# Switching Residues: A Platform for the Synthesis of Fidaxomicin Antibiotics

Erik Jung, Tizian Griesser, Jordan Costafrolaz, Ondine Duverger, Yves Mattenberger, Silvia Dittmann, Andrea Dorst, Alexander Major, David Dailler, Daniel Schäfle, et al.

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Erik Jung, Tizian Griesser, Jordan Costafrolaz, Ondine Duverger, Yves Mattenberger, et al., Switching Residues: A Platform for the Synthesis of Fidaxomicin Antibiotics. Angewandte Chemie International Edition, 2024, 64 (7), pp.e202419095. 10.1002/anie.202419095. [hal-04951671](https://hal.science/hal-04951671v1)

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# Switching Residues: A Platform for the Synthesis of Fidaxomicin Antibiotics

Erik Jung, Tizian Griesser, Jordan Costafrolaz, Ondine Duverger, Yves Mattenberger, Silvia Dittmann, Andrea Dorst, Alexander Major, David Dailler, Daniel Schäfle, Susanne Sievers, Konstantin Brodolin, Patrick H. Viollier, Peter Sander, and Karl Gademann*

**Abstract:** Peripheral modification is often the main approach to optimize natural products for improved biological activity or desired physicochemical properties. This procedure inevitably increases molecular weight, often accompanied by undesired increased lipophilicity. Removing structural elements from natural products is not always tolerated. This is also the case for the antibiotic fidaxomicin (Fdx), where every structural component has been shown to be crucial for antibiotic activity. In this work, we demonstrate how the residue switching approach can maintain biological activity of Fdx derivatives by replacing the rhamnoside-dichlorohomoorstellinate moiety of Fdx with smaller, more polar building blocks. We used palladium-catalysed allylic substitution to selectively install *N*-nucleophiles on the core of Fdx. The new derivatives were designed to mimic the binding of Fdx to the bacterial RNA polymerase. Evaluation against *Mycobacterium tuberculosis*, *Clostridioides difficile*, and the Gram-negative model organism *Caulobacter crescentus* demonstrated that the newly introduced residues can restore antibiotic activity, which was further supported by on-target RNA polymerase assays. We combined the allylic substitution with an organocatalysed novioside acylation protocol to enable the functionalisation of two vectors on Fdx in one pot. This platform greatly expands the accessible chemical space for Fdx derivatives and enables the future development of systemic Fdx antibiotics.

## Introduction

For decades, natural products have served as a crucial source of lead structures for drug development. The natural product itself can turn out to be sufficiently potent, safe, and bioavailable to treat disease (e.g. vancomycin, paclitaxel, all-trans retinoic acid, dextran, among many others).[1] However, modification of the natural product structure is often necessary to improve physicochemical properties, spectrum of activity, or counter resistance (Figure 1A). Antibiotic discovery relies heavily on natural products and great efforts to modify their structure have led to dozens of approved drugs such as the cephalosporins or tetracyclines.[1,2] The structural complexity of a natural product dictates which types of modifications are feasible for drug development. Often, the most accessible strategy is peripheral modification,[3] wherein new moieties are added to existing reactive functional groups (recently approved examples include Lefamulin[4] and Plazomicin[5]). In contrast, de novo synthesis is only possible after significant investment into the development of practical synthetic routes (e.g. Eravacycline).[6,7] Complexity reduction of natural products can be a powerful approach if parts of the structure are known not to contribute to biological activity. The resulting structurally less complex targets are then much more synthetically accessible (e.g. Eribulin).[8,9] If complexity reduction diminishes activity, then new substituents have to be introduced at the site of truncation to recover activity and modulate physicochemical properties. We term this less common approach residue switching. A notable example of residue switching is Docetaxel, which was prepared either

<figure><img src="image_5.png" /></figure>

<footnote>[*] E. Jung, Dr. A. Dorst, A. Major, Dr. D. Dailler, Prof. Dr. K. Gademann
Department of Chemistry, University of Zurich, 8057 Zürich,
Switzerland
E-mail: karl.gademann@uzh.ch</footnote>

<footnote>T. Griesser, Dr. D. Schäfle
Institute of Medical Microbiology, University of Zurich, Zurich,
Switzerland</footnote>

<footnote>Dr. J. Costafrolaz, Dr. Y. Mattenberger, Prof. Dr. P. H. Viollier
Department of Microbiology and Molecular Medicine, Faculty of
Medicine, University of Geneva, Geneva, Switzerland</footnote>

<footnote>O. Duverger, Dr. K. Brodolin
Institut de Recherche en Infectiologie de Montpellier, Univ.
Montpellier, CNRS, Montpellier, 34293, France</footnote>

<footnote>S. Dittmann, Dr. S. Sievers
Department of Microbial Physiology and Molecular Biology,
Institute of Microbiology, Center for Functional Genomics of
Microbes, University of Greifswald, Greifswald, Germany</footnote>

<footnote>Prof. Dr. P. Sander
Institute of Medical Microbiology, University of Zurich, Zurich,
Switzerland; National Reference Laboratory for Mycobacteria,
University of Zurich, Zurich, Switzerland</footnote>

<footnote>© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>
A Approaches to natural product optimisation

<figure><img src="image_6.png" /></figure>

Unmodified
natural product

<figure><img src="image_7.png" /></figure>

**Peripheral**
**Modification**

Lefamulin
Plazomicin

<figure><img src="image_8.png" /></figure>

**Skeletal**
**Editing**

Azithromycin

<figure><img src="image_9.png" /></figure>

**De novo**
**Synthesis**

Cephalosporins
Tetracyclines

<figure><img src="image_10.png" /></figure>

**Complexity**
**Reduction**

Eribulin
Bryolog

<figure><img src="image_11.png" /></figure>

**Residue**
**Switching**

less common
**This work**

B Multi-step residue switching

<figure><img src="image_12.png" /></figure>

D Fidaxomicin complexity reduction is not tolerated

<figure><img src="image_13.png" /></figure>

Fidaxomicin
in *M. tuberculosis* RNAP

**Potent activity**
Rhamnose-arene moiety engages key residues

C The antibiotic fidaxomicin

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

FdxG2-IBu
Shunt metabolite

**Activity lost**
Core inactive without additional substituents

E One-step residue switching: **this work**

<figure><img src="image_16.png" /></figure>

F Unlocking previously inaccessible chemical space

<figure><img src="image_17.png" /></figure>

**Figure 1.** (A) Examples for different approaches to natural product optimisation (B) Switching residues on natural products usually requires multiple steps. (C) Structure of the glycosylated polyketide antibiotic fidaxomicin. (D) Complexity reduction is not a viable strategy for Fdx derivatisation due to loss of activity. Simplified representation of cryo-EM structure of Fdx bound to RNAP (PDB: 6FBV). (12) (E) A one-step residue switching platform to access Fdx derivatives with reduced weight and lipophilicity. (F) Derivatives obtained through residue switching can reach previously inaccessible physicochemical space. Compounds synthesised in this work (orange), acyl derivatives (13) (light blue), arene alkylation (14,15) (dark blue). clogP was calculated using OSIRIS DataWarrior 6.1.0. (16)

from Paclitaxel in 4 steps or from 10-deacetylbaccatin III in 5 steps, switching the naturally occurring Bz-group to a Boc-group (Figure 1B). (10,11)

The natural product antibiotic fidaxomicin (Fdx, **1**, tiacumicin B, lipiarmycin A3) is approved for the treatment of gastrointestinal *Clostridioides difficile* infections (Figure 1C). (17–19) Fdx also shows potent in vitro activity against

other Gram-positive bacteria such as *Mycobacterium tuberculosis*. (20) However, poor systemic absorption of Fdx precludes treatment of systemic infections by pathogens such as *M. tuberculosis*. (21) Therefore, rationally designed derivatives with improved physicochemical properties that are informed by a detailed structural understanding are needed to unlock fidaxomicin's potential to treat tuber-
culosis (TB). The groups of Ebright and Campbell inde-
pendently elucidated cryo-EM structures of Fdx in complex
with *M. tuberculosis* (*Mtb*) RNA polymerase (RNAP).[12,22]
These structures revealed that the dichlorohomoorsellinate
engages in hydrogen bonding with β-Lys1101 and a cation-π
interaction with β'-Arg84. The rhamnoside is engaged in
hydrogen bonding with β'-Arg89. The latter interaction was
shown to be essential in a transcription assay, since the IC₅₀
of Fdx against the mutant RNAP<sup>R89A</sup> increased > 400-fold.
Removal of the dichlorohomoorsellinate moiety also re-
duced RNAP inhibition > 400-fold.[12] Biosynthetic inter-
mediates lacking the rhamnoside-arene fragment, such as
the shunt metabolite FdxG2-iBu, were also inactive against
*Staphylococcus aureus*, underscoring the importance of this
structural element (Figure 1D).[23]

A likely reason for the lack of new Fdx derivatives in the
first four decades since its discovery in 1972 could lie in its
structural complexity. Fdx contains two reactive phenolic
hydroxy groups, five aliphatic hydroxy groups, two allylic β-
glucosides, and a labile isobutyrate moiety. Therefore, both
total synthesis and selective semisynthetic modification of
Fdx are extremely challenging.[19] In 2015, the groups of
Altmann, Gademann, and Zhu independently described
total syntheses of the Fdx aglycon.[24–26] In the same year, the
Gademann group achieved the first total synthesis of the
fully glycosylated natural product.[25,27] Since then, Roulland
and co-workers have also achieved the total synthesis of
Fdx.[28] The lessons learned in total synthesis have been
paramount for the development of semisynthetic methods
for the modification of Fdx.[27]

Previous efforts in our group have focused on site-
selective peripheral modification, which can retain antibiotic
activity.[13–15] However, the addition of new substituents leads
to an undesirable increase in molecular mass and little
control over the overall physicochemical properties of the
derivatives. Only major changes to the structure of Fdx
could lead to the drastic physicochemical property shifts
needed to achieve systemic availability. Extensive studies on
the biosynthesis of Fdx, the biological activity of shunt
metabolites, and synthetic studies revealed that every
structural component of Fdx is essential for antimicrobial
activity.[13,23,29–31] Consequently, reduction of complexity is
not a feasible strategy for the development of new Fdx
antibiotics (Figure 1D). *Only removal of structural compo-
nents with concomitant introduction of new moieties (residue
switching) could affect binding and physicochemical proper-
ties while also reducing or maintaining molecular mass.*
Distinct from previous multi-step residue switching ap-
proaches for other natural products, we developed a method
to replace the rhamnoside-dichlorohomoorsellinate residue
of unprotected Fdx directly in one step through selective
palladium catalysis (Figure 1E). By using various *N*-nucleo-
philes, we synthesised a total of 43 new Fdx derivatives.

To illustrate how the residue switching approach unlocks previously inaccessible physicochemical space, we plotted calculated lipophilicity (clogP) against molecular weight (MW) of Fdx (grey), FdxG2-iBu (grey), previously synthesised Fdx derivatives via the peripheral modification approach (acyl derivatives[13] in light blue, arene alkylation[14,15]

in dark blue), and the new compounds derived from the
residue switching approach (orange) (Figure 1F). It is
immediately apparent that peripheral modification generally
leads to an increase in molecular weight, while also
increasing lipophilicity. In contrast, residue switching deliv-
ers derivatives that are generally smaller and less lipophilic
than parent Fdx, which is desirable for our future goal of
developing systemically available Fdx antibiotics.[32]

**Results and Discussion**

We set out to develop a synthetic platform that allows the
facile introduction of *N*-nucleophiles to displace the rham-
noside-dichlorohomoorsellinate fragment of Fdx. *O*-Allyl
glycosides are usually seen as protected sugars,[33–35] yet
those glycosides can also be seen as leaving groups and
functional handles (Scheme 1A). The two allylic glycosides
of Fdx could therefore serve as derivatisation points through
formal nucleophilic displacement. Our group previously
demonstrated the palladium-mediated substitution of the
rhamnoside-dichlorohomoorsellinate fragment by activated
*C*-nucleophiles, with limited diversity of accessible deriva-
tives (3 nucleophiles).[13] The observed perfect selectivity for
displacement of the rhamnoside over the novioside stems
from the preference of Pd to operate via an *anti-anti*
mechanism.[36] The backside of the π-system adjacent to the
noviose is shielded from *anti*-attack of palladium by the rigid
macrocycle. The rhamnoside is attached to the primary C20-
OH group, which allows σ-bond rotation to position the
glycosidic bond *anti* to the peripheral attack of palladium
(see Scheme 1B, *Supplementary Scheme* 1). Exploiting this
selectivity and expanding the method to *N*-nucleophiles
would allow the use of the vast range of easily accessible *N*-
nucleophile building blocks, rapidly accessing a library of
new Fdx derivatives.

In developing our method, we considered that Fdx is
prone to decomposition (loss of noviose) and rearrange-
ments (ester migration, double bond isomerisation) upon
exposure to heat or even mildly basic or acidic conditions
(see *Supplementary Scheme* 2).[27] Due to their close struc-
tural similarity, the resulting side products are extremely
difficult to separate chromatographically. The displacement
of challenging allylic leaving groups such as alcohols
(including glycosides) by Pd can be promoted by polar
solvents, which served as a starting point for our
optimization.[37–39] After extensive screening, we found that
the use of THF or alcohol/water mixtures, in combination
with Pd(PPh₃)₄ or [Pd(cinnamyl)Cl]₂/dppf at 40–60°C effi-
ciently displaces the rhamnoside-dichlorohomoorsellinate
residue of Fdx with the model nucleophiles *N*-meth-
ylpiperazine and 2-aminobenzothiazole (see *Supplementary
Scheme* 3, *Supporting Information Table* 1). The use of
benzylamine resulted in the unexpected formation of Bn-*N*-
(Fdx)₂ dimers, as substitution of the resulting secondary
amine with another equivalent of Fdx is possible. Increasing
the equivalents of benzylamine to 5.0 eq. and diluting the
reaction to 38 mM significantly reduced the formation of
this side product (see *Supporting Information Table* 2). The
A The concept

<figure><img src="image_18.png" /></figure>

B Mechanism-based selectivity

<figure><img src="image_19.png" /></figure>

D Scope of N-nucleophiles

<figure><img src="image_20.png" /></figure>

Conditions A

Nucleophile (3.0 eq.), Pd(PPh₃)₄ (5-50 mol%), i-PrOH/water 5:1, 40-50 °C, or MeOH, 60 °C

Conditions B

Nucleophile (3.0 eq.), [Pd(cinnamyl)Cl]₂ (5-20 mol%) or [Pd(allyl)Cl]₂ (50 mol%), dppf (10-100 mol%), THF, 60 °C

Conditions C

Nucleophile (5.0 eq.), [Pd(cinnamyl)Cl]₂ (10 mol%), dppf (20 mol%), THF (38 mM), 60 °C

C Computational design of nucleophiles

<figure><img src="image_21.png" /></figure>

Secondary amines

Conditions A

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

Primary amines

Conditions C

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

2-Aminothiazoles

Conditions B

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>

Anilines

Conditions B

<figure><img src="image_49.png" /></figure>

2-Aminopyridines

Conditions B

<figure><img src="image_50.png" /></figure>

**Scheme 1.** (A) Allylic glycosides can be functional handles for natural product derivatisation. (B) Mechanistic explanation for the excellent selectivity for substitution of the rhamnose. (C) Three-dimensional representations of energy-minimised structures of 2-aminothiazole **13** (orange) and aminomethyl thiadiazole **20** (purple), each with Fdx (blue) in the cryo-EM structure of *M. tuberculosis* RNAP by Ebright and co-workers (PDB: 6FBV).[12] (D) Scope of *N*-nucleophiles for the Pd-catalysed allylic substitution of the allylic rhamnoside of Fdx. [a] Mixture with 2'-isobutyrate. [b] Obtained as the formate salt.

formation of the linear substitution product and the identity
of common by-products were assigned based on 2D NMR
spectroscopy (see *Supplementary* Figures 1–3). Prior to

purification, the reaction mixtures were treated with 3-
mercaptopropyl-functionalised silica gel, scavenging palladi-
A Mimicking the binding mode of Fdx by computational design

<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

B Protocol for amide-linked derivatives

<figure><img src="image_53.png" /></figure>

C Protocol for sulfamate- or sulfate-linked derivatives

<figure><img src="image_54.png" /><figcaption>**Scheme 2.** (A) Design of derivatives to mimic the arene of Fdx leads to structural evolution of aniline **24**. Three-dimensional representation of energy-minimised structure of aniline **31** (beige) with Fdx (blue) in the cryo-EM structure of *Mtb* RNAp by Ebright and co-workers (PDB: 6FBV).<sup>[12]</sup> (B) Synthesis of amides **28–31** via late-stage amidation of primary amine **27**. (C) Synthesis of sulfamate- and sulfate-linked derivatives **34–38** via late-stage SuFEx with fluorosulfate **33**. AISF = [4-(acetylamino)phenyl]imidodisulfuryl difluoride.</figcaption></figure>

um to prevent potential interference in the biological
evaluation.[40,41]

We selected further *N*-nucleophiles based on their
predicted ability to mimic crucial RNAP contacts of Fdx,
such as that of the rhamnose to β'-Arg89, based on a cryo-
EM structure of Fdx bound to RNAP.[12] Using energy
minimisation with the MAB force field in moloc,[42] we
designed different linker structures such as 4-substituted 2-
aminothiazoles, aminomethyl-substituted heterocycles, ani-
lines, and 2-aminopyridines (Scheme 1C, Scheme 1D). Ana-
logues of derivatives, such as the aminomethyl oxadiazole
series, were synthesised via multi-step synthesis (see *Supple-
mentary Schemes* 4–8). The allylic substitution on Fdx was
also achieved on gram scale using 5 mol % Pd(PPh₃)₄,
demonstrating the scalability of this method. Thus, a robust
protocol for the introduction of *N*-nucleophiles into Fdx was
established and the synthesis of 24 Fdx derivatives was
achieved (Scheme 1D).

The synthesis of building blocks and the separation of
closely eluting side products were the main bottlenecks of
our allylic displacement protocol. To circumvent this, we
envisioned the installation of a reactive handle through
allylic substitution to readily introduce new substituents at a
late stage and simplify purification. Two approaches were
established: Amide bond formation from a primary amine
and sulfamate/sulfate formation from a fluorosulfate. For
amide formation, an aniline was chosen as a linker since the
aniline 24 showed activity against M. tuberculosis. Computa-
tional design suggested that an aminomethyl moiety *meta* to
the aniline would position benzamides in the RNAP in the
same location as the arene residue of Fdx (Scheme 2A).
Several benzylamine protecting groups were evaluated for
compatibility with Fdx and the reaction conditions.

2-(Trimethylsilyl)ethyloxycarbonyl- (Teoc) and nitrover-
atryl-oxycarbonyl- (Nvoc) protected amines were introduced
to Fdx and successfully deprotected, however separation of
the benzylamine **27** from deprotection reagents proved
Flexible dual functionalisation strategy

<figure><img src="image_55.png" /></figure>

Scope of dual functionalisation

<figure><img src="image_56.png" /><figcaption>via Path A for nucleophile diversification</figcaption></figure>

41ª
39% (from 39)

<figure><img src="image_57.png" /></figure>

42
14% (from 39)

<figure><img src="image_58.png" /></figure>

43
40% (from 39)

<figure><img src="image_59.png" /><figcaption>via Path B for ester diversification</figcaption></figure>

44ª
49% (from 40)

<figure><img src="image_60.png" /></figure>

45ª
22% (from 40)

<figure><img src="image_61.png" /></figure>

46ª
35% (from 40)

**Scheme 3.** One-pot dual functionalisation strategy. [a] Obtained as the formate salt.

difficult. In the end, using the mesylate salt of 3-(aminomethyl)aniline was sufficient to suppress primary amine reactivity and yielded **27** in one step. Succinamide **28** and the tetrazole **29** were chosen for their potential to form salt bridges with the basic residues at the entrance of the Fdx binding site.[14,43] The catechol **31** mimics the dichlorohomoorsellinate moiety of Fdx and corresponds to the siderophore moiety of the 5th generation cephalosporin antibiotic Cefiderocol.[44] Succinamide **28** was obtained rapidly with succinic anhydride in 70% yield. Amides **29–31** were obtained quickly after HATU-mediated coupling with the respective carboxylic acids (Scheme 2B).

S¹'-F Electrophiles have been established as connecting hubs for the SuFEx (Sulfur Fluoride Exchange) click reaction framework.[45–46] Through reaction with an SO₂F-transfer reagent, phenols can be turned into fluorosulfate electrophiles for late-stage diversification. As free phenols are compatible with our allylic substitution protocol, we selected 3-(2-aminothiazol-4-yl)phenol as the nucleophile, resulting in phenol **32**. After screening SO₂F transfer reagents, base, solvent, and temperature, the use of AISF in combination with DBU in THF at –20 °C gave fluorosulfate **33** in 56% yield.[47] Subsequently, we obtained the sulfamates **34–37** from fluorosulfate **33** and various amines through HOBt-mediated SuFEx in 59–80% yield.[48] The sulfate **38** was synthesized in 52% yield through reaction with phenol, HMDS, and BTMG (Scheme 2C).[49] With these approaches we have demonstrated the utility and flexibility of reactive handles for rapid access to Fdx derivatives.

To gain better control over the physicochemical properties of the derivatives, it would be desirable to modify more than one site of Fdx at once. To achieve this, we combined our established selective acylation protocol[13] with the newly developed allylic substitution. Using Shimada's boronic acid

catalyst,[50] it is possible to selectively introduce acyl groups to the 3'-OH group of OP1118 (the deacylated metabolite of Fdx, **39**) from readily available acid chlorides. We found that allylic substitution in MeOH, followed by a solvent swap to 1,4-dioxane, enabled the introduction of both a secondary amine at the C20-position as well as an acyl group on the 3'-OH of the novioside in one pot in 14–40% yield over both steps for the furans **41–43** (Scheme 3, path A, *Supplementary Scheme* 9). Alternatively, allylic substitution of Fdx on gram scale in MeOH, followed by methanolysis of the isobutyrate in one-pot through addition of potassium carbonate, gave the acylation precursor **40** in 50% yield over two steps. Precursor **40** could then be acylated to yield the three pyrazoles **44–46** in 22–49% yield (Scheme 3, path B). If diversification of the nucleophile is desired, path A should be taken, and path B for diversification of the ester moiety. The combination of the palladium-catalysed site-selective substitution and the organocatalysed site-selective acylation is a powerful method to gain control over two functionalisation vectors on Fdx at the same time. In total, we synthesised 43 new Fdx derivatives for evaluation of their biological activities.

With robust synthetic access to new Fdx derivatives, we investigated if these compounds of reduced complexity could still retain antibiotic activity. The shunt metabolite FdxG2-iBu (**26**), which can be obtained from a glycosyl-transferase knock-out producer strain, represents complexity reduction of Fdx without replacement substituents on the core.[23,51] We first determined the ability of Fdx, its shunt metabolite FdxG2-iBu, and representative derivatives to inhibit *M. tuberculosis* (*Mtb*) RNAP using a fluorescent run-off transcription assay (Figure 2A). Truncation of the rhamnoside-dichlorohomoorsellinic fragment leads to complete loss of inhibitory activity against *M. tuberculosis* RNAP (Fdx IC₅₀ 0.04±0.01 µM vs. FdxG2-iBu IC₅₀ >
A **In vitro inhibition of M. tuberculosis RNA polymerase**

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>

B **Activity against cultured M. tuberculosis**

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

Concentration µg/mL

D **Reducing molecular weight and clogP**

<figure><img src="image_72.png" /></figure>

C **Activity against Rif-resistant Mtb**

Rif-resistant Mtb isolate

Mtb H37Rv poly-g-GFP

<figure><img src="image_73.png" /></figure>

MIC in µg/mL - determined by turbidity read-out

E **Gram-negative C. crescentus mutants**

<figure><img src="image_74.png" /></figure>

**Figure 2.** (A) $IC_{50}$ determination by fluorescent run-off transcription assay with *Mtb* RNA. Inhibitor concentration plotted against normalised response and fitted using non-linear regression with three parameters and shared top and bottom values for all data sets (GraphPad Prism 10.1.2). Performed in triplicate ($n=3$), centerline and error bars represent mean ± standard deviation (s.d.). $IC_{50}$ values given in µM ± s.d. (B) Growth inhibition of *M. tuberculosis* H37Rv poly-g-GFP determined by fluorescent read-out. Inhibitor concentration plotted against normalised response and fitted using non-linear regression with variable slope and four parameters, lower bound was constrained to 0 (GraphPad Prism 10.1.2). Performed in triplicate ($n=3$), centerline and error bars represent mean ± s.d. (C) Minimal inhibitory concentrations against a Rif-resistant *Mtb* isolate and *M. tuberculosis* H37Rv poly-g-GFP determined by turbidity read-out. (D) Physicochemical property plot (clogP vs. MW). clogP was calculated using *OSIRIS DataWarrior 6.1.0*.¹⁶ Colour coding corresponds to potency against *Mtb* ($IC_{90}$) as determined in panel B. (E) Activity against *C. crescentus* pfhuA\_AcrAB-nodT and *C. crescentus* pfhuA determined by Kirby-Bauer disk diffusion method. Inhibition zone was measured with ImageJ 1.8.0. Performed in triplicate for *Fdx*, **16**, **18**, and **20** ($n=3$) and in duplicate for **8** and *FdxG2-iBu* ($n=2$), centerline and error bars represent mean ± s.d. [a] The shunt metabolite *FdxG2-iBu* also lacks the C18-OH group of *Fdx* as it is installed later in the biosynthesis.

100 µM). Therefore, the main challenge we faced was
regaining activity by mimicking how the rhamnoside-arene
fragment binds to RNAP through careful design of the
nucleophiles. Because even if the potency of Fdx is not
entirely restored, this may be outweighed by the potential
improvements in physicochemical properties owed to a
smaller molecular size of the derivatives. We were delighted
to see that 2-aminothiazoles **13** ($IC_{50}$ 0.13±0.05 µM) and **10**
($IC_{50}$ 0.17±0.06 µM) inhibit RNAP within an order of
magnitude of Fdx, which is remarkable considering the 2-
aminothiazoles are drastically less complex than the rham-

noside-dichlorohomoorsellinate moiety. Notable is also the
increase in RNAP inhibition from amine **27** ($IC_{50}$ > 100 µM)
to benzamide **30** ($IC_{50}$ 0.62±0.29 µM), which may be due to
additional interactions created by the dichlorohomoorselli-
nate mimic.

Next, we investigated whether the observed on-target
activity also translated to cultured *M. tuberculosis*. Growth
inhibition was determined by fluorescent read-out of a

*M. tuberculosis* strain expressing GFP.[52] Within the
aminomethyl oxa-/thiadiazole series, an increase in activity
against *Mtb* was observed with increasing length of the
introduced nucleophile (Figure 2B). Swapping phenyl for
methyl substitution on the oxadiazole (19→16) improved
activity against Mtb (IC90 4 µg/mL (inhibitory concentration
needed for 90% inhibition of fluorescence as proxy for
growth inhibition)). The thiadiazole 20, which features
increased substituent angles on the heterocycle, leads to
further improved antibiotic activity (IC90 2 µg/mL). For the
most potent 2-aminothiazole 10 an IC90 value of 8 µg/mL
was observed. Fdx derivatives could substitute Rifampicin
(Rif) in the treatment of infections caused by Rif-resistant
M. tuberculosis strains, since Fdx is also an RNAP inhibitor
with no Rif binding site overlap. Encouragingly, the
previously observed activities also translated to a Rif-
resistant Mtb isolate, as representative aminomethyl oxa-
thiadiazole derivatives (20–23) showed MIC values of up to
4 µg/mL (compared to Fdx MIC 1-2 µg/mL) (Figure 2C).
Taken together, these derivatives restore activity lost by Fdx
truncation to the same order of magnitude as Fdx (e.g. Fdx
IC90 0.5 µg/mL vs. 20 IC90 2 µg/mL). Potent derivatives were
obtained with a molecular weight reduction of more than
200 g/mol and lipophilicity reduction of more than two clogP
units (Figure 2D). With further development, these deriva-
tives could therefore fulfil physicochemical requirements
that are not met by Fdx or other previously described
derivatives. In addition, we evaluated the derivatives against
Clostridioides difficile, revealing 2-aminothiazole 10 (MIC
1 µg/mL) and 2-aminopyridine 25 (MIC 1 µg/mL) as the
most potent derivatives (see Supporting Information Ta-
ble 3).

Since Fdx is not active against most Gram-negative
(GN) bacteria, one of our goals is expanding its spectrum of
activity. For entry into GN bacteria, generally, smaller and
more polar compounds are preferred.[32,53] The presence of
primary amines can also be beneficial to compound accumu-
lation in GN bacteria.[54] Previously, it was established that
Fdx lacks GN activity due to poor cell permeation and
partially due to efflux.[14,55] This was elucidated using mutant
strains of the GN model organism *Caulobacter crescentus*
that harbours an aqueous (ungated) protein pore in the
outer membrane (*pfhuA*<sup>hyp</sup>) leading to a permeable enve-
lope and/or deletion in the *acrAB-nodT* genes (*ΔacrAB-
nodT*) which impairs antibiotic efflux.[56–59] We evaluated our
new derivatives against all three mutant *C. crescentus* strains
as well as the wild type. Fdx displayed an inhibition zone of
311 ± 4.0 mm² against Δ*acrAB-nodT* + *pfhuA*<sup>hyp</sup> cells. To our
delight, the oxadiazole **16** (322 ± 26 mm²) was as active as
Fdx against Δ*acrAB-nodT* + *pfhuA*<sup>hyp</sup> cells, suggesting a
similar level of *C. crescentus* RNAP inhibition (Figure 2E,
see *Supporting Information Table 3* for full data). The
derivatives obtainable via the residue switching platform
approach the “600 Dalton” threshold that has been observed
for compounds that are able to traverse the outer membrane
of GN bacteria.[32,60,61] Evaluation against the Δ*acrAB-nodT*,
*pfhuA*<sup>hyp</sup>, and WT strains of *C. crescentus* showed, that
despite the reduced size and increased polarity of the
derivatives, envelope permeation, and to a smaller extent
efflux, is still limiting activity against GN bacteria. As some
of our derivatives (**16**, **20**, and **18**, see Figure 2E and
*Supporting Information Table 3*) are nearly or equally as

potent as Fdx against permeable and efflux-deficient strains
of the GN *C. crescentus*, we hope that further optimisation
to improve uptake will expand the spectrum of Fdx anti-
biotics to encompass GN bacteria.

Conclusions

We developed a platform for the synthesis of new
fidaxomicin derivatives. Using site-selective palladium catal-
ysis, it is now possible to replace the rhamnoside-dichlor-
ohomoorsellinate residue of Fdx by primary amines, secon-
dary amines,

2-aminothiazoles, anilines, and 2-aminopyridines in one step from the unprotected natural product. This was demonstrated on gram scale, which underscores the suitability of this method to produce large amounts of the desired derivatives for biological characterisation. The introduction of reactive handles with the allylic substitution enabled rapid introduction of different carboxylic acids via amide bond formation and the introduction of different amines via SuFEx to the scaffold. Two vectors on Fdx were functionalized at once by combining the allylic substitution protocol with organocatalytic site-selective acylation. In total, 43 new Fdx derivatives were synthesised and evaluated for their biological activities. Every structural element of Fdx has been shown to be essential for biological activity. Therefore, restoring antibiotic activity after it has been lost through truncation of the structure is a great challenge. We synthesized derivatives based on computational design to mimic Fdx binding to RNAP. Several of the new derivatives are potent inhibitors of *M. tuberculosis* RNAP and partially or fully restore activity against *M. tuberculosis*, Rif-resistant *M. tuberculosis*, *C. difficile*, and mutant strains of *C. crescentus*. The new derivatives are less lipophilic and have lower molecular weight. As a result, they occupy previously inaccessible physicochemical space that is closer to the properties of approved antibiotics. The newly developed platform may therefore enable future development of the next generation of Fdx antibiotics.

CRediT Statement

Erik Jung: Conceptualization, Methodology, Investigation, Formal analysis, Writing – Original Draft, Visualization, Project administration. Tizian Griesser: Methodology, Investigation, Formal analysis. Jordan Costafrolaz: Methodology, Investigation. Ondine Duverger: Investigation, Formal analysis. Yves Mattenberger: Methodology. Silvia Dittmann: Investigation, Formal analysis. Andrea Dorst: Investigation, Conceptualization. Alexander Major: Investigation. David Dailler: Investigation, Conceptualization. Daniel Schäfle: Methodology, Investigation, Formal analysis. Susanne Sivers: Formal analysis, Resources, Supervision, Project administration, Funding acquisition. Konstantin Brodolin: Formal analysis, Resources, Supervision, Project administration, Funding acquisition. Patrick H. Viollier: Resources, Supervision, Project administration, Funding acquisition.
Peter Sander: Resources, Supervision, Project administra-
tion, Funding acquisition. Karl Gademann: Conceptualiza-
tion, Formal analysis, Resources, Writing – Review &
Editing, Supervision, Project administration, Funding ac-
quisition.

Supporting Information

Supporting Information contains supplementary Schemes,
Figures, and Tables, experimental procedures, compound
characterization, and NMR spectra. The authors have cited
additional references in the *Supporting Information*
(Ref. [62–74]).

Acknowledgements

E. J. is grateful to the SCS Foundation for an Alfred Werner Scholarship and Hoffmann - La Roche for financial support during master studies. We thank Prof. Liviu Movileanu (Syracuse University, USA) for providing us with the *fhuA* hyp sequence. We thank Laurence Degeorges for the help in cloning the hyperpore construct. We thank Dr. Felix Hartrampf for fruitful discussions. We gratefully acknowledge the Swiss National Science Foundation for financial support (212603 to KG). Research in the laboratory of P. Sander is supported by the Swiss National Science Foundation (310030_197699), the Federal Office of Public Health (3632001500), the Joint Program Initiative Antimicrobial Resistance (JPIAMR-ACOMa-2022-050), and University of Zurich. Research in the K.B. laboratory was supported by the French National Research Agency (ANR-20-CE44-0020-01).

Conflict of Interest

The authors declare no competing financial interest.

Data Availability Statement

Additional data were deposited at zenodo (10.5281/zenodo.13884496).

**Keywords:** Natural products • antibiotics • semisynthesis • site-selective catalysis • fidaxomicin

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Manuscript received: October 3, 2024

Accepted manuscript online: November 25, 2024

Version of record online: December 4, 2024