Dual-acting hybrid antibiotics: a promising strategy to ...teaching.ncl.ac.uk/antimicrobials/Lectures/Reading... · Antibiotic resistance (both endogenous and acquired) has been an

1. Introduction

2. Fluoroquinolone-containing

hybrid compounds

3. Aminoglycoside-containing

hybrid compounds

4. Other hybrid compounds

5. Expert opinion

Review

Dual-acting hybrid antibiotics: apromising strategy to combatbacterial resistanceVarvara Pokrovskaya & Timor Baasov†

Technion -- Israel Institute of Technology, The Edith and Joseph Fischer Enzyme Inhibitors

Laboratory, Schulich Faculty of Chemistry, Haifa, Israel

Importance of the field: The emerging and sustained resistance to currently

available antibiotics and the poor pipeline of new antibacterials urgently

call for the development of new strategies that can address the problem of

growing antibacterial resistance. One such strategy is the development of

dual-action hybrid antibiotics: two antibiotics that inhibit dissimilar targets

in a bacterial cell covalently linked into one molecule. The possible benefits

include: i) activity against drug-resistant bacteria, ii) expanded spectrum of

activity and iii) reduced potential for generating bacterial resistance.

Areas covered in this review: In this article, we detail the recent activity

in the design and development of dual-action hybrid drugs with a non-

cleavable linker. We explore newly developed synergistic and antagonistic

hybrid compounds with emphases on their potential to reduce

resistance development.

What the reader will gain: Recently developed synergistic and antagonistic

antibacterial drug--drug interactions and the impact of such interactions on

the evolution of antibiotic drug resistance are described. Additionally, we dis-

cuss the implications of the latter observations on the development of hybrid

antibiotics with the emphases on whether their synergistic or antagonistic

effect will be more efficient at forestalling/reducing the development of

new resistances.

Take home message: The approach of dual-acting hybrid antibiotics holds sig-

nificant current promise in overcoming existing resistance mechanisms, as

three of such compounds are entering clinical trials. However, the key chal-

lenge in this area should be a broader experimental demonstration of

whether the “synergistic effect” or the “antagonistic effect” of the developed

hybrid drug is better at preventing/reducing the evolution of resistance. This

fundamental challenge must be overcome before yielding a successful drug.

Keywords: bacterial resistance delay, dual-action drugs, heterodimer antibiotics,

hybrid antibiotics

Expert Opin. Drug Discov. (2010) 5(9):883-902

1. Introduction

Antibiotic resistance (both endogenous and acquired) has been an important deter-mining factor in the historical development of antibiotics as indispensable therapeu-tic agents for the treatment of infectious diseases [1]. Only a year after penicillin (afungal product) was introduced for use by the general public, the first report ofpenicillin-resistant strains of Staphylococcus aureus appeared [2]. At that time the bio-chemical mechanism of resistance was not known. This alarming situation, unprec-edented for those times, stimulated broad research in different scientific fields ofantibacterial therapy. A major breakthrough came with the discovery of thesynthetic derivative of penicillin, methicillin, less susceptible to hydrolysis by

10.1517/17460441.2010.508069 © 2010 Informa UK, Ltd. ISSN 1746-0441 883All rights reserved: reproduction in whole or in part not permitted

Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

b-lactamases, introduced in 1961 for the treatment ofpenicillin-resistant S. aureus. But again, only a year later, themethicillin-resistant Staphylococcus aureus (MRSA) strainsappeared [3]. Streptomycin, discovered by Selman Waksmanin 1944, was the first aminoglycoside antibiotic to be isolatedfrom a bacterial source and was the first effective therapeuticfor tuberculosis (TB), caused by Mycobacterium tuberculosis.Soon after streptomycin was introduced for the treatment ofTB, it was found that bacterial resistance often developeddue to spontaneous mutants of M. tuberculosis arising duringthe course of the therapy with an antibiotic [1]. Kanamycin,another member of aminoglycoside family of antibiotics,was isolated in Japan in 1957 and rapidly became theantibiotic of choice in that country. The first example onenzyme-mediated kanamycin resistance was reported in1967 [4]. Because such resistance is transferable and largelyspreads via R-plasmids, transposons and integrons, ahigh level of aminoglycoside resistance rapidly spreadinternationally. The natural aminoglycosides (tobramycinand gentamicins) and the semi-synthetic derivative ofkanamycin, amikacin, all extremely effective antibioticsagainst kanamycin-resistant bacteria, were introduced in

the early 1970s [1]. Consequently, novel enzyme-mediatedresistance mechanisms that conferred high-level resistance tothis newest generation of aminoglycosides began to appearon the scene very soon after their introduction in clinicaluse [5,6]. Finally, the most recent incidence relates to linezolid;the first novel antibiotic chemical class to be marketed for38 years and the first oxazolidinone antibacterial agent to bedeveloped for clinical use. It was approved by the US FDAin 2000 as an active antibiotic against most Gram-positive bacteria that cause disease, including streptococci,MRSA and vancomycin-resistant enterococci (VRE). Line-zolid was licensed in the UK in early 2001 and, already by2002, the first three examples of resistant enterococci wereisolated in the UK, obtained from patients who had receivedlinezolid [7].

Although the above-mentioned examples illustrate howdevelopment of resistance to antibiotics has had a profoundimpact on the clinical utility and medicinal chemistry of anti-biotics, they also demonstrate that once a new antibiotic isintroduced into the clinic, whether it is a novel chemicalentity acting at distinct bacterial targets or a semi-synthetic derivative that counters the resistance to its parentdrug, it is only a short matter of time until new resistancewill yet again emerge and create a public health problem [8].Thus, even though this continuous battle between humansand bacteria has resulted in several millstone drugs, the situa-tion today is more severe due to the emergence of seriousmultidrug-resistant (MDR) bacterial strains that are highlyresistant to the majority of currently available antibiotics [9,10].Gram-positive pathogens of particular concern includeMRSA, VRE, vancomycin-resistant S. aureus (VRSA) andpenicillin-resistant Streptococcus pneumoniae. Several Gram-negative pathogens, such as Klebsiella pneumoniae, Antibacterbaumannii and Pseudomonas aeruginosa, that are resistant toextended-spectrum b-lactam antibiotics (aminoglycosides,fluoroquinolones and other classical antibacterials) are alsoon the rise. Nosocomal infections associated with these latterorganisms are usually hard to treat and are often associatedwith considerable morbidity and mortality. Of equal concernis the spread of MDR strains of M. tuberculosis and the risk ofdissemination of such resistant pathogens is a serious diseasecontrol threat [11]. Thus, there is a crucial and urgent needto develop novel antibacterial agents and more advance strat-egies that can combat the problems of growingbacterial resistance.

1.1 Combination therapy as a solution to resistance

developmentOne approach that has been used to address the growing bac-terial resistance problem with some clinical success is a combi-nation therapy (Figure 1) [12]. Four principal modes of actionby which two compounds in combination (a cocktail) canenhance the activity of each other are classified where a secondcompound (an adjuvant): i) prevents the degradation ormodification of the primary drug (an antibiotic); ii) allows

Article highlights.

. There is a crucial and urgent need to develop novelantibacterial agents and more advanced strategies thatcan address the concerns of growingbacterial resistance.

. Combination therapy/cocktail of drugs has been usedwith some clinical success to address the growingbacterial resistance problem.

. Kishony and co-workers showed that while the“synergistic” antibacterial drug combinations havedeveloped multidrug resistance by sequentialsingle-resistance steps, suppressive or “antagonistic”drug combinations have slowed the evolutionof resistance.

. “Hybrid (heterodimer) antibiotics” strategy has severaladvantages versus simple cocktails including greatlyreduced potential for generating bacterial resistance.

. Three hybrid molecules currently investigated in humanclinical trials, TD-1792, MCB-3837 and CBR-2092, havebeen reported as compounds with impressive activityagainst MDR strains exhibiting resistance to both ofpartner drugs, and superior antibacterial efficacy relativeto a simple combination of partner drugs.

. Although several synthesized hybrids overcome theexisting resistance mechanisms of MDR pathogens byaddressing two different targets, none of these“synergistic” antibacterials have delayed evolution ofbacterial resistance.

. Fluoroquinolone--aminoglycoside “antagonistic”hybrids provided the first demonstration of the abilityof a hybrid structure to delay the emergence ofresistance development in both Gram-positive andGram-negative bacteria.

This box summarizes key points contained in the article.

Dual-acting hybrid antibiotics: a promising strategy to combat bacterial resistance

884 Expert Opin. Drug Discov. (2010) 5(9)

Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

the accumulation and retention of the primary drug (an anti-biotic) by inhibiting the efflux pumps; iii) inhibits the intrin-sic repair pathway or tolerance mechanism of cells to theprimary drug (an antibiotic); iv) is itself an antibiotic thattargets a similar or different pathway that is inhibited by thefirst antibiotic drug. In general, all these modes of a combina-torial approach can resurrect and enhance the antibacterialactivity of known and effective antibiotics. In addition, thisapproach might lead to shorter/lower dosing regiments, whichhas the potential to reduce the rate of acquirement of resis-tance by pathogens. The financial incentive, for expandingthe therapeutic use of an approved drug, is another importantadvantage to this approach. Because of these advantages, com-bination therapy has long been a common clinical practice,but the development in more recent years has been the ratio-nal design of different combinations, some of which haveachieved commercial success [12,13].

The clinical effectiveness of combination therapy to slowthe emergence of new resistance has been proven with partic-ular success in the treatment of TB-causing M. tuberculosis.Currently, a standard course treatment for TB is a combina-tion of isoniazid, rifampicin, pyrazinamide and ethanbutolor streptomycin for 2 months, then isoniazid and rifampicinfor a few further months [14]. The drugs in this particularcombination are acting in synergy by weakening the cellmembrane and disrupting protein synthesis. Isoniazid is neverused as a monotherapy since resistance emerges rapidly [11].Thus, apart from the synergistic action of multiple drugs incombination for the treatment of TB, such cocktails alsoslow the emergence of resistance. However, whether the syn-ergistic drug combinations are “always” associated with thedelay in development of resistance or not, is not definitive.Of particular concern in this regard are recent reports suggest-ing that synergistic drug combinations can actually enhancethe development of resistance rather than slowing itdown [15,16]. To clarify this issue, we use different models ofdrug--drug interactions, outlined in Figure 2.

Generally, two drugs interacting in combination can beclassified into three main types: additive (no interaction),synergistic (greater than the additive effect) and antagonistic(lesser than the additive effect) [17]. Since synergisticcombinations generate increased efficiency at lower doses,clinicians have for a long time taken advantage of the clinical

and biological benefits of synergy, whereas antagonistic com-binations have been completely ignored [18]. Recently, math-ematical models that predict and explain the emergence ofresistance have been formulated to explain and promotecombination therapy [16,19,20]. Establishing the basic con-cepts of drug interactions within a particular cocktail,Kishony and co-workers showed that some antagonisticcombinations have a potential advantage to delay and evenreverse development of bacterial resistance [16]. The labora-tory experiments and theoretical modeling used in theirstudy are selected for doxycycline resistance. Using a directcompetition assay between doxycycline-resistant and doxy-cycline-sensitive Escherichia coli, it was shown that in thepresence of either doxycycline alone or the synergistic pairdoxycycline--erythromycin, the resistant strain always out-competes its wild-type strain. By contrast, in the environ-ment of an antagonistic pair of doxycycline--ciprofloxacin,the sensitive strain wins under certain drug ratios, indicatingselection against mutants resistant to one of the two drugs.The following explanation was provided: when the combina-tion of two drugs (A + B) is less inhibitory to the bacterialgrowth than each drug alone (A and B separately,Figure 2C, antagonistic drug--drug interaction), then thereis no benefit for the bacteria to develop resistance againsteither A or B alone. In such a situation the resistant strainwill face a more hostile environment than in the case ofA + B; the strain that acquires resistance to one drug (eitheragainst A or B) in the cocktail loses in competition with thesensitive strains because either drug alone is a stronger anti-biotic than the combination. Consequently, a large delayin resistance development occurs. On the contrary, synergis-tic combinations (Figure 2A), while increasing antibacterialpotency against both sensitive and resistant strains,also increases the relative selection for resistance, becauseresistance mutations to either drug (A or B) are morefavorable for the bacterium [15]. However, one importantconsideration against the use of antagonistic drug combina-tions, also mentioned by the authors, is the reduced efficacy.This may lead to significantly longer treatments for infec-tious clearance and subsequently increase the chance forthe resistance to develop. Nevertheless, this critical discrep-ancy, between the current thinking in antibiotic drugdiscovery toward synergy and the above-mentioned recent

Combination therapy Hybrid antibiotics

Drug A

Target 1 Target 2

Drug B Drug A

Target 1 Target 2

Drug B

Covalent linker

Strategies to fight againstbacterial resistance

+

Figure 1. Two distinct strategies, combination therapy and hybrid antibiotics, to combat bacterial resistance.

Pokrovskaya & Baasov

Expert Opin. Drug Discov. (2010) 5(9) 885

Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

reports of Kishony and co-workers stressing the benefits ofantagonism, should definitely be weighed out whencombination therapy is being considered with the goalof minimizing the emergence of resistance. The limited suc-cess of combination therapy to date within the antibioticfield for only the treatment of TB further supportsthis statement.

1.2 Hybrid antibiotics as an emerging solution to

resistance developmentUnfortunately, combination therapy has several serious disad-vantages; the effect of a particular combination therapyin vitro does not always lead to a clear response in vivo dueto different pharmacokinetic properties of the drugs in combi-nation [12,21]. Consequently, there is the necessity of fine tun-ing the formulation to ensure that the in vivo concentrationsare the same as in the tablet. Furthermore, this strategy cannotaddress the problem of those MDR strains exhibiting thedeveloped resistance for both drug families in combination,and thus necessitates employment of other families of drugsin combination. The small number of powerful antibacterialdrug families largely limits the application of this approachfor that purpose. An emerging strategy, with the potential toaddress some of the above limitations of combination therapy,is the “hybrid (heterodimer) antibiotics” strategy. Accordingto this strategy, two pharmacophores that inhibit dissimilartargets in a bacterial cell are covalently linked into one mole-cule (Figure 1). Covalent connection between two drugsmakes the pharmacokinetics of the resultant hybrid moleculemore predictable. It is also possible to use the penetrationcapacity of one antibiotic moiety in a hybrid molecule toboost the bioactivity of the second antibiotic, and impor-tantly, by a rationally designed linkage between the twodrug motifs, better inhibit both targets, thus overcoming theexisting resistance mechanisms against either or both drugs.Furthermore, a potential reduction in the toxicity of a drugdue to hybridization with another drug might be observed.Finally, the rational design, syntheses, and systematic

development of novel hybrid entities should be much moreinspiring and beneficial for the medicinal chemist (the impor-tant player in drug design and development) both academi-cally and practically than configuring “ideal” drug cocktails.All these possible advantages of hybrids versus simple cock-tails, in conjunction to their greatly reduced potential forgenerating new resistance, surely make the development ofdual-action hybrid drugs a highly promising strategy tocombat hostile MDR bacterial strains.

A covalent linkage between two drugs in a hybrid can be acleavable moiety, which can actually be considered a prodrugapproach. Alternatively, the linkage can be a chemically andmetabolically stable entity, making the hybrid structure actas a dual-action drug. Similar to cocktails, the partner drugsin a hybrid can exhibit either a synergistic or an antagonisticantibacterial effect. It is apparent to date, that the main inspi-ration in hybrid drug research has/is toward the developmentof novel chemical entities with synergistic activity (superior tothe sum of the constituent agents) [22]. Additional importantadvantages to hybrid drugs that have been addressed in the lit-erature include: activity against drug-resistant bacteria, anexpanded spectrum of activity and increased duration afterthe onset of resistance, and reduced likelihood for generatingbacterial resistance [13,23,24]. These new and challenging taskshave introduced fresh research avenues into the field of antibi-otics research. In this article, we discuss recent examples ofdual-action hybrid drugs with a non-cleavable linker. Addi-tionally, we explore newly developed synergistic and antago-nistic hybrid compounds with emphases on their potentialto reduce resistance development.

2. Fluoroquinolone-containing hybridcompounds

Undoubtedly the most comprehensively represented hybridcompounds to date are those that contain the fluoroquinoloneclass of antibiotic linked to another antibacterial agent. Toexplain such a broad utilization of fluoroquinolones, several

Resistanceto A

Resistanceto B

Synergy

DrugA

DrugB

A + B

100

75

50

25

0

Resistanceto A

Resistanceto B

Additivity

DrugA

DrugB

A + B

100

75

50

25

0

Resistanceto A

Resistanceto B

Antagonism

DrugA

DrugB

A + B

100

75

50

25

0

A. B. C.

Gro

wth

inh

ibit

ion

(%

)

Figure 2. Bacterial growth inhibition by two drugs A and B due to their (A) synergistic, (B) additive, and (C) antagonistic

combination. Solid arrows illustrate favorable single-drug resistance steps and dashed arrows illustrate unfavorable

single-drug resistance steps.



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

reasons might be mentioned. In general, fluoroquinolones tar-get two enzymes inside the bacterial cell, topoisomerase IVand DNA gyrase, thereby inhibiting DNA replication andtranscription. Since these compounds target two dissimilarbut essential enzymes in bacteria. Some disturbance in bind-ing to one of the targets, for example, due to steric hindranceimposed by a linker or a second partner, might be compen-sated by targeting the other enzyme without a significantreduction in antibacterial potential. Another major benefit isthe widely studied and well-defined structure--activity rela-tionship of fluoroquinolones that emphasized the basic aminegroup in the C-7 piperidino moiety as a readily acceptableand tolerant point for attachment of various bulky substitu-ents through carbamate formation, N-alkylation, quaternarysalt formation and more. Fluoroquinolones are also stableunder a wide variety of synthetic conditions making themideal candidates for hybrid formation and development. It isnoteworthy, however, that because of their very strong bacte-ricidal activity (very low MIC values) and the spontaneousmutations of targeted enzymes as the major resistance mecha-nism, very fast rates of resistance development have beenreported for this type of antibiotic [25,26].

One of the most successful series of fluoroquinolone-based dual-action hybrid compounds, which has been exten-sively described in both the scientific and patent literature, isthe fluoroquinolone--oxazolidinone (eperezolide) hybrids [27-31].The oxazolidinone class of antibiotics inhibit protein synthesisby binding at the P-site of the ribosomal 50S subunit [32].Due to their limited ability to penetrate Gram-negative mem-brane, oxazolidinones exhibit relatively lower activity againstGram-negative bacteria. Therefore, the linking of a lipophilicfluoroquinolone (ciprofloxacin) to an oxazolidinone (eperezo-lide) in a single entity was expected to be highly beneficial.Since both eperezolide and ciprofloxacin possess a piperazinesubstituent at positions 4 and 7, respectively, they were mergedat these two positions with a variety of heterocyclic linkers(including piperazinyl, pyrrolidinyl, azetidinyl, piperidinyland others) to give a library of hybrid compounds (exemplifiedby compound 1, Figure 3). Several hybrids showed superioractivity than both the parent drugs and their 1:1 combinationas measured against both resistant and susceptible strains ofS. aureus (MRSA and methicillin-sensitive S. aureus [MSSA]),S. pneumoniae, Enterococcus faecalis and Enterococcus faecium.Both enantiomers of the most active hybrid (containing a3-hydroxymethyl pyrrolidinyl linker) exhibited improved anti-bacterial activity against bacterial strains resistant to eitherciprofloxacin or oxazolidinone alone and showed a balanceddual-mode of action on both biological targets in vitro [29].However, while the authors clearly mentioned the potentialof the newly developed hybrids toward a lowered propensityfor the development of bacterial resistance, no data have beenshown to justify this hypothesis. In the more recent patentliterature, another hybrid compound from this series contain-ing a 4-hydroxy piperidine linker (MCB-3681) was highlightedas the most active fluoroquinolone--oxazolidinone hybrid

and claimed activity against Bacillus anthracis bacteria (MICvalue of 0.03 µg/ml), the causative of anthrax (Figure 3) [28].In addition, MCB-3681 exhibited synergistic antibacterialactivity against various bacterial strains, including linezolide-and fluoroquinolone-resistant Gram-positive (MRSA, VRE,methicillin-resistant S. pneumoniae, glycopeptide intermediateS. aureus) and Gram-negative (but not P. aeruginosa) bacteria.MCB-3681 has progressed into human clinical trials underthe guise of a more water soluble phosphate ester prodrug(MCB-3837) that hydrolyzes in vivo to give the activeagent [31].

The fluoroquinolone pharmacophore has also been utilizedfor the synthesis of fluoroquinolone--anilinouracil hybrid com-pounds [33-35]. 6-Anilinouracils are non-traditional antibioticsthat selectively bind and inhibit the bacterial DNA polymeraseIIIC that is absolutely essential for the DNA replication processin Gram-positive bacteria and mycoplasmas. In addition, theinhibitors of the bacterial DNA polymerase IIIC have demon-strated bactericidal activity. However, anilinouracils alone arenot always effective as DNA polymerase inhibitors, probablydue to lack of penetration, removal of compound by effluxpumps, or alteration of the sensitivity of the target enzyme [36].The connection of the fluoroquinolonemoiety to this promisingclass of antibacterial agents via its secondary amine group atpiperazine ring, could improve their penetration, and broadenand improve its antibacterial spectrum by targeting two distinctsteps in the DNA replication process. Various fluoroquinolonecompounds were linked to the N-3 position of 6-anilinouracilesto afford a library of new hybrid compounds that demonstratedup to 64-fold improvement in inhibition of DNA polymer-ase and Gram-positive bacterial growth (exemplified bycompound 2, Figure 3) [34]. But, the hybrids showed a lack ofactivity against Gram-negative bacteria (E. coli) and exhibitedsignificantly reduced DNA gyrase and topoisomerase IV inhib-itory activities in comparison to parent fluoroquinolone com-pounds. More recently, one of the best representatives of thisseries, compound 2 (251D) (Figure 3) was tested in a varietyof in vitro assays, including DNA polymerase and topoisomer-ase/DNA gyrase enzyme assays, antibacterial, bactericidal,and mammalian cytotoxicity assays [33]. The activity ofcompound 2 generally was significantly higher than that of theparent 6-anilinouracil component in a wide variety of Gram-positive organisms tested, including both sensitive and resistantstrains (e.g., MRSA, VRE and others). This hybrid structurewas however significantly less potent against most sensitiveGram-positive organisms (fourfold less active against Bacillussubtilis and E. faecalis, and twofold less active against Bacilluscereus, Bacillus thuringiensis, S. aureus) in comparison to the flu-oroquinolone component alone, yet, the hybrid maintainedpotent activity against those organisms that were resistant toeither fluoroquinolone or to both fluoroquinolone and anili-nouracil components (more than 20-fold improvement againstMRSA and vancomycin-resistant E. faecalis (VREF)). Thus, interms of the synergistic effect of this particular hybrid compoundagainst Gram-positive bacteria, the synergy was only clearly



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

N N

F

O O

OH

FNN

NH

O

ONH

2 (251D)Anilinouracil-fluoroquinolone hybrid

O

HOHO

NH

OH

O

O

OH

H3CO O

OH

O

O

NN

NN

NF

O O

OH

3 (CBR-2092)Rifamycin-fluoroquinolone hybrid

N(CH3)2

OH O HO O

OH

N(CH3)2

OHO

HN N

N N

C2H5

F

O O

OH

F

4Tetracycline-fluoroquinolone hybrid

N N N

F

O O

OH

NO

OCH3

N

N

NH2

H2N

5 (BP-4Q-002)Benzyl pyrimidine-fluoroquinolone hybrid

Oxazolidinone-fluoroquinolone hybrids

N

F

O

HN

O

O

N N

F

O O

OH

O

OR

1 MCB-3681, R = H MCB-3837, R = P(O)(OH)2

OCH3

Figure 3. Structures of fluoroquinolone-containing hybrid compounds.



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

demonstrated in those instances where the organism was alreadyhighly resistant to the fluoroquinolone component. Sinceanilinouracil compounds do not significantly inhibit the growthof Gram-negative bacteria, an attempt to enhance such activityby the preparation of anilinouracil--fluoroquinolone hybridswas not particularly successful; the hybrid 2 was only moder-ately active against most Gram-negative strains tested (e.g.,P. aeruginosa, K. pneumaniae, E. coli), but displayed 32-foldgreater activity in comparison to the fluoroquinolone partneragainst the ciprofloxacin-resistant E. coli strain. Again, the syner-gistic effect of the hybrid 2 has been demonstrated only underconditions where the organism is resistant (poorly active) toeach partner alone. Furthermore, unlike hybrid 2, no synergisticeffect is observed when an equimolar combination of each of thepartner compounds alone were tested either against sensitive orresistant Gram-positive organisms. It has been suggested thatthe greater uptake of the hybrid 2 or its enhanced inhibitoryactivity againstDNApolymerase IIIC due to the increased bind-ing provided by the fluoroquinolone moiety, could explain theobserved synergistic effect of 2 versus the lack of synergy in thecocktail. Finally, when the frequency of spontaneous (singlestep) resistance development was tested in S. aureus, the individ-ual components developed resistance, but not the hybrid com-pound, within a single passage. In multi-passage experiments,however, the hybrid 2 developed resistance at a rate comparableto those of the partner components (16-fold increase in MICvalues after 17 successive passages). Thus, even though thehybrid 2 displayed synergistic effects against resistant strains,the expected propensity to delay the resistance development isnot truly demonstrated. Note that the latter resistance develop-ment experiments were performed on the S. aureus (Smith13709) strain against which the hybrid 2 neither demonstrateda clear synergistic nor an antagonistic effect (the MIC values of0.313, 5 and 0.078 µg/ml for the hybrid 2, anilinouracil, andfluoroquinolone components, respectively). Importantly, thehybrid 2 demonstrated a lack of in vitro toxicity and good inhib-itory activity of the targeted enzymes. In a separate study theauthors also demonstrated the in vivo efficacy of this class ofhybrids when given intravenously in a murine staphylococcalinfection model, confirming its potential as novel anti-infective agent [34]. A recent patent application fromMicrobiot-ics (MA, USA) highlights several prodrugs of this type ofhybrid where the carboxylic acid group at position 3 ofthe fluoroquinolone moiety has been esterified [37]. Thecompounds were tested in vivo against lethal infections. Thelead morpholino-ester prodrug showed a half effectivedose (ED50) of 18 mg/kg (intravenous dose) for protectionof mice from the intraperitoneal injection of S. aureus(Smith strain) infection.

Another type of novel hybrid design involves an analog of thefluoroquinolone class linked to macrocyclic core of rifamy-cin [38,39]. The biological activity of rifamycins relies on theinhibition of DNA-dependent RNA polymerase leading to asuppression of RNA synthesis and cell death [40]. Rifamycinsare utilized globally for the treatment of TB in combinations

with other agents and against a variety of Gram-positivebacteria. However, the liability of high resistance developmentobserved in bacteria treated with rifamycins, limits theirapproved use to combination regimes [41]. Antibiotic therapywith a combination of rifampicin and ciprofloxacin has beenshown to be a reasonable treatment option for biofilm-associated staphyloccocal infection [42]. In order to improve theantibacterial spectrum and bacterial resistance developmentproperties of rifamycin, workers at Cumbre Pharmaceuticalsprepared a series of hybrids in which rifamycin SV wascovalently connected to a quinolone pharmacophore derivedfrom the 4H-4-oxo-quinolizine subfamily of fluoroquinolones.4H-4-oxo-quinolizines were recently designed by replacingnitrogen with a carbon between ring carbons C-4 and C-5 toovercome bacterial resistance to fluoroquinolones. The leadcompound 3 (CBR-2092) contains a fluorinated 4H-4-oxo-quinolizine at the 3-position of the rifamycin core, linked via ahydrazone linkage (Figure 3). The detailed biochemical evalua-tion of compound 3 shows its excellent antibacterial activityagainst Gram-positive pathogens, including rifamycin- andciprofloxacin-resistant strains, and that it is equipotent to theparent compounds’ inhibitory activities against targetedenzymes [39]. However, antibacterial activity against Gram-negative bacteria (E. coli ATCC 25922) was only comparableto that of rifampin (rifamycin), and was significantly lowerthan that of ciprofloxacin [43]. In its favor, compound 3 displayedsuperior efficacy in multiple in vitro models of staphylococcalbiofilm states and in an in vivo standard rodent infection andrabbit endocarditis models, caused byMRSA. Although the fre-quency of spontaneous mutation in the presence of 3 is at a lowlevel, the comparative multistep passage resistance selectionsbetween 3 and the parent antibacterials have resulted in the iso-lation of S. aureus strains with enormous resistance levels afterthe course of 2 -- 15 passages in all compounds (MIC valueof 3 increased up to 500-fold after 15 passages). In contrast tofluoroquinolone compounds, the obtained resistant strainsshowed that mutational activation of efflux pumps was not acontributory factor in the development of resistance to thehybrid 3. In light of the increasing prevalence of efflux-mediated resistance traits in Gram-positive cocci, the authorspointed to the observed data as a promising advantage of hybridagents over cocktail combinations.

Recent work by Sriram et al. reveals a series of hybrids con-taining various fluoroquinolones linked to tetracycline deriva-tives as promising anti-HIV and antimycobacterial agents, andas inhibitors of HIV-1 integrase [44]. Tetracyclines inhibit theprotein synthesis in bacterial cells by binding to the 30S ribo-somal subunit and preventing the docking of amino-acylated tRNA. In addition, tetracyclines have demonstratedpromising anti-HIV inhibitory activity, especially those contain-ing bulky substituents at C-2 position of carboxamine moi-ety [45]. The design of tetracycline--fluroquinolone hybridswith a robust, non-cleavable linker at this position was envi-sioned as a potential strategy to ensure that the pharmacokinetcs,pharmacodynamics, and tissue distribution of the composite



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

pharmacophores are matched. As such, bulky aryl fluoroquino-lones were linked to several tetracycline derivatives to give alibrary of 12 hybrid compounds. Two pharmacophores wereconnected by reacting appropriate tetracyclines, formaldehydeand secondary amino (piperazino) group of fluoroquinolonesusing microwave irradiation (exemplified by compound 4,Figure 3). One of the lead hybrids, compound 4, was also foundto be active against the HIV-1 replication process and non-toxic to the CEM cells. Importantly, the MIC value of hybrid 4

wasmore than 30-fold lower than the parent compounds againsttetracycline-resistant M. tuberculosis bacterium. This increasedantimycobacterial potency of the hybrid compound has beenexplained as a possible synergistic dual-mode of action byinhibiting both biological targets of the parent compounds.One of the most recent patents described a new series

of hybrid compounds in which either 4-quinolones or4H-4-oxo-quinolizines have connected with benzyl pyrimidinesas potential antimicrobial agents [46]. Benzyl pyrimidines targetthe production of tetrahydrofolic acid by inhibiting the dihydro-folate reductase enzyme that reduces dihydrofolate to tetrahy-drofolate in bacterial, parasitic and epithelial cells. One of therepresentatives of this class, trimethoprim is a widely used anti-biotic with a broad spectrum of action. A combination therapycomprising the administration of a trimethoprim and ciproflox-acin cocktail has not been successful due to the different pharma-cokinetic properties of the two antibacterial agents [47]. Toaddress this problem a series of hybrid compounds, inwhich benzyl pyrimidines connected at the C-7 position of4-quinolones or 4H-4-oxo-quinolizines with different linkers,were designed and synthesized (exemplified by compound 5, -Figure 3). This approach of targeting two individual steps inthe same target (DNA synthesis) offered several possible bene-fits, such as a synergistic effect in terms of efficacy, lowered resis-tance selection propensity, activity against resistant bacteria, andreduced susceptibility to efflux pumps and toxicity in compari-son to a cocktail of the two drugs. Indeed, the lead compound 5

(BP-4Q-002) was 30-times more potent than ciprofloxacinalone or its 1:1 cocktail with trimethoprim, and four-times more potent than trimethoprim against S. aureus NRS19 (resistant to ciprofloxacin). The antibacterial activity of 5against susceptible E. coli and B. subtilis bacteria was similar totrimethoprim, but significantly lower than ciprofloxacin. Addi-tional analogs showed antibacterial activity lower than ciproflox-acin and similar to or better than trimethoprim. Unfortunately,no bacterial resistance development tests have been reported forthese compounds.

3. Aminoglycoside-containing hybridcompounds

Aminoglycoside antibiotics are long-known antibacterialagents, which are active against a wide variety of Gram-positive and Gram-negative bacterial strains. The mainmolecular target of aminoglycosides is the decoding site(A-site) of 16S rRNA in the 30S bacterial ribosome subunit.

Binding of aminoglycosides at this site decreases the fidelityof protein synthesis during the translation process that leadsto bacterial cell death. However, the appearance of bacterialstrains resistant to these drugs and their relative toxicity arecritical problems that largely limit their intensive clinicaluse [48-50]. Generally, aminoglycosides suffer from resistanceenzymes that modify their hydroxyl or amino groupsrendering the resulting products inactive. One of therecent efforts to solve these problems attaches another phar-macophore to the aminoglycoside molecule resulting inheterodimer/hybrid compounds.

The first example of an aminoglycoside-based hybrid struc-ture was published by Mobashery and co-workers in 2001 [51].The b-lactam moiety of cephalosporin was exploited todevelop a dual-action compound by linking an aminoglyco-side at the C-3 position of the b-lactam partner (exemplifiedby isepamicin--deacetylcephalothin hybrid 6, Figure 4). Severalprevious studies have indicated that a combination of amino-glycoside and b-lactam antibiotics is frequently synergis-tic [52,53]. Accordingly, the design principle involved the“expulsion” of aminoglycoside on the C-3 position of cepha-losporin antibiotic in the presence of b-lactamases, or syner-gistic action on two biological targets as a hybrid antibioticin the absence of b-lactamases. Significant reduction in toxic-ity and better uptake of the highly polar aminoglycoside wereexpected after its fusion with the cephalosporin moiety.Although the synthesis of such hybrid compounds waspointed out to be very challenging due to the complicatedchemistries of both b-lactams and aminoglycosides, severalhybrid compounds, such as 6 were synthesized (Figure 4). Pre-liminary biological evaluation of compound 6 indicated goodsubstrate activity with b-lactamase and reduced toxicity to themammalian host in comparison to the aminoglycoside com-ponent, isepamicin [51]. However, no antibacterial activity ofthe resultant compounds has been reported.

More recently, Yu and co-workers reported on the develop-ment of a series of hybrids containing an aminoglycoside(neomycin B) linked to chloramphenicol or oxazolidinone (line-zolid) partners with different linkers [54-57] (Figure 4). Chloram-phenicol and oxazolidinone antibiotics bind to the 23S rRNA ofthe 50S ribosomal subunit; chloramphenicol inhibits the pep-tidyl transferase activity and oxazolidinone inhibits the initiationstep of protein synthesis. Similar to aminoglycosides, these anti-bacterial agents have broad spectrum of activity against Gram-positive and Gram-negative bacterial strains. In order toimprove RNA binding and specificity of aminoglycosidesand to reduce their toxicity, the loop-binding compoundschloramphenicol and linezolid were linked to the stem-binding neomycin B, providing heterodimers with a potentialto recognize both RNA stem and loop motifs present on theribosome of the pathogenic organisms. Some of the designedhybrids displayed enhanced affinities to specific RNAs with dis-sociation constants significantly lower than that of neomycin B.In addition, the results of foot-printing and mutation studiessuggested that the affinity of hybrids is RNA sequence-specific.



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

The neomycin--linezolid heterodimer 7 showed a more than20 times improvement in affinity to models of bacterial 16Sand human 18S A-sites in comparison to neomycin B, whereasthe neomycin--chloramphenicol 8 showed the highest discrimi-nation factor between bacterial and human A-sites. Unfortu-nately, antimicrobial activities of 7 and 8 against a panel of20 standard pathogenic strains, as well as their IC50 values intranslation inhibition assays in vitro, have not correlated wellwith their dissociation constants and were lower than those ofneomycin B in all cases.

The recent development of aminoglycoside--fluoroquino-lone hybrids by Pokrovskaya et al. highlights the potential ofantagonistic heterodimer compounds to delay the emergenceof bacterial resistance [58]. By linking an aminoglycoside (neo-mycin B) and a fluoroquinolone (ciprofloxacin) via different1,2,3-triazole containing spacers a series of 17 hybrid com-pounds with a common structure 9 were prepared (Figure 4).Although none of the synthesized compounds showed higheractivity than ciprofloxacin, the majority of hybrids wassignificantly more potent than neomycin B against a panel

SHN

N

S

OHNO

O

O OHO

O

O

H3N

OHOH

OH

NH3

OHHN

O

OOH

NH2MeOH

Me

OH

O

6Cephalosporin-aminoglycoside hybrid

OO

NH2

OH

OH

H2N

O

NH2

HO

HOH2N

O

O

NH2

OH

NH2

OS

OH

SN

6

N

F

N

O

AcHN

O

O

7Oxazolidinone (linezolid)-aminoglycoside hybrid

OO

NH2

OH

OH

H2N

O

NH2

HO

HOH2N

O

O

NH2

OH

NH2

OS

OH

S 6

O

HN

O2N

OH

HO

8Chloramphenicol-aminoglycoside hybrid

Figure 4. Structures of aminoglycoside-containing hybrid compounds.



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

of Gram-positive (e.g., MRSA resistant to neomycin B) andGram-negative bacteria (neomycin-susceptible and neomy-cin-resistant E. coli strains). Furthermore, selected hybridcompounds were also able to overcome most prevalent typeof aminoglycoside resistance associated with APH(3¢)-Ia,APH(3¢)-IIIa, and AAC(6¢)/APH(2¢¢) resistance enzymes.Selected hybrids inhibited bacterial protein synthesis with

potencies similar to or better than that of neomycin B, andwere up to 32-fold more potent as inhibitors than ciprofloxa-cin for the fluoroquinolone targets, DNA gyrase and topo-isomerase IV, indicating a balanced dual-mode of action.Perhaps the most important discovery of this study was thecase of multi-passage experiments of one of the hybridsshowing a significant delay of resistance development in

OHOHO

RHN

NN

N

NH

BnHN

O

FmocHN

ONH

O O

OHO

HO

OH

NHR

NHRRHN

HO

OO

NHR

OH

OH

RHN

O

NHR

HO

HORHN

O

O

NHR

OH

NHR

O

OH

NN

N

NH

BnHN

O

FmocHN

ONH

R = -C = NH(NH2)

Bn = benzyl

OO

NH2

OH

OH

H2N

O

O

OH

NH2

NH2

OH

O

O

NH2

HOHO

H2N

N

O

F

N

HOOC

NN

NN

Spacer 2

9Fluoroquinolone-aminoglycoside hybrid

10Peptide-aminoglycoside (neomycin B) hybrid

11Peptide-aminoglycoside (kanamycin A) hybrid

R = -C = NH(NH2)

Spacer 1

Figure 4. Structures of aminoglycoside-containing hybrid compounds (continued).



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

both Gram-negative (E. coli ATCC 35218) and Gram-positive (B. subtilis ATCC 6633) bacteria in comparison tothat of each parent antibiotic separately or their 1:1 mixture;although the MIC value of the tested hybrid remainedunchanged (3 µg/ml) in both bacteria after the 1st and 15thpassages, the MIC values for ciprofloxacin, neomycinB and their 1:1 combination increased significantly corre-sponding to 75-, 4- and 20-fold resistance development inE. coli, respectively, and 37.5-, 8- and 7.6-fold resistancedevelopment in B. subtilis, respectively. To our knowledge,this study provided the first demonstration of the ability ofa hybrid structure to delay the emergence of resistance devel-opment in both Gram-positive and Gram-negative bacteria. Itis of particular note that the MIC values, as measured againstE. coli for the hybrid, ciprofloxacin and neomycin B, were 3,0.01, and 12 µg/ml, respectively, and against B. subtilis were3, 0.02 and 0.75 µg/ml, respectively, demonstrating antago-nistic activity of the tested hybrid structure in comparisonwith each partner drug separately. Therefore, the observeddata in this study of the hybrid antibiotic correspond well toKishony and co-workers results [15,16] with antibacterialdrug combinations, and support the idea that the antago-nistic effect of drug combinations, whether as a simplecocktail or as a hybrid structure, can lead to the delay ofresistance development.

Very recently, novel aminoglycoside--peptide conjugateshave been reported with a 1,2,3-triazole-containing linker(Figure 4) [59]. Short amphiphilic peptides were linked to anaminoglycoside (neomycin B and kanamycin A) partner viaclick chemistry to afford a variety of hybrid structures. Thehypothesis was that conversion of aminoglycoside antibioticsinto cationic amphiphiles would enhance antibacterial activityagainst resistant strains and slowdown the development ofresistance rate. In addition, the aminoglycoside amino groups oftwo hybrid compounds were also converted to guanidines inorder to explore the effects of basicity. Evaluation of theantibacterial activity in this series of hybrids showed a stronginfluence dependant on the nature of the peptidic component.Although the majority of compounds showed enhancedantibacterial activity against neomycin B-, kanamycin A-resistantMRSA, kanamycin A-resistant methicillin-resistant Streptococcusepidermitis (MRSE) and gentamicin-resistant P. aeruginosa, themost potent compounds were conjugates of dipeptides with atryptophan unit where the aminoglycoside part contained a gua-nidine instead of amine (compounds 10 and 11, Figure 4). How-ever, reduced antibacterial activity against neomycin B- andkanamycin A-susceptible strains has been demonstrated. In vitrotoxicity measurements show little hemolytic activity againstmammalian erythrocytes at low MIC concentrations, but signifi-cant hemolytic activity at higher concentrations. The observedconcentration-dependent hemolytic activities of the aminoglyco-side--peptide hybrids and previous studies with other cationicamphiphiles have confirmed a membranolytic mode of actionof the resultant compounds. Unfortunately, no resistancedevelopment tests have been reported.

4. Other hybrid compounds

In this section we combined a series of hybrid structuresreported recently that do not contain fluoroquinolone andaminoglycoside as partner pharmacophores. One family ofsuch novel hybrid molecules was created by merging similararomatic motifs of oxazolidinone and chalcone, which mayrepresent the simplest/smallest hybrid structures reported todate (exemplified by compound 12, Figure 5) [60]. Chalconesare products of condensation of simple or substituted aro-matic aldehydes with acetophenones in the presence of alkali,possessing a wide range of biological activities including anti-microbial potency. It was found that a, b-unsaturated keto-functional group is the structural feature responsible forantibacterial activity of chalcones. Numerous regioisomericchalcone--oxazolidinone hybrids were synthesized with thegoal to obtain compounds with synergistic antibacterial activ-ity. To achieve this goal, a series of different substituents thatspan from strong electron donating to strong electron with-drawing natures, were introduced onto the aromatic ringof the chalcone part. However, synthesized hybrids exhibitedonly trace or no activity against a panel of Gram-positive organisms tested (MSSA, MRSA, VREF, E. faecalis).By introducing a fluorine atom onto the aromatic ring andconverting the acetamido group to the corresponding thiocar-bamate, several more active compounds were designed andsynthesized. One of them, the hybrid 12, showed activitiessimilar to linezolid and vancomycin against susceptibleS. aureus and MRSA bacterial strains, and exhibited a morethan 30-fold improvement against VREF and E. faecium incomparison to vancomycin. Since no antibacterial activity ofthe chalcone component has been reported, it is difficult todraw a conclusion about the synergistic properties of theresultant hybrid 12.

In another study, Breukink and co-workers describedhybrid compounds containing vancomycin and nisinfragments connected via different spacers [61]. Even thoughthese two antibiotics have different modes of action, theyboth target the essential cell wall precursor lipid II. Vancomy-cin is a glycopeptide that inhibits cell wall biosynthesisin Gram-positive bacteria by forming a tight complex withD-Ala-D-Ala-containing peptidoglycan precursors thatpresents in lipid intermediate II. Nisin (1 -- 12) demonstrateshigh-affinity binding to lipid II leading to pore formation andthus disrupting the cell wall synthesis pathway. Subsequently,connection of these two into one molecule was claimed toincrease affinity to lipid II through a bivalency or chelateeffect that could restore vancomycin activity against resistantstrains, such as VRE. The lengths of the spacers and theattachment site(s) were predicted by computer modeling. Byusing click-chemistry reactions, three hybrid compoundswere synthesized and evaluated for their antibacterial potencyagainst susceptible and vancomycin-resistant strains of entero-cocci and K. pneumoniae, and against Moraxella catarrhalisresistant to both vancomycin and nisin (exemplified by



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

O

O

O

N H

H N

O

O

H2N

H N

O

N HOH

OH N

Cl

NH

NH

OO

OH

HO

HO

N H

Cl

O

O

OO

HO

HO

NH

2

OH

OH

O

OH

ON H

O

3

NN

NN H

H NN H

O

O

S

O

HN

O

NO

H2N

HNH

N

O

HN

N H

O HN

O

S

O

O

NH

H N

NH

2

O

O

13N

isin

(1-1

2)-v

anco

myc

in h

ybrid

F

N

N

O

O

H N

O

O

S

12O

xazo

lidin

one-

chal

cone

hyb

rid

Figure

5.Structuresofvarioushybridco

mpounds.



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

compound 13, Figure 5). The lead compound 13 exhibitedhigher antibacterial activity than parental agents against allresistant strains, but was slightly less active than vancomycinagainst susceptible enterococci. Thus, the synergistic activityof the hybrid was only demonstrated against the resistantstrains, being ~ 40 times more active than its compo-nents against VRE. It was suggested that, since in thevancomycin-resistant strains the vancomycin part of the

hybrid should lose most of its affinity for the availableD-Ala-D-Lac binding sites, the nisin part that retains affinityfor lipid II in these resistant strains allows the hybrid todisrupt the cell wall biosynthesis pathway.

A similar strategy was applied to the design of vancomycinand b-lactam hybrid compounds [62,63]. These hybridstructures were developed to inhibit Gram-positive bacterialcell wall biosynthesis by simultaneously targeting both

15 (TD-1792)Cephalosporin-vancomycin hybrid

O

O

O

NH

HN

O

O

H2N

HN

O

NH

OHO

HN

Cl

NH

NH

O

O

OH

HO

HO

NH

Cl

O

O

O OHO

HO

NH2

OH

OH

O

OHO

N

O

NH

N

S

O

OHO

N

N

SCl

NH2

14Cephalosporin-vancomycin hybrid

ON

O

HN

N

S

O

O OH

N

N

S

Cl

H2N

O

O

O

NH

HN

O

O

H2N

HN

O

NH

OHO

HN

Cl

NH

HO

O

O

OH

HO

HO

NH

Cl

O

O

O OHO

HONH2

OH

OH

O

OH

NHNH

O

NH

O

4

C3

4′

Figure 5. Structures of various hybrid compounds (continued).



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

cellular targets of vancomycin (lipid intermediate II) andb-lactams (transpeptidase domain of penicillin bindingprotein). Due to the physical proximity of two targets andtheir sequential role in the cell wall biosynthetic pathway,enhanced potency and bactericidal activity were expected.A series of vancomycin--cephalosporin heterodimers were syn-thesized by connecting two antibacterial agents at severalattachment positions with a range of linking moieties. Thecephalosporin core was derivatized at three positions: atthe C3 pyridinium substituent by a methyl amino moiety(exemplified by compound 14, Figure 5), at the oxime

(exemplified by compound 15, Figure 5) and at aminothiazole(exemplified by compound 16, Figure 5). In addition, threedifferent attachment points of vancomycin were explored viathe amide bond formation: the vancosamine amino group ofthe terminal sugar (compound 16), the carboxyl terminus(compound 15), and the 4¢ resorcinol-like position on the aro-matic side chain of amino acid 7 (compound 14). The combi-nation of vancomycin and cephalosporin attachment pointsresulted in the preparation of nine amide-linked hybrids.The compounds were screened against a panel of susceptibleand resistant Gram-positive human pathogens. Interestingly,

O

O

O

NH

HN

O

O

H2N

HN

O

NH

OHO

HN

Cl

NH

HO

O

O

OH

HO

HO

NH

Cl

O

O

O OHO

HONH

OH

OH

O

OH

HN

ON

O

HN

N

S

O

O OH

N

N

S

Cl

HN

C7

HN

OOO

4

16Cephalosporin-vancomycin hybrid

O

OHO

NH

O

O

OH

H3COO

OH

O

O

N

NH

N

N

N

NO2

17Rifamycin-metronidazole hybrid

Figure 5. Structures of various hybrid compounds (continued).



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

all the tested hybrids exhibited synergistic antibacterial activ-ity (> 15 times higher activity than the parent antibiotics)against tested bacterial strains, including MRSA and VREF.In view of the fact that the specific attachment position ofthe two drugs’ moieties appeared to have little effect on thepotency profile, it was concluded that the two active compo-nents of the hybrids do not bind simultaneously at both cellu-lar targets but synergistically inhibit cell wall synthesis. Inaddition, compounds 14 and 15 exhibited superior in vivoactivity in a mouse model of MRSA infection. Compound 15

was selected as a clinical candidate (TD-1792) that success-fully completed the Phase II clinical trial for the treatmentof complicated Gram-positive skin and skin structure infec-tions. Additional patents from Theravance, Inc. (CA, USA)describe a series of other hybrids of different b-lactams andglycopeptids [64,65]. The compounds of this invention, consistof 2 -- 10 ligands of the b-lactam and glycopeptide classes ofantibacterial agents covalently connected by a variety of link-ers, were claimed to interfere with the synthesis/metabolism ofthe cell wall. The lead compound from this series showed sig-nificantly higher antibacterial activity than vancomycinagainst MRSA and MRSE (MIC value of < 0.1 µg/ml). Invivo studies with neutropenic mice showed a half effectivedose (ED50) of < 0.20 mg/kg (intravenous dose) for the pro-tection of mice from S. aureus (MRSA 33591) infection,whereas the ED50 of vancomycin was 9 mg/kg.

Cumbre Pharmaceuticals (TX, USA) has recently pub-lished a patent that describes rifamycin--metronidazole hybridcompounds with potent synergistic activity against Gram-positive and Gram-negative pathogens (exemplified bycompound 17, Figure 5) [66]. Metronidazole is a nitroimida-zole antibiotic used particularly for anaerobic bacteria andprotozoa (Trichomonas infections). After permeation by diffu-sion, metronidazole undergoes reduction in vivo and theresultant intermediate exhibiting cytotoxicity interacts withthe host cell DNA, resulting in DNA strand breakage andfatal destabilization of the DNA helix [67]. In order to achievesynergy, minimize the resistance development to these antibi-otics and make the pharmacokinetic profile more predictable,rifamycin was covalently connected to an antibacterialpharmacophore from the nitroimidazole, nitrothiazole, ornitrofuran class of compounds. The hybrids demonstratedsignificant enhancement of activity in comparison to theirsimple (unlinked) combinations. The lead compound ofthis series, hybrid 17, exhibited antibacterial activity higherthan the parental agents against Gram-positive bacteriaincluding susceptible Clostridium difficile, metronidazole-resistant S. aureus, metronidazole-resistant M. tuberculosis,and M. tuberculosis resistant to both parental agents. Inaddition, 17 showed synergistic activity (higher than bothmetronidazole and rifampin alone) against Gram-negativeorganisms, including susceptible and resistant to rifampinHelicobacter pylori and susceptible Bacteroides fragilis.Although no resistance development tests have beenperformed, the data of the study clearly indicated desired

synergistic character of the hybrid 17 with both susceptibleand resistant bacterial strains.

A large number of aminoquinoline-based hybrids wererecently described in a patent application by Palumed SA(France) and CNRS (France) [68]. 4-Aminoquinoline and8-aminoquinoline compounds were covalently combinedwith penicillin (represented by compound 18), cephalosporin(represented by compound 19), fluoroquinolone (representedby compound 20), streptogramine (represented by com-pound 21), macrolide (represented by compound 22), glyco-peptide (vancomycin) (represented by compound 23) andoxazolidinone (represented by compound 24) (Figure 6). Ami-noquinolines are primarily known as antimalarial agents [69],but in addition they are known as inhibitors of efflux pumpsin MDR Gram-negative bacterial clinical isolates [70]. It hasbeen shown that aminoquinolines, by inhibiting theAcrAB--TolC efflux pump, restore the susceptibility ofMDR clinical strains to structurally unrelated antibioticssuch as norfloxacin, tetracycline and chloramphenicol [70,71].Therefore, the covalent connection between aminoquinolineand different antibacterial agents could overcome an inhibi-tory effect on the efflux pumps of resistant bacteria andenhanced activity of a hybrid could be observed. The resultanthybrids were tested against a panel of susceptible and resistantbacterial strains. The hybrid 18, designed as a prodrug, con-tains a penicillin moiety with a protected carboxylic acidand was expected to convert to the free carboxylate in vivo.However, it exhibited twofold lower antibacterial activitythan that of penicillin G against the MSSA bacterial strainin vitro; no MIC data were reported for the free carboxylatederivative. More extensive data were reported for theaminoquinoline--cephalosporin hybrid compounds. The leadcompound 19 exhibited similar or slightly lower activitythan cephalosporin ceftriaxone against a panel of suscep-tible strains, including S. aureus, S. pyogenes, S. pneumoniaebacteria, and showed high synergistic activity against S. aureusand S. pneumoniae resistant to both parental agents and their1:1 cocktail. In addition, 19 demonstrated low oral toxicityand low binding to human serum, and exhibited enhancedin vivo activity in a murine model of septicemia due toMSSA. Aminoquinoline--fluoroquinolone heterodimer 20

demonstrated improved antibacterial activity against sensitiveS. aureus, E. faecalis, B. subtilis, B. thuringiensis Gram-positive bacteria, and ciprofloxacin-resistant S. aureus. How-ever, lower potency than ciprofloxacin was reported againstall tested Gram-negative bacteria, such as sensitive E. coli,H. influenza, and P. aeruginosa. The lead compounds of ami-noquinoline hybrids with streptogramin (compound 21),macrolide (compound 22), vancomycin (compound 23)and oxazolidinone (compound 24) exhibited higher antibacte-rial activity than parental agents against a panel of susceptibleand resistant Gram-positive bacterial strains, includingMSSA, and S. pneumoniae. To conclude, most of the preparedhybrids showed high synergistic antibacterial activity againstsusceptible and resistant Gram-positive bacteria. However,



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

their activity was lower when tested against Gram-negative bacteria, probably due to the fact that aminoquino-lines do not exhibit antibacterial activity by themselvesbut exhibited inhibitory properties with efflux pumps inGram-negative E. aerogenes and K. pneumoniae bacteria only.

5. Expert opinion

This abbreviated overview illustrates that the combined effortsover the past several years between academy and industry havesignificantly advanced our understanding of how chemical

N

S

O

O

N

N

Cl

OO

O

tBu

O N

S

OHO

OAc

O

O

HN

NCl

N N

F

O O

OH

N

N

Cl

18Aminoquinoline-penicillin hybrid

19Aminoquinoline-cephalosporine hybrid

20Aminoquinoline-fluoroquinolone hybrid

O

HN

HN

O O

N S

HNO

N

O

N

O

O

NHO

NOH

N

N

Cl

Et

O

OHO

HO

O

N

OHN

NCl

O

O

O

N

HO

OHO

21Aminoquinoline-streptogramine hybrid

22Aminoquinoline-macrolide hybrid

Figure 6. Structures of aminoquinoline-containing hybrid compounds.



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

redesign of the existing antibacterial drugs can evade the resis-tance mechanisms that have evolved in pathogenic bacteria tothese drugs. This knowledgebase has prompted the develop-ment of hybrid antibiotics as an emerging option in antibiotictherapy with the goal to control the growing army of resistantpathogens. The primary goal of the hybrid antibioticsapproach to overcoming the existing resistance mechanismsof MDR pathogens, by addressing two different targets eitherin parallel or subsequently, has been accomplished in a num-ber of examples; the three hybrid molecules currently investi-gated in human clinical trials, TD-1792, MCB-3837 andCBR-2092, have been reported with impressive activityagainst MDR strains exhibiting resistance to both of the part-ner drugs alone, and with superior efficacy relative to a simplecombination of partner drugs. Therefore, we would like toencourage the continuation of hybrid antibiotics develop-ment, even though the optimization course toward clinicallysuccessful hybrids might be more complex.

The other scene of hybrid antibiotics development, how-ever, that relies on their anticipated ability to avoid/reduce the emergence of new resistance development stillawaits further elucidation and validation. Although the strat-egy of hybrid antibiotics development is principally basedon the “multivalent therapy” approach [23,72], a strategy witha hypothesized advantage in slowing down the emergence ofspontaneous target-related resistance development relative to“monovalent therapy”, the reality is that this issue in the

antibiotics field has recently faced some controversial observa-tions [15,16,19], requiring further careful revision, especiallywhen drug--drug combinations are considered. Thus,although the primary goal of the hybrid antibiotics approach,to justify their development for clinical use, has been a dem-onstration of synergy superior to that provided by the simplecombination of partner drugs, whether such synergistichybrids can avoid/reduce the emergence of new resistancedevelopment remains to be demonstrated conclusively.Indeed, surprisingly, none of the synergistic hybrids reportedeither in the scientific or patent literature have beentested for their ability to delay, or if they even enhance, resis-tance development in those resistant strains on which theirsuperior antibacterial efficacy have been demonstrated.Only in a few examples, were the hybrids that mainly demon-strated the synergy in resistant bacteria tested for resistancedevelopment in wild-type bacteria by using multi-passage experiments. Although the results obtained in theseexperiments were not particularly encouraging, no clear expla-nation has been provided as to whether the contested behaviorof the hybrid drugs -- the reduced propensity for resistancedevelopment -- can in principle be achieved by the “synergistichybrids” or if that challenge ought to be directed in anotherdirection, e.g., “antagonistic hybrids”.

The most recently developed system biology approaches,equipped with theoretical models and subsequent in vitroexperimental data, have provided very clear and justified

O

O

O

NH

HN

O

O

H2N

HN

O

NH

OHO

HN

Cl

NH

HO

O

O

OH

HO

HO

NH

Cl

O

O

O OHO

HO

HN

OH

OH

O

OH

O

NH

N

Cl

N Cl

NHNH

O

O

N

O

FN

O

23Aminoquinoline-glycopeptide hybrid

24Aminoquinoline-oxazolidinone hybrid

Figure 6. Structures of aminoquinoline-containing hybrid compounds (continued).



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

arguments on this issue: while the “synergistic” antibacterialdrugs combinations have developed multidrug resistance bysequential single-resistance steps, suppressive or “antagonistic”drug combinations have selected against resistant bacteria andslowed the evolution of resistance [15,16,19]. Even though thisfundamental concept of drug--drug interaction has only beendemonstrated in vitro and it is still unclear if it will also beoperational in vivo, this fresh and extremely intriguing obser-vation in the antibiotic arena, together with a recent report inwhich an aminoglycoside--fluoroquinolone “antagonistichybrid” (compound 9, Figure 4) that exhibited considerableantibacterial efficacy also demonstrated significant reductionin resistance development in comparison to partner drugsand their cocktail, clearly support the above notion that themain challenge with regard to hybrid antibiotics developmentshould be carefully revised.After all, and in with applause to the medicinal

chemists, while the main challenge toward the hybrid drugdevelopment has been designated the development of“synergistic hybrid drugs” due to reduced potential for generat-ing bacterial resistance, the main motivation, ambition, andencouragement have been directed toward the achievement of“synergy” rather than the essentiality of demonstrating the

delay of resistance development. Therefore, the key challengeshould definitely be a clear and broader experimental demon-stration whether the “synergistic effect” or “antagonistic effect”of the developed hybrid drug is better at preventing/reducing the evolution of resistance. Althoughmany additionalissues can be counted as limiting factors for successful hybriddrug development for use in a clinical setting [23,24,73], it is clearthat this fundamental issue of drug--drug interaction must besolved before these drugs will find their way into the clinic.The hybrid antibiotics should provide excellent model systemsfor answering this question at both in vitro and in vivo levels.

Acknowledgment

We thank Yael Balazs for reading the manuscript andfruitful comments.

Declaration of interest

This work supported by research grants from the IsraelScience Foundation founded by the Israel Academy of Scien-ces and Humanities (grant no. 515/07) and by the TechnionV.P.R. Fund.

Bibliography

1. Davies J. In the beginning there was

streptomycin. In: Arya DP, editor,

Aminoglycoside antibiotics.

John Wiley & Sons, Inc., Hoboken,

New Jersey; 2007. p. 1-15

2. Gale EF. Correlation between penicillin

resistance and assimilation affinity in

Staphylococcus aureus. Nature

1947;160(4064):407

3. Palumbi SR. Evolution -- humans as the

world’s greatest evolutionary force.

Science 2001;293(5536):1786-90

4. Umezawa H, Okanishi M, Kondo S,

et al. Phosphorylative inactivation of

aminoglycosidic antibiotics by

Escherichia coli carrying R factor.

Science 1967;157(796):1559-61

5. Martin CM, Ikari NS, Zimmerman J,

et al. A virulent nosocomial Klebsiella

with a transferable R factor for

gentamicin: emergence and

suppression. J Infect Dis

1971;124(Suppl):S24-9

6. Benveniste R, Davies J. R-factor

mediated gentamicin resistance: a new

enzyme which modifies aminoglycoside

antibiotics. FEBS Lett 1971;14(5):293-96

7. Auckland C, Teare L, Cooke F, et al.

Linezolid-resistant enterococci: report of

the first isolates in the United Kingdom.

J Antimicrob Chemother

2002;50(5):743-6

8. Miller JR, Waldrop GL. Discovery of

novel antibacterials. Expert Opin

Drug Discov 2010;5(2):145-54

9. Giamarellou H, Poulakou G.

Multidrug-resistant gram-negative

infections: what are the treatment

options? Drugs 2009;69(14):1879-901

10. Arias CA, Murray BE.

Antibiotic-resistant bugs in the 21st

century -- a clinical super-challenge.

N Engl J Med 2009;360(5):439-43

11. Gillespie SH. Evolution of drug

resistance in Mycobacterium tuberculosis:

clinical and molecular perspective.

Antimicrob Agents Chemother

2002;46(2):267-74

12. Cottarel G, Wierzbowski J.

Combination drugs, an emerging option

for antibacterial therapy.

Trends Biotechnol 2007;25(12):547-55

13. Bremner JB, Ambrus JI, Samosorn S.

Dual action-based approaches to

antibacterial agents. Curr Med Chem

2007;14(13):1459-77

14. Mitchison DA. Treatment of

tuberculosis. The Mitchell lecture 1979.

J R Coll Physicians Lond

1980;14(2):91-5, 98-9

15. Yeh PJ, Hegreness MJ, Aiden AP, et al.

Drug interactions and the evolution of

antibiotic resistance. Nat Rev Microbiol

2009;7(6):460-6

16. Chait R, Craney A, Kishony R.

Antibiotic interactions that select against

resistance. Nature

2007;446(7136):668-71

17. Loewe S. The problem of synergism and

antagonism of combined drugs.

Arzneimittelforschung 1953;3(6):285-90

18. Pillai SK, Moellering RC,

Eliopoulos GM. In: Lorian V, editor,

Antibiotics in laboratory medicine.

Lippincott Williams & Wilkins,

Philadelphia; 2005. p. 365-440

19. Michel JB, Yeh PJ, Chait R, et al.

Drug interactions modulate the

potential for evolution of resistance.

Proc Natl Acad Sci USA

2008;105(39):14918-23

20. Hegreness M, Shoresh N, Damian D,

et al. Accelerated evolution of resistance

in multidrug environments. Proc Natl

Acad Sci USA 2008;105(37):13977-81

21. Bliziotis IA, Samonis G, Vardakas KZ,

et al. Effect of aminoglycoside and

beta-lactam combination therapy

versus beta-lactam monotherapy on

the emergence of antimicrobial



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

resistance: a meta-analysis of randomized,

controlled trials. Clin Infect Dis

2005;41(2):149-58

22. Long DD, Marquess DG.

Novel heterodimer antibiotics: a

review of recent patent literature.

Future Med Chem 2009;1(6):1037-50

23. Brotz-Oesterhelt H, Brunner NA.

How many modes of action should an

antibiotic have? Curr Opin Pharmacol

2008;8(5):564-73

24. Barbachyn MR. Recent advances in the

discovery of hybrid antibacterial agents.

Annu Rep Med Chem 2008;43:281-90

25. Radzishevsky IS, Rotem S,

Bourdetsky D, et al. Improved

antimicrobial peptides based on

acyl-lysine oligomers. Nat Biotechnol

2007;25(6):657-9

26. Komp LP, Karlsson A, Hughes D.

Mutation rate and evolution of

fluoroquinolone resistance in

Escherichia coli isolates from patients

with urinary tract infections.


2003;47(10):3222-32

27. Hubschwerlen C, Specklin JL,

Baeschlin D, et al.

Oxazolidinone-quinolone hybrid

antibiotics. US2008027040; 2008


Baeschlin DK, et al. Use of

oxazolidinone-quinoline hybrid

antibiotics for the treatment of

anthrax and other infections.

WO2004096221; 2004


Baeschlin DK, et al.

Structure-activity relationship in the

oxazolidinone-quinolone hybrid series:

influence of the central spacer on the

antibacterial activity and the mode of

action. Bioorg Med Chem Lett

2003;13(23):4229-33

30. Hubschwerlen C, Specklin JL, Sigwalt C,

et al. Design, synthesis and biological

evaluation of oxazolidinone-quinolone

hybrids. Bioorg Med Chem

2003;11(10):2313-19


Baeschlin D, et al.

Oxazolidinone-quinolone hybrid

antibiotics. WO2005058888; 2005

32. Bozdogan B, Appelbaum PC.

Oxazolidinones: activity, mode of action,

and mechanism of resistance. Int J

Antimicrob Agents 2004;23(2):113-19

33. Butler MM, Lamarr WA,

Foster KA, et al. Antibacterial

activity and mechanism of action of a

novel anilinouracil-fluoroquinolone

hybrid compound.


2007;51(1):119-27

34. Zhi C, Long ZY, Manikowski A,

et al. Hybrid antibacterials.

DNA polymerase-topoisomerase inhibi-

tors. J Med Chem 2006;49(4):1455-65

35. Zhi C, Wright GE.

Novel heterocyclic antibacterial

compounds. WO02102792; 2002

36. Zhi C, Long ZY, Gambino J,

et al. Synthesis of substituted

6-anilinouracils and their

inhibition of DNA polymerase IIIC

and Gram-positive bacterial growth.

J Med Chem 2003;46(13):2731-9

37. Yanachkova MI, Wright GE. Polar ester

prodrugs of heterocyclic hybrid

antibacterial compounds and salts

thereof. WO2008094203; 2008

38. Robertson GT, Bonventre EJ, Doyle TB,

et al. In vitro evaluation of CBR-2092, a

novel rifamycin-quinolone hybrid

antibiotic: studies of the mode of action

in Staphylococcus aureus.


2008;52(7):2313-23

39. Robertson GT, Bonventre EJ, Doyle TB,

et al. In vitro evaluation of CBR-2092, a

novel rifamycin-quinolone hybrid

antibiotic: microbiology profiling studies

with staphylococci and streptococci.


2008;52(7):2324-34

40. Floss HG, Yu TW. Rifamycin-mode of

action, resistance, and biosynthesis.

Chem Rev 2005;105(2):621-32

41. Chaisson RE. Treatment of chronic

infections with rifamycins: is

resistance likely to follow?


2003;47(10):3037-9

42. Konig DP, Schierholz JM, Munnich U,

et al. Treatment of staphylococcal

implant infection with

rifampicin-ciprofloxacin in stable

implants. Arch Orthop Trauma Surg

2001;121(5):297-9

43. Ding CZ, Jin Y, Combrink K, Kim IH.

Quinolone carboxylic acid-substituted

rifamycin derivatives. WO2009064792;

2009

44. Sriram D, Yogeeswari P, Senchani G,

et al. Newer tetracycline derivatives:

synthesis, anti-HIV, antimycobacterial

activities and inhibition of

HIV-1 integrase. Bioorg Med Chem Lett

2007;17(8):2372-5

45. Neamati N, Hong H, Sunder S, et al.

Potent inhibitors of human

immunodeficiency virus type 1 integrase:

identification of a novel four-point

pharmacophore and tetracyclines as novel

inhibitors. Mol Pharmacol

1997;52(6):1041-55

46. Labischinski H, Cherian J, Calanasan C,

Boyce RS. Hybrid antimicrobial

compounds and their use.

WO2010025906; 2010

47. Huovinen P, Wolfson JS, Hooper DC.

Synergism of trimethoprim and

ciprofloxacin in vitro against clinical

bacterial isolates. Eur J Clin Microbiol

Infect Dis 1992;11(3):255-7

48. Wright GD, Berghuis AM, Mobashery S.

Aminoglycoside antibiotics. Structures,

functions, and resistance. Adv Exp

Med Biol 1998;456:27-69

49. Mingeot-Leclercq MP, Glupczynski Y,

Tulkens PM. Aminoglycosides:

activity and resistance.


1999;43(4):727-37

50. Wright GD. Mechanisms of

aminoglycoside antibiotic resistance.

In: Lewis K, Salyers AA, Taber HW,

Wax RG, editors, Bacterial resistance to

antimicrobials. 2nd edition. CRC press,

Taylor & Francis group;

2008. p. 71-101

51. Grapsas I, Lerner SA, Mobashery S.

Conjoint molecules of cephalosporins

and aminoglycosides.

Arch Pharm (Weinheim)

2001;334(8-9):295-301

52. Giamarellou H. Aminoglycosides plus

beta-lactams against gram-negative

organisms. Evaluation of in vitro synergy

and chemical interactions. Am J Med

1986;80(6B):126-37

53. Clark RB, Pakiz CB, Hostetter MK.

Synergistic activity of

aminoglycoside-beta-lactam combinations

against Pseudomonas aeruginosa with an

unusual aminoglycoside antibiogram.

Med Microbiol Immunol

1990;179(2):77-86

54. Lee J, Kwon M, Lee KH, et al.

An approach to enhance specificity



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

against RNA targets using

heteroconjugates of aminoglycosides and

chloramphenicol (or linezolid). J Am

Chem Soc 2004;126(7):1956-7

55. Kwon M, Kim HJ, Lee J, et al.

Enhanced binding affinity of

neomycin-chloramphenicol (or linezolid)

conjugates to A-site model of 16S

ribosomal RNA. Bull Kor Chem Soc

2006;27(10):1664-66

56. Yu J, Lee J, Kwon M, Shin K.

Heterodimeric conjugates of

neomycin-chloramphenicol having

an enhanced specificity against

RNA targets and its preparation.

US2005009765; 2005

57. Yu J, Lee J, Kwon M, et al.

Heterodimeric conjugates of

neomycin-oxazolidinone, their

preparation and their use.

US2005222055; 2005

58. Pokrovskaya V, Belakhov V,

Hainrichson M, et al. Design,

synthesis, and evaluation of novel

fluoroquinolone-aminoglycoside hybrid

antibiotics. J Med Chem

2009;52(8):2243-54

59. Bera S, Zhanel GG, Schweizer F.

Evaluation of amphiphilic

aminoglycoside-peptide triazole

conjugates as antibacterial agents.

Bioorg Med Chem Lett

2010;20(10):3031-5

60. Selvakumar N, Kumar GS, Azhagan AM,

et al. Synthesis, SAR and antibacterial

studies on novel chalcone oxazolidinone

hybrids. Eur J Med Chem

2007;42(4):538-43

61. Arnusch CJ, Bonvin AMJJ, Verel AM,

et al. The vancomycin-nisin(1-12) hybrid

restores activity against vancomycin

resistant Enterococci. Biochemistry

2008;47(48):12661-63

62. Long DD, Aggen JB, Chinn J, et al.

Exploring the positional attachment of

glycopeptide/beta-lactam heterodimers.

J Antibiot 2008;61(10):603-14

63. Long DD, Aggen JB, Christensen BG,

et al. A multivalent approach to drug

Discovery for novel antibiotics.

J Antibiot 2008;61(10):595-602

64. Christensen B, Moran EJ, Griffin JH,

et al. Novel antibacterial agents.

US20070134729; 2007

65. Christensen B, Moran EJ, Griffin JH,

et al. Novel antibacterial agents.

US2009111737; 2009

66. Ding CZ, Kim IH.

Nitroheteroaryl-containing rifamycin

derivatives. WO2008008480; 2008

67. Edwards DI. Nitroimidazole drugs --

action and resistance mechanisms. II.

Mechanisms of resistance.

J Antimicrob Chemother

1993;31(2):201-10

68. Sanchez M, Meunier B. Hybrid

molecules QA where Q is an

aminoquinoline and A is an antibiotic

residue, the synthesis and uses thereof as

antibacterial agents. US2007060558;

2007

69. Acharya BN, Thavaselvam D,

Kaushik MP. Synthesis and antimalarial

evaluation of novel pyridine quinoline

hybrids. Med Chem Res

2008;17(8):487-94

70. Mallea M, Mahamoud A, Chevalier J,

et al. Alkylaminoquinolines inhibit the

bacterial antibiotic efflux pump in

multidrug-resistant clinical isolates.

Biochem J 2003;376(Pt 3):801-5

71. Chevalier J, Bredin J, Mahamoud A,

et al. Inhibitors of antibiotic efflux in

resistant Enterobacter aerogenes and

Klebsiella pneumoniae strains.


2004;48(3):1043-46

72. Morphy R, Rankovic Z.

Designed multiple ligands. An emerging

drug discovery paradigm. J Med Chem

2005;48(21):6523-43

73. Gediya LK, Njar VCO. Promise and

challenges in drug discovery and

development of hybrid anticancer drugs.

Expert Opin Drug Discov

2009;4(11):1099-111

AffiliationVarvara Pokrovskaya & Timor Baasov†

†Author for correspondence

Technion -- Israel Institute of Technology,

The Edith and Joseph Fischer

Enzyme Inhibitors Laboratory,

Schulich Faculty of Chemistry,

Haifa 32000, Israel

Tel: +972 4 829 2590; Fax: +972 4 829 5703;

E-mail: [email protected]



Exp

ert O

pin.

Dru

g D

isco

v. D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

03/

14/1

1Fo

r pe

rson

al u

se o

nly.

Dual-acting hybrid antibiotics: a promising strategy to ...teaching.ncl.ac.uk/antimicrobials/Lectures/Reading... · Antibiotic resistance (both endogenous and acquired) has been an

Documents