Simplified Novel Muraymycin Analogues; using a Serine Template Strategy for Linking Key Pharmacophores

Article abstract of DOI:10.1002/cmdc.202000033

The present status of antibiotic research requires the urgent invention of novel agents that act on multidrug-resistant bacteria. The World Health Organization has classified antibiotic-resistant bacteria into critical, high and medium priority according to the urgency of need for new antibiotics. Naturally occurring uridine-derived “nucleoside antibiotics” have shown promising activity against numerous priority resistant organisms by inhibiting the transmembrane protein MraY (translocase I), which is yet to be explored in a clinical context. The catalytic activity of MraY is an essential process for bacterial cell viability and growth including that of priority organisms. Muraymycins are one subclass of naturally occurring MraY inhibitors. Despite having potent antibiotic properties, the structural complexity of muraymycins advocates for simplified analogues as potential lead structures. Herein, we report a systematic structure-activity relationship (SAR) study of serine template-linked, simplified muraymycin-type analogues. This preliminary SAR lead study of serine template analogues successfully revealed that the complex structure of naturally occurring muraymycins could be easily simplified to afford bioactive scaffolds against resistant priority organisms. This study will pave the way for the development of novel antibacterial lead compounds based on a simplified serine template.

Full text of DOI:10.1002/cmdc.202000033

Accepted Article
Title: Simplified Novel Muraymycin Analogues; using a Serine
Template Strategy for Linking Key Pharmacophores
Authors: Santosh Rudrawar, Bhautikkumar Patel, Rachel V Kerr,
Alpeshkumar K Malde, Matthew Zunk, Timothy DH Bugg, and
Gary Grant
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000033
Link to VoR: https://doi.org/10.1002/cmdc.202000033
01/2020

10.1002/cmdc.202000033
ChemMedChem
FULL PAPER
Simplified Novel Muraymycin Analogues; using a Serine Template
Strategy for Linking Key Pharmacophores
Bhautikkumar Patel,[a,b,c] Rachel V Kerr,[d] Dr Alpeshkumar K Malde,[e,f] Dr Matthew Zunk,[a,b,c] Prof
Timothy DH Bugg,[d] A/Prof Gary Grant,[a,b,c] and Dr Santosh Rudrawar*[a,b,c]
Abstract: The present status of antibiotic research requires an urgent
invention of novel agents that act on multidrug resistant bacteria. The
World Health Organization (WHO) has classified antibiotic-resistant
priority bacterium into critical, high and medium priority according to
the urgency of need for new antibiotics. Naturally occurring uridine-
derived ‘nucleoside antibiotics’ have shown promising activity against
numerous priority resistant organisms via inhibition of the
transmembrane protein MraY (translocase I), which is yet to be
explored in a clinical context. The catalytic activity of MraY is an
essential process for bacterial cell viability and growth including that
of priority organisms. Muraymycins are one subclass of naturally
occurring MraY inhibitors. Despite having potent antibiotic properties,
the structural complexity of muraymycins advocates for simplified
analogues as potential lead structures. Herein we report the
systematic structure-activity relationship (SAR) study of serine
template linked simplified muraymycin-type analogues. This
preliminary SAR lead study of serine template analogues has
successfully revealed that the complex structure of naturally occurring
muraymycins could be easily simplified to afford bioactive scaffolds
against resistant priority organisms. This study will pave the way for
the development of novel antibacterial lead compounds based on a
simplified serine template.
peptidoglycan synthesis are not well explored in clinical context.
Therefore development of antibacterial agents targeting enzymes
involved in these initial stages could provide an exciting
opportunity to meet the current need for novel targets.6 The
transmembrane enzyme MraY (phospho-N-acetylmuramoyl-
pentapeptide-transferase; translocase I) is one such enzyme with
its active site located on the cytosolic side of the membrane,
which fulfils the requirement of a novel target.7 MraY catalyses the
first membrane-associated step of peptidoglycan formation which
involves transfer of an UDP-N-acetylmuramoyl (UDP-MurNAc)-
pentapeptide (-L-Ala-g-D-Glu-diaminopimelic acid/L-Lys-D-Ala-D-
Ala) (Park’s nucleotide) to the lipid carrier [C55 isoprenoid
phosphate (C55-P)/C50 decaprenyl phosphate (C50-P)], resulting in
the formation of lipid I (undecaprenyl-pyrophosphoryl-MurNAc-
pentapeptide) (Figure 1).8
Introduction
Bacterial resistance against clinically used antibiotics is an
emerging health concern in contemporary healthcare.1,2 To
address growing global resistance, the World Health Organization
(WHO) has published
a list of antibiotic-resistant priority
pathogens.3 The major objective of publishing this list was to
guide and promote research and development (R&D) of new
unexplored classes of antibiotics aimed at treating multidrug
resistant pathogens.4 Ideally, these novel antibiotics should target
clinically unexploited modes of action to avoid cross resistance
with existing drugs. Bacterial cell-wall peptidoglycan layer
biosynthesis has been a promising antibacterial target for
decades led by the discovery of β-lactam (penicillin) and
glycopeptide (vancomycin) classes of antibiotics.5 However,
these cell wall inhibitors target the late extracellular steps of
peptidoglycan synthesis. The early intracellular steps of
Figure 1. Reaction catalysed by the bacterial transmembrane enzyme MraY.8
The MraY enzyme (translocase I) has been identified as the
target of bacterial nucleoside antibiotics, a class of bacterial
derived natural products containing
a
nucleoside core
structure.9,10 Due to their promising antibacterial activity against a
broad spectrum of Gram-positive bacteria, nucleoside antibiotics
have been considered as promising lead structures for the
development of novel antibacterial agents. Based on their
structural characteristics, nucleoside antibiotics have been
classified into four classes: i) ribosamino-uridines, ii)
uridylpeptides, iii) tunicamycins, and iv) capuramycin.11,12
Muraymycins belong to the family of ribosamino-uridine type
nucleoside antibiotics, which were first discovered as a collection
of 19 structurally related secondary metabolites from a broth of a
Streptomyces sp. in 2002.13 More recently novel members of the
muraymycins class of natural products have been isolated from
the same bacterial strains.14 Muraymycin A1 has been reported
active against Gram-positive Staphylococcus (MIC: 2-16 µg/mL),
Enterococcus (MIC: 16-64 µg/mL), as well as Gram-negative E.
coli (MIC: <0.03 µg/mL).15
[a]
[b]
Menzies Health Institute Queensland, Griffith University,
Gold Coast, QLD 4222, Australia.
School of Pharmacy and Pharmacology, Griffith University,
Gold Coast, QLD 4222, Australia.
E-mail: s.rudrawar@griffith.edu.au
[c]
[d]
[e]
[f]
Quality Use of Medicines Network, Griffith University,
Gold Coast, QLD 4222, Australia.
Department of Chemistry, University of Warwick
Coventry CV4 7AL, United Kingdom
Institute for Glycomics, Griffith University,
Gold Coast, QLD 4222, Australia.
MaldE Scientific, https://maldescientific.com, Australia
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000033
ChemMedChem
FULL PAPER
In general, the muraymycin scaffold contains a (5¢S, 6¢S)-
introduced.18d Kristin et al. reported solid phase-based synthetic
strategy for accessing muraymycin analogues.18e Recently, Anna
et al. explored variations in the nucleoside unit of muraymycin
antibiotics.18f
glycyluridine (GlyU) core structure linked to a urea peptide moiety
by an alkyl linker.16 The urea peptide contains the non-
proteinogenic amino acid epicapreomycidine derived from a cyclic
arginine derivative. Based on the structural diversity of the central
L-leucine (Leu) unit of the peptide moiety, muraymycins have
been classified into four sub-classes, being series A to D (Figure
2).17 Subclasses A, B and C contain a (3S)-3-hydroxyl-L-leucine,
which are further distinguished between each other by varying
substitutions at the 3-hydroxyl position. Series A muraymycins
(e.g., A4 1) are 3-O-acylated with w-functionalised fatty acids
To address this synthetic complexity of muraymycin-based
natural products, the prime goal of our research is to investigate
bioactive, structurally simplified muraymycin analogues, which
are active against a range of bacterial strains. Herein, we describe
the full details of our efforts to simplify the complex structure of
muraymycins based on a serine template strategy for linking key
pharmacophores. This study includes a preliminary SAR for our
serine template linked simplified muraymycin analogues toward
discovery of novel structures effective against a range of drug-
resistant bacterial strains.
terminating with
a
guanidinium moiety. While series
B
muraymycins (e.g. B5 2) are 3-O-acylated with unfunctionalised
terminally branched fatty acids. The C-type muraymycins (e.g. C2
3) are not O-acylated at the 3-hydroxy-L-leucine, and the D-series
of muraymycin D2 4) contain proteinogenic non-hydroxylated L-
leucine at this position (Figure 2).
Results and Discussion
Design
To achieve the primary aim of synthesising simplified
analogues of muraymycins, it was essential to understand the
structural orientation of the key pharmacophores present in the
muraymycins. To start, key interactions reported for the co-crystal
structure of MraY (Aquifex aeolicus) in complex with the
muraymycin D2
4
(Figure 3), were investigated.21 This
crystallographic study revealed that the 5²-aminoribosyl and uracil
moieties of muraymycin D2 (MD2) bind to the nucleoside-binding
pocket of MraY like a two-pronged electrical plug inserted into a
socket.21 Moreover, these interactions were found to be the most
critical for binding of muraymycin D2 with the MraY enzyme.21
Specifically, the 5²-amino group of the aminoribose moiety
interacts with Asp-193 through a hydrogen bond and an
electrostatic interaction.21 The importance of the aminoribose
moiety has been substantiated. A 5’-defunctionalised (5’-deoxy)
muraymycin analogue was essentially inactive.22 Another very
specific key interaction found was for the uracil nucleobase, which
binds in a neighboring pocket interacting with Asn-255, Asp-196,
Lys-70 through hydrogen bonding as well as with Phe-262 via p-
stacking.21 These crystallographic findings were found to be in line
with reported literature while evaluating the core aminoribosylated
glycyluridine (GlyU) against MraY (from Escherichia coli).23 It was
found that the core aminoribosylated glycyluridine unit exhibit
significant anti-MraY activity (IC50 = 50 µM), reflecting the minimal
structural requirement responsible for antibacterial activity against
MraY (from Escherichia coli).23 After having sufficient literature
reports supporting the aminoribose and uridine moieties as an
essential structural requirement for interaction in the active site of
MraY,21-23 it was decided to retain both the pharmacophores
within our intended simplified muraymycin scaffold.
Figure 2. Chemical diversity of naturally occurring muraymycin sub-classes A-
D
A
significant challenge in order to create drug-like
compounds is muracycin’s complex and synthetically challenging
structure.18a-f,19,20a-b To date, two approaches are reported in the
literature towards the total synthesis of naturally occurring
muraymycins.18a,b Tanino et al. reported the first total synthesis of
muraymycin D2 in total 18 steps with a 4.3% overall yield.18b While
the second report for stereo-controlled total synthesis of
muraymycin D1 was published by Kurosu et al. in 19 steps with
overall yield of only 15.9%.18a Moreover, Kurosu et al. evaluated
muraymycin D1 and its amide derivatives for antibacterial
activity.18a These muraymycin analogues exhibited growth
inhibitory activity against Mycobacterium tuberculosis (MIC50
1.56-6.25 µg/mL) and strong enzyme inhibitory activities against
the bacterial phosphotransferases (MurX and WecA) (IC50
=
0.096-0.69 µM).18a Furthermore, Kurosu et al. created an
anticancer DPAGT1 inhibitor from a muraymycin biosynthetic
intermediate.18c Another group of scientists from Wyeth also
worked on the semisynthetic access and have synthesised a total
of 16 derivatives of muraymycin C1.18d Structure-activity
relationship (SAR) studies of these synthesised analogues
suggested that the mono-substituted derivatives demonstrated
good inhibitory activity against MraY or MurG (6.25 µg/mL), which
importantly, correlated with the lipophilicity of the substituents
2
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10.1002/cmdc.202000033
ChemMedChem
FULL PAPER
R
Serine template
R Asp193
R
Thr75
R
Asn190
R
R
O
R
Gln305
R
R
O
O
H
O
Phe262
HO
Lipophilic
side chain
O
H2N
H3
C
OH
O
N
NH2
R
N
NH
N
H
n
O
H2N
5''
O
HO
HO
OH
3''
4''
O
O
2''
1''
HO
O
H2N
Uridine moiety
O
O
H
H
H
H
N
H
Asn255
R
H2N
O
N
N
N
5
O
HO
N
5
HO
OH
6
2
R
4
O
H
5'
1
N
3
O
O
O
NH
HO
4'
1'
H
O
HO
NH
NH
3' 2'
5-Aminoribosyl moiety
4
OH
O
O
OH
N
Figure 4. The proposed design of our simplified serine template-based
R
H
Asp196
muraymycin analogous.
R
H
N
H2N
N
R
Lys70
R
His325
Chemistry
R
R
Figure 3. Key interactions of muraymycin D2 with the MraY enzyme (PDB:
5CKR).21
Investigation of the serine template basic scaffold
The SAR study of the serine template linked truncated
muraymycins analogues was initiated by synthesis and biological
evaluation of the core serine template (L-serine) linked structure
6, comprising uridine, 5¢-aminoribose and octanoic acid as key
pharmacophores. This core serine template linked molecule 6
could be synthesised from their synthetic equivalents, including
the isopropylidene protected 5¢-amino uridine 7, the ribose
building block 8 (glycosyl donor) and the serine template (glycosyl
acceptor) 13 as described in retrosynthetic analysis (Figure 5).
Next, regarding substitutions on the core pharmacophore,
the literature reports that there is strong hydrophobic interaction
between muraymycin D1 and the MraY hydrophobic tunnel.24 The
significant antibacterial activity of lipophilic side chain containing
molecules suggests that the side chain itself might be contributing
to the membrane permeability of the molecules as MraY is an
integral membrane protein and its active site exists on the
cytoplasmic side of the membrane.24 As the peptidoglycan lipid
bilayer is a common barrier for bacteria, incorporating a lipophilic
group appears necessary in simplified analogues to afford any
antibacterial activity.24 Following the trend reported for inclusion
of a lipophilic side chain,24 it was decided to retain this
characteristic while designing our simplified muraymycin scaffold.
O
6
O
H
N
O
N
NH
N
H
O
O
O
O
HO
OH
In this work, the principle objective was to link key
pharmacophores with a simplified linker to afford rapid access to
biologically active muraymycins analogues. Having encountered
the significant contribution of the 5²-aminoribose,21-23 uridine21,23
and lipophilic side chain,24 we turned our attention to link these
pharmacophores together to achieve bioactive muraymycins.
Naturally occurring muraymycins contain a 5¢-glycyluridine core
unit with specific stereochemistry to hold key pharmacophores in
a specific orientation. Synthetic access to this 5¢-glycyluridine
(GlyU) core unit is not trivial and involves multi-step challenging
synthetic routes.25 Henceforth, to achieve the prime objective of
chemically simplified muraymycins analogues, we have selected
a serine template as a linker to which the desired key
pharmacophores can be linked via conventional amide-coupling
methodologies (5, Figure 4). Additionally, we also aimed to vary
the lipophilic side chain by investigating the linear carbon chain
length to achieve optimised antibacterial activity. Moreover, as
revealed in the X-ray co-crystal structure,21 orientation of both 5²-
aminoribosyl and uracil moieties of muraymycin is crucial for
binding with the MraY enzyme. So, in this proof-of-principle study,
we aimed to investigate the stereochemical outcome of the
proposed serine template by using both enantiomers L and D-
serine amino acids as starting points, while performing total
synthesis of serine template analogues.
H2N
HO
OH
O
O
O
O
F
N
H
NH
H2N
N3
N
Fmoc
O
O
O
O
O
O
HO
Protected 5'-Amino
Glycosyl donor
8
Glycosyl acceptor
13
Uridine
7
Figure 5. The retrosynthetic analysis of core serine template (L-serine) linked
structure 6.
Synthesis of the serine template based muraymycin
analogue 6 was started by independently synthesising each of
building blocks via modified literature reported methods. The
isopropylidene protected 5¢-amino uridine 7 was synthesised from
commercially available uridine in four synthetic steps in
quantitative yields (58%).26 After protection of the secondary
hydroxyl groups (2¢,3¢-diol) of uridine with isopropylidene unit, the
primary hydroxyl group (5¢-hydroxyl) was activated through
addition of a p-toluenesulfonyl ester to produce a tosylate product.
Subsequent nucleophilic substitution of the tosyl group with NaN3
gave the azido derivative in high yield (95%). The building block
7 was prepared by reduction of the corresponding isopropylidene
protected 5'-azido uridine readily obtained from commercially
available uridine in four steps, according to known routes.26 To
avoid the undesired Michael addition already described in
Staudinger conditions for this reaction,27 a catalytic hydrogenation
3
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10.1002/cmdc.202000033
ChemMedChem
FULL PAPER
O
O
O
was preferred and its duration was optimised to limit the formation
of over reduced side product. In these optimised conditions (Pd/C,
MeOH, 1 hour) the expected amine 7 was obtained in 90% yield,
it was used without further purification in the next reaction due to
its inherent instability
a
b
HO
O
O
OH
Fmoc
O
O
HN
HN
HN
Fmoc
Fmoc
13,14
9,10
11,12
O
F
N3
c
O
O
Synthesis of the ribose building block 8 as a glycosyl donor
was carried out concurrently. Prior to synthesis, a thorough
literature search related to key structural features that
aminoribose linkage should contain to have antibacterial active
was undertaken.28 Importantly, ribosyl building blocks must be β-
glycosides to retain antibacterial activity, in naturally occurring
muraymycins.28a The introduction of a 5¢-aminoribose moiety in a
β-selective manner was a major challenge towards the total
synthesis of our simplified serine template analogues.28a Hirano
et al. have reported an optimised protocol towards this desired
outcome.28a They established that to achieve β-selectivity for the
8
O
O
O
O
OH
Fmoc
O
d
O
O
N3
HN
N3
HN
Fmoc
O
O
O
O
17,18
15,16
Scheme 1. Reagents and conditions: a) allyl bromide, DIPEA, acetonitrile, 40°C,
4 h, 90% (for comd. 11), 87% (for comd. 12) b) TFA:DCM (1:1), rt, 1 h, 85% (for
comd. 13), 84% (for comd. 14), c) BF3·OEt2, CH2Cl2, MS 4Å, 0°C, 1 h, 62% (for
comd. 15), 61% (for comd. 16), d) Pd(PPh3)4, CH2Cl2, N-methylaniline, rt, 1 h,
84% (for comd. L-17), 82% (for comd. D-18).
glycosylated product, the ribofuranoside must have
a 3-
After the successful linking of the ribose intermediate 8 with
the serine template (either in 15 or 16), we planned to introduce
the uridine pharmacophore 7 on to the serine template. The plan
was executed by reacting either acid intermediates L-17 or D-18
pentylidene protecting group across the 2,3-diol, a fluoride leaving
group at C-1 and an azide group at the C-5 position.28a Therefore,
based on this report we decided to use the optimised ribose
building block 8 as our glycosyl donor,28a starting the synthesis
from commercially available D-ribose.
with the previously synthesised 5¢-amino uridine
7 via a
conventional amide coupling reaction (Scheme 2). The resulting
amide product L-19 or D-20 was further reacted for Fmoc
deprotection to yield the corresponding amino compound L-21 or
D-22 (Scheme 2). This amine product (21 or 22 Scheme 2) is an
advanced precursor for the synthesis of our target serine template
analogues.
In order to assemble the various synthetic fragments
together, we also required synthesis of the serine template
(glycosyl acceptor) (Scheme 1). For the preparation of this
compound, commercially available Fmoc-Ser(tBu)-OH 9 (L-
isomer) or 10 (D-isomer) was first converted into the allyl ester L-
11 or D-12. The tert-butyl ether was then deprotected with TFA to
afford the desired serine intermediate (glycosyl acceptor) L-13 or
D-14 ready for the glycosylation reaction with 8. Having all the
building blocks in hand, we first planned to couple the uridine 7
and ribose 8 together with the serine template L-13 or D-14
followed by linking of the lipophilic side chain. The Hirano et al.28
had evaluated different reaction conditions for this transformation
such as using various activators like BF3·OEt2, AgOTf/Cp2HfCl2,
AgOTf/SnCl2 (or) AgClO4/SnCl2 under varying temperature
conditions. They observed the use of BF3·OEt2 as an activating
agent at 0°C was the best outcome in comparison with other
conditions.28 The ribosylation reaction was followed as reported
by the Hirano et al.28 and reproduced successfully yielding the
glycosylated product with only β-anomer L-15 or D-16. After
purification, the glycosylated product L-15 or D-16 were reacted
for deallylation to afford the corresponding acid compounds.
However, initial deallylation attempts by using Pd(PPh3)2Cl2 and
phenylsilane resulted in undesired side product with reduction of
the azide functionality. This was assumed due to the inherent
reduction properties of organosilane (phenylsilane) to act as a
hydride donor. The issue was resolved by replacing phenylsilane
with N-methylaniline for deallylation of the glycosylated products
L-15 or D-16, which afforded the desired corresponding acid
intermediates L-17 or D-18 efficiently (Scheme 1).
O
O
N
NH
H2N
O
O
O
O
H
N
O
O
O
N
NH
O
Fmoc
O
N
O
OH
Fmoc
H
N3
O
HN
7
O
O
O
N3
O
O
a
O
O
19,20
17,18
b
O
O
O
H2N
O
N
NH
N
H
O
O
O
O
N3
O
O
21,22
Scheme 2. Reagents and conditions: a) EDC·HCl, HOBt, DMF, rt, overnight,
84% (for comd. 19), 82% (for comd. 20), b) piperidine (20%), DMF, rt, 1 h.
After optimisation of the conditions toward the advanced
stage amine intermediate 21 and 22, we focused on preparing our
target structures. This work began by optimisation towards the
western zone of muraymycins, preparing the synthetically
accessible core structure 6, having three building blocks including
the uridine, the aminoribose and lipophilic side chain (octanoic
functional group) linked together onto the serine template. The
designed target molecule 6 was synthesised starting from 21 in
three reaction steps (Scheme 3). Firstly, the free amino group of
the amine product 21 was reacted for amide coupling with
octanoic acid. Next the azide group of ribose was subsequently
reduced to the amine under Staudinger reaction conditions.
Finally, the target compound 6 was produced by a global acidic
deprotection of the amino intermediate with 80% TFA in water
(Scheme 3), followed by its isolation through preparative HPLC.
4
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10.1002/cmdc.202000033
ChemMedChem
FULL PAPER
O
O
O
O
O
O
H2N
O
N
H2N
O
N
NH
NH
R =
N
H
N
H
24
25
26
O
O
O
O
O
O
O
O
N3
N3
R =
R =
O
O
O
O
21
21
a,b,c
a,b,c
O
O
O
O
27
28
R =
R =
H
H
O
O
N
N
N
N
NH
N
NH
R
N
H
H
O
O
O
O
HO
OH
O
HO
OH
O
O
O
H2N
H2N
HO
OH
HO
OH
6
Scheme 4. Reagents and conditions: a) EDC·HCl, HOBt, DMF, rt, overnight, b)
PPh3, H2O, THF/toluene (1:1), 50°C, 18 h, c) aqueous TFA (80%), rt, 5-6 h, 37%
(for comd. 24), 31% (for comd. 25), 36% (for comd. 26), 27% (for comd. 27),
29% (for comd. 28).
Scheme 3. Reagents and conditions: a) EDC·HCl, HOBt, DMF, rt, overnight, b)
PPh3, H2O, THF/toluene (1:1), 50°C, 18 h, c) aqueous TFA (80%), rt, 6 h, 26%
in 4 steps.
Investigation of the lipophilic side chain
Investigation of the amino acid spacer
Owing to the lipophilic side chain contribution toward the
membrane permeability of muraymycins, further work was carried
out to investigate the importance of this moiety. We wanted to
evaluate the effect of increasing lipophilicity of the synthetically
accessible core structure 6, and how that impacted on
antibacterial properties. Therefore, different target molecules
were designed with varied lipophilicity in the side chain (23, Figure
6).
After the successful creation of the lipophilic side chain
intermediates 24-28, we next focused on investigating the
simplified peptide unit in place of synthetically challenging urea-
peptide moiety of the western zone. Previously, Tanino et al.
performed a systematic SAR study of the accessory urea-peptide
moiety.24 In this study, diverse muraymycin analogues were
synthesised by replacing the native L-epi-capreomycidine with
simple amino acids such as L-arginine, L-ornithine, L-methionine
and L-valine.24 The results from biological evaluation suggested
that the synthetically challenging western zone could be simplified
by using amino acids with a lipophilic side chain.24 Inspired by this
SAR study24 which was carried out towards simplification of the
western zone of the muraymycin structure, we planned to
introduce a simplified amino acid spacer between the serine
template and the lipophilic side chain. The reasoning behind the
introduction of a spacer in between the serine template and
lipophilic side chain was to mimic the alkyl linker which is present
between the core 5¢-glycyluridine and lipophilic side chain of
naturally occurring muraymycins.13,15 It was envisaged that
introduction of the spacer molecule would orientate the introduced
pharmacophores in order to best retain antibacterial activity. The
approach employed uses different amino acids as a spacer,
selecting them based on their varied polarity (29, Figure 7). We
selected non-polar aromatic amino acids such as phenylalanine
and tryptophan, a polar aromatic amino acid such as tyrosine and
a polar aliphatic amino acid such as lysine.
Lipophilic
side chain
O
O
H
O
N
N
NH
N
n
H
O
O
HO
OH
O
O
H2N
Uridine moiety
HO
OH
5ʹ-Aminoribosyl
moiety
23
Figure 6. The proposed serine template (L-serine) linked scaffold with varied
lipophilic side chains 23
Previous SAR studies have reported that addition of a
lipophilic side chain beyond octanoic acid to nonanoic 24,
undecanoic 26 and tridecanoic 28 functional groups helped
increase antimicrobial activity. Therefore, we began work on
inducing increased chain length intermediates to mimic the most
active natural product muraymycin A1 (C13 carbon length).
Amino acid linker part
Furthermore,
introduction
of
unsaturation
increases
hydrophobicity of the molecule, which should enhance membrane
permeability. With this idea in mind, we also synthesised
extended target molecules having linear lipophilic side chain with
unsaturation, such as 8-nonenoic 25 and undecylenic 27
functional groups (Scheme 4). At this stage, all of the synthesised
target molecules (24-28) were evaluated for antibacterial activity
(see biological evaluation section).
Lipophilic side chain
O
O
O
H
N
O
N
NH
N
N
H
H
O
O
HO
OH
O
O
H2N
Uridine moiety
HO
OH
5-Aminoribosyl
moiety
29
Figure 7. The proposed serine template (L/D-serine) linked scaffold having
different amino acids as spacer unit 29.
Synthesis of target molecules with the amino acid spacer
was initiated by independent synthesis of acid intermediates 30-
5
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10.1002/cmdc.202000033
ChemMedChem
FULL PAPER
33 followed by linking them on to core structure of the serine
template 21 (Scheme 5). The acid intermediates, which all contain
different amino acids (tyrosine 30, tryptophan 31, phenylalanine
32 and lysine 33) and a tridecanoic lipophilic side chain
(mimicking the natural product muraymycin A1), were prepared
by a conventional four step synthetic protocol as summarised in
SI. With the successful synthesis of the acid intermediates 30-33,
each was efficiently transferred to the core serine template
structure 21 in a similar manner to the creation of compound 6.
Following on from the successful amide coupling reaction of 21
with the acid intermediates 30-33, the resulting amide coupled
products were further reacted under Staudinger reaction
conditions to produce the amino intermediates, which were all
globally deprotected under acidic conditions to afford our final
target molecules 34-37 (Scheme 5).
O
R1
O
R
O
O
O
O
O
H
N
H2N
O
N
O
NH
N
N
H
OH
NH
N
H
N
N
H
H
O
O
O
O
O
O
O
N3
O
HO
OH
O
H2N
a, b, c
O
O
HO
OH
O
21
30 R =
HO
=
=
32 R
34 R1
=
=
36
37
=
=
R1
R1
Boc
N
H
N
HN
31
33
R
R =
Boc
H2N
35
R1
Scheme 5. Reagents and conditions: a) EDC·HCl, HOBt, DMF, rt, overnight, b) PPh3, H2O, THF/toluene (1:1), 50°C, 18 h, c) aqueous TFA (80%), rt, 5-6 h, 39%
(for compd. 34), 44% (for compd. 35), 44% (for compd. 36), 42% (for compd. 37).
chain, except having opposite stereochemistry of serine template
by using
D
-serine as a linker. Synthesis of the target molecules 38
At this stage of our SAR study of simplified serine template
analogues of muraymycins, we were interested in investigating
stereochemical constraints related to the serine template itself
and how this may contribute toward antibacterial activity.
Therefore, we designed target molecules by using tyrosine and
tryptophan as spacer molecule and tridecanoic lipophilic side
and 39 was performed exactly in the same fashion except starting
from the advanced stage precursor 22 (R, by using
linker) (Scheme 6).
D
-serine as
O
R1
O
R
O
O
O
O
O
H
N
H2N
O
N
O
NH
N
N
H
OH
NH
N
H
N
N
H
H
O
O
O
O
O
O
O
N3
O
HO
OH
O
H2N
a, b, c
O
O
Boc
N
HO
OH
O
22
HN
HO
30
R =
31 R =
38 R1
=
39
R1
=
Scheme 6. Reagents and conditions: a) EDC·HCl, HOBt, DMF, rt, overnight, b) PPh3, H2O, THF/toluene (1:1), 50°C, 18 h, c) aqueous TFA (80%), rt, 5-6 h, 48%
(for compd. 38), 31% (for compd. 39).
.
Biological Evaluation
the core serine template compound 6 did not show any
antibacterial activity against all the screened bacterial pathogens
(MIC > 32 µg/mL). As the most active analogue of naturally
occurring muraymycins A1 contains a lipophilic side chain of n =
13, we were anxious to investigate the effect of increasing
lipophilicity on antibacterial activity, comparable to that of the
natural product. Therefore, we have synthesised and screened
compounds 24-28 having a longer linear lipophilic side chain such
as nonanoic 24, undecanoic 26, and tridecanoic 28 functional
groups. We also explored molecules with higher lipophilicity,
which contained unsaturated lipophilic side chains, such as
undecylenic 27 and 8-nonenoic 25 functional groups. However,
none of the lipophilic side chain analogues 24-28 showed any
antibacterial activity against resistant bacterial pathogens (MIC >
32 µg/mL). This set of biological results suggested that increasing
lipophilicity equivalent to that of the naturally occurring
muraymycin A1 was also not enough to generate a significant
antibacterial agent. Therefore, it was clear that linking only three
key units was not sufficient to generate the bioactive scaffold. We
All newly synthesised serine template linked muraymycins
analogues 6,24-28,34-37,38-39 were evaluated for antibacterial
activity against a range of clinically relevant bacterial pathogens,
including S. aureus ATCC 43300 (methicillin-resistant), E. coli
ATCC 25922, K. pneumoniae ATCC 700603, P. aeruginosa
ATCC 27853, A. baumannii ATCC 19606. Colistin and
vancomycin were used as positive bacterial inhibitor standards for
Gram-negative and Gram-positive bacteria, respectively (MIC
values are depicted in Table 1). Interestingly, these
microorganisms are all priority 1 (critical) or 2 (high) listed
pathogens according to WHO classification.3 The results obtained
from a dose response assay to determine the minimum inhibitory
concentration (MIC) are summarised in Table 1. Each sample
concentration was prepared in 384-well, non-binding surface
plates (NBS; Corning 3640) for each bacterial strain. We first
screened the core serine template analogue 6 linked with three
pharmacophores, including uridine, aminoribose and the lipophilic
side chain (octanoic acid). However, contrary to our expectation,
6
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10.1002/cmdc.202000033
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had envisaged that further optimisation of the western zone was
therefore necessary.
39 lead to an increase in antibacterial activity against S. aureus
ATCC 43300 (methicillin-resistant) in comparison with the natural
To optimise western zone of the serine template analogues,
we planned to introduce an amino acid spacer in between the
serine template and lipophilic side chain. As we wanted to mimic
the lipophilicity of the natural product, we planned to retain
linearity of n = 13 carbon chain (tridecanoic functional group) as
present in muraymycin A1.13,15 We then selected representative
amino acids with varying polarity such as non-polar aromatic
(phenylalanine 36, tryptophan 35), polar aromatic (tyrosine 34),
and polar aliphatic (lysine 37) amino acids. The antibacterial
activity of this series of compounds was then evaluated, and the
results are summarised in Table 1. Interestingly, compounds
possessing tryptophan 35 and phenylalanine 36 amino acid linker
were found to be moderately active against multiple WHO priority
resistant strains. The phenylalanine linker containing molecule 36
was active against S. aureus ATCC 43300 (methicillin-resistant)
(MIC = 32 µg/mL), E. coli ATCC 25922 (MIC = 32 µg/mL) and
P. aeruginosa ATCC 27853 (MIC = 32 µg/mL). The tryptophan
linker containing molecule 35 was also active against multiple
bacterial strains, including S. aureus ATCC 43300 (methicillin-
resistant) (MIC = 32 µg/mL) and A. baumannii ATCC 19606 (MIC
= 32 µg/mL). However, the tyrosine linker analogue 34, exhibited
no antibacterial activity (MIC > 32 µg/mL). Additionally, we
investigated a polar aliphatic amino acid (lysine amino acid linker
37) which was also found inactive. Based on the results we had
obtained the serine template with a non-polar aromatic amino acid
(phenylalanine and tryptophan) linker appeared to be essential for
antibacterial activity. Conversely, the presence of a polar aromatic
amino acid (tyrosine) or polar aliphatic amino acid (lysine) linker
resulted in loss of antibacterial properties. Therefore, the
antibacterial activity of our simplified serine template analogues
was well-correlated with the presence of lipophilicity in the linker
part of serine template scaffold. This is consistent with the
observation by Mansour and co-workers, where lipophilicity of
semi-synthetic muraymycin C1 analogues directly related to the
biological activity.18d
amino acid template (
were observed against A. baumannii ATCC 19606 in both cases
-serine 39 and -serine 35). These results clearly show that a
presence of unnatural amino acid template ( -serine) replacing
natural amino acid template -serine) could form either
L
-serine) 35. However, equivalent results
(
D
L
D
(
L
equipotent or more potent antibacterial scaffold.
Next we turned to the screening of the serine template
analogues for MraY inhibitory activity (Table 2). This
radiochemical MraY assay was performed at 100 µM final
concentration against membranes containing overexpressed E.
coli MraY by a previously described method.29 Interpretation of
these results reveal that the inhibition of the MraY enzymatic
activity does not correlate with the antibacterial activity. The most
active hit compounds 35, 36 and 39 showed MraY inhibition of
only 3.0%, 0.0% and 5.7%, respectively. However, in contrast the
compounds 34 and 37 have shown significant MraY inhibition of
25.3% and 19.7%, respectively. We believe that this contradiction
between in vitro antibacterial activity and MraY enzyme inhibition
could be possible due partly to uptake issues, and the possibility
of other targets. Also, there are minor differences in the
sequences of the MraY enzymes across different bacterial
species,10b so it is possible that compounds 35,36 and 39 are
more active against MraY enzymes from different bacteria.
Table 2. The inhibitory activity of structurally simplified serine template
analogues of muraymycins against MraY enzymea
Compound
% MraY Inhibition
6
9.3
11.3
9.0
24
25
26
27
28
34
35
36
37
38
39
3.3
6.4
7.1
After optimising the minimum structural requirement of
serine template scaffold, we further investigated its
stereochemical requirement towards antibacterial activity. This
was achieved by screening two more compounds, which
25.3
3.0
contained the -serine template with a tryptophan 39 and tyrosine
D
0.0
38 as spacers, along with the lipophilic side chain mimicking the
natural product muraymycin A1 (n = 13 carbon length, tridecanoic
functional group). Though having similar structural characteristics,
these analogues 38 and 39 were designed by replacing the
19.7
4.4
natural
Interestingly, antimicrobial screening results for molecules 38 and
39 were similar to that observed for the -serine template
L
-serine template with the unnatural amino acid -serine.
D
5.7
aAssay was performed at 100 µM final concentration against membranes
containing overexpressed E. coli MraY. This MraY enzyme inhibitory assay was
performed with controls which included a no inhibitor negative control, and a
positive inhibitor control tunicamycin (100 µM). IC50 was measured from a plot
of enzyme activity vs. inhibitor concentration where the negative control was
defined as 100% activity.
L
analogues 34 and 35. The tryptophan linker containing molecule
39 showed activity against multiple bacterial strains, including S.
aureus ATCC 43300 (methicillin-resistant) (MIC = 16 µg/mL) and
A. baumannii ATCC 19606 (MIC = 32 µg/mL). However, analogue
38, the tyrosine linker, molecule had lost antibacterial activity (MIC
> 32 µg/mL). These results were in line with the previous
observation that non-polar aromatic amino acid linkers were
active antimicrobials, whilst polar aromatic amino acid linkers
reduced this observed activity (MIC > 32 µg/mL). These results
also allowed us to summarise that the presence of an unnatural
Selective toxicity towards bacterial strains is a key concern
in the development of chemotherapeutics for infectious disease.
Subsequently, determining the specificity of our synthesised
compounds towards bacterial cells and determination of their
toxicity towards mammalian cell is an important aspect of potential
clinical drug development. Accordingly, the lead serine template
analogues 35, 36 and 39 were evaluated via in-vitro cytotoxicity
assay against human embryonic kidney cells (HEK-293, ATCC
amino acid template containing -serine, as seen for compound
D
7
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10.1002/cmdc.202000033
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FULL PAPER
CRL-1573) as well as for haemolytic assay against human red
blood cells (RBC). The three screened serine template analogues
35, 36 and 39 were found to be free from any significant
cytotoxicity. However, in contrast all of them 35, 36 and 39 were
found to be haematotoxic against human red blood cells (RBC).
Table 1. Antibacterial and cytotoxicity values (µg/mL) of structurally simplified serine template analogues of muraymycins 35,36,39
Cytotoxicity
HEK-293c
MIC (µg/mL)
RBCd
(HC50)
Gram-positive bacteriaa
Sa
Gram-negative bacteriab
Kp
Compound
(CC50)
Ec
Pa
Ab
36
35
32
32
16
1
32
>32
>32
-
>32
>32
>32
-
32
>32
>32
-
>32
32
>32
>32
>32
-
11.26
11.54
39
32
<=0.25
Vancomycin
Colistin
-
-
-
-
0.125
0.25
0.25
0.25
-
a Sa (Staphylococcus aureus MRSA ATCC 43300).
b Ec (Escherichia coli ATCC 25922); Kp (Klebsiella pneumoniae ATCC 700603); Pa (Pseudomonas aeruginosa ATCC 27853); Ab (Acinetobacter baumannii
ATCC 19606).
c
HEK-293, Human embryonic kidney cells (ATCC CRL-1573, CC50).
d RBC, Human red blood cells (HC50).
Proposed binding mode of serine template analogues
three complexes. The crystal structure-derived bound
conformation of Muramycin D2 4 shows the lowest RMSD
amongst all ligands, and validates the MD simulation protocol and
parameters. Molecules 34 and 37 show slightly higher RMSD
values indicating the re-arrangement of the docked conformations
With exact MraY inhibition data of serine template
compounds in hand, we were interested if the relative inhibition
potencies of our serine template analogues could be correlated to
their structural characteristics by in silico modelling. To predict the
putative binding mode of the most active molecules 34 and 37
against a MraY enzyme, a combination of semi-automated
docking and molecular dynamics (MD) simulations was employed.
The initial structures of muraymycin D2 4 (from the crystal
structure), molecules 34 and 37 (derived from the guided docking)
in complex with the MraY enzyme are given in Figure S1. To
mimic the cell membrane environment, each protein-ligand
complex was inserted into a model lipid bilayer. The stability of
these complexes was evaluated using the root mean square
deviation (RMSD) of protein backbone and ligand atoms from two
independent 50 ns MD simulations for each of the complexes. The
RMSD plots (Figure S2) indicates that the protein is stable in all
to reach
a more stable state. The representative bound
conformations of ligands obtained after 50 ns of MD simulations
are shown in Figure 8. The key pharmacophores (GlyU and 5-
aminoribose) of 34 and 37 bind to the negatively charged cavity
as observed for Muramycin D2 4. The rest of the pharmacophores
(including the aliphatic chain) can hop around and bind to the
protein surface around the cavity. The aliphatic chain inserts into
a hydrophobic cavity at the junction of TM5 and TM9 helices. The
aliphatic chain plays a crucial role of being an anchor and we
believe this proposed binding of the serine template analogues
could be a guide to the rational design of selective inhibitors for
MraY.
Figure 8. Representative structure of muraymycin D2 4 (left) and serine template analogue 34 and 37 (middle and right, respectively) in complex with MraY from
Aquifex aeolicus, after 50 ns of MD simulations with the position of 4 derived from the previously reported X-ray co-crystal structure21 and the position of 34 and 37
derived from the guided docking. The electrostatic surface of the protein is shown with the colour spectrum red (negative) to white (neutral) to blue (positive) regions.
8
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10.1002/cmdc.202000033
ChemMedChem
FULL PAPER
scholarship (GUPRS and GUIPRS) from Griffith University. The
antimicrobial screening performed by CO-ADD (The Community
for Antimicrobial Drug Discovery) was funded by the Wellcome
Trust (UK) and The University of Queensland (Australia). The
computational work was funded with the assistance of high-
performance computing resources provided through the National
Computational Merit Allocation Scheme supported by the
Australian Government (Project cj47) and Queensland Cyber
Infrastructure Foundation (QCIF, Project fi49).
Conclusion
In conclusion, a systematic SAR study of simplified serine
template analogues was investigated. The simplified analogues
of muraymycins, having their key pharmacophores linked on a
serine template were synthesised through a multistep protocol.
Interestingly, these compounds have shown their antibacterial
potential against WHO priority organisms classified as priority 1
(critical) and priority 2 (high). Initial antimicrobial screening results
revealed that three pharmacophores, including uridine, 5-
aminoribose and the lipophilic side chain, were not enough to fulfil
minimum structural requirements for activity. Moreover, an
increment of linearity or unsaturation in the lipophilic side chain
was also not efficient for generating an active scaffold. However,
Keywords: Antibiotics • Nucleoside natural product • MraY •
Muraymycins • Structure-activity relationship
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9
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Entry for the Table of Contents
Systematic SAR study of 12 structurally simplified serine template linked muraymycin analogues were conducted. Our lead compounds
are active against WHO priority organisms classified as priority 1 (critical) and priority 2 (high), including one Gram-positive and four
Gram-negative bacterial strains. This preliminary investigation of serine template analogues has successfully revealed that the complex
structure of naturally occurring muraymycins could be easily simplified to afford bioactive scaffolds active against resistant priority
organisms and has set the stage for development of novel class of antibacterial agents.
10
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