistoefficiently
interrogate wide areas of chemical space simultaneously; this
may include known bioactive regions of chemical space and
unexploredregionsofchemicalspace.9,31,32,85Thehopeisthat
by sampling a greater total area of chemical space the
functional (biological) diversity of the library will be greater,
thusincreasingthechancesofidentifyingacompoundwiththe
Fig.3 Someexamplesofcompoundswithantibacterialactivitybased
desiredproperties,forexampleantibacterialactivitythrougha
around privileged scaffolds. Compound 1 was found to act as an
novelmodeofaction.33–35
inhibitoroftheMycobacteriumtuberculosisenoylacylcarrierprotein
reductaseInhA.751isbasedaroundapiperazinescaffold(highlighted AcomparisonofthesyntheticstrategiesusedinaDOSand
in red) which is frequently found in biologically active compounds a ‘traditional’ combinatorial approach is given in Fig. 4.
acrossanumberofdifferenttherapeuticareas,includingantifungals, A DOS pathway is analysed in the forward sense; simple
antidepressantsandantivirals.75,76Compound2wasfoundtoexhibit starting materials (in this case, a single compound) are
antibacterialactivityagainstarangeofGram-positivebacteria.772is converted into a collection of structurally diverse small
basedaroundabiarylscaffold(highlightedinblue)whichhasshown molecules in no more then five synthetic steps (in order to
activityagainstacrossawiderangeoftherapeuticclasses.62 maximise synthetic efficiency).9,32,86 The overall aim is the
broad coverage of chemical space, which can be contrasted
familyandcarriesthatimplicationthatthesesubstructuresare
withtheoutcomesofmoretraditionalcombinatorialsyntheses.
specific to that target family.78 However a library based
A successful DOS must address the four main types of
around a ‘promiscuous privileged scaffold’ may be advanta-
structural diversitymentioned previously;thatis,appendage,
geous if the compounds are to be screened against a wide functional group, stereochemical and skeletal.9,25,26,86,87 The
variety of targets.78 It has been argued that selectivity could
most challenging facet of DOS, and of central importance to
possibly be introduced at a later date through appropriate
itssuccess,istheabilitytoincorporateskeletaldiversityintoa
substitutionofthemolecularframework.62,78 compoundcollection.9,47,85,88Makingmoleculescosts,bothin
Related approaches include ‘natural-product guided’ meth-
terms of time and money. Therefore the ideal synthesis of a
ods such as ‘biologically oriented synthesis’79,80 and ‘biology
structurally diverse small molecule collection is one in which
inspiredsynthesis’26whichinvolvethegenerationofcompound
this diversity is achieved in the most efficient manner. The
libraries based around the core structures of natural product
efficientgenerationofmultiplemolecularscaffoldsisregarded
templates with proven biological relevance (i.e. in some sense
as one of the most effective methods of increasing the
‘traditional’combinatorialdiversityaroundanaturalproduct
overall structural diversity of a collection of molecules and
scaffold or fragment thereof, with the aim of providing
has been reported to increase the odds of addressing a
screening collections with high biological relevance).81 These broadrangeofbiologicaltargets34(relativetoasingle-scaffold
methods have had success in the identification of novel bio- library).25,35,47,48
active molecules, including antibacterials.81–83 However,
There is a clear distinction between DOS and traditional
conservation of a significant portion of the core structure of
combinatorialmethods;DOSlibrariesareusuallysmallerbut
aparentantibacterialcompoundmaybeapotentialdrawback
consist of molecules that are typically structurally more
inthecontextofantibioticdevelopment;theantibacterial‘hits’
complex, have a greater variety of core structures (skeletons)
from such a collection are likely to share a similar mode of and possess richer stereochemical variation.58 However, the
actionastheparentcompoundandthusbemoresusceptibleto
boundary between modern, more considered combinatorial
existing resistance mechanisms (vide supra). That is, this
methods and DOS is less clear-cut, and the terms DOS and
approachmaybelesslikelytoleadtonovelantibacterialsthat
combinatorialchemistryareoftenusedinterchangeablyinthe
exerttheirbiologicaleffectthroughauniquemechanism.
literature. Indeed, since DOS incorporates many of the
Clearly, such ‘biased’ synthesis methods are particularly
principles of combinatorial chemistry, it is probably best to
relevant when a specific biological target is being considered.
consider DOS as a more evolved version of traditional
When a less focused approach is required, for example the combinatorialmethodologies.zRecentlyourgrouphasintro-
biological target is unknown or we are hoping to find novel
duced the concept of the ‘molecular diversity spectrum’ as a
biologicallyactivemolecules,suchasantibacterialagents,the
usefulqualitativemeansforcomparingthestructuraldiversity
useofnon-biasedsyntheticapproaches,whichaimtoaccessa
associated with a particular molecular collection (Fig. 5). It
widerrangeofchemicaldescriptorspace,maybemoreuseful.
should therefore be the goal of a DOS to synthesize, in a
Non-biased approaches: diversity-oriented synthesis
zIt is widely accepted in the literature that the use of a traditional
as a source of skeletally diverse small molecules
combinatorialapproach(diversityaroundasinglescaffold)asameans
forstructuraloptimisationonceabiologicalactivemolecularskeleton
Diversity-oriented synthesis (DOS)hasrecently emergedas a
hasbeenidentifiediswithoutpar;theprinciplebenefitofDOSisinthe
new syntheticapproachtowardslibrarysynthesis whichaims initialdiscoveryof(potentiallynovel)biologicallyactiveskeletons.56
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Fig.4 AcomparisonoftheplanningstrategiesusedinatraditionalcombinatorialsynthesisandaDOStogetherwithavisualrepresentationof
thechemicalspacecoverageachievedinbothcases(i.e.focusedaroundaspecificpointordiversecoverage).
Fig.5 The‘moleculardiversityspectrum’.‘Diversity’canbeviewedasaspectrumrangingfromatarget-orientedsynthesis(TOS)ofaspecific
moleculetothesynthesisofallpossiblecompounds(i.e.totalchemicalspacecoverage);atraditionalcombinatorialapproachandaDOSproduce
compoundcollectionsthatsitbetweenthesetwoextremes.Belowthediversityspectrumaretwodiagrams,createdusingprincipalcomponent
analysis, which show the distribution of representative chemical collections produced by a traditional combinatorial synthesis and a DOS in
chemicaldescriptorspace.Alsoincludedisadiagramshowingthedistributionofallknowndrugmoleculesinchemicaldescriptorspace.These
diagramsillustratetheabilityofaDOSlibrarytocoverawiderareaofchemicalspacethanatraditionalcombinatoriallibrary.TheDOSlibrary
also achieves a better coverage of known bioactive chemical space. The nature of these libraries is discussed in more detail later in the text
(seesectionson‘AchievingskeletaldiversityinaDOS’,and‘Assessingthediversityofasmallmoleculelibrary’).Moredetailregardingtheuseof
principalcomponentanalysisisgiveninFig.8.
qualitative sense, collections of small molecules which are as areneverthelesscertainchemicalfeaturesthatarerequiredina
nearaspossibletotherighthandsideofthisspectrum.25 moleculeinorderforittobeabletointeractwithabiological
InisimportanttonotethatalthoughaDOSaimstoaccessa system in a useful manner. In the context of antibacterial
wideareaofchemicalspace,thereisastillacertaindegreeof discovery using DOS, it is important to note that such
biasassociatedwiththecompoundsproduced.Thisisbecause synthesisdesigncriteriadonotincludelimiting thelibraryto
thegoalofaDOSistogeneratecompoundsthatarecapable compounds that span the physiochemical property space
ofinteractingwithbiologicalmacromolecules;thatis,aDOS occupied by know antibiotic compounds (vide supra). There
is designed to access biologically relevant space. Though the are,however,molecularfeaturesthataredesirableintermsof
boundaries of biological relevant chemical space are not any further development of the molecule into a therapeutic
known,andthusthereisundoubtedvalueingeneratingnovel agent (for example, favourable ADME (absorption, distribu-
molecules from unexplored regions of chemical space, there tion, metabolism and elimination) properties as well as low
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toxicity). Consequently, a DOS is not simply a random
generation of compounds, but a carefully considered process
that aims to generate small molecules with diverse molecular
structuresthatarenaturalproduct-likeanddrug-likeinterms
of their capability to interact with, and modulate, biological
systems.
DOS and antibacterial discovery
Since Schreiber’s seminal works in the field9,47,84,89,90 the use
of DOS as a method for the generation of small molecule
libraries has increased dramatically. Many different general
strategies have been developed which aim to address all of Fig. 6 An illustration of the substrate-based approach to skeletal
these aspects of diversity and the reader is directed to recent diversity. A collection of substrates which are each based around a
review articles prepared by our group and others for a more similarcoreskeletonbutwhichcarrydifferentappendages(so-called
detaileddiscussionofthistopic.25,46,86,91Therearenumerous s-elements,labelleds1–s3)isshown.Underacommonsetofreaction
examples of novel, biologically useful small molecules conditions, each substrate is converted into a product having a
different molecular skeleton, with the skeletal outcome dependent
which have been discovered through the screening of DOS
libraries.66,72,90,92–94 However DOS is arguably an under- uponthenatureofthes-elementspresentinthesubstrate;e.g.‘red’
s2 elements react in such a fashion so as to generate the molecular
exploitedtoolinthefieldofantibacterialdiscoveryandthere
skeletonhighlightedinred.
areveryfewreportsintheliteraturepertainingtothescreening
of DOS collections (which incorporate skeletal diversity)
Thereagent-basedapproachtoskeletaldiversity
against bacterial targets. Therefore for the purposes of this
discussion we will highlight some examples from our own Thereagent-basedapproachtoskeletaldiversityisabranching
laboratories on the useof DOSto discover new antibacterial synthetic strategy which involves a short series of divergent,
agents. complexity generating reactions from a common starting
The overall aim of a DOS is the generation of a small material to generate a collection of compounds with distinct
molecule collection that spans as much bioactive chemical molecularskeletons(Fig.7).9
space as possible as this should increase the probability of In practise, reagent-based skeletal diversity is achieved via
discoveringsmallmoleculeswiththedesiredbiologicalproper- twomainmethods:9,25
ties(inthiscase,antibacterialactivity).Asoutlinedpreviously, 1. The use of a densely functionalised molecule where
this functional diversity is most efficiently achieved through differentfunctionalitiesinthesamemoleculearetransformed
the efficient incorporation of skeletal (scaffold) diversity in a bydifferentreagents;
smallmoleculelibrary. 2.Theuseofapluripotentfunctionalgroup(i.e.onethatcan
participateinanumberofdifferentreactions)whereexposure
of a given molecule to different reagents results in different
Achieving skeletal diversity in a DOS
reactionsoccurringatthesamepart(functionalgroup)ofthe
There are two main methods that have been employed in a molecule.
DOS context for the generation of skeletal diversity, based
upon either the reagent (the reagent-based approach) or the Theuseofapluripotentfunctionalgroupstrategy
substrate(thesubstrate-basedapproach).9,95,96
Within the Spring group, we have developed several reagent-
based DOS approaches that utilise starting materials with a
Thesubstrate-basedapproachtoskeletaldiversity
pluripotent functionality. Critical to the success of a pluri-
The substrate-based approach to skeletal diversity is based potent DOS is the choice of a synthetically versatile starting
around a folding process. It involves the conversion of a material that is capable of undergoing a wide variety of
collection of substrates, which contain appendages with
suitable ‘pre-encoded’ skeletal information (so-called s
elements), into products have distinct molecular skeletons
using a common set of conditions (Fig. 6).9,85,95 In practice,
such methods are usually based around intramolecular
reactionsthat‘pair’strategicallypositionedfunctionalgroups
in the substrates, resulting in compounds with diverse
skeletons.96 This approach is exemplified in a recent DOS
pathwaydevelopedbyMortonandco-workerswhichresulted
inthegenerationofatotalof84distinctmolecularskeletons,
representing a significant landmark in the degree of skeletal
diversity incorporated in a synthetically-derived small
moleculelibrary.97 Fig.7 Thereagent-basedapproachtogeneratingskeletaldiversity.
2454 | Chem.Commun., 2009, 2446–2462 Thisjournalis(cid:3)c TheRoyalSocietyofChemistry2009
different chemicaltransformationsandthushasthe potential transamidation and ester reduction; R replaced by R11,
tobeconvertedintoseveralproductswithdifferentmolecular R12 etc.). Various substrates were used in the branching
skeletons through the variation of reagents alone. These synthetic routes outlined in Scheme 1 to synthesize
products should themselves be suitable for further diver- a library of 223 compounds based on 30 different core
sification, preferably in further complexity-generating and molecular skeletons in no more then four linear synthetic
branching reaction sequences; this provides a means to steps.
augmenttheskeletaldiversityofthelibraryfurtherandideally Severalofthesemolecularskeletonshaveprovenbiological
offer a route for the introduction of stereochemical diversity. relevance. Consequently, the inclusion of molecules based
Variation in the substrates used in these reactions provides around such scaffolds should allow the DOS library to span
scope for the introduction of building block and functional regions of (and close to) known bioactive chemical space
groupdiversityintothefinallibrary. (videinfra).Forexample,4isanecgonineanalogue;ecgonine
These considerations are illustrated by two representative itself is a member of the tropane alkaloid family of natural
casestudiesfromourgroup.Inbothcasestheoverallaimwas products and known bioactive molecules based on similar
thegenerationofasmallmoleculecollectionthatspannedas scaffolds include cocaine and the molecules b-CIT and
muchbioactivechemicalspaceaspossibleinordertoincrease b-CPT (radiolabelled forms of which have been used to help
the probability of discovering small molecules with anti- investigatedopamineuptakeinvivo).100,101Compounds5are
bacterial activity. Towards this end, we wanted the libraries basedaroundacoumarinskeleton.Thisprivilegedscaffoldis
to cover known regions of bioactive space, as by definition found in a wide variety of synthetic and naturally occurring
thesearefruitfulareasforthediscoveryofbiologicallyactive compounds that display a range of different biological
molecules. Additionally, we wanted to access regions of activities, for example the anti-coagulant warfarin102 and the
chemical space that are not covered by known natural pro- rodenticidebrodifacoum.103Furthermore,thedihydropyrimi-
ducts or pharmacologically active compounds, as bioactive done framework present in 6 is a well-established privileged
moleculesfromsuchregionsmaybemorelikelytoexerttheir sub-structure; compounds with this core scaffold have been
biologicalactivitythroughanovelmodeofaction.However, reported as anti-hypertensives and anti-cancer leads and this
the two DOS approaches differed in terms of the relative class of compounds also exhibits anti-viral and anti-
degreeofemphasisthatwasplacedonexploringthesedifferent inflammatory activity.62 Compounds based around these
regionsofchemicalspace. privileged scaffolds are desirable to include in a structurally
The first of these methods involved the use of fluorous diverse compound collection for use in chemical genetics
taggeddiazaoacetate3asastartingunitinabranchingDOS screens.
strategy(Scheme1).48InthisDOSthemainfocuswasonthe ThesecondexampleofabranchingDOSstrategyfromour
efficient generation of compounds that, while structurally own group involves the generation of a library of small
novel, were also based around a variety of natural product- molecules starting from a solid-supported phosphonate 7
like scaffolds; that is, we aimed to explore areas of chemical (Scheme 2).104 In this DOS, an emphasis was placed upon
space ‘close’ to those occupied by known bioactive small the exploration of uncharted regions of chemical space
molecules through the incorporation of compounds based through the development of routes to unnatural-product-like
known bioactive frameworks. Diazoacetate 3 was identified (i.e.completelynovel)molecularscaffolds.Inthefirststepof
as an attractive starting unit for two main reasons; the DOS (Step 1, Scheme 2), 7 was reacted with a variety of
(i) diazoacetate compounds exhibit enormous synthetic aldehyde building blocks in an E-selective Horner–
versatility, permitting the use of a wide variety of different Wadsworth–Emmons reaction (building block diversity) to
synthetic transformations and (ii) polyfluorocarbon tag deliver twelve a,b-unsaturated acyl-imidazolidinones 8. The
technologyallowedstandardsolutionphaseparallelsynthesis pluripotentnatureof8wasexploitedinthesecondstepofthe
methods to be coupled with the benefits of fluorous-based DOS (Step 2, Scheme 2) which involved three catalytic
purification protocols,98,99 thus simplifying the isolation and enantioselective divergent reaction pathways (stereochemical
purificationofthelibrarycompounds.Themainpathwaysare diversity):(i)[2+3]cycloaddition(reactionb);(ii)dihydroxyl-
summarised in Scheme 1. In the first stage of the DOS the ation (reactionc);and(iii)[4+2]cycloaddition(reactiond)
reactivediazoacetatefunctionalitywasexploitedinfourmain to yield a collection of molecules based on three molecular
branchingreactions(Step1,Scheme1,reactionsathroughtog). frameworks (skeletal diversity). The next step of the DOS
ThesecondstageoftheDOSinvolvedaseriesofcomplexity- (Step 3, Scheme 2) involved a series of branching reactions
generating reactions to diversify these molecular frameworks to diversify these key branch-point substrates further. For
further,increasingtheskeletaldiversityofthelibrary(Step2, example, the norbornene derivatives 9 (formed in step d)
Scheme 1,reactions ithroughtoq).Inselectedcases,athird served as versatile intermediates for a series of branching
stage of reactions was carried out (reactions r, s and t) to reactions(reactionsltoo)togeneratefivedifferentmolecular
introduceadditionalcomplexityanddiversity.Variationinthe scaffolds (skeletal diversity). Of particular note was an
substrates used in these branching reactions (i.e. variation interesting tandem ring-closing–opening–closing metathesis
in R1(cid:2)10) introduced appendage and functional group reaction (reaction o) which created skeletally diverse tricyclic
diversity into the library. Inaddition, further appendage and products 10a (7-5-7) and 10b (7-5-8). In some cases, a fourth
functional group diversity was incorporated into the stage of reactions was carried out (Step 4, Scheme 2)
products from these pathways via the use of different ester to introduce additional complexity and diversity. The
cleavage mechanisms (i.e ester hydrolysis, transesterification, imidazolidinone portion of 7 (R group in Scheme 2) allowed
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