The Evolving Role of Chemical Synthesis in Antibacterial Drug

30 Pages·2014·5.08 MB·English

Angew.andte
A.G. Myers etal.
Reviews
DOI:10.1002/anie.201310843
Antibiotic Development
The Evolving Role of Chemical Synthesis in
Antibacterial Drug Discovery
Peter M. Wright, Ian B. Seiple, and Andrew G. Myers*
Keywords:
antibiotics·chemicalsynthesis·
drugdiscovery·semisynthesis
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Chemie
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T
hediscoveryandimplementationofantibioticsintheearlytwentieth From the Contents
century transformed human health andwellbeing. Chemical synthesis
1.Introduction 8841
enabled the development of the first antibacterial substances, orga-
noarsenicalsandsulfadrugs,buttheseweresoonoutshonebyahostof
2.ChemicalSynthesis Ushers in
more powerful and vastly more complex antibiotics from nature:
theGolden Age ofAntibiotics
penicillin,streptomycin,tetracycline,anderythromycin,amongothers. Discovery 8842
These primary defences are now significantly less effective as an
3.Semisynthesis: APowerful
unavoidable consequence of rapid evolution of resistance within
Postwar Enginefor
pathogenic bacteria, made worse by widespread misuse of antibiotics.
AntibacterialDiscovery 8845
For decades medicinal chemists replenished the arsenal of antibiotics
by semisynthetic and to a lesser degree fully synthetic routes, but 4.FullySynthetic Antibacterials,
economic factors have led to a subsidence of this effort, which places 1940-Present 8851
society on the precipice of a disaster. We believe that the strategic
5.ChemicalSynthesis asa Path
application of modern chemical synthesis to antibacterial drug
Forward 8857
discoverymustplayacriticalroleifacrisisofglobalproportionsisto
be averted.
mechanismsareemerging,[5]andinfec-
tions caused by multidrug-resistant
Gram-negative bacteria are becoming
1.Introduction particularlydifficulttotreat.Theproblemisexacerbatedby
the ease of international travel and increasing global pop-
The emergence of pathogenic bacteria resistant to many ulationdensities.Ourcurrentarsenalofantibioticsissteadily
orallcurrentantibioticsisamajorpublichealthconcernand losing its efficacy and there is little sign that it will be
one of particular importance in clinical settings. The World adequately replenished in the near future.[3,6] The develop-
Economic Forum recently identified antibiotic resistance as ment of bacterial resistance is an inevitable consequence of
oneofthegreatestthreatstohumanhealthinitsGlobalRisks evolution, and without continued replenishment of our
2013report.[1]TheCenterforDiseaseControlandPrevention arsenal of antibacterial agents, humanity runs the risk of
released a summary of antibiotic resistance threats in the returningtoapre-antibioticera.
UnitedStatesin2013,outliningthe“potentiallycatastrophic In this Review we examine the 100-year history of
consequences of inaction.”[2] Natural selection, assisted by antibiotics discovery and development from its dawning
globalmisuseofexistingantibiotics,andtheslowingpaceof with the synthesis of the first arsenical agent to those few
discovery of new antibiotics conspire to place society at or antibiotic candidates that are currently in late-stage clinical
nearacrisispoint.Theinnovationdeficitisinlargemeasure evaluation,[6] highlighting the essential and evolving role of
due to the fact that many major pharmaceutical companies chemical synthesis throughout. Our objectives are to recog-
have abandoned antibacterial research and development, nize select key contributions of the thousands of scientists
atrendwhichhascreatedorattheveryleastcontributedto who have provided the modern antibacterial pharmacopeia
the steep decline in the number of new antibacterials and to make the point that the clearest path forward to
launched inthe last 30years(Figure1).[3] Meanwhile, resist- discover future generations of life-saving medicines will
ance rates around the world are rising,[4] new resistance involvechemicalsynthesisasitscoreactivity.
More specifically, we suggest that the development of
practical, diversifiable, fully synthetic routes to antibiotic
natural product scaffolds that are not yet accessible in this
waypresentsthegreatestopportunityforrapiddiscoveryand
developmentofnewantibioticsinthenearterm(5–20years).
By this analysis, many of the natural product classes that
emerged during and defined the golden era of antibiotics
discovery (ca. 1940–1960) represent underutilized resources.
As we argue in this Review, the development of practical,
fullysyntheticroutestoantibacterialmoleculesisatried-and-
tested strategy whose perceived constraints (molecular size
andcomplexity,scalability)needtobereevaluatedinlightof
[*] Dr.P.M.Wright,Dr.I.B.Seiple,Prof.Dr.A.G.Myers
DepartmentofChemistryandChemicalBiology,HarvardUniversity
Figure1. NumberofnewantibacterialsapprovedbytheFDAin5-year Cambridge,MA02138(USA)
periodsfrom1983topresent.[3] E-mail:[email protected]
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advances in modern chemical synthesis, both strategic and sectionof thisReview, weidentify specificopportunitiesfor
methodological. We believe that ambitious, translational chemical innovation to fuel future antibiotic drug develop-
chemical synthesis must be a core activity of antibiotics ment.Lastly,wehastentonotethatwhileourexpertiseand
researchmovingforward,asithasbeensincetheinceptionof emphasisischemistry-centered,werecognizethatthefieldof
thefield. antibiotic discovery would not exist nor could it advance
withouttheessentialcontributionsofindividualsfrommany
disciplines: isolation scientists, microbiologists, crystallogra-
1.1.ScopeandFocusofthisReview phers, clinicians, geneticists, toxicologists, and formulations
experts,amongothers.
The vast literature of antibiotics includes several fine
reviewarticles,[7]manyofthempublishedinthisjournal.[8]For
clearandcomprehensiveaccountsofallaspectsofthisfield— 1.2.NomenclatureofthisReview
includingresistance,mechanismsofaction,microbialscreen-
ingforantibioticnaturalproducts,antibioticbiosynthesis,and All antibiotics used in human therapy since the dawn of
drugdevelopment—wedirectreaderstotwoexcellenttexts, theantibioticseraintheearly1900scanbedividedintothree
oneauthoredbyChristopherWalsh[9]andtheothereditedby distinct categories according to how they were ultimately
Thomas Dougherty and Michael Pucci.[10] A detailed under- manufacturedonlargescale.Thesecategorizationsfollow.
standing of the molecular basis for antibiotic activity and Natural products: compounds manufactured directly by
resistance is critical to the success of any drug development large-scalefermentationofbacteriaorfungi.
program, but these factors are not the focus of this Review. Semisyntheticantibacterials:compoundsmanufacturedby
Withtheexceptionofanoverviewofrifampicin,thecomplex, chemical synthesis using as starting material a natural prod-
extraordinarilychallenging,andimportantproblemofdevel- uct.
oping drugs to treat tuberculosis is also not covered here. Fully synthetic antibacterials: compounds that are manu-
While others have previously articulated the importance of facturedbyfullysyntheticroutes.
chemical synthesis in antibiotics drug discovery,[7f,8c,d] our Throughout this article we have attempted to adopt the
focal point is the development of platform technologies to green,blue,andredcolor-coding,respectively,inFiguresand
access natural product scaffolds (broadly defined) by con- Schemes to clarify the means of manufacture of the anti-
vergent,component-based,fullysyntheticroutes.Ourinten- bioticspresented.Whilenewcategoriesmayyetemerge,such
tionistoilluminatetheevolvingrolechemicalsynthesishas as antibiotics produced by metabolic engineering, here we
playedinthediscoveryanddevelopmentofnewantibacterial focus on processes that have already yielded clinical agents.
agents so as to make clear its potential to contribute to the
alleviation of the current innovation deficit. In the final
2.Chemical Synthesis Ushers in the Golden Age of
Antibiotics Discovery
PeterM.Wrightwasbornandraisedin
Southport,U.K.HestudiedchemistryatSt.
John’sCollege,UniversityofOxford,under 2.1.DiscoveryandDevelopmentoftheFirstAntibiotics
theguidanceofProf.GeorgeW.J.Fleet,and
conductedresearchwithProf.TimothyJ. Thefirsteffectivetreatmentforabacterialinfectionarose
Donohoe.HethenmovedtotheU.S.to fromaconvergenceofdisparateadvances,includinganearly
pursuegraduatestudieswithProf. chemical synthesis of aniline, Paul Ehrlich(cid:2)s “magic bullet”
AndrewG.MyersatHarvardUniversity,
hypothesis, and the development of the first treatments for
workingonthesynthesisoftetracyclineanti-
Africansleepingsickness.In1854theFrenchchemistAntoine
biotics.HeisnowanAssociateatMcKinsey
&CompanyinNewYorkCity. B(cid:3)champachievedthefirsteconomicalsynthesisofanilineby
reduction of nitrobenzene with iron in the presence of
IanB.SeiplereceivedhisB.Sc.fromthe AndrewG.MyersreceivedhisB.Sc.fromthe
UniversityofCaliforniaatBerkeleyunder MassachusettsInstituteofTechnologyin
thetutelageofProf.DirkTrauner,andin 1981,andwentontopursuegraduate
2006joinedthelaboratoryofProf.PhilS. studieswithProf.EliasJ.CoreyatHarvard
BaranattheScrippsResearchInstituteas University.Hestartedhisindependentcareer
anNSFPredoctoralFellow.Duringhis attheCaliforniaInstituteofTechnologyin
graduatestudieshedevelopedfullysynthetic 1987,andmovedtoHarvardUniversityin
routestopyrrole-imidazolealkaloids,includ- 1998,whereheiscurrentlyAmory
ingpalau’amine.HeiscurrentlyanNIH HoughtonProfessorofChemistry.His
postdoctoralfellowinthelaboratoryofProf. researchprograminvolvesthesynthesisand
AndrewG.MyersatHarvardUniversity. studyofcomplexmoleculesofimportancein
biologyandhumanmedicine.Heandhis
studentshavealsodevelopedmethodsof
generalutilityintheconstructionofcomplex
molecules.
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hydrochloricacid,adiscoverythatcatalyzedthegrowthofthe a simple composition and extraordinary reactivity, which
synthetic dye industry.[11] Subsequent efforts to prepare permitted a wide variety of modifications.” According to
aniline derivatives led B(cid:3)champ to synthesize a compound Ehrlich,atoxyl“enabledchemotherapytodistanceitselffrom
knownasatoxylin1859bythereactionofanilinewitharsenic purely empirical trial and error testing and to introduce
acid.ThechemicalstructureofatoxylproposedbyB(cid:3)champ chemicalsynthesis.”[17]
waslaterrevised(seeScheme1). Bertheim, Ehrlich and co-workers proceeded to synthe-
Inthelatterpartofthe19thcentury,PaulEhrlichbegan size hundreds of structurally related organoarsenic com-
his prodigious search for a “magic bullet,” a molecule that pounds based on atoxyl and test them for activity against
could combat disease-causing organisms.[12] Ehrlich was trypanosomes. Following a suggestion by Erich Hoffmann,
broadly interested in fully synthetic dyes, their apparent theseorganoarseniccompoundswerealsotestedagainstthe
selective affinities for living tissues, and their therapeutic microorganismfoundin1905byHoffmannandSchaudinnto
potential. He hypothesized that the affinity of specific cell bethecausalagentofsyphilis.[18]Thisworkculminatedinthe
types for dye molecules could be harnessed to selectively discovery of salvarsan (Figure2),[19] the first effective treat-
destroymicroorganismsinthebodywithoutdamaginghuman ment for syphilis and the first antibacterial drug. Salvarsan
cells.Anearlybreakthroughcamein1891whenEhrlichand wasalsoknownas“Compound606,”enumeratingitsplacein
Paul Guttmann reported that two patients suffering from thesequenceofapproximately2000fullysyntheticmolecules
malariahadbeensuccessfullytreatedwiththefullysynthetic evaluated during Ehrlich(cid:2)s investigations, and it rapidly
thiazinedyemethyleneblue,[13]possiblythefirstexample of became the most widely prescribed drug in the world. By
afullysyntheticdrugbeingusedinhumanmedicine.Ehrlich 1920, 2million doses of salvarsan and neosalvarsan (“Com-
wasalsoactivelyinvolvedinthedevelopmentofsyntheticdye pound 914,” a more water-soluble derivative of salvarsan)
therapeutics for African sleeping sickness, which ravaged werebeingproducedannuallyintheU.S.alone.[20]Salvarsan
equatorial Africa around the turn of the 19th century in an was very difficult to administer and had terrible side effects
epidemic that claimed between 300000 and 500000 lives.[14] (including deafness), and chemotherapy remained a highly
HisinterestwaspiquedbyapaperbyH.W.Thomasin1905 controversialidea.[21]
demonstrating that B(cid:3)champ(cid:2)s atoxyl exhibited activity Intheearly1900sHeinrichHçrlein,directorofpharma-
against trypanosomes, including the causative organism of ceuticalresearchattheGermanchemicalconglomerateI.G.
sleepingsickness.[15] Farben,initiatedamajorefforttofindchemotherapeuticsfor
Working under Ehrlich(cid:2)s direction, Alfred Bertheim bacterialinfections.[22]Hçrleinandchemistcollaboratorshad
determined in 1907 that the chemical structure of atoxyl previously discovered that addition of sulfonamide substitu-
had been incorrectly assigned: atoxyl was p-aminophenyl ents to synthetic dyes often strengthened their binding to
arsenic acid (containing both amine and arsenic acid func- wool and silk fibers. They reasoned that the search for
tionalgroups,seeFigure2),notanarsenicacidanilide(aless achemotherapeuticagentcouldbuilduponthisinsightfrom
easily derivatized structural isomer containing a nitrogen- dyechemistry,inthatastructuralmodificationthatenhanced
arsenicbond),ashadbeensuggestedbyB(cid:3)champ.[16]Thiswas amolecule(cid:2)saffinityforfiberscouldalsoincreaseitsaffinity
a momentous discovery, as noted later by Bertheim: “Prob- fortheprotoplasmofbacteria.[23]
ablyforthefirsttime,abiologicallyeffectivesubstanceexisted Around 1927, I.G. Farben chemists Fritz Mietzsch and
whosestructurewasnotonlyknownpreciselybutalsowasof Joseph Klarer began to synthesize azo dyes for biological
Figure2. Earlyhistoryofantibioticsdiscoveryanddevelopment.
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testing. Several factors led them to investigate azo dyes: functional group nor the dye character of prontosil were
numerous azo compounds with promising activity against responsible for its therapeutic effect. During the ensuing
trypanosomes had been discovered during earlier efforts to decade chemists synthesized more than 5000 structural
find therapeutics for sleeping sickness;[14] the azo dye variants of sulfanilamide, and a number of them were
chrysoidine had been found to exhibit in vitro bactericidal launched as drugs.[23] One of these, sulfapyridine (known
effects in 1913;[23] I.G. Farben dominated the global market familiarly as “M&B” after the British manufacturer May &
for synthetic dyes, so the expertise and facilities required to Baker),wasusedtotreatWinstonChurchillduringaboutof
prepare azo dyes were available in-house; and azo com- pneumoniainthewinterof1943.[24]Somesulfadrugssuchas
pounds of wide structural variability were chemically acces- sulfamethoxazole are still used today, but problematic side
sible(akeyreasonthiscompoundclasswasappealing).[22]By effectsandthespreadofresistancedrovemanyantibacterials
1932,MietzschandKlarerhadsynthesizedmorethan300azo from this class out of favor.[25] It is noteworthy that in the
compounds, including a series containing sulfonamide sub- historyofhumanmedicinethefirsttwoantibioticsclassesof
stituents,andprovidedthesefortestingtoGerhardDomagk clinicalutilitywerenotnaturalproduct-based,butwerefully
andotherswhohaddevelopedasuiteofinvitroandinvivo synthetic substances that arose from extensive chemical
biological assays to determine utility against streptococcal synthesisandserendipity.
infections(amongothers).[22]
Domagk discovered that the red dye prontosil produced
incredible curative effects in mice previously injected with 2.2.WorldWarIICatalyzesProductionofPenicillinby
lethal doses of streptococci. In the years that followed Fermentation,butnotChemicalSynthesis[23]
prontosil saved the lives of a 10-month-old baby suffering
fromstaphylococcalsepticemiaand,famously,Domagk(cid:2)sown Oneofthekeyscientificbreakthroughsofthe20thcentury
6-year-olddaughter.In1935,thesameyearasthecommercial occurred when Alexander Fleming discovered in 1928 that
launch of prontosil, it was revealed by researchers at the asubstanceproducedbythefungusPenicilliumchrysogenum
PasteurInstituteinParisthattheactiveprincipleofthefirst (formerly known as Penicillium notatum) exhibited antibac-
“sulfa drug” was the simpler substance known as sulfanila- terialactivity.[26]Althoughthisfindingwasmadepriortothe
mide(Figure2,Scheme1),acompoundveryeasilyprepared key achievements of Domagk and collaborators, the fully
inthelaboratoryevenbytherelativelyprimitivemethodsof synthetic sulfa drugs found widespread clinical use many
the day. This work demonstrated that neither the azo yearsbeforepenicillinbecameavailableforthetreatmentof
bacterialinfections.NearlyadecadepassedfollowingFlem-
ing(cid:2)s famous discovery before Howard Florey and Ernst
Chain received a grant from the Rockefeller Foundation to
isolate penicillin and investigate its biological properties. In
1940, the Oxford team member Norman Heatley demon-
strated that treatment with crude penicillin significantly
extended the lives of mice previously injected with a lethal
strain of Streptococcus.[27] The landmark 1940 report in The
Lancet begins as follows: “In recent years interest in chemo-
therapeuticeffectshasbeenalmostexclusivelyfocusedonthe
sulphonamidesandtheirderivatives.Thereare,however,other
possibilities,notablythoseconnectedwithnaturallyoccurring
substances.” In February 1941, multiple doses of partially-
purified penicillin broth were administered to an Oxford
policeman suffering from a staphylococcal infection.[28] The
policeman(cid:2)sconditionimproveddramaticallyfollowingtreat-
mentwithpenicillin,butafterfivedaysthelimitedsupplyhad
been exhausted and the policeman succumbed to the
resurgent infection. Florey and Chain needed much larger
quantities.
By1941,Britishindustrywasengrossedinthewareffort
andlackedtheresourcestotacklealarge-scaleexperimental
project.UsinghisRockefellerconnections,Floreycrossedthe
AtlanticandpetitionedAmericanpharmaceuticalcompanies
to consider mass-production of their therapeutic compound
by fermentation. His timing was propitious. In June 1941,
President Roosevelt established the Office of Scientific
Research and Development (OSRD), a federal agency
responsible for coordinating scientific and medical research
relating to national defense. The Allies urgently needed to
Scheme1. Chemicalsynthesisofsalvarsanandprontosil. find new treatments for the vast number of troops with
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disease and wound infections. Sulfa drugs were a hugely carbodiimidereagents.[36]Thistransformationbecamethekey
important medical breakthrough but also had significant step in the first fully synthetic route to a natural penicillin
limitations—theirspectrumofactivitywasnarrow,andsome (penicillinV), published in 1957 by Sheehan and Henery-
bacteriaacquiredresistancerapidly.Furthermore,production Logan(Scheme2).[37]
of these antibiotics was concentrated in Nazi Germany. The
CommitteeonMedicalResearchofOSRDinitiatedamassive
projecttoproducepenicillin:onearmoftheprojectaimedto
maximizeproductionofpenicillinbyfermentation,whilethe
othersoughttodevelopafullysyntheticroute.
This unprecedented convergence of governments, phar-
maceuticalcompaniesandacademicscientistssparkedrapid
scientific innovation. Regulatory barriers were knocked
down—even impure penicillin had curative effects—and
intellectual property concerns were temporarily cast
aside.[29] Pfizer scientists James Currie and Jasper Cane
achieved a landmark advance by the implementation of
deep-tankfermentationtechniquesforpenicillinproduction,
dramatically increasing the production of this life-saving
drug.[30]Meanwhile,thesynthesiseffortinvolvingmorethan
1000 chemists and 39 major laboratories failed to produce
aviablechemicalsynthesisofpenicillinandwasterminatedin
1945.[23] Disagreement over the true chemical structure of
penicillinmeantthatdifferentgroupsweretryingtosynthe-
sizedifferentmolecules.Ironically,ateamofchemistsledby
Vincent du Vigneaud did manage to synthesize a minute
quantity of penicillinG in spite of the fact that they were
targetingastructurethatlaterprovedtobeincorrect.[31]All
Scheme2. FullysyntheticapproachestopenicilinVand6-aminopeni-
efforts to synthesize what turned out to be the correct cillanicacid.[23]
structure of penicillin, containing a so-called b-lactam or 4-
membered cyclic amide function, failed due to the dual
challenges of strain and sensitivity posed by the critical b- InMarch1958,Sheehanreportedatasymposiumthathis
lactam ring.[32] This “diabolical concatenation of reactive group had prepared a compound known as 6-aminopenicil-
groups”[33] at the core of the penicillin molecule remained lanicacid(6-APA)bybothfullysyntheticandsemisynthetic
essentiallyinaccessibleafterperhapsthelargestcoordinated routes, the first public disclosure of a compound that would
project (albeit a fairly short-lived one) in the history of prove to be critically important to the future discovery of
organicsynthesis. dozensofnewb-lactamantibiotics,allwith modificationsof
theC6sidechain.[38]Thefollowingyear,scientistsatBeecham
ResearchLaboratoriesintheU.K.reportedtheisolationof6-
2.3.AGlimmerofHopeforaPractical,FullySyntheticPathway APA from penicillin fermentation broths (having submitted
toPenicillins a patent application in 1957),[39] and soon thereafter this
intermediate, now produced by fermentation, became the
The failure of the penicillin synthesis project during dominant precursor for production of semisynthetic penicil-
WWII led Ernst Chain to declare in 1946 that the molecule lins.Duetothenumberofstepsinvolvedandthelowoverall
would remain inaccessible by fully synthetic means “unless yield Sheehan(cid:2)s fully synthetic route to penicillin was not
someone invents an entirely new technique unknown to competitivewithmanufacturebyfermentation-semisynthesis,
chemistry.”[34] Shortly thereafter, despite a precipitous drop buthispioneeringsyntheticeffortshadledtothediscoveryof
inresearchefforts(andresearchfunding)directedtowardthe 6-APA and thereby the preparation by semisynthesis of
development of a fully synthetic route to penicillins, John structuralanalogsthatcouldnothavebeenpreparedbyother
Sheehan began making progress toward his landmark labo- means.
ratorysynthesisofpenicillinV.Hisresearchinthisareawas
made possible by the long-term support of the Bristol
company. In 1950, Sheehan reported the total synthesis of 3.Semisynthesis: A Powerful Postwar Engine for
apenicillinderivativebearinganovel5-phenylsubstituent.[35] Antibacterial Discovery
Althoughthisnon-natural,fullysyntheticanalogwasinactive,
theworkmarkedanimportantstepforward.Assuggestedby Bacteria and fungi have continuously evolved over
Chain, a fully synthetic route to penicillins only became approximately 109years, producing compounds that confer
accessible following a transformative chemical innovation. anevolutionaryadvantagebykilling(other)bacteria,notby
Sheehan brought forward this innovation by inventing an theirefficacyintreatinghumanswithopportunisticbacterial
extremely mild method for formation of amide bonds using infections. The evolutionary pressures of human pharmaco-
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kinetics, safety, oral bioavailability, and efficacy only came tetracycline (Terramycin).[44] Chlorotetracycline and oxyte-
into play in the 1940s when medicinal chemists began to tracycline were found to be active against a wide range of
modifyfermentationproductswiththeobjectiveofobtaining Gram-positive and Gram-negative bacteria—together with
safer,moreefficacious(andproprietary)antibiotics,aprocess chloramphenicol(seeSection4.1),theywerethefirst“broad-
we refer to as “human chemical evolution.” A primary spectrum” antibiotics. As a brief aside, Gram-positive and
methodbywhichhumanshavediscoveredanddevelopednew Gram-negativebacteriaaresocalledbecauseoftheirdiffer-
antibacterial therapies for more than 60years has been ent responses to a common staining protocol developed by
semisynthesis: chemical synthesis using natural products as Hans Christian Gram. All bacterial cells are bounded by
startingpoints. a cytoplasmic membrane, a lipid bilayer that tends to be
permeabletouncharged,lipophilicmolecules.Gram-negative
bacteria(suchasEscherichiacoli,Klebsiellapneumoniae,and
3.1.OriginsofAntibacterialSemisynthesis Pseudomonasaeruginosa)alsohaveanoutermembranethat
is significantly less permeable to lipophilic molecules. In
Semisynthesis came to the forefront of antibacterial addition, Gram-negative bacteria often possess multidrug
discovery efforts following innovative chemical alterations efflux pumps that expel many antibiotics. As a result, the
ofnaturallyoccurringaminoglycosidesandtetracyclines.The development of antibiotics with activity against Gram-neg-
firstaminoglycosideantibioticwasdiscoveredin1943,when ativebacteriaisparticularlychallengingandcurrentlyrepre-
Albert Schatz, a graduate student working with Selman sentsanurgentunmetclinicalneed.[3,7i,45]
Waksman, isolated streptomycin from the soil microbe Soon after the discovery of chlorotetracycline and oxy-
Streptomyces griseus. Just as they had done with penicillin tetracycline, Lloyd Conover at Pfizer discovered that the
afewyearspreviously,pharmaceuticalchemistsimmediately carbon-chlorinebondofchlorotetracyclinecouldbecleaved
begantoprobethestructureandpropertiesofstreptomycin. bycatalytichydrogenolysis,producingthefirstsemisynthetic
In1946,RobertPeck,CharlesHoffhine,andKarlFolkersat tetracyclineantibiotic—tetracyclineitself(Scheme3).[46]The
Merck[40]andQuentinBartz,JohnControulis,HarryCrooks, name “tetracycline” is derived from the four linearly fused,
and Mildred Rebstock at Park, Davis & Co.[41] separately six-memberedringsthatarecommontoallmoleculesinthis
discovered that catalytic hydrogenation of streptomycin family. Subsequently, tetracycline was found to be a natural
produced a new compound, dihydrostreptomycin, which product,[47]andbytheendofthe1950stetracyclinewasthe
exhibitedsimilarantibacterialpropertiesbutgreaterchemical mostprescribedbroad-spectrumantibioticintheU.S.
stability (Scheme3). In 1950, U.S. pharmaceutical firms Although the chemical innovations that enabled the
producedalmost100tonsofstreptomycinanddihydrostrep- discovery of dihydrostreptomycin and tetracycline may
tomycin combined, as both antibiotics rapidly found clinical appeartrivialtoday,theyhadaseismicimpactonthestrategic
applications.[42]Clinicaluseofthesedrugsinhumanswaslater mindsetofantibioticsdiscoveryandpharmaceuticaldevelop-
discontinued as a result of their ototoxicity, though they ment more broadly. These innovations demonstrated that
continuetobeusedinveterinarymedicine. naturalproductscouldbeconsideredasstartingpointsforthe
discovery process—extremely useful but not necessarily
optimal molecular scaffolds—and henceforth scientists in
industryandacademiapursuedantibioticresearchwithequal
vigor on two fronts: screening of soil samples for new
antibacterial natural products, and chemical modification of
natural antibiotics to find semisynthetic derivatives with
improved therapeutic properties and patentable chemical
structures.[29] Semisynthetic innovations have enabled dra-
matic improvements in antibiotic therapy across all major
families of natural antibiotics—here we will discuss the key
events and chemical insights that helped overcome the
numerous (and constantly evolving) limitations of cephalo-
sporin,tetracycline,andmacrolideantibacterials.
3.2.Semisynthesisofb-LactamAntibiotics
Scheme3. Theoriginsofantibacterialsemisynthesis. Theproducingstrainofthefirstcephalosporinantibiotics
was discovered in 1948 by Giuseppi Brotzu, Professor of
Hygiene at the University of Cagliari. Brotzu observed the
The first tetracycline antibiotic was discovered in 1948, propensityoflocalsewageforself-purificationandhypothe-
when Benjamin Duggar of Lederle Laboratories isolated sized that microorganisms were responsible. He studied the
chlorotetracycline (Aureomycin) from the culture broth of microorganisms present at the outlet of a sewage pipe and
Streptomyces aureofaciens,[43] and within two years Pfizer discovered that cultures of the mold Cephalosporium acre-
scientists had isolated a second natural tetracycline, oxy- monium contained one or more substances that were
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antagonistictobacteria.Brotzufailedtoarouseinterestinhis discovered a compound that was soon to become (and
discoveryintheItalianpharmaceuticalindustry,andhisdata remains to this day) the key semisynthetic intermediate for
and a sample of Cephalosporium acremonium eventually production of cephalosporin antibiotics. A few years later,
madetheirwaytoEdwardAbrahamatOxford.[21] RobertMorinandBillJacksonatEliLillydevelopedanovel
In1955,EdwardAbrahamandGuyNewton,twochemists chemical method to remove the side chain of cephalospor-
whoworkedwithFlorey,succeededinpurifyingcephalospor- inC,providingsemisynthetic7-ACAinacommerciallyviable
inC from the Cephalosporium culture.[48] Abraham, like yield(40%).[50]
others,hadobservedthesubtlyormarkedlydifferentproper- The synthesis of 6-aminopenicillanic acid (6-APA, Shee-
ties of structurally distinct natural b-lactams and was han,1958)andsemisynthesisof7-aminocephalosporanicacid
extremely interested in making chemical modifications to (7-ACA, Abraham & Newton, 1959), and the ensuing
cephalosporins. Regarding cephalosporinC, he later development of practical methods for their preparation by
recounted: “There was a great incentive to modify the fermentation (6-APA, Beecham Research Laboratories,
molecule chemically with a view to increasing its intrinsic 1959)andbysemisynthesis(7-ACA,EliLilly,1962),respec-
activity without affecting its resistance to staphylococcal tively, opened up the richest treasure trove of antibiotics in
penicillinase.”[38c] (Penicillinase is a type of b-lactamase with human history. More than fifty commercial antibiotics have
specificity for penicillins; b-lactamases are a collection of beendiscoveredandmanufacturedbychemicalmodifications
bacterialenzymesthathydrolyticallyopentheb-lactamring, of semisynthetic 6-APA and 7-ACA. Here we will limit our
producing inactive molecules). By 1959 Abraham and discussiontocephalosporins,describingthefavorableproper-
Newtonhadsynthesizedsmallquantitiesof7-aminocephalo- ties that have been engineered into successive generations
sporanic acid (7-ACA) by hydrolysis of cephalosporinC through 50years of human chemical evolution. This evolu-
underacidicconditions(Scheme4).[49]Althoughtheyieldof tionary process began with cephalosporins that exhibited
thisreactionwastoolowforcommercialproduction,theyhad usefulactivityagainstGram-positivebacteriaaloneandledto
the development of compounds that are active against both
Gram-positiveandGram-negativeorganisms(Figure3).This
transitionishighlynoteworthyandisdiscussedagainlaterin
thisReview.
First-generation parenteral cephalosporins such as ceph-
alothin(EliLilly,approved1964)[51]exhibitedpotentactivity
against Gram-positive organisms but only moderate activity
against Gram-negative bacteria. Since the 1960s, chemists
have been able to synthesize compounds that possess
a broader spectrum of activity, better pharmacological
properties, as well as lower susceptibility to resistance
mechanisms by introducing innovative side chains at just
two modifiable sites of 7-ACA—the amine function at C7,
and C3’ (see Figure3). The expanded-spectrum, second-
generation cephalosporins tended to be somewhat less
effective against Gram-positive bacteria but significantly
Scheme4. Semisynthesisof7-aminocephalosporanicacid(7-ACA) moreactiveagainstGram-negativebacteria,owingtobetter
fromcephalosporinC. cell penetration and resistance to b-lactamases. Importantly,
Figure3. Humanchemicalevolutionofsemisyntheticcephalosporinantibiotics(definingstructuralfeaturesofeachgenerationarehighlightedin
blue).
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Angew.andte
A.G. Myers etal.
Reviews
the a-methoxyimino group first introduced in cefuroxime 3.3.SemisynthesisofTetracyclineAntibiotics
(Glaxo, approved 1983)[52] reduced susceptibility to b-lacta-
mases by sterically blocking cleavage of the b-lactam ring. Beginning with Conover(cid:2)s landmark semisynthesis of
Gram-negative activity was further improved in third-gen- tetracycline from chlorotetracycline, the development of
erationcephalosporinssuchasceftazidime(Glaxo,approved semisynthetic tetracyclines has been marked by a series of
1985).[53] Ceftazidime incorporated an aminothiazole oxime specific, impactful discoveries. Charles Stephens and collab-
with a charged carboxylate side chain, a combination that oratorsatPfizerachievedamajorenablingadvanceapprox-
enhanced penetration through the porins embedded in the imately10yearsaftertheclasshadbeenidentifiedwhenthey
outermembraneofGram-negativebacteriaandhelpedretain demonstrated in 1958 that the C6-hydroxy group of the
high affinity for the bacterial target (penicillin binding natural products oxytetracycline, tetracycline and 6-deme-
proteins). The emergence of b-lactamases that cleaved thyltetracyclinecouldberemovedreductively(Scheme5).[57]
third-generation cephalosporins led to the development of The 6-deoxytetracyclines that arose as a consequence were
fourth-generationmoleculessuchascefipime(Bristol-Myers found to be more stable than the parent compounds, yet
Squibb, 1994),[54] which were more active than many third- retained broad-spectrum antibacterial activity. Their
generation cephalosporins against both Gram-positive and enhancedchemicalstabilityenabledfurtherstructuralmodi-
Gram-negative pathogens, including Pseudomonas aerugi- fications that had not been possible with acid- and base-
nosa.[55] The fifth-generation cephalosporin ceftobiprole sensitive natural tetracyclines, leading to the discovery of
received approval for use in Europe in 2013 for treatment minocyclinein1967byMichaelMartell,Jr.andJamesBoothe
ofhospital-acquiredpneumonia.[56] at Lederle laboratories.[57b,58] Minocycline was synthesized
The human chemical evolutionof cephalosporins vividly from6-deoxy-6-demethyltetracycline(sancycline)byanelec-
illustrates the ability of medicinal chemists to continuously trophilicaromaticsubstitutionreactionatC7,anditexhibited
tailorthepropertiesofantibacterialstomeetspecificclinical a broader spectrum of activity than prior tetracyclines
needs.Widespreadclinicaluseofcephalosporinsandotherb- (including activity against some tetracycline-resistant staph-
lactam antibiotics has selected for bacteria with fierce ylococci).Likeothermembersofthefamily,theclinicalutility
collections of resistance determinants, but cephalosporins of minocycline declined in the ensuing decades due to
remaincriticalcomponentsofourantibioticarmamentarium. increasinglywidespreadresistance.
Of the seven drugs currently in advanced clinical develop- Aiming to overcome tetracycline resistance in the late
ment(phaseIIorIII)forthetreatmentofinfectionscaused 1990s, a team of Wyeth scientists led by Frank Tally
by Gram-negative bacilli, three are combinations of a ceph- synthesized7,9-disubstitutedtetracyclinederivatives,leading
alosporin and a b-lactamase inhibitor.[3] Only one of these to the discovery of the life-saving antibiotic tigecycline (US
new combination therapies (ceftolozane/tazobactam, Cubist approval 2005, Scheme5).[59] Tigecycline is the defining
Pharmaceuticals) incorporates a novel cephalosporin anti- member of a new class of tetracyclines known as glycylcy-
biotic, suggesting that the development of new b-lactams is clines, which greatly extend the spectrum of tetracyclines,
becomingincreasinglydifficult. especiallytowardtetracycline-resistantmicroorganisms.Tige-
Scheme5. Chemicalinnovationsintetracyclinesemisynthesis(importantnewstructuralfeaturesofeachgenerationarehighlightedinblue).
8848 www.angewandte.org (cid:2)2014Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim Angew.Chem.Int.Ed.2014,53,8840–8869
Angewandte
Antibacterial DrugDiscovery Chemie
cycline has become a last line of defense against multidrug- metabolicproductsfromStreptomycesthatcontainamacro-
resistantbacteria;forexample,itisoneofonlytwoapproved lactone ring.[62] Erythromycin was approved for use against
antibioticsthatareactiveagainstsomecarbapenem-resistant a variety of Gram-positive bacterial infections, but upon
bacteria carrying New Delhi metallo-b-lactamase enzymes widespread clinical implementation several limitations were
(NDMs, see Section4.3 for discussion of carbapenem anti- quickly identified. Erythromycin displayed poor oral bio-
biotics).[5] The other is colistin, which can cause damage to availabilityandashortinvivohalf-life,andmostimportantly
kidneys and nerves. Some of the benefits of tigecycline are itwasfoundtobeunstableunderacidicconditions,givingrise
attributabletothefactthatitbindsmorestronglythanolder to side effects such as stomach pain. Administration of the
tetracyclines to the small subunit of the bacterial ribosome antibiotic as an enteric-coated tablet helped sidestep insta-
(the biological target of all tetracyclines).[60] Its drawbacks bilitytogastricacid;however,innovativechemicalsolutions
include dose-limiting tolerability (nausea, vomiting) and were much desired. Studies of chemical instability under
alackoforalbioavailability.Thehumanchemicalevolution acidicconditionsrevealedthaterythromycindecomposesby
of semisynthetic tetracyclines has provided antibacterial intramolecular cyclization reactions beginning with addition
therapies that have overcome many limitations of their of the C6 hydroxy group to the C9 ketone, leading to
predecessors, but the slowing pace of discovery in this area formation of both anhydrohemiketal and spiroketal deriva-
isevident(Figure4). tives (Scheme6).[63] Knowledge of the chemical basis for
instability catalyzed the discovery of semisynthetic macro-
lidesthatlackedthissignificantlimitation.
3.4.SemisynthesisofMacrolideAntibiotics One solution was provided by Sadafumi Omura and
collaborators at Taisho Pharmaceutical Co. in Japan who
Macrolide antibiotics have undergone serial human developed a 6-stepsequence from erythromycin resulting in
chemical evolutionary advances that in many ways parallel selectivecappingoftheC6hydroxysubstituentwithamethyl
those that occurred within tetracycline antibiotics: each group, affording the antibiotic clarithromycin (Scheme6).
semisynthetic advance has built upon prior innovations, and ProtectionoftheC9ketoneoferythromycinasanoximewas
each new (successful) semisynthetic antibiotic has become critical to this work, providing an intermediate whose
a starting material for further chemical modification conformation enabled selective methylation at C6-OH.[64]
(Figure4).[61] This strategy is sensible, since it enables Clarithromycin displayed a slightly expanded spectrum of
favorable characteristics to be carried forward, but it also activityrelativetoerythromycin,anditwasfoundtobeboth
inevitably leads to a gradual increase in the number of acid-stableandorallyactive.
chemical operations required to synthesize new derivatives Another innovative semisynthetic solution to the chem-
fromtheoriginalnaturalproduct. ical instability of erythromycin was developed in 1980 by
Erythromycin, the first macrolide antibiotic, was discov- GorjanaLazarevzkiandco-workersatPlivainCroatia.Inthis
ered in 1949 when scientists at Eli Lilly isolated the natural case, the C9 ketone was completely removed from the
product from the culture broth of the soil-dwelling fungus erythromycin scaffold by a sequence comprising oxime
Saccharopolyspora erythrea. The term “macrolide” was formation, Beckmann rearrangement (ring expansion), and
originallyintroducedbyR.B.Woodwardin1957todescribe thenhydrogenolysisoftheresultingiminoetherintermediate
Figure4. Humanchemicalevolutionoftetracyclineandmacrolideantibioticsbysemisynthesis(importantnewstructuralfeaturesofeach
generationarehighlightedinblue).
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