c2np20080e

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From natural prod

aLaboratoire de Chimie Organique, ESPCI, 1

France. E-mail: [email protected] of Chemical Sciences and Engine

Lausanne, EPFL-SB-ISIC-LSPN, CH-1015

[email protected]

Cite this: Nat. Prod. Rep., 2013, 30,161

Received 24th July 2012

DOI: 10.1039/c2np20080e

www.rsc.org/npr

This journal is ª The Royal Society of

uct to marketed drug: the tiacumicinodyssey

William Erb*a and Jieping Zhu*b

Covering: 1975 to 2012

The first members of the tiacumicin family of antibiotics, encompassing more than 40 compounds, were

isolated in 1975. Structurally, the core aglycon is an 18-membered macrolactone having two conjugated

diene units, one isolated double bond, 5 stereogenic centers and most often, at least one glycosidic

linkage. Tiacumicin B, a RNA synthesis inhibitor, is a narrow-spectrum antibiotic against clostridia. For

the treatment of Clostridium difficile infection (CDI), it has the same cure rate as vancomycin but with

lower relapse rate and was approved by the FDA in May 2011. The aim of this review is to present an

overview of the chemistry and biology of tiacumicins since their discovery.

1 Introduction2 Isolation and characterization2.1 Lipiarmycin from Actinoplanes deccanensis2.2 Clostomicins from Micromonospora echinospora2.3 Tiacumicins from Dactylosporangium aurantiacum2.4 Lipiarmycin from Catellatospora3 Biosynthesis4 Biological activity4.1 Biological activity4.2 Mechanism of action5 SAR studies6 Synthesis6.1 Homodichloro-orsellinic acid6.2 2-O-Methyl-b-D-rhamnose6.3 5-Methyl-b-rhamnose7 Clinical application8 Summary and outlook9 Acknowledgements10 References

1 Introduction

Microbial diversity represents an almost innite pool for thediscovery of novel compounds. There are more than 23 000known microbial secondary metabolites, close to 60% of themproduced by bacteria, and their usefulness in drug developmentis well established.1–4

0 rue Vauquelin, 75231, Paris Cedex 05,

ering, Ecole Polytechniques Federale de

Lausanne, Switzerland. E-mail: jieping.

Chemistry 2013

Actinomycetes are among the most morphologically diverseprokaryotes and are widely distributed all around the Earth.5,6

They arouse the attention of the scientic community due to thegreat diversity and biological activities associated with the cor-responding metabolites: antimicrobial, antifungal, immuno-suppressive, antitumor, etc. Compounds such as erythromycin,streptomycin, amphotericin B and rapamycin, all sold as drugs,came from such strains,7,8 and they are still considered as apromising source of new antibiotics.9–12

In 1975, Parenti and co-workers identied a new substance,lipiarmycin, which exhibited a strong activity against gram-positive bacteria from actinoplanaceae strains (a sub-class ofActinomycetes). A few years later, related compounds wereisolated from parent strains and were named clostomicin andtiacumicin. This eld remained broadly unexplored until thelate 90’s when Optimer Pharmaceuticals began the commercialdevelopment of one tiacumicin for the treatment of Clos-tridium difficile infection (CDI). C. difficile is an importantnosocomial pathogen frequently diagnosed in infectioushospital-acquired diarrhoeas whose cost is estimated from433–797 million dollars annually in the USA.13 The research inthis eld turned out to be highly rewarding since tiacumicin Bhas recently been approved by the FDA for the treatment of C.difficile infection. The aim of this review is to present anoverview of the chemistry and biology of tiacumicincompounds and their application to the treatment of C. difficileassociated infection.14–18

2 Isolation and characterization2.1 Lipiarmycin from Actinoplanes deccanensis

In the 1970s, Parenti and co-workers reported the isolation of anovel antibiotic from a new strain, isolated from a soil samplecollected in India, named Actinoplanes deccanensis ATCC

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http://dx.doi.org/10.1039/c2np20080e

http://pubs.rsc.org/en/journals/journal/NP

http://pubs.rsc.org/en/journals/journal/NP?issueid=NP030001

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21983.19–21 Growth of the soil extract on agar led to the forma-tion of sporangia that liberated spores by rupture of the wall(Fig. 1). The major compound isolated from this strain wasnamed lipiarmycin, from leap year, because the strain wasisolated on February 29th 1972.

Initial analysis of lipiarmycin revealed the presence of twochlorine atoms, at least one phenolic hydroxyl group, threecarbonyls (one saturated and two conjugated to double bonds),a probable sugar moiety, and an aromatic nucleus, which wasdetermined to be homodichloro-orsellinic acid by degradationstudies.

A few years later, scientists from Gruppo Lepetit reported theisolation of 2-O-methyl-4-O-homodichloroorsellinate-b-rham-noside upon acid methanolysis of lipiarmycin.22 This sugar wasalso found in other natural antibiotics with either an a- or b-glycosidic linkage.23–25 They also identied the second sugar inlipiarmycin as 5-methyl-b-rhamnose. Note that the absolutecongurations of both sugars have not been established andwere shown articially in their L form.

Finally, in 1987, Nasini and co-workers reported that thelipiarmycin known at that time was a mixture of two products,lipiarmycin A3 (1) and A4 (2), in a 3 : 1 ratio, separable byash chromatography.26 Chemical degradation and extensiveNMR studies allowed them to elucidate the structure of the

Fig. 1 Sporangium obtained on soil extract-agar. Magnification �800.

William Erb studied chemistryat the University of Paris-Sud XI.He received his PhD in organicchemistry under the guidance ofPr. Jieping Zhu (Institut de Chi-mie des Substances Naturelles)in 2010. He then joined thegroup of Pr. Varinder Aggarwalat the University of Bristol,working on organocatalysis andits application to the synthesis ofnatural products. He is currentlyAttache Temporaire d’Enseigne-

ment et de Recherche in the group of Janine Cossy in Paris, workingon total synthesis and metal-catalyzed reactions.

162 | Nat. Prod. Rep., 2013, 30, 161–174

two lipiarmycins (Fig. 2). These molecules feature a 18-membered macrolactone incorporating four stereogeniccenters (unkown conguration), two conjugated dienes andone tri-substituted double bond. The macrolactone is glyco-sylated by 2-O-methyl-b-D-rhamnose esteried in the 4thposition either by homodichloro-orsellinic acid (lipiarmycinA3) or dichloro-orsellinic acid (lipiarmycin A4). The secondsugar link to the macrolactone is 4-O-isobutyrate-5-methyl-b-rhamnose, whose absolute conguration was not determinedbut shown as D form.

One year later, two novel lipiarmycins were isolated from thesame strain:27 lipiarmycins B3 (3) and B4 (4), which differ fromthe corresponding A3 and A4 by the position of the isobutyricester on the 2-O-methyl-b-D-rhamnose moiety (position 20 0 forlipiarmycin B and 40 0 for A). Lipiarmycins B3 and B4 differthrough the substituent (methyl or ethyl) of the aromatic ring.

Fig. 2 The structure of lipiarmycins.

Jieping Zhu received his B. Scfrom Hangzhou Normal Univer-sity and his M.Sc. degree fromLanzhou University (P. R. China)under the guidance of ProfessorLi Yulin. He got his Ph.D. degreefrom University Paris XI, Franceunder the supervision ofProfessor H.-P. Husson and Pr. J.C. Quirion. Aer 18 monthspost-doctoral stay with ProfessorSir D. H. R. Barton at Texas A &M University in USA, he joined

in the “Institut de Chimie des Substances Naturelles”, CNRS,France as Charge de Recherche and was promoted to Director ofResearch 2nd class in 2000 and then 1st class in 2006. He moved toEcole Polytechnique Federale de Lausanne (Swiss Federal Instituteof Technology Lausanne), Switzerland in September 2010 as a fullprofessor.

This journal is ª The Royal Society of Chemistry 2013


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2.2 Clostomicins from Micromonospora echinospora

In 1985, ~Omura and co-workers isolated a strain of Micro-monospora echinospora from a soil sample collected in a riceeld in Japan.28,29 Based on taxonomic studies, it appeared thatit was a new strain, subsequently named Micromonospora echi-nospora subsp. armeniaca subsp. nov. KMR-593.

Fermentation yielded ve compounds: clostomicins A (5),B1, B2 (6), C (7) and D (8). NMR studies revealed that closto-micin B1 was identical to lipiarmycin A3 previously isolated andthat the difference between clostomicins A and B2 resided inthe substitution pattern of the 5-methyl-b-rhamnose sugar: theisobutyric ester substituent was proposed to be in the C3

0 0

position for clostomicin A and in the C20 0 for clostomicin B2.

Therefore it was concluded that the structure of clostomicin B2is identical to lipiarmycin B3.

The IR spectrum of clostomicins C and D showed the pres-ence of an additional carbonyl group compared to lipiarmycinA3, which was attributed to a ketone at the C18 position. The

13CNMR spectra of clostomicin C also reveals the absence of onemethyl carbon at 28.6 ppm assigned to the equatorial methylgroup of the 5-methyl-b-rhamnose sugar. Therefore, the struc-ture of clostomicins C and D was assigned as shown in Fig. 3.

2.3 Tiacumicins from Dactylosporangium aurantiacum

In 1986, McAlpine and co-workers reported a new strain isolatedfrom a soil sample collected in Connecticut, which was namedDactylosporangium aurantiacum subsp. hamdenensis subsp. nov.AB718C-41 (NRRL 18085).30–32 A rst fermentation experimentusing 20 litres of broth yielded three compounds named tia-cumicins A (9), B (10) and C (11) (10 mg, 35 mg and 24 mg,respectively). A second study using a much bigger broth (4500litres) led to the isolation of three additional tiacumicins (D(12), E (13) and F (14)) in very low yields (7 mg, 20mg and 13mg,respectively) compared to tiacumicin B (3.82 g).

Extensive NMR studies allowed scientists to propose thestructure of tiacumicin B, which was found to be identical tolipiarmycin A3 and clostomicin B1. Tiacumicins C and F are

Fig. 3 The structure of clostomicins.


different from tiacumicin B in the position of the isobutyricester on the 5-methyl-b-rhamnose moiety (respectively at C20 0

and C30 0). Tiacumicin D is another isomer of tiacumicin B inwhich the 2-O-methyl-b-D-rhamnose is esteried in the C30

position by homodichloro-orsellinic acid (Fig. 4). Tiacumicin Eis almost identical to tiacumicin C except for the ester moiety onposition C20 0, which is not an isobutyric ester but a propionateone. Finally, tiacumicin A is a simpler analog without the 2-O-methyl-b-D-rhamnose moiety and with an acetate ester on the 5-methyl-b-rhamnose sugar. Note that although described withthe right conguration (2R, 3S, 4S, 5S, 6R) in the text, the 2-O-methyl-b-D-rhamnose was written as its enantiomer in the nalstructure of tiacumicins.31

The absolute conguration of the macrolactone wasassigned in 2005 by X-ray crystal structure analysis of tiacumicinB. It was subsequently established that C18 has the (R) cong-uration for tiacumicin B and (S) conguration for lipiarmycinA4.33,34 Even though we cannot conclude denitively, it could beassumed the conguration of C18 to be (R) for other tiacumicinsand (S) for lipiarmycins. The C18 conguration remainedunassigned for clostomicins.

Therefore, without taking into account the C18 congura-tion, lipiarmycin A3, clostomicin B1 and tiacumicin B seem tobe identical. So are lipiarmycin B3, clostomicin B2 and tiacu-micin C or clostomicin A and tiacumicin F.

In the late 90’s, with the aim of producing novel tiacumicinanalogs, McAlpine and co-workers replaced the potassiumchloride added to the broth with potassium bromide.35,36 Usingthe same Dactylosporangium aurantiacum strain as before, theyisolated four new compounds (Fig. 5, 15–18) incorporatingbromide on the aromatic ring, whose structure have beenelucidated by mass spectroscopy and NMR studies.

In 2007, scientists from Optimer Pharmaceuticals, Inc.reported seven new members of tiacumicin family (Fig. 6, 19–25), present in very low concentration in the fermentationbroth, as judged by the HPLC prole of the mixture and thecorresponding integrations (Fig. 6).37,38 During formulationstudies, Optimer scientists also identied the new tiacumicinderivative 26. It is identical to tiacumicn B except for the pres-ence of carbonyl function at C7.39 Although the conguration ofthese new compounds has not been elucidated, they areassumed to be the same as for tiacumicin B.

The most important library of tiacumicin analogs wasgenerated by Zhang and co-workers while working on theelucidation of the biosynthesis of tiacumicin B.40–44 Indeed,from different mutants of the Dactylosporangium aurantiacumstrain, they have been able to characterize 37 new analogs(Fig. 7). We can note, based on published data, that 50 is the C18

epimer of lipiarmycin A4 (2).It is interesting to note that the yield of tiacumicin B

production by D. aurantiacum has been greatly improved overthe years by Optimer scientists.45 Using a growth mediummainly composed of sh powder, glucose, casamino acid, yeastextract and some inorganic salts to support microorganismgrowth, it is possible to obtain 100–500 mg of crude tiacumicinper litre of broth, much higher than the 18.8 mg L�1 initiallyreported by McAlpine. The introduction of a resin able to trap

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Fig. 4 The structure of tiacumicins.

Fig. 5 The structure of brominated tiacumicins.

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macrocycles as they are formed in the broth increased the yieldof tiacumicin and facilitated the recovery of product by sievingfrom the broth and elution with organic solvents. Purication ismainly achieved by reversed-phase medium-pressure liquidchromatography.

2.4 Lipiarmycin from Catellatospora

In 2008, during the course of a screening programme to identifynew anti-tuberculosis agents, scientists from Novartis reportedthe isolation of lipiarmycin A3 from Catellatospora sp. Bp3323-81, a strain from the company’s screening library.46

Fig. 6 The structure of tiacumicin analogs.

3 Biosynthesis

Despite more than 30 years of history, little was known aboutthe biosynthesis of tiacumicin until the work of Zhang and co-workers in 2011.40,41 In early studies, Parenti noted that thesource of chlorine could be the meat extract used in the broth orthe added sodium chloride and that omission of both chlorinesources greatly reduce the yield of lipiarmycin.19 Furthermore,McAlpine has shown that addition of potassium bromide to thebroth allows the formation of brominated tiacumicinanalogs.35,36 Apart from feeding experiments, the biosynthesis oftiacumicin remained undetermined.

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In 2011, Zhang and co-workers reported their studies onthe biosynthesis of tiacumicin based on a genetic approachon Dactylosporangium aurantiacum hamdenensis NRRL18085.47–50 Firstly, they targeted polyketide synthase (PKS) andhalogenase using probes to identify genes involved in thebiosynthesis of tiacumicin. They have been able to identifythe complete tia-gene cluster which comprised 50 orfs (OpenReading Frame) and 110 633 bp (base pairing). In a rstattempt, the putative functions of orfs have been deduced bycomparison with protein databases and a further renement



Fig. 7 The structure of tiacumicin analogs.

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allowed them to eliminate some orfs, which are not involvedin tiacumicin biosynthesis. Thus the nal gene cluster con-tained 31 orfs, from gene TiaG1 to TiaR2 for approximately83 kb.

Genes TiaA1 to TiaA4 were predicted as modular polyketidesynthases, and inactivation experiments led to the conclusionthat they are responsible for the tiacumicin aglycone synthesis(Fig. 8, A). The biosynthesis involved propionyl-CoA, malonyl-CoA, (2S)-methylmalonyl-CoA and (2S)-ethylmalonyl-CoA.

Bioinformatic analysis of the TiaB gene revealed somesimilarity to 6-methyl-salicylic acid synthase and thus it isprobably involved in the biosynthesis of the homo-orsellinicacid part 69 from a propionyl-CoA starting unit (Fig. 8, B). Thearomatic moiety could then be transferred to the 2-O-methyl-D-rhamnose residue by TiaF due to similarity to acyltransferases,responsible for incorporation of aromatic parts into secondarymetabolites. Finally, the gene TiaM showed some similarity toother halogenases and its selective inactivation led to theformation of tiacumicin analogs lacking chlorine atoms,therefore allowing the assignment of its function. It has alsobeen shown that instead of chlorine (from NaCl), TiaM is able


to transfer bromine atoms to the homo-orsellinic acid moiety(from NaBr). However, F� and I� are not halide donors forTiaM.

The genes TiaS1, S3 and S4 are probably involved in thebiosynthesis of the D-rhamnose derivatives 70 from GDP-D-mannose 71 but the precise sequence is not currently known(Fig. 8, C). Bioinformatic analysis allowed the researchers topropose the role of TiaS2, TiaS5, TiaS6, TiaG1 and G2, whichwere further veried by selective inactivation. TiaG1 andTiaG2 are 5-C-methyl-D-rhamnosyl-transferase and 2-O-methyl-D-rhamnosyl-transferase, respectively, used to attachsugar moieties to the aglycone. TiaS2 and TiaS6 are a sugarC-methyltransferase and an acyltransferase, respectively,responsible for incorporation of the methyl and the iso-butyryl moiety of the 4-O-isobutyrate-5-methyl-b-rhamnose.TiaS6 showed a relaxed substrate specicity for rhamnosederivatives but a great regioselectivity, being unable tomethylate the C2 position of 5-C-methylrhamnose and othersugars.

The TiaS5 gene is the 20-O-methyltransferase involved in thesynthesis of the 2-O-methyl-b-D-rhamnose. Further experiments

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Fig. 8 The proposed biosynthesis of tiacumicin B.

Fig. 9 The roles of the principal genes in tiacumicin B (10) biosynthesis.

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revealed that this methyltransferase was divalent metal-cationdependant: its activity is higher with Mg2+ and Mn2+, moderatewith Co2+, Ni2+, Fe2+, weak with Zn2+, Cu2+ and inactive in theabsence of any metal cation and in the presence of EDTA orCa2+. It is also a pH-dependant enzyme displaying its bestactivity at pH 8. The two genes TiaP1 and TiaP2 encode forcytochrome P450 hydroxylase, responsible for natural productoxygenation. Inactivation experiments allowed determinationof their roles as follows: TiaP1 catalyzes the hydroxylation at C18

and TiaP2 at C20.Some others genes are involved in the precursor supply: TiaC

and TiaDmay be involved in isobutyryl-CoA, propionyl-CoA andacyl-CoA generation. TiaL probably catalyses the formation of(2S)-methylmalonyl-CoA from propionyl-CoA. TiaJ, TiaN andTiaK are probably involved in the ethylmalonyl-CoA pathwayand TiaE, showing some similarity with thioesterases, maypromote the accuracy and efficiency of the polyketide synthase.TiaR1 could be a transcription activator in bacteria, TiaR2seems to be a negative regulator of tiacumicin biosynthesis.Genes TiaT1–T4 may constitute a system to transport thesynthesized metabolites out of the cell whereas the role of TiaIis actually unknown.

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In spite of this great achievement, some points remainedunclear about tiacumicin biosynthesis. The right timingbetween genes TiaP1 and TiaS5 (hydroxylation of the C18 posi-tion and methylation of C20 hydroxyl, respectively) is stillunclear even though it seemed that TiaP1 should be the laststep, directly preceded by TiaS5. Furthermore, the acylation ofthe C40 0 position by TiaS6, even if shown as anterior to glyco-sylation, could be a later step in the biosynthesis.

Fig. 9 shows the action of the most importants genesinvolved in the tiacumicin biosynthesis.



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4 Biological activity4.1 Biological activity

The initial report of the discovery of lipiarmycin mentioned, forthe mixture of lipiarmycin A3 and A4, a fairly good activityagainst some Staphylococcus aureus strains and other gram-positive bacteria. A good activity against strains of cariogenicSteptococcus mutans, suggesting possible application as anantiplaque agent, was also discovered.20 A few years later,Sonenshein and co-workers reported the inhibition of bactero-phage growth in Bacillus subtilis.51 During the early studies ontiacumicins, researchers from Abbott Laboratories reported agood activity of tiacumicin B against different aerobic bacteria(including S. aureus and Enterococcus faecium) but a loweractivity against anaerobic bacteria.30 Tiacumicin B is also activeagainst Staphylococcus epidermidis biolms formed at thesurface of medical devices (a major cause of nosocomial infec-tions)52 and on some drug-resistant strains of Mycobacteriumtuberculosis.46 Another study, reported by JMI Laboratories,shows a limited bacterial activity of 10 against S. aureus, CoNS,E. faecalis and E. faecium with minimal bactericidal concentra-tions/MIC ratios (0.5–16 mg mL�1).53

In 2006 Optimer mentioned that the tiacumicins may ndapplication in the treatment of gastrointestinal cancers, butwithout reporting any more detailed information.34 The rstand only study for such an application of tiacumicin came fromEchem Hightech Co.54 A series of tiacumicin benzylidene acetalderivatives have shown interesting activity against breast cancercells with similar IC50 values to Tamoxifen�, a drug usuallyused for this type of cancer (Table 1).55 Note that the moleculesdrawn in this patent are tiacumicin C derivatives (with isopropylester on C20 0), although they are claimed to be tiacumicin Bderivatives in the text (with isopropyl ester on C40 0).

Table 1 Biological activity of Tiacumicin B derivatives against various cancer cells(mg mL�1)

CompoundsIC50 forMCF7 breast cancer cells

IC50 forT-47D breast cancer cells

Tamoxifen� 6.35 � 0.45 5.50 � 0.21Tiacumicin B (10) 8.39 � 1.00 5.56 � 0.5774 7.06 � 0.83 3.99 � 0.5875 6.90 � 0.36 4.24 � 0.0576 5.78 � 0.81 4.04 � 1.09


Notwithstanding these biological activities, it is in the ghtagainst Clostridium difficile associated infection (CDI) that tia-cumicin B was developed as a drug.

Clostridium difficile, a Gram-positive anaerobic bacteria, isthe causative agent of between 20% and 25% of all cases ofantibiotic-dependant diarrheas.56–60 Indeed, the gastrointestinaltract microbiota protects the host against most infections bypathogenic microorganisms through mechanisms known as“colonization resistance”.61 The use of broad spectrum antibi-otics leads to severe perturbations of the gut microbiota,creating opportunities for the growth of bacteria usuallyrestricted by microbial competition. C. difficile is recognized assuch an opportunistic pathogen. The most encountered clinicalmanifestations of CDI are diarrhea (mild to moderate), pseu-domembranous colitis, fever, and abdominal pain. Approxi-mately 3% of patients will develop a fulminant colitis, withserious complications, such as colonic perforation, toxic meg-acolon and death.62

The treatment of these infections involves the use ofmetronidazole and/or vancomycin depending on the clinicalpresentation of the disease. However, these two broad-spectrumantibiotics have some drawbacks: a) metronidazole is easilyabsorbed along the gastrointestinal tract, resulting in the use ofmassives doses of the antibiotic and is not as efficient as van-comycin for the treatment of severe cases,63–65 b) both metro-nidazole and vancomycin promote the development ofvancomycin-resistant Enterococci,66 c) the risk of relapse,between 15% and 35%.67,68

The fast development of tiacumicin B in CDI treatment isdue to its good in vitro bioactivity and interesting characteris-tics:69–75 a) it is taken orally, b) tiacumicin B stays in thegastrointestinal tract and is only detectable in the blood at verylow concentrations (nanomolar range), c) it shows a rate ofclinical cure almost identical to vancomycin (91.7% to 90.6%)but a lower rate of recurrence of CDI (12.8% compared to25.3%).

These results can be explained by the bactericidal action ofthe tiacumicins, killing C. difficile strains whereas vancomycin,as a bacteriostatic, only inhibits the development of bacteria.76

Furthermore, the narrow spectrum of tiacumicin respects thenon-pathogenic species of guts,77–79 limiting the development ofpathogenic microorganisms like C. difficile.80 On the otherhand, vancomycin is a broad-spectrum antibiotic, facilitatingthe recurrence of CDI.

4.2 Mechanism of action

The synthesis of RNA from DNA, a process known as tran-scription, occurs in RNA polymerase (RNAP), an essentialenzyme present in most organisms.81–84 The bacterial RNAP iscomposed of ve core subunits (2a, b, b0, u) and a s co-factor.The two subunits b and b0 have a pincer structure, forming achannel in which the incoming DNA strand ts. The active siteof the enzyme, complexing a Mg2+ ion is also in this channel.The subunit b0 is a mobile clamp, serving as a lock of the active-site, a docking site for the s subunit and helps to position DNAtemplate into the active site. The s co-factor is a subunit which

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binds to the enzyme core to form a new complex: the holoen-zyme. When formed, the holoenzyme binds to the DNApromoter, leading to the formation of a closed-complex. Initi-ation of transcription requires the formation of an open-complex of RNAP in which promoter DNA are melted to formthe transcription bubble.

The rst studies directed toward the elucidation of lip-iarmycin mechanism of action were conducted soon aer itsdiscovery by Lepetit’s scientists.85 In vivo studies on Bacillussubtilis revealed that at a low concentration, lipiarmycin couldinhibit RNA synthesis and that at a higher concentration, DNAsynthesis itself could be depressed. On Escherichia coli, they alsonoted that the inhibitory action of lipiarmycin was higher whenit was added prior to the association of RNAP and DNA. If lip-iarmycin is added aer polymerisation has started, it doesgradually reduce the extent of RNA synthesis whereas it is totallysuppressed when lipiarmicin is present at the beginning. It hasbeen proposed that lipiarmicin could bind to RNAP and thusinteracts with the early steps of RNA synthesis. Similar resultswere obtained by Talpaert and co-workers on E. coli whoproposed that lipiarmycin could interfere with initiation stepsrather than elongation of RNA.86

In 1977, using B. subtilis mutants resistant to lipiarmycin,Sonenshein and co-workers showed that the core enzyme ofRNAP (2a, b, b0 and u units) is relatively resistant to the anti-biotic but addition of the s co-factor restores sensitivity.87

Mapping data revealed that mutations leading to antibiotic-resistant strains were likely to reside in the gene coding for the bsubunit of the core.

Although it has been established that at least one unit of theRNAP is targeted by lipiarmycin, the mechanism of actionremained unclear. The antibiotic could bind to the core enzymeand thus prevent the binding of the s subunit or it could bind tothe polymerase only aer formation of the holoenzyme. In 1979,the same group reported the inhibition preference of lip-iarmycin for RNAP in the holoenzyme form, an action probablynot due to an interaction with the s subunit, but with prefer-ential interactions with the holoenzyme.88 It was still suggestedthat both the core and s subunit could each provide a part ofthe binding site and proposed that the antibiotic could inhibitthe formation of the rst phosphodiester bond.

In 2006, using DNA sequencing, Leonetti and co-workersshowed that mutation of the gene coding for the b0 subunit ofthe core confers resistance to B. subtilis.89 Other studies bySambandamurthy and co-workers and Leonetti and co-workersalso revealed that spontaneous mutations of genes coding forthe RNA exit channel (b and b0 subunits) could confer resistanceto lipiarmycin in Mycobacterium tuberculosis and Enterococcusfaecalis.46,90

Brodolin and colleagues reported in 2011 a study aimed atestablishing the mechanism of action of this antibiotic onE. coli.91 Using biochemical and genetic approaches, theyproposed that lipiarmycin acts as an inhibitor of transcription,docking to both the s70 3.2 region and the mobile-clampdomain b0 of the core, thus trapping RNAP at one of the closed(and inactive) intermediates of the enzyme. In this closed state,even if the promoter DNA remains bounded to RNAP, the single-

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stranded DNA template cannot t into the active site and thetranscription is inhibited.

Further studies by Ebright and colleagues, while suggestingno interaction between lipiarmycin and the s70 subunit, stillsuggested that tiacumicin could interfere with the RNAP switch-region (b0), the RNA-exit channel (b and b0), or both.92

All of the studies discussed above were performed usinglipiarmycin from Actinoplanes or Catellatospora strains as theantibiotic. The only study on the action of (R)-tiacumicin B(known as daxomicin) was reported by Artsimovitch andcolleagues in 2012.93 They showed that daxomicin can inhibitRNAP from both C. difficile and E. coli aer formation of theholoenzyme. However, the antibiotic was not able to stop RNAsynthesis aer formation of the open-complex of RNAP, sug-gesting the same mechanism of action as for lipairmycin. Inaddition, they showed that both lipiarmycin and daxomicininduce changes in the downstream DNA interactions withRNAP. However, only daxomicin was able to alter the DNAconformation at other regions of the RNAP–DNA complex andthat its action was not dependant on the s unit (contrary to thework of Brodolin and colleagues91 but in agreement with thework of Ebright and colleagues92). The source of theses differ-ences remains to be determined, even if it has been suggestedthat the use of different promoters in these studies might beone. The source of RNAP or the structure of the antibiotics(conguration of C18 assumed to be (S) for lipiarmycin andproved to be (R) for daxomicin) could also play a role.

5 SAR studies

Only a few reports have dealt with the structure–activity rela-tionship of this family of antibiotics. However, the work ofZhang directed toward the elucidation of its biosynthesisgenerated various analogs whose antibacterial activities havebeen evaluated.40,41

One of the rst parts of tiacumicin B which has beenmodied is the aromatic ring.94 The methylation of phenolsafforded a di-O-methyltiacumicin B, which was 8- to 16- fold lessactive than tiacumicin B on various aerobic bacteria, such asS. aureus, S. epidermidis or E. faecium. In addition, the activity onC. perfringens or C. difficile strains is also lower than tiacumicinB (2- to 8- fold) but remains equal or higher than vancomycin.

The isobutyric ester at the C40 0 position on the 5-methyl-b-rhamnose sugar is important for the bioactivity as its hydrolysisled to a knownmetabolite (OP-1118), characterized by a 8- to 16-fold lost in activity on various strains (Table 2 – entry 2).95

Interestingly, the conguration of the C18 hydroxyl group onthe macrocycle has a great inuence on the bioactivity as itsepimerisation to the (S) conguration or its oxidation to theketone leads to a dramatic increase of MIC on various bacteria(C. difficile, S. aureus or E. faecalis, resistant or not – Table 2,entries 3 and 4).54,96,97 However, lipiarmycin A4 with the (S)conguration at C18 and a methyl instead of an ethyl group onaromatic ring displayed almost equal activity to (S)-tiacumicin B(entry 5).

Removal of the C18 hydroxyl group was made possible withZhang’s mutants.40,41 The compound thus obtained showed a



Table 2 The structure–activity relationship of tiacumicin derivatives against various bacteria

Substitution pattern Minimum inhibitory concentration (mg mL�1)

Entry R1 R2 R3 R4C. difficileATCC 43255

E. faecalisATCC 29212

S. aureusATCC 29213

1 Z (R)–OH Me Et 0.5 2–4 82 H (R)–OH Me Et 4 16–64 >643 Z (S)–OH Me Et 1 8 644 Z ]O Me Et 0.5 — —5 Z (S)–OH Me Me 0.5 2 86 Z H Me Et — 4 87 H H Me Et — 32 1288 X H H Et — 4 89 Y H H Et — 2 410 Z H H Et — 1 211 Z Me H Et — 1 112 Z (R)–OH H Et — 4 16

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slight decrease of activity (2- to 4-fold), which still remainedgood (entry 6). Further removal of the isobutyric ester dramat-ically decrease the activity from 4- to 16-fold, results consistentwith OP-1118 (entry 7). It is interesting to note that analogues inwhich the C18 hydroxyl group was replaced by either a hydrogenatom or a methyl group and lacking the methoxy group at C20

showed similar or even increased bioactivities (entries 8–11).However, a compound only demethylated at C20 was character-ized by a slightly reduced activity (entries 1 vs. 12).

Table 3 Structure–activity relationship of tiacumicin derivatives against various ba

Substitution pattern Minimum inhibitory concentratio

Entry R1 R2S. aureus ATCC29213

S. aureusATCC 65

1 Cl Cl 8 0.782 H H 8 —3 Cl Br — 6.24 Br H — 6.2


The presence and the nature of halogens on the aromaticring also impacted the activity (Table 3).35,40 The replacement ofboth chlorines by hydrogens led to a slight increase ofminimum inhibitory concentration against some Gram-positivebacteria (entry 2). The replacement of the C60 0 0 chlorine atomwith bromine (entry 3) or just having bromine at C40 0 0 (entry 4)led to a variable decrease of activity against S. aureus or E. fae-cium whereas a good activity against C. difficile bacteriaremained.

cteria

n (mg mL�1)

38PE. faecalis ATCC29212

E. faeciumATCC 8043

C. difficileATCC 9689

2 1.56 0.064 — —— 6.2 0.06— 12.5 0.12

Nat. Prod. Rep., 2013, 30, 161–174 | 169


Table 4 Structure–activity relationships of tiacumicin derivatives against variousbacteria

Substitution patternMinimum inhibitory concentration(mg mL�1)

Entry R1 R2 R3 R4 R5S. aureusATCC 6538P

S. epidermidis3519

E. faeciumATCC 8043

1 Z H H H Ar 6.2 12.5 6.22 H Z H H Ar 12.5 12.5 6.23 H H Z H Ar 50 25 1004 H H Y H Ar 25 12.5 12.55 H H Z Ar H 25 >25 >25

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The nature and the position of subsituents on the sugar partcan also impact the bioactivities and this is readily seen bycomparing the bioactivities of different tiacumicins. (Table 4).30

Tiacumicin F and C differ from B by the position of the iso-butyric ester on 5-methyl-rhamnose, at C30 0, C20 0 and C40 0,respectively. Tiacumicin C shows a slightly reduced activity (2-to 16-fold) against all bacterias. The trend is more pronouncedfor tiacumicin F (entries 2 and 3). Change of isobutyric ester atC20 0 for propanoic ester at the same position on tiacumicin Eincreases the activity 2- to 4-fold (entry 4) and moving thearomatic ester from C40 to C30 (tiacumicin D, entry 5) reduces theactivity against S. epidermidis 3519.

Zhang’s investigation has shown that major modications,such as removing one sugar, both sugars or the aromatic ringfrom tiacumicin B, whatever the substitution pattern was, led toa drastic reduction of the antimicrobial activity.

Fig. 10 The synthesis of homodichloro-orsellinic acid. (a) (E)-Ethyl pent-2-enoate, Na, EtOH, reflux. (b) HCl aq.,�10 �C to 0 �C. (c) Br2, AcOH, 40 �C. (d) Ni/Al,NaOH aq., 0 �C. (e) SO2Cl2, Et2O, reflux. (f) H2SO4 conc.

170 | Nat. Prod. Rep., 2013, 30, 161–174

It is known that the antimicrobial activity of somecompounds can be inuenced by various parameters, such aspH, the concentration of divalent cations or bacterial density. Ithas been shown that, contrary to vancomycin, the inoculumdensity is not a signicant factor affecting the activity of tiacu-micin B.37,38,98 Similarly, the concentration of divalent cations,such as calcium and magnesium, didn’t affect the activity oftiacumicin B. However, it has been disclosed that the minimuminhibitory concentration values for tiacumicin B increased withthe increase of pH, with values at pH 8.1 being 8- to 16-foldhigher than those obtained at pH 6. To explain these results, ithas been proposed that at basic pH, phenolic hydroxyl groupscould be deprotonated, thus forming charged species, whichare expected to be less able to permeate bacterial cells.

6 Synthesis

To the best of our knowledge no total synthesis of the tiacu-micins has ever been reported in the literature in spite of thegreat interest in these compounds. However, syntheses of thesugar and aromatic parts of tiacumicin have been reported.

6.1 Homodichloro-orsellinic acid

In the early 90’s, in order to validate the proposed structure ofthe aromatic part of lipiarmycin, Scharf and co-workersdescribed the rst synthesis of homodichloro-orsellinic acid(Fig. 10).99 Condensation of ethyl acetoacetate 77 with (E)-ethylpent-2-enoate under basic conditions afforded the cyclo-hexenone 78. Aromatization took place during bromination togive the product 79. Product 81 was accessible through a twostep protocol involving removal of bromine with RANEY�nickel and subsequent chlorination using sulfuryl chloride in85% yield. Due to the easy decarboxylation of b-resorcyclic acidderivatives in basic media, the acid 82 is nally generated byhydrolysis of ethyl ester in concentrated sulfuric acid. Thehomodichloro-orsellinic acid 82 was thus obtained with a good41% overall yield. The spectroscopic data of this compoundwere in good agreement with those published for the aromaticpart of lipiarmcycin A3.

Fig. 11 The synthesis of 2-O-methyl-b-D-rhamnose. (a) a,a-Dimethoxytoluene,PTSA, DMF, 65–75 �C. (b) LiAlH4, AlCl3, Et2O, DCM. (c) MeI, Ag2O, DMF. (d) LiAlH4,AlCl3, Et2O, DCM. (e) TsCl. (f) LiAlH4, benzene, Et2O, reflux. (g) H2, Pd/C, EtOH,AcOH. (h) H2SO4 (1 M), 100 �C.



Fig. 12 5-Methyl-rhamnose and analogs.

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6.2 2-O-Methyl-b-D-rhamnose

In 1982, Liptak and co-workers reported the rst and onlysynthesis of 2-O-methyl-D-rhamnose 83 (Fig. 11).100,101 Themethyl a-D-mannopyranoside 84 was treated with a,a-dime-thoxytoluene in the presence of PTSA to give the bis-acetal 85 asa single diastereoisomer aer recrystallization in a 64% yield.86 was obtained by a selective opening of the dioxolane byhydrogenolysis followed by methylation of the C2 hydroxylgroup. The 1,3-dioxane opening by lithium aluminium hydrideallowed the formation of 87 in a 67% yield. The C6 position wasdeoxygenated by a tosylation/reduction sequence, affording 88which was deprotected to give the desired sugar. The 2-O-methyl-b-D-rhamnose 83 was thus obtained with a 15% overallyield.

Note that Zdorovenko and co-workers have previouslyprepared this sugar by cultivating a strain of Bacillus faecalisalcaligenes.102

6.3 5-Methyl-b-rhamnose

The sugar 89, with the absolute stereochemistry correspondingto tiacumicin B has never been reported in the literature(Fig. 12). However, its enantiomer 90 has been prepared byKlemer and Waldmann, and Walton (drawn as the pyranoseform) during studies directed toward the preparation of noviosederivatives.103,104 Note that the methylated analog on theC3 position showing the right stereochemistry was known asD-(�)-noviose (91).105–108

7 Clinical application

(R)-Tiacumicin B was developed by Optimer PharmaceuticalsInc. under the generic name daxomicin and the trade nameDicid�.

Due to its physicochemical properties (high molecularweight 1058.04 g mol�1, large number of hydrogen bonddonors and acceptors, 7 and 18, respectively), Lipinski’srules predict a poor absorption from the gastrointestinaltract.109 Indeed, both daxomicin and its major metaboliteOP-1118 (lacking the isobutyric ester at C40 0 and which isalso bactericidal110) displayed good pharmacokinetic prolewith minimal systemic absorption (plasma concentrationtypically in the nanomolar range) and high faecal druglevels.71,73

Interactions of daxomicin and OP-1118 with other biolog-ical function and drugs were also assessed. Minimal inhibitionof cytochrome P450 has been observed at a concentration up to10 mg mL�1. Both daxomicin and OP-1118 are substrates forefflux pumps, which could be inhibited by cyclosporine, an


immunosuppressant drug. Indeed, when cyclosporine is co-administrated with Dicid�, plasma concentration of dax-omicin and OP-1118 are increased, but still remain in the ngmL�1 range.

Even if guidelines recommend discontinuation of all anti-biotic therapy in the case of C. difficile infection, patientsfrequently require another antibiotic treatment to manageconcurrent infections. The effect of other antibiotics, such ascarbapenem, cephalosporin, uoroquinoline, penicilin orstreptogramin, on the efficacy of daxomicin has been recentlyevaluated by Mullane and co-workers.111 They have shown thatthe concomitant use of antibiotics were associated with aslightly lower cure rate (90.0% vs. 92.3%) and an extended timeto resolution of diarrheas. In addition, more recurrences wereobserved in patients taking other antibiotics (16.8% vs. 11.9%).But even in the case of concomitant antibiotic treatment of C.difficile infection, daxomicin remained a better choice thanvancomycin.

Fidaxomicin is also characterised by a low frequency ofspontaneous resistance development by C. difficile strains (from<1.4 � 10�9 to 12.8 � 10�9). Most known mutants resistant totiacumicin have been grown in the laboratory for researchpurposes. Nevertheless, a subject with recurrence of CDI hasdeveloped such resistance during clinical trials. However, nocross-resistance of daxomicin with other antibacterial drugshas been noted and a synergistic effect has even been revealedwith the rifamycin class of compounds, ampicillin andmetronidazole.

Note that both daxomicin and its major metabolite OP-1118 show a long post-antibiotic effect (PAE).112 Indeed on twoC. difficile strains, the PAE of daxomicin was about 10 h and 5 hon a clinical isolate (3 h for OP-1180), whereas vancomycinshows a PAE of 0 and 1.5 h, respectively, for strains and clinicalisolate. It has been proposed that this unusually long suppres-sion of bacterial growth by daxomicin may be due to thespecic binding of the drug to RNA polymerase or to the non-specic binding to other bacterial cell components. By thesemeans, daxomicin could remain inside the cell to exert itsantibacterial activity and only a slow dissociation and diffusionof the drug out of the bacteria could allow the microorganism togrow again.

In addition, it has been recently shown that daxomicininhibits sporulation by C. difficile, a mechanism which maycontribute to the better cure rate of this drug compared tovancomycin.113

Taking into account of all these properties and the results ofphase III clinical trials in Canada and the USA (later extended toEurope), which conrm the efficacy and safety of dax-omicin,114–117 the US Food & Drug Administration (FDA)approved its use in the treatment of CDI as a good alternative tovancomycin and metronidazole.118 However, the phase III studydid not include any pediatric patients, and additional studiesare therefore needed to ascertain the efficacy and safety ofdaxomicin in children.119 Note that Optimer Pharmaceuticalsis currently working on new indications of daxomicin, such asvancomycin-resistant enterococcal infection and meticillin-resistant Staphylococcus aureus prophylaxis.120

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8 Summary and outlook

The tiacumicin family encompass more than 40 molecules withthe same macrolactone framework. They are isolated from thebacterial culture of different strains of the Actinomycetes group.A recent study indicated the direct involvement of 17 genes andcorresponding enzymes with an unusual tailoring dihalogenasein its biosynthesis. These compounds and their derivativesdisplayed interesting bioactivity against pathogenic bacteriaand promising anti-cancer activity.

(R)-Tiacumicin B is a RNA synthesis inhibitor which acts bydocking to two domains of the closed RNA polymerase enzyme,preventing its opening and thus activation. For the treatment ofClostridium difficile infection, it has the same cure rate as van-comycin but with a lower relapse rate. It was approved by theFDA in May 2011 as a marketed drug for the treatment of CDI.

It is rather surprising that in spite of its interesting structureand biological activities, no total synthesis of this macrolide hasbeen reported in the literature. Such a synthesis could afford aninteresting alternative to the actual source of supply, allowingthe generation of new derivatives with new or improvedactivities.

9 Acknowledgements

W.E. would like to thank G. Coulthard for critically reviewingthis document and for making valuable suggestions.

10 References

1 D. J. Newman and G. M. Cragg, J. Nat. Prod., 2007, 70, 461–477.

2 M. S. Butler and A. D. Buss, Biochem. Pharmacol., 2006, 71,919–929.

3 F. Pelaez, Biochem. Pharmacol., 2006, 71, 981–990.4 J. Clardy, M. A. Fischbach and C. T. Walsh, Nat. Biotechnol.,2006, 24, 1541–1550.

5 T. M. Embley and E. Stackebrandt, Annu. Rev. Microbiol.,1994, 48, 257–289.

6 W. B. Whitman, D. C. Coleman and W. J. Wiebe, Proc. Natl.Acad. Sci. U. S. A., 1998, 95, 6578–6583.

7 T. R. P. Kekuda, K. S. Shobha and R. Onkarappa, J. Pharm.Res., 2010, 3, 250–256.

8 R. Solanki, M. Khanna and R. Lal, Indian J. Microbiol., 2008,48, 410–431.

9 R. H. Baltz, Microbe, 2007, 2, 125–131.10 R. H. Baltz, Curr. Opin. Pharmacol., 2008, 8, 557–563.11 S. Dharmaraj, World J. Microbiol. Biotechnol., 2010, 26,

2123–2139.12 O. Genilloud, I. Gonzalez, O. Salazar, J. Martın, J. R. Tormo

and F. Vicente, J. Ind. Microbiol. Biotechnol., 2011, 38, 375–389.

13 S. S. Ghantoji, K. Sail, D. R. Lairson, H. L. Dupont andK. W. Garey, J. Hosp. Infect., 2010, 74, 309–318.

14 W. Erb and J. Zhu, L’Act. Chim., 2012, 360–361, 83–89.15 M. Gerber and G. Ackermann, Expert Opin. Invest. Drugs,

2008, 17, 547–553.

172 | Nat. Prod. Rep., 2013, 30, 161–174

16 M. Miller, Expert Opin. Invest. Drugs, 2010, 11, 1569–1578.17 J. S. Hardesty and P. Juang, Pharmacotherapy, 2011, 31, 877–

886.18 J. W. Lancaster and S. J. Matthews, Clin. Ther., 2012, 34, 1–

13.19 F. Parenti, H. Pagani and G. Beretta, J. Antibiot., 1975, 28,

247–252.20 C. Coronelli, R. J. White, G. C. Lancini and F. Parenti, J.

Antibiot., 1975, 28, 253–259.21 C. Coronelli, F. Parenti, R. White and H. Pagani, GB

1458512, 1973, Gruppo Lepetit.22 E. Martinelli, L. Faniuolo, G. Tuan, G. G. Gallo and

B. Cavalleri, J. Antibiot., 1983, 36, 1312–1322.23 K. Schmidt-Base, M. Noltemeyer, E. Egert, E. Eigelt and

A. Zeeck, Acta. Cryst., 1993, C49, 250–253.24 A. Isogai, S. Sakuda, S. Matsumoto, M. Ogura, K. Furihata,

H. Seto and A. Suzuki, Agric. Biol. Chem., 1984, 48, 1379–1381.

25 J. B. McAlpine, J. W. Corcoran and R. S. Egan, J. Antibiot.,1971, 24, 51–56.

26 A. Arnone, G. Nasini and B. Cavalleri, J. Chem. Soc., PerkinTrans. 1, 1987, 1353–1359.

27 B. Cavalleri, A. Arnone, E. Di Modugno, G. Nasini andB. P. Goldstein, J. Antibiot., 1988, 41, 308–315.

28 S. ~Omura, N. Imamura, R. Oiwa, H. Kuga, R. Iwata,R. Masuma and Y. Iwai, J. Antibiot., 1985, 39, 1407–1412.

29 Y. Takahashi, I. Yuzuru and S. ~Omura, J. Antibiot., 1985, 39,1413–1418.

30 R. J. Theriault, J. P. Karwowski, M. Jackson, R. L. Girolami,G. N. Sunga, C. M. Vojtko and L. J. Coen, J. Antibiot., 1987,40, 567–574.

31 J. E. Hochlowski, S. J. Swanson, L. M. Ranfranz,D. N. Whittern, A. M. Buko and J. B. McAlpine, J.Antibiot., 1987, 40, 575–588.

32 J. B. McAlpine, M. Jackson and J. Karwowski, US 4918174,1990, Abbott Laboratories.

33 Y.-K. Shue, F. K. Babakhani, F. W. Okumu, P. S. Sears,S. L. Miller-Shangle and R. B. Walsh, WO 2005112990,2005, Optimer Pharmaceuticals, Inc.

34 Y.-K. Shue, C.-K. Hwang, Y.-H. Chiu, A. Romero,F. Babakhani, P. Sears and F. Okumu, WO 2006085838A1, 2006, Optimer Pharmaceuticals, Inc.

35 J. E. Hochlowski, M. Jackson, R. R. Rasmussen, A. M. Buko,J. J. Clement, D. N. Whittern and J. B. McAlpine, J. Antibiot.,1997, 50, 201–205.

36 J. E. Hochlowski, M. Jackson, J. B. McAlpine andR. R. Rasmussen, US 5767096, 1998, Abbott Laboratories.

37 P. Sears, Y.-K. Shue, S. L. Miller-Shangle, R. B. Walsh,F. Babakhani, Y.-H. Chiu, A. Romero, S. Gorbach andT. J. Louie, WO 2007048059 A2, 2007, OptimerPharmaceuticals, Inc.

38 P. Sears, S. L. Miller-Shangle, R. B. Walsh, Y.-K. Shue,F. Babakhani, T. J. Louie, Y.-H. Chiu, A. Romero andS. Gorbach, US 20070105791, 2007, OptimerPharmaceuticals, Inc.

39 S. Sanghvi, M. Roach, J. F. Zhou, E. M. Mittleberg and P. He,US 20080176927 A1, 2008, Optimer Pharmaceuticals, Inc.



Review NPR

Publ

ishe

d on

30

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2. D

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21:5

6:53


40 Y. Xiao, S. Li, S. Niu, L. Ma, G. Zhang, H. Zhang, G. Zhang,J. Ju and C. Zhang, J. Am. Chem. Soc., 2011, 133, 1092–1105.

41 S. Nui, T. Hu, S. Li, Y. Xiao, L. Ma, G. Zhang, H. Zhang,X. Yang, J. Ju and C. Zhang, ChemBioChem, 2011, 12,1740–1748.

42 C. Zhang, Y. Xiao, S. Li, S. Niu, G. Zhang, H. Zhang, T. Huand J. Ju, Patent CN 2010–10592416, 2011.

43 C. Zhang, S. Li, Y. Xiao, S. Niu and J. Ju, Patent CN 2010–10526416, 2011.

44 C. Zhang, Y. Xiao, L. Sumei, S. Niu, G. Zhang, H. Zhang,T. Hu and J. Ju, CN 102115757, 2011, Faming ZhuanliShenqing.

45 Y.-K. Shue, C.-J. F. Du, M.-H. Chiou, M.-C. Wu, Y.-T. Chen,F. W. Okumu and J. J. Duffield, WO 2004014295 A2, 2004,Optimer Pharmaceuticals, Inc.

46 M. Kurabachew, S. H. J. Lu, P. Krastel, E. K. Schmitt,B. L. Suresh, A. Goh, J. E. Knox, N. L. Ma, J. Jiricek,D. Beer, M. Cynamon, F. Petersen, V. Dartois, T. Keller,T. Dick and V. K. Sambandamurthy, J. Antimicrob.Chemother., 2008, 62, 713–719.

47 T.Weber, K. Welzel, S. Pelzer, A. Vente andW.Wohlleben, J.Biotechnol., 2003, 106, 221–232.

48 C. T. Walsh and M. A. Fischbach, J. Am. Chem. Soc., 2010,132, 2469–2493.

49 C. Olano, C. Mendez and J. A. Salas, Microb. Biotechnol.,2011, 4, 144–164.

50 A. L. Lane and B. S. Moore, Nat. Prod. Rep., 2011, 28, 411–428.

51 M. S. Osburne and A. L. Sonenshein, J. Virol., 1980, 33, 945–953.

52 P. Villain-Guillot, M. Gualtieri, L. Bastide and J.-P. Leoneti,Antimicrob. Agents Chemother., 2007, 51, 3117–3121.

53 D. J. Biedenbach, J. E. Ross, S. D. Putnam and R. N. Jones,Antimicrob. Agents Chemother., 2010, 54, 2273–2275.

54 Y.-K. Shue, C.-K. Hwang, Y.-H. Chiu, A. Romero,F. Babakhani, P. Sears and F. Okumu, WO 2006085838A1, 2006, Optimer Pharmaceuticals, Inc.; M.-C. Wu,C.-C. Huang, Y.-C. Lu and W.-J. Fan, US 20090110718 A1,2009, Echem Hightech Co. Ltd.

55 M. Clemons, S. Danson and A. Howell, Cancer Treat. Rev.,2002, 28, 165–180.

56 J. G. Bartlett, Clin. Infect. Dis., 2008, 46(Suppl 1), S4–S11.57 Clostridium difficile, ed. K. Aktories and T. C. Wilkins,

Springer, 2000.58 M. Kachrimanidou and N. Malisiovas, Crit. Rev. Microbiol.,

2011, 37, 178–187.59 G. P. Carter, J. I Rood and D. Lyras, Trends Microbiol., 2012,

20, 21.60 F. C. Lessa, C. V. Gould and L. C. McDonald, Clin. Infect.

Dis., 2012, 55(Suppl 2), S65–S70.61 E. J. Vollaard and H. A. Clasener, Antimicrob. Agents

Chemother., 1994, 38, 409–414.62 M. S. Rubin, L. E. Bodenstein and K. C. Kent, Dis. Colon

Rectum, 1995, 38, 350–354.63 R. P. Bolton and M. A. Culshaw, Gut, 1986, 27, 1169–1172.64 F. A. Zar, S. R. Bakkanagari, K. M. L. S. T. Moorthi and

M. B. Davis, Clin. Infect. Dis., 2007, 45, 302–307.


65 J. G. Bartlett, Clin. Infect. Dis., 2008, 46, 1489–1492.

66 W. N. Al-Nassir, A. K. Sethi, Y. Li, M. J. Pultz, M. M. Riggsand C. J. Donskey, Antimicrob. Agents Chemother., 2008,52, 2403–2406.

67 F. Barbut, A. Richard, K. Hamadi, V. Chomette,B. Burghoffer and J. -C. Petit, J. Clin. Microbiol., 2000, 38,2386–2388.

68 K. Z. Vardakas, K. A. Polyzos, K. Patouni, P. I. Rafailidis,G. Samonis and M. E. Falagas, Int. J. Antimicrob. Agents,2012, 40, 1.

69 G. Ackermann, B. Loffler, D. Adler and A. C. Rodloff,Antimicrob. Agents Chemother., 2004, 48, 2280–2282.

70 D. W. Hecht, M. A. Galang, S. P. Sambol, J. R. Osmolski,S. Johnson and D. N. Gerding, Antimicrob. AgentsChemother., 2007, 51, 2716–2719.

71 Y. K. Shue, P. S. Sears, S. Shangle, R. B. Walsh, C. Lee,S. L. Gorbach, F. Okumu and R. A. Preston, Antimicrob.Agents Chemother., 2008, 52, 1391–1395.

72 J. Karlowsky, N. M. Laing and G. G. Zhanel, Antimicrob.Agents Chemother., 2008, 52, 4163–4165.

73 T. Louie, M. Miller, C. Donskey, K. Mullane andE. J. C. Goldstein, Antimicrob. Agents Chemother., 2009, 53,223–228.

74 News and analysis in Nature Rev. Drug Discovery, 2010, 9,260.

75 For a systematic review on the comparative effectiveness ofC. difficile treatments, see: D. M. Drekonja, M. Butler,R. MacDonald, D. Bliss, G. A. Filice, T. S. Rector andT. J. Wilt, Ann. Intern. Med., 2011, 155, 839–847.

76 R. N. Swanson, D. J. Hardy, N. L. Shipkowitz, C. W. Hanson,N. C. Ramer, P. B. Fernandes and J. J. Clement, Antimicrob.Agents Chemother., 1991, 35, 1108–1111.

77 K. L. Credito and P. C. Appelbaum, Antimicrob. AgentsChemother., 2004, 48, 4430–4434.

78 S. M. Finegold, D. Molitoris, M.-L. Vaisanen, Y. Song, C. Liuand M. Bolanos, Antimicrob. Agents Chemother., 2004, 48,4898–4902.

79 T. J. Louie, J. Emery, W. Krulicki, B. Byrne and M. Mah,Antimicrob. Agents Chemother., 2009, 53, 261–263.

80 G. W. Tannock, K. Munro, C. Taylor, B. Lawley, W. Young,B. Byrne, J. Emery and T. Louie, Microbiology, 2010, 156,3354–3359.

81 K. S. Murakami, S. Masuda and S. A. Darst, Science, 2002,296, 1280–1284.

82 K. S. Murakami, S. Masuda, E. A. Campbell, O. Muzzin andS. A. Darst, Science, 2002, 296, 1285–1290.

83 D. G. Vassylyev, M. N. Vassylyeva, A. Perederina,T. H. Tahirov and I. Artsimovitch, Nature, 2007, 448, 157–162.

84 P. Villain-Guillot, L. Bastide, M. Gualtieru and J.-P. Leonetti,Drug Discovery Today, 2007, 12, 200–208 and referencescited therein.

85 S. Sergio, G. Pirali, R. White and F. Parenti, J. Antibiot., 1975,28, 543–549.

86 M. Talpaert, F. Campagnari and L. Clerici, Biochem.Biophys. Res. Commun., 1975, 63, 328–334.

Nat. Prod. Rep., 2013, 30, 161–174 | 173


NPR Review

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ishe

d on

30

Oct

ober

201

2. D

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oade

d on

21/

04/2

014

21:5

6:53


87 A. L. Sonenshein, H. B. Alexander, D. M. Rothstein andS. H. Fisher, J. Bacteriol., 1977, 132, 73–79.

88 A. L. Sonenshein and H. B. Alexander, J. Mol. Biol., 1979,127, 55–72.

89 M. Gualtieri, P. Villain-Guillot, J. Latouche, J.-P. Leonettiand L. Bastide, Antimicrob. Agents Chemother., 2006, 50,401–402.

90 M. Gualtieri, A. Tupin, K. Brodolin and J.-P. Leonetti, Int. J.Antimicrob. Agents, 2009, 34, 605–606.

91 A. Tupin, M. Gualtieri, J.-P. Leonetti and K. Brodolin, EMBOJ., 2010, 29, 2527–2537.

92 A. Srivastava, M. Talaue, S. Liu, D. Degen, R. Y. Ebright,E. Sineva, A. Chakraborty, S. Y. Druzhinin, S. Chatterjee,J. Mukhopadhyay, Y. W. Ebright, A. Zozula, J. Shen,S. Sengupta, R. R. Niedfeldt, C. Xin, T. Kaneko, H. Irschik,R. Jansen, S. Donadio, N. Connell and R. H. Ebright, Curr.Opin. Microbiol., 2011, 14, 532–543 and references therein.

93 I. Artsimovitch, J. Seddon and P. Sears, Clin. Infect. Dis.,2012, 55(Suppl 2), S127–S131.

94 J. B. McAlpine and J. E. Hochlowski, WO 9635702 A1, 1996,Abbott Laboratories.

95 Y. Ichikawa, Y.-H. Chiu, Y.-K. Shue and F. K. Babakhani,WO 2009070779 A1, 2009, Optimer Pharmaceuticals, Inc.

96 Y.-K. Shue, C.-K. Hwang, Y.-H. Chiu, A. Romero,F. Babakhani, P. Sears and F. Okumu, US 20080269145A1, 2008, Optimer Pharmaceuticals, Inc.

97 Y.-K. Shue, C.-K. Hwang, Y.-H. Chiu, A. Romero,F. Babakhani, P. Sears and F. Okumu, US 20100008825A1, 2010, Optimer Pharmaceuticals, Inc.

98 F. Babakhani, J. Seddon, N. Robert, Y.-K. Shue and P. Sears,Antimicrob. Agents Chemother., 2010, 54, 2674–2676.

99 M. Alexy and H.-D. Scharf, Liebigs Ann. Chem., 1991, 1363–1364.

100 A. Liptak, Carbohydr. Res., 1982, 107, 300–302.101 A. Liptak, I. Czegeny, J. Harangi and P. Nanasi, Carbohydr.

Res., 1970, 73, 327–331.102 G.M. Zdorovenko andE. Y. Rashba, SU 1977–2498635, 1977.103 A. Klemer and M. Waldmann, Liebigs Ann. Chem., 1986, 2,

221–225.

174 | Nat. Prod. Rep., 2013, 30, 161–174

104 E. Walton, US 2938900, 1960, Merck & Co.105 W. M. Pankau and W. Kreiser, Helv. Chim. Acta, 1998, 81,

1997–2004.106 W. M. Pankau and W. Kreiser, Tetrahedron Lett., 1998, 39,

2089–2090.107 X. M. Yu, G. Shen and B. S. J. Blagg, J. Org. Chem., 2004, 69,

7375–7378.108 D. S. Reddy, G. Srinivas, B. M. Rajesh, M. Kannan,

T. V. Rajale and J. Iqbal, Tetrahedron Lett., 2006, 47, 6373–6375.

109 C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney,Adv. Drug Delivery Rev., 2001, 46, 3–26.

110 F. Babakhani, A. Gomez, N. Robert and P. Sears, J. Med.Microbiol., 2011, 60, 1213–1217.

111 K. M. Mullane, M. A. Miller, K. Weiss, A. Lentnek, Y. Golan,P. S. Sears, Y.-K. Shue, T. J. Louie and S. L. Gorbach, Clin.Infect. Dis., 2011, 53, 440–447 and erratum: Clin. Infect.Dis., 2011, 53, 1312.

112 F. Babakhani, A. Gomez, N. Robert and P. Sears, Antimicrob.Agents Chemother., 2011, 55, 4427–4429.

113 F. Babakhani, L. Bouillaut, A. Gomez, P. Sears, L. Nguyenand A. L. Sonenshein, Clin. Infect. Dis., 2012, 55(Suppl 2),S162–S169.

114 T. J. Louie, M. A. Miller, K. M. Mullane, K. Weiss,A. Lentnek, Y. Golan, S. Gorbach, P. Sears and Y.-K. Shue,N. Engl. J. Med., 2011, 364, 422–431.

115 O. A. Cornely, D. W. Crook, R. Esposito, A. Poirier,M. S. Somero, K. Weiss, P. Sears and S. Gorbach, LancetInfect. Dis., 2012, 1, 281–289.

116 D. W. Crook, A. S. Walker, Y. Kean, K. Weiss, O. A. Cornely,M. A. Miller, R. Esposito, T. J. Louie, N. E. Stoesser,B. C. Young, B. J. Angus, S. L. Gorbach and T. E. A. Peto,Clin. Infect. Dis., 2012, 55(Suppl 2), S93–S103.

117 K. Weiss, R. L. Allgren and S. Sellers, Clin. Infect. Dis., 2012,55(Suppl 2), S110–S115.

118 http://www.fda.gov/NewsEvents/Newsroom/PressAnnoun-cements/ucm257024.htm.

119 T. Daniels and T.-Y. So, Gastroen. Res., 2011, 4, 93–96.120 News in Drugs, 2010, 10, 37–45.



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