From natural product to marketed drug: the tiacumicin

odyssey
William Erb
*
a
and Jieping Zhu
*
b
Covering: 1975 to 2012
The rst 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 dicile 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 Introduction
2 Isolation and characterization
2.1 Lipiarmycin from Actinoplanes deccanensis
2.2 Clostomicins from Micromonospora echinospora
2.3 Tiacumicins from Dactylosporangium aurantiacum
2.4 Lipiarmycin from Catellatospora
3 Biosynthesis
4 Biological activity
4.1 Biological activity
4.2 Mechanism of action
5 SAR studies
6 Synthesis
6.1 Homodichloro-orsellinic acid
6.2 2-O-Methyl-b-D-rhamnose
6.3 5-Methyl-b-rhamnose
7 Clinical application
8 Summary and outlook
9 Acknowledgements
10 References
1 Introduction
Microbial diversity represents an almost innite pool for the
discovery of novel compounds. There are more than 23 000
known microbial secondary metabolites, close to 60% of them
produced by bacteria, and their usefulness in drug development
is well established.
14
Actinomycetes are among the most morphologically diverse
prokaryotes and are widely distributed all around the Earth.
5,6
They arouse the attention of the scientic community due to the
great 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 a
promising source of new antibiotics.
912
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 of
Actinomycetes). A few years later, related compounds were
isolated from parent strains and were named clostomicin and
tiacumicin. This eld remained broadly unexplored until the
late 90s when Optimer Pharmaceuticals began the commercial
development of one tiacumicin for the treatment of Clos-
tridium dicile infection (CDI). C. dicile is an important
nosocomial pathogen frequently diagnosed in infectious
hospital-acquired diarrhoeas whose cost is estimated from
433797 million dollars annually in the USA.
13
The research in
this eld turned out to be highly rewarding since tiacumicin B
has recently been approved by the FDA for the treatment of C.
dicile infection. The aim of this review is to present an
overview of the chemistry and biology of tiacumicin
compounds and their application to the treatment of C. dicile
associated infection.
1418
2 Isolation and characterization
2.1 Lipiarmycin from Actinoplanes deccanensis
In the 1970s, Parenti and co-workers reported the isolation of a
novel antibiotic from a new strain, isolated from a soil sample
collected in India, named Actinoplanes deccanensis ATCC
a
Laboratoire de Chimie Organique, ESPCI, 10 rue Vauquelin, 75231, Paris Cedex 05,
France. E-mail: w.erb@exchem.fr
b
Institut of Chemical Sciences and Engineering,

Ecole Polytechniques Federale de
Lausanne, EPFL-SB-ISIC-LSPN, CH-1015 Lausanne, Switzerland. E-mail: jieping.
zhu@ep.ch
Cite this: Nat. Prod. Rep., 2013, 30,
161
Received 24th July 2012
DOI: 10.1039/c2np20080e
www.rsc.org/npr
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21983.
1921
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 was
named lipiarmycin, from leap year, because the strain was
isolated on February 29th 1972.
Initial analysis of lipiarmycin revealed the presence of two
chlorine atoms, at least one phenolic hydroxyl group, three
carbonyls (one saturated and two conjugated to double bonds),
a probable sugar moiety, and an aromatic nucleus, which was
determined to be homodichloro-orsellinic acid by degradation
studies.
A few years later, scientists from Gruppo Lepetit reported the
isolation of 2-O-methyl-4-O-homodichloroorsellinate-b-rham-
noside upon acid methanolysis of lipiarmycin.
22
This sugar was
also found in other natural antibiotics with either an a- or b-
glycosidic linkage.
2325
They also identied the second sugar in
lipiarmycin as 5-methyl-b-rhamnose. Note that the absolute
congurations of both sugars have not been established and
were shown articially in their L form.
Finally, in 1987, Nasini and co-workers reported that the
lipiarmycin known at that time was a mixture of two products,
lipiarmycin A3 (1) and A4 (2), in a 3 : 1 ratio, separable by
ash chromatography.
26
Chemical degradation and extensive
NMR studies allowed them to elucidate the structure of the
two lipiarmycins (Fig. 2). These molecules feature a 18-
membered macrolactone incorporating four stereogenic
centers (unkown conguration), two conjugated dienes and
one tri-substituted double bond. The macrolactone is glyco-
sylated by 2-O-methyl-b-D-rhamnose esteried in the 4th
position either by homodichloro-orsellinic acid (lipiarmycin
A3) or dichloro-orsellinic acid (lipiarmycin A4). The second
sugar link to the macrolactone is 4-O-isobutyrate-5-methyl-b-
rhamnose, whose absolute conguration was not determined
but shown as D form.
One year later, two novel lipiarmycins were isolated from the
same strain:
27
lipiarmycins B3 (3) and B4 (4), which dier from
the corresponding A3 and A4 by the position of the isobutyric
ester on the 2-O-methyl-b-D-rhamnose moiety (position 2
0 0
for
lipiarmycin B and 4
0 0
for A). Lipiarmycins B3 and B4 dier
through the substituent (methyl or ethyl) of the aromatic ring.
Fig. 1 Sporangium obtained on soil extract-agar. Magnication 800. Fig. 2 The structure of lipiarmycins.
William Erb studied chemistry
at the University of Paris-Sud XI.
He received his PhD in organic
chemistry under the guidance of
Pr. Jieping Zhu (Institut de Chi-
mie des Substances Naturelles)
in 2010. He then joined the
group of Pr. Varinder Aggarwal
at the University of Bristol,
working on organocatalysis and
its application to the synthesis of
natural products. He is currently
Attache Temporaire dEnseigne-
ment et de Recherche in the group of Janine Cossy in Paris, working
on total synthesis and metal-catalyzed reactions.
Jieping Zhu received his B. Sc
from Hangzhou Normal Univer-
sity and his M.Sc. degree from
Lanzhou University (P. R. China)
under the guidance of Professor
Li Yulin. He got his Ph.D. degree
from University Paris XI, France
under the supervision of
Professor H.-P. Husson and Pr. J.
C. Quirion. Aer 18 months
post-doctoral stay with Professor
Sir 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 of
Research 2nd class in 2000 and then 1st class in 2006. He moved to
Ecole Polytechnique Federale de Lausanne (Swiss Federal Institute
of Technology Lausanne), Switzerland in September 2010 as a full
professor.
162 | Nat. Prod. Rep., 2013, 30, 161174 This journal is 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 rice
eld in Japan.
28,29
Based on taxonomic studies, it appeared that
it 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 and
that the dierence between clostomicins A and B2 resided in
the substitution pattern of the 5-methyl-b-rhamnose sugar: the
isobutyric ester substituent was proposed to be in the C
3
0 0
position for clostomicin A and in the C
2
0 0
for clostomicin B2.
Therefore it was concluded that the structure of clostomicin B2
is identical to lipiarmycin B3.
The IR spectrum of clostomicins C and D showed the pres-
ence of an additional carbonyl group compared to lipiarmycin
A3, which was attributed to a ketone at the C
18
position. The
13
C
NMR spectra of clostomicin C also reveals the absence of one
methyl carbon at 28.6 ppm assigned to the equatorial methyl
group 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 newstrain isolated
from a soil sample collected in Connecticut, which was named
Dactylosporangium aurantiacum subsp. hamdenensis subsp. nov.
AB718C-41 (NRRL 18085).
3032
A rst fermentation experiment
using 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 (4500
litres) led to the isolation of three additional tiacumicins (D
(12), E (13) and F (14)) in very low yields (7 mg, 20 mg and 13 mg,
respectively) compared to tiacumicin B (3.82 g).
Extensive NMR studies allowed scientists to propose the
structure of tiacumicin B, which was found to be identical to
lipiarmycin A3 and clostomicin B1. Tiacumicins C and F are
dierent from tiacumicin B in the position of the isobutyric
ester on the 5-methyl-b-rhamnose moiety (respectively at C
2
0 0
and C
3
0 0 ). Tiacumicin D is another isomer of tiacumicin B in
which the 2-O-methyl-b-D-rhamnose is esteried in the C
3
0
position by homodichloro-orsellinic acid (Fig. 4). Tiacumicin E
is almost identical to tiacumicin C except for the ester moiety on
position C
2
0 0 , which is not an isobutyric ester but a propionate
one. 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 with
the right conguration (2R, 3S, 4S, 5S, 6R) in the text, the 2-O-
methyl-b-D-rhamnose was written as its enantiomer in the nal
structure of tiacumicins.
31
The absolute conguration of the macrolactone was
assigned in 2005 by X-ray crystal structure analysis of tiacumicin
B. It was subsequently established that C
18
has the (R) cong-
uration for tiacumicin B and (S) conguration for lipiarmycin
A4.
33,34
Even though we cannot conclude denitively, it could be
assumed the conguration of C
18
to be (R) for other tiacumicins
and (S) for lipiarmycins. The C
18
conguration remained
unassigned for clostomicins.
Therefore, without taking into account the C
18
congura-
tion, lipiarmycin A3, clostomicin B1 and tiacumicin B seem to
be identical. So are lipiarmycin B3, clostomicin B2 and tiacu-
micin C or clostomicin A and tiacumicin F.
In the late 90s, with the aim of producing novel tiacumicin
analogs, McAlpine and co-workers replaced the potassium
chloride added to the broth with potassium bromide.
35,36
Using
the same Dactylosporangium aurantiacum strain as before, they
isolated four new compounds (Fig. 5, 1518) incorporating
bromide on the aromatic ring, whose structure have been
elucidated 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 fermentation
broth, as judged by the HPLC prole of the mixture and the
corresponding integrations (Fig. 6).
37,38
During formulation
studies, Optimer scientists also identied the new tiacumicin
derivative 26. It is identical to tiacumicn B except for the pres-
ence of carbonyl function at C
7
.
39
Although the conguration of
these new compounds has not been elucidated, they are
assumed to be the same as for tiacumicin B.
The most important library of tiacumicin analogs was
generated by Zhang and co-workers while working on the
elucidation of the biosynthesis of tiacumicin B.
4044
Indeed,
from dierent mutants of the Dactylosporangium aurantiacum
strain, they have been able to characterize 37 new analogs
(Fig. 7). We can note, based on published data, that 50 is the C
18
epimer of lipiarmycin A4 (2).
It is interesting to note that the yield of tiacumicin B
production by D. aurantiacum has been greatly improved over
the years by Optimer scientists.
45
Using a growth medium
mainly composed of sh powder, glucose, casamino acid, yeast
extract and some inorganic salts to support microorganism
growth, it is possible to obtain 100500 mg of crude tiacumicin
per litre of broth, much higher than the 18.8 mg L
1
initially
reported by McAlpine. The introduction of a resin able to trap Fig. 3 The structure of clostomicins.
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macrocycles as they are formed in the broth increased the yield
of tiacumicin and facilitated the recovery of product by sieving
from the broth and elution with organic solvents. Purication is
mainly achieved by reversed-phase medium-pressure liquid
chromatography.
2.4 Lipiarmycin from Catellatospora
In 2008, during the course of a screening programme to identify
new anti-tuberculosis agents, scientists from Novartis reported
the isolation of lipiarmycin A3 from Catellatospora sp. Bp3323-
81, a strain from the companys screening library.
46
3 Biosynthesis
Despite more than 30 years of history, little was known about
the biosynthesis of tiacumicin until the work of Zhang and co-
workers in 2011.
40,41
In early studies, Parenti noted that the
source of chlorine could be the meat extract used in the broth or
the added sodium chloride and that omission of both chlorine
sources greatly reduce the yield of lipiarmycin.
19
Furthermore,
McAlpine has shown that addition of potassium bromide to the
broth allows the formation of brominated tiacumicin
analogs.
35,36
Apart fromfeeding experiments, the biosynthesis of
tiacumicin remained undetermined.
In 2011, Zhang and co-workers reported their studies on
the biosynthesis of tiacumicin based on a genetic approach
on Dactylosporangium aurantiacum hamdenensis NRRL
18085.
4750
Firstly, they targeted polyketide synthase (PKS) and
halogenase using probes to identify genes involved in the
biosynthesis of tiacumicin. They have been able to identify
the complete tia-gene cluster which comprised 50 orfs (Open
Reading Frame) and 110 633 bp (base pairing). In a rst
attempt, the putative functions of orfs have been deduced by
comparison with protein databases and a further renement
Fig. 4 The structure of tiacumicins.
Fig. 5 The structure of brominated tiacumicins.
Fig. 6 The structure of tiacumicin analogs.
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allowed them to eliminate some orfs, which are not involved
in tiacumicin biosynthesis. Thus the nal gene cluster con-
tained 31 orfs, from gene TiaG1 to TiaR2 for approximately
83 kb.
Genes TiaA1 to TiaA4 were predicted as modular polyketide
synthases, and inactivation experiments led to the conclusion
that 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 some
similarity to 6-methyl-salicylic acid synthase and thus it is
probably involved in the biosynthesis of the homo-orsellinic
acid part 69 from a propionyl-CoA starting unit (Fig. 8, B). The
aromatic 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 secondary
metabolites. Finally, the gene TiaM showed some similarity to
other halogenases and its selective inactivation led to the
formation of tiacumicin analogs lacking chlorine atoms,
therefore allowing the assignment of its function. It has also
been 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 for

TiaM.
The genes TiaS1, S3 and S4 are probably involved in the
biosynthesis 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 to
propose the role of TiaS2, TiaS5, TiaS6, TiaG1 and G2, which
were further veried by selective inactivation. TiaG1 and
TiaG2 are 5-C-methyl-D-rhamnosyl-transferase and 2-O-
methyl-D-rhamnosyl-transferase, respectively, used to attach
sugar moieties to the aglycone. TiaS2 and TiaS6 are a sugar
C-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 rhamnose
derivatives but a great regioselectivity, being unable to
methylate the C
2
position of 5-C-methylrhamnose and other
sugars.
The TiaS5 gene is the 2
0
-O-methyltransferase involved in the
synthesis of the 2-O-methyl-b-D-rhamnose. Further experiments
Fig. 7 The structure of tiacumicin analogs.
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revealed that this methyltransferase was divalent metal-cation
dependant: its activity is higher with Mg
2+
and Mn
2+
, moderate
with Co
2+
, Ni
2+
, Fe
2+
, weak with Zn
2+
, Cu
2+
and inactive in the
absence of any metal cation and in the presence of EDTA or
Ca
2+
. It is also a pH-dependant enzyme displaying its best
activity at pH 8. The two genes TiaP1 and TiaP2 encode for
cytochrome P450 hydroxylase, responsible for natural product
oxygenation. Inactivation experiments allowed determination
of their roles as follows: TiaP1 catalyzes the hydroxylation at C
18
and TiaP2 at C
20
.
Some others genes are involved in the precursor supply: TiaC
and TiaD may be involved in isobutyryl-CoA, propionyl-CoA and
acyl-CoA generation. TiaL probably catalyses the formation of
(2S)-methylmalonyl-CoA from propionyl-CoA. TiaJ, TiaN and
TiaK are probably involved in the ethylmalonyl-CoA pathway
and TiaE, showing some similarity with thioesterases, may
promote the accuracy and eciency of the polyketide synthase.
TiaR1 could be a transcription activator in bacteria, TiaR2
seems to be a negative regulator of tiacumicin biosynthesis.
Genes TiaT1T4 may constitute a system to transport the
synthesized metabolites out of the cell whereas the role of TiaI
is actually unknown.
In spite of this great achievement, some points remained
unclear about tiacumicin biosynthesis. The right timing
between genes TiaP1 and TiaS5 (hydroxylation of the C
18
posi-
tion and methylation of C
2
0 hydroxyl, respectively) is still
unclear even though it seemed that TiaP1 should be the last
step, directly preceded by TiaS5. Furthermore, the acylation of
the C
4
0 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 genes
involved in the tiacumicin biosynthesis.
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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4 Biological activity
4.1 Biological activity
The initial report of the discovery of lipiarmycin mentioned, for
the mixture of lipiarmycin A3 and A4, a fairly good activity
against some Staphylococcus aureus strains and other gram-
positive bacteria. A good activity against strains of cariogenic
Steptococcus mutans, suggesting possible application as an
antiplaque 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 on
tiacumicins, researchers from Abbott Laboratories reported a
good activity of tiacumicin B against dierent aerobic bacteria
(including S. aureus and Enterococcus faecium) but a lower
activity against anaerobic bacteria.
30
Tiacumicin B is also active
against Staphylococcus epidermidis biolms formed at the
surface of medical devices (a major cause of nosocomial infec-
tions)
52
and on some drug-resistant strains of Mycobacterium
tuberculosis.
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 (0.516 mg mL
1
).
53
In 2006 Optimer mentioned that the tiacumicins may nd
application in the treatment of gastrointestinal cancers, but
without reporting any more detailed information.
34
The rst
and only study for such an application of tiacumicin came from
Echem Hightech Co.
54
A series of tiacumicin benzylidene acetal
derivatives have shown interesting activity against breast cancer
cells with similar IC
50
values to Tamoxifen, a drug usually
used for this type of cancer (Table 1).
55
Note that the molecules
drawn in this patent are tiacumicin C derivatives (with isopropyl
ester on C
2
0 0 ), although they are claimed to be tiacumicin B
derivatives in the text (with isopropyl ester on C
4
0 0 ).
Notwithstanding these biological activities, it is in the ght
against Clostridium dicile associated infection (CDI) that tia-
cumicin B was developed as a drug.
Clostridium dicile, a Gram-positive anaerobic bacteria, is
the causative agent of between 20% and 25% of all cases of
antibiotic-dependant diarrheas.
5660
Indeed, the gastrointestinal
tract microbiota protects the host against most infections by
pathogenic 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 usually
restricted by microbial competition. C. dicile is recognized as
such an opportunistic pathogen. The most encountered clinical
manifestations of CDI are diarrhea (mild to moderate), pseu-
domembranous colitis, fever, and abdominal pain. Approxi-
mately 3% of patients will develop a fulminant colitis, with
serious complications, such as colonic perforation, toxic meg-
acolon and death.
62
The treatment of these infections involves the use of
metronidazole and/or vancomycin depending on the clinical
presentation of the disease. However, these two broad-spectrum
antibiotics have some drawbacks: a) metronidazole is easily
absorbed along the gastrointestinal tract, resulting in the use of
massives doses of the antibiotic and is not as ecient as van-
comycin for the treatment of severe cases,
6365
b) both metro-
nidazole and vancomycin promote the development of
vancomycin-resistant Enterococci,
66
c) the risk of relapse,
between 15% and 35%.
67,68
The fast development of tiacumicin B in CDI treatment is
due to its good in vitro bioactivity and interesting characteris-
tics:
6975
a) it is taken orally, b) tiacumicin B stays in the
gastrointestinal tract and is only detectable in the blood at very
low concentrations (nanomolar range), c) it shows a rate of
clinical cure almost identical to vancomycin (91.7% to 90.6%)
but a lower rate of recurrence of CDI (12.8% compared to
25.3%).
These results can be explained by the bactericidal action of
the tiacumicins, killing C. dicile strains whereas vancomycin,
as a bacteriostatic, only inhibits the development of bacteria.
76
Furthermore, the narrow spectrum of tiacumicin respects the
non-pathogenic species of guts,
7779
limiting the development of
pathogenic microorganisms like C. dicile.
80
On the other
hand, vancomycin is a broad-spectrum antibiotic, facilitating
the 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 essential
enzyme present in most organisms.
8184
The bacterial RNAP is
composed of ve core subunits (2a, b, b
0
, u) and a s co-factor.
The two subunits b and b
0
have a pincer structure, forming a
channel in which the incoming DNA strand ts. The active site
of the enzyme, complexing a Mg
2+
ion is also in this channel.
The subunit b
0
is a mobile clamp, serving as a lock of the active-
site, a docking site for the s subunit and helps to position DNA
template into the active site. The s co-factor is a subunit which
Table 1 Biological activity of Tiacumicin B derivatives against various cancer cells
(mg mL
1
)
Compounds
IC
50
for
MCF7 breast cancer cells
IC
50
for
T-47D breast cancer cells
Tamoxifen 6.35 0.45 5.50 0.21
Tiacumicin B (10) 8.39 1.00 5.56 0.57
74 7.06 0.83 3.99 0.58
75 6.90 0.36 4.24 0.05
76 5.78 0.81 4.04 1.09
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binds to the enzyme core to form a new complex: the holoen-
zyme. When formed, the holoenzyme binds to the DNA
promoter, 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 form
the transcription bubble.
The rst studies directed toward the elucidation of lip-
iarmycin mechanism of action were conducted soon aer its
discovery by Lepetits scientists.
85
In vivo studies on Bacillus
subtilis revealed that at a low concentration, lipiarmycin could
inhibit RNA synthesis and that at a higher concentration, DNA
synthesis itself could be depressed. On Escherichia coli, they also
noted that the inhibitory action of lipiarmycin was higher when
it was added prior to the association of RNAP and DNA. If lip-
iarmycin is added aer polymerisation has started, it does
gradually reduce the extent of RNA synthesis whereas it is totally
suppressed when lipiarmicin is present at the beginning. It has
been proposed that lipiarmicin could bind to RNAP and thus
interacts with the early steps of RNA synthesis. Similar results
were obtained by Talpaert and co-workers on E. coli who
proposed that lipiarmycin could interfere with initiation steps
rather than elongation of RNA.
86
In 1977, using B. subtilis mutants resistant to lipiarmycin,
Sonenshein and co-workers showed that the core enzyme of
RNAP (2a, b, b
0
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 b
subunit of the core.
Although it has been established that at least one unit of the
RNAP is targeted by lipiarmycin, the mechanism of action
remained unclear. The antibiotic could bind to the core enzyme
and thus prevent the binding of the s subunit or it could bind to
the 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 probably
not due to an interaction with the s subunit, but with prefer-
ential interactions with the holoenzyme.
88
It was still suggested
that both the core and s subunit could each provide a part of
the binding site and proposed that the antibiotic could inhibit
the formation of the rst phosphodiester bond.
In 2006, using DNA sequencing, Leonetti and co-workers
showed that mutation of the gene coding for the b
0
subunit of
the core confers resistance to B. subtilis.
89
Other studies by
Sambandamurthy and co-workers and Leonetti and co-workers
also revealed that spontaneous mutations of genes coding for
the RNA exit channel (b and b
0
subunits) could confer resistance
to lipiarmycin in Mycobacterium tuberculosis and Enterococcus
faecalis.
46,90
Brodolin and colleagues reported in 2011 a study aimed at
establishing the mechanism of action of this antibiotic on
E. coli.
91
Using biochemical and genetic approaches, they
proposed that lipiarmycin acts as an inhibitor of transcription,
docking to both the s
70
3.2 region and the mobile-clamp
domain b
0
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-
stranded DNA template cannot t into the active site and the
transcription is inhibited.
Further studies by Ebright and colleagues, while suggesting
no interaction between lipiarmycin and the s
70
subunit, still
suggested that tiacumicin could interfere with the RNAP switch-
region (b
0
), the RNA-exit channel (b and b
0
), or both.
92
All of the studies discussed above were performed using
lipiarmycin from Actinoplanes or Catellatospora strains as the
antibiotic. The only study on the action of (R)-tiacumicin B
(known as daxomicin) was reported by Artsimovitch and
colleagues in 2012.
93
They showed that daxomicin can inhibit
RNAP from both C. dicile and E. coli aer formation of the
holoenzyme. However, the antibiotic was not able to stop RNA
synthesis aer formation of the open-complex of RNAP, sug-
gesting the same mechanism of action as for lipairmycin. In
addition, they showed that both lipiarmycin and daxomicin
induce changes in the downstream DNA interactions with
RNAP. However, only daxomicin was able to alter the DNA
conformation at other regions of the RNAPDNA complex and
that its action was not dependant on the s unit (contrary to the
work of Brodolin and colleagues
91
but in agreement with the
work of Ebright and colleagues
92
). The source of theses dier-
ences remains to be determined, even if it has been suggested
that the use of dierent promoters in these studies might be
one. The source of RNAP or the structure of the antibiotics
(conguration of C
18
assumed to be (S) for lipiarmycin and
proved to be (R) for daxomicin) could also play a role.
5 SAR studies
Only a few reports have dealt with the structureactivity rela-
tionship of this family of antibiotics. However, the work of
Zhang directed toward the elucidation of its biosynthesis
generated various analogs whose antibacterial activities have
been evaluated.
40,41
One of the rst parts of tiacumicin B which has been
modied is the aromatic ring.
94
The methylation of phenols
aorded a di-O-methyltiacumicin B, which was 8- to 16- fold less
active than tiacumicin B on various aerobic bacteria, such as
S. aureus, S. epidermidis or E. faecium. In addition, the activity on
C. perfringens or C. dicile strains is also lower than tiacumicin
B (2- to 8- fold) but remains equal or higher than vancomycin.
The isobutyric ester at the C
4
0 0 position on the 5-methyl-b-
rhamnose sugar is important for the bioactivity as its hydrolysis
led to a known metabolite (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 C
18
hydroxyl group on
the macrocycle has a great inuence on the bioactivity as its
epimerisation to the (S) conguration or its oxidation to the
ketone leads to a dramatic increase of MIC on various bacteria
(C. dicile, 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 C
18
and a methyl instead of an ethyl group on
aromatic ring displayed almost equal activity to (S)-tiacumicin B
(entry 5).
Removal of the C
18
hydroxyl group was made possible with
Zhangs mutants.
40,41
The compound thus obtained showed a
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slight decrease of activity (2- to 4-fold), which still remained
good (entry 6). Further removal of the isobutyric ester dramat-
ically decrease the activity from 4- to 16-fold, results consistent
with OP-1118 (entry 7). It is interesting to note that analogues in
which the C
18
hydroxyl group was replaced by either a hydrogen
atom or a methyl group and lacking the methoxy group at C
2
0
showed similar or even increased bioactivities (entries 811).
However, a compound only demethylated at C
2
0 was character-
ized by a slightly reduced activity (entries 1 vs. 12).
The presence and the nature of halogens on the aromatic
ring also impacted the activity (Table 3).
35,40
The replacement of
both chlorines by hydrogens led to a slight increase of
minimum inhibitory concentration against some Gram-positive
bacteria (entry 2). The replacement of the C
6
0 0 0 chlorine atom
with bromine (entry 3) or just having bromine at C
4
0 0 0 (entry 4)
led to a variable decrease of activity against S. aureus or E. fae-
cium whereas a good activity against C. dicile bacteria
remained.
Table 2 The structureactivity relationship of tiacumicin derivatives against various bacteria
Substitution pattern Minimum inhibitory concentration (mg mL
1
)
Entry R
1
R
2
R
3
R
4
C. dicile
ATCC 43255
E. faecalis
ATCC 29212
S. aureus
ATCC 29213
1 Z (R)OH Me Et 0.5 24 8
2 H (R)OH Me Et 4 1664 >64
3 Z (S)OH Me Et 1 8 64
4 Z ]O Me Et 0.5
5 Z (S)OH Me Me 0.5 2 8
6 Z H Me Et 4 8
7 H H Me Et 32 128
8 X H H Et 4 8
9 Y H H Et 2 4
10 Z H H Et 1 2
11 Z Me H Et 1 1
12 Z (R)OH H Et 4 16
Table 3 Structureactivity relationship of tiacumicin derivatives against various bacteria
Substitution pattern Minimum inhibitory concentration (mg mL
1
)
Entry R
1
R
2
S. aureus ATCC
29213
S. aureus
ATCC 6538P
E. faecalis ATCC
29212
E. faecium
ATCC 8043
C. dicile
ATCC 9689
1 Cl Cl 8 0.78 2 1.56 0.06
2 H H 8 4
3 Cl Br 6.2 6.2 0.06
4 Br H 6.2 12.5 0.12
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The nature and the position of subsituents on the sugar part
can also impact the bioactivities and this is readily seen by
comparing the bioactivities of dierent tiacumicins. (Table 4).
30
Tiacumicin F and C dier from B by the position of the iso-
butyric ester on 5-methyl-rhamnose, at C
3
0 0 , C
2
0 0 and C
4
0 0 ,
respectively. Tiacumicin C shows a slightly reduced activity (2-
to 16-fold) against all bacterias. The trend is more pronounced
for tiacumicin F (entries 2 and 3). Change of isobutyric ester at
C
2
0 0 for propanoic ester at the same position on tiacumicin E
increases the activity 2- to 4-fold (entry 4) and moving the
aromatic ester from C
4
0 to C
3
0 (tiacumicin D, entry 5) reduces the
activity against S. epidermidis 3519.
Zhangs investigation has shown that major modications,
such as removing one sugar, both sugars or the aromatic ring
from tiacumicin B, whatever the substitution pattern was, led to
a drastic reduction of the antimicrobial activity.
It is known that the antimicrobial activity of some
compounds can be inuenced by various parameters, such as
pH, the concentration of divalent cations or bacterial density. It
has been shown that, contrary to vancomycin, the inoculum
density is not a signicant factor aecting the activity of tiacu-
micin B.
37,38,98
Similarly, the concentration of divalent cations,
such as calcium and magnesium, didnt aect the activity of
tiacumicin B. However, it has been disclosed that the minimum
inhibitory concentration values for tiacumicin B increased with
the increase of pH, with values at pH 8.1 being 8- to 16-fold
higher than those obtained at pH 6. To explain these results, it
has been proposed that at basic pH, phenolic hydroxyl groups
could be deprotonated, thus forming charged species, which
are 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 the
great interest in these compounds. However, syntheses of the
sugar and aromatic parts of tiacumicin have been reported.
6.1 Homodichloro-orsellinic acid
In the early 90s, in order to validate the proposed structure of
the aromatic part of lipiarmycin, Scharf and co-workers
described the rst synthesis of homodichloro-orsellinic acid
(Fig. 10).
99
Condensation of ethyl acetoacetate 77 with (E)-ethyl
pent-2-enoate under basic conditions aorded the cyclo-
hexenone 78. Aromatization took place during bromination to
give the product 79. Product 81 was accessible through a two
step protocol involving removal of bromine with RANEY
nickel and subsequent chlorination using sulfuryl chloride in
85% yield. Due to the easy decarboxylation of b-resorcyclic acid
derivatives in basic media, the acid 82 is nally generated by
hydrolysis of ethyl ester in concentrated sulfuric acid. The
homodichloro-orsellinic acid 82 was thus obtained with a good
41% overall yield. The spectroscopic data of this compound
were in good agreement with those published for the aromatic
part of lipiarmcycin A3.
Table 4 Structureactivity relationships of tiacumicin derivatives against various
bacteria
Substitution pattern
Minimum inhibitory concentration
(mg mL
1
)
Entry R
1
R
2
R
3
R
4
R
5
S. aureus
ATCC 6538P
S. epidermidis
3519
E. faecium
ATCC 8043
1 Z H H H Ar 6.2 12.5 6.2
2 H Z H H Ar 12.5 12.5 6.2
3 H H Z H Ar 50 25 100
4 H H Y H Ar 25 12.5 12.5
5 H H Z Ar H 25 >25 >25
Fig. 10 The synthesis of homodichloro-orsellinic acid. (a) (E)-Ethyl pent-2-
enoate, Na, EtOH, reux. (b) HCl aq., 10

C to 0

C. (c) Br
2
, AcOH, 40

C. (d) Ni/Al,
NaOH aq., 0

C. (e) SO
2
Cl
2
, Et
2
O, reux. (f) H
2
SO
4
conc.
Fig. 11 The synthesis of 2-O-methyl-b-D-rhamnose. (a) a,a-Dimethoxytoluene,
PTSA, DMF, 6575

C. (b) LiAlH
4
, AlCl
3
, Et
2
O, DCM. (c) MeI, Ag
2
O, DMF. (d) LiAlH
4
,
AlCl
3
, Et
2
O, DCM. (e) TsCl. (f) LiAlH
4
, benzene, Et
2
O, reux. (g) H
2
, Pd/C, EtOH,
AcOH. (h) H
2
SO
4
(1 M), 100

C.
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6.2 2-O-Methyl-b-D-rhamnose
In 1982, Liptak and co-workers reported the rst and only
synthesis of 2-O-methyl-D-rhamnose 83 (Fig. 11).
100,101
The
methyl a-D-mannopyranoside 84 was treated with a,a-dime-
thoxytoluene in the presence of PTSA to give the bis-acetal 85 as
a single diastereoisomer aer recrystallization in a 64% yield.
86 was obtained by a selective opening of the dioxolane by
hydrogenolysis followed by methylation of the C
2
hydroxyl
group. The 1,3-dioxane opening by lithium aluminium hydride
allowed the formation of 87 in a 67% yield. The C
6
position was
deoxygenated by a tosylation/reduction sequence, aording 88
which was deprotected to give the desired sugar. The 2-O-
methyl-b-D-rhamnose 83 was thus obtained with a 15% overall
yield.
Note that Zdorovenko and co-workers have previously
prepared this sugar by cultivating a strain of Bacillus faecalis
alcaligenes.
102
6.3 5-Methyl-b-rhamnose
The sugar 89, with the absolute stereochemistry corresponding
to tiacumicin B has never been reported in the literature
(Fig. 12). However, its enantiomer 90 has been prepared by
Klemer and Waldmann, and Walton (drawn as the pyranose
form) during studies directed toward the preparation of noviose
derivatives.
103,104
Note that the methylated analog on the
C
3
position showing the right stereochemistry was known as
D-()-noviose (91).
105108
7 Clinical application
(R)-Tiacumicin B was developed by Optimer Pharmaceuticals
Inc. under the generic name daxomicin and the trade name
Dicid.
Due to its physicochemical properties (high molecular
weight 1058.04 g mol
1
, large number of hydrogen bond
donors and acceptors, 7 and 18, respectively), Lipinskis
rules predict a poor absorption from the gastrointestinal
tract.
109
Indeed, both daxomicin and its major metabolite
OP-1118 (lacking the isobutyric ester at C
4
0 0 and which is
also bactericidal
110
) displayed good pharmacokinetic prole
with minimal systemic absorption (plasma concentration
typically in the nanomolar range) and high faecal drug
levels.
71,73
Interactions of daxomicin and OP-1118 with other biolog-
ical function and drugs were also assessed. Minimal inhibition
of cytochrome P450 has been observed at a concentration up to
10 mg mL
1
. Both daxomicin and OP-1118 are substrates for
eux 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 ng
mL
1
range.
Even if guidelines recommend discontinuation of all anti-
biotic therapy in the case of C. dicile infection, patients
frequently require another antibiotic treatment to manage
concurrent infections. The eect of other antibiotics, such as
carbapenem, cephalosporin, uoroquinoline, penicilin or
streptogramin, on the ecacy of daxomicin has been recently
evaluated by Mullane and co-workers.
111
They have shown that
the concomitant use of antibiotics were associated with a
slightly lower cure rate (90.0% vs. 92.3%) and an extended time
to resolution of diarrheas. In addition, more recurrences were
observed in patients taking other antibiotics (16.8% vs. 11.9%).
But even in the case of concomitant antibiotic treatment of C.
dicile infection, daxomicin remained a better choice than
vancomycin.
Fidaxomicin is also characterised by a low frequency of
spontaneous resistance development by C. dicile strains (from
<1.4 10
9
to 12.8 10
9
). Most known mutants resistant to
tiacumicin have been grown in the laboratory for research
purposes. Nevertheless, a subject with recurrence of CDI has
developed such resistance during clinical trials. However, no
cross-resistance of daxomicin with other antibacterial drugs
has been noted and a synergistic eect has even been revealed
with the rifamycin class of compounds, ampicillin and
metronidazole.
Note that both daxomicin and its major metabolite OP-
1118 show a long post-antibiotic eect (PAE).
112
Indeed on two
C. dicile strains, the PAE of daxomicin was about 10 h and 5 h
on a clinical isolate (3 h for OP-1180), whereas vancomycin
shows a PAE of 0 and 1.5 h, respectively, for strains and clinical
isolate. It has been proposed that this unusually long suppres-
sion of bacterial growth by daxomicin may be due to the
specic binding of the drug to RNA polymerase or to the non-
specic binding to other bacterial cell components. By these
means, daxomicin could remain inside the cell to exert its
antibacterial activity and only a slow dissociation and diusion
of the drug out of the bacteria could allow the microorganism to
grow again.
In addition, it has been recently shown that daxomicin
inhibits sporulation by C. dicile, a mechanism which may
contribute to the better cure rate of this drug compared to
vancomycin.
113
Taking into account of all these properties and the results of
phase III clinical trials in Canada and the USA (later extended to
Europe), which conrm the ecacy and safety of dax-
omicin,
114117
the US Food & Drug Administration (FDA)
approved its use in the treatment of CDI as a good alternative to
vancomycin and metronidazole.
118
However, the phase III study
did not include any pediatric patients, and additional studies
are therefore needed to ascertain the ecacy and safety of
daxomicin in children.
119
Note that Optimer Pharmaceuticals
is currently working on new indications of daxomicin, such as
vancomycin-resistant enterococcal infection and meticillin-
resistant Staphylococcus aureus prophylaxis.
120
Fig. 12 5-Methyl-rhamnose and analogs.
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8 Summary and outlook
The tiacumicin family encompass more than 40 molecules with
the same macrolactone framework. They are isolated from the
bacterial culture of dierent strains of the Actinomycetes group.
A recent study indicated the direct involvement of 17 genes and
corresponding enzymes with an unusual tailoring dihalogenase
in its biosynthesis. These compounds and their derivatives
displayed interesting bioactivity against pathogenic bacteria
and promising anti-cancer activity.
(R)-Tiacumicin B is a RNA synthesis inhibitor which acts by
docking to two domains of the closed RNA polymerase enzyme,
preventing its opening and thus activation. For the treatment of
Clostridium dicile infection, it has the same cure rate as van-
comycin but with a lower relapse rate. It was approved by the
FDA in May 2011 as a marketed drug for the treatment of CDI.
It is rather surprising that in spite of its interesting structure
and biological activities, no total synthesis of this macrolide has
been reported in the literature. Such a synthesis could aord an
interesting alternative to the actual source of supply, allowing
the generation of new derivatives with new or improved
activities.
9 Acknowledgements
W.E. would like to thank G. Coulthard for critically reviewing
this document and for making valuable suggestions.
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