Total Synthesis of the Glycosylated Macrolide Antibiotic Fidaxomicin

Article abstract of DOI:10.1021/acs.orglett.5b01602

The first enantioselective total synthesis of fidaxomicin, also known as tiacumicin B or lipiarmycin A3, is reported. This novel glycosylated macrolide antibiotic is used in the clinic for the treatment of Clostridium difficile infections. Key features of

Full text of DOI:10.1021/acs.orglett.5b01602

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Letter
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Total Synthesis of the Glycosylated Macrolide Antibiotic Fidaxomicin
Elias Kaufmann, Hiromu Hattori, Hideki Miyatake-Ondozabal, and Karl Gademann*
Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056 Basel, Switzerland
S
* Supporting Information
ABSTRACT: The first enantioselective total synthesis of
fidaxomicin, also known as tiacumicin B or lipiarmycin A3, is
reported. This novel glycosylated macrolide antibiotic is used in
the clinic for the treatment of Clostridium difficile infections. Key
features of the synthesis involve a rapid and high-yielding access
to the noviose, rhamnose, and orsellinic acid precursors; the first
example of a β-selective noviosylation; an effective Suzuki
coupling of highly functionalized substrates; and a ring-closing
metathesis reaction of a noviosylated dienoate precursor. Careful
selection of protecting groups allowed for a complete deprotection yielding totally synthetic fidaxomicin.
ntibacterial resistance constitutes a growing concern in
diol β-mannose problem);14 and (3) a suitable protecting
group strategy allowing for selective deprotection. In this study,
we report on the first total synthesis of fidaxomicin (1). Our
highly convergent synthetic route assembles five main frag-
ments by a β-selective noviosylation, a Suzuki cross-coupling, a
ring-closing metathesis (RCM), and a β-rhamnosylation.
1
Apublic health for many societies all over the world, and
many antibiotics have become less effective if not ineffective
against many pathogens.2 This is particularly striking given that
the last discovery of a new class of antibiotics dates back to the
1980s. Tuberculosis (TB) has become a threatening example
among the diseases that evolved to be resistant to common
antibiotic therapy. In 2012 alone, out of 8.7 million new cases
of TB, 450 000 patients were infected by multidrug resistant-
TB.1 With increased frequency, frontline antibiotics (such as
rifampicin or isoniazid) fail in therapeutic treatment. All these
issues call for new antibiotics to fill this gap.3 To this end, we
became interested in fidaxomicin (1, tiacumicin B, lipiarmycin
A3),4 which inhibits RNA polymerase in bacteria and shows
high activity against drug resistant strains of Mycobacterium
tuberculosis.5 Fidaxomicin (1) was FDA-approved in 2011 for
the treatment of Clostridium difficile infections involved in
nosocomial (hospital acquired) diarrhea. Even though advanta-
geous for the therapy of CDI, the low bioavailability of
fidaxomicin (1) in the plasma by oral application prohibits its
use as a therapeutic against systemic diseases such as TB.
In addition to the interesting bioactivity, the 18-membered
macrolactone constitutes an architecturally complex synthetic
target featuring multiple stereogenic centers and a high degree
of unsaturation. The central macrolide is β-linked to a unique
D-noviose and D-rhamose, which is complemented with a
dichloro homoorselinic acid unit. Despite the fact that
fidaxomicin (1) has been isolated in the 1970s and is clinically
used to date, no total synthesis of a member of this class has
been reported in the literature.7 Coincidentally, syntheses of the
core aglycon have been recently published back-to-back-to-back
by Altmann et al.,8 Zhu et al.,9 and our group.10 Furthermore,
synthetic studies have been performed on 4-OMe D-noviose11
carbohydrates12 and a resorcinol unit.13 The challenges of
fidaxomicin (1) concerning its total synthesis reside in (1)
rapid access to the carbohydrate and orsellinate building blocks;
(2) two challenging β-selective glycosylations (cf. the cis-1,2-
The synthesis commenced with the preparation of the
carbohydrate and orsellinic acid precursors. For the preparation
of the protected resorcylate 4, we chose the biomimetic
aromatization strategy established by Barrett et al.15 (Scheme
1). Therefore, the dianion of the known keto dioxinone 216,17
was reacted with freshly prepared propionyl imidazole18 to
furnish the corresponding diketo dioxinone. The subsequent
aromatization under basic conditions directly yielded the
resorcylate 3 (57%). Chlorination with sulfuryl chloride19,20
Scheme 1. Synthesis of the Protected Resorcylate 4
Received: June 1, 2015
© XXXX American Chemical Society
A
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proceeded in quantitative yield, and the allylation gave rise to
the protected homoorsellinic acid 4 in excellent yield (95%).
The preparation of the rhamnose unit 9 originated in the
known D-rhamnoside 521 (Scheme 2), which was synthesized
Scheme 3. Synthesis of Noviosylbromide 16
Scheme 2. Synthesis of the Rhamnosyl Donor 9
at high temperature, first to hydrolyze the acetonide and second
to isomerize the furanoside to the pyranoside. As the
equilibrium of the 5- vs 6-membered ring was found to be
only 1:2 in favor of the desired pyranoside, the furanoside was
recovered and resubmitted twice to the same reaction
conditions. In this way, the unfunctionalized novioside was
obtained as an inseparable anomeric mixture in good overall
yield (81%, α/β = 2:1). Based on the previous studies in the
area of β-rhamnosylation,14 we opted for introducing the
electron-withdrawing 2,3-carbonate protecting group with CDI,
which was followed by esterification with isobutyryl chloride to
yield the two separable anomers of 15 (67% over two steps).
Finally, the acetal 15 (either epimer) was converted to the
desired glycosyl bromide donor 16 using HBr in acetic acid.
Due to the instability of glycosyl bromides, the intermediate
was used in the next step without purification.
At this point we aimed for the assembly of the three building
blocks 9, 16, and the previously prepared aglycon.10 Even
though we achieved satisfactory β-rhamnosylation on the
primary alcohol (not shown), the β-noviosylation on the
complete macrolide proved exceptionally challenging. Tedious
experimentation using different glycosidation methods con-
sistently resulted only in α-selective glycosylation on the
protected macrolide.
Hypothesizing that the alcohol in the rigid ring system is
poorly accessible for an attack from the sterically hindered face
of the glycoside (cf. the β-mannose problem),14 we sought to
introduce the noviose unit on a linear, more flexible
intermediate at an early stage. Therefore, we prepared the
alcohol 18 by reductive cleavage of the PNB group of fragment
17 (99%, Scheme 4), which constituted an intermediate in our
aglycon synthesis.10 We were pleased to find that the
glycosylation with the sterically demanding, yet more flexible
secondary alcohol 18, using Helferich’s conditions,30 furnished
the noviosylated fragment 19 with a good α/β ratio (63%, α/β
= 1:3, 48% of β-anomer isolated). The relative configuration of
the β-glycosidic linkage was assigned by NMR studies
(NOESY). To the best of our knowledge, this experiment
constitutes the first example of a β-selective noviosylation that
has been reported in the literature. To assemble the remaining
parts of the macrocycle, fragment 22 was prepared from the
known precursor 218 by a Yamaguchi esterification (61%) with
from the corresponding methyl-α-D-mannopyrannoside by a
Garegg Samuelsson iodination and subsequent Pd-catalyzed
hydrogenation.22,23 Butane-2,3-diacetal protection of the trans-
3,4-hydroxy groups allowed for the selective installation of the
methyl group at O-2, and subsequent deprotection furnished
the diol 6 in 72% yield over three steps. Next, the thiophenyl
group was installed by ZnI2 mediated trans-acetalization with
TMSSPh to afford the thioglycoside 7.24
To our delight, the coupling reaction between the resorcylate
4 and the diol 7 proceeded with excellent regioselectivity at O-4
to yield the ester 8 (66%). Interestingly, the O-3 esterified
regioisomer was initially formed exclusively and the ester
migration occurred with a prolonged reaction time to give rise
to the desired product 8. Next, allylation of the phenolic OH
group (96%) and hydrolysis of the thioacetal with NBS in
acetone/H2O 10:1 was performed with good yields and
selectivity (95%, α/β = 5:1). Finally, the lactol was function-
alized with N-phenyl trifluoroacetimidoyl chloride to give the
rhamnosyl donor 9.25 The switch of the leaving group was
crucial in order to achieve good yields as well as anomeric
selectivity in the rhamnosylation step (vide infra).
The known 6-deoxy-6-iodopyranoside 1026 served as starting
material for the preparation of the novioside unit (Scheme 3).
First, a Vasella ring contraction by a modified procedure27,28
furnished the olefinic furanose 11 (91%). Treatment of lactol
11 with CSA in methanol and 2,2′-dimethoxypropane gave the
furanoside 12 without cleavage of the acetonide. Ozonolysis of
the terminal olefin using Marshall’s protocol29 in methanolic
sodium hydroxide gave direct access to the ester 13 (62%). The
major side product in this oxidative cleavage was the
corresponding aldehyde (11%), which could be transformed
to the desired ester under the same reaction conditions.
Next, the gem-dimethyl groups were introduced using
MeMgBr to afford the tertiary alcohol 14 in excellent yield
(99%). The alcohol 14 was then treated with TFA in methanol
B
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Scheme 4. Completion of the Total Synthesis
the dienoic acid 20.10 The following Suzuki cross-coupling of
the boronate 22 and iodide 19 was highly effective and
furnished product 23 in good yield. Fortunately, the basic
conditions (Pd(PPh3)4, TlOEt in THF−H2O), first used by
Altmann and Glaus in their aglycon synthesis,8 did not affect
the base labile carbonate and ester moiety, which can be
attributed to the very short reaction time (less than 30 min).31
The subsequent ring-closing metathesis reaction by treatment
of the linear fragment 23 with the second generation Grubbs
catalyst (20 mol %) for 1 h at 100 °C gave the macrolide with
an (E/Z) ratio of 2:1 in 75% yield (54% of E-24). The
chromatographic separation of the E/Z isomers, allowed for a
subsequent recycling of the (Z) isomer. As a result, the overall
yield of (E)-24 in this transformation was increased to 63%.
Selective deprotection of the primary TBS ether was achieved
with trihydrogenfluoride triethylamine in moderate yield
(49%). As anticipated, the β-selective installation of the
rhamnosyl side chain turned out to be challenging.14 After an
extensive screening, we were able to obtain high β-selectivity
and a good yield with the trifluoroacetimidate donor 9 in its 4C1
conformation (α/β = 1:4, estimated by 1H NMR analysis of the
crude reaction mixture, 62% of 25).25,14e Conveniently, the
protection of the O-3 hydroxy group was unnecessary.
The final task for the completion of the total synthesis was
the deprotection of the TBS, carbonate, and allyl groups. First,
the two TBS groups were removed by treatment with
trihydrogenfluoride triethylamine at 50 °C (60%). The
following carbonate deprotection proved to be troublesome
due to competitive isobutyrate ester hydrolysis. Nevertheless,
Barton’s base in wet CH2Cl2 gave satisfactory results (good
conversion and purity, as judged by NMR analysis). Finally, Pd-
catalyzed allyl-deprotection and two-stage purification (prepa-
rative TLC followed by HPLC) gave fully synthetic fidaxomicin
(1, 10% over two steps). The observed overall yield for these
transformations can be explained by difficulties in the
purification of the final product on a small scale. The identity
of the synthetic compound was confirmed by coinjection of
synthetic and authentic material on reversed-phase HPLC.
Interestingly, as the synthetic material contained residual
1
formate from the HPLC purification, the H NMR spectrum
did at first not fully match with the spectra of the natural
product. Again, by mixing equimolar amounts of synthetic and
authentic samples, the 1H NMR spectrum clearly confirmed the
identity of the synthetic material to authentic fidaxomicin (1).
In this letter, we report the first, enantioselective total
synthesis of the glycosylated macrolide antibiotic fidaxomicin
(1), which has been isolated over 40 years ago and constitutes a
clinically used drug. Key features of the synthetic route include
(1) a rapid access to the rhamnosyl side chain, (2) the first β-
selective noviosylation followed by Suzuki cross-coupling and
ring-closing metathesis of complex noviosylated precursors, and
(3) a β-selective rhamnosylation. In particular, the stereo-
selective installment of both 1,2-cis diols in the β-linked
carbohydrate units constitutes a notable feature of the
approach. This successful total synthesis contributes to a
detailed understanding of the chemistry of fidaxomicin and
paves the way for the generation of analogs addressing some of
the shortcoming of the natural product.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, characterization data, NMR spectra
and HPLC chromatogram of fidaxomicin (1). The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/acs.orglett.5b01602.
C
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Letter
van der Marel, G. A.; Codee
́
, J. D. C. Chem. Commun. 2012, 48, 2686.
AUTHOR INFORMATION
Corresponding Author
■
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the investigations of β-rhamnopyranosides using the 2,3-carbonate-
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*E-mail: karl.gademann@unibas.ch.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge partial financial support by the
NCCR Molecular Systems Engineering, the Latsis Prize (to
K.G.), and the Novartis Early Career Award (to K.G.). We
thank Dominik Lotter and Reto Witzig at University of Basel
for skillful technical support and Priv.-Doz. Dr. D. Haussinger
(University of Basel) for assistance with NMR spectroscopy.
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