Synthesis of Tetramic Acid Fragments Derived from Vancoresmycin Showing Inhibitory Effects towards S. aureus

Article abstract of DOI:10.1002/cmdc.202000241

An efficient route to various vancoresmycin-type tetramic acids has been developed. The modular route is based on an effective Fries-type rearrangement to introduce various appending acetyl residues. The minimum inhibitory concentration (MIC) values of the new tetramic acids against Staphylococcus aureus and Escherichia coli were determined, revealing that three of the new compounds exhibit antimicrobial activity against S. aureus. These bioactive compounds were structurally most closely related to the authentic vancoresmycin building block. Additionally, the compounds induced a lial-lux bioreporter, which responds to cell wall stress induced by antibiotics that interfere with the lipid II biosynthesis cycle. These data suggest the tetramic acid moiety to be a part of the vancoresmycin pharmacophore.

Full text of DOI:10.1002/cmdc.202000241

Accepted Article
Title: Synthesis of Tetramic Acid Fragments Derived from
Vancoresmycin Showing Inhibitory Effects Towards S. aureus
Authors: Lukas Martin Wingen, Marvin Rausch, Tanja Schneider, and
Dirk Menche
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To be cited as: ChemMedChem 10.1002/cmdc.202000241
Link to VoR: https://doi.org/10.1002/cmdc.202000241
01/2020

10.1002/cmdc.202000241
ChemMedChem
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Synthesis of Tetramic Acid Fragments Derived from
Vancoresmycin Showing Inhibitory Effects Towards S. aureus
Lukas Martin Wingen,[a] Marvin Rausch,[b,c] Tanja Schneider,[b] and Dirk Menche*[a]
[a]
[b]
[c]
M. Sc. L. M. Wingen, Prof. Dr. D. Menche
Kekulé-Institut für Organische Chemie und Biochemie
Rheinische Friedrich-Wilhelms-Universität Bonn
Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
E-mail: dirk.menche@uni-bonn.de
Dr. M. Rausch, Prof. Dr. T. Schneider
Institut für Pharmazeutische Mikrobiologie
Rheinische Friedrich-Wilhelms-Universität Bonn
Meckenheimer Allee 168, 53115 Bonn, Germany
E-mail: tschneider@uni-bonn.de
Dr. M. Rausch
German Center for Infection Research (DZIF), partner site Bonn-Cologne, Bonn.
Supporting information for this article is given via a link at the end of the document.
Abstract: An efficient route to various vancoresmycin type tetramic
acids has been developed. The modular route is based on an
effective Fries type rearrangement to introduce various appending
acetyl residues. The minimum inhibitory concentration (MIC) values
of the new tetramic acids against S. aureus and E. coli were
determined, revealing three of the new compounds to exhibit
antimicrobial activity against S. aureus. These bioactive compounds
were structurally most closely related to the authentic vancoresmycin
Introduction
Vancoresmycin (1, Figure 1) presents a structurally unique linear
metabolite that is characterized by a functionalized acetyl
tetramic acid connected to an extended polyketide chain with a
plethora of hydroxyl- and methyl-bearing stereogenic centers
and also contains an attached aminoglycoside moiety. It was
first isolated in 2002 from the fermentation broth of the
actinomycete Amycolatopsis sp. ST 101170.[1] This polyketide
shows extremely potent MICs values ranging from 0.125 to
2 µg/mL against a variety of pathogens, including multi-resistant
gram-positive bacteria. In 2017 the mode of action was
investigated and it was proposed that vancoresmycin is involved
in a non-pore-forming, concentration-dependent depolarization
of bacterial membranes.[2]
building block. Additionally, the compounds induced
a lial-lux
bioreporter, which responds to cell wall stress induced by antibiotics
that interfere with the lipid II biosynthesis cycle. These data suggest
the tetramic acid moiety to be
pharmocophore.
a part of vancoresmycin
NH2
HO
O
OH
O
OH
O
OH OH
OH OH
OH OH
O
OH OH
OH OH
OH OH
N
OH
O
1
Figure 1. Structure of vancoresmycin (1) with the proposed stereoinformation.[2]
Additionally, a stereochemical assignment based on domain
analysis of the ketoreductase domains was proposed for most of
the configurations, but was not proven yet.[2] As part of our
studies with potent polyketide antibiotics bearing extended
polyene segments[3–7] we became interested in developing a
synthetic route to the vancoresmycin type acetylated tetramic
acids bearing characteristic labile diene segments and to
evaluate their antibacterial properties. In general, tetramic acids
including their 3-acyl derivatives found in a variety of terrestrial
and marine species have been studied and reviewed
these compounds. Their intriguing structures enables complex
formation with various metal ions like Mg2+, Fe2+, Zn2+ and
Cu2+[13] and in some cases this type of chelation has been shown
to be essential for the biological activity.[14] Furthermore, these
features are characterized by extended tautomerism. As shown
in Scheme 1, 3-acyltetramic acids may occur in solution in four
forms (a, b, c, d), which may be classified as a pair of ‘external’
(ab/cd) and a pair of ‘internal’ (a/b; c/d) tautomers. Due to the
C-C bond rotation of the 3-acyl group, the interconversion
between the ‘external’ isomers is a rather slow process
compared to the NMR time scale resulting in separate NMR
signals in non-polar solvents (e.g. CD2Cl2).[15,16] Complexation as
previously.[8,9]
A
range of bioactivities like antibacterial,[10]
antiviral,[11] and antitumoral[12] potencies have been reported for
1
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10.1002/cmdc.202000241
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isobutyr-
aldehyde,
NaOH
O
O
well as tautomerism renders tetramic acids fascinating
heterocycles but also complicate synthesis and analysis.[8,9]
NaH, MeI
HN
HN
83%
96%
O
O
O
O
2
3
H
H
O
O
O
O
O
O
FAST
5
3
R
R
4
HCl
HN
HN
O
N
N
99%
a
b
O
4
5
SLOW
O
O
Scheme 2. Synthesis of compound 5.
R
R
O
O
HN
HN
H
FAST
H
O
O
For the elaboration of structures 12/13 and 17/18 (Scheme 3),
compound 8 was required. For synthesis of 8 a modification of a
reported procedure from propanediol (6) was applied.[18] In detail,
a Parikh-Doering oxidation was used instead of a Swern
reaction and a different Horner-Wadsworth-Emmons (HWE)
procedure was applied for better E/Z selectivity. Finally,
saponification gave desired 8, which was used for coupling with
compound 5. Various esters at the 4-position of compound 5 are
easily accessible using acid chlorides or esterification reactions
like the Steglich protocol using N,N’-dicyclohexylcarbodiimide
(DCC) and 4-(dimethyl-amino)-pyridine (DMAP) giving
compounds 9-12 (Scheme 3) in good to excellent yields.[15,17] For
realization of the required oxygen to carbon transfer all novel
tetramic acid esters 9-13 were submitted to a literature known
rearrangement using CaCl2 in presence of DMAP and
triethylamine.[19,20] This process efficiently realized the synthesis
of 3-acyl derivatives 14-18 in high yields. Acylation reagents
were chosen to be authentic to vancoresmycin and also to allow
further modifications like aldol condensation, cross metathesis or
a nucleophilic attack of the ester at the 3-position. The TBS
protecting group of compounds 12 and 17 can be cleaved either
at the ester stage to furnish 13 or at the final stage to give 18.
Notably, preparation of compound 18 represents the successful
synthesis of a vancoresmycin fragment, which is also the largest
part of the natural product that bears no chiral center.
c
d
Scheme 1. Tautomerism of 3-acyltetramic acids.
Results and Discussion
As shown in Scheme 2, our successful route to vancoresmycin
type tetramic acids started with introduction of the 5-substituent
to commercially available 4-methoxy-3-pyrrolin-2-on (2). As
previously reported, isobutyraldehyde was added in basic
conditions to give compound 3 in high yield.[17] After methylation
of the ring nitrogen with iodomethane, the methoxy group was
cleaved under acidic conditions yielding compound 5 in 79%
over three steps, without the need of column chromatography. If
required, any intermediate may also be purified using column
chromatography.
O
O
1.
P(OEt)2
EtO
1. imidazole,
TBSCl, 90%
Ba(OH)2, 81%,
E/Z = 16:1
O
OTBS
OTBS
OH
HO
HO
O
R =
10
15 (86%)
11
(87%)
16 (quant.)
9 (86%)
14
(99%)
O
2. SO3*py, DMSO,
DIPEA, 98%
2. KOH, 75%
(quant.)
6
7
8
HCl
quant.
O
R
OH
12
13
18
(89%)
(99%)
OTBS
O
NEt3,
O
O
OH
O
CaCl2, DMAP, NEt3
HCl
Cl
R
17
quant.
R
or
N
N
N
8 for compound 12,
DCC, DMAP
O
O
O
5
9-13
14-18
Scheme 3. Synthetic route to vancoresmycin derived 3-acyltetramic acids (14-18).
In CD2Cl2 tautomerism was observed for all tetramic acids 14-18, broadening of the signals. For compound 15 even more data
resulting in double data sets of NMR measurements and also in sets could be observed in the NMR, due to the extended
2
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10.1002/cmdc.202000241
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COMMUNICATION
polyene system, emerging from the additional conjugated double
bond. On the other hand, compound 16 showed only traces of a
second data set, which indicates a stabilization of the double
bond system by the introduced ester group. Several trials to
protect and trap one of the tautomers of 14 as a silyl ether failed.
Therefore, protection of 16 was evaluated as this representative
is characterized by a lower degree of such isomerization. Here,
an allylic protection was chosen as silyl enol ethers would be
expected to be more labile. However, likewise the hydroxyl
group could not be protected in initial experiments. Instead, the
allyl group was attached to the 3-position yielding compound 19
as a racemic mixture, which was proven by HPLC on chiral
phase. Future investigations of suitable reaction conditions will
show whether the alkylation may be tuned with respect to the
oxygen to carbon regioselectivity.
compared to 15 (MIC = 32 μg/mL), even though 18 is the longest
and most authentic compound. A possible reason for this
discrepancy may be due to lower degrees of stability, as
evidenced by NMR studies showing that 18 is not stable in
DMSO or acetonitrile. Additionally, 18 decomposes when stored
over weeks (under argon, at −15 °C). No activities were
observed against E. coli, which is in agreement with the reported
data for vancoresmycin.[2]
To see if the synthesized compounds interact with the bacterial
cell wall machinery they were further screened in a lial-lux
bioreporter assay, which responds to cell wall stress induced by
antibiotics that interfere with the lipid II biosynthesis cycle
substantiating again antimicrobial effects of the compounds, in
line with the MIC values.[21] As shown in Figure 2, 14, 15 and 18
induce the B. subtilis lial-lux bioreporter, resulting in measurable
luminescence and revealing interference with cell wall
biosynthesis. A figure including the lial-lux bioreporter assay
outcome of additional novel tetramic acids can be found in the
supporting information.
OH
O
O
O
O
K2CO3, allyl bromide
82%
O
N
N
O
O
16
19 rac.
O
O
O
Br
N
O
Scheme 4. Synthesis of compound 19 and the proposed mechanism.
In total, 14 tetramic acid derivatives, i.e. 3-5 and 9-19, have
been synthesized with 13 not reported before. Only 3 has been
described previously.[17] For these compounds the MIC values
against gram-positive and gram-negative bacteria, i.e. E. coli
K12 and S. aureus SG511 were determined and summarized in
Table 1.
Figure 2. Results of the lial-lux bioreporter assay. VAN = Vancomycin. In the
negative control no antibiotic compound was added to the assay.
Table 1. MIC values of the synthesized compounds.
MIC
[μg/mL]
E. coli
K12
MIC
MIC
[μg/mL]
E. coli
K12
MIC
[μg/mL]
S. aureus
SG511
[μg/mL]
S. aureus
SG511
No
No
Conclusion
In summary, we have reported an efficient synthetic route to
novel vancoresmycin-type 3-acyl tetramic acids. The successful
route utilizes an aldol condensation to efficiently attach the
alkyliden substituent at C-5 and a Fries-type rearrangement for
introduction of the acetyl residue. Additionally, we determined
the MIC values against E. coli and S. aureus identifying three
compounds with antimicrobial activities against the gram-
positive pathogen. Among all tested compounds the active
heterocycles are structurally most closely related to
vancoresmycin, indicating that this segment is part of the
vancoresmycin pharmacophore. These structures are also
characterized by high degrees of tautomerism, which may
suggest that polyene shifts are important for biological activity.
The active tetramic acids also induced the B. subtilis lial-lux
bioreporter, revealing their interference with cell wall
biosynthesis.
3
4
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
13
14
15
16
17
18
19
>128
>128
>128
>128
>128
>128
>128
128
64
5
32
9
128
>128
>128
64
10
11
12
128
>128
>128
>128
As shown in Table 1, some of the synthesized tetramic acids
demonstrated inhibitory effects against S. aureus. The more
potent compounds were compounds 14, 15 and 18, which are
also most similar as compared to vancoresmycin. Among these
compounds 18 was a little less potent (MIC: 64 μg/mL) as
3
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Acknowledgements
Financial support by the DFG (TRR261) is gratefully acknowl-
edged. Furthermore, we thank Andreas J. Schneider (University
of Bonn) for excellent HPLC support.
Keywords: Vancoresmycin • tetramic acids • antibiotic •
tautomerism
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4
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Entry for the Table of Contents
An efficient procedure for the synthesis of various tetramic acid fragments related to vancoresmycin, a highly potent polyketide
antibiotic, is reported. Activities against S. aureus and results of the lial-lux bioreporter assay suggest the tetramic acid moiety to be
part of the pharmacophore of this complex polyketide.
5
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