Semisynthetic Analogs of the Antibiotic Fidaxomicin - Design, Synthesis, and Biological Evaluation

Article abstract of DOI:10.1021/acsmedchemlett.0c00381

The glycoslated macrocyclic antibiotic fidaxomicin (1, tiacumicin B, lipiarmycin A3) displays good to excellent activity against Gram-positive bacteria and was approved for the treatment of Clostridium difficile infections (CDI). Among the main limitations for this compound, its low water solubility impacts further clinical uses. We report on the synthesis of new fidaxomicin derivatives based on structural design and utilizing an operationally simple one-step protecting group-free preparative approach from the natural product. An increase in solubility of up to 25-fold with largely retained activity was observed. Furthermore, hybrid antibiotics were prepared that show improved antibiotic activities.

Full text of DOI:10.1021/acsmedchemlett.0c00381

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Letter
Semisynthetic Analogs of the Antibiotic FidaxomicinDesign,
Synthesis, and Biological Evaluation
⊥
⊥
̈
Andrea Dorst, Regina Berg, Christoph G. W. Gertzen, Daniel Schafle, Katja Zerbe, Myriam Gwerder,
Simon D. Schnell, Peter Sander, Holger Gohlke, and Karl Gademann*
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ABSTRACT: The glycoslated macrocyclic antibiotic fidaxomicin (1, tiacumicin B, lipiarmycin A3) displays good to excellent
activity against Gram-positive bacteria and was approved for the treatment of Clostridium difficile infections (CDI). Among the main
limitations for this compound, its low water solubility impacts further clinical uses. We report on the synthesis of new fidaxomicin
derivatives based on structural design and utilizing an operationally simple one-step protecting group-free preparative approach from
the natural product. An increase in solubility of up to 25-fold with largely retained activity was observed. Furthermore, hybrid
antibiotics were prepared that show improved antibiotic activities.
KEYWORDS: Fidaxomicin, antibiotics, semisynthesis, homology modeling, water solubility
idaxomicin (1, tiacumicin B, lipiarmycin A3) is a
glycosylated macrocyclic lactone produced by actino-
rhamnose substituent, which is esterified to a dichlorohomoor-
sellinic acid subunit. The complex structure and the remarkable
biological properties of fidaxomicin (1) aroused the interest of
many research groups and led to several synthetic studies toward
the preparation of the aglycon16−20 and finally the first total
synthesis of this natural product reported by some of us21 and
subsequently by others.22 Furthermore, investigations of the
mechanism of action revealed that fidaxomicin, in contrast to
other macrolide antibiotics, does not affect ribosomal protein
synthesis, but rather inhibits the transcription process catalyzed
by bacterial RNA polymerase (RNAP).8,23−30 In order to
identify promising semisynthetic target molecules based on
structural considerations, docking calculations of fidaxomicin on
a homology model of M. tuberculosis RNAP as well as stability
assays31 under various conditions were carried out first.
F
mycetes and has been isolated from four different soil
bacteria.1−7 Fidaxomicin shows good antibiotic activity in
vitro against many Gram-positive bacteria, with minimum
inhibitory concentration (MIC) values between 0.012 μg/mL
and 0.25 μg/mL for Clostridium difficile,8,9 a pathogen causing
nosocomial diarrhea. Since 2011, fidaxomicin has been
approved for the treatment of inflammations of the intestine
caused by C. difficile.10−12 Furthermore, excellent activity
against multiresistant Mycobacterium tuberculosis (MIC values
<0.008−0.045 μg/mL) and Staphylococcus aureus (MIC values
2−16 μg/mL) has been reported.7,13 Due to the low water
solubility of fidaxomicin (1) and in consequence its poor
systemic absorption, its application for the treatment of systemic
infections has not yet been achieved. The development of
semisynthetic derivatives is therefore a promising approach to
render this class of antibiotics available for such treatments of
systemic infections, in particular by improving water solubility.
With regard to its aglycon ring system, fidaxomicin (1)
structurally belongs to the macrocyclic lactone antibiotics
(Figure 1), but features a larger 18-membered ring.14,15 The
aglycon is connected to a modified noviose (isobutyl ester
instead of a methoxy group in 4″-position) as well as to a
Received: July 9, 2020
Accepted: October 1, 2020
https://dx.doi.org/10.1021/acsmedchemlett.0c00381
© XXXX American Chemical Society
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Figure 1. Structure of fidaxomicin (tiacumicin B, lipiarmycin A3).
Figure 2. Interaction diagram of the predicted fidaxomicin binding mode in M. tuberculosis RNAP. The protein subunits are given in Greek letters. Gray
circles around atoms show exposition to solvent. Orange circles show residues that lead to resistance when mutated; indirectly affected residues are
displayed as hexagons.
We started our work with the prediction of the binding mode
of fidaxomicin in a structural model of M. tuberculosis RNAP. We
applied multitemplate homology modeling and a multimodel
docking approach.32,33 In the predicted complex, fidaxomicin
forms one direct and three indirect interactions (mediated by
water) with an RNAP residue whose mutation triggered
resistance (Figure 2).7,26,34−36 The dichlorohomoorsellinic
acid is located outside the binding pocket and its phenolic
hydroxy groups do not take part in any of these interactions.
This led to the hypothesis that modifications at these positions
represent a promising strategy when aiming to improve water
solubility. Furthermore, these hydroxy groups are in proximity
to the binding pocket of the antibiotic rifampicin (Figure S1)
and we used our binding mode prediction to calculate the
optimal linker length (24 atoms of a polyethylene glycol chain)
to covalently connect the two antibiotics in order to target both
binding sites at the same time. The actual binding mode of
fidaxomicin in M. tuberculosis RNAP was recently elucidated
using cryo-electron microscopy (cryo-EM).29,30 A comparison
with our binding mode prediction revealed that the macrocycles
are overlapping, but the resorcinol moieties point in opposite
directions (Figure S2). However, the cryo-EM structure also
revealed a solvent pocket around the dichlorohomoorsellinic
acid subunit, thus further corroborating the hypothesis that this
part is a suitable site for modifications.
According to our binding mode prediction, 3′′′- and 5′′′-
positions displayed promising sites for modifications as they are
exposed to the solvent. Furthermore, functionalizations in these
positions turned out to be synthetically feasible without the use
of any protecting group; thus, analogs were accessible with only
one preparative step.37 Therefore, fidaxomicin (1) was treated
with different electrophiles under slightly basic conditions in
DMF at elevated temperatures (Scheme 1). The products 2−10
were obtained as separable mixtures of mono- and disubstituted
compounds (except 9b38). It is noteworthy that the
monosubstitutions were exclusively observed at 5′′′-hydroxy
B
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Scheme 1. Functionalization at Positions 3′′′ and 5′′′ of the Resorcinol Unit (Ns = Nosyl)
Scheme 2. Synthesis of a Fidaxomicin−Ciprofloxacin Hybrid
group since electronic effects might render this hydroxy group
more nucleophilic. Under basic conditions, formation of trace
amounts of isomers, which proved to be the transacylated
isobutyric ester, could not be prevented.31 After purification by
preparative HPLC, the desired compounds were obtained in
moderate to good yields (28−81%).
The substituents in compounds 2−7 were introduced for
their high polarity and their common use in medicinal chemistry.
C
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Scheme 3. Synthesis of the Fidaxomicin−Rifampicin Hybrid 19
The sulfonyl fluorides 8a/8b were synthesized for their ability to
form covalent bonds with the enzyme of interest (SuFEx-Click
chemistry),39 which might also be useful for MS-based studies
on the binding site. In the context of a study on labeling of
macromolecules with tetrazine moieties, we synthesized
tetrazine-fidaxomicin 9b.38 Furthermore, we reacted the
nosylated azidopropanol to obtain azide derivatives 10a/b,
which were used as a platform to connect several structurally
diverse alkynes to the resorcinol moiety using Click chemistry
(CuAAC) with a dicopper catalyst developed by Straub and co-
workers40 or commercially available Cu(I)OAc. Using azide 10a
as starting material, anilines 11, PEG5-acid 12a, and piperidin-
amine 13a/b were obtained, which showed improved water
solubility in comparison with fidaxomicin (see below).
In addition to the derivatives discussed so far, hybrids of
fidaxomicin with other antibiotics were synthesized. These
either have different targets or interact with different binding
sites within a common target. Fluoroquinolones are common
components of hybrid antibiotics due to their stability in a great
variety of reaction conditions and their broad spectrum of
activity.41,42 Modifications on the amine of the piperazine
moiety were shown to barely influence their biological activity.43
Therefore, this position was chosen for covalently connecting
ciprofloxacin to fidaxomicin, thus expecting only minor effects
on both molecules’ biological properties. For the synthesis of the
fidaxomicin-ciprofloxacin hybrid 15, ciprofloxacin was first
transformed into the corresponding bromoacetyl-ciprofloxacin
14 using bromoacetyl bromide (Scheme 2). Subsequent
exposure to basic conditions (K2CO3 in DMF) together with
fidaxomicin resulted in the desired hybrid 15.
As fidaxomicin and rifampicin share the same target enzyme
(RNAP), but interact with different binding sites (Figure S1),8
we synthesized a fidaxomicin−rifampicin hybrid. Based on our
calculations on the predicted binding mode, an octaethylene
glycol linker was deemed suitable in order to covalently connect
the two antibiotics while retaining their ability to interact with
their respective binding sites at the same time. Starting from
fidaxomicin (1) and nosylated octaethylene glycol azide 16, the
octaethlyene glycol linker was introduced and azide 17 was then
connected with literature-known alkynylated rifampicin 1844
using a Cu(I)-catalyst40 to give the desired fidaxomicin-
rifampicin hybrid 19 (Scheme 3).
We investigated the antibiotic activity of the synthesized
derivatives on different bacterial strains such as Bacillus subtilis
(DSM3256), S. aureus (ATCC29213), and M. tuberculosis by
evaluation of the minimum inhibitory concentration (MIC) (see
SI). Derivatives featuring promising antibiotic activity in these
tests were further assessed for their antibiotic activity against
several isolates of C. difficile (Table 1). Although some of the
derivatives did not retain their antibiotic activity, it was
demonstrated that large substituents on the resorcinol do not
necessarily impair biological activity, which is shown by the good
biological activities of derivatives 4a/b, 5a, 9b, 13a, and hybrids
15a and 19. Although derivatives 4a/b and 13a/b display
decreased activity against C. difficile, some promising activity
was observed against M. tuberculosis, S. aureus, and B. subtilis.
D
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Table 1. Summary of the Minimum Inhibitory Concentrations (MIC) of Selected Derivatives
Moreover, compounds 5a and 9b retained excellent activity
against C. difficile.
The fidaxomicin−rifampicin hybrid 19 shows an improved
activity compared to fidaxomicin against all investigated strains
and similar activity against C. difficile when compared to
rifampicin. Interestingly, hybrid 19 retains its biological activity
even against strains which are not susceptible to rifampicin (C.
difficile 8260 and 8282). Though no antibiotic activity against S.
aureus and B. subtilis has been observed, the fidaxomicin-
ciprofloxacin hybrid 15a retains its excellent activity against all
C. difficile strains even though ciprofloxacin itself is inactive
against the latter.
Additionally, we investigated the water solubility of the
obtained derivatives at pH = 7 by HPLC. For this purpose,
saturated solutions of the derivatives in phosphate buffer were
Figure 3. Solubility of selected derivatives in phosphate buffer (pH =
prepared and the concentration was determined after filtration
of the resulting suspensions. The results (Figure 3) provide
evidence for higher water solubility of PEG-derivatives 7b and
12a, piperidinol 6a/b, and piperidinamine 13b. Thus, PEG5-
acid 12a displays 25-fold and amine 13b 5-fold increase in water
solubility, though its antibiotic activity is reduced.
7).
water solubility. Some of these compounds showed improved
water solubility with maintained antibiotic activity. The
synthesized hybrid antibiotics 15 and 19 show improved activity
compared to fidaxomicin, while also retaining activity against
strains that show resistance against the attached antibiotic itself.
Access to a great variety of derivatives of this complex natural
product was achieved by using easy and reliable synthetic
methods. These derivatives could provide an important
contribution in ongoing efforts to reduce the rate of antibiotic
resistance development in bacteria and broaden the scope of
application of fidaxomicin.
In conclusion, apart from methylations45 and benzylations30
at the phenolic hydroxy groups, the new analogs presented here
are, to our knowledge, the first examples of complex semi-
synthetic derivatives of fidaxomicin obtained via phenolic
modifications. Other derivatives recently reported are obtained
by semisynthesis,46−48 fermentation,49,50 and fermentation of
knockout mutants.51−54 Based on our predictions of a binding
mode of fidaxomicin in a homology model of M. tuberculosis
RNAP, which suggested that modifications on the dichlor-
ohomoorsellinic acid would be promising, several analogs have
been synthesized and tested for their biological activity and
E
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Lung Association (2018-02). We acknowledge partial financial
support by the DFG (GRK 2158, P4 for H.G.). We gratefully
acknowledge the NMR and MS services of the University of
Zurich and F. Fazzi and G. Uzunidis for skillful laboratory
assistance. We further thank A. Siegle and O. Trapp (LMU
̈
Munchen) for using the HPLC in their research groups and R.
Wehlauch and J. Michel for proofreading the manuscript.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00381.
■
sı
Supplementary Figures S1 and S2, experimental proce-
dures and methods, characterization data, H and 13C
1
NMR spectra, supplementary MIC tables, and computa-
tional details (PDF)
ABBREVIATIONS
■
AUTHOR INFORMATION
Corresponding Author
CDI C. difficile infection; MIC minimum inhibitory concen-
tration; RNAP RNA polymerase; cryo-EM cryo-electron
microscopy; HPLC high performance liquid chromatography;
PEG polyethylene glycol.
■
Karl Gademann − Department of Chemistry, University of Zurich,
8057 Zurich, Switzerland; orcid.org/0000-0003-3053-
̈
0689; Email: karl.gademann@chem.uzh.ch
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Regina Berg − Department of Chemistry, University of Zurich,
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Daniel Schafle − Institute of Medical Microbiology, University of
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̈
Holger Gohlke − Institute for Pharmaceutical and Medicinal
Chemistry, Heinrich Heine University Dusseldorf, 40225
̈
Dusseldorf, Germany; orcid.org/0000-0001-8613-1447
̈
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00381
Author Contributions
⊥A.D. and R.B. contributed equally. The manuscript was written
through contributions of all authors. A.D., R.B., and K.G.
designed the study. A.D., R.B., and S.D.S. carried out the
synthesis and characterization of the derivatives. C.G.W.G., and
H.G. performed the homology modeling and computational
studies. D.S. and P.S. developed the MIC tests for M. tuberculosis
and performed the biological evaluation of the derivatives. K.Z.
and M.G. performed the MIC tests against S. aureus and B.
subtilis. A.D., R.B., and K.G. analyzed and discussed the results.
A.D.,R.B., and K.G. wrote the manuscript. All authors have given
approval to the final version of the manuscript.
Notes
The authors declare the following competing financial
interest(s): A patent application (WO2019135010A1,
EP18150671.8A) was filed Jan 8th, 2018 that includes
antibiotics presented in this work.
ACKNOWLEDGMENTS
■
We thank the Swiss National Science Foundation for financial
support (200021_182043 (K.G.) and 31003A_153349/1
(P.S.)). Work in the laboratory of P.S. is supported by Swiss
F
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