Cystobactamid 507: Concise Synthesis, Mode of Action, and Optimization toward More Potent Antibiotics

Article abstract of DOI:10.1002/chem.202000117

Lack of new antibiotics and increasing antimicrobial resistance are among the main concerns of healthcare communities nowadays, and these concerns necessitate the search for novel antibacterial agents. Recently, we discovered the cystobactamids—a novel natural class of antibiotics with broad-spectrum antibacterial activity. In this work, we describe 1) a concise total synthesis of cystobactamid 507, 2) the identification of the bioactive conformation using noncovalently bonded rigid analogues, and 3) the first structure–activity relationship (SAR) study for cystobactamid 507 leading to new analogues with high metabolic stability, superior topoisomerase IIA inhibition, antibacterial activity and, importantly, stability toward the resistant factor AlbD. Deeper insight into the mode of action revealed that the cystobactamids employ DNA minor-groove binding as part of the drug–target interaction without showing significant intercalation. By designing a new analogue of cystobactamid 919-2, we finally demonstrated that these findings could be further exploited to obtain more potent hexapeptides against Gram-negative bacteria.

Full text of DOI:10.1002/chem.202000117

A Journal of
Accepted Article
Title: Cystobactamid 507: Concise Synthesis, Mode of Action and
Optimization toward More Potent Antibiotics
Authors: Walid A. M. Elgaher, Mostafa M. Hamed, Sascha Baumann,
Jennifer Herrmann, Lorenz Siebenbürger, Jana Krull, Katarina
Cirnski, Andreas Kirschning, Mark Brönstrup, Rolf Müller, and
Rolf W. Hartmann
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using the Digital Object Identifier (DOI) given below. The VoR will be
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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: Chem. Eur. J. 10.1002/chem.202000117
Link to VoR: http://dx.doi.org/10.1002/chem.202000117
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10.1002/chem.202000117
Chemistry - A European Journal
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Cystobactamid 507: Concise Synthesis, Mode of Action and
Optimization toward More Potent Antibiotics
Walid A. M. Elgaher,+ Mostafa M. Hamed,+ Sascha Baumann, Jennifer Herrmann, Lorenz Siebenbürger,
Jana Krull, Katarina Cirnski, Andreas Kirschning, Mark Brönstrup, Rolf Müller, and Rolf W. Hartmann*
Abstract: Lack of new antibiotics and increasing antimicrobial
resistance are the main concerns of healthcare community nowadays,
which necessitate the search for novel antibacterial agents. Recently,
we discovered the cystobactamids - a novel natural class of antibiotics
with broad-spectrum antibacterial activity. In this work, we describe a
concise total synthesis of cystobactamid 507, the identification of the
bioactive conformation using non-covalently bonded rigid analogs, the
first structure–activity relationship (SAR) study for cystobactamid 507
leading to new analogs with high metabolic stability, superior
topoisomerase IIA inhibition, antibacterial activity and, importantly,
stability toward the resistant factor AlbD. Deeper insight into the mode
of action revealed that the cystobactamids employ DNA minor groove
binding as part of the drug–target interaction without showing
significant intercalation. By designing a new analog of cystobactamid
919-2 we finally demonstrated that these findings could be further
exploited to obtain more potent hexapeptides against Gram-negative
bacteria.
507 (2), is a tripeptide representing the eastern part of most of the
hexapeptides (1a–1f) (Figure 1). The cystobactamids display
broad-spectrum antibacterial activity through inhibition of
topoisomerases type IIA, namely DNA gyrase and topoisomerase
IV.[5] Here we report studies on the optimization of 2 as it is the
only compound with appropriate physicochemical properties for
oral absorption according to Lipiniski.[7]
Introduction
The ongoing prevalence of antibiotic-resistant bacteria poses
an imminent threat to humanity.[1,2] Therefore, the need to
discover novel antibiotics with new chemical scaffolds has been
established. Nature represents a rich repository of antibiotics,
however the major part of these natural products (NPs) have to
be modified in order to optimize pharmacokinetic and
pharmacodynamic properties.[3,4]
Recently, we reported the discovery of the cystobactamids –
a new family of antibiotics – isolated from Cystobacter sp. (Figure
1).[5,6] Cystobactamids (1a–1f) have hexapeptidic structures
comprised of three p-aminobenzoic acid motifs (eastern part) and
two p-nitro/aminobenzoic acids (western part) connected through
different linkers. The structurally simplest natural cystobactamid
Figure 1. Examples of the natural cystobactamids (1a–1f, 2).[5,6]
Results and Discussion
We used gyrase inhibition for establishing the SAR, as it is the
primary target of cystobactamids in Escherichia coli.[5] The assay
evaluates the effect on the DNA supercoiling activity of gyrase
using a relaxed circular plasmid as a substrate. Cystobactamids
have been shown to inhibit the gyrase similarly to quinolones
through stabilization of the covalent enzyme–DNA complex with
double-stranded DNA breaks resulting in an accumulation of
linear DNA.[5] Previously, we reported that the methyl homolog 3
(Figure 2 and Table 1) showed only a slight decrease in activity
suggesting that the natural antibiotic 2 could be amenable to
structural modification.[8]
[*]
Dr. W. A. M. Elgaher,[+] Dr. M. M. Hamed,[+] Prof. Dr. R. W. Hartmann
Department of Drug Design and Optimization,
Helmholtz Institute for Pharmaceutical Research Saarland,
Saarland University, Campus E8.1, 66123 Saarbrꢀcken
E-mail: rolf.hartmann@helmholtz-hzi.de
Dr. S. Baumann, Dr. J. Herrmann, K. Cirnski, Prof. Dr. R. Müller
Department of Microbial Natural Products,
Helmholtz Institute for Pharmaceutical Research Saarland,
Saarland University, Campus E8.1, 66123 Saarbrꢀcken
L. Siebenbürger
PharmBioTec GmbH, 66123 Saarbrꢀcken
J. Krull, Prof. Dr. M. Bronstrüp
Department of Chemical Biology, Helmholtz Centre for Infection
Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany
Prof. Dr. A. Kirschning
Figure 2. General structure of cystobactamid 507 analogs.
Institute of Organic Chemistry, Leibniz University of Hannover,
Schneiderberg 1B, 30167 Hannover, Germany
These authors contributed equally to this work
[+]
Supporting information for this article is given via a link at the end of
the document.
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Table 1. In vitro inhibitory activities of compounds 2–16 in the gyrase
A
B
supercoiling assay and topoisomerase IV relaxation assay.
Compd.
R1
R2
R3
R4
X
IC50 (µM)
gyrase[a]
IC50 (µM)
topo. IV[b]
C4
C4
C3
C2
C3
C1
C1
C3
C3
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
CP[d]
iPrO
MeO
iPrO
iPrO
iPrO
iPrO
-
OH
OH
OH
OH
MeO
iPrO
iPrO
H
OH
H
H
OH
H
OH
-
iPrO
MeO
MeO
Cl
iPrO
iPrO
H
iPrO
H
iPrO
iPrO
H
H
H
H
H
H
H
-
-
H
H
H
H
H
H
-
355 ± 25
463 ± 28
360 ± 26
> 1000
115 ± 18
60 ± 10
195 ± 20
50 ± 10
> 1000
165 ± 18
85 ± 12
101 ± 15
110 ± 20
106 ± 18
3.6
> 500
n.d.[c]
n.d.
n.d.
n.d.
175 ± 10
n.d.
147 ± 10
n.d.
n.d.
255 ± 14
n.d.
C2
C4
C4
Figure 3. Conformational analysis of cystobactamid 507 (2): A) anti-form
(lowest energy conformation); B) syn-form (ΔE 0.4 kcalmol-1).
-
-
iPrO
iPrO
iPrO
iPrO
iPrO
iPrO
-
H
H
H
H
H
H
O
O
-
OH
iPrO
OH
iPrO
-
iPrO
H
-
n.d.
n.d.
n.d.
n.d.
-
-
-
-
-
0.4 ± 0.05
[a] E. coli gyrase (1 unit, 20.5 nM). [b] E. coli topoisomerase IV (1 unit, 20.5
nM). [c] Not determined. [d] Ciprofloxacin.
We therefore investigated the role of the substituents and the
conformation of 2 for activity. Replacement of the isopropoxy side
chain in the middle ring of 2 by a methoxy group (4) did not impact
activity, while substitution with chlorine (5) resulted in a significant
loss of gyrase inhibition (Table 1). This result highlights the
importance of the alkoxy side chains for ligand–target interaction.
On the other hand, the isopropoxy of the C-terminal ring showed
stronger inhibition than the methoxy in this position (4 vs 3)
probably due to its steric and hydrophobic properties.
Consequently, we kept it in the optimization process.
As a next step, we performed in silico conformational analyses
of compounds 2–4 using molecular dynamics (MD) to identify the
potential conformations of the molecules and their relative
energies. Generally, the compounds adopt linear conformations
with a backbone curvature[9] of about 158° (Figure S31 and S32).
They can adopt two constrained conformations (anti and syn) with
respect to the alkoxy side chains that are controlled by the
hydroxy group at the middle ring. The lowest energy conformation
is the anti-form (Figure 3A), which is stabilized by three
intramolecular hydrogen bonds (IMHBs). An IMHB between C4-
NH and C3-alkoxy group restricts rotation of the C-terminal ring
around the Ar–NH axis. Another IMBH between C1ʹ-CO and C2ʹ-
OH restricts rotation of the middle ring around the Ar-CO axis. The
third IMHB between C4ʹ–NH and C3ʹ–alkoxy group restricts
rotation of the middle ring around Ar–NH axis. The syn conformer
is also stabilized by three IMHBs similar to the anti-form except
that C2ʹ–OH switches from HB donor to HB acceptor and forms a
six-membered ring with C4–NH (restricting rotation of the middle
ring around the Ar–CO axis) (Figure 3B). The energy difference
(ΔE) between the anti and syn forms is 0.4–0.7 kcalmol-1. Such a
minor energy difference could allow the interconversion between
both conformations at ambient temperature.
Figure 4. 2D-NOESY spectrum of compound 26 in a cryoprotective mixture
(20% H2O/DMSO-d6) adopting anti-conformation.
We then investigated the preferred conformation for 2–4, their
esters and nitro precursors experimentally in solution by a
NOESY study in a biomimetic solvent (20% H2O/DMSO-d6).[10] In
agreement with MD calculations, all compounds show a strong
cross peak between C4–NH and C6ʹ–H, and a weak or no cross
peak with C2ʹ–OH, indicating that these compounds
predominantly exist in the anti-conformation (Figure 4 and S8).
Using standard NMR solvents, the same results were obtained
(Figure S1–S10).
Subsequently, we verified whether the syn-conformer could
exist under physiological conditions by a 1H NMR experiment for
the cystobactamid 507 isopropyl ester (26) at 20 and 37 °C. We
found that the chemical shift of the C2ʹ–OH proton moved upfield
from 12.40 ppm at 20 °C (mainly HB donor form, anti) to 12.35
ppm at 37 °C (pushing the conformational equilibrium to HB
acceptor form, syn) (Figure S29). In addition, we observed the
syn-conformation in the solid state by determining the crystal
structure for the dipeptide precursor of 3 (Figure S37E), whereas
in solution there was a prevalence of the anti-conformation
(Figure S11). These results demonstrate that these compounds
can easily interconvert from anti to syn at physiological
temperature.
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Since the anti-form was the predominant conformation in
solution for 2 and all derivatives so far, we designed compounds
that preferably adopt the syn-form via blocking the ambiguous
hydroxy motif by methylation, thus converting it into a HB acceptor
group only (compound 6). MD calculations indicated that 6 adopts
typically an IMHB-stabilized syn-conformation with a large ΔE
compared to the anti-form (3.8 kcalmol-1). NOESY studies
showed a cross peak between C4–NH and C2ʹ–OMe, and no
cross peak with C6ʹ–H indicating that the syn-conformation is
predominant (Figure S15). Compound 6 showed a 3-fold higher
activity than 2 (Table 1), indicating that the hydroxy group is not
essential for activity.
Another important achievement could be that hopping of the
cystobactamid 507 scaffold to the novel pyridine-based
chemotype (compound 9) might circumvent the cystobactamids’
cleavage through AlbD, a known resistance protein inactivating
the structurally related antibiotics albicidins.[13] The AlbD
endopeptidase hydrolyses the amide bond between the middle
and the N-terminal ring of the eastern part of
albicidins/cystobactamids and the tripeptidic derivatives.[13] By
incubation of compound
9 with AlbD under the reported
conditions,[13] our hypothesis turned out to be correct as no
cleavage of 9 in the presence of AlbD was observed (Figure 6).
To clarify whether this enhancement was due to the induction
of the syn-conformation or due to an additional hydrophobic
interaction, we first designed compound 7 bearing an isopropoxy
group in place of the methoxy (6). This modification resulted in a
6-fold improvement in activity compared to 2 indicating that
besides restricting the conformation to the syn-form, alkoxy
groups at position 2 of the middle ring also contribute to target
interactions.
For investigating the syn-conformation as a decisive factor,
we then designed two rigid cystobactamid 507 analogs, 8 and 9
with a pyridine scaffold adopting only one conformation, anti or
syn, respectively (Figure 2). Rigidity was achieved via a bifurcated
IMHB between C4–NH and oxygen atom of C3–isopropoxy as
well as the nitrogen atom of the pyridine ring. We confirmed
stability of 8 and 9 by MD calculations showing only the desired
conformation in an energy window ΔE of 7.0 kcalmol-1. In NOESY
experiments, no cross peak was observed between C4–NH and
pyridine C3–H at 27 °C and at higher temperatures up to 67 °C
(Figure S21–S27). Moreover, X-ray crystal structures of the
compounds were as expected (Figure 5, S37C and S37D).
Results revealed that 9 is 4-fold and 7-fold more potent than 8 and
2, respectively (Table 1), indicating that the syn-form is indeed the
more active conformation of cystobactamid 507. Noteworthy, the
slightly better activity of 8 than 2 suggests that the introduced
pyridine ring might contribute to the interaction with gyrase and
the inhibitory effect.
Figure 6. Stability of 9 against AlbD: HPLC-MS analysis of 9 (12 µM) in
phosphate buffer as control (A) and incubated with AlbD (24 µM) for 3 h at 28 °C
showing only traces of the cleavage product (B).
For further SAR exploration, compound 2 was simplified by
omitting either the isopropoxy or the hydroxy group from the
central ring in 10 and 11, respectively (Table 1). Removal of the
former resulted in a loss of activity emphasizing the importance of
alkoxy substituents for activity. Interestingly, removing the
hydroxy group increased the activity 2-fold compared to 2. This
confirms that the hydroxy group is not essential for activity. Its
removal permits free rotation of the middle ring as evidenced in
the NOESY spectrum of 11 showing two cross peaks of equal
intensities between C4–NH and C2ʹ–H (syn) and C6ʹ–H (anti)
(Figure S28).
Introduction of an isopropoxy or hydroxy at the N-terminal ring
of 10 and 11, to maintain the beneficial free rotation, resulted in
restoration of activity with a 4-fold enhancement compared to 2
(13 and 12, respectively) (Table 1). These results demonstrate
the usefulness of the alkoxy motifs (2 and 13 vs. 10), and indicate
that varying the distance between them can be tolerated owing to
the flexibility of the new analogs.
A
B
Esterification of 4 and the most potent compounds 6, 7, 9 and
11–13 decreased activity strongly revealing that the terminal
carboxyl group is important for activity (Table S2). Replacing the
amino moiety of 12 and 13 by nitro groups, as in the hexapeptidic
cystobactamids, resulted in similar inhibitory activities (14 and 15,
respectively) (Table 1). This indicates that substitution in 4″
position of cystobactamid 507 analogs provides only little
contribution to activity.
Figure 5. X-ray crystal structures of the nitro ester precursors of 8 (82, A) and
9 (83, B) adopting anti and syn conformation, respectively via IMHB.
The newly designed compound 9 is clearly advantageous
compared to the natural compound 2. Introduction of a polar
pyridine ring in lieu of the benzene enhances water solubility and
ligand-lipophilicity efficiency[11] (SolpH 7.4: 59.4 µmolmL-1, LLE: 3.54
for 9 vs. 9.6 µmolmL-1, 1.75 for 2, respectively). In addition,
removal of the hydroxy group, while keeping the right
conformation, increases ligand efficiency[11] (LE: 0.17 for 9 vs.
0.13 for 2). Moreover, the enhancement of the binding affinity of
the rigid ligand 9 is at least in part due to the reduction of the
unfavourable entropic contribution to the Gibbs free energy of
binding.[12]
For the synthesis of 2 and its analogs, retrosynthetic analysis
revealed three units of either p-aminobenzoic acid or 5-
aminopicolinic acid derivatives linked by amide bonds.
Accordingly, the individual middle, C- and N-terminal rings were
prepared followed by the coupling of the constituents. We
established brief and efficient synthetic pathways for novel as well
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as reported amino acids, which are also precursors of other
NPs[14–17] (Scheme S1). Comparing to previous methods,[6,8,18] the
middle ring of 2 (20) was prepared in only four steps using
catechol as a starting material. Nitration of the catechol to the 3-
nitro derivative 17 followed by regioselective isopropylation at the
site of the cystobactamids is most probably located at the gyrase–
DNA interface overlapping that of the quinolone antibiotics.[5] To
gain a deeper insight into the mode of action, we investigated
whether and eventually how cystobactamids and their analogs
bind to the DNA part of the target complex. Mainly, there are two
binding modes of small molecules to DNA, minor groove binding
or intercalation.[20] Intercalation is of particular concern, as
compounds utilizing this binding mode may trigger genotoxic
effects in eukaryotes.[21] We carried out displacement titration
experiments using fluorescent dyes that show increased
fluorescence upon DNA binding: Hoechst 33342 for DNA minor
groove binding and ethidium bromide (EtBr) for intercalation.
Titration of calf thymus DNA bound Hoechst 33342 with
cystobactamids 1a, 1b, and 2–15 induced a concentration-
dependent loss of fluorescence (Figure 7A and S39). No
compound-induced fluorescence quenching was observed in
absence of DNA. In the presence of EtBr, no compound showed
significant reduction in fluorescence (Figure 7B and S39). These
results indicate that the cystobactamids are able to bind to DNA
utilizing the minor groove without significant intercalation.
Moreover, they reveal the cystobactamids/analogs as a new
chemical frame for minor groove recognition besides the known
family of five-membered fused and non-fused heterocyclic
polyamides.[22]
2-hydroxy position using
a stoichiometric amount of 2-
bromopropane provided 18. Ortho formylation of 18 with
paraformaldehyde in MgCl2/TEA/MeCN mixture under strictly dry
conditions produced the p-nitrobenzaldehyde 19. The latter was
finally oxidized using AgNO3 under basic condition to yield the
corresponding acid 20 (Scheme 1), the structure of which was
confirmed by X-ray crystallography (Figure S37A). The C-terminal
ring 22 was prepared by isopropylation of 3-hydroxy-4-
nitrobenzoic acid followed by reduction of the resulting nitro
derivative 21 via heating with iron in ethanol (Scheme 1).
CHO
COOH
OH
OH
OH
OH
OH
OH
O
OH
O
i-PrBr
HNO3
HO(CH2O)n
H
AgNO3
K2CO3/DMF
60%
MgCl2/TEA
40%
NaOH
55%
Et2O, rt
50%
O
NO2
NO2
NO2
NO2
catechol
17
18
19
20
COOH
X2
N
O
O
H
N
X2
N
OH
OH
O
COOH
COOi-Pr
20
i-PrBr
, Cl2PPh3
NO2 , Cl2PPh3
O
O
K2CO3/DMF
95%
CHCl3, 80 °C
87%
CHCl3, 80 °C
62%
HN
HN
OH
O
NO2
NX2
O
COOi-Pr
O
COOR
Fe/NH4Cl,
EtOH, 90 C
90%
Fe/NH4Cl,
Fe/NH4Cl, EtOH,
21: X = O
22: X = H
23: X = O
24
25: X = O; R = i-Pr
26
A
B


EtOH, 90
90%
C
90 C, 85%
: X = H
: X = H; R = i-Pr
1 N NaOH, MeOH/THF,
rt, 96%
2: X = H; R = H
Scheme 1. Synthesis of cystobactamid 507 (2).
For amide coupling three obstacles were encountered: The
free OH as an interfering group, the non-reactive carboxylic acids,
and the weakly reactive aromatic amines.[6,8,16,18] We developed a
straightforward strategy that could overcome these difficulties and
bypass the activation of carboxylic acids and phenolic OH
protection/deprotection.[6,8,16,18] The total synthesis of 2, for
example, was accomplished in overall 11 steps instead of 21 and
13, respectively.[8,18] Coupling of the central ring 20 to the C-
protected C-terminal ring 22 was achieved by heating the mixture
with dichlorotriphenylphosphorane in dry chloroform to afford
dipeptide 23 (Scheme 1). After reduction, the corresponding
amine 24 was coupled with p-nitrobenzoic acid using the same
procedure as described above. Reduction of the nitro substituted
tripeptide 25 followed by C-deprotection via ester saponification
yielded the amino acid 2 (Scheme 1). This synthetic procedure
was broadly applicable and enabled us to prepare a large series
of peptidomimetics in short time with good to excellent yields
(Scheme S2–S4).
Figure 7. DNA interaction of cystobactamid 507 (2): A) Concentration
dependent decrease in fluorescence of Hoechst 33342 bound to calf thymus
DNA (ct-DNA, 15 µM each) upon titration with 2; B) No change in fluorescence
of the ct-DNA bound EtBr upon titration with 2.
Evaluation of the antibacterial activity against a panel of
Gram-positive and Gram-negative bacteria revealed that the new
compounds show up to 8–16-fold enhanced activities against
Gram-positive strains compared to the parent antibiotic 2 (Table
2). A good correlation between the antibacterial effects and
topoisomerases IIA inhibitory activities was observed. In contrast
to 1a and 1b,[5,6] compound 2 and its analogs did not show activity
against E. coli wild-type, but they were active against the efflux-
deficient E. coli tolC3 mutant. This implies that efflux is
responsible for the inactivity toward E. coli wild-type. Some
compounds, like 7 and 12, suffered from penetration issues
through the Gram-negative outer membrane as indicated by the
enhanced MIC values (8-fold) in the presence of a permeability
enhancer.
We evaluated the inhibitory activity of 2 and the most potent
gyrase inhibitors 7, 9, and 12 on the second bacterial target of
cystobactamids (topoisomerase IV) using a relaxation assay,
which assesses the effect on the conversion of a supercoiled
plasmid to a relaxed form.[5] Results indicated that 2 was not
active up to 500 µM, whereas the new analogs displayed
moderate inhibitory activities (Table 1). This demonstrates that
the modifications applied to improve the gyrase inhibitory activity
are also valid for topoisomerase IV, which is not surprising due to
the high homology between both enzymes.[19]
Moreover, the same activity trend of the compounds on both
targets suggests a similar mode of action. The primary binding
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X2
N
Table 2. Antibacterial activities of cystobactamids 1b, 2 and synthetic analogs
COOH
OH
O
COOH
X2
N
HN
O
MIC (µgmL-1)
O
O
O
COOi-Pr
COOR
NO2
O
Compd.
S.
M. luteus E. faecalis E. coli
E. coli
DSM-
E. coli
OH HN
O
OH HN
O
30
, Cl2PPh3
, Cl2PPh3
1 N NaOH
NO2
CHCl3, 80 °C
S. aureus
Newman
pneumoniae
DSM-20566
DSM-
1790
ATCC-
29212
DSM-
1116
DSM-26863
26863[b]
+ PMBN[c]
CHCl3, 80 °C
90%
O
MeOH, rt
93%
O
COOt-Bu
COOt-Bu
80%
NO2
NX2
27
2
4
6
7
9
12
13
16
1b
CP[d]
n.d.[a]
64
32
8
8
32
8
n.d.
n.d.
0.1
64
16–32
16
8–16
> 64
8
128
> 64
64
64–128 > 128 > 128
32–64
> 64
2
4
4
4
4
n.d.
n.d.
n.d.
Boc2O, DCM,
TEA/DMAP, 95%
Fe/NH4Cl, EtOH,
Fe/NH4Cl
EtOH, 90 C
92%
Fe/NH4Cl
EtOH, 90 C
21
31
32: X = H
33
: X = O; R = H
28: X = O; R = t-Bu
: X = O
: X = O
34: X = H
> 64
32
16–32
> 64
64
> 64
> 64
> 64
> 64
> 64
> 64
1
> 64
4
32
16
32
16
0.25
0.5
90%
29
: X = H; R = t-Bu

90 C, 92%
32
O
Ph
Ph
Ph
O
Ph
8–16
> 64
64
n.d.
n.d.
0.8
O
O
N
Ph
Ph
H
N
H
NH2
Ph3COH
PNBC
PNBC
N
COOH
H
H2SO4/(Ac)2
O
H2
NaOH, dioxane
65%
NaOH, dioxane
75%
4-8
32
N
COOH
H2N
COOH
L-Asn
X2
N
77%
n.d.
n.d.
0.8
n.d.
n.d.
0.8
Fe/NH4Cl
35
36: X = O
: X = H
37

C
EtOH, 90
2
85%
R1
0.013 0.003
HN
[a] Not determined. [b] ΔtolC3 genotype. [c] Polymyxin B nonapeptide (3 µgmL-
1). [d] Ciprofloxacin.
O
O
H
N
O
Ph
N
H
Ph
Ph
O
O
N
HN
O
O
H
Additionally, we investigated compounds 2, 7, 9, and 12 for
their phase І and ІІ biotransformation using human liver S9
fraction. Cystobactamid 507 and all synthetic compounds
displayed an extraordinary high metabolic stability (t1/2 > 240 min,
Figure S42). Surprisingly, stability was observed for both amide
groups against peptidases as well as for the hydroxy groups of 2
and 12 toward conjugating enzymes. Moreover, conformational
modification anti to syn, i.e., 2 to 7 and 9 maintained the
outstanding metabolic stability of the natural compound.
HN
O
N
COOH
H
O
O2N
HN
34
, Cl2PPh3
OH HN
O
CHCl3, 80 °C
75%
: R = t-Bu; R1 = Trt
O
COOR
O2
N
38
39
TFA/DCM
97%
1
16
: R = H; R = H
Scheme 2. Synthesis of cystobactamid 919-2 analog 16.
Conclusion
Finally, in order to demonstrate that structure optimization of
2 can be translated into potent hexapeptides, we picked an analog
with a hydroxy group at the middle ring to keep the general
features of isolated cystobactamids.[6] Accordingly, compound 4
was connected to the western part of the natural compounds
In summary we described a brief synthetic route for the natural
antibiotic cystobactamid 507 (2) and the design of new derivatives
with improved topoisomerases IIA inhibition, and antibacterial
activity. SAR study revealed importance of the alkoxy side chains
and the irrelevance of the hydroxy group for gyrase inhibition. The
terminal carboxy and amino/nitro moieties were found to be
necessary for activity. The molecular conformation in solution as
well as in solid state was investigated to deduce the
conformation–activity relationship. We pioneered the design of
non-covalently bonded rigid structures via IMHBs to disclose the
bioactive conformation of 2 as syn-form without using the classical
methods of cyclization or introduction of sterically demanding
moieties.[24] Increase of chemical diversity by modification of
cystobactamid 507 scaffold into a pyridine-based structure
(compound 9) turned out to make the molecule stable toward the
albicidin resistance factor AlbD. It was shown that the
cystobactamids’ mode of action could be at least in part mediated
by DNA minor groove binding and not intercalation. An important
advantage of 2 and the novel analogs is the high metabolic
stability. Ultimately, we demonstrated that optimization of the
tripeptides could be translated into hexapeptidic cystobactamids
with improved gyrase inhibition and antibacterial activity against
Gram-negative strains. It is worth mentioning that compounds
could be designed with better pharmacokinetic properties
compared to the natural compounds. Thus, this work would be
useful for the development of cystobactamids and similar natural
compounds[14–16] toward better antibiotics.
through L-asparagine as
a simplified linker to form the
cystobactamid 919-2 analog 16 (Scheme 2). Compound 16
showed potent gyrase inhibition and antibacterial activity on the
Gram-negative E. coli wild type (Table 1 and 2). This indicates
that modification of the natural cystobactamid 919-2 using the
tripeptidic cystobactamid 507 analogs is an appropriate strategy
to improve activity. Moreover, removal of the methoxy group at
the linker was tolerated offering a new space for further
optimization.
The hexapeptide 16 was prepared via convergent synthesis
including a coupling of two tripeptide fragments, i.e., the eastern
and the western part with a linker.[23] The eastern part was
prepared as mentioned above as C-Boc protected amino acid 34
(Scheme 2). Synthesis of the western fragment started with trityl
protection of L-asparagine at the amide residue 35 (Scheme 2).
Reaction of 35 with p-nitrobenzoylchloride (PNBC) under
Schotten-Baumann conditions afforded the dipeptide 36.
Reduction of the nitro group to the amine 37 followed by
benzoylation yielded the tripeptide 38. Coupling of the latter to the
amine 34 using Cl2PPh3 afforded the hexapeptide 39. General
deprotection of 39 using TFA 15% (v/v) in DCM yielded 16
(Scheme 2).
Experimental Section
Experimental procedures for the synthesis of the final compounds and
intermediates as well as their characterization, NMR spectra,
computational work, biological experiments and X-ray crystallographic
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10.1002/chem.202000117
Chemistry - A European Journal
FULL PAPER
data (Deposition numbers: CCDC 1958329–1958335) are described in
details in the Supporting Information.
[21] L. R. Ferguson, W. A. Denny, Mutat. Res. 2007, 623, 14–
23.
[22] P. B. Dervan, B. S. Edelson, Curr. Opin. Struct. Biol. 2003,
13, 284–299.
Acknowledgements
[23] T. Planke, M. Moreno, S. Hüttel, J. Fohrer, F. Gille, M. D.
Norris, M. Siebke, L. Wang, R. Müller, A. Kirschning, Org.
Lett. 2019, 21, 1359–1363.
[24] D. W. Carney, K. R. Schmitz, J. V. Truong, R. T. Sauer, J.
K. Sello, J. Am. Chem. Soc. 2014, 136, 1922–1929.
We thank Prof. Andreas Speicher for supporting the
conformational study, Dr. Josef Zapp for NMR measurements and
Dr. Volker Huch for X-ray structure determinations. Walid A. M.
Elgaher gratefully acknowledges a scholarship from the German
Academic Exchange Service (DAAD).
Keywords: Bioactive conformation • Cystobactamids • Drug
design • Intramolecular hydrogen bond • Total synthesis
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10.1002/chem.202000117
Chemistry - A European Journal
FULL PAPER
Entry for the Table of Contents
NH2
Translation into
Hexapeptidic
Cystobactamids
O
O
H2
N
H
N
N
O
H
O
O
R4
N
HN
H
N
O
H
O
O
O
X
O
R3
N
O2N
O
R2
H
HO
N
O
H
COOH
O
R1
COOH
Optimized Cystobactamid 507 Analogs
New Cystobactamid 919-2 Analog
Minor groove
DNA
Gyrase
Optimized tripeptides for superior hexapeptidic cystobactamids: MedChem optimization of the antibiotic cystobactamid 507
targeting DNA gyrase resulted in analogs with improved activity. Bioactive conformation was disclosed and the shortest synthetic route
established. Mode of action studies revealed specific binding at the DNA minor groove. Incorporation of a tripeptidic cystobactamid 507
analog into a hexapeptide resulted in superior activity.
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